                         SEQUENCE LISTING

<110>  Consejo Superior de Investigaciones Cientficas (CSIC)
 
<120>  ETANERCEPT VARIANTS WITH IMPROVED THERAPEUTICAL EFFECT

<130>  PCT1641.1422

<150>  EP18382812
<151>  2018-11-16

<160>  12    

<170>  PatentIn version 3.5

<210>  1
<211>  235
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Ligand binding domain of the human TNF receptor 2 (TNFR2)

<400>  1

Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser 
1               5                   10                  15      


Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys 
            20                  25                  30          


Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr 
        35                  40                  45              


Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu 
    50                  55                  60                  


Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser 
65                  70                  75                  80  


Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys 
                85                  90                  95      


Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys 
            100                 105                 110         


Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala 
        115                 120                 125             


Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro 
    130                 135                 140                 


Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 
145                 150                 155                 160 


Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala 
                165                 170                 175     


Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val 
            180                 185                 190         


His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr 
        195                 200                 205             


Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly 
    210                 215                 220                 


Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp 
225                 230                 235 


<210>  2
<211>  235
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  SEQ ID NO: 1 comprising the amino acid substitutions A105E and 
       L106F

<400>  2

Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser 
1               5                   10                  15      


Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys 
            20                  25                  30          


Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr 
        35                  40                  45              


Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu 
    50                  55                  60                  


Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser 
65                  70                  75                  80  


Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys 
                85                  90                  95      


Thr Cys Arg Pro Gly Trp Tyr Cys Glu Phe Ser Lys Gln Glu Gly Cys 
            100                 105                 110         


Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala 
        115                 120                 125             


Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro 
    130                 135                 140                 


Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 
145                 150                 155                 160 


Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala 
                165                 170                 175     


Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val 
            180                 185                 190         


His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr 
        195                 200                 205             


Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly 
    210                 215                 220                 


Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp 
225                 230                 235 


<210>  3
<211>  467
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Etanercept original sequence

<400>  3

Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser 
1               5                   10                  15      


Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys 
            20                  25                  30          


Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr 
        35                  40                  45              


Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu 
    50                  55                  60                  


Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser 
65                  70                  75                  80  


Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys 
                85                  90                  95      


Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys 
            100                 105                 110         


Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala 
        115                 120                 125             


Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro 
    130                 135                 140                 


Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 
145                 150                 155                 160 


Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala 
                165                 170                 175     


Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val 
            180                 185                 190         


His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr 
        195                 200                 205             


Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly 
    210                 215                 220                 


Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro Lys Ser Cys 
225                 230                 235                 240 


Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 
                245                 250                 255     


Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 
            260                 265                 270         


Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 
        275                 280                 285             


Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 
    290                 295                 300                 


His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 
305                 310                 315                 320 


Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 
                325                 330                 335     


Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 
            340                 345                 350         


Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 
        355                 360                 365             


Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 
    370                 375                 380                 


Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 
385                 390                 395                 400 


Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 
                405                 410                 415     


Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 
            420                 425                 430         


Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 
        435                 440                 445             


His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 
    450                 455                 460                 


Pro Gly Lys 
465         


<210>  4
<211>  467
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  SEQ ID NO: 1 comprising the amino acid substitutions A105E and 
       L106F (i. e. SEQ ID NO: 2) linked to the Fc fragment of a human 
       IgG1 shown in SEQ ID N: 11

<400>  4

Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser 
1               5                   10                  15      


Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys 
            20                  25                  30          


Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr 
        35                  40                  45              


Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu 
    50                  55                  60                  


Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser 
65                  70                  75                  80  


Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys 
                85                  90                  95      


Thr Cys Arg Pro Gly Trp Tyr Cys Glu Phe Ser Lys Gln Glu Gly Cys 
            100                 105                 110         


Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala 
        115                 120                 125             


Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro 
    130                 135                 140                 


Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 
145                 150                 155                 160 


Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala 
                165                 170                 175     


Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val 
            180                 185                 190         


His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr 
        195                 200                 205             


Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly 
    210                 215                 220                 


Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro Lys Ser Cys 
225                 230                 235                 240 


Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 
                245                 250                 255     


Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 
            260                 265                 270         


Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 
        275                 280                 285             


Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 
    290                 295                 300                 


His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 
305                 310                 315                 320 


Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 
                325                 330                 335     


Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 
            340                 345                 350         


Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 
        355                 360                 365             


Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 
    370                 375                 380                 


Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 
385                 390                 395                 400 


Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 
                405                 410                 415     


Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 
            420                 425                 430         


Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 
        435                 440                 445             


His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 
    450                 455                 460                 


Pro Gly Lys 
465         


<210>  5
<211>  36
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Primer Crm34

<400>  5
gcgggatccg atgttccgta tacacccatt aatggg                                 36


<210>  6
<211>  32
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Primer CrmD33

<400>  6
gcgctcgagg catctctttc acaatcattt gg                                     32


<210>  7
<211>  31
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Primer RM6mut2F

<400>  7
gctggtactg cgcgttcagc aagcaggagg g                                      31


<210>  8
<211>  31
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Primer RM6mut2R

<400>  8
ccctcctgct tgctgaacgc gcagtaccag c                                      31


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

<220>
<223>  Primer RM6mut3F

<400>  9
cggctggtac tgcgagttca gcaagcagga ggg                                    33


<210>  10
<211>  33
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Primer RM6mut3R

<400>  10
ccctcctgct tgctgaactc gcagtaccag ccg                                    33


<210>  11
<211>  232
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Fc fragment of a human IgG1

<400>  11

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 
1               5                   10                  15      


Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 
            20                  25                  30          


Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 
        35                  40                  45              


Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 
    50                  55                  60                  


Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 
65                  70                  75                  80  


Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 
                85                  90                  95      


Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 
            100                 105                 110         


Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 
        115                 120                 125             


Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 
    130                 135                 140                 


Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 
145                 150                 155                 160 


Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 
                165                 170                 175     


Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 
            180                 185                 190         


Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 
        195                 200                 205             


Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 
    210                 215                 220                 


Ser Leu Ser Leu Ser Pro Gly Lys 
225                 230         


<210>  12
<211>  22
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Signal peptide

<400>  12

Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu 
1               5                   10                  15      


Trp Ala Ala Ala His Ala 
            20          


