Patent Application: US-47441583-A

Abstract:
a panel of monoclonal antibodies produced from human gastrointestinal tumors as immunogen is used to diagnose the presence of colon cancer . the antibody panel subsets the human gastrointestinal tract in its reactivity vis - a - vis esophagus , stomach , small intestine and colon . the panel is useful as a diagnostic probe for cancer .

Description:
a preferred embodiment of the present invention is to test a human speciman as for example human body wastes , fluids and exudates with each of the monoclonal antibodies of the panel . the cells are tested or contacted separately with each of the monoclonal antibodies in a series of dilutions . thus , an assay for cancer is possible with minimal patent disruption . indeed , the present invention permits testing of human urine specimans for cell fragments containing antigenic markers for the monoclonal antibodies . entire cells need not be present . cytohistological methods requiring whole cells are not always successful . the monoclonal antibodies of the present invention were prepared by an improved kohler - millstein procedure wherein spleen cells from a mouse ( or other mammal ) immunized with human cancerous colon cells from established human tumor cell lines were fused with mouse myeloma to form hybridomas . by serological screening , antibodies from these hybridomas were found which recognize differentiation antigens on normal bladder and / or cancerous bladder . other tissues , both normal and cancerous , may be recognized as well by some of these monoclonal antibodies . a system of classification of normal as well as cancerous colon based on these differentiation antigens is now possible , and serological assays for tumors of the colon have been developed . these assays are of special use in the early diagnosis of gastrointestinal cancer especially colon cancer . the assay of the present invention comprises contacting a tissue containing colon cells with the antibody recognizing colon cell antigens , preferably monoclonal antibodies to one or more cell antigens of the gastrointestinal antigenic system , and observing the immunoserological or immunopathological antigenic reaction between said monoclonal antibody and said antigen . in a preferred embodiment of the invention , the tissue sample to be contacted is gastrointestinal tissue and the antigenic reaction of the contacted tissue is observed by well known techniques such as immunofluorescence , rosette formation with sheep or human red blood cells linked to protein a or anti - ig direct absorption and the like . in another embodiment of the present invention , the tissue to be assayed is first excised and is then either freshly , or after being frozen or embedded in paraffin by methods well - known in the art , contacted with the monoclonal antibodies of the invention . observation of the reaction is as before . in another preferred embodiment of the present invention , the tissue to be assayed comprises the intact body of an individual or a whole portion thereof . the antibody , tagged with a radioactive or other energy - producing element , is administered to the individual , and the whole body or part thereof is scanned externally for localization of radioactivity at the site of cancerous gastrointestinal cells . in another preferred embodiment cancerous colon cells are located . the present invention also makes possible the treatment of gastrointestinal tumors in a patient wherein the monoclonal antibody recognizing the cell antigen of cancerous colon or other cancerous gi cells , is administered to the patient in an amount effective to inhibit the growth or proliferation of cancer cells . in a preferred embodiment of this method , the antibody is tagged with a potentially tissue destructive agent which causes destruction of the cancer cells . examples of tissue destructive agents comprise chemotoxic agents , chemotherapeutic agents including vaccines , radionuclides , toxins , complement activators , clotting activators and the like . the above examples are for illustrative purposes only and are not meant to limit the scope of the invention . the following examples are intended to illustrate the invention without limiting same in any manner especially with respect to substantially functional equivalents of hybridomas , monoclonal antibodies and cell lines described and claimed herein which equivalents can be produced in accordance with the invention following the procedures outlined in the specification of this application . the monoclonal antibodies selected for use in the present invention were derived from spleen cells of mice immunized with whole cells of colon carcinoma cell lines such as tallevi and ht - 29 by fusion methods well known in the art . a group of monoclonal antibodies which were found to recognize specific cell antigens of gastrointestinal cells , was selected as the grastrointestinal panel . this panel and the antigenic systems recognized are given in tables i & amp ; ii . heterogeneity of human colon carcinoma is therein noted . the table data point out and define the heterogeneity of colon carcinomas . gastrointestinal antigenic systems are defined by these mabs as determined by serological analysis with over 70 tumor cell lines ; 18 colon cancers , over 50 non - gastrointestinal cancers as well as immunopathology on frozen sections of normal adult and normal fetal tissue . ( see table i & amp ; ii ) several of the antigens , as defined by the monoclonal antibodies of the panel , are expressed differentially by cell populations within the adult gi system . clt152 antigen is expressed by epithelial cells of the gi mucosa of esophagus , stomach , small intestine and colon , but is not found in other adult tissues . clh70 , clt307 , clt86 and clh68 antigens are expressed by adult stomach , small intestine and colon . clt218 is expressed by adult small intestine and colon . ht29 / 26 is expressed by colon and some cells of small intestine in the adult . clt15 also is expressed by normal colon epithelium as well as some upper gi cells except stomach in adult tissues . thus the mabs antigens ht29 / 26 , clt15 , clt218 , clh70 , clt307 , clt86 and clh68 occur in adult colon epithelial cells ; they vary among themselves in their pattern of distribution within the rest of the gi tract . there is some limited expression of these antigens in epithelial cells of other tissues as well see table ii . thus , for example , clt218 , clt86 , ht29 / 26 antigens are expressed on bronchial epithelium whereas clh6 , ht29 / 36 and ht29 / 15 are not . thus , the panel antibodies differ in their expression on normal cells even as to similar cells of other tissues . it is important that the mabs clh6 , clt85 , and 29 / 36 do not react with normal adult tissue in immunopathology of frozen tissue sections but do react with distinct subsets of colon adenocarcenomas . serologically clt85 reacts with approximately 11 of 17 colon cancer lines , and clh6 with approximately 8 out of 17 colon cancer cell lines . clt85 and clh6 show no reaction with normal adult cells in serology . thus normal versus neoplastic cells of the colon can be differentiated and assayed by contacting a speciman from a human patient with each of the 14 monoclonal antibodies of the panel in serial dilution , and observing any antigen antibody reaction by any of the methods cited . although 14 hybridomas producing monoclonal antibody against gastrointestinal cell antigens are presented , it is obvious that the present invention encompasses all the mabs exhibiting the characteristics described therein , especially the embodiment describing reaction with normal as well as tumor cell antigens of the gi tract . changes in cell antigens are associated with different stages of differentiation and different stages of cancer . thus this invention technique defined cell antigens associated with differentiation and cancer of the gi tract . the following hybridoma cell lines are maintained on deposit at sloan - kettering institute , 1275 york avenue , new york , ny 10021 under designations corresponding to the mab produced by each hybridoma as follows : clh 6 , clt 85 , clt 479 , clt 174 , clh 68 , clt 152 , clh 70 , ht 29 / 15 , ht 29 / 26 , ht 29 / 36 , clt 218 , clt 15 , clt 307 and clt 86 . said hybridoma cell lines have been deposited on mar . 11 , 1983 with the american type culture collection , 1230 parklawn drive , rockville , md . 20852 under atcc designations corresponding to the above sloan - kettering designations as follows : ______________________________________sloan - kettering corresponding atccdeposit # deposit accession # ______________________________________clh 6 hb 8232clt 85 hb 8240clt 479 hb 8241clt 174 hb 8242clt 68 hb 8243clt 152 hb 8244clh 70 hb 8245ht 29 / 15 hb 8246ht 29 / 26 hb 8247ht 29 / 36 hb 8248clt 218 hb 8249clt 15 hb 8250clt 307 hb 8251clt 86 hb 8252______________________________________ serological reaction of colon panel monoclonal antibodies with human tumor cell lines of various tissues by rosette formation with human red blood cells conjugated with rabbit anti - ig , dippold supra if there is no rosette reaction , the absorption test was done . thus if a mab was negative for the rosette reaction but absorbed onto the test antigen system it was deemed to be a positive reaction such that 1 = positive reaction by the absorption test though mab gives a negative test for rosette formation i . e . 0 test for rosette reaction is further tested by the absorption test . therefore 0 on this table indicates no reaction by either absorption or rosette reactions . for comparison , mab 19 . 9 was obtained from h . kaprowski and assayed as well alongside the mabs of the present invention atkinson , b . f . et al ., cancer research , 42 : 4820 - 4823 ( 1982 ). immunogen for clt series is tallevi , for ht and clh antibodies the immunogen is ht - 29 . immunopathological reaction of colon panel monoclonal antibodies with fetal and adult normal human tissue in frozen section by indirect immunofluorescence . for comparison , mab 19 . 9 was obtained from h . kaprowski and assayed as well alongside the mabs of the present invention atkinson , b . f . et al ., cancer research , 42 : 4820 - 4823 ( 1982 ). table i__________________________________________________________________________ serologyserological reaction of monoclonal antibodiesproduced from human colon tumor immunogen with varioushuman cancer cell linesimmunizing tumor : colon clh clt clt clt clh clt clh ht29 ht29 ht29 clt clt clt cltantibody 6 85 479 174 68 152 70 - 15 - 26 - 36 218 15 307 86 19 . 9__________________________________________________________________________ig class of antibody : μ γ1 γ1 γ1 γ1 γ1 γ1 γ2a γ3 μ μ μ μgp antigen gp95 p52 gp25 gp29detectedcells testedcolon ca . : ht 29 3 3 3 3 3 3 3 3 3 3 0 0 3 3 10 . sup . 3sw 48 3 3 2 3 0 3 0 0 3 3 2 2 0 0 10 . sup . 3sw 403 0 3 3 3 2 3 0 3 3 2 3 0 3 3 10 . sup . 6sw 480 3 3 3 3 3 3 3 3 3 3 3 3 3 10sw 620 3 3 2 0 0 3 3 3 3 3 2 3 2 0 10 . sup . 2sw 837 0 0 0 1 0 0 0 0 3 2 0 0 0 0sw 1083 2 0 0 0 0 0 0 0 3 0 3 0 0 0 0sw 1116 0 1 3 3 3 3 0 3 3 3 3 0 3 3 10 . sup . 2sw 1222 0 3 3 3 3 3 3 3 3 3 0 0 3 3 10 . sup . 3sw 1417 1 1 3 3 3 3 3 3 3 0 0 2 3 0caco2 2 3 0 0 0 2 3 3 3 0 3 0 0 0 0sk - co - 1 0 0 0 0 0 0 2 3 3 0 0 0 0tallevi 0 3 3 3 3 3 3 3 3 0 3 3 3 0 10 . sup . 3moutsiatzos 1 0 0 0 0 0 0 0 0 0 0 0 0 0kolraga 0 3 3 3 3 3 3 3 3 3 2 0 3 10friedland 0 0 0 3 3 2 3 0 3 0 3 0 3 0 0redmond 1 0 0 0 0 0 0 3 3 0 2 0 0 0 1hso7o3tpancreas ca . : capan 1 3 0 0 1 0 0 3 0 0 0 3 0 0capan 2 0 3 3 3 3 3 0 3 3 3 0 3 10 . sup . 2sw 850 0 0 0 0 0 0 0 0 0 0 0 0sw 979 0 0 0 0 0 0 0 0 0 0liver ca . : sk - hep - 1 1 0 0 0 0 0 2 3 3 0 0 0 0 0 0biliary duct : charles 0 0 2 0 0 0 0 3 3 0 0 0 0astrocytoma : goodstein 0 0 0 0 0 0 0 0 0 0 0 0 0u251 0 0 0 0 0 0 0 0 0 0 0 0becker 0 0 0 0 0 0 0 0 0 0 0 0machino 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0jones 0 0 0 0 0 0 0 0 0 0 0 0aj 0 0 0 0 0 0 0 0 0 0 0 0 0lear 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0healy 0 0 0 0 0 0 0 0 0 0 0 0 0 0t98 0 0 0 0 0 0 0 0 0 0 0 0 0u373 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0melanoma : sk - mel - 31 ( 3 ) 0 0 0 0 0 0 0 2 0 0 0 0 0 0 023 0 0 0 0 0 0 0 0 0 0 0 0 0 013 0 0 0 0 0 0 0 0 0 0 0 0 0 0 037 0 0 0 0 0 0 0 2 0 0 0 0 0 0 093 ( 2 ) 0 0 0 0 3 0 0 3 2 0 0 0 0 0 028 0 0 0 0 0 0 0 0 0 3 0 0 0 0 0mewo 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0neuroblastoma : sk - n - mc 0 0 0 0 0 0 3 0 0 0 0 0 0 0 0sh 0 0 0 0 0 0 3 0 0 0 0 0 0 0 0be2 0 0 0 0 0 0 3 0 0 0 0 0 0 0 0lan - 1 - 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0chp - 234nbreast ca . : mda mb 361 0 0 0 0 0 0 3 3 0 0 0 0 0 0 0mda mb 231 0 0 0 0 0 0 2 3 3 0 0 0 3 0 0bt 20 0 0 0 0 0 0 0 3 3 0 0 0 0 0 0cama 0 0 1 1 0 0 0 3 0 3 3 3 3 0sk - br - 3 0 0 0 0 0 0 0 3 0 0 0 0 0 0alab 0 0 0 0 0 0 3 3 0 0 0 0 0mcf - 7 0 0 0 0 0 0 0 3 3 3 3 0 0 3 0kidney ca : sk - rc - 6 0 0 0 0 0 0 0 0 3 0 0 0 0 07 0 0 0 0 0 0 0 0 3 0 029 0 0 0 0 0 3 0 0 3 0 0 0 0 04 0 0 0 0 0 0 0 0 3 0 0 0 0ovary ca : sk - ov - 3 0 0 0 0 0 0 0 0 3 0 0 0 0 0 0roac 0 0 0 0 0 0 3 0 3 0 0 0 0 02774 0 0 0 0 0 0 0 0 3 0 0 0 0 0sw 626 0 0 3 3 0 0 0 3 3 3 0 3 3 0shustak 0 0 0 0 0 3 0 3 3 3 3 3 3 0turanek 0 2 0 0 3 2 3 3 0 0 0 0 3 10uterine ca : me180 0 0 0 0 0 0 0 0 3 3 0 0 0 0 0chorioepithelium : svcc 0 0 2 0 0 0 0 3 0 0 3 0 0lung ca : sk - lc - 3 0 0 2 0 0 0 3 3 3 0 0 0 0 0 04 0 0 0 0 0 0 0 3 3 2 0 0 0 0 105 1 0 0 0 0 0 0 3 3 0 0 0 0 0 06 0 0 0 0 0 0 3 2 0 0 0 0 2 0 07 0 0 0 0 0 0 0 0 3 2 0 0 0 0 08 0 0 0 2 0 0 0 0 3 3 0 0 0 0 013 0 0 0 0 0 0 2 0 3 2 0 0 0 0 0lawson 0 1 1 1 0 3 2 3 3 2 3 0 0 3 10 . sup . 3bladder ca : t - 24 0 0 0 0 0 0 0 0 0 3 0 0 0 0 0tcc sup 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0253j 0 0 0 0 0 0 0 0 0 0 0 3 0 0 0639v 0 0 0 0 0 0 0 0 3 3 0 0 0 0 0486p 0 0 0 0 0 0 3 3 3 0 0 0 0 0 0__________________________________________________________________________ table ii__________________________________________________________________________ immunopathologynormal tissue distribution of the monoclonalantibodies produced from humancolon tumor immunogen clt clh ht ht clt clt clt clt clh clt clt clt ht clh 85 28 6 29 / 36 29 / 15 479 15 174 86 70 152 19 . 9 307 218 29 / 26 68__________________________________________________________________________a . fetal tissueslung ± 0 0 0 + + + + + + + + + 0bronchial ± 0 0 0 ± ± ± + + + + + + 0epitheliumcartilage 0 0 0 0 0 0 0 0 0 0 0 0 0 0pneumocytes 0 0 0 0 0 0 0 0 0 0 0 0 0 0connect . tis 0 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