Patent Application: US-57326305-A

Abstract:
it is disclosed that the αvβ5 integrin mediates the proliferative signal provided by cd23 to pre - b cells . the region of cd23 which interacts with αvβ5 has been defined , and found to interact with a site on the integrin distinct from that which binds rgd . the invention provides methods for disrupting the interaction between cd23 and αvβ5 and methods of screening for chemical entities capable of disrupting this interaction .

Description:
anti - cd47 ( bric 126 , igg2b ), anti - αvβ3 ( lm609 , igg1 ), anti - αvβ5 ( p1f6 , igg1 ; 15f11 , igg2a ), and rabbit polyclonal anti - peptide antibodies specific for integrin α v and β 5 subunits were from chemicon , uk . anti - αv / cd51 ( amf7 , igg1 ) was obtained from beckman coulter , high wycombe , uk . radiochemicals and materials for enhanced chemiluminescence ( ecl ) were obtained from amersham international plc , amersham , england , and fine chemicals , including streptavidin - quantum red ( sa - qr ), horseradish peroxidase ( hrp )- coupled protein a , cyanogen bromide - activate sepharose beads , and octyl - β - d - glucopyranoside ( ogp ), were supplied by sigma , poole , england . normal peripheral blood mononuclear cells ( pbmc ) were obtained from volunteers , and b - cll leukaemic samples from patients attending the haematology clinic , western infirmary , glasgow , with appropriate ethical permissions . archival all and aml diagnostic samples ( blood or bone marrow ), collected with ethical permission in connection with earlier mrc clinical trials , were drawn from the lrf centre leukaemia bank ( institute of cancer research , london ). the sms - sb cell line was derived from a female patient presenting with all 39 , and the nalm - 6 and blin - 1 cell lines were from laboratory stocks . human bone marrow stromal cells were immortalised by retroviral transduction of the human telomerase ( htert ) gene 40 . cell lines were maintained in rpmi - 1640 medium supplemented with 10 % ( v / v ) heat - inactivated foetal calf serum ( fcs ), 2 mm fresh glutamine , and penicillin and streptomycin , at 37 ° c . in a 5 % co 2 in air in a humidified atmosphere . cytokines , obtained from r & amp ; d laboratories , were used at 5 - 10 ng / ml , but had no effect over a wide dose - response range . recombinant 25 kda scd23 , encompassing residues met 151 - ser 231 with an n - terminal his 6 - tag , was expressed in e . coli and affinity - purified by nickel chelate chromatography . sms - sb cells were also propagated in protein - free hybridoma medium - ii ( pfhm , gibco - brl , paisley , scotland ), at & gt ; 10 5 cells / ml (“ normal cell density ”— ncd ). telomerised stromal cells were cultured in dmem supplemented with 10 % fcs and 2 mm fresh glutamine , and were sub - cultured once per week . in the experiments described , the passage number for the stromal cells was between 80 and 95 , and cells were allowed to adhere for 24 hr prior to addition of lymphoid cells in co - culture . for stimulation experiments , sms - sb cells were cultured at 2500 cells / 100 μl culture ( low cell density , lcd ) a seeding density at which the cells are prone to apoptose 15 . nalm - 6 cells were washed extensively in pfhm prior to culture at 2 , 500 cells / 100 μl culture . cultures were propagated , in the presence or absence of cytokines , mabs or peptides , at 37 ° c . for 72 hours followed by addition of 0 . 3 μci / well tritiated thymidine ([ 3 h ]- tdr ) for 18 hours prior to harvest ; incorporation was determined by liquid scintillation spectrometry . apoptosis was determined by staining harvested cells with propidium iodide ( pi ) and hoechst 33342 for 1 minute prior to analysis on a coulter elite flow cytometer as previously described 15 . for stromal cell / sms - sb co - cultures , 6000 stromal cells were seeded into individual wells of a 96 - well tray and allowed to adhere for 24 hr ; anti - cd23 mab or isotype matched control and sms - sb cells were added to the wells to give a total culture volume of 100 μl . sms - sb cells were added at 5 , 25 or 50 cells per well and cultures were allowed to expand over a 2 - 4 week period . ba / f03 cells were routinely cultured in rpmi - 1640 medium supplemented with 10 % ( v / v ) fcs , 1 mm fresh glutamine , antibiotics ( penicillin and streptomycin ) and 50 μm 2 - mercaptoethanol (‘ complete medium ’; cm ). for stimulation experiments , cells were either washed in this medium and used or were extensively washed in protein - free hybridoma medium prior to use in short - term assays . for the assays , cells were cultured at 2500 - 3000 cells / 100 μl culture ( low cell density , lcd ) and propagated in the presence or absence of cytokines , mabs or peptides , at 37 ° c . for 24 - 72 hours followed by addition of 0 . 3 μci / well tritiated thymidine ([ 3 h ]- tdr ) for 18 hours prior to harvest ; incorporation was determined by liquid scintillation spectrometry . in cm , we ( like many others ) found that a sub - nanomolar dose of il - 3 was sufficient to sustain ba / f03 growth ( ec 50 ˜ 0 . 5 nm ). il - 3 is believed to activate the akt pathway leading to phosphorylation and inactivation of the pro - apoptotic bad protein . 5 × 10 5 cells , or 20 - 50 μl whole blood ( pre - treated with erythrocyte lysis buffer : − 0 . 17 m ammonium chloride , 10 mm potassium bicarbonate , 0 . 1 mm edta ), were stained with either fluorescein -( fitc )- conjugated or unlabelled primary mab for 30 - 60 minutes ; unlabelled primary antibody was visualised using a secondary fitc - conjugated anti - mouse igg or , in the case of biotinylated anti - αvβ5 , using sa - qr . phycoerythrin -( pe )- labelled anti - cd19 was used to identify b cells . cells were analysed on a facscan flow cytometer , collecting 10 , 000 events per sample and the data analysed using cellquest software . 10 7 sms - sb cells were harvested , washed twice with serum - free rpmi 1640 medium , suspended in 0 . 5 ml of labelling medium ( dmem lacking cold methionine and supplemented with 100 μci of [ 35 s ]- methionine ) and incubated at 37 ° c . for 3 hours . five millilitres of labelling medium supplemented with 10 % ( v / v ) fcs were added and the culture incubated overnight at 37 ° c . the cells were washed twice in ice - cold pbs , suspended in 1 . 5 ml ice - cold ogp extraction buffer ( 1 % ( w / v ) ogp in 50 mm hepes / koh ph 7 . 4 , 5 mm cacl 2 , 140 mm nacl , 1 mm pmsf , 1 mm aprotonin , 1 mm leupeptin ) and lysed with 40 strokes of a chilled glass homogeniser . the homogenate was centrifuged at 1000 × g for 10 minutes at 4 ° c ., and the resulting supernatant further centrifuged at 35 , 000 × g for 45 minutes at 4 ° c . in experiments employing unlabelled cells , extracts were prepared from 10 8 cells in ogp buffer . cellular extracts were added to bsa - affigel pre - equilibrated with ogp extraction buffer and incubated at 4 ° c . for 6 hours . the matrix was pelleted , unbound proteins recovered and added to pre - equilibrated scd23 - affigel and incubated at 4 ° c . overnight . the scd23 - affigel was pelleted and the unbound fraction retained . both matrices were exhaustively washed , specifically bound material eluted by boiling in sample buffer and subjected to sds - page under reducing conditions on a 10 % ( w / v ) acrylamide gel . radiolabelled proteins were visualised by fluorography 58 . unlabelled protein eluates were transferred to nitrocellulose membranes and probed with anti - vnr component antibodies , followed by an hrp - labelled secondary antibody and ecl . 10 5 sms - sb cells were added to 15 μl cd23 - or bsa - coupled sepharose beads and mixed gently for 30 minutes in the presence or absence of 0 . 5 μg anti - cd23 or anti - integrin mab , or isotype control antibody , and the number of cells associated with each bead in 10 fields per slide was determined under the light microscope . data are shown as fold - increase in cell binding to cd23 - sepharose compared to bsa - sepharose , normalised to binding reactions in the absence of any immunoglobulin . a library of 83 overlapping nonapeptides encompassing residues 151 - 321 of the 25 kda scd23 sequence was custom synthesised by mimotopes inc ( chester , uk ); mutant peptides were synthesised by the same firm . each peptide was synthesised as tridecapeptide comprising a unique cd23 - derived nonapeptide sequence , plus a common n - terminal tetrapeptide extension ( sgsg ) to which a biotin moiety was attached . each unique nonapeptide sequence had a two - residue c - terminal offset relative to its immediate neighbour . aliquots of biotinylated peptides were captured on individual wells of a 96 - well streptavidin - coated elisa tray ( mimotopes , chester , uk ); binding of integrin was determined by addition of 0 . 2 μg of purified αvβ5 integrin to each well followed by addition of the p1f6 mab and hrp - anti - mouse igg to quantitate binding . for ab binding , aliquots of ab were added and binding scored by addition of appropriate hrp - conjugated secondary ab . binding of peptides to cells was visualised by treating cells exposed to biotinylated peptides with fluorochrome - conjugated streptavidin and scoring binding by flow cytometry ; mean fluorescence intensity data were obtained using the cellquest programme . in culture experiments , peptides were used in the 10 nm to 100 mm concentration range and appropriate solvent vehicle controls ( e . g ., acetonitrile , dmso ) were always performed . 5 × 10 5 human u937 or murine raw 264 . 7 monocytic cells were treated with biotinylated peptide at concentrations between 1 ng / ml and 1 μg / ml . most experiments used 0 . 2 μg / ml . peptide was added to 100 μl of cells for 30 - 60 minutes on ice , washed with phosphate - buffered saline ( pbs ), then resuspended in 300 μl pbs and exposed to 1 μg strepatavidin - phycoerythrin ( or pe - cy5 - streptavidin in some experiments ). after a further incubation on ice ( 30 - 60 minutes ), the cells were washed with pbs and then analysed on a facscan flow cytometer , collecting 10 , 000 events per sample . data was analysed using cellquest software . for competition experiments , non - biotinylated peptide was present at approximately a 10 - fold molar excess relative to the biotinylated probe peptide . note that the probe peptides are of the form biotin - sgsg - x 9 ( where x 9 is the unique nonapeptide sequence based on cd23 and sgsg is a tetrapeptide linker ), while the competitors are of the form x 9 ( i . e ., contain only the cd23 - derived sequence ). approximately 0 . 5 μg of purified integrin protein ( αvβ3 , αvβ5 and α5β1 , all purified from human placenta and purchased from chemicon ) was electrophoresed on 10 % ( w / v ) acrylamide gels using the laemlli discontinuous buffer system . the stacking and separating gel and sample buffer mixes contained sds , but lacked any reducing agent ( i . e ., had no dithiothreitol or 2 - mercaptoethanol ). this configuration ensures that the two component chains of the non - covalent heterodimeric integrin complexes are separated ( e . g ., to αv and p5 ), but that any intrachain disulphide bonds are not hydrolysed . thus , in this system , the αv chain will migrate at ˜ 150 kda since the 125 kda and 25 kda elements of the mature αv chain derived from the single large precursor protein will remain disulphide - bonded . after electrophoresis , the proteins were transferred to nitrocellulose filters and , after blocking ( with 10 % milk powder in appropriate buffer ), the filters were treated for 2 - 3 hours with 50 μg of probe peptide ( e . g ., biotinylated peptide # 9 ). the filters were washed with 4 - 5 changes of buffer over a 2 - 3 hour period on a rocking platform , then treated with hrp - streptavidin for 60 minutes and again washed 4 - 5 times . binding was visualised using the supersignal west enhanced chemiluminescence system . for competition experiments , non - biotinylated peptides were included at a two - to five fold molar excess at the step where the probe peptide was added to the filters . the filters were pre - incubated with unlabelled peptide for one hour prior to exposure to biotinylated peptide ; the latter was added to the buffer already containing the unlabelled peptide so that the non - biotinylated peptide was continuously present . we have previously shown 15 that recombinant soluble cd23 ( scd23 ) sustains growth and blocks apoptosis in a dose - dependent manner in low cell density ( lcd ) cultures of a human pre - b cell - like cell line , sms - sb 39 , derived from a female acute lymphoblastic leukaemia ( all ) patient ( fig1 a ). sms - sb cells do not express cd21 , the p2 integrins cd11b - cd18 or cd11c - cd18 15 , or the vnr αvβ3 ( fig1 ciii ). in order to identify the cd23 binding structure , lysates of [ 35 s ]- methionine - labelled sms - sb cells were passed over a scd23 - affigel column and bands of mr ˜ 120 kda and ˜ 80 kda , consistent with those of mature α v and β 5 integrin chains , respectively , are specifically enriched in the eluates ( fig1 bi ). western blots of sms - sb cellular proteins specifically eluted from scd23 - affigel matrices probed with anti - α v and anti - β 5 antibodies ( fig1 bii and 1 biii , respectively ), confirm that both α v and β 5 species bound to the matrix . note that the polyclonal anti - αv antibody binds to a c - terminal epitope on αv that is located on the 25 kda fragment generated during biosynthetic maturation of αv 41 . no β3 integrin protein is detected in whole cell extracts of sms - sb cells or in eluates from scd23 affinity columns ( data not shown ). sms - sb cells stain with both the p1f6 mab ( fig1 ci ) that recognises the assembled αvβ5 heterodimer 42 , and anti - cd47 vnr - associated protein mabs ( fig1 cii ). the cells do not stain with the αvβ3 - specific lm609 mab ( fig1 ciii ) 42 , 43 . rt - pcr analysis of sms - sb rna yields amplicons of the appropriate sizes for cd47 , cd51 / α v , β1 and β 5 , but no correctly - sized pcr products are detected for β 3 , β 6 or β 8 coding sequences ( data not shown ). the data demonstrate that cd23 binds to the αvβ5 integrin in sms - sb cells and that this interaction regulates survival and growth of a model pre - b cell line . the data of fig1 demonstrate that cd23 binds the αvβ5 vnr and suggest this integrin sustains cell growth . the anti - αv mab amf7 induces a strong dose - dependent increase in thymidine incorporation in both sms - sb cells ( fig2 a ) and , importantly , in a second pre - b cell line , nalm - 6 ( fig2 b ). we next used as stimulants the p1f6 and 15f11 mabs that recognise distinct epitopes dependent upon complete assembly of the αvβ5 heterodimer . the 15f11 mab , whose binding to αvβ5 is insensitive to ligand 44 , sustains sms - sb cell survival ; however , the p1f6 reagent ( that blocks binding to vn ) fails to enhance growth of sms - sb cells ( fig2 c ). the data confirm that the αvβ5 integrin regulates cell survival in pre - b cells . the observation that the 15f11 and p1f6 mabs also elicit different responses in sms - sb cells ( fig2 c ) suggests the pro - survival effect is ligand - selective , an interpretation supported by the fact that neither vn nor fn sustain the growth of sms - sb cells ( fig2 d ). the finding that neither vn nor fn stimulate pro - survival responses suggests that cd23 interacts with αvβ5 at a site distinct from that used by the integrin to bind rgd - containing matrix proteins . elisa - type assays demonstrate dose - dependent binding of recombinant 25 kda scd23 to immobilised αvβ5 ( fig3 ai ) and vice versa ( data not shown ), and binding of sms - sb cells to agarose beads coated with 25 kda scd23 is inhibited by mabs directed against either αvβ5 or cd23 ( fig3 a ii ). we next used purified αvβ5 protein to probe a library of 83 biotinylated peptides containing overlapping nonapeptide sequences based on the 25 kda scd23 sequence . purified recombinant αvβ5 protein binds specifically to a group of four peptides ( peptides # 9 -# 12 ) near the n - terminus of 25 kda scd23 and to a further peptide (# 17 ) ( fig3 bi ). in multiple experiments , the αvβ5 protein bound only to peptides # 9 -# 12 , with no other peptide showing a consistent binding to the integrin . the conformation - dependent m - l233 anti - cd23 mab bound to none of the 83 peptides , but a goat polyclonal ab directed against the c - terminus of cd23 binds strongly to peptide # 82 ( data not shown ). flow cytomety shows that peptides # 9 and # 11 bind strongly to sms - sb cells but peptide # 17 , which gives a minor signal in the in vitro binding assay to integrin , does not bind to cells ( fig3 ci ). the sequences of the peptides used are shown on fig3 cii and the sole feature shared by all peptides with αvβ5 binding activity is a tripeptide motif of arg - lys - cys ( rkc , embolded on the figure ). peptides # 9 -# 12 also display strong binding to two other pre - b cell lines , blin - 1 ( fig3 c iii ) and nalm - 6 ( fig3 civ ). four other cd23 - derived peptides , chosen either randomly (# 57 ), on the basis of being immediately adjacent to (# 8 and # 13 ), or having a similar charge (# 15 ) to peptides with binding activity fail to bind any cell line ( fig3 c ii ). peptides # 61 -# 63 , which contain an rkl sequence , or peptides # 78 -# 80 , which possess the ‘ inverse rgd ’ sequence , do not bind purified αvβ5 ( fig3 b ) or cells ( data not shown ). rkc is the minimum requirement for binding to the αvβ5 integrin . in proliferation assays , peptides # 9 and # 12 stimulate thymidine incorporation by sms - sb cells ( fig4 a ); peptides # 10 and # 11 have minimal effects . the data show that the peptides derived from the region of the cd23 protein recognised by αvβ5 integrin not only bind specifically to cells , but also , in some instances , mimic the effect of cd23 itself in promoting growth of sms - sb cells . inverting the sequence of peptide # 9 ( 9 - inv ) reduces substantially , but does not ablate , its ability to elevate thymidine incorporation ( fig4 a ). the biological activity of sequence - inverted peptide # 11 was minimally altered ( fig4 a ). since peptides # 10 and # 11 bind to cells but do not stimulate thymidine incorporation , we next probed the potential antagonistic function of peptides # 10 and # 11 by pre - treating sms - sb cells with one of these peptides before addition of either peptide # 9 or # 12 , both of which drive sms - sb cell growth . each of peptide # 10 and # 11 reduces the growth stimulatory effect of either peptide # 9 ( fig4 bi ) or # 12 ( fig4 bii ) to background levels , but an irrelevant peptide , # 57 , is without significant antagonistic effect . neither peptide # 10 or # 11 had any positive or negative effect on the ability of peptide # 57 to influence sms - sb cell growth ( fig4 b ii ). these data indicate that peptides # 10 and # 11 can antagonise specifically the growth - promoting activities of peptides # 9 and # 12 . the sequence encompassed by the rkc - containing peptides # 9 -# 12 is equivalent to residues lys 166 - gly 180 in the full length cd23 protein , with the rkc motif located at residues 172 - 174 . comparison of available cd23 sequences indicates that the presence of arginine at position 172 ( arg 172 ) is unique to human cd23 , suggesting that this residue was critical for binding . inversion of the peptide sequence of peptides # 9 and # 11 ( 9 - inv and 11 - inv ) does not reduce greatly binding of peptides to sms - sb cells , although substitution of arg172 with gln ( r172q ) in both peptides does impair binding ( fig4 ci and ii ). similar data are obtained for binding to nalm - 6 and blin - 1 cells ( data not shown ). strikingly , the capacity of peptide # 9 to promote thymidine incorporation in sms - sb cells is not significantly reduced by the r172q substitution ( fig4 d ). substitution of arg in peptide # 11 to either lys or gln had no effect on its inability to sustain sms - sb cell growth . these data suggest that although binding of peptide # 9 to cells is reduced by replacement of arg 172 with gln , the mutant peptide retains the capacity to sustain the growth of sms - sb cells . the data of fig4 show that arg172 is not required for peptide biological activity , suggesting that the αvβ5 integrin recognises the rkc motif in cd23 via binding site distinct from that used to capture rgd - containing ligands . consistent with this hypothesis , an excess of a tetrapeptide ( rgds ) containing the prototypic integrin binding tripeptide motif , rgd , neither impedes peptide binding to sms - sb cells ( fig5 ai and ii ), nor inhibits thymidine incorporation induced by peptides # 9 and # 12 ( fig5 a ii ). these data confirm that arg172 is dispensable for growth - sustaining activity of peptides # 9 and # 12 and that αvβ5 integrin does not recognise cd23 using the rgd - binding site . the minimal rkc sequence recognised by the αvβ5 integrin resides in a region that is basic in character ; the human cd23 sequence is qrkc while the murine equivalent is qqkc ( and the r172q variant of peptide # 9 both binds cells and stimulates thymidine incorporation ). to test the hypothesis that αvβ5 integrin recognises a basic region in cd23 , we substituted arg172 and lys173 in the agonist peptides # 9 and # 12 with alanine either singly or together . single alanine substitutions reduce peptide binding to different extents ( fig5 bi and ii ); the r172a variant of peptide # 9 retains more binding ability than the k173a equivalent , but the reverse is true for variants of peptide # 12 . double alanine substitutions reduce peptide binding to sms - sb cells significantly for both peptide , but particularly so in peptide # 9 ( fig5 bi and ii ). in proliferation assays , the r172a and k173a variants of both peptides # 9 and # 12 promote sms - sb cell growth less effectively than wild type peptides ; the k173a variants are consistently less growth promoting than the r172a equivalents ( fig5 b iii ). the double alanine substitution variants do not promote growth to a significant level ; proliferation levels are close to that driven by peptide # 58 that does not bind to sms - sb cells ( fig5 b iii ). these data are entirely consistent with the interpretation that αvβ5 recognises the basic character of the rkc motif using a binding site distinct from the rgd - binding site , and also help to explain ligand - selective signalling via the αvβ5 integrin in b cell precursors . the αvβ5 staining pattern of lymphocytes derived from peripheral blood , bone marrow and two representative all patients show that although subsets of total , cd4 + and cd8 + peripheral t cells express αvβ5 , there is essentially no αvβ5 expression on cd19 + b lymphocytes in normal individuals ( fig6 a ). normal human bone marrow contains cd19 + cells that are αvβ5 + ( fig6 b ), and two all samples display substantial populations of αvβ5 + cells in peripheral blood ( fig6 ci and ii ). the finding of high levels of αvβ5 +/ cd19 + b cells in the peripheral blood of all patients contrasts strikingly with the absence of such cells in the blood of normal subjects . analysis of the expression patterns of cd23 receptors on cells from representative all and b - chronic lymphoblastic leukaemia ( b - cll ) patients demonstrates that αvβ5 is the only cd23 receptor expressed on all cells ; αvβ3 is not present ( fig6 d ). in contrast to all , no αvβ5 ( or αvβ3 ) expression is detected in any b - cll sample ( fig6 e ); the cytometric data for b - cll are totally supported by analysis of cd23 receptor transcripts ( data not shown ). we next compared proportions of αvβ5 + cells in three distinct all types with cohorts of ˜ 20 b - cll and acute myeloblastic leukaemia ( aml ) samples . the data demonstrate that αvβ5 is universally expressed on all - derived samples ( regardless of the lineage of the tumour cells , or age of the patient ) and is present on the majority of aml samples at variable levels ; αvβ5 is consistently absent from b - cll cells ( fig6 f ). the patterns of expression of αvβ5 in all and b - cll reflects those found in non - malignant b cells ( fig6 a - c ), with αvβ5 being found exclusively on precursor cell - derived leukaemias . competition experiments were performed to investigate whether binding of peptides containing an rkc motif to αv integrins can be specifically inhibited by similar peptides . peptides # 9 - 12 derived from 25 kda scd23 contain the rkc motif required for binding to the αv integrin family . binding of biotinylated peptide # 9 to pre - b cell lines ( as exemplified by sms - sb cells ) can be inhibited by inclusion of non - biotinylated forms of peptide # 10 in the assays ; this tallies well with the ability of peptide # 10 to inhibit the capacity of peptide # 9 to stimulate sms - sb cell growth and survival . similar inhibition of binding of biotinylated ( probe ) peptide # 9 by non - biotinylated variants is also observed in monocytic cell line models ( e . g ., the u937 cell line ). in vitro cell culture experiments show that inclusion of an rgds tetrapeptide in cultures containing biotinylated peptide # 9 fails to block binding of peptide # 9 to sms - sb cells or the ability of peptide # 9 to promote cell growth . these data suggest the αv integrin uses a site distinct from the well - understood “ rgd - binding site ” to capture the basic rkc motif . this conclusion is supported by the observation that monoclonal antibodies that neutralise rgd - dependent adhesion do not mimic the ability of cd23 to sustain cell growth , while mabs directed to sites not linked to adhesion do mimic cd23 activity in sms - sb cells . finally , the crystallographic model of αvβ3 structure illustrates unequivocally that an rkc motif could not be accommodated in the rgd binding site . in order to investigate which part of the αv integrin molecule interacts with peptides containing an rkc motif , purified αvβ5 , αvβ3 and α5β1 integrin proteins were electrophoresed under non - reducing conditions , transferred to nitrocellulose membranes and probed with biotinylated peptide # 9 using hrp - streptavidin and enhanced chemiluminescence to visualise binding . peptide binding to β1 , β3 and β5 chains was readily detectable , but binding to the α5 or αv subunits was weak or absent . binding of biotinylated peptide # 9 to β3 and β5 chains was blocked by inclusion of excess non - biotinylated , rkc - containing peptides in the binding reactions . these data suggest that rkc - containing peptides interact specifically with the β chains of the αv integrin family and not with the αv subunit . moreover , since the rgd - binding site for recognition of adhesion ligands requires elements from both the αv and β ( 3 ) subunit to form the binding site , our western blotting data further demonstrate that the αv integrins have a second ligand binding site that is distinct from the rgd - binding site . the data also suggest that free β chains might be used in in vitro assays for ligands interacting with this second integrin binding site or for screening small molecules that might impede the interaction of our rkc - containing probe peptides with isolated β chains . in vivo exploitation of compounds that block the prototype cd23 - αvβ5 interaction will require a good animal model for validation . we used the il - 3 - dependent murine pro - b cell line , ba / f03 cell line as a model equivalent to the human sms - sb cell line . peptides # 9 -# 12 bind well to ba / f03 cells and peptide # 9 can sustain ba / f03 cell survival in the absence of il - 3 , but only at high doses and even then only rather weakly . however , if the cells are treated with a sub - optimal dose of il - 3 , peptide # 9 overtly stimulates growth . murine cd23 does not possess an rkc motif at the position equivalent to that found in human cd23 but rather has a qkc ( gln - lys - cys ) sequence . a peptide # 9 variant containing the murine sequence (“ peptide # 9 r172q ”) fails to bind ba / f03 cells and fails to have any growth - sustaining effect either by itself or in combination with il - 3 . peptide # 11 r172q also fails to bind to ba / f03 cells . the r172q substitution also greatly reduces binding of peptide # 9 to the raw murine macrophage cell line . murine cd23 is believed to lack cytokine - like activity . these binding data indicate that the presence of a non - basic amino acid , glutamine , at the position equivalent to 172 of human cd23 , may partly explain this lack of cytokine activity mediated via αv integrins . in vivo models for analysis of agents perturbing the cd23 - αvβ5 interactions ( and others like it ) may therefore require cell lines expressing cd23 molecules having a positive charge at the position equivalent to r172 in human cd23 . murine cells transfected with cd23 containing a q to r substitution , or with human cd23 , may suffice . a transgenic line expressing human cd23 or murine cd23 having a q to r substitution may enable human conditions in which the αvβ5 - cd23 is implicated to be mimicked in an animal model in vivo , allowing better validation of potential drug molecules than is currently possible . these data demonstrate that the αvβ5 integrin is a cd23 binding protein that regulates growth of pre - b cell lines . the observation that anti - αvβ5 mabs that block adhesion to matrix proteins cannot mimic cd23 action , while mabs directed to other αvβ5 epitopes can sustain cell growth , supports the interpretation that the αvβ5 integrin mediates distinct responses depending on the ligand encountered . the αvβ5 integrin recognises an rkc tripeptide motif that resides in a small basic region of the cd23 protein using a site that is distinct from the rgd - binding structure , and sensitive to basic character , rather than precise sequence . this explains why cd23 , but not vn or fn , drive pro - survival responses in pre - b cell lines . the data of fig2 demonstrate that different αvβ5 ligands ( cd23 , vn and fn ) and mabs directed to distinct epitopes of αvβ5 itself elicit different characteristic responses in pre - b cell lines , potentially by acting via distinct binding sites on αvβ5 . there are precedents for ligand - selective - signalling via αvβ3 . in monocytes , cd23 promotes pro - inflammatory cytokine synthesis while vn drives cell spreading but no cytokine production 17 . similarly , in k562 cells , αvβ3 adheres with different affinities to fn and vn via processes regulated by , and resulting in activation of , distinct signalling pathways 45 . in our system , the finding that rgd - containing peptides fail to inhibit either peptide # 9 or # 12 binding to cells or their ability to sustain cell growth ( fig4 and 5 ) confirms that αvβ5 binds cd23 using a structure distinct from the rgd - binding site . the x - ray crystallographic model of αvβ3 integrin in association with an rgd - containing cyclic pentapeptide fully supports this interpretation . the rgd - peptide arg is secured by a bidentate salt link with asp 218 and a second such link with asp 150 from the αv chain 37 , while the asp side chain is secured by contacts with tyr 122 , arg 214 and asn 215 from the β3 chain , plus contact with a mn 2 + ion 37 . the peptide gly residue resides at the interface between the αv and β3 domains making multiple hydrophobic interactions , including a dominant contact with the carbonyl oxygen of arg 216 of αv 37 . the long side chain of the rkc peptide lysine would clash seriously with this carbonyl moiety and so preclude stable insertion of the rkc motif into the rgd - binding site . integrins recognise sequences other than rgd , and the αvβ5 integrin recognises the hiv tat protein via a non - rgd motif that is basic in character 38 recognition of basic domains has been established for other integrins including α2β1 integrin which binds a basic tetrapeptide sequence ( rkkh ) derived from the snake venom metalloprotease , jararhagin , a potent inhibitor of platelet binding to collagen 46 . the data of fig4 and 5 illustrate that substitution of arg172 to gln ( the latter being found in murine cd23 ) can reduce binding of peptide # 9 to sms - sb cells slightly but has no striking effect on stimulation of thymidine incorporation . moreover , replacement of arg172 alone with ala only slightly reduces both peptide binding to cells and growth sustaining capacity ; conversion of lys173 to ala gives a more marked reduction in binding and stimulation , but activity remains . it is only when both basic residues are substituted with non - polar alanine that both cell binding and growth sustaining ability is lost . these data argue strongly that the αvβ5 integrin recognises the basic nature of the rkc - containing region of cd23 and does so via a binding site distinct from that used to capture rgd - containing ligands . recognition of the rkc - containing region is not sequence - specific , since peptides containing the qkc sequence based on murine cd23 bind and retain activity ; moreover , the inverse sequence of peptide # 9 ( ckrqfniwk ) preserves basic character but not sequence and retains some residual binding and growth - stimulating properties . this is entirely in keeping with data from the binding of αvβ5 to the hiv tat protein that showed neither poly - lysine or poly - arginine peptides bind αvβ5 affinity columns as well as the tat or vn basic domain peptides . the rkc motif recognised by the αvβ5 integrin is located at positions 172 - 174 of the human cd23 protein . models of the cd23 structure based on the mannose binding protein c - type lectin 49 , 50 do not contain the full rkc motif , but suggest it is located just beneath the lectin head domain and at the upper reaches of the coiled - coil stalk region of the cd23 protein . the precise structure of this region remains to be resolved . the regions of cd23 needed for binding to ige and cd21 reside in the lectin domain , and are known to overlap but be distinct 10 ; trimeric scd23 is capable of binding both cd21 and ige simultaneously . the location of the rkc motif for αv integrin binding is , therefore , distinct again from the cd23 structures required for ige and cd21 engagement . it is therefore possible that cd23 could interact simultaneously with three different ligands ; ige and cd21 will bind at the lectin domain and αv integrins via the stalk region . the rkc motif is also distinct from the sequence in the stalk region reported to bind to mhc class ii molecules 51 . the proteolytic processing pathways that generate soluble cd23 proteins are well understood . importantly , the rkc motif is preserved in all forms of scd23 that possess cytokine activity , including the 16 kda form . the absence of αvβ5 from peripheral b cells indicates the integrin has no role as a cd23 receptor in these cells , and that its function ( s ) is limited to the interaction of b cell precursors with bone marrow stroma . the expression of αvβ5 on a subset of cd19 + / cd9 + / cd10 + bone marrow - derived cells , and on b cell all cells ( including cd10 − leukaemias ), indicates that αvβ5 is expressed from a very early stage in b lymphopoiesis . the large population of αvβ5 + non - adherent marrow cells that is cd19 − is likely to include precursors of t and myelomonocytic cells , since both t - all and aml cells are also αvβ5 + ; thus , expression of αvβ5 may also be functionally important for haematopoietic cells other than b cells . surprisingly , since elevated plasma levels of scd23 in b - cll patients are correlated with poor prognosis 52 , there was no detectable expression of αvβ3 or αvβ5 in any b - cll sample . this argues in favour of active silencing of β5 expression in mature b cells and restriction of αvβ5 pro - survival function to precursor cells . the importance of adhesion interactions between pre - b cells and all cells with stromal cells , for survival of lymphoid cells is widely appreciated , and the vla - 4 / vcam - 1 interaction is particularly prominent . the data of fig1 b indicate that the cd23 - αvβ5 interaction is also critical for promoting growth of sms - sb cells on cd23 + stromal cells in vitro . thus , in the well - described interactions of all blasts and pre - b - like cell lines with stromal elements 53 - 55 , in an environment where all αv ligands ( fn , vn , 56 and cd23 57 ) are present , the availability of ligand - selective responses , mediated via two distinct binding sites on the same integrin , may be important . if engagement of the rgd - binding site was linked to cell survival , then vn and fn in the bone marrow would sustain growth of precursor cells in a non - selective manner ; this could preclude elimination of precursors with non - productive rearrangements of antigen receptor genes or malignant precursors . however , the αvβ5 integrin may allow b cell precursors to adhere to the stromal matrix via vn in an interaction that is neutral with respect to cell survival . signals for inhibition of apoptosis and promotion of cell growth would then be delivered via the second , non - rgd binding site on the αvβ5 integrin . whether αvβ5 integrin delivers a growth - sustaining signal to cell types other than lymphoid precursors remains to be established . this study demonstrates a new role for cd23 , regulation of human b cell precursor growth , and defines a further cd23 receptor , the αvβ5 integrin , and its point of contact on the cd23 protein . expression of the integrin on b cell precursors and all cells suggests that αvβ5 may have a role in sustaining normal and leukaemic b cell growth . the data demonstrate that survival signalling via the integrin is both ligand - selective and mediated via a structure on the integrin distinct from the rgd - binding site that recognises a basic domain on cd23 . this study underscores the complex roles of cd23 in regulating human b cell function and suggests that the distinct biological functions of cd23 are programmed by discrete structural motifs on the protein . a high - resolution structural model of cd23 will be valuable in elucidating the relationships of such structural motifs . while the invention has been described in conjunction with the exemplary embodiments described above , many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure . accordingly , the exemplary embodiments of the invention set forth are considered to be illustrative and not limiting . various changes to the described embodiments may be made without departing from the spirit and scope of the invention . all documents cited herein are expressly incorporated by reference . 1 . gould , h . j . et al . the biology of ige and the basis of allergic disease . annu . rev . immunol . 21 , 579 - 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