3824
R. K. Goswami et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3821–3824
Thus, the Ab conjugates 38C2-2 and 38C2-3 clearly target integrin
vb3, and shows some reactivity with the related integrin vb5.
a comparative study of one of the efficient cpAb, such as cp84G3,
to cp38C2 should provide useful information.
a
a
Even though the integrins expressed by the tumor cells were fully
activated by exogenous addition of Mn2+, the unconjugated Ab
38C2 showed no binding activity, clearly demonstrating the bind-
ing specificity of the Ab conjugate. To assess the ability of the Ab
conjugates to recognize the integrin at its endogenous activity le-
In conclusion, we confirmed that, like Ab 38C2, most aldolase
Abs were programmed using a TA equipped with a diketone linker,
such as 1. Highly active Abs, 84G3, 85H6, and 90G8, were also pro-
grammed using the targeting agent equipped with the pro-vinyl
ketone function, such as 2. Many conjugates showed high binding
vel, binding of 38C2-2 and 38C2-3 to
a
vb3 positive M21 and BMS
to cells expressing integrin avb3, thus preserving the binding spec-
cells was analyzed in the presence of Ca2+, which does not activate
integrins but supports cation dependent integrin ligand binding.
Figure 4B shows that the Ab conjugate clearly and specifically rec-
ificity of the conjugated compound, and underlining their impor-
tance as the therapeutic Abs. Further studies with the selected
Abs are in progress, and will be reported in due course.
ognized
avb3 on the tumor cells without a requirement of exoge-
nous activation, thus indicating a high binding affinity to the
integrin which is expressed by M21 and BMS tumor cells in an
intrinsically activated functional state. ‘The cp38C2s bound only
to the activated integrins and in the cation dependent manner’
was further confirmed by repeating the binding experiments in
the absence of metal ions, which did not show binding to any cells,
including M21, BMS, BCM1, and UCLA-P3 (data not shown).
Subsequently, the binding properties of the new cpAbs were
determined on M21 and M21-L cells using the same flow cytome-
try approach. The experiments were conducted using one set of
cpAbs, including 33F12-1, 84G3-1, 84G11-2, 85A2-2, 85C7-1,
85H6-1, 90G8-2, 92F9-1 and 93F3-1, which were prepared from
the respective antibodies and either compound 1 or 2. Only those
cpAbs from compound 2 were used, which were fully conjugated
as observed by determining their catalytic activity. All cpAbs
showed positive binding to M21 cells and were negative on M21-
L cells, except 92F9-1 which did not bind to either cell line. Shifts
in the flow cytometry histogram with the positive binders were
noted in the following order: 84G3-1 ꢀ 85H6-1 ꢀ 90G8-2 > 85A2-
2 > 33F12-1 ꢀ 84G11-2 > 85C7-1 ꢀ 93F3-1. At least three cpAbs,
including 84G3-1, 85H6-1, and 90G8-2 showed the largest shifts
in the flow cytometry histogram indicating the best binders of cells
Acknowledgements
We thank Drs. Richard A. Lerner and Carlos Barbas of Scripps
and C. Rader from NCI, for helpful comments, and the NCI (R01
CA120289) and DOD (W81XWH-07-1-0421) for financial support.
References and notes
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expressing integrin avb3 (Fig. 5).
There are several important points that could not be addressed
in this Letter, and would be worth finding out in the future. For
example, all aldolase Abs could react with other reactive functional
groups, including b-lactam and c
-lactone, like Ab 38C2.15 Second,
Ab 93F3 possessed two lysine residues in each binding sites, and
we did not confirm whether both lysine residues could take part
in conjugation. Also, the conjugate prepared from 93F3 and the
TAs with diketone function did not show binding comparable to
other Abs. We wonder whether cp93F3 undergoes a rapid disas-
sembly to release TA during the experiments. Third, we have con-
structed numerous cp38C2s with high affinities for cells expressing
the avb3 integrin, and determined their inhibitory effects on tumor
growth and metastasis in a number of cancer models.6,16 One can
anticipate different levels of Ab-mediated immune response from
one Ab to another, and similarly of one cpAb to other. Therefore,