                         SEQUENCE LISTING

<110>  Board of Trustees of the University of Illinois
       Fred Hutchinson Cancer Research Center
       Smith, Sheena N.
       Harris, Daniel T.
       Kranz, David M.
       Greenberg, Philip D.
       Schmitt, Thomas M.
 
<120>  ENGINEERED HIGH-AFFINITY HUMAN T CELL RECEPTORS

<130>  IMMU-003/02WO

<150>  US 61/907,887
<151>  2013-11-22

<160>  34    

<170>  PatentIn version 3.5

<210>  1
<211>  115
<212>  PRT
<213>  Homo sapiens

<400>  1

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly 
1               5                   10                  15      


Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His Glu Asn Met 
            20                  25                  30          


Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe 
        35                  40                  45              


Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr 
    50                  55                  60                  


Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser 
65                  70                  75                  80  


Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Ser Ile 
                85                  90                  95      


Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Asp 
            100                 105                 110         


Leu Lys Asn 
        115 


<210>  2
<211>  112
<212>  PRT
<213>  Homo sapiens

<400>  2

Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly 
1               5                   10                  15      


Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Val Ser Gln Ser 
            20                  25                  30          


Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met 
        35                  40                  45              


Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln 
    50                  55                  60                  


Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln 
65                  70                  75                  80  


Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Ala Asn Asn Ala Gly Asn 
                85                  90                  95      


Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro His Ile 
            100                 105                 110         


<210>  3
<211>  115
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Modified variable beta region of the TCR with high affinity to 
       WT1/HLA-A2

<400>  3

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly 
1               5                   10                  15      


Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His Glu Asn Met 
            20                  25                  30          


Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Ser 
        35                  40                  45              


Ser Tyr Gly Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr 
    50                  55                  60                  


Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser 
65                  70                  75                  80  


Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Thr Ser Asn Tyr 
                85                  90                  95      


Leu Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Asp 
            100                 105                 110         


Leu Lys Asn 
        115 


<210>  4
<211>  112
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Modified variable alpha region of the TCR with high affinity to 
       WT1/HLA-A2

<400>  4

Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly 
1               5                   10                  15      


Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Asp Leu Gly Ser 
            20                  25                  30          


Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met 
        35                  40                  45              


Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln 
    50                  55                  60                  


Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln 
65                  70                  75                  80  


Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Ala Asn Asn Ala Gly Asn 
                85                  90                  95      


Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro His Ile 
            100                 105                 110         


<210>  5
<211>  244
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Single-chain TCR that binds with high-affinity to WT1/HLA-A2

<400>  5

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly 
1               5                   10                  15      


Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His Glu Asn Met 
            20                  25                  30          


Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Ser 
        35                  40                  45              


Ser Tyr Gly Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr 
    50                  55                  60                  


Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser 
65                  70                  75                  80  


Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Thr Ser Asn Tyr 
                85                  90                  95      


Leu Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Asp 
            100                 105                 110         


Leu Lys Asn Gly Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys 
        115                 120                 125             


Asp Gly Lys Ser Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser 
    130                 135                 140                 


Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg 
145                 150                 155                 160 


Asp Leu Gly Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro 
                165                 170                 175     


Glu Leu Ile Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg 
            180                 185                 190         


Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile 
        195                 200                 205             


Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Ala Asn 
    210                 215                 220                 


Asn Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val 
225                 230                 235                 240 


Lys Pro His Ile 
                


<210>  6
<211>  9
<212>  PRT
<213>  Homo sapiens

<400>  6

Arg Met Phe Pro Asn Ala Pro Tyr Leu 
1               5                   


<210>  7
<211>  9
<212>  PRT
<213>  Human T cell lymphotrophic virus

<400>  7

Leu Leu Phe Gly Tyr Pro Val Tyr Val 
1               5                   


<210>  8
<211>  17
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Amino acid linker sequence

<400>  8

Gly Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys Asp Gly Lys 
1               5                   10                  15      


Ser 
    


<210>  9
<211>  24
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Primer Splice 4L

<400>  9
ggcagcccca taaacacaca gtat                                              24


<210>  10
<211>  63
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Reverse primer used to generate the WT1-D13 CDR1alpha library

<400>  10
acgatcgcta taggtgcagt tcaatgatgc aatagcacct tccgggacac ttaatgggcc       60

gct                                                                     63


<210>  11
<211>  81
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Forward primer used to generate the WT1-D13 CDR1alpha library


<220>
<221>  misc_feature
<222>  (34)..(35)
<223>  n = any base

<220>
<221>  misc_feature
<222>  (37)..(38)
<223>  n = any base

<220>
<221>  misc_feature
<222>  (40)..(41)
<223>  n = any base

<220>
<221>  misc_feature
<222>  (43)..(44)
<223>  n = any base

<400>  11
attgcatcat tgaactgcac ctatagcgat cgtnnsnnsn nsnnsttctt ttggtataga       60

cagtacagtg gcaaatcccc g                                                 81


<210>  12
<211>  20
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Primer T7

<400>  12
taatacgact cactataggg                                                   20


<210>  13
<211>  66
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Reverse primer used to generate the WT1-D13.1 CDR3 beta1 library

<400>  13
tgcacacagg tacatggaag tttgattggt actagcgctt tccagaatca aactgaaacg       60

ttcttt                                                                  66


<210>  14
<211>  84
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Forward primer used to generate the WT1-D13.1 CDR3 beta1 library


<220>
<221>  misc_feature
<222>  (34)..(35)
<223>  n = any base

<220>
<221>  misc_feature
<222>  (37)..(38)
<223>  n = any base

<220>
<221>  misc_feature
<222>  (40)..(41)
<223>  n = any base

<220>
<221>  misc_feature
<222>  (43)..(44)
<223>  n = any base

<220>
<221>  misc_feature
<222>  (46)..(47)
<223>  n is a, c, g, or t

<400>  14
agtaccaatc aaacttccat gtacctgtgt gcannsnnsn nsnnsnnsga acagtttttc       60

ggcccaggta caagattaac ggtg                                              84


<210>  15
<211>  66
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Reverse primer used to generate the WT1-D13.1 CDR3 beta2 library

<400>  15
tgcacacagg tacatggaag tttgattggt actagcgctt tccagaatca aactgaaacg       60

ttcttt                                                                  66


<210>  16
<211>  84
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Forward primer used to generate theWT1-D13.1 CDR3 beta2 library


<220>
<221>  misc_feature
<222>  (46)..(47)
<223>  n = any base

<220>
<221>  misc_feature
<222>  (49)..(50)
<223>  n = any base

<220>
<221>  misc_feature
<222>  (52)..(53)
<223>  n = any base

<220>
<221>  misc_feature
<222>  (55)..(56)
<223>  n = any base

<220>
<221>  misc_feature
<222>  (58)..(59)
<223>  n is a, c, g, or t

<400>  16
agtaccaatc aaacttccat gtacctgtgt gcaagcagtt ccatcnnsnn snnsnnsnns       60

ggcccaggta caagattaac ggtg                                              84


<210>  17
<211>  66
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Reverse primer used to generate the WT1-D13.1 CDR3  alpha1 
       library

<400>  17
ggcgcacagg taagtggcgc tatctgacgg ttggctatca cggattaaca gagagacata       60

ctggga                                                                  66


<210>  18
<211>  84
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Forward primer used to generate theWT1-D13.1 CDR3  alpha1 library


<220>
<221>  misc_feature
<222>  (34)..(35)
<223>  n = any base

<220>
<221>  misc_feature
<222>  (37)..(38)
<223>  n = any base

<220>
<221>  misc_feature
<222>  (40)..(41)
<223>  n = any base

<220>
<221>  misc_feature
<222>  (43)..(44)
<223>  n = any base

<220>
<221>  misc_feature
<222>  (46)..(47)
<223>  n is a, c, g, or t

<400>  18
caaccgtcag atagcgccac ttacctgtgc gccnnsnnsn nsnnsnnsaa tatgctgacc       60

ttcggtggcg gtactcgctt aatg                                              84


<210>  19
<211>  66
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Reverse primer used to generate the WT1-D13.1 CDR3  alpha2 
       library

<400>  19
ggcgcacagg taagtggcgc tatctgacgg ttggctatca cggattaaca gagagacata       60

ctggga                                                                  66


<210>  20
<211>  84
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Forward primer used to generate the WT1-D13.1 CDR3  alpha2 
       library


<220>
<221>  misc_feature
<222>  (46)..(47)
<223>  n = any base

<220>
<221>  misc_feature
<222>  (49)..(50)
<223>  n = any base

<220>
<221>  misc_feature
<222>  (52)..(53)
<223>  n = any base

<220>
<221>  misc_feature
<222>  (55)..(56)
<223>  n = any base

<220>
<221>  misc_feature
<222>  (58)..(59)
<223>  n is a, c, g, or t

<400>  20
caaccgtcag atagcgccac ttacctgtgc gccgcgaata acgcgnnsnn snnsnnsnns       60

ttcggtggcg gtactcgctt aatg                                              84


<210>  21
<211>  115
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Modified variable beta region of the TCR with high affinity to 
       WT1/HLA-A2

<400>  21

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly 
1               5                   10                  15      


Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His Glu Asn Met 
            20                  25                  30          


Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Ser 
        35                  40                  45              


Ser Tyr Gly Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr 
    50                  55                  60                  


Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser 
65                  70                  75                  80  


Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Ser Ile 
                85                  90                  95      


Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Asp 
            100                 105                 110         


Leu Lys Asn 
        115 


<210>  22
<211>  112
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Modified variable alpha region of the TCR with high affinity to 
       WT1/HLA-A2

<400>  22

Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly 
1               5                   10                  15      


Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Val Ser Gln Pro 
            20                  25                  30          


Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met 
        35                  40                  45              


Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln 
    50                  55                  60                  


Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln 
65                  70                  75                  80  


Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Ala Asn Asn Ala Gly Asn 
                85                  90                  95      


Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro His Ile 
            100                 105                 110         


<210>  23
<211>  9
<212>  PRT
<213>  Influenza A

<400>  23

Ala Ser Asn Glu Asn Met Asp Ala Met 
1               5                   


<210>  24
<211>  9
<212>  PRT
<213>  Influenza A

<400>  24

Ala Ser Asn Glu Asn Met Glu Thr Met 
1               5                   


<210>  25
<211>  9
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  WT1 structurally similar human peptide

<400>  25

Lys Leu Phe Ser Gln Leu Ser Tyr Leu 
1               5                   


<210>  26
<211>  9
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  WT1 structurally similar human peptide

<400>  26

Lys Leu Phe Ser Thr Ala Met Phe Leu 
1               5                   


<210>  27
<211>  9
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  WT1 structurally similar human peptide

<400>  27

His Met Trp Gly Gln Ser Leu Tyr Ile 
1               5                   


<210>  28
<211>  9
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  WT1 structurally similar human peptide

<400>  28

Lys Ile Phe Gly Ser Leu Ala Phe Leu 
1               5                   


<210>  29
<211>  9
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  WT1 structurally similar human peptide

<400>  29

Lys Ile Phe Pro Ser Cys Leu Phe Val 
1               5                   


<210>  30
<211>  9
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  WT1 structurally similar human peptide

<400>  30

Lys Leu Trp Pro Ser Val Ala Phe Ala 
1               5                   


<210>  31
<211>  9
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  WT1 structurally similar human peptide

<400>  31

His Leu Trp Gly Gln Ser Leu Tyr Ile 
1               5                   


<210>  32
<211>  9
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  WT1 structurally similar human peptide

<400>  32

Arg Ile Trp Leu Ser Val Val Phe Val 
1               5                   


<210>  33
<211>  9
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  WT1 structurally similar human peptide

<400>  33

His Leu Phe Ser Ser Leu Pro Phe Ala 
1               5                   


<210>  34
<211>  9
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  WT1 structurally similar human peptide

<400>  34

Arg Ile Phe Ser Thr Ala Leu Phe Val 
1               5                   


