Patent Application: US-79121704-A

Abstract:
a peptide sequence of the so - called minor h antigen . the minor h antigens are associated with graft versus host disease . the peptide and its derivatives find many uses in bone marrow transplantation , organ transplantation , and in the treatment of leukemia . the peptide and its derivatives can be incorporated into vaccines and pharmaceutical formulations , and they can be used in diagnostic test kits . the peptide is derived from the ha - 1 minor antigen , and has the sequence vlxddllea , wherein x represents a histidine or arginine residue . both donors and recipients in bone marrow transplantation can be treated with the peptides , optionally in combination with other peptides , coupled to carriers , and with suitable excipients and / or adjuvants .

Description:
graft - versus - host disease ( gvhd ) is a frequent and life - threatening complication after allogeneic hla - identical bone marrow transplantation ( bmt ). recipients of hla - identical bone marrow develop acute or chronic graft - versus - host - disease in respectively 36 % and 49 % 1 . 2 . disparities in genes other than the mhc , referred to as minor histocompatibility ( mh ) antigens , are clearly involved in the development of gvhd after hla - identical bmt . a recent retrospective analysis revealed the significant association between mismatching for the mh antigen ha - 1 and the induction of gvhd after hla - identical bmt 3 . minor histocompatibility antigens are recognized by mhc restricted t cells and were shown to be peptides derived from intracellular proteins presented by mhc molecules 4 . 6 . here we report the first identification of a polymorphic gene encoding an human mh antigen . the gvhd associated mh antigen ha - 1 is a nonapeptide derived from the di - allelic kiaa0223 gene . the ha - 1 allelic counterpart encoded by the kiaa0223 gene differs only at one amino acid from the mh antigen ha - 1 . family studies demonstrated an exact correlation between the - kiaa0223 gene polymorphism and the ha - 1 phenotype as was previously determined by recognition by the ha - 1 specific ctl clones . the elucidation of the ha - 1 encoding gene enables prospective ha - 1 dna typing of bmt donors and recipients to improve donor selection and prevention of gvhd . cytotoxic t cell clones specific for the mh antigen ha - 1 have been isolated from three different patients with severe gvhd 7 . the mh antigen ha - 1 is presented in the context of hla - a2 . 1 and present in 69 % of the hla - a2 . 1 positive population 7 . ha - 1 expression was demonstrated to be tissue specific and limited to cells of hematopoietic origin , including dendritic cells , langerhans cells and leukemic cells 8 - 10 . family analysis indicated a mendelian mode of inheritance for ha - 1 and segregation independent from the mhc complex 11 . comparison of the t cell receptor ( tcr ) sequences of different ha - 1 specific t cell clones derived from different individuals revealed conserved usage of the tcr vb6 . 9 and conserved amino acids in the cdr3 region 12 . in a retrospective study , mismatching for a number of mh antigens was evaluated with regard to the association with gvhd after hla - identical bmt . a single ha - 1 mismatch between donor and recipient was significantly correlated with the induction of gvhd after hla - identical bmt 3 . to identify the mh antigen ha - 1 , hla - a2 . 1 , molecules were purified from two ha - 1 expressing ebv - transformed b lymphoblastoid cell lines ( ebv - blcl ) rp and blk . the hla - a2 . 1 bound peptides were isolated by acid treatment and fractionation of the peptides was performed by multiple rounds of reverse phase hplc . the fractions were analyzed for their capacity of inducing ha - 1 specific lysis using t2 cells as target cells and an ha - 1 specific ctl clone as effector cells in a 51 cr - release assay ( fig1 - a ). fraction 24 contained ha - 1 activity and was two times further fractionated with reverse phase hplc using a different organic modifier ( fig1 b . and c .). fraction 33 and 34 of the third hplc fractionation showed ha - 1 activity 51 cr - release assay and were analyzed by tandem mass spectrometry . because over 100 different peptides were present in these fractions , around 40 % of fractions 33 and 34 was chromatographed with an on - line microcapillary column effluent splitter . the fractions were simultaneously analyzed by tandem mass spectrometry and 51 cr - release assay ( fig1 d .). five peptide species ( at m / z 550 , 520 , 513 , 585 and 502 ) were specifically present in active fractions and absent in fractions without activity in the cml assay . collision activated dissociation analysis of peptide candidate m / z 550 revealed the sequence yxtdrvmtv . ( seq id no : 3 ). x stands for isoleucine or leucine that cannot be discriminated with this type of mass spectrometer . however , a synthetic peptide with this sequence was not able to reconstitute the ha - 1 epitope ( results not shown ). to determine which of the four remaining candidates was the ha - 1 peptide , the second ha - 1 purification of the ebv - blcl blk was evaluated . ha - 1 positive peptide fraction 33 of the second reverse phase hplc fractionation was further chromatographed by microcapillary hplc with a third organic modifier . a single peak of reconstituting activity was observed in a 51 cr - release assay ( results not shown ). mass spectral analysis of these fractions revealed that only peptide candidate m / z 513 was present . this peptide was analyzed with collision activated dissociation analysis and sequenced as vxhddxxea ( seq id no : 4 ) ( fig2 a ). isoleucine and leucine variants of the peptide were synthesized and run on the microcapillary hplc column . only peptide vlhddllea ( seq id no : 2 ) coeluted with the naturally processed peptide 513 ( results not shown ). next , synthetic vlhddllea ( seq id no : 2 ) added in different concentration to a cml assay with 3 different ha - 1 specific ctl clones revealed recognition by all three clones of the peptide with a half maximal activity at 150 - 200 pm for all three clones ( fig2 a ). this demonstrated that the mh antigen ha - 1 is represented by the nonapeptide vlhddllea ( seq id no : 2 ). database searches performed to identify the gene encoding ha - 1 , revealed that the ha - 1 peptide vlhdllea ( seq id no : 2 ) was identical for 8 out of 9 amino acids with the peptide vlrddllea ( seq id no : 5 ) from the kiaa0223 partial complementary dna ( cdna ) sequence , derived from the acute myelogenous leukemia kg - 1cell line . because ha - 1 has a population frequency of69 %, we reasoned that the vlrddllea ( seq id no : 5 ) peptide sequence might represent the ha - 1 allelic counterpart present in the remaining 31 % of the population . to elaborate on this assumption , we performed cdna sequence analysis of the putative ha - 1 encoding region of kiaa0223 in ebv - blcl derived from a presumed ha - 1 homozygous positive ( vr ), from a presumed ha - 1 negative individual ( dh ) and from the kg - 1 cell line ( table 1 .). the ha - 1 encoding region of kiaa0223 of the ha - 1 +/+ individual ( vr ) displayed two nucleotides differences from the kiaa0223 sequence in the databank , leading to the amino acid sequence vlhddllea ( seq id no : 2 ) ( designated ha - 1 h ). the ha - 1 encoding region of kiaa0223 of the ha - 1 −/− individual ( dh ) showed 100 % homology with the reported kiaa0223 sequence ( designated ha - 1 r ). the kg - 1 cell line expressed both kiaa0223 alleles . because kg - 1 does not express the restriction molecule hla - a2 . 1 necessary for t cell recognition , we transfected kg - 1 with hla - a2 . 1 and used these cells as target cells in a 51 cr - release assay with the ha - 1 specific t cell clone as effector cells . according to the cdna sequence analysis results , the kg - 1 cells were recognized by the ha - 1 specific t cell clone ( data not shown ). this result suggested that the kiaa0223 gene forms a di - allelic system of which the ha - 1 h allele leads to recognition by the mh antigen ha - 1 specific t cell clones . two families , who were previously typed for ha - 1 with ha - 1 specific ctl , were studied on the cdna level for their kiaa0223 polymorphism . the family members of family 1 were screened for their kiaa0223 sequence polymorphism by sequencing the ha - 1 encoding sequence region . all ha - 1 negative members displayed the ha - 1 r sequence , whereas all ha - 1 positive members turned out to be heterozygous , thus carrying both ha - 1 alleles ( fig3 a ). we subsequently designed ha - 1 allele specific pcr primers to screen another family previously cellularly typed for ha - 1 . both parents and one child were determined as heterozygous for ha - 1 , two ha - 1 negative children homozygous for the ha - 1 r allele and one child homozygous for the ha - 1 h allele ( fig3 b ). the screening of both families showed an exact correlation of the ha - 1 phenotype as determined by recognition by the ha - 1 specific t cell clones and the kiaa0223 gene polymorphism . to definitely prove that the kiaa0223 gene encodes the mh antigen ha - 1 , the ha - 1 encoding sequence region of kiaa0223 of both the ha - 1 h and the ha - 1 r alleles were cloned in a eukaryotic expression vector and transiently transfected in ha - 1 negative hela cells in combination with hla - a2 . 1 . ha - 1 specific t cell recognition of these transfected hela cells was assayed using a tnf - α release assay . the hela cells transfected with the ha - 1 h sequence containing vector were recognized by two ha - 1 specific t cell clones ( fig3 c ). in contrast transfection of the ha - 1 r sequence containing vector did not lead to recognition . in conclusion , our results clearly demonstrate that the mh antigen ha - 1 is encoded by the ha - 1 h allele of the kiaa023 gene . reconstitution and hla - a2 . 1 binding assays were performed to determine the capacity of ha - 1 r peptide vlrddllea ( seq id no : 5 ) to bind to hla - a2 . 1 and to be recognized by the ha - 1 specific t cell clones . the concentration of the ha - 1 r peptide that inhibited the binding of a fluorescent standard peptide to hla - a2 . 1 by 50 % ( ic50 ) was 365 nm , falling in the intermediate binders , whereas the ic50 of the ha - 1 h peptide was 30 nm , which is in the range of high affinity binders ( fig4 a ) 13 . 14 . different concentrations of vlrddllea ( seq id no : 5 ) were tested in a 51 cr - release assay with three ha - 1 specific t cell clones . one out of three clones ( 3ha15 ) tested showed recognition of the ha - 1 r peptide , but only at 1000 times higher peptide concentration than that necessary for the recognition of the ha - 1 h peptide ( fig4 b ). as the binding affinity of the two peptides to hla - a2 . 1 differs only 10 - fold , it can be concluded that all the t cell clones specifically recognize the ha - 1 h peptide . the 3ha15 t cell clone , recognizing the ha - 1 r peptide at high concentrations , does not recognize ha - 1 r homozygous individuals . this suggests that the vlrddllea ( seq id no : 5 ) peptide is not presented by hla - a2 . 1 or presented below the detection limit of the t cell . to determine whether the ha - 1 r peptide vlrddllea ( seq id no : 5 ) was presented by hla - a2 . 1 , hla - a2 . 1 bound peptides were eluted from a ha - 1 r homozygous ebv - blcl and fractionated with reverse phase hplc . the synthetic ha - 1 - peptide vlrddllea ( seq id no : 5 ) was run on reverse hplc to determine at which fraction this peptide eluted . the corresponding hplc fractions derived from the ha - 1 r expressing ebv - blcl were analyzed using mass spectrometry . presence of peptide vlrddllea ( seq id no : 5 ) could not be detected ( results not shown ), indicating that this peptide is not present or presented by hla - a2 . 1 in very low amounts on the cell surface . this is most likely due to the 10 - fold lower binding affinity of the peptide for hla - a2 . 1 . the supposed absence of the ha - 1 r peptide in hla - a2 . 1 indicates that this allele must be considered as a null allele with regard to t cell reactivity . this implicates that only bmt from an ha - 1 r / r ( ha - 1 −) donor to ha - 1 h / h or ha - 1 r / h ( ha - 1 +) recipient direction and not the reverse would be significantly associated with gvhd . this is indeed observed in a retrospective study in which hla - 2 . 1 positive bmt pairs were typed for ha - 1 3 . however , ha - 1 r derived peptides may bind to other hla alleles and possibly be recognized by t cells . if the latter peptides are not generated and presented by the ha - 1 h allele , then t cell reactivity towards the ha - 1 r allele may be envisaged and gvhd in that direction may occur . only a few murine and human mh antigens have been identified so far on the peptide and gene level . two murine mh antigens are encoded by mitochondrial proteins , leading to respectively four and two alleles 15 - 17 . in addition , two murine h - y mh antigens were shown to be peptides encoded by y - chromosome located genes 18 - 21 . the human smcy gene , located on the y chromosome , encodes the hla - b7 and the hla - a2 . 1 restricted h - y mh antigens 5 , 6 . of the human non - sex linked mh antigens , only the mh antigen ha - 2 has been sequenced on the peptide level , but the ha - 2 encoding gene remained unknown 4 . the identification of the gene encoding the mh antigen ha - 1 is the first demonstration that human mh antigens are derived from polymorphic genes . the ha - 1 encoding kiaa0223 gene has two alleles differing in two nucleotides leading to one single amino acid difference . however , because the kiaa0223 gene has not been fully sequenced yet , it remains to be established whether additional amino acid polymorphisms between the two alleles of this gene are present . because the ha - 1 mh antigen is the only known human mh antigen that is correlated with the development of gvhd after bmt the results of our study are of significant clinical relevance 3 . although the numbers of different human mh antigens is probably high , it is envisaged that only few immunodominant mh antigens can account for the risk for gvhd 22 . identification of those human immunodominant mh antigens and screening for those antigens may result in a significant decrease in gvhd after bmt . here we describe the first elucidation of a polymorphic gene encoding the immunodominant mh antigen ha - 1 . this enabled us to design ha - 1 allele specific pcr primers for pre - transplant donor and recipient typing to improve donor selection and thereby prevention of ha - 1 induced gvhd development . it also enabled us to start targeting leukemic cells carrying minor antigens present on hematopoietic cells . one way of arriving at agents targeting leukemic cells , is the ex vivo preparation of ctl &# 39 ; s . this is explained herein . allogeneic bone marrow transplantation ( bmt ) is a common treatment of hematological malignancies 29 . recurrence of the underlying malignancy is a major cause of treatment failure 30 , 31 . relapsed cml patients can be successfully treated by donor lymphocyte infusions ( dli ) 32 , 33 , but the treatment is less effective for relapsed aml and all 32 , 33 , and is frequently complicated with graft versus host disease ( gvhd ) 32 - 34 . donor derived ctls specific for patients &# 39 ; minor histocompatibility antigens ( mhags ) play an important role in both gvhd and gvl reactivities 10 , 35 - 38 . mhags ha - 1 and ha - 2 induce hla - a2 restricted ctls in vivo . mhags ha - 1 and ha - 2 are exclusively expressed on hematopoietic cells including leukemic cells 10 , 36 and leukemic precursors 37 , 38 , but not on cells of the gvhd target organs such as skin fibroblasts , keratinocytes or liver cells 8 . recently the chemical nature of the mhags ha - 1 and ha - 2 was unraveled 4 , 39 . the feasibility of ex vivo generation of mmag ha - 1 and ha - 2 specific ctls from unprimed mhag ha - 1 and / or ha - 2 negative healthy blood donors with the purpose of adoptive immunotherapy of relapsed leukemia with a low risk of gvhd is reported . to define the optimal antigen presenting cell ( apc ) for ex vivo generation of ha - 1 and ha - 2 specific ctls , we prepared peripheral blood mononuclear cells ( pbmc ), monocytes , peripheral blood circulating dendritic cells ( pbdc ) or dendritic cells derived from bone marrow cd34 + progenitor cells ( bmdc ) from fifteen hla - a2 positive , ha - 1 or ha - 2 negative healthy blood donors . these apcs were pulsed with ha - 1 and / or ha - 2 synthetic peptides and used to stimulate autologous unprimed cd8 + cells . the attempts to induce ha - 1 or ha - 2 specific ctls using monocytes or pbmc were not very successful . pbmc induced in only one out of three attempts ha - 2 specific ctls . using monocytes , we generated two ha - 1 peptide specific ctls , but these ctls did not lyse ha - 1 positive target cells in our experiments ( data not shown ). it is possible that these “ peptide specific ” ctls have a lower affinity for the naturally expressed ha - 1 antigen , but this does not mean that these cells cannot be used for generating ctl &# 39 ; s against minor antigens . pbdc were enriched from nine individuals to induce ha - 1 or ha - 2 specific ctls . in the four cases where the preparations had a purity of less than 30 % the ctls lysed peptide loaded target cells but not mhag positive target cells ( data not shown ). in contrast , in all cases ( n = 5 ) where pbdc purity was 30 % or more , the ctls not only recognized mhag negative , peptide pulsed target cells , but also mhag positive ebv - lcl , demonstrating the recognition of the naturally expressed ligand ( fig1 ). these results underscore the superior capacity of dc to induce t cell responses from naive precursors and confirm the current opinion 40 . similarly , two bmdc induced ctls that recognized both peptide pulsed target cells and ha - 1 positive target cells ( fig5 ). no cytotoxic activity was observed against autologous pha stimulated t cell blasts ( pha blasts ) or against mhag negative ebv - lcl . thus , neither autoreactivity nor “ third - party ” alloantigen reactivity was observed . several ha - 1 or ha - 2 specific ctl clones isolated from these ctls did not react against autologous cells either . these results show that ha - 1 and ha - 2 specific ctls can be safely transferred to patients after bmt . the ex vivo induced ha - 1 and ha - 2 specific ctls were tested for their hematopoietic cell restricted reactivity and compared with the in vivo induced ha - 1 and ha - 2 specific ctls ( fig6 ). pha blasts , but not fibroblasts ( neither after ifn - γ / tnf - α stimulation ) were recognized by both ex vivo and in vivo induced ha - 1 and ha - 2 specific ctls . fibroblasts , were only lysed after pulsing with the mhag peptides , demonstrating their susceptibility to ctl mediated lysis . these data not only confirm that the ha - 1 and ha - 2 antigens are functionally expressed solely on hematopoietic cells 8 , but also show that adoptive transfer of ha - 1 or ha - 2 specific ctls to ha - 1 or ha - 2 positive patients will spare the patient &# 39 ; s non - hematopoietic tissues and cells . thus , upon adoptive transfer of ha - 1 and ha - 2 specific ctls , a low risk of gvhd is to be expected . some precaution may be necessary since we have previously demonstrated that ha - 1 disparity between patient and donor is associated with the development of gvhd in adults 3 . therefore , we do not transfer the ctls before 50 - 60 days post bmt . it is assumed that most recipient hematopoietic cells are then replaced by donor cells . alternatively , one may transduce the ha - 1 and ha - 2 specific ctls with a suicide gene which will make the in vivo elimination of cells possible if adverse effects occur 41 . the ex vivo induced ha - 1 and ha - 2 specific ctls were subsequently analyzed for cytolytic activity against , for this study most relevant target cells , leukemic cells . in vivo induced ha - 1 and ha - 2 specific ctls and an hla - a2 specific alloreactive ctl were used as control effector cells . as shown in fig7 aml and all cells were lysed by hla - a2 specific alloreactive ctl , and by in vivo induced ha - 1 and ha - 2 specific ctls , indicating that the leukemic cells were positive for hla - a2 and expressed ha - 1 or ha - 2 antigens . as expected the ex vivo induced ctls lysed the leukemic cells comparable to the control effector cells . these results show that ha - 1 and ha - 2 specific ctls can also be used as therapy for relapsed aml or all , which are resistant to dli treatment . the level of cytotoxicity could be significantly enhanced following ifn - γand tnf - α treatment of the leukemic cells indicating that cytokines upregulated hla class i expression on the leukemic cells . ha - 1 and ha - 2 specific ctl clones produce ifn - γ and tnf - α ex vivo . it is possible that cytokine production by ha - 1 and ha - 2 specific ctls occurs in vivo as well . alternatively the efficacy of adoptive immunotherapy with ha - 1 and ha - 2 specific ctls may be enhanced by co - administration of ifn - α in resistant cases . the feasibility of adoptive immunotherapy with ex vivo generated ctls depends also on their expandability to sufficient numbers . we therefore scored the expansion rates of ha - 1 and ha - 2 specific ctls generated by dc . the results indicate that sufficient numbers of ctls for adoptive immunotherapy can be obtained if t cell cultures will be started with 5 × 10 7 responder cells . for instance , two ha - 2 specific ctls induced by pbdc showed expansion rates of above 9 , 25 and 8 fold at the second , third , and fourth week , respectively . these expansion rates translate into an estimated total yield of 3 × 10 9 - 10 10 ctls at the end of the fourth week . the expansion kinetics of the ha - 1 specific ctls were slower , but the cells expanded consistently with doubling times of 2 - 3 days during each restimulation . it is estimated that 10 9 ha - 1 specific ctls can be obtained after five weeks of culture . in conclusion , our results show for the first time that mhag ha - 1 and ha - 2 specific ctls can reproducibly be generated ex vivo from hla - a2 positive , mhag ha - 1 and / or ha - 2 negative healthy blood donors using dendritic cells pulsed with synthetic peptides . after the successful application of ebv - specific ctls as specific adoptive immunotherapy of ebv - related malignancies 42 , our results now provide a new possibility for the treatment of relapsed , ha - 1 and / or ha - 2 positive leukemia patients with ha - 1 or ha - 2 specific ctls induced ex vivo from their hla identical , mhag negative bone marrow donors . table 1 . kiaa0223 sequence polymorphism in mh ha - 1 positive and ha - 1 negative individuals . sequencing of ha - 1 region in kiaa0223 gene in ha - 1 +/+ and ha - 1 −/− homozygous individuals and kg - 1 revealed two alleles differing in two nucleotides resulting in a one amino acid difference ( h to r ) and designated ha - 1 h and ha - 1 r . for dh and vr 6 independent pcr products were sequenced . for kg - 1 8 pcr products were sequenced . cell culture the cd8 + hla - a2 . 1 restricted ha - 1 specific cytotoxic t cell clones 3ha15 , clone 15 and 5w38 were derived from pbmc of two patients who had undergone hla identical bone marrow transplantation 7 , 23 . the clones were cultured by weekly stimulation with irradiated allogeneic pbmc and blcl in rpmi - 1640 medium containing 15 % human serum , 3 mm 1 - glutamin , 1 % leucoagglutinin - a and 20 u / ml ril - 2 . the hla - a2 . 1 positive ha - 1 expressing ebv transformed b cell lines ( blcl ) rp and blk were maintained in imdm containing 5 % fcs . the kg - 1 and t2 cell lines were cultured in 1640 medium containing 3 mm 1 - glutamin and 10 % fcs . [ 0055 ] 51 cr - release assay . hplc fractions and synthetic peptides were tested in a 51 cr - release assay as described 24 . 2500 51 cr labeled t2 cells in 25 ml were incubated with 25 ml peptide dissolved in hanks 50 mm hepes for 30 minutes at 37 ° c . cytotoxic t cells were added in an end volume of 150 ml . when hplc peptide fractions were tested , t2 was incubated with 2 mg / ml ma2 . 1 during the 51 cr labeling . after 4 hours at 37 ° c . the supernatants were harvested . peptide purification . peptides were eluted out of purified hla - a2 . 1 molecules as earlier described 24 . briefly , hla - a2 . 1 molecules were purified two times from 90 . 10 9 hla - a2 . 1 positive ebv - blcl by affinity chromatography with bb7 . 2 coupled cnbr - activated sepharose 4b beads ( pharmacia lkb ) and extensively washed . peptides were eluted from the hla - a2 . 1 with treatment with 10 % acetic acid , further acidified by 1 % tfa and separated from the hla - a2 . 1 heavy chain and b2 - microglobulin by filtration over a 10 kd centricon ( amicon ) filter . peptides were fractionated using reverse phase micro hplc ( smart system , pharmacia ). for the first purification , three rounds of hplc fractionation were used to purify the hla - a2 . 1 restricted ha - 1 active peptide fractions from 90 . 10 9 rp cells . the first fractionation consisted of buffer a : 0 . 1 % hfba in h2o , buffer b : 0 . 1 % hfba in acetonitrile . the gradient was 100 % buffer a ( 0 to 20 min ), 0 to 15 % buffer b ( 20 to 25 min ) and 15 to 70 % buffer b ( 25 to 80 min ) at a flow rate of 100 ml / min . fractions of 100 ml were collected . fraction 24 of the first gradient was further fractionated . the second fractionation consisted of buffer a : 0 . 1 % tfa in h2o , buffer b : 0 . 1 % tfa in acetonitrile . the gradient was 100 % buffer a ( 0 to 20 min ), 0 to 12 % buffer b ( 20 to 25 min ), and 12 to 50 % buffer b ( 25 to 80 min ) at a flow rate of 100 ml / min . fractions of 100 ml were collected . a shallower third gradient was used to further purify fraction 27 that contained ha - 1 activity . the gradient was 100 % buffer a ( 0 to 29 min ), 0 to 18 % buffer b ( 29 to 34 min ), 18 % buffer b ( 34 to 39 min ), 18 to 23 . 9 % buffer b ( 39 to 98 min ) at a flowrate of 100 ml / min . 1 / 180 to 1 / 45 of the starting material was used to test for positive fractions in the 51 cr - release assay . comparable hplc fractionations were used for the second purification of hla - a2 . 1 restricted ha - 1 active peptide fractions from 90 . 10 9 blk . 40 % of the ha - 1 containing fraction 33 of the second ha - 1 purification was used for an additional reverse phase microcapillary hplc fractionation . buffer a was 0 . 1 % triethyl amine ( tea ) in water buffered to ph 6 . 0 with acetic acid and buffer b was 0 . 085 % tea in 60 % acetonitrile buffered to ph 6 . 0 with acetic acid . the gradient was 100 % buffer a ( 0 to 5 min ), 0 to 100 % b ( 5 to 45 min ) at a flow rate of 0 . 5 ml / min . fractions were collected in 50 ml of 0 . 1 % acetic acid every minute for 5 to 15 minutes , every 30 seconds from 15 to 20 minutes , every 20 seconds from 20 to 40 minutes , and every 30 seconds from 40 to 45 minutes . for each fraction collected , 20 % was used to test for ha - 1 activity and 80 % was used to obtain mass spectral data . mass spectrometry . fractions from third dimension hplc separation of the rp purification that contained the ha - 1 activity were analyzed by microcapillary hplc - electrospray ionization mass spectrometry 25 . peptides were loaded onto a c18 microcapillary column ( 75 mm i . d . × 10 cm ) and eluted with a 34 minute gradient of 0 to 60 % b , where solvent a was 0 . 1 m acetic acid in water and solvent b was acetonitrile at a flow - rate of 0 . 5 ml / min . one - fifth of the effluent was deposited into the wells of a 96 - well plate containing 100 ml of culture media in each well ( 10 seconds fractions ), while the remaining four - fifths was directed into the elctrospray source of the tsq - 70u . mass spectra and cad mass spectra were recorded on a finnigan - mat tsq - 7000 ( san jose , calif .) triple quadruple mass spectrometer equipped with an electrospray ion source . hla - a2 . 1 peptide binding assay . a quantitative assay for hla - a2 . 1 binding peptides based on the inhibition of binding of the fluorescent labeled standard peptide hbc 18 - 27 f to c6 ( flpsdcfpsv ) ( seq id no : 17 ) to recombinant hla - a2 . 1 protein and b2 - microglobulin was used 26 , 27 . in short , hla - a2 . 1 concentrations yielding approximately 40 - 60 % bound fluorescent standard peptide were used with 15 pmol / well ( 150 nm ) b2 - microglobulin ( sigma ). various doses of the test peptides were coincubated with 100 fmol / well ( 1 nm ) fluorescent standard peptide , hla - a2 . 1 and b2 - microglobulin for 1 day at room temperature in the dark in a volume of 100 ml in assay . buffer . the percent of mhc - bound fluorescence was determined by gel filtration and the 50 % inhibitory dose was deduced for each peptide using one - site competition non - linear regression analysis with the prismgraph software . synthetic peptides were manufactured on a abimed 422 multiple peptide synthesizer ( abimed , langenfeld , germany ) and were more than 90 % pure as checked by reverse phase hplc . total or mrna was prepared from blcl using the rnazol method ( cinaa / biotecx laboratories , houston , tex .) or according to manufacturer &# 39 ; s instructions ( quickprep mrna purification kit , pharmacia biotech ). cdna was synthesized with 1 mg rna as template and with kiaa0223 based reverse primer 5 ′- gctcctgcatgacgctctgtctgca - 3 ′ ( seq id no : 6 ). to amplify the ha - 1 region of kiaa0223 the following primers were used : forward primer 5 ′- gacgtcgtcgaggacatctcccat - 3 ′ ( seq id no : 7 ) and reverse primer 5 ′- gaaggccacagcaatcgtctccagg - 3 ′ ( seq id no : 8 ). cycle parameters used were denaturation 95 ° c ., 1 min , annealing 58 ° c ., 1 min and extension 72 ° c ., 1 min ( 25 cycles ). the pcr - products were purified using the magic pcr - preps dna purification system ( promega ) and direct cloned using the pmosblue t - vector kit ( amersham life science ). six independent colonies from each individual were sequenced using the t7 - sequencing kit ( pharmacia biotech ). in the case of ha - 1 allele specific pcr amplification , cdna was synthesized as described above . a pcr amplification was performed with allele specific forward primers : for the ha - 1 h allele primer h1 : 5 ′- cct - tga - gaa - act - taa - gga - gtg - tgt - gct - gca - 3 ′ ( seq id no : 9 ), for the ha - 1 r allele primer r1 : 5 ′- cct - tga - gaa - act - taa - gga - gtg - tgt - gtt - gcg - 3 ′ ( seq id no : 10 ) 0 and for both reaction the reverse primer as described above was used . cycle parameters used were denaturation 95 ° c ., 1 min , annealing 67 ° c ., 1 min and extension 72 ° c ., 1 min ( 25 cycles ). cloning and expression of ha - 1 h and ha - 1 r allelic region of kiaa0223 . a forward kiaa 00223 based pcr primer containing an atg start codon ( 5 ′- ccg - gca - tgg - acg - tcg - tcg - agg - aca - tct - ccc - atc - 3 ′) ( seq id no : 11 ) and a reverse kiaa0223 based pcr primer containing a translational stop signal ( 5 ′- cta - ctt - cag - gcc - aca - gca - atc - gtc - tcc - agg - 3 ′) ( seq id no : 12 ) were designed and used in a rt - pcr reaction with cdna derived from an homozygous ha - 1 h and a homozygous ha - 1 r blcl . cycle parameters used were denaturation 95 ° c ., 1 min , annealing 60 ° c ., 1 min and extension 72 ° c ., 1 min ( 25 cycles ). the desired pcr - products were purified using the magic pcr - preps dna purification system ( promega ). the purified dna was direct cloned using the pmosblue t - vector kit ( amersham life science ) and recloned in the eukaryotic pcdna3 . 1 (+) vector under the control of a cmv promotor . transient cotransfections were performed with hla - a2 . 1 in hela cells using deae - dextran coprecipitation . after 3 days of culture ha - 1 specific t cells were added and after 24 hours the tnf - α release was measured in the supernatant using wehi cells 28 . peptides : ha - 1 and ha - 2 peptides were synthesized using a semi automatic multiple peptide synthesizer 4 , 3 . the purity of the peptides was checked by reversed phase high pressure liquid chromatography ( hplc ). pbmc were isolated by ficoll - hypaque density gradient separation of blood collected with manual hemapheresis . pbdc were enriched from pbmc by depletion of t cells , monocytes , b and nk cells as described earlier . briefly , t cells were depleted by sheep red blood erythrocyte rosetting . non - t cells were cultured 36 h at 37 ° c . in rpmi + 10 % autologous plasma . after depleting monocytes non adherent cells were layered on 14 . 5 % metrizamide gradients and centrifugated . the light density pbdc were recovered from the interphase . pbdc were identified by facs being negative for cd3 , cd14 , cd16 and cd19 and positive for hla - dr . the preparations contained 2 - 6 × 10 6 cells with a dc content of 20 - 50 %. in some cases the light density cells were further depleted from cd14 and cd19 cells using antibody coated magnetic beads . bmdc were differentiated from bone marrow cd34 + cells ( isolated using cd34 + isolation kit , macs , bergisch gladbach , germany ) by culturing with 100 ng / ml flt3 - ligand ( genzyme , leuven ; belgium ), 30 ng / ml il - 3 , 25 ng / ml scf ( genzyme ) 50 u / ml tnf - α ( genzyme ), 250 u / ml gm - csf ( genzyme ) for 10 to 14 days . the cultures contained 20 - 60 % dc as detected by high levels of dr and negative expression of cd3 / cd14 / cd16 / cd19 . ex vivo induction of ha - 1 and ha - 2 specific ctls : apc were pulsed with ha - 1 or ha - 2 peptides ( both 10 mg / ml ) for 90 min . at 37 ° c . in serum free aim - v medium . after washing , apc and 10 - 15 × 10 6 responder cells ( cd4 depleted autologous pbmc ) were cultured at different apc responder cell ratios depending on the type of apc ( 5 : 1 , 1 : 3 and 1 : 10 for pbmc , mo and dc , respectively ) in 24 well culture plates . culture medium was rpmi supplemented with 10 % autologous plasma , 1 u / ml il - 2 ( cetus ), 1 u / ml il - 12 ( genzyme ). the cells were kept at 37 ° c . in a humidified , 5 % co 2 − air mixture . at day 5 , 10 u / ml of il - 2 was added . starting from day seven , the t cell cultures were restimulated weekly with peptide pulsed autologous monocytes . 10 u / ml of il - 2 was added 24 h . after each restimulation . the t cell lines were expanded with 10 - 20 u / ml il - 2 containing culture medium . cytotoxicity ( 51 cr release ) assays : standard 4 h 51 cr release assays using pha - blasts , ebv - blcl and fibroblasts and leukemic cells as target cells were performed as described before 8 . the percent specific lysis was calculated using the following formula : 100 ×( cpm experimental release - cpm spontaneous release )/( cpm maximal release - cpm spontaneous release ). target cells : ebv - blcl were generated as described before 8 and cultured in rpmi plus 10 % fcs . pha activated t cell blasts ( pha - blasts ) were obtained by stimulation of pbmc with 0 . 1 mg / ml pha ( wellcome ) during 72 h . pha - blasts were expanded with medium containing 20 u / ml il - 2 . skin fibroblasts of an hla - a2 +, ha - 1 +, ha - 2 + healthy individual were isolated , cultured and tested as described before 8 . in short , fibroblasts were trypsinized and cultured in the wells of 96 well flat bottomed microtiter culture plates at a concentration of 3 × 10 3 cells / well with or without addition of ifn - γ and tnf - α ( both 300 u / ml ) during 72 h . when indicated , target cells were pulsed with ha - 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