orientation of the variable regions, allowing each
region to interact with its target ( 23). Several
known bnAbs, including VRC01, 10E8, PGT121,
and PGT128 [reviewed in (1)], were evaluated for
their ability to neutralize a select panel of viruses
with known resistance or sensitivity to these antibodies (fig. S1). Initially, we determined whether
the position of the variable regions from VRC01
and 10E8 in the proximal or distal positions (Fig.
1A) could affect neutralization breadth and potency. Inclusion of both variable regions in either
orientation in the bispecific antibody reduced
the number of resistant strains relative to the
parental antibodies alone (Fig. 1B). Better potency was observed when VRC01 was proximal
and 10E8 distal, although neither bispecific antibody was as potent as a mixture of the two antibodies alone.
To explore whether other bnAbs could perform
better in the bispecific format, we evaluated two
different combinations: VRC01 plus PGT121, or
VRC01 plus PGT128. For PG T121, expression was
observed only with VRC01 in the distal position.
When this antibody was compared to the parental antibodies alone, it provided marginally better
neutralization (Fig. 2A). In contrast, VRC01 could
be expressed with PGT128 in both positions, with
greater breadth observed when VRC01 was distal
(Fig. 2B). Together, these data indicate that improvements in breadth could be achieved with a
bispecific format; however, in this case, the potency was not consistently improved relative to
each Ab alone. We therefore sought an alternative format to improve the potency and breadth
Generation and comparison of broad
and potent trispecific antibodies
To achieve our goal, we used a previously undescribed trispecific Ab format. Three specificities were combined by using knob-in-hole
heterodimerization ( 24) to pair a single arm derived from a normal immunoglobulin (IgG) with
a double arm generated in the CODV-Ig. A panel
of bnAbs was evaluated, including those directed
against the CD4bs that included VRC01 and N6,
as well as PGT121, PGDM1400, and 10E8 (fig. S1).
A modified version of the latter, termed 10E8v4,
was used because of its greater solubility ( 25).
We first determined which bispecific arms showed
the best potency, breadth, and yield. This screening analysis revealed that combinations containing PGDM1400, CD4bs, and 10E8v4 showed the
Fig. 1. CODV-Ig bispecific antibody design and neutralization titers of the VRC01/10E8
bispecific antibodies. (A) CODV-Ig bispecific antibody design with two different orientations of 10E8
and VRC01. (B) Neutralization titers (IC50), in mg/ml, of VRC01/10E8 bispecific Abs and parental Abs
against a select panel of 19 circulating HIV-1 strains; values highlighted in red, orange, and yellow
indicate highest, medium, and lowest potency, respectively.
Fig. 2. Neutralization titers of VRC01/PGT121- and VRC01/PGT128-based bispecific antibodies.
(A and B) Neutralization titers (IC50), in mg/ml, of the VRC01/PGT121 (A) and VRC01/PGT128 (B)
bispecific Abs against a select panel of 20 circulating HIV-1 strains, with colors as in Fig. 1.
1Sanofi, 640 Memorial Drive, Cambridge, MA 02139, USA.
2Vaccine Research Center, National Institute of Allergy and
Infectious Diseases, Bethesda, MD 20892, USA. 3Center for
Virology and Vaccine Research, Beth Israel Deaconess
Medical Center, Harvard Medical School, Boston, MA 02215,
USA. 4Ragon Institute of MGH, MIT, and Harvard, Cambridge,
MA 02139, USA. 5Department of Immunology and
Microbiology, International AIDS Vaccine Initiative (IAVI)
Neutralizing Antibody Center, Center for HIV/AIDS Vaccine
Immunology and Immunogen Discovery, The Scripps
Research Institute, La Jolla, CA 92037, USA. 6National
Institute of Allergy and Infectious Diseases, Bethesda, MD
*These authors contributed equally to this work.
†Corresponding author. Email: firstname.lastname@example.org (G.J.N.);