Patent Application
20080261243
Currently only 25 to 30% of breast cancer patients receive HERCEPTIN therapy based on the findings of elevated levels of the Her-2/neu (also called Her2/neu; HER2; c-erbB-2 and erbB2) protein or gene in biopsies of their primary tumor. The data in the literature suggest that a significant number of women (11 of 26 tested) with negative results for Her2/neu in their primary tumor biopsy go on to develop Her2/neu positivity on their circulating cancer cells (Hayes DF, et al., Int J Oncol 21:1111-7; Meng S et al., 2004 Proc Natl Acad Sci USA 101:9393-98). Also, a considerable number of women with breast cancer do not have biopsy material readily available for testing for Her-2-neu status. The approaches used in the papers by Hayes et al and by Meng et al are cumbersome and time-consuming and a rapid more convenient test is needed, especially one that can be done in the physician's office. The method of Hayes et al. requires flow cytometric analysis and the method of Meng et al. requires fluorescence in situ hybridization (FISH) which are more complicated and time-consuming than direct detection (e.g., by ECL) of Her-2/neu protein as noted in this invention.
Her2/neu and HERCEPTIN treatment: Overexpression of Her2/neu oncogene is observed in approximately 25% of biopsy samples from women with breast cancer and is associated with a poor prognosis. Trastuzumab (HERCEPTIN) is a humanized monoclonal antibody that is directed against the extracellular domain (ECD) of the Her2/neu receptor and inhibits the proliferation of human breast cancer cells overexpressing this receptor (see Esteve FJ 2004, The Oncologist 9(Suppl 3):pp 4-9 for a recent review). Protein expression of Her-2/neu on breast cancer cells can easily reach levels of 500,000 molecules or more per cell as is the case of the Her-2/neu overexpressing human breast cancer cell line called SK-BR-3. Current tests for Her2/neu rely on testing tissue sections of the patients biopsy for overexpression of the protein or for gene amplification. In those women with gene amplification or at least 2+ immunostaining for the protein, significant responses have been demonstrated with single agent trastuzumab or with trastuzumab in combination with a chemotherapeutic such as paclitaxel. Other agents which can target Her-2/neu are in development and have a similar applicability as HERCEPTIN for this invention. These include but are not limited to: OMNITARG (pertuzumab) being developed by Genentech; GW-572016 being developed by GlaxoSmithKline (Xia et al., 2004, Oncogene 23:646-653); CP-654577 being developed by Pfizer (Barbacci et al., 2003, Cancer Res 63:4450-4459); HKI-272 being developed by Wyeth (Rabindran et al., 2004, Cancer Res 2004, 64:3958-65). Other anticancer agents that include Her-2/neu among their specificity are described in Janmaat and Giaccone, 2003 (The Oncologist 8:576-86). Besides breast cancer, Her-2/neu is overexpressed on other carcinoma cells including ovarian carcinoma.
ECL: Electrochemiluminescence is a process which uses labels designed to emit light when electrochemically stimulated (for a review on ECL see Yang et al., 1994, Biotechnology 12:193-194; also see Blackburn et al., 1991, Clin Chem 37:1534-1539). These labels, together with the appropriate instrumentation (such as developed by BioVeris) provide for a highly sensitive method of detecting a variety of biological molecules such as proteins, mRNA, and DNA. Martin et al., (2003, U.S. Pat. No. 6,524,865) describes an ECL-based enzyme immunoassay; however application to the detection of proteins such as Her-2/neu on circulating cancer cells in blood is not described.
ECL methods using whole eukaryotic cells have been disclosed by Chinn et al (U.S. Pat. No. 6,300,143 B1). However the method by Chinn et al. is for measuring binding affinity of cell surface antibodies rather than the determination of the relative number of receptor molecules on the cell surface. Also the method by Chinn begins with a purified cell population of a large number of cells (167,000 cells per ml) as opposed to first isolating the selected cell population which can be in small numbers (typically 1 to 200 cells per ml; Hayes et al., 2002) from an impure mixture found in whole blood.
The current US Food and Drug Administration (FDA)-approved assays for selecting patients for treatment with HERCEPTIN (trastuzumab) are (1) immunohistochemistry for Her-2/neu protein overexpression (HERCEPTEST by DAKO) and (2) the FISH (fluorescence in-situ hybridization) assay for amplification of the Her-2/neu gene (PATHVISION kit by VYSIS). These tests have been shown to be predictive of the patient response and benefit to HERCEPTIN (Formier M, et al., 2002. HER2 testing and correlation with efficacy of trastuzumab therapy. Oncology 16:1340-58). However, both of these assays have the following four limitations:
Koski, 1998 (U.S. Pat. No. 5,783,404) describes immunohistochemical assays of breast cancer cells and breast cancer tissue for the Her-2/neu protein using monoclonal antibodies that recognize a denatured epitope of a specific portion of the Her-2/neu protein. However these assays are in the setting of a pure population of breast cancer cells or, as in the breast cancer tissue, a very high percentage of breast cancer cells. Koski does not address the issue of detecting small numbers of Her-2/neu-expressing breast cancer cells in a complex matrix such as blood.
Slamon et al, 1990 (U.S. Pat. No. 4,968,603) describes an assay for amplification of the Her-2/neu gene and uses in screening patients. No examples are given of an assay for detecting the Her-2/neu protein. Furthermore, this patent does not address the issue of detecting small numbers of Her-2/neu-expressing breast cancer cells in a complex matrix such as blood. Application toward identifying patients for treatment of breast cancer patients with a monoclonal antibody against Her-2/neu protein such as HERCEPTIN is not indicated.
Carney et al., 1995 (U.S. Pat. No. 5,401,638) describes an immunoassay of human serum or plasma for the detection of a neu related protein p100 consisting of a truncated version of the full-length Her-2/neu protein consisting of the extracellular domain (ECD) of the human neu gene product. This Carney patent does not address detection of cell-associated or full-length Her-2/neu protein, especially in a complex mixture such as whole blood. A commercially based assay (from ONCOGENE) based on Carney has a sensitivity (1.5 ng of truncated Her-2/neu per mL of serum) that is too low to be used for detecting the potentially small numbers of Her-2/neu protein (pg amounts) that can be detected and is required for detection in accordance with the instant invention. Assays, as in this invention, for cell-associated Her-2/neu are distinctly advantageous since immunohistochemistry of patient biopsy tissue has demonstrated a predictability of cell-associated expression for response to HERCEPTIN treatment. A recent report by Burstein et al. (2003, J Clin Oncol 21:2889-2895) indicates the limited usefulness of a serum assay for truncated p100 protein (HER2 ECD). Burstein et al report that “Neither the baseline level of HER2 ECD nor a decrease in HER2 ECD with therapy predicted clinical response to trastuzumab after one cycle.” In a study of breast cancer patients treated with HERCEPTIN, Cobleigh et al (1999, J Clin Oncol 9:2639) reached a similar conclusion that there was no significant correlations demonstrable between serum ECD levels and HERCEPTIN response status.
Carney et al., 1997 (U.S. Pat. No. 5,604,107) describe an immunoassay for detecting the full-length pl 85 Her-2/neu protein in cell lysates; however a commercially based ELISA assay based on Carney has a sensitivity that is orders of magnitude too low to be used for detecting the small numbers of Her-2/neu protein (pg amounts) required for the application described here. As noted above, the sensitivity of this assay for quantification of Her-2/neu proteins is reported as 1.5 ng of truncated Her-2/neu per mL of serum; it would be presumed to be much worse in a more complex mixture such as whole blood. Furthermore, Carney et al. does not address a Her-2/neu assay using whole blood. Instead the level of a truncated Her-2/neu called p100 is detected in the plasma component of blood rather than the cell-associated component. The rationale for preferring an assay measuring cell-associated full-length protein as in this invention rather than an assay measuring plasma levels of the truncated protein are discussed above. Also Carney et al. does not indicate use of whole cells but rather the use of a cell lysate.
Hudziak et al., 1998 (U.S. Pat. No. 5,720,937) claims an in vivo assay by exposing cells within the body of a mammal to a monoclonal antibody to determine overexpression of the Her-2/neu protein. However, methods of assaying tumor cells outside the body are not described.
Terstappen et al., 2002 (U.S. Pat. No. 6,365,362) describes use of immunomagnetic beads with antibodies against epithelial cell adhesion molecule (EpCAM) for the isolation of breast cancer cells from patient blood samples. The Terstappen assay combines immunomagnetic enrichment with flow cytometric and immunocytometric analysis. However, flow cytometry and immunocytometry suffer from being cumbersome and time-consuming techniques.
Consequently, there is a need in medical practice for a rapid and convenient assay that is sensitive enough to use with whole blood samples to identify those women with breast cancer who have overexpression of Her-2/neu protein on circulating breast cancer cells and who therefore are likely to benefit from Her-2/neu-targeting therapy, e.g. trastuzumab.
This invention provides a method of detecting the expression of Her-2/neu protein on circulating cancer cells in a blood sample comprising isolating the cancer cells from the blood sample followed by performing on the isolated cancer cells an immunoassay capable of detecting cancer cell-associated Her-2/neu, in which a positive immunoassay result indicates the presence of Her-2/neu on the cancer cells. The assay in accordance with this invention has a sensitivity defined by: a) being capable of detecting cancer cell-associated Her-2/neu at a level of between one-tenth picogram and twenty picograms of Her-2/neu per milliliter of the blood sample; or b) being capable of detecting Her-2/neu from SK-BR-3 breast cancer cells when spiked into blood at a concentration of less than or equal to 100 SK-BR-3 cells per milliliter of blood.
This invention provides a method of identifying a cancer patient likely to benefit from treatment with an anticancer agent that targets Her-2/neu, comprising the detection method described above. When used to identify such patients, the cancer cell-containing blood sample is drawn from the patient. This invention provides a method of treating cancer patients so-identified, which method comprises administering a Her-2/neu-targeting anticancer agent to the patient.
As used herein the transitional term “comprising” is open-ended. A claim utilizing this term can contain elements in addition to those recited in such claim. Thus, for example, the claims can read on methods that also include other steps not specifically recited therein, as long as the recited elements or their equivalent are present.
This invention provides methods sensitive enough for quantifying the levels of Her-2/neu protein on circulating breast cancer cells in blood samples and provides methods for identifying those women with breast cancer who are likely to benefit from therapy using HERCEPTIN or another agent targeted to Her-2/neu. A convenient, highly sensitive and rapid means to test blood samples to identify additional patients who would benefit from HERCEPTIN therapy would be an important advance in the breast cancer treatment field. As indicated below, a rapid and highly sensitive immunological assay to detect Her-2/neu protein on the surface of circulating breast cancer cells such as using electochemiluminescence (ECL)-detection is a preferred means to accomplish this.
This invention is based on combining the high specificity of procedures used to isolate circulating carcinoma cells from blood with the high sensitivity of certain immunologically based assays such as ECL. Preferably and advantageously, immunomagnetic beads are used for both aspects of the invention, thus serving a novel dual function: Immunomagnetic beads are used for isolating and purifying the circulating cancer cells from blood and either different beads or these same beads can then be used as a support phase for performing the ECL allowing a magnet to capture them and concentrate target antigen along with tagged antibody (e.g., ruthenium tag labeled antibody).
In embodiments of the detection method of this invention the sensitivity level in a) above is between one and twenty picograms, between one and ten picograms, or between one and five picograms, of Her-2/neu per milliliter of the blood sample. In another embodiment of the detection method of this invention the sensitivity level in b) above is being capable of detecting Her-2/neu from SK-BR-3 breast cancer cells when spiked into blood at a concentration of less than or equal to ten SK-BR-3 cells per milliliter of blood. In further embodiments the sensitivity level in b) above is being capable of detecting Her-2/neu from SK-BR-3 breast cancer cells when spiked into blood at a concentration of less than or equal to three SK-BR-3 cells per milliliter of blood, and even less than or equal to one SK-BR-3 cells per milliliter of blood.
A sample (usually in the range of approximately 8 to 20 ml) of blood from a patient with cancer, especially breast cancer, is taken. Steps include as detailed below:
A variety of methods are available to remove red cells including but not limited to separation based on density (such as collection of blood directly into the BECTON DICKINSON BD Vacutainer CPT tubes) followed by centrifugation) and commercial lysing buffers such as PURESCRIPT RBC lysis buffer (GENTRA, Minneapolis), FACS lysing solution (BDIS), IMMUNOLYSE (COULTER), OPTILYSE B (IMMUNOTECH), and ACK lysing buffer (BIOSOURCE, Rockville, Md.).
A preferred method uses the BD Vacutainer CPT tubes with anticoagulant (EDTA or citrate). These tubes contains a material that upon correct centrifugation (1,100×g for 10 minutes, swing-out bucket rotor) allows for elimination of red blood cells and neutrophils. After centrifugation, the bottom of the tube contains a cell pellet of erythrocytes (red blood cells) and neutrophils. Above the cell pellet is a gel barrier and above the gel barrier are tumor cells, lymphocytes and monocytes as a band at the bottom of the plasma. The tumor cells, lymphocytes and monocytes can then be readily collected from the top above the gel barrier. This method is preferred as it removes not only the red blood cells but also the neutrophils.
A preferred embodiment of this invention uses of negative selection step for isolation of tumor cells. Negative selection allows for further depletion of leukocytes especially the lymphocytes and monocytes. This step comprises the use of antibodies that are bispecific for both leukocyte antigens, especially CD45, the common leukocyte antigen, and for a red blood cell antigen such as glycophorin A. A commercially available cocktail of such bispecific antibodies is available from STEMCELL TECHNOLOGIES (Rosettesep Catalog #15127 and #15167). This cocktail includes bispecific antibodies against glycophorin A and against a variety of cell surface antigens on human hematopoietic cells (CD2, CD16, CD19, CD36, CD38, CD45, CD66b). One or more of these bispecific antibodies are added to the BD Vacutainer CPT tubes before blood collection. In the preferred embodiment, the cocktail of bispecific antibodies against more than one leukocyte-associated CD molecule is used. When the blood is introduced into the CPT vacutainer tube, the bispecific antibodies form immunorosettes each consisting of leukocytes plus many red blood cells. These immunorosettes have a density approximately that of red blood cells and when centrifuged are found in the red blood cell pellet, thus further removing leukocytes from the tumor cell fraction found above the cell pellet and gel barrier. The fraction with the tumor cells in plasma is collected for further processing.
The preferred method of isolating circulating carcinoma cells uses immunomagnetic beads. Other methods of isolation of circulating cancer cells include filtration (Vona G et al., 2000, Am J. Pathol. 2000 156:57-63). In the preferred embodiment, the immunomagnetic beads have antibodies against antigens found selectively on the surface of carcinoma cells such as epithelial cell adhesion molecule (EpCAM), cytokeratins such as cytokeratin-19 and especially a cocktail of antibodies against cytokeratins and other surface markers. Immunomagnetic beads with antibodies against Her2/neu may also be used. The immunomagnetic beads may be of various sizes (50 microns to less than 200 nm) and include DYNAL beads (>1.5 microns to about 50 microns) with antibodies against EpCAM (which are ommercially available) or against Her2/neu. In the preferred embodiment nanoparticle beads are used as it will allow for faster and more efficient binding of tumor cells to the beads. In an embodiment of the invention, EasySep™ human EpCAM positive selection cocktail and EasySep™ Magnetic nanoparticles (STEMCELL TECHNOLOGIES) are added to the fraction with the tumor cells in plasma from the previous step. A magnet is then used to separate tumor cells from the rest of the material and the tumor cells are washed with an aqueous solution. Purified tumor cells are then ready for detection of antigens in the next step.
4. Detection and Quantification of the Her-2/neu Protein from Circulating Carcinoma Cells.
Detection of Her-2/neu can then be accomplished by use of a monoclonal antibody (niAb) such as HERCEPTIN or mAb 191924 (R&D systems Catalog number MAB 1129) or a polyclonal antibody against Her-2/neu (e.g., Goat polyclonal antibody catalog number AF 1129 from R&D systems) that are linked to a detecting molecule. In the case of electrochemiluminescence (ECL), the detecting molecule is ruthenium. There is abundant literature in the public domain provides amply useful methods for linking ruthenium to antibodies (eg., Lee et al., Am J Trop Med Hyg 2001, 65:1-9) followed by ECL detection of antigens on magnetic beads in a solution containing tripropylamine. With application of an electric potential, the ruthenium label is excited and light is emitted and detected using an ECL detecting instrument (such as the ORIGEN analyzer or a commercially available instrument like the M-Series® 384 from BIOVERIS Corporation, Gaithersburg, Md.)
The immunoassay utilized in accordance with this invention can be either a polyclonal or a monoclonal antibody against Her-2/neu. Preferably the monoclonal antibody is a humanized mouse monoclonal antibody, e.g. trastuzumab. Trastuzumab is preferred for the immunoassay and treatment methods in accordance with this invention.
For purposes of detection of Her-2/neu, a variety of monoclonal and polyclonal antibodies against Her-2/neu and include antibodies against the extracellular domain and against the cytoplasmic domain are commercially available from such sources as R&D Systems (Minneapolis, Minn. Biosource (Camarillo, Calif.) and BD Biosciences, San Diego, Calif.). Rabbit polyclonal antibodies are also available from LABVISION Corp, Fremont. CA; such as neu Ab-21) and from UPSTATE CELL SIGNALING SOLUTIONS (Lake Placid, N.Y.; such as Catolog number 06-562). A goat polyclonal antibody against the extracellular domain from Her-2/neu is available from R&D systems (catalog number AF1129). A goat polyclonal antibody against full-length recombinant Her-2/neu is available from EXALPHA BIOLOGICS (Rosedale, Mass.; catalog number M100P). Such polyclonal antibody against full-length Her-2/neu would be expected to be able to bind to extracellular and cytoplasmic domains of Her-2/neu and not to be specific for the extracellular domain. Monoclonal antibodies are available against both the extracellular domain (e.g., R&D Systems Catalog number MAB1129) and the cytoplasmic domain (e.g., LABVISION neuAB-8) including against the C-terminal peptide (e.g., LABVISION neuAB-15). Monoclonal antibodies against Her-2/neu are also disclosed in Hudziak et al (1997, U.S. Pat. No. 5,677,171). One improved embodiment uses HERCEPTIN since binding with this antibody is best able to predict binding of HERCEPTIN as a treatment in the patient. Another advantageous embodiment uses a rabbit polyclonal antibody or a cocktail of antibodies binding to many epitopes on the Her-2/neu protein allows for higher sensitivity.
In one embodiment of this invention the immunoassay is performed on intact cancer cells and utilizes an antibody that binds selectively to the extracellular domain of Her-2/neu. Altenatively, the isolated cancer cells can be lysed prior to the immunoassay and the immunoassay is performed on the cell lysate. In this case the immunoassay can utilize antibodies that bind selectively either to the extracellular or cytoplasmic domain of Her-2/neu. In a more specific embodiment of this invention the immunoassay uses one or two antibodies that bind selectively to the cytoplasmic domain of Her-2/neu.
The immunoassay of this invention is more rapid and has a significantly greater sensitivity than any previously developed immunoassay for Her-2/neu. The immunoassay of this invention is capable of detecting Her-2/neu expression from 100 or less of SK-BR-3 breast cancer cells added per ml of blood from a human volunteer without cancer. The immunoassay of this invention is capable of detecting cancer cell-associated Her-2/neu at a level of twenty picograms or less of Her-2/neu per milliliter of a blood sample
Besides electrochemiluminesence, other immunoassays that can yield a high sensitivity required for this application include, but are not limited to:
In a preferred embodiment, the relative quantity of breast cancer cells used in the assay is estimated. This allows for a ratio of total Her-2/neu protein per cell to be obtained and can be compared to control standards of breast cancer cells with high, moderate, and low levels of Her-2/neu protein per cell. This is the preferred embodiment since it eliminates a false positive situation in which there are many circulating breast cancer cells that have a low level of Her-2/neu protein expression that may give a signal that mimics that obtained from a small number of breast cancer cells with a high level expression. In this embodiment, a variety of approaches can be used to estimate relative cell numbers including flow cytometic analysis, quantification of total DNA or DNA related antigens such as histones from lysed cells (there is 6 pg DNA per diploid cell), and turbidity or absorbance measurements.
Due to its sensitivity the method according to this invention for identifying patients likely to benefit from treatment with an anticancer agent that targets Her-2/neu can be fruitfully applied to patients from whom a tumor biopsy tissue had been previously determined (e.g. by immunohistochemistry or FISH analysis) to be negative for Her-2/neu expression by a tissue assay for Her-2/neu.
The invention will be better understood by reference to the following examples, which illustrate but do not limit the invention described herein.
A patient with metastatic breast cancer comes into the office and a blood sample (8 to 40 mL) is withdrawn directly into BD Vacutainer CPT tubes containing an anticoagulant such as citrate as well as an added negative selection product: ROSETTESEP (from STEMCELL TECHNOLOGIES) containing bispecific antibodies toward erythrocytes antigens as well as toward leukocyte surface antigens. The material is centrifuged for 20 minutes at 1500 to 1800 RCF (relative centrifugal force). The cell layer above the gel barrier is removed. EasySep™ human EpCAM positive selection cocktail and EasySep™ Magnetic nanoparticles (StemCell Technologies) are added and the tumor cells isolated and washed using a magnetic field. Ruthenium-labeled polyclonal antibody against Her-2/neu is added along with a solution of tripropylamine to the tumor cells attached to the magnetic beads bound to an electrode. Routine methods of ruthenium labeling the antibody are described in the art such as Lee et al., Am J Trop Med Hyg 2001, 65:1-9. An electric current is applied and electrochemiluminescence (ECL) is detected using an ECL detection device such as one commercially available (BIOVERIS Corporation). Within these instruments is a photomultiplier tube (PMT) placed just above the working electrode for efficient light capture. Under the working electrode, a magnet is in place for capturing the beads coated with the target antigen. The signal is proportional to the amount of Her-2/neu found bound on the surface of the circulating tumor cells.
Methods identical to that used in example 1 are provided except that a monoclonal antibody rather than a polyclonal antibody to Her-2/Neu is used for detection.
Methods identical to that used in examples 1-2, except that isolated breast cancer cells are lysed either before addition of an antibody against Her-2/neu linked to a magnetic bead. Lysis can be achieved with any number of cell lysis reagents described in the art such as, but not limited to Lysis Buffer A [1% NP-40, 20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 10 ug/mL Aprotinin, 10 Ug/mL Leupeptin] and RIPA buffer (Papetti and Herman, 2001, Am J Pathology 159:165-178). In this sandwich type ECL (see Yang et al., 1994, for an illustration of a ‘sandwich’ immunoassay using ECL), two sets of antibodies against Her-2/neu are used: one antibody is biotinylated and attached to strepavidin-coated magnetic beads while the second antibody is ruthenium-labeled.
Methods identical to that used in examples 1 through 3 are provided except that the patient had a prior negative result for Her2/neu based on analysis of her primary tumor or does not have tumor tissue readily available for analysis.
Methods as that used in examples 1 through 4 are provided with the addition of a step to quantify the relative number of breast cancer cells are used in the analysis An ELISA to quantify cells by DNA content (there are 6 pg of DNA per diploid nucleus (www.gentra.com/calcularing.asp) has described by Friis et al (2003; APMIS 111:658-68) but lacks the sensitivity required for this invention. A higher sensitivity assay required for the detection of the small number of cells required in this invention is achieved using a highly-sensitive immunoassay, e.g., by using ECL and the same instrument used to quantify Her-2/neu.
In order to quantify cells by ECL, they are first lyzed (e.g., with lysis buffers such as, but not limited to, Lysis Buffer A as detailed above) and then two different antibodies directed against double stranded DNA (e.g., Mouse monoclonal antibody HYB 33-01 available from STATENS SERUM INSTITUT (Copenhagen, Denmark); Mouse monoclonal antibody MAB3032 available from CHEMICON (Temecula, Calif., USA); Mouse Monoclonal Antibody catalog number DNA11-M from ALPHA DIAGNOSTICS INTERNATIONAL (San Antonio, Tex., USA) are added; one has been labeled with ruthenium (routine methods of ruthenium labeling the antibody are described in the art such as Lee et al., Am J Trop Med Hyg 2001, 65:1-9) and the other labeled with biotin for attachment to strepavidin-coated magnetic beads. Quantification of antigen [in this case dsDNA (double-stranded DNA)] is achieved by ECL with a solution of tripropylamine and the magnetic beads bound to an electrode by use of a magnetic field. An electric current is applied and electrochemiluminescence (ECL) is detected using an ECL detection device such as one commercially available (BIOVERIS Corporation). Within these instruments is a photomultiplier tube (PMT) placed just above the working electrode for efficient light capture. The signal is proportional to the amount of dsDNA and, therefore, is proportional to the cell number. This then allows for a ratio of Her-2/neu per cell number to be obtained. This ratio, rather than an absolute number of tumor cell-associated Her-2/neu per ml blood, is advantageous in this invention for determining the patient's sensitivity toward HERCEPTIN treatment.
A patient with a level of Her-2/neu above control samples as indicated in Examples 1-5 is deemed to have tumor cells positive for Her-2/neu and then treated with a regimen containing a monoclonal antibody against Her2/neu such as HERCEPTIN. A preferred treatment consists of HERCEPTIN at an initial loading dose of 4 mg/kg administered as a 90 minute infusion with a weekly maintenance dose of 2 mg/kg as a 30 minute infusion.
In this example, purified recombinant Her-2/neu (extracellular domain) was used as a standard to examine the sensitivity of a sandwich immunoassay using electrochemiluminescence.
Four different PBS assay buffers were prepared:
Her-2/neu standard (recombinant Her-2/neu extracellular domain) was obtained from DakoCytomation (Carpinteria, Calif. 93013 USA; Product EL541). Goat anti-human Her-2/neu polyclonal antibody was obtained in both biotinylated and non-biotinylated forms (catalog numbers BAF1129 and AF1129, respectively) from R&D Systems, Inc. (Minneapolis, Minn. 55413 USA) as was the monoclonal antibody MAB1129 (R&D Systems catalog number 191924). The polyclonal antibody AF1129 and monoclonal antibody MAB1129 were ruthenium labeled (“TAG-labeled”) as follows:
The ruthenium-labeled polyclonal antibody AF1129 and the biotinylated polyclonal antibody BAF1129 are referred hereafter in this example and subsequent examples as “TAG-pAb” and “Biotin-pAb”. The ruthenium-labeled monoclonal antibody MAB1129 is referred hereafter in this example as “TAG-mAb”.
An electrochemiluminsence assay was performed as follows:
Results showed that all tested levels of recombinant Her-2/neu extracellular domain (16, 160 and 1600 pg/well) were detectable and above baseline using all four different assay buffers using the sandwich immunoassay with TAG-pAb and Biotin-pAb (Table 1). Recombinant Her-2/neu extracellular domain was also detectable using the sandwich immunoassay with TAG-mAb and Biotin-pAb (Table 2).
Methods as that used in example 7 were used except that:
Results showed that all tested levels of recombinant Her-2/neu extracellular (4, 16, and 64 pg/well) were clearly detectable and above baseline (see
Methods as that used in example 8 except that:
The ability to detect Her-2/neu was tested in the presence or absence of 0.02 μl/well of cell lysis buffer. Pierce Lysis Buffer [M-PER® Extraction Reagent (Product number 78501 from Pierce Biotechnology, Inc., Rockford, Ill.)] and Sigma Lysis Buffer [Sigma Cellytic™-M (Sigma Product Nubmer C 2978, Sigma-Aldrich, Inc., St. Louis, Mo. 63103)] were tested in separate wells.
The result are presented in Table 3. All amounts of Her-2/neu in this experiment including the lowest amount of 16 pg/well were detectable and above baseline (Table 3). This finding was observed in the presence or absence of each cell lysis buffer (Pierce Lysis Buffer or Sigma Lysis Buffer).
Immunoassay methods were as that used in example 8 except that:
SK-BR-3 cells (from ATCC, Manassas, Va.) were grown in 6-well tissue culture plates as per ATCC recommended conditions, washed two times with PBS, and an aliquot counted using a hemacytometer. Lysis of SK-BR-3 cells was performed using either the Pierce Lysis Buffer or Sigma Lysis Buffer. These two lysis buffers are described in Experiment 9 above. In order to lyze SK-BR-3 cells, 200 μl lysis buffer were added per 1 million cells. Cell lysis were performed as per each manufacture's recommendation with the addition of 5 minutes of vigorous vortexing prior to cell debris removal. Cell debris was removed from the cell lysate by centrifugation at 14,000 rpm for 30 minutes in an Eppendorf Centrifuge (Model 5415C). The amount of lysate supernatant per well was varied from that extracted from 10 to 1000 SK-BR-3 cells and analyzed for Her-2/neu using the immunoassay described in Experiment 7 with PBS Assay Buffer 1.
The results from this experiment are presented in Table 4. Her-2/neu was detectable and above baseline from lysates from SK-BR-3 cells in this experiment including those wells using the lowest amount of SK-BR-3 lysate in this experiment (lysate from 10 cells added per well; Table 4). This result was observed regardless of the cell lysis buffer (Pierce Lysis Buffer or Sigma Lysis Buffer) used.
In this experiment, immunoassay methods were as that used in example 10, except that lysates using Sigma Lysis Buffer from the following cells were examined:
Results in this experiment are presented in
In this experiment, immunoassay methods were as that used in example 11, except that the concentration for both Tag-pAb and Biotin-pAb added in 50 μl was 0.5 μg/ml each instead of 1 μg/ml each. Again the cell lysate from SK-BR-3 cells was compared in terms of Her-2/neu expression to the cell lysate from MDA-MB-468 cells. Results in this experiment are presented in
In this experiment, immunoassay methods were as that used in example 12, except that an additional cell lysate (mouse peripheral blood mononuclear cells, PBMCs) was obtained using the Sigma Lysis Buffer. PMBCs are composed of lymphocyes and monocytes and may co-purify during the initial steps leading to the isolation of circulating breast cancer cells from blood.
Four ml of mouse blood was collected in a 4-ml BECTON DICKINSON BD Vacutainer CPT tube and centrifuged at 3000 rpm for 30 minutes in a Jouan CR412 centrifuge. PBMCs were collected in the cell fraction above the gel and washed 4 times with PBS. A total of 1 million PBMCs were collected from the 4 ml of blood. Cell lysis was performed as for SK-BR-3 cells. Cell debris was removed from the cell lysate by centrifugation at 14,000 rpm for 30 minutes in an Eppendorf Centrifuge (Model 5415C). The supernatant was then used for analysis in this experiment.
Samples for testing were prepared by adding into each well the following:
Results in this experiment are presented in Tables 5 and 6 and in
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US05/35894 | 10/6/2005 | WO | 00 | 3/22/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60616332 | Oct 2004 | US |
