When advancing a new therapeutic or imaging agent toward human clinical trials, teams must consider not only efficacy, pharmacokinetics, pharmacodynamics and toxicity, but also practical issues that could compromise regulatory approval or increase manufacturing costs.20, 21
In the case of Tumor Paint, the original bioconjugate posed a manufacturing challenge, as it was a mixture of mono-, di- and tri-labeled CTX. In this manuscript, we report three new chemical entities that are functionally equivalent to CTX for targeting NIRF molecules to cancer, yet conjugate to only a single NIRF molecule.
We recently mapped CTX conjugation sites in CTX:Cy5.5 using arginase cleavage coupled with proteomic analyses (M. Veiseh, B. Bahrami, J. Olson, unpublished data). This analysis showed that typically >80% of product was mono-labeled at Lys27 and smaller amounts were also conjugated at Lys 15 or Lys 23. In an analysis of CTX conjugated to four other NIRF dyes, similar patterns of predominantly mono-labeled peptide with smaller amounts of di- and tri-labeled peptide were observed, with the exception of Dylight 750, a NIRF dye that creates only mono-labeled species when bound to CTX (S. Hansen, M. Stroud, J. Olson, unpublished data). The fact that Dylight 750 binds in a monomeric fashion to unmodified CTX suggests that access to the other two lysines is limited in a way that is dependent on the nature of the dye. Here we show that cyclization is also able to moderate the reactivity because cyclized CTX binds only one Cy5.5 molecule per peptide despite the fact that all three lysine residues remain intact in the cyclized version.
Other examples where macrocyclic disulfide-rich peptides have been labeled include the studies of Greenwood et al. on biotin labeled cyclotide MCoTI-II and that of Herrmann et al. on the chemical modification of Lys/Arg residues in cycloviolacin O2 to probe structure-activity relationships.22, 23
Both of these peptides have multiple labeling sites which are accessible even in the cyclic form, in contrast to the current study. Combined with the results reported here for cyclic chlorotoxin, these studies highlight the practicality of labeling cyclic peptides despite the lack of an N-terminus.
None of the lysine residues in CTX seem to be involved in the active binding of CTX to its target on cancer cells. This conclusion is based on the observations that target binding is preserved despite substitution of Lys15 or Lys23 with Ala or Arg and that addition of bulky Cy5.5 or other NIRF dyes to Lys27 does not preclude binding to the active site. The target of CTX was earlier reported to be matrix metalloproteinase-2 (MMP2).24
Recently, it was reported that a protein called annexin A2, which is expressed on the cell surface of human tumor cells,25
is a novel target of TM601, a synthetic version of CTX.26
Moving toward human clinical trials, we now have three new chemical entities that could be used for targeting purposes. Since Tumor Paint is intended to be administered through a single intravenous injection, it is not necessary to develop an orally available version of the active molecule and hence this potential advantage of the cyclization is less important in this imaging application than it is in drug development. Also, cyclization did not improve the serum half-life of the peptide. The serum stability was improved by cyclization from 70% intact peptide to 90% intact peptide following 24 h of incubation in human plasma at 37°C, suggesting proteolytic cleavage was reduced following cyclization but in vivo
excretion and metabolism was not improved. Thus, in the presence of Cy5.5 conjugation, which is known to markedly increase the serum half-life of CTX, the additional benefit gained by cyclization does not outweigh the potential added manufacture costs associated with using cyclic versus linear peptides in the clinical setting. Nevertheless, the benefits of cyclization might be useful in future analogs of chlorotoxin or other imaging agents where stability and bioavailability considerations are more important. Both chemical27
approaches have been developed in recent years for the synthesis of macrocyclic disulfide-rich peptides.
Of the linear peptides we synthesized, the signal in tumors from bioconjugates made with linear Ala substitutions was slightly higher than that of Arg substitutions. Both molecules will be studied further and one will be selected to move toward human clinical trials. Thus, the work presented in this manuscript successfully identified two mono-labeled peptide conjugates that retain the targeting activity of native CTX, while circumventing the regulatory and manufacturing problems of multiply conjugated CTX. These data provide a clear path forward for translating Tumor Paint from mouse trials to human clinical practice.