Method of classifying antibody, method of identifying antigen, method of obtaining antibody or antibody set, method of constructing antibody panel and antibody or antibody set and use of the same

Abstract

It is intended to provide a method whereby a plural number of antibodies against cell surface antigens are quickly classified and to provide a method whereby antigens of the thus classified antibodies are quickly identified. Further, it is intended to provide a method of promoting the utilization of the useful data obtained by the above methods. Furthermore, it is intended to provide an antibody which is effective in treating or diagnosing cancer. Namely, a method of classifying antibodies which comprises: (1) the step of preparing a plural number of antibodies respectively recognizing cell surface antigens; (2) the step of bringing each of these antibodies into contact with a cell of the same species; (3) the step of analyzing each of the cells having been treated in the step (2) by flow cytometry and thus obtaining data indicating the reactivity of each antibody with its cell surface antigen; and (4) the step of comparing the thus obtained data and classifying the individual antibodies depending on the similarity. A method of identifying antigens which further comprises: (5) the step of selecting one to several antibodies from each antibody group formed in the step (4) and identifying antigens thereof; and (6) on the assumption that antigens of the antibodies belonging to a single antibody group are the same or highly related to one another, making relations between the antigens having been identified in the step (5) and the antibody groups to thereby identify the antigens. An antibody against HER1, an antibody against HER2, an antibody against CD46, an antibody against ITGA3, an antibody against ICAM1, an antibody against ALCAM, an antibody against CD147, an antibody against C1qR, an antibody against CD44, an antibody against CD73, an antibody against EpCAM and an antibody against HGFR, each obtained by using the above methods.

1 . A method of classifying antibody including the following steps:
 (1) preparing a plurality of antibodies recognizing cell surface antigen;   (2) bringing each of the antibodies into contact with cells of the same kinds;   (3) analyzing each cell after step (2) by flow cytometry so as to obtain data showing reactivity between the antibody and the cell surface; and   (4) comparing the obtained data and classifying antibodies based on the similarity of the data.   
     
     
         2 . The method of classifying antibody according to  claim 1 , wherein the cell surface antigen is an intact cell surface antigen. 
     
     
         3 . The classifying method according to  claim 1 , wherein the cell surface antigen is a cell surface antigen of a cancer cell. 
     
     
         4 . The classifying method according to  claim 1 , wherein the plurality of antibodies recognize cell surface antigen are composed of an assembly of antibodies derived from antibody clones selected as being capable of recognizing a cell surface antigen, from an antibody library. 
     
     
         5 . The classifying method according to  claim 4 , wherein the antibody library is a phage antibody library. 
     
     
         6 . The classifying method according to  claim 1 , wherein the antibody is an antibody to which a label material is bound or fused. 
     
     
         7 . The classifying method according to  claim 1 , wherein the antibody does not include a label material and the method includes a step of labeling the antibody bound to the cell after step (2). 
     
     
         8 . The classifying method according to  claim 1 , wherein the cell is an established cell line. 
     
     
         9 . The classifying method according to  claim 1 , wherein the cell is an established cancer cell line. 
     
     
         10 . The classifying method according to  claim 1 , wherein the data are shown in a histogram showing a relationship between a binding amount of antibodies and a number of cells, and the similarity of the data is determined by comparing the shapes of the histograms. 
     
     
         11 . The classifying method according to  claim 1 , wherein the data are shown in a histogram showing a relationship between a binding amount of antibodies and a number of cells, and the similarity of the data is determined based on one or more values selected from the group consisting of a median value, a mode, a maximum value, a range, a standard deviation, a kurtosis and a skewness of the histogram. 
     
     
         12 . The classifying method according to  claim 11 , wherein the similarity of the data is determined based on the values of the median value, the mode, and the kurtosis and a skewness of the histogram. 
     
     
         13 . The classifying method according to  claim 10 , wherein the binding amount of antibody is shown by a fluorescence intensity. 
     
     
         14 . The classifying method according to  claim 1 , wherein in step (4), a plurality of antibodies having the identical or high similar data are classified into one antibody group. 
     
     
         15 . The classifying method according to  claim 1 , wherein two or more kinds of cells are prepared and each kind of cell is subjected to steps (2) to (4). 
     
     
         16 . The classifying method according to  claim 15 , wherein a plurality of antibodies having the identical or high similar data with respect to two or more kinds of cells in the cells are classified into one antibody group. 
     
     
         17 . The classifying method according to  claim 1 , wherein an antibody that has been determined to have a low reactivity with respect to the cell surface antigen during classification or after classification is excluded. 
     
     
         18 . The classifying method according to  claim 1 , wherein classification results of antibodies are displayed as a panel. 
     
     
         19 . The classifying method according to  claim 1 , wherein after step (4), the following steps are carried out:
 (i) associating the classified antibodies to a combination of n pieces of parameters including a first parameter, a second parameter, . . . , and an n-th parameter (wherein, n represents an integer of 2 or more, each parameter has two or more parameter values and the same parameter value is given to two or more antibodies in each parameter);   (ii) with respect to each parameter, preparing antibody mixtures of the antibodies having the same parameter value;   (iii) examining a reactivity of each of the antibody mixtures with a target antigen by an enzyme linked immunosorbent assay (ELISA) so as to specify the antibody mixture which shows reactivity;   (iv) specifying a combination of a parameter name and a parameter value that are common to the antibody group contained in the specified antibody mixture;   (v) selecting an antibody corresponding to the combination specified in the step (iv) in terms of all parameters among the antibodies subjected to step (i); and   (vi) classifying the selected antibodies into one antibody group.   
     
     
         20 . The classifying method according to  claim 19 , wherein the steps (i) to (v) are repeated several times under the conditions in which the combination of parameters is different in each trial; an antibody in which results of all trials are not contradictory is selected; and the antibody is subjected to the step (vi). 
     
     
         21 . The classifying method according to  claim 19 , further including the following steps between the step (v) and the step (vi);
 (v-1) newly associating the classified antibodies selected in step (v) with a combination of n pieces of parameters in a same manner as in the step (i);   (v-2) with respect to each parameter, preparing the antibody mixture of antibodies having the same parameter value for each parameter;   (v-3) examining a reactivity of each of the antibody mixtures with a target antigen by an enzyme linked immunosorbent assay (ELISA) so as to specify the antibody mixture showing the reactivity;   (v-4) determining a combination of a parameter name and a parameter value that are common to the antibody group contained in the specified antibody mixture; and   (v-5) selecting an antibody having the combination specified in the step (v-4) in terms of all parameters among the antibodies subjected to the step (v-1).   
     
     
         22 . The classifying method according to  claim 21 , wherein the steps (v-1) to (v-4) are repeated twice or more. 
     
     
         23 . The classifying method according to  claim 19 , wherein n is 3. 
     
     
         24 . The classifying method according to  claim 19 , wherein two or more kinds of target antigens are prepared and the steps (iii) to (vi) are carried out by using each target antigen. 
     
     
         25 . The classifying method according to  claim 19 , wherein the target antigen is an antigen selected from the group consisting of HER1, HER2, CD46, ITGA3, ICAM1, ALCAM, CD147, IgSF4, BCAM, C1qR, CD44, CD73, LAR, EpCAM and HGFR. 
     
     
         26 . An identifying method of an antigen including the following steps:
 (1) preparing a plurality of antibodies recognizing cell surface antigen;   (2) bringing each of the antibodies into contact with cells of the same kind;   (3) analyzing each cell after step (2) by flow cytometry so as to obtain data showing the reactivity between the antibody and the cell surface;   (4) comparing the obtained data and classifying antibodies based on the similarity of the data;   (5) selecting one or several antibodies from each antibody group formed in the step (4) and identifying an antigen thereof; and   (6) associating the antigens identified in the step (5) with an antibody group, based on the estimation that antigens to antibodies belonging to the same antibody group are identical or have high relationship.   
     
     
         27 . The identification method according to  claim 26 , wherein in the step (5), one antibody is selected from each antibody group. 
     
     
         28 . The identification method according to  claim 26 , wherein in the step (5), from the results of a flow cytometry analysis, an antibody that is determined to have a high reactivity with respect to an antigen is selected. 
     
     
         29 . The identification method according to  claim 26 , wherein in the step (5), the identification of an antigen is carried out by one or more methods selected from the group consisting of an immunoprecipitation test, Western blotting, affinity chromatography, proteomics techniques (electrophoresis, mass spectrometry, genome data base retrieve, and analysis by bioinformatics), and an expression analysis of corresponding gene. 
     
     
         30 . The identification method according to  claim 26 , further including a step of examining a reactivity between an antigen identified in the step (5) and an antibody belonging to an antibody group with which the antigen is associated in the step (6) so as to confirm that the estimation is correct. 
     
     
         31 . The identification method according to  claim 26 , wherein an identification result of antigen is displayed as a panel. 
     
     
         32 . The identification method according to  claim 31 , wherein the panel is any of the following (a) to (c):
 (a) a panel displaying a plurality of antibodies showing identical or high similar data in the flow cytometry analysis in the step (3) as one antibody group in which each antibody group is associated with its antigen;   (b) a panel displaying a plurality of antibodies showing identical or high similar data in the flow cytometry analysis in the step (3) as one antibody group in which each antibody group is associated with a cell expressing a cell surface antigen recognized by the each antibody group; and   (c) a panel displaying a plurality of antibodies showing identical or high similar data in the flow cytometry analysis in the step (3) as one antibody group in which each antibody group, its antigen and a cell expressing a cell surface antigen recognized by the antibody group are associated with each other.   
     
     
         33 . A method of obtaining an antibody having a relationship with respect to a certain disease, the method comprising the following steps:
 (1) selecting one or two or more of antibody groups from the plurality of antibody groups classified by the classifying method according to  claim 1 ;   (2) with respect to one kind or two or more kinds of diseases, examining a reactivity between an antibody in each of the selected antibody groups and a certain disease; and   (3) selecting an antibody in the antibody group, to which an antibody having a specific reactivity to any of diseases belongs, as a useful antibody.   
     
     
         34 . A method of obtaining an antibody having a relationship with respect to a certain disease, the method comprising the following steps:
 (1) selecting one or two or more of antibody groups from the plurality of antibody groups classified by the classifying method according to  claim 19 ;   (2) with respect to one kind or two or more kinds of diseases, examining a reactivity between an antibody in each of the selected antibody groups and a certain disease; and   (3) selecting an antibody in the antibody group, to which an antibody having a specific reactivity to any of diseases belongs, as a useful antibody.   
     
     
         35 . A method of obtaining an antibody set having a relationship with respect to a certain disease, the method comprising the following steps:
 (1) selecting one or two or more of antibody groups from the plurality of antibody groups classified by the classifying method according to  claim 1 ;   (2) with respect to one kind or two or more kinds of diseases, examining a reactivity between an antibody in each of the selected antibody groups and a certain disease; and   (3′) selecting a disease to which two or more antibodies show a specific reactivity, then selecting antibodies from the antibody group, to which the antibody having a specific reactivity to the disease belongs, and combining the selected antibodies.   
     
     
         36 . A method of obtaining an antibody set having a relationship with respect to a certain disease, the method comprising the following steps:
 (1) selecting two or more antibody groups recognizing different antigens from the plurality of antibody groups classified by the classifying method according to  claim 1 ;   (2) with respect to two kinds or more diseases, examining a reactivity between an antibody in each of the selected antibody groups and a certain disease; and   (3) selecting antibodies from the antibody group, to which the antibody having a specific reactivity to any of disease belongs, and combining the selected antibodies.   
     
     
         37 . A method of obtaining an antibody set having a relationship with respect to a certain disease, the method comprising the following steps:
 (1) selecting two or more antibody groups recognizing different antigens from the plurality of antibody groups classified by the classifying method according to  claim 1 ;   (2) with respect to one kind or two or more kinds of diseases, examining a reactivity between an antibody in each of the selected antibody groups and a certain disease; and   (3) selecting an antibody from the antibody group to which the antibody having a specific reactivity to any of diseases belongs, and an antibody belonging to other antibody group whose antigen is common to that of the antibody group, and combining the selected antibodies.   
     
     
         38 . A method of obtaining an antibody set having a relationship with respect to a certain disease, the method comprising the following steps:
 (1) selecting two or more antibody groups recognizing the common antigen from the plurality of antibody groups classified by the classifying method according to  claim 1 ;   (2) with respect to one kind or two or more kinds of pathologic conditions, examining a reactivity between an antibody in each of the selected antibody groups and a pathologic condition; and   (3) connecting information about the reactivity and then combining the antibodies in the antibody groups.   
     
     
         39 . A method of obtaining an antibody set having a relationship with respect to a certain disease, the method comprising the following steps:
 (1) selecting one or two or more antibody groups from the plurality of antibody groups classified by the classifying method according to  claim 19 ;   (2) with respect to one kind or two or more kinds of diseases, examining a reactivity between an antibody in each of the selected antibody groups and a certain disease; and   (3′) selecting a disease to which two or more antibodies show a specific reactivity, then selecting antibodies from an antibody group which the antibodies showing a specific reactivity to the disease belong to, and combining the selected antibodies.   
     
     
         40 . A method of obtaining an antibody set having a relationship with respect to a certain disease, the method comprising the following steps:
 (1) selecting two or more antibody groups recognizing different antigens from the plurality of antibody groups classified by the classifying method according to  claim 19 ;   (2) with respect to two or more kinds of diseases, examining a reactivity between an antibody in each of the selected antibody groups and a certain disease in two or more kinds of diseases; and   (3) selecting antibodies from the antibody group to which the antibody having a specific reactivity to any of diseases belong, and combining the selected antibodies.   
     
     
         41 . A method of obtaining an antibody set having a relationship with respect to a certain disease, the method comprising the following steps:
 (1) selecting two or more antibody groups recognizing different antigens from the plurality of antibody groups classified by the classifying method according to  claim 19 ;   (2) with respect to one kind or two or more kinds of diseases, examining a reactivity between an antibody in each of the selected antibody groups and a certain disease; and   (3) selecting an antibody from the antibody group to which the antibody having a specific reactivity to any of disease belongs, and an antibody belonging to other antibody group whose antigen is common to that of the antibody group, and combining the selected antibodies.   
     
     
         42 . A method of obtaining an antibody set having a relationship with respect to a certain disease, the method comprising the following steps:
 (1) selecting two or more antibody groups recognizing the common antigen from the plurality of antibody groups classified by the classifying method according to  claim 19 ;   (2) with respect to one kind or two or more kinds of pathologic conditions, examining a reactivity between an antibody in each of the selected antibody groups and a pathologic condition; and   (3) associating information about the reactivity and then combining the antibodies in the antibody groups.   
     
     
         43 . The obtaining method according to  claim 33 , wherein the disease is selected from the group consisting of: kidney cancer, hepatic cell carcinoma, gallbladder and liver cancer, alveolar cell carcinoma, lung squamous cell cancer, pulmonary adenocarcinoma, pancreas cancer, adenocarcinoma, and ovarian cancer. 
     
     
         44 . The obtaining method according to  claim 33 , wherein in the step (2), the reactivity is examined by one or more methods selected from the group consisting of an immunostaining procedure, an immunoprecipitation method, a flow cytometry analysis, cell ELISA, an intermolecular interactive analysis between a disease-related molecule (disease causative gene product and the like) and an antibody, and application test to a disease model cell (or animal). 
     
     
         45 . An isolated antibody obtained by the method according to  claim 33 . 
     
     
         46 . An antibody set obtained by the method described in  claim 35 . 
     
     
         47 . A production method of a panel displaying a relationship between an antibody and a disease, the method comprising the following steps:
 (1) selecting one or two or more of antibody groups from the plurality of antibody groups classified by the classifying method according to  claim 1 ;   (2) with respect to one kind or two or more kinds of diseases, examining a reactivity between an antibody in each of the selected antibody groups and a certain disease; and   (3) associating the results of the step (2) with each antibody and displaying by using a drawing or a tabular format.   
     
     
         48 . A production method of a panel displaying a relationship between an antibody and a disease, the method comprising the following steps:
 (1) selecting two or more of antibody groups recognizing different antigens from the plurality of antibody groups classified by the classifying method according to  claim 1 ;   (2) with respect to one kind or two or more kinds of diseases, examining a reactivity between an antibody in each of the selected antibody groups and a certain disease; and   (3) associating the results of the step (2) with each antibody and displaying by using a drawing or a tabular format.   
     
     
         49 . A production method of a panel displaying a relationship between an antibody and a pathologic condition, the method comprising the following steps:
 (1) selecting two or more of antibody groups recognizing a common antigen from the plurality of antibody groups classified by the classifying method according to  claim 1 ;   (2) with respect to one kind or two or more kinds of pathologic condition, examining a reactivity between an antibody in each of the selected antibody groups and a certain pathologic condition of disease; and   (3) associating the results of the step (2) with each antibody and displaying by using a drawing or a tabular format.   
     
     
         50 . A production method of a panel displaying a relationship between an antibody and a disease, the method comprising the following steps:
 (1) selecting one or two or more of antibody groups from the plurality of antibody groups classified by the classifying method according to  claim 19 ;   (2) with respect to one kind or two or more kinds of diseases, examining a reactivity between an antibody in each of the selected antibody groups and a certain disease; and   (3) associating the results of the step (2) with each antibody and displaying by using a drawing or a tabular format.   
     
     
         51 . A production method of a panel displaying a relationship between an antibody and a disease, the method comprising the following steps:
 (1) selecting two or more of antibody groups recognizing different antigens from the plurality of antibody groups classified by the classifying method according to  claim 19 ;   (2) with respect to one kind or two or more kinds of diseases, examining a reactivity between an antibody in each of the selected antibody groups and a certain disease; and   (3) associating the results of the step (2) with each antibody and displaying by using a drawing or a tabular format.   
     
     
         52 . A production method of a panel displaying a relationship between an antibody and a pathologic condition, the method comprising the following steps:
 (1) selecting two or more of antibody groups recognizing a common antigen from the plurality of antibody groups classified by the classifying method according to  claim 19 ;   (2) with respect to one kind or two or more kinds of pathologic condition, examining a reactivity between an antibody in each of the selected antibody groups and a certain pathologic condition of disease; and   (3) associating the results of the step (2) with each antibody and displaying by using a drawing or a tabular format.   
     
     
         53 . A panel produced by the method according to  claim 47 . 
     
     
         54 . (canceled) 
     
     
         55 . A method of testing a disease in which a cell surface antigen is an indicator, the method comprising the following steps:
 (1) preparing a cell or a tissue separated from a subject;   (2) examining a reactivity between the cell or the tissue and each antibody displayed on the panel according to  claim 53 ; and   (3) collating the results in the step (2) with the panel.   
     
     
         56 . A method of selecting an optimum treatment method for a certain disease, the method comprising the following steps:
 (1) preparing a cell or a tissue separated from a subject;   (2) examining a reactivity between the cell or the tissue and each antibody displayed on the panel according to  claim 53 ;   (3) collating the results in the step (2) with the panel, and   (4) selecting an effective antibody according to the results of collating.   
     
     
         57 . The method according to  claim 56 , wherein the effective antibody is an antibody showing a specific reactivity in the step (2) or an antibody equivalent thereto. 
     
     
         58 . The method according to  claim 56 , wherein the certain disease is a disease in which a cell surface antigen selected from the group consisting of HER1, HER2, CD46, ITGA3, ICAM1, ALCAM, CD147, IgSF4, BCAM, C1qR, CD44, CD73, LAR, EpCAM and HGFR is an indicator. 
     
     
         59 . The method according to  claim 56 , wherein the panel displays two or more antibodies selected from the group consisting of 048-006 antibody, 057-091 antibody, 059-152 antibody, 048-040 antibody, 054-101 antibody, 055-147 antibody, 059-173 antibody, 067-149 antibody, 067-176 antibody, 015-126 antibody, 015-044 antibody, 015-102 antibody, 015-136 antibody, 015-143 antibody, 015-209 antibody, 039-016 antibody, 053-216 antibody, 075-024 antibody, 075-110 antibody, 086-032 antibody, 086-035 antibody, 086-036 antibody, 086-061 antibody, 086-138 antibody, 086-182 antibody, 035-224 antibody, 045-011 antibody, 051-144 antibody, 052-053 antibody, 052-073 antibody, 053-049 antibody, 3172-120 antibody, 066-069 antibody, 015-003 antibody, 064-002 antibody, 064-006 antibody, 064-012a antibody, 064-012b antibody, 064-014 antibody, 064-054 antibody, 064-085 antibody, 064-093 antibody, 064-116 antibody, 065-183 antibody, 067-142 antibody, 068-007 antibody, 052-033 antibody, 053-042 antibody, 053-051 antibody, 053-059 antibody, 053-085 antibody, 035-234 antibody, 040-107 antibody, 041-118 antibody, 066-174 antibody, 083-040 antibody, 029-143 antibody, 045-134 antibody, 062-101 antibody, 062-109 antibody, 084-103 antibody, 052-274 antibody, 029-067 antibody, 083-131 antibody, 059-053 antibody, 064-003 antibody, 067-213 antibody, 067-153 antibody, 067-126 antibody, 067-133 antibody, 067-287 antibody, 064-044 antibody, 065-030 antibody, 065-358 antibody, 066-019 antibody, 079-085 antibody, 067-024 antibody and 076-048 antibody. 
     
     
         60 . A method of selecting an optimum treatment method of a certain disease, the method comprising the following steps:
 (1) preparing a panel displaying a reactivity between one or more antibodies selected from the group consisting of 048-006 antibody, 015-126 antibody, 067-133 antibody, 064-044 antibody, 076-048 antibody and 059-053 antibody, and a clinical cancer tissue of one or more diseases selected from the group consisting of squamous carcinoma, adenosquamous carcinoma, alveolar adenocarcinoma, adenocarcinoma, and large cell carcinoma, and a cell or tissue separated from a subject;   (2) examining a reactivity between the cell or the tissue and each antibody displayed on the panel;   (3) collating the results in the step (2) with the panel, and   (4) selecting an effective antibody according to the results of collating.   
     
     
         61 . The method according to  claim 60 , wherein the effective antibody is an antibody showing a specific reactivity in the step (2) or an antibody equivalent thereto. 
     
     
         62 . The method according to  claim 60 , wherein the certain disease is a disease selected from the group consisting of squamous carcinoma, adenosquamous carcinoma, alveolar adenocarcinoma, adenocarcinoma, and large cell carcinoma. 
     
     
         63 - 85 . (canceled)