Patent Application: US-12478508-A

Abstract:
the invention relates to a vaccine which comprises at least one antigen and a peptide comprising a sequence r 1 — xzxz n xzx — r 2 , whereby n is a whole number between 3 and 7 , preferably 5 , x is a positively charged natural and / or non - natural amino acid residue , z is an amino acid residue selected from the group consisting of l , v , i , f and / or w , and r 1 and r 2 are selected independently one from the other from the group consisting of — h , — nh 2 , — coch 3 , — coh , a peptide with up to 20 amino acid residues or a peptide reactive group or a peptide linker with or without a peptide ; x — r 2 may also be an amide , ester or thioester of the c - terminal amino acid residue , as well as the use of said peptide for enhancing a patient &# 39 ; s adaptive immune response to an antigen .

Description:
transloading murine macrophages with a synthetic antimicrobial peptide as “ carrier peptide ” to test if the ( synthetic antimicrobial ) peptide klklllllklk ( seq id no : 1 ) is able to function as “ carrier - peptide ” for antigens , to transload apcs in vitro , which means enhancing the antigen uptake into apcs , a fluorescently labelled peptide was used as antigenic peptide . it was mixed with diverse concentrations of klklllllklk ( seq id no : 1 ) and other previously described “ carrier - peptides ” as indicated . to compare the efficiency of peptide delivery of these diverse “ carrier - peptides ”, the amount of peptide uptake into apcs was monitored by incubating p388d1 cells ( murine monocyte - macrophage antigen presenting cell line ; purchased from atcc ( tib - 63 )) for 1 h at 37 ° c . with a constant amount of fluorescein - tagged peptide alone or in combination with diverse “ carrier - peptides ” at concentrations indicated . before analysing the cells by flow cytometry , the cells were washed extensively to remove free peptide . the relative amount of fluorescein - tagged peptide taken up by the cells was measured by flow cytometry . the antigenic peptide used is an influenza - haemagglutinin - derived mhc class i ( kd ) binding peptide ( buschle , m . ( 1997 )). 2 μg of this fluorescein - tagged antigenic peptide ( lfeaiegfi ) ( seq id no : 22 ) were mixed with 3 different amounts of each carrier peptide tested at concentrations representing 101 . 7 , 50 . 9 and 5 . 09 nmol positive charges . ( fig1 shows the fold increase in enhanced peptide uptake compared to peptide alone ): ( 1 )+ poly - l - arginine ( pr 60 ; 60 mer ) ( 2 )+ murine cathelicidin - derived antimicrobial peptide ( mcramp ); seq id . no . 2 ( 3 )+ ll - 37 ; seq id . no . 3 ( 4 )+ l - indolicidin ; seq id . no 4 ( 5 )+ klklllllklk ( free c - terminus ); seq id . no . 1 ( 6 )+ linear bovine dodecapeptide ; seq id . no . 5 ( 7 )+ cyclized bovine dodecapeptide whereas fluorescence is known to be sparse in cells treated with peptide alone ( as shown previously ), intense fluorescence of “ transloaded ” cells was especially found in cells which were transloaded with the ( synthetic antimicrobial ) peptide klklllllklk ( seq id no : 1 ) as “ carrier peptide ”, indicating that it is able to pulse apcs with an antigenic peptide very efficiently . transloading murine macrophages with diverse synthetic antimicrobial peptides as “ carrier peptides ” diverse synthetic antimicrobial peptides comprising peptide a sequences were tested to function as “ carrier - peptide ” for antigens , to transload apcs in vitro , which means enhancing the antigen uptake into apcs . for that purpose , a fluorescent labeled peptide was used as antigenic peptide . it was mixed with diverse concentrations of peptides comprising peptide a sequences and other previously described “ carrier - peptides ” as indicated . to compare the efficiency of peptide delivery of these diverse “ carrier - peptides ”, the amount of peptide uptake into apcs was monitored by incubating p388d1 cells ( murine monocyte - macrophage antigen presenting cell line ; purchased from atcc ( tib - 63 ) for 1 h at 37 ° c . with a constant amount of fluorescein - tagged peptide alone or in combination with diverse “ carrier - peptides ” at concentrations indicated . before analysing the cells by flow cytometry , the cells were washed extensively to remove free peptide . the relative amount of fluorescein - tagged peptide taken up by the cells was measured by flow cytometry . the antigenic peptide used is an influenza - haemagglutinin - derived mhc class i ( kd ) binding peptide ( buschle , m . ( 1997 )). 3 μg of this fluorescein - tagged antigenic peptide ( lfeaiegfi ) ( seq id no : 22 ) were mixed with 3 different amounts of each carrier peptide tested at concentrations representing 101 . 7 , 50 . 9 , and 5 . 09 nmol positive charges . ( fig2 shows the fold increase in enhanced peptide uptake compared to peptide alone ): ( 1 ) poly - l - arginine ( 60 mer ) ( 2 ) hp ( 2 - 20 ), a cecropin - like antibacterial peptide derived from the ribosomal protein l1 of helicobacter pylori ; seq id . no : 6 ( 3 ) lalf - peptide : seq id no : 7 ( 4 ) murine cathelicidine - derived antimicrobial peptide ; seq id no : 2 ( 5 ) kakaaaaakak - nh 2 ; seq id . no : 8 ( 6 ) kgkgggggkgk - nh 2 ; seq id . no : 9 ( 7 ) ktktttttktk - nh 2 ; seq id . no : 10 ( 8 ) klklvifwklk - nh 2 ; seq id . no : 11 ( 9 ) kvkvvvvvkvk - nh 2 ; seq id . no : 12 ( 10 ) kwkwwwwwkwk - nh 2 ; seq id . no : 13 ( 11 ) kfkfffffkfk - nh 2 ; seq id . no : 14 ( 12 ) rlklllllklr - nh 2 ; seq id . no : 15 ( 13 ) rlrllllltrlr - nh 2 ; seq id . no : 16 ( 14 ) klklllllklk - nh 2 ; seq id . no : 17 ( 15 ) klklllllklk - cooh ( free c - terminus ); seq id . no . 1 whereas fluorescence is known to be sparse in cells treated with peptide alone ( as shown previously ), intense fluorescence of “ transloaded ” cells was especially found in cells which were transloaded with the peptide comprising a peptide a sequence ( including the above mentioned preferred embodiments ) as “ carrier peptide ”, indicating that the peptides according to the present invention are able to pulse apcs with an antigenic peptide very efficiently . testing the ability to enhance the induction of peptide - specific t cell responses in vivo for testing the ability of the ( synthetic antimicrobial ) peptide klklllllklk ( seq id no : 1 ) to enhance the induction of peptide - specific t cell responses in vivo , groups of 4 mice ( c57bl / 6 , female , 8 weeks of age , h - 2b ) were injected subcutaneously into the flank 3 times ( days 0 , 28 , and 56 ), with an antigenic melanoma peptide ( 100 μg ) derived from trp - 2 ( mouse tyrosinase related protein - 2 ) alone or in combination with either poly - larginine or the ( synthetic antimicrobial ) peptide klklll . lklk ( seq id no : 1 ) as “ carrier peptide ”. the amounts of the ( synthetic antimicrobial ) peptide klklllllklk ( seq id no : 1 ) used represent four different amounts at concentrations representing the equal amount ( 100 μg ) of poly - l - arginine in terms of μg , the equal ( 168 μg ), the double ( 336 μg ) and the triple ( 504 μg ) amount of poly - l - arginine in terms of positive charges . the groups of mice were injected as follows ( amounts indicated / per mouse ). ( 1 ) 100 μg peptide ( 2 ) 100 μg peptide + 100 μg poly - l - arginine ( pr 60 ) ( 3 ) 100 μg peptide + 100 μg klklllllklk ( seq id no : 1 ) ( 4 ) 100 μg peptide + 168 μg klklllllklk ( seq id no : 1 ) ( 5 ) 100 μg peptide + 336 μg klklllllklk ( seq id no : 1 ) ( 6 ) 100 μg peptide + 504 μg klklllllklk ( seq id no : 1 ) 12 days after the 3 rd vaccination , draining ( inguinal ) lymph nodes were removed and lymph node cells ( fig3 ) were activated ex vivo with trp - 2 - derived ( mouse tyrosinase related protein - 2 ) peptide to determine ifn - γ - producing specific cells in an elispot assay ( number of ifn - γ - elispots per million lymph node cells ). fig3 shows that injection of mice with peptide plus increasing amounts of klklllllklk ( seq id no : 1 ) resulted in many more ifn - γ - producing specific cells than injection of mice with peptide alone or in combination with poly - l - arginine . it has also been confirmed that the peptide klklllllklk ( seq id no : 1 ) does not elicit ifn - γ - producing peptide - specific t cells ( as confirmed by elispot - assay ), i . e . that only non klkllllklk ( seq id no : 1 ) specific t - cells have been obtained in the present experiments . this example clearly demonstrates that the ( synthetic antimicrobial ) peptide klklllllklk ( seq id no : 1 ) enhances the induction of peptide - specific t cell responses in vivo . in summary , the ( synthetic antimicrobial ) peptide klklllllklk ( seq id no : 1 ) showed a high “ transloading ” and immunostimulating efficiency , indicating that peptides alpha are able to pulse apcs with antigenic peptides in vitro and in vivo very efficiently and are good adjuvants /“ carrier - peptides ” for antigenic peptides in inducing adaptive immune responses . banchereau , et al . 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