The major contributions of this manuscript are the development of a novel targeted toxin, dCD133KDEL, which targets CD133 tumor-initiating cells and its efficacy against two head and neck carcinoma lines. dCD133KDEL is a new and powerful reagent for three main reasons. The drug 1) targets only a small subpopulation of the total tumor, 2) has been successfully deimmunized bypassing a major clinical problem with targeted toxins, and 3) has a mechanism of action unlike typical chemotherapy agents. Targeted toxins function by binding to cell surface receptors, internalizing, and enzymatically inhibiting protein synthesis (30
). CD133 is readily internalized rendering it an excellent marker for a targeted toxin (14
). The fact that we observed heterogenous peaks in CD133 expression in transfected HEK cells in supports the argument that CD133 cycles and internalizes.
Bonnet and Dick reported the first CSC subpopulation in leukemia in 1997 (31
). Since then investigators have validated the presence of CSC subpopulations not only in leukemia, but in many carcinomas as well. CD133 in particular has been identified as a CSC marker in these carcinomas (6
). Wei et al showed in their work that CD133 is a CSC marker for the head and neck laryngeal cancer line HEP-2. In their study, they showed that CD133+ sorted cells initiated tumors and uniquely possessed clonogenic capacity when compared with CD133− cells (16
). We were able to confirm CD133 as a marker for tumor initiation cells in the current study with a different head and neck cell line UMSCC-11B.
Also unique to our study is the use of a new monoclonal antibody (clone 7) that binds all forms of the CD133 receptor (18
). This is important because commercial antibodies currently available recognize an epitope that can be masked upon differentiation (32
). In contrast, the scFV from clone 7 was used in our construction of dCD133KDEL recognizes only the CD133 peptide backbone, avoiding the issue of epitope masking or differential glycosylation. Specificity was determined by showing that our anti-CD133 scFV recognized cells transfected with DNA encoding the CD133 receptor. Furthermore, two head and neck cell lines contained subpopulations of CD133 expressing cells at similar levels to other head and neck cell lines described in the literature (16
). When dCD133KDEL was tested in vitro, we discovered that it selectively inhibited cancer cell expansion in both cell lines. “We found that about 5% of the UMSCC-11B head and neck cancer cells used in the study were Annexin V positive following CD133KDEL treatment indicating apoptotic death. Since flow studies also showed that about 5% of the UMSCC-11B population were CD133+, this suggests an apoptotic death for CD133 cells. We also attempted dual staining to confirm that the same population that was Annexin positive was CD133 positive. However, these mechanistic studies proved complicated since earlier treatment with dCD133KDEL interfered with our ability to later recognize and quantitate CD133+ cells. We attributed this difficulty to either blocking interference or the high modulation rate of CD133.”
Since tumor initiation is a hallmark of CSC, UMSCC-11B tumor cells were pretreated with dCD133KDEL and transplanted into nude mice. These cells formed the lowest incidence of tumors when compared with controls, while cells enriched for CD133+ expression formed the largest tumors at the fastest rate. Furthermore, in two separate studies tumor cells were injected into the flanks of nude mice and when palpable were directly treated with dCD133KDEL. All treated tumors regressed over time, unlike control tumors and all treated animals were impressively tumor free after 79 days. We favor the explanation that dCD133KDEL acts to inhibit the self-renewing ability of CD133+ CSC. However, it is possible that dCD133KDEL has a bystander killing effect but this was not supported by our in vitro studies showing that dCD133KDEL did not kill CD133 negative cancer cells.
Several studies suggest the existence of another CSC population in some tumors that is CD133− (34
). For example, Chen et al discovered CD133− cells within glioblastoma primary tumors that had the capability to self-renew and support long-term growth in vitro and initiate tumors in vivo (35
). dCD133KDEL may be a helpful biological tool in validating the existence of these cells and in helping to develop more inclusive therapies that may target all CSC and progenitors simultaneously.
The development of a novel TT that selectively targets CSC may have unique implications for the ubiquitous problem of carcinoma drug resistance and subsequent relapse. Several studies have shown that CSC are resistant to current chemotherapeutic agents (5
). Since TT's work by a different mechanism and because it selectively kills CSC, dCD133KDEL may possess the unique ability to target the very cells responsible for drug resistance. Adding a TT as an adjunct to chemotherapy has been shown to be more effective than using either therapy alone (38
). Studies are underway to determine whether dCD133KDEL may be a useful adjunct to chemotherapy.
One of the major limitations of TT in clinical therapy is immunogenicity. Patients will start developing antibodies to the toxin and thus the number of treatments is limited. To address this problem, we mutated 7 immunogenic epitopes that account for the majority of antibodies produced against this form of PE toxin. We then used this mutated construct to create our fusion protein. As a result, we showed that even after 9 immunizations there is very little antibody produced against the toxin moiety of dCD133KDEL and 40% of the animals had no antibody response at all.
Drug safety issues regarding the affects of dCD133KDEL on normal human hematopoietic progenitor cells are important and need to be addressed prior to any therapeutic consideration. Therefore, we isolated CD34+ cells from UCB an established source of normal human hematopoietic stem cells, cultured them with dCD133KDEL and then determined their ability to form various hematopoietic colonies in established CFU (colony-forming unit) assays. To be sure, we changed media containing drug weekly and cultured the cells in long-term colony initiation assays for 5 weeks. Only toxin alone inhibited survival of hematopoietic progenitor cells. These results indicate that although the UCB CD34+ cells co-express CD133, these cells are resistant to treatment with dCD133KDEL These findings could be explained by multiple normal stem cell populations that are CD133− since 24% of the starting stem cells were CD34+CD133−. Rutella et al discovered a stem cell population in human cord blood that was CD133− and still capable of differentiating into multiple cell types (40
). Also, Surronen et al showed that CD133+ cells can be generated from normal CD133− cells (41
). Perhaps dCD133KDEL destroyed normal CD133+ cells and replacements emerged from the CD133− fraction. Alternatively, CD34+CD133+ cells may be metabolically quiescent enough or have other drug resistance mechanism to prevent significant killing by dCD133KDEL. Finally, the expression level of CD133 on tumor cells may be greater than the expression level on normal progenitors (42
). This could allow for the observed selective killing of CSC over normal stem cells. Sehl et al, used mathematical modeling to conclude that in order for a treatment to be safe it must be highly selective and able to target quiescent cancer stem cells (43
) and since dCD133KDEL is selective and because cells do not have to be actively cycling for targeted toxins to be taken up and effectively cause apoptosis, dCD133KDEL warrants further investigation.
Several cell populations in the body express CD133 and therefore may be potential targets for the toxin, causing either short-term or long-term health effects. Thus, extensive toxicity studies will be necessary to validate a CD133 targeted drug for clinical use. These studies will not be trivial. CD133 expression on normal cells has proven somewhat complicated since CD133 has been shown to undergo post-translational modulation such that mRNA and internal protein expression does not correlate with the surface expression (44
). Also, the commercial antibodies such as AC133 only bind certain forms of the receptor (18
). Furthermore, studies in an “ontarget” model closer to humans than rodents may be necessary.
In summary, we developed a novel deimmunized TT that selectively binds the cancer stem cell marker CD133. dCD133KDEL impressively inhibited cell proliferation in vitro, decreased tumor initiation, does not kill normal human hematopoietic progenitor cells, and caused complete tumor regression in a an accepted model of human head and neck cancer which is an xenotransplant flank model. This work represents the development of a new cancer therapeutic that function by selectively targeting the minority cancer stem cell subpopulation within the tumor. We believe dCD133KDEL warrants further study as a possible solution for drug resistant relapse in human carcinoma.