Classifications

• • 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
• • 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
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
  • G01N33/54326—Magnetic particles

Definitions

• the invention generally relates to cell proliferative disorders and more particularly to enriching cells having a cell proliferative disorder in a biological sample.
• Cell proliferative disorders can be characterized by a number of cellular changes, including expression of growth factors, growth factor receptors, adhesion molecules, and other cellular determinants which are readily identifiable to those of skill in the art.
• the detection of cell proliferative disorders is important in detecting, diagnosing and treating neoplasms, and cancers.
• the detection limits of many assays are not sufficient to detect cells having proliferative disorders because the number of cells present in a sample are too few to provide a detectable signal.
• the present invention provides methods and compositions useful in enriching a desired cell type contained in a sample.
• Such cells include, for example, those that can be characterized as having a cell proliferative disorder.
• the invention provides a method for enriching the number of neoplastic cells from a biological sample.
• the method is applicable to any number of cell proliferative disorders including, for example, neoplasms, and cancer.
• the method of the invention can be used to enrich carcinoma cells from peripheral blood.
• the invention provides a method of obtaining a composition substantially enriched in cells having a cell proliferative disorder, by obtaining a source of cells; contacting the source with a binding agent specific for an cell surface marker indicative of a cell proliferative disorder wherein the binding agent is bound to a magnetic bead and wherein the binding agent binds to cells in the source expressing the cell surface marker; and separating cells bound by the binding agent from the source thereby obtaining a sub-population of cells having a cell proliferative disorder.
• the binding agent is typically an antibody (e.g., monoclonal or polyclonal antibodies) .
• the method encompasses both positive and negative selection processes.
• the invention provides a method of obtaining a composition substantially enriched in cells having a cell proliferative disorder, by obtaining a source of cells; contacting the source with a binding agent specific for a cell surface marker indicative of a cell proliferative disorder wherein the binding agent is bound to a magnetic bead and wherein the binding agent binds to the cells expressing the cell surface marker in the source; separating the cells that are bound by the binding agent from the source thereby obtaining a sub-population of cells having a cell proliferative disorder; contacting the source with a second binding agent specific for a cell surface marker indicative of cells in the source which are not affected by a cell proliferative disorder, such that the second binding agent binds to the cells in the source; separating the cells that are bound by the second binding agent from the source thereby obtaining a second sub- population of cells having a cell proliferative disorder.
• Mutation is the process whereby changes occur in the quantity or structure of the genetic material of an organism. Mutations are permanent alterations in the genetic material which may lead to changes in phenotype . Mutations can involve modifications of the nucleotide sequence of a single gene, blocks of genes or whole chromosomes. Changes in single genes may be the consequence of point mutations, which involve the removal, addition, or substitution of a single nucleotide base within a DNA sequence, or they can be the consequence of changes involving the insertion or deletion of large number of nucleotides. Modifications of whole chromosomes include both changes in number or structural changes involving chromosome abnormalities.
• Numerical chromosome mutations can involve multiples of the complete karyotype, termed “polyploidy, " or they may involve deviations from the normal number of chromosomes, termed “aneuploidy. " Mutations can arise spontaneously as a result of events such as errors in the fidelity of DNA replication or the movement of transposable genetic elements within genomes. They are also induced following exposure to chemical or physical mutagens . Such mutation-inducing agents include ionizing radiations, ultraviolet light and a diverse range of chemicals such as the alkylating agents, and polycyclic aromatic hydrocarbons, all of which are capable of interacting either directly or indirectly (generally following some metabolic biotransformations) with nucleic acids.
• somatic mutations may accumulate in the genomes of previously normal cells, some of which may then demonstrate the phenotypes associated with malignant growth.
• Such oncogenic mutations may include a number of different types of alterations in DNA structure, including deletions, translocations, and single nucleotide alterations. The latter, also known as point mutations, may frequently intervene in carcinogenesis, since a variety of mutagenic chemicals induce such mutations. In addition, such mutations may occur spontaneously as a result of mistakes in DNA replication.
• mutant or mutated as applied to a target neoplastic nucleotide sequence shall be understood to encompass a mutation, a restriction fragment length polymorphism, a nucleic acid deletion, or a nucleic acid substitution.
• a point mutation constitutes a single base change in a DNA strand, for example a G residue altered to a T. Such a mutation may alter the identity of the codon in which it lies thereby creating a missense mutation or nonsense mutation.
• Transition mutations involve the substitution of one purine in the DNA by another purine or one pyrimidine by another pyrimidine, that is A by G or vice versa, or T by C and vice versa.
• a missense mutation is a point mutation in which a codon is changed into one encoding amino acid other than that normally found at a particular position.
• a nonsense mutation is any mutation that converts a codon specifying an amino acid into one coding for termination of translation. Such nonsense changes are usually accompanied by the loss of function of the gene product. However, regardless of the type of change, a change in the amino acid sequence is potentially detectable by antibodies, such as monoclonal antibodies developed against a particular peptide sequence.
• a splicing mutation is any mutation affecting gene expression by affecting correct RNA splicing.
• Splicing mutations may be due to mutations at intron-exon boundaries which alter splice sites.
• a polyadenylation site mutant is a mutation of the consensus sequence required for addition of poly (A) to the 3 ' end of mature mRNA and which results in premature mRNA degradation.
• An insertion is any mutation caused by the insertion of a nucleotide or stretch of nucleotides into a gene. For example, naturally occurring insertion mutations can be the result of the transposition of transposable genetic elements.
• Mutations that occur in somatic cells are not transmitted to the sexually produced offspring. However, such somatic mutations may be transferred to descendant daughter cells and mutations in some genes have been implicated in cancer. It is now clear that mutations may lead to the induction of cancer when they occur in one or more of a battery of normal genes referred to as the proto-oncogenes .
• Proto-oncogenes may be modified by a variety of mutational changes to produce the cancer- causing oncogenes.
• Proto-oncogenes play an essential part in the control of cell growth and differentiation and disruption of their normal activity by mutational events may lead to the aberrant growth characteristics observed in cancer cells.
• carcinomas encompasses any carcinoma in a tissue of a subject. Such carcinomas would include, for example, carcinoma of the mouth, esophagus, throat, larynx, thyroid gland, tongue, lips, salivary glands, nose, paranasal sinuses, nasopharynx, superior nasal valut and sinus tumors, esthesioneuroblastoma, squamous call cancer, malignant melanoma, sinonasal undifferentiated carcinoma (SNUC) , or blood neoplasia.
• SNUC sinonasal undifferentiated carcinoma
• carcinomas of the regional lymph nodes including cervical lymph nodes, prelaryngeal lymph nodes, pulmonary juxtaesophageal lymph nodes, and submandibular lymph nodes.
• Other carcinomas include carcinomas of the breast tissue or ducts.
• subject is meant any mammal such as bovine, canine, feline, porcine and humans.
• the invention allows for diagnosis of cell proliferative disorders in biological samples containing a small percentage of cells having a disorder compared to the total number of cell in the sample (i.e., rare cells) .
• the invention provides a method whereby cells in a sample eliciting markers of a cell proliferative disorder can be efficiently concentrated from total cell content of the sample. Accordingly, by identifying and concentrating these "rare" cells the invention provides the ability to more accurately diagnose a cell proliferative disorder in a subject from a small sample or in samples where cancerous cells are rare.
• the invention provides methods, compositions, and kits that use antibodies (as described more fully below) , which recognize makers on cells indicative of a cell proliferative disorder.
• Monoclonal Antibodies are capable of binding to markers on a cell having a cell proliferative disorder.
• the antibodies themselves are bound to magnetic beads that are then used to separate the antibody-bound cells to concentrate them from the sample (e.g., by creating a "sub-sample”).
• the hybridomas secreted a single type of immunoglobulin specific to the antigen; moreover, like the myeloma cells, the hybrid cells had the potential for indefinite cell division.
• antisera derived from vaccinated animals are variable mixture of polyclonal antibodies which never can be reproduced identically, monoclonal antibodies are highly specific immunoglobulins of single type.
• the single type of immunoglobulin secreted by a hybridoma is specific to one and only one antigenic determinant, or epitope, on the antigen, a complex molecule having a multiplicity of antigenic determinants.
• an antigenic determinant may be one of the many peptide sequences, generally 6-7 or more amino acids in length (M. Z. Atassi, Molec. Cell. Biochem., 32:21-43 (1980)), within the entire protein molecule.
• monoclonal antibodies raised against a single antigen may be distinct from each other, depending on the determinant that induced their formation; but for any given clone, all of the antibodies it produces are identical.
• the hybridoma cell line can be reproduced indefinitely, is easily propagated in vi tro or in vivo, and yields monoclonal antibodies in extremely high concentration.
• Monoclonal antibodies are presently being applied by investigators to the diagnosis and treatment of cancer (for a general discussion of the topic, see Hybridomas in Cancer Diagnosis and Treatment, Mitchell, M. S. and Oettgen, H. F., (eds.), Progress in Cancer Research and Therapy, Vol. 21, Raven Press, New York (1982)). Monoclonal antibodies have been raised against tumor cells (U.S. Pat. No. 4,196,265), carcinoembryonic antigen (U.S. Pat. No. 4,349,528), and thymocytes, prothymocytes, monocytes, and suppressor T cells (U.S. Pat. Nos.
• Ciocca D.R. et al . , Cancer Res., 42;4256-4258 (1982); Colcher, D. et al . , Proc. Natl. Acad. Sci. U.S.A., 78:3199-3203 (1981); Foster, C. S., et al . , Virchows Arch. Pathol. Anat., 394:279-293 (1982); Greene, G. L. et al . , Proc. Natl. Acad. Sci. U.S.A., 77:5115-5119 (1980); McGee, JO'D. et al . , Lancet, 2:7-11(1982); Nuti, M. et al . , Int. J. Cancer, 291:539-545 (1982); and
• the invention provides methods and compositions for enriching cancer cells in a sample.
• One method employs positive selection and utilizes the binding affinity of antibodies directed to cell surface markers indicative of a cancer phenotype to purify these cells from non-cancer cells. Such techniques may employ column fractionation or affinity purification protocols.
• An alternative carcinoma cell enrichment method is based on the depletion of non-tumor cells present in a sample.
• This method utilizes antibodies directed to one or several cell surface markers expressed by non-carcinoma cells, such as CD45 expressed by white blood cells.
• the negative selection method offers the advantage of not relying on the presence of a carcinoma cell surface marker. These markers can have a wide range of expression due to the diversity of tumor cell prototypes .
• the Applicant has developed a method of enriching the number of neoplastic cells in a sample, using both positive and negative selection sequentially, to maximize the sensitivity of the carcinoma cell detection.
• each method positive and negative selections
• the method is described in the protocol outlined below, which is meant to illustrate, but not limit, the present invention.
• Example I 20 ml of peripheral blood was drawn and anticoagulated with EDTA.
• the red blood cells were lysed for 5 minutes at room temperature with a red blood cell lysis buffer at a final concentration of 155 mM NH 4 C1, 10 mK KHC0 3 , 0.1 mM EDTA, at pH 7.2.
• Whole cells were separated from lysed red blood cells by centrifugation at 300 RCF for 5 minutes at room temperature. The supernatant was carefully aspirated and the RBC lysis step was repeated a second time with fresh lysis buffer.
• Carcinoma cells expressing the human Epithelial Antigen were enriched with magnetic beads by adding 0.1 ml of HAE125-microbeads (Miltenyi Biotec) to the 0.9 ml of cell suspension in the 1.5 ml microcentrifuge tube and incubated for 30 minutes at room temperature on an orbital shaker or a rotisserie.
• LS+ column on a midiMACS magnet (Miltenyi Biotec) was mounted and prepared with 3 ml of PEB.
• the cell suspension was loaded on the column, followed by 2 ml of PEB and 4 ml of PBS.
• the flow through was collected in a tube for the negative selection step.
• the column was eluted by removing the midiMACS magnet from the column and placing the column over a large capacity cytospin chamber (Hettich #1666) .
• 3 ml of PBS buffer was added to the column and collected in the chamber by gravity elution.
• a second 3 ml volume of PBS was added to the column and eluted by positive pressure (i.e., gently pushed through) .
• the eluant was collected in the chamber and mixed with the 5 ⁇ l aliquot of cell suspension taken before the positive selection.
• the cells from the eluant and the aliquot were spun together onto a slide in a cytocentrifuge at 500 RPM with a Hettich Universal 16A centrifuge (RevPro) for 15 minutes at room temperature.
• the slides were removed and allowed to dry for at least 1 hour at room temperature.
• the cell/magnetic bead ratio of the negative selection step can be optimized by one skilled in the art.
• the total cell number should not exceed the capacity (100 million cells) of the column used in the negative selection.
• the number of WBC originally present in the sample was calculated. Using this number, the volume of the flow through containing 100 million WBC was then determined and transferred to a separate tube and spun at 300 RCF for 5 minutes. The cells in the pellet were resuspended in 0.9 ml PEB and transferred in a microcentrifuge tube. A 5 ⁇ l aliquot of the cell suspension was kept with 0.5 ml PEB in the tube that was used to collect the flow through from the negative selection. The purpose of this aliquot was to provide a minimum of amount of cells at the end of the procedure. A volume of 150 ⁇ l of CD45-microbeads (Miltenyi Biotec, Inc.
• the microfuge tube that contained the cell suspension was washed with 1 ml PEB, which was added to the column. When the top of the column was empty, the column was washed with 5 ml PEB. The flow through was clear, indicating that the negative selection worked properly.
• the tube containing the flow through from the negative selection was spun at 300 RCF for 5 minutes.
• the slides were removed and allowed to dry for at least 1 hour at room temperature.
• the slides were fixed with 400 ⁇ l 0.5% formalin for 10 minutes at room temperature in a moist chamber and washed two times in PBS for 3 minutes each. Permeabilization was performed by using standard buffers in Coplin jars. Antibodies specifically recognizing cytokeratins were mixed and incubated with the slides for 45 minutes or more at room temperature. The slides were washed and stained with standard buffers and chromogens for 10 minutes at room temperature. The slides were washed once in PBS and once in deionized water.
• PEB PBS, EDTA, BSA; lxPBS, 0.1 mM EDTA, and 0.5% BSA.
• the cell pellet was washed one time with an additional centrifugation and resuspension step as described above.
• the final pellet was resuspended in 0.9 ml of PEB and transferred to a 1.5 ml microcentrifuge tube.
• Carcinoma cells were enriched with magnetic beads by adding 0.2 ml of HAE125-microbeads (Miltenyi Biotec Inc.) to the 0.9 ml of cell suspension in the 1.5 ml microcentrifuge tube and incubated for 30 minutes at room temperature on an orbital or rotary shaker.
• HAE125-microbeads Miltenyi Biotec Inc.
• LS+ column on a midiMACS magnet (Miltenyi Biotec) was mounted and washed with 3 ml of PEB.
• the cell suspension was loaded on the column followed by 2 ml of PEB and 4 ml of PBS.
• the column was eluted by removing the midiMACS magnet from the column and placing the column over a Hettich #1666 chamber.
• 3 ml of PBS buffer was added to the column and collected in the chamber by gravity elution.
• a second 3 ml volume was added to the column and eluted by positive pressure (i.e., gently pushed through) .
• the eluant was collected in the chamber and spun onto slides in a cytocentrifuge at 500 RPM with a Hettich 16A centrifuge (RevPro) for 15 minutes at room temperature .
• the slides were removed and allowed to dry for at least 1 hour at room temperature.
• the slides were fixed in 400 ⁇ l 0.5% formalin for 10 minutes at room temperature in a moist chamber and washed 2x in PBS for 3 minutes each. Permeabilization was performed by using standard buffers in Coplin Jars. Antibodies were mixed and incubated with the slides for 45 minutes at room temperature.
• the slides were washed and stained with standard buffers for 10 to 15 minutes at room temperature.
• the slides were washed once in PBS and once in deionized water.
• the following protocol is designed to enrich for carcinoma cells expressing the human Epithelial Antigen (recognized by the monoclonal antibody HAE125) , starting with 20 ml of peripherial blood containing EDTA. In order to keep a precise schedule, it is recommended not to use more than three samples simultaneously.
• Red Blood Cells Lysis a. Prepare lx lysis buffer (see stock solutions) out of a lOx stock solution with deionized or distilled water. We need 80 ml lx lysis buffer for each 20 ml blood sample. b. Prepare PEB (see stock solutions) . c. 20 ml fresh blood is equally split in 2 disposable 50 ml conical tubes (label tubes) . d. In each conical tube, add 40 ml lx lysis buffer mix by inverting the tubes; keep the tubes at room temperature for 5 minutes. e. Spin at 300 RCF for 10 minutes at room temperature. Remove the supernatant carefully by aspiration. f.
• Cytospin a. Cytospin - the cells are spun at the lowest speed (-250 RMP) with a Hettich 16A centrifuge (RevPro) for 15 minutes at room temperature. b. Eliminate the supernatant with a vacuum pump linked to a Pasteur pipet having a disposable conical tip at its end. Avoid scratching the spot. c. Disassemble the chamber and dry the slid for at least 1 hour at room temperature. The slid is ready to be stained for sytokeratin.
• lx Lysis Buffer 155 mM NHRCl, 10 mM KHC03, 0.1 mM EDTA pH 7.2.
• lOx lysis stock solution for 1 liter 82.9 g NH4C1 (SIGMA #A 0171); 10.0 g KHC03 (SIGMA #P 7682); 370 mg Na2 EDTA (SIGMA #E 5134) .
• PBS Phosphate-Buffered Saline
• PBS is either: 0.144 g/L KH2P04; 0.795 g/L NA2HP04-7H20; 9 g/L
• PEB PBS, EDTA, BSA
• PEB is lx PBS, 0.1 mM EDTA, 0.5% BSA.
• Bovine Serum Albumin, Fraction V (SIGMA #A 8022) ; 10 ⁇ l EDTA 0.5 M; and 50 ml PBS.
• the slide should be dried about 1 hour to overnight. Unstained slides should be conserved at -20°C in a dry environment.
• the slide After cytospin, the slide is dried for about 1 hour at room temperature.
• the spot containing the cytospun cells is circled with a hydrophobic pen
• Permound (Fisher #SP15-100) . Press the coverslip gently to eliminate the surplus of mounting medium and to minimize the distance between the coverslip and the slid. This medium is permanent and does not need additional sealing. 2.
• the sample can be covered with a cellulose film from SAKURA Tissue-Tek automated coverslipper, using xylene . This method is permanent and does not introduce bubbles. This method can also be used manually.

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