Patent Application: US-39916204-A

Abstract:
this invention describes the design and therapeutic use of antisense peptides for immunomodulation . specifically applications include blocking the interaction of immunoglobulin g with fcγ receptors and blocking the interaction of immunoglobulin e with fcεri or iga with fcαri . this invention also describes the application of antisense peptides to target vaccines to fcγr to induce or suppress immunity , and also describes the application of antisense peptide specific for fcγr or fcαri to direct cytotoxic effector cells to target cells or pathogens .

Description:
the present invention refers to peptides whose sequence has been defined in such a way that they are antisense in sequence to the target protein . such peptides have been shown to bind to their target protein . this approach has been applied to the immunoglobulin binding regions of the following receptors : recently the structures of several fcγ receptors complexed with their ligands have been described ( garman et al ., 2000 , maxwell et al ., 1999 ; sondermann et al ., 1999 ; sondermann et al ., 2000 ). these show similar structure and mutational studies have implicated a number of regions the b / c , c ′/ e , f / g and cc ′ loops of ec2 and the ec1 - ec2 linker region as important for binding to the igg molecule . the ec1 domain of fcαri has been implicated as important for binding iga ( wines et al . 1999 ). antisense peptides were designed to the binding regions of fcγri , fcγriia , fcγriib , fcγriii , fcεri and fcαri on the basis that a peptide designed from the antisense strand of a receptor will bind to its corresponding sense peptide . the peptides of the invention are of an integral number of amino acids in the range of 5 to 20 amino acid . describes the design of antisense peptides to the loop regions of fcγri and their use as inhibitors of fcγri activity . peptides of 6 amino acids in length were designed to be antisense in sequence to the corresponding loop region of fcγri . peptides could , however , be any length ranging from 5 up to 20 amino acids in length . * numbers in brackets refer to the amino acid position in the full - length protein . two separate assays were used to assess the binding and biological activity of these peptides . rosetting is a measure of the ability of sheep red blood cells ( sbrcs ) to attach to the surface of cos - 7 cells . binding could be via the fcγri or other cell surface proteins . thus the inhibition of rosetting by antisense peptides designed to fcγri shows inhibition of binding only and not the ability of a peptide to block phagocytosis that is triggered by a specific interaction of iag with its receptor . the phagocytosis / rosetting assay described can be used to assess the ability of antisense peptides to specifically block the interaction of fcγri expressed on the surface of transfected cos 7 cells with igg expressed on the surface of srbcs . following binding srbcs are opsonized and phagocytosed by the cos 7 cells only through binding to fcγri . any inhibition of phagocytosis by an antisense peptide must therefore be via a specific interaction of the antisense peptide with its receptor . both phagocytosis and rosetting require the expression of fcγri on the surface of cos 7 cells as described below . cos - 7 cells were maintained at 10 5 cells / ml . for transfection cells were seeded at 3 - 4 × 10 5 cells / ml in growth medium and incubated overnight in 3 ml / 60 mm petri dishes . cells were transfected using the deae - dextran method with 10 ug of a fusion construct fcγri - ii dna ( comprising the extracellular domain of ri and the intracellular itam containing domain of rii ) and allowed to recover at 37 ° c . overnight . cells were then detached from the plates , resuspended at 10 5 cells / ml and plated at 1 ml / well on coverslips in 24 well plates . the cells were left to re - adhere overnight . in order for srbcs to adhere to and subsequently be phagocytosed by fcγri expressing cos 7 cells they require surface - bound igg1 ( opsonization ). srbcs were resuspended at 10 5 / ml in pbs ± 3 ul / ml of rabbit ( igg1 fraction ) anti — srbc stroma antibody ( sigma ). they were then rolled for 60 minutes at 4 ° c . after 60 minutes they were washed three times with pbs and resuspended at 4 × 10 6 and 20 × 10 6 in pbs . unopsonized ( i . e . no surface bound igg1 ) were used as controls . unopsonized cells will not adhere to fcγri expressing cos 7 cells . peptides were dissolved in dh 2 o and made up to a concentration of 20 ug / ml and 200 ug / ml in pbs . the medium was removed from each plate of cells , and 0 . 25 ml of opsonised or unopsonised , washed srbcs were added to the wells of cells , also 250 μl / well of peptides 1 - 3 were added to their respective wells . plates were incubated for 2½ hours at 37 ° c , and were washed 3 times with warm pbs . cells were fixed for 5 minutes . positive cells were identified by light microscopy . rosetted cells ( i . e . cos7 cells with more than one srbc attached to their surface ) were scored ++ ( approximately 75 % of cells totally surrounded by srbcs ),+( approximately 40 % of cells with many srbcs ),±( approximately 10 % of cells with few srbcs attached ) and —( no attachment of srbcs ). the medium was removed from each plate of cells , and 0 . 25 ml of opsonised or unopsonised , washed srbcs were added to the wells of cells , also 250 μl / well of peptides 1 - 3 were added to their respective wells . plates were incubated for 2½ hours at 37 ° c ., and were washed 3 times with warm pbs . to assess phagocytosis any surface bound srbcs were first lysed with hypotonic shock buffer ( 150 ml dh20 , 1 ml pbs and 50 μl of conc hcl for exactly 2 mins ). cells were then fixed for 5 mins and stained for 15 minutes at room temperature with o - dianisidine stain : “ stain and go ”: 8 . 1 ml of 0 . 2m nah 2 po 4 [ 1 . 2 g / 50 ml ], 1 . 9 ml of 0 . 2m na 2 hpo 4 [ 1 . 42 g / 50 ml ], 12 mls hanks buffer ( sigma ), 1 ml o - dianisidine hcl ( at 1 . 25 mg / ml h 2 o , sigma ) and 15 μl of h 2 o 2 ( 30 %). the cell - coated coverslips were removed and mounted on slides . positive cells i . e . cos 7 cells containing srbcs were identified by light microscopy . cells were counted within several randomly chosen fields of view and the number of cos 7 cells containing phagocytosed srbcs expressed as a percentage of the total number of cells . the number of srbcs was counted and expressed as percentage positive cells or phagocytic index ( the number of phagocytosed srbcs / 100 cos 7 cells ). the peptides listed in example 1 ( seq id nos 1 - 3 ) have been shown to inhibit both rosetting and phagocytosis carried out as described . [ 0077 ] fig1 shows an example of the inhibition of phagocytosis by antisense peptides 1 - 3 at a concentration of 100 μg / ml . a similar effect was observed with antisense peptides 1 - 3 at a concentration of 10 μg / ml . given the high degree of sequence similarity between the fc receptors as illustrated in sondermann et al ., 2000 , it is predicted that antisense peptides designed to the binding regions of fcγriia , fcγriib , fcγriii , fcεri and fcαr would show biological activity in similar or related bioassays . antisense peptides have been designed to these regions and are listed in the sequence listing ( seq id nos 4 - 17 ). the c ′ e loop of fcγriia has a polymorphism which has been shown to be important for igg2 binding ( h167 ) and igg1 binding ( r167 ) ( clark et al ., 1989 ). antisense peptides have been designed to both forms of the protein ( seq id nos 5 and 6 respectively ). nb where the antisense peptide sequence contains a stop codon this has been replaced with a glycine residue . chang , t . w . 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( 2000 ). the 3 . 2 - a crystal structure of the human igg1 fc fragment - fc gammariii complex . nature 406 ( 6793 ): 267 - 73 . van spriel , a . b ., van den herik - oudijk , i . e . et al . ( 1999 ) effective phagocytosis and killing of candida albicans via targeting fcgamma ri ( cd64 ) or fcalphari ( cd89 ) on neutrophils . j infect dis 179 ( 3 ): 661 - 669 . wines , b . d ., hulett , m . d ., jamieson , g . p ., trist , h . m ., spratt , j . m . & amp ; hogarth , p . m . ( 1999 ). identification of residues in the first domain of human fcα receptor essential for interaction with iga . j immunol 162 : 2146 - 2153 . 5 ′ gga aag cat cgc tac aca ( seq id no . 25 ) 5 ′ tgt gta gcg atg ctt tcc ( seq id no . 26 )