Science - 6 December 2019

further examination of the biochemical proper-
ties and specificities of the naïve Abs. Twenty-
three HMP Fabs bound to N332-GT1 and/or
N332-GT2 by SPR and did not bind detect-
ably to the MD39 native-like trimer, demon-

strating proper N332 glycan supersite epitope

specificity (Fig. 3, E and F). These SPR-

validated epitope-specific clones were highly

enriched for VL3-25, VL3-1, or the closely related

VL3-10 (Fig. 3E). Thus, the protein design

strategy resulted in Envtrimersthatcould

successfully bind human naïve B cells with

BG18-like LCs.

All N332-supersite bnAbs identified to date

require a long HCDR3 [≥20 amino acids (aa)],

Steichenet al.,Science 366 , eaax4380 (2019) 6 December 2019 5of13

45

N332-GT2 AF647

N332-GT2 AF488

N332-KO AF568

N332-GT2 A488

0.059

12.4

79.0

95.8

B220+ GT2++

BG18gH

WT

Non-immunized

VH

genes

VL

genes IGKV6-32*01

IGKV4-80*01

IGKV4-79*01

IGKV4-69*01

IGKV4-68*01

IGKV4-55*01

IGKV3-4*01

IGKV3-10*01

IGKV4-61*01

IGKV3-5*01

IGKV3-1*01

IGKV12-41*01

IGKV4-57-1*01

IGKV10-96*01

IGKV12-46*01

IGKV10-94*01

IGKV12-44*01

48

48

20.8%

20.9%

12.5%

10.

5%

Frequency of B cells (%)

GT2++KO-

Day8

post-immunization

Day42

post-immunization

VH

genes

VL

genes Tree scale: 0.01

GT2 MD39

0.0

12.5

25.0

GT2 MD39

0.00

0.63

1.25

*

**

CD45.2 GT2++KO- CD45.1 GT2++KO-

Frequency of B cells [%] Frequency of B cells [%]

GC formation CD45.2

IgG ED

50

Immunogen:

GT2

GT2KOGT2KOGT2KOGT2KO

1×10-1

1×10^0

1×10^1

1×10^2

1×10^3

1×10^4

1×10^5

***

Immunogen:

MD39

0

5

10

15

BG18gH

WT

134

134

Day8

post-immunization

Day42

post-immunization

GT2-AF488 GT2-AF488

GT2-AF647 GT2-AF647

ELISA

GT2 MD39

0.00

0.40

0.80

*

**

GT2 MD39

0.00

0.06

0.12

*

ns

IGKV12-41*01

IGKV4-57-1*01

IGKV12-46*01

IGKV12-44*01

IGHV4-4*02

IGHV4-4*02

45

BC

F

A

HI

D E

G

J K

IC50

(μg/mL)

<0.01

0.01 - 0.1

0.1 - 1

1 - 50

>50

BG505_V1

mod

SF162P3_V

1 mod

AC10_V

1 mod

AD8_V1

mod

BG505_7mutBG505_MD39N332-GT2_KO

1×10^1

1×10^2

1×10^3

1×10^4

1×10^5

KD

(nM) d42_iGL

d42

naive

7m

ut

BG505_MD39N332-GT2_KO

d42_iGL

d42

naive

KD

(nM)

Ab BG505 T332N BG505-V1mod

d42.4 >50 3.3

d42.8 >50 0.06

d42.13 >50 1.61

d42.11 >50 0.45

d42.12 >50 0.1

d42.10 >50 ND

d42.iGL1 >50 >50

d42.iGL2 >50 >50

d42.iGL3 >50 >50

d42.iGL4 >50 >50

d42.iGL5 >50 >50

naive1 >50 >50

naive2 >50 >50

naive3 >50 >50

naive4 >50 >50

naive5 >50 >50

BG18 0.005 0.006

RESEARCH | RESEARCH ARTICLE

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