Purpose: To develop a targeted biological drug that when systemically injected can penetrate to metastatic breast cancer tumors, one needs a drug of high potency and reduced immunogenicity. Thus, we bioengineered a novel bispecific ligand-directed toxin (BLT) targeted by dual high-affinity cytokines with a PE 38 KDEL COOH terminus. Our purpose was to reduce toxin immunogenicity using mutagenesis, measure the ability of mutated drug to elicit B-cell antitoxin antibody responses, and show that mutated drug was effective against systemic breast cancer in vivo. Experimental Design: A new BLT was created in which both human epidermal growth factor (EGF) and interleukin 4 cytokines were cloned onto the same single-chain molecule with truncated Pseudomonas exotoxin (PE 38 ). Site-specific mutagenesis was used to mutate amino acids in seven key epitopic toxin regions that dictate B-cell generation of neutralizing antitoxin antibodies. Bioassays were used to determine whether mutation reduced potency, and ELISA studies were done to determine whether antitoxin antibodies were reduced. Finally, a genetically altered luciferase xenograft model was used; this model could be imaged in real time to determine the effect on the systemic malignant human breast cancer MDA-MB-231. Results: EGF4KDEL 7mut was significantly effective against established systemic human breast cancer and prevented metastatic spread. Mutagenesis reduced immunogenicity by ∼90% with no apparent loss in in vitro or in vivo activity. Conclusions: Because EGF4KDEL 7mut was highly effective even when we waited 26 days to begin therapy and because immunogenicity was significantly reduced, we can now give multiple drug treatments for chemotherapy-refractory breast cancer in clinical trials. (Clin Cancer Res 2009;15(19):6137-47)
Purpose: Overexpressed cytokine receptors are considered valid targets for new biologicals targeting prostate cancer. However, current reagents are limited in efficacy. Our goal was to determine the advantages of simultaneously targeting two established targets, epidermal growth factor receptor and interleukin-13 (IL-13) receptor, with a new bispecific cytotoxin in which both EGF and IL-13 cytokines were cloned onto the same single-chain molecule with truncated diphtheria toxin (DT 390 ). Experimental Design: In vitro experiments measured the potency of bispecific DTEGF13 and compared its activity to its monospecific counterparts, DTEGF and DTIL13. We determined whether the presence of both cytokine ligands on the same molecule was responsible for its superior activity. In vivo, DTEGF13 was given i.t. to athymic nude mice with established PC-3 human prostate cancer tumor xenografts on their flanks. Results: In vitro, DTEGF13 was more potent than the monospecific cytotoxins against human prostate cancer lines. Enhanced activity was related to the presence of both cytokines on the same single-chain molecule and was not attributed to enhanced binding capacity. Killing was receptor specific. Cytotoxicity could be blocked with anti-EGF and anti^IL-13 antibodies. In vivo, DTEGF13, but not monospecific DTEGF or DTIL13, significantly inhibited the growth of established PC-3 tumors in nude mice (P < 0.0001).Conclusions: These data show for the first time that simultaneous targeting of cytokine receptors with two ligands on the same molecule has pronounced anticancer advantages. In an animal model in which human DTEGF13 is cross-reactive with mouse, DTEGF13 was highly effective in checking aggressive prostate tumor progression and was reasonably tolerated.
Purpose: erbB2, the product of the Her2-neu gene, is a well-established therapeutic target for antibody-based biologicals, but anti-erbB2 antibody-toxin fusion proteins are limited in their activity. The goal of this study was to determine if genetically adding an sFv targeting epithelial cell adhesion molecule (EpCAM) to an anti-Her2 sFv immunotoxin would result in enhanced antitumor activity. Experimental Design: In vitro studies were done in which the new bispecific immunotoxin DTEpCAM23 was compared with monospecific immunotoxins (DTEpCAM and DT23) to quantitate immunotoxin activity. Mixtures of monospecific immunotoxins were tested to determine if they were as effective as the bispecific immunotoxin. Binding and internalization studies were also done. In vivo, bispecific immunotoxins were given i.t. to athymic nude mice bearing HT-29 human colon cancer flank tumors and i.p. to mice with i.p. tumors. Results: DTEpCAM23 bispecific immunotoxins showed far greater activity than monospecific immunotoxin (sometimes over 2,000-fold) against most tumor lines. Bispecific immunotoxin was superior and selective in its activity against different carcinoma cell lines. Bispecific immunotoxin had greater activity than monospecific immunotoxin indicating an advantage of having both sFv on the same single-chain molecule. Binding and internalization studies did not explain the differences between bispecific immunotoxin and monospecific immunotoxin activity. Orientation of the sFvs on the molecule had a significant effect on in vitro and in vivo properties. The bispecific immunotoxins were more effective than the monospecific immunotoxin in the flank tumor mouse model. Conclusions: The synthesis of bispecific immunotoxin created a new biological agent with superior in vitro and in vivo activity (over monospecific immunotoxin), more broad reactivity, more efficacy against tumors in vivo, and diminished toxic effects in mice.
BACKGROUND: Potency, immunogenicity, and toxicity are three problems that limit the use of targeted toxins in solid tumour therapy. METHODS: To address potency, we used genetic engineering to develop a novel bispecific ligand-directed toxin (BLT) called EGF4KDEL, a novel recombinant anti-mesothelioma agent created by linking human epidermal growth factor (EGF) and interleukin-4 (IL-4) to truncated pseudomonas exotoxin (PE38) on the same single-chain molecule. Immunogenicity was reduced by mutating seven immunodominant B-cell epitopes on the PE38 molecule to create a new agent, EGF4KDEL 7Mut. RESULTS: In vitro, bispecific EGF4KDEL showed superior anti-mesothelioma activity compared with its monospecific counterparts. Toxicity in mice was diminished by having both ligands on the same molecule, allowing administration of a 10-fold greater dose of BLT than a mixture of monomeric IL4KDEL and EGFKDEL. EGF4KDEL 7Mut, retained all of its functional activity and induced about 87% fewer anti-toxin antibodies than mice given the parental, non-mutated form. In vivo, intraperitoneal (IP) injection of the BLT showed significant (Po0.01) and impressive effects against two aggressive, malignant IP mesothelioma models when treatment was begun 14-16 days post tumour innoculation. CONCLUSION: These data show that EGF4KDEL 7Mut is a promising new anti-mesothelioma agent that was developed to specifically address the obstacles facing clinical utility of targeted toxins.
To improve activity of a recombinant IL-13 cytotoxin (CT) comprised of IL-13 spliced to truncated diphtheria toxin (DT(390)), epidermal growth factor (EGF) was added to the same single chain protein. This new recombinant bispecific CT, called DTEGF13, enhanced the killing potency against the human glioblastoma lines, U87MG (0.015 nM) and U118MG (0.02 nM). A similar enhancement was observed against the lung carcinoma cell line, Calu-3 (0.0018 nM). Enhanced activity could not be explained by an increased number of cytokines available for binding since a combination of monospecific DTEGF and DTIL13 did not cause the same enhanced activity. Enhanced activity was dependent on the presence of both cytokines on the same single chain molecule and killing was receptor specific since target receptor negative leukemia cells were unaffected by the highly selective DTEGF13 and cytotoxicity could be blocked with anti-EGFR and anti-IL-13 antibodies. In a xenograft flank tumor model, intratumoral injection of DTEGF13, but not monospecific DTEGF or DTIL13, significantly inhibited the growth of established U87 tumors in nude mice (P < 0.04). In this model, the human EGF and IL-13 components of DTEGF13 are reactive with mouse EGFR and IL-13R, respectively. These studies show that a new co-targeting agent that simultaneously recognizes EGFR and IL-13R is more effective than its monospecific counterparts and that DTEGF13 has therapeutic advantages for glioblastoma.
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