The original contribution of this research is the development of EGFATFKDEL 7mut, a promising new anti-glioblastoma agent with potential for clinical development. Our laboratory has designed and published BLTs which simultaneously bind to dual independent receptors on the surface of tumor cells [6
]. However, EGFATFKDEL is the first targeted toxin that simultaneously attacks tumor cells and the blood supply that feeds them. Angiogenesis, or generation of neovasculature, is a complex process which involves several elements including endothelial cells, stromal cells, and factors from the extracellular matrix. There has been tremendous interest in drugs which target the neovasculature [36
]. For example, bevacizumab and other anti-angiogenic drugs currently in clinical trials have demonstrated that the vasculature can be successfully targeted with antibodies and other neovasculature targeting strategies [36
]. Although angiogenesis inhibitors possess impressive potential, their success has been limited mostly by the fact that tumor regressions are often only partial [36
]. Since EGFATFKDEL directly attacks the most prominent cells in the tumor (vascular cells and tumor cells), we believe that effects will be magnified because the toxic effect of PE is catalytic and irreversible. Our in vitro data with HUVECs shows that our drug can potentially impact tumor neovasculature. While we acknowledge that HUVECs are not an optimal model of tumor neovasculature, our studies show that the ATF portion of our drug effectively targeted uPAR on endothelial cells. Our in vivo data supports this because we have demonstrated that EGFATFKDEL 7mut is highly effective in a mouse model in which flank tumors were induced by the injection of the human glioblastoma line U87. Tumor-free survivors were observed beyond day 100, despite the fact that treatment ended on day 40, indicating that drug responses were durable.
Other properties that set EGFATFKDEL 7mut apart from other biological targeted toxins are its toxin modifications. First, the KDEL sequence was added to the c-terminus to enhance potency. The KDEL modification does not necessarily result in an increased therapeutic window and it is possible that the advantage of increased potency may be negated by enhanced toxicity. To address this issue KDEL-modified EGFATF 7mut could be compared to non-KDEL modified EGFATF 7mut in future studies.
Another, important toxin modification was the “deimmunization” of EGFATFKDEL 7mut. Kreitman et al. showed that the clinical efficacy of treatment with targeted toxins against solid tumors hinges on the ability to give multiple treatments or sustained treatment which enables the drug to penetrate a solid tumor [37
]. Toxins may be administered locally to treat tumors in sensitive organs, but targeted toxins must still be used repeatedly or via sustained delivery to achieve positive results. The major problem with this is that neutralizing antibodies will be generated that significantly reduce efficacy over time. To address this issue, investigators used an expansive library of anti-PE monoclonal antibodies to epitope map prominent molecular regions which elicited the strongest antibody response. Fortunately, the immunogenic regions of PE were mapped in seven distinct epitotic areas, and not distributed throughout the molecule. We constructed our PE-based BLT and mutated key amino acids in each of the 7 regions without compromising toxin activity. The immune response to the resultant second-generation drug was reduced by 80–90% in a validated mouse model [34
Our immunization experiments are designed to evoke gradual immune responses by administering low concentrations of drug on a weekly basis. Initial responses are mild, but they grow exponentially, as seen in . Responses likely could be expedited by using a more aggressive immunization regimen employing greater dosages and more immunizations. However, from other studies we know that neutralizing antibodies are present when antitoxin levels reach about 500 μg/ml. Consequently, analysis of early and low antibody production is important. In our experiments, anti-toxin levels in some mice treated with the parental drug exceeded this threshold after only four injections (day 26). After eight injections, none of the mice treated with the mutant drug had reached 20% of this threshold. However, it is possible that extending the experiment beyond day 62 could reveal that the response to the mutant is delayed and not eliminated.
Another concern of our mouse immunogenicity studies is that major histocompatibility complex (MHC) haplotypes differ in their presentation of peptide fragments in the MHC groove and a different haplotype might present different peptides. In other words, basic immunology dictates that a danger of mutating B cell recognizing regions of the toxin is that peptides regarded as immunogenic by one MHC polymorphism, may not be regarded as immunogenic by a different polymorphism. Thus, we used two mice strains, BALB/c and C57BL/6, with entirely different MHC haplotypes. We observed significant anti-toxin reductions in both strains, indicating that the strength of mutating all 7 regions was enough to overcome differences in MHC polymorphisms. However, if the remaining anti-toxin response cannot be eliminated by future mutation of other B cell recognizing amino acids, we may need to pursue mutation of the T cell recognizing toxin regions.
A final concern of the immunogenicity studies is whether mice are useful for studying human anti-toxin responses. Kreitman and Pastan have treated over 300 patients with non-mutated PE targeted toxins [33
]. Nagata studied the antibodies in patients with high titers of anti-PE antibodies and found they bind to the same seven epitopes which regulate toxin B cell recognition in mice indicating that mice are a useful model for human anti-PE responses [33
Of course, there are other potential issues when applying findings from mouse studies to humans. In humans, we do not know whether treatments will be separated by days or weeks or how the disruption of the blood-brain barrier (BBB) by tumor and surgery may expedite immunologic recognition. We do know that antitoxin antibodies developed in a surprisingly high proportion of 73% of CED patients in phase 1 clinical studies where patients received intracranial IL-13 spliced to the same pseudomonas exotoxin used in our studies [26
]. Also, now that we know PE can be deimmunized, some will argue that any additional clinical trials should be performed with deimmunized PE. (The same way that most clinical trials are no longer performed without humanizing antibodies.) Knowing that we can now reduce the anti-toxin response at least 80%, we believe that deimmunization will be necessary in future trials.
In this paper, we showed that in a mouse flank tumor model, EGFATFKDEL 7mut was impressively effective against GBM tumors. Although flank tumor models are useful for determining drug efficacy against vascularized tumors, they are not ideal. For our study, drug was injected directly into small, palpable, and established tumors because targeted toxins have been vigorously pursued for IC therapy in which they are delivered directly into the tumor. A more sophisticated model would use controlled IC delivery via CED in which drug is pumped through a catheter directly into the brain tumor [2
]. This is an established model in our laboratory and these studies are underway [38
]. Another delivery option that will require further exploration is systemic delivery. Due to its vascular reactivity, the drug may be highly effective systemically because of its ability to disrupt the BBB. Additionally, treatment in conjunction with hyperosmolar mannitol may enhance the drug’s BBB disruptive capabilities.
We have noticed that toxin mutation (deimmunization) increases the activity of EGFATFKDEL 7mut, but not other BLT [6
]. Onda et al. noted that certain toxin mutations enhanced the activity of one targeted toxin as well [34
]. In these instances, different toxin mutation combinations were compared, but only one ligand was explored. Several explanations are possible, but we favor binding change as a possible reason. Binding has a major impact on the activity of targeted toxins [39
]. Any mutation can affect positions of alpha helices, beta strands, and turns. This consequentially impacts configuration and these shifts can improve binding. Sequence variances can also affect refolding quality which can also affect binding. This could explain why mutation of EGFATFKDEL enhances activity, but mutation of EGF4KDEL (a PE38KDEL, EGF and interleukin-4 containing targeted toxin) does not [35
Regarding binding, our flow cytometry data indicated that the bispecific drug was superior to its monospecific counterparts because it bound better. These data correlated with our in vivo studies which showed that the monospecific forms were not as effective at dosages comparable to the bispecific drug. ATFKDEL did not induce complete remissions in 4 of 5 or 80% of the mice, and EGFKDEL killed all of the mice. We have observed that monospecific EGFKDEL is at least a log more toxic than bispecific EGFATFKDEL and we are currently determining whether this is related to the smaller size of EGFKDEL and consequent kidney filtration.
In summary, we have shown that EGFATFKDEL 7mut is effective in inducing complete remissions against GBM tumors. In vitro, the drug is effective in the picomolar range against tumor cells and against HUVEC cells which proves that it binds vascular cells. Attempts at deimmunization of the drug have been successful with a reduction of 80–90% anti-toxin antibodies. Based on its reduced immunogenicity and in vivo efficacy, the drug warrants further consideration for clinical development.