Patent Application: US-201715458893-A

Abstract:
the present description relates to peptides , proteins , nucleic acids and cells for use in immunotherapeutic methods . in particular , the present description relates to the immunotherapy of cancer . the present description further relates to tumor - associated t - cell peptide epitopes , alone or in combination with other tumor - associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti - tumor immune responses , or to stimulate t - cells ex vivo and transfer into patients . peptides bound to molecules of the major histocompatibility complex , or peptides as such , can also be targets of antibodies , soluble t - cell receptors , and other binding molecules .

Description:
the syfpeithi routine ( rammensee et al ., 1997 ; rammensee et al ., 1999 ) predicts binding of kiqeiltqv ( seq id no : 1 ) to a * 02 : 01 with an absolute score of 27 and a relative score of 0 . 74 . the peptide igf2bp3 - 001 was presented on cancer cells as follows : igf2bp3 - 001 was quantified in & gt ; 380 hla - a * 02 positive tumor samples including 16 different cancer types and & gt ; 240 normal tissue samples of different origin covering all risk categories with a focus on high and medium risk organs ( status : august 2015 ). fig1 shows the relative peptide presentation levels of igf2bp3 - 001 in these tissues . igf2bp3 - 001 was frequently and exclusively found on primary tumor samples of different entities and not on normal tissues . thus , the igf2bp3 - 001 target is presented in the context of hla - a * 02 in a highly tumor - specific manner . the data shown in fig5 further provides evidence that igf2bp3 - 001 is a peptide with very good binding to hla - a * 02 : 01 . allo - reactive settings can be used to circumvent self - tolerance and yield t - cells with a higher avidity when compared to t - cells derived from autologous settings , i . e ., patients . examples of such settings include in vitro generation of allo - hla reactive , peptide - specific t - cells ( sadovnikova et al . 1998 ; savage et al . 2004 ; wilde et al . 2012 ), and immunization of mice transgenic for human - mhc or human tcr ( stanislawski et al . 2001 ; li et al . 2010 ). in vitro generation of allo - hla reactive , peptide - specific t - cells ( savage et al . 2004 ) pbmcs from hla - a * 02 - positive and hla - a * 02 - negative healthy donors were used after obtaining informed consent . recombinant biotinylated hla - a2 class i monomers and a2 fluorescent tetramers containing igf2bp3 - 001 were obtained from mbli ( woburn , mass .). pbmcs were incubated with anti - cd20sa diluted in phosphate buffered saline ( pbs ) for 1 hour at room temperature , washed , and incubated with the biotinylated a2 / igf2bp3 - 001 monomers for 30 minutes at room temperature , washed , and plated at 3 × 10 6 cells / well in 24 - well plates in rpmi with 10 % human ab serum . interleukin 7 ( il - 7 ; r & amp ; d systems , minneapolis , minn .) was added on day 1 at 10 ng / ml and il - 2 ( chiron , harefield , united kingdom ) was added at 10 u / ml on day 4 . over a 5 - week period cells were restimulated weekly with fresh pbmcs , mixed with responder cells at a 1 : 1 ratio , and plated at 3 × 10 6 / well in 24 - well plates . to obtain high avidity t - cells , approximately 10 6 pbmcs with hla - a2 / igf2bp3 - 001 tetramer - phycoerythrin ( pe ) ( obtained from mbli ) were incubated for 30 minutes at 37 ° c ., followed by anti - cd8 - fluorescein isothiocyanate ( fitc )/ allophycocyanin ( apc ) for 20 minutes at 4 ° c ., followed by fluorescence activated cell sorting ( facs )- calibur analysis . sorting was done with a facs - vantage ( becton dickinson , cowley , oxford , united kingdom ). sorted tetramer - positive cells were expanded in 24 - well plates using , per well , 2 × 10 5 sorted cells , 2 × 10 6 irradiated a2 - negative pbmcs as feeders , 2 × 10 4 cd3 / cd28 beads / ml ( dynal , oslo norway ), and il - 2 ( 1000 u / ml ). the high avidity t - cells , thus obtained , were then used to identify and isolate tcrs using techniques known in the art , such as single cell 5 ′ race ( rapid amplification of cdna ends ). non - redundant tcr dnas were then analyzed for amino acid / dna sequences determination and cloning into expression vectors using methods well known in the art . methods of cloning tcrs are known in the art , for example , as described in u . s . pat . no . 8 , 519 , 100 , which is hereby incorporated by reference in its entirety for said methods . the alpha chain variable region sequence specific oligonucleotide a1 ( ggaattccatatgagtcaacaaggagaagaagatcc seq id no : 11 ) which encodes the restriction site ndei , an introduced methionine for efficient initiation of expression in bacteria , and an alpha chain constant region sequence specific oligonucleotide a2 ( ttgtcagtcgacttagagtctctcagctggtacacg seq id no : 12 ) which encodes the restriction site sali are used to amplify the alpha chain variable region . in the case of the beta chain , a beta chain variable region sequence specific oligonucleotide b1 ( tctctcatatggatggtggaattactcaatccccaa seq id no : 13 ) which encodes the restriction site ndei , an introduced methionine for efficient initiation of expression in bacteria , and a beta chain constant region sequence specific oligonucleotide b2 ( tagaaaccggtggccaggcacaccagtgtggc seq id no : 14 ) which encodes the restriction site agei are used to amplify the beta chain variable region . three tcrs ( r10p1a7 , r13p1c6 , and r18p1c12 ), each encoding tumor specific tcr - alpha and tcr - beta chains , were isolated and cloned from t - cells of three healthy donors . tcr r18p1c12 was derived from hla - a2 positive donor and tcr r10p1a7 and tcr r13p1c6 were derived from hla - a2 negative donors . the alpha and beta variable regions of the tcrs were sequenced . the tcr alpha and beta variable regions were then cloned into pgmt7 - based expression plasmids containing either cα or cβ ( respectively ) by standard methods described in ( molecular cloning a laboratory manual third edition by sambrook and russell ). plasmids were sequenced using an applied biosystems 3730 × 1 dna analyzer . the dna sequences encoding the tcr alpha chain cut with ndei and sali were ligated into pgmt7 + cα vector , which was cut with ndei and xhoi . the dna sequences encoding the tcr beta chain cut with ndei and agei was ligated into separate pgmt7 + cβ vector , which was also cut with ndei and agei . ligated plasmids are transformed into competent escherichia coli strain xl1 - blue cells and plated out on lb / agar plates containing 100 μg / ml ampicillin . following incubation overnight at 37 ° c ., single colonies are picked and grown in 10 ml lb containing 100 μg / ml ampicillin overnight at 37 ° c . with shaking . cloned plasmids are purified using a miniprep kit ( qiagen ) and the insert is sequenced using an automated dna sequencer ( lark technologies ). phage display can be used to generate libraries of tcr variants to identify high affinity mutants . the tcr phage display and screening methods described in ( li et al , ( 2005 ) nature biotech 23 ( 3 ): 349 - 354 ) can be applied to a reference tcr . for example , all three cdr regions of the alpha chain sequence and all three cdr regions of the beta chain sequence can be targeted by mutagenesis , and each cdr library panned and screened separately . accordingly , tcrs with affinities and / or binding half - lives at least twice that of the reference tcr ( and therefore impliedly at least twice that of the native tcr ) can be identified . tcr heterodimers are refolded using the method including the introduced cysteines in the constant regions to provide the artificial inter - chain disulphide bond . in that way tcrs are prepared , consisting of ( a ) the reference tcr beta chain , together with mutated alpha chains ; ( b ) the reference tcr alpha chain together with mutated beta chains ; and ( c ) various combinations of beta and alpha chains including the mutant variable domains . the interaction between high affinity soluble disulfide - linked tcrs , and tcr variants , and the native peptide kiqeiltqv ( seq id no : 1 ) hla - a * 02 complex can be analyzed using the biacore method . high avidity tcr variants can also be selected from a library of cdr mutants by yeast , or t - cell display ( holler et al . 2003 ; chervin et al . 2008 ). candidate tcr variants , thus , provide guidance to design mutations of the tcr &# 39 ; s cdrs to obtain high avidity tcr variants ( robbins et al . 2008 ; zoete et al . 2007 ). t - cells can be engineered to express high avidity tcrs ( so - called tcr therapies ) or protein - fusion derived chimeric antigen receptors ( cars ) that have enhanced antigen specificity to mhc i / igf2bp3 - 001 complex or mhc ii / igf2bp3 - 001 complex . in an aspect , this approach overcomes some of the limitations associated with central and peripheral tolerance , and generate t - cells that will be more efficient at targeting tumors without the requirement for de novo t - cell activation in the patient . in one aspect , to obtain t - cells expressing tcrs of the present description , nucleic acids encoding the tumor specific tcr - alpha and / or tcr - beta chains identified and isolated , as described in examples 1 - 2 , are cloned into expression vectors , such as gamma retrovirus or lentivirus . the recombinant viruses are generated and then tested for functionality , such as antigen specificity and functional avidity . an aliquot of the final product is then used to transduce the target t - cell population ( generally purified from patient pbmcs ), which is expanded before infusion into the patient . in another aspect , to obtain t - cells expressing tcrs of the present description , tcr rnas were synthesized by techniques known in the art , e . g ., in vitro transcription systems . the in vitro - synthesized tcr rnas were then introduced into primary cd8 + t - cells obtained from healthy donors by electroporation to re - express tumor specific tcr - alpha and / or tcr - beta chains . to test whether the exogenous tcrs were functionally expressed on cell surface of the transformed t - cells , a tetramer staining technique was used to detect mhc / igf2bp3 - 001 - binding t - cells . as shown in fig5 and table 6 , a higher percentage of cd3 - positive specific t - cell population , i . e ., 6 . 78 % ( tcr r10p1a7 ) and 16 . 54 % ( tcr r13p1c6 ), was observed in tcr - expressing cd8 + t - cells by fluorescent - labeled mhc / igf2bp3 - 001 tetramer staining than that with mhc / unrelated peptide ( e . g ., nyeso1 - 001 ) tetramers , e . g ., 1 . 53 %, or mock control , e . g ., 1 . 73 %. as a control , primary cd8 + t - cells transformed with unrelated tcrs , such as 1g4 tcr , which is known to bind specifically to mhc / nyeso1 - 001 complex , was readily detected by mhc / nyeso1 - 001 tetramer , i . e ., 17 . 69 %. these results indicate that tcr r10p1a7 and tcr r13p1c6 are expressed on t - cell surface and can bind specifically to mhc / igf2bp3 - 001 complex . to determine whether the tcrs induce mhc / igf2bp3 - 001 - specific cytotoxic activity , the transformed cd8 + t - cells were co - incubated with igf2bp3 - 001 - loaded target cells or with target cells loaded with similar but unrelated peptide , or with the controls , e . g ., unloaded target cells and cd8 + t - cells only , followed by ifn - γ release assay . ifn - γ secretion from cd8 + t - cells is indicative of t - cell activation with cytotoxic activity . it was found that all primary cd8 + t - cells transformed with tcrs of the present disclosure , after co - incubation with igf2bp3 - 001 - loaded target cells , released much higher levels of ifn - γ than that stimulated by unrelated peptide - loaded target cells , and the controls . target peptide titration analysis showed ec50 at ˜ 1 nm ( tcr r10p1a7 ) and ˜ 0 . 8 nm ( tcr r13p1c6 ). these results suggest that tcrs of the present invention can activate cytotoxic t - cell activity , e . g ., ifn - γ release , through specific interaction with the mhc / igf2bp3 - 001 complex . to determine the binding motif of the tcrs for the mhc / igf2bp3 - 001 complex , positional alanine scanning analysis was performed at each of the 9 amino acids of the igf2bp3 - 001 peptide . alanine - substituted igf2bp3 - 001 peptides are shown in table 7 . a genome - wide screen for a * 02 - binding peptides with an identical motif revealed no potentially cross - reactive peptides . these results suggest that igf2bp3 - 001 positions 3 - 6 may be important for tcr r10p1a7 and tcr r13p1c6 binding . to determine efficacy of t - cells expressing tcrs described herein , primary cd8 + t - cells transformed with tcr r10p1a7 were co - incubated with human cancer cell lines , e . g ., a - 375 ( human melanoma cell line ) and t98g ( human glioblastoma cell line ), which are hla - a2 - positive and igf2bp3 - 001 ( target )- positive , and sk - br - 3 ( human breast cancer cell line ), which is hla - a2 - negative and igf2bp3 - 001 - negative , followed by ifnγ release assay . ifnγ release was observed in both a - 375 and t98g cells , which are hla - a2 - positive and igf2bp3 - 001 - positive , but not in sk - br - 3 cells , which have basal levels of ifnγ release that is comparable to that of effector cell only control . these results indicate that t - cells expressing tcr r10p1a7 can specifically induce cytotoxic activity targeting cancer cells in a hla - a2 / igf2bp3 - 001 specific manner . the present description provides tcrs that are useful in treating cancers / tumors , preferably melanoma and glioblastoma that over - or exclusively present igf2bp3 - 001 . gamma delta ( γδ ) t cells , which are non - conventional t lymphocyte effectors implicated in the first line of defense against pathogens , can interact with and eradicate tumor cells in a mhc - independent manner through activating receptors , among others , tcr - gamma and tcr - delta chains . these γδ t cells display a preactivated phenotype that allows rapid cytokine production ( ifn - γ , tnf - α ) and strong cytotoxic response upon activation . these t - cells have anti - tumor activity against many cancers and suggest that γδ t cell - mediated immunotherapy is feasible and can induce objective tumor responses . ( braza et al . 2013 ). recent advances using immobilized antigens , agonistic monoclonal antibodies ( mabs ), tumor - derived artificial antigen presenting cells ( aapc ), or combinations of activating mabs and aapc have been successful in expanding gamma delta t - cells with oligoclonal or polyclonal tcr repertoires . for example , immobilized major histocompatibility complex class - i chain - related a was a stimulus for γδ t - cells expressing tcrδ1 isotypes , and plate - bound activating antibodies have expanded vδ1 and vδ2 cells ex vivo . clinically sufficient quantities of tcrδ1 , tcrδ2 , and tcrδ1 neg tcrδ2 neg have been produced following co - culture on aapc , and these subsets displayed differences in memory phenotype and reactivity to tumors in vitro and in vivo . ( deniger et al . 2014 ). in addition , γδ t - cells are amenable to genetic modification as evidenced by introduction of tcr - alpha and tcr - beta chains . ( hiasa et al . 2009 ). another aspect of the present description relates to production of γδ t - cells expressing tcr - alpha and tcr - beta that bind to igf2bp3 - 001 . to do so , γδ t - cells are expanded by methods described by deniger et al . 2014 , followed by transducing the recombinant viruses expressing the tcrs that bind to igf2bp3 - 001 ( as described in example 3 ) into the expanded γδ t - cells . the virus - transduced γδ t - cells are then infused into the patient . in situ hybridization ( ish ) was used to detect mrna expression directly in formalin - fixed or frozen tissue sections . due to its high sensitivity and its spatial resolution , it is a suitable method to determine cell type specific target expression and the distribution or frequency of target expression within cancer tissue sections . ish has been performed to detect igf2bp3 mrna using the rnascope ® technology developed by advanced cell diagnostics ( acd ). the rnascope ® technology is based on the hybridization of around 20 pairs of z - shaped oligonucleotide probes to the target sequence . signal amplification is achieved by branched dna amplification , which is based on multiple hybridization steps of oligonucleotides , ultimately building up a branched dna ( bdna ) tree . finally , a great number of label probes hybridize to the branches of the bdna tree and the enhanced signal can be detected . the chromogenic rnascope ® detection kit ( red ) includes label probes which are linked to an enzyme ( alkaline phosphatase ). signal detection depends on the enzymatic conversion of the chromogenic substrate fastred , which additionally amplifies the original signal . rnascope ® is a very sensitive technology , which is due to the efficient process of signal amplification , paired with the high sensitivity and the robust binding of the z probe pairs to the target mrna , even if it is partially crosslinked or degraded . according to acd , binding of three out of 20 probe pairs to each single rna molecule is enough to generate a detectable ish signal . each ish experiment is subdivided into two methodological processes : 1 ) tissue pretreatment for target retrieval , and 2 ) target hybridization , signal amplification and detection . optimal pretreatment conditions are critical for successful target detection in ffpe tissue sections . the fixation process induces crosslinking of proteins , dna and rna in cells and tissues and thereby masks hybridization sites . thus , to assure accessibility of the target mrna and proper binding of the probe set , these crosslinks have to be removed prior to target hybridization . tissue pretreatment includes three discrete steps : 1 ) blocking of endogenous alkaline phosphatase by hydrogen peroxide treatment , 2 ) target retrieval by boiling in target retrieval reagent , and 3 ) target retrieval by protease digestion . as the extent of fixation and crosslinking may vary between different ffpe blocks , the optimal target retrieval conditions have to be determined experimentally for each individual ffpe block . therefore , tissue sections were exposed to different boiling and protease digestion times followed by hybridization with a positive and a negative control probe set . the optimal conditions were determined by microscopic evaluation of specific signal intensity in the positive control , unspecific background in the negative control and tissue morphology . tissue pretreatment was performed according to the manufacturer &# 39 ; 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