                         SEQUENCE LISTING

<110>  Neogene Therapeutics B.V.
 
<120>  METHOD TO ISOLATE TCR GENES

<130>  NTBV.001WO

<150>  US 62/874125
<151>  2019-07-15

<150>  US 62/975924
<151>  2020-02-13

<150>  US 63/024341
<151>  2020-05-13

<150>  US 63/034157
<151>  2020-06-03

<150>  US 63/039346
<151>  2020-06-15

<160>  4     

<170>  PatentIn version 3.5

<210>  1
<211>  819
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Synthetic; Amino acid sequence for a TCR gene expressed as a 
       TCRbeta-P2A-TCRalpha-T2A-Puromycin resistance expression cassette

<400>  1

Met Gly Thr Arg Leu Leu Cys Trp Ala Ala Leu Cys Leu Leu Gly Ala 
1               5                   10                  15      


Glu Leu Thr Glu Ala Gly Val Ala Gln Ser Pro Arg Tyr Lys Ile Ile 
            20                  25                  30          


Glu Lys Arg Gln Ser Val Ala Phe Trp Cys Asn Pro Ile Ser Gly His 
        35                  40                  45              


Ala Thr Leu Tyr Trp Tyr Gln Gln Ile Leu Gly Gln Gly Pro Lys Leu 
    50                  55                  60                  


Leu Ile Gln Phe Gln Asn Asn Gly Val Val Asp Asp Ser Gln Leu Pro 
65                  70                  75                  80  


Lys Asp Arg Phe Ser Ala Glu Arg Leu Lys Gly Val Asp Ser Thr Leu 
                85                  90                  95      


Lys Ile Gln Pro Ala Lys Leu Glu Asp Ser Ala Val Tyr Leu Cys Ala 
            100                 105                 110         


Ser Ser Leu Tyr Pro Tyr Asp Gly Arg Ser Ser Tyr Glu Gln Tyr Phe 
        115                 120                 125             


Gly Pro Gly Thr Arg Leu Thr Val Thr Glu Asp Leu Arg Asn Val Thr 
    130                 135                 140                 


Pro Pro Lys Val Ser Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn 
145                 150                 155                 160 


Lys Gln Lys Ala Thr Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp 
                165                 170                 175     


His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly 
            180                 185                 190         


Val Cys Thr Asp Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys 
        195                 200                 205             


Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg 
    210                 215                 220                 


Asn His Phe Arg Cys Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp 
225                 230                 235                 240 


Lys Trp Pro Glu Gly Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala 
                245                 250                 255     


Glu Ala Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His 
            260                 265                 270         


Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys 
        275                 280                 285             


Ala Thr Leu Tyr Ala Val Leu Val Ser Gly Leu Val Leu Met Ala Met 
    290                 295                 300                 


Val Lys Lys Lys Asn Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu 
305                 310                 315                 320 


Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Thr Ser Ile 
                325                 330                 335     


Arg Ala Val Phe Ile Phe Leu Trp Leu Gln Leu Asp Leu Val Asn Gly 
            340                 345                 350         


Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser Val Gln Glu Gly Asp 
        355                 360                 365             


Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser Ala Ser Asn Tyr Phe 
    370                 375                 380                 


Pro Trp Tyr Lys Gln Glu Leu Gly Lys Gly Pro Gln Leu Ile Ile Asp 
385                 390                 395                 400 


Ile Arg Ser Asn Val Gly Glu Lys Lys Asp Gln Arg Ile Ala Val Thr 
                405                 410                 415     


Leu Asn Lys Thr Ala Lys His Phe Ser Leu His Ile Thr Glu Thr Gln 
            420                 425                 430         


Pro Glu Asp Ser Ala Val Tyr Phe Cys Ala Ala Tyr Asn Gln Gly Gly 
        435                 440                 445             


Lys Leu Ile Phe Gly Gln Gly Thr Glu Leu Ser Val Lys Pro Asp Ile 
    450                 455                 460                 


Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln 
465                 470                 475                 480 


Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile Asn Val 
                485                 490                 495     


Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Cys Val Leu 
            500                 505                 510         


Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile Ala Trp Ser 
        515                 520                 525             


Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala 
    530                 535                 540                 


Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr Glu Lys 
545                 550                 555                 560 


Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Ser Val Met 
                565                 570                 575     


Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met 
            580                 585                 590         


Thr Leu Arg Leu Trp Ser Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu 
        595                 600                 605             


Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Thr Glu Tyr 
    610                 615                 620                 


Lys Pro Thr Val Arg Leu Ala Thr Arg Asp Asp Val Pro Arg Ala Val 
625                 630                 635                 640 


Arg Thr Leu Ala Ala Ala Phe Ala Asp Tyr Pro Ala Thr Arg His Thr 
                645                 650                 655     


Val Asp Pro Asp Arg His Ile Glu Arg Val Thr Glu Leu Gln Glu Leu 
            660                 665                 670         


Phe Leu Thr Arg Val Gly Leu Asp Ile Gly Lys Val Trp Val Ala Asp 
        675                 680                 685             


Asp Gly Ala Ala Val Ala Val Trp Thr Thr Pro Glu Ser Val Glu Ala 
    690                 695                 700                 


Gly Ala Val Phe Ala Glu Ile Gly Pro Arg Met Ala Glu Leu Ser Gly 
705                 710                 715                 720 


Ser Arg Leu Ala Ala Gln Gln Gln Met Glu Gly Leu Leu Ala Pro His 
                725                 730                 735     


Arg Pro Lys Glu Pro Ala Trp Phe Leu Ala Thr Val Gly Val Ser Pro 
            740                 745                 750         


Asp His Gln Gly Lys Gly Leu Gly Ser Ala Val Val Leu Pro Gly Val 
        755                 760                 765             


Glu Ala Ala Glu Arg Ala Gly Val Pro Ala Phe Leu Glu Thr Ser Ala 
    770                 775                 780                 


Pro Arg Asn Leu Pro Phe Tyr Glu Arg Leu Gly Phe Thr Val Thr Ala 
785                 790                 795                 800 


Asp Val Glu Val Pro Glu Gly Pro Arg Thr Trp Cys Met Thr Arg Lys 
                805                 810                 815     


Pro Gly Ala 
            


<210>  2
<211>  467
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Synthetic; Amino acid sequence for a CD8alpha-P2A-CD8beta 
       transgene

<400>  2

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 
1               5                   10                  15      


His Ala Ala Arg Pro Ser Gln Phe Arg Val Ser Pro Leu Asp Arg Thr 
            20                  25                  30          


Trp Asn Leu Gly Glu Thr Val Glu Leu Lys Cys Gln Val Leu Leu Ser 
        35                  40                  45              


Asn Pro Thr Ser Gly Cys Ser Trp Leu Phe Gln Pro Arg Gly Ala Ala 
    50                  55                  60                  


Ala Ser Pro Thr Phe Leu Leu Tyr Leu Ser Gln Asn Lys Pro Lys Ala 
65                  70                  75                  80  


Ala Glu Gly Leu Asp Thr Gln Arg Phe Ser Gly Lys Arg Leu Gly Asp 
                85                  90                  95      


Thr Phe Val Leu Thr Leu Ser Asp Phe Arg Arg Glu Asn Glu Gly Tyr 
            100                 105                 110         


Tyr Phe Cys Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe 
        115                 120                 125             


Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg 
    130                 135                 140                 


Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 
145                 150                 155                 160 


Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 
                165                 170                 175     


Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 
            180                 185                 190         


Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His 
        195                 200                 205             


Arg Asn Arg Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val Lys Ser 
    210                 215                 220                 


Gly Asp Lys Pro Ser Leu Ser Ala Arg Tyr Val Gly Ser Gly Ala Thr 
225                 230                 235                 240 


Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly 
                245                 250                 255     


Pro Met Arg Pro Arg Leu Trp Leu Leu Leu Ala Ala Gln Leu Thr Val 
            260                 265                 270         


Leu His Gly Asn Ser Val Leu Gln Gln Thr Pro Ala Tyr Ile Lys Val 
        275                 280                 285             


Gln Thr Asn Lys Met Val Met Leu Ser Cys Glu Ala Lys Ile Ser Leu 
    290                 295                 300                 


Ser Asn Met Arg Ile Tyr Trp Leu Arg Gln Arg Gln Ala Pro Ser Ser 
305                 310                 315                 320 


Asp Ser His His Glu Phe Leu Ala Leu Trp Asp Ser Ala Lys Gly Thr 
                325                 330                 335     


Ile His Gly Glu Glu Val Glu Gln Glu Lys Ile Ala Val Phe Arg Asp 
            340                 345                 350         


Ala Ser Arg Phe Ile Leu Asn Leu Thr Ser Val Lys Pro Glu Asp Ser 
        355                 360                 365             


Gly Ile Tyr Phe Cys Met Ile Val Gly Ser Pro Glu Leu Thr Phe Gly 
    370                 375                 380                 


Lys Gly Thr Gln Leu Ser Val Val Asp Phe Leu Pro Thr Thr Ala Gln 
385                 390                 395                 400 


Pro Thr Lys Lys Ser Thr Leu Lys Lys Arg Val Cys Arg Leu Pro Arg 
                405                 410                 415     


Pro Glu Thr Gln Lys Gly Pro Leu Cys Ser Pro Ile Thr Leu Gly Leu 
            420                 425                 430         


Leu Val Ala Gly Val Leu Val Leu Leu Val Ser Leu Gly Val Ala Ile 
        435                 440                 445             


His Leu Cys Cys Arg Arg Arg Arg Ala Arg Leu Arg Phe Met Lys Gln 
    450                 455                 460                 


Phe Tyr Lys 
465         


<210>  3
<211>  2405
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Synthetic; Nucleotide sequence for K562-HLA-A*02:01-IRES-FusionRed

<400>  3
atggctgtta tggcccctag aacactggtg ctgctgctgt ctggtgccct ggctctgaca       60

caaacatggg ccggcagcca cagcatgcgg tactttttca ccagcgtgtc cagacctggc      120

agaggcgagc ctagattcat tgccgtgggc tacgtggacg acacccagtt cgtcagattc      180

gattccgatg ccgccagcca gcggatggaa cctagagcac cttggatcga gcaagagggc      240

cccgagtatt gggacggcga gacaagaaaa gtgaaggccc acagccagac acacagagtg      300

gatctgggaa ccctgcgggg ctactacaat cagtctgagg ccggctctca caccgtgcag      360

aggatgtatg gctgtgacgt gggcagcgat tggcggttcc tgagaggcta tcaccagtac      420

gcctacgacg gcaaggacta tatcgccctg aaagaggacc tgcggtcttg gacagccgcc      480

gatatggctg cccagaccac aaagcacaag tgggaagccg ctcacgtggc cgaacagctg      540

agagcttatc tggaaggcac ctgtgtggaa tggctgcgga gatacctgga aaacggcaaa      600

gagacactgc agcggacaga cgcccctaag acacacatga cacaccacgc cgtgtccgac      660

cacgaagcca cacttagatg ttgggccctg agcttctacc ccgccgagat cacactgaca      720

tggcagagag atggcgagga tcagacccag gataccgagc tggtggaaac aagacctgcc      780

ggcgacggca ccttccagaa atgggctgct gtggtggtgc ctagcggcca agagcagaga      840

tacacctgtc acgtgcagca cgagggcctg cctaagcctc ttacactgag atgggagccc      900

agcagccagc ctacaatccc catcgtggga atcattgccg gcctggtgct gtttggcgcc      960

gtgattacag gtgcagtggt ggccgctgtt atgtggcgga gaaagagcag cgacagaaaa     1020

ggcggcagct actctcaggc cgccagctct gattctgccc agggctctga tgtgtctctg     1080

accgcctgca aagtctgagc ggccgctacg taaattccgc ccccccccct cccccccccc     1140

taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt     1200

ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt     1260

gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt     1320

cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct     1380

ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt     1440

ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt     1500

ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa     1560

ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta     1620

gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa     1680

aacacgatga taatatggcc acaaccatgg tgtccgagct gatcaaagaa aacatgccca     1740

tgaagctgta catggaaggg accgtgaaca accaccactt caagtgcacc agcgaaggcg     1800

agggcaagcc ttatgagggc acccagacca tgcggatcaa ggtggtggaa ggtggccctc     1860

tgcctttcgc ctttgatatc ctggccacca gctttatgta cggcagccgg accttcatca     1920

agcaccctcc aggcatcccc gatttcttca agcagagctt tcccgagggc ttcacctggg     1980

agagagtgac cacatacgag gatggcggcg tgctgacagc cacacaggat acaagtctgc     2040

aggacggctg cctgatctac aacgtgaaag tgcggggcgt gaacttcccc gccaatggac     2100

ctgtgatgca gaagaaaacc ctcggctggg aagccagcac cgagacaatg tatcctgccg     2160

acggtggact ggaaggggcc tgtgatatgg ccctgaagct tgttggcggc ggacacctga     2220

tctgcaacct ggaaaccacc tacagaagca agaagcccgc caccaacctg aagatgcccg     2280

gcgtgtacaa cgtggaccac cggctggaaa ggatcaaaga ggccgacgac gagacatacg     2340

tggaacagca cgaagtggcc gtggccagat actctacagg cggagctggc gacggcggca     2400

aatga                                                                 2405


<210>  4
<211>  755
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Synthetic; Amino acid sequence for a TCR gene expressed as a 
       TCRbeta-P2A-TCRalpha-T2A-Blasticidin resistance expression 
       cassette

<400>  4

Met Gly Pro Gln Leu Leu Gly Tyr Val Val Leu Cys Leu Leu Gly Ala 
1               5                   10                  15      


Gly Pro Leu Glu Ala Gln Val Thr Gln Asn Pro Arg Tyr Leu Ile Thr 
            20                  25                  30          


Val Thr Gly Lys Lys Leu Thr Val Thr Cys Ser Gln Asn Met Asn His 
        35                  40                  45              


Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Gln 
    50                  55                  60                  


Ile Tyr Tyr Ser Met Asn Val Glu Val Thr Asp Lys Gly Asp Val Pro 
65                  70                  75                  80  


Glu Gly Tyr Lys Val Ser Arg Lys Glu Lys Arg Asn Phe Pro Leu Ile 
                85                  90                  95      


Leu Glu Ser Pro Ser Pro Asn Gln Thr Ser Leu Tyr Phe Cys Ala Ser 
            100                 105                 110         


Arg Ala Ser Gly Arg Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr 
        115                 120                 125             


Val Leu Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe 
    130                 135                 140                 


Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val 
145                 150                 155                 160 


Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp 
                165                 170                 175     


Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Ala 
            180                 185                 190         


Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val 
        195                 200                 205             


Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val 
    210                 215                 220                 


Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro 
225                 230                 235                 240 


Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp 
                245                 250                 255     


Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu Ser Ala Thr 
            260                 265                 270         


Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu 
        275                 280                 285             


Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys Lys Asn Ser Gly 
    290                 295                 300                 


Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu 
305                 310                 315                 320 


Glu Asn Pro Gly Pro Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu 
                325                 330                 335     


Trp Leu Gln Leu Ser Trp Val Trp Ser Gln Asp Gln Gln Val Lys Gln 
            340                 345                 350         


Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn 
        355                 360                 365             


Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys 
    370                 375                 380                 


Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys 
385                 390                 395                 400 


Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala 
                405                 410                 415     


Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala 
            420                 425                 430         


Val Tyr Phe Cys Ala Ala Phe Leu Gln Ser Asn Asp Met Arg Phe Gly 
        435                 440                 445             


Ala Gly Thr Arg Leu Thr Val Lys Pro Asp Ile Gln Asn Pro Glu Pro 
    450                 455                 460                 


Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys 
465                 470                 475                 480 


Leu Phe Thr Asp Phe Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu 
                485                 490                 495     


Ser Gly Thr Phe Ile Thr Asp Lys Cys Val Leu Asp Met Lys Ala Met 
            500                 505                 510         


Asp Ser Lys Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe 
        515                 520                 525             


Thr Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser 
    530                 535                 540                 


Asp Val Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp 
545                 550                 555                 560 


Met Asn Leu Asn Phe Gln Asn Leu Ser Val Met Gly Leu Arg Ile Leu 
                565                 570                 575     


Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp 
            580                 585                 590         


Ser Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp 
        595                 600                 605             


Val Glu Glu Asn Pro Gly Pro Met Lys Thr Phe Asn Ile Ser Gln Gln 
    610                 615                 620                 


Asp Leu Glu Leu Val Glu Val Ala Thr Glu Lys Ile Thr Met Leu Tyr 
625                 630                 635                 640 


Glu Asp Asn Lys His His Val Gly Ala Ala Ile Arg Thr Lys Thr Gly 
                645                 650                 655     


Glu Ile Ile Ser Ala Val His Ile Glu Ala Tyr Ile Gly Arg Val Thr 
            660                 665                 670         


Val Cys Ala Glu Ala Ile Ala Ile Gly Ser Ala Val Ser Asn Gly Gln 
        675                 680                 685             


Lys Asp Phe Asp Thr Ile Val Ala Val Arg His Pro Tyr Ser Asp Glu 
    690                 695                 700                 


Val Asp Arg Ser Ile Arg Val Val Ser Pro Cys Gly Met Cys Arg Glu 
705                 710                 715                 720 


Leu Ile Ser Asp Tyr Ala Pro Asp Cys Phe Val Leu Ile Glu Met Asn 
                725                 730                 735     


Gly Lys Leu Val Lys Thr Thr Ile Glu Glu Leu Ile Pro Leu Lys Tyr 
            740                 745                 750         


Thr Arg Asn 
        755 


