The promise of affinity enhancement pretargeting is the combination of improved target/nontarget ratios by conventional pretargeting with the higher binding affinities provided by affinity enhancement. We previously confirmed by SPR that the phenomenon of affinity enhancement is real and can be achieved with bivalent MORF effectors.20,21
We have also demonstrated with MORF effectors that MORFs with different molecular spacings may be readily synthesized and that spacing is a critical variable.21
The objective of this investigation was to synthesize a bivalent MORF capable of being radiolabeled and compare its properties to the radiolabeled monovalent version in cell culture and in normal and pretargeted tumored mice.
In this investigation, our standard 18 mer MORF sequence was again used, now for the synthesis of a bivalent MORF capable of chelating 99m
Tc and capable of hybridizing with a 12 mer cMORF conjugated to the monoclonal antibody MN14. The 12 mer cMORF chain length was selected based on our previous SPR results showing the largest affinity enhancement (i.e., ratio of affinity constants of the bivalent over the monovalent MORF) of almost 3 orders of magnitude for an 18 mer bivalent MORF hybridizing with a 12 mer cMORF presumably because the shorter cMORF lowered the equilibrium constant for monomolecular binding. The SPR results also showed that using bivalent effectors can greatly improve MORF pretargeting and that bivalent MORFs with reduced equilibrium constants may actually provide higher affinity enhancement.20,21
The cMORF sequence was complementary to only 12 of the 18 bases of MORF. Thus after bridging two cMORF conjugated antibodies, the bivalent MORF will have a flexible single strand six bases long on either side furthest from the antibodies that should favor bimolecular binding.
To facilitate the synthesis of a MAG3 conjugated bivalent MORF, the Fmoc-HGA-OtBu with two carboxylate groups and one protected amine was selected. After deprotection with TFA, the tBu protected carboxylate groups were readily conjugated with the amine derivatized MORFs without affecting the Fmoc protected amine that was subsequently deprotected before reacting with MAG3-NHS.
presents the results of the tumor animal pretargeting study and shows that tumor accumulation was significantly higher in animals receiving the same MORF dosage as the bivalent compared to the monovalent MORF effector. However, a fraction of the higher tumor accumulation may be the result of pharmacokinetic influences since the accumulation of the bivalent MORF in muscle and blood was also higher than that of the monovalent MORF both in normal mice () and in tumored mice (). The differences in size and/or base sequence between the two MORFs may explain the observed differences in pharmacokinetics. Nevertheless, the tumor to normal tissue ratios are moderately in favor of the bivalent MORF as evidence of affinity enhancement pretargeting. The evidence for affinity enhancement is more definitive in the cell binding studies shown in where the radiolabeled bivalent MORF showed enhanced tumor cell binding compared to the monovalent MORF added at the same MORF dosage.
It is important to emphasize that the tumored mouse pretargeting study of this investigation was intentionally designed with a dosage of the radiolabeled bivalent MORF effector much lower than that required to saturate the accessible cMORF in tumor and therefore much lower than optimum.23
In principle, the pretargeting results for the bivalent compared to the monovalent MORFs should have been compared with both at their optimum dosages (i.e., at their maximum percent tumor accumulations).4
The blood accumulations suggest that the lower binding affinity of bivalent effectors to the untethered MN14–cMORF in circulation may not have been observed in this case as was expected.7
The large difference in blood levels between the bivalent and monovalent MORFs in the tumored animals with circulating MN14–cMORF (2.38% vs 0.78%) would be expected to be smaller than the difference in the normal mice without circulating MN14–cMORF (0.25% vs 0.11%). Instead the opposite was observed. Although different pharmacokinetics certainly contributed, the cell binding results suggest that affinity enhancement is a likely explanation for the higher tumor uptake for bivalent MORF in vivo.
As previously reported, there are many factors such as target density that contribute to affinity enhancement.21
While our SPR experiments have provided clear evidence of affinity enhancement, unfortunately, the cMORF target density that is easily defined and controlled on SPR microchips cannot be accurately defined or reproduced in living cells in vitro and even less so in xenografts in which pharmacokinetic factors are involved. Thus while the dosage of MN14–cMORF may be a critical parameter since this can define the concentration of the available cMORF in the tumor, it may be difficult or even impossible to predict the number and spacing of the pretargeting antibody following administration. Accordingly, affinity enhancement may prove to be of more value in connection with amplification pretargeting in which polymeric cMORF as the pretargeted entity is used since, in that case, the number and spacing of the cMORFs on the polymer can be more easily controlled especially in patients.
In conclusion, a bivalent MAG3–MORF was successfully synthesized and radiolabeled with 99mTc. The bivalent MORF behaved similarly in vivo to the monovalent MORF in that both showed the rapid clearance required for pretargeting applications. Although it is not possible to attribute the higher tumor accumulation in mice entirely to affinity enhancement because of differences in the pharmacokinetics between the bivalent and monovalent MORF, both the in vitro and in vivo studies showed evidence of improved binding of the bivalent compared to the monovalent MORF certainly in part due to affinity enhancement. As an obvious advantage of affinity enhancement pretargeting with oligomers, the sequences and length of the MORF of this investigation can be optimized to minimize unfavorable pharmacokinetics and to more definitively favor affinity enhancement pretargeting. Thus bivalent MORFs merit further attention as radiolabeled effectors in MORF pretargeting studies.