METHODS OF USING ANTIBODIES AGAINST HUMAN CHORIONIC GONADOTROPIN-RELATED DETERMINANTS TO LYSE, MEASURE AND ENRICH MALIGNANT CELLS

Abstract: The invention provides a method of lysing malignant cells, which comprises contacting the malignant cells with an antibody which specifically reacts with an epitope present on the beta subunit of human chorionic gonadotropin and/or beta subunit fragment, under conditions such that the malignant cells lyse. This invention further provides a method of quantitatively measuring malignant cells or a method of obtaining an enriched population of malignant cells, which comprises contacting live malignant cells to be measured or contacting a population of cells comprising live malignant cells with an antibody directed to an epitope of human chorionic gonadotropin, its subunits and/or subunit fragments, under conditions such that the antibody is capable of forming a complex with the epitope if present on the cell surface, and detecting the presence of any complex formed using fluorescence activated cell sorting technology, the presence of such complex on the surface of the cells indicating that the cells are malignant, and isolating the cells which form a complex with the antibody.

Description: METHODSOFUSINGANTIBODIESAGAINSTHUMANCHORIONICGONADOTROPI RELATEDDETERMINANTSTOLYSE, MEASUREANDENRICHMALIGNANTCEL This invention was made with support under Grant No. P01 HD 15454 from the National Institute of Health. Accordingly, the U.S. government has certain rights in the invention. Background of the Invention Throughout this application various publications are referenced by Arabic numerals. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the art to which this invention pertains. The hormone of pregnancy, human chorionic gonadotropin (hCG) , is a dimeric sialoglycoprotein composed of two noncovalently linked dissimilar subunits known as α and β. The single α-subunit (hCGα) gene is located in chromosome 6 (1-3). A cluster of at least six genes (4, 5) located in chromosome 19 (3) encode individually for the β subunit (hCGβ) . Within species, hCGα is identical to the α-subunit of the three other members that constitute the family of glycoprotein hormones arising from the anterior pituitary, that is lutropin, follitropin and thyrotropin. The unigue β-subunits determine the biological specificity for each one of these hormones (6) . Under normal nonpregnant conditions, only sperm cells in male humans (7, 8) and cells from some fetal tissues (9) synthesize the subunits de novo. Expression of the subunits, as well as translatable levels of hCGβ mRNA, have been detected early after fertilization (10-13) . The dimer, which is the intact or whole compound (hCG- holo) , is the only bioactive molecule. Its formation depends not only on the different factors regulating the expression of the α-subunit and β-subunit genes (14, 15) but also on the synthesis of competent subunits (16-19) . Therefore, in cells that synthesize hCG, free subunits can also be found. Another important chemical characteristic of these highly glycosylated proteins (the carbohydrate component of intact hCG comprises approximately one third of its 37 KD molecular weight) is their high degree of sialylation. Thus, hCGβ has up to 12 sialic acid residues, 8 of them in the carboxy terminal peptide (CTP) (residues 111 to 145) , and hCGα has 4, resulting in molecules having a high negative charge. In addition, hCG-holo and its subunits are present in the cell in two pools, a secretory pool (hydrophilic, also known as the soluble pool) and a membrane pool, also described as the insoluble pool. In the latter pool, the dimer and its subunits become an intrinsic part of the cell membrane and cannot be extracted unless the membranes are destroyed by sonication and treated with detergents to solubilize the material—hence the name insoluble pool (20) . During the last 5 years, several pieces of information pertaining to the chemistry and biology of hCG have been published. The first was the elucidation of the structure of the urinary hCGβ core fragment, a dimer formed by peptides lacking 72 of the 145 amino acid residues of hCGβ, including the highly glycosylated (and sialylated) CTP of hCGβ (hCGβ-CTP) . The latter does not combine with the α-subunit, cross-reacts with practically all hCGβ antisera and most monoclonal antibodies (MoAb) (21, 22) , and contaminates in various degrees all hCG standard preparations. This is one of the main reasons for the heterogeneity and differences between immunoreactivity and bioactivity of these preparations (23) . The second was the finding, using high-resolution high- performance liquid chromatography fingerprinting of purified hCG together with analysis of selected peptides by amino acid sequencing and mass spectroscopy, that oxidation, not carbohydrate variability, is another cause of the chromatographic heterogeneity of the hCG isolated from pregnancy urine, the source of all existing hCG preparations (24) . The third was a recent report that this glycopeptide may circulate in blood associated with other macromolecules that mask its epitopes to existing hCGβ antibodies (25) . This new information (added to the potential for producing alterations in the biophysical and physiological characteristics of the cell membrane as a result of the existence of the membrane-associated hCG pool) and the reports that the metastatic potential of cultured murine tumor cell lines correlated with the amount of cell membrane sialylation (26) and increased negative charge (27) created the need for a practical quantitative method sensitive enough to measure this pool. The possibility of using flow cytometry for the analysis of membrane-associated hCG, it subunits, and fragments was demonstrated in 1982 (28) . This report was followed by feasibility studies on the use of a Coulter Epics-C flow cyto eter (Hialeah, FL) for determining chorionic gonadotropin-like material on the cell surface of malignant cells (29) . However, there was still absent any method of accurately quantitatively analyzing hGC, its subunits and fragments as membrane-associated material. fliiTnmwrγ o the Invention This invention provides a method of lysing malignant cells, which comprises contacting the malignant cells with an antibody which specifically reacts with an epitope present on the beta subunit of human chorionic gonadotropin and/or beta subunit fragment, under conditions such that the malignant cells lyse. This invention further provides a method of quantitatively measuring malignant cells, which comprises contacting live malignant cells to be measured with an antibody directed to an epitope of human chorionic gonadotropin, its subunits and/or subunit fragments, under conditions such that the antibody is capable of forming a complex with the epitope if present on the surface of the cells being measured, and detecting the presence of any complex formed using fluorescence activated cell sorting technology, the presence of such complex on the surface of the cells indicating that the cells are malignant. Finally, this invention provides a method of obtaining an enriched population of malignant cells, which comprises contacting a population of cells comprising live malignant cells with an antibody directed to an epitope of human chorionic gonadotropin, its subunits and/or subunit fragments, under conditions such that the antibody is capable of forming a complex with the epitope if present on the cell surface, the formation of such complex indicating that the cells are malignant, and isolating the cells which form a complex with the antibody. Brief Description of the Figures Figure 1 Fluorescence profiles illustrated by two-dimensional and three-dimensional histograms (left and right panels, respectivley) of an experiment demonstrating that background fluorescence due to nonspecific binding of the Ms IgG x Coulter clone control to the ATCC (American Type Culture Collection, Rockville, MD) CCL 2 HeLa cells (passage 109; viability 98%) was no different than that of PBS used as solvent for the reagents and carrier for the cells to illustrate the specific binding of MoAb ASH (same isotype as the nonspecific control MoAb) to an epitope of hCGβ-CTP (right and left lower panels) . In all two-dimensional histograms, the antibody code number is ii the upper left corner and the percent of cells showing reactivity is in the upper right corner. Unless otherwise indicated, antibody concentration is 200μg/lO 6 cells and 10,000 events are counted in each sample. All other MoAb showed similar profiles. Figure 2 Fluorescence profiles, illustrtated by two-dimensional and three-dimensionmal histograms of the competition study using a MoAb to an epitope of hCGβ (AS12) . Because the antigen used in this experiment was intact hCG, the results demonstrate that the membrane-associated antigenic determinant is identical to that of the human trophoblastic hormone. Figure 3 Flow cytometry fluorescence profiles in 2-dimensional and 3-dimensional histograms showing expression of hCGβ epitopes by cells from passage 150 of the retinoblastoma ATTC HTB 169 cell line. Viability was 77.5% and the cells were treated with propidium iodide. Figure 4 Histograms obtained from exact duplicate of cells from Figure 3. In this figure, the cells were not treated with propidium iodide. Figure 5 Expression of membrane-associated epitopes of intact hCG(MoAb B109) and hCGβ-CTP(ASH) by cultured human malignant cells. Group 1 - ; melanocarcinomas; X: adenocarcinomas of lung; : carcinomas of breast; : carcinomas of prostate. Group 2 - : carcinomas of urinary bladder; : carcinomas of colon. Group 3 - : carcinomas of nasopharynx; : small cell carcinomas of lung; : carcinomas of endometrium; : retinoblastomas. Vertical cross bar: mean ± SE. P values of 0.05 or less were considered significant. Detailed Description of the Invention Specifically, this invention provides a method of lysing malignant cells, which comprises contacting the malignant cells with an antibody which specifically reacts with an epitope present on the beta subunit of human chorionic gonadotropin and/or beta subunit fragment, under conditions such that the malignant cells lyse. As used in the subject invention, the malignant cells to be lysed include, but are in no way limited to, sarcoma cells, lymphoma cells and retinoblastoma cells. As used in the subject invention, the term "antibody" includes, but is not limited to, both naturally occurring and non-naturally occurring antibodies. Specifically, the term "antibody" includes polyclonal and monoclonal antibodies, and binding fragments thereof. Furthermore, the term "antibody" includes chimeric antibodies and wholely synthetic antibodies, and fragments thereof. The antibody may be a monoclonal antibody. In one embodiment of the invention, the monoclonal antibody is the monoclonal antibody designated B201. In another embodiment of the invention, the monoclonal antibody is designated B205. The method of the subject invention may be used to treat a patient suffering from a malignant form of cancer. Specifically, treating the patient may comprise administering to the patient an amount of antibody which specifically reacts with an epitope present on the beta subunit of human chorionic gonadotropin and/or beta subunit fragment effective to lyse malignant cells in the patient's body. The administration may comprise intravenous, subcutaneous, or intraperitoneal administration, or any other route of administration well known to those skilled in the art. Furthermore, the antibody may be administered using a pharmaceutical carrier well known to those skilled in the art. This invention further provides a method of quantitatively measuring malignant cells, which comprises contacting live malignant cells to be measured with an antibody directed to an epitope of human chorionic gonadotropin, its subunits and/or subunit fragments, under conditions such that the antibody is capable of forming a complex with the epitope if present on the surface of the cells being measured, and detecting the presence of any complex formed using fluorescence activated cell sorting technology, the presence of such complex on the surface of the cells indicating that the cells are malignant. As used in the subject invention, the malignant cells to be detected include, but are in no way limited to, melanocarcinoma cells, nasopharyngial carcinoma cells, lung non-small cell carcinoma cells, lung small cell carcinoma cells, breast cancer cells, urinary bladder carcinoma cells, uterine cervix squamous cell carcinoma cells, endometrial carcinoma cells, colonic carcinoma cells, prostate carcinoma cells, osteocarcinoma cells, rhabdomyosarcoma cells, leukemia cells, lympho a cells, retinoblastoma cells and choriocarcinoma cells. The antibody may be a monoclonal antibody. In one embodiment of the invention, the monoclonal antibody is the monoclonal antibody designated B201. In another embodiment of the invention, the monoclonal antibody is designated B205. Finally, this invention provides a method of obtaining an enriched population of malignant cells, which comprises contacting a population of cells comprising live malignant cells with an antibody directed to an epitope of human chorionic gonadotropin, its subunits and/or subunit fragments, under conditions such that the antibody is capable of forming a complex with the epitope if present on the cell surface, the formation of such complex indicating that the cells are malignant, and isolating the cells which form a complex with the antibody. As used in the subject invention, the malignant cells to be enriched include, but are in no way limited to, melanocarcinoma cells, nasopharyngial carcinoma cells, lung non-small cell carcinoma cells, lung small cell carcinoma cells, breast cancer cells, urinary bladder carcinoma cells, uterine cervix squamous cell carcinoma cells, endometrial carcinoma cells, colonic carcinoma cells, prostate carcinoma cells, osteocarcinoma cells, rhabdomyosarcoma cells, leukemia cells, lymphoma cells, retinoblastoma cells and choriocarcinoma cells. The antibody may be bound to an insoluble matrix such as that used in affinity chromatography. As used in the subject invention, isolating the cells which form a complex with the immobilized monoclonal antibody may be achieved by standard methods well known to those skilled in the art. For example, isolating may comprise affinity chromatography using immobilized antibody. The antibody may be a free antibody. In this case, isolating may comprise cell sorting using free, labeled primary or secindary antibody. Such cell sorting methods are standard and are well known to those skilled in the art. The antibody may be a monoclonal antibody. In one embodiment of the invention, the monoclonal antibody is the monoclonal antibody designated B201. In another embodiment of the invention, the monoclonal antibody is designated B205. This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter. Experimental Details I. Flow Cγtometrv Method For The Analysis Of M«mhιr»τ>a- Associated Human Chorionic Gonadotropin. Its Introduction The combination of flow cytometry and MoAb binding to different epitopes located in different parts of the hCG molecule has produced a method that has three advantages: 1) the use of live cells, 2) a high degree of sensitivity that can detect as few as 10 3 molecules of fluorochrome per cell (30) , and 3) a good supply of standardized reagents. Presented here is a detailed description of this new method, its reliability and reproducibility, and some examples of its application to the measurement of membrane-associated hCG in cultured human malignant cells. Materials and Methods Cell Cultures HeLa cells (adenocarcino a of uterine cervix) and two of its variants, HeLa 229, and HeLa S3 were obtained from the American Type Culture Collection (ATCC; ATCC CCL 2, CCL 2.1, and CCL 2.2, respectively). The origin, characteristics and references of these cell lines are given in the ATCC catalog (31) . JEG-3 cells, a clone 3 of the choriocarcinoma BeWo cells (32-34) are available from ATCC (ATCC HTB 36) . All cells were grown in a humidified incubator at 37°C and 5% C0 2 in RPMI-1640 medium (JRH Biosciences, Lenexa, KS) with 10% defined iron-supplemented bovine calf serum (HyClone, Logan, UT) , 2 mmol/1 L-glutamine (Fisher Scientific, Fairlawn, NJ) , 1 mmol/1 sodium pyruvate (JRH Biosciences) , and 20 mg/1 of gentamicin sulfate (Sigma, St. Louis, MO) . In one experiment, JEG-3 cells were grown in minimum essential medium (MEM) , alpha modification (Sigma) with 4% fetal bovine serum (HyClone) and 20 mg/1 of gentamicin sulfate. The cells were cultured in 25-cm 2 and 75-cm 2 tissue culture flasks. For passage or analysis, the cells were detached using trypsin 0.25% with ethylenediamine tetracetic acid 0.1% (JRH Biosciences) or a cell scraper (Costar, Cambridge, MA) . All cells were harvested at confluence for analysis. A hemacytometer was used to count the cells for all experiments, and the viability was determined by dye exclusion with trypan blue. Cell Controls Because ectopic de novo synthesis, cell membrane localization, and secretion of subunits and bioactive hCG by HeLa cells has been well established (35-37) , the ATCC CCL 2 HeLa cell line was selected for use as a cell control for membrane-associated hCG, its subunits, and fragments and for overall quality control of the method. Antibodies The development of the method using the indirect or double-antibody technique required the testing of numerous antibodies to select the most appropriate ones to be used as first (or primary) antibodies. Immunoglobulin (Ig) G fractions of three polyclonal rabbit antisera (Clinetics, Tustin, CA) were used with the following: 1) an antiwhole hCG-hLH (human luteinizing hormone) IgG, reacting with intact hCG-hLH and the free subunits; 2) an anti-hCGβ-hLHβ IgG, reacting with dimer and the free β-subunits; and 3) an anti-hCGα IgG, reacting with the dimer and the free α-subunit. Of 22 MoAb studied initially, 12 were selected for additional testing. These are listed in Table 1, which Table 1 Nomenclature of Antibodies, Their Binding Sites, and Relative Specificities Toward Huma Chorionic Gonadotropin, Its Subunits and Fragments hCG hCGβ hCGβ Antibody Immunogen holo hCGα hCGβ CTP frgmt ASH hCG + NT + + NT + + + + + + +: Antibody binding to antigen with Ka, no less than 1 x 10 7 /mol; -: no measurable binding of antibody to antigen; NT: not tested; hCG: human chorionic gonadotropin; holo: intact; α: alpha subunit; β: beta subunit; CTP: carboxy terminal peptide. ♦Synthetic hCGβ-CTP, residues 109-145, conjugated with diphtheria toxoid. fPrefusion boost with hCGβ. All measurements were done by liquid-phase radioimmunoassays. —: Bibliographic reference also gives the nomenclature used for their identification, the im unogens used for their production, and their relative specificities. Details of the production and the characteristics of A105, B107, B108, B109, B201, B203 and B204 have been published (22, 38). The three CTP. antibodies (CTP101, CTP102 and CTP103) are antibodies directed against epitopes of a synthetic hCGβ- CTP. They were obtained by using (as an immunogen) an anti-hCG vaccine consisting of a synthetic hCGβ-CTP, residues 109 to 145, conjugated to diphtheria toxoid (39) . Details of the vaccine's preparation and the results of its effects and productions of anti-hCGβ-CTP antibodies in Phase 1 studies in humans, have been published (40-42) . Secondary antibodies for all these reagents were goat anti-rabbit fluorescein isothiocyanate (FITC)-conjugated F(ab) 2 IgG (Tago, Burlingame, CA) for the polyclonal IgG, and goat anti-mouse FITC-conjugated F(ab) 2 IgG (Caltag, San Francisco, CA) for the MoAb. Preparation Of Cultured Cells For Flow Cvtometry 1. Harvest the cells at confluence, using trypsin 0.25% with ethylenediamine tetracetic acid 0.1% when necessary, and collect them in a 15-ml or 50-ml tube. 2. Determine the number of cells per ml and the viability using a hemacytometer and trypan blue dye exclusion. 3. Pipette IO 6 cells into a series of 12 x 75-mm polypropylene tubes, the number of tubes depending on the number of cells available and the number of antibodies to be used. 4. Pellet the cells by centrifuging at 200 x g for 7 minutes and discard the supernatant. 5. Add lOOμl normal goat serum 3% in PBS to each tube and vortex gently for approximately 10-15 seconds. PBS was prepared from a stock solution of PBS 10X concentration (Whittaker, Walkersville, MD) . 6. Incubate the tubes in ice for 30 minutes. 7. Suspend the cells in 3 to 3.5ml of PBS, vortex gently, and centrifuge suspension at 200 x g for 7 minutes. Discard the supernatant. 8. Resuspend the cells in PBS, vortex gently, and centrifuge them at 200 x g for 7 minutes. Discard the supernatant. 9. Add each primary antibody to its respective tube, 0 following the plan of the experiment and using appropriate controls. a. The protein concentration (mg/ml) of each antibody is determined by the Bradford method (58) using the Bio-Rad protein assay according 5 to the manufacturers instructions. b. The amount of antibody in micrograms of protein per IO 6 cells must be determined experimentally by titration of the antibody with IO 6 cells of the ATCC CCL 2 HeLa cell control. 0 10. Vortex the tubes gently and briefly. 11. Incubate overnight at 4 to 8°C on a dimensional rotator. 12. Wash the cells two times with PBS as in steps 7 and 8. 5 13. Add lOOμl of appropriately diluted florescein isothiocyanate-conjugated species-specific second antibody. The concentration must be determined by titration. 14. Vortex the tubes gently and briefly. " 15. Incubate in ice for 30 minutes. 16. Wash .the cells twice with PBS as in steps 7 and 8. 5 17. Resuspend the cells in 0.5 ml paraformaldehyde 1% ή PBS and vortex each tube gently. The cells can be stored at 4 to 8°C for one week at this point. 18. Use flow cytometry to determine the percent of cells binding with each antibody. Flow Cytometry To determine the percentage of cells expressing hCG, its subunits and fragments, a Coulter Epics-C flow cytometer with a 2-W argon laser and standard settings and filters for FITC use was used. Instrument performance was controlled by daily calibration using the Coulter Immuno- Check system (fluorescence beads specially prepared and calibrated for fluorescence analysis) . The percent of cells binding with each MoAb was determined using Coulter Immuno software that subtracts a control fluorescence profile from the test fluorescence profile when the two flow cytometry runs are superimposed. To select the first antibodies to be used with this technique, the MoAb were tested for effects of antibody concentration on test sensitivity by titration and for effects of variability of epitope expression. The polyclonal IgG were tested only for effects of antibody concentration because, as a result of their polyclonality, they carry IgG recognizing different available determinants of hCG, thus making them more sensitive and relatively free of the effects of variability of epitope expression. The specificity of the polyclonal IgG was ascertained by using rabbit IgG as a control reagent for nonspecific binding and to obtain the control fluorescence profile. The control fluorescence profile for the MoAb was obtained by testing two unrelated MoAb of the same isotype for nonspecific binding, a mouse IgGl Coulter Clone Control and an - 18 - antileukocyte antigen (HLe-1) MoAb (Becton Dickinson, Mountain View, CA) . These control reagents were used at the same concentration as the MoAb tested. The method was also tested for reproducibility at different levels of epitope expression and for effects of incubation time of the viability of the cells and on the percentage of cells reacting. Finally, to ascertain the specificity of the antigen-MoAb reaction, a competition experiment was done by reacting IO 6 HeLa cells with 150 μg of MoAb AS12 and different amounts of pure hCG. The ATCC CCL 2 HeLa cultured cells were used for all these tests with phosphate-buffered saline as the carrier for the cells and as the solvent for all antibodies. Results The histograms generated by analyzing cell surface proteins using the Coulter Epics-C flow cytometer and the Coulter Immuno software, depict 1) the frequency of fluorescent cells relative to the number of cells in the sample, expressed as a percent on the ordinate, and 2) the log of the fluorescence intensity on the abscissa (Fig. 1, left panels) . Three-dimensional histograms cannot give quantitative results. In general, there were three different levels of hCG- related determinants measurable by the panel of primary antibodies tested; a high level of expression, with over 60% of cells presenting a high fluorescence intensity; an intermediate level; and a low level with less than 15% of cells showing low fluorescence intensity. Undetectable levels or traces were considered when less than 10% of the cells showed a very low fluorescence intensity, down to a computer value of zero (Figure 1, second panel, left) . - 19 - Effects Of Antibody Concentration (Titration) These tests were done to determine the sensitivity of the assay with a MoAb to a given epitope and to determine the optimal concentration of the different MoAb to be used because, for flow cytometric analysis, the best results are obtained using antibodies at saturating levels (43). The results, using the MoAb against epitopes located in the previously described regions of the intact hCG and its subunits (Table 1) , showed that the saturating concentrations for all MoAb (except those raised to the synthetic hCGβ-CTP) were approximately 200 μg/10 6 cells. The decreased sensitivity of this type of MoAb has been reported previously (44) . The results of the experiment illustrated in Figure 1 show that, at the concentrations used, nonspecific binding by the second antibody (FITC-tagged goat anti- mouse F(ab 1 ) 2 IgG) and by the first antibody, MoAb MsIgGl (the Coulter clone control) was practically nonexistent (upper right panel and second left and right panels) . When the second control (the antileukocyte antigen MoAb HLe-1) was used, similar results were obtained (data not shown) . However, the reactivity of the cells with MoAb ASH, directed to an epitope of hCGB-CTP was notable, as illustrated by the two bottom panels, right and left, in Figure 1. Reproducibility Two sets of six aliquots of IO 6 cells/tube, with the same amount of MoAb, were used to test for reproducibility, employing two MoAb against hCGβ-holo (hCGB-free and as part of the dimer). The first, MoAb B108, showed a low level of epitope expression in this particular passage (less than 15% of cells and low fluorescence intensity) . The second, MoAb AS12, demonstrated a high level of epitope expression (greater than 60% of cells and high fluorescence intensity) . The results show that the reproducibility of the assay was excellent; in each test, almost identical histograms were found for each aliquot (standard deviation [DS], ± 0.82; standard error of the mean [SE] , ± 0.33; and coefficient of variation [CV] , 6.5; mean, 12.6% of cells expressing the epitope in the first test. SD ± 1.09; SE ± 0.44; CV 1.5; mean, 72.05% of cells expressing the epitope in the second test) . Effects of Variability of Epitope Expression This test used, in most cases, those antibodies that were the most sensitive by titration. There was low variability (CV, 1.58 to 6.42) when cells of the same passage were tested on the same day, on consecutive days, or even within a six-day interval. However, significant variability in the expression of any single related immunologic determinant was seen when the reagents were tested at different passages. As an example, in four different passages, the range of expression of the hCGfi- CTP (as given by its epitope reacting with MoAb ASH) was 44.1% to 90.9% (mean ± SD, 58.1 ± 22.05; CV, 37.9). Effects of Incubation Time on Cell Viability To investigate the effects of incubation with the first antibody on the viability of the cells, incubations were done by different technicians using MoAb AS12, duplicates of IO 5 cells, and incubation times of 2 hours and overnight (19 to 22 hours) with and without the first antibody. All cell counts (trypan blue exclusion) were done in duplicate, and the experiments were repeated 1 week later using cells from the same passage. The viability of the cells incubated without the first antibody ranged from 90.4% to 95.3%; the viability of the cells incubated with the first antibcάy for the same periods ranged from 90.1% to 93.8%. It was concluded that, under the conditions used, the length of the incubation time with the first antibody did not have any effect on the viability of the cells being studied. Effect of Incubation on the Reaction The results of these experiments using three different MoAb (Table 2) show that overnight incubation produced a significant increase in the percentage of cells expressing a given epitope. Using Students's t test for paired data, the difference between the means of the results of these experiments was statistically significant in favor of the overnight incubation (P = 0.02 for MoAb AS12, P = 0.01 for MoAb B108, and P = 0.008 for MoAb A105) . Competition Study The two-dimensional and three-dimensional histograms in Figure 2 show that incubation of the same number of cells (106 cells) with the same amount (150 μg) of MoAb against hCGβ (AS12) and increasing amounts of pure antigen (5 x IO "8 to IO "4 mol/1 of hCG) resulted in a significant decrease in the fluorescence intensity and the percent of cells expressing hCGβ. Therefore, specific competition for the binding of the soluble MoAb was found between epitopes on the molecule of soluble free antigen (hCG CR- 127) and epitopes on the molecules of membrane-associated antigen. Application of the Method The three polyclonal IgG and MoAb B109 (or B107) , AS12 (or B108) , B201 (or B204) , ASH (or CTP103) , and A105, were selected for use as first antibodies to validate the method on cultured human malignant cells. When the Table 2. Mean Percent of cells 2 hours 19-22 hours Difference 10.2 29.1 17.9 13.2 17.6 19.1 13.7 16.3 24.5 24.1 19.2 8.4 10.5 8.0 21.8 15.0 12.7 ATCC: American Type Culture Collectin; P: passage No.; %V: percent viability; MoAb: monoclonal antibody. purified IgG fractions from the polyclonal antisera were used, they permitted a general determination of the total expression of the membrane-associated glycoproteins. MoAb binding showed the various epitopes in the hCG molecule (Table 1) . According to the titration studies, the amounts of antibody to be used were 100 μg/10 6 cells for the anti-hCG-holo polyclonal IgG and 200 μg/10 6 cells for the other antibodies (except MoAb CTP103, which was used at 300 μg/10 6 cells) . To standardize conditions, protein analysis, maintenance of the pH of the antibody solution between 6.0 and 7.5, and titration of the antibodies using ATCC CCL2 HeLa cells all were done with each new batch or lot of antibody. This method, applied under our controlled conditions, is being used in a systematic investigation of the expression of these sialoglycoproteins on the membranes of cultured human cancer cells of every origin and type that can be obtained, mainly from the ATCC The first results of serial flow cytometric analyses of different passages of JEG-3 cells and ATCC HeLa CCL2 and its two variants, HeLa S3 and HeLa 229, using the selected panel of antibodies are presented in Tables 3 and 4, respectively. JEG-3 cells, the choriocarcinoma clone, represent the classic eutopic producer of hCG, its subunits and fragments. HeLa cells represent the classic ectopic producer. The importance of maintaining the same conditions of culture (45) is shown by the results of the concomitant analyses of two aliquots of Passage 4 JEG-3 cells. One was grown in MEM with 4% fetal calf serum instead of the selected RPMI medium with 10% defined bovine serum (Table 3) , and the results of the expression of hCG, its subunits and fragments were significantly different. The two cell lines expressed different Table 3. Choriocarcinoma hCG: human chorionic gonadotropin; holo: intact; α: alpha subunit; β: beta subunit; CTP: carboxy terminal peptide; P: passage No.; %V: percent viability; MEM: minimum essential medium; FBS: fetal bovine serum. *Our passage. tGrown in MEM, 4% FBS. tGrown in RPMI-1640, 10% defined bovine calf serum. SMoAb CTP103 was used. NT: not tested; Ig: immunoglobulin; P: passage No.; %V: percent viability; MoAb: monoclonal antibody; hCG: human chorionic gonadotropin; holo: intact; α: alpha subunit; β: beta subunit; ATCC: American Type Culture Collection; CTP: carboxy terminal peptide. *Our passage. fMoAb CTGP103 was used. epitopes for antibodies raised against hCG-holo, hCGα, hCGβ and hCGβ-CTP, by a high percentage of cells. Discussion It is stressed that there are fundamental differences between the flow cytometric method used here and all other techniques for detection and quantitation of hCG, its subunits and fragments. In the other types of analyses (liquid-phase or solid-phase radioimmunoassay and im unoradiometric assays) , the antigen (hCG, its subunits or its fragments) is tested outside of its site of production (solubilized) , and the results are expressed by the amount of antigen per unit of volume or weight. The flow cytometric technique used here uses live cells, and the results show the percent of cells analyzed which express the antigen on their cell membranes. The relative amount of antigen being expressed was given by its fluorescence intensity. These characteristics make the method presented here unique, and there is no comparison or relationship between the results obtained here and those using any of the other analytical techniques. For the same reasons, all sensitivity values and characteristics of the antibodies used were related solely to their application in this technique. The application of flow cytometry in concert with the use of well-defined standardized reagents resulted in a quantitative method for analyzing hCG, its subunits and fragments as membrane-associated material or, in other words, as a phenotypic characteristic of cultured human cancer cells. The method included external and internal quality control, i.e., zero values obtained when the antigenic determinant was not available or was not being expressed in the cell surface, thus providing additional proof of the specificity of the antibody reaction (see Results and Tables 7 and 9 in part II of Experimental Details, infra) and the highest attainable degree of sensitivity. Because the technique is commercially available, it can be used anywhere. The specificity and quality assurance, which were superior to the best obtained by flow cytometric analysis of phenotypic markers (46) , was caused in part by the fact that the antigen is one of the most widely studied glycoprotein hormones and is available with a high degree of purity (44) . The types of studies done to ascertain the specificity of the MoAb against phenotypic markers commonly used in flow cytometric analyses are, at best, blocking experiments because pure antigens are not available in most cases. The selection of the ATCC CCL 2 line as a malignant cell control and for standardization of the reagents was based on the following considerations: 1) it is a cell line which has been studied extensively for its ectopic synthesis and secretion of hCG and its subunits by biologic, biochemical, and molecular biologic methods (35-37) ; 2) differential expression of hCGα genes in ectopic and eutopic hCG-producing cells has been demonstrated (47) ; 3) it is the original strain from which all other existing HeLa variants have been derived (31); 4) it has the advantage of being easily accessible worldwide; 5) it provides a low-cost quality control system; and 6) it permits comparison of results from different laboratories because different antibodies can be tested and standardized under the same conditions described here. The results of experiments using other IgGl MoAb as control antibodies for nonspecific attachment (Figure 1) showed not only the specificity of the reaction with this panel of well-characterized MoAb (23-38) , but also permitted, during analysis of cultured human cells, the use of inexpensive phosphate-buffered saline for the control fluorescence profile instead of expensive MoAb directed to unrelated antigens. With respect to this latter type of control reagent, it is important to remember that transformed and malignant cells have multipotential capabilities with respect to protein synthesis and phenotypic characteristics. For example, expression of lymphocyte markers by small cell carcinomas of lung has been reported (48) as has expression of epithelial cell antigen by histocytic lymphomas (31) . Therefore, when using an unrelated MoAb as a control for nonspecific binding, there is always the possibility that binding may occur because some cancer cells may express epitopes of that particular protein (49) . One or more of the MoAb used here might cross react with an equal determinant of another type of protein. All these possibilities were minimized by the simultaneous use of 1) MoAb against epitopes located in five distinct zones of the hCG molecule, and 2) MoAb reacting with different epitopes of the same zone of the molecule that were close enough to compete with each other, such as BIO8 and AS12 and B204 and B201. In the competition experiment, MoAb amounts of less than the saturation levels employed for phenotypic analyses (43) were used to avoid an excess of soluble antibody that would bind to the soluble added antigen (hCG) easily, thus decreasing the amount available for displacement. The MoAb specific for the intact hormone (B109) was not used for the same reason. Nevertheless, the results showed that binding displacement of the MoAb AS12 to the cancer cell occurred, thus providing additional evidence of the specificity of the reaction. This experiment and its results are the first to demonstrate the use of competition in a system completely different from that of radioimmunoassay and its variations, a system that uses live cells with variable or unstable expression of epitopes of a relatively small peptide. This variability was found in the serial studies of the JEG-3 cells and HeLa cells and its variants (Tables 3 and 4) . The extreme variability, and sometimes low degree of expression, of the conformational epitopes of intact hCG- -as in the cases of Passage 118 HeLa CCL 2 and Passage 13 HeLa CC1 2.2 (HeLa S3 variant), where only 7.6% and 7.3% of the cells, respectively, reacted with MoAb B109, while a high percentage showed expression of epitopes of hCGB and hCG —may occur for two reasons. First, there is a possibility that, during these particular passages, the determinant was not accessible in the cell surface. Second, incompetent hCGα might have been produced by the cancer cell caused by posttranslational modifications that led to altered subunit synthesis, rendering it unable to combine with hCGβ to form hCG-holo (50) . The initial studies using this new technique, which included testing of 40 carcinomas and 8 leukemias and lymphomas (51) , have shown that epitopes for the intact trophoblastic hormone, its subunits, and/or fragments are expressed in the cell membrane of all cancers currently studied. Conclusion A quantitative flow cytometry method for the analysis of membrane-associated human chorionic gonadotropin (hCG) , its subunits, and fragments on human cancer cells was developed using a double-antibody reaction; a flow cytometer with a 2-W argon laser, standard settings, and filters for fluorescein isothiocyanate use; commercially available software; and the ectopic hCG producer CCL 2 HeLa cells from the American Type Culture Collection (ATCC) as a cell control to standardize the reagents and for overall quality control. Twenty-two monoclonal antibodies (MoAb) and immunoglobulin G fractions from three rabbit polyclonal antisera were tested for effects of antibody concentration (titration) , reproducibility at different levels of epitope expression, and variability of epitope expression to select appropriate primary antibodies. Based on the results of the various tests, three polyclonal immunoglobulin G antibodies and a panel of nine MoAb directed to epitopes located in five different regions on the hCG molecule were selected as first antibodies. Their specificity was determined by using two unrelated MoAb of the same isotype at the same concentration to replace the primary MoAb and by a competition experiment. The unrelated MoAb were also used for the selection of the appropriate control fluorescence profile needed for the software. The unique characteristics of this method are the use of living cells, standardized reagents, internal and external quality control, and the high sensitivity, capable of allowing the detection of as few as IO 3 molecules of fluorochrome per cell. Serial analyses of the ATCC CCL 2 HeLa cells and two of its variants of the eutopic hCG producer JEG-3 choriocarcinoma cells revealed the expression of membrane-associated epitopes of intact hCG, its subunits, and fragments by a high percentage of the cells, indicating that the expression of these sialoglycoproteins by these two different types of cancer cells is a common phenotypic characteristic. II. Expression Of Maniivrane-Associatefl wιwι»n Chorionic Gonadotropin, Its Subunits, And Fragments Bv Cultivated Human Cancer cells Introduction Because of the heterogeneity of hCG standard preparations and the great differences if immunoreactivity versus bioactivity reported in the literature (23) , a reassessment of hCG, its biochemistry, and its association with malignant transformation and cancer was needed. Reported here are the results of a systematic study of the expression of membrane-associated hCG, its subunits, and fragments by a wide variety of established, well- characterized cultured human malignant cell lines. Through this assessment, the following basic questions are addressed: 1) is the expression of membrane- associated hCG, its subunits, or fragments a phenotypic characteristic of some or all cultured cancer cells; 2) are there quantitative or qualitative differences in the expression of membrane-associated hCG-holo, its subunits, or fragments; 3) is the unique CTP of hCBB always expressed in the cell membrane; and 4) do any of the MoAb used as reagents have any cytolytic or cytotoxic activity toward cancer cells in culture? To answer these guestions, the flow cytometric procedure described supra was used which uses live cells with specific standardized reagents to achieve both the highest specificity and unsurpassed sensitivity characteristic of flow cytometry, with a level of detection of as little as IO 3 molecules of fluorochrome per cell (30) . Materials and Methods Cell Cultures Seventy-four cell lines were studied. There were a total of 52 carcinomas, including 4 melanocarcinomas, 2 carcinomas of nasopharynx, 13 carcinomas of the lung (5 adenocarcinomas, 3 squamous cell, and 5 small cell) , 6 carcinomas of breast, 7 carcinomas of urinary bladder, 8 squamous cell carcinomas of uterine cervix, 2 carcinomas of endometrium, 7 carcinomas of colon, and 3 carcinomas of prostate; 10 sarcomas (6 osteosarcomas and 4 rhabdomyosarcomas) ; 4 leukemias; 6 lymphomas; and 2 retinoblastomas. All cells were grown in a humidified incubator at 37°C and 5% C0 2 , in RPMI 1640 medium (JRH Biosciences, Lenexa, KS) with 10% defined iron-supplemented bovine calf serum (HyClone, Logan, UT) , 2 mmol/1 L-glutamine (Fisher, Pittsburgh, PA) , 1 mmol/1 sodium pyruvate (JRH Biosciences) , and 20 mg/1 of gentamicin sulfate (Sigma, St. Louis, MO) . The 10% defined bovine calf serum was replaced with 1% Nutrido a (Boehringer Mannheim, Indianapolis, IN) in one experiment and with 10% decomplemented defined bovine calf serum (HyClone) in another. The cells were grown in 25-cm 2 tissue culture flasks and were detached using trypsin 0.25% with ethylene-diamine tetraacetic acid 0.1% (JRH Biosciences) or a cell scraper (Costar, Cambridge, MA) for passage or analysis. A hemacytometer was used to count the cells, and viability was determined by dye exclusion with trypan blue. Flow Cytometry Expression of membrane-associated hCG, its subunits and fragments was determined using our flow cytometric technique. Briefly, the method involves a double- antibody reaction and the use of a Coulter Epics C flow cytometer (Hialeah, FL) with an argon laser and standard settings and filters for fluorescein isothiocyanate (FITC) . Daily calibration for control of instrument performance was done using the Coulter Immuno-Check system. Coulter Immuno software, which subtracts a control fluorescence profile from the test fluorescence profile when the two flow cytometric runs are superimposed, was used to determine quantitatively the presence of cells binding with each antibody. ATCC CCL 2 HeLa cells were used as a positive cell control for hCG and its subunits. They were also used to standardize the polyclonal antibodies and MoAb. Polyclonal antisera, immunoglobulin (Ig) G fractions raised against the hCG dimer and both subunits, and a panel of MoAb recognizing different epitopes on the hCG-holo molecule, its subunits and fragments, were used as first antibodies, and goat anti-rabbit and goat anti-mouse FITC-conjugated F(ab) 2 IgG, respectively, as second antibodies. The purified IgG fractions from the polyclonal antisera, when used, gave a general assessment of the total expression of these particular membrane-associated glycoproteins. The MoAb were used to detect the expression of specific epitopes located at five different sites of the hCG molecule (22) on the membrane of the cultured cancer cells. These MoAb were directed against different conformational epitopes of hCG-holo (B109 or B107) , hCGβ (AS12 or B108), hCGβ-free (B204 or B201) , hCGβ-CTP (ASH or CTP103), and an epitope of hCGα (A105) . Unless indicated otherwise, polyclonal IgG against hCG-hLH was used at a concentration of 100 μg/10 6 viable cells. All other polyclonal antibodies and MoAb were used at a concentration of 200 μg/10 6 viable cells (except MoAb CTP103 which was used at a concentration of 300 μg/10 6 cells) . These saturating concentrations were defined by titrations of the antibodies using CCL 2 HeLa cells. To study the effects of a relatively low viability (under the same conditions of culture) on the flow cytometric analysis, cells from a retmoblastoma cell culture were analyzed simultaneously with and without propidium iodide (PI) treatment (10 6 cells treated with 10 μl of 0.5 mg/ml PI in phosphate-buffered saline for 5 minutes) . Although PI and FITC are excited by the argon laser at the same wavelength (488 nm) , their emission wavelengths are different, i.e., 520 nm (green) for FITC and 615 nm (orange-red) for PI. This permits exclusion of the dead Pi-containing cells (30) . Results and Discussion Results obtained from analyses of the 74 cancer cell lines are given in Tables 5 to 19. All cell cultures were grown in the same media under the same parameters to eliminate or minimize effects of the conditions of culture, an important factor which must be considered (45) . The viabilities obtained in each passage of each cell line, using the same culture conditions, were notable considering the great diversity in type and origin of the cultured cells. Thus, viability ranged from 90% to 100% in 137 passages from 67 of the cell lines and from 70% to 89% in 18 passages of 13 cell lines. Five passages of four of these cultures had viabilities of 70% to 79%, permitting study of the effects of a relatively low viability on the result? of the flow cytometric analyses. It was not possible t- do this during the development of the method because of the consistently high viability of the HeLa cells used to standardize the reagents and technique. Table 5. Melanocarcinomas MoAb NT: not tested; Ig: immunoglobulin; hCG: human chorionic gonadotropin; holo: intact; α: alpha subunit; β: beta subunit; CTP: carboxy terminal peptide; ATCC: American Type Culture Collection; MoAb: monoclonal antibody *High % of cells lysed tl00% of cells lysed φOur passage Table 6, NT: not tested; Ig: immunoglobulin; hCG: human chorionic gonadotropin; holo: intact; α: alpha subunit; B: beta subunit; CTP: carboxy terminal peptide; ATCC: American Type Culture Collection; MoAb: monoclonal antibody ♦Passage from different vial of cells tl00% of cells lysed $High percentage of cells lysed § MoAb used was B201 instead of B204 - 38 - Table 7. Non-Small Cell Carcinomas of Lung MoAb Calu-1 32 99 95.3 NT 7.7 23.4 NT: not tested; Ig: immunoglobulin; hCG: human chorionic gonadotropin; holo: intact; α: alpha subunit; β: beta subunit; CTP: carboxy terminal peptide; ATCC: American Type Culture Collection; MoAb: monoclonal antibody ♦Passage form a different vial of cells tMoAb used was B107 instead of B109 *MoAb used was B201 instead of B204 SHigh % of cells lysed Table 8. NT: not tested; Ig: immunoglobulin; hCG: human chorionic gonadotropin; holo: intact; α: alpha subunit; β: beta subunit; CTP: carboxy terminal peptide; ATCC: American Type Culture Collection; MoAb: monoclonal antibody *Our Passage tl00% cells lysed *MoAb used was B201 instead of B204 NT: not tested; Ig: immunoglobulin; hCG: human chorionic gonadotropin; holo: intact; α: alpha subunit; β: beta subunit; CTP: carboxy terminal peptide; ATCC: American Type Culture Collection; MoAb: monoclonal antibody. * MoAb used was CTP103 instead of ASH. tMoAb used was B201 instead of B204. *High % of cells lysed. Table 10. NT: not tested; Ig: immunoglobulin; hCG: human chorionic gonadotropin; holo: intact; α: alpha subunit; β: beta subunit; CTP: carboxy terminal peptide; ATCC: American Type Culture Collection; MoAb: monoclonal antibody. *High % of cells lysed. * 6.1 53.1 35.0 Table 11. (Continued) MoAb NT: not tested; Ig: immunoglobulin; hCG: human chorionic gonadotropin; holo: intact; α: alpha subunit; β: beta subunit; CTP: carboxy terminal peptide; ATCC: American Type Culture Collection; MoAb: monoclonal antibody. *Our passage. tMoAb used was CTP103 instead of ASH *MoAb used was B201 instead of B204. SMoAb used was B108 instead of AS12. Ig: immunoglobulin; hCG: human chorionic gonadotropin; holo: intact; α: alpha subunit; β: beta subunit; CTP: carboxy terminal peptide; ATCC: American Type Culture Collection; MoAb: monoclonal antibody. *MoAb used was B201 instead of B204 ATCC/CCL 187 Primary tumor Duke's type B ATCC/CCL 218 Primary tumor Sigmoid colon ATCC/CCL 220 Primary tumor Sigmoid colon ATCC/CCL 222 Ascites fluid ATCC/CCL 227 Lymph node metastasis ATCC/CCL 229 Metastic nodule left supraclavicular region ATCC/CCL 248 Lung metastasis Ig: immunoglobulin; hCG: human chorionic gonadotropin; holo: intact; α: alpha subunit; β: beta subunit; CTP: carboxy terminal peptide; ATCC: American Type Culture Collection; MoAb: monoclonal antibody. *High % of cells lysed. tMoAb used was B201 instead of B204. *100% of cells lysed. Ig: immunoglobulin; hCG: human chorionic gonadotropin; holo: intact; α: alpha subunit; B: beta subunit; CTP: carboxy terminal peptide; ATCC: American Type Culture Collection; MoAb: monoclonal antibody. *MoAb used was B108 instead of AS12. tHigh % of cells lysed. *MoAb was B204 instead of B201. SMoAb used was CTP-103 blf ?. Osteosarcomas B204 ASH A105 %hCGB %hCGB %hCGα Cell line free CTP total Source G-292 5.1 12.7 NT ATCC/CRL 1423 Primary tumor, 9- year old girl ATCC/CRL 1427 Primary tumor, 14- year old boy ATCC/HTB 96 Primary tumor, 15- year old girl ATCC/CRL 1543 Primary tumor, 13- year old girl ATCC/CRL 1544 Kirstein urine sarcoma virus- transformedcells ATCC/CRL 1547 Chemically transformedhuman osteosarcoma Ig: immunoglobulin; hCG: human chorionic gonadotropin; holo: intact; a : alpha subunit; B: beta subunit; CTP: carboxy terminal peptide; ATCC: American Type Culture Collection; MoAb: monoclonal antibody. *Decomplemented serum was used. tHigh % of cells lysed. *MoAb used was B201 instead of B204 SPassage from a different vial of cells. 1100% of cells lysed. Ig: immunoglobulin; hCG: human chorionic gonadotropin; holo: intact; α: alpha subunit; β: beta subunit; CTP: carboxy terminal peptide; ATCC: American Type Culture Collection; MoAb: monoclonal antibody. ♦Decomplemented serum was used tHigh % of cells lysed. *MoAb used was B204 instead of B201 SD: 100% of cells lysed Table 17 . emcemias Ig: immunoglobulin; hCG: human chorionic gonadotropin; holo: intact; α: alpha subunit; β: beta subunit; CTP: 0 carboxy terminal peptide; ATCC: American Type Culture Collection; MoAb: monoclonal antibody. *Our passage. tMoAb used was B107 instead of B109 *100% of cells lysed SMoAb used was B201 instead of B204 5 Table is. Lymphomas MoAb NT: not tested; EBV: Epstein-Barr virus; Ig: immunoglobulin; hCG: human chorionic gonadotropin; holo: intact; α: alpha subunit; B: beta subunit; CTP: carboxy terminal peptide; ATCC: American Type Culture Collection; MoAb: monoclonal antibody. *Our passage. tHigh % of cells lysed. *100% of cells lysed. SMoAb used was B201 instead of B204. ICalf serum replaced by Nutridoma. NT: not tested; EBV: Epstein-Barr virus; Ig: immunoglobulin; hCG: human chorionic gonadotropin; holo: intact; α: alpha subunit; B: beta subunit; CTP: carboxy terminal peptide; ATCC: American Type Culture Collection; MoAb: monoclonal antibody. *MoAb used was B108 instead of AS12. tl00% of cells lysed. *MoAb used was CTP-103. Figures 3 and 4 show the flow cytometric fluorescence profiles in two-dimensional and three-dimensional histograms (left and right panels, respectively) of the findings using aliquots (IO 6 viable cells) from Passage 150 of the retinoblasto a cell line WERI-Rb-1, with and without PI treatment. These indicate that, in cultured cells with relatively low viability, PI treatment did not produce any significant difference in the percent of cells expressing the epitopes or in the intensity of the fluorescence. Therefore, around the 75% viability range, the gating (90% light scatter versus forward light scatter) was adequate to differentiate between the viable and nonviable cells in every sample of cultured cells (30) . It is stressed that, when applying this technique to the analysis of cells isolated from human tumor tissues, it is possible that viabilities of less than 75% may be observed and PI treatment may be necessary in such instances. The results of serial analyses done in all cancer cell lines (Tables 5 to 19) showed expression of epitopes of hCG-holo, its subunits, and fragments by cells from all cell lines studied. Variable degrees of reactivity were seen. These variations in the expression of some of the membrane-associated hCG epitopes ranged from more than 90% of the cells to 0%, as in the case of one of the determinants of the free hCGβ recognized by MoAb B204 and illustrated by Passage 1 of the histiocytic lymphoma cell line U-937, ATCC CRL 1593 (Table 18). Here, reactivity was found in 91.1% of cells, while 0% values were shown by Passage 37, one of three passages studied of the adenocarcinoma of lung cell line ERT-17 (Table 7) , and by Passage 48, one of four passages studied of the adenocarcinoma of breast cell line BT-549, ATCC HTB 122 (Table 9). All these values, obtained with cells grown under the established standard conditions of culture, indicate that expression of the membrane-associated epitopes of hCG, its subunits and fragments was a phenotypic characteristic of the cell and not a product of growth conditions. Moreover, although in this technique a value of less than 10% with low fluorescence intensity was considered to be 0%, an instrument value of 0% for the expression of a particular epitope in one of several passages of the same cell line gave additional proof of the specificity of the reagents considering that all MoAb used (except B201) are of the same isotype, IgGl. The MoAb B201 has an IgG2 isotype. The conformational epitope of the intact hCG recognized by MoAb B109 was expressed by a variable percent of cells from all cell lines in the 151 passages in which this determinant was investigated. Because this epitope is expressed only in hCG-holo, these results give strong evidence for the synthesis of both competent subunits (17-19) by all human cancer cells in culture. There was also a striking quantitative difference in the expression of hCG-holo by different types of cancers. In 34 passages of 18 lines of melanocarcinomas, adenocarcinomas of lung, adenocarcinomas of breast, and carcinomas of prostate (Tables 5, 7, 9 and 14, respectively), identified as Group 1 hereafter, the means of the percent of cells of each type of cancer expressing the determinant were 19.83% ± 4.39 (mean ± standard error of the mean; range, 5.8% to 32.4%), 13.54% ± 2.56 (range, 5.6% to 31.4%), 14.87% ± 2.25 (range, 6.5% to 26.3%), and 17.77% ± 4.61 (range, 8.5% to 35.8%), respectively. None of these means were statistically different from one another based on analysis of variance followed by Scheffe's test for pairwise comparisons (56) . P values of 0.05 or less were considered significant. A second group consisting of 11 passages of seven carcinomas of colon cell lines (Table 13) and 11 passages of seven carcinomas of urinary bladder cell lines (Table 10) showed a mean expression of 32.81% ± 3.18 and 29.24% ± 4.93 within a relatively narrow range of expression of this determinant, 17.4% to 51.1% and 5.0% to 39.1%, respectively, with only one value, 70.2% (Table 10), falling outside this range. As in Group 1, their mean values were not significantly different. By contrast, in 28 passages of 11 cultures of carcinomas of nasopharynx (Table 6) , small cell carcinomas of lung (Table 8) , carcinomas of endometrium (Table 12) , and retinoblasto as (Table 19) , identified as Group 3 hereafter, the mean values of the percent of cells of each type of cancer expressing the conformational determinant were 63.33% ± 9.97 (range, 31.9% to 95.3%), 67.3 ± 6.98 (range, 35.1% to 88.6%), 58.4% ± 4.64 (range, 38.9% to 71.3%), and 62.72% ± 6.20 (range, 30.3% to 78.8%) respectively, demonstrating the highest degree of expression of hCG-holo. As in groups 1 and 2, none of the means in Group 3 were significantly different from each other. The distribution of all experimental values of Groups 1 to 3, the mean, values of the percent of cells of each type of cancer expressing the conformational epitope of hCG-holo, and the corresponding mean of these mean values are illustrated graphically in Figure 5. Analysis of variance and Scheffe's test for pairwise comparison done on the means of the mean values of the three groups, 15.91% ± 1.59 (Group 1), 31.02% ± 2.89 (Group 2), and 63.41% ± 3.46 (Group 3), demonstrated that the differences among these means were statistically significant. In the other 31 cell lines studied, corresponding to six types of cancer (Tables 7, 11 and 15 to 18) , the variability of expression of the conformational epitope in the different passages of each of the cell lines of each cancer was too great to permit any grouping and comparison (e.g., range, 5.6% to 87.5%). The expression of any single epitope on the surface of a cell depends on several factors. Because conditions of growth were eliminated or minimized as a variable, the only other factors to be considered in our studies (in addition to the inherent phenotypic variabilities from the kinetics of cell membrane synthesis) were the different kinetics of hCGα and hCGβ synthesis. In the particular case of the specific conformational epitope of hCG-holo, the need for the biosynthesis of the two competent subunits was also a factor (16-19) . The different kinetics of the transcription of the genes encoding hCGα and hCGB (14, 15) were not sufficient to explain the low expression of the conformational determinant of hCG-holo in the cancers in Group 1 (melanocarcinoma (Table 5) , carcinoma of breast (Table 9), and carcinoma of prostate (Table 14)) because epitopes of hCGα and hCGβ-CTP were expressed by a relatively high percentage of cells in all corresponding passages. This was demonstrated by the reactivity of these cells with MoAb A105 and ASH, respectively. The only exception in this group was the carcinoma of lung cell lines (Table 7) , which also had a low expression of the epitope of hCGα (A105) . These results showed that it was logical to study the distribution of the expression of the specific epitopes of hCGB and hCGα. The distribution of the mean values of expression of the determinant recognized by MoAb ASH for each of these groups of cancer is also illustrated in Figure 5. In Group l, the difference of the means of the expression of each determinant was significantly different for each type of cancer, indicating that the cells of the cancers in this group, with the exception of the adenocarcinomas of lung, produced a significant amount of modified (incompetent) hCGα and hCGB because of the elevated values of hCGβ and hCGα. By contrast, in the adenocarcinomas of lung cells, the limiting factor for the formation of hCG-holo appeared to be the low synthesis of hCGα. The values for Passage 55 of the carcinoma of breast line BT-549 (Table 9) were excluded from statistical calculation because the MoAb CTP103 (against the synthetic hCGB-CtP) was used instead of MoAb ASH. Carcinomas of colon in Group 2 and carcinomas of endometrium and retinoblastomas in Group 3, as in these carcinomas of Group 1, also appeared to produce considerable quantities of incompetent subunits. By contrast, the cells from carcinomas of nasopharynx and small cell carcinoma of lung in Group 3 and carcinoma of urinary bladder in Group 2 appeared to produce a significantly lower amount of incompetent subunits. The expression of the epitope of the unique hCGβ-CTP, recognized by MoAb ASH by all cell lines in all passages investigated, was higher than the expression of hCGα in most cases, providing additional evidence that the material being recognized in the cell membrane of the cultured cancer cells was related to hCBB, free or as part of hCG-holo. Additional evidence was also provided by the expression of the epitope recognized by MoAb CTP103 in the four instances in which this MoAb against a synthetic hCGβ-CTP (residues 109 to 145) was used: a carcinoma of breast (BT-549, ATCC HTB 122, Table 9), two squamous cell carcinomas of uterine cervix (C33A, ATCC HTB 31, and SiHa, ATCC HTB 35, Table 11), and a prostatic carcinoma (DU-145, ATCC HTB 81, Table 14). Others (37) recently reported that they were unable to detect hCGα mRNA in CaSki cells (ATCC CRL 1550, Table 11) , a cell line obtained from mesenteric metastases of a patient with a squamous cell carcinoma of the uterine cervix, in which synthesis and secretion of hCGB and bioactive hCG was reported several years ago. The results presented here demonstrate expression of membrane-associated conformational epitopes of hCG-holo (range, 15.9% to 56.0% of the cells in three passages) and expression of the epitope of hCGα recognized by MoAb A105 (by 79.7% of the cells) , with a high expression (range, 40.6% to 75.3%) of hCGβ-CTP determinants. These authors (37) admitted that some of their results may have only reflected conditions of cell culture or, more probably, the lack of sensitivity of the methology because no poly-A mRNA was analyzed nor was the polymerase chain reaction technique. An unexpected finding was the observation that MoAb directed to epitopes of hCGβ-free (B201 or B204) are able to lyse cells. These effects were remarkable in the sarcomas (Tables 14 and 15) , lymphomas (Table 17) , and retinoblastomas (Table 18) . In several instances, there were no cells left to be analyzed by flow cytometry (indicated by W D" in the tables) . These effects were also observed in some passages of carcinoma cell lines, melanocarcinomas, carcinomas of nasopharynx, small cell carcinomas of lung, and carcinomas of urinary bladder, colon, and prostate (Tables 5, 6, 8, 10, 13, and 15, respectively) . The RD rhabdomyosarcoma cell line (ATCC 136, Table 16) grown in medium with defined bovine calf serum with and without complement and analyzed conco itantly in a duplicate experiment, and in another experiment, the Jiyoye Burkitt lymphoma cell line (ATCC CCL 87, Table 18) grown in seru less medium (Nutridoma) , showed that the cytolytic activity appeared to be independent of complement. The technique and results presented here cannot be compared with those of others also working with human cell lines, not only because of the different in specificity and sensitivity of the flow cytometric method, but also because their analyses were done on extracts of media (58, 59), reflecting only the secretory pool, or on extracts on media and cell lysates (60, 61) , reflecting the secretory pool and the total cell pool (i.e., cytosol and membrane pools alone or in combination with the secretory pool) . In this respect, it was stated that these types of glycoproteins can be found in the cytosol extracts, although they are not detectable in the media, a finding which may invalidate the negative results obtained by analyzing culture media alone (or blood serum) and the conclusions based on such results (61). In summary, our studies thus far have demonstrated several things. First, determinants of membrane- associated hCG-holo, its subunits and fragments in cultured human cancer cells are not a product of growth conditions but are a phenotypic characteristic of all cell lines investigated, irrespective of type or origin. Second, the cultured cells from a defined group of carcinomas and the retinoblastomas showed quantitative and qualitative differences in the expression of the membrane-associated hCG-holo, its subunits and fragments. Thus, melanocarcinomas, adenocarcinomas of lung, and carcinomas of breast and prostate cell lines (Group l) appear to express a relatively low amount of membrane- associated hCG-holo. Concomitantly, but with the exception of the adenocarcinoma of lung cells, large amounts of modified free hCGα and/or hCGβ are expressed, as indicated by the high percentage of cells reacting with subunit-recognizing MoAb. Adenocarcinomas of colon cell lines in Group 2 and retinoblastomas and carcinomas of endometrium in Group 3 appeared to follow the same trend of expression, by contrast with the other cell lines in Group 2 and retinoblastomas and carcinomas of endometrium in Group 3 appeared to follow the same trend of expression, by contrast with the other cell lines studied. Adenocarcinoma (non-small cell carcinoma) of lung appeared to be the only type of cultured cancer cells in which the limiting factor for the expression of intact hCG was the low degree of hCGα synthesis. Third, the unique CTP was always expressed in the membrane of a high percentage of cells of all cancer cell lines studied, indicating that the membrane-associated material being recognized by both MoAb ASH and CTP103, was hCGB, free or as part of hCG-holo. Fourth, the experiments in which MoAb directed against hCGβ-free determinants were used, revealed an in vitro complement-independent cytolytic activity for some types of the cultured cancer cells used. Conclusion The expression of human chorionic gonadotropin (hCG) , its subunits, and fragments on the cell membrane of cultured human cancer cells was investigated using a flow cytometric method. This method uses living cells; a double-antibody reaction; a flow cytometer with an argon laser, standard settings, and filters for fluorescein isothiocyanate; commercially available software; the American Type Culture Collection (ATCC) CCL 2 HeLa cell line as a cell control and overall quality control; polyclonal rabbit antisera raised against the hCG dimer, its α subunit (hCGα) , and its β subunit (hCGB) ; and a panel of monoclonal antibodies (MoAb) recognizing different epitopes on the intact hCG molecule, its subunits, and fragments. The purified immunoglobulin G fractions from the polyclonal antisera were used to estimate the total expression of the membrane-associated glycoproteins; the MoAb were used to detect the expression of epitopes of the hCG dimer, it subunits and fragments. The results of the analyses done on cells from 74 established cancer cell lines of different types and origins (including 52 carcinomas, 10 sarcomas, 4 leukemias, 6 lymphomas, and 2 retinoblastomas) showed variable degrees of reactivity in a great percentage of cells in all cell lines studied with MoAb directed against different conformational epitopes of intact hCG (hCG-holo) , hCGB, hCGB-free, the carboxy terminal peptide (CTP) of hCGβ, and an epitope of hCGα. The expression of the membrane-associated epitopes of hCG and its subunits was found to be a phenotypic marker characteristic of all evaluated cultured human cancer cell lines, irrespective of their type or origin. There were, however, quantitative and qualitative differences in the expression of the different epitopes. Thus, hCGβ, free and as part of hCG-holo, recognized by the MoAb against hCGB-CTP, was expressed by a high percentage of cells in most lines. There was a great variability in the expression of hCG-holo, recognized by the MoAb B109. For this reason some groups of cancers expressed larger amounts of incompetent hCGα and/or hCGB than others. Cell lines derived from adenocarcinomas of the lung were the only exception to this general finding. The expression of small amounts of hCG-holo was caused by a low degree of hCGα synthesis. III. In Vivo Expression Of Membrane-Associated Human Chorionic Gonadotropin Bγ Cells Isolated From Cancerous Human Tissues The expression of hCG, its subunits and fragments by live cancer cells isolated from 15 malignant human neoplasms (6 lung neoplasms, 3 breast neoplasms, 3 colon neoplasms, one kidney neoplasm, one urinary bladder neoplasm, and one testicular neoplasm) was demonstrated using a flow cytometry method and a panel of monoclonal antibodies specific for epitopes from 5 different sites on the hCG molecule (59) . In contrast to the cells isolated from the malignant neoplasms, cells isolated from a benign neoplasm (uterine leiomyoma) showed no measurable hCG- related determinants. These results, similar to those seen with cultured human cancer cells (60) , indicate that the in vivo expression of hCG, its subunits and fragments is a common phenotypic characteristic of cancer cells of different types and origins. References 1. Fiddes, J.C., Goodman HM. 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Claims: What is claimed is: 1. A method of lysing malignant cells, which comprises contacting the malignant cells with an antibody which specifically reacts with an epitope present on the beta subunit of human chorionic gonadotropin and/or beta subunit fragment, under conditions such that the malignant cells lyse. 2. The method of claim 1, wherein the antibody is a monoclonal antibody. 3. The method of claim 2, wherein the monoclonal antibody is designated B201. 4. The method of claim 2, wherein the monoclonal antibody is designated B205. 5. A method of quantitatively measuring malignant cells, which comprises contacting live malignant cells to be measured with an antibody directed to an epitope of human chorionic gonadotropin, its subunits and/or subunit fragments, under conditions such that the antibody is capable of forming a complex with the epitope if present on the surface of the cells being measured, and detecting the presence of any complex formed using fluorescence activated cell sorting technology, the presence of such complex on the surface of the cells indicating that the cells are malignant. 6. The method of claim 5, wherein the antibody is a monoclonal antibody. 7. The method of claim 6, wherein the monoclonal antibody is designated B201. 8. The method of claim 6, wherein the monoclonal antibody is designated B205. 9. A method of obtaining an enriched population of malignant cells, which comprises contacting a population of cells comprising live malignant cells with an antibody directed to an epitope of human chorionic gonadotropin, its subunits and/or subunit fragments, under conditions such that the antibody is capable of forming a complex with the epitope if present on the cell surface, the formation of such complex indicating that the cells are malignant, and isolating the cells which form a complex with the antibody. 10. The method of claim 9, wherein the antibody is a monoclonal antibody. 11. The method of claim 10, wherein the monoclonal antibody is designated B201. 12. The method of claim 10, wherein the monoclonal antibody is designated B205.

Inventors: ACEVEDO HERNAN, KRICHEVSKY ALEXANDER, ACEVEDO, HERNAN, KRICHEVSKY, ALEXANDER
Applicants: ALLEGHENY SINGER RES INST, UNIV COLUMBIA, ALLEGHENY-SINGER RESEARCH INSTITUTE, THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF
Application year: 1993
Main classification: C07K15
IPC codes: A61K38, C07K14, C07K16, G01N33