Patent Application: US-38885295-A

Abstract:
a melanoma associated antigen known as gp100 . furthermore , peptides derived from the antigen are described . gp100 and its peptides can be used in vaccines for the treatment of melanoma . another aspect of the invention is host cells capable of expressing gp100 for the gp100 - derived peptides . furthermore , tumor infiltrating lymphocytes specifically recognizing gp100 are described , as are vaccines with these til &# 39 ; s . also disclosed are diagnostics for the detection of melanoma and for the monitoring of vaccination .

Description:
the melanoma cell lines mel - 2a , m14 , mewo , blm ( vennegoor et al ., 1988 ; van muijen et al ., 1991 ; bean et al ., 1975 ; katano et al ., 1984 ) and the uveal melanoma cell line mel 202 ( ksander et al ., 1991 ) have been previously described . isolation of normal human melanocytes from breast or foreskin was performed by the method of eisinger and marko ( 1982 ) with modifications by ( smit et al ., 1993 ). mabs nki - beteb and hmb - 50 have been described previously ( vennegoor et al ., 1988 ; vogel and esclamado , 1988 ). mab hmb - 45 was purchased from enzo biochem . the 2 . 2 kb eco ri fragment containing gp100 cdna was blunt - ended by filling in the ends with klenow dna polymerase and then cloned in both orientations ( psvlgp100 + and psvlgp100 −) in the sma i site of the eukaryotic expression vector psvl ( pharmacia ). psvl contains the sv40 late promoter and polyadenylation site as well as the sv40 origin of replication , allowing a very high copy number during transient expression in cos - 7 cells . for the construction of the 3 ′ truncated gp100 transcription unit psvlgp100 +(@ bs ) we deleted the sequence between the bgl ii site in the 3 ′ part of gp100 cdna and the sac i site in the multiple cloning site of the vector . the resulting construct encodes a truncated gp100 protein in which the carboxy - terminal 133 amino acids of gp100 are replaced by 4 amino acids ( arg - ile - gln - thr ) seq . id . no . 32 encoded by vector sequences . transient expression of the constructs in cos - 7 cells was performed by using 40 μg / ml lipofectin reagent from brl ( felgner et al ., 1987 ) and 7 . 5 μg dna as described previously ( loenen et al ., 1991 ). transfected cos - 7 cells were prepared for immunofluorescence 48 hours after the addition of the lipofectin / dna mixture as described previously ( vennegoor et al ., 1988 ). after incubation with the primary antibody for 45 minutes , cells were washed and incubated with fluorescein isothiocyanate ( fitc )- labeled goat f ( ab )′ 2 anti - mouse igg ( nordic ) for 30 minutes . preparations were examined using a confocal laser scanning microscope at 488 nm ( biorad mrc 600 ). immunoprecipitation experiments were performed on metabolically labeled ( l -[ 35 s ]- methionine / cysteine ; amersham ) cells as described by vennegoor et al . ( 1988 ) using either mab nki - beteb or hmb - 50 covalently linked to protein a - cl 4b sepharose beads ( pharmacia ). in some experiments tunicamycin ( 75 μg / ml , calbiochem ) was added during the pre - labeling period and remained present during the metabolic labeling reaction ( 12 . 5 minutes ). immunoprecipitates were analyzed under reducing conditions ( 5 % β - mercaptoethanol in sds - sample buffer ) by sds - page using 5 - 17 . 5 % polyacrylamide gradient gels . the relative molecular weight of the proteins was determined using co - electrophorised , pre - stained molecular weight markers ( brl ). gels were treated with 1 m sodium salicylate ( ph 5 . 4 ) prior to autoradiography ( kodak xar ). v8 protease mapping was performed using the digestion for proteins in gel slices procedure described by cleveland et al . ( 1977 ). briefly , gel slices containing the 100 kd proteins were placed in the wells of a second sds - gel ( 10 %) and overlayed with staphylococcus aureus v8 protease ( 2 . 5 μg / sample , miles laboratories ). after electrophoresis gels were treated as described above . part of the gp100 / pmel17 gene was amplified by pcr ( taq dna polymerase was from gibco ) on human genomic dna isolated from peripheral blood lymphocytes ( pbl &# 39 ; s ) using the following primers : 1497 / 1516 : 5 ′- tattgaaagtgccgagatcc - 3 ′ seq id no : 26 and 1839 / 1857 : 5 ′- tgcaaggaccacagccatc - 3 ′ seq id no : 27 as described previously ( adema and baas , 1991 ). the pcr products were subsequently amplified using a nested set of primers containing an additional eco ri site ( 5 ′- tatctagaattctgcaccagatactgaag - 3 ′ seq id no : 11 and 5 ′- tatctagaattctgcaagatgcccacgatcag - 3 ′) seq id no : 12 . the underlined eco ri sites in these primers were used to clone the pcr product in the eco ri site of puc 18 . total rna was isolated using the guanidine thiocyanate procedure and centrifugation through a cushion of cesium chloride ( chirgwin et al ., 1979 ). cdna was prepared using the geneamp rna pcr kit ( perkin elmer cetus ) as indicated by the manufacturer . pcr analysis of the cdnas was performed for 35 cycles in the presence of 3 mm mgcl 2 using primers 1497 / 1516 and 1839 / 1857 ( see above ) as described previously ( adema and baas , 1991 ). the reaction products were size - fractionated on an agarose gel , blotted onto a nylon membrane ( hybond - n , amersham ) and hybridized to [ 32 p ]- labeled oligonucleotide probes as described previously ( adema and baas , 1991 ). as probes we used either a gp100 - specific exon / exon junction oligonucleotide ( 5 ′- cttcttgaccaggcatgata - 3 ′) seq id no : 19 or a pmel17 - specific oligonucleotide ( 5 ′- tgtgagaagaatcccaggca - 3 ′) seq id no : 14 which corresponds to 20 of the additional 21 nucleotides present in pmel17 cdna . in every hybridization experiment a spot blot containing an oligonucleotide comprising the pmel17 exon / exon junction ( 5 ′- gcttatcatgcctgtgcctggattcttctcacaggt - 3 ′) seq id no : 15 was included as a control . gp100 cdna and genomic dna clones were sequenced by the dideoxy - nucleotide sequencing method ( sanger et al ., 1977 ) using t7 dna polymerase ( pharmacia ). the sequence of both strands was determined in each case . since the genomic dna clones were obtained after pcr , the sequence of four independent clones was determined . analysis of the dna sequence was performed using the university of winconsin genetics computing group sequence analysis programs ( devereux et al ., 1984 ). expression of gp100 cdna in non - pigmented cos - 7 cells results in immunoreactivity with mabs nki - beteb , hmb - 50 and hmb - 45 . expression of gp100 cdna in gp100 - negative blm melanoma cells results in immunoreactivity with the melanocyte lineage - specific mabs , nki - beteb , hmb - 50 and hmb - 45 . to determine whether expression of gp100 - c1 cdna in non - melanocytic cells also results in immunoreactivity with these mabs , we performed transient expression experiments in cos - 7 cells ( monkey kidney fibroblasts ) with constructs containing gp100 cdna in the coding or non - coding orientation . only cos - 7 cells transfected with the construct containing the cdna in the coding orientation ( cos - 7 / psvlgp100 +) react with all three mabs . these data demonstrate that immunoreactivity with mabs nki - beteb , hmb - 50 and hmb - 45 after expression of gp100 cdna is not restricted to melanocytic cells . in addition , these data show that the cos expression system can be used for further biochemical characterization of the proteins encoded by gp100 cdna . to characterize the proteins encoded by gp100 cdna , cos - 7 / psvlgp100 + cells were metabolically labeled and subjected to immunoprecipitation with mab nki - beteb or hmb - 50 . moabs nki - beteb and hmb - 50 specifically immunoprecipitate proteins of approximately 100 kd ( 95 - 110 kd ) from extracts of cos - 7 / psvlgp100 + cells . the molecular weight of these proteins is similar ( see also below ) to those immunoprecipitated from extracts of metabolically labeled mewo cells which express the antigens endogenously ( vennegoor et al ., 1988 ). consistent with p revious reports ( vennegoor et al ., 1988 ; vogel and esclamado , 1988 ), both mabs also recognize a protein of 10 kd in extracts of mewo melanoma cells . a protein of the same size reacts with mab nki - beteb in cos - 7 / psvlgp100 + cells and can be discerned with mab hmb - 50 after prolonged exposure ( not shown ). we note that the amount of the 10 kd protein varied considerably between experiments . no specific proteins are immunoprecipitated by either of the mabs from extracts prepared from cos - 7 cells transfected with the construct containing the dna in the non - coding orientation . glycoproteins of approximately 100 kd reacting with mabs nki - beteb and hmb - 50 have also been found in culture medium of melanoma cells ( vennegoor et al ., 1988 ; vogel and esclamado , 1988 ). comparison of the culture medium of metabolically labeled cos - 7 / psvlgp100 + cells and mewo cells reveals that both mabs also recognize proteins of about 100 kd ( see also below ) in the culture medium of these cells . no proteins of 10 kd are immunoprecipitated by the mabs from the culture medium of cos - 7 / psvlgp100 + cells , as has been shown for melanoma cells . these data demonstrate that , as in melanoma cells , the proteins of about 100 kd recognized by mabs nki - beteb and hmb - 50 in cos - 7 / psvlgp100 + cells are secreted . to exclude the possibility that the proteins detected by the mabs are derived from endogenous genes induced after transfection with gp100 cdna , we performed immunoprecipitation experiments with cos - 7 cells expressing a 3 ′ truncated gp100 transcription unit ( see materials & amp ; methods for details ). proteins of approximately 85 kd are immunoprecipitated by both mabs from cos - 7 cells expressing this construct , consistent with a deletion of 129 amino acids . this finding provides direct evidence that the 100 kd protein recognized by mabs nki - beteb and hmb - 50 in cos - 7 / psvlgp100 + cells is encoded by gp100 cdna . the 100 kd protein encoded by gp100 cdna is identical to gp100 the proteins of about 100 kd identified by mabs nki - beteb and hmb - 50 in cos - 7 / psvlgp100 + cells versus mewo cells have a slightly different mobility when analyzed by sds - page . since the proteins reacting with these mabs have been shown to be glycosylated in melanoma cells ( vennegoor et al ., 1988 ; vogel and esclamado , 1988 ), these differences could be due to altered glycosylation , an event frequently observed in the cos expression system . to confirm this , mab nki - beteb was used to immunoprecipitate proteins from mewo cells and cos - 7 / psvlgp100 + cells cultured in the presence of the glycosylation inhibitor tunicamycin . in both cos - 7 / psvlgp100 + cells and mewo cells the size of the proteins of about 100 kd is reduced to two protein bands of 90 kd and 85 kd , confirming that the observed difference in mobility is due to altered glycosylation . to provide further evidence that the proteins recognized by mab nki - beteb in cos - 7 / psvlgp100 + cells and mewo cells are identical , we performed a v8 protease mapping experiment . the same protein fragments are obtained after v8 protease digestion of the major 100 kd protein isolated from cos - 7 / psvlgp100 + cells or mewo cells . we conclude from these data that gp100 cdna encodes the melanocyte lineage - specific glycoprotein gp100 recognized by mabs nki - beteb and hmb - 50 in melanoma cells . gp100 is a type i transmembrane protein highly homologous to pmel17 the nucleotide sequence of gp100 cdna was determined . it contains 2115 base pairs ( bp ) and terminates with a poly ( a ) tract of 15 nucleotides which is preceded by the consensus polyadenylation sequence aataaa seq . id . no : 31 ( proudfoot and brownlee , 1976 ). an open reading frame ( orf ) extending from nucleotide 22 through 2007 is present in gp100 cdna . this orf starts with an atg codon within the appropriate sequence context for translation initiation ( kozak , 1987 ) and codes for a protein of 661 amino acids ( seq id no : 1 ). the amino - terminal 20 amino acids fit all criteria for signal sequences , including a potential cleavage site after ala at position 20 ( von heyne , 1986 ), which would indicate that mature gp100 contains 641 amino acids ( approximately 70 kd ). based on hydrophobicity plot analysis ( kyte and doolittle , 1982 ), a single transmembrane domain bordered by charged residues is present in the carboxy - terminal part ( amino acids 591 - 611 ) of gp100 . the predicted cytoplasmic domain is 45 amino - acids long . five putative n - linked glycosylation sites are present , consistent with gp100 being a glycoprotein . furthermore , a histidine - rich domain ( amino acids 182 - 313 ), a threonine - rich domain ( amino acids 309 - 427 ) containing repetitive amino acid sequences , and a cysteine - rich domain ( 475 - 566 amino acids ) are present . a data base search ( pearson and lipman , 1988 ; altschul et al ., 1990 ) revealed that gp100 is almost identical to pmel17 , another melanocyte - specific protein ( kwon et al ., 1991 ). the amino acid differences between gp100 and pmel17 consist of substitutions at position 274 ( t - c / pro - leu ) and 597 ( c - g / arg - pro ) and a stretch of 7 amino acid absent in gp100 at position 587 ( see also fig2 ). a single nucleotide difference at position 782 ( c - t ) does not result in an amino acid substitution . gp100 is also 80 % homologous to a putative protein deduced from a partial cdna clone ( rpe - 1 ) isolated from a bovine retinal cdna library ( kim and wistow , 1992 ) and 42 % homologous to a chicken melanosomal matrix protein , mmp115 ( mochii et al ., 1991 ). the most striking difference between gp100 and pmel17 cdnas is the inframe deletion of 21 bp in gp100 cdna . one possible explanation for this difference is the existence of two closely related genes . however , since both cdnas have identical nucleotide sequences in their 3 ′ untranslated regions this explanation is not likely . another possibility is that both cdnas correspond to transcripts generated by alternative splicing of a single primary transcript . to test this hypothesis , we used pcr to analyze the genomic dna corresponding to the part of the gp100 gene surrounding the putative alternative splice site . comparison of the nucleotide sequence of this genomic dna with the sequence of gp100 - c1 cdna revealed the presence of an intron ( 102 bp ) just at the position of the 21 bp insertion in pmel17 cdna ( fig1 ). the exon / intron boundaries nicely fit the consensus 5 ′ donor and 3 ′ acceptor splice site sequences ( padgett et al ., 1986 ). in the genomic dna , the sequence comprising the additional 21 bp in pmel17 cdna is located directly upstream of the 3 ′ cleavage site used to generate gp100 rna and is preceded by an alternative 3 ′ acceptor splice site ( fig1 ). whereas the gp100 - specific 3 ′ acceptor splice site fits the consensus sequence , the pmel17 - specific 3 ′ acceptor splice site appears to be sub - optimal in that it lacks a pyrimidine - rich region ( fig1 ). sub - optimal rna processing sites are present in many alternatively processed messenger rna precursors and have been implicated to function in regulation of alternative rna processing ( reviewed by green , 1991 ). collectively , these data prove that the transcripts corresponding to gp100 and pmel17 cdnas are generated by alternative splicing of a single primary transcript and thus originate from a single gene . expression of gp100 and pmel17 rnas in cells of the melanocytic lineage the finding that gp100 and pmel17 rnas arise by alternative splicing of a single primary transcript , raises the question whether this occurs in a developmentally regulated manner . an rna species of 2 . 5 kb is the major rna product detected by gp100 cdna on northern blots containing rna isolated from melanocytic cells . the same results were obtained by kwon et al . ( 1987 ) using pmel17 - 1 cdna as a probe . however , neither of the probes discriminate between gp100 and pmel17 rnas . to investigate the expression of gp100 and pmel17 rnas in cells of the melanocytic lineage , we performed a reverse transcriptase / polymerase chain reaction ( rt / pcr ) assay followed by southern blotting and hybridization to either a gp100 specific exon / exon junction - or a pmel17 - specific oligonucleotide probe ( see materials & amp ; methods ). gp100 and pmel17 spliced products are both detected in 3 out of 4 cutanous melanoma cells , in uveal melanoma cells as well as in neonatal and adult melanocytes . no products are detected with either probe in gp100 - negative blm melanoma cells . these results demonstrate that in all melanocytic cells examined , gp100 and pmel17 rnas are expressed simultaneously . til &# 39 ; s were generated by growth of single cell suspensions of metastatic melanomas with 1 , 000 u / ml il - 2 ( cetus corp ., emeryville , calif .) and were grown as described previously ( kawakami , 1992 ). melanoma cell lines mel 397 and mel 624 were obtained and grown as reported previously ( kawakami , 1992 ). hla - a2 . 1 + melanoma cell lines mewo ( bean , 1975 ) and blm ( katano , 1984 ) and murine p815 transfectants were grown in dmem ( gibco , paisley , scotland , uk ) plus 7 . 5 % heat inactivated fcs ( gibco ). jy , k562 , and murine el4 transfectants were cultured in iscoves medium ( gibco ) plus 7 . 5 % fcs . murine cells were grown in the presence of 5 · 10 5 m β - me , and all media contained antibiotics . isolation of normal melanocytes from foreskin was performed by the method of eisinger and marko ( 1982 ) with modifications as described previously ( smit , 1989 ). melanocytes from passages two to three were used in chromium release assays . plasmid pbj1gp100neo was obtained by cloning the ecori fragment of a lambda gp100 cdna clone in the coding orientation in the polylinker pbj1 - neo ( lin , 1990 ). plasmid pba2 containing a genomic fragment encoding hla - a2 . 1 and human β - 2 microglobulin was kindly provided by e . j . baas ( the netherlands cancer institute , division of biochemistry , amsterdam , the netherlands ). plasmid pgk - hyg contains the hygromycin phosphotransferase gene ( te riele , 1990 ). for the introduction of the hla - a2 . 1 and human β - 2 microglobulin genes , el4 cells were transfected with 18 μg of pba2 and 2 μg of pgk - hyg dna according to the calcium phosphate coprecipitation procedure ( graham , 1973 ) using calciumphosphate transfection systems ( gibco brl , gaithersburg , md .). 24 h after transfection , 500 μg / ml hygromycin b ( calbiochem - novabiochem corp ., la jolla , calif .) was added to the medium for the selection of stable transfectants . hla - a2 . 1 + gp100 + el4 cells were obtained by transfection of stable hla - a2 . 1 + el4 clones with 20 μg of pbj1 - gp100neo dna by calcium phosphate coprecipitation and were selected with 1 mg / ml g418 . p815 a2 . 1 and p815 a2 . 1 / gp100 cells were kindly provided by p . coulie ( ludwig ins ., brussels , belgium ). phenotypic analysis of melanomas , transfectants , and normal melanocytes was performed by indirect immunofluorescence followed by flow cytometry using a facscan r ( becton dickinson & amp ; co ., mountain view , calif .). purified anti - gp100 mab nki - beteb ( vennegoor , 1988 ) and anti - hla - a2 mabs bb7 . 2 ( culture supernatant ; parham , 1981 ) and ma2 . 1 ( ascites 1 : 500 dilution ; parham , 1978 ) were used as primary reagents . fitc conjugated gam - igg - f ( ab ′) 2 ( zymed laboratories , inc . s . san francisco , calif .) was used for the second incubation . for the detection of the intracellular gp100 antigen cells were permeabilized in 0 . 01 % digitonin and were subsequently fixed in 1 % paraformaldehyde . chromium release assays were performed as described previously ( kawakami , 1992 ). briefly , 10 6 target cells were incubated with 100 μci na 51 cro 4 ( amersham int ., bucks , uk ) for 1 hour . various amounts of effector cells were then added to 2 · 10 3 target cells in triplicate wells of u - bottomed microtiter plates ( costar , badhoevedorp , the netherlands ) in a final volume of 150 μl . after 5 hours of incubation , part of the supernatant was harvested and its radioactive content measured . target cells were incubated for 48 hours with 50 u / ml human ( boehringer , ingelheim , germany ) or mouse recombinant ifn - ( tno , rijswijk , the netherlands ) before use in chromium release assays . in search of gp - 100 specific cytotoxic t lymphocytes ( ctls ) we focused on hla - a2 . 1 as a restriction element because of its widespread occurrence in caucasians and its presumptive dominant role in ctl reactivity against melanoma . a hla - a2 . 1 + til line , til 1200 ( shilyansky , j . et al , 1994 ), was used for this study . this til line expresses tcr α / β , cd3 and cd8 . hla - a2 . 1 - restricted killing of melanoma tumor cells by til 1200 corresponds to gp100 expression . cytolytic activity of til 1200 was analyzed using a panel of human melanoma cell lines . til 1200 efficiently lysed hla - a2 . 1 + mel 624 and mewo melanoma tumor cells , which both express gp100 , whereas no reactivity towards hla - a2 . 1 − gp100 + mel 397 cells was seen . it is interesting to note that we observed that hla - a2 . 1 + blm melanoma cells are also resistant to lysis by til 1200 . furthermore , hla - a2 . 1 + ebv - transformed b cells ( jy ), which also lack gp100 expression , and k562 cells , were not lysed by til 1200 . together , these data demonstrate that til 1200 displays hla - a2 . 1 - restricted killing which correlates with gp100 expression . el4 cells cotransfected with a genomic fragment encoding hla - a2 . 1 together with a plasmid conferring hygromycine resistance were selected and analyzed by flow cytometry . hla - a2 . 1 expressing cells were subsequently transfected with pbj1 - gp100neo , which encodes gp100 and confers resistance to g418 . stable transfectants were selected and were screened for gp100 expression using mab nki / beteb . in collaboration with p . coulie a similar panel of transfectants was generated in murine p815 cells ( p815 a2 . 1 and p815 a2 . 1 / gp100 ). using these murine transfectants as target cells in chromium release assay , we clearly observed gp100 specific lysis by til 1200 . the percent specific lysis ( 25 - 35 %, e / t 30 : 1 ) of murine el4 a2 . 1 / gp100 and p815 a2 . 1 / gp100 transfectants by til 1200 was somewhat lower compared with that observed with hla - a2 . 1 + gp100 + human melanoma cells ( 45 - 60 %, e / t 30 : 1 ). this difference may be explained by nonmatched accessory molecules between human til &# 39 ; s and murine transfectants . to overcome this we introduced the gp100 antigen into human hla - a2 . 1 + gp100 − blm melanoma cells by transfection of pbj1 - gp100neo . stable blm gp100 clones were tested in chromium release assays using til 1200 . blm gp100 clones proved to be as sensitive to lysis by til 1200 as mel 624 and mewo cells which express the gp100 antigen endogenously . the gp100 specificity of til 1200 was further demonstrated by the absence of lysis of g418 - resistant blm cells not expressing gp100 , excluding the possibility that neomycin - derived peptides are recognized . napping of gp100 epitopes recognized by til 1200 using gp100 deletion mutants . basically , two methods are commonly used in the art to map epitopes recognized by anti - tumor ctl . 1 . according to the hla binding motifs peptides can be synthesized that reside in the target protein . these peptides can then be loaded onto cells bearing the appropriate restriction element , and used as targets for ctl . 2 . generation of deletion mutants and expression of these deletion mutants in for example cos - 7 cells together with the appropriate restriction element . these transfected cells are then co - cultured with ctl and target cell lysis or tnf - α / ifnγ production by the ctl are measured . transfectants not recognized by the ctl do not express the peptide . both methods have been done in search for the epitopes of the invention . we have chemically synthesized gp100 peptides potentially recognized by til 1200 . peptides were synthesized by a solid phase strategy on an automated multiple peptide synthesizer ( abimed ams 422 ) using fmoc - chemistry ( nijman , 1993 ). actual binding of the peptides to hla - a2 . 1 was established with a recently described peptide binding assay making use of processing defective t2 cells ( nijman , 1993 ). this analysis resulted in the identification of gp100 derived peptides that strongly bind to hla - a2 . 1 . subsequently , t2 cells loaded with the peptides that strongly bind to hla - a2 . 1 were subjected to lysis by til 1200 using a standard chromium release assay . in this way the peptide l - l - d - g - t - a - t - l - r - l seq id no : 4 has been identified according to this procedure .. gp100 cdna was inserted into expression vectors pbj1neo , pcmvneo ( baker et al , 1990 ) and psvl . for the generation of a gp100 cdna lacking the coding sequences for the peptide 457 - 466 , pcr reactions were performed with the following combinations of oligonucleotides : 5 ′- catggaagtgactgtctacc - 3 ′ seq id no : 16 / 5 ′- ctgagcgaattcggaacctgtaatactttccg - 3 ′ seq id no : 17 , and 5 ′- ctgagcgaattcgtgaagagacaagtccccc - 3 ′ seq id no : 18 / 5 ′- tcacagcatcatatgagagtac - 3 ′ seq id no : 19 using the full length gp100 cdna as atemplate . pcr products were digested with eco ri , ligated and served as a template for a nested pcr using the following primers : 5 ′- gcacaggccaactgcaga - 3 ′ seq id no : 28 / 5 ′- ttcagtatctggtgcagaac - 3 ′ seq id no : 29 . the kpn i - cla i fragment from this pcr product was then exchanged with the corresponding fragment in pcmvgp100neo to generate pcmvgp100del454 - 481neo . gp100 cdna mutants del149 - 654 and del454 - 654 were obtained by deletion of the 1 . 7 kb hind iii and the 0 . 8 kb eco ri fragments from pbj1gp100del454 - 481neo , respectively . gp100 cdna mutants del100 - 654 , del194 - 528 and del167 - 508 were obtained by deletion of the bgl i - sac i , bamh hi - bgl ii and apa i - nsi i fragmnets from psvlgp100 respectively . blm cells were transfected with 20 μg of pcmvgp100del454 - 481neo dna according to the clacium phosphate coprecipitation procedure ( graham and van der eb , 1973 ) using calciumphosphate transfection systems ( brl , gaithersburg , md .) and were slected with 1 mg / ml g418 ( gibco , paisley , scotland uk ). cos - 7 cells were contransfected with 5 μg of pbj1hla - a2 . 1neo and 5 μg of pbj1 or psvl plasmids containing either full length or deleted gp100 cdnas using the deae - dextran / chloroquine method ( seed and aruffo , 1987 ). after 48 hours of transfection cos - 7 cells were used as stimulator cells in ifn - γ release experiments . for ifn - release assya 10 5 til 1200 responder cells were incubated together with 5 · 10 4 transiently transfected cos - 7 stimulator cells in 300 μl medium in the presence of 100 u / ml il - 2 in a flat bottom 96 well microtiter plate . after 24 hours of incubation , 100 μl of supernatant was harvested and was screened for the presence of ifn - γ using a hifn - γ - irma immunoradiometric assay kit ( megenix diagnostics sa , fleurus , belgium ). fig3 a shows the gp100 cdna deletion mutants that were generated . as shown in fig3 b , til 1200 specifically secreted ifn - γ when stimulated with cos - 7 cells transfected with hla - a2 . 1 and the full length gp100 cdna . again til 1200 reactivity was observed against the gp100del454 - 481 mutant . from the other gp100 deletion mutants , only the dekl100 - 661 and del149 - 661 constructs were not recognized , thereby excluding the possibility that til 1200 was reactive with a peptide located n - terminal from amino acid position 148 in the gp100 protein . also the c - terminal region of the gp100 protein protein could be excluded , because til 1200 reactivity could be observed using a mutant construct , del454 - 661 , encoding the first 453 amino acids of gp100 . from the observation that a construct coding within this n - terminal region upto amino acid 166 was able to stimulate til 1200 ( del167 - 508 ), it was concluded that the epitope recognized was located between amino acids 148 - 166 of the gp100 protein . several motifs have been described for 9 - mer or 10 - mer peptides binding to hla - a2 . 1 ( falk et al ., 1991 ; hunt et al ., 1992 ; ruppert et al ., 1993 ) that were deduced from naturally processed and synthetic hla - a2 . 1 binding peptides . the 148 - 166 region of the gp100 protein was screened against these motifs and a number of peptides were synthesized that fitted into a somewhat broader motif , including threonine residues at position two . these peptides were loaded onto hla - a2 . 1 + t2 cells and tested for their ability to induce til 1200 mediated target cell lysis ( fig4 a ). the five tested peptides were all able to sensitize t2 cells for lysis by til 1200 when used at a concentration of 10 μg / ml . all these peptides contain the 8 - mer peptide twgqywqv seq . id . no . 8 , corresponding to gp100 amino acids 155 - 162 . all peptides were titrated to evaluate their relative ability to sensitize t2 target cells for lysis by til 1200 . fig4 b shows that the 9 - mer peptide ktwgqywqv seq . id . no . 22 can be recognized by til 1200 when applied at a concentration of 3 ng / ml , whereas the other peptides had to be applied at higher concentrations . a comparison was made of the peptides ktwgqywqv seq id no : 30 ( gp100 amino acids 155 - 162 ), lldgtatlrl seq id no : 4 ( gp100 amino acids 457 - 466 ) and ylepgpvta seq id no : 31 ( gp100 amino acids 280 - 288 , identified by cox et al ., 1994 ) with three known viral epitopes presented in hla - a2 . 1 : the influenza matrix 58 - 66 peptide ( gotch et al ., 1987 ), the hiv polymerase 510 - 518 peptide ( tsomides et al ., 1991 ) and the hiv gp120 197 - 205 peptide ( dadaglio et al ., 1991 ). the hla - a2 . 1 binding capacity of the above mentioned epitopes was analyzed by means of an indirect binding assay using the processing defective cell line t2 ( nijman et al ., 1993 ). shortly : t2 cells were incubated with 12 . 5 μg of the epitopes . hla - a2 . 1 stabilization at the cell surface was determined by flow cytometry using mab bb7 . 2 . the fluorescence index is expressed as the experimental mean fluorescence divided by the mean fluorescence that is obtained when t2 cells are incubated with a hla - a2 . 1 non - binding peptide at a similar concentration . using this assay , a similar hla - a2 . 1 stabilization with the gp100 280 - 288 epitope and the tested viral epitopes . both epitopes of the invention ( ktwgqywqv seq id no : 22 and lldgtatlrl seq id no : 4 ) bind with a somewhat lower affinity to hla - a2 . 1 ( fig5 ). from this it is concluded that the gp100 epitopes bind to hla - a2 . 1 with distinct affinities . adema , g . j . and baas , p . d . 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