the hybridoma cell line h460 - 16 - 2 was deposited , in accordance with the budapest treaty , with the american type culture collection , 10801 university blvd ., manassas , va . 20110 - 2209 on sep . 4 , 2002 , under accession number pta - 4621 . in accordance with 37 cfr 1 . 808 , the depositors assure that all restrictions imposed on the availability to the public of the deposited materials will be irrevocably removed upon the granting of a patent . h460 - 16 - 2 monoclonal antibody was produced by culturing the hybridoma in cl - 1000 flasks ( bd biosciences , oakville , on ) with collections and reseeding occurring twice / week . the antibody was purified according to standard antibody purification procedures with protein g sepharose 4 fast flow ( amersham biosciences , baie d &# 39 ; urfë , qc ). it is within the scope of this invention to utilize monoclonal antibodies that are human , humanized , chimerized or murine antibodies . ihc studies were previously conducted to characterize h460 - 16 - 2 antigen distribution in humans ( ser . no . 10 / 603 , 000 ) and in comparison to l178 ( ser . no . 10 / 647 , 818 ). the current studies compare h460 - 16 - 2 to another antibody directed against cd44 ( bu75 ) since the h460 - 16 - 2 antigen may be a cancer variant of cd44 as determined previously by biochemical methods . binding of antibodies to 59 normal human tissues was performed using a human normal organ tissue array ( imgenex , san diego , calif .). all primary antibodies ( h460 - 16 - 2 ; bu75 anti - cd44 ( biocan scientific inc ., mississauga , on ); and mouse igg 1 negative control ( dako , toronto , on )) were diluted in antibody dilution buffer ( dako , toronto , on ) to a concentration of 5 μg / ml ( found to be the optimal concentration in previous optimization steps ). the negative control antibody has been shown to be negative to all mammalian tissues by the manufacturer . the procedure for ihc is as follows . tissue sections were deparaffinized by drying in an oven at 58 ° c . for 1 hr and dewaxed by immersing in xylene 5 times for 4 min each in coplin jars . following treatment through a series of graded ethanol washes ( 100 %- 75 %) the sections were re - hydrated in water . the slides were immersed in 10 mm citrate buffer at ph 6 ( dako , toronto , ontario ) then microwaved at high , medium , and low power settings for 5 min each and finally immersed in cold pbs . slides were then immersed in 3 percent hydrogen peroxide solution for 6 min , washed with pbs 3 times for 5 min each , dried , incubated with universal blocking solution ( dako , toronto , ontario ) for 5 min at room temperature . h460 - 16 - 2 , bu75 or isotype control antibody ( directed towards aspergillus niger glucose oxidase , an enzyme which is neither present nor inducible in mammalian tissues ) were diluted in antibody dilution buffer ( dako , toronto , ontario ) to its working concentration ( 5 μg / ml for each antibody ) and incubated for 1 hr at room temperature . the slides were washed with pbs 3 times for 5 min each . immunoreactivity of the primary antibodies was detected / visualized with hrp conjugated secondary antibodies as supplied ( dako envision system , toronto , ontario ) for 30 min at room temperature . following this step the slides were washed with pbs 3 times for 5 min each and a color reaction developed by adding dab ( 3 , 3 ′- diaminobenzidine tetrahydrachloride , dako , toronto , ontario ) chromogen substrate solution for immunoperoxidase staining for 10 min at room temperature . washing the slides in tap water terminated the chromogenic reaction . following counterstaining with meyer &# 39 ; s hematoxylin ( sigma diagnostics , oakville , on ), the slides were dehyrdated with graded ethanols ( 75 - 100 %) and cleared with xylene . using mounting media ( dako faramount , toronto , ontario ) the slides were coverslipped . slides were microscopically examined using an axiovert 200 ( zeiss canada , toronto , on ) and digital images acquired and stored using northern eclipse imaging software ( mississauga , on ). results were read , scored and interpreted by a pathologist . table 1 presents a summary of the results of h460 - 16 - 2 and bu75 anti - cd44 staining of an array of normal human tissues . the staining of tissues with h460 - 16 - 2 is similar to that described previously ( ser . no . 10 / 603 , 000 ). it should be again noted that the antigen is generally not present on cells in the vital organs , including the liver , kidney , heart and lung . the h460 - 16 - 2 antibody does bind to macrophages and lymphocytes , and their presence is observed in some of the organs in these sections . however , there was a higher intensity of staining of lymphocytes seen with the bu75 anti - cd44 antibody ( fig1 ). tissues that were positive for h460 - 16 - 2 were also usually positive for bu75 anti - cd44 ( sometimes to a greater intensity ). tissues that were negative for h460 - 16 - 2 were also generally negative for bu75 anti - cd44 albeit there are a few exceptions such as one sample of esophagus and lymph node . these results demonstrate that h460 - 16 - 2 binds to a smaller subset of the tissues recognized by the bu75 anti - cd44 antibody and within tissues the intensity of staining is also sometimes less . these results show that the antigen for h460 - 16 - 2 is not widely expressed on normal tissues , and that the antibody binds specifically to a limited number of tissues in humans . it also supports the biochemical data in that h460 - 16 - 2 is directed against an epitope of cd44 , that is a different variant than the one recognized by the bu75 used for these ihc studies . previous ihc studies were undertaken to determine the cancer association of the h460 - 16 - 2 antigen with human breast cancers and whether the h460 - 16 - 2 antibody was likely to recognize human cancers ( ser . no . 10 / 603 , 000 ) and how it compared to anti - cd44 staining with l178 ( ser . no . 10 / 647 , 818 ). currently , a comparison was made for bu75 anti - cd44 staining , c - erbb - 2 anti - her2 and an antibody directed towards aspergillus niger glucose oxidase , an enzyme which is neither present nor inducible in mammalian tissues ( negative control ). a breast cancer tissue array derived from 50 breast cancer patients and 10 samples derived from non - neoplastic breast tissue in breast cancer patients was used ( imgenex corporation , san diego , calif .). the following information was provided for each patient : age , sex , american joint committee on cancer ( ajcc ) tumor stage , lymph node , estrogen receptor ( er ) and projesterone receptor ( pr ) status . the procedure for ihc from example 5 was followed . all antibodies were used at a working concentration of 5 μg / ml except for anti - her2 which was used at a concentration of 1 . 5 μg / ml . tables 2 and 3 provide summaries of h460 - 16 - 2 and bu75 anti - cd44 antibody staining of breast cancer tissue arrays respectively . each array contained tumor samples from 50 individual patients . overall , 62 percent of the 50 patients tested were positive for h460 - 16 - 2 antigen compared to 73 percent for bu75 anti - cd44 . in cases where both h460 - 16 - 2 and bu75 anti - cd44 stained the same tissue , 45 percent of the samples had higher intensity staining with the bu75 anti - cd44 in comparison to h460 - 16 - 2 . for the h460 - 16 - 2 and bu75 antigen , 4 and 8 out of 10 normal breast tissue samples from breast cancer patients were positive , respectively . no clear correlation between estrogen and progesterone receptor status was evident . it also did not appear to be a trend to greater positive expression of the h460 - 16 - 2 and cd44 antigen with higher tumor stage . the h460 - 16 - 2 staining was specific for cancerous cells in comparison to normal cells as demonstrated in fig2 where stromal cells were clearly negative and sheets of malignant cells were highly positive . the cellular localization pattern seen with the h460 - 16 - 2 antigen was confined to the cell membrane in the majority of cases . the bu75 cd44 antibody stained more breast cancer samples and showed a higher degree of membrane than cytoplasmic localization compared to h460 - 16 - 2 ( table 4 ). bu75 anti - cd44 also stained malignant cells of paget &# 39 ; s disease , which was not the case for h460 - 16 - 2 ( fig3 ). the samples of normal tissue from breast cancer patients that were positive for h460 - 16 - 2 staining were also positive for bu75 anti - cd44 staining . in comparison to c - erbb - 2 , h460 - 16 - 2 showed a completely different staining profile where 16 out of the 31 breast tumor tissue samples that were positive for the h460 - 16 - 2 antigen were negative for her2 expression indicating a yet unmet targeted therapeutic need for breast cancer patients ( table 5 , fig4 ). there were also differences in the intensity of staining between the breast tumor tissue sections that were positive for both h460 - 16 - 2 and her2 ; some breast tumor tissue sections that were highly positive for the h460 - 16 - 2 antigen were only mildly positive for her2 and vice versa again illustrating that h460 - 16 - 2 would therapeutically target a different cohort of breast cancer patients . the c - erbb - 2 antibody also positively stained one of the normal breast tissue sections . these results suggest the antigen for h460 - 16 - 2 may be expressed by almost two thirds of breast cancer patients . in addition , the majority of those suitable for h460 - 16 - 2 treatment would not have been suitable for anti - her2 treatment . the staining pattern showed that in patient samples , the antibody is highly specific for malignant cells and the h460 - 16 - 2 antigen is localized to the cell membrane thereby making it an attractive drugable target . the similar albeit more limited staining of h460 - 16 - 2 versus bu75 anti - cd44 antibody again demonstrates the likelihood of the h460 - 16 - 2 epitope being a more restricted variant of cd44 . with reference to fig5 and 6 , 6 to 8 week old male scid mice were implanted with 1 million pc - 3 human prostate cancer cells in 100 microlitres saline injected subcutaneously in the scruff of the neck . tumor growth was measured with calipers every week . when the majority of the cohort reached a tumor volume of 80 mm 3 ( range 50 - 130 mm 3 ) at 21 days post - implantation , 8 mice were randomly assigned into each of 4 treatment groups . h460 - 16 - 2 antibody , the chemotherapeutic drug cisplatin , the combination of h460 - 16 - 2 and cisplatin or buffer control was administered intraperitoneally with 15 or 6 mg / kg of antibody or cisplatin respectively at a volume of 300 microliters after dilution from the stock concentration with a diluent that contained 2 . 7 mm kcl , 1 mm kh 2 po 4 , 137 mm nacl and 20 mm na 2 hpo 4 . h460 - 16 - 2 or buffer control was then administered 4 times per week for the first week followed by 3 times per week for 11 doses in total in the same fashion until day 41 post - implantation . cisplatin was administered on days 0 , 5 , 10 and 15 of the antibody treatment period . tumor growth was measured about every seventh day with calipers until day 48 post - implantation or until individual animals reached the ccac end - points . body weights of the animals were recorded for the duration of the study . at the end of the study all animals were euthanised according to ccac guidelines . using a paired t - test , there was a post - treatment tumor burden reduction ( fig5 ) associated with treatment with either cisplatin or the combination of h460 - 16 - 2 and cisplatin . at day 48 ( 7 days post - treatment ) cisplatin and h460 - 16 - 2 treatment was significantly more effective in suppressing tumor growth shortly after the treatment period than buffer control ( p & lt ; 0 . 0001 ), cisplatin treatment alone ( p = 0 . 004 ) or h460 - 16 - 2 treatment alone ( p & lt ; 0 . 0001 ). pc - 3 is a cachexic model of prostate cancer , in which increased tumor burden and disease progression in the xenograft model is accompanied by weight loss . as demonstrated by the mean weights shown in fig6 , the mice in all the groups experienced severe weight loss . in this study , mice in all groups showed a weight loss of approximately 23 to 35 percent by the end of the treatment period . the group treated with h460 - 16 - 2 showed the smallest degree of weight loss ( 21 . 7 percent ). shortly after the end of treatment , there was no additional significant loss of body weight associated with h460 - 16 - 2 plus cisplatin treatment in comparison to the buffer control ( p = 0 . 5042 ). therefore h460 - 16 - 2 plus cisplatin lowered the tumor burden in comparison to a buffer control in a well - recognized model of human breast cancer disease . these results suggest pharmacologic and pharmaceutical benefits of this antibody for cancer therapy in mammals , including man . mda - mb - 231 cells ( shown previously to express the h460 - 16 - 2 antigen ( cd44 ) by facs analysis ) were dissociated after aspirating the spent media from the tissue culture plates , washing the plates with pbs , adding 5 ml of dissociation buffer to each plate and incubating the plates at 37 ° c . until the cells detached . cells were then counted and collected into 50 ml tube ( s ). cells were spun at 1200 rpm for 5 min and resuspended in media to yield 1 - 5 million cells / ml . one ml was then added to each well of a 2 ml deep well . cells were pelleted by spinning the plate at 1200 rpm for 5 min and excess supernatant was removed by inverting the plate onto paper towels . deep well plates were then gently vortexed to dislodge and break up the cell pellets . one ml of h460 - 16 - 2 , bu75 ( positive control , biocan scientific inc ., mississauga , on ) or isotype negative control ( 107 . 3 , bd biosciences , oakville , on ) antibody was added to each well and then mixed by gentle vortexing . plates were then incubated at 37 ° c . for 2 hrs . meanwhile , 48 - well plates were coated with ha by incubating 300 μl of 4 mg / ml ha stock solution / well for 1 - 2 hrs at 37 ° c . after incubation , excess ha was aspirated off and the plates ( s ) were allowed to completely air dry in the laminar flow hood . after antibody - cell incubation was completed , the cells were again pelleted at 1200 rpm for 5 min . supernatant was removed by inverting the deep well on paper towels . the deep well was again vortexed to dissociate the cell pellets followed by addition of 1 . 2 ml of 2 μm calcein - am , in pbs containing mgcl 2 and cacl 2 , to each well . cells were resuspended and 250 μl / well was transferred to the ha coated plate . ha coated plates were then incubated at 37 ° c . for 2 hrs to allow adhesion . after incubation , unattached cells were removed through aspiration . each well was then washed 2 - 3 times with pbs containing mgcl 2 and cacl 2 in order to remove any unattached cells or cell clumps . the plates were read in a perkin - elmer hts7000 fluorescence plate reader and the data was analyzed in microsoft excel and the results were tabulated in table 6 or fig7 . the results from an average of 6 separate experiments revealed that an average of 1 . 87 (+/− 1 . 01 ) μg / ml of h460 - 16 - 2 was required in order to cause a 50 percent reduction in the binding of mda - mb - 231 cells to ha ( table 6 ). the effect of h460 - 16 - 2 on mda - mb - 231 cell binding to ha was dose dependent ; 20 μg / ml of h460 - 16 - 2 resulted in over a 60 percent reduction in cell binding to ha ( fig7 ). these results indicated that h460 - 16 - 2interacts with , at least in part , the region ( s ) on cd44 that are responsible for binding to ha and consequently could be elucidating its anti - cancer effects through down regulation of angiogenesis or tumor invasiveness through the ecm . the effect of h460 - 16 - 2 on the cell cycle of mda - mb - 231 breast cancer cells was evaluated using facs analysis . h460 - 16 - 2 antibody ( 0 , 0 . 2 , 2 . 0 and 20 μg / ml ) or isotype control ( clone 107 . 3 , bd biosciences , oakville , on ) was incubated with mda - mb - 231 breast cancer cells for 24 , 48 and 72 hrs . treated and untreated cells were stained with propidium iodide and single cells were analyzed by flow cytometry to assess relative dna content . the acquired data set was analyzed using bd cellquest , by gating on the single cells population as well as cells showing hypo - diploid staining . after this analysis , cells treated with h460 - 16 - 2 for 24 hrs showed an overall decrease in the percentage of cycling cells as well as a dose dependent increase in the sub - g 1 population . cells that appeared in the sub - g 1 population are cells that have lost dna due to the loss of cell membrane integrity and may represent the apoptotic cell population ( fig8 ). this data demonstrated that h460 - 16 - 2 had an effect on mda - mb - 231 cell cycling and this effect led to a dose dependent increase in the number of apoptotic cells . the preponderance of evidence shows that h460 - 16 - 2 mediates anti - cancer effects through ligation of a carbohydrate dependent conformational epitope present on a variant of cd44 and that this epitope is at least partially involved in the binding of cd44 to ha . there is also evidence that binding of h460 - 16 - 2 to this epitope can lead to apoptosis on the corresponding cell . it has been shown , in ser . no . 10 / 713 , 451 , h460 - 16 - 2 antibody can be used to immunoprecipitate the cognate antigen from expressing cells such as mda - mb - 231 cells . further it could be shown that the h460 - 16 - 2 antibody could be used in detection of cells and / or tissues which express a cd44 antigenic moiety which specifically binds thereto , utilizing techniques illustrated by , but not limited to facs , cell elisa or ihc . thus , it could be shown that the immunoprecipitated h460 - 16 - 2 antigen can inhibit the binding of h460 - 16 - 2 to such cells or tissues using such facs , cell elisa or ihc assays . further , as with the h460 - 16 - 2 antibody , other anti - cd44 antibodies could be used to immunoprecipitate and isolate other forms of cd44 antigen , and the antigen can also be used to inhibit the binding of those antibodies to the cells or tissues that express the antigen using the same types of assays . it could also be shown that if an anti - cd44 antibody that recognizes all forms of cd44 ( i . e . a pan - cd44 antibody ) were used to isolate its cognate antigen , then that antigen could also inhibit the binding of h460 - 16 - 2 antigen to cells or tissues that express that antigen , thus also demonstrating the binding of h460 - 16 - 2 to an epitope of cd44 on cells and tissues expressing that antigen . alternatively , a comparison of h460 - 16 - 2 and pan - cd44 antibody in assays such as competitive binding assays . elisa , cell elisa , facs or the like , where both antibodies are present can also demonstrate the binding of h460 - 16 - 2 to an epitope of cd44 on cells and tissues expressing that antigen . all patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains . all patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference . it is to be understood that while a certain form of the invention is illustrated , it is not to be limited to the specific form or arrangement of parts herein described and shown . it will be apparent to those skilled in the art that various changes may be made without departing from the scope of the invention and the invention is not to be considered limited to what is shown and described in the specification . one skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned , as well as those inherent therein . any oligonucleotides , peptides , polypeptides , biologically related compounds , methods , procedures and techniques described herein are presently representative of the preferred embodiments , are intended to be exemplary and are not intended as limitations on the scope . changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the appended claims . although the invention has been described in connection with specific preferred embodiments , it should be understood that the invention as claimed should not be unduly limited to such specific embodiments . indeed , various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims .
this invention relates to the diagnosis and treatment of cancerous diseases , particularly to the mediation of cytotoxicity of tumor cells ; and most particularly to the use of cancerous disease modifying antibodies or derivatives thereof , optionally in combination with one or more chemotherapeutic agents , as a means for initiating the cytotoxic response . the invention further relates to binding assays which utilize the cdmabs of the instant invention .