The self-activation the viridin Wm undergoes when modified at the C20 position by N-methyl hexanoic acid is shown in . An intramolecular attack of the C6 OH generates Wm, while the carboxyl group can be reacted with amines. WmC20 derivatives featuring a secondary amine at C20 (2a
) and a tertiary amine at C20 (2b
) were converted to NHS esters and attached to the amino groups of amino-dextran, to yield 5a
, or to the amino groups of cetuximab to yield 7a
. The compound designations shown in are from our previous communications, with our new cetuximab based compounds designated as 7a
. Thus 2a
are the same as in our earlier publications, with the “a” designation referring to an N(Me) hexanoic acid, self-activating linker reacted with Wm's C20. The “b” designation refers to N(H)hexanoic acid based compounds which do not self-activate due to an extra hydrogen bond stabilization shown in (3
To determine whether the attachment of Wm to cetuximab (as in 7a
) resulted in a reduced immunoreactivity, a self-displacement assay () was employed. However, we first characterized the binding of the fluorescein labeled 8
to cells as shown in . Binding of 8
was determined as the relative cell fluorescence (RCF), that is the mean fluorescence intensity determined by FACS of cells incubated with 8
divided the mean fluorescence intensity of control cells. Preliminary experiments indicated the binding of 8
to cells was rapid and independent of incubation times between 1 and 24 hours. We then varied the concentration of 8
, and determined a Kd of 0.40 nM as shown in . The amount of 8
bound per cell at a saturating concentration of 10 nM was determined by a fluorescein hapten immunoassay to be 56,400 ± 14,200 molecules per cell. Our estimate of the number of binding sites per cell was about 10 fold higher than Mukohara who used a FACS based bead method(12
), while we measured immunoreactive fluorescein from 8
after cell lysis.
Binding of Fl-cetuximab (8) and Wm-cetuximab (7a) to cells
With the concentration dependence and number of binding sites for 8 binding to cells in hand, we determined the immunoreactivity of the Wm modified centuximab 7a by its ability to displace the binding of 8 (10 nM) from cells (). The similar decrease (p>0.05 for all concentrations) in RCF produced by 7a and cetuximab indicated the attachment of Wm to cetuximab, as occurred with 7a, did not impair its immunoreactivity.
A surprising feature of the binding of 8 to A549 cells was lack of time dependence of this binding, which suggested that 8 remained as a stable complex bound to its ErbB1 target receptor on the cell surface. To verify that this was in fact the case, we incubated cells with cetuximab (10 nM, 1 h), washed cells, and detected cell surface cetuximab by the addition of a fluorescein labeled goat anti-Human IgG. As shown in , the RCF was essentially constant for 6 hours and decreased to 50.6 % of control at 24 h. A 50% decrease in cell fluorescence would be expected at 22.9 hours based on the doubling time of A549 cells, see above. Thus cetuximab appears not to be internalized by A549 cells.
We next assessed the antiproliferative activity of the Wm-cetuximab conjugates (7a
), with the dextran conjugates featuring the same linkages (5a
) serving as non-receptor binding Wm controls. Representative curves for the decrease in cell mass as a function of concentration are shown in with IC50's provided in . Cetuximab had no effect on the proliferation of A549 cells, consistent with the observations of others(12
), though cetuximab has been shown to inhibit the growth of A549 xenografts (10
), an effect that may reflect immune mediated killing, see above. Based on the similar IC50's of 5a
release Wm into the media in a similar manner (). Based on the lack of internalization of cetuximab (), 7a
was not internalized ().
Antiproliferative Activity Of Wortmannin-Cetuximab Conjugates
The antiproliferative activity of cetuximab was greatly enhanced by the attachment of Wm with a self-releasing linkage. That compound, 7a, exhibited an IC50 of 153 nM on a per mole cetuximab basis (690 nM on a Wm basis at 4.5 Wm/per mole of cetuximab), while cetuximab lacked activity in this assay. This improvement in antiproliferative activity of 7a relative to cetuximab was not due an immune mediated binding of 7a, since the IC50 of the self-activating dextran (5a) was 700 nM on a wortmannin basis, and not different from the IC50 of 7a (p>0.05). With both cetuximab and dextran carriers, the configuration at C20 was critical for activity, with non-releasing C20 configurations of 5b and 7b showing no antiproliferative activity. The potency of Wm was greatly increased by its attachment to cetuximab with the self-releasing linkage. Wm has an IC50 of 11.4 μM against A549 cells, while 7a had an IC50 of 690 nM on a per mole of Wm basis.
There are two conclusions from these studies. First, Wm could be attached to cetuximab at levels that did not affect its immunoreactivity but increased its antiproliferative activity. The experimental approach developed here, attachment of Wm followed by verification of immunoreactivity, could be employed to obtain slowly Wm releasing, immunologically active, monoclonal antibodies. Second, the attachment of Wm to cetuximab increased the antiproliferative activity of both the antibody and Wm. This improvement occurred through the slow release of Wm into culture media, based on the similar antiproliferative IC50 of 5a and 7a.
Though we did not demonstrate the immune mediated targeting of Wm to A549 cells in culture, this may still be possible in vivo with appropriate antibody/tumor combinations. The A549 we employed is a Ras driven tumor(21
), and the amount of Wm that must targeted to achieve a pharmacological effect might be considerably higher than for PI3 kinase driven tumors, see(23
). In addition, our Wm-cetuximab was not internalized by A549 cells, a fate common with cetuximab and other cell lines(25
). Internalization of Wm-monoclonal antibodies and receptor recycling might lead to higher intracellular levels of Wm. Finally, a self-releasing Wm antibody might be attached to a monoclonal antibody or protein with an immune suppressive activity, which would then act as a pharmacologically active (but non-targeting) carrier for a slow Wm release. The slow release of Wm from 5a
is immune suppressive in an animal models of lung inflammation and arthritis(5
). Wm is a potent antiangiogenic agent(26
), offering a mechanism for immune suppression or inhibiting tumor growth without targeting.
From 1980 to 2005 some 206 monoclonal antibodies were studied in clinical trials with 12 being approved(27
), offering a wide array of potential Wm-antibody double drugs. The conjugation of Wm with a self-activating linker to an approved monoclonal antibody offers a potentially general chemistry for the design of double drugs, the pharmacological effectiveness of which will have to be evaluated for each antibody considered.