NOVEL APPLICATIONS OF BISPECIFIC DART PROTEINS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 531

when combined with the appropriate fluoresceinated mAb
was equivalent in its in vitro biological activity to a DART 1
directly linking CD32B and CD79B.

2. Weiner LM, Clark JI, Davey M, Li WS, Garcia de Palazzo I,
Ring DB, et al. (1995) Phase I trial of 2B1, a bispecific
monoclonal antibody targeting c-erbB-2 and Fc gamma
RIII. Cancer Res. 55(20), 4586-4593.
3. Weiner LM, Alpaugh RK, Amoroso AR, Adams GP, Ring DB,
Barth MW. (1996) Human neutrophil interactions of a bispe-
cific monoclonal antibody targeting tumor and human Fc
gamma RIII. Cancer Immunol. Immunother. 42(3), 141-150.
4. Pullarkat V, Deo Y, Link J, Spears L, Marty V, Curnow R, et al.
(1999) A Phase I study of a HER2/neu bispecific antibody with
granulocyte-colony-stimulating factor in patients with meta-
static breast cancer that overexpresses HER2/neu. Cancer
Immunol. Immunother. 48(1), 9-21.
5. Lanzavecchia A, Scheidegger D. (1987) The use of hybrid
hybridomas to target human cytotoxic T lymphocytes. Eur. J.
Immunol. 17(1), 105-111.
6. Tutt A, Stevenson GT, Glennie MJ. (1991) Trispecific F(ab')3
derivatives that use cooperative signaling via the TCR/CD3
complex and CD2 to activate and redirect resting cytotoxic T
cells. J. Immunol. 147(1), 60-69.
7. Nitta T, Ishizawa A, Ito M, Sato K, Yagita H, Kumura K.
(1990) [Induction of cytotoxicity from human lymphocytes
coated with bispecific antibody against human glioma cells].
No Shinkei Geka 18(11), 1001-1006.
8. Lu D, Zhang H, Ludwig D, Persaud A, Jimenez X, Burtrum D,
et al. (2004) Simultaneous blockade of both the epidermal growth
factor receptor and the insulin-like growth factor receptor signal-
ing pathways in cancer cells with a fully human recombinant
bispecific antibody. J. Biol. Chem. 279(4), 2856-2865.
9. Lu D, Jimenez X, Zhang H, Wu Y, Bohlen P, Witte L, et al.
(2001) Complete inhibition of vascular endothelial growth
factor (VEGF) activities with a bifunctional diabody directed
against both VEGF kinase receptors, fms-like tyrosine kinase
receptor and kinase insert domain-containing receptor. Cancer
Res. 61(19), 7002-7008.
10. Robinson MK, Hodge KM, Horak E, Sundberg AL, Russeva
M, Shaller CC, et al. (2008) Targeting ErbB2 and ErbB3 with a
bispecific single-chain Fv enhances targeting selectivity
and induces a therapeutic effect in vitro. Br. J. Cancer 99
(9), 1415-1425.
11. Bird RE, Hardman KD, Jacobson JW, Johnson S, Kaufman
BM, Lee SM, et al. (1988) Single-chain antigen-binding
proteins. Science 242 (4877), 423-426.
12. Huston JS, Levinson D, Mudgett-Hunter M, Tai MS, Novotny
J, Margolies MN, et al. (1988) Protein engineering of antibody
binding sites: recovery of specific activity in an anti-digoxin
single-chain Fv analogue produced in Escherichia coli. Proc.
Natl. Acad. Sci. USA 85(16), 5879-5883.
13. Olafsen T, Cheung C-W, Yazaki PJ, Li L, Sundaresan G,
Gambhir SS, et al. (2004) Covalent disulfide-linked anti-
CEA diabody allows site-specific conjugation and radiolabel-
ing for tumor targeting applications. Protein Eng. Des. Sel.
17(1), 21-27.
14. Holliger P, Prospero T, Winter G. (1993) “Diabodies”: small
bivalent and bispecific antibody fragments. Proc. Natl. Acad.
Sci. USA 90(14), 6444-6448.

36.7 CONCLUSIONS AND FUTURE
PERSPECTIVES

DART 1 proteins are highly potent, stable, and manufactur-
able fragment-based bispecific antibodies. They are mono-
valent for each antigen; this is advantageous for redirected
killing, which requires activation of the effector cell only
upon engagement of the target cell. The DART 1 size of

50 kDa, similar to a Fab fragment, is close to the threshold
for renal clearance, and thus the half-life of these proteins
should be in the same order of magnitude. It may be
desirable in certain indications to extend the half-life so
that at least weekly dosing can be contemplated in a clinical
setting. This can be accomplished by fusion to an albumin-
binding domain that has been deimmunized by removing
potential T-cell helper epitopes, while retaining binding to
serum albumin. Albumin binds to FcRn on a site separate
from that of IgG, nonetheless resulting in similar recycling
and reduced renal clearance.
In an alternate approach, the DART 1 structure can be
combined in an Ig-like bispecific molecule, such that one or
two of the DART 1 bispecific units is assembled with an Fc.
This approach results in both increasing the molecular size
and, more importantly, allowing binding to FcRn with
ensuing recycling and extended half-life. In the case where
two binding sites for each target are produced, complete
retention of antigen binding has been observed compared to
the parent mAbs. This structure has utility in applications
where receptor agonism may be advantageous or is not a
concern. In cases where agonism is a potential concern,
structures monovalent for each antigen but containing an Fc
have been expressed in several alternative configurations.
Just as the multiple applications of bispecific antibodies
are now being realized through more robust configurations
such as DART 1 , new possibilities both in research and for
clinical application can be conceived. While challenges
remain to their clinical use in a binary or “pretargeting”
mode, the U-DART system has considerable utility in
selecting the appropriate candidate antibodies for making
the bispecific clinical product, be it a T-DART 1 , I-DART 1 ,
or Fc-engineered IgG.

REFERENCES

1. Seimetz D, Lindhofer H, Bokemeyer C. (2010) Development
and approval of the trifunctional antibody catumaxomab (anti-
EpCAM x anti-CD3) as a targeted cancer immunotherapy.
Cancer Treat. Rev. 36(6), 458-467.