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WO2008097229A1 - Procédé de quantification en spectroscopie d'her-2 dans des échantillons biologiques - Google Patents

Procédé de quantification en spectroscopie d'her-2 dans des échantillons biologiques Download PDF

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Publication number
WO2008097229A1
WO2008097229A1 PCT/US2007/003478 US2007003478W WO2008097229A1 WO 2008097229 A1 WO2008097229 A1 WO 2008097229A1 US 2007003478 W US2007003478 W US 2007003478W WO 2008097229 A1 WO2008097229 A1 WO 2008097229A1
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seq
peptide
cysteine
free
fragment
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Thomas P. Conrads
Timothy D. Veenstra
Brian L. Hood
David B. Krizman
Marlene M. Darfler
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US Department of Health and Human Services
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US Department of Health and Human Services
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Definitions

  • FIELD This disclosure relates, to the field of diagnostic testing. More specifically, this disclosure relates to methods of detecting and determining certain characteristics of cancer using mass spectrometric detection of HER-2 peptides. This disclosure also relates to peptide standards and their use in quantitative mass spectrometric analyses.
  • Cancer is the second leading cause of death in the United States, only exceeded by heart disease. Cancer is characterized as an abnormal state in which uncontrolled proliferation of one or more cell populations interferes with normal biological functioning. The transformation of a normal cell into a cancer cell can result from amplification in the number of copies of a proto-oncogene, which in turn can result in overproduction of the protein and its concomitant effects. Amplified proto-oncogenes have been found in subjects with cancer.
  • the proto-oncogene encoding the human epidermal growth factor receptor-2 (HER- 2 or ErbB2) has been found to be amplified in a range of tumor types including colon, prostate, stomach, thyroid, ovarian, bladder, salivary gland, endometrial, pancreatic, renal, and non-small-cell lung cancer (NSCLC).
  • NSCLC non-small-cell lung cancer
  • HER-2 is a transmembrane protein of approximately 185 kilodaltons and is a member of the ErbB family of receptor tyrosine kinases. This family of receptor tyrosine kinases are important mediators of cell growth, differentiation, and survival. It is believed that HER-2 overexpression leads to tumor growth via ligand-independent activation of the HER-2 intracellular kinase domain.
  • anti-HER-2 monoclonal antibodies have been produced to specifically antagonize the function of the HER-2 receptor in HER-2-positive tumors.
  • the most notable of these anti-HER-2 antibodies is the recombinant humanized version of the murine anti-HER-2 antibody 4D5 (trastuzumab, huMAb4D5-8, rhuMAb HER-2, and the subject of U.S. Patent. Nos. 5,821,337 and 5,720,954, the specifications of which are incorporated herein by reference in their entirety) marketed under the trade name HERCEPTINTM.
  • Trastuzumab has been shown to be clinically active in patients with HER- 2 overexpressing metastatic breast cancers that have received extensive prior anti-cancer therapy.
  • anti-HER-2 MAbs such as trastuzumab
  • trastuzumab suppress the proliferation of ovarian, gastric and NSCLC cell lines that overexpress the HER-2 receptor.
  • the prevalence of HER-2 overexpression and/or gene amplification in various tumor types raises the possibility of using anti-HER-2 MAbs to antagonize the abnormal function of overexpressed HER-2 receptors in HER-2-positive tumors other than breast.
  • trastuzumab offers tremendous therapeutic benefits to cancer subjects diagnosed with HER-2 positive tumors.
  • FISH fluorescent in situ hybridization
  • IHC immunohistochemistry
  • serum HER-2 tests measure circulating levels of the shed extracellular domain of HER-2, not gene amplification or overexpression on the surface of tumor cells, and therefore may not be a true measure of the amount of cellular HER-2. Due to the difficulties in the current methods used to diagnosis HER-2 positive cancers, there is a need for improved methods of detecting HER-2 in biological samples, especially histopathologically processed tumor tissue.
  • the disclosed methods are useful in determining if a subject has HER-2 associated cancer.
  • the disclosed methods include obtaining a biological sample from a subject, digesting the biological sample with a protein cleavage agent (such as a serine protease, for example trypsin), optionally chromatographing the sample, and detecting a cysteine-free HER-2 fragment peptide in the protein digest.
  • a protein cleavage agent such as a serine protease, for example trypsin
  • chromatographing the sample optionally chromatographing the sample
  • detecting a cysteine-free HER-2 fragment peptide in the protein digest indicates the presence of HER-2 associated cancer.
  • the cysteine-free HER-2 fragment peptide is detected by mass spectrometry.
  • the cysteine-free HER-2 fragment peptide is detected by the detection of fragment ions of the cysteine-free HER-2 fragment peptide, for example using tandem mass spectrometry.
  • Cysteine residues are relatively chemically reactive and can undergo oxidation prior to detection by mass spectrometry leading to peptides of varying masses.
  • cysteine residues readily undergo side reactions such as disulfide formation that results in products having differing masses.
  • cysteine- free HER-2 fragment peptides are particularly useful for determining the presence of a HER-2 in a sample by mass spectrometry.
  • such methods include comparing an amount of the cysteine-free HER-2 fragment peptide to an optionally isotopically labeled cysteine-free peptide standard of known amount.
  • Peptide standards for use in quantitating HER-2 in a biological sample are also disclosed.
  • Such peptide standards contain a cysteine- free peptide corresponding to about 8 to about 45 amino acid residues of HER-2 and are optionally isotopically labeled.
  • the disclosed methods can be used to determine the progression of the cancer, for example by correlating the detected amount of the cysteine-free HER-2 fragment peptide to progression of the cancer.
  • the disclosed methods can be used to select a treatment based the presence or absence of the cysteine-free HER-2 fragment peptide in the protein digest.
  • the presence of a cysteine-free HER-2 fragment peptide in the protein digest indicates that the subject can be treated with anti-HER-2 therapy, such as an anti-HER-2 antibody therapy.
  • Fig. 1 is a set of four representative mass spectra obtained from a tryptic digest of HER-2.
  • Fig. 2 is a representative tandem mass spectrum showing the fragment ions of the HER-2 fragment peptide of interest (SGGDLTLGLEPS EEEAPR, SEQ ID NO: 14) at m/z 914.4, 1043.5, and 1213.6.
  • Fig. 3 is a set of four representative mass spectra obtained from a tryptic digest of HER-2 showing that the HER-2 fragment peptide SGGDLTLGLEPSEEEAPR (SEQ ID NO: 14) can be accurately measured without ambiguity using a combination of parent and/or fragment ions.
  • Fig. 4 is a set of three representative mass spectra of a tryptic digest of a sample obtained from a subject's tumor biopsy after LC/MS/MS analysts.
  • Fig. 5 is a representative tandem mass spectrum of a tryptic digest of a sample obtained from a subject's tumor biopsy showing the fragment ions from HER-2 fragment peptide SGGDLTLGLEPSEEEAPR (SEQ ID NO: 14).
  • Fig 6 is a set of three tandem mass spectra showing the intact HER-2 fragment peptide SGGDLTLGLEPSEEEAPR (SEQ ID NO: 14) and fragment ions of the HER-2 fragment peptide SGGDLTLGLEPSEEEAPR (SEQ ID NO.14).
  • amino acid sequences listed in the accompanying sequence listing are shown using standard three letter abbreviations for amino acids, as defined in 37 C.F.R. ⁇ 1.822.
  • SEQ ID NOs: 1—18 are exemplary amino acid sequences corresponding to portions of the amino acid sequence of HER-2.
  • SEQ ID NOs: 19-20 are exemplary amino acid sequences of human HER-2.
  • AMU atomic mass unit
  • CAD/CAM computer-aided design/computer-aided machining
  • CCD charge-coupled device
  • EI electron-impact ionization
  • ESI electrospray ionization
  • FISH fluorescent in situ hybridization
  • FT-ICR Fourier-transform ion cyclotron resonance
  • HER-2 or ErbB2 Human epidermal growth factor receptor 2 HPLC: high performance liquid chromatography
  • MAbs monoclonal antibodies
  • MALDI matrix-assisted laser desorption-ionization
  • MARS multiple affinity removal system
  • MS/MS tandem mass spectrometry
  • nano-RPLC nano-reversed-phase liquid chromatography
  • NSCLC non-small-cell lung cancer
  • PAGE poly acrylamide gel electrophoresis
  • Q quadrupole mass analyzer
  • Administering refers to the introduction of a composition into a subject by a chosen route. For example, if the chosen route is intravenous, the composition is administered by introducing the composition into a vein of the subject.
  • Biological sample Any solid or fluid sample obtained from, excreted by or secreted by any living organism, including without limitation, multicellular organisms
  • a biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a normal joint or a joint affected by disease such as a rheumatoid arthritis, osteoarthritis, gout or septic arthritis).
  • a biological sample can also be a sample obtained from any organ or tissue (including a biopsy or autopsy specimen, such as a tumor biopsy) or can comprise a cell (whether a primary cell or cultured cell) or medium conditioned by any cell, tissue or organ.
  • a biological sample can also be a sample which has been chemically treated, for example a sample which is, fixed (such as fixed in formalin or any other chemical fixative known in the art) and/or paraffin embedded.
  • Chromatographing The process of separating a mixture. It involves passing a mixture through a stationary phase, which separates molecules of interest from other molecules in the mixture and allows it to be isolated.
  • methods of chromatographic separation include capillary-action chromatography such as paper chromatography, thin layer chromatography (TLC), column chromatography, fast protein liquid chromatography (FPLC), nano-reversed phase liquid chromatography, ion exchange chromatography, gel chromatography such as gel filtration chromatography, size exclusion chromatography, affinity chromatography, high performance liquid chromatography (HPLC), and reverse phase high performance liquid chromatography (RP-HPLC) amongst others.
  • TLC thin layer chromatography
  • FPLC fast protein liquid chromatography
  • ion exchange chromatography ion exchange chromatography
  • gel chromatography such as gel filtration chromatography, size exclusion chromatography, affinity chromatography, high performance liquid chromatography (HPLC), and reverse phase high performance liquid chromatography (RP-HPLC)
  • corresponding is a relative term indicating similarity in position, purpose or structure.
  • a peptide standard "corresponding" to a HER-2 amino acid sequence has an amino acid sequence identical to a portion of HER-2 (regardless of whether or not they have the same mass).
  • Such a "peptide standard” can be used to quantitate the amount of a fragment peptide of identical sequence, such as a HER-2 fragment peptide.
  • mass spectral signals in a mass spectrum that are due to corresponding peptides of identical structure but differing masses are "corresponding" mass spectral signals.
  • a mass spectral signal due to a particular peptide is also referred to as a signal corresponding to the peptide.
  • DNA deoxyribonucleic acid
  • DNA is a long chain polymer which comprises the genetic material of most living organisms (some viruses have genes comprising ribonucleic acid (RNA)).
  • the repeating units in DNA polymers are four different nucleotides, each of which comprises one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached.
  • Triplets of nucleotides (referred to as codons) code for each amino acid in a polypeptide, or for a stop signal.
  • codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.
  • any reference to a DNA molecule is intended to include the reverse complement of that DNA molecule.
  • Expression The process whereby the genetic information contained in a nucleotide sequence is converted into other cellular components, such as mRNA and protein. Generally, expression of a nucleotide sequence takes place within a cell, but can also take place in a cell-free system.
  • Fragment peptide A peptide generated by proteolytic cleavage of a protein or polypeptide with a protein cleavage agent, for example in a protein digest.
  • proteolytic peptides include peptides produced by treatment of a protein with one or more endoproteases such as trypsin, chymotrypsin, endoprotease ArgC, endoprotease aspN, endoprotease gluC, and endoprotease lysC, as well as peptides produced by cleavage using chemical agents, such as cyanogen bromide, formic acid, and thiotrifluoroacetic acid.
  • One or more cleavage peptides from a particular protein can be mass identifiers for the protein.
  • a fragment peptide is a HER-2 fragment peptide.
  • HER-2 (also known as neu and ErbB2) is a member of the epidermal growth factor receptor (EGFR) family and is implicated in the pathogenesis of cancer. It is a cell membrane surface-bound tyrosine kinase and is involved in the signal transduction pathways leading to cell growth and differentiation. Exemplary amino acid sequences of HER-2 can be found at GENBANK® accession numbers NP_004439 and NM_001005862 as available November 18, 2006.
  • HER-2 is composed of an extracellular portion from about residue number 23 to about residue number 653 of SEQ ID NO: 19, a transmembrane portion from about residue number 653 to about residue number 675 of SEQ ID NO: 19, and a cytoplasmic portion from about residue number 676 to about residue number 1255 of SEQ ID NO: 19.
  • the kinase domain is contained within the cytoplasmic portion of HER-2 and includes from about residue number 720 to about residue number 987 of SEQ ID NO: 19.
  • the amino acid sequence given as GENBANK® accession number NP_004439 is set forth below as SEQ ID NO: 19.
  • amino acid sequence given as GENBANK® accession number NM OO 1005862 is set forth below as SEQ ID NO:20.
  • a HER-2 fragment peptide includes an amino acid sequence as set forth as SEQ ID NOs: 1-29.
  • HER-2 fragment peptides can be used as mass identifiers for HER-2 to identify the presence of HER-2 in a sample, such as a sample obtained from a subject.
  • Host cell A host cell is a cell that is used to express a nucleic acid sequence coding for a peptide standard. Examples of host cells include microorganisms such as bacteria, protozoans, yeast, viruses and algae, and cultured cells such as cultured human, porcine and murine cell lines.
  • Isolated An "isolated" biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs or is transgenically expressed, that is, other chromosomal and extrachromosomal DNA and RNA, and proteins.
  • Nucleic acids, peptides and proteins which have been "isolated” thus include nucleic acids and proteins purified by standard or non-standard purification methods.
  • the term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized peptides and nucleic acids.
  • Isotopic analog refers to a molecule that differs from another molecule in the relative isotopic abundance of an atom it contains. For example, peptide sequences containing identical sequences of amino acids, but differing in the isotopic abundance of an atom, are isotopic analogs of each other.
  • the term "isotopic analog” is a relative term that does necessarily not imply that the isotopic analog necessarily contains an isotope that is present in less or greater abundance in nature. For example, a mass identifier containing a natural abundance of 12 C and 13 C is an isotopic analog of a corresponding mass identifier having non-natural abundances of these isotopes, and vice versa.
  • Isotopically-labeled or labeled “Isotopically-labeled” or “labeled” refer to a molecule that includes one or more stable heavy isotopes in a greater-than-natural abundance. Heavy stable isotopes include, for example 2 H, 13 C, 15 N, 34 S, 17 O, and 18 O.
  • Mass spectrometry is a method wherein, a sample is analyzed by generating gas phase ions from the sample, which are then separated according to their mass-to-charge ratio (m/z) and detected.
  • Methods of generating gas phase ions from a sample include electrospray ionization (ESI), matrix-assisted laser desorption-ionization (MALDI), surface-enhanced laser desorption-ionization (SELDI), chemical ionization, and electron-impact ionization (EI).
  • ESI electrospray ionization
  • MALDI matrix-assisted laser desorption-ionization
  • SELDI surface-enhanced laser desorption-ionization
  • EI electron-impact ionization
  • Separation of ions according to their m/z ratio can be accomplished with any type of mass analyzer, including quadrupole mass analyzers (Q), time-of-flight (TOF) mass analyzers, magnetic sector mass analyzers, 3D and linear ion traps (IT), Fourier-transform ion cyclotron resonance (FT-ICR) analyzers, and combinations thereof (for example, a quadrupole-time-of-flight analyzer, or Q-TOF analyzer).
  • Q quadrupole mass analyzers
  • TOF time-of-flight
  • IT linear ion traps
  • FT-ICR Fourier-transform ion cyclotron resonance
  • the sample Prior to separation, the sample may be subjected to one or more dimensions of chromatographic separation, for example, one or more dimensions of liquid or size exclusion chromatography.
  • Nucleotide A base, such as a pyrimidine, purine, or synthetic analogs thereof, linked to a sugar, plus a phosphate, which forms one monomer in a polynucleotide.
  • a nucleotide sequence refers to the sequence of bases in a polynucleotide.
  • Oligonucleotide or "oligo” Multiple nucleotides (that is, molecules comprising a sugar (for example, ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (Py) (for example, cytosine (C), thymine (T) or uracil (U)) or a substituted purine (Pu) (for example, adenine (A) or guanine (G)).
  • oligonucleotide refers to both oligoribonucleotides and oligodeoxyribonucleotides. Oligonucleotides can be obtained from existing nucleic acid sources (for example, genomic or cDNA), but are preferably synthetic (that is, produced by oligonucleotide synthesis).
  • Peptide/Protei ⁇ /Polypeptide All of these terms refer to a polymer of amino acids and/or amino acid analogs that are joined by peptide bonds or peptide bond mimetics.
  • the twenty naturally-occurring amino acids and their single-letter and three-letter designations are as follows:
  • Predictable mass difference is a difference in the molecular mass of two molecules or ions (such as two peptides, peptide ions) that can be calculated from the molecular formulas and isotopic contents of the two molecules or ions. Although predictable mass differences exist between molecules or ions of differing molecular formulas, they also can exist between two molecules or ions that have the same molecular formula but include different isotopes of their constituent atoms. A predictable mass difference is present between two molecules or ions of the same formula when a known number of atoms of one or more type in one molecule or ion are replaced by lighter or heavier isotopes of those atoms in the other molecule or ion.
  • replacement of a 12 C atom in a molecule with a 13 C atom provides a predictable mass difference of about 1 atomic mass unit (amu)
  • replacement of a 14 N atom with a 15 N atom provides a predictable mass difference of about 1 amu
  • replacement of a 1 H atom with a 2 H provides a predictable mass difference of about 1 amu.
  • Such differences between the masses of particular atoms in two different molecules or ions are summed over all of the atoms in the two molecules or ions to provide a predictable mass difference between the two molecules or ions.
  • the predictable mass difference between the two molecules is about 6 amu (1 amu difference/carbon atom).
  • Prognosis The probable course or outcome of a disease process.
  • the prognosis of a subject with cancer can indicate the likelihood of survival and/or the likelihood of metastasis.
  • the prognosis of a subject with cancer can indicate the likelihood that the subject will survive for a period of time, such as about one, about two, about three, about four, about five or about ten years.
  • the prognosis of a subject with cancer can also indicate the likelihood of a cure, of the likelihood that the subject will remain disease-free following treatment for a period of time, such as about one, about two, about three, about four, about five or about ten years.
  • proteolytic enzymes include endoproteases such as trypsin, chymotrypsin, e ⁇ doprotease ArgC, endoprotease aspN, endoprotease gluC, and endoprotease lysC.
  • chemical protein cleavage agents include cyanogen bromide, formic acid, and thiotrifluoroacetic acid. The specific bonds cleaved by an endoprotease or a chemical protein cleavage agents may be more specifically referred to as "endoprotease cleavage sites" and "chemical protein cleavage agent sites,” respectively.
  • Proteins typically contain one or more intrinsic protein cleavage agent sites that are recognized by one or more protein cleavage agents by virtue of the amino acid sequence of the protein.
  • Standard A standard is a substance or solution of a substance of known amount, purity or concentration. A standard can be compared (such as by spectrometric, chromatographic, or spectrophotometric analysis) to an unknown sample (of the same or similar substance) to determine the presence of the substance in the sample and/or determine the amount, purity or concentration of the unknown sample.
  • a standard is a peptide standard.
  • An internal standard is a compound that is added in a known amount to a sample prior to sample preparation and/or analysis and serves as a reference for calculating the concentrations of the components of the sample.
  • Isotopically-labeled peptides are particularly useful as internal standards for peptide analysis since the chemical properties of the labeled peptide standards are almost identical to their non-labeled counterparts. Thus, during chemical sample preparation steps (such as chromatography, for example, HPLC) any loss of the non-labeled peptides is reflected in a similar loss of the labeled peptides.
  • Therapeutically effective amount A quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to inhibit or suppress growth of a tumor. In one embodiment, a therapeutically effective amount is the amount necessary to eliminate a tumor.
  • a dosage When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in tumors) that has been shown to achieve a desired in vitro effect.
  • Treating Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as cancer.
  • Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.
  • the term "ameliorating,” with reference to a disease or pathological condition refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to a particular cancer.
  • a "prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.
  • Tumor The product of neoplasia is a neoplasm (a tumor), which is an abnormal growth of tissue that results from excessive cell division.
  • a tumor that does not metastasize is referred to as "benign.”
  • a tumor that invades the surrounding tissue and/or can metastasize is referred to as "malignant.”
  • a tumor can be a primary tumor meaning it has originated in the same organ in which it is present, and has not metastasized to it.
  • a tumor can be a secondary tumor, meaning that it has migrated away from the original organ (site of a primary tumor). Secondary tumors are also referred to as metastatic tumors.
  • a malignant tumor is generally classified as cancer.
  • hematological cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblasts, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, and myelodysplasia.
  • acute leukemias such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia
  • solid cancers such as sarcomas and carcinomas
  • solid cancers include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, bladder carcinoma, and CNS tumors
  • HER-2 Human epidermal growth factor receptor 2
  • HER-2 positive breast cancer is a more aggressive disease with a greater likelihood of recurrence, a poorer prognosis, and a decreased chance of survival. It has been estimated that HER-2 positive breast cancer cases account for approximately 25—30% of patients with primary or metastatic breast cancer and has been shown to be predictive of breast cancer outcome. In addition, many other cancers are known to be associated with HER-2 amplification and/or overexpression.
  • cancers associated with HER-2 include some cancers of the bladder, breast, colon, endometrial, kidney, lung, ovarian, pancreas, prostate, salivary gland, stomach, and thyroid.
  • Targeted therapies have been developed to combat HER-2 associated cancers. However, to determine if a subject will benefit from such therapies the HER-2 status of the subjects should be known.
  • This disclosure relates to methods of detecting HER-2 in a biological sample, for example in a sample obtained from a subject. Among the methods described herein are those that allow the direct detection and/or quantification of HER-2 from biological samples, such as tissue samples. Accordingly, disclosed embodiments include methods for quantitating HER-2 in subjects with cancer. For example, to determine if a subject would benefit from HER-2 targeted therapy, such as antibody-based anti-tumor therapy with anti-HER-2 antibodies.
  • the methods include obtaining a biological sample from a subject and screening the biological sample for the presence of HER-2.
  • the presence of HER-2 in a biological sample is determined by obtaining a protein digest from the biological sample and detecting the presence of a cysteine-free HER-2 fragment peptide in the protein digest.
  • the presence of a cysteine-free HER-2 fragment peptide in the protein digest indicates that the subject has HER-2 associated cancer.
  • cancers known to be associated with HER-2 amplification and/or overexpression include certain cancers of the bladder, the breast, the colon, the endometrium, the kidney, the lung, the ovaries, the pancreas, the prostate, the salivary gland, the stomach, and the thyroid.
  • a biological sample obtained from the subject is a blood sample, a urine sample, a serum sample, an ascites sample, a saliva sample, a cell, or portion of tissue, although any biological sample of interest can be used.
  • Tissue samples such as a portion of a tissue, can be obtained by a variety of invasive, minimally invasive, and/or non-invasive methods.
  • tissue samples that can be used include, but are not limited to, bladder, breast, colon, endometrium, kidney, lung, ovarian, pancreas, prostate, salivary gland, stomach, or thyroid tissue samples.
  • the tissue sample can be obtained by a variety of procedures including, but not limited to, surgical excision, aspiration, or biopsy.
  • the tissue sample is obtained from a tumor from a subject.
  • the tumor is a primary tumor.
  • the tumor is a secondary tumor, for example when the cancer is a metastatic cancer.
  • the cancer is cancer of the bladder, breast, colon, endometrium, kidney, lung, ovarian, pancreas, prostate, salivary gland, stomach, or thyroid.
  • Fragment peptides such as HER-2 fragment peptides
  • HER-2 fragment peptides can be obtained by proteolytic cleavage of the biological sample with a protein or polypeptide with a protein cleavage agent, such as in a protein digest.
  • the HER-2 fragment peptide can be excised from an intracellular or extracellular portion of HER-2.
  • Such fragment peptides are excised from the full length protein.
  • a HER-2 fragment peptide is removed from the full length protein such that it does not include the full length HER-2 protein.
  • Proteolytic peptides include peptides produced by treatment of a protein with one or more endoproteases such as trypsin, chymotrypsin, endoprotease ArgC, endoprotease aspN, endoprotease gluC, and endoprotease lysC, as well as peptides produced by chemical cleavage reactions, such as those that employ cyanogen bromide, formic acid, and thiotrtfluoroacetic acid as is well known to those of skill in the art.
  • the proteolytic cleavage agent is serine protease.
  • the proteolytic cleavage agent is trypsin
  • the resulting digest is a trypsin digest.
  • HER-2 fragment peptides derived from a full length HER-2 protein can be uniquely associated with the full length HER-2 protein sequence.
  • these peptides can be used to determine the presence of HER-2 in a biological sample, such as a biological sample obtained from a subject.
  • Identification of the peptide sequence that is uniquely associated with the larger peptide sequence in a sample identifies the larger peptide sequence in the sample.
  • a HER-2 fragment peptide that is uniquely associated with a full length HER-2 protein is a mass identifier that contains enough sequence information to discriminate between the HER-2 protein and other proteins in the sample.
  • Mass identifiers are peptides (or a set of peptides) having a particular sequence(s) that is (are) uniquely generated from a protein of interest (such as HER-2) by treatment with a particular protein cleavage agent. Detection of a mass identifier for a protein of interest in a sample unambiguously identifies the presence of the protein of interest in a sample treated with the protein cleavage agent, and determination of the concentration or amount of the mass identifier in a sample also determines the concentration or amount of the protein of interest in the sample either directly or after multiplying the concentration of the mass identifier by the number of such mass identifier generated per protein of interest.
  • Mass identifiers can be identified by treating proteins with a protein cleavage agent in vivo, in vitro or in silico.
  • Various methods and algorithms for determining a mass identifier for a protein of interest are known, but all have in common that peptide sequences obtained by digestion (actual or theoretical) of a protein of interest with a protein cleavage agent (such as an endoprotease or a model of an endoprotease' s cleavage specificity) are compared to peptide sequences obtained by digestion of other known proteins with the same cleavage agent to determine one or more peptide sequences that are uniquely produced from the protein of interest (such as HER-2).
  • a protein cleavage agent such as an endoprotease or a model of an endoprotease' s cleavage specificity
  • cysteine-free cysteine-free cysteine residues can become oxidized and form cystine or even cysteic acid.
  • the multiple oxidation states of cysteine produce peptides with unpredictable masses, which can present problems for the identification and/or quantification of peptides containing cysteines.
  • HER-2 fragment peptides useful in the disclosed examples do not contain cysteine residues. Similar to cysteine, the sulfur atom present in methionine residues can become oxidized, thereby complicating the determination of the HER-2 presence in a sample by detecting methionine containing peptide fragments of HER-2.
  • the cysteine-free cysteine residues can become oxidized, thereby complicating the determination of the HER-2 presence in a sample by detecting methionine containing peptide fragments of HER-2.
  • HER-2 fragment peptide also is a methionine-free peptide. Tryptophan is an another amino acid that is oxidation sensitive, thus hampering the determination of HER-2 presence in a sample by detecting tryptophan containing peptide fragments of HER-2.
  • the cysteine-free HER-2 fragment peptide also is a tryptophan-free peptide.
  • the cysteine-free HER-2 fragment peptide also is a methionine- and tryptophan-free peptide. Shi et al. (Shi et al, J. Proteome Res.
  • Cysteine-free HER-2 fragment peptides useful in the disclosed examples are from about 8 to about 45 amino acid residues in length, such as about 9 amino acids, about 10 amino acids, about 1 1 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, about 20 amino acids, about 21 amino acids, about 22 amino acids, about 25 amino acids, about 30 amino acids, about 35 amino acids, about 40 amino acids, or about 45 amino acids in length.
  • the cysteine-free HER-2 fragment peptide corresponds to an amino acid sequence in the extracellular portion of HER-2.
  • the cysteine-free HER-2 fragment peptide corresponds to an amino acid sequence in the intracellular portion of HER-2.
  • the cysteine-free HER-2 fragment peptide includes an amino acid sequence as set forth as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 17, or SEQ ID NO: 18.
  • the cysteine-free HER-2 fragment peptide includes the amino acid sequence as set forth as SEQ ID NO: 14, which is both methionine- and tryptophan-free.
  • cysteine-free HER-2 fragment peptide consist of an amino acid sequence as set forth as one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:1 1,
  • cysteine-free HER-2 fragment peptide consists of an amino acid sequence as set forth as SEQ ID NO: 14.
  • the cysteine-free HER-2 fragment peptides can be detected by any method that allows for the detection and identification of peptides. Methods particularly suited to the detection and identification of peptides, such as HER-2 fragment peptides, are mass spectrometric methods. In certain embodiments, the cysteine-free HER-2 fragment peptides are detected with mass spectrometry. In certain embodiments, the cysteine-free HER-2 fragment peptides are detected with tandem mass spectrometry. It some embodiments, the cysteine-free HER-2 fragment peptides are detected by detection of ion fragments generated from the cysteine-free HER-2 fragment peptides (for example by collision using tandem mass spectrometry).
  • Methods of fractionation of a protein sample include without limitation paper chromatography, thin layer chromatography (TLC), liquid chromatography, column chromatography, fast protein liquid chromatography (FPLC), ion exchange chromatography, size exclusion chromatography, affinity chromatography, high performance liquid chromatography (HPLC), poly acrylamide gel electrophoresis (PAGE), capillary electrophoresis (CE) and reverse phase high performance liquid chromatography (RP-HPLC) amongst others.
  • TLC thin layer chromatography
  • FPLC fast protein liquid chromatography
  • ion exchange chromatography size exclusion chromatography
  • HPLC high performance liquid chromatography
  • PAGE poly acrylamide gel electrophoresis
  • CE capillary electrophoresis
  • RP-HPLC reverse phase high performance liquid chromatography
  • aspects of the disclosed methods relate to quantitating the amount of cysteine-free HER-2 fragment peptide present in the biological sample.
  • the quantity of cysteine-free HER-2 fragment peptide present in the biological sample is proportional to the amount of HER-2 present in the sample prior to digestion, thus the disclosed method allows for the quantitation of HER-2.
  • Protein expression levels can be quantified by mass spectrometry if peptide standards of known concentration are available.
  • Methods for quantitating a cysteine-free HER-2 fragment peptide include comparing an amount of the cysteine-free HER-2 fragment peptide to a cysteine-free peptide standard of known amount.
  • the peptide standards are isotopically labeled peptides, and these are added in known amounts to a non-labeled protein digest.
  • non-isotopically labeled peptide standards also can be used.
  • the change in relative peak intensity before and after the addition of a peptide standard can be used to calculate the amount of a cysteine-free HER-2 fragment peptide present in a biological sample, thus providing quantification of HER-2 in the sample.
  • a mass spectrum of the protein digest is obtained without addition of the non- isotopically labeled peptide standard and mass spectrum of the protein digest is obtained with the addition of the non-isotopically labeled peptide standard.
  • the ratio of the intensity of the signals with and without the addition of the non-isotopically labeled peptide standard reflects the relative amounts (or concentrations) of the cysteine-free HER-2 fragment peptide present in a biological sample, and thus the amount of HER-2 present in the sample. It is understood that the spectra with and without the peptide standard can be obtained in any order.
  • an isotopically labeled peptide standard typically the combined sample (peptide standard plus protein digest) is analyzed by mass spectrometry, and the ratios of the mass spectral signal intensities for the labeled peptide standard and the sample peptides are measured.
  • the peptide standard is added to the biological sample prior to the protein digest, however in some circumstances it may be advantageous to add the peptide standard after proteolytic digest.
  • a mass spectrum of a sample containing both sample peptides and the added peptide standard typically includes one or more pairs of separated signals that are due to a sample peptide and its corresponding peptide standard. The ratio of the intensity of the signals in each pair reflects the relative amounts (or concentrations) of each peptide present in the sample.
  • the amount (or concentration) of the sample peptide can be calculated by multiplying the ratio of the intensity of the signal for the sample peptide to the intensity of the signal for the peptide standard by the known amount (or concentration) of the peptide standard. Furthermore, since the sample peptides are present in amounts (or concentrations) that are the same as (or related by a known ratio to) the amounts (or concentrations) of the proteins originally in the sample, a determination of the amounts (or concentrations) of the sample peptides also permits a determination of the amounts (or concentrations) of the proteins in the sample. Since the concentrations of the peptide standards are known, the concentration of the sample peptides (and the proteins they are derived from, such as full length HER-2) can be calculated using the ratios.
  • Peptide standards useful in the disclosed method correspond to an amino acid sequence of about 8 to about 45 amino acid residues of HER-2, such as about 9 amino acids, about 10 amino acids, about 1 1 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, about 20 amino acids, about 21 amino acids, about 22 amino acids, about 25 amino acids, about 30 amino acids, about 35 amino acids, about 40 amino acids, or about 45 amino acids of HER-2.
  • the peptide standard corresponds to an amino acid sequence in the extracellular portion of HER- 2.
  • the peptide standard corresponds to an amino acid sequence in the intracellular portion of HER-2.
  • the peptide standard also is a methionine-free peptide. In certain embodiments, the peptide standard also is atryptophan- free peptide. In some embodiments, the peptide standard includes an amino acid sequence as set forth as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 17, or SEQ ID NO: 18. In particular embodiments, the peptide standard includes an amino acid sequence set forth as SEQ ID NO: 14.
  • the peptide standard consists of an amino acid sequence as set forth as one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 1 1, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 17, or SEQ ID NO: 18.
  • the peptide standard consists of an amino acid sequence set forth as SEQ ED NO: 14.
  • the peptide standard is labeled with an isotope, such as a heavy stable isotope. Examples of particularly useful heavy stable isotopes are 18 0, 17 0, 34 S, 15 N, 13 C, and 2 H.
  • Peptide standards can be labeled with one ore more isotopes, for example a labeled peptide can contain 18 O 7 17 O, 15 N, 34 S, 13 C, and 2 H or any combination thereof.
  • Methods of labeling peptides with heavy isotopes are well known in the art and exemplary methods are given below in section D.
  • One advantage of the present technique is that it can be used to detect HER-2 in fixed and paraffin embedded tissues. Because most pathology laboratories use formalin fixation and paraffin embedding to store tissues, this approach is particularly useful for tracking HER-2 tumor status over time. Tissue samples can be fresh, frozen, or fixed (i.e., preserved), for example in formalin, such as buffered formalin.
  • the disclosed methods accommodate the use of paraffin embedded tissue samples, such as archived paraffin embedded biopsy samples.
  • the tissue sample can be fixed by conventional methodology (see for example, Manual of Histological Staining Method of the Armed Forces Institute of Pathology, 3 rd Edition Lee G. Luna, H.T. (ASCP) Editor, The Blakston Division McGraw- Hill Book Company: New York; (1960); The Armed Forces Institute of Pathology Advanced Laboratory Methods in Histology and Pathology ( 1994) Ulreka V. Mikel, Editor, Armed Forces Institute of Pathology, American Registry of Pathology, Washington, D.C.).
  • the choice of a fixative is determined by the purpose for which the tissue is to be histologically stained or otherwise analyzed.
  • tissue samples can be fixed with neutral buffered formalin, Bouin's fluid, paraformaldehyde, and the like.
  • the tissue sample is fixed and optionally embedded in paraffin or the like.
  • tissue sample is first fixed and is then dehydrated through an ascending series of alcohols, infiltrated, and embedded with paraffin or other sectioning media so that the tissue sample can be sectioned.
  • Paraffin is readily obtained from commercial sources such as
  • Tissue samples can be embedded and processed in paraffin by conventional methodology. Once the tissue sample is embedded, the sample can be sectioned by a microtome or the like. By way of example, sections typically range from about three micrometers to about five micrometers in thickness, although the disclosed methods can accommodate larger sections, such as sections about ten micrometers are greater in thickness. Paraffin processing can be performed via a routine overnight or accelerated cycle in an automated tissue processor. Once embedded, thin sections typically are mounted onto slides (such as glass or quartz slides).
  • the tissue sections are generally deparaff ⁇ nized and rehydrated in water.
  • the tissue sections can be deparaff ⁇ nized by several conventional standard methodologies. For example, xylenes and a gradually descending series of alcohols can be used. Alternatively, commercially available deparaffinizing agents such as HEMO-DETM (Scientific Safety Solvents, Keller, TX) or HISTOCLEARTM (East Wales, England) can be used.
  • the sections can be stained by standard techniques such as hematoxylin and eosin, methylene green nuclear stain, fluorescent in situ hybridization, or immunohistochemistry for identification of tissue morphology and cell populations of interest.
  • the tissue sample is subjected to laser-mediated dissection (such as laser capture dissection or laser microdissection and pressure catapulting) prior to protein digestion.
  • laser-mediated dissection can be used when the tissue sample contains both cancerous and non-cancerous cells.
  • laser mediated dissection is used to select only cancer cells for protein digestion.
  • the disclosed methods are particularly suited for monitoring disease progression in a subject. Such methods involve detecting an amount of HER-2 in a biological sample from a subject at a first time point, detecting an amount of HER-2 in a biological sample from a subject at a second time point, and comparing the amount of HER-2 at the two time points. It has been found that the expression level of HER-2 in a tumor correlates with the severity of the cancer. Thus, a decrease in the amount of HER-2 present in a biological sample, for example as measured by the presence of a cysteine-free HER-2 fragment peptide, would correlate with regression of the cancer. Conversely, an increase in the amount of cysteine- free HER-2 fragment peptide (indicating an increase in HER-2) could correlate to a progression of the cancer, for example progression to a metastatic form of cancer.
  • the disclosed methods are particularly useful for selecting a treatment for a subject having a cancer in which HER-2 expression is correlated with tumor development or severity.
  • Such methods involve detecting the presence of an amount of cysteine-free HER-2 fragment peptide in a biological sample from a subject, such as in protein digest obtained from the sample. The presence of the cysteine-free HER-2 fragment peptide indicates that a treatment can be selected that specifically targets HER-2 positive cancer.
  • an antibody treatment specific for HER-2 positive cancers is selected, such as treatment with trastuzumab.
  • the cysteine-free HER-2 fragment peptide is present, the subject is treated with a therapeutically effective amount of an anti- HER-2 antibody, such as trastuzumab.
  • a HER-2 kinase inhibitor is selected.
  • the biological sample is HER-2 negative, in such a case a therapy specific for HER-2 positive cancer would not be selected.
  • a treatment such as the use of chemotherapeutic agents, immunotherapeutic agents, radiotherapy, or surgical intervention can be selected.
  • the disclosed methods are also useful for determining if a subject, such as a subject with cancer, will benefit from treatment with a HER-2 targeted therapy, such as an anti- HER-2 antibody therapy, for example trastuzumab therapy.
  • a HER-2 targeted therapy such as an anti- HER-2 antibody therapy, for example trastuzumab therapy.
  • Such methods include selecting a subject for evaluation of their HER-2 status, such as the HER-2 status of their tumor(s).
  • the presence of an amount of cysteine-free HER-2 fragment peptide in a biological sample obtained from the subject, such as in a protein digest obtained from the sample, is determined.
  • the presence of the cysteine-free HER-2 fragment, and thus a HER-2 associated cancer indicates that the subject will benefit from treatment with the anti-HER-2 therapy, such as an anti-HER-2 antibody therapy, for example trastuzumab therapy.
  • suitable anti-HER-2 antibodies for administration to a subject with HER-2 associated cancer.
  • suitable anti-HER-2 antibodies that can be administered to a subject with HER-2 associated cancer can be found in U.S. Patent Nos. 5,821,337, 5,720,954, 5,783,186, and 6,627,196; U.S. Patent Publication No. 2006/0018899; and International Patent Publication Nos. WO 94/00136, WO 89/06692, and amongst others.
  • Additional anti-HER2 antibodies that can be used with the disclosed methods have been described in Tagliabue et al. Int. J. Cancer 47:933— 937 (1991); McKenzie et al.
  • Mass spectrometry is particularly suited to the identification of peptides from biological samples, such as peptides excised from HER-2. Mass spectrometry also is particularly useful in the quantitation of peptides in a biological sample, for example using isotopically labeled peptide standards.
  • the application of mass spectrometric techniques to identify proteins in biological samples is known in the art and is described for example in Akhilesh et al, Nature, 405:837-846, 2000; Dutt et al, Curr. Opin. Biotechnol., 1 1 -.176- 179, 2000; Gygi et al, Curr. Opin. Chem.
  • mass spectrometers generate gas phase ions from a sample (such as a sample containing HER-2 fragment peptides and/or peptide standards).
  • the gas phase ions are then separated according to their mass-to-charge ratio (m/z) and detected.
  • Suitable techniques for producing vapor phase ions for use in the disclosed methods include without limitation electrospray ionization (ESI), matrix-assisted laser desorption-ionization (MALDI), surface-enhanced laser desorption-ionization (SELDI), chemical ionization, and electron-impact ionization (EI).
  • Separation of ions according to their m/z ratio can be accomplished with any type of mass analyzer, including quadrupole mass analyzers (Q), time-of-flight (TOF) mass analyzers (for example linear or reflecting) analyzers, magnetic sector mass analyzers, 3D and linear ion traps (IT), Fourier-transform ion cyclotron resonance (FT-ICR) analyzers, and combinations thereof (for example, a quadrupole-time-of-flight analyzer, or Q-TOF analyzer).
  • the mass spectrometric technique is tandem mass spectrometry (MS/MS) and the presence of a HER-2 fragment peptide is detected.
  • a HER-2 fragment peptide entering the tandem mass spectrometer is selected and subjected to collision induced dissociation (CID).
  • CID collision induced dissociation
  • the spectra of the resulting fragment ion is recorded in the second stage of the mass spectrometry, as a so-called CID spectrum.
  • a CID spectrum alone often provides enough information to determine the presence of a peptide such as a HER-2 fragment peptide.
  • Suitable mass spectrometer systems for MS/MS include an ion fragmentor and one, two, or more mass spectrometers, such as those described above.
  • Suitable ion fragmentors include, but are not limited to, collision cells (in which ions are fragmented by causing them to collide with neutral gas molecules), photo dissociation cells (in which ions are fragmented by irradiating them with a beam of photons), and surface dissociation fragmentor (in which ions are fragmented by colliding them with a solid or a liquid surface).
  • Suitable mass spectrometer systems can also include ion reflectors.
  • the sample Prior to mass spectrometry the sample can be subjected to one or more dimensions of chromatographic separation, for example, one or more dimensions of liquid or size exclusion chromatography.
  • chromatographic separation include paper chromatography, thin layer chromatography (TLC), liquid chromatography, column chromatography, fast protein liquid chromatography (FPLC), ion exchange chromatography, size exclusion chromatography, affinity chromatography, high performance liquid chromatography (HPLC), nano-reverse phase liquid chromatography (nano-RPLC), poly acrylamide gel electrophoresis (PAGE), capillary electrophoresis (CE), reverse phase high performance liquid chromatography (RP-HPLC) or other suitable chromatographic techniques.
  • TLC thin layer chromatography
  • FPLC fast protein liquid chromatography
  • ion exchange chromatography size exclusion chromatography
  • affinity chromatography affinity chromatography
  • HPLC high performance liquid chromatography
  • nano-RPLC nano-reverse phase liquid chromatography
  • PAGE poly
  • the mass spectrometry technique is directly or indirectly coupled with a liquid chromatography technique, such as column chromatography, fast protein liquid chromatography (FPLC), ion exchange chromatography, size exclusion chromatography, affinity chromatography, high performance liquid chromatography (HPLC), nano-reverse phase liquid chromatography (nano-RPLC), poly acrylamide gel electrophoresis (PAGE), capillary electrophoresis (CE) or reverse phase high performance liquid chromatography (RP-HPLC) to further resolve the biological sample prior to mass spectrometric analysis.
  • FPLC fast protein liquid chromatography
  • HPLC high performance liquid chromatography
  • nano-RPLC nano-reverse phase liquid chromatography
  • PAGE poly acrylamide gel electrophoresis
  • CE capillary electrophoresis
  • RP-HPLC reverse phase high performance liquid chromatography
  • the reagents (such as buffers and the like), and particularly LIQUID TISSUE ® reagents, used in accordance with the disclosed methods are preferably chosen such as to not significantly interfere with mass spectral analysis, such as tandem mass spectrometric methods.
  • the reagents are selected so as to impart desirable characteristics to the analysis. Examples of such characteristics include for example decreasing the energy required to volatilize the peptide, facilitating ionization, creating predominantly singly charged ions, reducing the peak width, and increasing the sensitivity and/or selectivity of the desired analysis product.
  • the peptide standard is labeled with an isotope, such as a heavy stable isotope.
  • an isotope such as a heavy stable isotope.
  • particularly useful heavy stable isotopes are 18 0, 17 0, 34 S, 15 N, 13 C, and 2 H.
  • Useful isotopically labeled peptide standards have a predictable number of sites where the heavy isotope replaces a non-heavy isotope yielding a peptide with a predictable mass difference from a peptide that does not have any heavy isotopes incorporated.
  • the isotopic- label ing of the peptide standards yields separate distinct mass spectrometric signals from peptides obtained from the biological samples. These isotopically labeled peptide standards can be used to quantify proteins present in biological samples.
  • Stable isotopes can be incorporated into peptides either biologically ⁇ in vivo) or chemically ⁇ in vitro).
  • Methods for providing isotopically-labeled peptide standards are to express the peptides in a host cell (such as E. col ⁇ ) grown on an isotopically-altered medium.
  • a host cell such as E. col ⁇
  • peptides can be expressed in cell free systems.
  • Isotopically-altered medium is a growth medium that is enriched in one or more stable heavy isotopes of an element or elements relative to their natural isotopic abundances.
  • a growth medium including greater amounts of 2 H, 13 C, 34 S, 15 N, 17 O, and/or 18 O than are found in nature is an isotopically-altered medium.
  • Enrichment of the medium with stable heavy isotopes can be partial (where both heavy and light isotopes of a particular element are present in the medium), or uniform (where substantially only heavy isotopes of a particular element are present, such as greater than 90%, 95%, 98% or 99% of the atoms of an element are the heavy isotope).
  • Stable heavy isotopes can be added to the medium in any form.
  • the isotopes can be added in the form of a simple chemical substance such as 15 NH 3 , u C-glucose, 2 H 2 O, or can be added in the form of a more complex substance such as an isotopically-altered amino acid (for example, amino acids labeled with 2 H, 13 C, 34 S, 15 N, 17 O, and/or 18 O, such as deuterium-enriched leucine, serine, and/or tyrosine).
  • Uniform labeling media refers to a growth medium wherein substantially ali atoms (such as greater than 90%, 95%, 98% or 99% of all atoms) of a particular element present in the medium are present in the form of a particular isotope of the element.
  • a uniformly-labeled growth medium provides a particular type of atom substantially in the form of a single isotope of the atom.
  • a medium that provides nitrogen in the form Of 15 NH 3 as the sole nitrogen source for host cells grown on the medium is a uniformly-labeled media.
  • a peptide standard expressed in host cells grown in media with 15 NH 3 as the sole nitrogen source will be uniformly isotopically labeled with 15 N.
  • Biological isotopic labeling schemes for quantitative mass spectrometry include stable isotope labeling by amino acids in cell culture (SILAC) and related techniques (see, for example, Ong et ah, Molecular & Cellular Proteomics, 1.5:376-386, 2002).
  • SILAC stable isotope labeling by amino acids in cell culture
  • isotopically-labeled amino acids are added to an amino acid deficient cell culture, and are incorporated into proteins or peptides during cell growth.
  • peptides or proteins can be expressed in host cells grown in media with a stable heavy isotope as the sole source for a particular element.
  • peptides expressed in a host cell are unstable and prone to degradation (see, for example, Lindhout et al., Protein Science, 12: 1786—1791, 2003).
  • One solution to the stability problem is to express peptides in a fusion construct with a larger fusion protein partner.
  • the fusion protein approach has been applied to produce a single peptide or a few copies of a single peptide (a homopolymer of peptides) as part of the fusion protein (see, Jones et al., Biochemistry, 39: 1870-1878, 2000; Majerle et al., J. Biomol. NMR, 18: 145-151, 2000; Sharon et al, Protein Expr.
  • peptide standard is then excised from the fusion construct, for example by proteolytic cleavage with an endopeptidase such as trypsin.
  • Peptide standards can also be produced from HER-2 or a portion thereof that is expressed in an isotopically-altered medium, (for example in a host cell).
  • the resulting HER-2 protein or a portion thereof can be digested with an endoprotease (such as trypsin) to excise HER-2 fragment peptides that can be used as peptide standards.
  • Peptide standards such as a peptide corresponding to a portion of HER- 2, can be subsequently purified and quantitated for use as a peptide standard of known molecular weight and concentration.
  • Isotopically-labeled peptide standards of known concentration and molecular weight can be synthesized from isotopically labeled amino acids.
  • Peptide synthesis is well known in the art (see for example, Atherton and Sheppard, Solid Phase Peptide Synthesis: a Practical Approach published by published by Oxford University Press, USA, and Chan and White Fmoc Solid Phase Peptide Synthesis: A Practical Approach, published by Oxford University Press, USA).
  • Peptide standards useful in the disclosed method corresponds to an amino acid sequence of about 8 to about 45 amino acid residues of HER-2, such as about 9 amino acids, about 10 amino acids, about 1 1 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, about 20 amino acids, about 21 amino acids, about 22 amino acids, about 25 amino acids, about 30 amino acids, about 35 amino acids, about 40 amino acids, or about 45 amino acids of HER-2.
  • the peptide standard comprises an amino acid sequence as set forth as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ED NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 17, or SEQ ID NO: 18.
  • the peptide standard comprises an amino acid sequence set forth as SEQ ID NO: 14.
  • the peptide standard consists of an amino acid sequence as set forth as one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO.8, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 17, or SEQ ID NO: 18.
  • the peptide standard consists of an amino acid sequence set forth as SEQ ID NO: 14.
  • Laser mediated microdissection such as laser capture microdissection, laser microdissection and pressure catapulting (LMPC) as well as other technical approaches, are a methods for procuring pure cells from specific microscopic regions of tissue sections.
  • tissue samples obtained from a subject such as tissue biopsies
  • tissue biopsies include heterogeneous cell populations, with different cell types exhibiting strong adhesive interactions with adjacent cells, connective stroma, blood vessels, glandular and muscle components, adipose cells, inflammatory, or immune cells amongst others.
  • the diseased cells of interest such as precancerous cells, cancer cells, or invading groups of cancer cells, are surrounded by these heterogeneous tissue elements.
  • the cell types undergoing similar molecular changes, such as cancer cells may constitute less than 5% of the volume of the tissue biopsy sample.
  • Laser-mediated microdissection can be applied to obtain pure cell populations, such as populations from homogenous tissue samples.
  • Laser capture microdissection typically uses a laser beam that focally activates a transfer film capable of specifically bonding to cells identified and targeted by microscopy within the tissue sample.
  • the transfer film with the bonded cells is then lifted off the thin tissue section, leaving all unwanted cells behind (which would contaminate the molecular purity of subsequent analysis).
  • the transparent transfer film (such as ethylene vinyl acetate polymer film containing a naphthalocyanine near infrared (IR) absorbing dye) is applied to the surface of the thin tissue section overlying the target.
  • the operator such as diagnostic pathologist
  • views the thin tissue section through the slide such as a glass or quartz slide) on which it is mounted and chooses microscopic clusters of cells to study.
  • the operator activates a laser (such as a near ER. laser diode) integral with the microscope optics.
  • the pulsed laser beam activates a precise spot on the transfer film immediately above the cells of interest in order to heat the polymer to its melting point.
  • the film melts and the melted polymer expands downward making contact with the cell surface and flowing into microscopic air spaces in the desiccated tissue section and fuses with the underlying cells of choice.
  • the heat is rapidly conducted away in the slide within a couple of milliseconds of the end of the pulse causing the cooled polymer to rapidly resolidify and bond to the targeted tissue.
  • the strong bond formed allows the bonded cell(s) to be ripped out of the surrounding tissue when the film is subsequently lifted off the tissue.
  • the region of polymer melting, expansion, and bonding can be confined to an individual cell.
  • the chosen cell(s) are tightly held within the focally expanded polymer while the rest of the tissue is left behind. This allows multiple homogeneous samples within the tissue section or cytological preparation to be targeted and pooled for extraction of proteins and or peptides for analysis.
  • the strength of the bond between polymer film and the targeted tissue must be stronger than that between the tissue and the underlying slide. Therefore, for most tissue types, techniques which increase the adhesion of the tissue section to the slide should be avoided. In a commercial system, such as the instruments produced by Arcturus (Mountain
  • the film is permanently bonded to the underside of a transparent vial cap.
  • a mechanical arm precisely positions the transfer surface onto the tissue.
  • the microscope focuses the laser beam to discrete sizes (presently either 30 or 60 micrometer diameters), delivering precise pulsed doses to the targeted film.
  • Targeted cells are transferred to the cap surface, and the cap is placed directly onto a vial for molecular processing.
  • the size of the targeting pulses is selected by the operator. Cells adherent to the film retain their morphologic features, and the operator can verify that the correct cells have been procured.
  • a pulsed ultra-violet (UV-A) laser of high beam quality is interfaced into the microscope and focused through an objective to a beam spot size of less than 1 micrometer in diameter for to excise tissues of interest (such as cells of interest) from the surrounding tissue.
  • Laser mediated excision is a locally restricted ablative process that occurs without heating of the adjacent tissue and results in a clear cut gap between the tissue of interest and the surrounding tissue.
  • the isolated specimens are ejected out of the object plane and catapulted directly into the cap of a common microfuge tube. This is performed in an entirely non- contact manner with the help of a single defocused laser pulse.
  • tissue slides such as DIRECTORTM slides
  • computer software is used to control the tissue microdissection process and to store data records, including digital images of the microdissected cells before and after transfer. These records can be integrated with subsequent molecular analysis results.
  • This example describes representative methods for the preparation, extraction, and trypsin digests of peptides from formalin fixed paraffin embedded (FFPE) tissues.
  • FFPE whole-mount tissue samples were histologically analyzed by standard methods. Sections were cut and placed on glass slides and stained with hematoxylin and eosin for identification of histologically distinct tissue regions. Histological analysis was performed on an Olympus BX51 microscope, and images (10 X 20 magnification) were taken with an Olympus Q color camera mounted on the microscope.
  • the tissue microdissection instrument is based on an optical setup designed to direct a pulsed laser onto a target slide holding a ten micrometer thick tissue section.
  • Target slides are made of optically transparent quartz with an energy transfer coating with the exact dimensions of a standard histology glass slide.
  • the slide stage is a computer controlled, XY translation stage with a maximum translation speed of 200 millimeter/second.
  • the laser is split by a 1/8 beam splitter to an energy meter, and the remaining beam travels to an UV reflective mirror and directed down (-Z) to a 10x microscope objective (LMU-IOx-UVR, OFR), which focuses the laser onto the slide and allows for observation of the dissection process via a confocally aligned CCD camera.
  • Microdissection was performed by software directed laser pulses to strike at a constant velocity and rate throughput over the previously defined and mapped cellular regions to achieve complete transfer of cells within the selected area into a 1.5 milliliter low binding microcentrifuge receiving tube.
  • This example describes illustrative procedures for the extraction and digestion of peptides from microdissected tissue.
  • Microdissected tissue was suspended in 20 microliters of LT-MS reaction buffer, incubated at 95 0 C for 90 minutes, then cooled on ice for 3 minutes at which time 1 microliter of trypsin (15—18 U) was added followed by incubation at 37 0 C overnight. DTT was added to a final concentration of 10 millimolar, and the samples were heated for 5 minutes at 95°C to reduce cysteine residues. Digestates were stored at — 20 0 C until analysis.
  • This example describes illustrative methods for the immunodepletion of major plasma proteins from serum prior to trypsin digestion.
  • serum is immunodepletion of the six highest abundance proteins albumin, IgG, antitrypsin, IgA, transferrin and haptoglobin (representing 85-90% of total serum protein mass) according to manufacturer's protocols.
  • Depleted samples are exchanged in to 50 millimolar ammonium bicarbonate using a vivaspinconcentrator (5000 molecular weight cut off, Vivascience).
  • Immunodepleted samples are denatured and reduced by incubation in 5% SDS and 5 millimolar tris-(2-carboxyethyl)phosphine at 60 0 C for 15 minutes. Trypsin is then added and the samples digested for 5 hours. Digestates are stored at -20 0 C until analysis.
  • This example describes illustrative methods for the analysis of peptides using nano reverse phase liquid chromatography followed by mass spectrometric identification of peptides.
  • Nano reverse phase liquid chromatography was performed using an Agilent 1 100 capillary LC system (Agilent Technologies, Palo Alto, CA) coupled on-line to either a linear ion trap (LIT) mass spectrometer (LTQ 5 Thermo Electron, San Jose, CA) or a hybrid LIT-FT-ICR MS.
  • LIT linear ion trap
  • LTQ 5 Thermo Electron San Jose, CA
  • LTQ 5 Thermo Electron Thermo Electron, San Jose, CA
  • hybrid LIT-FT-ICR MS hybrid LIT-FT-ICR MS.
  • Nano- RPLC separations of each sample were performed using 75 micrometer inner diameter X 360 outer diameter X 10 centimeter long fused silica capillary columns (Polymicro Technologies, Phoenix, AZ) that were slurry packed in-house with 3 micrometer, 300 A pore size C-18 silica bonded stationary phase (Vydac, Hysperia, CA).
  • the LIT-MS was operated in a data-dependent MS/MS mode in which each full MS scan is followed by five MS/MS scans where peptide molecular ions are selected for collision-induced dissociation (CID) using a normalized collision energy of 35%. Dynamic exclusion was utilized to minimize redundant selection of peptides previously selected for CID.
  • the heated capillary temperature and electrospray voltage were set at 160 0 C and 1.5 kV, respectively. Data were collected over a broad mass to charge (jnlz) precursor ion selection scan range.
  • This example describes the methodology used to select exemplary peptide standards for use in the quantitation of HER-2 in biological samples.
  • HER-2 was digested with trypsin and analyzed by nano-RPLC-MS/MS to facilitate selection of a readily detectable peptide with the best signal intensity and chromatographic peak shape. Eighteen identifiable HER-2 fragment peptides were observed from a 100 nanogram injection of a HER-2 tryptic digest (see Table 1). Table 1. HER-2 Fragment Peptides Identified.
  • # is the number of peptides, xC, cross-correlation value; dCN, delta correlation value.
  • a tryptic peptide spanning amino acid residues 1054 to 1072 of HER-2 (SGGGDLTLGLEPSEEEAPR, SEQ ID NO: 14) was selected for use as a peptide standard for the quantitation of HER-2 in a biological sample.
  • spectrum A shows a representative base peak spectrum obtained from a tryptic digest of HER-2 centered on m/z ration 957 to 958, which is roughly the m/z of HER-2 fragment peptide SGGDLTLGLEPSEEEAPR (SEQ ID NO:14).
  • the spectrum is shown as relative abundance versus retention time (minutes). The spectrum was collected with a gradient of 0-40 minutes at 1% mobile phase B/minute, and 250 nanoliters/minute. Individual peaks in the spectrum are indicated by retention time. Three observable fragment ions of this peptide could also be detected using spectral windows of 913.9— 914.9 m/z, 1043-1044 m/z, and 1213-1214 m/z (see Fig.
  • HER-2 fragment peptide SGGGDLTLGLEPSEEEAPR SEQ ID NO: 14 fragments under tandem mass spectrometric analysis to give a distinctive triplet of peaks at m/z 914.4, 1043.5, and 1213.6, as shown in Fig. 2.
  • the spectrum shown in Fig. 2 is given as relative abundance versus m/z ratio over the m/z ratio from of 250 to 1930. The positions of the three readily monitored transition ions are indicated. Because the selected peptide provides a distinctive, readily recognized fragmentation pattern rather than a single peak, it can be detected with increased selectivity.
  • the mass signal of the selected peptide and/or the fragmentation ions observed the presence and amount of HER-2 can be accurately measured without ambiguity.
  • the fragment ions can be used to quantitate the amount of HER-2 in a biological sample.
  • the first spectrum labeled A is a mass spectrum with an expanded scale roughly centered on the doubly charged intact peptide (m/z 957).
  • spectrum B, C, and D is a tandem mass spectrum with an expanded scale roughly centered on the HER-2 fragment peptide SGGDLTLGLEPSEEEAPR (SEQ ID NO: 14) fragment ion at m/z 914, the fragment ion at m/z 1043, and the fragment ion at m/z 1213, respectively.
  • the set of spectra shown in Fig. 3 demonstrates that the HER-2 fragment peptide SGGDLTLGLEPSEEEAPR (SEQ ID NO: 14) can be accurately measured without ambiguity using a combination of parent and/or fragment ions.
  • a synthetic peptide corresponding to residues 1054 to 1072 of HER-2 (SGGGDLTLGLEPSEEEAPR, SEQ ID NO: 14) was synthesized with 13 C labeling to serve as an internal standard for quantification of HER-2 in biological samples.
  • the linear range and limits of detection and quantitation were evaluated by analyzing mixtures of the peptide isotopomers by nanoflow reversed-phase liquid chromatography (nano-RPLC) coupled online with a linear ion trap mass spectrometer (MS) operation in tandem MS mode operating in a selected reaction monitoring (SRM) mode.
  • nano-RPLC nanoflow reversed-phase liquid chromatography
  • MS linear ion trap mass spectrometer
  • This example describes the determination of the presence of HER-2 fragment peptides in biological samples obtained from tumor biopsies. Trypsin peptide digests were obtained from formalin fixed breast cancer biopsy samples using the LIQUID TISSUE ® reagents and protocols as described. The peptide digests were subjected to LC/MS/MS analysis to determine if the HER-2 fragment peptide corresponding to amino acid residues 1054 to 1072 of HER-2 (SGGGDLTLGLEPSEEEAPR, SEQ ID NO: 14) was detectable in a complex biological sample, as shown the set of three spectra in Fig. 4. With reference to Fig. 4, spectrum A shows the spectrum obtained with a spectral window of 956.1— 959.1 m/z.
  • This spectral window corresponds to the m/z of the doubly charged HER-2 fragment peptide SGGGDLTLGLEPSEEEAPR (SEQ ID NO: 14).
  • the HER-2 fragment peptide is observable at a retention time of 85.19 minutes.
  • MS/MS selection and analysis of peptides at m/z 957.5 reduced the number of additional peaks as shown in Fig. 4, spectrum B. Further refinement yielded a reproducible peak corresponding to the ion fragments of the HER-2 fragment peptide SGGGDLTLGLEPSEEEAPR (SEQ ID NO: 14), (see Fig. 4, spectrum C and Fig 5).
  • This example describes exemplary methods for the determination of the amount of HER-2 protein in tissue samples obtained from formalin fixed human breast cancer tissue.
  • Breast carcinoma cells were microdissected from formalin fixed paraffin embedded (FFPE) breast cancer tissue using DIRECTORTM slides as described.
  • FFPE formalin fixed paraffin embedded
  • Known concentrations of HER-2 peptide standard (SGGGDLTLGLEPSEEEAPR, SEQ ID NO: 14) were added to the samples obtained via tissue microdissection.
  • the samples were processed using the LIQUID TISSUE ® reagents and protocol according to manufacturer's recommendations (Expression Pathology Inc).
  • Known concentrations of a HER-2 peptide standard (SGGGDLTLGLEPSEEEAPR, SEQ ID NO: 14) were added to the microdissected tissue samples.
  • the digests were analyzed by nano-RPLC-MS/MS.
  • the amount of HER-2 was quantified by calculating relative peak areas generated from reconstructed ion chromatograms of selected transition ions directly detected from the SRM analysis of the selected peptide ion (see Fig. 6, spectra A-C). With reference to Fig.
  • spectrum A is a mass spectrum of an m/z window of 956.01 to 959,01, showing the presence of the HER-2 fragment peptide (SGGGDLTLGLEPSEEEAPR, SEQ ID NO: 14)
  • Fig 6 spectrum B shows a tandem mass spectrum of the intact HER-2 fragment peptide SGGDLTLGLEP S EEEAPR (SEQ ID NO: 14)
  • Fig 6 spectrum C is a tandem mass spectrum of the fragment ions of the HER-2 fragment peptide SGGDLTLGLEPSEEEAPR (SEQ ID NO: 14).

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Abstract

La présente invention concerne des procédés de détection de HER-2 dans un échantillon biologique mettant en œuvre la spectrométrie de masse. D'une manière spécifique, ces procédés détectent un fragment peptidique d'HER-2 exempt de cystéine dans l'échantillon biologique. La présence d'HER-2 dans un échantillon biologique peut être utilisée pour déterminer si un sujet présente un cancer associé à l'HER-2. La présence de cancer associé à l'HER-2 peut être utilisée pour déterminer si un sujet pourrait bénéficier d'une thérapie ciblée d'HER-2, telle qu'un anticorps anti-HER-2. Selon un aspect, ces procédés peuvent être utilisés pour quantifier l'HER-2 dans un échantillon biologique par la spectrométrie de masse à l'aide d'un peptide standard éventuellement marqué isotopiquement.
PCT/US2007/003478 2007-02-09 2007-02-09 Procédé de quantification en spectroscopie d'her-2 dans des échantillons biologiques Ceased WO2008097229A1 (fr)

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US9970943B2 (en) * 2010-03-15 2018-05-15 Anderson Forschung Group, Llc Mass spectrometric assays for peptides
US9714294B2 (en) 2010-05-27 2017-07-25 Genmab A/S Monoclonal antibodies against HER2 epitope
US11091553B2 (en) 2010-05-27 2021-08-17 Genmab A/S Monoclonal antibodies against HER2
US11046771B2 (en) 2010-05-27 2021-06-29 Genmab A/S Monoclonal antibodies against HER2
US9862769B2 (en) 2010-05-27 2018-01-09 Genmab A/S Monoclonal antibodies against HER2
US10793640B2 (en) 2010-05-27 2020-10-06 Genmab A/S Monoclonal antibodies against HER2 epitope
EP2649192A4 (fr) * 2010-12-08 2014-05-14 Expression Pathology Inc Dosage de her2 srm/mrm tronqué
EP3144394A1 (fr) * 2010-12-08 2017-03-22 Expression Pathology, Inc. Dosage de her2 srm/mrm tronqué
US10577641B2 (en) 2010-12-08 2020-03-03 Expression Pathology, Inc. Truncated Her2 SRM/MRM assay
US9765380B2 (en) 2010-12-08 2017-09-19 Expression Pathology, Inc. Truncated HER2 SRM/MRM assay
JP2018013492A (ja) * 2010-12-08 2018-01-25 エクスプレッション、パソロジー、インコーポレイテッドＥｘｐｒｅｓｓｉｏｎ Ｐａｔｈｏｌｏｇｙ， Ｉｎｃ． 切断型Ｈｅｒ２ ＳＲＭ／ＭＲＭアッセイ
JP2014503811A (ja) * 2010-12-08 2014-02-13 エクスプレッション、パソロジー、インコーポレイテッド 切断型Ｈｅｒ２ＳＲＭ／ＭＲＭアッセイ
AU2016259294B2 (en) * 2010-12-08 2018-11-01 Expression Pathology, Inc. Truncated Her2 SRM/MRM assay
US10302656B2 (en) 2010-12-27 2019-05-28 Expression Pathology, Inc. CMET protein SRM/MRM assay
US20140005282A1 (en) * 2010-12-27 2014-01-02 Expression Pathology, Inc. Cmet protein srm/mrm assay
US9372195B2 (en) * 2010-12-27 2016-06-21 Expression Pathology, Inc. cMET protein SRM/MRM assay
US11578141B2 (en) 2011-04-20 2023-02-14 Genmab A/S Bispecific antibodies against HER2 and CD3
US10208130B2 (en) 2015-05-29 2019-02-19 Expression Pathology, Inc. Quantifying Her2 protein for optimal cancer therapy
AU2016270686B2 (en) * 2015-05-29 2019-12-19 Expression Pathology, Inc. Quantifying Her2 protein for optimal cancer therapy
EP3304072A4 (fr) * 2015-05-29 2018-12-05 Expression Pathology, Inc. Quantification de la protéine her2 pour une thérapie anticancéreuse optimale
CN108027360A (zh) * 2015-05-29 2018-05-11 爱科谱迅病理研究公司 用于最优癌症治疗的定量Her2蛋白质
WO2016196523A1 (fr) * 2015-05-29 2016-12-08 Expression Pathology, Inc. Quantification de la protéine her2 pour une thérapie anticancéreuse optimale
US10537576B2 (en) 2015-12-09 2020-01-21 Expression Pathology, Inc. Methods for treating Her2-positive breast cancer

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