
                                SEQUENCE LISTING

<110> DUKE UNIVERSITY

<120> CERTAIN IMPROVED HUMAN BISPECIFIC
  EGFRViii ANTIBODY ENGAGING MOLECULES
  

<130> 000250.00174

<160> 10

<170> FastSEQ for Windows Version 4.0

<210> 1
<211> 1506
<212> DNA
<213> Artificial Sequence

<220> 
<223> modified human sequence construct; codon optimized
      for expression in CHO cells

<400> 1
atgaagtggg tgacctttat tagcctgctg ttcctgttct cctccgccta ttccgacatc      60
cagatgactc agagcccttc ttccctgtca gcttccgtgg gcgacagggt caccatcaca     120
tgccgggctt cccagggaat tagaaacaat ctggcatggt accagcagaa gccaggcaaa     180
gcccccaagc gcctgatcta tgccgcttct aacctgcaga gtggagtgcc ctcacgattc     240
acaggcagcg gatctgggac agagtttact ctgattgtct ccagcctgca gccagaagat     300
ttcgccactt actattgcct gcagcaccat tcctaccccc tgacaagcgg cggagggact     360
aaagtggaga tcaagggtgg aggaggatct ggtggaggag gaagtggtgg aggaggatca     420
gaggtgcagg tcctggaaag cggtggagga ctggtgcagc caggaggttc cctgcgtctg     480
agctgtgcag cctctggctt caccttttct agttatgcaa tgtcctgggt gcgccaggca     540
cctggcaagg gactggaatg ggtcagcgca atcagtggct caggcggaag tacaaactac     600
gccgactcag tgaaaggaag gttcaccatt agtcgcgata actcaaagaa tactctgtat     660
ctgcagatga atagcctgcg ggccgaggac accgctgtgt actattgcgc tggctcatcc     720
ggatggtctg aatactgggg acaggggacc ctggtgacag tcagctctgg gggtggcgga     780
tctcaggtgc agctggtcga gagtggaggt ggagtggtcc agccaggaag gtccctgcga     840
ctgagctgtg ctgcatctgg tttcaaattt tctggttacg gcatgcactg ggtgagacag     900
gctcccggaa aggggctgga atgggtggca gtcatctggt atgacggaag caagaaatac     960
tatgtggatt ctgtcaaagg gcgattcacc attagtcgtg ataactcaaa gaatacactg    1020
tacctgcaaa tgaatagctt acgggcagag gacactgccg tgtactattg cgctagacag    1080
atgggctatt ggcattttga tctgtggggt cgcggcactc tggtgaccgt cagttctgga    1140
ggaggtggat ccggaggagg tggaagcgga gggggtggct ctgagatcgt gctgacccag    1200
tctccagcaa cactgtccct gagccctgga gaacgcgcca cactgtcctg tcgagcttct    1260
cagagtgtgt ccagctacct ggcctggtat cagcagaagc ctggccaggc tccacgactg    1320
ctgatctacg acgcttccaa ccgtgcaact ggcattcctg ctaggttctc aggatccggg    1380
agcggtaccg actttactct gaccatctct agtctggagc cagaagattt cgcagtgtac    1440
tattgtcagc agaggagcaa ttggccccct ctgacttttg gagggggtac caaagtcgag    1500
attaag                                                               1506

<210> 2
<211> 502
<212> PRT
<213> Artificial Sequence

<220> 
<223> modified human sequence construct; complete amino
      acid sequence including  signal peptide

<400> 2
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala
 1               5                  10                  15
Tyr Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
            20                  25                  30
Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg
        35                  40                  45
Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg
    50                  55                  60
Leu Ile Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe
65                  70                  75                  80
Thr Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Ile Val Ser Ser Leu
                85                  90                  95
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His His Ser Tyr
            100                 105                 110
Pro Leu Thr Ser Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly
        115                 120                 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Val
    130                 135                 140
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
145                 150                 155                 160
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp
                165                 170                 175
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser
            180                 185                 190
Gly Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe
        195                 200                 205
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn
    210                 215                 220
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Gly Ser Ser
225                 230                 235                 240
Gly Trp Ser Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                245                 250                 255
Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
            260                 265                 270
Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
        275                 280                 285
Lys Phe Ser Gly Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys
    290                 295                 300
Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr
305                 310                 315                 320
Tyr Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
                325                 330                 335
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
            340                 345                 350
Ala Val Tyr Tyr Cys Ala Arg Gln Met Gly Tyr Trp His Phe Asp Leu
        355                 360                 365
Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
    370                 375                 380
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln
385                 390                 395                 400
Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser
                405                 410                 415
Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln
            420                 425                 430
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg
        435                 440                 445
Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
    450                 455                 460
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr
465                 470                 475                 480
Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro Leu Thr Phe Gly Gly Gly
                485                 490                 495
Thr Lys Val Glu Ile Lys
            500

<210> 3
<211> 484
<212> PRT
<213> Artificial Sequence

<220> 
<223> modified human sequence construct; mature amino
      acid seuqence

<400> 3
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
 1               5                  10                  15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asn
            20                  25                  30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile
        35                  40                  45
Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Thr Gly
    50                  55                  60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Ile Val Ser Ser Leu Gln Pro
65                  70                  75                  80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His His Ser Tyr Pro Leu
                85                  90                  95
Thr Ser Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser
            100                 105                 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Val Leu Glu
        115                 120                 125
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
    130                 135                 140
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg
145                 150                 155                 160
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser
                165                 170                 175
Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
            180                 185                 190
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
        195                 200                 205
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Gly Ser Ser Gly Trp
    210                 215                 220
Ser Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly
225                 230                 235                 240
Gly Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln
                245                 250                 255
Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe
            260                 265                 270
Ser Gly Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
        275                 280                 285
Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Val
    290                 295                 300
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
305                 310                 315                 320
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
                325                 330                 335
Tyr Tyr Cys Ala Arg Gln Met Gly Tyr Trp His Phe Asp Leu Trp Gly
            340                 345                 350
Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
        355                 360                 365
Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro
    370                 375                 380
Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg
385                 390                 395                 400
Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
                405                 410                 415
Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr
            420                 425                 430
Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
        435                 440                 445
Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
    450                 455                 460
Gln Gln Arg Ser Asn Trp Pro Pro Leu Thr Phe Gly Gly Gly Thr Lys
465                 470                 475                 480
Val Glu Ile Lys


<210> 4
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> signal peptide

<400> 4
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala
 1               5                  10                  15
Tyr Ser


<210> 5
<211> 107
<212> PRT
<213> Artificial Sequence

<220> 
<223> modified human sequence construct; anti-EGFRvIII
      VL sequence

<400> 5
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
 1               5                  10                  15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asn
            20                  25                  30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile
        35                  40                  45
Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Thr Gly
    50                  55                  60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Ile Val Ser Ser Leu Gln Pro
65                  70                  75                  80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His His Ser Tyr Pro Leu
                85                  90                  95
Thr Ser Gly Gly Gly Thr Lys Val Glu Ile Lys
            100                 105

<210> 6
<211> 116
<212> PRT
<213> Artificial Sequence

<220> 
<223> modified human sequence construct; anti-EGFRvIII
      VH sequence

<400> 6
Glu Val Gln Val Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
 1               5                  10                  15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
            20                  25                  30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
        35                  40                  45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val
    50                  55                  60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65                  70                  75                  80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                85                  90                  95
Ala Gly Ser Ser Gly Trp Ser Glu Tyr Trp Gly Gln Gly Thr Leu Val
            100                 105                 110
Thr Val Ser Ser
        115

<210> 7
<211> 118
<212> PRT
<213> Artificial Sequence

<220> 
<223> modified human sequence construct; anti-CD3VH
      sequence

<400> 7
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
 1               5                  10                  15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe Ser Gly Tyr
            20                  25                  30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
        35                  40                  45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Val Asp Ser Val
    50                  55                  60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65                  70                  75                  80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                85                  90                  95
Ala Arg Gln Met Gly Tyr Trp His Phe Asp Leu Trp Gly Arg Gly Thr
            100                 105                 110
Leu Val Thr Val Ser Ser
        115

<210> 8
<211> 108
<212> PRT
<213> Artificial Sequence

<220> 
<223> modified human sequence construct; anti-CD3 VL
      sequence

<400> 8
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
 1               5                  10                  15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
            20                  25                  30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
        35                  40                  45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
    50                  55                  60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65                  70                  75                  80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro
                85                  90                  95
Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
            100                 105

<210> 9
<211> 15
<212> PRT
<213> Artificial Sequence

<220> 
<223> linker 1

<400> 9
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
 1               5                  10                  15

<210> 10
<211> 5
<212> PRT
<213> Artificial Sequence

<220> 
<223> linker 2

<400> 10
Gly Gly Gly Gly Ser
 1               5
