                         SEQUENCE LISTING

<110>  Regeneron Pharmaceuticals Inc.
       WU, Jiaxi
       ZHANG, Tong
       DAVIS, Samuel
       BLOCH, Nicolin
 
<120>  IL-2 VARIANTS WITH REDUCED BINDING TO IL-2 RECEPTOR ALPHA AND USES THEREOF

<130>  179227.01801/10729US01; 179227.01802/10729WO01

<160>  12    

<170>  PatentIn version 3.5

<210>  1
<211>  153
<212>  PRT
<213>  Homo sapiens

<400>  1

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 
1               5                   10                  15      


Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 
            20                  25                  30          


Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 
        35                  40                  45              


Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 
    50                  55                  60                  


Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 
65                  70                  75                  80  


Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 
                85                  90                  95      


Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 
            100                 105                 110         


Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 
        115                 120                 125             


Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 
    130                 135                 140                 


Cys Gln Ser Ile Ile Ser Thr Leu Thr 
145                 150             


<210>  2
<211>  161
<212>  PRT
<213>  Artificial

<220>
<223>  with a C-terminal myc-myc-hexahistidine (mmh) tag

<400>  2

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 
1               5                   10                  15      


Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 
            20                  25                  30          


Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 
        35                  40                  45              


Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 
    50                  55                  60                  


Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 
65                  70                  75                  80  


Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 
                85                  90                  95      


Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 
            100                 105                 110         


Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 
        115                 120                 125             


Ile Ser Thr Leu Thr Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gly 
    130                 135                 140                 


Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu His His His His His 
145                 150                 155                 160 


His 
    


<210>  3
<211>  161
<212>  PRT
<213>  Artificial

<220>
<223>  hIL2(E68C)-mmh

<400>  3

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 
1               5                   10                  15      


Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 
            20                  25                  30          


Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 
        35                  40                  45              


Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 
    50                  55                  60                  


Pro Leu Glu Cys Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 
65                  70                  75                  80  


Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 
                85                  90                  95      


Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 
            100                 105                 110         


Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 
        115                 120                 125             


Ile Ser Thr Leu Thr Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gly 
    130                 135                 140                 


Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu His His His His His 
145                 150                 155                 160 


His 
    


<210>  4
<211>  133
<212>  PRT
<213>  Artificial

<220>
<223>  hIL-2(E68C)

<400>  4

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 
1               5                   10                  15      


Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 
            20                  25                  30          


Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 
        35                  40                  45              


Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 
    50                  55                  60                  


Pro Leu Glu Cys Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 
65                  70                  75                  80  


Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 
                85                  90                  95      


Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 
            100                 105                 110         


Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 
        115                 120                 125             


Ile Ser Thr Leu Thr 
    130             


<210>  5
<211>  133
<212>  PRT
<213>  Artificial

<220>
<223>  hIL-2(P34C)

<400>  5

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 
1               5                   10                  15      


Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 
            20                  25                  30          


Asn Cys Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 
        35                  40                  45              


Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 
    50                  55                  60                  


Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 
65                  70                  75                  80  


Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 
                85                  90                  95      


Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 
            100                 105                 110         


Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 
        115                 120                 125             


Ile Ser Thr Leu Thr 
    130             


<210>  6
<211>  133
<212>  PRT
<213>  Artificial

<220>
<223>  hIL-2(F42C)

<400>  6

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 
1               5                   10                  15      


Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 
            20                  25                  30          


Asn Pro Lys Leu Thr Arg Met Leu Thr Cys Lys Phe Tyr Met Pro Lys 
        35                  40                  45              


Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 
    50                  55                  60                  


Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 
65                  70                  75                  80  


Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 
                85                  90                  95      


Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 
            100                 105                 110         


Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 
        115                 120                 125             


Ile Ser Thr Leu Thr 
    130             


<210>  7
<211>  133
<212>  PRT
<213>  Artificial

<220>
<223>  hIL-2(E61C)

<400>  7

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 
1               5                   10                  15      


Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 
            20                  25                  30          


Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 
        35                  40                  45              


Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Cys Glu Leu Lys 
    50                  55                  60                  


Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 
65                  70                  75                  80  


Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 
                85                  90                  95      


Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 
            100                 105                 110         


Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 
        115                 120                 125             


Ile Ser Thr Leu Thr 
    130             


<210>  8
<211>  133
<212>  PRT
<213>  Artificial

<220>
<223>  hIL-2(K64C)

<400>  8

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 
1               5                   10                  15      


Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 
            20                  25                  30          


Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 
        35                  40                  45              


Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Cys 
    50                  55                  60                  


Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 
65                  70                  75                  80  


Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 
                85                  90                  95      


Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 
            100                 105                 110         


Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 
        115                 120                 125             


Ile Ser Thr Leu Thr 
    130             


<210>  9
<211>  133
<212>  PRT
<213>  Artificial

<220>
<223>  hIL-2(P65C)

<400>  9

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 
1               5                   10                  15      


Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 
            20                  25                  30          


Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 
        35                  40                  45              


Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 
    50                  55                  60                  


Cys Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 
65                  70                  75                  80  


Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 
                85                  90                  95      


Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 
            100                 105                 110         


Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 
        115                 120                 125             


Ile Ser Thr Leu Thr 
    130             


<210>  10
<211>  402
<212>  DNA
<213>  Artificial

<220>
<223>  cDNA sequence of hIL-2(E68C)

<400>  10
gcacctactt caagttctac aaagaaaaca cagctacaac tggagcattt actgctggat       60

ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc      120

acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa      180

gaagaactca aacctctgga gtgtgtgcta aatttagctc aaagcaaaaa ctttcactta      240

agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa      300

acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga      360

tggattacct tttgtcaaag catcatctca acactgactt ga                         402


<210>  11
<211>  366
<212>  PRT
<213>  Artificial

<220>
<223>  Synthetic

<400>  11

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 
1               5                   10                  15      


Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 
            20                  25                  30          


Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 
        35                  40                  45              


Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 
    50                  55                  60                  


Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 
65                  70                  75                  80  


Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 
                85                  90                  95      


Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 
            100                 105                 110         


Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 
        115                 120                 125             


Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly Pro 
    130                 135                 140                 


Pro Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe 
145                 150                 155                 160 


Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 
                165                 170                 175     


Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 
            180                 185                 190         


Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 
        195                 200                 205             


Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 
    210                 215                 220                 


Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 
225                 230                 235                 240 


Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 
                245                 250                 255     


Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 
            260                 265                 270         


Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 
        275                 280                 285             


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


Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 
305                 310                 315                 320 


Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 
                325                 330                 335     


Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 
            340                 345                 350         


Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 
        355                 360                 365     


<210>  12
<211>  366
<212>  PRT
<213>  Artificial

<220>
<223>  Synthetic

<400>  12

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 
1               5                   10                  15      


Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 
            20                  25                  30          


Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Ala Met Pro Lys 
        35                  40                  45              


Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 
    50                  55                  60                  


Pro Leu Glu Glu Val Leu Asn Gly Ala Gln Ser Lys Asn Phe His Leu 
65                  70                  75                  80  


Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 
                85                  90                  95      


Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 
            100                 105                 110         


Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 
        115                 120                 125             


Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly Pro 
    130                 135                 140                 


Pro Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe 
145                 150                 155                 160 


Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 
                165                 170                 175     


Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 
            180                 185                 190         


Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 
        195                 200                 205             


Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 
    210                 215                 220                 


Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 
225                 230                 235                 240 


Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 
                245                 250                 255     


Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 
            260                 265                 270         


Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 
        275                 280                 285             


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


Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 
305                 310                 315                 320 


Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 
                325                 330                 335     


Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 
            340                 345                 350         


Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 
        355                 360                 365     


