Application of a trifunctional reactive linker for the construction of antibody-drug hybrid conjugates

Article abstract of DOI:10.1016/j.bmcl.2008.09.078

A flexible, trifunctional poly(ethylene glycol)-succinamide-Lysine-Lysine-maleimide (PEG-SU-Lys-Lys-mal) linker was employed to simultaneously allow biotin tagging and cell-surface targeting through an integrin α₄β₁-binding peptidomimetic that was regiospecifically conjugated to an IgG1-derived Fc fragment with an engineered C-terminal selenocysteine residue. The resulting antibody derivative mediates Fc receptor binding by virtue of the Fc protein and selectively targets cancer cells expressing human integrin α₄β₁. The PEG-SU-Lys-Lys-mal linker may have general utility as an organic tether for the construction of antibody-drug conjugates.

Full text of DOI:10.1016/j.bmcl.2008.09.078

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5785–5788
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Application of a trifunctional reactive linker for the construction
of antibody–drug hybrid conjugates
Joshua D. Thomas a, Thomas Hofer b, Christoph Rader b, Terrence R. Burke Jr. a,
*
a Laboratory of Medicinal Chemistry, CCR, NCI, NIH, Frederick, MD 21702, USA
b Experimental Transplantation and Immunology Branch, CCR, NCI, NIH, Bethesda, MD 20892, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A flexible, trifunctional poly(ethylene glycol)-succinamide-Lysine-Lysine-maleimide (PEG-SU-Lys-Lys-
mal) linker was employed to simultaneously allow biotin tagging and cell-surface targeting through an
integrin a4b1-binding peptidomimetic that was regiospecifically conjugated to an IgG1-derived Fc frag-
ment with an engineered C-terminal selenocysteine residue. The resulting antibody derivative mediates
Fc receptor binding by virtue of the Fc protein and selectively targets cancer cells expressing human inte-
Received 1 August 2008
Revised 16 September 2008
Accepted 17 September 2008
Available online 14 October 2008
grin
a4b1. The PEG-SU-Lys-Lys-mal linker may have general utility as an organic tether for the construc-
Keywords:
LLP2A
Immunoconjugate
Fc
tion of antibody–drug conjugates.
Ó 2008 Elsevier Ltd. All rights reserved.
IgG1
Peptidomimetic
Integrin
The development of antibody–drug conjugates has been
undertaken for the treatment of cancer, in part because this ap-
proach may improve selectively and pharmacokinetics (PK).1,2
Tissue-specificity and increased serum half-life are typically
governed by the antibody component, while the cytotoxic or
radioactive drug cargo provides the therapeutic effect. Alternate
immunoconjugates, termed ‘chemically programmed antibodies’
(cpAbs), have also been described that employ cell-targeting by
the drug cargo rather than by the antibody.3–5 An important
advantage of cpAbs over traditional immunoconjugates is that
a single antibody can be directed to multiple targets via conjuga-
tion to different antigen-specific peptides or small molecules.
Such an approach expands the versatility of a given antibody
while endowing the small molecule with the effector functions
and PK characteristics of an antibody.
To broaden the scope of immunoconjugate-based chemother-
apy, we recently reported a genre of cpAbs that does not require
antibody-variable domains.6 Instead, while the antigen-specific
small molecule provides target specificity, an IgG1-derived Fc frag-
ment improves the PK properties of the small molecule and allows
alternative routes of administration such as interaction with the
neonatal Fc receptor (FcRn).6 In addition, an engineered C-terminal
selenocysteine (Sec) residue on the Fc protein (Fc–Sec) insures
site-specific attachment of a single drug molecule. To achieve this,
we designed a flexible trifunctional poly(ethylene glycol)-succina-
mide-Lysine-Lysine-maleimide (PEG-SU-Lys-Lys-mal) linker that
simultaneously allows cell-targeting, regiospecific conjugation to
the Fc protein and conjugate detection. For cell-targeting we
employed LLP2A 1 (Fig. 1) a recently developed peptidomimetic
that binds with high affinity and specificity to the cell-surface
protein integrin
Integrin 4b1 has been shown to promote metastasis and
a
4b1 (IC50 = 2 pM).7
a
angiogenesis in a variety of cancers, and it plays a key role in
the onset of drug-resistance that can lead to relapse following
chemotherapy for acute myelogenous leukemia (AML).8–10
Although targeting integrin
ies suggest that integrin
a
4b1 is not without its risks,11 stud-
4b1 antagonists may be particularly
a
valuable therapeutic agents for the treatment of hematologic
malignancies, such as multiple myeloma and AML.10,12 We
wondered whether conjugation of LLP2A to Fc–Sec could over-
come undesirable PK characteristics of LLP2A while maintaining
its potency and selectivity.6,13
In conjugating 1 to Fc–Sec the linking segment needed to be
sufficiently long to allow 1 to bind to integrin
a4b1 without ste-
ric interference from the relatively large Fc protein. For this rea-
son PEG-SU was chosen because it can be extended in a modular
fashion depending on the number of PEG-SU units employed.
Additionally, the N-Fmoc and N-Boc protected forms of PEG-SU
can be used in solid-phase syntheses.14 A PEG-SU dimer was uti-
lized in the Fc–Sec–LLP2A conjugate, since it had been previously
* Corresponding author.
shown that 1 retains its affinity for integrin
a4b1 when indirectly
E-mail address: tburke@helix.nih.gov (T.R. Burke).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.078

5786
J. D. Thomas et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5785–5788
O
O
H
N
LLP2A
O
O
HN
O
O
NH
CO2H
O
O
H
N
O
O
O
H
N
H
N
HN
PEG-SU
O
N
H
NH2
O
O
O
O
N
H
N
H
NH
Biotin
H2N
N
H
N
NH
HN
2
R
LLP2A
1
d R = COCH2CH2-Maleimide
e R = COCH3
a R = H
b R = COCH2I
c R = COCH2Br
Figure 1. Structure of LLP2A (1) and trifunctional linker 2 showing sites of attachment for biotin and LLP2A, with R showing the intended site of Fc–Sec attachment.
e
coupled to streptavidin through a biotin attached via this linker.7
The versatility of PEG-lysine-based linkers is also known.7,15 For
our purposes, two lysine residues were introduced at the C-ter-
minal end of the PEG-SU spacer to provide primary amines as
attachment points for (1) auxiliary functionality and (2) an alkyl-
ating agent that could be used for conjugation to the Sec residue
of the Fc protein. Inclusion of biotin as the auxiliary functionality
Lys(N -R)-amide (2), where R would be the nucleophile acceptor
suitable for conjugation with the Sec residue of the Fc protein
(Fig. 1).6,16
The solid-phase preparation of final products 2b–2e (Fig. 1)
required the previously reported acids 3–5 (Fig. 2).7 Coupling of
e
a
N -Mmt-N -Fmoc-L-Lys to Rink amide MBHA resin followed by
N -biotin-N -Fmoc-L-Lys using standard Fmoc protocols provided
the intermediate 6 (Scheme 1). Two subsequent coupling cycles
e
a
e
yielded the prototype construct LLP2A-(PEG-SU)2-Lys(N -Biotin)-
O
HO
H
O
Fmoc
O
N
HO
N
N
O
OH
O
N
N
H
O
H
H
O
3
5
4
Figure 2. Structures of reagents used in the solid-phase synthesis of 2a–2e.
NH2
O
H
O
O
O
NH
H
N
Fmoc
a, c
a, b
NH
N
N
H
N
H
H
S
H
Rink Amide MBHA Resin
6
HN
Mmt
O
H
N
O
O
H2N
O
O
NH
O
O
H
N
O
HN
NH
d, e, a
d, e, a
O
O
H
O
O
NH
H
N
N
N
H
H
S
H
HN
7
Mmt
H
N
N
O
O
O
O
O
H
N
O
O
N
H
O
N
H
N
H
NH
j
f, e, a
g, e, a
h, e, a
O
O
i
2b - 2e
H
N
O
O
N
H
O
NH
O
O
H
N
HN
O
O
O
H
O
O
H
N
NH
N
H
N
H
NH
S
H
HN
8
Mmt
Scheme 1. Reagents and conditions: (a) 20% piperidine in DMF; (b) Fmoc-Lys(Mmt)-OH, HOBt, DIC; (c) Fmoc-Lys(Biotin)-OH, HOBt, DIC; (d) 3, HOBt, DIC; (e) 10% (w/v) 1-
acetylimidazole in DMF; (f) Fmoc-Ac6c-OH, HATU, DIEA; (g) Fmoc-Aad(OBut)-OH, HATU, DIEA; (h) Fmoc-Lys(Dde)-OH, HOBt, DIC; (i) i—4, HOBt, DIC; ii—2% NH2NH2 in DMF;
iii—5, HOBt, DIC; (j) i—1% TFA in CH2Cl2; ii—3-maleimidopropionic acid, HOBt, DIC; iii—TFA/i-Pr3Si/H2O (98:2.5:2.5).

J. D. Thomas et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5785–5788
5787
with N-Fmoc-PEG-SU (3) gave the resin-bound intermediate 7,
onto which was constructed the LLP2A sequence.7 Treatment of
the resulting resin 8 with 1% TFA in CH2Cl2 removed the acid-labile
showed that conjugation only occurred when the Sec residue was
present in the Fc protein (Fig. 3A).6,20 The Fc–Sec–2d conjugate was
then incubated with integrin a4b1-expressing cells. The cells were
e
N -Mmt-Lys group without cleavage from the resin. Subsequent
washed and then treated with Cy5-labeled rabbit anti-human IgG.
Under these conditions, cells would only fluoresce if both the Fc
and LLP2A components of the hybrid construct were present. As
shown in Figure 3B, cells treated with the Fc–Sec–2d conjugate
were detected by flow cytometry while cells treated with Fc–Stop
(Fc protein minus the Sec residue) or 2e alone, did not exhibit such
dual specificity (Fig. 3B).21
Competition experiments with an anti-integrin a4 mAb (pur-
chased from Serotec) indicated that the Fc–Sec–2d hybrid and
the mAb exhibit identical/overlapping epitopes as evidenced by
the diminished binding of Fc–Sec–2d to lymphoma cells in the
presence of the Serotec mAb (Fig. 3C).22 In contrast, the binding
of an anti-integrin a4 mAb from a different source (R&D Systems)
could not be competed with the Serotec mAb, suggesting that the
acylation of the resulting free amine followed by resin cleavage
(95% TFA) yielded the peptides 2a–2e. However, it was found that
the N-haloacetamide acylation products proved difficult to obtain
in pure form. Iodoacetamide-containing 2b could not be prepared
using standard protocols,17,18 and although 2c could be prepared
in low yield, it could not be obtained in pure form, even following
preparative reverse-phase HPLC.
In contrast to 2b and 2c, peptide 2d was obtained with rela-
tively few side products. One significant side product resulted
from deletion of the Aad residue from 2d (approximately 30%)
due to incomplete coupling of residues amino-proximal to the
sterically hindered aminocyclohexanecarboxylic acid (Ac6c).
However, pure 2d could be obtained through a two-stage proto-
col that involved the initial solid-phase synthesis of 2a followed
by a final solution-phase coupling step to yield 2d. This two-
stage route provided pure 2d in sufficient quantity for cell-based
assays. A more efficient solid-phase route resulting in improved
coupling of residues amino-proximal to the Ac6c, was achieved
using 2-(1H-7-azabenzotriazol-1-yl)-tetramethyluronium hexa-
fluorophosphate (HATU).19 Introducing a capping cycle (1-acetyl-
imidazole) also facilitated the separation of deletion by-products
(Scheme 1).
two mAbs recognize different epitopes on integrin
a4b1. These
results indicate that the binding of the Fc–Sec–2d hybrid to the
cells examined is mediated by the peptidomimetic LLP2A (1) and
not by the Fc protein portion, and that the targeting specificity of
the parent peptidomimetic for integrin a4 is retained in the
Fc–Sec–2d hybrid.
e
In conclusion, the trifunctional linker A–(PEG-SU)nLys(N –
e
B)Lys(N –C), where A is a targeting moiety, B is auxiliary function-
ality such as a fluorescent tag and C is a nucleophile acceptor, is
well-suited for the preparation of immunoconjugates in which a
small synthetic molecule governs cell-targeting.3,5 In the current
study, this linker allowed the conjugation of a LLP2A-biotin con-
struct to Fc–Sec in a chemoselective manner. Subsequent in vitro
The chemoselective alkylation of Sec residues by 2d was exam-
ined using a variety of Fc constructs. Inclusion of a biotin handle
within 2d allowed facile detection of Fc covalent adducts by avidin
pull-down experiments followed by ELISA visualization. This
Figure 3. In vitro evaluation of Fc–Sec–2d conjugate. (A) ELISA assay examining conjugation of Fc proteins to reagent 2d as detected by HRP-coupled streptavidin (left
column) and with HRP-coupled donkey anti-human IgG polyclonal antibodies as control (right column). Fc–Sec, Fc protein with C-terminal Sec; Fc–Stop, Fc protein without
C-terminal Sec; Fc–Cys, Fc protein in which the Sec residue has been replaced with Cys. Fc*–Sec–His, Fc protein with mutation Asn297Ala that reduces binding to the Fc
receptor; BSA, bovine serum albumin negative control. (B) Flow cytometry histogram. Following reaction with 2d, Fc–Sec and Fc–Stop were incubated with cells expressing
integrin
a4b1 (HEK 293F cells), followed by staining with Cy5-conjugated rabbit anti-human IgG. Compound 2e (approximately 70% pure) was used as a negative control. (C)
Flow cytometry histogram. Fc–Sec–2d or a biotinylated mouse anti-human integrin
a
4b1 antibody (R&D Systems) were incubated with cells from the human Burkitt’s
lymphoma cell line Raji that were pre-incubated with either a monoclonal mouse anti-human integrin a4 (Serotec Ab) or with flow cytometry buffer alone. Streptavidin-
coupled phycoerythrin (Strep-PE) was used for detection and as a negative control (blue line).

5788
J. D. Thomas et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5785–5788
overnight or 2ꢀ 2 h). The resin was Fmoc-deprotected and treated with a
analysis revealed that all three components of the linker system—
targeting agent (1), tag (biotin), and antibody fragment (Fc–Sec),
were fully functional, with the affinities of the parent 1 and Fc pro-
solution of Fmoc-Ac6c-OH (0.274 g, 0.750 mmol), HATU (0.285 g, 0.750 mmol),
and DIEA (209 lL, 0.155 g, 1.20 mmol) (2 h, overnight), and then capped with
1-acetylimidazole as described above. The resin then underwent additional
cycles of deprotection, coupling, and capping (1-acetylimidazole) with Fmoc-
Aad(OBut)-OH (HATU/DIEA protocol), followed by Fmoc-Lys(Dde)-OH (HOBt/
DIC protocol). The resin was Fmoc-deprotected and then reacted with 5 (2-(4-
(3-o-tolylureido)phenyl)acetic acid [0.426 g, 1.5 mmol (see Ref. 7)], HOBt
tein for integrin a
4b1 and Fc receptor, respectively, being retained.6
The trifunctional PEG-SU-Lys-Lys-maleimide linker may have more
general utility as an organic tether for the construction and evalu-
ation of antibody–drug conjugates.
(0.230 g, 1.5 mmol), and DIC (232
treated with hydrazine hydrate (8 mL, 2% in DMF, 5 min, 10 min) to remove the
Dde protecting group, and then it was reacted with (trans-3-(3-
lL, 1.5 mmol) (overnight). The resin was
6
Acknowledgments
pyridyl)acrylic acid) (HOBt/DIC protocol, 5 h, overnight). The resin was then
washed well with DMF, MeOH, and DCM, and swollen in DCM for 2–3 h. The
Mmt protecting group was removed with 1% TFA in DCM (8 mL, 3ꢀ 5 min, TFA/
TIS/DCM 1:5:94), and the resin was allowed to swell in DMF for 1.5 h. A
solution of 3-maleimidopropionic acid (0.254 g, 1.5 mmol), HOBt (0.230 g,
This work was supported by the Intramural Research Program
of the Center for Cancer Research, NCI-Frederick and NCI, NIH.
1.5 mmol), and DIC (232 lL, 1.5 mmol) was added to the resin, and the
resulting suspension was shaken (3 h). The resin was filtered, washed
sequentially with DMF, MeOH, DCM, diethyl ether, and then dried under
high vacuum (overnight). The resin was cleaved with 95% TFA (10 mL, TFA/TIS/
H2O 95:2.5:2.5) (2 h), precipitated with diethyl ether, and purified by RP-HPLC
[80% H2O (0.1% TFA) to 90% MeCN (0.1% TFA), using a concave gradient over
35 min]. Product 2d was obtained as a white solid (95% pure by LC–MS): 1H
NMR (DMSO-d6), 8.99 (s, 1H), 8.75 (s, 1H), 8.55 (d, J = 4.2 Hz, 1H), 8.20–8.12 (m,
3 H), 8.02–7.98 (m, 2H), 7.91 (s, 1H), 7.88–7.82 (m, 3H), 7.74–7.72 (m, 2H),
7.47–7.43 (m, 2H), 7.36 (d, J = 8.5 Hz, 2H), 7.16 (d, J = 8.5 Hz, 2H), 7.12 (d,
J = 8.0 Hz, 2H), 6.99 (s, 1H), 6.98 (bs, 1H), 6.91 (t, J = 6.4 Hz, 1H), 6.72 (d,
J = 15.9 Hz, 1H), 6.41 (bs, 1H), 6.35 (bs, 1H), 4.31–4.26 (m, 1H), 4.26–4.20 (m,
1H), 4.20–4.10 (m, 2H), 4.07–4.01 (m, 2H), 3.65–3.32 (m, 40H), 3.25–3.05 (m,
8H), 3.04–2.91 (m, 4H), 2.82 (dd, J = 5.0 Hz, J = 12.4 Hz, 1H), 2.67 (m, 1H), 2.57
(d, J = 11.5 Hz, 1H), 2.42–2.35 (m, 2H), 2.34–2.27 (m, 4H), 2.23 (s, 3H), 2.17–
2.13 (m, 1H), 2.04 (t, J = 7.4 Hz, 2H), 1.87–1.82 (m, 1H), 1.70–1.20 (m, 26H) MS
(ESI), m/z: 1904.7 (MH+); 953.4 (M+2H)2+, 635.8 (M+3H)3+. HRMS (ESI-TOF)
Calcd for C92H134N19O23S: 1904.9621 (MH+). Found: 1904.9622.
References and notes
1. Wu, A. M.; Senter, P. D. Nat. Biotechnol. 2005, 23, 1137.
2. Ricart, A. D.; Tolcher, A. W. Nat. Clin. Pract. Oncol. 2007, 4, 245.
3. Rader, C.; Sinha, S. C.; Popkov, M.; Lerner, R. A.; Barbas, C. F., III Proc. Natl. Acad.
Sci. U.S.A. 2003, 100, 5396.
4. Popkov, M.; Rader, C.; Gonzalez, B.; Sinha, S.; Barbas, C., III Int. J. Cancer 2006,
119, 1194.
5. Doppalapudi, V.; Tryder, N.; Li, L.; Aja, T.; Griffith, D.; Liao, F.; Roxas, G.;
Ramprasad, M.; Bradshaw, C.; Barbas, C., III Bioorg. Med. Chem. Lett. 2007, 17,
501.
6. Hofer, T.; Thomas, J. D.; Burke, T. R., Jr.; Rader, C. Proc. Natl. Acad. Sci. U.S.A. 2008,
105, 12451.
7. Peng, L.; Liu, R.; Marik, J.; Wang, X.; Takada, Y.; Lam, K. S. Nat. Chem. Biol. 2006,
2, 381.
8. Holzmann, B.; Gosslar, U.; Bittner, M. Curr. Top. Microbiol. Immunol. 1998, 231,
125.
9. Jin, H.; Su, J.; Garmy-Susini, B.; Kleeman, J.; Varner, J. Cancer Res. 2006, 66, 2146.
10. Matsunaga, T.; Takemoto, N.; Sato, T.; Takimoto, R.; Tanaka, I.; Fujimi, A.;
Akiyama, T.; Kuroda, H.; Kawano, Y.; Kobune, M.; Kato, J.; Hirayama, Y.;
Sakamaki, S.; Kohda, K.; Miyake, K.; Niitsu, Y. Nat. Med. 2003, 9, 1158.
11. (a) Langer-Gould, A.; Atlas, S.; Green, A.; Bollen, A.; Pelletier, D. N. Engl. J. Med.
2005, 353, 375; (b) Berger, J.; Koralnik, I. N. Engl. J. Med. 2005, 353, 414; (c)
Drazen, J. N. Engl. J. Med. 2005, 353, 417.
12. Olson, D. L.; Burkly, L. C.; Leone, D. R.; Dolinski, B. M.; Lobb, R. R. Mol. Cancer
Ther. 2005, 4, 91.
13. Carpenter, R. D.; Andrei, M.; Lau, E. Y.; Lightstone, F. C.; Liu, R.; Lam, K. S.; Kurth,
M. J. J. Med. Chem. 2007, 50, 5863.
14. Song, A.; Wang, X.; Zhang, J.; Marik, J.; Lebrilla, C. B.; Lam, K. S. Bioorg. Med.
Chem. Lett. 2004, 14, 161.
15. Thushim, G.; Hersel, U.; Goodman, S. L.; Kessler, H. Chem. Eur. J. 2003, 9, 2717.
16. A biotin label was required for determining the half-life of free and conjugated
LLP2A in mice (see Ref. 6).
17. Robey, F. A.; Fields, R. L. Anal. Biochem. 1989, 177, 373.
18. Zuckermann, R. N.; Kerr, J. M.; Kent, S. B.; Moos, W. H. J. Am. Chem. Soc. 1992,
114, 10646.
19. Solid-phase synthesis of LLP2A-(PEG-SU)2-Lys(N -Biotin)-Lys(N -CO(CH2)2-
maleimide)-amide (2d). Rink amide MBHA resin (0.259 g, 0.150 mmol,
0.58 mmol/g loading) was allowed to swell in DMF for 6 h, and then
deprotected with 20% piperidine in DMF. A solution of Fmoc-Lys(Mmt)-OH
20. Procedures for generating ELISA data shown in Figure 3A. All incubations
were for 1 h at 37 °C. To confirm selective biotinylation with 2d at the Sec
interface, each well of a 96-well Costar 3690 plate (Corning) was incubated
*
with 200 ng of conjugated Fc–Sec protein or derivatives thereof (Fc –Sec
protein, Fc–Stop protein and Fc–Cys protein that had been exposed to the
same conjugation conditions) in 25 lL PBS. Note: conjugation with 2d was
carried out as previously described in Ref. 6 After blocking with 3% (w/v)
BSA/PBS, the plate was incubated with either HRP-coupled streptavidin
(50 ng/well) or a 1:1000 dilution of HRP-coupled donkey anti-human IgG
polyclonal antibodies in 1% (w/v) BSA/PBS. After washing with H2O (10ꢀ
200 l
L/well), colorimetric detection was performed using 2,20-azino-bis(3-
ethylbenzthiazoline)-6-sulfonic acid (Roche) as substrate according to the
manufacturer’s directions.
21. Procedures for functional analysis of Fc–Sec–LLP2A shown in Figure 3B.
Following incubation in 10% (v/v) FCS/PBS (1 h, 0 °C), HEK 293F cells were
centrifuged, resuspended in 1% (v/v) FCS/PBS, and aliquots (50 lL)
containing 5 ꢀ 105 cells were distributed into wells of a V-bottom 96-well
plate (Corning). The cells were then incubated with Fc–Sec–LLP2A, Fc–Stop,
and 2e (1 h, 0 °C). After washing twice with 1% (v/v) FCS/PBS, the cells were
incubated with
a 1:20 dilution of Cy5-conjugated rabbit anti-human IgG
e
e
(1 h, 0 °C). After washing twice as above, the cells were resuspended in
FCS/PBS (400
Dickinson).
lL 1% v/v) and analyzed using a FACScan instrument (Becton-
22. Procedures for monoclonal antibody competition data shown in Figure 3C.
(0.469 g, 0.750 mmol), HOBt (0.115 g, 0.750 mmol), and DIC (116
0.750 mmol) was added to suspension of the resin in DMF, and the
resulting mixture was agitated (overnight). The resin was filtered, washed,
deprotected, and then reacted (overnight) with Fmoc-Lys(Biotin)-OH (0.446 g,
lL,
Following incubation in 10% (v/v) FCS/PBS (1 h, 0 °C), Raji cells were
a
centrifuged, resuspended in 1% (v/v) FCS/PBS, and aliquots (50 lL) containing
5 ꢀ 105 cells were distributed into wells of a V-bottom 96-well plate (Corning).
The cells were then incubated with either a monoclonal mouse anti-human
0.750 mmol), HOBt (0.115 g, 0.750 mmol), and DIC (116
DMF as described above. Incomplete coupling (as indicated by a Kaiser test)
resulted in second cycle of coupling (1.5 h) with PyBrop (0.210 g,
0.450 mmol), DIEA (156 L, 0.90 mmol), and Fmoc-Lys(Biotin)-OH (0.281 g,
lL, 0.750 mmol) in
integrin a4 (Serotec, 10
with 1% (v/v) FCS/PBS, the cells were incubated with Fc–Sec–LLP2A or
biotinylated mouse anti-human integrin 4b1 antibody (R&D Systems) (both
10 g/mL) (1 h, 0 °C). Cells were washed and a 1:25 dilution of PE-coupled
streptavidin (BD Biosciences) was added. After washing twice as before, the
lg/mL) or with FCS (1 h, 0 °C). After washing twice
a
a
l
l
0.450 mmol) in DMF. Two additional cycles of deprotection and coupling were
then carried out with 4 [Fmoc-PEG-SU (see Ref. 11)] using the HOBt/DIC
protocol described above. After each coupling step, unreacted amino groups on
the resin were capped with 1-acetylimidazole (8 mL, 10% w/v in DMF,
cells were resuspended in FCS/PBS (400
FACScan instrument (Becton-Dickinson).
lL 1% v/v) and analyzed using a