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
			R
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
3a. - Contact
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
																			W
																			G
3b. - Contact
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
																			G
Single round affinity maturation:
DBR (Kabat def.)
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
DBR (Contact def.)
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.