Patent Application: US-30344805-A

Abstract:
the present invention relates to a peptide which is immunologically recognizable as a t cell epitope of the minor histocompatibility antigen h - y . the peptide comprises amino acid sequence spsvdkarael or fidsyicqv . the peptide is obtainable from the minor histocompatibility antigen h - y . providing a toxic moiety to the peptide eliminates t cells having specific binding affinity for the peptide . the peptide induces tolerance for transplantations when administered to h - y - negative recipients .

Description:
as with other mhag , the recognition of h - y by t lymphocytes is mhc - restricted ( 3 , 24 , 25 ), and it has been shown that some h - y antigens are peptides derived from cellular proteins that are presented on the cell surface in association with mhc class i molecules ( 26 ). we have developed a technique for the identification of individual peptides that are bound to mhc molecules and recognized as antigens by t cells . by combining microcapillary liquid chromatography / electrospray ionization mass spectrometry with t cell epitope reconstitution assays , we previously identified peptide antigens recognized by t cells specific for human melanoma ( 27 ), human xenografts ( 28 ), and a non - sex - linked human mhag ( 22 ). we now report the identification of a peptide antigen recognized by a human cytotoxic t lymphocyte ( ctl ) clone that is h - y specific and restricted by the class i mhc molecule hla - b7 , as well as a peptide antigen that is recognized by two hla - a2 . 1 restricted ctl clones . to isolate endogenously processed h - y peptides , hla - b7 molecules were purified by affinity chromatography from the h - y positive , b lymphoblastoid cell line , jy ( 29 ). the associated peptides were extracted in acid and separated from high molecular weight material by ultrafiltration as previously described ( 31 ), and subsequently fractionated by reverse - phase high - performance liquid chromatography ( hplc ) ( 27 ). aliquots of each fraction were incubated with hla - b7 positive , h - y negative t2 - b7 target cells in order to assay for the ability to reconstitute the epitope recognized by an hla - b7 - restricted , h - y specific ctl clone , 5w4 ( ref . 12 ). a single peak of reconstituting activity was observed ( fig1 a , fraction 28 and 29 ), which was rechromatographed using a different organic modifier . although a single active peak of reconstituting activity was also observed from this separation ( fig1 b , fraction 14 , 15 and 16 ), it still contained more than 100 distinct peptide species , as assessed by electrospray ionization tandem mass spectrometry . to identify active h - y peptides in this mixture , we applied each active fraction separately to a microcapillary hplc column and split the effluent following the separation ( 11 ): four - fifths of the effluent was directed into the mass spectrometer for analysis , while one - fifth was simultaneously directed into a 96 - well microtiter plate for a subsequent epitope reconstitution assay . the amount of the h - y sensitizing activity in each well was correlated to signals observed in the mass spectrum , and therefore to the abundance of different peptide species . by comparing the profile of h - y activity and the ion abundance data ( fig2 ), we were able to identify an ( m + 3h )+ 3 ion at a mass - to - charge ratio ( m / z ) of 391 ( neutral molecular mass = 1171 ), whose abundance correlated with the amount of h - y epitope reconstituting activity . further confirmation of the importance of peptide 1171 was provided by the demonstration that a peptide with an identical mass and collision - activated dissociation ( cad ) spectrum was also present in hla - b7 associated peptides extracted from a second h - y positive b lymphoblastoid line , dm , but absent from a spontaneous h - y antigen loss variant of this cell , dm (−) assignment of a complete amino acid sequence to the 1171 peptide from the cad mass spectrum recorded at the 20 fmol level proved difficult due to the absence of high mass fragment ions containing the amine terminus ( b - type ions ). a series of single and / or doubly charged fragment ions containing the amine terminus ( b - type ions ). a series of single and / or doubly charged fragment ions containing the carboxyl terminus ( y - type ions ) identified the c - terminal residue as either l or i and the first six amino acids as spsvdk ( seq id no : 7 ). the difference in molecular mass between this partial sequence and that of the full length peptide suggested the presence of four additional residues , for a total length of 11 . since the candidate peptide existed exclusively in the gas phase as an ( m + 3h )+ 3 ion , and underwent mass shifts of 42 and 84 da on conversion to the corresponding methyl ester and acetylated derivative , respectively , two of the remaining residues were assigned as r and either d or e . only two combinations of four residues ( area ( seq id no : 8 ) and grdv ( seq id no : 9 )) meet the above criteria and satisfy the missing mass of 427 da . cad spectra recorded on synthetic peptides suggested that r could not be located at either position 7 or 10 . data bases were searched for proteins containing peptides with these characteristics , and a sequence consistent at 9 out of 11 positions was found in residues 909 - 919 of the protein encoded by a gene called xe169 or smcx ( 34 ), which is located on the x chromosome . a homolog of smcx , called smcy , is located on the y chromosome ( 20 ). this protein ( 35 ) contains a sequence ( residues 902 - 912 ) that is consistent at 11 out of 11 positions , and has the expected mass of 1171 da . a cad mass spectrum recorded on the naturally processed material after conversion of the r residue to ornithine confirmed that its sequence was identical to that found in smcy protein ( fig3 ). in the same manner as described above for the hla - b7 restricted t - cell clone , the peptide recognized by two hla - a2 . 1 t - cell clones was identified . in short the hla - a2 . 1 restricted h - y specific t cell clone r416 recognizes hplc fraction 34 , the hla - a2 . 1 restricted h - y specific t clone 1r35 recognizes hplc fractions 36 and 39 ( fig6 ). the amino acid sequence analyses and h - y reconstitution assays demonstrate that both hla - a2 . 1 restricted h - y specific t cell clones recognize peptide sequence fidsyicqv ( seq id no : 2 ) with a m / z ratio of 544 or the cystinylated form of the same peptide with a m / z ratio of 604 . a synthetic peptide corresponding to the 11 residue smcy sequence ( spsvdkarael ( seq id no : 1 )) was found to sensitize t2 - b7 cells for recognition by the h - y specific ctl clone . half - maximal lysis was achieved at a peptide concentration of 10 pm ( fig4 ). the corresponding peptide derived from the sequence of the x chromosomal homolog , smcx , has substitutions of a for s at position 3 and q for r at position 8 . although this peptide also was able to sensitize t2 - b7 cells for recognition , comparable levels of killing were only achieved by using a 10 , 000 - fold higher peptide concentration . binding studies showed that the concentration of the smcy peptide that inhibited the binding of an iodinated standard peptide to purified hla - b7 by 50 % ( ic50 ) was 34 nm , while the ic50 for the smcx peptide was 140 nm ( fig5 ). thus , the significant difference in the ability of the smcy and smcx peptides to sensitize targets for t cell recognition is almost entirely due to the fine specificity of the t cell receptor , rather than to differences in mhc binding affinities . the smcx peptide is also present in naturally processed peptide extracts of hla - b7 , although its abundance is only 25 % of that of the smcy peptide ( 33 ). based on all of this information , it is concluded that the peptide epitopes representing the hla - b7 restricted h - y antigen is derived from the protein encoded by smcy , which is also true for the hla - a2 . 1 recognized peptide , also encoded by smcy . the location of the smcy gene and the control of its expression fit well with those expected of the h - y antigen based on previous work . deletion mapping in humans has placed the hy locus to a portion of interval 6 on the long arm of the human y chromosome ( 18 ), and smcy maps to this same interval ( 20 ). h - y antigens are expressed ubiquitously in different tissues ( 5 , 15 ), and expression of smcy has been detected in all male tissues tested ( 20 ). one interesting issue is whether the h - y epitope peptides presented by other mhc molecules will also be derived from smcy . smcy and smcx are 85 % identical at the amino acid sequence level , and the smcx gene is expressed ubiquitously from both the active and the inactive x chromosomes in both mice and human ( 34 , 36 ). therefore , self - tolerance to smcx will limit the number of smcy peptides that could give rise to h - y epitopes in association with different mhc molecules . on the other hand , smcy contains almost 1500 residues , and the over 200 amino acid sequence differences between it and smcx are scattered relatively uniformly throughout its length . thus , there is the potential to generate a large number of distinct smcy - specific peptides as h - y epitopes . it is still an open question whether the h - y epitope peptides presented by other mhc molecules are also derived from smcy . genetic mapping of the mouse y chromosome has suggested at least two and up to five distinct loci encoding h - y antigens ( 37 ). interestingly , a murine h - y epitope restricted by h - 2kk has also been shown to be derived from the murine smcy protein ( 38 ). the demonstration that two h - y epitopes from either mouse or human are derived from the same protein makes smcy the prime target in searching other h - y epitopes . the identification of the protein that gives rise to an h - y antigen culminates 40 years of uncertainty regarding its origin . however , the function of smcy , as well as the homologous smcx , remains unclear . both proteins share significant sequence homology to retinoblastoma binding protein 2 , which has been suggested to be a transcription factor ( 39 ). nonetheless , this and other h - y specific peptides are candidates for immunomodulatory approaches in bone marrow transplantation . they may also form the basis for genetic probes to be used for prenatal diagnosis in sex - linked congenital abnormalities , as well as for investigating minimal residual disease and chimerism . 1 . e . j . eichwald , c . r . silmser . : transplant bull ; 148 - 149 , 1955 . 2 . r . e . billingham , w . k . silvers : studies on tolerance of the y chromosome antigen in mice . j . immunol . 85 : 14 - 26 , 1960 . 3 . e . goulmy , a . termijtelen , b . a . bradley , j . j . van rood . alloimmunity to human h - y . lancet ii : 1206 , 1976 . 4 . e . goulmy , a . termijtelen , b . a . bradley , j . j . van rood . y - 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