US20150079003A1 - Methods and Compositions for Cancer Diagnosis - Google Patents

Methods and Compositions for Cancer Diagnosis Download PDF

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US20150079003A1
US20150079003A1 US14/391,750 US201314391750A US2015079003A1 US 20150079003 A1 US20150079003 A1 US 20150079003A1 US 201314391750 A US201314391750 A US 201314391750A US 2015079003 A1 US2015079003 A1 US 2015079003A1
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binding
patient
binding molecules
cancer
thy1
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Teri Brentnall
Juergen Karl Willmann
Sheng Pang
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Leland Stanford Junior University
University of Washington Center for Commercialization
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Leland Stanford Junior University
University of Washington Center for Commercialization
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Assigned to THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY, A NON-PROFIT ORGANIZATION DULY ORGANIZED AND PURSUANT TO THE LAWS OF CALIFORNIA reassignment THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY, A NON-PROFIT ORGANIZATION DULY ORGANIZED AND PURSUANT TO THE LAWS OF CALIFORNIA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WILLMANN, JUERGEN KARL
Publication of US20150079003A1 publication Critical patent/US20150079003A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/221—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by the targeting agent or modifying agent linked to the acoustically-active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
    • A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney

Definitions

  • Pancreatic cancer is the fourth leading cause of cancer-related death in both women and men in the USA with an estimated 36,000 deaths in 2012 and 43,000 new diagnoses. Survival from pancreas cancer is stage dependent and currently the disease is most frequently detected at advanced tumor stages. Patients diagnosed with advanced pancreatic cancer have a median survival time of less than one year and are considered incurable at the time of diagnosis. Populations who are at elevated risk of pancreatic cancer include: adult-onset diabetics have a 1:300 lifetime risk, individuals who inherit a genetic predisposition to the disease (such as BRCA2 carriers) and individuals who have 2 or more family members with the disease.
  • BRCA2 carriers genetic predisposition to the disease
  • the present invention provides methods for detecting pancreatic cancer in a patient, or for determining a risk for pancreatic cancer development in a patient comprising:
  • the invention provides methods for detecting pancreatic cancer in a patient, or for determining a risk for pancreatic cancer development in a patient comprising:
  • a detectable binding molecule to a patient at risk of having or developing pancreatic cancer, wherein the binding molecule is selected from the group consisting of (a) MMRN1 binding molecules, (b) MRC2 binding molecules, (c) NRP1 binding molecules, and/or (d) VCAM1 binding molecules, under conditions suitable to promote binding complex formation between the binding molecule and a binding molecule target present in pancreatic tumor neovasculature or a precancerous lesion; and (b) detecting the presence or absence of the binding complexes; wherein the presence of an increased number of the binding complexes compared to control is indicative of the presence of pancreatic cancer in the patient or indicates a risk of pancreatic cancer development in the patient.
  • the binding molecule is selected from the group consisting of (a) MMRN1 binding molecules, (b) MRC2 binding molecules, (c) NRP1 binding molecules, and/or (d) VCAM1 binding molecules, under conditions suitable to promote binding complex formation between the binding molecule and a binding
  • the invention provides methods for determining efficacy of pancreatic cancer therapy in a patient, comprising:
  • the present invention provides methods for determining efficacy of pancreatic cancer therapy in a patient, comprising:
  • a detectable binding molecule selected from the group consisting of (i) MMRN1 binding molecules, (ii) MRC2 binding molecules, (iii) NRP1 binding molecules, and (iv) VCAM1 binding molecules, to a patient undergoing or who has previously undergone pancreatic cancer therapy, under conditions suitable to promote binding complex formation between the binding molecule and its target present in pancreatic tumor neovasculature to form a binding complex; and (b) detecting the presence or absence of the binding complexes; wherein the presence or absence of binding complexes is indicative of the efficacy of the anti-cancer therapy in the patient.
  • compositions comprising:
  • the first microbubble may further comprise a plurality of other binding molecules attached to a surface of the first microbubble, wherein the other binding molecules are selected from the group consisting of (a) VEGFR2 binding molecules, (b) MMRN1 binding molecules, (c) MRC2 binding molecules, (d) NRP1 binding molecules, and/or (e) VCAM1 binding molecules.
  • compositions may further comprise a second microbubble, wherein the second microbubble has attached to its surface a plurality of other binding molecules, wherein the other binding molecules are selected from the group consisting of (a) VEGFR2 binding molecules, (b) MMRN1 binding molecules, (c) MRC2 binding molecules, (d) NRP1 binding molecules, and/or (e) VCAM1 binding molecules.
  • compositions comprising
  • binding molecules attached to a surface of the first microbubble are selected from the group consisting of (a) MMRN1 binding molecules, (b) MRC2 binding molecules, (c) NRP1 binding molecules, and/or (d) VCAM1 binding molecules.
  • the invention provides methods for detecting cancer in a patient, or for determining a risk for cancer development in a patient comprising:
  • composition of any embodiment of the invention comprising: (a) administering the composition of any embodiment of the invention to a patient at risk of having or developing cancer, under conditions suitable to promote binding complex formation between the binding molecules and targets of the binding molecules present in neovasculature of the tumor or a precancerous lesion; and
  • FIG. 1 Overview of study design, from identification of novel human PDAC-neovasculature-associated imaging biomarker Thy1, to target validation, creation of human Thy1-targeted ultrasound contrast agent, generation of a novel orthotopic PDAC xenograft model expressing human Thy1 on its neovasculature, and in vivo testing of imaging properties of Thy1-targeted ultrasound contrast agent.
  • FIG. 2 Immunohistochemistry (IHC) analysis of Thy1 staining in human pancreatic tissue samples. Examples of normal pancreas (A) and primary chronic pancreatitis (B) with no Thy1 staining on vasculature. (C). Example of positive Thy1-staining on vessels associated with PDAC. D. Summary of IHC scores on Thy1 stained tissues from normal pancreas (normal), chronic pancreatitis (CP), and pancreatic cancer (PC).
  • IHC Immunohistochemistry
  • FIG. 3 Evaluation of Thy1 expression on vascular endothelial cells.
  • A Stably transfected cells and wild-type cells were assessed for human Thy1 expression by immunofluorescence staining Clone 1 showed strong Thy1 staining, clone 2 showed low staining, and wild-type cells showed no Thy1 staining.
  • B Human Thy1 expression levels on different cell types were quantitatively assessed by FACS analysis. Histogram overlay of signals from cells with different levels of Thy1 expression is shown.
  • C Mean fluorescence intensity values are shown in bar graph. Error bars are ⁇ standard deviations.
  • FIG. 4 Dynamic cell culture binding assay of microbubbles (MB) in a parallel plate flow chamber setting. Phase-contrast bright-field micrographs show binding of MBThy1 and MBControl (white spheres, arrowheads) to different cell types; binding could be substantially blocked by incubation of cells beforehand with an anti-human Thy1 antibody (quantitative date in Table 5).
  • FIG. 5 In vivo ultrasound molecular imaging of orthotopic PDAC xenografts in mice and corresponding ex vivo immunofluorescence analysis.
  • amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; 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).
  • the present invention provides novel compositions, comprising
  • Thy-1 is present in newly created micro-blood vessels in cancer, but is not present in normal blood vessels.
  • Thy-1 specific binding molecules are shown herein for the first time to stain cancer-related blood vessels in pre-cancer as well as cancer.
  • the compositions of the invention find wide use as, for example, imaging agents to enhance early stage cancer detection, as well as pre-cancerous states in individuals at risk for developing cancer, including but not limited to pancreatic cancer.
  • Human Thy-1 or CD90 (Cluster of Differentiation 90) is a 25-37 kDa N-glycosylated, glycophosphatidylinositol (GPI) anchored conserved cell surface protein. Its amino acid sequence is provided in SEQ ID NO:1.
  • microbubbles refer to micron-sized contrast agents composed of a shell and a gas core, as is well known to those of skill in the art. Microbubbles are commercially available from a number of sources.
  • the shell may be formed from any suitable material, including but not limited to albumin, polysaccharides (such as galactose), lipids (such as phospholipids), polymers and combinations thereof.
  • Any suitable gas core can be used in the microbubbles of the invention, including but not limited to air, octafluoropropane, perfluorocarbon, sulfur hexafluoride or nitrogen. The gas core determines the echogenecity of the microbubble.
  • Gas cores can be composed of air, or heavy gases like octafluoropropane, perfluorocarbon, sulfur hexafluoride or nitrogen. Heavy gases are less water-soluble so they are less likely to leak out from the microbubble to impair echogenecity.
  • the average diameter of the microbubble can be between 1 ⁇ m and 25 ⁇ m.
  • the microbubbles have a diameter of about 1 ⁇ m and about 10 ⁇ m on average, and more preferably between about 1 ⁇ m and 5 ⁇ m, 1 ⁇ m and 4 ⁇ m, 1 ⁇ m and 3 ⁇ m, 1 ⁇ m and about 2 ⁇ m, 2 ⁇ m and 5 ⁇ m, 2 ⁇ m and 4 ⁇ m, 2 ⁇ m and 3 ⁇ m, 3 ⁇ m and 5 ⁇ m, 3 ⁇ m and 4 ⁇ m, or about as 1 ⁇ m, 2 ⁇ m, 2.5 ⁇ m, 3 ⁇ m, 3.5 ⁇ m or 4 ⁇ m on average.
  • OPTISON® is preferably between about 1 ⁇ m and 5 ⁇ m, 1 ⁇ m and 4 ⁇ m, 1 ⁇ m and 3 ⁇ m, 1 ⁇ m and about 2 ⁇ m, 2 ⁇ m and 5 ⁇ m, 2 ⁇ m and 4 ⁇ m, 2 ⁇ m and 3 ⁇ m, 3 ⁇ m and 5 ⁇ m, 3 ⁇ m
  • ALBUNEX® (made by Molecular Biosystems), SONOVUE® (made by Bracco Diagnostics, Inc.), SONOZOID® (made by Schering AG), SONOVIST® (made by Schering AG), and DEFINITY® (made by DuPont Pharmaceuticals).
  • ALBUNEX® has an albumin shell and an air core.
  • SONOZOID® is another microbubble preparation containing a perfluorocarbon gas core and a lipid shell.
  • DEFINITY® is another FDA-approved microbubble that contains a lipid shell and an octafluoropropane (C 3 F 8 ) gas core.
  • microbubbles of the present invention comprise a lipid shell and perfluorocarbon gas core of between about 1 ⁇ m and about 5 ⁇ m, 1 ⁇ m and about 4 ⁇ m diameter, 1 ⁇ m and about 3 ⁇ m, or 1 ⁇ m and about 2 ⁇ m, on average.
  • microbubbles of the invention can be used, for example, as a contrast agent for ultrasound imaging.
  • Microbubbles have a high degree of echogenicity (i.e.: the ability of an object to reflect ultrasound waves).
  • the echogenicity difference between the gas in the microbubbles and the soft tissue surroundings of the body is large.
  • ultrasonic imaging using microbubble contrast agents enhances the ultrasound backscatter, or reflection of the ultrasound waves, to produce a unique sonogram with increased contrast due to the high echogenicity difference.
  • the microbubbles can be functionalized in any suitable manner for binding of the Thy-1 binding molecules (or other binding molecules, as described herein). Such techniques are well known to those of skill in the art, such as those for functionalizing the surface of a microbubble to permit binding of a protein.
  • the microbubble surface if functionalized to permit direct attachment of the binding agent to the microbubble surface.
  • the microbubble surface is functionalized to permit indirect attachment of the binding molecule to the microbubble surface.
  • the microbubble surface can be coated with streptavidin, to which biotinylated binding molecules can be bound. Any other suitable binding pair can be similarly used, as will be apparent to those of skill in the art.
  • the targeted microbubbles Following administration to the patient (such as by intravenous injection), the targeted microbubbles accumulate at tissue sites that over-express Thy-1 (or other markers discussed herein), causing a local increase in the ultrasound imaging signal. Due to their small size, the microbubbles stay predominantly within the vascular compartment after intravenous injection. Thus, the microbubbles can be used, for example, to exclusively detect vascular endothelial cell associated molecular markers that are present in early stage cancers or precancerous lesions.
  • Thy-1 binding molecule is any molecular entity capable of selectively binding to human Thy-1.
  • exemplary specific binding agents include, but are not limited to, Thy-1 itself (SEQ ID NO:1), anti-Thy-1 antibodies or fragments thereof, aptamers selective for Thy-1, the I-domain (SEQ ID NO:3) of ⁇ X (CD11c) integrin domain, or proteins comprising this I domain, including but not limited to ⁇ X (SEQ ID NO:2, noting that AA 1-19 constitutes the signal peptide, and thus can be deleted), and ⁇ X- ⁇ 2 integrin; the I-domain (SEQ ID NO:5) of ⁇ M integrin domain, or proteins comprising this I domain, including but not limited to ⁇ M (SEQ ID NO:4) and ⁇ M- ⁇ 2 integrin (see Choi et al., Biochemical and Biophysical Research Communications; 07/2005; 331(2):557-61.
  • the first microbubble further comprises a plurality of other binding molecules attached to a surface of the first microbubble, wherein the other binding molecules are selected from the group consisting of (a) VEGFR2 binding molecules, (b) multimerin-1 (MMRN1) binding molecules, (c) mannose receptor type 2 (MRC2) binding molecules, (d) neuropilin 1 (NRP1) binding molecules, and/or (e) vascular cell adhesion molecule 1 (VCAM1) binding molecules.
  • the other binding molecules are selected from the group consisting of (a) VEGFR2 binding molecules, (b) multimerin-1 (MMRN1) binding molecules, (c) mannose receptor type 2 (MRC2) binding molecules, (d) neuropilin 1 (NRP1) binding molecules, and/or (e) vascular cell adhesion molecule 1 (VCAM1) binding molecules.
  • the inventors have identified MMRN1 (SEQ ID NO:21), MRC2 (SEQ ID NO:55), NRP1 (SEQ ID NO:57), and VCAM1 (SEQ ID NO:90) as additional vascular endothelial cell markers that can be used in identifying tumor neovasculature, and which thus can be used in combination with Thy-1 (as can VEGFR2) as markers for early stage cancer and precancerous lesion detection.
  • the first microbubble such as a population of first microbubbles, would each comprise both Thy-1 binding molecules and “other” binding molecules for one or more of VEGFR2, MMRN1, MRC2, NRP1, and VCAM1.
  • the first population of microbubbles contains other binding molecules for 1, 2, 3, or all 4 of MMRN1, MRC2, NRP1, and VCAM1.
  • the composition further comprises a second microbubble, wherein the second microbubble has attached to its surface a plurality of other binding molecules, wherein the other binding molecules are selected from the group consisting of (a) VEGFR2 binding molecules, (b) MMRN1 binding molecules, (c) MRC2 binding molecules, (d) NRP1 binding molecules, and/or (e) VCAM1 binding molecules.
  • the other binding molecules are selected from the group consisting of (a) VEGFR2 binding molecules, (b) MMRN1 binding molecules, (c) MRC2 binding molecules, (d) NRP1 binding molecules, and/or (e) VCAM1 binding molecules.
  • composition would comprise different subsets of microbubbles: a first microbubble (such as a population of first microbubbles) that comprises Thy-1 binding molecules, and at least a second microbubble (such as a population of second microbubbles) that includes binding molecules for one or more of VEGFR2, MMRN1, MRC2, NRP1, and VCAM1.
  • the second population of microbubbles may each comprise VEGFR2, MMRN1, MRC2, NRP1, and VCAM1.
  • the second population of microbubbles may each comprise other binding molecules for only one of VEGFR2, MMRN1, MRC2, NRP1, and VCAM1.
  • the second population of microbubbles may comprise a second population of microbubbles that each have VEGFR2 binding molecules, a third population of microbubbles that each have MMRN binding molecules, a fourth population of binding molecules that each have MRC2 binding molecules, a fifth population of microbubbles that each have NRP binding molecules, and a sixth population of microbubbles that each have VCAM1 binding molecules.
  • the second population of microbubbles may comprise a second population that each have binding molecules for two of MMRN1, MRC2, NRP1, and VCAM1, and a third population of microbubbles comprising binding molecules for the other two of MMRN1, MRC2, NRP1, and VCAM1.
  • the other binding molecules are binding molecules for one or more of MMRN1, MRC2, NRP1, and VCAM1.
  • the different populations of microbubbles i.e.: first microbubble, second microbubble, etc.
  • first microbubble, second microbubble, etc. can be prepared so as to be distinguishable from each other, though this is not a requirement for use of multiple populations of microbubbles in the methods of the invention.
  • Any suitable means to distinguish the microbubbles can be used, including but not limited to, differentially labeling each population with a separate detectable label (fluorescent, radioactive, etc.) and using different sized microbubbles for each different microbubble population.
  • Multimerin-1 is a soluble human protein found in platelets and in the endothelium of blood vessels. It is composed of linked subunits to form large, variably sized homomultimer. MMR1 has a number of identified ligands.
  • a “MMR1 binding molecule” is any molecular entity capable of selectively binding to human MMR1. Exemplary specific binding molecules include, but are not limited to, MMR1 itself (SEQ ID NO:21), anti-MMR1 antibodies, and a protein selected from the group consisting of SEQ ID NOS: 22 to 54 (see Table 2 below).
  • a “MRC2 binding molecule” is any molecular entity capable of selectively binding to human MRC2.
  • Exemplary specific binding molecules include, but are not limited to, MRC2 itself (SEQ ID NO:55), anti-MRC2 antibodies, and the NDEL1 protein comprising the amino acid SEQ ID NO: 56, an identified ligand for MRC2.
  • a “NRP1 binding molecule” is any molecular entity capable of selectively binding to human NRP 1.
  • Exemplary specific binding molecules include, but are not limited to, NRP 1 itself (SEQ ID NO:57), anti-NRP1 antibodies, and a protein selected from the group consisting of SEQ ID NOS:58-89, identified ligands for NRP11 (see table below).
  • VCAM1 binding molecule is any molecular entity capable of selectively binding to human VCAM 1.
  • exemplary specific binding molecules include, but are not limited to, VCAM1 itself (SEQ ID NO:90), anti-VCAM1 antibodies, and a protein selected from the group consisting of SEQ ID NOS: 91 to 130, identified ligands for VCAM1 (see table below).
  • VCAM1 ITGB7 integrin beta-7 (SEQ ID NO: 91) IL13 interleukin-13 (SEQ ID NO: 92) EZR ezrin (SEQ ID NO: 93) MSN moesin (SEQ ID NO: 94) CCL17 c-c motif chemokine 17 (SEQ ID NO: 95) CTSG cathepsin G (SEQ ID NO: 96) ELANE neutrophil; elastase (SEQ ID NO: 97) ITGAD integrin alpha-D (SEQ ID NO: 98) ITGB1 integrin beta-1 (SEQ ID NO: 99) CCL22 C-C motif chemokine 22 (SEQ ID NO: 100) IRF1 interferon regulatory factor 1 (SEQ ID NO: 101) NFKB1 nuclear factor NF-kappa-B p105 subunit (SEQ ID NO: 102) LGALS3 Galectin-3 (SEQ ID NO: 103) RELB transcription factor Re
  • a “VEGFR2 binding molecule” is any molecular entity capable of selectively binding to human VEGFR2.
  • Exemplary specific binding molecules include, but are not limited to, VEGFR2 itself (SEQ ID NO:131), and anti-VEGFR2 antibodies.
  • compositions comprising
  • binding molecules attached to a surface of the first microbubble are selected from the group consisting of (a) MMRN1 binding molecules, (b) MRC2 binding molecules, (c) NRP1 binding molecules, and/or (d) VCAM1 binding molecules.
  • compositions of the invention find wide use as, for example, imaging agents to enhance early stage cancer detection, as well as pre-cancerous states in individuals at risk for developing cancer, including but not limited to pancreatic cancer
  • antibodies that can be used as binding molecules mean an immunoglobulin molecule immunologically reactive with the recited target, and includes polyclonal and monoclonal antibodies.
  • Various isotypes of antibodies exist, for example IgG1, IgG2, IgG3, IgG4, and other Ig, e.g., IgM, IgA, IgE isotypes.
  • the term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies), and fully humanized antibodies.
  • antibody includes fragments with antigen-binding capability (e.g., Fab′, F(ab′) 2 , Fab, Fv and rIgG, as are well known in the art.
  • the term also refers to recombinant single chain Fv fragments (scFv).
  • the term also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies, as are known in the art. Antibodies for many if not all of the proteins disclosed herein are commercially available.
  • the binding molecules on the surface of the microbubbles can be at any density that is effective for targeting of the microbubble to target-containing neovasculature in a target tumor or precancerous lesion.
  • the average number of binding molecules per square micrometer of the microbubble surface in a microbubble population is at least 1,000/cm 2 ; in various further embodiments, it is at least 2,000/cm 2 , 3,000/cm 2 , 4,000/cm 2 , 5,000/cm 2 , 6,000/cm 2 , 7,000/cm 2 , 7,500/cm 2 , or 7,600/cm 2 .
  • compositions may comprise any number of microbubbles in the composition that can be detected once targeted to the target-containing neovasculature in a target tumor or precancerous lesion.
  • the compositions comprise at least 10 6 microbubbles (i.e.: combined total of all microbubbles, whether all first microbubbles, or a combination of first and second microbubbles.
  • the compositions comprises at least 2 ⁇ 10 6 , 3 ⁇ 10 6 5 ⁇ 10 6 , 7.5 ⁇ 10 6 , 10 7 , 2 ⁇ 10 7 , 3 ⁇ 10 7 , 5 ⁇ 10 7 , 7.5 ⁇ 10 7 , 10 8 , 2 ⁇ 10 8 , 3 ⁇ 10 8 , 5 ⁇ 10 8 , 7.5 ⁇ 10 8 , or at least 10 9 microbubbles.
  • the microbubbles of the invention target tumor or precancerous lesion neovasculature upon administration, they can also be used as a drug delivery device.
  • the composition of any embodiment or combination of embodiments of the present invention further comprises one or more anti-cancer therapeutics on or in the first microbubble and/or the second microbubble.
  • any suitable anti-cancer therapeutic can be loaded onto or into the microbubbles, including but not limited to alkylating agents such as busulfan, cis-platin, mitomycin C, and carboplatin; antimitotic agents such as colchicine, vinblastine, paclitaxel, and docetaxel; topo I inhibitors such as camptothecin and topotecan; topo II inhibitors such as doxorubicin and etoposide; RNA/DNA antimetabolites such as 5-azacytidine, 5-fluorouracil and methotrexate; DNA antimetabolites such as 5-fluoro-2′-deoxy-uridine, ara-C, hydroxyurea and thioguanine; antibodies such as Herceptin®.
  • alkylating agents such as busulfan, cis-platin, mitomycin C, and carboplatin
  • antimitotic agents such as colchicine, vinblastine, paclitaxel, and docetaxel
  • topo I inhibitors
  • melphalan chlorambucil, cyclophosamide, ifosfamide, vincristine, mitoguazone, epirubicin, aclarubicin, bleomycin, mitoxantrone, elliptinium, fludarabine, octreotide, retinoic acid, tamoxifen and alanosine.
  • the present invention provides methods for detecting cancer in a patient, or for determining a risk for cancer development in a patient comprising:
  • composition of any embodiment or combination of embodiments of the first or second aspect of the invention to a patient at risk of having or developing cancer, under conditions suitable to promote binding complex formation between the binding molecules and targets of the binding molecules present in neovasculature of the tumor or a precancerous lesion;
  • the microbubbles of the present invention can be used as a general tool for detection of tumor neovasculature, such as in early stage tumors and precancerous lesions.
  • neovasculature means the vasculature of tumors.
  • the methods according to this aspect of the invention can be used with any patient at risk of having or developing cancer.
  • the patient may be any mammal, such as a human.
  • cancer is intended to mean any cellular malignancy whose unique trait is the loss of normal controls which results in unregulated growth, lack of differentiation and ability to invade local tissues and metastasize. Cancer can develop in any tissue of any organ.
  • cancer is intended to include, without limitation, prostate cancer, leukemia, hormone dependent cancers, breast cancer, colon cancer, epidermal cancer, liver cancer, esophageal cancer, stomach cancer, hepatic carcinoma, melanoma, epidermoid carcinoma, pancreatic cancer, brain malignancies (such as neuroblastoma, glioblastoma, glioma, medulloblastoma, astrocytoma, acoustic neuroma, oligodendroglioma and meningioma), lung cancer (such as small cell lung and non-small cell lung cancer) ovarian adenocarcinoma, bladder cancer, and renal cancer.
  • brain malignancies such as neuroblastoma, glioblastoma, glioma, medulloblastoma, astrocytoma, acoustic neuroma, oligodendroglioma and meningioma
  • lung cancer such as small cell lung and non-
  • Thy-1 and the other recited markers are expressed in pancreatic neovasculature but not in normal pancreatic vasculature, and thus can be used to detect cancer.
  • the inventors have also demonstrated use of the microbubbles of the invention to selectively target tumor neovasculature.
  • the methods of this aspect of the invention thus provide early cancer detection, thus allowing earlier clinical intervention in treatment and substantially improved clinical outcome.
  • the methods may further comprise treating the patient based on the presence of an increased number of binding complexes compared to control.
  • Any suitable control can be used, including but not limited to comparison to a number of binding complexes identified in a subject or population of subjects known to not have cancer. Any amount of Thy-1 or the other markers above control levels may indicate presence of cancer in the patient or indicates a risk of cancer development in the patient.
  • the increase is binding complexes is at least 10% above control, and preferably at least 25%, 50%, 100%, or more above control.
  • the methods can be used with patients that are at risk of having cancer, based on symptoms they presently have.
  • the methods can also be used with patients who are at risk of developing cancer.
  • risk factors include, but are not limited to, a family history of cancer and genetic disorders indicating a propensity to develop cancer.
  • the microbubbles can be administered by any suitable technique, including but not limited to parenterally, transmucosally (orally, nasally, or rectally) or transdermally.
  • Parenteral administration includes, but is not limited to, intravenous, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration.
  • administration of the microbubbles is parenteral, via intravenous injection. Following administration to the patient (such as by intravenous injection), the targeted microbubbles accumulate at tumor sites that over-express Thy-1 or other marker, causing a local increase in the ultrasound imaging signal.
  • microbubbles with an average diameter of between about 1 ⁇ m and 5 ⁇ m, 1 ⁇ m and 4 ⁇ m, 1 ⁇ m and 3 ⁇ m, 1 ⁇ m and about 2 ⁇ m, 2 ⁇ m and 5 ⁇ m, 2 ⁇ m and 4 ⁇ m, 2 ⁇ m and 3 ⁇ m, 3 ⁇ m and 5 ⁇ m, 3 ⁇ m and 4 ⁇ m, or about as 1 ⁇ m, 2 ⁇ m, 2.5 ⁇ m, 3 ⁇ m, 3.5 ⁇ m or 4 ⁇ m on average are intravenously injected. Due to their small size, the microbubbles stay predominantly within the vascular compartment after intravenous injection. Thus, the microbubbles can be used, for example, to exclusively detect vascular endothelial cell associated molecular markers that are present in early stage cancers or precancerous lesions.
  • condition suitable to promote binding complex formation used in the methods of the invention will depend on the means by which the binding molecule is labeled, the type of assay (i.e.: in vitro or in vivo), and all other relevant factors, and can be determined by one of skill in the art based on the teachings herein).
  • the present invention provides methods for detecting pancreatic cancer in a patient, or for determining a risk for pancreatic cancer development in a patient comprising:
  • the present invention provides methods for detecting pancreatic cancer in a patient, or for determining a risk for pancreatic cancer development in a patient comprising:
  • a detectable binding molecule selected from the group consisting of (i) MMRN1 binding molecules, (ii) MRC2 binding molecules, (iii) NRP1 binding molecules, and (iv) VAM1 binding molecules, to a patient at risk of having or developing pancreatic cancer, under conditions suitable to promote binding complex formation between the binding molecule and its target present in pancreatic tumor neovasculature or a precancerous lesion; and
  • Thy-1 and the other recited markers are expressed in pancreatic neovasculature but not in normal pancreatic vasculature, and thus can be used to detect pancreatic cancer.
  • the methods of the invention provide early detection of pancreatic cancer or precancerous lesions, thus allowing earlier clinical intervention in treatment and substantially improved clinical outcome.
  • the methods may further comprise treating the patient based on the presence of an increased number of binding complexes compared to control.
  • Any suitable control can be used, including but not limited to comparison to a number of binding complexes identified in a subject or population of subjects known to not have pancreatic cancer or precancerous lesions. Any amount of Thy-1 or the other markers above control levels may indicate presence of pancreatic cancer in the patient or indicates a risk of pancreatic cancer development in the patient.
  • the increase is binding complexes is at least 10% above control, and preferably at least 25%, 50%, 100%, or more above control.
  • the methods can be used with patients that are at risk of having pancreatic cancer, based on symptoms they presently have, including but not limited to abdominal pain, lower back pain, heartburn, significant weight loss, Trousseau sign, pulmonary embolisms, new onset of diabetes in elderly individuals, and jaundice.
  • the methods can also be used with patients who are at risk of developing pancreatic cancer.
  • Such risk factors include, but are not limited to, a family history of pancreatic cancer; genetic disorders including but not limited to autosomal recessive ataxia-telangiectasia, autosomal dominantly inherited mutations in the BRCA2 gene and/or PALB2 gene, Peutz-Jeghers syndrome due to mutations in the STK11 tumor suppressor gene, hereditary non-polyposis colon cancer (Lynch syndrome), familial adenomatous polyposis, familial atypical multiple mole melanoma-pancreatic cancer syndrome (FAMMM-PC) due to mutations in the CDKN2A tumor suppressor gene; cigarette smoking, age 60 or above, and obesity.
  • genetic disorders including but not limited to autosomal recessive ataxia-telangiectasia, autosomal dominantly inherited mutations in the BRCA2 gene and/or PALB2 gene, Peutz-Jeghers syndrome due to mutations in the STK11 tumor suppressor gene, hereditary non-polyposis
  • the Thy-1 or other binding molecule is detectable.
  • a detectable imaging agent selected from the group consisting of a radioactive agent (e.g., radioiodine (125I, 131I); technetium; yttrium; 35S or 3H) or other radioisotope or radiopharmaceutical; a contrast agent (e.g., gadolinium; manganese; barium sulfate; an iodinated or noniodinated agent; an ionic agent or nonionic agent); a magnetic agent or a paramagnetic agent (e.g., gadolinium, iron-oxide chelate); liposomes (e.g., carrying radioactive agents, contrast agents, or other imaging agents); nanoparticles; a positron emitting isotope for PET scanner, MRI contrast agents, and ultrasound agents (e.
  • a radioactive agent e.g., radioiodine (125I, 131I); technetium; yttrium
  • the binding molecules can be administered by any suitable technique, including but not limited to parenterally, transmucosally (orally, nasally, or rectally) or transdermally.
  • Parenteral administration includes, but is not limited to, intravenous, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration.
  • administration of the binding molecules is parenteral, via intravenous injection. This embodiment is particularly preferred when the binding molecules are present on microbubbles, such as the microbubbles of the present invention.
  • the targeted microbubbles accumulate at pancreatic sites that over-express Thy-1 or other marker, causing a local increase in the ultrasound imaging signal.
  • microbubbles with an average diameter of between about 1 ⁇ m and 5 ⁇ m, 1 ⁇ m and 4 ⁇ m, 1 ⁇ m and 3 ⁇ m, 1 ⁇ m and about 2 ⁇ m, 2 ⁇ m and 5 ⁇ m, 2 ⁇ m and 4 ⁇ m, 2 ⁇ m and 3 ⁇ m, 3 ⁇ m and 5 ⁇ m, 3 ⁇ m and 4 ⁇ m, or about as 1 ⁇ m, 2 ⁇ m, 2.5 ⁇ m, 3 ⁇ m, 3.5 ⁇ m or 4 ⁇ m on average are intravenously injected. Due to their small size, the microbubbles stay predominantly within the vascular compartment after intravenous injection. Thus, the microbubbles can be used, for example, to exclusively detect vascular endothelial cell associated molecular markers that are present in early stage cancers or precancerous lesions.
  • condition suitable to promote binding complex formation used in the methods of the invention will depend on the means by which the binding molecule is labeled, the type of assay (i.e.: in vitro or in vivo), and all other relevant factors, and can be determined by one of skill in the art based on the teachings herein.
  • Detection of binding molecule binding complexes with present in pancreatic neovasculature can be by any suitable means, and will depend at least in part on the means by which the binding molecule is made detectable.
  • pancreatic biopsies can be obtained and detectably labeled binding molecules can be contacted to a biopsy sample under conditions suitable to promote binding complex formation to target in the biopsy sample, and binding complexes can be detected via immunohistochemistry or other suitable technique.
  • the detection means is non-invasive, meaning that detection of the binding molecules does not require obtaining any type of sample (blood, tissue, bone, urine, or saliva) from the patient.
  • Methods for non-invasive detection of binding molecules include magnetic resonance imaging (MRI), positron-emission tomography (PET), single photon emission tomography (SPECT) and ultrasound imaging, including, high-intensity focused ultrasound (HIFU) and contrast-enhanced ultrasound (CEUS).
  • MRI magnetic resonance imaging
  • PET positron-emission tomography
  • SPECT single photon emission tomography
  • ultrasound imaging including, high-intensity focused ultrasound (HIFU) and contrast-enhanced ultrasound (CEUS).
  • HIFU high-intensity focused ultrasound
  • CEUS contrast-enhanced ultrasound
  • binding molecules are present on the surface of microbubbles and are administered to a patient under conditions suitable to promote binding complex formation between target in the pancreas and the binding molecules.
  • Detection then comprises using non-invasive contrast-enhanced ultrasound, to determine the presence or absence of the binding complexes, wherein the binding complex is only detectable in the presence of target and wherein the presence of binding complexes is indicative of the presence of pancreatic cancer, or a risk of pancreatic cancer in the patient.
  • Non-invasive contrast-enhanced ultrasound (as well as other non-invasive detection means) can be performed so as to localize detection to the pancreas. Specifics of methods for performing the non-invasive detection techniques disclosed herein are well known to those of skill in the art.
  • an increased number of the binding complexes compared to control is indicative of the presence of pancreatic cancer in the patient.
  • the results can be used to help direct patient treatment, including but not limited to aggressive chemotherapy and/or radiation treatments and/or surgical resection of the tumor.
  • the pancreatic cancer is pancreatic ductal adenocarcinoma. Median survival of patients with pancreatic ductal adenocarcinoma (PDAC) is less than one year; and thus the earlier detection provided by the methods of the present invention allows earlier surgical resection, which offers the best hope for longer patient survival.
  • PDAC pancreatic ductal adenocarcinoma
  • the presence of the binding complexes is indicative of the presence of precancerous lesions, and thus indicative of a risk of pancreatic cancer development in the patient.
  • Pre-cancerous lesions are associated with a significantly increased risk of cancer.
  • Non-limiting examples include cervical squamous intraepithelial lesion, ductal carcinoma in situ, Bowen's disease, colon polyps, prostatic intraepithelial neoplasia, and pancreatic intraepithelial neoplasia.
  • the pre-cancerous cells are pre-cancerous pancreatic cells, including but not limited to pancreatic intraepithelial neoplasia form (PanIN) 1A-B, PanIN 2, and PanIN 3. These terms as defined as follows:
  • PanIN-1A Pyloric gland metaplasia, goblet cell metaplasia, mucinous hypertrophy, flat duct lesion without atypia, mucinous ductal hyperplasia, simple hyperplasia, mucinous cell hyperplasia, flat ductal hyperplasia, non-papillary epithelial hypertrophy.
  • PanIN-1B Papillary hyperplasia, papillary duct lesion without atypia, and ductal hyperplasia.
  • PanIN-2 Atypical hyperplasia, papillary duct lesion with atypia, low-grade dysplasia, and some cases of moderate dysplasia. Mucous metaplasia and pyloric gland metaplasia commonly involve small branch ducts or extend into lobules surrounding PanIN in ducts. Such involvement has been called adenomatoid or adenomatous hyperplasia, especially when the change dominates involvement of ductal epithelium. It is regarded as part of the spectrum of PanIN-1.
  • PanIN-3 Carcinoma in situ, intraductal carcinoma, high-grade dysplasia, severe dysplasia, and some cases of moderate dysplasia.
  • the results can be used to help direct patient treatment, including but not limited to aggressive chemotherapy and/or radiation treatments to limit development of the lesion into a tumor.
  • an increased number of binding complexes compared to control wherein the binding complexes are multifocal (i.e.: scattered throughout the pancreas) indicates the presence of a precancerous lesion in the patient.
  • an increased number of binding complexes compared to control wherein the binding complexes are focused in the pancreas (i.e.: predominately in a single location within the pancreas) indicates the presence of pancreatic cancer, such as PDAC.
  • the present invention provides methods for determining efficacy of pancreatic cancer therapy in a patient, comprising:
  • the present invention provides methods for determining efficacy of pancreatic cancer therapy in a patient, comprising:
  • a detectable binding molecule selected from the group consisting of (i) MMRN1 binding molecules. (ii) MRC2 binding molecules, (iii) NRP1 binding molecules, and (iv) VCAM1 binding molecules, to a patient undergoing or who has previously undergone pancreatic cancer therapy, under conditions suitable to promote binding complex formation between the binding molecule and its target present in pancreatic tumor neovasculature to form a binding complex; and
  • All embodiments and combinations of embodiments of the first through fifth aspects of the invention can be used in these sixth and seventh aspects.
  • the methods of these aspects of the invention can be used, for example, to assess the growth, regression, or metastasis of the pancreatic tumor, and thus whether a patient being treated for pancreatic cancer is benefitting from therapy, or for monitoring patients who have completed treatment for recurrence of cancer.
  • the Thy-1 or other binding molecule is detectable.
  • a detectable imaging agent selected from the group consisting of a radioactive agent (e.g., radioiodine (125I, 131I); technetium; yttrium; 35S or 3H) or other radioisotope or radiopharmaceutical; a contrast agent (e.g., gadolinium; manganese; barium sulfate; an iodinated or noniodinated agent; an ionic agent or nonionic agent); a magnetic agent or a paramagnetic agent (e.g., gadolinium, iron-oxide chelate); liposomes (e.g., carrying radioactive agents, contrast agents, or other imaging agents); nanoparticles; a positron emitting isotope for PET scanner, MRI contrast agents, and ultrasound agents (e.
  • a radioactive agent e.g., radioiodine (125I, 131I); technetium; yttrium
  • the binding molecules can be administered by any suitable technique, including but not limited to parenterally, transmucosally (orally, nasally, or rectally) or transdermally.
  • Parenteral administration includes, but is not limited to, intravenous, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration.
  • administration of the binding molecules is parenteral, via intravenous injection. This embodiment is particularly preferred when the binding molecules are present on microbubbles, such as the microbubbles of the present invention.
  • the targeted microbubbles accumulate at pancreatic sites that over-express Thy-1 or other marker, causing a local increase in the ultrasound imaging signal.
  • microbubbles with an average diameter of between about 1 ⁇ m and 5 ⁇ m, 1 ⁇ m and 4 ⁇ m, 1 ⁇ m and 3 ⁇ m, 1 ⁇ m and about 2 ⁇ m, 2 ⁇ m and 5 ⁇ m, 2 ⁇ m and 4 ⁇ m, 2 ⁇ m and 3 ⁇ m, 3 ⁇ m and 5 ⁇ m, 3 ⁇ m and 4 ⁇ m, or about as 1 ⁇ m, 2 ⁇ m, 2.5 ⁇ m, 3 ⁇ m, 3.5 ⁇ m or 4 ⁇ m on average are intravenously injected. Due to their small size, the microbubbles stay predominantly within the vascular compartment after intravenous injection. Thus, the microbubbles can be used, for example, to exclusively detect vascular endothelial cell associated molecular markers that are present in early stage cancers or precancerous lesions.
  • condition suitable to promote binding complex formation used in the methods of the invention will depend on the means by which the binding molecule is labeled, the type of assay (i.e.: in vitro or in vivo), and all other relevant factors, and can be determined by one of skill in the art based on the teachings herein).
  • Detection of binding molecule binding complexes with present in pancreatic neovasculature can be by any suitable means, and will depend at least in part on the means by which the binding molecule is made detectable.
  • pancreatic biopsies can be obtained and detectably labeled binding molecules can be contacted to a biopsy sample under conditions suitable to promote binding complex formation to target in the biopsy sample, and binding complexes can be detected via immunohistochemistry or other suitable technique.
  • the detection means is non-invasive, meaning that detection of the binding molecules does not require obtaining any type of sample (blood, tissue, bone, urine, or saliva) from the patient.
  • Methods for non-invasive detection of binding molecules include magnetic resonance imaging (MRI), positron-emission tomography (PET), single photon emission tomography (SPECT) and ultrasound imaging, including, high-intensity focused ultrasound (HIFU) and contrast-enhanced ultrasound (CEUS).
  • MRI magnetic resonance imaging
  • PET positron-emission tomography
  • SPECT single photon emission tomography
  • ultrasound imaging including, high-intensity focused ultrasound (HIFU) and contrast-enhanced ultrasound (CEUS).
  • HIFU high-intensity focused ultrasound
  • CEUS contrast-enhanced ultrasound
  • binding molecules are present on the surface of microbubbles and are administered to a patient under conditions suitable to promote binding complex formation between target in the pancreas and the binding molecules.
  • Detection then comprises using non-invasive contrast-enhanced ultrasound, to determine the presence or absence of the binding complexes, wherein the binding complex is only detectable in the presence of target and wherein the presence of binding complexes is indicative of the presence of pancreatic cancer, or a risk of pancreatic cancer in the patient.
  • Non-invasive contrast-enhanced ultrasound (as well as other non-invasive detection means) can be performed so as to localize detection to the pancreas. Specifics of methods for performing the non-invasive detection techniques disclosed herein are well known to those of skill in the art.
  • PDAC pancreatic ductal adenocarcinoma
  • Thy1 Differentiation Antigen 1
  • Thy1-targeted ultrasound contrast agent was tested in cell culture in a flow chamber assay and in vivo in a novel orthotopic PDAC xenograft model in mice expressing human Thy1 on its neovasculature.
  • Thy1-targeted ultrasound molecular imaging signal significantly increased in human Thy1-positive PDAC xenografts.
  • Thy1 neoangiogenesis target
  • Pancreatic ductal adenocarcinoma is the fourth leading cause of cancer death in both women and men in the USA (1).
  • the American Cancer Society estimated 43,920 new diagnoses of PDAC and 37,390 deaths from this cancer in 2012 in the USA. Survival from pancreas cancer is stage dependent. Because the disease is most frequently detected at advanced tumor stages, patients diagnosed with PDAC have a median survival of less than one year and only 5% of patients survive five years after diagnosis (2, 3). Unfortunately, current chemotherapy and radiotherapy approaches offer only moderate survival benefits and surgery for localized disease is only possible in 15-20% of patients at the time of diagnosis (4, 5). Therefore, earlier detection of PDAC that allows potentially curable surgical resection offers our best hope to improve patient survival (6).
  • One potential strategy for earlier detection of cancer involves screening moderate and high risk patients with a highly accurate and inexpensive blood biomarker test (or combination of biomarkers) followed by a second-level, imaging-based test to confirm a positive biomarker result and anatomically localize the cancer (7).
  • Serum CA-19-9 currently the only clinically used blood biomarker, lacks the sensitivity and, more importantly, the specificity needed to detect early-stage PDAC; active research to discover more accurate blood-based or saliva-based biomarkers is underway (8). Imaging tests are also limited in accuracy for early detection of pancreatic cancer.
  • neovasculature differs from normal blood vessels at the molecular and protein level (11).
  • Molecularly-targeted contrast-enhanced ultrasound is a promising new imaging technique with the potential to detect molecular markers overexpressed in the neovasculature of cancer (12, 13) and potentially increase the sensitivity and specificity of ultrasound in detecting early cancer.
  • micro-sized gas-filled contrast agents microbubbles; MB
  • MB gas-filled contrast agents
  • contrast MB are several microns in size, they remain in the intravascular space and can thus be used to exclusively detect and visualize molecular markers over-expressed on the neovasculature of precursor lesions or early stage cancer.
  • the challenge for ultrasound molecular imaging is to discover and validate imaging targets that are differentially expressed on the vasculature of PDAC versus normal pancreatic tissue and benign diseases, such as chronic pancreatitis, to maximize diagnostic accuracy in early cancer detection (13).
  • Thy1 thymocyte differentiation antigen 1
  • FIG. 1 The overall experimental set up of this study is summarized in FIG. 1 .
  • LTQ-OrbitrapTM hybrid mass spectrometer Thermo Fisher Scientific, Waltham, Mass.
  • nano-flow HPLC Eksigent Technologies, Dublin, Calif.
  • neovascular proteins were further triaged based on 1) the highest expression in cancer; 2) lack of or low expression in chronic pancreatitis tissue; 3) membrane association of the protein; and 4) assessment of protein expression in normal tissues using the Human Protein Atlas and/or published literature in PubMed, with lack of expression in normal organs being preferred.
  • the highest rated candidate was the membrane protein Thy1 (17). Selection of Thy1 was further supported by its association with tumor vascular endothelium as previously described (18, 19).
  • pancreatic tissue microarray was purchased from USBioMaxTM (Rockville, Md.) Immunohistochemical (IHC) analysis of Thy1 expression was performed in pancreatic tissue obtained from 4 normal patients; 15 primary chronic pancreatitis tissues (defined as chronic pancreatitis not associated with PDAC); 21 PDAC; and in a commercial tissue microarray with 24 normal pancreatic tissues and 175 PDAC cases. Consecutive tissue sections were stained for the vascular endothelial cell marker CD31 and for Thy1 using standard techniques (Supplementary Materials and Methods). All slides were reviewed and graded by a pathologist, experienced in pancreatic pathology.
  • Vascular endothelial cell staining of Thy1 was scored with a semi-quantitative IHC score from 0 to 3+ as previously described 20.
  • cases with Thy1-staining of less than 5%, 5-32%, 33-67%, and greater than 67% of CD31 positive vessels were scored as 0, 1+, 2+, and 3+, respectively.
  • Murine vascular endothelial (MS1) cells stably expressing human Thy1 on the cell surface were generated using standard protocols (Supplementary Materials and Methods). Stably-transfected cells (selected by incubation with 5 ⁇ g/ml puromycin; Sigma, St. Louis, Mo.) were confirmed for Thy1 expression by flow cytometry analysis and by immunofluorescence staining using standard techniques (Supplementary Materials and Methods). For subsequent flow chamber experiments (see below), two clones with high (clone 1) and low (clone 2) human Thy1 expression were selected. Clone 1 was also used for the generation of a novel human PDAC xenograft model in mice expressing human Thy1 on its neovasculature (see below).
  • Human Thy1-targeted (MBThy1) and control (MBControl) contrast MB were prepared by attaching anti-human Thy1 antibody or isotype-matched control IgG antibody onto the surface of perfluorocarbon-containing, lipid-shelled MB as described previously 21 (Supplementary Materials and Methods).
  • Flow cytometry analysis incubation of targeted MB with Fluorescein-conjugated anti-biotin antibody; Jackson ImmunoResearch, 1:200) showed that the average number of attached antibodies per square micrometer of the MB surface was approximately 7,600 for both MB types.
  • MBThy1 binding to clones 1 and 2 was assessed in cell culture experiments under flow shear stress conditions, simulating flow in tumor capillaries, using a previously described protocol (22, 23) (Supplementary Materials and Methods).
  • pancreas of the mice was exposed, and AsPC1 cells along with clone 1 cells at 1:5 ratio (total of 6 ⁇ 10 6 cells, dissolved in 25 ⁇ l of MatrigelTM containing epidermal growth factor (0.7 ng/mL), insulin-like growth factor (16 ng/ml), and transforming growth factor-beta (2.3 ng/ml); BD Biosciences, San Jose, Calif.) co-injected into the body or tail of the pancreas in 25 female nude mice (6-8 weeks old; Charles River, Wilmington, Mass.) into the body or tail of the pancreas.
  • Ultrasound molecular imaging was analyzed offline using dedicated software with motion compensation capabilities (VevoCQ, Visualsonics; Toronto, Canada).
  • the imaging signal (expressed in arbitrary units, a.u.) from attached MB was defined as the difference between pre- and post-destruction imaging signals as described 23. Regions of interest were draws over the different tumors and over adjacent normal pancreas tissue by one reader, blinded to the types of MB (MBThy1 vs. MBControl) and tumor (positive vs. control tumors).
  • Thy1 IHC scores were performed using GraphPad Prism (La Jolla, Calif.); the remainder of the statistical analyses was performed with R 2.10.1 with a significance level of 0.05.
  • Thy1 Expression in Human Pancreatic Tissues
  • the expression of Thy1 was essentially restricted to the neovasculature associated with PDAC, with occasional staining of the peritumoral stromal compartment. There was no expression within the neoplastic epithelium in most of the cases; therefore, only vascular staining (guided by CD31 staining) was evaluated.
  • IHC scores of 2+ or 3+ as positive staining
  • Mouse neovascular endothelial cells do not express human Thy1, thus to recapitulate the protein expression of Thy1 in human PDAC neovasculature, we created two mouse vascular endothelial cell lines with different expression levels of human Thy1 (clone 1 and clone 2) and confirmed the Thy1 expression levels by fluorescence microscopy ( FIG. 3A ), and FACS analysis ( FIG. 3B , C).
  • Table 5 summarizes binding of MBThy1 and MBControl to clone 1 (high human Thy1 expression), clone 2 (low human Thy1 expression) and to wild-type vascular endothelial cells (no Thy1 expression) in flow chamber experiments.
  • MBControl only demonstrated background attachment to all three cell types without statistically significant difference of MBControl attachment among the three different cell types (P>0.5).
  • AsPC1 only control tumors 1.7 ⁇ 1.6 1.4 ⁇ 0.6 0.68
  • Ultrasound fulfills many prerequisites for becoming a promising imaging tool for early cancer detection: It is noninvasive and inexpensive compared to other imaging modalities; does not use ionizing irradiation; has a very high spatial and temporal (real-time exam) resolution (26-28); provides deep tissue penetration (e.g., compared to optical based approaches); and is routinely available in almost all clinical imaging departments worldwide.
  • Several morphological imaging criteria of pre-invasive or early PDAC have been described on endoscopic ultrasound, including parenchymal heterogeneity, echogenic foci, and hypoechogenic nodules (29).
  • the goal of this proof-of-principle study was to discover and validate a PDAC associated protein that can be used as an imaging target for ultrasound molecular imaging of the pancreas and that may increase diagnostic accuracy of ultrasound in earlier detection of PDAC. Since most current ultrasound contrast agents remain exclusively within the vascular compartment, molecular imaging targets for ultrasound need to be expressed on the luminal site of vascular endothelial cells of the pancreatic neovasculature. Through quantitative proteomic analysis and several prioritizing steps, we identified Thy1, a membrane protein, as a promising new tissue marker for ultrasound molecular imaging expressed on PDAC-associated neovasculature.
  • Thy1 also known as cluster of differentiation 90 (CD90) is a cell-surface glycoprotein that belongs to the immunoglobulin-like supergene family (31). While Thy1 was originally described as a marker for thymocyte differentiation in mice (32), it was later found to be expressed on various other tissues 15 and up-regulated on the surface of newly formed blood vessels (33).
  • Our immunohistochemical validation study demonstrated positive Thy1 expression on the neovasculature of 81% of human pancreatic adenocarcinoma samples while there was minimal Thy1 staining in normal pancreatic tissue. Chronic pancreatitis samples showed marginally higher Thy1 staining than normal pancreas, but significantly lower Thy1 staining than PDAC.
  • Thy1 staining in chronic pancreatitis cases is expected because Thy1 expression had been associated with inflammatory tissues in previous studies. For example, in rat models of inflammation including a renal ischemia model following renal artery ligation, and in a balloon injury model of the carotid arteries, Thy1 has been shown to be overexpressed on angiogenic vessels (37).
  • Thy1 vascular microvessel exposure with necrosis factor- ⁇ and interleukin-1 ⁇ stimulated cellular Thy1 expression, suggesting that inflammatory cytokines may increase Thy1 expression (37).
  • Chronic pancreatitis is characterized by fibroinflammatory changes to the pancreatic tissue with variable extents of superimposed acute inflammatory changes depending on the etiology and the stage of the disease (38). Such active inflammation may have stimulated Thy1 expression in some of our chronic pancreatic cases.
  • Thy1 After identification and validation of Thy1 as a promising molecular imaging target for PDAC, we designed a new contrast MB targeted to human Thy1.
  • Embedding spheroids containing human umbilical vein endothelial cells in a Matrigel-fibrin matrix has been previously shown to generate a functional three-dimensional human vascular network in severe combined immunodeficiency mice (39, 40).
  • MS1 cells are murine pancreatic microvasculature cells transduced with a temperature-sensitive simian virus 40 large T antigen (41). When injected into immunocompromised mice, these cells form benign, maximum 2-mm3 hemangiomas (41).
  • this new mouse model may provide versatility for in vivo testing of neoangiogenesis-targeted ultrasound contrast agents in mice by simply replacing the human Thy1 gene by any other human-specific receptor gene.
  • Thy1-positive tumors in the pancreas injection of MBThy1 resulted in an about 4-5.5-fold higher imaging signal compared to control conditions. While the goal of our study was not to determine the smallest detectable size of PDAC, Thy1 expressing tumors down to a size of 100 mm 3 could be visualized with ultrasound molecular imaging.
  • Immunohistochemistry was performed on standard serial sections of paraffin-embedded pancreatic tissue slices and microarrays. Briefly, prior to incubation with the primary antibody, sections were pre-treated with CC1 antigen retrieval solution (Ventana, Arlington, Ariz.). Thereafter, primary antibody to human CD31 (no dilution; Ventana Medical Systems, catalog 760-4378) and human Thy1 (1:100 dilution; Novus Biologicals, catalog NBP1-42068) was applied at 37° C. for 16 min followed by counter staining by Hematoxylin. Labeling was performed on an ES automatic immunohistochemical stainer (Ventana Medical Systems). Slides were imaged using Aperio Imagescope.
  • the human Thy1 DNA sequence (gi
  • Thy1 was then subcloned into pcDNA3.1 (Invitrogen), modified by substitution of CMV for an ubiquitin promoter and neomycin for puromycin resistance selection gene.
  • Thy1-pcDNA was amplified in Escherichia coli and isolated using the Pure-YieldTM Maxi Prep Kit (Promega, Madison, Wis.) according to manufacturer instructions.
  • Murine pancreatic vascular endothelial cells [MS 1; ATCC, Manassas, Va.; cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 ⁇ g/mL; Invitrogen)] were transfected with 10 ⁇ g of Thy1-pcDNA3.1 ubi/puro-plasmid or with empty control vector (no human Thy1 gene; henceforth control wild-type cells) using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions.
  • DMEM Dulbecco's modified Eagle's medium
  • Thy1 primary antibody eBioscience; at 1:100
  • Jackson ImmunoResearch 1:200
  • Thy1 expression on vascular endothelial MS1 cells immunofluorescence staining of the cells was performed using standard techniques.
  • wild-type and Thy-1 expressing cells were grown on cover slips under standard conditions in DMEM complete growth media for 24 hours; after the media was removed, cells were washed in PBS and fixed in 4% paraformaldehyde in PBS solution for 30 min at room temperature. Cells were then washed in PBS, and 1% bovine serum albumin (BSA) blocking solution was applied for one hour.
  • Primary antibody (rabbit anti-human Thy1, Sigma, 1:100) incubation was performed overnight at 4′C.
  • Perfluorocarbon-filled, lipid-shelled, streptavidin-coated contrast microbubbles were reconstituted in 1 mL sterile saline (0.9% sodium chloride) according to the manufacturer's protocol.
  • the mean diameter of the microbubbles as assessed by a cell counter and sizer was 1.5 ⁇ 0.1 ⁇ m (range, 1-2 ⁇ m).
  • Targeted MB were prepared by mixing 6 ⁇ g of biotinylated antibodies to 5 ⁇ 10 7 streptavidin-coated MB for 10 min at room temperature.
  • Microbubbles targeted to human Thy1 MB Thy1
  • biotinylated mouse anti-human Thy1 monoclonal antibodies eBiosciences; San Diego, Calif.
  • control MB MB Control
  • IgG antibody eBioscience, San Diego, Calif.
  • the three cell types (clone 1 and 2 as well as the wild-type negative control vascular endothelial cells) were grown on neutral-charged glass microscope slides triple-coated with Sigmacote® (Sigma, Mo.) for 48 hours, then mounted on a parallel plate flow chamber (GlycoTech Corporation, Rockville, Md.).
  • a syringe infusion/withdrawal pump (Genie PlusTM, Kent Scientific Corporation, Torrington, Conn.) was used to pass solutions over the cells at a flow rate of 0.6 ml/min, corresponding to a wall shear stress rate of 100 sec ⁇ 1 similar to that in capillaries 2 .
  • Thy1-positive cells were pre-incubating Thy1-positive cells with mouse anti-human Thy1 monoclonal antibody (eBiosciences, San Diego, Calif.; 30 ⁇ g/ml growth medium) for 30 min at 37° C. All experiments performed under different conditions were performed in triplicates.
  • mice were anesthetized with 2% isoflurane in room air (2 L/min) during scanning.
  • Ultrasound molecular imaging was performed using a dedicated small animal ultrasound machine (Vevo 2100; VisualSonics, Toronto, Canada). Images were acquired in a transverse plane with a high-frequency transducer (MS250; center frequency of 21 MHz; lateral and axial resolution of 165 ⁇ m and 75 ⁇ m, respectively; focal length, 10 mm; transmit power, 4%; mechanical index, 0.2; dynamic range, 40 dB).
  • the acoustic focus was centered at the level of the PDAC xenografts with the imaging plane aligned in the center of the tumor. The same imaging setting was used in all imaging sessions.
  • mice Prior to tumor excision, mice were perfused in vivo with 4% paraformaldehyde at a rate of 4 mL/min for 5 minutes. Mice were then sacrificed and the tumors excised, fixed in 4% paraformaldehyde at 4° C. for 24 hours and transferred into 30% sucrose solution at 4° C. for another 24 hours. Tumors were cryosectioned (slice thickness of 10 ⁇ m) and sections were analyzed from the center of the tumor that approximated the corresponding imaging plane from ultrasound imaging. Incubation in 5% normal goat serum in PBS for 30 min was performed to block nonspecific proteins.
  • Sections were simultaneously incubated for 12 hours (4° C.) with 1:100 each of primary antibodies: rabbit anti-human Thy1 antibody (Sigma) and rat anti-mouse CD31 (BD Pharmingen). Secondary antibodies (goat anti-rabbit AlexaTM Fluor 488 antibody and donkey anti-rat AlexaTM Fluor 594 antibody; Invitrogen) were simultaneously applied at 1:600 dilutions in PBS for 30 minutes at room temperature to confirm Thy1 expression on the tumor neovasculature.
  • Fluorescent images were acquired using MetamorphTM software (Universal Imaging Corp., West Chester, Pa.) and a Zeiss AxioscopeTM microscope (Axiophot, Carl Weiss AG, Thornwood, N.Y.) attached to a digital camera (AxioCamTM MRc, Bernried, Germany).
  • MMRN1 multimerin 1
  • MRC2 mannose receptor type C2
  • NPP1 neuropilin 1
  • VCAM1 VCAM1

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Publication number Priority date Publication date Assignee Title
US10159711B2 (en) 2010-04-16 2018-12-25 Salk Institute For Biological Studies Methods for treating metabolic disorders using FGF
US10293027B2 (en) 2010-04-16 2019-05-21 Salk Institute For Biological Studies Methods for treating metabolic disorders using FGF
US10398759B2 (en) 2010-04-16 2019-09-03 Salk Institute For Biological Studies Methods for treating metabolic disorders using FGF
US10695404B2 (en) 2015-10-30 2020-06-30 Salk Institute For Biological Studies Treatment of steroid-induced hyperglycemia with fibroblast growth factor (FGF) 1 analogs
WO2017214050A1 (fr) * 2016-06-08 2017-12-14 The Board Of Trustees Of The Leland Stanford Junior University Dépistage du cancer du pancréas à l'aide d'un anticorps à chaîne unique anti-thy1 modifié
WO2018018010A1 (fr) * 2016-07-21 2018-01-25 Metacrine, Inc. Mutants de fgf et leurs utilisations
US11542309B2 (en) 2019-07-31 2023-01-03 Salk Institute For Biological Studies Fibroblast growth factor 1 (FGF1) mutant proteins that selectively activate FGFR1B to reduce blood glucose

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