               SEQUENCE LISTING

<110> FONDAZIONE IRCCS ISTITUTO NAZIONALE DEI TUMORI

<120> Bispecific antibodies for use in cancer immunotherapy

<130> 13278PTWO

<150> EP15174741.7
<151> 2015-07-01

<160> 22

<170> BiSSAP 1.3

<210> 1
<211> 121
<212> PRT
<213> Homo sapiens

<220> 
<223> variable domain of a heavy chain of an immunoglobulin (VH): VHa
      of 16e2 (Class IGHV 3-20*01)

<400> 1
Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Glu Arg Pro Gly Gly 
1               5                   10                  15      
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 
            20                  25                  30          
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 
        35                  40                  45              
Ser Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val 
    50                  55                  60                  
Lys Gly Arg Val Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Lys Ile Leu Gly Ala Gly Arg Gly Trp Tyr Phe Asp Leu Trp Gly 
            100                 105                 110         
Lys Gly Thr Thr Val Thr Val Ser Ser 
        115                 120     

<210> 2
<211> 363
<212> DNA
<213> Homo sapiens

<220> 
<223> variable domain of a heavy chain of an immunoglobulin (VH): VHa;

<400> 2
gaggtacagc tggtgcaatc aggtggtggg gttgagagac ccggagggag tctccggttg      60

tcttgtgctg ccagcggctt tacgttcgat gattacggga tgtcgtgggt taggcaagct     120

ccaggcaagg gcctagaatg ggtatctggg ataaattgga acggcgggtc aacagggtat     180

gctgatagtg tgaaaggcag ggttactatt tccagggata atgccaaaaa ttcgctgtat     240

cttcagatga attcgctgcg agcggaggat actgcagtct actactgtgc taagatcctt     300

ggagcaggcc gtggatggta ttttgactta tgggggaagg gtaccaccgt gacggtctcg     360

tct                                                                   363


<210> 3
<211> 108
<212> PRT
<213> Artificial Sequence

<220> 
<223> variable domain of a light chain of an immunoglobulin (VL): VLb
      (UCTH1)
      Humanized antibody with murine CDR

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

<210> 4
<211> 324
<212> DNA
<213> Artificial Sequence

<220> 
<223> /note="amino acid sequence of a variable domain of a light chain
      of an immunoglobulin (VL): VLb (UCTH1)"
      /note="Humanized antibody with murine CDR"

<400> 4
gatattcaaa tgactcagtc cccatcctca ctctcggcat cagttggtga tcgagtgacg      60

ataacatgta gggcctcgca ggacatccgc aactatctta actggtacca acaaaaacca     120

gggaaggctc caaaattact aatctattac acctcacgtc tcgaatcagg tgttcctagt     180

cgcttcagcg gatcaggttc cggaacagat tataccctta ctatatcgtc cctccaacca     240

gaagactttg ccacttatta ctgtcaacag ggcaacacct taccctggac ttttggacag     300

gggacaaaag tcgagattaa gcgc                                            324


<210> 5
<211> 122
<212> PRT
<213> Artificial Sequence

<220> 
<223> variable domain of a heavy chain of an immunoglobulin (VH): VHb
      (UCTH1)
      Humanized antibody with murine CDR

<400> 5
Glu Val Gln Leu Val 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 Tyr Ser Phe Thr Gly Tyr 
            20                  25                  30          
Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 
        35                  40                  45              
Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe 
    50                  55                  60                  
Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala 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 Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp 
            100                 105                 110         
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 
        115                 120         

<210> 6
<211> 366
<212> DNA
<213> Homo sapiens

<220> 
<223> variable domain of a heavy chain of an immunoglobulin (VH): VHb

<400> 6
gaagtacaac tggttgagag tgggggcggt ttagttcaac cgggcggaag cctgagactt      60

tcgtgcgccg cgagtggata ttcttttacg ggctacacaa tgaactgggt ccggcaggca     120

ccaggaaagg ggcttgaatg ggtcgcttta ataaacccat ataagggcgt aagcacctat     180

aaccagaaat ttaaagatcg tttcacaata tcagtcgata aatctaagaa taccgcctat     240

ctccaaatga attccctcag agctgaagat accgccgtgt attactgtgc aaggagcggt     300

tactatggcg attcagattg gtacttcgat gtgtggggac aggggacgct tgtaactgtg     360

tcgtcc                                                                366


<210> 7
<211> 107
<212> PRT
<213> Homo sapiens

<220> 
<223> variable domain of a light chain of an immunoglobulin (VL): VLa
      of 16e2 (Class IGLV3-19*01)

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

<210> 8
<211> 321
<212> DNA
<213> Homo sapiens

<220> 
<223> variable domain of a light chain of an immunoglobulin (VL): VLa
      of 16e2 (Class IGLV3-19*01)

<400> 8
agtgaactaa ctcaggaccc cgctgtgtct gtcgcgcttg gccaaacggt gcggataacc      60

tgtcaaggtg acagtctaag gtcctattat gcatcttggt atcaacagaa acccggacaa     120

gcaccggttc tggttatcta cggtaaaaac aatcgccctt cgggtatacc tgatcgcttc     180

tccgggtcga gctccgggaa tacagcctct ctaacgataa cgggggctca agcagaagat     240

gaagcagact attattgcaa ttcaagggac agctctggca accacgtcgt ctttggggga     300

ggcacaaagt tgacagtcct t                                               321


<210> 9
<211> 21
<212> DNA
<213> Homo sapiens

<220> 
<223> (sequence which comes before VHA domain, Sfi I restriction site)

<400> 9
gcggcccagc cggccatggc c                                              21


<210> 10
<211> 18
<212> DNA
<213> Homo sapiens

<220> 
<223> (linker between VHA and VLB domains, Age I restriction site)

<400> 10
accggtggag gcggttca                                                  18


<210> 11
<211> 45
<212> DNA
<213> Homo sapiens

<220> 
<223> (linker between VLB and VHB domains)

<400> 11
ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcg                     45


<210> 12
<211> 18
<212> DNA
<213> Homo sapiens

<220> 
<223> (linker between VHB and VLA domains, Xba I restriction site)

<400> 12
ggtggaggcg gttctaga                                                  18


<210> 13
<211> 487
<212> PRT
<213> Homo sapiens

<220> 
<223> single-chain bispecific antibody

<400> 13
Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Glu Arg Pro Gly Gly 
1               5                   10                  15      
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 
            20                  25                  30          
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 
        35                  40                  45              
Ser Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val 
    50                  55                  60                  
Lys Gly Arg Val Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Lys Ile Leu Gly Ala Gly Arg Gly Trp Tyr Phe Asp Leu Trp Gly 
            100                 105                 110         
Lys Gly Thr Thr Val Thr Val Ser Ser Thr Gly Gly Gly Gly Ser Asp 
        115                 120                 125             
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp 
    130                 135                 140                 
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu 
145                 150                 155                 160 
Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 
                165                 170                 175     
Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 
            180                 185                 190         
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 
        195                 200                 205             
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr 
    210                 215                 220                 
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly Ser 
225                 230                 235                 240 
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu 
                245                 250                 255     
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 
            260                 265                 270         
Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg 
        275                 280                 285             
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro Tyr 
    290                 295                 300                 
Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile 
305                 310                 315                 320 
Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu 
                325                 330                 335     
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr 
            340                 345                 350         
Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val 
        355                 360                 365             
Thr Val Ser Ser Gly Gly Gly Gly Ser Arg Ser Glu Leu Thr Gln Asp 
    370                 375                 380                 
Pro Ala Val Ser Val Ala Leu Gly Gln Thr Val Arg Ile Thr Cys Gln 
385                 390                 395                 400 
Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro 
                405                 410                 415     
Gly Gln Ala Pro Val Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser 
            420                 425                 430         
Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser 
        435                 440                 445             
Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 
    450                 455                 460                 
Asn Ser Arg Asp Ser Ser Gly Asn His Val Val Phe Gly Gly Gly Thr 
465                 470                 475                 480 
Lys Leu Thr Val Leu Ala Ala 
                485         

<210> 14
<211> 1484
<212> DNA
<213> Homo sapiens

<220> 
<223> single-chain bispecific antibody with vector term. upstream

<400> 14
gcggcccagc cggccatggc cgaggtacag ctggtgcaat caggtggtgg ggttgagaga      60

cccggaggga gtctccggtt gtcttgtgct gccagcggct ttacgttcga tgattacggg     120

atgtcgtggg ttaggcaagc tccaggcaag ggcctagaat gggtatctgg gataaattgg     180

aacggcgggt caacagggta tgctgatagt gtgaaaggca gggttactat ttccagggat     240

aatgccaaaa attcgctgta tcttcagatg aattcgctgc gagcggagga tactgcagtc     300

tactactgtg ctaagatcct tggagcaggc cgtggatggt attttgactt atgggggaag     360

ggtaccaccg tgacggtctc gtctaccggt ggaggcggtt cagatattca aatgactcag     420

tccccatcct cactctcggc atcagttggt gatcgagtga cgataacatg tagggcctcg     480

caggacatcc gcaactatct taactggtac caacaaaaac cagggaaggc tccaaaatta     540

ctaatctatt acacctcacg tctcgaatca ggtgttccta gtcgcttcag cggatcaggt     600

tccggaacag attataccct tactatatcg tccctccaac cagaagactt tgccacttat     660

tactgtcaac agggcaacac cttaccctgg acttttggac aggggacaaa agtcgagatt     720

aagcgcggtg gaggcggttc aggcggaggt ggctctggcg gtggcggatc ggaagtacaa     780

ctggttgaga gtgggggcgg tttagttcaa ccgggcggaa gcctgagact ttcgtgcgcc     840

gcgagtggat attcttttac gggctacaca atgaactggg tccggcaggc accaggaaag     900

gggcttgaat gggtcgcttt aataaaccca tataagggcg taagcaccta taaccagaaa     960

tttaaagatc gtttcacaat atcagtcgat aaatctaaga ataccgccta tctccaaatg    1020

aattccctca gagctgaaga taccgccgtg tattactgtg caaggagcgg ttactatggc    1080

gattcagatt ggtacttcga tgtgtgggga caggggacgc ttgtaactgt gtcgtccggt    1140

ggaggcggtt ctagaagtga actaactcag gaccccgctg tgtctgtcgc gcttggccaa    1200

acggtgcgga taacctgtca aggtgacagt ctaaggtcct attatgcatc ttggtatcaa    1260

cagaaacccg gacaagcacc tgttctggtt atctacggta aaaacaatcg cccttcgggt    1320

atacctgatc gcttctccgg gtcgagctcc gggaatacag cctctctaac gataacgggg    1380

gctcaagcag aagatgaagc agactattat tgcaattcaa gggacagctc tggcaaccac    1440

gtcgtctttg ggggaggcac aaagttgaca gtccttgcgg ccgc                     1484


<210> 15
<211> 15
<212> PRT
<213> Homo sapiens

<220> 
<223> (alternative linker between VLB and VHB domains)

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

<210> 16
<211> 511
<212> PRT
<213> Homo sapiens

<220> 
<223> single-chain bispecific antibody with His and Myc tags

<400> 16
Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Glu Arg Pro Gly Gly 
1               5                   10                  15      
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 
            20                  25                  30          
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 
        35                  40                  45              
Ser Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val 
    50                  55                  60                  
Lys Gly Arg Val Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Lys Ile Leu Gly Ala Gly Arg Gly Trp Tyr Phe Asp Leu Trp Gly 
            100                 105                 110         
Lys Gly Thr Thr Val Thr Val Ser Ser Thr Gly Gly Gly Gly Ser Asp 
        115                 120                 125             
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp 
    130                 135                 140                 
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu 
145                 150                 155                 160 
Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 
                165                 170                 175     
Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 
            180                 185                 190         
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 
        195                 200                 205             
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr 
    210                 215                 220                 
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly Ser 
225                 230                 235                 240 
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu 
                245                 250                 255     
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 
            260                 265                 270         
Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg 
        275                 280                 285             
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro Tyr 
    290                 295                 300                 
Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile 
305                 310                 315                 320 
Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu 
                325                 330                 335     
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr 
            340                 345                 350         
Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val 
        355                 360                 365             
Thr Val Ser Ser Gly Gly Gly Gly Ser Arg Ser Glu Leu Thr Gln Asp 
    370                 375                 380                 
Pro Ala Val Ser Val Ala Leu Gly Gln Thr Val Arg Ile Thr Cys Gln 
385                 390                 395                 400 
Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro 
                405                 410                 415     
Gly Gln Ala Pro Val Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser 
            420                 425                 430         
Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser 
        435                 440                 445             
Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 
    450                 455                 460                 
Asn Ser Arg Asp Ser Ser Gly Asn His Val Val Phe Gly Gly Gly Thr 
465                 470                 475                 480 
Lys Leu Thr Val Leu Ala Ala Ala His His His His His His Gly Ala 
                485                 490                 495     
Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala Ala 
            500                 505                 510     

<210> 17
<211> 254
<212> PRT
<213> Homo sapiens

<220> 
<223> Clone 7 anti-TRAIL-R2 ScFv isolated with Phage Display
      (VH-LINKER-VL)

<400> 17
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Ser Pro Gly Glu 
1               5                   10                  15      
Ser Leu Lys Ile Ser Cys Lys Gly Leu Gly Tyr Asp Phe Thr Thr Tyr 
            20                  25                  30          
Trp Ile Ala Leu Val Arg Gln Met Pro Gly Lys Gly Leu Glu Tyr Met 
        35                  40                  45              
Gly Leu Ile Tyr Pro Gly Asp Ser Asp Thr Lys Tyr Ser Pro Ser Phe 
    50                  55                  60                  
Gln Gly Gln Val Thr Ile Ser Val Asp Lys Ser Val Ser Thr Ala Tyr 
65                  70                  75                  80  
Leu Gln Trp Ser Ser Leu Arg Pro Ser Asp Ser Ala Val Tyr Phe Cys 
                85                  90                  95      
Ala Arg His Asp Val Gly Tyr Cys Ser Ser Ser Asn Cys Ala Lys Arg 
            100                 105                 110         
Pro Glu Tyr Phe Gln His Trp Gly Gln Gly Thr Leu Val Thr Val Ser 
        115                 120                 125             
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 
    130                 135                 140                 
Asp Ile Val Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 
145                 150                 155                 160 
Asp Arg Ile Thr Ile Thr Cys Arg Ala Ser His Gly Ile Ser Ser Tyr 
                165                 170                 175     
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 
            180                 185                 190         
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Gly Gly 
        195                 200                 205             
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Val Ser Gly Leu Gln Pro 
    210                 215                 220                 
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Leu Asn Ser Tyr Pro Leu 
225                 230                 235                 240 
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Ala 
                245                 250                 

<210> 18
<211> 242
<212> PRT
<213> Homo sapiens

<220> 
<223> Clone 8 anti-TRAIL-R2 ScFv isolated with Phage Display
      (VH-LINKER-VL)

<400> 18
Glu Val Gln Leu 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 Gln Gln Gly Gln Val Thr Asp 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 Lys Arg Trp Asn Arg Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 
            100                 105                 110         
Ile Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 
        115                 120                 125             
Gly Gly Ser Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 
    130                 135                 140                 
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser 
145                 150                 155                 160 
Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 
                165                 170                 175     
Lys Leu Leu Ile Tyr Ala Ala Ser Gly Leu Gln Ser Gly Val Pro Ser 
            180                 185                 190         
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 
        195                 200                 205             
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Ser 
    210                 215                 220                 
Met Ser Pro Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 
225                 230                 235                 240 
Ala Ala 
        

<210> 19
<211> 250
<212> PRT
<213> Homo sapiens

<220> 
<223> Clone 44 anti-TRAIL-R2 ScFv isolated with Phage Display
      (VH-LINKER-VL)

<400> 19
Gln Val His Leu Arg Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 
1               5                   10                  15      
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 
            20                  25                  30          
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 
        35                  40                  45              
Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Ser Tyr Ala Asp Ser Val 
    50                  55                  60                  
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 
65                  70                  75                  80  
Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 
                85                  90                  95      
Ala Arg Asp Pro Asn His Gly Gly Asp Arg Arg Asp Asp Ala Leu Asp 
            100                 105                 110         
Met Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly 
        115                 120                 125             
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Glu Leu Thr Gln 
    130                 135                 140                 
Asp Pro Ala Val Ser Val Ala Leu Gly Gln Thr Val Arg Ile Thr Cys 
145                 150                 155                 160 
Gln Gly His Ser Leu Arg Ser Tyr Phe Ala Ser Trp Tyr Gln Gln Arg 
                165                 170                 175     
Pro Arg Gln Ala Pro Thr Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro 
            180                 185                 190         
Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala 
        195                 200                 205             
Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr 
    210                 215                 220                 
Cys Gly Ser Arg Asp Ser Ser Ser Thr His Arg Gly Val Phe Gly Gly 
225                 230                 235                 240 
Gly Thr Lys Leu Thr Val Leu Gly Ala Ala 
                245                 250 

<210> 20
<211> 231
<212> PRT
<213> Homo sapiens

<220> 
<223> Clone 56 anti-TRAIL-R2 ScFv isolated with Phage Display
      (VH-LINKER-VL)

<400> 20
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 
1               5                   10                  15      
Ser Leu Lys 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 Ser Ile Ala Asn Gly Lys Met Thr Gln Tyr Ala Asp Ser Val Lys 
    50                  55                  60                  
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 
65                  70                  75                  80  
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 
                85                  90                  95      
Lys Arg Trp Asn Arg Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Ile 
            100                 105                 110         
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 
        115                 120                 125             
Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser 
    130                 135                 140                 
Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser 
145                 150                 155                 160 
Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp 
                165                 170                 175     
Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser 
            180                 185                 190         
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 
        195                 200                 205             
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Gly Gln 
    210                 215                 220                 
Gly Thr Lys Leu Glu Ile Lys 
225                 230     

<210> 21
<211> 1463
<212> DNA
<213> Homo sapiens

<220> 
<223> single-chain bispecific antibody

<400> 21
gaggtacagc tggtgcaatc aggtggtggg gttgagagac ccggagggag tctccggttg      60

tcttgtgctg ccagcggctt tacgttcgat gattacggga tgtcgtgggt taggcaagct     120

ccaggcaagg gcctagaatg ggtatctggg ataaattgga acggcgggtc aacagggtat     180

gctgatagtg tgaaaggcag ggttactatt tccagggata atgccaaaaa ttcgctgtat     240

cttcagatga attcgctgcg agcggaggat actgcagtct actactgtgc taagatcctt     300

ggagcaggcc gtggatggta ttttgactta tgggggaagg gtaccaccgt gacggtctcg     360

tctaccggtg gaggcggttc agatattcaa atgactcagt ccccatcctc actctcggca     420

tcagttggtg atcgagtgac gataacatgt agggcctcgc aggacatccg caactatctt     480

aactggtacc aacaaaaacc agggaaggct ccaaaattac taatctatta cacctcacgt     540

ctcgaatcag gtgttcctag tcgcttcagc ggatcaggtt ccggaacaga ttataccctt     600

actatatcgt ccctccaacc agaagacttt gccacttatt actgtcaaca gggcaacacc     660

ttaccctgga cttttggaca ggggacaaaa gtcgagatta agcgcggtgg aggcggttca     720

ggcggaggtg gctctggcgg tggcggatcg gaagtacaac tggttgagag tgggggcggt     780

ttagttcaac cgggcggaag cctgagactt tcgtgcgccg cgagtggata ttcttttacg     840

ggctacacaa tgaactgggt ccggcaggca ccaggaaagg ggcttgaatg ggtcgcttta     900

ataaacccat ataagggcgt aagcacctat aaccagaaat ttaaagatcg tttcacaata     960

tcagtcgata aatctaagaa taccgcctat ctccaaatga attccctcag agctgaagat    1020

accgccgtgt attactgtgc aaggagcggt tactatggcg attcagattg gtacttcgat    1080

gtgtggggac aggggacgct tgtaactgtg tcgtccggtg gaggcggttc tagaagtgaa    1140

ctaactcagg accccgctgt gtctgtcgcg cttggccaaa cggtgcggat aacctgtcaa    1200

ggtgacagtc taaggtccta ttatgcatct tggtatcaac agaaacccgg acaagcacct    1260

gttctggtta tctacggtaa aaacaatcgc ccttcgggta tacctgatcg cttctccggg    1320

tcgagctccg ggaatacagc ctctctaacg ataacggggg ctcaagcaga agatgaagca    1380

gactattatt gcaattcaag ggacagctct ggcaaccacg tcgtctttgg gggaggcaca    1440

aagttgacag tccttgcggc cgc                                            1463


<210> 22
<211> 15
<212> DNA
<213> Homo sapiens

<220> 
<223> linker between VHA and VLB domains

<220> 
<221> variation
<222> 15
<223> /replace="A may be substituted by T"

<400> 22
ggtggaggcg gttca                                                     15


