subject 1 , a 53-year old female with extensive refractory metastatic melanoma to the lung and lower extremity received autologous til ( # 2098 ) in 2003 and underwent a complete regression of all metastatic melanoma that was sustained until her death from an unrelated ovarian cancer six years later . subject 2 , a 32-year old male with metastatic melanoma to the brain , liver and periportal lymph nodes , refractory to prior treatment , received autologous til ( # 2369 ) in 2005 and experienced a complete cancer regression that is ongoing 6 - 1/2 years later . subject 3 , a 27 year old female with metastatic melanoma to the brain , lung , thigh and popliteal fossa , received autologous til ( # 3309 ) in august of 2009 and had a nearly complete regression of all lesions but then developed recurrent thigh and chest wall lesions six months following therapy . all patients signed an informed consent form approved by the institutional review board of the us national cancer institute . genomic dna purification , library construction , exome capture of approximately 20,000 coding genes , and next generation sequencing of tumor and normal samples were performed at personal genome diagnostics ( baltimore , md ) , while bioinformatic analyses were carried out by personal genome diagnostics and the genome technology access center , genomics and pathology services of the washington university school of medicine . in brief , genomic dna from tumor and normal samples were fragmented and used for illumina truseq library construction ( illumina , san diego , ca ) . exonic regions were captured in solution using the agilent sureselect 50 mb kit ( version 3 ) according to the manufacturer s instructions ( agilent , santa clara , ca ) . paired - end sequencing , resulting in 100 bases from each end of the fragments , was performed using a hiseq 2000 genome analyzer ( illumina , san diego , ca ) . sequence data were mapped to the reference human genome sequence and sequence alterations were determined by comparison of over 50 million bases of tumor and normal dna . over 8 billion bases of sequence data were obtained for each sample , with a high fraction from the captured coding regions . over 43 million bases of target dna were analyzed in the tumor and normal samples , and an average of 42 to 51 reads were obtained at each base in the normal and tumor dna samples . the tags were aligned to the human genome reference sequence ( hg18 ) using the eland algorithm of casava 1.6 software ( illumina , san diego , ca ) . the chastity filter of the basecall software of illumina was used to select sequence reads for subsequent analysis . the elandv2 algorithm of casava 1.6 software ( illumina , san diego , ca ) was then a applied to identify point mutations and small insertions and deletions . the dna isolated from the 2098 mel , 2369 mel and 3309 mel cell lines possessed 264 , 574 and 278 non - synonymous single and di - nucleotide substitutions , respectively . between 80 and 90% of the non - synonymous point mutations identified in the 2098 , 2369 and 3309 tumor cell linesrepresented c / g to t / a transitions , consistent with the role of uv in generating the majority of mutations found in non - acral melanomas . oligonucleotide primers were designed to amplify fragments of gene products ranging between approximately 100 and 600 nucleotides encompassing the mutated epitopes in 2098 , 2369 and 3309 mel ( supplementary methods primer list ) . these primers sets were used to carry out rt - pcrs , as previously described , from approximately 1 g of rna that was isolated from the appropriate tumor cell lines using rnaeasy ( qiagen inc . amplified cdna transcripts were either directly sequenced using after purification of the rt - pcr products using the purelink pcr purification kit ( life technologies , grand island , ny 14072 ) or cloned into pcdna3.1 topo ta ( life technologies ) and sequenced using the t7 oligonucleotide primer 5- taatacgactcactataggg-3. the residues surrounding amino acids resulting from non - synonymous mutations were scanned to identify candidate nonamer and decamer peptides that were predicted to bind with high affinity to individual hla class i alleles using the netmhcpan2.4 binding algorithm . peptides were obtained from piproteomics ( huntsville , al , 35816 ) or peptide 2.0 ( chantilly , va 20151 ) . mutated peptides identified by this approach were then purified to greater than 95% homogeneity by hplc and re - evaluated to confirm their ability to stimulate t cell responses . the til 2098 , 2369 and 3309 were expanded from fresh tumor digests as previously described . reactivity of til 2098 was evaluated by incubating hla - a*02:01 t2 cells that possess antigen processing defects that allow efficient loading of exogenous peptides   with candidate hla - a*02:01 binding peptides at a concentration of 10 m for two hours at 37c followed by two washing steps . endogenous processing of the epitopes recognized by til 2098 was evaluated in 293 cells that were co - transfected with 200 ng of a recombinant pcdna3.1 plasmid ( life technologies , grand island , ny ) encoding candidate epitopes and 50 ng of a recombinant pcdna3.1 plasmid encoding hla - a*02:01 , and the levels of soluble ifn- released from til that were cultured overnight with either peptide pulsed target cells , transfectants or tumor cells were measured , as previously described . evaluation of responses of til 2098 against the gas7 and c4orf15 mutated and wild type constructs were carried out in 293 cells expressing the 1i and 5i immunoproteasomal subunits . the hla - a*01:01 tumor cell line 537 mel , which is not recognized by til 2369 , and the hla - a*02:01 tumor cell line 624 mel , that is not recognized by til 2098 , served as negative controls for t cell reactivity . evaluation of peptide reactivity of til 2369 was carried out by pulsing 293 human embryonic kidney cells that were transduced with a recombinant retroviral expression plasmid encoding hla - a*01 ( 293-a1 ) with candidate hla - a*01:01 binding peptides at a concentration of 10 m for two hours at 37c , followed by two washing steps . to determine if the epitopes identified as targets of til 2369 were endogenously processed and presented , 293-a1 target cells were transiently transfected with either the genes encoding the mutated candidates or the corresponding wild type genes . evaluation of the reactivity of til 3309 was carried out by pulsing cos cells that had been stably transduced with retroviral constructs encoding either hla - a*0101 or hla - a*1101 with candidate hla - a*01 or a*11-binding peptides at a concentration of 10 m for two hours at 37c , followed by two washing steps . to determine if the epitopes identified as targets of til 3309 were endogenously processed and presented , cos - a11 target cells were transiently transfected with either the genes encoding the mutated candidates or the corresponding wild type genes , and the levels of soluble ifn- released from til that were cultured overnight with target cells were measured as previously described . quantitation of responses directed against tumor cell lines and peptide pulsed target cells was evaluated in an ifn- elispot assay using a monoclonal antibody pair obtained from mabtech . assays were carried out by initially thawing t cells overnight in the absence of exogenous cytokines , followed by culturing with tumor cells or cos-7 cells expressing either hla - a*0201 , a*0101 or a*1101 that were pulsed with candidate peptides for two hours at 37c . cells were also incubated in parallel overnight in complete media containing 50 ng / ml phorbol 12-myristate 13-acetate plus 1 m ionomycin ( pma / i ) to provide an estimate of the maximum number of ifn--secreting cells present in these populations . subject 1 , a 53-year old female with extensive refractory metastatic melanoma to the lung and lower extremity received autologous til ( # 2098 ) in 2003 and underwent a complete regression of all metastatic melanoma that was sustained until her death from an unrelated ovarian cancer six years later . subject 2 , a 32-year old male with metastatic melanoma to the brain , liver and periportal lymph nodes , refractory to prior treatment , received autologous til ( # 2369 ) in 2005 and experienced a complete cancer regression that is ongoing 6 - 1/2 years later . subject 3 , a 27 year old female with metastatic melanoma to the brain , lung , thigh and popliteal fossa , received autologous til ( # 3309 ) in august of 2009 and had a nearly complete regression of all lesions but then developed recurrent thigh and chest wall lesions six months following therapy . all patients signed an informed consent form approved by the institutional review board of the us national cancer institute . genomic dna purification , library construction , exome capture of approximately 20,000 coding genes , and next generation sequencing of tumor and normal samples were performed at personal genome diagnostics ( baltimore , md ) , while bioinformatic analyses were carried out by personal genome diagnostics and the genome technology access center , genomics and pathology services of the washington university school of medicine . in brief , genomic dna from tumor and normal samples were fragmented and used for illumina truseq library construction ( illumina , san diego , ca ) . exonic regions were captured in solution using the agilent sureselect 50 mb kit ( version 3 ) according to the manufacturer s instructions ( agilent , santa clara , ca ) . paired - end sequencing , resulting in 100 bases from each end of the fragments , was performed using a hiseq 2000 genome analyzer ( illumina , san diego , ca ) . sequence data were mapped to the reference human genome sequence and sequence alterations were determined by comparison of over 50 million bases of tumor and normal dna . over 8 billion bases of sequence data were obtained for each sample , with a high fraction from the captured coding regions . over 43 million bases of target dna were analyzed in the tumor and normal samples , and an average of 42 to 51 reads were obtained at each base in the normal and tumor dna samples . the tags were aligned to the human genome reference sequence ( hg18 ) using the eland algorithm of casava 1.6 software ( illumina , san diego , ca ) . the chastity filter of the basecall software of illumina was used to select sequence reads for subsequent analysis . the elandv2 algorithm of casava 1.6 software ( illumina , san diego , ca ) was then a applied to identify point mutations and small insertions and deletions . the dna isolated from the 2098 mel , 2369 mel and 3309 mel cell lines possessed 264 , 574 and 278 non - synonymous single and di - nucleotide substitutions , respectively . between 80 and 90% of the non - synonymous point mutations identified in the 2098 , 2369 and 3309 tumor cell linesrepresented c / g to t / a transitions , consistent with the role of uv in generating the majority of mutations found in non - acral melanomas . oligonucleotide primers were designed to amplify fragments of gene products ranging between approximately 100 and 600 nucleotides encompassing the mutated epitopes in 2098 , 2369 and 3309 mel ( supplementary methods primer list ) . these primers sets were used to carry out rt - pcrs , as previously described , from approximately 1 g of rna that was isolated from the appropriate tumor cell lines using rnaeasy ( qiagen inc . , amplified cdna transcripts were either directly sequenced using after purification of the rt - pcr products using the purelink pcr purification kit ( life technologies , grand island , ny 14072 ) or cloned into pcdna3.1 topo ta ( life technologies ) and sequenced using the t7 oligonucleotide primer 5- taatacgactcactataggg-3. the residues surrounding amino acids resulting from non - synonymous mutations were scanned to identify candidate nonamer and decamer peptides that were predicted to bind with high affinity to individual hla class i alleles using the netmhcpan2.4 binding algorithm . peptides were obtained from piproteomics ( huntsville , al , 35816 ) or peptide 2.0 ( chantilly , va 20151 ) . mutated peptides identified by this approach were then purified to greater than 95% homogeneity by hplc and re - evaluated to confirm their ability to stimulate t cell responses . the til 2098 , 2369 and 3309 were expanded from fresh tumor digests as previously described . reactivity of til 2098 was evaluated by incubating hla - a*02:01 t2 cells that possess antigen processing defects that allow efficient loading of exogenous peptides   with candidate hla - a*02:01 binding peptides at a concentration of 10 m for two hours at 37c followed by two washing steps . endogenous processing of the epitopes recognized by til 2098 was evaluated in 293 cells that were co - transfected with 200 ng of a recombinant pcdna3.1 plasmid ( life technologies , grand island , ny ) encoding candidate epitopes and 50 ng of a recombinant pcdna3.1 plasmid encoding hla - a*02:01 , and the levels of soluble ifn- released from til that were cultured overnight with either peptide pulsed target cells , transfectants or tumor cells were measured , as previously described . evaluation of responses of til 2098 against the gas7 and c4orf15 mutated and wild type constructs were carried out in 293 cells expressing the 1i and 5i immunoproteasomal subunits . the hla - a*01:01 tumor cell line 537 mel , which is not recognized by til 2369 , and the hla - a*02:01 tumor cell line 624 mel , that is not recognized by til 2098 , served as negative controls for t cell reactivity . evaluation of peptide reactivity of til 2369 was carried out by pulsing 293 human embryonic kidney cells that were transduced with a recombinant retroviral expression plasmid encoding hla - a*01 ( 293-a1 ) with candidate hla - a*01:01 binding peptides at a concentration of 10 m for two hours at 37c , followed by two washing steps . to determine if the epitopes identified as targets of til 2369 were endogenously processed and presented , 293-a1 target cells were transiently transfected with either the genes encoding the mutated candidates or the corresponding wild type genes . evaluation of the reactivity of til 3309 was carried out by pulsing cos cells that had been stably transduced with retroviral constructs encoding either hla - a*0101 or hla - a*1101 with candidate hla - a*01 or a*11-binding peptides at a concentration of 10 m for two hours at 37c , followed by two washing steps . to determine if the epitopes identified as targets of til 3309 were endogenously processed and presented , cos - a11 target cells were transiently transfected with either the genes encoding the mutated candidates or the corresponding wild type genes , and the levels of soluble ifn- released from til that were cultured overnight with target cells were measured as previously described . quantitation of responses directed against tumor cell lines and peptide pulsed target cells was evaluated in an ifn- elispot assay using a monoclonal antibody pair obtained from mabtech . assays were carried out by initially thawing t cells overnight in the absence of exogenous cytokines , followed by culturing with tumor cells or cos-7 cells expressing either hla - a*0201 , a*0101 or a*1101 that were pulsed with candidate peptides for two hours at 37c . cells were also incubated in parallel overnight in complete media containing 50 ng / ml phorbol 12-myristate 13-acetate plus 1 m ionomycin ( pma / i ) to provide an estimate of the maximum number of ifn--secreting cells present in these populations .
significant tumor regressions have been observed in up to 70% of patients receiving adoptively transferred autologous melanoma - reactive tumor infiltrating lymphocytes ( til ) 1,2 , and in pilot trials , 40% of treated patients experienced complete regressions of all measurable lesions for at least five years following treatment 3 . 
 to evaluate the potential association between the ability of til to mediate durable regressions and their ability to recognize potent antigens that presumably include mutated gene products , a novel screening approach was developed that involved mining whole exome sequence data to identify the mutated proteins that were expressed in patient tumors . 
 candidate mutated t cell epitopes that were identified using an mhc binding algorithm 4 were then synthesized and evaluated for recognition by til . using this approach , mutated antigens expressed on autologous tumor cells 
 were identified as targets of three til that were associated with objective tumor regressions following adoptive transfer . 
 this simplified approach , which avoids the need to generate and laboriously screen cdna libraries from tumors , may represent a generally applicable method for identifying mutated t cell antigens expressed in melanoma as well as other tumor types .