Patent Application: US-73359308-A

Abstract:
peptide - lipid constructs of the structure l - s - f are disclosed , where f is a peptide , s is a spacer covalently linking f to l via an oligomer of ethylene glycol , and l is a diacyl - or dialkyl - glycerolipid . the spacer ideally has 6 to 14 ethylene glycol repeats , corresponding to peg with a molecular weight of approximately 250 to 600 . also disclosed is a method of detecting reactive antibodies in serum by contacting serum with cells modified to incorporate a peptide - lipid construct , where the peptide is an epitope of the antibody , and determining the degree of aggluthination of the cells .

Description:
in general terms the invention provides peptide - lipid constructs of the structure ( l - s -) i f (- s - l ) j where : f is a peptide ; s is a spacer covalently linking f to l ; l is a lipid selected from the group consisting of diacyl - and dialkyl - glycerolipids , including glycerophospholipids ; i and j are independently 0 or 1 ; and the use of these peptide - lipid constructs in diagnostic and therapeutic applications . where i is 0 and j is 1 the peptide - lipid constructs are of the structure : where i is 1 and j is 0 the peptide - lipid constructs are of the structure : where s is linked to f via the amino terminus of the peptide the construct is represented by the structure or substructure l - s - f . where s is linked to f via the carboxyl terminus of the peptide the construct is represented by the structure or substructure f - s - l . where s is linked to f via a sulphide bond formed via the sulfhydryl group of a cys residue of the peptide the residue is identified with an underscore ( cys ). where s is linked to f via a sulphide bond formed with one or more cys residues of the peptide , the representation of the peptide - lipid construct by the structure l - s - f - s - l , l - s - f or f - s - l is not intended to imply the sulphide bond is formed exclusively with terminal cys residues . the use of the peptide - lipid constructs in diagnostic applications is illustrated with reference to the use of constructs including the substructure : where m is a monovalent cation ( m + ), n is 6 to 14 , * is other than h , and the peptide is selected from the group of peptides consisting of peptides included in the list of peptides provided on the following pages where z is an integer from 0 to 6 . the amino acid residues of peptides are identified according to table 3 of appendix 2 of annex c of the administrative instructions under the patent cooperation treaty dated 7 feb . 2007 and in accordance with the convention : there is a need for inexpensive and low level sensitivity test systems for a range of diagnostic markers in donated blood , in transfusion recipients , or in antenatal patients ( where the unborn child may be at risk of haemolytic disease ), e . g . syphilis markers and markers of the mns blood group system . a particular advantage provided by the invention is the opportunity to employ established blood typing platforms to detect a range of peptide antigen - antibody interactions . the capital costs associated with establishing a new diagnostic assay may therefore be avoided . some clinically significant blood group antigens are rare ( or rare in some populations ). for example mutations of the mns blood group system resulting in miltenberger antigens are rare in europeans , but common in asians . being able to create antibody detection and identification panels requires that these antigens be present on the diagnostic screening cells . obtaining cells suitable for antibody screening / identification having rare antigens is therefore problematic . being able to add to cells rare antigens prepared exogenously is therefore a major advantage . according to the method of the invention epitope containing peptide sequences for a range of diagnostic markers , such as specific reacting antibodies , can be localized to the surface of red blood cells ( rbcs ). these modified rbcs may then be used on existing blood typing platforms to detect blood antobodies or pathologies . although the invention is illustrated with reference to the modification of red blood cells and embryos the outer surface of other cells and multi - cellular structures is contemplated . however , red blood cells are preferred for use in diagnostic assays because of the facility with which these modified cells could be used in blood typing laboratories . the level of peptide - lipid construct incorporated into the cell membrane of red blood cells is controlled by the concentration of the construct in the dispersion contacted with the suspension . the presence of diagnostic markers may then be assessed by agglutination whether direct ( induced by centrifugation of cells ) or indirect ( induced by adding an antibody directed against the immunoglobulins of the subject ). other methods of assessment may be employed including , for example , rosetting ( indiveri et al 1979 ) and enzyme linked immunosorbant assays ( elisa ). in contrast with the preparation of constructs where the function ( f ) is a carbohydrate , the preparation of constructs where f is a peptide presents a combination of technical difficulties . firstly , it is desirable for the peptide ( f ) ligated to the l - s or s - l moiety to be dispersible in the solvents used for the ligation chemistry . overcoming this difficulty may require the selection of a proximal terminal sequence ( pts ) to promote solubility without modifying the desired biological properties of the construct . secondly , it is r for the construct ( l - s - f - s - l , l - s - f or f - s - l ) to be dispersible in water , or at least a biocompatible medium such as buffered saline , according to the requirements of the proposed application ( i . e . it is desirable for the construct to be “ water soluble ” as defined herein ). overcoming this difficulty requires the selection of a spacer ( s ) to promote solubility of the construct . thirdly , where the proposed application is the modification of cells such as red blood cells ( rbcs ) for use in diagnostic applications , including use as quality controls in blood group typing or detection of diagnostic antibodies present in patient serum , it is required for the construct to be dispersible without participating in antigen - antibody cross reactivity not specific to the diagnostic marker . satisfying this requirement requires the identification of suitable structural motifs for the spacer ( s ) and proximal terminal sequence ( pts ) when the latter is present , or the development of sample preparation procedures that neutralize or at least substantially mitigate the undesired cross reactivity and likelihood of false positives . it should also be noted that where the application is for use in the modification of the surface of cells or multi - cellular structures ( e . g . an embryo ) with a view to promoting the association of the modified cell or modified multi - cellular structure with a target surface ( e . g . the endometrium ) exposing the cell or multi - cellular structure to solvents is incompatible with maintaining the cells or multicellular structures in a viable state . the ability to localise peptides to the surface of cells or multi - cellular structures via a residue proximal to either the n - or c - terminus of the peptide may also allow the naturally occurring configuration of the peptide sequence relative to the cell surface to be approximated . the presentation of the peptide sequence in the tertiary ( or quaternary ) structure of the parent polypeptide ( or protein ) may therefore be mimicked . although not demonstrated here it is contemplated that peptides may be localised to the surface of cells via multiple residues . for example , where both a residue proximal to the amino terminus and a residue proximal to the carboxyl terminus are used to localize the peptide , a “ looped ” configuration of the peptide may be promoted at the surface . the use of polyethylene glycol ( peg ) as a spacer to promote solubility is known . however , polymers of peg may interfere with the expression and function of the peptide at the surface . in the peptide - lipid constructs of the invention an oligomer of ethylene glycol ( 6 to 14 mer ) is selected as a component ( s 1 ) of the spacer ( s ) linking the lipid ( l ) and peptide ( f ). oligomers of ethylene glycol impart less solubility to peptide - lipid constructs of the structure l - s - f than polymers of peg . the difficulty referred to above therefore arises when it is desired to obtain peptide - lipid constructs that are dispersible in biocompatible solvents and can be used in methods of effecting qualitative and quantitative changes in the levels of peptide expressed at the surface of cells and multi - cellular structures . the properties of the peptide - lipid constructs must be such that they can be readily dispersed in biologically compatible media in the absence of solvents or detergents , but incorporate into the lipid bilayer of a membrane when a solution of the construct is contacted with a suspension of cells or multi - cellular structures . peptide - lipid constructs with these potentially conflicting properties are prepared by adopting the combination of structural motifs described here . the preparation of the peptide - lipid constructs where s is linked to f via a sulphide bond formed with a terminal cys residue of the peptide at the carboxy - terminus of the peptide is preferred as the peptide is less prone to oxidation . adopting the combinations of structural motifs in accordance with the description provided here a range of peptides may be prepared as peptide - lipid constructs for use in methods of effecting qualitative and quantitative changes in the levels of peptide expressed at the surface of cells and multi - cellular structures . it will be understood that for a non - specific interaction , such as the interaction between diacyl - or dialkyl - glycerolipids or glycerophospholipids and a membrane , structural and stereo - isomers of naturally occurring lipids can be functionally equivalent . for example , it is contemplated that diacylglycerol 2 - phosphate could be substituted for phosphatidate ( diacylglycerol 3 - phosphate ). furthermore it is contemplated that the absolute configuration of phosphatidate can be either r or s . dope - peg 6 - nh 2 ( l - s 1 - nh 2 ) ( 7 , 800 mg ) was prepared by the method of scheme 1 . to a stirred solution of dope ( 5 ) ( 36 mg , 0 . 0484 mmol ) in dry chcl 3 ( 3 ml ) a solution of fmoc - peg - nos ( 4 ) ( 237 mg , 0 . 0697 mmol ( containing about 80 % of active n - oxysuccinimide ester )) in dry chcl 3 ( 1 ml ) and et 3 nh ( 30 ml ) was added . the solution was stirred for 15 h at 20 ° c ., then et 3 nh ( 3 ml ) was added , and the mixture was maintained for at 8 h at 20 ° c . the solution was then diluted with toluene ( 10 ml ), evaporated under reduced pressure ( 10 to 15 torr ) and dried under vacuum . the crude residue was dissolved in h 2 o / meoh / acoh mixture ( 10 : 5 : 1 ( v / v / v ), 3 ml ) and the solution was slowly applied to a reverse phase c 16 column ( 15 ml , water ). salts , n - hydroxysuccinimide and h 2 n - peg - dope ( 7 ) were eluted from the column with meoh / h 2 o 1 : 2 ( v / v ) ( 30 ml ), 1 : 1 ( v / v ) ( 15 ml ) and 2 : 1 ( v / v ) ( 15 ml ). target h 2 n - peg - dope ( 7 ) was eluted from the column with meoh ( 30 ml ) and then with meoh to meoh / chcl 3 mixtures ( 4 : 1 ( v / v ), 3 : 1 ( v / v ), 2 : 1 ( v / v ) and 1 : 1 ( v / v ); 30 ml each ). fractions containing h 2 n - peg - dope ( 7 ) were combined , evaporated under reduced pressure ( 10 to 15 torr ) and dried under vacuum . the residue obtained as a thin film on the flask walls was extracted twice with hexane ( 2 × 5 ml ) and dried under vacuum to yield 143 mg of h 2 n - peg - dope ( 7 ) ( 78 % on dope ) as a white solid . tlc : r f = 0 . 62 ( ethanol / water / pyridine / acoh ; 3 : 1 : 1 : 1 ( v / v / v / v )). 1 h - nmr ( 500 mhz , cd 3 od , 30 ° c . ): δ = 5 . 541 ( m , 4h ; 2 — c h ═ c h —), 5 . 416 ( m , 1h ; och 2 c h ch 2 o ), 4 . 624 ( dd , j = 12 hz , j = 3 . 2 hz , 1h ; co — oc h chch 2 ), 4 . 373 ( dd , j = 12 hz , j = 6 . 6 hz , 1h ; co — oc h chch 2 ), 4 . 195 ( t , j = 5 . 6 hz , 2h ; poc h 2 ch 2 n ), 4 . 117 ( m , 2h ; poc h chch 2 ), 3 . 968 ( m , 4h ; oc h 2 ch 2 o , oc h 2 ch 2 n ), 3 . 932 ( t , j = 6 . 2 hz , 2h ; oc h 2 ch 2 co ), 3 . 827 ( m , 272 h ; (— oc h 2 ch 2 —) n , n = 68 ), 3 . 683 ( m , 2h ; oc h 2 oh 2 o ), 3 . 622 ( t , j = 5 . 6 hz , 2h ; och 2 c h 2 n ), 3 . 397 ( t , j = 5 . 0 hz , 2h ; och 2 ch 2 n ), 2 . 678 ( t , j = 6 . 2 hz , 2h ; och 2 c h 2 co ), 2 . 519 ( m , 4h ; 2 ch 2 co ), 2 . 228 ( m , 8h ; 2 ch 2 ch ═ chch 2 ), 1 . 801 ( m , 4h ; 2 ch 2 ch 2 co ), 1 . 508 ( m , 40h ; — ch 2 —), 1 . 096 (˜ t , 6h ; 2 ch 3 ) ppm . maleimido - derivatives of dope - peg 6 - nh 2 were used for the preparation of peptide - lipid constructs ( l - s - f ) by the method of scheme 2 via the maleimide - thiol michael addition reaction . synthesis via the maleimido - derivatives of dope - peg 6 - nh 2 has particular advantages over synthesis via iodoacetate derivatives as difficulties and low yields as a consequence of oxidation of the sulfhydryl residues of the peptide and subsequent dimer formation . reducing agents ( e . g . tertiary phosphines ) may be used during conjugation . maleimido - derivatives were synthesized with 65 to 70 % yields starting from n - oxysuccinimid esters of maleimidobutyric and maleimidopropionic acids ( 8a , 8b ). an unexpected complication arose due to the presence of excess bu 3 p which appeared to be highly reactive towards the maleimide function . phosphine was therefore used only in sub - equivalent amounts ( 0 . 1 to 0 . 2 equivalents ). trifluoroethanol used as a co - solvent in the preparation of 10bc where the peptide was glnthrasnaspmethislysargaspthrtyrglyserglyserglycys appeared to be highly efficient for solubilization of both reactants . however , the solvent also caused unwanted acidification of the reaction medium which may inhibit the michael reaction . the isolated yield of 10bc in this experiment was ˜ 25 %. preparation of 10ac where the peptide was glnthrasnaspmethislysargaspthrtyrglyserglyserglycys ( dope - peg 6 - βala - mal - 3mutm ( m3 )) carried out using dmso as co - solvent was more successful and provided a 43 % yield . the same solvent strategy in the preparation of 10bc where the peptide was glnthrasnasplyshislysargaspthrtyrserserglnthrasnaspmethislysargaspthrtyralaalaalaalacys ( dope - peg 6 - βala - mal - pts - milt ( k , m )) failed because the peptide supplied appeared to be very acidic and caused solubilization problems . the yield of 10bc in this experiment was only 23 % and about half of the peptide was recovered . molecular weights for the peptide lipid constructs were determined to be : as expected for peptides bearing the glutamine residue at the n - terminus , all preparations contain variable amounts of related pyroglytamyl derivatives , m - 17 in ms , due to loss of nh 3 . the formation of related pyroglytamyl derivatives was mitigated in peptides with n - terminal ser residues . the use of the peptide - lipid constructs in methods for effecting qualitative and quantitative changes in the levels of peptide expressed at the surface of cells and multi - cellular structures is illustrated with reference to the serodiagnosis . red blood cells are modified by mixing 1 part by volume of washed packed red blood cells with 1 part by volume of peptide - lipid construct dispersed at a concentration of 10 to 1000 μg / ml in cell media ( celpresol ™). 1 . incubated for 2 hours at 37 ° c . before being washed and suspended in a cell medium for serological analysis at a concentration of 0 . 8 to 3 % ( method 1 ); or 2 . incubated for 3 to 4 hours at room temperature ( circa 25 ° c .) followed by 18 hours at 4 ° c . before being washed and suspended in a cell medium for serological analysis at a concentration of 0 . 8 to 3 % ( method 2 ). 4 . 7 mg of the lipid - peptide construct dope - peg 6 - βala - mal - milt ( k ) ( m00 ) was reconstituted in 0 . 47 ml of celpresol ™ by sonicating for 10 min and allowing to stand for 1 hour to provide a clear 10 mg / ml stock solution . the stock solution was diluted two - fold to provide a solution of 5 mg / ml and a dilution series then prepared for the peptide - lipid construct at the following concentrations : 1 mg / ml ( 1 : 5 dilution in celpresol ™) 0 . 5 mg / ml ( 1 : 10 dilution in celpresol ™) 0 . 25 mg / ml ( 1 : 20 dilution in celpresol ™) 200 μl of miltenberger negative red blood cells ( milt − rbcs ) were washed two times with pbs and one time with celpresol ™. 40 μl of a washed packed volume of milt − rbcs were mixed with 40 μl of a dilution of the peptide - lipid construct and incubated for 2 hours at 37 ° c . the modified rbcs were then washed with celpresol ™ and stored in celpresol ™ until used in tube serology testing ( 3 days and 24 days ). serological reactions are graded or scored by either of two established systems ( 0 or ‘−’= no agglutination , 1 + or 3 = very weak agglutination , 2 + or 5 = weak agglutination , 3 + or 8 = moderate strong agglutination , 4 + or 10 / 12 = strong agglutination ) serological platforms used are tube ( addition of reagents and reactants into plastic or glass serology tubes and after appropriate incubations , washing and centrifugation observing reactions macroscopically by eye and a 10 × magnification eyepiece and scoring ) and biovue ™ ( addition of reactants into cassettes containing beads ( including some reactants ) and after appropriate incubations and centrifugation observing the reaction patterns traped within the gel matrix ). biovue is the serological column agglutination platform of ortho - clinical diagnostics . serum samples were available from 47 blood donors of negative antibody screen status . these samples were designated “ negative samples ”, but not determined not to have anti - miltenberger antibodies ). three serum samples known to have miltenberger related antibodies t217 , t6025 , t5896 . these samples were designated “ positive samples ”, but not determined to have anti - antibodies against the peptide of the peptide of the construct designated dope - peg 6 - βala - mal - milt ( k ) ( m00 ). a suspension of 3 % modified rbcs was prepared in pbs and 30 μl of the suspension mixed with 30 μl serum sample . the mixtures were then incubated for 45 min at 37 ° c . following incubation the rbcs were centrifuged for 10 s in an immufuge ™ ( setting : “ high ”) and observed for agglutination before being washed 3 times with pbs . after washing one drop of epiclone ™ anti - human globulin ( ahg ) was added and the tubes then centrifuged for 10 s in an immufuge ™ ( setting : “ high ”). tubes were then read and serology scores recorded . score results show sample reactivity by the anti - human globulin test , 1 + = weak , 2 + = medium , 3 + = medium / strong , 4 + = strong , — means sample is unreactive . rbcs were modified with the peptide - lipid construct at the concentrations indicated . sera were tested against modified rbcs following 3 days and 24 days storage . ( n . t .— not tested ). score results show sample reactivity by the anti - human globulin test , 1 + = weak , 2 + = medium , 3 + = medium / strong , 4 + = strong , — means sample is unreactive . rbcs were modified with the peptide - lipid construct at the concentrations indicated . sera were tested against modified rbcs following 3 days and 24 days storage . a 5 mg / ml stock solution of the peptide glnthrasnasplyshislysargaspthrtyrcys dissolved in celpresol ™ was prepared . a 4 μl ( 20 μg peptide ) volume of the stock solution was added to a 30 μl volume of each serum sample ( test ). a 4 μl volume of celpresol ™ was added to 30 μl of each serum sample ( control ). serum samples ( test and control ) were then incubated at room temperature ( rt ) for 10 min . a 30 μl volume of a 5 % suspension of the modified rbcs was added to each sample and incubated at 37 ° c . for 45 min . the incubated rbcs were then washed 3 times with pbs in an immufuge ™. one drop of epiclone ™ anti - human globulin ( ahg ) reagent was then added to each sample and the tubes centrifuged for 10 s in an immufuge ™ ( setting : “ high ”). tubes were read and serology scores recorded . cys denotes the cysteine residue via the sulfhydryl residue of which the spacer ( s ) is covalently linked to the peptide or pts - peptide ( f ). * where cys is absent the spacer ( s ) is covalently linked to the peptide ( f ) via the terminal amino residue . all peptide - lipid constructs ( f - s - l or l - s - f ) were prepared as the dope ( l ) variant . where false positives were observed these could be substantially eliminated by pre - treatment of the sample of serum with the peptide of the peptide - lipid constructs ( table 10 and 11 ). sera were neutralized by adding 10 μl of 1 mg / ml solution of peptide to a 50 μl volume of sera and incubating for 30 minutes at 37 ° c . testing was performed using biovue ™ cards . the zona pellucida of day 3 . 5 embryos prepared as microdrops were removed by incubation in 0 . 5 % pronase solution for circa 5 minutes at 37 ° c . the zona pellucida removed embryos were transferred to microdrops containing media alone and contacted with a dispersion of the peptide - lipid construct designated dope - peg6 - βala - mal - pts - milt ( k ) ( m2 ) at a concentration of 1 mg / ml for 2 hours . the dispersion of the peptide - lipid construct contained azide as an anti - microbial agent . the incubated embryos were washed four times in handling media and transferred to microdrops containing the gam monoclonal antibody ( see table 8 ) and incubated at 37 ° c . for 40 min . the embryos were then washed four times in handling media and transferred to microdrops containing secondary antibody ( fitc anti - mouse ) at a 1 : 50 dilution . the microdrops were incubated at room temperature in the dark for 30 minutes before being washed four times in handling media , placed on microscope slides , and overlaid with mineral oil . the embryos were visualized using an olympus ™ bx51 fluorescent microscope at 200 × magnification with wib filter 550 nm emission wavelength . the scale used for grading fluorescence was 0 to 4 +, where 0 is no fluorescence and 4 + is very bright fluorescence . the mean fluorescence of the modified embryos was 2 + versus zero for unmodified embryos . the grading of fluorescence is recorded in table 12 . a mean fluorescence of 2 + was observed for the zona pellucida removed embryos modified to incorporate the peptide - lipid construct designated dope - peg6 - βala - mal - pts - milt ( k ) ( m2 ). no fluorescence was observed for control embryos . the de - compaction of treated embryos was attributed to the presence of azide in the dispersion of the peptide - lipid construct . although the invention has been described by way of exemplifying embodiments it should be appreciated that variations and modifications may be made without departing from the scope of the invention . furthermore where known equivalents exist to specific features , such equivalents are incorporated as if specifically referred to in this specification . blume et al ( 1993 ) specific targeting with poly ( thylene glycol )- modified liposomes coupling of homing devices to the ends of the polymeric chains combines effective target binding with long circulation times biochimica et biophysica acto , 1149 : 180 - 184 chung et al ( 2004 ) casual cell surface remodeling using biocompatible lipid - poly ( ethylene glycol )( n ): development of stealth cells and monitoring of cell membrane behaviour in serum - 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