                         SEQUENCE LISTING

<110>   National University Of Singapore
 
<120>  GENETICALLY MODIFIED NK CELLS AND USES THEREOF

<130>  10104SG1198

<160>  20    

<170>  PatentIn version 3.5

<210>  1
<211>  1053
<212>  DNA
<213>  Homo sapiens

<400>  1
atgtcaaata ttacagatcc acagatgtgg gattttgatg atctaaattt cactggcatg       60

ccacctgcag atgaagatta cagcccctgt atgctagaaa ctgagacact caacaagtat      120

gttgtgatca tcgcctatgc cctagtgttc ctgctgagcc tgctgggaaa ctccctggtg      180

atgctggtca tcttatacag cagggtcggc cgctccgtca ctgatgtcta cctgctgaac      240

ctggccttgg ccgacctact ctttgccctg accttgccca tctgggccgc ctccaaggtg      300

aatggctgga tttttggcac attcctgtgc aaggtggtct cactcctgaa ggaagtcaac      360

ttctacagtg gcatcctgct gttggcctgc atcagtgtgg accgttacct ggccattgtc      420

catgccacac gcacactgac ccagaagcgt cacttggtca agtttgtttg tcttggctgc      480

tggggactgt ctatgaatct gtccctgccc ttcttccttt tccgccaggc ttaccatcca      540

aacaattcca gtccagtttg ctatgaggtc ctgggaaatg acacagcaaa atggcggatg      600

gtgttgcgga tcctgcctca cacctttggc ttcatcgtgc cgctgtttgt catgctgttc      660

tgctatggat tcaccctgcg tacactgttt aaggcccaca tggggcagaa gcaccgagcc      720

atgagggtca tctttgctgt cgtcctcatc ttcctgcttt gctggctgcc ctacaacctg      780

gtcctgctgg cagacaccct catgaggacc caggtgatcc aggagagctg tgagcgccgc      840

aacaacatcg gccgggccct ggatgccact gagattctgg gatttctcca tagctgcctc      900

aaccccatca tctacgcctt catcggccaa aattttcgcc atggattcct caagatcctg      960

gctatgcatg gcctggtcag caaggagttc ttggcacgtc atcgtgttac ctcctacact     1020

tcttcgtctg tcaatgtctc ttccaacctc tga                                  1053


<210>  2
<211>  350
<212>  PRT
<213>  Homo sapiens

<400>  2

Met Ser Asn Ile Thr Asp Pro Gln Met Trp Asp Phe Asp Asp Leu Asn 
1               5                   10                  15      


Phe Thr Gly Met Pro Pro Ala Asp Glu Asp Tyr Ser Pro Cys Met Leu 
            20                  25                  30          


Glu Thr Glu Thr Leu Asn Lys Tyr Val Val Ile Ile Ala Tyr Ala Leu 
        35                  40                  45              


Val Phe Leu Leu Ser Leu Leu Gly Asn Ser Leu Val Met Leu Val Ile 
    50                  55                  60                  


Leu Tyr Ser Arg Val Gly Arg Ser Val Thr Asp Val Tyr Leu Leu Asn 
65                  70                  75                  80  


Leu Ala Leu Ala Asp Leu Leu Phe Ala Leu Thr Leu Pro Ile Trp Ala 
                85                  90                  95      


Ala Ser Lys Val Asn Gly Trp Ile Phe Gly Thr Phe Leu Cys Lys Val 
            100                 105                 110         


Val Ser Leu Leu Lys Glu Val Asn Phe Tyr Ser Gly Ile Leu Leu Leu 
        115                 120                 125             


Ala Cys Ile Ser Val Asp Arg Tyr Leu Ala Ile Val His Ala Thr Arg 
    130                 135                 140                 


Thr Leu Thr Gln Lys Arg His Leu Val Lys Phe Val Cys Leu Gly Cys 
145                 150                 155                 160 


Trp Gly Leu Ser Met Asn Leu Ser Leu Pro Phe Phe Leu Phe Arg Gln 
                165                 170                 175     


Ala Tyr His Pro Asn Asn Ser Ser Pro Val Cys Tyr Glu Val Leu Gly 
            180                 185                 190         


Asn Asp Thr Ala Lys Trp Arg Met Val Leu Arg Ile Leu Pro His Thr 
        195                 200                 205             


Phe Gly Phe Ile Val Pro Leu Phe Val Met Leu Phe Cys Tyr Gly Phe 
    210                 215                 220                 


Thr Leu Arg Thr Leu Phe Lys Ala His Met Gly Gln Lys His Arg Ala 
225                 230                 235                 240 


Met Arg Val Ile Phe Ala Val Val Leu Ile Phe Leu Leu Cys Trp Leu 
                245                 250                 255     


Pro Tyr Asn Leu Val Leu Leu Ala Asp Thr Leu Met Arg Thr Gln Val 
            260                 265                 270         


Ile Gln Glu Ser Cys Glu Arg Arg Asn Asn Ile Gly Arg Ala Leu Asp 
        275                 280                 285             


Ala Thr Glu Ile Leu Gly Phe Leu His Ser Cys Leu Asn Pro Ile Ile 
    290                 295                 300                 


Tyr Ala Phe Ile Gly Gln Asn Phe Arg His Gly Phe Leu Lys Ile Leu 
305                 310                 315                 320 


Ala Met His Gly Leu Val Ser Lys Glu Phe Leu Ala Arg His Arg Val 
                325                 330                 335     


Thr Ser Tyr Thr Ser Ser Ser Val Asn Val Ser Ser Asn Leu 
            340                 345                 350 


<210>  3
<211>  20
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  T7 promoter

<400>  3
taatacgact cactataggg                                                   20


<210>  4
<211>  93
<212>  DNA
<213>  Artificial Sequence

<220>
<223>   alpha-globin 3' UTR

<400>  4
gctgccttct gcggggcttg ccttctggcc atgcccttct tctctccctt gcacctgtac       60

ctcttggtct ttgaataaag cctgagtagg aag                                    93


<210>  5
<211>  1230
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  CXCR1 expression cassette comprising a T7 promoter, a CXCR1 
       coding sequence and an alpha globin 3'UTR

<400>  5
taatacgact cactataggg aaataagaga gaaaagaaga gtaagaagaa atataagaac       60

cggtgccacc atgtcaaata ttacagatcc acagatgtgg gattttgatg atctaaattt      120

cactggcatg ccacctgcag atgaagatta cagcccctgt atgctagaaa ctgagacact      180

caacaagtat gttgtgatca tcgcctatgc cctagtgttc ctgctgagcc tgctgggaaa      240

ctccctggtg atgctggtca tcttatacag cagggtcggc cgctccgtca ctgatgtcta      300

cctgctgaac ctggccttgg ccgacctact ctttgccctg accttgccca tctgggccgc      360

ctccaaggtg aatggctgga tttttggcac attcctgtgc aaggtggtct cactcctgaa      420

ggaagtcaac ttctacagtg gcatcctgct gttggcctgc atcagtgtgg accgttacct      480

ggccattgtc catgccacac gcacactgac ccagaagcgt cacttggtca agtttgtttg      540

tcttggctgc tggggactgt ctatgaatct gtccctgccc ttcttccttt tccgccaggc      600

ttaccatcca aacaattcca gtccagtttg ctatgaggtc ctgggaaatg acacagcaaa      660

atggcggatg gtgttgcgga tcctgcctca cacctttggc ttcatcgtgc cgctgtttgt      720

catgctgttc tgctatggat tcaccctgcg tacactgttt aaggcccaca tggggcagaa      780

gcaccgagcc atgagggtca tctttgctgt cgtcctcatc ttcctgcttt gctggctgcc      840

ctacaacctg gtcctgctgg cagacaccct catgaggacc caggtgatcc aggagagctg      900

tgagcgccgc aacaacatcg gccgggccct ggatgccact gagattctgg gatttctcca      960

tagctgcctc aaccccatca tctacgcctt catcggccaa aattttcgcc atggattcct     1020

caagatcctg gctatgcatg gcctggtcag caaggagttc ttggcacgtc atcgtgttac     1080

ctcctacact tcttcgtctg tcaatgtctc ttccaacctc tgagtcgacc caagcttgct     1140

gccttctgcg gggcttgcct tctggccatg cccttcttct ctcccttgca cctgtacctc     1200

ttggtctttg aataaagcct gagtaggaag                                      1230


<210>  6
<211>  43
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  CXCR1 primer 1

<400>  6
aataaccggt gccaccatgt caaatattac agatccacag atg                         43


<210>  7
<211>  33
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  CXCR1 primer 2

<400>  7
taaagtcgac tcagaggttg gaagagacat tga                                    33


<210>  8
<211>  134
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  extracellular domain of NKG2D

<400>  8

Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro 
1               5                   10                  15      


Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe 
            20                  25                  30          


Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln 
        35                  40                  45              


Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu 
    50                  55                  60                  


Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro Thr 
65                  70                  75                  80  


Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu 
                85                  90                  95      


Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser 
            100                 105                 110         


Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile 
        115                 120                 125             


Cys Met Gln Arg Thr Val 
    130                 


<210>  9
<211>  402
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  extracellular domain of NKG2D

<400>  9
ttcaaccaag aagttcaaat tcccttgacc gaaagttact gtggcccatg tcctaaaaac       60

tggatatgtt acaaaaataa ctgctaccaa ttttttgatg agagtaaaaa ctggtatgag      120

agccaggctt cttgtatgtc tcaaaatgcc agccttctga aagtatacag caaagaggac      180

caggatttac ttaaactggt gaagtcatat cattggatgg gactagtaca cattccaaca      240

aatggatctt ggcagtggga agatggctcc attctctcac ccaacctact aacaataatt      300

gaaatgcaga agggagactg tgcactctat gcctcgagct ttaaaggcta tatagaaaac      360

tgttcaactc caaatacgta catctgtatg caaaggactg tg                         402


<210>  10
<211>  41
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  CD8 transmembrane domain (protein)

<400>  10

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile 
1               5                   10                  15      


Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val 
            20                  25                  30          


Ile Thr Leu Tyr Cys Asn His Arg Asn 
        35                  40      


<210>  11
<211>  112
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  CD3z intracellular receptor signaling domain

<400>  11

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 
1               5                   10                  15      


Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 
            20                  25                  30          


Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 
        35                  40                  45              


Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 
    50                  55                  60                  


Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 
65                  70                  75                  80  


Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 
                85                  90                  95      


Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 
            100                 105                 110         


<210>  12
<211>  1249
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  NKG2D CAR

<400>  12
taatacgact cactataggg aaataagaga gaaaagaaga gtaagaagaa atataagagc       60

caccatgctt ctcctggtga caagccttct gctctgtgag ttaccacacc cagcattcct      120

cctgatccca ggcgcgcatg ccttcaacca agaagttcaa attcccttga ccgaaagtta      180

ctgtggccca tgtcctaaaa actggatatg ttacaaaaat aactgctacc aattttttga      240

tgagagtaaa aactggtatg agagccaggc ttcttgtatg tctcaaaatg ccagccttct      300

gaaagtatac agcaaagagg accaggattt acttaaactg gtgaagtcat atcattggat      360

gggactagta cacattccaa caaatggatc ttggcagtgg gaagatggct ccattctctc      420

acccaaccta ctaacaataa ttgaaatgca gaagggagac tgtgcactct atgcctcgag      480

ctttaaaggc tatatagaaa actgttcaac tccaaatacg tacatctgta tgcaaaggac      540

tgtggctagc ttcgtgccgg tcttcctgcc agcgaagccc accacgacgc cagcgccgcg      600

accaccaaca ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg      660

ccggccagcg gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgatatcta      720

catctgggcg cccttggccg ggacttgtgg ggtccttctc ctgtcactgg ttatcaccct      780

ttactgcaac cacaggaaca gagtgaagtt cagcaggagc gcagacgccc ccgcgtacca      840

gcagggccag aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt      900

tttggacaag agacgtggcc gggaccctga gatgggggga aagccgagaa ggaagaaccc      960

tcaggaaggc ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat     1020

tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag     1080

tacagccacc aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgctgaac     1140

cggtcgaccc aagcttgctg ccttctgcgg ggcttgcctt ctggccatgc ccttcttctc     1200

tcccttgcac ctgtacctct tggtctttga ataaagcctg agtaggaag                 1249


<210>  13
<211>  126
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  CD8 hinge region

<400>  13
ttcgtgccgg tcttcctgcc agcgaagccc accacgacgc cagcgccgcg accaccaaca       60

ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg      120

gcgggg                                                                 126


<210>  14
<211>  42
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  CD8 hinge (protein)

<400>  14

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro 
1               5                   10                  15      


Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu 
            20                  25                  30          


Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 
        35                  40          


<210>  15
<211>  123
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  CD8 transmembrane domain

<400>  15
ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg       60

gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg caaccacagg      120

aac                                                                    123


<210>  16
<211>  336
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  CD3z intracellular receptor signaling domain

<400>  16
agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc       60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc      120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat      180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc      240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc      300

tacgacgccc ttcacatgca ggccctgccc cctcgc                                336


<210>  17
<211>  41
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  CMV-F primer

<400>  17
atccgctcga gtagttatta atagtaatca attacggggt c                           41


<210>  18
<211>  762
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  EpCAM scFv

<400>  18
gatatccaga tgacccagtc cccgtcctcc ctgagtgctt ctgttggtga ccgtgttacc       60

atcacctgcc gttccaccaa atccctcctg cactccaacg gtatcaccta cctttattgg      120

tatcaacaga aaccgggtaa agctccgaaa cttctgatct accagatgtc caacctggct      180

tccggtgttc cgtctcgttt ctccagttct ggttctggta ccgacttcac cctgaccatc      240

tcttctctgc agccggaaga cttcgctacc tactactgcg ctcagaacct ggaaatcccg      300

cgtaccttcg gtcagggtac caaagttgaa cttaagcgcg ctaccccgtc tcacaactcc      360

caccaggttc catccgcagg cggtccgact gctaactctg gaactagtgg atccgaagta      420

cagctggttc agtccggccc gggtcttgtt caaccgggtg gttccgttcg tatctcttgc      480

gctgcttctg gttacacgtt caccaactac ggcatgaact gggtcaaaca ggctccgggt      540

aaaggcctgg aatggatggg ctggatcaac acctacaccg gtgaatccac ctacgctgac      600

tccttcaaag gtcgcttcac tttctccctc gacacaagtg ctagtgctgc atacctccaa      660

atcaactcgc tgcgtgcaga ggatacagca gtctattact gcgcccgttt cgctatcaaa      720

ggtgactact ggggtcaagg cacgctgctg accgtttcct cg                         762


<210>  19
<211>  254
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  EpCAM scFv (protein)

<400>  19

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 Ser Thr Lys Ser Leu Leu His Ser 
            20                  25                  30          


Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala 
        35                  40                  45              


Pro Lys Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 
    50                  55                  60                  


Ser Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 
65                  70                  75                  80  


Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Asn 
                85                  90                  95      


Leu Glu Ile Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Leu Lys 
            100                 105                 110         


Arg Ala Thr Pro Ser His Asn Ser His Gln Val Pro Ser Ala Gly Gly 
        115                 120                 125             


Pro Thr Ala Asn Ser Gly Thr Ser Gly Ser Glu Val Gln Leu Val Gln 
    130                 135                 140                 


Ser Gly Pro Gly Leu Val Gln Pro Gly Gly Ser Val Arg Ile Ser Cys 
145                 150                 155                 160 


Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Lys 
                165                 170                 175     


Gln Ala Pro Gly Lys Gly Leu Glu Trp Met Gly Trp Ile Asn Thr Tyr 
            180                 185                 190         


Thr Gly Glu Ser Thr Tyr Ala Asp Ser Phe Lys Gly Arg Phe Thr Phe 
        195                 200                 205             


Ser Leu Asp Thr Ser Ala Ser Ala Ala Tyr Leu Gln Ile Asn Ser Leu 
    210                 215                 220                 


Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Ala Ile Lys 
225                 230                 235                 240 


Gly Asp Tyr Trp Gly Gln Gly Thr Leu Leu Thr Val Ser Ser 
                245                 250                 


<210>  20
<211>  1759
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  EpCAM CAR

<400>  20
taatacgact cactataggg aaataagaga gaaaagaaga gtaagaagaa atataagagc       60

caccatgctt ctcctggtga caagccttct gctctgtgag ttaccacacc cagcattcct      120

cctgatccca ggcgcgcatg ccttcaacca agaagttcaa attcccttga ccgaaagtta      180

ctgtggccca tgtcctaaaa actggatatg ttacaaaaat aactgctacc aattttttga      240

tgagagtaaa aactggtatg agagccaggc ttcttgtatg tctcaaaatg ccgatatcca      300

gatgacccag tccccgtcct ccctgagtgc ttctgttggt gaccgtgtta ccatcacctg      360

ccgttccacc aaatccctcc tgcactccaa cggtatcacc tacctttatt ggtatcaaca      420

gaaaccgggt aaagctccga aacttctgat ctaccagatg tccaacctgg cttccggtgt      480

tccgtctcgt ttctccagtt ctggttctgg taccgacttc accctgacca tctcttctct      540

gcagccggaa gacttcgcta cctactactg cgctcagaac ctggaaatcc cgcgtacctt      600

cggtcagggt accaaagttg aacttaagcg cgctaccccg tctcacaact cccaccaggt      660

tccatccgca ggcggtccga ctgctaactc tggaactagt ggatccgaag tacagctggt      720

tcagtccggc ccgggtcttg ttcaaccggg tggttccgtt cgtatctctt gcgctgcttc      780

tggttacacg ttcaccaact acggcatgaa ctgggtcaaa caggctccgg gtaaaggcct      840

ggaatggatg ggctggatca acacctacac cggtgaatcc acctacgctg actccttcaa      900

aggtcgcttc actttctccc tcgacacaag tgctagtgct gcatacctcc aaatcaactc      960

gctgcgtgca gaggatacag cagtctatta ctgcgcccgt ttcgctatca aaggtgacta     1020

ctggggtcaa ggcacgctgc tgaccgtttc ctcggctagc ttcgtgccgg tcttcctgcc     1080

agcgaagccc accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc     1140

gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac     1200

gagggggctg gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg     1260

ggtccttctc ctgtcactgg ttatcaccct ttactgcaac cacaggaaca gagtgaagtt     1320

cagcaggagc gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct     1380

caatctagga cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga     1440

gatgggggga aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa     1500

agataagatg gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa     1560

ggggcacgat ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct     1620

tcacatgcag gccctgcccc ctcgctgaac cggtcgaccc aagcttgctg ccttctgcgg     1680

ggcttgcctt ctggccatgc ccttcttctc tcccttgcac ctgtacctct tggtctttga     1740

ataaagcctg agtaggaag                                                  1759


