A novel bispecific single-chain fusion protein, DT2219, was assembled consisting of the catalytic and translocation domains of diphtheria toxin (DT 390 ) fused to two repeating sFv subunits recognizing CD19 and CD22 and expressed in Escherichia coli. Problems with yield, purity, and aggregation in the refolding step were solved by incorporating a segment of human muscle aldolase and by using a sodium N-lauroyl-sarcosine detergent-based refolding procedure. Problems with reduced efficacy were addressed by combining the anti-CD19 and anti-CD22 on the same singlechain molecule. DT2219 had greater anticancer activity than monomeric or bivalent immunotoxins made with anti-CD19 and anti-CD22 sFv alone and it showed a higher level of binding to patient leukemia cells and to CD19 + CD22 + Daudi or Raji cells than did anti-CD19 and anti-CD22 parental monoclonal antibodies. The resulting DT2219, mutated to enhance its avidity, was cytotoxic to Daudi cells in vitro (IC 50 = 0.3 nmol/L). In vivo, DT2219 was effective in a flank tumor therapy model in which it significantly inhibited tumor growth (P < 0.05) and in a systemic model in which it significantly prolonged survival of severe combined immunodeficient mice with established Daudi (P < 0.008) compared with controls. DT2219 has broader reactivity in recognizing B-cell malignancies, has more killing power, and requires less toxin than using individual immunotoxin, which warrants further investigation as a new drug for treating B leukemia/lymphoma.
IL-1 is a key inflammatory and immune mediator in many diseases, including dry-eye disease, and its inhibition is clinically efficacious in rheumatoid arthritis and cryopyrin-associated periodic syndromes. To treat ocular surface disease with a topical biotherapeutic, the uniqueness of the site necessitates consideration of the agent's size, target location, binding kinetics, and thermal stability. Here we chimerized two IL-1 receptor ligands, IL-1β and IL-1Ra, to create an optimized receptor antagonist, EBI-005, for topical ocular administration. EBI-005 binds its target, IL-1R1, 85-fold more tightly than IL-1Ra, and this increase translates to an ∼100-fold increase in potency in vivo. EBI-005 preserves the affinity bias of IL-1Ra for IL-1R1 over the decoy receptor (IL-1R2), and, surprisingly, is also more thermally stable than either parental molecule. This rationally designed antagonist represents a unique approach to therapeutic design that can potentially be exploited for other β-trefoil family proteins in the IL-1 and FGF families.T he IL-1 cytokines (IL-1α and IL-1β) are master mediators of inflammatory responses (1). IL-1β also regulates immune function through its role in T helper 17 (Th17) cell differentiation and maintenance (2, 3). IL-1 action has been implicated in numerous human diseases, including rheumatoid arthritis, MuckleWells syndrome, gout, type 2 diabetes, and stroke (4). Several natural mechanisms directly oppose the actions of IL-1, including a soluble and cell surface decoy receptor (IL-1R2), a natural antagonist (IL-1Ra), and a soluble signaling receptor (IL-1R1) (5). Therapeutics that block IL-1 based on these mechanisms have been developed (6-8).Recently, a nonoptimized formulation of anakinra (methionyl-IL-1Ra; Kineret) was shown to provide clinical benefit in dry-eye disease (DED) (9). Moderate to severe DED is a chronic inflammatory condition of the corneal surface that results in pain, discomfort, and epitheliopathy (as measured by fluorescein staining). Inability to maintain a proper tear film over the cornea (owing to a variety of etiologies) results in desiccating stress, which drives an inflammatory cascade (10, 11). IL-1 plays a central role in the initiation and maintenance of this cascade, as well as in the pain mediated by the corneal neural plexus. IL-1α and IL-1β protein are elevated in the lacrimal gland, tears, and the ocular surface in all forms of dry-eye disease (12), and their mRNA is increased in both humans and in rodent disease models (13,14). Genetic ablation of IL-1R1, the primary receptor for IL-1α and IL-1β, can block the development of corneal staining in a Sjögren syndrome corneal epitheliopathy model (15), and topically administered anakinra can improve surface epithliopathy in a mouse dry-eye model (14). IL-1β is essential for Th17 cell differentiation and maintenance, and Th17 cells are likely the main effector cells that induce epithelial damage (2, 3). Genetic and pharmacologic studies have shown that IL-1β mediates, and IL-1Ra blocks, normal, inflamm...
A bispecific ligand-directed toxin (BLT), called EGFATFKDEL, consisting of human epidermal growth factor, a fragment of urokinase, and truncated pseudomonas exotoxin (PE38) was assembled in order to target human glioblastoma. Immunogenicity was reduced by mutating seven immunodominant B-cell epitopes on the PE38 molecule to create a new agent, EGFATFKDEL 7mut. In vitro, the drug selectively killed several human glioblastoma cell lines. EGFATFKDEL is our first BLT designed to simultaneously target EGFR on solid tumors and uPAR on the tumor neovasculature. In vitro assays revealed that the agent is effective against glioblastoma cell lines as well as human umbilical vein endothelial cells (HUVEC). Additionally, the bispecific drug displayed enhanced binding to overexpressed epidermal growth factor receptor and urokinase receptor when compared to similar monospecific drugs, EGFKDEL and ATFKDEL. In vivo, an aggressive human glioblastoma cell line was genetically marked with a firefly luciferase reporter Correspondence to: Daniel A. Vallera, valle001@umn.edu. NIH Public AccessAuthor Manuscript J Neurooncol. Author manuscript; available in PMC 2012 June 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript gene and administered to the flanks of nude mice. Treatment with intratumoral injections of EGFATFKDEL 7mut eradicated small tumors in over half of the treated mice, which survived with tumor free status at least 100 days post tumor inoculation. ATFKDEL, which primarily targets the tumor neovasculature, prevented tumor growth but did not result in tumor-free mice in most cases. Specificity was shown by treating with an irrelevant BLT control which did not protect mice. Finally, immunization experiments in immunocompetent mice revealed significantly reduced anti-toxin production in EGFATFKDEL 7mut treated groups. Thus, EGFATFKDEL 7mut is an effective drug for glioblastoma therapy in this murine model and warrants further study.
A bispecific immunotoxin (IT) called DTAT13 was synthesized in order to target simultaneously the urokinase-type plasminogen activator receptor (uPAR)-expressing tumor neovasculature and IL-13 receptor expressing glioblastoma cells with the goal of intratumoral administration for brain tumors. The recombinant hybrid was created using the non-internalizing N-terminal fragment (ATF) of uPA and the IL-13 molecule for binding plus the catalytic and translocation portion of diphtheria toxin (DT) for killing. The 71 kDa protein was highly selective for human glioblastoma in vitro showing no loss on binding compared with DTAT and DTIL13 controls. In vivo, DTAT13 caused the regression of small tumors when administered at 10 micro g/day given on a five-dose schedule every other day. DTAT13 was able to target both overexpressed uPAR and the vasculature, as demonstrated by its ability to kill HUVEC cells. Also, mortality studies indicated that DTAT13 was less toxic than DTAT or DTIL13. These findings indicate that bispecific IT may allow treatment of a broader subset of antigenically diverse patients while simultaneously reducing the exposure to toxin required than if two separate agents were employed.
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.
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