Naive Mouse Antibody
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germline gene 186.2 VH
QVQLQQPGAELVKPGASVKMS
CKASGYTFTSYNMHWVKQTPG
RGLEWIGAIYPGNGYTSYNQKF
KGKATLTADKSSSTAYMQLSSL
TSEDSAVYYCARXXXX(X)(X)FD
YWGQGTTVTVSS
linker
GGGGS GGGGS GGGG
germline gene kv4-72
QIVLSQSPAILSASPGEKVTMTC
RASSSVSYMHWFQQKPGSSPK
PWIYATSNLASGVPVRFSGSGS
GTSYSLTISRVEAEDAATYYCQQ
XXXXPXTFGGGTKLEIKRTVAA
BALB/c
VH
C C
gtgcagctgcaggagtctggggctgagctggtgaagcctggggcctcagtgaagatgtcc
V Q L Q E S G A E L V K P G A S V K M S
V Q L Q Q P G A E L V K P G A S V K M S
tgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacct
C K A S G Y T F T S Y N M H W V K Q T P
C K A S G Y T F T S Y N M H W V K Q T P
G G T
ggacagggcctggaatggattggaactatttatccaggaaatggtgatacttcctacaat
G Q G L E W I G T I Y P G N G D T S Y N
G R G L E W I G A I Y P G N G Y T S Y N
cagaagttcaaaggcaaggccacattgactgcagacaaatcctccagcacagcctacatg
Q K F K G K A T L T A D K S S S T A Y M
Q K F K G K A T L T A D K S S S T A Y M
cagctcagcagcctgacatctgaggactctgcggtctattactgtgcaagacggggtaac
Q L S S L T S E D S A V Y Y C A R R G N
Q L S S L T S E D S A V Y Y C A R
ccctatgctatggactactggggtcaaggaacctcagtcaccgtctcctca
P Y A M D Y W G Q G T S V T V S S
F D Y W G Q G T T V T V S S
Vk
caaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcaca
Q I V L S Q S P A I L S A S P G E K V T
Q I V L S Q S P A I L S A S P G E K V T
T
atgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccagga
M T C R A S S S V S Y M H W Y Q Q K P G
M T C R A S S S V S Y M H W F Q Q K P G
T
tcctcccccaaaccctggatttatgccacatccaacctggcttctggagtccctgctcgc
S S P K P W I Y A T S N L A S G V P A R
S S P K P W I Y A T S N L A S G V P V R
C
ttcagtggcagtgggtctgggacctcttattttctcacaatcagcagagtggaggctgaa
F S G S G S G T S Y F L T I S R V E A E
F S G S G S G T S Y S L T I S R V E A E
gatgctgccacttattactgccagcagtggagtaataacccacgagcgctcacgttcggt
D A A T Y Y C Q Q W S N N P R A L T F G
D A A T Y Y C Q Q X X X X P X X X T F G
gctgggaccaagctggagctgaaac
A G T K L E L K
G G T K L E I K R T V A A
PHILOtop library construction primers
a LMB3long 5'-CAGGAAACAGCTATGACCATGATTAC-3'
b1 186_CDR304ba 5'-GTCCCTTGGCCCCAGTAGTCAAAMNNMNNMNNMNNTCTTGCACAATAATAGACCGCA-3'
b2 186_CDR305ba 5'-GTCCCTTGGCCCCAGTAGTCAAAMNNMNNMNNMNNMNNTCTTGCACAATAATAGACCGCA-3'
b3 186_CDR306ba 5'-GTCCCTTGGCCCCAGTAGTCAAAMNNMNNMNNMNNMNNMNNTCTTGCACAATAATAGACCGCA-3'
c 186_CDR3fo 5'-TTTGACTACTGGGGCCAAGGGAC-3'
d kv4-72_CDR3ba 5'-CGAGCTTGGTCCCCCCTCCGAACGTMNNCGGMNNMNNMNNMNNCTGCTGGCAGTAATAAGTGGC-3'
e kv4-72_FR4NotIba 5'-TTTTCCTTTTGCGGCCGCTGCAGCCACAGTCCGTTTGATCTCGAGCTTGGTCCCCCCTCC-3'
fdseqlong 5'-GACGTTAGTAAATGAATTTTCTGTATGAGG-3'
PHILOtop affinity maturation library primers
a LMB3long 5'-CAGGAAACAGCTATGACCATGATTAC-3'
f CDR1VH 1aa ba 5'-GTGTCTGCTTTACCCAGTGCATMNNGTAGCTGGTGAAGGTGTAGC-3'
g CDR2 VH 5aa fo 5'-CAGGGGTCTGGAATGGATCGGGNNMATTNNMCCTGGANNMGGTNNMACTNNMTACAATCAGAAGTTCAA
GGGCAAG -3'
h Ext FR1/2 ba 5'-CCGATCCATTCCAGACCCCTGCCAGGTGTCTGCTTTACCCAGTG -3'
fdseqlong 5'-GACGTTAGTAAATGAATTTTCTGTATGAGG-3'
(M=A/C, N=A/C/G/T)
PHILOtop library construction primers
a LMB3long 5'-CAGGAAACAGCTATGACCATGATTAC-3'
reverse primers for heavy CDR3
b1 186_CDR304ba 5'-GTCCCTTGGCCCCAGTAGTCAAAMNNMNNMNNMNNTCTTGCACAATAATAGACCGCA-3'
(
tgcggtctattattgtgcaagannmnnmnnmnnmtttgactactggggccaagggac
A V Y Y C A R X X X X F D Y W G Q G
)
b2 186_CDR305ba 5'-GTCCCTTGGCCCCAGTAGTCAAAMNNMNNMNNMNNMNNTCTTGCACAATAATAGACCGCA-3'
b3 186_CDR306ba 5'-GTCCCTTGGCCCCAGTAGTCAAAMNNMNNMNNMNNMNNMNNTCTTGCACAATAATAGACCGCA-3'
forward primer complement to reverse primers for heavy CDR3
c 186_CDR3fo 5'-TTTGACTACTGGGGCCAAGGGAC-3'
F D Y W G Q G
reverse primer for light chain CDR3
d kv4-72_CDR3ba 5'-CGAGCTTGGTCCCCCCTCCGAACGTMNNCGGMNNMNNMNNMNNCTGCTGGCAGTAATAAGTGGC-3'
( gccacttattactgccagcagnnmnnmnnmnnmccgnnmacgttcggaggggggaccaagctcg
A T Y Y C Q Q X X X X P X T F G G G T K L
)
reverse primer for light chain
e kv4-72_FR4NotIba 5'-TTTTCCTTTTGCGGCCGCTGCAGCCACAGTCCGTTTGATCTCGAGCTTGGTCCCCCCTCC-3'
( Not1
ggaggggggaccaagctcgagatcaaacggactgtggctgca gcggccgc aaaaggaaaa
G G G T K L E I K R T V A A A A A K G K
)
fdseqlong 5'-GACGTTAGTAAATGAATTTTCTGTATGAGG-3'
========
PHILOtop affinity maturation library primers
a LMB3long 5'-CAGGAAACAGCTATGACCATGATTAC-3'
f CDR1VH 1aa ba 5'-GTGTCTGCTTTACCCAGTGCATMNNGTAGCTGGTGAAGGTGTAGC-3'
g CDR2 VH 5aa fo 5'-CAGGGGTCTGGAATGGATCGGGNNMATTNNMCCTGGANNMGGTNNMACTNNMTACAATCAGAAGTTCAA
GGGCAAG -3'
h Ext FR1/2 ba 5'-CCGATCCATTCCAGACCCCTGCCAGGTGTCTGCTTTACCCAGTG -3'
fdseqlong 5'-GACGTTAGTAAATGAATTTTCTGTATGAGG-3'
(M=A/C, N=A/C/G/T)
Synthetic genes (GenScript Corporation; NJ, USA) were used as templates for
the PCR amplification of the variable heavy chain (VH) gene 186.2 (Bothwell,
Paskind et al. 1981; Williams, Martinez et al. 2001; Chang and Mohan 2005) and
for the amplification of the light variable chains (Vk) gene kv4-72 (Kirschbaum,
Roschenthaler et al. 1999). The amplified fragments were used for the
construction in the scFv format of the PHILOtop library. the linker
Gly4SerGly4SerGly4 was introduced between the heavy and the light variable
chain amplified genes (Table 3.1 and Figure 3.2). The resulting scFv segments
were used for library construction as follows.
Antibody residues are numbered according to (Chothia and Lesk 1987;
Tomlinson, Cox et al. 1995) and are indicated in Figure 3.2. Sequence variability
in the variable heavy chains component of the libraries was introduced by PCR
using partially degenerated primers (Table 3.6 and Figure 3.2), in a process that
generate random mutations at position 95-99 of the VH CDR3. The variable light
chain components of the libraries were generated in a similar fashion, introducing
random mutations at position 91, 92, 93, 94 and 96 in the Vk CDR3 (Table 3.6
and Figure 3.2). VH/Vk combinations were assembled in scFv format by PCR
assembly, using gel purified VH and Vk segments as templates. The assembled
VH/Vk fragments were doubly-digested with NcoI/NotI (New England Biolabs;
MA, USA) and cloned (T4 DNA ligase, New England Biolabs; MA, USA) into
NcoI/NotI-digested pHEN1 phagemid vector (Hoogenboom, Griffiths et al. 1991).