Patent Application: US-201715472043-A

Abstract:
the invention provides methods for identifying a hla - b * 0702 - restricted cryptic epitope in an antigen , as well as methods for increasing the immunogenicity of hla - b * 0702 - restricted cryptic epitopes . the hla - b * 0702 - restricted cryptic epitopes and their cognate immunogenic epitopes are useful for stimulating an immune reaction against the cryptic epitopes in a subject . accordingly , the invention further provides pharmaceutical compositions comprising a hla - b * 0702 - restricted cryptic epitope or a cognate immunogenic epitope thereof , and vaccination kits comprising such epitopes . the novel materials of the invention are particularly useful for efficiently treating patients having an hla - b * 0702 phenotype .

Description:
the examples have been performed using the following materials and methods : transgenic mice . the hla - b7 h - 2 class - i knockout mice were previously described ( rohrlich et al ., 2003 ). cells . hla - b * 0702 transfected murine rma - b7 and human t2 - b7 cells were previously described ( rohrlich et al ., 2003 ). cos - 7 and wehi - 164 clone 13 cells were provided by f . jotereau ( inserm 463 , nantes , france ). the hla - b * 0702 positive sk - mes - 1 ( lung cancer ), hbl - 100 ( breast cancer ), and the hla - b * 0702 negative sw - 480 ( colon cancer ) and hss ( myeloma ) cell lines were used as targets of human ctl . all cell lines were grown in fcs 10 % supplemented rpmi1640 culture medium . peptides and plasmids . peptides were synthesized by epytop ( nimes , france ). hla - b * 0702 plasmid was provided by dr . lemonnier ( institut pasteur , paris , france ) ( rohrlich et al ., 2003 ) and tert plasmid was provided by dr weinberg ( mit , boston , mass .) ( meyerson et al , 1997 ). measurement of peptide relative affinity to hla - b * 0702 . the protocol used has been described previously ( rohrlich et al ., 2003 ). briefly , t2 - b7 cells were incubated at 37 ° c . for 16 hours with peptides concentrations ranging from 100 μm to 0 . 1 μm , and then stained with me - 1 monoclonal antibody ( mab ) to quantify the surface expression of hla - b * 0702 . for each peptide concentration , the hla - b * 0702 specific staining was calculated as the percentage of staining obtained with 100 μm of the reference peptide cmv 265 - 274 ( r10v ; rpherngftv , seq id no : 9 ). the relative affinity ( ra ) was determined as : ra =( concentration of each peptide that induces 20 % of hla - b * 0702 - expression / concentration of the reference peptide that induces 20 % of hla - b * 0702 expression ). ctl induction in vivo in hla - b * 0702 transgenic mice . mice were injected subcutaneously with 100 μg of peptide emulsified in incomplete freund &# 39 ; s adjuvant ( ifa ) in the presence of 150 μg of the i - a b restricted hbvcore 128 t helper epitope ( tppayrppnapil , seq id no : 10 ). after 11 days , 5 × 10 7 spleen cells were stimulated in vitro with peptide ( 10 μm ). on day 6 of culture , the bulk responder populations were tested for specific cytotoxicity . peptide processing assay on cos - 7 transfected cells . 2 . 2 × 10 4 simian cos - 7 cells were plated in flat - bottomed 96 - well plates in dmem + 10 % fcs , in triplicate for each condition . eighteen hours later , cells were transfected with 100 ng of each dna plasmid with deae dextran . after 4 hours , pbs + 10 % dmso was added for 2 minutes . transfected cos cells were incubated in dmem + 10 % fcs during 40 hours and then used to stimulate murine ctl in a tnfα secretion assay . tnfα secretion assay . transfected cos - 7 cells at day 4 were suspended in 500 of rpmi + 10 % fcs and used as stimulating cells . 5 × 10 4 murine t cells were then added in 50 μl rpmi 10 % fcs and incubated for 6 hours . each condition was tested in triplicate . 50 μl of the supernatant was collected to measure tnfα . standard dilutions were prepared in 50 μl with final doses of tnfα ranging from 104 to 0 pg / ml . on both the supernatants and the standard dilutions , 3 × 10 4 tnfα sensitive wehi - 164c13 cells in 50 μl were added . they were incubated for 16 h at 37 ° c . inhibition of cell proliferation was evaluated by the mtt colorimetric method ( espevik and nissen - meyer , 1986 ). generation of ctl from human pbmc . pbmc were collected by leukapheresis from healthy hla - b * 0702 volunteers . dendritic cells ( dc ) were produced from adherent cells cultured for seven days ( 2 × 10 6 cells / ml ) in the presence of 500 iu / ml gm - csf and 500 iu / ml il - 4 ( r & amp ; d systems , minneapolis , minn .) in complete medium ( rpmi - 1640 supplemented with 10 % heat inactivated human ab serum , 2 μm l - glutamine and antibiotics ). on day seven , dc were pulsed with 10 μm peptides for 2 hrs ; maturation agents poly i : c ( sigma , oakville , canada ) at 100 ng / ml and anti - cd40 mab ( clone g28 - 5 , atcc , manassas , va .) at 2 μg / ml were added in the culture and dcs were incubated at 37 ° c . overnight or up to 48 hours . mature dc were then irradiated ( 3500 rads ). cd8 + cells were purified by positive selection with cd8 microbeads ( miltenyi biotec , auburn , calif .) according to the manufacturer &# 39 ; s instructions . 2 × 10 5 cd8 + cells + 6 × 10 4 cd8 − cells were stimulated with 2 × 10 4 peptide pulsed dc in complete culture medium supplemented with 1000 iu / ml il - 6 and 5 iu / ml il - 12 ( r & amp ; d systems , minneapolis , minn .) in round - bottomed 96 well plates . from day seven , cultures were weekly restimulated with peptide - loaded dc in the presence of 20 iu / ml il - 2 ( proleukin , chiron corp ., emeryville , calif .) and 10 ng / ml il - 7 ( r & amp ; d systems , minneapolis , minn .). after the third in vitro restimulation , bulk cell cultures were tested for cytotoxicity ( tert 4 ) or for ifnγ intracellular staining ( tert 444a1 ). cytotoxic assay . targets were labelled with 100 μci of cr 51 for 60 min , plated in 96 - well v - bottomed plates ( 3 × 10 3 cell / well in 100 μl of rpmi 1640 medium ) and , when necessary , pulsed with peptides ( 1 μm ) at 37 ° c . for 2 hours . effectors were then added in the wells and incubated at 37 ° c . for 4 hours . percentage of specific lysis was determined as : % lysis =( experimental release − spontaneous release )/( maximal release − spontaneous release )× 100 . ifnγ intracellular staining . t cells ( 10 5 ) were incubated with 2 × 10 5 t2 cells loaded with stimulating peptide in the presence of 20 μg / ml brefeldin - a ( sigma , oakville , canada ). six hours later they were washed , stained with r - phycoerythrin - conjugated anti - cd8 antibody ( caltag laboratories , burlingame , calif ., usa ) in pbs for 25 min at 4 ° c ., washed again , and fixed with 4 % pfa . the cells were then permeabilized with pbs , 0 . 5 % bsa , 0 . 2 % saponin ( sigma , oakville , canada ), and labeled with an allophycocyanin - conjugated anti - ifnγ mab ( pharmingen , mississauga , canada ) for 25 min at 4 ° c . before analysis with a facscalibur ® flow cytometer . eight peptides with the hla - b * 0702 specific anchor motifs , i . e . p2 and preferentially l / v at c - terminal position ( sidney et al ., 1996 ) belonging to hsp70 ( hsp70 115 , hsp70 137 , hsp70 397 ), tert ( tert 4 and tert 444 ), and mage - a ( mage - a 121 . 1 , mage - a 121 . 2 and mage - a 121 . 4 ) antigens were tested for binding to the hla - b * 0702 molecule . only tert 4 bound to hla - b * 0702 with a high affinity , the remaining seven peptides were very weak or non binders ( table ii ). this demonstrates that the presence of anchor motifs is not sufficient to ensure a high binding affinity to hla - b * 0702 . given their low affinity , peptides hsp70 115 , hsp70 137 , hsp70 397 , tert 444 , mage - a 121 . 1 , mage - a 121 . 2 , mage - a 121 . 4 , are considered cryptic peptides . the low affinity hsp 137 , hsp 115 , hsp 397 , tert 444 and the high affinity tert 4 peptides have been tested for their capacity to induce a specific ctl immune response in hla - b * 0702 transgenic mice . only the high affinity tert 4 was immunogenic confirming that immunogenicity of peptides is strongly related to their affinity for hla ( fig1 ). since all these cryptic peptides had favourable primary anchor motifs , enhancement of their affinity is a prerequisite for them to be immunogenic . it required the identification of unfavourable secondary anchor motifs and their substitution with favourable motifs . these substitutions should however preserve the conformation of the peptide segment that interacts with the tcr ( position 4 to position 8 ). the interest was , therefore , focused on secondary anchor positions 1 and 3 : aliphatic amino acids are favourable motifs at position 1 ( sidney , southwood et al ., 1996 ). however , peptides hsp70 115 and hsp70 137 that have a y ( tyrosine ) at position 1 are non binders . moreover , the substitution of the amino acid at position 1 by an a ( alanine ) that is also favourable at this position . ( parker et al , 1994 ) enhances the affinity of the tert 444 but not of the hsp70 115 and the mage - a 121 . 1 peptides ( table ii ). this indicates that the presence of favourable amino acids at position 1 and anchor positions 2 and 9 / 10 cannot ensure by itself a high binding affinity of all peptides . in the other hand , positively charged peptides ( r / h / k ) have been described to be favourable at position 3 ( sidney et al ., 1996 ) and ten out of 26 identified tumor and hiv derived immunogenic peptides have an r / k / h at position 3 ( table iii ). according to all these observations , peptides with the sequence apx 3 x 4 x 5 x 6 x 7 x 8 x 9 x 10 x 11 ( seq id no : 61 ) should have a high affinity for hla - b * 0702 . this is confirmed by results shown in table iv . all eighteen peptides with the above cited sequence had a high affinity and / or were immunogenic in hla - b * 0702 transgenic mice . affinity and immunogenicity of apx 3 x 4 x 5 x 6 x 7 x 8 x 9 x 10 x 11 example 4 : in vivo immunogenicity of peptides with enhanced affinity and recognition of the native counterpart hla - b7 transgenic mice were vaccinated with the selected peptides , and eleven days later , their spleen cells were in vitro stimulated with the peptide . in this context , hsp70 115 , hsp70 397 and tert 444 , were therefore modified at position 1 ( substitution of the amino acid by an a ) and / or position 3 ( substitution of the amino acid by an r ). for peptide hsp70 397 an additional modification at c - terminal position ( substitution of the t by an l ) has been introduced . modified peptides i . e . hsp70 115a1r3 ( seq id no : 55 ), hsp70 397r3l9 ( seq id no : 56 ), tert 444a1 ( seq id no : 5 ) exhibited a strong affinity for hla - b * 0702 ( table iv ) and induced an immune response in the majority of vaccinated mice ( fig2 ). however , for all peptides but tert 444a1 , generated ctl recognized the optimized peptide but not the corresponding native peptide ( fig2 ). this strongly suggests that substitution of the amino acid at position 3 by an r may change the conformation of the peptide segment that interacts with the tcr and guarantees tcr cross - recognition . since a ) all tested peptides with apx 3 x 4 x 5 x 6 x 7 x 8 x 9 x 10 x 11 have a high affinity and are immunogenic ( table iv and fig1 ) and b ) substitution of the amino acid at position 3 by an r may break the cross - recognition of the native peptide , the inventors selected native peptides having a p and r at positions 2 and 3 respectively , and they substituted the amino acid at position 1 by an a if the last amino acid was favourable ( l , a , i , v or m ). given the high importance of position 3 in both affinity and ctl recognition of hla - b * 0702 restricted peptides inventors selected peptides with the sequence x 1 px 3 ( wherein x 1 is any amino acid and x 3 is k , r , h or m ; these amino acids have been described as being favourable residues at position 3 ) and a favourable amino acid ( a / i / l / v ) at c - terminal position . peptides with this sequence and low affinity for hla - b * 0702 have been modified by substitution of the first residue by an a . this is the case of tert 444 , her - 2 / neu 760 and her - 2 / neu 246 . inventors also selected peptides with the sequence apx 3 ( wherein x 3 is k , r , h or m ) and a non favourable residue at c - terminal position ( i . e ., an amino acid other than l , a , i , v or m ). peptides with this sequence and low affinity for hla - b * 0702 have been modified by substituting residue at c - terminal position with a l . this is the case of her - 2 / neu 1069 . all these modified peptides had a strong affinity for hla - b * 0702 . example 5 : immunogenicity of optimized peptides and cross - recognition of the native counterpart native her2 / neu 246 , her2 / neu 760 , her2 / neu 1069 and tert 444 peptides were not immunogenic , whereas the optimized peptides were immunogenic in hla - b * 0702 transgenic mice . moreover , ctl induced by all these optimized peptides were able to cross - react with the corresponding native peptide ( fig3 and table v ). in conclusion , the inventors have described a method to optimize immunogenicity ( and also affinity ) of hla - b * 0702 restricted cryptic peptides . it consists in a ) substituting the residue at position 1 with an a in all peptides comprising the sequence x 1 px 3 ( wherein x 1 is any amino acid except a and x 3 is r or k or h or m ), a favourable amino acid at c - terminal position ( i . e ., l or a or i or v or m ), and a low affinity for hla - b * 0702 , or b ) substituting the residue at c - terminal position with a l in peptides comprising the sequence apx 3 ( x 3 being defined as above ), a non favourable residue at c - terminal position ( i . e ., an amino acid other that l or a or i or v or m ), and a low affinity for hla - b * 0702 . hla - b7 transgenic mice were then immunized with the tert 4 ( seq id no : 15 ) and eleven days later their spleen cells were in vitro stimulated with the peptide . generated ctl killed rma - b7 targets loaded with decreasing concentrations of tert 4 peptide ( fig4 a ). the half maximal lysis of tert 4 loaded targets was obtained with 1 . 5 nm ( fig4 a ). ctl were then tested for their capacity to recognize cos - 7 cells expressing hla - b * 0702 and endogenous tert . results presented in fig4 b show that ctl recognized cos - 7 cells transfected with both hla - b * 0702 and tert but not cos - 7 cells transfected with either hla - b * 0702 or tert , demonstrating that tert 4 dominant peptide is an hla - b * 0702 restricted epitope naturally processed from endogenous tert . moreover , cd8 cells from healthy donors were in vitro stimulated with autologous dendritic cells loaded with tert 4 peptide . after four stimulations , ctl were tested for cytotoxicity against tert 4 loaded t2 - b7 targets . three donors were tested and ctl were induced in two of them . results from one responding donor are presented in fig4 c . ctl killed t2 - b7 targets presenting tert 4 but not t2 - b7 cells presenting the irrelevant nef peptide ( left graph ). interestingly , ctl killed the hla - b * 0702 tert + sk - mes - 1 and hbl - 100 but not the hla - b * 0702 - tert + sw - 480 and hss human tumor cell lines confirming the hla - b * 0702 restricted presentation and the endogenous processing of the tert 4 epitope ( right graph ). example 7 : ctl induced by tert 444a1 peptide recognize endogenous tert tert 444a1 ( seq id no : 5 ) was tested for its ability to induce ctl able to recognize endogenous tert and to induce ctl in healthy donors ( example 6 ). hla - b * 0702 transgenic mice were then immunized with the tert 444a1 and eleven days later their spleen cells were in vitro stimulated with the native tert 444 peptide ( seq id no : 1 ). generated ctl killed rma - b7 targets loaded with decreasing concentrations of tert 444a1 and tert 444 peptides . the half maximal lysis of tert 444 loaded and tert 444a1 loaded targets was obtained with 5 . 5 nm and 1 nm respectively ( fig5 a ). ctl were then tested for their capacity to recognize cos - 7 cells expressing hla - b * 0702 and endogenous tert . results presented in fig5 b show that ctl recognized cos - 7 cells transfected with both hla - b * 0702 and tert but not cos - 7 cells transfected with either hla - b * 0702 or tert demonstrating that tert 444 is an hla - b * 0702 restricted cryptic epitope naturally processed from endogenous tert . cd8 cells from healthy donors were in vitro stimulated with autologous dendritic cells loaded with tert 444a1 peptide . after four stimulations , proliferating cells were divided into 4 pools . each pool was then tested for intracellular ifng production upon stimulation with t2 - b7 cells loaded with optimized tert 444a1 or native tert 444 . results from d5609 responding donor are presented in fig6 . ifng producing ctl were detected in pools 2 and 4 after stimulation with either tert 444 or tert 444a1 loaded t2b7 cells ( fig6 ). anderton , s . m ., viner , n . j ., matharu , p ., lowrey , p . a ., and wraith , d . c . 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