Journal of the American Chemical Society
Communication
pulmonary inflammation resulting in death within about two
weeks, but this outcome can be prevented by dosing twice daily
with 5 μmol/kg intraperitoneal CGS for four days after adoptive
transfer.3 Anticipating enhanced pharmacokinetic stability of Fc-
CGS vs CGS, we designed a related pneumonitis therapeutic trial
in which treatment with Fc-CGS involved two intraperitoneal
injections total (day 1 and day 3) of 50 nmol/kg. Control arms of
the study involved treating mice with vehicle, 50 nmol/kg Fc, or
5000 nmol/kg CGS, also on days 1 and 3. As shown in Figure 3B,
mice treated with Fc-CGS showed significantly enhanced
survival over animals injected with vehicle, CGS, or Fc. Necropsy
of the animals that succumbed showed a lymphocytic infiltrate in
the lungs that appeared less pronounced in surviving mice treated
with Fc-CGS (Figure 3D). Immunocytochemistry revealed that
Fc-CGS could be detected in the pulmonary tissue on day 11 and
at a lower level on day 21 of the experiment, 8 and 18 days after
the Fc-CGS second treatment (Figure 3C). These images
establish the tremendous stabilization of the Fc-containing
conjugate at the critical site of action versus that previously
established for the untethered small molecule CGS. It is
noteworthy that mice receiving Fc-CGS showed an improved
outcome relative to CGS, even though a 100-fold lower dose of
the protein−small molecule conjugate was administered.
It is not yet determined the extent to which the various FcR
isoforms (FcRn, Fc gamma receptor I) are important for Fc-CGS
pharmacology, nor the relative importance of pharmacokinetic
stabilization versus immune cellular targeting conferred by the Fc
domain. FcRn would be expected to be more important to the Fc
stabilizing functions whereas Fc gamma receptor I might have
more influence on immuno-targeting.9−11 Fc-CGS was readily
detected in the heart by immunohistochemistry but was barely
detectable in the brain of mice 5 days after treatment (Figure
S12). Low detection of Fc-CGS in the brain may be related to the
presence of the blood-brain barrier to large molecules.19
Interestingly, the level of Fc-CGS observed in cardiac tissue
appeared to be reduced in the mice with pneumonitis compared
with healthy controls (Figure S12). In contrast, Fc-CGS
appeared to be more abundant in the lung tissue of mice with
pneumonitis compared with healthy controls (Figure S13).
Taken together, these data suggest that the lung immune
response facilitated recruitment of Fc-CGS to the site of
inflammation, although further studies will be needed to fully
explore these mechanisms.
ASSOCIATED CONTENT
* Supporting Information
Experimental methods and supplementary figures are included in
the Supporting Information. This material is available free of
■
S
AUTHOR INFORMATION
■
Corresponding Authors
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the NIH and FAMRI Foundation for financial support.
We thank T. Jackson, X. Kuo, H. Weng, C. Patel, Y. Lo, Y. Li, J.
Kavran, M. Ward, C. Nirschl, C. Drake, and J. Zhang for helpful
advice and/or experimental assistance.
REFERENCES
■
(1) Shen, B.-Q.; Xu, K.; Liu, L.; Raab, H.; Bhakta, S.; Kenrick, M.;
Parsons-Reponte, K. L.; Tien, J.; Yu, S.-F.; Mai, E.; Li, D.; Tibbitts, J.;
Baudys, J.; Saad, O. M.; Scales, S. J.; McDonald, P. J.; Hass, P. E.;
Eigenbrot, C.; Nguyen, T.; Solis, W. A.; Fuji, R. N.; Flagella, K. M.; Patel,
D.; Spencer, S. D.; Khawli, L. A.; Ebens, A.; Wong, W. L.; Vandlen, R.;
Kaur, S.; Sliwkowski, M. X.; Scheller, R. H.; Polakis, P.; Junutula, J. R.
Nat. Biotechnol. 2012, 30, 184.
(2) Hurwitz, E.; Arnon, R.; Sahar, E.; Danon, Y.; Ann, N. Y. Acad. Sci.
1983, 417, 125.
(3) Perez, H. L.; Cardarelli, P. M.; Desphpande, S.; Gangwar, S.;
Schroeder, G. M.; Vite, G. D.; Borzilleri, R. M. Drug Discovery Today,
2013, in press, 10.1016/j.drudis.2013.11.004
(4) Ohta, A.; Sitkovsky, M. Nature 2001, 414, 916.
(5) Zarek, P. E.; Huang, C.-T.; Lutz, E. R.; Kowalski, J.; Horton, M. R.;
Linden, J.; Drake, C. G.; Powell, J. D. Blood 2008, 111, 251.
(6) Naganuma, M.; Wiznerowicz, E. B.; Lappas, C. M.; Linden, J.;
Worthington, M. T.; Ernst, P. B. J. Immunol. 2006, 177, 2765.
(7) de Lera Ruiz, M.; Lim, Y.-H.; Zheng, J. J. Med. Chem. 2013,
DOI: 10.1021/jm4011669.
(8) Chovan, J. P.; Zane, P. A.; Greenberg, G. E. J. Chromatogr. 1992,
578, 77.
(9) Czajkowsky, D. M.; Hu, J.; Shao, Z.; Pleass, R. EMBO Mol. Med.
2012, 4, 1015.
(10) Jefferis, R. Arch. Biochem. Biophys. 2012, 526, 159.
(11) Bumbaca, D.; Boswell, C. A.; Fielder, P. J.; Khawli, L. A. AAPS
2012, 14, 554.
(12) Xiao, J.; Chen, R.; Pawlicki, M. A.; Tolbert, T. J. J. Am. Chem. Soc.
2009, 131, 13616.
In summary, we have successfully generated an Fc-small
molecule conjugate that retains the agonist properties of the
attached A2AR small molecule agonist but shows enhanced
pharmacokinetic and pharmacodynamic performance in a mouse
model of inflammatory pneumonitis. Conjugating a small
molecule to the immunologically relevant Fc domain may
prove to be a general method to enhance small molecule delivery
to areas of inflammation. The bivalency of such Fc conjugates
may also be beneficial for receptor binding. Expressed protein
ligation with Sf9 cell secreted proteins thus offers a
straightforward and efficient technique to generate such Fc
conjugates in functional, glycosylated form, placing the chemical
modification at the terminus of the natural antibody domain.
This approach may be broadly applicable for improving the
pharmacokinetic properties of small molecule therapeutics and
the production of next generation bivalent protein-based drugs.
(13) Hofer, T.; Thomas, J. D.; Burke, T. R., Jr.; Rader, C. Proc. Natl.
Acad. Sci. U.S.A. 2008, 105, 12451.
(14) Barbuto, S.; Idoyaga, J.; Vila-Perello, M.; Longhi, M. P.; Breton,
G.; Steinman, R. M.; Muir, T. W. Nat. Chem. Biol. 2013, 9, 250.
(15) Muir, T. W.; Sondhi, D.; Cole, P. A. Proc. Natl. Acad. Sci. U.S.A.
1998, 95, 6705.
(16) Bolduc, D.; Rahdar, M.; Tu-Sekine, B.; Sivakumaren, S. C.; Raben,
D.; Amzel, L. M.; Devreotes, P.; Gabelli, S. B.; Cole, P. A. Elife 2013, 2,
e00691.
(17) Hutchison, A. J.; Williams, M.; de Jesus, R.; Yokoyama, R.; Oei, H.
H.; Ghai, G. R.; Webb, R. L.; Zoganas, H. C.; Stone, G. A.; Jarvis, M. F. J.
Med. Chem. 1990, 33, 1919.
(18) Nimmerjahn, F.; Ravetch, J. V. Nat. Rev. Immunol. 2008, 8, 34.
(19) Daneman, R. Ann. Neurol. 2012, 72, 648.
3373
dx.doi.org/10.1021/ja5006674 | J. Am. Chem. Soc. 2014, 136, 3370−3373