WO2008076788A2 - Ligands pan-her optimisés - Google Patents

Ligands pan-her optimisés Download PDF

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Publication number
WO2008076788A2
WO2008076788A2 PCT/US2007/087355 US2007087355W WO2008076788A2 WO 2008076788 A2 WO2008076788 A2 WO 2008076788A2 US 2007087355 W US2007087355 W US 2007087355W WO 2008076788 A2 WO2008076788 A2 WO 2008076788A2
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amino acid
lysine
ligand variant
ligand
peg
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PCT/US2007/087355
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WO2008076788A3 (fr
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Philip T. Pienkos
Daniel J. Monticello
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Molecular Logix, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • ACL anti-cancer ligand
  • DNL dominant negative ligand
  • EGF epidermal growth factor
  • EGFR epidermal growth factor receptor
  • GPCR G-protein coupled receptor
  • HER human epidermal receptor
  • HERl human epidermal receptor 1
  • hGH human growth hormone
  • IFN interferon
  • IGF insulin-like growth factor
  • IR insulin receptor
  • mPEG methyl polyethylene glycol
  • NGF nerve growth factor
  • Pan-HER antagonist panoramic human epidermal receptor antagonist
  • PEG polyethylene glycol
  • TNF tumor necrosis factor
  • VEGF vascular endothelial growth factor
  • HER Human Epidermal Receptors
  • EGFR epidermal growth factor receptors
  • HER signaling such as the overexpression of the genes coding for HER family members has been implicated in a number of pathologies, especially cancers of the breast, ovary, head and neck.
  • Epidermal growth factor a cognate HER ligand
  • EGF Epidermal growth factor
  • HER ligand a 53 amino acid cytokine which plays an important role in the growth control of mammalian cells. It is proteolytically cleaved from a large integral membrane protein precursor.
  • the amino acid and nucleotide sequences of human EGF (EGF) are, for example, disclosed In Hollenberg, "Epidermal Growth Factor-Urogastrone, A Polypeptide
  • Pan-HER antagonists are disclosed in copending application Serial Number 11/172,611 filed June 30, 2005 and incorporated herein reference in its entirety.
  • EGFR dominant negative ligands (DNLs) and anti-cancer ligands (ACLs) which may function as pan-HER antagonists, and methods of designing them, are disclosed in copending applications; Serial Numbers 60/818,735 (Attorney docket Number 3530.3004US) and 60/818,736 (Attorney
  • One strategy for optimizing ligands, especially protein based ligands involves modification of the protein with bulking moieties to improve or impart certain therapeutically beneficial properties to the protein.
  • the most common bulking moiety is the polymer or derivative of the polymer, polyethylene glycol
  • PEG Polymers, and particularly polyethylene glycol (PEG), are highly flexible and soluble and have gained widespread scientific and regulatory acceptance as a chemical modification for therapeutic proteins.
  • Polyethylene glycol (PEG) is a hydrophilic, biocompatible and non-toxic water-soluble polymer of general formula H-(OCH 2 CH 2 )J 1 — OH. In typical form n ranges from about 10 to about 2000. Its molecular weight varies from 300 to 40,000 Daltons.
  • PEG is useful in biological applications because it has properties that are highly desirable and is generally approved for biological or biotechnical applications.
  • PEG typically is clear, colorless, odorless, soluble in water, stable to heat, inert to many chemical agents, does not hydro lyze or deteriorate, and is generally nontoxic. It is considered to be biocompatible, which is to say that PEG is capable of coexistence with living tissues or organisms without causing harm.
  • PEG polyethylene glycol
  • PEGylated protein therapeutics have significantly reduced specific activity relative to the unmodified proteins.
  • developers of PEGylated therapeutics are often faced with the difficult challenge of seeking PEG attachment sites that minimally impact the specific activity of the modified protein.
  • Many of the successful derivatization strategies rely on the chemically reactive epsilon amino group of lysine residues as a target for attachment. This is the most common target for both PEGylation and acylation strategies. There are at least three major issues with this target.
  • compositions of the present invention function as Pan-HER antagonists and are capable of being selectively derivatized with bulking moieties, such as PEG, which impart or improve, among other things, the pharmacodynamic and pharmacokinetic properties without loss of activity.
  • the resulting optimized pan-HER antagonists are useful therapeutics for the treatment of diseases, disorders or conditions which implicate multiple human epidermal receptors.
  • a HER ligand variant comprising (a) one or more amino acid substitutions in the B-loop (amino acids 21-30) of the wild- type human epidermal growth factor (EGF), and (b) a polyethylene glycol (PEG) moiety attached to a lysine of the HER ligand variant of (a) wherein the lysine is either lysine 28 (K28) or lysine 48 (K48).
  • the HER ligand variants of the present invention preferably act as Pan-HER antagonists.
  • the HER ligand variants of the present invention comprise one or more amino acid substitutions in the B-loop of wild type EGF.
  • substitutions are in the second half of the B-loop (amino acids 26-30) and in other embodiments the substitutions are in the first half of the B- loop (amino acids 21-25).
  • a PEG moiety is attached to lysine 48 (K48) of the variants having one or more substitutions in the B-loop.
  • substitutions are made in the B-loop and comprise replacing the amino acids of the second half of the B-loop (LDKYA) with amino acids EPQRG or QPQRG.
  • LDKYA second half of the B-loop
  • EPQRG or QPQRG amino acids EPQRG or QPQRG.
  • These variants may also be pegylated on any conjugating amino acid.
  • These variants may be pegylated at particularly lysine 48 (K48).
  • the HER ligand variant is SEQ ID No. 10 or SEQ ID No. 9 or SEQ ID No. 13.
  • the HER ligand variants of the present invention are modified or conjugated with a PEG derivative.
  • the PEG or PEG derivatives of the present invention may be substantially linear and have a molecular weight from about 10,000 to about 40,000 Daltons.
  • a HER ligand variant of human wild-type epidermal growth factor comprising: (a) substitution of arginine (R45) with tyrosine resulting in (R45Y), (b) substitution of lysine (K48) with leucine resulting in (K48L), and (c) a polyelthylene glycol (PEG) moiety attached to a wherein the lysine is either lysine 28 (K28).
  • This variant may further comprise (a) substitution of tyrosine (Y22) with aspartic acid resulting in (Y22D), and (b) substitution of leucine (L26) with glycine resulting in (L26G).
  • the HER ligand variants comprise one or more amino acid substitutions in the B-loop comprise replacing the amino acids of the first half of the B-loop (LDKYA) with amino acids EPQRG, and a PEG moiety attached to lysine 48 (K48).
  • This variant may further comprise: (a) substitution of lysine (K28) with leucine resulting in (K28L), (b) substitution of serine (S2) with tryptophan resulting in (S2W), and (c) substitution of aspartic acid (D3) with valine resulting in (D3V).
  • a HER ligand variant comprises (a) substitution of lysine (K28) and lysine (K48) of the wild-type human epidermal growth factor (EGF), with a non-reactive amino acid, and (b) substitution of one of any of the amino acids in the sequence forming the Domain I binding face, said Domain I binding face consisting of residues 1-5 (NSDSE) and residues 20-33 (MYIEALDKYACNC), with a conjugating amino acid that can be selectively conjugated with a bulking agent.
  • the non-reactive amino acid is leucine.
  • the HER ligand variants described above may further comprise one or more amino acid substitutions in residues 34-48 (VVGYIGERCQYRDLK) of the wild-type human epidermal growth factor (EGF), and optionally, one or more further substitutions in residues 7-19 (PLSHDGYCLHDGV).
  • variants may further comprise attachment of a polyelthylene glycol (PEG) moiety to the conjugating amino acid in the Domain I binding face.
  • PEG polyelthylene glycol
  • the HER ligand variants comprise (a) substitution of lysine (K28) and lysine (K48) of the wild-type human epidermal growth factor (EGF) with a non-reactive amino acid and (b) substitution of one of any of the amino acids in the sequence forming the Domain III binding face, said Domain III binding face consisting of residues 34-48 (VVGYIGERCQYRDLK) and residues 7- 19 (PLSHDGYCLHDGV) of the wild-type human epidermal growth factor (EGF) with a conjugating amino acid.
  • variants may further comprise one or more amino acid substitutions in a first half of the B-loop (amino acids 21-25) of the wild-type human epidermal growth factor (EGF), and optionally, one or more further substitutions in a second half of the B-loop (amino acids 26-30).
  • the HER ligand variants of the present invention comprise a polyelthylene glycol (PEG) moiety attached or conjugated to the conjugating amino acid in the Domain III binding face.
  • PEG polyelthylene glycol
  • the present invention also provides pharmaceutical compositions of HER ligand variants and a pharmaceutically acceptable carrier. Also provide are methods of treating a patient with a disease characterized by overexpression of HER comprising, administering to the patient, a therapeutically effective amount of a pharmaceutical composition of the present invention comprising a HER ligand variant of the present invention. Included in the HER ligand variants of the present invention comprising pharmaceutical compositions are any modifications or conjugations or substitutions which may have been made to the wild type EGF starting molecule.
  • the diseases and compositions of the present invention are useful in methods for treating diseases or disorders.
  • the disease is cancer.
  • the cancer is selected from the group consisting of gliomas, squamous cell carcinomas, breast carcinomas, melanomas, invasive bladder carcinomas, colorectal carcinomas and esophageal cancers.
  • methods of treating a patient with a disease characterized by overexpression of HER or a HER-mediated pathology comprising, administering to the patient, a therapeutically effective amount of a pharmaceutical composition of the present invention.
  • Pan-HER antagonists novel therapeutic HER ligand variants termed Pan-HER antagonists, so named because of their capacity to antagonize signaling mediated by two or more human epidermal receptors (HERs).
  • HERs human epidermal receptors
  • ligands that bind human epidermal receptors can be grouped into three categories: 1) Those that bind to HERl alone (EGF, TGF- a, amphiregulin), 2) those that bind to HER3 and/or HER4 (heregulins and neuregulins) and 3) those that bind to HER-I and HER-4 (betacellulin, heparin- binding EGF, NRG3, epigen, and epiregulin) (Riese and Stern 1998 Bioessays 20:41). Each human epidermal receptor exists as a monomer in the inactive state.
  • Ligand binding promotes either homodimerization or heterodimerization between the bound receptor and other members of the HER family.
  • the various EGF-like growth factors bind with high affinity to ErbB receptors except for HER2, which has no known ligand and has the constitutive ability to form homodimers and heterodimers.
  • HER2 homodimers have been implicated in tumor cell growth, but also are important for cardiac muscle development and repair. (Dougall et al 1994 Oncogene 9:2109, Hynes and Stern 1994, Biochim Biophys Acta 1198:165, Tzahar and Yarden 1998 Biochim Biophys Acta 1377:M25, Negro et al. 2004, Recent Prog Horm Res. 59:1.).
  • HER2 is the preferred heterodimeric partner of the other HER receptors (Tzahar et al. 1996, MoI Cell Biol, 16:5276, Beerli et al. 1995 MoI Cell Biol 15:6496, Karunagaran et al.1996 EMBO J 15:254, Wang et al. 1998, PNAS, 95 :6809).
  • HER3 differs from the other HER family members in that it has a deficient tyrosine kinase domain (Guy et al 1994 PNAS 91 :8132) and must associate with another HER-family receptor to trigger signaling.
  • Examples of HER ligands include mammalian EGF (e.g.
  • HER ligands include transforming growth factor- ⁇ (TGF ⁇ ), betacellulin, heparin-binding EGF-like growth factor (HB-EGF), neuregulins, heregulin (HRG) including HRG ⁇ , HRG ⁇ l, HRG ⁇ 2 and HRG-factor (NDF), amphiregulin (AR), epigen and epiregulin.
  • TGF ⁇ transforming growth factor- ⁇
  • HB-EGF heparin-binding EGF-like growth factor
  • HRG heregulin
  • HRG including HRG ⁇ , HRG ⁇ l, HRG ⁇ 2 and HRG-factor (NDF)
  • AR amphiregulin
  • epigen and epiregulin epigen and epiregulin.
  • the HER ligand variants of the present invention are ligand variants which bind HERs.
  • Preferred HER ligand variants of the invention are based on HER ligands which are capable of selectively inhibiting HER-mediated biological activity.
  • the term "Pan-HER antagonist" encompasses any amino-acid based molecule that inhibits, suppresses or causes the cessation of at least one HER-mediated biological activity by reducing, interfering with, blocking, supplanting or otherwise preventing the interaction or binding of a native or active HER ligand to more than one human epidermal receptor (HER) thereby attenuating or inhibiting signaling via a human epidermal receptor.
  • amino-acid based means that the molecule is predominantly protein in nature. It is understood that amino-acid based molecules may have non-protein moieties attached or linked to them. Specifically excluded from this definition are antibodies to a receptor and noncovalent conjugates of an antibody and an antigen for that antibody.
  • HERs include HERl, HER2, HER3, and/or HER4 and variant forms of these receptors. It is understood that direct interference with HERl, HER3 and HER4 can provide indirect interference with HER2, by blockading the dimerization partners implicated in much of HER2's role in cancer. It is not necessary that the HER ligand variant of the invention target HER2, thereby suppressing HER2 biological activity that may be undesirable. It is advantageous to avoid suppression of HER2 homodimerization because such activity has been shown to cause cardiomyopathy, a life threatening side effect.
  • the term "antagonist” means any molecule that blocks the ability of a given chemical to bind to its receptor, thereby preventing a biological response.
  • antagonist can be used in a functional sense and is not intended to limit the invention to compounds having a particular mechanism of action.
  • antagonist includes, but is not limited to, molecules that function as competitive antagonists.
  • competitive antagonist is one which binds the receptor but does not trigger the biological activity of the receptor.
  • HER-mediated biological activity means the intrinsic protein-tyrosine kinase activity of the HER and/or its downstream signal transduction cascade.
  • HER-mediated biological activities include reducing or inhibiting HER kinase activation, signaling, regulation, dimerization, HER-regulated cell proliferation or phosphorylation as well as any HER-mediated pathology or phenotypic manifestation evidenced as HER-mediated.
  • the HER ligand variants of the invention are designed to act as Pan-HER antagonists. Such HER ligand variants, and nucleic acids encoding these variants, can be used therapeutically in situations in which inhibition of HER biological activity is indicated, e.g. cancer, inflammation and the like. As such the present invention encompasses therapeutic Pan-HER antagonists and variants thereof and methods for their design and use in medicine, diagnostics and drug discovery.
  • Pan-HER Dominant Negative Ligands with only a single lysine, and then to produce, purify and PEGylate these variants Binding properties and antagonist properties (in vitro) and their serum half-lives in mice are then investigated.
  • the present invention is directed to at least two classes of Pan-HER DNLs: "EGFDI” variants that bind only to Domain I of HER receptors and "EGFDIII” variants that bind only to Domain III.
  • EGFDI variants comprise, in addition to the other constructs disclosed herein, variants in which both lysines at position 28 and 48 of the native EGF are eliminated and substitutions of any one of the residues in the sequence at the Domain III binding face are made to allow modification with a bulking group (e.g., pegylation).
  • a bulking group e.g., pegylation
  • HER ligand variants in which both native lysines have been substituted and in which further substitutions have been performed to introduce a lysine (or another residue that can be conjugated to a bulking agent) at any amino acid residue in the sequence on the Domain III binding face. This would be evident as it is shown herein that residues on the Domain III binding face may be changed without a loss of antagonist activity.
  • EGFDIII variants are designed having both native lysines substituted and with substitutions of one of any of the amino acids in the sequence at the Domain I binding face of the molecule which will allow modification with a bulking group (e.g. pegylation).
  • Non-reactive residue or non-reactive amino acid is any amino acid which is not readily derivatized with a bulking agent.
  • conjugating residue or conjugating amino acid is one which can be conjugated with or to a bulking group.
  • Both of these variants currently posses lysines at positions 28 (in the Domain I binding region) and 48 (in Domain III binding region). Both of these lysines have been substituted with leucines in EGF and a Pan-HER EGF variant with little or no loss of activity (Groenen, L et al. 1994, Growth Factors 11:253-257). This will allow monoPEGylation at the remaining lysine, further disrupting binding to the desired region of the receptor.
  • Other HER ligands TGF- ⁇ , heregulin, etc.
  • TGF- ⁇ heregulin, etc.
  • the variants, K28L in the EGFDI class of antagonist and the K48L substitution in the EGFDIII class are studied.
  • the starting ligand variant is the R45Y mutation of panerbin disclosed by Van der Woning, S. P. et al. (2006. Negative constraints underlie the ErbB specificity of EGF-like growth factors. J. Biological Science. JBC Papers in Press: Published on line October 10, 2006 as manuscript M6031682000).
  • Residues S and D at positions 2 and 3 are retained because they prevent binding of EGF to HER3 and HER4 and Y22D and L26G substitutions are made to further ablate binding to Domain I of EGFR.
  • the K28L mutation is created in the EGFDI variant so that PEGylation will further disrupt binding to Domain III and the K48L mutation in the EGFDIII variant so that PEGylation will further disrupt binding to Domain I.
  • the present invention contemplates the use and investigation of EGF homologs, analogs and fragments of the EGF in the creation of HER ligand variants or Pan-HER antagonists.
  • the term "homolog” refers to the corresponding polypeptides of HER ligands from other species having substantial identity to human wild-type HER ligands. These homologs may be modified and optimized according to the present invention to produce Pan-HER antagonists.
  • homologs of EGF polypeptide sequences from various mammalian species are disclosed in Table 1. Table 1
  • analog refers to compounds whose structure is related to that of another compound but whose chemical and biological properties may be quite different.
  • ligand is used to designate an amino acid-based molecule capable of specific binding to a receptor as herein defined.
  • the definition includes any native (cognate) ligand for a receptor or any region or derivative thereof retaining at least a qualitative receptor binding ability.
  • antibodies to a receptor and noncovalent conjugates of an antibody and an antigen for that antibody are included in this definition.
  • mutant ligand and wild-type ligand are used interchangeably and refer to the amino acid sequence of a ligand occurring in nature ("native sequence ligand"), including mature, pre-pro and pro forms of such ligands, purified from natural source, chemically synthesized or recombinantly produced. Native ligands that can activate receptors are well known in the art or can be prepared by art known methods.
  • the mature wild-type human EGF protein sequence is represented by SEQ ID. NO. 1.
  • the HER ligand variants or Pan-HER antagonists act as dominant negative ligands (DNLs).
  • DNLs dominant negative ligands
  • the term "dominant negative” is used to describe that type of ligand, when altered or modif ⁇ ed to differ from the native or wild-type ligand in any respect, results in a ligand that retains binding affinity for a wild-type binding partner (e.g., a receptor) but inhibits the function or signaling of the wild-type binding partner.
  • a wild-type binding partner e.g., a receptor
  • dominant negative ligand activity refers to the functions associated with dominant negative ligands (e.g., binding a receptor but inhibiting a function of the receptor).
  • Amino acid sequences of the HER ligand variants and Pan-HER antagonists of the invention may be obtained through various means such as chemical synthesis, phage display, cleavage of proteins or polypeptides into fragments, or by any means in which amino acid sequences of sufficient length to possess selected properties may be made or obtained.
  • the HER ligand variants and Pan-HER antagonists of the invention are produced by expression in a suitable host of a gene coding for the relevant HER ligand variant or Pan-HER antagonist.
  • a gene is most readily prepared by site-directed mutagenesis of the wild-type gene, a technique well known in the art.
  • the present invention also provides nucleic acid molecules encoding a HER ligand variant or Pan-HER antagonist of the invention.
  • the nucleic acid molecules of the present invention can be RNA, for example, mRNA, or DNA.
  • DNA molecules can be double-stranded or single-stranded.
  • the nucleic acid molecule can also be fused to a marker sequence, for example, a sequence that encodes a polypeptide to assist in isolation or purification of the polypeptide.
  • a marker sequence for example, a sequence that encodes a polypeptide to assist in isolation or purification of the polypeptide.
  • sequences include, but are not limited to, those that encode a glutathione- S- transferase (GST) fusion protein, those that encode a hemagglutinin A (HA) polypeptide marker from influenza, and sequences encoding a His tag.
  • GST glutathione- S- transferase
  • HA hemagglutinin A
  • the design of the expression vector can depend on such factors as the choice of the host cell to be transformed and the level of expression of HER ligand variant or Pan-HER antagonist desired.
  • the expression vectors of the invention can be introduced into host cells to thereby produce the modified polypeptides of the invention, including fusion polypeptides, encoded by nucleic acid molecules as described herein.
  • Molecular biology techniques for carrying out recombinant production of the modif ⁇ ed polypeptides of the invention are well known in the art and are described for example, in, Sambrook, et al, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab Press; 3 rd ed., 2000).
  • the HER ligand variant or Pan-HER antagonist of the invention may be produced in whole or in part by chemical synthetic techniques such as by a Merrifield-type synthesis (J. Am. Chem. Soc. 85:2149 (1963), although other equivalent chemical syntheses known in the art may be used. Solid-phase synthesis is initiated from the C-terminus of the peptide by coupling a protected alpha-amino acid to a suitable resin. The amino acids are coupled the peptide chain using techniques well known in the art for the formation of peptide bonds.
  • Chemical synthesis of all or a portion of a HER ligand variant or Pan-HER antagonist of the invention may be particularly desirable in the case of the use of a non-naturally occurring amino acid substituent in the HER ligand variant or Pan-HER antagonist.
  • the present invention contemplates the design of HER ligand variants, Pan- HER antagonists and dominant negative ligands, which have as their design reference point, other HER ligand variants, Pan-HER antagonists and dominant negative ligands. These further designed HER ligand variants, Pan-HER antagonists and dominant negative ligands may be the result of further optimization of properties in addition to or beyond binding and signal inhibition.
  • a HER ligand variant, Pan-HER antagonist or dominant negative ligand may then be the starting point for further optimization meaning that, in the design scheme, the resultant compound would then become the starting compound. Therefore, a "HER ligand” or "Pan-HER antagonist” can, in certain contexts, be construed as a "HER ligand variant” or "Pan-HER antagonist variant", respectively, and vice versa.
  • a HER ligand variant, Pan-HER antagonist or dominant negative ligand may also be referred to or considered a druggable ligand.
  • the HER ligand variants and Pan-HER antagonists of the present invention are amino acid-based molecules. These molecules may be "peptides,” “polypeptides,” or “proteins.”
  • amino acid and “amino acids” refer to all naturally occurring L- alpha-amino acids.
  • amino acids are identified by either the one-letter or three- letter designations as listed in Table 2.
  • amino acid sequences of the HER ligand variants and Pan-HER antagonists of the invention may comprise naturally occurring amino acids and as such may be considered to be proteins, peptides, polypeptides, or fragments thereof.
  • the HER ligand variants and Pan-HER antagonists may comprise non-naturally occurring amino acids or both naturally and non-naturally occurring amino acids.
  • amino acid sequence variant refers to molecules with some differences in their amino acid sequences as compared to a native sequence.
  • the amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence of a native ligand.
  • variants will possess at least about 70% homology to a native ligand, and preferably, they will be at least about 80%, more preferably at least about 90% homologous to a native ligand.
  • “Homology” as it applies to amino acid sequences is defined as the percentage of residues in the candidate amino acid sequence that are identical with the residues in the amino acid sequence of a native ligand after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for the alignment are well known in the art. It is understood that homology depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation.
  • the HER ligand variants and Pan-HER antagonists produced by the methods of the present invention may have substantial sequence identity to wild-type ligands.
  • substantial sequence identity is not a single defining feature of the compounds of the present invention.
  • Structural components are also factors when considering identity of a variant to the parent molecule.
  • substantially sequence identity means at least 60% sequence identity, preferably at least 70% identity, preferably at least 80% and more preferably at least 90% sequence identity to the amino acid sequence of starting ligand (or domains thereof in the instance where the variant is a chimera produced by swapping domains), while maintaining HER-mediated biological activity.
  • the HER ligand variants and Pan-HER antagonists of the present invention have at least 91%, at least 92%, at least 93%, at least 94%, at least 95% at least 96%, at least 97%, or at least 98% sequence identity to the amino acid sequence of wild-type human ligand, while maintaining HER-mediated biological activity.
  • the actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm.
  • a preferred, non- limiting example of such a mathematical algorithm is described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993). Such an algorithm is incorporated into the BLASTN and BLASTX programs (version 2.2) as described in Schaffer et al. , Nucleic Acids Res., 29:2994-3005 (2001).
  • derivative is used synonymously with the term “variant” and refers to a molecule that has been modified or changed in any way relative to a reference molecule or starting molecule.
  • derivative and variant HER ligands or Pan-HER antagonists are amino acid-based molecules which are modified, altered, improved or optimized relative to a starting parent molecule.
  • the present invention contemplates several types of HER ligand and Pan- HER antagonist variants and derivatives. These modifications can be useful to alter receptor specificity (either broaden specificity such that the variant binds to additional receptors or target specificity towards a specific receptor or narrow specificity to less than all of the family of receptors but not less than two) or to alter phenotypic outcomes. Also included in the modifications of the compounds of the invention are domain swapping and/or domain modifications.
  • amino acid-based molecules containing substitutions, insertions and/or additions, deletions or covalently modifications.
  • sequence tags or amino acids such as one or more lysines
  • Sequence tags can be used for peptide purification or localization.
  • Lysines can be used to increase peptide solubility or to provide sites for biotinylation or pegylation.
  • amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences.
  • Certain amino acids e.g., C-terminal or N-terminal residues
  • substitutional variants are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position.
  • the substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule. Substitutions may be, therefore, one or more.
  • conservative amino acid substitution refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue.
  • conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions.
  • non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.
  • a non-polar amino acid residue such as isoleucine, valine, leucine, alanine, methionine
  • a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.
  • the present invention features a HER ligand variant or Pan-
  • HER antagonist designed from a naturally occurring HER ligand that has at least one amino acid substitution at amino acid position that corresponds to any one or more amino acids selected from the B-loop (amino acids 21-30) of wild type EGF.
  • amino acid substitutions may occur in only a portion of the B-loop such as in the first half (amino acids 21-25) or in the second half (amino acids 26-30).
  • the entire B-loop, or either half may be replaced.
  • the replacement of half of the B-loop would represent five amino acid substitutions.
  • amino acid position that corresponds to means that when the starting HER ligand is aligned with the variant for optimal comparison, the amino acids that appear at or near the positions identified may be substituted with another amino acid. See U.S. Utility Application bearing Attorney Docket Number 3530.1002 US2 entitled “Epidermal Growth Factor Receptor Antagonists and Methods of Use,” filed on June 30, 2005, incorporated herein by reference.
  • “Insertional variants” are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native or starting sequence. "Immediately adjacent" to an amino acid means connected to either the alpha-carboxy or alpha-amino functional group of the amino acid at the particular position.
  • deletional variants are those with one or more amino acids in the native or starting amino acid sequence removed. Ordinarily, deletional variants will have one or more amino acids deleted in a particular region of the molecule.
  • Covalent derivatives include modifications of a native or starting ligand (which may be a molecule already validated as a HER ligand variant or Pan-HER antagonist) with an organic proteinaceous or non-proteinaceous derivatizing agent, or post-translational modifications. Covalent modifications are traditionally introduced by reacting targeted amino acid residues of the ligand with an organic derivatizing agent that is capable of reacting with selected side-chains or terminal residues, or by harnessing mechanisms of post-translational modifications that function in selected recombinant host cells. Covalent derivatives specifically include fusion molecules in which ligands of the invention are covalently bonded to a nonproteinaceous polymer.
  • the nonproteinaceous polymer ordinarily is a hydrophilic synthetic polymer, i.e. a polymer not otherwise found in nature.
  • polymers which exist in nature and are produced by recombinant or in vitro methods are useful, as are polymers which are isolated from nature.
  • Hydrophilic polyvinyl polymers fall within the scope of this invention, e.g. polyvinylalcohol and polyvinylpyrrolidone. Particularly useful are polyvinylalkylene ethers such a polyethylene glycol (PEG), polypropylene glycol.
  • the ligands may be linked to various nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
  • non-covalent derivatives may be attached to the HER ligand variants or Pan-HER antagonists and these can be labile (both internally labile or labily attached).
  • the serum half- life of the HER ligand variants and Pan-HER antagonists of the present invention is expected to be about the same as unmodified EGF (about 10 minutes). This may be acceptable or even desirable for some applications, where rapid clearing of the drug is beneficial. In order to be considered for significant oncology applications, a half-life of 5-7 days is desirable.
  • acyl (fatty acid) side chains or albumin binding domains can enhance the binding of the drug to other circulating proteins (primarily albumin), allowing for slow release back into the serum (Home, P. and P. Kurtzhals, 2006,
  • reversible (labile) PEG attachment it is desirable to use reversible (labile) PEG attachment to temporarily inactivate a protein while improving its half-life in vivo.
  • This concept can be used, for example, to generate a slow-release version of a therapeutic protein or a protein that bypasses some biological systems at early time points after administration.
  • Labile attachments will also be desirable such as where it is impossible to add a bulking agent big enough to the excluded from the kidney filtering system without also blocking important binding sites.
  • the linkage between the PEG moieties described herein and the therapeutic protein are preferably covalent and optionally but preferably reversible. That is, under physiological conditions, the linkage between the PEG and the protein is preferably labile.
  • Using reversible chemistry for PEG attachment allows regeneration of active therapeutic protein over time, preferably following absorption from the site of administration. For such situations, the present invention finds use for the optimization of reversible PEG attachment sites. In some cases it is desirably that biological activity is lost when the PEG is attached; in other cases, the activity is stable or even increased.
  • PEG conjugation means and refers to modifying a protein by covalently attaching polyethylene glycol (PEG) to the protein, with "PEGylated” referring to a protein having a PEG attached.
  • PEG polyethylene glycol
  • a range of PEG, or PEG derivative sizes with optional ranges of from about 10,000 Daltons to about 40,000 Daltons may be attached to proteins using a variety of chemistries.
  • the molecular weight of the poly(ethylene glycol) used in the present invention may be between about 10,000 and about 40,000 Dalton.
  • PEGs with low polydispersity. Normally, a PEG with molecular weight of between about 10,000 and about 40,000 is used.
  • a specific PEG molecular weight range of the present invention is from about 10,000 to about 30,000. In another specific embodiment the PEG molecular weight is greater than about 15,000 to about 40,000. In another specific embodiment the PEG molecular weight is about 20,000 to about 40,000.
  • the polyethylene glycol may have an average molecular weight of about 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000 or 50,000 Dalton.
  • PEG conjugation is an established methodology for peptide and protein modification pioneered by the work of Davis and Abuchowski [Abuchowski, A. et al, J. Biol. Chem., 252, 3571 (1977) and J. Biol. Chem., 252, 3582 (1977)].
  • PEG conjugation to peptides or proteins generally involved the activation of PEG and coupling of the activated PEG-intermediates directly to target proteins/peptides or to a linker, which is subsequently activated and coupled to target proteins/peptides.
  • One of the key issues with the conjugation is the chemistry used to activate PEG and PEG-linker, which in turn will determine the coupling efficiency and specificity of the activated PEG or PEG-linker to its targets.
  • PEG For PEG a variety of means have been used to attach the PEG molecules to the protein. Generally, PEG molecules are connected to the protein via a reactive group found on the protein. Linkages are typically formed between PEG and primary amines (lysine side chains or the protein N-terminus), thiols (cysteine residues), or histidines. Linkages can also be formed between PEG and nonnatural amino acids, especially those involving an acetyl moiety, for instance, p-acetyl-L- phenylalanine.
  • lysine at position 28 is modified to any of the naturally occurring amino acids.
  • this modification comprises K28R or K28L.
  • variants containing a K28 modification may further be derivatized at lysine 48 with a non-amino acid moiety, such as polyethelyene glycol (PEG).
  • PEG polyethelyene glycol
  • cysteines naturally occurring or engineered are commonly targeted for site-specific PEGylation.
  • the present invention finds use for replacing specific lysine or histidine residues with alternative amino acids, such that PEGylation at such residues is no longer possible.
  • HER ligand variants or Pan-HER antagonists are conjugated to PEG moieties or PEG derivatives. Conjugation or derivatization of PEG to the compositions of the present invention may be selective as the compositions have been designed to minimize the number of PEGylation sites while still retaining biologic activity.
  • PEG is commonly used as methoxy PEG-OH, or mPEG in brief, in which one terminus is the relatively inert methoxy group, while the other terminus is a hydroxyl group that is subject to ready chemical modification.
  • the PEG moiety may be a branched PEG having more than one PEG moiety attached thereto (see U.S. Pat. No. 5,932,462; U.S. Pat. No. 5,342,940; U.S. Pat. No. 5,643,575; U.S. Pat. No. 5,919,455; U.S. Pat. No.
  • Branched polymer backbones are generally known in the art.
  • a branched polymer has a central branch core moiety and a plurality of linear polymer chains linked to the central branch core.
  • PEG is commonly used in branched forms that can be prepared by addition of ethylene oxide to various polyols, such as glycerol, pentaerythritol and sorbitol.
  • the central branch moiety can also be derived from several amino acids, e.g., lysine.
  • Suitable branched PEGs can be prepared in accordance with International Publication No. WO 96/21469, entitled Multi-Armed, Monofunctional, and Hydrolytically Stable Derivatives of Poly(Ethylene Glycol) and Related Polymers For Modification of surfaces and Molecules, which was filed Jan. 11, 1996, the contents of which are incorporated herein in their entirety by reference (which corresponds to U.S. Pat. No. 5,932,462, which is also incorporated by reference). These branched PEGs can then be modified in accordance with the teachings herein.
  • the branched PEGs can be represented in general form as R(— PEG— 0H)n in which R represents the central "core" molecule, such as glycerol or pentaerythritol, and n represents the number of arms.
  • Branched PEGs can also be prepared in which two PEG "arms" are attached to a central linking moiety having a single functional group capable of joining to other molecules; e.g., Matsushima et ah, (Chem. Lett., 773, 1980) have coupled two PEGs to a central cyanuric chloride moiety.
  • Matsushima et ah (Chem. Lett., 773, 1980) have coupled two PEGs to a central cyanuric chloride moiety.
  • activated the PEG to prepare a derivative of the PEG having a functional group at the terminus.
  • the functional group can react with certain moieties on the protein such as an amino group, thus forming a PEG-protein conjugate.
  • Many activated derivatives of PEG have been described.
  • An example of such an activated derivative is the succinimidyl succinate "active ester.”
  • the amino acid sequence of the active HER ligand variants can be modified without compromising antagonist properties. This is an important and novel finding that enables the development of several alternative, superior forms of HER ligand variants or Pan-HER antagonists as the active molecules may be further derivatized thereby optimizing other characteristics of the molecule, e.g., pharmacodynamic and pharmacokinetic properties.
  • Pan-HER antagonist for use as an anti-cancer ligand (ACL) that can be derivatized through its lysine residues.
  • EGF has two lysines (abbreviated in sequence nomenclature "K") at positions 28 and 48. It has been shown that PEGylation of either of these lysines significantly increases the serum half- life of the protein (Lee, H. and G. Park, 2002, Pharmaceutical Research, 19(6), 845-851). PEGylation of K28 greatly reduced the biological activity of the protein while PEGylation at K48 appears to totally destroy activity (Lee, H. and G. Park, 2002, Pharmaceutical
  • N-terminal PEGylation was not nearly as detrimental but is a much harder reaction to control (Lee, H., et al., 2003, Pharmaceutical Research, 20(5), 818-825).
  • Another embodiment of the invention relates to methods for the prevention and/or treatment of a disease or disorder in which use of a HER ligand variant or pan-HER antagonist, is beneficial, comprising administering to a patient in need thereof a therapeutically effective amount of a PEG conjugated or modified pan- HER antagonist of the invention or variant thereof, alone or in combination with another therapeutic agent.
  • the invention also relates to the use of a PEG modified pan-HER antagonist of the invention or variant thereof in the manufacture of a medicament for the prevention and/or treatment of a disease or disorder in which use of a pan-HER antagonist is beneficial.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a PEG modified pan-HER antagonist of the invention or variant thereof for the prevention and/or treatment of a disease or disorder in which use of pan-HER antagonist is beneficial.
  • compositions designed by making modifications to one or more features of the HER ligand variants to alter one or more properties, said properties selected from the group consisting of optimal pH or pH-activity, digestibility, antigenicity, half- life, bioavailability, the amphipathic properties, ligand-receptor interactions, thermal or kinetic stability, solubility, folding, posttranslational modification, hydrophobicity, hydrophilicity, and combinations thereof.
  • properties selected from the group consisting of optimal pH or pH-activity, digestibility, antigenicity, half- life, bioavailability, the amphipathic properties, ligand-receptor interactions, thermal or kinetic stability, solubility, folding, posttranslational modification, hydrophobicity, hydrophilicity, and combinations thereof.
  • the term "optimized or optimization” refers to the modification or alteration of a molecule such that one or more characteristics of the molecule are improved for a particular purpose as compared to a starting molecule. "Modification” is the result of modifying wherein the thing being modified is changed in form or character.
  • the molecules of the present invention being optimized via modifications include HER ligand variants and Pan-HER antagonists. For the purposes of the instant invention, these molecules are being optimized for the purpose of creating therapeutic, diagnostic or research reagents.
  • the modifications of the present invention are herein made to one or more features of the druggable ligands, HER ligand variants or Pan-HER antagonists.
  • Features are defined as distinct amino acid sequence-based components of a molecule.
  • Features of the HER ligand variants or Pan-HER antagonists of the present invention include surface manifestations, local conformational shape, folds, loops, half-loops, domains, half-domains, sites, termini or any combination thereof.
  • surface manifestation refers to an amino acid- based component of a HER ligand variant or Pan-HER antagonist appearing on an outermost surface.
  • local conformational shape means an amino acid- based structural manifestation of a HER ligand variant or Pan-HER antagonist which is located within a definable space of the HER ligand variant or Pan-HER antagonist.
  • fold means the resultant conformation of an amino acid sequence upon energy minimization.
  • a fold may occur at the secondary or tertiary level of the folding process.
  • secondary level folds include beta sheets and alpha helices.
  • tertiary folds include domains and regions formed due to aggregation or separation of energetic forces. Regions formed in this way include hydrophobic and hydrophilic pockets, and the like.
  • the term "turn” as it relates to protein conformation means a bend which alters the direction of the backbone of a peptide or polypeptide and may involve one, two, three or more amino acid residues.
  • the term “loop” refers to a structural feature of a peptide or polypeptide which reverses the direction of the backbone of a peptide or polypeptide and comprises four or more amino acid residues. Oliva et al. have identified at least 5 classes of protein loops (JMoI Biol, 266 (4): 814-830; 1997).
  • domain refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions).
  • sub-domains may be identified within domains or half-domains, these subdomains possessing less than all of the structural or functional properties identified in the domains or half domains from which they were derived.
  • amino acids that comprise any of the domain types herein need not be contiguous along the backbone of the polypeptide (i.e., nonadjacent amino acids may fold structurally to produce a domain, half-domain or subdomain).
  • site is used synonymous with "amino acid residue” and "amino acid side chain” and "residue”.
  • a site represents a position within a peptide or polypeptide that may be modified, manipulated, altered, derivatized or varied within the amino acid-based molecules of the present invention.
  • terminal or terminus refers to an extremity of a peptide or polypeptide. Such extremity is not limited only to the first or final site of the peptide or polypeptide but may include additional amino acids in the terminal regions.
  • the polypeptide based molecules of the present invention may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)).
  • Druggable ligands are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces (multimers, oligomers). These sorts of ligands will have multiple N- and C-termini.
  • the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non-polypeptide based moiety such as an organic conjugate.
  • any of the features have been identified or defined as a component of a molecule of the invention, any of several manipulations and/or modifications of these features may be performed by moving, swapping, substituting, inverting, deleting, randomizing or duplicating. Furthermore, it is understood that manipulation of features may result in the same outcome as a modification to the molecules of the invention. For example, a manipulation which involved deleting a domain would result in the alteration of the length of a molecule just as modification of a nucleic acid to encode less than a full length molecule would.
  • Modifications and manipulations can be accomplished by methods known in the art such as site directed mutagenesis.
  • the resulting modified molecules may then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.
  • domain binding optimization of the druggable ligand may be performed. Domain binding optimization is described in detail in copending applications; Serial Numbers 60/818,735 (Attorney docket Number 3530.3004US) and 60/818,736 (Attorney Docket Number 3530.3006US), both filed July 6, 2006, each of which is incorporated herein by reference in its entirety.
  • binding includes the formation of one or more ionic, covalent, hydrophobic, electrostatic, or hydrogen bonds between a receptor binding surface of the HER ligand variants and Pan-HER antagonists of the invention and one or more amino acids of a target receptor domain of a target receptor. Binding can be considered "tight" if the HER ligand variant or Pan-HER antagonist is not substantially displaced in an in vitro assay. The HER ligand variant or Pan-HER antagonist is not substantially displaced if at least 50%, preferably at least 70%, more preferably at least about 90%, such as 100%, of the HER ligand variant or Pan-HER antagonist remains bound to a receptor or receptor moiety when competitively challenged with a native ligand.
  • the HER ligand variants of the present invention may still be therapeutically relevant even if binding is 10-100 times poorer than native ligand. Binding can also be considered tight if the HER ligand variant or Pan-HER antagonist substantially displaces the native ligand from the receptor.
  • the HER ligand variant or Pan-HER antagonist substantially displaces the native ligand if at least 50%, preferably at least 70%, more preferably at least about 90%, such as 100%, of the native ligand is displaced from the receptor.
  • binding or bioactive activity of a HER ligand variant or Pan-HER antagonist of the invention can further be assessed by any other suitable assay or other method, wherein the results or activity of such assay are compared to the binding or receptor activity from an assay which measures the binding or receptor activity of wild-type human ligands and receptors.
  • binding studies are performed on libraries of compounds of the invention.
  • Methods of library production can also be used to create the starting molecules of the invention.
  • the modifications made to the HER ligand variants or Pan-HER antagonists result in or from the production of a library of modified polypeptides.
  • the library of modified polypeptides may comprise a phage library or any other selection or grouping of polypeptide sequences independent of the manner in which they were generated.
  • the term "library” means a collection of molecules.
  • a library can contain a few or a large number of different molecules, varying from about two to about 10 15 molecules or more.
  • the chemical structure of the molecules of a library can be related to each other or be diverse. If desired, the molecules constituting the library can be linked to a common or unique tag, which can facilitate recovery and/or identification of the molecule.
  • the HER ligand variants and Pan-HER antagonists of the present invention can be assayed for inhibition of HER-mediated bioactivity in one or more cell lines using a number of known methods, assays, devices and kits well known in the art.
  • the one or more cell lines comprises a cancer cell line. Cancer cell lines include, but are not limited to lung, breast, liver, heart, bone, blood, colon, brain, skin, kidney, pancreatic, ovarian, uterine and prostate or any cells isolated from tissues or tumors of the cancers listed herein.
  • identifying anticancer agents or anticancer ligands comprising assaying therapeutic Pan-HER antagonists and HER ligand variants designed by the methods described herein in a tumor xenograft system wherein a measured reduction in tumor growth rate, tumor size or tumor metastasis represents a positive hit as a candidate cancer therapeutic.
  • the disease associated with HER-mediated biological activity is a tumor.
  • the tumor is a solid tumor and/or blood or lymphatic node cancer.
  • tumors which can be of epithelial or mesodermal origin, can be benign or malignant types of tumors in organs such as lungs, prostate, urinary bladder, kidneys, esophagus, stomach, pancreas, brain, ovaries, skeletal system, with adenocarcinoma of breast, prostate, lungs and intestine, bone marrow cancer, melanoma, hepatoma, ear-nose-throat tumors in particular being explicitly preferred as members of so-called malignant tumors.
  • organs such as lungs, prostate, urinary bladder, kidneys, esophagus, stomach, pancreas, brain, ovaries, skeletal system, with adenocarcinoma of breast, prostate, lungs and intestine, bone marrow cancer, melanoma, hepatoma, ear-nose-throat tumors in particular being explicitly preferred as members of so-called malignant tumors.
  • the group of blood or lymphatic node cancer types includes all forms of leukemias (e.g. in connection with B cell leukemia, mixed-cell leukemia, null cell leukemia, T cell leukemia, chronic T cell leukemia, HTLV-II-associated leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, mast cell leukemia, and myeloid leukemia) and lymphomas.
  • leukemias e.g. in connection with B cell leukemia, mixed-cell leukemia, null cell leukemia, T cell leukemia, chronic T cell leukemia, HTLV-II-associated leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, mast cell leukemia, and myeloid leukemia
  • lymphomas e.g. in connection with B cell leukemia, mixed-cell leukemia, null cell leukemia, T cell leukemia, chronic T cell leukemia, HTLV-I
  • mesenchymal malignant tumors are: fibrosarcoma; malignant histiocytoma; liposarcoma; hemangiosarcoma; chondrosarcoma and osteosarcoma; Ewing sarcoma; leio- and rhabdomyosarcoma, synovialsarcoma; carcinosarcoma.
  • fibrosarcoma malignant histiocytoma
  • liposarcoma hemangiosarcoma
  • chondrosarcoma and osteosarcoma chondrosarcoma and osteosarcoma
  • Ewing sarcoma leio- and rhabdomyosarcoma, synovialsarcoma
  • carcinosarcoma are also contemplated within the scope of the invention.
  • neoplasms are also contemplated within the scope of the invention.
  • Neoplasms include: bone neoplasms, breast neoplasms, neoplasms of the digestive system, colorectal neoplasms, liver neoplasms, pancreas neoplasms, hypophysis neoplasms, testicle neoplasms, orbital neoplasms, neoplasms of head and throat, of the central nervous system, neoplasms of the hearing organ, pelvis, respiratory tract and urogenital tract.
  • the cancerous disease or tumor being treated or prevented is selected from the group of: tumors of the ear-nose-throat region, comprising tumors of the inner nose, nasal sinus, nasopharynx, lips, oral cavity, oropharynx, larynx, hypopharynx, ear, salivary glands, and paragangliomas, tumors of the lungs, comprising non-parvicellular bronchial carcinomas, parvicellular bronchial carcinomas, tumors of the mediastinum, tumors of the gastrointestinal tract, comprising tumors of the esophagus, stomach, pancreas, liver, gallbladder and biliary tract, small intestine, colon and rectal carcinomas and anal carcinomas, urogenital tumors comprising tumors of the kidneys, ureter, bladder, prostate gland, urethra, penis and testicles, gynecological tumors comprising tumors of the cervix, vagina, vulva, uterine
  • the biological activity being assayed includes, but is not limited to; a receptor-mediated pathology such as any of the diseases or conditions noted herein, receptor-mediated cell signaling, phosphorylation, cell growth, cell proliferation and tumor growth.
  • a receptor-mediated pathology such as any of the diseases or conditions noted herein, receptor-mediated cell signaling, phosphorylation, cell growth, cell proliferation and tumor growth.
  • the term "receptor-mediated” refers to any phenomenon or condition, the occurrence of which can be linked or traced to the function or activity of a receptor, as that term is defined herein.
  • the inhibited biological activity is a receptor-mediated pathology selected from the group consisting of cancer (including all those identified hereinabove), inflammation, cardiovascular disease, hyperlipidemia, glucose dysregulation, epilepsy, allergies, Alzheimers disease, metabolic syndrome, Cortisol resistance, Crohn's disease and Huntington disease.
  • the inhibited biological activity is receptor-mediated cell signaling.
  • This inhibition of receptor-mediated cell signaling may result in ablation of downstream signaling by a receptor and this effect can be determined by measuring altered phosphorylation states of one or more proteins.
  • inhibition of receptor-mediated cell signaling can be measured using autophosphorylation assays or gene expression assays. Methods of measuring and quantifying cell signaling cascades are known in the art as are methods to measure gene expression either by measuring mRNA (e.g., RT-PCR) or measuring protein levels (e.g., Western blot analysis).
  • Pan-HER antagonists that are capable of activity which is panoramic (i.e., has an effect of the same kind on multiple receptors) over two or more receptors. Further, the level or degree panoramic inhibition of biological activity may be or is substantially the same against said two or more HERs.
  • Identification of panoramic capacity of any Pan-HER antagonist or HER ligand variant simply involves assaying the Pan-HER antagonist or HER ligand variant for inhibition of biological activity against the two or more receptors of interest.
  • the Pan-HER antagonists and HER ligand variants of the invention possess a number of uses.
  • the Pan-HER antagonists of the present invention can be used to treat patients wherein dysregulation of cell signaling is implicated in the pathological process of disease (e.g. cancer, inflammation).
  • the molecules of the present invention be administered as amino-acid based molecules, they may also be administered as nucleic acid molecules in the context of gene therapy. Furthermore, these molecules may be used in diagnostic applications as well as to further basic research.
  • the present invention also embraces pharmaceutical compositions comprising the therapeutic Pan-HER antagonists described herein.
  • a Pan-HER antagonist of the invention can be formulated with a pharmaceutically acceptable carrier or excipient to prepare a pharmaceutical composition.
  • the carrier and composition can be sterile.
  • the formulation should suit the mode of administration.
  • pharmaceutically acceptable “physiologically tolerable” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
  • Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylase or starch, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, etc., as well as combinations thereof.
  • salt solutions e.g., NaCl
  • saline e.g., buffered saline
  • alcohols e.glycerol
  • ethanol e.glycerol
  • gum arabic e.glycerol
  • vegetable oils e.glycerol
  • benzyl alcohols e.glycerol
  • polyethylene glycols e.glyce
  • Pan-HER antagonists of the invention may also be covalently attached to a protein carrier such as albumin, or a polymer, such as polyethylene glycol so as to minimize premature clearing of the polypeptides.
  • the pharmaceutical preparations can, if desired, be mixed with auxiliary agents, e.g. lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like that do not deleteriously react with the active agent in the composition (i.e., a polypeptide and/or nucleic acid molecule of the invention).
  • the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium saccharine, cellulose, magnesium carbonate, etc.
  • compositions include, but are not limited to, transdermal, intramuscular, intraperitoneal, intraocular, intravenous, subcutaneous, pulmonary, topical, oral and intranasal.
  • topical applications include those for treating conditions such as scarring, skin cancer and psoriasis.
  • compositions of this invention can also be administered as part of a combination therapy with other Pan-HER antagonists or other compounds.
  • compositions for intravenous administration typically are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentration in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active compound (polypeptide and/or nucleic acid).
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • Pan-HER antagonists described herein can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • Pan-HER antagonists of the invention are administered in a therapeutically effective amount.
  • the amount of Pan-HER antagonist that will be therapeutically effective in the treatment of a particular disorder or conditions will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
  • in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the symptoms of the disease or condition, and should be decided according to the judgment of a practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the present invention also pertains to methods of treatment (prophylactic, diagnostic, and/or therapeutic) for conditions characterized by HER-mediated pathology, HER overexpression, or dysregulation of cell signaling.
  • a "condition characterized by dysregulation of cell signaling" is a condition in which the presence of a Pan-HER antagonist of the invention is therapeutic. Such conditions include many types of cancer. Dysregulation of cell signaling has also been implicated in a variety of other disorders.
  • the present invention also features a method of treating a condition characterized by HER over-expression or HER ligand-mediated pathology in a patient, comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising at least one Pan-HER antagonist of the invention.
  • a single Pan-HER antagonist specific for HERl, HER3 and HER4 is very desirable as it provides a powerful therapeutic for targeting and treating diseases (such as cancer) in which undesirable HER overexpression, overexpression of HER ligands or other HER-mediated biological activity of one or more HER family members is implicated.
  • treatment refers not only to ameliorating symptoms associated with the disease or condition, but also preventing or delaying the onset of the disease, and also lessening the severity or frequency of symptoms of the disease or condition.
  • More than one Pan-HER antagonist of the present invention can be used concurrently as a co-therapeutic treatment regimen, if desired.
  • a "co-therapeutic treatment regimen” means a treatment regimen wherein two drugs are administered simultaneously, in either separate or combined formulations, or sequentially at different times separated by minutes, hours or days, but in some way act together to provide the desired therapeutic response.
  • the Pan- HER antagonists of the invention may also be used in conjunction with other drugs that inhibit various aberrant activities of HER-mediated pathologies or dysregulated cell signaling. Such additional drugs include but are not limited to receptor specific antibodies, small molecule receptor inhibitors, and traditional chemotherapeutic agents.
  • the therapeutic compound(s) of the present invention are administered in a therapeutically effective amount (i.e., an amount that is sufficient to treat the disease or condition, such as by ameliorating symptoms associated with the disease or condition, preventing or delaying the onset of the disease or condition, and/or also lessening the severity or frequency of symptoms of the disease or condition).
  • a therapeutically effective amount i.e., an amount that is sufficient to treat the disease or condition, such as by ameliorating symptoms associated with the disease or condition, preventing or delaying the onset of the disease or condition, and/or also lessening the severity or frequency of symptoms of the disease or condition.
  • the amount that will be therapeutically effective in the treatment of a particular individual's disease or condition will depend on the symptoms and severity of the disease, and can be determined by standard clinical techniques.
  • in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • a therapeutically effective amount of a Pan-HER antagonist of this invention is typically an amount of Pan-HER antagonist such that when administered in a physiologically tolerable composition is sufficient to achieve a plasma concentration of from about 0.1 microgram (ug) per milliliter (ml) to about 100 ug/ml, preferably from about 1 ug/ml to about 5 ug/ml, and usually about 5 ug/ml.
  • the dosage can vary from about 0.1 mg/kg to about 300 mg/kg, preferably from about 0.2 mg/kg to about 200 mg/kg, most preferably from about 0.5 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or several days.
  • Dosages recited are on a protein basis as the bulking agent (e.g., PEG) might be much bigger than the therapeutically active agent.
  • PEG bulking agent
  • EGF weighs approximately 5,000 Daltons
  • attachment of a PEG might add 50,000 Daltons to the total weight of the composition.
  • Dosages may also be based on the range of serum levels of EGF (0.1-1 ng/ml) and/or relative to the affinity for the ACL. Using this starting point, compounds of the invention may be administered in doses up to ten- fold these measurements. For example, if the ACL affinity is 1OnM and the affinity of EGF is InM, then the dosing range would be between about 10 ng/mL and about 100 ng/mL.
  • the therapeutic compositions containing a Pan-HER antagonist or a polyeptide of this invention may be administered via a unit dose.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
  • the therapeutic compounds of the present invention can be used either alone or in a pharmaceutical composition as described above.
  • the gene for a Pan-HER antagonist or HER ligand variant of the present invention can be introduced into cells (either in vitro or in vivo) such that the cells produce the desired Pan-HER antagonist polypeptide.
  • cells that have been trans fected with the nucleic acid molecule of the present invention can be introduced (or re-introduced) into an individual affected with the disease.
  • the therapeutic Pan-HER antagonists of the invention may also be contained within a kit. As such, the invention also relates to a kit comprising the therapeutic Pan-HER antagonist and/or the pharmaceutical composition.
  • kits and arrays can be used in the diagnosis and/or therapy of diseases associated with the dysregulation of cell signaling.
  • the invention also relates to the use of said therapeutic Pan-HER antagonist, said kit, said array in the diagnosis, prophylaxis, reduction, therapy, follow-up and/or aftercare of diseases associated with an HER-mediated pathology or dysregulation of cell signaling.
  • the human epidermal growth factor gene (EGF) was synthesized chemically and ligated into the Pet-9a vector (Novagen) at the Ndel and BamHI cloning sites.
  • the EGF gene contained the OmpA leader sequence followed by an N-terminal 6x- his tag (underlined) and a factor Xa cleavage site for future his-tag removal, (BOLDED: IEGR) if necessary, and corresponds to the following amino acid sequence:
  • This original clone designated pMLPPl, was used as a basis for cloning all Pan HER ligand variants (including substitution, deletion, insertion and domain swap variants) using the QuickChange mutagenesis kit (Stratagene).
  • the EGF plasmids were transformed into E. coli strain BL21 (DE3) pLysS (Novagen). Production of ligand variants.
  • Human EGF gene was synthesized chemically and ligated into PET9a (Novagen) at the Ndel and BamHI cloning sites.
  • the gene contained the OmpA leader sequence followed by an N-terminal HiS 6 tag and a factor Xa cleavage site.
  • the plasmid, pMLPP was then mutagenized with the Quick Change mutagenesis kit (Stratagene) and selected plasmids used to transform E. coli BL21 (DE3) pLysS (Novagen). 1 Single colonies of transformants were inoculated into shake flasks cultures containing 15mL LB plus kanamycin 25ug/mL plus chloramphenicol
  • A431 cells and MDA-MB-453 cells were used as a source of EGFR and HER3, respectively.
  • A431 cells were grown in DMEM with 10% FBS in an atmosphere containing 5% CO 2 ;
  • MDA-MB-453 cells were grown in Liebovitz's L-15 with 10% FBS in ambient CO 2 .
  • Cells were trypsinized, and transferred to 96 well plates at 10 5 cells per well for A431 and 3 X 10 5 cells per well for MDA-MB-453.
  • Each well contains a fixed amount of biotinylated panerbin and varied amounts of the competing ligand. Plates were incubated for 1 hour on ice. Unbound ligand was washed off and bound panerbin detected with horse radish peroxidase conjugated to streptavidin (Pierce), using 1-Step Ultra TMB-ELISA (Pierce).
  • DuoSet ELISA kits (R&D Systems) are used to measure total and phosphorylated levels of EGFR, HER3 and HER4 in T47D cell lysates. It has been observed that this cell line produces measurable amount of all four HER receptors and that these receptors can be phosphorylated in cells treated with the appropriate ligand.
  • the cells are grown in complete medium and then serum starved overnight. They are then treated with ligand in serum- free medium containing 1 mM Na 3 VO 4 at 37° for 15 minutes. Cells are washed and then treated with PBS containing 1 mM Na 3 VO 4 on ice for 15 minutes.
  • Cells are resuspended by scraping and lysed with Cellytic-M (Sigma) containing 1 mM Na 3 VO 4 plus protease inhibitors. Lysates are stored at -80° prior to analysis. Protein concentrate and buffer exchange.
  • Phage panning is performed according to the teachings of Rodi and Malowski, (Curr. Opin. BiotechnoL, 10:87-93; 1999). Briefly, genes encoding HER ligand variants were cloned into the pentavalent M 13 phage display system (New England Biolabs). Sequences may include those coding for pan-HER agonists (TIE, WVS, and BiR); those coding for HER ligand variants having the agonist modifications in addition to modifications that reduce binding to HER receptor domains (e.g.
  • phage are used to measure binding affinity of the HER ligand variants as well as biological activity by stimulation of HER receptor dependent cell proliferation or phosphorylation.
  • the phage binding assay system is disclosed in co- pending applications; Serial Numbers 60/818,735 (Attorney docket Number 3530.3004US) and 60/818,736 (Attorney Docket Number 3530.3006US), both filed July 6, 2006, each of which is incorporated herein by reference in its entirety.
  • A431 cells for EGFR binding or T47D cells for HER3 binding are grown as monolayers in tissue culture flasks in media containing fetal bovine serum. Cells are trypsinized, neutralized with growth medium, washed twice with DPBS and resuspended in ice-cold PBS-GIu-T. IO5 cells are transferred to 96 well plates and incubated on ice for 1 hour in the presence of varied concentrations of phage. Cells are centrifuged and washed 5X with PBS-T then incubated for one hour at room temperature with anti-M13 pVIII coat protein antibody conjugated with horseradish peroxidase (HRP).
  • HRP horseradish peroxidase
  • Phage particles displaying ligand variants are evaluated for binding affinity to the HERl receptor (EGFR) in T47D whole cell suspension by measuring absorbance at Abs450. Theoretical estimates may also be performed. Additional phage particles displaying HER ligand variants may be evaluated for binding affinity to the HER3 receptor in A431 whole cell suspensions by measuring absorbance at Abs450.
  • EGFR HERl receptor
  • Additional phage particles displaying HER ligand variants may be evaluated for inhibition of phosphorlation of HER3 receptor in A431 whole cell suspensions by measuring absorbance at Abs450.
  • the HER5 cell line a murine fibroblast line (derived from the NR-6 line; mouse fibroblast cells that overexpress human EGFR) that has been stably transfected to express the human EGF receptor was provided by Dr. M. C. Hung (MD Anderson Cancer Center).
  • Stock cultures of HER5 are propagated in D-MEM/F12 medium containing 10% fetal bovine serum, 100 units/ml of penicillin and 100 ug/ml of streptomycin in a water-jacketed incubator at 37 0 C in a humidified 5% CO 2 atmosphere.
  • HER5 proliferation assays the cells are changed into DMEM/F12 without serum for 24 hours. Cells are then trypsinized and suspended at 1E5 cells/ml. Serial dilutions of EGF (PeproTech, Rocky Hill, NJ), and HER ligand polypeptide variants are prepared in serum- free DMEM/F12 at 2-fold the final concentration and plated into the wells of 96-well plates. Fifty microliters of cell suspension (5000 cells) is added to appropriate wells bringing the total volume to 100 ul at the desired concentrations. Plates are incubated for a 48 hour proliferation period.
  • WST-I is a tetrazolium salt that is cleaved to formazan dye by mitochondrial dehydrogenases in viable cells. The amount of formazan is measured at 450 nm using a microplate reader (Dynex Technologies) with MRX Revelation software.
  • MCF-7 cells human breast cancer cell lines that express HER2 and HER3 are obtained from the American Type Culture Collection (ATCC). Stock cultures of MCF-7 are maintained in Eagle's MEM supplemented with 1% ITS-X (Invitrogen) and 10% fetal bovine serum.
  • MCF-7 cells are transferred to serum- free medium (SFM) for 24 hours and then trypsinized and suspended at 10 5 cells/mL in SFM. Fifty microliters of cell suspension (5000 cells) is plated per well in 96 well microtiter plates. Serial dilutions of HER ligands or mutant proteins are prepared at twice the final concentration in SFM and 50 ul is added to wells, bringing the final volume to 100 ul at the desired final concentration. Plates are incubated for 72 hours at 37C in a humidified 5% CO2 atmosphere. Cell proliferation is determined by addition of 10 ul/well of WST- 1 Cell Proliferation Reagent (Roche Applied Sciences, Indianapolis, IN) for the last three hours of the proliferation period.
  • SFM serum- free medium
  • the human epidermoid carcinoma line, A431 is obtained from ATCC. Stock cultures of A-431 are propagated in DMEM medium containing 10% fetal bovine serum. A431 cells are used to evaluate EGFR receptor binding. T 47 D cells
  • Human ductal carcinoma cells are obtained from ATCC. They are maintained in RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate and supplemented with 0.2 Units/ml bovine insulin, 90%; fetal bovine serum, 10%. T47-D cells are used to evaluate HER3 receptor binding.
  • EXAMPLE 2 Identification of Pan-HER antagonist Using the phage panning assay described herein, phage libraries of the two halves of the B-loop, amino acids 21-25 and amino acids 26-30 were created. Each library, comprising over 1 billion variants, was panned against HERl targets and HER3 targets.
  • HER ligand variant sequences including the "EPQRG motif in the first half of the B-loop should represent a superior starting point for PEGylation, based on the expectation that it will be a better binder than the variant with the changes in residue 26-30.
  • variant EGFDI was designed and tested. Substitutions were made to the EPQRG-first half sequence to further improve the properties of the variant. To this end the N-terminus included substitutions of amino acids 2-3 from SD to WV as well as a substitution of lysine at position 28 (K28) to leucine (K28L). This variant was also found to possess excellent binding properties.
  • variant EGFDIII was designed and tested. Starting with an R45Y mutation to enhance binding to Domain III of HER3 and HER4, residues S and D at positions 2 and 3 were retained (because they prevent binding of EGF to HER3 and HER4 at Domain I) and substitutions Y22D and L26G were incorporated to further ablate binding to Domain I of EGFR.
  • the EGFDIII variant is useful when PEGylation at the 28 position is desired. Once pegylated, this variant should not interact with Domain I.
  • Table 4 summarizes the B-loop (first half) HER ligand variants designed and tested. Substitutions are BOLDED and underlined.
  • residue at amino acid position 28 is not particularly conserved in the HER ligands, it is conserved in the EGFR ligands EGF and TGF-alpha, and the others have a positively charged residue (such as lysine) nearby.
  • polymers such as poly(alkylene oxide) are converted into activated forms, as such term is known to those of ordinary skill in the art.
  • the reactive group for example, is a terminal reactive group, which mediates a bond between chemical moieties on the protein and poly(ethylene glycol).
  • one or both of the terminal polymer hydroxyl end-groups, i.e. the alpha and omega terminal hydroxyl groups
  • one of the terminal polymer hydroxyl end-groups is converted or capped with a non-reactive group. In one embodiment one of the terminal polymer hydroxyl end-groups is converted or capped with a methyl group.
  • mPEG refers to a PEG, which is capped at one end with a methyl group.
  • the activated polymers are thus suitable for mediating a bond between chemical moieties on the protein, such as alpha- or epsilon-amino, carboxyl or thiol groups, and poly(ethylene glycol).
  • Bis-activated polymers can react in this manner with two protein molecules or one protein molecule and a reactive small molecule in another embodiment to effectively form protein polymers or protein-small molecule conjugates through cross linkages.
  • Secondary amine or amide linkages are formed using the epsilon-amino groups of lysine of a pan-HER antagonists or HER ligand variant and the activated PEG.
  • a secondary amine linkage may also be formed between the lysine epsilon- amino group of a pan-HER antagonists or HER ligand variant and single or branched chain PEG aldehyde by reductive alkylation with a suitable reducing agent such as NaCNBH.3, NaBH.3, pyridine borane etc. as described in Chamow et al., Bioconjugate Chem. 5: 133-140 (1994), U.S. Pat. No. 4,002,531, WO 90/05534, and U.S. Pat. No. 5,824,784.
  • a suitable reducing agent such as NaCNBH.3, NaBH.3, pyridine borane etc.
  • the chemical modification through a covalent bond may be performed under any suitable condition generally adopted in a reaction of a biologically active substance with the activated poly(ethylene glycol).
  • PEGylation reagents There are several suppliers of PEGylation reagents. In one embodiment, para-nitrophenyloxycarbonyl-PEG derivatives for the PEGylation studies may be used.
  • PNP-PEG is a well established starting material and generates a higher percentage of conjugated and stable product compared to other linkers (such as NHS esters). They are also easy to make (Conditions: borate-phosphate buffer (pH 8.0- 8.3), room temperature with gentle stirring, overnight (Sartore, L. et al. 1991. Appl. Biochem. Biotechnol. 27(l):45-54).
  • Dow Pharma supplies very high purity material (narrow polydispersity and very low levels of PEG diol).
  • a poly(ethylene glycol)-modif ⁇ ed pan-HER antagonist or HER ligand variant may be purified from a reaction mixture by conventional methods which are used for purification of proteins, such as dialysis, salting-out, ultrafiltration, ion-exchange chromatography, hydrophobic interaction chromatography (HIC), gel chromatography and electrophoresis.
  • dialysis, salting-out, ultrafiltration, ion-exchange chromatography, hydrophobic interaction chromatography (HIC), gel chromatography and electrophoresis such as dialysis, salting-out, ultrafiltration, ion-exchange chromatography, hydrophobic interaction chromatography (HIC), gel chromatography and electrophoresis.
  • HIC hydrophobic interaction chromatography
  • the HER ligand variants are produced and the proteins purified for PEGylation.
  • Pan-HER optimized EGF variants can then be conjugated with PEG molecules big enough to provide a benefit of PEGylation (extended serum half life) while still retaining clinically relevant Pan-HER binding affinity.
  • Pegylation is performed using standard methods, using a relatively small PEG (5000 mol wt.) and a large PEG (40,000 mol wt). Evaluation of the 5K- PEGylated material will allow comparison to published experiments with PEGylated EGF, and the effect of a small PEG on Pan-HER antagonist activity. Testing the larger (40K-PEGylated form) will allow testing of the central hypothesis.
  • the EGFDI and EGFDIII with and without PEGylation can be tested for their abilities to displace panerbin in competition binding assays and to antagonize panerbin-dependent phosphorylation of EGFR, HER3 and HER4 in T47D cells using the assays described herin.
  • the PEG5K (HER ligand variant modified with a 5K PEG moiety) proteins should bind with an affinity similar to that reported for non-PEGylated proteins. If the binding of the PEG40K (HER ligand variant modified with a 4OK PEG moiety) proteins is significantly lower than that of the 5K material, it is likely that the larger PEG is interfering with the single domain binding and further optimization may be necessary. If the binding of the PEGylated proteins is similar to that of the non- PEGylated proteins, it is expected that they will demonstrate equivalent or improved antagonism towards HER receptor phosphorylation because the PEG groups are designed to add to the interference with the conformational shift that is necessary for receptor dimerization and activation.
  • mice mice (5/group) are injected intravenously with lOO ⁇ L per mouse (0.2 mg protein/kg) of the authentic protein or the PEGylated conjugates. Following sedation with 0.09% avertin, sampling of blood will be undertaken via the retro- orbital sinus into vials containing EDTA. At 2, 15, 30, and 60 min, the mice are bled 100 ⁇ L, and at 4, 24, 48, 72, and 96 h, mice are terminally bled by cardiac puncture. The plasma is collected following centrifugation of the blood at 5000 rpm at 4 0 C for 5 min and immediately frozen on dry ice. The concentrations of the compounds are analyzed by EGF- and His-tag ELISA. The data are modeled to determine pharmacokinetic parameters using a two compartment, bolus, first-order elimination model for the intravenous samples.
  • the objective of this exercise is to block binding to one of the HER ligand binding domains with the PEG unit, thus creating an antagonist while also benefiting from the new desirable therapeutic properties bestowed by the PEG.
  • Antagonist properties of the compounds are measured using the phosphorylation assay disclosed herein.

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Abstract

La présente invention concerne des antagonistes du récepteur épidermique humain panoramique (HER) appelés antagonistes pan-HER. Les antagonistes pan-HER selon la présente invention sont des ligands de variants polypeptidiques de HER qui peuvent être sélectivement chimiquement dérivés, avec par exemple des fractions polyéthylène glycol, sans aucune perte de l'activité biologique antagoniste. Par conséquent, ces antagonistes pan-HER optimisés par PEG sont conçus pour présenter les propriétés antagonistes pan-HER associées à une liaison améliorée et à des profils pharmacocinétiques et pharmacodynamiques accrus.
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EP3707166A4 (fr) * 2017-11-06 2021-11-24 Daniel J. Monticello Systèmes à récepteur antigénique chimérique avec ligand dominant négatif

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Publication number Priority date Publication date Assignee Title
EP3707166A4 (fr) * 2017-11-06 2021-11-24 Daniel J. Monticello Systèmes à récepteur antigénique chimérique avec ligand dominant négatif

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