HK1185659B - Methods and kits for the detection of circulating tumor cells in pancreatic patients using polyspecific capture and cocktail detection reagents - Google Patents

Description

Methods and kits for detecting circulating tumor cells in pancreatic patients using multi-specific capture and cocktail detection reagents

Related patent application

This patent application claims the benefit of priority from U.S. provisional application No. 61/393,036 filed on 14/10/2010, the entire contents of which are hereby incorporated by reference.

Technical Field

The present invention relates to the field of oncology and diagnostic testing. The invention is useful for cancer screening, staging, monitoring of chemotherapy treatment response, cancer recurrence, and the like. More specifically, the invention provides reagents, methods and test kits that facilitate the analysis and enumeration of tumor cells or other rare cells isolated from a biological sample.

Background

Counting Circulating Tumor Cells (CTCs) in patients with metastatic breast, prostate and colon cancers using the CellSearchCTC assay predicts patient survival and renders monitoring of treatment response possible. In addition, CTCs can be characterized with respect to a variety of molecular markers that have been proposed as a means for dynamically studying tumor biology in patients. However, there have been few studies on CTC detection in patients with pancreatic cancer, and preliminary studies suggest that the initial assay configuration may not be optimal for detecting these cells (see US7,332,288 incorporated by reference). The initial CellSearchCTC assay used anti-EpCAM (EpCAM ferrofluid) conjugated to paramagnetic nanoparticles to capture CTCs. CTCs captured by EpCAM ferrofluid were stained by anti-cytokeratin antibodies (CK8, 18 and 19) conjugated to phycoerythrin to detect CTCs.

In addition to the problem that cells from different cancers may express different tumor antigens compared to those of breast, prostate or colorectal cancer, there is literature describing the increase in cellular heterogeneity within primary tumors. Recent progress has shown that Tumor Progenitor Cells (TPC) may be critically important in explaining why some cancers recover after chemotherapy. These TPCs appear to be highly resistant to many conventional therapies and are capable of reconstituting tumors at some future time. Recently, the literature has also identified epithelial mesenchymal transformed cells (EMTs) that may play a major role in the metastatic process. TPC and EMT are considered to express antigens different from those of CTC. CTC capture is dependent on EpCAM expression on CTCs; as CTC capture has been limited to a single capture antigen. The biology of EpCAM on CTCs is not well understood and it is likely that EpCAM antigen may be down-regulated or negative in some CTCs. In such cases, CTCs will not be captured by EpCAM ferrofluid and will result in zero CTCs in the assay. Another possibility is that CTCs captured by them are not detectable due to the absence of cytokeratin markers used in the assay. Thus, although many CTCs are successfully captured and detected by current techniques, the presence of undetectable CTCs in the blood of cancer patients is possible because they fail to express the markers used in current assays. Finally, it is unknown whether TPC and EMT circulate in the blood and can be detected. However, the fact that current capture and detection techniques have been limited to the targeting of an antigen or class of antigens means that TPC or EMT cannot be detected with current techniques.

Based on the above, it is clear that methods for identifying these cells in circulation before establishment of secondary tumors are highly desirable, especially in early stages of cancer. Many laboratory and clinical procedures employ biospecific affinity reactions for isolating rare cells from biological samples. Such reactions are commonly used in diagnostic tests, or for isolating a wide range of target substances, in particular biological entities such as cells, proteins, bacteria, viruses, nucleic acid sequences and the like.

Various methods are available for analyzing or isolating a target substance based on complex formation between the target substance and another substance to which the target substance specifically binds. Separation of the complex from unbound material may be accomplished gravimetrically, for example, by sedimentation of finely divided particles or beads coupled to the target substance or by centrifugation. Such particles or beads may be prepared to be magnetic, if desired, to facilitate the binding/free separation step. Magnetic particles are well known in the art as are their use in immunological and other biospecific affinity reactions. See, for example, U.S. Pat. No. 4,554,088 and immunoassays for clinical chemistry (immunoassays), p.147-162, edited by Hunter et al, ChurchillLivingston, Edinburgh, of Edinburgh (1983). Generally, any material that facilitates magnetic or gravitational separation may be used for this purpose. However, it has become clear that the magnetic separation method is the method of choice.

Magnetic particles can be classified on the basis of size as large (1.5 to about 50 microns), small (0.7-1.5 microns), or colloidal (< 200nm), also known as nanoparticles. The latter, also known as ferrofluid or ferrofluid-like materials and having many of the properties of conventional ferrofluids, are sometimes referred to herein as colloidal, superparamagnetic particles.

Small magnetic particles of the above type are quite useful in assays involving biospecific affinity reactions, as they are conveniently coated with biofunctional polymers (e.g. proteins), provide extremely high surface area and give reasonable reaction kinetics. Magnetic particles in the range of 0.7 to 1.5 microns have been described in the patent literature, including, for example, U.S. patent nos. 3,970,518; 4,018,886, respectively; 4,230,685; 4,267,234, respectively; 4,452,773; 4,554,088, respectively; and 4,659,678. Certain of these particles are disclosed as useful solid supports for immunological reagents.

As with the small magnetic particles mentioned above, large magnetic particles (> 1.5 to 50 microns) can also exhibit paramagnetic behavior. Such materials are generally those described by Ugelstad in U.S. patent No. 4,654267 and manufactured by Dynal (Oslo, Norway, Oslo, Norway). The Ugelstad process involves the synthesis of polymer particles that cause swelling, and magnetic crystals are embedded in the swollen particles. Other materials in the same size range are prepared by synthesizing polymer particles in the presence of dispersed magnetic crystals. This results in the capture of magnetic crystals in the polymer matrix, making the resulting material magnetic. In both cases, the particles produced have a superparamagnetic behaviour, which is manifested by the ability to disperse easily after removal of the magnetic field. Unlike the magnetic colloids or nanoparticles mentioned previously and discussed in further detail below, these materials, as well as the small magnetic particles, are easily separated by simple laboratory magnetics due to the mass/particles of the magnetic material. Thus, separation is achieved in a gradient as low as several hundred gauss/cm to at most about 1.5 kilogauss/cm. Colloidal magnetic particles (below about 200nm), on the other hand, require substantially higher magnetic gradients due to their dispersed energy, small magnetic mass/particle and stokes' resistance.

U.S. Pat. No. 4,795,698 to Owen et al relates to polymer-coated, colloidal, paramagnetic particles prepared by reacting Fe with a polymer in the presence of a carrier⁺²/Fe⁺³Magnetite formation of salt occurs. U.S. Pat. No. 4,452,773 to Molday describes materials similar in nature to those described in Owen et al made from Fe by addition via alkali in the presence of very high concentrations of dextran⁺²/Fe⁺³Formation of magnetite and other iron oxides. The particles obtained from both procedures showed no suitable tendency to settle from aqueous suspensions for observation periods up to several months. The material so produced has colloidal properties and has proven to be very useful in cell separation. The Molday technique has been commercialized by the American day and whirlpool incorporated of Bergistadarbach, Germany (Miltenyi Biotec, Bergisch Gladbach, Germany) and by the Tary Tomas incorporated of Vancouver, Canada (TerryThomas, Vancouver, Canada).

Another method for producing paramagnetic, colloidal particles is described in U.S. patent No. 5,597,531. In contrast to the particles described in Owen et al or the Molday patent, these latter particles are produced by coating a biofunctional polymer directly onto preformed superparamagnetic crystals that have been dispersed by high power sonic energy into quasi-stable crystalline clusters in the range of 25 to 120 nm. The resulting particles, referred to herein as direct coated particles, exhibit a magnetic moment that is significantly greater than that of colloidal particles of the same overall size, such as those described by Molday or Owen et al.

Magnetic separation techniques are known in which a magnetic field is applied to a fluid medium in order to separate a ferromagnetic body from the fluid medium. In contrast, the tendency of colloidal, superparamagnetic particles to remain in suspension, coupled with their relatively weak magnetic responsiveness, requires the use of High Gradient Magnetic Separation (HGMS) techniques to separate such particles from the nonmagnetic fluid medium in which they are suspended. In HGMS systems, the gradient, i.e. the spatial derivative, of the magnetic field exerts a greater influence on the behaviour of the suspended particles than at a given point by the field strength.

HGMS systems can be divided into two broad categories. One such category includes magnetic separation systems that employ magnetic circuits that are completely external to the separation chamber or vessel. An example of such an external separator is described in U.S. patent No. 5,186,827 to Liberti et al. In several embodiments described in this patent, the necessary magnetic field gradients are generated by placing permanent magnets near the periphery of a nonmagnetic vessel such that the same poles of the magnets are in a field-opposed configuration. The degree of magnetic field gradient within the test medium that can be achieved in such systems is limited by the strength of the magnets and the separation distance between the magnets. Thus, there is a limited limit to the gradient that can be achieved by an external gradient system.

Another class of HGMS splitters utilizes a ferromagnetic collection structure disposed within the test medium to 1) strengthen the applied magnetic field and 2) generate a magnetic field gradient within the test medium. In one type of known internal HGMS system, a fine steel wool or gauze is packed into a column located adjacent to the magnet. The applied magnetic field is concentrated near the steel wire so that the suspended magnetic particles will be attracted towards and adhere to the wire surface. The gradient produced on such wires is inversely proportional to the wire diameter, such that the magnetic range decreases as the diameter increases. Thus, very high gradients can be generated.

One disadvantage of internal gradient systems is that the use of steel wool, gauze material or steel microbeads can trap the non-magnetic components of the test medium by capillary action near the crossing wires or in the interstices between the crossing wires. Various coating procedures have been applied to such internal gradient columns (see, e.g., U.S. Pat. No. 5,693,539 to Miltenyi and 4,375,407 to Kronick), however, the large surface area in such systems still raises recovery concerns due to adsorption. Therefore, internal gradient systems are not desirable, especially when recovery of very low frequency capture entities is the purpose of separation. Furthermore, they make automation difficult and expensive. The materials described by Owen et al and Molday both require the use of such high gradient columns.

In contrast, the HGMS method using an external gradient for cell separation provides a number of conveniences. First, simple laboratory vessels such as test tubes, centrifuge tubes, or even vacutainers (for blood collection) may be employed. When the external gradient is of a kind that produces a monolayer of separated cells, washing or subsequent manipulation of the cells is facilitated as is the case with the quadrupole/hexapole device of U.S. patent No. 5,186,827 referred to above or the relative dipole arrangement described in U.S. patent No. 5,466,574 to Liberti et al. Further, cell recovery from a tube or similar container is a simple and efficient process. This is especially the case when compared to recovery from a high gradient column. Such separation vessels also provide another important feature, namely the ability to reduce the volume of the sample. For example, if a particular subset of human blood cells (e.g., magnetically labeled CD 34) is isolated from a 10ml blood sample diluted 50% with buffer to reduce viscosity⁺Cells), then a15 ml conical tube can be used as the separation vessel in a suitable quadrupole magnetic device. Starting from the 15mls solution, a first separation was performed and the recovered cells were resuspended in 3 mls. A second wash/separation was then performed and the separated cells were resuspended in a final volume of 200 ul.

CD34 after washing and/or separation and resuspension to remove unbound cells⁺Cells can be effectively resuspended in a volume of 200. mu.l. When used already for these separationsCell recovery in the range of 40-90% is quite effective when the machine optimized direct coated ferrofluid is carefully completed in properly treated vessels, depending on antigen density. Such techniques and reagents are necessary to achieve the degree of sensitivity required for the cancer test species mentioned above.

The efficiency with which magnetic separation can be accomplished, as well as the recovery and purity of magnetically labeled cells, will depend on a number of factors. These include such considerations as the number of cells to be separated, the receptor density of such cells, the magnetic load/cell, the non-specific binding (NSB) of the magnetic material, the technique employed, the nature of the vessel surface, the viscosity of the medium, and the magnetic separation device employed. If the level of nonspecific binding of the system is essentially constant, as is often the case, then the target population is reduced as is the purity. For example, a system with 0.8% NSB that recovers 80% of the population that is at 0.25% in the original mixture will have a purity of 25%. However, if the initial population is at 0.01% (at 10)⁶One target cell among bystander cells) and if NSB is 0.001%, the purity will be 8%. The greater the purity, the easier and better the analysis. Thus, it is clear that very low non-specific binding is required to perform meaningful rare cell analysis.

Less obvious is the fact that the smaller the target cell population, the more difficult it is to magnetically label and recover. Furthermore, labeling and recovery will depend significantly on the nature of the magnetic particles employed. For example, when cells are incubated with large magnetic particles such as Dynal beads, the cells are labeled by collisions resulting from the systematic mixing because the beads are too large to disperse effectively. Thus, if cells are present in the population at 1 cell/ml blood or even less frequently, as is the case with tumor cells in very early cancers, the probability of labeling the target cells will be related to the number of magnetic particles added to the system and the length of time of mixing. Since it would be detrimental for cells to be mixed with such particles for a large period of time, it becomes necessary to increase the particle concentration as much as possible. However, there is a limit as to the number of magnetic particles that can be added, because rare cells mixed with a large number of magnetic particles after separation can be replaced with rare cells mixed with other blood cells. The latter condition does not significantly improve the ability to count or examine cells of interest.

There is another disadvantage of using large particles to separate cells at rare frequencies (1 to 50 cells/ml blood). Despite the fact that large magnetic particles allow the use of very simple designed external gradients and relatively low magnetic gradients, large particles tend to cluster around cells in a cage-like fashion, making them difficult to see or analyze. Therefore, the magnetic particles must be released from the target cell prior to analysis, and releasing the particles obviously introduces other complications.

Based on the foregoing, high gradient magnetic separation with an external field device employing high magnetic, low non-specific binding, colloidal magnetic particles is a selection method for separating subsets of cells of interest from a mixed population of eukaryotic cells, especially when the subsets of interest comprise a small fraction of the entire population. Due to their dispersive nature, such materials are easily found and magnetically label rare events, such as tumor cells in blood. Such isolation typically relies on the identification of cell surface antigens that are unique to the specific subset of cells of interest, which in the case of tumor cells may be tumor antigens to which a ferrofluid conjugated to a suitable monoclonal antibody may be targeted. Alternatively, when examining a blood sample, determinants on cell classes, such as epithelial cells, not normally found in blood may provide suitable receptors.

There are other good reasons for using colloidal magnetic materials for such separations, provided that the magnetic load of recovery can be achieved. With the appropriate load, sufficient force is applied to the cells so that separation can be achieved even in media as viscous as that of moderately diluted whole blood. As noted, colloidal magnetic materials below about 200 nanometers will exhibit brownian motion that significantly enhances their ability to collide with and magnetically label rare cells. This is demonstrated in us patent No. 5,541,072, which describes the results of a very effective tumor cell decontamination experiment using colloidal magnetic particles or ferrofluids with an average diameter of 100 nm. Equally important, colloidal materials having particle sizes at or below this size range generally do not interfere with the examination of cells. The cells so retrieved can be examined by flow cytometry, by laser scanning microscopy or by microscopy using visual or fluorescent techniques.

Disclosure of Invention

The present invention provides a rapid and efficient screening method for characterizing not only tumor cells but also rare cells or other biological entities from a biological sample. The methods of the invention provide highly sensitive analytical techniques that enable efficient enrichment of entities of interest. This two-stage approach to ensure enrichment of the target biological entity while eliminating large amounts of debris and other interfering substances prior to analysis allows examination of sample sizes that would otherwise be impractical. The methods described herein combine immunomagnetically enriched elements with multi-parameter flow cytometry, microscopy, and immunocytochemistry analysis in a unique approach. Other enrichment methods, such as density gradient centrifugation or panning or altering the target cell density by appropriate labeling, can also be used. According to a preferred embodiment, the method of the invention enables the determination of whole blood for cancer staging, monitoring and screening. The sensitive nature of the assay facilitates the detection of residual disease, thereby enabling the monitoring of cancer recurrence.

The present invention incorporates a method of conjugating different antibodies to the same ferrofluid. This has the effect of making the ferrofluid bi-, tri-or multispecific with respect to the ferrofluid bound antigens. The multiple antibodies present on the same ferrofluid do not appear to block or otherwise interfere with each other. Such ferrofluids have highly desirable effects capable of specific binding to more than one type of cell, making possible the ability to capture CTCs with low EpCAM expression but high expression of other tumor markers;

in one embodiment of the invention, a biological sample comprising a mixed cell population suspected of containing rare cells of interest is obtained from a patient. The immunomagnetic sample is then prepared by mixing the biological sample with: (i) magnetic particles coupled to biospecific ligands specifically reactive with rare cell determinants or determinant classes other than those found on blood cells to the substantial exclusion of other sample components, and (ii) at least one biospecific reagent that labels rare cells. The resulting immunomagnetic sample is subjected to a magnetic field that effectively separates the sample into an unlabeled fraction and a labeled magnetic fraction comprising rare cells of interest (if present in the sample). The cell populations thus isolated are then analyzed to determine the presence and number of rare cells. In a preferred embodiment, the particles used in such a method are colloidal nanoparticles.

The present invention allows for improved detection of CTCs in pancreatic cancer patients. Instead of using an anti-EpCAM agent alone, a multispecific agent such as, but not limited to, anti-EpCAM used as a control along with several other antibodies that recognize different antigens on CTCs is used to detect circulating cancer cells that were not previously detected in the blood of a patient. The capture reagent is referred to as a multispecific capture reagent because it recognizes several antigens. Thus, CTC capture is independent of EpCAM antigen alone, and CTCs are captured even if EpCAM antigen is not expressed, provided that the other antigens selected for capture are present on CTCs. The ability to generate cross-blocked multispecific ferrofluids with sufficient binding capacity, low non-specific binding and no antibodies, along with the use of an appropriate specific detection antibody mix, renders capture of all multiple types of CTCs possible. In the present invention, a mixture of antibodies specific for CTCs is conjugated to ferrofluid for capture instead of a single antibody to minimize CTC capture dependence on a single target. In addition, the additional antibodies detected cover a wider range of antigens. Although the present invention relates to the field of circulating tumor cells, other forms of rare cell analysis in blood are contemplated.

Drawings

FIG. 1(A) shows the staining intensity of four cell lines based on the expression of target antigen on the cell surface. All antibodies were tested at 2ug/ml (n-2). (B) The average percent positive population of four cell lines for target antigen expression is shown.

FIG. 2(A) shows the staining intensity of four cell lines based on intracellular target antigen expression. (B) The average percent positive population of four cell lines for target antigen expression is shown.

Figure 3(a) shows the recovery of spiked CAPAN1 tumor cells with different kit configurations. (B) Recovery of spiked BxPC3 tumor cells with different kit configurations is shown.

Detailed Description

According to preferred embodiments, the present invention provides compositions, methods and kits for the rapid and efficient separation of rare biological entities from biological samples using multispecific ferrofluids. The method can be used to efficiently isolate and characterize tumor cells present in a blood sample while minimizing the selection of non-specifically bound cells.

Using multiple capture and detection reagents as described herein, rare cell assays are further improved over previously used single target molecules. This modification improves the capture and detection of rare cells such as, but not limited to, pancreatic CTCs. In addition, non-epithelial markers such as mesenchymal markers (n-cadherin) may be used in conjunction with epithelial markers to capture epithelial and mesenchymal tumor cells. The invention enables the simultaneous detection of different populations of rare cells, for example different populations of CTCs and tumor cells.

The term "target bioentity" as used herein refers to a wide variety of materials of biological or medical interest. Examples include hormones, proteins, peptides, lectins, oligonucleotides, drugs, chemicals, nucleic acid molecules (e.g., RNA and/or DNA), and particulate analytes of biological origin, including biological particles such as cells, viruses, bacteria, and the like. In preferred embodiments of the invention, rare cells such as fetal cells in maternal circulation or circulating cancer cells can be effectively separated from non-target cells and/or other biological entities using the compositions, methods, and kits of the invention. The term "biological sample" includes, but is not limited to, cell-containing bodily fluids, peripheral blood, tissue homogenates, nipple aspirates, and any other source of rare cells obtainable from a human subject. Exemplary tissue homogenates may be obtained from sentinel knots in breast cancer patients. The term "determinant", when used in reference to any of the aforementioned target biological entities, may be specifically bound by a biospecific ligand or biospecific agent, and refers to that portion of the target biological entity involved in and responsible for selective binding with a specific binding substance whose presence is required for selective binding to occur. In fundamental terms, a determinant is a molecular contact region on a target biological entity that is recognized by a receptor in a specific binding pair reaction. The term "specific binding pair" as used herein includes antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, nucleic acid (RNA or DNA) hybridization sequences, Fc receptor or mouse IgG-protein a, avidin-biotin, streptavidin-biotin and virus-receptor interactions. Various other determinant-specific binding substance combinations are contemplated for use in practicing the methods of the present invention, such as would be apparent to one of skill in the art. The term "antibody" as used herein includes immunoglobulins, monoclonal or polyclonal antibodies, immunoreactive immunoglobulin fragments and single chain antibodies. Also contemplated for use in the present invention are peptides, oligonucleotides, or combinations thereof that specifically recognize determinants with specificity similar to that of conventionally generated antibodies. The term "detectable label" is used herein to refer to any substance whose direct or indirect detection or measurement by physical or chemical means is indicative of the presence of a target biological entity in a test sample. Representative examples of useful detectable labels include, but are not limited to, the following: molecules or ions that can be detected directly or indirectly based on light absorbance, fluorescence, reflectance, light scattering, phosphorescence, or luminescence properties; a molecule or ion detectable by its radioactive properties; molecules or ions that can be detected by their nuclear magnetic resonance or paramagnetic properties. Included among the group of molecules that can be indirectly detected based on light absorbance or fluorescence are, for example, a variety of enzymes that enzymatically convert a suitable substrate, for example, from non-absorbing to absorbing molecules, or from non-fluorescent to fluorescent molecules. The phrase "substantially excludes" refers to the specificity of the binding reaction between a biospecific ligand or biospecific agent and its corresponding target determinant. Biospecific ligands and reagents have specific binding activity for their target determinants and may still exhibit low levels of non-specific binding to other sample components. The term "early stage cancer" as used herein refers to those cancers that have been clinically identified as organ-restricted. Also included are tumors that are too small to be detected by conventional methods, such as mammography for breast cancer patients or X-ray for lung cancer patients. The term "enrichment" as used herein refers to the enrichment of monocytes from a biological sample. In the case where peripheral blood is used as a raw material, red blood cells are not counted when the degree of enrichment is evaluated. Preferred magnetic particles for use in carrying out the invention are particles that behave as colloids. Such particles are characterized by their submicron particle size, which is typically less than about 200 nanometers (nm) (0.20 microns), and by their stability to gravity separation from solution for extended periods of time. This size range makes it essentially invisible to the analytical techniques normally applied for cell analysis, among many other advantages. Particles in the range of 90-150nm and having a magnetic mass between 70-90% are contemplated for use in the present invention. Suitable magnetic particles consist of a crystalline core of superparamagnetic material surrounded by molecules which are bonded, e.g. physisorbed or covalently attached, to the magnetic core and which impart stable colloidal properties. The coating material should preferably be applied in an amount effective to prevent non-specific interactions between the biomacromolecules found in the sample and the magnetic core. Such biological macromolecules may include sialic acid residues, lectins, glycoproteins, and other membrane components on the surface of non-target cells. Furthermore, the material should contain as much magnetic mass per nanoparticle as possible. The size of the magnetic crystals comprising the core is small enough that they do not contain intact magnetic domains. The size of the nanoparticles is small enough that their brownian energy exceeds their magnetic moment. Thus, the north-south pole ratio and its subsequent mutual attraction/repulsion of colloidal magnetic particles do not appear to occur even in moderately strong magnetic fields, contributing to its solution stability. Finally, the magnetic particles should be separable in a high magnetic gradient external field separator. This feature facilitates sample handling and provides an economic advantage over more complex internal gradient columns loaded with ferromagnetic beads or steel wool. Magnetic particles having the above properties can be prepared by modifying the substrate materials described in U.S. patent nos. 4,795,698, 5,597,531, and 5,698,271. The preparation of which from those base materials is described below.

It should be noted that many different cellular analysis platforms can be used to identify and enumerate enriched samples. Examples of such analytical platforms are the CellSpotter system (magnetic cell holder for manual observation of cells) and the CellTracks system (automated optical scanning magnetic cell holder) described in U.S. patent applications 08/931,067 and 08/867,009, respectively. The above-mentioned us patent applications are all incorporated herein by reference as disclosing respective instruments and methods for manual or automated quantitative and qualitative cell analysis.

Other analytical platforms include laser scanning cytometry (Comucyte), brightfield-based image analysis (Chromavision), and capillary capacitance assay (biometricing).

Counting circulating epithelial cells in blood using the methods and compositions of the invention is achieved by immunomagnetic selection (enrichment) of epithelial cells from blood followed by analysis of the sample by multiparameter flow cytometry. Immunomagnetic sample preparation for reducing sample volume and obtaining target (epithelial) cells 10⁴Fold enrichment is important. Reagents for multiparameter flow cytometry analysis are optimized such that the target cell is localized to a unique location in the multidimensional space created by the tabular acquisition of two light diffusers and three fluorescence parameters.These include 1) antibodies against the leukocyte antigen CD45 to identify leukocytes (non-tumor cells); for cell types specific for nucleic acid dyes, the nucleic acid dye allows for the exclusion of residual red blood cells, and other non-nuclear events; and 3) a biospecific agent or antibody directed against cytokeratin or an antibody specific for an epitope of EpCAM different from that used for immunomagnetic selection of cells.

Capture target：

The antibodies used for capture were selected based on initial testing of tumor cells in tissue culture. This strategy selects cross-sections of cell lines that represent multiple differentiation states, as expression levels vary according to differentiation state. The data suggest that most primary site cell lines are poorly differentiated, whereas ascites and metastasis derived cell lines are moderately to well differentiated. Accordingly, BxPC3 (moderate), Panc-1 (poor), CAPAN-1 (good), and CAPAN-2 (good) tumor cells were selected for antibody estimation based on differentiation status. EpCAM, Mucin1, Mesothelin, Claudin-4, EGFR1 and CEACAM6 were identified as targets for capture antigens. Flow cytometry analysis strategies were used to determine antigen expression levels and percentage of positive cell population numbers. All antibodies used in the study were primary antibodies and staining was monitored using anti-mouse secondary antibodies conjugated to fluorescent dyes. FIG. 1(A) shows the intensity of cell staining with various markers indicating expression levels. FIG. 1(B) shows the percentage of positive cells with markers.

The data in figure 1 show that all targets screened are present on cell lines at different expression levels with respect to each other, and that the expression levels are not consistent for all cell lines tested. The data suggests that antigen expression is altered across cell lines, indicating that antigens can also be altered in CTCs, similar to tissue cultured tumor cells. Analysis revealed that EpCAM, Claudin-4, EGFR1 and to some extent Mucin-1 were ubiquitously expressed across all cell lines. On average, antigens are expressed in 65% to 100% of the cell population. Therefore, the use of multiple capture targets covering a wide range of tumor cells is important for efficient capture. Furthermore, these markers should be specific for tumor cells and should not be present on white blood cells. Candidate capture antibodies are tested with white blood cells. Most markers are expressed at low levels on white blood cells, except CEACAM6, which appears to be highly prevalent in granulocytes and to a lesser extent in monocytes. This result precluded the use of CECAM6 as a capture target for use in this assay. Based on the above data, EpCAM, Mucin1, Mesothelin, Claudin-4 and EGFR antigens are good targets for capturing tumor cells.

Detection target：

Various labeled antibodies were tested for the detection of tumor cells. The markers tested were cytokeratin 7, 8, 17, 18, 19 and c-Src. These markers were tested by flow cytometry and at a concentration of 2ug/ml across all four cell lines. Fig. 2(a) and 2(B) summarize these results. The cytokeratin family is ubiquitously expressed in all cell lines (79% to 100% of the population is positive for at least one of the targets). The c-Src family is also expressed in all cell lines tested, with different expression levels (66% to 100% positive population). Overall, all cytokeratin antibodies worked equally well with comparable staining patterns and results. c-Src from andy organisms (R & DSystems) appears to be a weak binder and is not very bright, although this may be due to the secondary level of FITC conjugation used as a detection reagent. However, both anti-cytokeratin 17 and anti-c-Src are positive on white blood cells (monocytes and granulocytes). Based on this data, cytokeratins 7, 8, 18 and 19 were selected for use as mixture detection antibodies.

Conjugation of capture targets to ferrofluids：

anti-EpCAM, anti-EGFR 1, anti-Muc 1 and anti-Claudin 4 were selected for capture based on initial estimates of tumor cell lines cultured with pancreatic tissue. Up to three antibodies were conjugated to ferrofluid in several combinations using Veridex. LLC conjugation chemistry and combinations are as follows:

A. anti-EpCAM/EGFR 1/Muc1 multi-specific ferrofluid

B. anti-EpCAM/EGFR 1/Claudin4 multi-specific ferrofluid

C. anti-EpCAM/Muc 1/Claudin4 multispecific ferrofluid

anti-EpCAM antibodies were used in all combinations.

Conjugation of detection targets to Phycoerythrin (PE)：

Anti-cytokeratin (C11 antibody that recognizes cytokeratin 8 and 18), anti-cytokeratin 7, anti-cytokeratin 18, and anti-cytokeratin 19 were all conjugated to PE using veridex llc standard conjugation chemistry. All cytokeratin antibodies conjugated to PE were combined with anti-CD 45-APC to generate a cocktail staining reagent.

One skilled in the art will recognize that the assay method for enriching a tumor cell population will depend on the intended use of the invention. For example, in screening for cancer or monitoring for disease recurrence, as described below, the number of circulating epithelial cells may be extremely low. In that case, analysis based on microscopy may prove to be the most accurate. Such examination may also include examination of morphology, identification of known tumor markers and or oncogenes. Alternatively, in disease states where the number of circulating epithelial cells far exceeds that observed in the normal population, an analytical method to count such cells should be sufficient. The determination of the patient's status according to the methods described herein is made based on a statistical average of the number of circulating rare cells present in the normal population. Levels of circulating epithelial cells in early cancer patients and patients with aggressive metastatic cancer can also be determined statistically as described herein.

As described above, the kit begins with reagents, devices and methods for enriching tumor cells from whole blood. The kit will contain reagents for testing breast cancer cells in a blood sample, which reagents evaluate six factors or indicators. The assay platform needs to be configured such that the reporter molecules DAPI, CY2, CY3, CY3.5, CY5 and CY5.5 are distinguished by suitable excitation and emission filters. The analysis platform in this example uses a fluorescence microscope equipped with a mercury arc lamp, and a suitable filter bank for evaluating the wavelength of the detection marker employed. All tags are introduced at once in this way.

The present invention is an improvement on the CellSearch epithelial cell kit (CellSearch epistalcellkit) (Veridex, LLC). Several combinations of multispecific ferrofluids and mixture detection reagents are included in the present invention to create a variety of kit configurations. The primary improvement is the incorporation of capture and detection reagent components. The different reagent kit configurations are as follows:

1. kit 1 CellSearch epithelial cell kit as control: EpCAM for capture; c11(CK8, 18) and CK19 for detection.

2. Kit 2 multiple capture and mixture detection: EpCAM/EGFR/Muc-1 for capture (E29); c11(CK8, 18) + CK19+ CD7+ CD18 for detection.

3. Kit 3 multiple capture and mixture detection: EpCAM/EGFR/Claudin4 for capture; c11(CK8, 18) + CK19+ CK7+ CK18 for detection.

4. Multiplex capture and mixture detection: EpCAM/Muc-1(E29)/Claudin4 for capture; c11(CK8, 18) + CK19+ CK7+ CK18 for detection.

Estimation of different kit configurations

The above kit configuration was first evaluated with a normal blood sample of tissue-cultured pancreatic tumor cell spiking to examine its performance. Pancreatic tumor cells (BxPC3 and CAPAN-1 cell line) were spiked into 7.5mls cellsave donor blood (n ═ 2). Samples were processed on a celltrackautoprep and analyzed on a CellTracks analyzer II.

Recovery of spiked Capan-1 cells with the modification kit was similar to that of the standard epithelial cell kit (approximately 60%), however, there was consistently higher recovery of BxPC3 cells (approximately 90%) with both versions of the modification kit (FIGS. 3a and 3 b).

Four kit configurations were evaluated with blood samples from normal healthy donors and metastatic breast cancer patients. The results from this estimation are shown in tables 1 and 2. Preliminary data show that CTC recovery is higher in clinical samples using all versions of the modifier kit compared to standard epithelial kit 1 (table 1). CTCs were not detected from normal healthy donor samples using the modified kit (table 2).

TABLE 1：

TABLE 2：

Examples of different types of cancers that can be detected using the compositions, methods and kits of the invention include amine precursor uptake decarboxylation cell tumors, labyrinthine tumors, gill's native tumors, malignant benign tumor syndromes, benign tumor heart disease, malignant tumors such as wacker's cancer, stromal cell carcinoma, stromal squamous cell carcinoma, bubber's cancer, ductal carcinoma, elishi tumors, carcinoma in situ, Krebs2 cancer, merck cell carcinoma, mucinous carcinoma, non-small cell lung cancer, oat cell carcinoma, papillary carcinoma, hard cancer, bronchiolar carcinoma, bronchogenic carcinoma, squamous cell carcinoma and transitional cell carcinoma, reticuloendothelial proliferation, melanoma, chondroblastoma, chondroma, fibrosarcoma, giant cell tumor, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, ewing's sarcoma, and combinations thereof, Synovioma, adenofibroma, adenolymphoma, carcinosarcoma, chordoma, phyllodes tumor, mesonephroma, myosarcoma, ameloboma, cementoma, odonoma, teratoma, trophoblastic tumor, adenocarcinoma, adenoma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, granulomatoma, amphoblastoma, hepatoma, sweat gland adenoma, islet cell tumor, ledixic cell tumor, papilloma, Sertoli cell tumor, theca cell tumor, leiomyoma, leiomyosarcoma, myoblastoma, myoma, myosarcoma, rhabdomyosarcoma, ependymoma, ganglioma, neuroblastoma, gliomas, medulloblastoma, meningioma, schwanoma, neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma, non-chromophilous paraneuroma, angiokeratoma, scleroma, hemangioblastoma, hemangioma, hemangioblastoma, hema, Angiomatosis, hemangioblastoma, hemangioendothelioma, hemangioblastoma, hemangiopericytoma, angiosarcoma, lymphangioma, lymphangiomyoma, lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma, phyllocystic sarcoma, fibrosarcoma, angiosarcoma, leiomyosarcoma, leukemic sarcoma, liposarcoma, lymphangiosarcoma, myosarcoma, myxosarcoma, ovarian cancer, rhabdomyosarcoma, sarcoma (kaposi's and mast cells), neoplasms (e.g., bone, digestive system, colorectal, liver, pancreas, pituitary, testis, orbit, head and neck, central nervous system, auditory, pelvic, respiratory and urogenital), neurofibromatosis, and cervical dysplasia.

The present invention is not limited to the detection of circulating epithelial cells. Endothelial cells have been observed in blood of patients with myocardial infarction. Endothelial cells, cardiomyocytes, and virus-infected cells such as epithelial cells have cell-type specific determinants that are recognized by available monoclonal antibodies. Accordingly, the methods and kits of the invention may be suitable for detecting such circulating endothelial cells. In addition, the present invention allows for the detection of bacterial cell load in peripheral blood of patients with infectious disease, who can also be evaluated using the compositions, methods and kits of the present invention.

Some references to journal articles, U.S. patents, and U.S. patent applications are provided above. Each of the above-referenced subject matter is incorporated by reference into this specification as if fully set forth herein.

While certain preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the present invention be limited to these embodiments. Various modifications may be made to these embodiments without departing from the spirit of the invention, the full scope of which is covered by the following claims.

Claims (23)

1. Use of components a), b) and c) in the preparation of a kit for detecting and enumerating rare cells in a mixed cell population, the presence of said rare cells in said population being indicative of a disease state,

a) a coated magnetic nanoparticle comprising a magnetic core material, a protein base coating material, and more than one type of antibody, wherein each antibody specifically binds to a determinant characteristic of the rare cell, the antibody being directly or indirectly coupled to the base coating material;

b) at least one antibody having binding specificity for a second characteristic determinant of said rare cell; and

c) a cell-specific dye for excluding sample components other than the rare cells from the analysis,

wherein the detecting and counting comprises:

1) obtaining a biological sample from a test subject, the sample comprising a mixed cell population suspected of containing the rare cells;

2) preparing an immunomagnetic sample, wherein the biological sample is mixed with the coated magnetic nanoparticles to substantially exclude other sample components;

3) contacting the immunomagnetic sample with the at least one antibody having binding specificity for the second characteristic determinant of rare cells; and

4) analyzing the labeled rare cells to determine the presence and number of any rare cells in the immunomagnetic sample, the greater the number of rare cells present in the sample, the greater the severity of the disease state.

2. The use of claim 1, wherein as an intermediate step between the preparation of the immunomagnetic sample and contacting the immunomagnetic sample with at least one antibody having binding specificity for the second characteristic determinant of rare cells, the immunomagnetic sample is subjected to a magnetic field to produce a rare cell-enriched cell suspension as the immunomagnetic sample.

3. The use of claim 2, wherein the volume of the immunomagnetic sample containing enriched rare cells is reduced.

4. The use of claim 1, wherein the immunomagnetic sample is separated into a fraction containing labeled rare cells and an unlabeled fraction prior to analysis.

5. The use of claim 4, wherein the magnetic nanoparticles are colloidal and separation is achieved by subjecting the immunomagnetic sample to a magnetic gradient field.

6. The use of claim 1, wherein the rare cell is selected from the group consisting of a fetal cell in maternal circulation, a bacterial cell, a cardiomyocyte, an epithelial cell, and a virally-infected cell.

7. The use of claim 1, wherein the rare cell is an endothelial cell.

8. Use of components a), b) and c) for the preparation of a kit for the detection and enumeration of cancer cells in a mixed cell population,

a) a coated magnetic nanoparticle comprising a magnetic core material, a protein substrate coating material, and a plurality of types of epithelial cell-specific antibodies, the antibodies being directly or indirectly coupled to the substrate coating material;

b) at least one antibody having binding specificity for a cancer cell determinant; and

c) a cell-specific dye for excluding sample components other than cancer cells from the analysis,

wherein the detecting and counting comprises:

1) obtaining a biological sample from a test subject, said sample comprising a mixed cell population suspected of containing said cancer cells;

2) preparing an immunomagnetic sample, wherein the biological sample is mixed with the coated magnetic nanoparticles to substantially exclude other sample components;

3) contacting the immunomagnetic sample with the at least one antibody having binding specificity for a cancer cell determinant; and

4) analyzing the labeled cancer cells to determine the presence and number of any cancer cells in the immunomagnetic sample, the greater the number of cancer cells present in the sample, the greater the severity of the cancer.

9. The use of claim 8, wherein the magnetic nanoparticles are colloidal and the immunomagnetic sample is separated into a labeled cancer cell-containing fraction and an unlabeled fraction prior to analysis.

10. The use of claim 9, wherein colloidal magnetic nanoparticles and the at least one antibody having binding specificity for a cancer cell determinant are mixed sequentially with the biological sample, and as an intermediate step, the immunomagnetic sample is subjected to a magnetic field to produce a cancer cell-enriched cell suspension as the immunomagnetic sample.

11. The use according to claim 10, wherein the immunomagnetic sample is separated into a labeled cancer cell-containing fraction and an unlabeled fraction prior to analysis.

12. The use of claim 11, wherein the fraction of cancer cells containing markers is analyzed by a method selected from the group consisting of: multiparameter flow cytometry, immunofluorescence microscopy, laser scanning cytometry, bright field-based image analysis, capillary capacitance measurements, spectral imaging analysis, manual cell analysis, and automated cell analysis.

13. A test kit for screening a patient sample for the presence of circulating rare cells comprising:

a) a coated magnetic nanoparticle comprising a magnetic core material, a protein base coating material, and more than one type of antibody, wherein each antibody specifically binds to a determinant characteristic of the rare cell, the antibody being directly or indirectly coupled to the base coating material;

b) at least one antibody having binding specificity for a second characteristic determinant of said rare cell; and

c) a cell-specific dye for excluding sample components other than the rare cells from the analysis.

14. The kit of claim 13, further comprising an antibody having binding affinity for non-target cells, a biological buffer, a permeabilization buffer, a protocol, and optionally an information sheet.

15. The kit of claim 13, wherein the rare cells are selected from the group consisting of fetal cells, bacterial cells, cardiomyocytes, epithelial cells, and virally-infected cells in maternal circulation.

16. The kit of claim 13, wherein the rare cells are endothelial cells.

17. A kit for screening a patient sample for the presence of circulating tumor cells comprising:

a) a coated magnetic nanoparticle comprising a magnetic core material, a protein substrate coating material, and a plurality of types of epithelial cell-specific antibodies, the antibodies being directly or indirectly coupled to the substrate coating material;

b) at least one antibody having binding specificity for a cancer cell determinant; and

c) a cell-specific dye for excluding sample components other than said tumor cells from the analysis.

18. The kit of claim 17, further comprising an antibody having binding affinity for non-tumor cells, a biological buffer, a permeabilization buffer, a protocol, and optionally an information sheet.

19. The kit of claim 17 for screening patients for breast cancer, wherein the at least one antibody having binding specificity for a cancer cell determinant specifically binds to a breast cancer cell determinant, said determinant being selected from the group consisting of MUC-1, estrogen, progesterone receptor, cathepsin D, p53, urokinase-type plasminogen activator, epidermal growth factor receptor, BRCA1, BRCA2, CA27.29, CA15.5, prostate specific antigen, plasminogen activator inhibitor and Her 2-neu.

20. The kit of claim 17 for screening patients for prostate cancer, wherein the at least one antibody having binding specificity for a cancer cell determinant specifically binds to a prostate cancer cell determinant, said determinant being selected from the group consisting of prostate specific antigen, prostatic acid phosphatase, thymosin b-15, p53, HPC1 alkaline prostate gene, creatine kinase, and prostate specific membrane antigen.

21. The kit of claim 17 for screening patients for colon cancer, wherein the at least one antibody having binding specificity for a cancer cell determinant specifically binds to a colon cancer cell determinant, the determinant being selected from the group consisting of carcinoembryonic antigen, C protein, APC gene, p53 and matrix metalloproteinase (MMP-9).

22. The kit of claim 17 for screening patients with bladder cancer, wherein the at least one antibody with binding specificity for a cancer cell determinant specifically binds to a bladder cancer cell determinant, said determinant being selected from the group consisting of nuclear matrix protein (NMP 22), Bard Bladder Tumor Antigen (BTA) and Fibrin Degradation Product (FDP).

23. The kit of claim 17, wherein the at least one antibody having binding specificity for a cancer cell determinant comprises a set of antibodies each having binding specificity for a different cancer cell characteristic determinant.