Table 1.
Sequential affinity maturation: | ||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
|
H1 |
|
|
H2 |
|
H3 |
|
|
|
L1 |
|
|
|
|
|
|
|
L2 |
|
|
|
|
|
L3 |
|
|
|
|
|
|
|
1. - APC |
30 |
31 |
32 |
|
58 |
|
96 |
99 |
100c |
|
24 |
25 |
26 |
27 |
28 |
29 |
|
49 |
52 |
53 |
54 |
55 |
56 |
|
92 |
93 |
94 |
95 |
|
104 |
Div. |
|
Scaffold |
D2E7 |
D |
D |
Y |
|
I |
|
S |
S |
S |
|
R |
A |
S |
Q |
G |
I |
|
Y |
S |
T |
L |
Q |
S |
|
N |
R |
A |
P |
|
V |
|
|
Substitutions |
|
Q |
F |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
N |
W |
|
|
|
|
A |
K |
P |
|
|
|
3840 |
|
|
H |
R |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
G |
|
|
|
|
|
I |
M |
|
|
|
|
||
|
|
G |
S |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
W |
|
|
|
|
|
|
Q |
|
|
|
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||
|
|
R |
|
|
|
|
|
|
|
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|
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||
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|
|
2. - DBR |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Scaffold |
2.1b_9.0_1 |
D |
Q |
F |
|
I |
|
S |
S |
S |
|
R |
A |
S |
Q |
G |
I |
|
Y |
S |
W |
L |
Q |
S |
|
I |
Q |
P |
P |
|
V |
|
|
Substitutions |
|
|
|
|
|
|
K |
P |
|
|
T |
|
|
M |
R |
L |
|
|
|
S |
T |
I |
R |
|
|
|
|
|
|
|
4096 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
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|
|
|
|
|
|
|
|
|
|
|
3a. - Contact |
|
|
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|
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|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Scaffold |
3.1d_6.1_2 |
D |
Q |
F |
|
I |
|
S |
S |
S |
|
R |
A |
S |
Q |
R |
L |
|
Y |
S |
S |
T |
I |
S |
|
I |
Q |
P |
P |
|
V |
|
|
Substitutions |
G |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
R |
|
|
|
|
|
|
|
|
|
|
|
|
8 |
|
|
|
|
|
|
|
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|
|
|
|
|
|
W |
|
|
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|
|
|
|
|
|
|
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||
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
G |
|
|
|
|
|
|
|
|
|
|
|
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||
|
|
|
|
|
|
|
|
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|
|
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|
|
|
|
|
3b. - Contact |
|
|
|
|
|
|
|
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|
|
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|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Scaffold |
3.1f_12.1_1 |
D |
Q |
F |
|
I |
|
K |
S |
S |
|
T |
A |
S |
Q |
R |
L |
|
Y |
S |
S |
T |
Q |
R |
|
I |
Q |
P |
P |
|
I |
|
|
Substitutions |
G |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
R |
|
|
|
|
|
|
|
|
|
|
|
|
8 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
W |
|
|
|
|
|
|
|
|
|
|
|
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||
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
G |
|
|
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|
|
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||
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|
Single round affinity maturation: |
|
|
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|||||||
DBR (Kabat def.) |
|
|
|
|
|
|
|
|
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|
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|
|
|
|
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|
|
|
|
|
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|
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|
Scaffold |
D2E7 |
D |
D |
Y |
|
I |
|
S |
S |
S |
|
R |
A |
S |
Q |
G |
I |
|
Y |
S |
T |
L |
Q |
S |
|
N |
R |
A |
P |
|
V |
|
|
Substitutions |
|
[HF] |
|
V |
|
K |
|
Q |
|
H |
[TSM] |
[RV] |
|
|
|
W |
F |
K |
K |
|
D |
|
P |
|
|
|
8192 |
||||
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|
DBR (Contact def.) |
|
|
|
|
|
|
|
|
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|
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|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Scaffold |
D2E7 |
D |
D |
Y |
|
I |
|
S |
S |
S |
|
R |
A |
S |
Q |
G |
I |
|
Y |
S |
T |
L |
Q |
S |
|
N |
R |
A |
P |
|
V |
|
|
Substitutions |
G |
[HF] |
|
V |
|
K |
|
Q |
|
H |
[TSM] |
[RV] |
|
R |
|
W |
|
K |
|
|
D |
|
P |
|
|
|
8192 |
||||
Affinity-improving single and double substitutions were co-optimized with alternative recombination strategies to screen for the best combinations of additive or synergistic mutations. Sequential affinity maturation: Multiple alternative residues at each of three focused “improvement anchor point” positions in the antibody were co-optimized (APC), followed by one round of dispersed binary recombination (DBR; see text). A third round (3a and 3b) was used to diversify and screen at a CDR-L2 and a CDR-H1 contact definition position. Single-round affinity maturation: One round of dispersed binary recombination (including three adjacent mutation loci [square brackets] that were each recombined as single, linked substitutions, respectively; see text). Div., Total library complexity; bold red highlighted amino acids, Substitutions in scaffolds compared with D2E7.