Patent Application: US-24216199-A

Abstract:
a method for regulating immunological interaction comprising inhibiting the binding between cells bearing cd55 protein and activated leucocytes bearing cd97 protein .

Description:
clb - cd97 / 1 ( igg2a ) is a new cd97 - specific mab generated by fusing mouse myeloma sp2 / 0 with spleen cells from a balb / c mouse which has been immunized with nih - 3t3 cells stably expressing cd97 . clb - cd97 / 1 inhibits binding of biotinylated bl - ac / f2 which indicates that both mabs are directed to the same cd97 epitope . the mab clb - cd97l / 1 ( igg1 ) was generated by fusing mouse myeloma sp2 / 0 with spleen cells from a balb / c mouse immunized with human erythrocytes . hybridoma supernatants were screened for the capacity to block the adhesion of erythrocytes to cos cells transfected with cd97 ( see below ) and replated into 96 - well culture plates . 125 i - labelled k562 cells were lysed in 1 % np - 40 buffer , precleared with mouse normal ig and incubated with clb - cd97l / 1 and ia10 , a cd55 mab derived from the fifth international leucocyte typing workshop ( 12 ). immune complexes were adsorbed onto protein a - sepharose ( pharmacia , uppsala , sweden ), eluted under reducing conditions , electrophoretically separated by 5 to 15 % sds - page , and visualized by autoradiography . blocking of clb - cd97l / 1 by cd55 mabs was tested by incubation of pbl with ia10 for 20 min prior to staining with biotinylated clb - cd97l / 1 , followed by pe - streptavidine . flow cytometric analysis was done on a facscan ( becton dickinson , mountain view , calif .). binding assays were performed with cos cells three days after transient transfection with cd97 cdna ( 2 ) using lipofectamine ( life technologies , inc ., gaithersburg , md .). typically , 30 % of cos cells expressed cd97 , as determined by immunoperoxidase staining with cd97 mabs . at day one , cos cells were replated into six - well culture plates . mock - transfection was performed by the same procedure , except that no cdna was added . to analyze binding , 10 × 10 6 pbl , obtained from human venous blood by isolation on a percoll density gradient followed by counterflow centrifugal elutriation , or 100 × 10 6 erythrocytes were suspended in 1 ml dmem and overlayed on the cos cells for 30 min at 20 ° c . non - adhering cells were removed by gentle washing with pbs prior to examination by microscopy . for blocking experiments , erythrocytes were labelled with 51 cr according to manufacturers recommendations ( amersham co ., buckinghamshire , uk ). binding assays were performed in 12 - well culture plates in the presence of 5 μg / ml of mabs . after removing non - adhering cells , the γemission of well contents lysed with 1 % triton x - 100 was determined . binding of erythrocytes to cd97 - transfected cos cells was analyzed as described ( see above ). to deplete cd55 - positive cells from erythrocytes of an pnh patient , cells were incubated with clb - cd97l / 1 prior to addition of saturating amounts of anti - mouse igg magnetic beads ( dynal , oslo , norway ) and immunomagnetic selection . expression of cd55 on the erythrocyte populations was determined by flow cytometry with clb - cd97l / 1 or a subclass control mab . the inab phenotype erythrocytes examined in this study are from a new , unpublished case of this extremely rare disorder ( dr . g . daniels , personal communication ). cd97 cdnas truncated for distinct egf domains were produced using the splice - overlap extension polymerase - chain reaction ( soe - pcr ) [ horton 89 ]. since the egf domains are encoded by individual exons , the sequences of these exons , separately or in combination , were deleted resulting in mutant cdnas which encoded the following recombinants : cd97 - degf1 , cd97 - degf2 , and cd97 - degf5 . cos cells were transiently transfected with equal amounts of full - length or mutant cd97 cdna using lipofectamine ( life technologies , inc ., gaithersburg , md .). three days after transfection , binding of seven cd97 mabs was tested by flow cytometric analysis on a facscan ( becton - dickinson , mountain view , calif .). none of the mabs stained mock - transfected cos cells . adherence of human pbl ( a ) and erythrocytes ( b ) to cos cells expressing cd97 ( see above ) both , b and t lymphocytes adhere to cd97 - transfected cos cells as revealed from experiments with purified cells ( data not shown ). no binding is detectable in the presence of cd97 mabs clb - cd97 / 1 ( shown ) or bl - ac / f2 ( c ), or when cells are overlayed on mock - transfected cos cells ( d ). clb - cd97l / 1 , a mab generated to the cellular ligand of cd97 ( see above ) is specific for cd55 . fig2 a , clb - cd97l / 1 and the cd55 mab ia10 immunoprecipitate the same major protein of 70 kd from the erythromyeloid cell line k562 ( see above ). notably , also a smaller band at 140 kd that represents dimeric cd55 ( 11 ) is detectable in the precipitate from clb - cd97l / 1 . the position of molecular size markers in kilodaltons are indicated on the left . fig2 b , the binding of biotinylated clb - cd97l / 1 to pbl ( dashed line ) is completely blocked by the cd55 mab ia10 ( solid line ) ( see above ). cd55 mabs inhibit the binding of erythrocytes to cd97 - transfected cos cells ( see the experimental part ). adhesion of 51 cr - labelled erythrocytes to cd97 - transfected cos cells was assessed in the presence of 5 μg / ml of mabs specific for cd97 ( clb - cd97 / 1 ), cd55 ( clb - cd97l / 1 , ia10 , bric 220 , 230 , 110 , 216 ) or a mouse igg1 control mab . the cd55 mabs used are directed to the first ( ia10 , bric220 , bric230 ), second ( bric110 ) or third ( bric216 ) scr domain ( 12 ). data are expressed as the mean percentage of cell binding (± s . e . m .) from duplicate wells of three independent experiments . cd55 - deficient erythrocytes are not able to adhere to cd97 - transfected cos cells ( see the experimental part ). fig4 a , erythrocytes from an pnh patient bind to cd97 - transfected cos cells ( upper right panel ) due to the presence of non - effected cells in this clonal disease ( 14 ) ( upper left panel ). after removing the cd55 - positive erythrocytes by immunomagnetic sorting ( lower left panel ), adherence was completely abolished ( lower right panel ). one representative experiment out of four is shown . fig4 b , the complete absence of cd55 expression in the inab phenotype ( 15 ) ( left panel ) prevents erythrocytes from binding to cd97 - transfected cos cells ( right panel ). the three cd97 isoforms have different binding capacities for cd55 . fig5 a , schematic structure of the cd97 isoforms possessing three ( egf1 , 2 , 5 ), four ( egf1 , 2 , 3 , 5 ), or five egf domains ( egf1 , 2 , 3 , 4 , 5 ). fig5 b , immunofluorescence analysis with a panel of cd97 mabs confirms expression of the cd97 isoforms on tranfected cos cells . shown is the staining with the clb - cd97 / 1 mab . fig5 c , adherence of erythrocytes to cos cells expressing the three cd97 isoforms . expression of cd97 ( egf1 , 2 , 3 , 5 ) and cd97 ( egf1 , 2 , 3 , 4 , 5 ) results in less and smaller rosettes . mock transfection or the presence of mabs to either cd97 ( clb - cd97 / 1 or bl - ac / f2 ) or cd55 ( clb - cd97l / 1 ) completely prevented adhesion ( data not shown ). fig5 d , erythrocytes bind with different affinity to cos cells expressing equal amounts of the three cd97 isoforms . results are expressed as the percent of erythrocyte binding , relative to cd97 ( egf1 , 2 , 5 ). data shown are mean ± sd of duplicate determinations in three independent experiments . epitope mapping of cd97 mabs . fig6 a , schematic structure of the cd97 - degf recombinants . systematic deletion of the egf domains was undertaken through deletion of the encoding exons by soe - pcr . fig6 b , immunofluorescence analysis with a panel of cd97 mabs confirms expression of the cd97 - degf recombinants in tranfected cos cells . the staining pattern correlates with binding of the mabs to either the first egf domain or outside the egf domains . 1 . w . eichler , g . aust , d . hamann , scand . j . immunol . 39 , 111 ( 1994 ). 2 . j . hamann et al ., j . immunol . 155 , 1942 ( 1995 ). 3 . t . ishihara et al ., embo j . 10 , 1635 ( 1991 ); g . v . segre and s . r . goldring , trends endocrinol . metab . 4 , 309 ( 1993 ). 5 . a . j . mcknight et al ., j . biol . chem . 271 , 486 ( 1996 ). 6 . j . hamann , e . hartmann , r . a . w . van lier , genomics 32 , 144 ( 1996 ). 8 . s . f . schlossman et al ., eds ., leucocyte typing v ( oxford university press , oxford , 1995 ). 9 . t . kinoshita , m . e . medof , r . silber , v . nussenzweig , j . exp . med . 162 , 75 ( 1985 ). 10 . d . m . lublin and j . p . atkinson , ann . rev . immunol . 7 , 35 ( 1989 ). 11 . m . w . nickells , j . i . alvarez , d . m . lublin , j . p . atkinson , j . immunol . 152 , 676 ( 1994 ). 12 . k . e . coyne et al ., j . immunol . 149 , 2906 ( 1992 ). 13 . w . g . brodbeck , d . liu , j . sperry , c . mold , m . e . medof , j . immunol . 156 , 2528 ( 1996 ). 14 . t . kinoshita , n . inoue , j . takeda , adv . immunol . 60 , 57 ( 1995 ); l . luzzatto , m . bessler , curr . opin . hematol . 3 , 101 ( 1996 ). 15 . d . m . lublin et al ., blood 84 , 1276 ( 1994 ); g . daniels , human blood groups ( blackwell science , oxford , 1995 ). 17 . j . m . baldwin , embo j . 12 , 1693 ( 1993 ); s . watson and s . arkinstall , the g - 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