               SEQUENCE LISTING

<110> FUNDACIÓN PARA LA INVESTIGACIÓN BIOMÉDICA HOSPITAL 12 DE OCTUBRE
      FUNDACIÓN CRIS DE INVESTIGACIÓN PARA VENCER AL CÁNCER
      HOSPITAL CLINIC DE BARCELONA
      INSTITUT D'INVESTIGACIONS BIOMÈDIQUES AUGUST PI I SUNYER

<120> Anti-CD19/anti-CD3 bispecific antibody, T cells secreting the same, method of preparation and use thereof

<130> PCT-08541

<150> EP20382113.7
<151> 2020-02-17

<160> 20

<170> BiSSAP 1.3.6

<210> 1
<211> 533
<212> PRT
<213> Artificial Sequence


<220> 
<221> SIGNAL
<222> 1..22
<223> Human kappa light chain signal peptide L1

<220> 
<223> A3B1-OKT3 bispecific antibody comprising A3B1 anti-CD19 scFv and
      OKT3 anti-CD3 scFv

<220> 
<221> DOMAIN
<222> 26..135
<223> A3B1 light chain variable domain

<220> 
<221> BINDING
<222> 51..60
<223> A3B1 light chain CDR1 region

<220> 
<221> BINDING
<222> 78..80
<223> A3B1 light chain CDR2 region

<220> 
<221> BINDING
<222> 117..125
<223> A3B1 light chain CDR3 region

<220> 
<221> DOMAIN
<222> 136..155
<223> Linker 1

<220> 
<221> DOMAIN
<222> 157..279
<223> A3B1 heavy chain variable domain

<220> 
<221> BINDING
<222> 183..190
<223> A3B1 heavy chain CDR1 region

<220> 
<221> BINDING
<222> 208..215
<223> A3B1 heavy chain CDR2 region

<220> 
<221> BINDING
<222> 254..268
<223> A3B1 heavy chain CDR3 region

<220> 
<221> DOMAIN
<222> 280..284
<223> Linker 2

<220> 
<221> DOMAIN
<222> 286..404
<223> OKT3 heavy chain variable domain

<220> 
<221> BINDING
<222> 311..318
<223> OKT3 heavy chain CDR1 region

<220> 
<221> BINDING
<222> 336..343
<223> OKT3 heavy chain CDR2 region

<220> 
<221> BINDING
<222> 382..393
<223> OKT3 heavy chain CDR3 region

<220> 
<221> DOMAIN
<222> 405..419
<223> Linker 3

<220> 
<221> DOMAIN
<222> 420..525
<223> OKT3 light chain variable domain

<220> 
<221> BINDING
<222> 446..450
<223> OKT3 light chain CDR1 region

<220> 
<221> BINDING
<222> 468..470
<223> OKT3 light chain CDR2 region

<220> 
<221> BINDING
<222> 507..515
<223> OKT3 light chain CDR3 region

<220> 
<221> DOMAIN
<222> 528..533
<223> Polyhistidine tag

<400> 1
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 
1               5                   10                  15      
Leu Ser Gly Ala Arg Cys Thr Gly Asn Ile Val Leu Thr Gln Ser Pro 
            20                  25                  30          
Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg 
        35                  40                  45              
Ala Ser Glu Ser Val Asp Asn Phe Gly Asn Ser Phe Met His Trp Tyr 
    50                  55                  60                  
Gln Gln Lys Ser Gly Gln Pro Pro Arg Leu Leu Ile Tyr Ile Ala Ser 
65                  70                  75                  80  
Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg 
                85                  90                  95      
Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala Asp Asp Ala Ala 
            100                 105                 110         
Thr Tyr Tyr Cys His Gln Asn Asn Glu Asp Pro Leu Thr Phe Gly Ala 
        115                 120                 125             
Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly 
    130                 135                 140                 
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Ser Gln Ile Gln 
145                 150                 155                 160 
Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser Ser Val Lys 
                165                 170                 175     
Ile Ser Cys Lys Ala Ser Gly Phe Ala Phe Ser Ser Tyr Trp Met Asn 
            180                 185                 190         
Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln Ile 
        195                 200                 205             
Tyr Pro Gly Asp Gly Asp Thr Lys Tyr Asn Val Lys Phe Arg Gly Lys 
    210                 215                 220                 
Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Ile Gln Leu 
225                 230                 235                 240 
Thr Ser Leu Thr Ser Glu Asp Ser Gly Val Tyr Phe Cys Ala Arg Lys 
                245                 250                 255     
Arg Ile Thr Ala Val Ile Thr Thr Val Phe Asp Val Trp Gly Ala Gly 
            260                 265                 270         
Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Arg Gln Val Gln 
        275                 280                 285             
Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys 
    290                 295                 300                 
Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His 
305                 310                 315                 320 
Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile 
                325                 330                 335     
Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys 
            340                 345                 350         
Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu 
        355                 360                 365             
Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr 
    370                 375                 380                 
Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu 
385                 390                 395                 400 
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 
                405                 410                 415     
Gly Gly Ser Asp Ile Asp Leu Thr Gln Ser Pro Ala Ile Met Ser Ala 
            420                 425                 430         
Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val 
        435                 440                 445             
Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg 
    450                 455                 460                 
Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe 
465                 470                 475                 480 
Arg Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met 
                485                 490                 495     
Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn 
            500                 505                 510         
Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Arg Ala His 
        515                 520                 525             
His His His His His 
    530             

<210> 2
<211> 25
<212> PRT
<213> Artificial Sequence


<220> 
<223> Human kappa light chain signal peptide L1

<400> 2
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 
1               5                   10                  15      
Leu Ser Gly Ala Arg Cys Thr Gly Asn 
            20                  25  

<210> 3
<211> 110
<212> PRT
<213> Artificial Sequence


<220> 
<223> A3B1 light chain variable domain

<400> 3
Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln 
1               5                   10                  15      
Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Phe Gly 
            20                  25                  30          
Asn Ser Phe Met His Trp Tyr Gln Gln Lys Ser Gly Gln Pro Pro Arg 
        35                  40                  45              
Leu Leu Ile Tyr Ile Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg 
    50                  55                  60                  
Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro 
65                  70                  75                  80  
Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys His Gln Asn Asn Glu 
                85                  90                  95      
Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 
            100                 105                 110 

<210> 4
<211> 20
<212> PRT
<213> Artificial Sequence


<220> 
<223> Linker 1

<400> 4
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 
1               5                   10                  15      
Gly Gly Gly Ser 
            20  

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


<220> 
<223> A3B1 heavy chain variable domain

<400> 5
Gln Ile Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 
1               5                   10                  15      
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Ala Phe Ser Ser Tyr 
            20                  25                  30          
Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 
        35                  40                  45              
Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Lys Tyr Asn Val Lys Phe 
    50                  55                  60                  
Arg Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 
65                  70                  75                  80  
Ile Gln Leu Thr Ser Leu Thr Ser Glu Asp Ser Gly Val Tyr Phe Cys 
                85                  90                  95      
Ala Arg Lys Arg Ile Thr Ala Val Ile Thr Thr Val Phe Asp Val Trp 
            100                 105                 110         
Gly Ala Gly Thr Thr Val Thr Val Ser Ser 
        115                 120         

<210> 6
<211> 5
<212> PRT
<213> Artificial Sequence


<220> 
<223> Linker 2

<400> 6
Gly Gly Gly Gly Ser 
1               5   

<210> 7
<211> 119
<212> PRT
<213> Artificial Sequence


<220> 
<223> OKT3 heavy chain variable domain

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

<210> 8
<211> 15
<212> PRT
<213> Artificial Sequence


<220> 
<223> Linker 3

<400> 8
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 
1               5                   10                  15  

<210> 9
<211> 106
<212> PRT
<213> Artificial Sequence


<220> 
<223> OKT3 light chain variable domain

<400> 9
Asp Ile Asp Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 
1               5                   10                  15      
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 
            20                  25                  30          
Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 
        35                  40                  45              
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser 
    50                  55                  60                  
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu 
65                  70                  75                  80  
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr 
                85                  90                  95      
Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn 
            100                 105     

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


<220> 
<223> A3B1 light chain CDR1 region

<400> 10
Glu Ser Val Asp Asn Phe Gly Asn Ser Phe 
1               5                   10  

<210> 11
<211> 9
<212> PRT
<213> Artificial Sequence


<220> 
<223> A3B1 light chain CDR3 region

<400> 11
His Gln Asn Asn Glu Asp Pro Leu Thr 
1               5                   

<210> 12
<211> 8
<212> PRT
<213> Artificial Sequence


<220> 
<223> A3B1 heavy chain CDR1 region

<400> 12
Gly Phe Ala Phe Ser Ser Tyr Trp 
1               5               

<210> 13
<211> 8
<212> PRT
<213> Artificial Sequence


<220> 
<223> A3B1 heavy chain CDR2 region

<400> 13
Ile Tyr Pro Gly Asp Gly Asp Thr 
1               5               

<210> 14
<211> 15
<212> PRT
<213> Artificial Sequence


<220> 
<223> A3B1 heavy chain CDR3 region

<400> 14
Ala Arg Lys Arg Ile Thr Ala Val Ile Thr Thr Val Phe Asp Val 
1               5                   10                  15  

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


<220> 
<223> A3B1 heavy chain CDR3 region

<400> 15
Ala Arg Lys Arg Ile Thr Ala Val Ile Thr Thr Val Phe Asp Val 
1               5                   10                  15  

<210> 16
<211> 8
<212> PRT
<213> Artificial Sequence


<220> 
<223> OKT3 heavy chain CDR2 region

<400> 16
Ile Asn Pro Ser Arg Gly Tyr Thr 
1               5               

<210> 17
<211> 12
<212> PRT
<213> Artificial Sequence


<220> 
<223> OKT3 heavy chain CDR3 region

<400> 17
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr 
1               5                   10          

<210> 18
<211> 5
<212> PRT
<213> Artificial Sequence


<220> 
<223> OKT3 light chain CDR1 region

<400> 18
Ser Ser Val Ser Tyr 
1               5   

<210> 19
<211> 9
<212> PRT
<213> Artificial Sequence


<220> 
<223> OKT3 light chain CDR3 region

<400> 19
Gln Gln Trp Ser Ser Asn Pro Phe Thr 
1               5                   

<210> 20
<211> 1605
<212> DNA
<213> Artificial Sequence


<220> 
<223> Artificial gene encoding for A3B1-OKT3 bispecific antibody

<400> 20
atggacatga gggtccctgc tcagctcctg gggctcctgc tgctctggct ctcaggtgcc      60

agatgtaccg gtaacatcgt gctgactcag agcccagcat ccctggccgt ctcactggga     120

cagcgggcta ccatcagctg cagagcaagc gagtccgtgg acaacttcgg aaattccttt     180

atgcactggt accagcagaa gtctggccag ccccctcgac tgctgatcta tattgcctct     240

aacctggaga gtggcgtgcc agctaggttc tctggcagtg ggtcacgcac agactttacc     300

ctgacaattg atcccgtcga agccgacgat gccgctacct actattgcca ccagaacaac     360

gaggacccac tgactttcgg agcagggaca aaactggaac tgaagggtgg aggcgggagc     420

ggaggaggcg ggtcgggagg aggaggatcg ggtggcggag gctcacattc tcagattcag     480

ctgcagcagt caggggcaga gctggtgcgg ccagggtcct cagtcaagat tagttgtaag     540

gcttcaggct ttgctttcag ctcctactgg atgaactggg tgaaacagcg accaggacag     600

ggactggagt ggatcggaca gatctacccc ggagacggcg atacaaagta taatgtcaaa     660

tttcggggca aggccaccct gacagctgac gagtctagtt caaccgccta catccagctg     720

acttcactga ccagcgaaga ttccggcgtg tatttctgcg ctcgaaagcg gatcactgca     780

gtcattacca cagtgtttga cgtgtggggg gcaggaacta ccgtgaccgt cagctccggc     840

ggaggtggca gtcgacaggt gcagctgcag cagagcggag ccgaactggc cagaccagga     900

gccagcgtga agatgagctg caaggctagc ggctacacct tcacccggta caccatgcac     960

tgggtgaaac agaggcctgg acagggcctg gaatggatcg gctacatcaa ccccagccgg    1020

ggctacacca actacaacca gaagttcaag gacaaggcca ccctgaccac cgacaagagc    1080

agcagcaccg cctacatgca gctgagcagc ctgaccagcg aggacagcgc cgtgtactac    1140

tgcgcccggt actacgacga ccactactgc ctggactact ggggccaggg caccaccctg    1200

acagtgtcta gcggcggagg cggaagtgga ggcggaggaa gcggaggcgg cggatctgac    1260

atcgacctga cccagagccc tgccatcatg tctgccagcc ctggcgagaa agtgaccatg    1320

acctgcagcg ccagcagcag cgtgtcctac atgaactggt atcagcagaa gtccggcacc    1380

agccccaagc ggtggatcta cgacaccagc aaactggcca gcggcgtgcc agcccacttt    1440

agaggctccg gcagcggcac cagctactcc ctgaccatca gcggcatgga agccgaggac    1500

gccgccacct actactgcca gcagtggagc agcaacccct tcaccttcgg ctccggcacc    1560

aagctggaaa tcaaccgggc ccaccaccac catcaccact gataa                    1605


