compositions and processes for the identification of tumor-specific neoantigens.

专利摘要:
  COMPOSITIONS AND PROCESSES FOR THE IDENTIFICATION OF TUMOR-SPECIFIC NEOANTIGENSThe present invention relates to immunotherapeutic peptides and their use in immunotherapy, in particular cancer immunotherapy. Specifically, the invention provides a process for identifying tumor-specific neoantigens that alone or in combination with other tumor-associated peptides serve as active pharmaceutical ingredients of vaccine compositions that stimulate antitumor responses.
公开号:BR112012029066A2
申请号:R112012029066-5
申请日:2011-05-16
公开日:2020-09-01
发明作者:Nir Hacohen；Cartherine Wu
申请人:The General Hospital Corporation；Dana-Farber Cancer Institute, Inc；
IPC主号:

专利说明:

[0001] [0001] Recently, several procedures for incorporating the guided nucleotide 30 by primer for testing polymorphic sites in DNA have been described (Komher, JS et al., Nucl. Acids. Res. 17: 7779-7784 (1989); Sokolov , B, P., Nucl. Acids Res. 18: 3671 (1990); Syvanen, A.-C., et al., Genomics 8: 684-692 (1990); Kuppuswamy, M- N. et al., Proc. Natl. Acad. Sci. (US.A-) 88: 1143-1147 (1991); Prezant, TR et al., Hum. Mutat. 1: 159-164 (1992); Ugozzoli, L. et al. , GATA 9: 107-112 (1992); Nyren, P. et al., Anal. Biochem. 35 208: 171-175 (1993)). These processes differ from GBA in that they are all based on the incorporation of deoxynucleotides marked for discrimination between bases in a polymorphic site. In such a format, since the sign is proportional to the number of deoxynu-
embedded cleotkieos, polymorphisms that occur in runs of the same nucleotide can result in signals that are proportional to the run length (Syvanen, A, -C., et al., Amer. j. Hum. Genet. 52: 46-59 (1993 )). A number of initiatives are currently being undertaken to obtain sequence information 5 directly from millions of individual DNA or RNA molecules in parallel. Sequencing technologies - through simple molecule synthesis in real time - rely on the detection of fluorescent nucleotides when they are incorporated into a nascent DNA strand that is complementary to the template being sequenced. In one process, oligonucleotides 30-50 bases in length are covalently anchored 10 at the 5 'end to glass cover strips. These anchored tapes serve two functions. First, they act as capture sites for target template tapes if the "templates are configured with complementary capture tails for surface-bound oligonucleotides. They also act as primers for the template-directed primer extension that forms the basis for reading The capture primers function as a fixed position site for sequence determination using multiple synthesis, detection, and chemical cleavage sites of the dye-to-dye to remove the dye. labeled nucleotide / polymerase, rinse, image formation and dye cleavage.In an alternative process, the polymerase is modified with a fluorescent donor molecule and immobilized on a glass "lamina", while each nucleotide is coded - color with a fluorescent receiving and log half bound to a gamma phosphate. The system detects the interaction between a fluorescently targeted polymerase and a fluorescently modified nucleotide when nucleotide 6 becomes incorporated into the strand again. There are also other sequencing-through-synthesis technologies.
. Preferably, any appropriate sequencing-through-synthesis platform can be used to identify mutations. As described above, four major sequencing-through-synthesis platforms are currently available: the Genome Sequencers from Roche / 454 Life sciences, the lL Illuminator / Solexa lg Analyzer, the SOL1D system from Applied BioSystems, and the Heliscope sytem from Helicos Biosciences . Sequencing-through-synthesis platforms have also been described by Pacific BioSciences and VisiGen Biotechnologies. Each of these platforms can be used in the processes of the invention. In some embodiments, a pIurality of nucleic acid molecules being sequenced is linked to a support (for example, solid support). To immobilize the nucleic acid on a support, a universal priming site / capture sequence can be added 35 at the 3 'end and / or 5' end of the mold. Nucleic acids can be linked to the support by capturing sequence hybridization to a complementary sequence covalently linked to the support. The capture sequence (also referred to as a
universal capture frequency) is a sequence of nucleic acids complementary to a sequence linked to a support that can serve as a universal primer. As an alternative to a capture sequence, a member of an ion coupling pair (such as, for example, antibody / antigen, receptor / ligand, or the avidin-5-biotin pair as described in, for example, US patent
N "2006/0252077) can; be connected to each fragment to be captured on a surface coated with a respective second member of that coupling pair.
Subsequent to capture, the sequence can be analyzed, for example, by detecting / sequencing simple molecules, for example, as described in Examples 10 and in the U.S. patent.
No. 7,283,337, including sequencing-through template-dependent synthesis.
In sequencing-through-synthesis, the "surface-binding" molecule is exposed to a plurality of labeled triphosphate nucleotides in the presence of polymerase.
The sequence of the template is determined by the number of labeled nucleotides incorporated at the 3 'end of the growing strand. This can be done in real time or can be done in a step-and-repeat mode.
For real-time analysis, different optical labels for each nucleotide can be incorporated and multiple lasers can be used for stimulation of incorporated nucleotides.
Any type of cell or tissue can be used to obtain samples of nucleic acid + for use in the diagnostics described here.
In a preferred embodiment, the sample "20 of DNA or RNA is obtained from a tumor or a body fluid, for example, blood, obtained ¶is through known techniques (for example, vein piercing) or saliva.
Alternatively, nucleic acid tests can be performed on dry samples (for example, hair or skin). Alternatively, protein mass spectrometry can be used to identify or validate the presence of mutated peptides linked to MHC proteins on tumor cells.
Peptides can be eluted with acid from tumor cells au HLA molecules that are immunoprecipitated from tumor, and then identified using mass spectrometry.
Neoantigenic Peptides 30 The invention further includes isolated peptides that comprise the specific tumor mutations identified by the processes of the invention, peptides that comprise known tumor-specific mutations, and mutant polypeptides or fragments thereof identified by the process of the invention.
These peptides and polypeptides are referred to herein as "neoantigenic peptides" or "neoantigenic polypeptides". The term "peptide" is used interchangeably with "mutant peptide" and "neoantigenic peptide" in this specification to designate a series of residues, typically L-amino acids, connected to each other, typically through peptide bonds between groups. alpha-amino
~ carboxyl of adjacent amino acids. Similarly, the term "polypeptide" is used interchangeably with "mutant polypeptide" and "neoantigenic polypeptide" in this descriptive report to designate a series of residues, typically L-amino acids, connects - from each other, typically by peptide bonds between the alpha amino and carbohydrate groups of adjacent amino acids.
Polypeptides or peptides can be of a variety of lengths, either in their neutral (uncharged) forms or in forms that are salts, and free of modifications such as g | jcosi | action, side chain oxidation, or phosphoryl - tion or containing these modifications, subject to the condition that the modification does not destroy the biological activity of the polypeptides as described herein. 10 In certain embodiments, the size of at least one neoanitogenic peptide molecule can understand, but not. littered at, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16 , about 17, about 18, about 18, about 19, about 20, about
- 21, about 22, about 23, about 24, about 25, about 26, about 27, about 15 28, about 29, about 30, about 31, about 32, about 32 33, about 34, about 35, about 36, about 37, about 38m about 39, about 40, about 41, about 42, about 43, about 44, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about '110, about 120 or more waste amino molecule, and any derivable band there.
In "20 specific embodiments, the neoantigenic molecules are equal to or less than 50 amino acids".
In some embodiments, the particular neoantigenic peptides and peptides "of the invention are: for MHC class 1 13 residues or less in length and usually consist of between about 8 and about 11 residues, particularly 9 or 10 residues; for '25 MHC Class ll, 15-24 residues.
A longer peptide can be designed in several ways.
In one case, when HLA-binding peptides are predicted or known, a longer peptide may consist of: (1) individual ligand peptides with 2-5 amino acid extensions towards the N- and C- terminus of each corresponding gene product : (2) a 30 concatenation of some or all of the peptide ligands with extended strings for each.
In another case, when sequencing reveals a long (> 10 residues) sequence of neoepitope present in the tumor (for example, due to a structure deviation, reading through or intron inclusion that leads to a new peptide sequence). a longer peptide may consist of: (3) the entire spine of new tumor-specific amino acids - thus 35 deflecting the need for computational prediction or in vitro peptide binding tests to HLA proteins.
In both cases, the use of a longer peptide allows endogenous processing through patient cells and can lead to more effective presentation.
antigen and induction of T cell responses.
Neoantigenic peptides and polypeptides bind an HLA protein.
In some respects, neoantigenic peptides and polypeptides bind an HLA protein with greater affinity than a wild-type peptide.
The neoantigenic peptide or polypeptide has an IC 50 of less than 5000 nM, at least less than 500 nM, at least less than 250 nM, p and less than 200 nM, at least less than 150 nM, at least less than 100 nM, at least less than 50 nM or less.
Neoangiogenic peptides and polypeptides do not induce an autoimmune response and / or invoke immunological tolerance when administered to a subject.
The invention also provides compositions comprising at least two or more neoantigenic peptides.
In some embodiments, the composition contains at least two distinct peptides.
Preferably, the at least two distinct peptides are derived from the same polypeptide.
By different polypeptides it is intended that the peptide varies through length, sequence of amino acids or both.
The peptides are derived from any known or verified pdipeptide using the processes of the invention to contain a specific tumor mutation.
Suitable polypeptides from which the anti-antigenic peptides can be derived can be found, for example, in the COSMlC database (htte: //www.sanqer.ac.uWcosmic) COSMlC adds comprehensive information on somatic mutations in human cancer.
The peptide contains the tumor-specific mutation.
In some respects, the specific tumor mutation is a conductive mutation for a particular type of cancer.
In some respects, the peptides are derived from an SF3B1 polypeptide, a MYD88 polypeptide, a TP53 polypeptide, an ATM polypeptide, an Abl polypeptide, an FBXW7 polypeptide, a DDX3X polypeptide, a MAPKI polypeptide of a GNBI polypeptide.
By an SF3E31 peptide it is intended that the peptide contains a portion of an SF3B1 polypeptide. Preferably, an SF3B1 peptide infuses a leucine at the amino acid position 625; a histidine at amino acid position 626: a glutamic acid at amino acid position 700; an aspartic acid at the amino acid position 742; or an arginine at amino acid position 903, when numbered according to wild type SF3B1.
A wild type SF3B1 is shown in Table A (SEQ ID NO: 1). '): Akiakthedjeaqireiqgkkaa | deaqgvgldstgyydqeiyggsdsr agyvtsiaate | edddddyssstsl | | gqkkpgyhapva ndipqsteq ydpfaehrppkiadredeykkhrrtmiispeddpfadggktpdpkmnar tymdvmreqh | tkeerejrqq | aekakage | kwngaaasqppsk "nw dqfadqtpgatpkklsswdqaetpghtpslrwdetpgrakgsetpgatpg skiwdptpshtpagaatpgrgdtpghatpghggatssarknrwdetpkte rdtpghgsgwaetpddrggdsigetptpgaskrksrwdetpasqmggst Pvltpgktpigtpamnmatptpghimsmtpeqlqawrwereidernrpls dee | damfpegykvlpppagyvpinparkltatptp | ggmtgfhmqted Rmksvndgpsgn | mp | kpddiqyfdk | vdvdest | sDeeakerkimkl!
| kikngtppmrkaa | rqitdkarefgagp | fnqijp | mspt | edqerh | vkvidri | yk | dd | vrpyvhki | wÍep | lidedyyamegreiisn | akaaglatmistmrpdidnmdeyvmttarafawasalgipsllpAka vckskkswqarhtgjkivqqiai | mgcai | ph | rs | vrtisa veiiehglvdeqqk | AIAA | aeaatpygiesfdsv | kplwkgirqhrgkg | aaflkai gy | ip mdaeyanmrevm |! ilirefqspdeemkkivlkwkqccgtdg veanyikteilppffkhMqhrmaldrrnyrqlvdtNelankvgaaeii srivddlkdeaeqyrkmvmetiekimgnlgaadidh% eeqlidgüyaf qeqttedsvm | ngf9Nvna | gkrvkpy | pqicgMwrlnnksakvrqq aad | isrtawmktcqeek | mgh | gw | yey | geeypev] gsi | g | kai vnvigmhkmtppikdúprltpilknrhekvqencidlvgriadrgaeyv sarewmricfe and | | kahkkairratvntfgyjakaigphdv | | at nn | kvqerqnNcttvaiaivaetcspftv | palmneyrvpe1nvqngv | ks | sf] feyigemgkdyiyavtpl | eda] MDRD | v | 1rqtasawqhms | gvy gfgcedsInh | nyvwpnvfetsphviqavmgaleg | wajgpcmlqyc | hq | fhparkvrdvywkiynsiyigsqdaliahypnynddkntyjrye | "'r.
dyil By a MYD88 peptide it is intended that the peptide contains a portion of a MYD88 polypeptide. Preferably, a MYD88 peptide includes a threonine at amino acid position 232: a leucine at amino acid position 258; or a proline at amino acid position 265, when numbered according to MYD88 wild type. A wild type MYD88 5 is shown in Table B (SEQ ID NO: 2).
a Table B: MYD88 Seivation Type (SEQ ID NO: 2) mrpdraeapgppamaaggpgagsaapvsstss | p | aa | nmNm | s | f | ·
W rwrtqvaadMalaeemdfeyleirqletqadptgrlldawqgrpgasvg rHelltklgrddvúelgpsieedcqkyilkqqqeeaekplqvaavdss d. vpdaelagittlddplghmperfdaficycpsdiqNqemirqleqtny & - rlklcvsdrdvlpgtcvwsiaseliekrcmnvwvsddylqskecdfqt kfalslspgahqkr | ipikykamkkefpsi | TPID | Preferably, the TP53 peptide includes both an arginine in position 'of amino acid 111; an arginine at amino acid position 215; a serine at amino acid position 238: a glutamine at amino acid position 248: a phenyl alanine at amino acid position 255; a cysteine at amino acid position 273 or an asparagine at amino acid position 281, when numbered according to TP53 wild type. A wild type TP53 is shown in Table C (SEQ ID NO: 3).
Table C: Wild Type P53 (SEQ ID NO: 3)! Meepqsdpsvepp | sqetfsd | vdk | pennvlsp | psqamdd | m | spddi eqwnedpgpdeaprmpeaappvapapaaptpaapapapswplsssypsq ktyqgsygfr | gf | hsgtaksvtcNspa | nkmfcq | aktcpvq | wvdst pppgtrvramaiykqsqhmtevvrrcphhercsdsdglappqhlimegn kvey | ddrntfrhswvpyeppevgsdcuihynymcnsscmggmnu i | tiit | edssgn | grnsfevrvcacpgrdrReeen | rkkgephhe | p pgstkra | pnntssspqpkkkp | dgeyft | qirgrerfemfre] nea! e | kdaqagkepggsrahssh | kskkgqstsrhkkímfktegpdsd By an ATM peptide, the peptide is intended to contain a portion of an SF3B1 polypeptide. Preferably, an ATM peptide includes a phenyl alanine at the position of '15 amino acid 1252; an arginine at the amino acid position 2038; a histidine at amino acid position 2522; or a cysteine at amino acid position 2954, when numbered according to wild type ATM.
A wild type ATM is shown in Table D (SEQ ID NO: 4). Table D: Wild Type ATM (SEQ ID NO: 4) mslvlndHiccrqlehdraterkkevekNrlirdpetikhldrhsdsk qgkylnwdavfmqkgqketeclriakpnvsastqasrqkkmqeiss | vkyfikcanrraprlkcqellnyimdtvkdssngaiygadcsnillkdil svrky Mceisqqqwle | fsvyfr | y | kpsqdvhrv | qtdg vanihadkgccs | nskndffskaiqcarqeksssglnhi | pa | tifjktfavnfnr vce | gdei] PTL] yiwtqhr [ndsIkeviieifq | qiyjhhpkgaktqek gayestkwrsilyn | | vneishigsrgkyssgfrniavken yd | ielma dichqnnedtrs | eisqsytttqressdysvpckrkkielgwevikdh | qksqndfd | vpwlqiatq | iskypas | pnce | sp |] mi | sq | lpqqrhge,. rtpyv | rc1teva | cqdkrsn | essqksd | k | wnkiwciÜrgisseqi, qaenfgIIgaijqgs | vevdreMklftgsacrpscpavcc | t | a | TTSI vpgtvkmgieqnmcevnrsfsIkesimkwhfyqlegdlenstevppilh snfph | vleki | vs | tmknckaamnffqsvpecehhqkdkee | sfsevee | flqttfdkmdf | tiwecgiekhqssigfsvhqn | kes | drcj | glseq | lnnysseitnseUvrcsr | psv | gcycymgviaeeeaykse | fqkak s [mqcagesit | fknktneefrigs | rnmmq | CTC | gff snctkkspnkias | r | tskImndiadickslasfjkkpfdrgevesmeddtngn | Meve dqssmn | $ fndypds vsdanepgesqstigainplaeey | skqd | f] dm LKF | c] cvttaqtntvsfraadirrk | lmIjdsstleptksIh | hmylml jke | pgeeyp | pmedv | e | lkplsnvcs | yrrdqdvcktjlnhv | hvvkn lgqsnmdsentrdaqgqfNigaMhltkerkyifsvrmalvnclktil eadpyskwailnvmgkdfpvnevftqfladnhhqvrmlaaesiMfqdt kgdssrl | k | p] k | qqtafenay | kaqegmremshsaenpet | deiynr ksvl | t | iavv | scspicekqa] fdcksvkeng | eph | fgyrr vkkvlekvset | edfmash | dylv | w | n | qdteynjssfpfil | nytniedfyrs cykv1iphlvirshfdevksianqiqedwksl | tdcfpki | NIV | pyfay egtrdsgmaqqretatkvydm | ksen | gkqidh] fisn | peivvel | mt vocepanssasqstdlcdfsgdldpapnpphfpshvikatfayisnchktk "| ksi | ei | skspdsyqki | aiceqaannvy | Kiyh | fvs1 | l vcqtavtyckda kdiksglggawafvlrdviytlihyinqrpscimdvslrsfslccdhsq | enh | hvivgt | IP | vyeqvevqkqvld | LKY | vidnk dnen | | yitik dpfpdhwfkd | ritqqkikysrgpfsl | svyda eeinhflsv | p | tr | eglkd | rrqle | hkdqmvdimrasqdnpqdgimvk | VVN | Q | skmainhtgekevleavgsc [gevgpidfstiaiqhskdasytka | k | fedkelqwtfimlN | nntIvedcvkvrsaavtclkni | atktghsfN ejykmttdpm | aylqpfRsrkknevprfdkenpfeg | ddin | wip | se nhdiwikt | tca¶dsggtkcei | q | kpmcevktdfcqdi lqdtneswrnl | sthvqgfftsc | rhfsqtsrsttpan | dsesehHrcc ldkksqrtmlavvdymrrqkrpssgtifndaMldlnylevakvaqscaa hfta | yaeiyadkksmddqekrs | afeegsqsttiss | lqdl sekskeetgis | eiyrsigepdslygcgggkm | qpitrlRyeheamwgka | vtyd | etaipsstrqagiiqa | qnlg | chilsvy | kgldyenkdwcpe | ee | hy qaawmmqwdhctsvskevegtsyhesiyna | qs | rdrefsÜyes | rvkeveemckrslesvys kya | YPT | sr | qaige | esige | fsrsdhrq | se vyikwqkhsq | kdsdfsfqepima | rtvi | ei | mekemdnsqrecikdi | tkh | ve | si | amntq1peraifqikqynsvscgvsewq | eeaqvfN akkeqs | alsi | kqmkgn enpavimqtylekavevagnydgessdelrngkmkaflslarfsdtqyqr ienymkssefenkqaIlkrakeevgl [rehkiqtnWtvkvqre | e | de | Alra | | kedrkmckavenyinc sqeehdmwvfr | cslw] ensavsev O, 24/57 ngmmkrdgmkiptykf 4 | p | myqlaarmgtkmmggIgfhev | nn | isrism dhphht | REF | a | ananrdef | tkpevarrsritknvpkqssq | dedrte aanriictirsrrpqmvrsvealcdayiilanldatqwktqrkginipad qpitklknledvwptmeikvdhtgeygnlvtiqsfkaefrlaggvnlpk iidcvgsdgkerrqlvkgrddlrqdavmqqvfqmcnthqrntetrkrkl) ticNkvvplsqrsgv | ewctgtvpigenvnnedgahkryrpndfsafq cqkkmmevqkksfeekyevfmdvcqnfqp 'ycmekHdpaiwfekr | aytrsvatssivgyi | g | gdrhvqnilineqsae (vhid | gvafeqgkj | v | ptpetvpfrltrdivdgmgitgvegvfrrccektmevmrnsqetlítive YDP | fdwtmnp | k | y | qqrpedete | hpt | naddqeckrnfsdidq sfnkvaervlmr | QEK | | kgveegtvlsvggqvn iqqaidpkn | srlfp, _ For a gwkawv Abl peptide is desired that the peptide contains a portion of an Abl polypeptide. Preferably, a Bcr-abl peptide includes a valine in an amino acid position 244; a valine in an amino acid position 248; a glutamic acid in an amino acid position 250; an alanine in an amino acid position 250; a histidine in position of 5 amino acid 252; an arginine in position of amino acid 252; a phenyl alanine in position of amino acid 253; a histidine in position of amino acid 253; a lysine in position of amino acid 255; a valine in position of amino acid 255; a glycine in position of amino acid 276; an isoleucine in position of amino acid 315; an asparagine in position of amino acid 315; a leucine in position of amino acid 317; a threonine in po- "" 10 position of amino acid 343; a threonine at amino acid position 351; a glycine in
W 355 amino acid position; a valine at amino acid position 359; an alanine at amino acid position 359; an isoleucine 'at amino acid position 379; a leucine at t: amino acid position 382; a methionine at amino acid position 387; a proline at amino acid position 396; an arginine at amino acid position 396; a tyrosine at the '15 position of amino acid 417; or a serine at amino acid position 486, when numbered according to wild type Abl. A wild type Abl is shown in Table E (SEQ "ID NO: 5).
)
· K 25/57 '- / d
J - ¥ 5 For an FBX N7 peptide it is intended that the peptide contains a portion of an FBXW7 polypeptide. Preferably, an FBXW7 peptide includes a leucine at amino acid position 280: a histidine at amino acid position465; a cysteine at amino acid position 505; or a glutamic acid in position of amino acid 597, when numbered according to wild type FBXW7. A wild type FBXW7 is shown in Table F (SEQ ID NO: 6).
it F: FBXW7 Wild type (SEQ ID NO: 6) mnqellsvgskrrrtggslrgnpsssqvdeeqmnrweeeqqqqlrqqee ehtamgevvgveprpggqndsqqgqleennnhisvdedssgnqeeqee deehageqdeedeeeeemdqesddfdqsddssredehthtnsvtnsssiv € dlpvhqisspfytkdkmkrkldhgsevrsfslgkkpckvseytsttglv pcsatptÜgd | raangqgqqrrntsvqpptg | QEW | kmfqswsgpek | lalde | idsceptqvkhmmqviepqfqrdnsi | pke | a | yv | sf1epkd | lqaaqtcrywri aednl |! wrekckeegideplhikrrkvjkpgnhsp and wksayjrqhridtnwrTge | kspkv | kghddhvitc | nt qfcgnnvsgsdd | geciht kvwsavtgkchmghtggvwssqmrdniiisgstdRlkvwnaet | yghtsNrcmhlhekrwsgsrdat | mwdietgqc | hv | mgh r vaavrcvqydgrmsgaydfmvkvwdpetetclhtlqghtnmyslqfd gihvvsgs | dtsiNwdvetgnciht {tghqsItsgme | kdni | vsgnad stvkiwdiktgqc | qt | qgpnkhqsavtc | qfnknfvitssddgNk | wd | ktgenrn | vt] exhaustgsggvvwrjrasntk | vcavgsrngteetk | v | dfdvdmk For a DDX3X peptide it is intended that the peptide contains a portion of a DDX3X polypeptide. A DDX3X peptide is a peptide that is the result of a non-synonymous mutation in amino acid position 24; a splice site at amino acid position 342 or a structure shift at amino acid position 410 when numbered according to wild type DDX3X. A wild type DD3X is shown in Table G (SEQ ID NO: 7). Table F: DDX3X Wild type (SEQ ID NO: 7) mshvavenalgldqqfag | d | nssdr1qsggstaskgryipph | rnreatk gfydkdssgwssskdkdayssfgsrsdsrgkssffsdrgsgsrgrfddrg rsdydgigsrgdrsgfgkferggnsrwcdksdeddwskplppserleqel fsggntginfekyddipveatgnncpphiesfsdvemgeiimgnieltry trptpvqkhaipiikekrdlmacaqtgsgktaaf1lpilsqiysdgpgea
~ Lramkengrygrrkqypislvlaptrelavqiyeearkfsyrswrpcw yggadigqqirdIergchl1vatpgdvdmmergkig dfckylvldead rmldmgfepqirriveqdtmppkgvrhtmmfsaHpkeiqmlardRdey if1avgNgstsenitqkvvwveesdkrsf | d | natgkds | T | vfvet kkgadsledflyhegyactsihgdrsqrdreealhqfrsgkspilvatav aarg | disnvkhvinfd | psdieeyvhrigrtgmgn | g | | d | atsKnerni nitkd veakqevpsw1enmayehhykgssrgrskssnsggfgar dyqssgassssfsssrasssrsgggghgssrgfggggyggfynsdgygg nynsqgvdwwgn For a MAPKI peptide is desired that the peptide contains a portion of a poIipeptide MAPKI. Preferably, a MAPKI peptide includes an asparagine at amino acid position 162; a glycine at amino acid position 291; or a phenyl alanine at amino acid position 316, when numbered according to wild type MAPKI. + .- 5 A wild type MAPKI is shown in Table H (SEQ ID NO: 8). Table F: MAPKI Wild Type (SEQ ID NO: 8) maaaaaagagpemvrgqddvgprytnlsyigegaygmvcsaydnvnkw vaikkispfehqtycqrUreikillrfrheniigindiiraptieqmkd vyivqdlmetd] yk | | kk | kpsn | Inttcd | kicdfglarvadpdhdhtgnteµatrwyrapejm | nskgytksidiwsvgcilaemlsnrpifpgkhyldqlnhilgiIgspsqe d [nciin | kamyl | s | phknkvpwnr | fpnadska | dl | dkmltfnphk rieveqa | ahpy1eqyydpsdepiaeap a | gn | Preferably, a GNBI peptide includes a threonine at the position of the amino acid 180, when numbered according to wild type GNBI. A GNBI type «
Wild W is shown in Table I (SEQ ID NO: 9).
t "Fssê ISII ==" "" _ " 'mtcayapsgnyvacggldnicsjynlktregnvrvsre |! aghtgy | SCCR f | ddnqivtssgdttca | wdietgqqtMtghtgdvmsls | apdtnN sgacdasaklwdvregmcrqthghesdinaicffpngnafatgsddatc r | fd | radqe | mtyshdniicgitsvsfsksgr [) lagyddfncnvwdal kadragvlaghdnrvsclgvtddgmavatgswds¶kiwn 10 neoantigênicos peptides and polypeptides having the desired activity can be modified when necessary to provide certain desired attributes, for example, improved pharmacological characteristics, while increasing or at least substantially retaining all the biological activity of the unmodified peptide to bind the desired MHC molecule and activate the appropriate T cell. For example , the neoantigenic peptide and polypeptides 15 can be subjected to various changes, such as substitutions, both conseNative and non-conservative, where such changes can provide certain advantages in their use, such as improved MHC binding. replacement of a waste amino acid with another that is biologically and / or chemically similar, for example, a hydrophobic residue on the other, or a polar residue 20 on the other. Substitutions include combinations such as Gly, Ala; Val, lle, Leu, Met; Asp,
&. Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. The effect of simple amino acid substitutions can also be probed using D-amino acids. Such modifications can be made using well-known peptide synthesis procedures, as described in, for example, Merrifield, Science 232: 341-347 (1986), Barany & Merrifield, The Peptides, Gross 5 & Meienhofer, eds. (N.Y., Academic Press), pp. 1-284 (1979); and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, |., Pierce), 2nd Ed. (1984). Neoantigenic peptides and polypeptides can also be modified by extending or decreasing the compound's amino acid sequence, for example, by adding or removing amino acids. Peptides, polypeptides or Arabia can also be modified by changing the order or composition of certain residues. P, duos, it is easily appreciated that certain residues give essential amino acids for biological activity, for example, those in critical contact sites or conserved residues, generally cannot be altered without an adverse effect on biological activity. Non-critical amino acids need not be limited to those naturally occurring in 15 proteins, such as L-alpha-amino acids, or their D-isomers, but they can also include unnatural amino acids, such as j3, y, õ-amino acids, as well like many derivatives of L-alpha-amino acids. "Typically, a series of peptides with simple amino acid substitutions" are employed to determine the effect of electrostatic charge, hydrophobicity, etc., on
V s 20 connection. For example, a series of positively charged + "(e.g. Lys or Arg) or negatively charged (e.g. Glu) amino acid substitutions are made along the length of the peptide revealing different patterns of sensitivity in the" direction "of various MHC molecules and T cell receptors. In addition, multiple substitutions using relatively neutral, small halves, such as Ala, Gly, Pro, (similar residues can be employed. Substitutions can be homo - oligomers Olj hetero - oligomers. O number and types of residues that are replaced or added depend on the necessary spacing between essential contact points and certain functional attributes that are sought (for example, hydrophobicity versus hydrophilicity). Increased binding affinity for an MHC molecule or T cell receptor 30 can also be obtained through such substitutions, compared to the affinity of the parent peptide. employ amino acid residues or other molecular fragments chosen to avoid, for example, stereo and charge interference that can disrupt the bond. Amino acid substitutions are typically simple residues. Substitutions, 35 deletions, insertions or any combination of them can be combined to arrive at a final peptide. Substitution variants are those where at least one residue of a peptide has been removed and a different residue inserted in its place. Such b
Ü 28/57 ^ substitutions are generally made according to the following Table when it is desired to finely modulate the characteristics of the peptide.
Original Residue Exemplary Substitution Ala Ser Arg Lys, His Asn Gln Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His,. Lys; Arg lle Leu; Val Leu lle; Val Lys Arg; His Met Leu: lle Phe Tyr; Trp Ser Thr Thr Ser Trp Tyr; Phe '"~ Tyr Trp; Phe · Val lle; Leu"% Pro Gly Substantial changes in function (eg affinity for MHC molecules or g T cell receptors) are made by selecting substitutions that are less con- than the ones in the Table above, that is, selection of residues that differ most significantly in their effect on maintaining (a) the structure of the peptide 7 'main chain in the substitution area, for example, as a leaf or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (C) the volume of the side chain. The substitutions that are generally expected to produce the greatest changes in peptide properties will be those in which (a) hydrophilic residue, for example, seryl, is a substitute for (or by) a 'hydrophobic residue, for example, leucyl, isdeucyl, phenyl alanyl, valyl or alanyl; (b) a residue having an electropositive side chain, for example, iisyl, argillite, or histidyl, is a substitute for (or by) an electronegative residue, for example, gutamyl or asparta; or (C) a residue having a bulky side chain, for example, phenyl alanine, is a substitute for (or by) one not having a side chain, for example glycine.
Peptides and polypeptides can also comprise isosteres of two or more residues in the neoantigenic peptides or polypeptides. An isostere as defined here is a sequence of two or more residues that can be replaced by a second sequence due to the stereo conformation of the first sequence to adapt to a specific binding site for the second sequence. The term specifically includes modifications
: 6.
main chain peptide reactions well known to those skilled in the art. Such modifications include modifications of nitrogen amide, alpha carbon, carbonyl amide, complete substitution of the amide bond, extensions, deletions or crosslinking of the main chain. See, generically, Spatola, Chemistry and Biochemistry of Amino Acids, Peptoides and Proteins, Vol. Vil (Weinstein ed., 1983). Modifications of peptides and polypeptides with mimetic caries of amino acids or unnatural amino acids are particularly useful in increasing the stability of the peptide and polypeptide in vivo. Stability can be tested in a number of ways. For example, peptidases and various biological media, such as plasma and human serum, have been used to test stability. See, for example, Verhoef et al., Eur. J. Drug Metab Pharmacokin. 11: 291-302 (1986). The half-life of the peptides of the present invention is conveniently determined using a 25% (V / v) human serum assay. The protocol is generally as follows. Collected human serum (Type AB, non-thermally inactivated) is delipidated by centrifugation before use. The serum is then diluted to 25% with RPMI tissue culture medium and used to test for peptide stability. At predetermined time intervals, a small amount of reaction solution is removed and added to 6% aqueous trichloroacetic acid or ethanol. The cloudy reaction sample is cooled (4 ° C) "for 15 minutes and then rotated to pellet precipitated whey proteins. The presence of the peptides is then determined by reverse phase HPLC using specific chromatography conditions - e Peptides and polypeptides can be modified to provide desired attributes other than improved serum half-life, for example, the ability of 'peptides to induce CTL activity can be enhanced by linking to a sequence that contains at least least one epitope that is capable of inducing a "25 helper T cell response. Particularly preferred helper T conjugates / immunogenic peptides are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. Spacers are typically selected from, for example, Ala, Gly, or other neutral spacers of 30 non-polar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer does not need to be comprised of the same residues and thus can be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues. Alternatively. the peptide can be attached to the helper peptide T without a spacer. 35 The neoantigenic peptide can be linked to the auxiliary T peptide either directly or via a spacer at the amino terminus or carboxy of the peptide. The amino terminus of the neoantigenic peptide or auxiliary T peptide can be acylated. T helper peptides
S 30/57 ± specimens include tetanus toxoid 830-843, influenza 307-319, circumsporozoic malaria 382-398 and 378-389. Proteins or peptides can be manufactured using any technique known to those versed, including the expression of proteins, polypeptides or peptides 5 using standard techniques of molecular biology, the isdation of proteins or peptides from natural sources, or the synthesis protein or peptide chemistry. The nucleotide and protein, polypeptide and peptide sequences corresponding to several genes have been previously shown, and can be found in computerized databases known to those skilled in the art. One such database is the National Center for Bioinformation Technology's Genbank and genpept databases located on the National Institutions of Health website. The coding regions for known genes may be: "am '' '- simplified and / or expressed using the techniques shown here or as may be known to those skilled in the art. Alternatively, several commercial preparations of proteins, polypeptides and peptides are known to those skilled in the art.15 In yet another aspect, the invention provides a nucleic acid (eg, polynucleotide) encoding a neoantigenic peptide of the invention The polynucleotide can be, for example, DNA, CDNA, PNA, CNA, RNA , either of double conio single tape, or native forms d
W or stabilized polynucleotides, such as, for example, polynucleotides with a phosphorothioate main chain, or combinations thereof, and it may or may not contain introns as much "20 as it encodes the peptide. Of course, only peptides containing amino residues , * naturally occurring naacids linked by naturally occurring peptide bonds are encodable by a polynucleotide. Another aspect of the invention provides an expression vector capable of expressing a polypeptide according to the invention. cells are well known in the art and can be selected without undue experimentation. Generically, the DNA is inserted into an expression vector, such as a plasmid, in its own orientation and correct reading frame for expression. If necessary, the DNA can be linked to the appropriate nucleotide sequences of translational and transcriptional regulatory control recognized by the desired host, although such controls are generally available in the expression vector. The vector is then introduced into the host using standard techniques. Guidance can be found, for example, in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratoy, Cold Spring Harbor, NY Vaccine Compositions The present invention is directed to an immunogenic composition, for example, a vaccine composition capable of elevating a specific T cell response The vaccine composition comprises mutant peptides and mutant polypeptides corresponding to tumor-specific neoantigens identified through the processes described herein.
& Those skilled in the art will be able to select preferred peptides, polypeptides or a combination of them through tests, for example, the generation of T cells in vitro as well as their efficiency and total presence, proliferation, affinity and expansion. are from certain T cells to certain peptides, and the functionality of T cells, for example, 5 through analysis of IFNy production or tumor death by T cells. Usually, the most efficient peptides are then combined as a vaccine. An appropriate vaccine preferably will contain between 1 and 20 peptides, more preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , or 20 different peptides, still preferred 6, 7, 8, 9, 10 11, 12, 13, Olj 14 different peptides, and more preferably 12, 13 or 14 different peptides. In one embodiment of the present invention, different peptides and / or polypeptides are selected so that a vaccine composition comprises peptides and / or polypeptides capable of association with different MHC molecules, such as different MHC class molecules I. Preferably, a vaccine composition comprises 15 most frequently occurring peptides and / or polypeptides capable of association with MHC class I molecules. Therefore vaccine compositions according to the invention comprise different fragments capable of association with at least 2 preferred, more & preferably at least 3 preferred, even more preferably at least 4 preferred. fast MHC class I molecules.
W "'20 The vaccine composition is capable of elevating a specific cyclic T - cell response and / or specific helper T cell response. The vaccine composition may further comprise an adjuvant and / or a carrier. 'Examples of useful adjuvants and carriers are given below. The peptides and / or polypeptides in the composition can be associated with a tai carrier, such as "25 a protein or a cell showing tat antigen, such as a dermal cell (DC) capable of presenting the peptide for a T cell. Adjuvants are any substances whose mixture in the vaccine composition increases or otherwise modifies the immune response to the mutant peptide. Carriers are tabulated structures, for example, a polypeptide or polysaccharide, to which the neoantigenic peptide is able to be associated Optionally, adjuvants are conjugated covalently or non-covalently to the peptides or polypeptides of the invention.The ability of an adjuvant to increase the immune response to an antigen is typically manifested by a significant increase in mediated reaction - immune, or reduction in disease symptoms, for example, an increase in humoral immunity is typically manifested by a significant increase in the titer of elevated antibodies to the antigen, and an increase in T cell activity is typically manifested in increased cell proliferation, Olj cell cytotoxicity, or cytokine secretion. An adjuvant can also
I '! "32157' i
P y ~
B alter an immune response, for example, by changing a Th response or primarily humoral to a Th response, or primarily cellular. Suitable adjuvants include but are not limited to 1018 lSS, aluminum salts, Amplivax, AS15, BCG, CP-870.893, CpG7909, CyaA, dSLIM, GM-CSF, lC30, IC31, lmi- 5 quimod, lmuFact IMP321, lS Patch, ISS, ISCOMATRIX, juvlmmune, LlpoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP- EC, ONTAK, PepTeLRTM. vector system, PLG mictoparticles, resiquimod, SRL172, Virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glycan, Pam3Cys, Aquila's QS21 stimulon (Aquila Biotech, 10 Worcester, Mass., USA) which is derived from saponin, mycobacterial extracts and synthetic bacterial cell wall mimetics, and other proprietary adjuvants such as Ribi's Detox. Quil or Superfos. Adjuvants such as incomplete Freund's or GM-CSF are preferred. Several immunological adjuvants (eg, MF59) specific for dendritic cells and their preparation have been previously described (Dupuis M, et al., Cell Immunol. 1998; 15 186 (1): 18-27; Allison AC; Dev Biol Stand. 1998 ; 92: 3-11). Cytokines can also be used. Several cytokines have been directly linked to influence migration of dendritic cells to lymphoid tissues (eg, TNF-alpha), accelerating the maturation of dendritic cells in efficient cells presenting antigen to T lymphocytes (eg,> "GM-CSF, IL-1 and IL-4) (US patent No. "5 849 589, specifically incorporated herein by" 20 reference in its entirety) and acting as immunoadjuvants (for example, IL-12) (Gabri- - "lovich DI, et al., lmmunother Emphasis Tumor lununol 1996 (6): 414-418). CpG immunostimulatory oligonucleotides have also been reported to improve the effects of adjuvants in a vaccine set. Without being trapped by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system via the "Toll-like 25 res (TLR), mainly TLR9. Activation of TLR9 triggered by CpG improves cellular and humoral responses antigen-specific for a wide variety of antigens, including peptide or protein antigens, live or dead viruses, dendritic cell vaccines, autologous cell vaccines and polysaccharide conjugates in both prophylactic and therapeutic vaccines. More importantly, it improves maturation and differentiation. dendritic cell association, resulting in improved THI cell activation and strong generation of cytotoxic T lymphocyte (CTL), even in the absence of CD4 T-cell aid. The THI trend induced by TLR9 stimulation is maintained even in presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA) that normally promotes a TH2 trend. CpG oligonucleotides even show i adjuvant activity when formulated or co-administered with other adjuvants or in formulations! such as microparticles, nanoparticles, lipid emulsions or similar formulations, which are especially necessary for inducing a strong response when the antigen is relatively weak.
They also accelerated the immune response and allowed antigen doses to be reduced by approximately two orders of magnitude, with comparable antibody responses to the full-dose vaccine without CpG in some experiments (Arthur M.
Krieg, Nature Reviews, Drug Discovery, 5, Jun. 2006, 471-484). U.S. Patent No. 6,406,705 B1 describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants and an antigen to induce an antigen-specific immune response.
A commercially available TLR9 CpG antagonist is dSLIM (double-stem loop immuno modulator) by Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other TLR binding molecules such as RNA ligand TLR 7, TLR 8 and / or TLR 9 can also be used- Other examples of useful adjuvants include, but are not limited to, chemically modified CpGs (eg, CpR, leader), poly (l: C) (for example, polii: Cl2U), non-CpG bacterial DNA or RNA as well as small immunoreactive molecules and antibodies such as phosphamide cycle, sunitinib, bevacizumab, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinibe, XL-999, CP- 547632, pazopanib, ZD2171, AZD21 71, ipilimumab, tremelimumab, and SC58175, which can act therapeutically and / or as an adjuvant.
The amounts and concentrations of adjuvants and additives useful in the context of the present invention can be easily determined by those skilled in the art without undue experimentation.
Additional adjuvants include colony stimulating factors, such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim). A vaccine composition according to the present invention can comprise more than one different adjuvant.
In addition, the invention encompasses a therapeutic composition comprising any adjuvant substance including any of the above or combinations thereof.
It is also contemplated that the peptide or polypeptide, and the adjuvant, can be administered separately in any appropriate sequence.
A carrier can be present regardless of an adjuvant.
The function of a carrier may, for example, be to increase the molecular weight of a particular mutant in order to increase its activity or immunogenicity, to confer stability, to increase biological activity, or to increase serum half-life.
In addition, a carrier can assist in the presentation of peptides to T-cells.
The carrier can be any suitable carrier known to those skilled in the art, for example, a cell containing protein or an antigen.
A carrier protein can be, but is not limited to, clean lock hemocyanin, whey proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulin
bulines, or hormones, such as insulin or palmitic acid.
For human immunization, the carrier must be a physiologically acceptable carrier for humans and safe.
However, tetanus toxoid and / or diphtheria toxoid are appropriate carriers in an
Use of the invention.
Alternatively, the carrier may be dextrans, for example, sefaro.
Cytotoxic T-cells (CTLs) recognize an antigen in the form of a peptide bond to an MHC molecule before the foreign antigen itself is intact.
The 5 MHC molecule itself is located on the cell surface of a cell showing antigen.
Thus, activation of CTLS is only possible if a trimeric complex of peptide antigen, MHC molecule, and APC is present.
Correspondingly, it can improve the immune response if not only the peptide is used for CTL activation, but additionally APCS with the respective MHC molecule are added.
Therefore, in some - 10 but embodiments, the vaccine composition according to the present invention "additionally contains at least one cell, showing antigen.
The cell showingÓ. , the antigen (or stimulator cell) typically has a Class I or II MHC molecule on its surface, and in one embodiment is substantially incapable of itself carrying the MHC class I or Il molecule with c) selected antigen.
As described in more detail below, 15 the class l or | MHC can be easily loaded with the selected antigen in vitro.
Preferably, the cells showing antigen are dendritic cells.
Appropri-. namely, dendritic cells are autologous dendritic cells that are pulsed with the neoantigenic peptide.
The peptide can be any appropriate peptide that elicits an appropriate T-cell response.
T-cell therapy using autologous dendritic cells. "%
"'pulsed with peptides from a tumor-associated antigen is shown in Murphy et al. (1996) The Prostate 29, 371-380 and Tjua et al. (1997) The Prostate 32, 272-278." Thus, in one embodiment of the present invention the vaccine composition containing at least one cell showing antigen is pulsed or loaded with one or more "25 peptides of the present invention.
Alternatively, peripheral blood mononuclear cells (PBMCS) isolated from a patient can be loaded with peptides ex vivo and injected back into the patient.
As an alternative, the antigen presenting cell comprises an expression construct encoding a peptide of the present invention.
The polynucleotide can be any appropriate polynucleotide and it is preferred that it is capable of transducing a dendritic cell, thus resulting in the presentation of a peptide and induction of immunity.
Therapeutic Processes The invention further provides a process for inducing a tumor specific immune response in a subject, vaccination against a tumor, treating and / or alleviating a cancer symptom in a subject by administering a neoantigid peptide to the subject. - single or vaccine composition of the invention.
The subject has been diagnosed with cancer or is at risk of developing cancer. O
The subject has an imatinib-resistant tumor.
The subject is a human, dog, cat, horse or any animal in which a tumor specific immune response is desired. the tumor is any solid tumor such as breast, ovarian, prostate, lung, kidney, gastric, colon, testicular, head and neck, pancreas, brain, melanoma, and other 5-organ tissue tumors and hematological tumors, such as lymphomas and leukemias, including acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, and B-cell lymphomas.
The peptide or composition of the invention is administered in an amount sufficient to induce a CTL response. . In specific embodiments, the invention provides processes for treating an imatinib resistant tumor by administration to a subject of one or more neoantigenic peptides that contain a bcr-abl mutation.
In some embodiments, the subject is HLA-A3. Bcr-abl mutations include, for example, T315l, E255K, M351T, Y253H, Q252H, F317L, F359V, G250E, Y253F, E355G, E255V, M244V, L248V, G250A, Q252R.
D276G, T315N, M343T, F359A, V379l, F382L, L387M, H396P, H396R, S417Y, F486S. The peptide, neoantigenic polypeptide or vaccine composition of the invention can be administered alone or in combination with other therapeutic agents.
The therapeutic agent is, for example, a chemotherapeutic agent, radiation, or immunotherapy.
Any- "any appropriate therapeutic treatment for a particular cancer may be administered." Examples of chemotherapeutic agents include, but are not limited to, aldesleukin, '20 altretamine, amifostine, asparaginase, bleomycin,' capecitabine, carboplatin, camustine, 2 cladribine, cisapride, cisplatin, phosphamide cycle, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, epoetin alfa, etoposide, filgrastim, fludarabine, fluorine "uracil, gemcitabine, urine, hydroxy, hydroxide, granisetron, hydroxy , irino-tecano, lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide, 25 mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (Taxol®), pilocarpine, procloroperazine, rituximab, rituximab, tamoxamine, rituximab, rituximab, rituximab topotecan, trastuzumab, vinblastine, vincristine and vinorelbine tartrate.
For treatment of prostate cancer, a preferred chemotherapeutic agent with which anti-CTLA-4 can be combined is paclitaxel (Taxol). In addition, the subject can still be administered with an immune stimulating / anti-immune suppressing agent.
For example, the subject is further administered with an anti-CTLA or anti-PD-1 or anti-PD-L1 antibody. Blocking CTLA-4 or PD-LI by antibodies can enhance the immune response to cancer cells in the patient.
In particular, CTLA-4 blockade has been shown to be effective when following a vaccination protocol. 35 The optimal amount of each peptide to be included in the vaccine composition and the optimal dosage regimen can be determined by those skilled in the art without undue experimentation.
For example, the peptide or its variant can be prepared for
P intravenous injection (i.v.), subcutaneous injection (S.C.), intradermal injection (i.d.), intraperitoneal injection (j-p.), Intramuscular injection (i.m.). Preferred peptide injection processes include sc, id, ip, im, and iv preferred DNA injection processes include id, im, sc, ip, and iv For example, doses between 1 and 500 mg, 50 micrograms and 1.5 mg, preferably 125 micrograms to 500 micrograms, of peptide or DNA can be given and will depend on the respective peptide or DNA. Doses in this range have been used successfully in previous experiments (Brunsvig PF, et al., Cancer lmmunol lmmunother. 2006; 55 (12): 1553-1564; M- Staehler, et al., ASCO meeting 2007: Abstract No 3017). Other methods of administering the vaccine composition are known to those skilled in the art. 10 The inventive pharmaceutical composition can be compiled so that the selection, number and / or quantity of peptides present in the composition is / are specific to tissue, cancer, and / or patient. For example, the exact selection of peptides can be guided by patterns of expression of related proteins in a given tissue to avoid side effects. The selection can be dependent on the specific type of cancer, the disease status, previous treatment regimens, the patient's immune status, and, of course, the patient's HLA haplotype. In addition, the vaccine according to the invention can contain individualized components, according to the personal needs of the particular patient. Examples include varying the amount of peptides according to the expression of the related neoantigen.
W in the particular patient, unwanted side effects due to personal or other allergies - '20 treatments, and adjustments for secondary treatments following a first round or es-. ¶F treatment breakdown. For a composition to be used as a cancer vaccine, peptides whose 'endogenous related proteins are expressed in high amounts in normal tissues will be avoided or will be present in low amounts in the composition of the invention. Per . '"25 on the other hand, if it is known that a patient's tumor expresses high amounts of a certain protein, the respective pharmaceutical composition for the treatment of this cancer may be present in high amounts and / or more than one specific peptide for this particular protein or pathway of this protein can be tilted Pharmaceutical compositions comprising the peptide of the invention can be administered to an individual already suffering from cancer In therapeutic applications, compositions are administered to a patient in an amount sufficient to elicit an effective response CTL for the tumor antigen and to cure or at least partially prevent siritomas and / or complications - An adequate amount to accomplish this is defined as a “therapeutically effective dose.” Effective amounts for this use will depend, for example, on peptide composition , the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient and the judgment of the attending physician, but generally varies for the initial immunization (that is, for
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Therapeutic or prophylactic administration) of about 1.0 microgram to about 50,000 micrograms of peptide for a 70 kg patient, followed by booster or about 1.0 microgram to about 10,000 micrograms. micrograms of peptide according to a booster regimen over weeks to months depending on the patient's response and condition 5 by measuring specific CTL activity in the patient's blood. It should be kept in mind that the peptide and compositions of the present invention can generally be employed in serious disease states, that is, life-threatening or potentially life-threatening situations, especially when the cancer has metastasized. In such cases, in view of the minimization of foreign substances and the relative non-toxic nature of the peptide, it is possible and can be felt desirable by the physician trying to administer substantial excesses of these peptide compositions. For therapeutic use, administration should begin with the detection or surgical removal of tL | mores. This is followed by booster doses until at least symptoms are substantially diminished and for a period to follow. 15 Pharmaceutical compositions (for example, vaccine compositions) for therapeutic treatment are intended for parenteral, topical, nasal, oral or local administration. Preferably, the pharmaceutical compositions are administered parenterally, e.g.
For example, intravenously, subcutaneously, intradermally, or intramuscularly. "The compositions can be administered to the surgical excision site to induce a 6" 20 local immune response to the tumor. The invention provides compositions for administration for-. ©, renteral that comprise a solution of the peptides and vaccine compositions are dispersed or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, for example, water, buffered water, 0.9% saline, 0.3 ° 6 glycine, hyaluronic acid and the like- These compositions can be sterilized using well-known, conventional sterilization techniques, or can be sterilized in filtration The resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. pharmaceutically acceptable auxiliaries as required to approximate physiological conditions, such as buffering and pFl adjusting agents, tonicity adjusting agents, wetting agents and the like, eg sodium acetate, sodium lactate, sodium chloride , potassium chloride, calcium chloride, sorbitan mono laurate, triethanolamine oleate, etc. The peptide concentration of the invention in pharmaceutical formulations can vary 35 riate widely, that is, from less than about 0.1%, usually at or at least about 2 ° / 0 to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., according to the particular mode of administration selected.
The peptide of the invention can also be administered via liposomes, which target the peptides to a particular cell tissue, such as lymphoid tissue - Liposomes are also useful in increasing the half-life of the peptides.
Liposomes include emulsions, 5 foams, micelles, insoluble monolayers, liquid crystals, dispersions of phospholipids, Iamel and similar layers.
In these preparations the peptide to be released is incorporated as part of a liposome, alone or in conjunction with a molecule that binds, for example, to a predominant receptor among lymphoid cells, such as monoclonal antibodies that bind to the CD45 antigen, or with other therapeutic or immunogenic compositions.
Thus, liposomes filled with a desired peptide of the invention can be targeted to the lymphoid cell site, where the liposomes were then the selected therapeutic / immunogenic peptide compositions.
Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a steroid, such as co-ester. 15 The selection of lipids is generally guided by considering, for example, liposome size, acid instability and stability of liposomes in the bloodstream.
A variety of processes are available for preparing liposomes, as described in, for example, Szoka et al., Ann.
Rev.
Biophys.
Bioeng. 9; 467 (1980), USAU.S.
Patent Nos. 4,235,871,4501728USA 4,501,728, 4,837,028, and 5,019,369. 20 To target immune cells, a ligand to be incorporated into the liposome may include, for example, antibodies or their specific fragments for cell surface determinants of the desired immune system cells.
A suspension of liposomes containing a peptide can be administered intravenously, locally, topically, etc., in a dose that varies according to, inter alia, the mode of administration, the peptide being released, and the stage of delivery. disease being treated.
For solid compositions, non-toxic nanoparticle or conventional solid carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate , and the like.
For oral administration, a pharmaceutically acceptable non-toxic composition! is formed by incorporating any of the excipients normally employed, such as those previously listed carriers, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably in a concentration of 25 ° /) -75 ° /. For aerosol administration, the immunogenic peptides are preferably supplied in finely divided form together with a surfactant and propellant.
Typical percentages of peptides are 0.01-20% by weight, preferably 1 ° / o-10 ° / o.
The surfactant must, of course, be non-toxic, and preferably soluble in the propellant.
Representatives of such
% 39/57
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agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as capróico, octanóico, lauric, paimitico, stearic, linoleic, lincdénico, olesterico, and oleic acids with an aliphatic polyhydric alcohol or its anhydride cyclical.
Mixed esters, such as mixed or natural glycerides can be employed.
The surfactant may constitute 0.1% -20 ° 6 by weight of the composition, preferably 0.25-5%. The composition's balance is commonly propellant.
A carrier can also be included when desired, such as, for example, lecithin for intranasal delivery.
For therapeutic or immunization purposes, nucleic acids encoding the peptide of the invention and optionally one or more of the peptides described herein can also be administered to the patient.
A number of processes are conveniently used to deliver nucleic acids to the patient.
For example, nucleic acid can be released directly, such as "naked DNA". This approach is described, for example, in Wolff et al., Science 247: 1465-1468 (1990) as well as US patents 5 580 859 and 5 589 466. Nucleic acids can also be administered using ballistic release as described, 15 by for example, in US patent 5,204,253. Particles comprised solely of DNA can be administered.
Alternatively, DNA can be attached to particles, such as gold particles. Nucleic acids can also be released complexed to cationic compounds. rials, such as cationic lipids - '20 lipid-mediated gene release processes are described, for example, in Q618372WOAWO 96/18372; 9324640WOAWO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6 (7): 682-691 (1988); 5279833USARose U.S.
Pat No. 5,279,833; 9106309WOAWO 91/06309; and Felgner et al., Proc.
Nat !. Acad.
Sci.
USA and 84: 7413-7414 (1987). The peptides and polypeptides of the invention can also be expressed via '25 attenuated viral hosts, such as vaccinia or contagious epithelioma.
This approach involves using vaccinia virus as a vector to express nucleotide sequences that encode the peptide of the invention.
Introduced in an acute or chronically infected host or in an uninfected host, the recombinant vaccinia virus expresses the immunogenic peptide, and therefore elicits a host CTL response.
Vaccine vectors 30 and processes useful in immunization protocols are described in, for example, U-S patent. 4,722,848. Another vector is BCG (Bacillus Calmette Guerin). BCG vectors are described in Stover et al- (Nature 351: 456-460 (1991)). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, for example, Salmonella typhi vectors and the like, will be visible to those skilled in the art from the present description.
A preferred means of administering nucleic acids encoding the peptide of the invention uses minigene constructs encoding multiple epitopes.
To create a sequence
DNA kit encoding the CTL epitopes (minigene) selected for expression in human cells, the amino acid sequences of the epitopes are translated in reverse.
A human codon utilization table is used to guide the choice of codori for each amino acid.
These epitope encoding DNA sequences are directly contiguous, creating a continuous polypeptide sequence.
To optimize expression and / or immunogenicity, additional elements can be incorporated into the minigene project.
Examples of the amino acid sequence that can be translated backwards and included in the mini-gene sequence include: auxiliary T lymphocyte, epitopes, a leader sequence (signal), and a retention signal in endoplasmic reticulum.
In addition, MHC presentation of CTL epitopes can be enhanced by including naturally occurring or synthetic flanking sequences (eg polyalanine) adjacent to CTL epitopes. The minigene sequence is converted to DNA by assembling oligonucleotides encoding the minigene pIus and minus tapes.
Overlapping oligonucleotides (30-100 bases of compresserite) are synthesized, phosphorylated, purified and annealed under appropriate conditions using well-known techniques.
The ends of the oligonucleotides are joined using T4 DNA ligase.
This synthetic minigene, encoding the CTL polypeptide, can then be cloned into a desired expression vector. 'Standard regulatory sequences well known to those skilled in the art' are included in the vector to ensure expression in target cells.
Several vector elements are required: a promoter with a downstream cIonation site for inserting a mini gene; a polyadenylation signal for efficient transcription termination; an E. coli replication source; and a selectable marker E. coli (eg, ampicillin · or kanamycin resistance). Numerous promoters can be used for this purpose, for example, the human cytomegalovkus (hCMV) promoter. See, US patents.
No. '"5 580 859 and 5 589 466' 25 for other appropriate promoter sequences.
Additional vector modifications may be desired to optimize minigene expression and immunogenicity.
In some cases, introns are required for efficient gene expression, and one or more naturally occurring or synthetic introns can be incorporated into the transcription region of the minigene.
The inclusion of mRNA stabilization sequences can also be considered for increased minigene expression.
It has recently been proposed that immunostimulatory sequences (lSSs or CpGs) play a role in the immunogenicity of DNA vaccines.
These sequences can be included in the vector, out of sequence encoding minigene, if found to improve immunogenicity.
In some embodiments, a bicistronic expression vector, to allow production of the encoded epitope-minigene and a second protein included to improve or decrease immunogenicity can be used.
Examples of proteins or polypeptides that can beneficially enhance the immune response if co-expressed include cytokines (for example,
For example, IL2, IL12, GM-CSF), cytokine-inducing molecules (e.g., LelF) or costimulatory molecules.
Auxiliary epitopes (HTL) can be linked to signals that have an intracellular target and expressed separately from CTL epitopes. This can allow targeting of HTL epitopes to a different cannula compartment from 5 CTL epitopes.
If required, this can facilitate more efficient entry of HTL epitopes into the MHC class ll pathway, thereby improving CTL induction.
In contrast, CTL induction, a specific decrease in immune response through the co-expression of immune suppressive molecules (eg, TGF-beta) can be beneficial in certain diseases.
Once an expression vector has been selected, the minigene is cloned into the poly-linker region 10 downstream of the promoter.
This plasmid is transformed into an appropriate strain of E. coli, and DNA is prepared using standard techniques.
The orientation and DNA sequence of the minigene, as well as all other elements in the vector, are confirmed using restriction mapping and DNA sequence analysis.
Bacterial cells harboring the correct plasmdehyde can be stored as a master cell bank and a working cell bank.
Purified plasmid DNA can be prepared for injection using a variety of formulations.
The simplest of these is reconstitution of lyophilized DNA in sterile phosphate buffered saline (PBS). A variety of processes have been described, and new, 'techniques may become available.
As noted above, nucleic acids are conveniently formulated with cationic lipids.
In addition, glycolipids, fusogenic liposomes. compounds, peptides and compounds collectively referred to as protective, interactive, non-condensates (PINC) can also be complexed to purified plasmid DNA to
· Variable influences such as stability, intramuscular dispersion, or traffic to specific organs or cell types. 25 Target cell sensitization can be used as a functional assay for expression and presentation of class 10 MHC of minigene-encoded CTL epitopes.
The pasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays.
The transfection process used will depend on the final formulation.
Electroporation can be used for "naked" DNA, while 30 cationic lipids allow direct transfection in vitro.
A plasmlide expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell classification (FACS). These cells are then labeled with chromium-51 and used as target cells for epitope-specific CTL lines.
Cytolysis, detected by chromium 51 release, indicates production of 35 MHC presentation of CTL epitopes encoded by minigene. Immunogenicity in vivo is a second approach to functional testing of minigene DNA formulations.
Transgenic mice expressing appropriate
human MHC molecules are immunized with the DNA product. The dose and route of administration are dependent on formulation (for example, IM for DNA in PBS, lP for DNA complexed with lipid). Twenty-one days after immunization, splenocytes are harvested and restimulated for 1 week in the presence of peptides encoding each epitope being tested. 5 These effector cells (CTLS) are assayed for cytolysis of target cells labeled with chromium-51, loaded with peptide using standard techniques. Lysis of target cells sensitized by MHC load of peptides corresponding to epitopes encoded by minigene demonstrates DNA vaccine function for in vivo induction of CTLS. Peptides can also be used to elicit CTL ex vivo. The resulting CTL, 10 can be used to treat chronic tumors in patients who do not respond to other conventional forms of therapy, or will not respond to a peptide vaccine therapy approach. Ex vivo CTL responses to a particular tumor antigen are induced by incubation in tissue culture of the patient's CTL precursor cells (CTLp) together with a source of cells presenting antigen (APC) and "the appropriate peptide. 15 After an appropriate incubation time (typically 1-4 weeks), in which CTLp are activated and mature and expand into effector CTL, the cells are infused back into the patient, where they will destroy their specific target cell (ie , a tumor cell) In order to optimize in vitro conditions for the generation of specific cytotoxic T cells, the culture of stimulator cells is maintained in an appropriate serum-free medium.
L 20 Before incubating the stimulator cells with the cells to be activated, for example, CD8 + precursor cells, an amount of antigenic peptide is added to the stimulator cell culture, of an amount sufficient to become loaded onto the CIasse 1 molecules. to be expressed on the surface of stimulator cells. In the present invention, a sufficient amount of peptide is an amount that will allow about 200, and preferably 200 or more, human MHC Class I molecules loaded with peptide to be expressed on the surface of each stimulator cell. Preferably, stimulator cells are incubated with> 2 µg / ml of peptide. For example, stimulator cells are incubated with> 3, 4, 5, 10, 15, or more µg / ml peptide. 30 precursor or remaining CD8 + cells are then incubated in culture with the appropriate stimulator cells for a sufficient time to activate the CD8 + cells. Preferably, CD8 + cells are activated in a specific antigen way. The ratio of precursor or remaining CD8 + (effector) cells to stimulated cells may vary from individual to individual and thus may depend on variables such as the individual's lymphocyte receptivity for culture conditions and the nature and severity of the disease condition. or another condition for which the described treatment modality is used. Preferably, however, the lymphocyte: stimulator cell ratio is in the range of
%%
about 30: 1 to 300: 1. Effector / stimulator care can be maintained for as long as is necessary to stimulate a number of therapeutically usable or effective CD8 + cells.
Induction of CTL in vitro requires specific recognition of peptides that are bound to allele-specific MHC class I molecules on APC.
The number of peptide / MHC complexes specified by APC is crucial for the stimulation of CTL, particularly in primary immune responses.
Although small amounts of peptide / MHC complexes per cell are sufficient to render a cell susceptible to CTL lysis, or to stimulate a secondary CTL response, the successful activation of a cn 10 precursor (pCTL) during primary response requires a significant number greater number of MHC / peptide complexes.
Peptide loading of primary empty histocompatibility complex molecules on cells allows the induction of primary cytotoxic T lymphocyte responses.
Since rion mutant cell lines exist for every human MHC allele, it is advantageous to use a technique to remove endogenous MHC-associated peptides from the APC surface, followed by loading of resulting empty MHC molecules with the immunogenic peptides of interest.
The use of uninfected, untransformed (non-tumorigenic) cells, and preferably autologous cells from patients such as APC is desirable .6 for the design of CTL induction protocols aimed at the development of "ex vivo CTL therapies .
This patent application shows processes for separation of associated peptides - endogenous MHC from the APC surface followed by loading of the desired peptides. A stable MHC molecule is a trimeric complex formed from the following elements: 1 ) a peptide of usually 8 - 10 residues, 2) a chain of
- heavy trans-membrane polymorphic protein that carries the peptide binding site in its a1 and a2 domains, and 3) a non-polymorphic light chain associated with non-25 covalently, l3-2-microglobulin.
Removal of bound peptides and / or dissociation of β-2-microglobulin from the complex renders class I MHC molecules non-functional and unstable, resulting in rapid degradation.
All class 1 MHC molecules isolated from PBMCs have endogenous peptides attached to them.
Therefore, the first step is to remove all endogenous peptides linked to MHC class I molecules on the APC without causing their degradation before exogenous peptides can be added to them.
Two possible ways to release MHC class I molecules from bound peptides include lowering the culture temperature from 37 ° C to 26 ° C overnight to destabilize β2-2-microglobulin and separate the endogenous peptides from the cell using a tract. - mild acid ment.
The processes release peptides previously bound in the ex-tracellular environment allowing new exogenous peptides to bind to empty class I molecules.
The cold temperature incubation process allows exogenous peptides to efficiently bind to the MHC complex, but requires an incubation overnight at 26 ° C which
- t 6 44/57 I '
È / may decrease the cell's metabolic rate. It is also likely that cells that are not actively synthesizing MHC molecules (eg, resting PBMC) will not be able to produce high amounts of empty surface MHC molecules through the cold temperature procedure. Severe acidic separation involves extraction of the peptides with trifluoroacetic acid, 5 pH 2, or acid denaturation of the peptide - class I complexes purified by immuno affinity. These processes are not feasible for inducing CTL, since it is important to remove endogenous peptides while preserving APC viability and an optimal metabolic state that is critical for antigen presentation. Mild pH 3 acid solutions such as glycine or citrate - phosphate buffers have been used to identify endogenous peptides and to identify tumor-associated T cell epitopes. The treatment is especially effective, where only MHC class 1 molecules are destabilized (and associated peptides released), while other surface antigens remain intact, including MHC class II molecules. Most importantly, cell treatment. them with mild acidic solutions does not affect cell viability or metabolic status.15 Gentle acid treatment is quick since the separation of endogenous peptides takes two minutes at 4 ° C and APC is ready to perform its function after appropriate peptides to be loaded. The technique is used here to obtain specific APCs - peptide for specific CTL generation - primary antigen, The resulting APCs "are efficient in inducing specific CD8 + CTL - peptide. "" 20 Activated CD8 + cells can be effectively separated from the stimulated cells using a variety of known processes. For example, specific monoclonal antibodies to the stimulated cells, to the peptides loaded on the cells stimulators, or for CD8 + cells (or a segment thereof) can be used to bind their appropriate complementary ligand Molecules labeled with antibody 25 can then be extracted from the effector-stimulator mixture via appropriate means, for example, via well-known immunoprecipitation processes Olj immunoassay.Cytotoxic, effective amounts of activated CD8 + cells may vary between, in vitro and in vivo uses, as well as the number and type of cells that are the ultimate target of these killer cells. it will also vary depending on the patient's condition and should be determined via consideration of all appropriate factors by the practitioner. however, however, about 1X106 to about 1X10'2, more preferably about 1X108 to about 1x10 ", and even more preferably, about 1x10 'to about 1x10' ° activated CD8 + cells are used for human adults, compared about 35 5X1O '- 5X1O' cells used in mice. Preferably, as discussed above, activated CD8 + cells are harvested from the cell culture prior to administration of the CD8 + cells to the subject being treated.
It is important to note, however, that unlike other treatment modalities proposed and present, the present process uses a cell culture system that is not tumorigenic.
Therefore, if complete separation of stimulator cells and activated CD8 + cells is not achieved, there is no inherent danger known to be associated with the administration of a small number of stimulating cells, whereas administration of mammalian tumor-promoting cells can be extremely dangerous.
Processes for reintroducing cellular components are known in the art and include procedures such as those exemplified in U.S. Patent 4,844,893 to Honsik, et al. and U.S. 4,690,915 to Rosenberg.
For example, administration of 10 activated CD8 + cells via intravenous infusion is appropriate.
The invention will be further described "in the examples which follow, which do not limit the scope of the invention described in the claims.
Examples Example 1: A strategy to identify neopeptides for vaccination 15 Our approach to identifying tumor-specific neoepitopes involves 3 steps- (1) identification of DNA mutations using whole genome or whole exome sequencing (ie, only captured exons ) of tumor versus paired germ line samples from each patient.
Our preliminary studies demonstrate that CLL cells contain many distinct genetic changes that alter the sequence of amino acids and may generate potential new T cell epitopes. (2) Application of highly validated MHC - peptide binding prediction algorithms to generate a set of Carididate T cell cpitopes based on non-silent mutations present in tumors.
We will confirm expression of genes that have mutated as RNA in CLL samples, and then confirm predictions of HLA - peptide binding using an experimental approach to quantify binding of candidate HLA allele peptides. (3) Generation of specific T cells - antigen against peptides that have undergone mutation.
Example 2: Sequencing of normal genome and tumor for the identification of mutant genes in tumors of patients with chronic lymphocytic leukemia (Step 1) To detect specific tumor mutations (which are not present in normal tissues), samples of tumors were collected and normal tissues for each patient.
For leukemias, tumors were purified using magnetic pearl isolation or fluorescence-activated cell classification using antibodies specific for tumor cells, for example, tumor cells from patients with chronic lymphocytic leukemia (CLL) express the markers of surface CD5 and CD19. Skin fibroblasts were used as a controie of normal tissue.
DNA or RNA for sequencing was purified from cells of normal tissue or tumor.
For melanoma, ovarian and other solid tumors (in which there is contamination with non-tumor cells), DNA and RNA were isolated
r "& ¢ 46/57 sides of relatively homogeneous short-term cultures of laser-captured tumor tumor cells. PBMCs were used as normal control cells. For all samples, PBMCS were cryopreserved until necessary for expansion of specific T cells - peptide mutants Finally, short-term cultures of tumor cells were also cryopreserved for later use as targets of expanded T cells. Isolated genomic DNA or RNA was tested for nucleic acid integrity and purity before For each DNA sample, entire genomic DNA was sheared and sequenced, or coding exons were captured by complementary digonucleotides using hybrid selection 10 and then sequenced (Gnirke et al., Nat Biotechnol. 2009, 27 (2): 182t9) DNA and RNA libraries were generated and sequenced using next-generation 1Lumina sequencing instmrhéntòs. Sequencing of 64 patients with chronic lymphocytic leukemia ( CLL) yielded an average of 23 non-silent mutations that alter amino acid sequences of 15 proteins (Figure 3) in the tumor in relation to the germ line DNA sequence. These non-stressful mutations fall into 5 distinct classes with the potential to generate neoeplopes: wrong direction, site amendment, structure deviation (indel, insertions and deletions), reading through and gene mergers (Figure 4). The frequencies of these mutations vary across 4 individual patients (Figure 5). All of these mutations provide potential neoepitopes for
D to 20 immunization, with structure deviation mutations; (reading through site splice (for example, with retained introns) generating longer stretches of new peptides, wrong sense mutations leading to short peptides with simpy amino acid changes! and finally, gene fusion generating hybrid peptides with new sequences Example 3: Identification of HLA-binding peptides derived from expressed proteins 25 harboring specific tumor mutations (Step 2) The next question is whether mutated genes can generate peptides that can be presented by HLA / MHC proteins from First, several algorithms were used to predict 30 and 137 HLA binding peptides with lC50 scores <500 nM from 10 patient 1 wrong sense mutations, and 53 indel 1 wrong sense 30 and 2 gene fusions from Patient 2. An example of a wrong sense mutation in a patient with 6 specific HLA alleles is shown with 2 predicted binding peptides from 54 combinations of 9-mer peptide the HLA alleles and alleles (Figure 6). To confirm that these genes are expressed in tumors, we measured levels of RNA for the mutated genes (using various approaches that depend on the class of mutation, Figure 35 7), and found that 98 ° / o of genes that mutated with HLA binding peptides were expressed. The HLA binding capacity of all predicted peptides that pass through
& 47/57 'expression of RNA expression are then experimentally validated by conducting competitive binding assays with test peptides versus known reference peptides that bind to the HLA- allele (Sidney et al. Curr Protoc Immuno). 2001, Chapter 18: Unit
18.3) (Figure 8A). Of the subset we submitted to experimental confirmation of HLA binding, 8 of 17 (47%) predicted peptides from Pt 1 wrong sense mutations were confirmed to have high binding affinities for HLA (| C50 "500) alleles (Figure 8B). For Pt 2, 25 of 49 predicted peptides were experimentally confirmed as HLA ligand (Figure 8B) - These results suggest that all peptides with | 150 nM | C50 "experimentally show HLA binding, while a < 500 nM generates 10 binding peptides, true 40-50% of the time (Figure 8C). Of note, 12 of the 25 confirmed peptides that underwent Pt 2 mutation have> 2-fold better binding affinity than the germline peptide (Figure 9). Although such peptides are preferable for incorporation into a tumor vaccine to reduce the chance that T cells will cross-react with the germline peptide, peptides that do not show differential binding may still provide specific tumor responses due to differential recognition germ line peptide vs. mutant via T cell receptor. Example 4: CD8 + T cell responses against mutated peptides' identified by sequencing patient samples CLL (Step 3) ", Based on the HLA binding mutations verified expe-" 20 experimentally or predicted, we can now determine whether T cells can be generated to recognize these 'tumor-specific mutated peptides. We thus synthesized peptides with binding scores of less than 1000 nM that are derived from $ genes with validated expression in tumor cells. To generate T cells of desired specificity, we stimulate patient T cells sequenced with autologous APCS "25 peptide pulsed (either using an individual peptide or a pool of peptides) (dendritic cells and expanded autologous B cells-CD40L) on a weekly basis, in the presence of IL-2 and IL-7. After 3-4 cycles of stimulation, the expanded CD8 + cells were tested on ELlSpot for evidence of reactivity against the peptide, based on lFNgama secretion. from Patient 1 (Figure 10), we detected IFN gamma secretion in T cells against autochthonous DCS pulsed with a mutated peptide from the TLK2 gene. Example 5: BCR-ABL gene that has mutated binds to patient MHC / HLA proteins and can elicit mutant peptide-specific CD8 + T cells We conducted a more complete study of T cell responses to 35 tumor-specific mutant peptides in patients with another type of leukemia, l chronic myeloid eukemia (CML). CML is defined by the expression of a specific tumor translocation, the product of the BCR-ABL gene fusion. BCR-ABL mutations develop in
K 48/57 »CML patients who develop drug resistance for front-line pharmacological therapy with imatinib mesylate, which targets BCR-ABL. Potentially, these mutations can generate neoepitopes that the host's T cells, or a normal grafted-donor, can recognize when bound to MHC proteins; these T cells are likely to be minimally tolerated. We consider the 20 most common mutations that evolve in patients with resistance to imatinib, and predict the binding of 9- and 1-mere roof peptides around each mutation. Using the prediction algorithms NetMHC (Nielsen et al. PLOS One. 2007, 2 (8): e796) or IEDB (Vita R et al. Nucleic Acids Res- 2010, 38: 0854-62), we predict 10 calls from 84 peptides from 20 common mutations for one or more 8 common HLA alleles (| C50 <1000), with many peptides derived from the three most common mutations, 24 of 84 peptides were predicted to be strong ligands (lC50 "50) ( Figure 14), 42 intermediate binding peptides (50 "| C50" 500), and 18 weak binding peptides (500 "IC ,, q" 1000). We have focused our attention on a mutant peptide generated from mutation 15 E255K (E255K-B255-263) (KVYEGVWKK) (SEQ ID NO: 10) which is expected to connect with high affinity to HLA-A3. (iC® = 33.1). Using a competitive MHC binding assay (Figure 8A), we experimentally confirmed the high binding affinity of E255K-B for HLA-A3 '(lC50 = 17 nM) with -10 times the stronger HLA binding of the mutant peptide compared to "parent peptide (wild type) (Figure 15A). E255K-B was also experimentally" 20 verified to bind to other members of the A3 super type family HLA-A ° 1101 and HLA-A "68. We next generate lines of T cells against E255K-B from a normal HLA-A3 + donor
V and 2 CML E255K + / HLA-A3 patients who each showed greater specificity against the mutated $ peptide than the relative (Figure 15 B, C). E255K-B appears to be endogenously processed and presented since T cells reactive to E255K-B also responded to APCS HLA-A3 + transfected with a minigene spanning 227 base pairs surrounding the E255K mutation. E255K in one patient developed only following curative allo-HSCT (Figure 15D) .These studies demonstrate that genetic changes driven by leukemia can provide new immunogenic tumor specific antigen targets that are associated with clinical response in vivo. for identification of immunogenic BCR-ABL T cell epitopes that have mutated thus illustrates an effective strategy for applying bio-informatics tools to discover T cell epitopes from mutated genes- Example 6: T cell clones of patients who recognize tumor epitopes can selectively kill cells presenting mutated epitopes - 35 Confirmation of specificity d and T-cell targeting is best addressed by characterizing individual T-cell clones. We therefore typically isolate mutated peptide-specific T cell clones by limiting dilution of reactive T cell lines and then using standard chromium release assays to select T cell cells that demonstrate differential death from autologous pulsed APCS with "germ line peptide vs. mutated ones. Using a standard dilution series for each peptide, we measure the required peptide concentration to 50% of death. If the ratio of wild type to mutant peptides needed to 50 ° 6 of death is greater than 10 times, we conclude that there is differential recognition of these peptides by T cells, as seen previously for mutated tumor antigens - WE performed this procedure for a CML tumor antigen, CML66. if CML66 peptide specific T cells recognize processed and displayed epitopes, CML66 peptide-reactive T cells were incubated with three autologous APCS instructions for expression of entire CML66 protein. We express CML66 through "- -." via nucleofection of pIasmid DNA, or RNA transcribed in vitro (in DCS, ex-"pandidas-CD40L B cells, or K562 cells with engineered HLA molecules). CML66-derived peptide 15 (peptide 66-72C). Since the entire CML66 protein was efficiently expressed when expanded B-cells CDCDL were nucleofected with CML66 mRNA (Figure 12B), we were able to use these cells (or cells pulsed with peptide as targets in a standard chromium release assay and we found that T cells effectively smoothed out these target cells (Figure 12C). Comparable assays, including lysis of
20 patient-paired tumor cells are being performed for each of the mutated peptide-specific T cell lines generated from each cancer patient (for example, using the T cell lines described in Examples 6 and 7). Example 7: Tumor propellants that mutated as potential tumor antigens 25 Of 1188 non-silent mutations across 64 patients, we identified 8 recurrent mutations, including SF3B1 (16 ° 6 CLL patients), TP53 (12.5%) , MYD88 (9 ° 6), ATM (9%), FBX N7 (6 ° 6), MAPKI (5%), GNBI (3 ° / o) and M6PR (3%) (Figure 11). These mutations (especially the most frequent ones: SF3B1, TP53, MYD88 and ATM) are predicted to drive mutations that are essential for tumor development or progression. These conducting genes represent promising tumor-specific antigens for inclusion in a vaccine. K SF3B1 is the gene that most frequently mutates in CLL, has mutated in related sites, is highly expressed in CLL patients (Figure 12), and has not been previously described. The most common SF3B1 mutation was K70OE (40 ° 6 of 35 SF3B1 mutations); genotyping of an additional 89 independent CLL patients discovered 6 more tumors from patients harboring this mutation. By applying HLA-peptide binding algorithms to SF3B1 mutations, we predict binding of peptides that
h 50/57 'mutate to the most common HLA-A2 allele (Figure 13). If a peptide harboring the most common CLL mutation (SF3B1 K70OE) binds the most common HLA class 1 allele (HLA-A2), then this peptide is an excellent candidate for inclusion in a CLL vaccine for many CLL patients. 5 References Albert, T. j., Molla, MN, Muzny, DM, Nazareth, L., Wheeler, D., Song, X., Richmond, TA, Middle, CM, Rodesch, M. j., Packard , CJ, et al. (2007). Direct seledion of human genomic loci by microarray hybridization. Nat Methods 4, 903-905. Alyea, EP, Soiffer, R. j., Canning, C., Neuberg, D., Schbssman, R., Pickett, C., 10 Collins, H., Wang, Y., Anderson, KC, and Ritz, j . (199Q) - Toxicity'and efficacy of defined doses of CD4 (+) donor lymphocytes for treatment of i'elapsè after ahogeneic bone marrow transplant. Blood 91, 3671-3680. Annunziata, C. M-, Davis, RE, Demchenko, Y., Bellamy, W., Gabrea, A., Zhan, F., Lenz, G., Hanamura, I., Wright, G., xiao, W. , et al. (2007). Frequent engagement of the clas- 15 sical and alternative NF-kappaB pathways by diverse genetic abnormalities in multiple myeloma. Cancer Cell12, 115-130. Attia, P., Phan, G. Q ,, Maker, A. V., Robinson, M. R., Quezado, M. M., Yang, j. C., Sherry, R. M., Topalian, S. L., Kammula, U. S., Royal, R. E., et al. (2005). Autoimmunity correlated with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic 20 T-lymphocyte antigen-4. J Clin Oncol 23, 6043-6053. ' Austeri, B., Powell, JE, Alvi, A., Edwards, L. Hooper, L., Starczynski, J. Taylor, AM, Fegan, C., Moss, P., and Stankovic, T. (2005 ). Mutations in the ATM gene lead to im- paired overall and treatment-free survival that is independent of ÍGVH mutation status in patients with B-CLL. Blood 106, 3175-3182. 25 Balakrishnan, A., Bleeker, FE, Lamba, S., Rodolfo, M., Daniotti, M., Scarpa, A., van Tilborg, AA, Leenstra, S., Zanon, C., and Bardelli, A. (2007). Novel somatic and germline mutations iri cancer candidate genes in glioblastoma, melanoma, and pancreatic carcinoma. Cancer Res 67, 3545-3550. Baskar, S., Kobrin, C. B., and Kwak, L. W. (2004). Autologous lymphoma vaccines 30 induce human T cell responses against multiple, unique epitopes. j Clin lnvest 113, 1498-
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1088.

权利要求:
Claims (39)
[1]
1. Method of identifying a plurality of specific neoantigen peptides for an individual, CHARACTERIZED by understanding: a. providing at least one sample of nucleic acid from the tumor, and at least one sample of non-tumor nucleic acid obtained from an individual having cancer; B. identify in the individual's sample a plurality of specific mutations of non-silent tumor in one or more expressed genes of an individual having cancer; by analyzing the tumor and non-tumor nucleic acid samples, where the tumor-specific mutations comprise mutations present in at least one tumor nucleic acid sample and not present in the non-tumor nucleic acid sample; ç. selecting a plurality of neoantigen specific peptides for an individual from a plurality of tumor specific mutations identified in step (b), where the neoantigen peptides have the following characteristics: i. bind to an HLA protein; and ii. where when the mutation is a point mutation, the neoantigenic peptides bind to the HLA protein with an IC 50 below 500 nm; d. where when the mutation identified in step (a) (b) is a point mutation; i. identifying a mutant peptide having the mutation identified in step (a), where the mutant peptide binds to an HLA class I protein with greater affinity than a wild-type peptide; and has an IC 50 of less than 500 nm; and is. where when the mutation identified in step (a) (b) is a slice site mutation, structure deviation, reading phase or gene fusion: i. identify a mutant polypeptide encoded by the mutation identified in step (a), where the mutant polypeptide binds to an HLA class I protein.
[2]
2. Method, according to claim 1, CHARACTERIZED by the fact that the mutant peptide is 8 to 10 amino acids in length.
[3]
3. Method, according to claim 1, CHARACTERIZED by the fact that the mutant peptide is greater than 10 amino acids in length.
[4]
4. Method, according to claim 3, CHARACTERIZED by the fact that the mutant peptide is greater than 15 amino acids in length.
[5]
5. Method, according to claim 4, CHARACTERIZED by the fact that the mutant peptide is greater than 20 amino acids in length.
[6]
6. Method, according to claim 5, CHARACTERIZED by the fact that the 5 mutant peptide is greater than 30 amino acids in length.
[7]
7. Method, according to claim 1, CHARACTERIZED by the fact that the mutant peptide is 8 to 50 amino acids in length.
[8]
8. Method, according to claim 1, CHARACTERIZED by the fact that the mutant peptide is 24 to 40 amino acids in length.
[9]
9. Method, according to claim 1, CHARACTERIZED by the fact that specific tumor mutations are identified through nucleic acid sequencing.
[10]
10. Method according to claim 1, characterized by further comprising selecting a peptide identified in step (b) or the polypeptide of step (c) that activates anti-tumor CD8 T cells.
[11]
11. Use of one or more peptides or polypeptides CHARACTERIZED because the peptides or polypeptides are identified according to claim 1 and an adjuvant for the manufacture of a drug to induce a specific immune response to a tumor.
[12]
12. Use, according to claim 11, CHARACTERIZED by the fact that the adjuvant is a TLR-based adjuvant.
[13]
13. Use according to claim 11, CHARACTERIZED by the fact that the peptide or polypeptide is emulsified with an adjuvant based on mineral oil.
[14]
14. Use according to claim 11, CHARACTERIZED by the fact that the peptide or polypeptide and a TLR-based adjuvant are emulsified with a mineral oil-based adjuvant.
[15]
15. Use, according to claim 11, CHARACTERIZED by still comprising administration of an anti-immune suppressive agent.
[16]
16. Use according to claim 15, CHARACTERIZED by the fact that the anti-immune suppressive agent is an anti-CTLA-4 antibody, an anti-PD1 antibody,
an anti-PD-L1 antibody, an anti-CD25 antibody or an IDO inhibitor.
[17]
17. Use of autologous dendritic cells or cells presenting antigen CHARACTERIZED by the fact that autologous dendritic cells or cells presenting antigen are pulsed with one or more of the peptides or 5 polypeptides identified according to claim 1 for the manufacture of a drug to induce a response tumor specific immune system.
[18]
18. Use according to claim 17, CHARACTERIZED by still comprising administration of an adjuvant.
[19]
19. Use according to claim 18, CHARACTERIZED by the fact that the adjuvant is a TLR-based adjuvant.
[20]
20. Use, according to claim 17, CHARACTERIZED by still comprising administration of an anti-immune suppressive agent.
[21]
21. Use according to claim 20, CHARACTERIZED by the fact that the anti-immune suppressive agent is an anti-CTLA-4 antibody, an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CD25 antibody or a IDO inhibitor.
[22]
22. Use of one or more peptides or polypeptides, autologous dendritic cells or cells presenting antigen pulsed with one or more peptides or polypeptides, CHARACTERIZED by the one or more mutant peptides or polypeptides to bind to a HLA class I protein and be able to activate antitumor CD8 T cells for the production of a drug in an individual with cancer where the individual has identified among a plurality of specific tumor mutations in an expressed gene of the individual, a mutation type: i. point mutation further identifying a mutant peptide having the point mutation; and / or i. splice site mutation, structure deviation, reading phase or gene fusion identifying a polypeptide encoded by the mutation.
[23]
23. Use, according to claim 22, CHARACTERIZED by still comprising administration to the individual of an adjuvant.
[24]
24. Use according to claim 23, CHARACTERIZED by the fact that the adjuvant is a TLR-based adjuvant.
[25]
25. Use according to claim 22, characterized in that it further comprises administration of an anti-immune suppressive agent.
[26]
26. Use according to claim 25, CHARACTERIZED by the fact that the anti-immune suppressive agent is an anti-CTLA-4 antibody, an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CD25 antibody or a IDO inhibitor. 5
[27]
27. Use according to claim 22, CHARACTERIZED by the fact that the mutant peptide is 8 to 10 amino acids in length.
[28]
28. Use according to claim 22, CHARACTERIZED by the fact that the mutant peptide is 8 to 50 amino acids in length.
[29]
29. Use according to claim 22, CHARACTERIZED by the fact that the mutant peptide is 24 to 40 amino acids in length.
[30]
30. Use, according to claim 22, CHARACTERIZED by the fact that the individual receives a hematopoietic stem cell transplant.
[31]
31. Use, according to claim 22, CHARACTERIZED by the fact that the individual is a human, dog, cat, or horse.
[32]
32. Use, according to claim 22, CHARACTERIZED by the fact that the cancer is breast cancer, ovarian cancer, prostate cancer, lung cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head cancer and neck, pancreatic cancer, brain cancer, melanoma lymphoma or leukemia.
[33]
33. Use according to claim 32, CHARACTERIZED by the fact that the lymphoma is a B cell lymphoma.
[34]
34. Use according to claim 32, CHARACTERIZED by the fact that leukemia is acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, or T-cell lymphocytic leukemia
[35]
35. Pharmaceutical composition CHARACTERIZED for comprising the identified peptide, as defined in claim 1, and a pharmaceutically acceptable carrier.
[36]
36. Composition CHARACTERIZED for comprising at least two distinct ones: a. SF3B1 peptides where each peptide is equal to or less than 50 amino acids in length and contains: i. a leucine at the amino acid position 625;
ii. a histidine at amino acid position 626; iii. a glutamic acid at amino acid position 700; iv. an aspartic acid at the amino acid position 742; or V. an arginine at amino acid position 903, when numbered according to wild type SF3B1; B. MYD88 peptides where each peptide is equal to or less than 50 amino acids in length and contains: i. a threonine at amino acid position 232; ii. a leucine at amino acid position 258; or iii. a proline at amino acid position 265, when numbered according to MYD88 wild-type; ç. TP53 peptides where each peptide is equal to or less than 50 amino acids in length and contains: i. an arginine at amino acid position 111; ii. an arginine at amino acid position 215; iii. a serine at amino acid position 238; iv. a glutamine at amino acid position 248; v. a phenyl alanine at amino acid position 255; saw. a cysteine at amino acid position 273 or vii. an asparagine at amino acid position 281, when numbered according to wild type TP53;
d. ATM peptides where each peptide is equal to or less than 50 amino acids in length and contains: i. a phenyl alanine at amino acid position 1252;
ii. an arginine at the amino acid position 2038; iii. a histidine at amino acid position 2522; or iv. a cysteine at amino acid position 2954, when numbered according to wild-type ATM; and. ab1 peptides where each peptide is equal to or less than 50 amino acids in length and contains:
i. a valine at the amino acid position 244;
ii. a valine at the amino acid position 248; iii. a glutamic acid at the amino acid position 250; iv. an alanine at amino acid position 250;
v. a histidine at amino acid position 252; Vi. an arginine at amino acid position 252; vii. a phenyl alanine at the amino acid position 253;
viii. a histidine at amino acid position 253; ix. a lysine at amino acid position 255; x. a valine at amino acid position 255;
xi. a glycine at amino acid position 276; xii. an isoleucine at amino acid position 315; xiii. an asparagine at the amino acid position 315;
xiv. a leucine at amino acid position 317; xv. a threonine at amino acid position 343; xvi. a threonine at amino acid position 351; xvii. a glycine at amino acid position 355; xviii. a valine at amino acid position 359; xix. an alanine at amino acid position 359; xx. an isoleucine at amino acid position 379; xxi. a leucine at amino acid position 382; xxii. a methionine at amino acid position 387;
xxiii. a proline at amino acid position 396; xxiv. an arginine at amino acid position 396; xxv. tyrosine at amino acid position 417; or xxvi. a serine at amino acid position 486, when numbered according to wild type ab1; f. FBXW7 peptides where each peptide is equal to or less than 50 amino acids in length and contains: i. a leucine at amino acid position 280; ii. a histidine at amino acid position 465;
iii. a cysteine at amino acid position 505; or iv. a glutamic acid at amino acid position 597, when numbered according to wild type FBXW7; g. MAPK1 peptides where each peptide is equal to or less than 50 amino acids in length and contains: 5 i. an asparagine at amino acid position 162; ii. a glycine at the amino acid position 291; or iii. a phenyl alanine at amino acid position 316, when numbered according to wild type MAPK1. H. GNB1 peptides where each peptide is equal to or less than 50 amino acids in length and contains a threonine at amino acid position 180, when numbered according to wild type GNB1.
[37]
37. Composition according to claim 36, further comprising an adjuvant.
[38]
38. Use of a composition CHARACTERIZED for being a Bcr-ab1 peptide equal to or less than 50 amino acids in length that contains a lysine at position 255, when numbered according to wild-type bcr-ab1, as it is in the manufacture of a medicine for treatment of an individual with an imatinib-resistant tumor.
[39]
39. Use of one or more peptides CHARACTERIZED by the fact that one or more peptides contains a bcr-ab1 mutation where said peptide is equal to or less than 50 amino acids and binds to a HLA class I protein with an IC50 of less than 500 nm, for the manufacture of a drug for the treatment of an individual with a tumor resistant to imatinib.

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法律状态:
2020-09-15| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-11-17| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2020-12-29| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-03-23| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-08-17| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-12-28| B09B| Patent application refused [chapter 9.2 patent gazette]|

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2022-03-03| B12B| Appeal against refusal [chapter 12.2 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US33486610P| true| 2010-05-14|2010-05-14|

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US61/334,866|2010-05-14|

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PCT/US2011/036665|WO2011143656A2|2010-05-14|2011-05-16|Compositions and methods of identifying tumor specific neoantigens|

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