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WO2016123575A1 - Compositions et méthodes pour moduler la liaison et la signalisation du récepteur eph - Google Patents

Compositions et méthodes pour moduler la liaison et la signalisation du récepteur eph Download PDF

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Publication number
WO2016123575A1
WO2016123575A1 PCT/US2016/015831 US2016015831W WO2016123575A1 WO 2016123575 A1 WO2016123575 A1 WO 2016123575A1 US 2016015831 W US2016015831 W US 2016015831W WO 2016123575 A1 WO2016123575 A1 WO 2016123575A1
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Prior art keywords
polypeptide
epha2
cell
amino acid
acid sequence
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PCT/US2016/015831
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English (en)
Inventor
Alexis KAUSHANSKY
D. Noah SATHER
Stefan H. I. Kappe
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Seattle Biomedical Research Institute
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Publication of WO2016123575A1 publication Critical patent/WO2016123575A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification.
  • the name of the text file containing the sequence listing is SBRI155178_ST25.txt.
  • the text file is 5 KB; was created on January 27, 2016; and is being submitted via EFS-Web with the filing of the specification.
  • Eph receptors and their corresponding natural ephrin ligands are membrane-bound proteins that require direct cell-cell interactions for Eph receptor activation.
  • Eph/ephrin signaling has been implicated in the regulation of a host of processes critical to embryonic development including axon guidance, formation of tissue boundaries, cell migration, and segmentation. Additionally, Eph/ephrin signaling has recently been identified to play a critical role in the maintenance of several processes during adulthood including long-term potentiation, angiogenesis, and stem cell differentiation and cancer.
  • the present disclosure is directed to an isolated polypeptide corresponding to or derived from a 6-Cys protein of Plasmodium.
  • the polypeptide comprises a first amino acid sequence corresponding to a 6-Cys domain from Plasmodium.
  • the polypeptide further comprises a second amino acid sequence corresponding to a 6-Cys domain from Plasmodium, wherein the second amino acid sequence is the same or different as the first amino acid sequence.
  • the present disclosure is directed to a pharmaceutical composition comprising the described polypeptide.
  • the present disclosure is directed to a nucleic acid encoding the described polypeptide.
  • the present disclosure is directed to a vector comprising the described nucleic acid.
  • the present disclosure is directed to a cell comprising the described vector.
  • the present disclosure is directed to a method of modulating binding of a natural ligand to an Eph receptor, comprising administering to a cell expressing the Eph receptor a sufficient amount of the polypeptide described herein.
  • the present disclosure is directed to a method of modulating signaling of an Eph receptor in a cell, comprising administering to a cell expressing the Eph receptor a sufficient amount of the polypeptide described herein.
  • the present disclosure is directed to a method of inhibiting the proliferation of a cancer cell, comprising contacting the cell with a sufficient amount of the polypeptide described herein.
  • the present disclosure is directed to a method of treating a disease characterized by elevated Eph receptor signaling, elevated Eph receptor expression, and/or elevated Eph receptor ligand expression, comprising administering to a subject in need an effective amount of the polypeptide described herein.
  • the disease is a cancer or cataract.
  • the disease is caused by hepatocyte infection by a Plasmodium parasite.
  • FIGURES 1A-1K illustrate that Plasmodium sporozoites invade hepatocytes with high EphA2 expression.
  • the upper panel shows EphA2 distribution in an exemplary Hepal-6 cell.
  • the middle panel shows UIS4 expression in the exemplary Hepal-6 cell.
  • the bottom panel is an overlay of the two individual images that combines the signals for EphA2 and UIS4 in the cell.
  • the scale bar is 5 mm.
  • FIGURES IB to ID Hepal-6 cells were infected with 10 5 P.
  • (IB) shows the distribution of EphA2 1.5 hours after infection (mEphA2, mouse EphA2); in (1C), EphA2 levels are compared between parasite-infected and uninfected cells; and (ID) shows parasite-infection rates in EphA2 hi ⁇ h and EphA2 low cells (Py, P. yoelii). The numbers in the bars are the percentages of infected cells within each subset.
  • FIGURES IE to 1G BALB/c mice were infected with 10 6 P. yoelii sporozoites by intravenous injection. Hepatocytes were analyzed as in (IB) to (ID).
  • FIGURES 1H to 1J HC-04 cells were infected with 10 5 P. falciparum sporozoites (hEphA2, human EphA2). Analyses were performed as in (IB) to (ID).
  • Each figure represents at least three independent experiments.
  • the bar graphs show means with standard deviations.
  • FIGURES 2A-2G illustrate that EphA2 affects PVM formation.
  • (2A) Time sequence showing the maintenance of P. yoelii infection in Hepal-6 cells. Maintenance of infection was defined as the infection rate at a given point divided by the infection rate at 1.5 hours after infection. Error bars indicate the standard deviation of biological replicates.
  • (2B) EphA2( ⁇ / ⁇ ) or age-matched wildtype (WT) mice were infected with 10 5 P. yoelii sporozoites. Infection was assessed by quantitative polymerase chain reaction 42 hours after infection (Py 18s, P.
  • yoelii 18S ribosomal RNA ribosomal RNA
  • mGAPDH mouse glyceraldehyde-3 -phosphate dehydrogenase
  • Error bars show SEM.
  • (2E and 2F) yUIS4-Myc parasites were used to infect Hepal-6 cells, and the cells were analyzed for the presence of the PVM (UIS4P 0S ) after 24 hours (MFI, median florescence intensity).
  • (2G) PyUIS4-Myc infected Hepal-6 cells were assessed for permeability after 48 hours. Each figure represents at least three independent experiments. (2E) to (2G) show means with standard deviations.
  • FIGURES 3A-3E illustrate that P36 interacts with EphA2.
  • 3A 2 ⁇ 10 5 WT or p52 ⁇ /p36- P. yoelii parasites were used to infect Hepal-6 cells. Levels of EphA2 were monitored in infected and uninfected cells.
  • 3C and 3D Hepal-6 cells were incubated with recombinant P52 or P36, alone or in combination with 10 min of EphrinAl treatment.
  • Immunoblots show levels of pEphA2 (pY772). (3E) 2 x 10 5 WT or p52 ' /p36 ⁇ /sapl ⁇ P. falciparum sporozoites were used to infect 6 x 10 5 HC-04 cells. EphA2 levels were measured in infected and uninfected cells. Each figure represents at least three independent experiments. The bar graphs show means with standard deviations.
  • FIGURES 4A-4C illustrate that EphA2 levels are increased in more susceptible hepatocytes.
  • FIGURES 5A and 5B illustrate that EphA2 levels vary more within a single culture than between cultures.
  • 5 A Image showing variability of EphA2 levels between hepatoma cells in a Hepal-6 culture. EphA2 staining is depicted in red (indicated with arrows), DNA is visualized by DAPI in blue. Scale bar is 10 ⁇ .
  • 5B Hepal-6 cells were passaged for one month and lysed every 1-3 passages. Levels of EphA2 were assessed by western blot and pActin was used as a loading control.
  • FIGURES 6A-6E The Gating strategy for identifying CSP-positive cells, EphA2high and EphA2 low cells.
  • MFI median fluorescence intensity
  • FIGURES 7A-7C illustrate that Dasatinib treatment does not impact initial parasite infection.
  • 7 A Schematic describing EphA2 structure.
  • 7B Hepal-6 cells were stimulated with 1 ⁇ g/mL EphrinAl-Fc for 10 min with or without 2 h pretreatment with Dasatinib. Cells were lysed and levels of activated EphA2 (pY772) was assessed by western blot.
  • 7C Untreated or Dasatinib treated Hepa 1-6 cells were infected with P. yoelii sporozoites. Infection rate was assessed by staining with an antibody against CSP and flow cytometry.
  • EphA2high cells Hepal-6 cells were stained for EphA2 and the cell cycle progression marker Ki-67 and assessed by flow cytometry. EphA2 hi sh cells have a greater proportion of cycling cells than EphA2 low cells.
  • EphA2 hi sh cells have a greater proportion of cycling cells than EphA2 low cells.
  • 8B Hepatocytes of EphA2( ⁇ / ⁇ ) mice have similar cell division rates when compared to hepatocytes of WT mice. Livers from three WT BALB/cByJ mice and three EphA2( _/ ⁇ ) mice were dissociated by collagenase-mediated perfusion. Hepatocytes were stained for the cell cycle marker Ki-67.
  • FIGURE 9 illustrates that p52Vp36- parasites are rapidly cleared from liver in vivo.
  • BALB/cJ mice were infected with 10 5 p52 ⁇ , p36 ⁇ or WT P. yoelii sporozoites. Mice were sacrificed 3 h after infection and assessed for liver stage burden by qRT-PCR.
  • FIGURE 10 illustrates the verification and characterization of recombinant protein produced in HEK293 cells.
  • Purified PyP52 ⁇ 160-kDa peak; indicated
  • PyP36 ⁇ 100-kDa peak; indicated
  • Eph receptor Ephrin receptor
  • Eph receptors are membrane-bound receptors that bind to various ephrin ligands.
  • Eph receptors There are currently about 10 different endogenous ephrin ligands for the 14 Eph receptors, which provide substantial cross-talk among the various Eph receptors. See, e.g., Tognolini, M., et al., "Structure-activity relationships and mechanism of action of Eph-ephrin antagonists: interaction of cholanic acid with the EphA2 receptor," ChemMedChem. 7(6): 1071-1083 (2012), incorporated herein by reference in its entirety.
  • Eph receptors and their corresponding ephrin ligands are membrane-bound proteins that require direct cell-cell interactions for Eph receptor activation.
  • Eph/ephrin signaling has been implicated in the regulation of a host of processes critical to embryonic development including axon guidance, formation of tissue boundaries, cell migration, and segmentation. Additionally, Eph/ephrin signaling has recently been identified to play a critical role in the maintenance of several processes during adulthood including long-term potentiation, angiogenesis, and stem cell differentiation and cancer.
  • Plasmodium 6-Cys proteins bind the EphA2 receptor on hepatocytes leading to the formation of the intracellular parasitophorous vacuole membrane (PVM) essential for successful infection.
  • PVM intracellular parasitophorous vacuole membrane
  • P36 does not activate the EphA2 receptor and, thus, provides means to prevent any human disease which is driven by EphA2 activation, including malaria disease.
  • the findings also have significance beyond possible application to the treatment of malaria.
  • High EphA2 receptor expression in cancer cells is correlated to a poor prognosis associated with recurrence due to enhanced metastasis (Tandon, M, et al., "Emerging strategies for EphA2 receptor targeting for cancer therapeutics," Expert Opinion Therapeutic Targets 75(1):31-51 (2011), incorporated herein by reference in its entirety).
  • the ability of P36 protein to block EphA2 receptor activation will be useful and clinically relevant, particularly, for the inhibition of formation of metastases or, generally for the treatment of cancer.
  • the parasitic ligands described herein have potential applicability to modulation of other Eph ligands to influence a host of other signaling pathways and resulting diseases.
  • the present disclosure provides a polypeptide comprising a first amino acid sequence corresponding to a 6-Cys protein from Plasmodium, or derivative thereof.
  • the polypeptide is isolated or purified from its natural environment in the Plasmodium.
  • the polypeptide is produced recombinantly from a non-Plasmodium cell according to standard protocols in the art.
  • the polypeptide comprises a first amino acid sequence with at least 10 contiguous amino acids from SEQ ID NO:2.
  • SEQ ID NO: 2 is an amino acid sequence of a truncated form of Plasmodium yoelii P36, which specifically corresponds to amino acid positions 74-356 of the full-length wild type P36 protein.
  • the indicated sequence lacks the wild-type transmembrane domains but retains two 6-Cys domains, as described in more detail below.
  • An exemplary nucleic acid encoding SEQ ID NO:2 is set forth in SEQ ID NO: l, which is modified from the wild-type encoding sequence for codon bias for optimal expression in human cells.
  • the polypeptide can comprise a first amino acid sequence with at least 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or more, or any range derivable therein, of contiguous amino acids of SEQ ID NO:2.
  • the first amino acid sequence comprises a domain that corresponds to a 6-Cys domain in a 6-Cys protein from Plasmodium.
  • the 6-Cys protein is P36.
  • the Plasmodium is Plasmodium yoelii .
  • the 6-Cys domains are believed to be unique to Plasmodium parasites and are involved in a variety functions throughout the parasite's life cycle.
  • the domains and Plasmodium proteins that contain them have been described elsewhere. See, e.g., Annoura, T., et al., "Two Plasmodium 6-Cys family-related proteins have distinct and critical roles in liver-stage development," The FASEB Journal 25:2158-2170 (2014)), incorporated herein by reference in its entirety.
  • various 6-Cys proteins are expressed on the surface of the sporozoite stage of the Plasmodium life cycle stage.
  • the inventors have discovered that the certain 6-Cys proteins on the surface on the sporozoite stage are critical for the selective and successful invasion of host hepatocyte cells due to their ability to bind to Eph receptors present on the hepatocyte surface.
  • 6-Cys proteins e.g., P52 and P36
  • the binding of the 6-Cys proteins to the Eph receptor is believed to be specifically due to the 6-Cys domains in the 6-Cys proteins, which are situated in the extracellular space of the sporozoite.
  • This is supported by the data described below that a truncated form of the P36 protein, as represented by the sequence set forth in SEQ ID NO:2, which contains two 6-Cys domains, retains the capacity to bind Eph receptor.
  • SEQ ID NO:2 which contains two 6-Cys domains
  • the polypeptide comprises a first amino acid sequence corresponding to a 6-Cys domain from Plasmodium.
  • the 6-Cys domain can be any 6-Cys domain known from the Plasmodium genus. See, e.g., Arredondo, S.A., et al., (2012).
  • the polypeptide further comprises a second amino acid sequence that also corresponds to a 6-Cys domain from Plasmodium.
  • the first and second polypeptides can have the same sequence or can have different or variant sequences.
  • Either 6-Cys domain can be any 6-Cys domain known from the Plasmodium genus. See, e.g., Arredondo, S.A., et al., (2012).
  • the first and second domains can be contiguous or can be separated by linker amino acids of variable length.
  • the linker amino acid sequence can be naturally occurring or synthetic (man-made).
  • the polypeptide is not naturally occurring, but is the result of human intervention. Accordingly, the polypeptide can be a purified or isolated form of a naturally occurring polypeptide.
  • isolated and purified refer the removal of the naturally occurring polypeptide from some, most or all of other molecular structures in its natural environment in the extracellular space of the Plasmodium sporozoite.
  • purified or isolated form of any polypeptide described herein possesses markedly different characteristics from the naturally occurring form of the polypeptide.
  • P52 and P36 might naturally form a complex in the Plasmodium sporozoite, which enables P36 to contact the EphA2 receptor on a host hepatocyte.
  • the contact is believed to lead to the formation of a parasitophorous vacuole membrane (PVM) that contributes to a successful and productive infection in the cell.
  • PVM parasitophorous vacuole membrane
  • the isolated or purified version of the polypeptide may possess the wild- type sequence, or be a truncated or otherwise altered version of a wild-type sequence. Being isolated or purified, the polypeptide encompassed by this description is not in a complex that would occur within the naturally occurring Plasmodium and, thus, does not trigger the formation of a PVM within a hepatocyte.
  • polypeptide can be produced using any techniques, such as recombinant protein expression, that are familiar and well-known in the art. An exemplary approach is illustrated in the description below.
  • the sequence of the polypeptide is not naturally occurring.
  • the sequence of the polypeptide may differ from a naturally occurring 6-Cys protein by virtue of lacking one or more amino acids that appear in the naturally occurring version.
  • the polypeptide may have a sequence that corresponds to a subsequence of a naturally occurring 6-Cys protein.
  • the polypeptide will not correspond precisely with a subsequence of a naturally occurring 6-Cys protein, but rather will contain at least one or more variations (e.g., one or more deletions, additions, and/or mutations) in the naturally occurring 6-Cys protein subsequence.
  • the first amino acid sequence is one of A) the amino acids sequence corresponding to residue positions 18 to 114 of SEQ ID NO:2, or an amino acid sequence with at least 80% identity thereto, and B) the amino acids sequence corresponding to residue positions 141 to 267 of SEQ ID NO:2, or an amino acid sequence with at least 80% identity thereto.
  • SEQ ID NO:2 sets forth the sequence of a P36 fragment that lacks the wild-type transmembrane domains. These amino acid subsequences of SEQ ID NO:2 indicated above correspond to the two separate 6-Cys domains within the P36 fragment. Accordingly, the polypeptide of the present disclosure can have a sequence corresponding to either one of these indicated domains, or have a sequence with at least 80% identity thereto.
  • the polypeptide has a first and second 6-Cys domain.
  • the first and second 6-Cys domains correspond to residue positions 18 to 114 of SEQ ID NO:2 or an amino acid sequence with at least 80% identity thereto.
  • the first and second 6-Cys domains correspond to residue positions 141 to 267 of SEQ ID NO:2, or an amino acid sequence with at least 80% identity thereto.
  • the polypeptide contains both of the amino acid sequences corresponding to residue positions 18 to 114 of SEQ ID NO:2, or an amino acid sequence with at least 80% identity thereto, as the first 6-Cys domain, and residue positions 141 to 267 of SEQ ID NO:2, or an amino acid sequence with at least 80% identity thereto, as the second 6-Cys domain.
  • the first and second 6-Cys domains can be contiguous or can be separated by linker amino acids of variable length.
  • the first and second 6-Cys domains are linked by a linker amino acid sequence that is naturally occurring or synthetic (man-made).
  • the polypeptide comprises the amino acid sequence corresponding to residue positions 18 to 267 of SEQ ID NO:2, or a sequence with at least 70%) identity thereto. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2, or a sequence with at least 60%> identity the amino acid sequence set forth in SEQ ID NO:2 (such as 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%), 98%), 99%), or any range therein, sequence identity thereto).
  • polypeptide generally refers to a macromolecule of multiple amino acids linked by peptide (amide) bonds.
  • amino acid refers to any of the naturally occurring amino acids found in proteins, D-stereoisomers of the naturally occurring amino acids (e.g., D-threonine), unnatural amino acids, and chemically modified amino acids. Each of these types of amino acids is not mutually exclusive.
  • a-Amino acids comprise a carbon atom to which is bonded an amino group, a carboxyl group, a hydrogen atom, and a distinctive group referred to as a "side chain.”
  • the side chains of naturally occurring amino acids are well-known in the art and include, for example, hydrogen (e.g., as in glycine), alkyl (e.g., as in alanine, valine, leucine, isoleucine, proline), substituted alkyl (e.g., as in threonine, serine, methionine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, and lysine), arylalkyl (e.g., as in phenylalanine and tryptophan), substituted arylalkyl (e.g., as in tyrosine), and heteroarylalkyl (e.g., as in histidine).
  • hydrogen e.g.
  • alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).
  • Unnatural amino acids that is, those that are not naturally found in proteins
  • ⁇ - and ⁇ -amino acids are known in the art and are also contemplated herein as unnatural amino acids.
  • the polypeptide can also have chemically modified amino acids, which refers to an amino acid whose side chain has been chemically modified.
  • a side chain may be modified to comprise a signaling moiety, such as a fluorophore or a radiolabel.
  • a side chain may be modified to comprise a new functional group, such as a thiol, carboxylic acid, or amino group.
  • Post-translationally modified amino acids are also included in the definition of chemically modified amino acids.
  • polypeptide can encompass a polypeptide derivative, such as a type of peptidomimetic where a canonical chemical aspect of the polypeptide is modified.
  • peptidomimetic refers to compounds whose essential elements (pharmacophore) mimic a natural peptide or polypeptide in 3D space, and which retain the ability to interact with the biological target (e.g., a receptor) and produce the same biological effect as an unmodified, canonical polypeptide structure.
  • peptidomimetics are designed to circumvent some of the problems associated with a natural peptide: e.g., stability against proteolysis (duration of activity) and poor bioavailability.
  • percent identity refers to the percentage of amino acid residues in a polypeptide sequence (or nucleotides in a nucleic acid sequence) that are identical with the amino acid sequence (or nucleic acid sequence) of a specified molecule, after aligning the sequences to achieve the maximum percent identity. Alignments can include the introduction of gaps in the sequences to be aligned to maximize the percent identity. In any embodiment described herein, described sequences can be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical, or have any range of identity derivable therein.
  • the present disclosure provides a pharmaceutical composition comprising any embodiment of the polypeptide described above.
  • Formulation strategies involving active polypeptides for treatments of various conditions are well-known in the art.
  • the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding any embodiment of the polypeptide described above.
  • nucleic acid refers to any polymer molecule that comprises multiple nucleotide subunits (i.e., a polynucleotide).
  • Nucleic acids encompassed by the present disclosure can include deoxyribonucleotide polymer (DNA), ribonucleotide polymer (RNA), cDNA or a synthetic nucleic acid known in the art.
  • DNA deoxyribonucleotide polymer
  • RNA ribonucleotide polymer
  • cDNA synthetic nucleic acid known in the art.
  • Nucleotide subunits of the nucleic acid polymers can be naturally occurring or artificial or modified.
  • a nucleotide typically contains a nucleobase, a sugar, and at least one phosphate group.
  • the nucleobase is typically heterocyclic.
  • Canonical nucleobases include purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T) (or typically in RNA, uracil (U) instead of thymine (T)), and cytosine (C)).
  • the sugar is typically a pentose sugar. Suitable sugars include, but are not limited to, ribose and deoxyribose.
  • the nucleotide is typically a ribonucleotide or deoxyribonucleotide.
  • the nucleotide typically contains a monophosphate, diphosphate, or triphosphate. These are generally referred to herein as nucleotides or nucleotide residues to indicate the subunit. Without specific identification, the general terms nucleotides, nucleotide residues, and the like, are not intended to imply any specific structure or identity.
  • the nucleotides can also be synthetic or modified.
  • Nucleotide subunits of the nucleic acid polymers can be naturally occurring or artificial or modified.
  • a nucleotide typically contains a nucleobase, a sugar, and at least one phosphate group.
  • the nucleobase is typically heterocyclic.
  • Canonical nucleobases include purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T) (or typically in RNA, uracil (U) instead of thymine (T)), and cytosine (C).
  • the sugar is typically a pentose sugar. Suitable sugars include, but are not limited to, ribose and deoxyribose.
  • the nucleotide is typically a ribonucleotide or deoxyribonucleotide.
  • the nucleotide typically contains a monophosphate, diphosphate, or triphosphate. These are generally referred to herein as nucleotides or nucleotide residues to indicate the subunit. Without specific identification, the general terms nucleotides, nucleotide residues, and the like, are not intended to imply any specific structure or identity.
  • the nucleotides can also be synthetic or modified.
  • the disclosure provides vectors comprising the nucleic acid sequences described herein, such as a vector comprising a nucleic acid sequence encoding the polypeptide described above.
  • Any vector described herein can further comprise a promoter sequence. Any vector described herein can further comprise a constitutive promoter or inducible promoter appropriate for the expression system to be used, as known in the art.
  • a promoter may comprise an inducible promoter.
  • An inducible promoter may comprise an acetamide-inducible promoter.
  • cultured cells transfected with any vector described herein, or progeny thereof wherein the cell is capable of expressing a polypeptide comprising a 6-Cys domain, as described above.
  • the cell can be prokaryotic or eukaryotic, such as insect or mammalian.
  • the present disclosure provides a method of modulating binding of a natural ligand to an Eph receptor.
  • the method comprises administering to a cell expressing the Eph receptor a sufficient amount of the polypeptide described herein above.
  • the Eph receptor can be any known Eph receptor, such as EphAl, EphA2, Eph A3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA9, EphAlO, EphBl, EphB2, EphB3, EphB4, EphB5, and EphB6.
  • the Eph receptor is an Eph2A receptor.
  • the Eph receptor is a human or rodent Eph receptor.
  • the natural ligand is any ephrin ligand. In some embodiments, the ligand is EphrinAl ligand. In some embodiments, the natural ligand is a 6-Cys protein expressed on the surface of a, Plasmodium sporozoite.
  • the term “modulating” refers to causing change in the binding of the natural ligand to the receptor.
  • the modulation is a prevention or inhibition.
  • the polypeptide may compete with or block binding of the natural ligand to the Eph receptor.
  • the disclosure also provides in another aspect a method of modulating Eph receptor signaling in a cell, comprising contacting the cell with a polypeptide described herein.
  • the Eph receptor can be any Eph receptor known in the art, for example human EphA2 receptor.
  • the modulation of Eph receptor signaling refers to changing the cell-signaling that normally results from binding of the Eph receptor to its cognate or natural ligand, as described above.
  • the application of a described polypeptide can cause a change in the signaling by modulating the availability of the Eph receptor for binding by the natural ligand.
  • the modulation of signaling can be observed using any known technique, such as described in more detail below.
  • the modulation of signaling is a reduction in Eph receptor signaling that would be observed by a natural ligand without the presence of the polypeptide described herein.
  • the cell can be a eukaryotic cell, including a mammalian cell, such as rodent or human.
  • the cell can be in vivo, ex vivo, or in vitro.
  • the present disclosure also provides a method of treating a disease characterized by elevated Eph receptor signaling, elevated Eph receptor expression, and/or elevated Eph receptor ligand expression, comprising administering to a subject in need an effective amount of the polypeptide described herein.
  • the method comprises administering to the subject an effective amount of a therapeutic formulation as described herein.
  • the term "treating a disease” indicates inducing any beneficial therapeutic effect on presence or progression of the disease. There is not necessarily any implication that treatment is required to result in a full cure.
  • the effects can include slowing or inhibiting the progression of any aspect of the disease, inhibiting or reducing the chance of the establishment of the disease, reducing or inhibiting the physical effects of the disease, and the like.
  • the disease is a cancer, such as any solid tumor cancer that is correlated with elevated Eph receptor signaling, Eph receptor expression, and/or Eph receptor ligand expression. See, e.g., Tandon, M, et al., "Emerging strategies for EphA2 receptor targeting for cancer therapeutics," Expert Opinion Therapeutic Targets 75(1):31- 51 (2011), incorporated herein by reference in its entirety.
  • the disease is caused by hepatocyte infection by Plasmodium.
  • the present disclosure provides a method for inhibiting tumor growth in a mammalian subject.
  • the method comprises administering a sufficient (or therapeutic) amount of the polypeptide described herein to the subject.
  • the administration of the polypeptide results in an inhibition or reduction in the angiogenesis in the immediate environment of the tumor.
  • the administration of the polypeptide results in an inhibition or reduction in the cell proliferation or cell survival of transformed cells in the tumor.
  • the administration of the polypeptide results in an inhibition or reduction in the tumor cell migration, which can also result in a reduction or inhibition in the potential for metastasis of the tumor.
  • the following describes the discovery that a, Plasmodium 6-Cys protein binds to a human Eph receptor to mediate successful hepatocyte infection by the sporozoite. This study demonstrated that Furthermore, the study demonstrates the surprising discovery that a truncated form of the 6-Cys protein comprising two 6-Cys domains can engage the human Eph receptor to block binding and signaling by the cognate human ligand, without causing signaling itself.
  • hepatocyte EphA2 receptor was critical for establishing a permissive intracellular replication compartment, the parasitophorous vacuole. Sporozoites productively infected hepatocytes with high EphA2 expression, and the deletion of EphA2 protected mice from liver infection. Lack of host EphA2 phenocopied the lack of the sporozoite proteins P52 and P36. Our data suggest that P36 engages EphA2, which is likely to be a key step in establishing the permissive replication compartment.
  • hepatocyte CD81 and scavenger receptor B l are important for hepatocyte infection (4-6). Beyond this, the molecular mechanisms underlying infection remain poorly understood.
  • Table 1 Assessment of the activity of 28 receptors in BALB/cJ and BALB/cByJ mouse livers.
  • TrkB-NTRK2 1.137 1.000 0.015
  • VEGFR2-KDR 1.211 1.000 0.022
  • EphB4 1.260 1.000 0.028
  • TrkA-NTRKl 1.193 1.000 0.074
  • Eph receptors and their cognate Ephrin ligands mediate cell-cell contact (9), making EphA2 a candidate to mediate the hepatocyte-sporozoite interaction.
  • Ephrin-like fold is present in the parasite's 6-Cys protein family (10).
  • Hepal-6 cells a murine hepatocyte line
  • EphA2 mediates sporozoite invasion, susceptibilities might vary within a culture of Hepal-6 cells.
  • EphA2 has an extracellular ligand-binding region and an intracellular kinase domain, which mediates downstream signaling.
  • EphA2 has an extracellular ligand-binding region and an intracellular kinase domain, which mediates downstream signaling.
  • EphA2 levels are important for liver-stage parasite survival and development.
  • the number of EphA2 hi sh infected cells was maintained throughout the course of infection, the number of EphA2 low infected cells decreased over time (FIGURE 2A). This difference could not be accounted for by division rates, because we observed lower levels of host cell division among EphA2 low cells.
  • our results may in fact underestimate the impact of EphA2 on infected cell survival (FIGURES 8A and 8B).
  • EphA2( ⁇ / ⁇ ) mice When we infected EphA2( ⁇ / ⁇ ) and wild-type mice with 10 5 P. yoelii sporozoites, we observed a large decrease in liver-stage burden after 42 hours in EphA2( ⁇ / ⁇ ) mice (FIGURE 2B). EphA2( ⁇ / ⁇ ) mice also exhibited a delay in the onset of blood-stage infection by 1 to 3 days (FIGURE 2C). Thus, without EphA2, the host is far less susceptible to productive parasite liver infection.
  • the parasitophorous vacuole membrane is critical for liver-stage development.
  • UIS4 liver stage PVM-resident protein
  • UIS4-Myc P. yoelii parasite line
  • FIGURE 2D endogenous UIS4 promoter
  • EphrinAl When we added EphrinAl in the presence of P36 to Hepal-6 cells, P36 blocked the activation of EphA2 (FIGURES 3C and 3D). P52, however, did not block EphrinAl -mediated activation of EphA2 (FIGURES 3C and 3D).
  • EphA2 and P36 To determine whether the interaction between EphA2 and P36 also occurs in human parasites, we assessed levels of EphA2 in P. falciparum wild-type or p52-/p36 ⁇ /sapl- parasite-infected HC-04 cells.
  • the P52-P36-deficient P. falciparum sporozoites exhibited partially reduced selectivity for EphA2 hi ⁇ h HC-04 cells compared with P. falciparum wild-type sporozoites (FIGURE 3E).
  • P36 engages EphA2 but does not trigger its activation in rodent and human parasites.
  • Hepal-6 Cells were purchased from ATCC. HC04 cells were a kind gift from Jetsumon Sattabongkot Prachumsri. Cells were maintained in DMEM complete media (Dulbecco's Modified Eagle Medium (Cellgro, Manassas, VA), supplemented with 10% FBS (Sigma-Aldrich, St. Louis, MO), 100 IU/mL penicillin (Cellgro), 100 ⁇ g/mL streptomycin (Cellgro), 2.5 ⁇ g/mL fungizone (HyClone/ Thermo Fisher, Waltham, MA) and split 1-2 times weekly.
  • DMEM complete media Dulbecco's Modified Eagle Medium (Cellgro, Manassas, VA)
  • FBS Sigma-Aldrich, St. Louis, MO
  • penicillin Cellgro
  • streptomycin Cellgro
  • fungizone HyClone/ Thermo Fisher, Waltham, MA
  • Cells were then detached from plate using trypsin, fixed in Cytoperm/Cytofix (BD Biosciences, Franklin Lake, NJ) on ice for 15 minutes, and stained for parasite CSP as described above [antibody 2A10, conjugated to Alexa-488]. After staining, cells were incubated with DNA staining buffer [1 ⁇ Sytox Blue (Thermo Fisher Scientific, Waltham, MA), 0.4 mg/mL RNAse A (Therm oFisher), and 5 mM EDTA in PBS] at room temperature for 30 minutes before flow cytometry on an LSRII (BD Biosciences), with analysis on FlowJo software (Tree Star, Ashland, OR).
  • DNA staining buffer [1 ⁇ Sytox Blue (Thermo Fisher Scientific, Waltham, MA), 0.4 mg/mL RNAse A (Therm oFisher), and 5 mM EDTA in PBS] at room temperature for 30 minutes before flow cytometry on an
  • Antibody Array Livers were collected from 7 BALB/cJ and 7 BALB/cByJ mice and flash frozen in liquid nitrogen. Frozen livers were then ground using RetschlOO Planetary Ball Mill. Cyroground liver powder was resuspended at 30 ⁇ g/mL in IX Cell Lysis buffer supplemented with ImM PMSF. Lysates were centrifuged for 10 minutes at 4°C and supernatant was used in the assay.
  • PathScan Antibody arrays Cell Signaling Technology
  • Signal was captured using GenePix 2000 microarray scanner. Spots were aligned and signal analyzed using Mapix software (Innopsys, Chicago, IL).
  • PyP52 UniProt:Q7K5V2
  • PyP36 UniProt:Q7RPW4
  • tPA -UniProt:P00750 tissue plasminogen activator
  • the transmembrane domain was removed (residues 458-480) and replaced by a GS linker and poly-Histidine tag (8X HIS).
  • the final expression construct containing PyP52 amino acid residues 25-457, was placed under CMV promotion for expression in mammalian cell culture.
  • the two amino-terminus transmembrane domains (residues 25-44 and 51-73) were replaced by the tPA signal, and a GS linker 8XHIS tag was added to the carboxyl- terminus.
  • the final PyP36 expression containing amino acids 74-356 was placed under the CMV promoter for expression in mammalian cells.
  • the expression constructs were used to transfect suspension HEK293 cells maintained in antibiotic free, serum free FreeStyle 293 Expression medium (Life Technologies) using 293 -Free transfection reagent (EMD Millipore, Billerica, MA, USA), according to manufacturer's instructions. Five to six days post-transfection, culture supernatants were harvested and clarified by centrifugation/filtration. The supernatants were then supplemented with 350 mM NaCl (final concentration) and 0.2% sodium azide (final concentration) prior to binding to Ni-NTA agarose.
  • the protein- bound resin was treated with the wash buffer (25 mM Tris pH 8, 300 mM NaCl, 20 mM imidazole), and the protein was eluted with the elution buffer (25 mM Tris pH 7.4, 300 mM NaCl, 200 mM imidazole).
  • Purified protein was further separated by size-exclusion chromatography using a HiLoad 16/600 Superdex-200 pg column (GE Healthcare) in HBS-E (10 mM Hepes pH 7, 150 mM NaCl, 2 mM EDTA) to remove contaminants and protein aggregates. Final purity was assessed by analytical size exclusion chromatography. Quality control analysis by Coomassie gel and size-exclusion trace is shown in FIGURE 10.
  • Hepal-6 cells were plated at 6 x 10 5 cells per well of a 12-well plate in DMEM complete media. Where indicated, cells were treated with complete media only, 10 ⁇ g/mL recombinant P52 (described above), 10 ⁇ g/mL recombinant P36 (described above), or a combination of both for 30 min.
  • EphrinAl-Fc R&D Systems, Minneapolis, MN, USA was supplemented at 1 ⁇ g/mL 10 min before cell lysis, alone or in combination with recombinant P52 or P36.
  • Dasatinib Cell Signaling Technology, Danvers, MA
  • SDS lysis buffer 2% SDS, 50mM Tris-HCl, 5% glycerol, 5 mM EDTA, 1 mM NaF, lOmM ⁇ - glycerophosphate, 1 mM PMSF, 1 mM activated Na 3 V0 4 , 1 mM DTT, 1% phosphatase inhibitor cocktail 2; Sigma-Aldrich, St. Louis, MO, USA), 1% PhosSTOP Phosphatase Inhibitor Cocktail Tablet (Roche, Indianapolis, IN, USA), filtered for 30 min at 4000 rpm through AcroPrep Advance Filter Plates (Pall Corporation, Port Washington, NY, USA) and stored at -80°C.
  • SDS lysis buffer 2% SDS, 50mM Tris-HCl, 5% glycerol, 5 mM EDTA, 1 mM NaF, lOmM ⁇ - glycerophosphate, 1 mM PMSF, 1 mM activate
  • EphA2 mice were purchased from Jackson labs. WT controls were crossed with EphA2 ("/_) mice, then F2 animals were generated by crossing heterozygotes and littermates were used as controls. Mice were infected i.v. with 10 5 P. yoelii sporozoites via tail-vein injection. Animals were sacrificed at 42 h post-infection and liver tissue was harvested in TRIzol (Life Technologies, Carlsbad, CA, USA). Animal handling was conducted according to Institutional Animal Care and Use Committee-approved protocols.
  • Mouse GAPDH was amplified using sequences 5'-CCTCAACTACATGGTTTACAT-3' (SEQ ID NO: 5) and 5 ' -GCTCCTGGAAGATGGTGATG-3 ' (SEQ ID NO:6).
  • qPCR quantitative PCR
  • EphA2 was monitored.
  • Surface EphA2 was measured using an antibody against EphA2 (R&D Systems, Minneapolis, MN, Clone #233720) conjugated to PE in 2% BSA prior to permeabilization, after fixation with 4% PFA.
  • Total mouse EphA2 was measured using an antibody against EphA2 conjugated to APC (R&D Systems, Minneapolis, MN, Clone #233720) following permeabilization with Perm/Fix solution (BD Biosciences) with 2% BSA in PBS.
  • Permeabilized cells were blocked in Perm/Wash buffer (BD Biosciences Franklin Lakes, NT, USA) supplemented with 2% BSA. Additional staining steps were performed in the same buffer.
  • Permeability dye from Invitrogen (LIVE/DEAD® Fixable Yellow Dead Cell Stain Kit, for 405 nm excitation) was used to manufacturer's specifications. All populations were identified by FACS. Analysis was performed on BD LSRII. Flow cytometric analysis was performed using FlowJo software (TreeStar). All experimental conditions were tested in biological triplicate. All data is representative of three independent experiments.

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Abstract

La présente invention concerne des compositions liées aux polypeptides à domaine 6 de Plasmodium et une méthode associés d'utilisation des compositions de l'invention pour moduler la liaison et la signalisation de récepteurs Eph.
PCT/US2016/015831 2015-01-30 2016-01-29 Compositions et méthodes pour moduler la liaison et la signalisation du récepteur eph WO2016123575A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060121042A1 (en) * 2004-10-27 2006-06-08 Medimmune, Inc. Modulation of antibody specificity by tailoring the affinity to cognate antigens
US20080107656A1 (en) * 2006-05-31 2008-05-08 The Walter And Eliza Hall Institute Of Medical Research Immunogenic compositions and methods of use thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060121042A1 (en) * 2004-10-27 2006-06-08 Medimmune, Inc. Modulation of antibody specificity by tailoring the affinity to cognate antigens
US20080107656A1 (en) * 2006-05-31 2008-05-08 The Walter And Eliza Hall Institute Of Medical Research Immunogenic compositions and methods of use thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ARREDONO ET AL.: "Structure of the Plasmodium 6-cysteine s48/45 domain.", PROC NATL ACAD SCI U S A., vol. 109, no. 17, 24 April 2012 (2012-04-24), pages 6692 - 6697 *
DATABASE UniProtKB [o] 29 October 2014 (2014-10-29), "Sporozoite surface protein P36", Database accession no. Q7RPW4 (PF36_PLAYO *

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