               SEQUENCE LISTING

<110> Miltenyi Biotec GmbH

<120> Chimeric antigen receptor specific for SSEA4 antigen

<130> Mil_073PCT

<160> 6

<170> BiSSAP 1.2

<210> 1
<211> 119
<212> PRT
<213> Artificial Sequence

<220> 
<223> SSEA4 VH

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

<210> 2
<211> 106
<212> PRT
<213> Artificial Sequence

<220> 
<223> SSEA4 VL

<400> 2
Glu Asn Val 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          
His Trp Tyr Gln Gln Lys Ser Ser Thr Ser Pro Lys Leu Trp Ile Tyr 
        35                  40                  45              
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser 
    50                  55                  60                  
Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 
65                  70                  75                  80  
Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Leu Thr 
                85                  90                  95      
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 
            100                 105     

<210> 3
<211> 240
<212> PRT
<213> Artificial Sequence

<220> 
<223> scFv VH-linker-VL

<400> 3
Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 
1               5                   10                  15      
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Gln 
            20                  25                  30          
Gly Val Tyr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 
        35                  40                  45              
Gly Ala Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met 
    50                  55                  60                  
Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 
65                  70                  75                  80  
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala 
                85                  90                  95      
Arg Val Asp Gly Tyr Arg Gly Tyr Asn Met Asp Tyr Trp Gly Gln Gly 
            100                 105                 110         
Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 
        115                 120                 125             
Ser Gly Gly Gly Gly Ser Glu Asn Val Leu Thr Gln Ser Pro Ala Ile 
    130                 135                 140                 
Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser 
145                 150                 155                 160 
Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Ser Thr Ser 
                165                 170                 175     
Pro Lys Leu Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro 
            180                 185                 190         
Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile 
        195                 200                 205             
Ser Ser Met Glu Ala Glu Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly 
    210                 215                 220                 
Ser Gly Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 
225                 230                 235                 240 



<210> 4
<211> 240
<212> PRT
<213> Artificial Sequence

<220> 
<223> scFv VL-linker-VH

<400> 4
Glu Asn Val 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          
His Trp Tyr Gln Gln Lys Ser Ser Thr Ser Pro Lys Leu Trp Ile Tyr 
        35                  40                  45              
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser 
    50                  55                  60                  
Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 
65                  70                  75                  80  
Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Leu Thr 
                85                  90                  95      
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly 
            100                 105                 110         
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Glu Ser 
        115                 120                 125             
Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr 
    130                 135                 140                 
Val Ser Gly Phe Ser Leu Ser Ser Gln Gly Val Tyr Trp Val Arg Gln 
145                 150                 155                 160 
Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Ala Ile Trp Ala Gly Gly 
                165                 170                 175     
Ser Thr Asn Tyr Asn Ser Ala Leu Met Ser Arg Leu Ser Ile Ser Lys 
            180                 185                 190         
Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr 
        195                 200                 205             
Asp Asp Thr Ala Met Tyr Tyr Cys Ala Arg Val Asp Gly Tyr Arg Gly 
    210                 215                 220                 
Tyr Asn Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 
225                 230                 235                 240 



<210> 5
<211> 679
<212> PRT
<213> Artificial Sequence

<220> 
<223> Full CAR sequence: SSEA4-CAR VH-linker-VL

<400> 5
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 
1               5                   10                  15      
Val Ile Met Ser Arg Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu 
            20                  25                  30          
Val Ala Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe 
        35                  40                  45              
Ser Leu Ser Ser Gln Gly Val Tyr Trp Val Arg Gln Pro Pro Gly Lys 
    50                  55                  60                  
Gly Leu Glu Trp Leu Gly Ala Ile Trp Ala Gly Gly Ser Thr Asn Tyr 
65                  70                  75                  80  
Asn Ser Ala Leu Met Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys 
                85                  90                  95      
Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala 
            100                 105                 110         
Met Tyr Tyr Cys Ala Arg Val Asp Gly Tyr Arg Gly Tyr Asn Met Asp 
        115                 120                 125             
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly 
    130                 135                 140                 
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Asn Val Leu Thr 
145                 150                 155                 160 
Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met 
                165                 170                 175     
Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln 
            180                 185                 190         
Lys Ser Ser Thr Ser Pro Lys Leu Trp Ile Tyr Asp Thr Ser Lys Leu 
        195                 200                 205             
Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser 
    210                 215                 220                 
Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Val Ala Thr Tyr 
225                 230                 235                 240 
Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Leu Thr Phe Gly Ala Gly Thr 
                245                 250                 255     
Lys Leu Glu Leu Lys Ala Ala Ala Leu Pro Ala Glu Pro Lys Ser Pro 
            260                 265                 270         
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly 
        275                 280                 285             
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 
    290                 295                 300                 
Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 
305                 310                 315                 320 
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 
                325                 330                 335     
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 
            340                 345                 350         
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 
        355                 360                 365             
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 
    370                 375                 380                 
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 
385                 390                 395                 400 
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 
                405                 410                 415     
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 
            420                 425                 430         
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 
        435                 440                 445             
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 
    450                 455                 460                 
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 
465                 470                 475                 480 
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Ser Leu Ser 
                485                 490                 495     
Pro Gly Lys Lys Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 
            500                 505                 510         
Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 
        515                 520                 525             
Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 
    530                 535                 540                 
Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 
545                 550                 555                 560 
Glu Gly Gly Cys Glu Leu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 
                565                 570                 575     
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 
            580                 585                 590         
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 
        595                 600                 605             
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly 
    610                 615                 620                 
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 
625                 630                 635                 640 
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 
                645                 650                 655     
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 
            660                 665                 670         
Met Gln Ala Leu Pro Pro Arg 
        675                 

<210> 6
<211> 679
<212> PRT
<213> Artificial Sequence

<220> 
<223> Full CAR sequence: SSEA4-CAR VL-linker- VH

<400> 6
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 
1               5                   10                  15      
Val Ile Met Ser Arg Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met 
            20                  25                  30          
Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser 
        35                  40                  45              
Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Ser Thr Ser Pro 
    50                  55                  60                  
Lys Leu Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly 
65                  70                  75                  80  
Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser 
                85                  90                  95      
Ser Met Glu Ala Glu Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly Ser 
            100                 105                 110         
Gly Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly 
        115                 120                 125             
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val 
    130                 135                 140                 
Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu 
145                 150                 155                 160 
Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Gln Gly Val 
                165                 170                 175     
Tyr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Ala 
            180                 185                 190         
Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met Ser Arg 
        195                 200                 205             
Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met 
    210                 215                 220                 
Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala Arg Val 
225                 230                 235                 240 
Asp Gly Tyr Arg Gly Tyr Asn Met Asp Tyr Trp Gly Gln Gly Thr Ser 
                245                 250                 255     
Val Thr Val Ser Ser Ala Ala Ala Leu Pro Ala Glu Pro Lys Ser Pro 
            260                 265                 270         
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly 
        275                 280                 285             
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 
    290                 295                 300                 
Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 
305                 310                 315                 320 
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 
                325                 330                 335     
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 
            340                 345                 350         
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 
        355                 360                 365             
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 
    370                 375                 380                 
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 
385                 390                 395                 400 
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 
                405                 410                 415     
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 
            420                 425                 430         
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 
        435                 440                 445             
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 
    450                 455                 460                 
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 
465                 470                 475                 480 
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Ser Leu Ser 
                485                 490                 495     
Pro Gly Lys Lys Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 
            500                 505                 510         
Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 
        515                 520                 525             
Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 
    530                 535                 540                 
Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 
545                 550                 555                 560 
Glu Gly Gly Cys Glu Leu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 
                565                 570                 575     
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 
            580                 585                 590         
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 
        595                 600                 605             
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly 
    610                 615                 620                 
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 
625                 630                 635                 640 
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 
                645                 650                 655     
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 
            660                 665                 670         
Met Gln Ala Leu Pro Pro Arg 
        675                 

