Receptor upregulation enhances cell surface receptor targeted therapies.
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Antibodies, Neoplasm↗
Biomedical subjects
Arthur E Frankel
Publications and source records attributed to Arthur E Frankel.
Malignant progenitors from patients with CD87+ acute myelogenous leukemia are sensitive to a diphtheria toxin-urokinase fusion protein.
In previous studies, we demonstrated that the diphtheria toxin-urokinase fusion protein DTAT was selectively toxic to acute myeloid leukemia (AML) cell lines overexpressing the CD87 urokinase receptor. In the present study, we analyzed the sensitivity of patient leukemic progenitors to DTAT and correlated the sensitivity with CD87 expression. We isolated leukemic blasts by density gradient centrifugation and performed immunophenotyping by flow cytometry and blast sensitivity measurements by inhibition of cell proliferation and colony formation in semisolid media. We found CD87 overexpression in 18 (25%) of 71 patient leukemic blast samples, including 18 (28%) of 64 myeloid malignancies and 0 (0%) of 7 lymphoid malignancies. DTAT was toxic to patient leukemic blasts by both proliferation inhibition (IC50 85% inhibition by 10 nM DTAT in 11/41 evaluable samples). Only AML and chronic myeloid leukemia (CML) blast crisis blasts (18/61 [30%]) were sensitive to DTAT by the proliferation inhibition assay. Lymphoid leukemia and chronic phase CML/chronic myelomonocytic leukemia (CMML) progenitors were insensitive to DTAT by the proliferation inhibition assay (n = 7 and n = 3, respectively). Similarly, normal marrow progenitors were insensitive to DTAT by both proliferation inhibition (n = 2) and colony inhibition (n = 5) assays. The DTAT toxicity measured by both proliferation inhibition assay and colony inhibition assay correlated with CD87 density (p < 0.0001 and p = 0.001, respectively). DTAT toxicity results were similar for leukemic blasts measured by either of the two assays (p = 0.0002). This study provides the first evidence that a urokinase receptor targeted diphtheria fusion protein is toxic to patient AML blasts. The work also suggests that blast proliferation assays yield similar responses to leukemia colony-forming cell colony assays.
Acute Disease↗
Effective targeted cytotoxicity of neuroblastoma cells.
BACKGROUND/PURPOSE: Despite aggressive treatment with surgery, chemotherapy, and radiotherapy, the prognosis for many children with neuroblastoma remains poor. Targeted toxins represent novel cancer therapeutics designed to selectively target and kill cancer cells. The authors have developed a novel fusion toxin, DT5F11, consisting of truncated diphtheria toxin (DT(A)) linked to a single chain antibody (sc5F11) targeting the GD(2) antigen found on most neuroblastoma cells. This report describes the construction, expression, and in vitro function of DT5F11. METHODS: Utilizing restriction enzyme digestion, polymerase chain reaction amplification, and gel electrophoresis, the prkDTL5F11 plasmid was created by the fusion of distinct coding sequences for a single-chain GD(2) targeting antibody (sc5F11) and truncated diphtheria toxin (DT(A)). DH5alpha Escherichi coli-competent cells were transformed with prkDTL5F11; DNA was amplified, isolated, and sequenced. The fusion protein was expressed and assayed by Western blot. Targeted cytotoxicity was analyzed on GD(2)-positive (SK-N-AS, IMR-32, SK-N-MC, LAN-1) and GD(2)-negative (HeLa) cells. RESULTS: Fluorescent dye-labeled cycle sequencing identified the constructed fusion toxin gene. Western blot analysis using a mouse antihuman DT(A) antibody showed a 69-kD band identifying the fusion toxin, DT5F11. Targeted cell killing with DT5F11 was seen only in GD(2) positive cells. CONCLUSIONS: This study demonstrates creation of a novel fusion toxin with effective GD(2)-targeted cellular toxicity. Further investigation of this fusion toxin as a therapeutic agent in the management of neuroblastoma is warranted.
Antibodies, Monoclonal↗
The majority of children and adolescents with acute myeloid leukemia have detectable anti-DT388-GMCSF IgG concentrations, but at concentrations that should not preclude in vivo activity.
PURPOSE: As a novel approach for the treatment of acute myeloid leukemia (AML), the authors are developing a fusion toxin (DT(388)-GMCSF) consisting of a truncated diphtheria toxin (DT(388)) linked to human granulocyte-macrophage colony-stimulating factor (GMCSF). A critical step in the development of DT(388)-GMCSF for clinical use in childhood and adolescent AML is to determine whether children and adolescents have preexisting antibodies to DT(388)-GMCSF due to childhood immunizations against diphtheria toxoid. PATIENTS AND METHODS: Sera from 33 children and adolescents with AML and one with juvenile myelomonocytic leukemia were collected. The median age was 11.8 years. All scheduled diphtheria toxoid vaccinations were current except for the child diagnosed at 4 months of age. Anti-DT(388)-GMCSF antibody concentrations were detected by an enzymoimmunoassay and by an in vitro bioassay. RESULTS: Thirty of 34 (88%) children and adolescents had detectable anti-DT(388)-GMCSF IgG antibody concentrations. The median concentration was 1.5 microg/mL, with a range from undetectable to 191.4 microg/mL. There was a positive correlation between the enzymoimmunoassay and bioassay. There was no difference between the anti-DT(388)-GMCSF IgG concentrations in these children and adolescents with AML and in 43 adults with AML. Preliminary results of the phase 1 trial of DT -GMCSF in adults with AML indicate that patients with baseline anti-DT(388)-GMCSF IgG concentrations of less than 2 microg/mL can achieve circulating DT(388)-GMCSF concentrations and can exhibit antileukemic activity. Twenty-three of 34 (67.6%) children and adolescents had anti-DT(388)-GMCSF IgG concentrations less than 2 microg/mL. CONCLUSIONS: Despite routine diphtheria toxoid vaccinations, most children and adolescents with AML do not have anti-DT -GMCSF IgG concentrations that preclude in vivo activity of DT -GMCSF.
Adolescent↗
Peptide toxins directed at the matrix dissolution systems of cancer cells.
Growth and spread of tumors requires a variety of membrane and extracellular proteases to modify membrane integrins, dissolve the surrounding matrix and release critical growth factors from both the tumor cell surface and surrounding structures. The two major protease systems involved in this process are the matrix metalloproteases and the serine proteases. Genes and gene products for both protease systems are overexpressed in a variety of neoplasms. Thus, these enzymes serve as excellent targets for the delivery of potent cytotoxic molecules to tumors. A number of peptide toxins have been engineered to bind to tumor cells with high levels of surface proteases and their receptors including anthrax toxins, Pseudomonas exotoxin, saporin and diphtheria toxin. These recombinant fusion proteins provide a novel class of anti-cancer agents that will enter clinical trials in the next several years.
Clinical Trials as Topic↗
Anthrax fusion protein therapy of cancer.
Most patients with cancer are treated with chemotherapy but die from progressive disease or toxicities of therapy. Current chemotherapy regimens primarily use cytotoxic drugs which damage cell DNA or impair cell proliferation in both malignant and normal tissues. After several treatment courses, the patients' tumor cells often overexpress multi-drug resistance genes which prevent further tumor cytoreduction. Novel agents which can kill such resistant tumor cells are needed. One such class of agents are targeted peptide toxins. Targeted peptide toxins consist of peptide toxins covalently linked to tumor selective peptide ligands. These molecules bind tumor cell surface receptors, internalize, and facilitate transfer of the toxin catalytic domains to the cytosol. Once in the cytosol, the enzyme activity leads to cell death. A number of plant, bacterial and fungal toxins have been used, and clinical trials with several of these have produced complete remissions in chemoresistant neoplasms. Nevertheless, there is a continuing need for novel targeted toxins. Many patients have pre-existing antibodies against the currently clinically used toxins and many toxins are inactive when used for myeloid malignancies where internalized proteins are rapidly routed and degraded in lysosomes. Anthrax toxins are the cytotoxic components of Bacillus anthracis. While the bacteria has been the source of serious illness, deaths and global anxieties related to past or future bioterrorism, the isolated toxins do not pose public health hazards. In fact, toxin treated patients will likely develop protective antibodies. Anthrax toxin is an excellent choice for tumor cell surface targeting. Other than U.S. military personnel immunized during the Gulf War, most people lack pre-existing antibodies. This may change in the future due to threats of additional terrorist acts, but for the present few patients will have antibodies to anthrax proteins. The separate subunits for binding, translocation and cell killing facilitate genetic engineering to yield tumor-specific cell killing. The toxins are more potent than most of the other peptide toxins and may yield highly efficacious targeted molecules. This essay will review anthrax toxin structure-function, preliminary experiments with re-targeted anthrax toxin and potential designs for new ligand-anthrax therapeutics.
Amino Acid Sequence↗
Diphtheria fusion protein therapy of chemoresistant malignancies.
Patients with widespread cancer respond initially to combination chemotherapy, immunotherapy, and/or radiotherapy, but most relapse with chemoresistant disease. Novel methods of killing resistant neoplastic stem cells are needed. One such approach is therapy with targeted toxins composed of tumor cell selective ligands covalently linked to group I peptide toxins (group II and III peptide toxins act on the cell surface). The targeted toxin is delivered to the cell by a tumor selective ligand. Once bound, the ligand-receptor complex is internalized. The catalytic domain escapes to the cytosol. The toxin then enzymatically modifies a critical cell function (protein synthesis, p21 Rho activity, protein kinase signaling, cyclic AMP signaling or others). The irreversibly damaged cells fails to divide and, eventually, undergoes lysis or programmed cell death. Targeted peptide toxins used to date in the treatment of chemotherapy refractory cancers include ricin toxin, Pseudomonas exotoxin, pokeweed antiviral protein, saporin, gelonin and diphtheria toxin. In this review, we have focused on the applications of genetically engineered diphtheria toxin for cancer therapy.
Animals↗
Increased sophistication of immunotoxins.
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Antibodies, Monoclonal↗
Phase I trial of a novel diphtheria toxin/granulocyte macrophage colony-stimulating factor fusion protein (DT388GMCSF) for refractory or relapsed acute myeloid leukemia.
PURPOSE: Patients with relapsed or refractory acute myeloid leukemia have a poor prognosis. We tested the safety and efficacy of a diphtheria fusion protein [diphtheria toxin (DT)388 granulocyte-macrophage colony-stimulating factor (GMCSF)] directed against the GMCSF receptor that is strongly expressed by leukemic blasts. EXPERIMENTAL DESIGN: DT388GMCSF fusion protein containing the catalytic and translocation domains of DT388 fused to human GMCSF was administered in an interpatient dose escalation trial by 15 min i.v. infusion daily for up to 5 days. RESULTS: The maximal tolerated dose was 4 microg/kg/day. The dose-limiting toxicity was liver injury and occurred at the 4.5-5-microg/kg/day dose level. Among nine treated patients at these doses, one patient developed liver failure, and one patient had transient hepatic encephalopathy. There was a positive correlation between peak serum DT388GMCSF levels and serum aspartate aminotransferase (P = 0.0002). DT388GMCSF did not damage hepatic cell lines in vitro; however, DT388GMCSF binds macrophages and induces cytokine release in vitro. Among the treated patients, we observed an early elevation in serum levels of interleukin (IL)-18 and a later rise in IL-8 but no significant changes in IL-1beta, IL-6, IFNgamma, macrophage inflammatory protein-1alpha, tumor necrosis factor alpha or IL-12. The IL-18 elevations occurred before elevations of liver enzymes and correlated with peak aspartate aminotransferase levels (P = 0.005). Of the 31 patients who were resistant to chemotherapy, 1 had a complete remission and 2 had partial remissions; all 3 of these patients were treated at or above the maximal tolerated dose, all 3 responding patients had baseline marrow blast percentage of <30%, whereas only 6 of the nonresponding 28 patients had less than 30% marrow blasts. Five of these six patients were treated with subtherapeutic doses. Eight (42%) of 19 patient courses at <4 microg/kg/day and 8 (40%) of 20 patient courses at 4-5 microg/kg/day showed marrow blast reductions at day 12. Patients with higher pretreatment anti-DT388GMCSF levels had significantly lower peak DT388GMCSF levels (P = 0.0001). CONCLUSIONS: DT388GMCSF can produce complete and partial remissions in patients with chemotherapy-resistant acute myeloid leukemia, but methods to prevent liver injury are needed before more widespread application of this novel agent.
Acute Disease↗
New HER2-directed therapies for breast cancer. Commentary re: C. I. Spiridon et al., Targeting multiple Her-2 epitopes with monoclonal antibodies results in improved antigrowth activity. Clin. Cancer Res., 8: 1720-1730, 2002.
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Animals↗
Diphtheria toxin conjugate therapy of cancer.
The approval by the FDA of ONTAK (DAB389IL2) for CTCL and the encouraging results described above with a number of DT conjugates for different neoplasms suggest that a niche is developing for these biologic agents. Moreover, practicing physicians are becoming more familiar with the side effect profile for these agents, increasing their comfort with using these unique drugs. Finally, the marked synergy observed between standard cytotoxic drugs and other biologics such as Herceptin and Rituximab suggests that similar supra-additive effects may be observed by combining DT conjugates with chemotherapy. In support of this hypothesis, in vitro synergy has been observed between DT388GMCSF and both cytarabine and doxorubicin [84,85]. The future appears to show expanded testing of DT conjugates with likely rapid clinical development, particularly for chemotherapy-resistant hematologic neoplasms and brain tumors.
Animals↗
Diphtheria toxin-epidermal growth factor fusion protein and Pseudomonas exotoxin-interleukin 13 fusion protein exert synergistic toxicity against human glioblastoma multiforme cells.
The cytotoxicity of combinations of a diphtheria toxin-human epidermal growth factor fusion protein (DAB(389)EGF) and a Pseudomonas exotoxin-human interleukin 13 fusion protein (IL13PE38QQR) was tested against 14 human glioma cell lines. After cells were cultured for 48 h with various concentrations of the fusion proteins, the percentage reductions in thymidine incorporation were determined. Seven of fourteen cell lines were highly sensitive to DAB(389)EGF alone, and six cell lines were highly sensitive to IL13PE38QQR alone with IC(90)'s < 100 pM. When combined, synergistic cell killing was observed for seven of the cell lines based upon concave isobolograms and combination indices (CI's) of 0.2 to 0.7. Supraadditive cytotoxicity was confirmed by measurements of induction of apoptosis. Receptor expression was assessed by flow cytometry and confocal microscopy. Marked heterogeneity of expression of EGFR and IL13Ralpha2 was seen on all the glioma cell lines. This heterogeneity may contribute to incomplete cell killing with the individual fusion proteins and synergistic cell kill with the combination. These results suggest that both fusion proteins may yield antitumor effects in patients with recurrent gliomas and that combination fusion protein intracranial therapy of malignant gliomas may yield an improved therapeutic index.
Antineoplastic Agents↗