Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 9 de 9
Filter
Add more filters










Database
Language
Publication year range
1.
Proc Natl Acad Sci U S A ; 110(10): 3913-8, 2013 Mar 05.
Article in English | MEDLINE | ID: mdl-23431173

ABSTRACT

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-1ß 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 ß-trefoil family proteins in the IL-1 and FGF families.


Subject(s)
Cytokines/antagonists & inhibitors , Drug Design , Administration, Topical , Amino Acid Sequence , Animals , Crystallography, X-Ray , Cytokines/chemistry , Drug Stability , Female , Humans , Interleukin 1 Receptor Antagonist Protein/antagonists & inhibitors , Interleukin 1 Receptor Antagonist Protein/chemistry , Interleukin 1 Receptor Antagonist Protein/genetics , Interleukin-1beta/antagonists & inhibitors , Interleukin-1beta/chemistry , Interleukin-1beta/genetics , Kinetics , Ligands , Mice , Mice, Inbred C57BL , Models, Molecular , Molecular Sequence Data , Ophthalmic Solutions , Protein Conformation , Receptors, Interleukin-1 Type I/antagonists & inhibitors , Receptors, Interleukin-1 Type I/chemistry , Receptors, Interleukin-1 Type I/genetics , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Sequence Homology, Amino Acid , Static Electricity
2.
J Neurooncol ; 103(2): 255-66, 2011 Jun.
Article in English | MEDLINE | ID: mdl-20830604

ABSTRACT

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 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.


Subject(s)
ADP Ribose Transferases/pharmacology , Antineoplastic Agents/pharmacology , Bacterial Toxins/pharmacology , Brain Neoplasms/drug therapy , ErbB Receptors , Exotoxins/pharmacology , Glioblastoma/drug therapy , Urokinase-Type Plasminogen Activator , Virulence Factors/pharmacology , Animals , Brain Neoplasms/metabolism , Endothelial Cells/drug effects , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , Flow Cytometry , Glioblastoma/metabolism , Humans , Ligands , Mice , Mice, Nude , Peptide Fragments/chemical synthesis , Recombinant Fusion Proteins/chemical synthesis , Recombinant Fusion Proteins/pharmacology , Xenograft Model Antitumor Assays , Pseudomonas aeruginosa Exotoxin A
3.
Mol Cancer Ther ; 9(6): 1872-83, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20530709

ABSTRACT

A drug of high potency and reduced immunogenicity is needed to develop a targeted biological drug that when injected systemically can penetrate to malignant B cells. Therefore, a novel deimmunized bispecific ligand-directed toxin targeted by dual high-affinity single-chain Fvs (scFv) spliced to PE38 with a KDEL COOH-terminus was genetically engineered. The aims were to reduce toxin immunogenicity using mutagenesis, measure the ability of mutated drug to elicit antitoxin antibody responses, and show that mutated drug was effective against systemic B-cell lymphoma in vivo. Both human anti-CD22 scFv and anti-CD19 scFv were cloned onto the same single-chain molecule with truncated pseudomonas exotoxin (PE38) to create the drug. 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 ELISAs were done to determine whether antitoxin antibodies were reduced. Finally, a powerful genetically altered luciferase xenograft model was used that could be imaged in real time to determine the effect on systemic malignant human B-cell lymphoma, Raji-luc. Patient B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B lymphoma were high in CD22 and CD19 expression. 2219KDEL7mut was significantly effective against systemic Raji-luc in mice and prevented metastatic spread. Mutagenesis reduced neutralizing antitoxin antibodies by approximately 80% with no apparent loss in in vitro or in vivo activity. Because 2219KDEL7mut immunogenicity was significantly reduced and the drug was highly effective in vivo, we can now give multiple drug treatments with targeted toxins in future clinical trials.


Subject(s)
ADP Ribose Transferases/immunology , Antibodies, Bispecific/immunology , Antigens, CD19/metabolism , B-Lymphocytes/pathology , Bacterial Toxins/immunology , Bioengineering/methods , Exotoxins/immunology , Neoplasm Metastasis/pathology , Sialic Acid Binding Ig-like Lectin 2/metabolism , Virulence Factors/immunology , ADP Ribose Transferases/pharmacology , Animals , Antibodies, Bispecific/pharmacology , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , Bacterial Toxins/pharmacology , Cell Death/drug effects , Cell Line, Tumor , Disease Models, Animal , Exotoxins/pharmacology , Humans , Mice , Mice, SCID , Models, Molecular , Mutation/genetics , Virulence Factors/pharmacology , Pseudomonas aeruginosa Exotoxin A
4.
Clin Cancer Res ; 13(21): 6486-93, 2007 Nov 01.
Article in English | MEDLINE | ID: mdl-17975161

ABSTRACT

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.


Subject(s)
Cytotoxins/chemistry , ErbB Receptors/metabolism , Interleukin-13/metabolism , Prostatic Neoplasms/drug therapy , Recombinant Fusion Proteins/pharmacology , Animals , Antineoplastic Agents/pharmacology , Biological Assay , Cell Line, Tumor , Disease Progression , Dose-Response Relationship, Drug , Humans , Inhibitory Concentration 50 , Male , Mice , Mice, Nude , Neoplasm Transplantation , Prostatic Neoplasms/pathology , Recombinant Fusion Proteins/chemistry , Treatment Outcome
5.
Clin Cancer Res ; 13(10): 3058-67, 2007 May 15.
Article in English | MEDLINE | ID: mdl-17505009

ABSTRACT

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.


Subject(s)
Carcinoma/drug therapy , Cell Adhesion Molecules/antagonists & inhibitors , Cell Adhesion Molecules/therapeutic use , Colonic Neoplasms/drug therapy , Immunotoxins/therapeutic use , Receptor, ErbB-2/antagonists & inhibitors , Recombinant Fusion Proteins/therapeutic use , Animals , Antigens, Neoplasm/genetics , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Epithelial Cell Adhesion Molecule , Humans , Immunotoxins/pharmacology , Mice , Mice, Nude , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/pharmacology
6.
Clin Cancer Res ; 11(10): 3879-88, 2005 May 15.
Article in English | MEDLINE | ID: mdl-15897589

ABSTRACT

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 single-chain 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.


Subject(s)
Antigens, CD19/immunology , Antigens, CD/immunology , Antigens, Differentiation, B-Lymphocyte/immunology , Cell Adhesion Molecules/immunology , Immunotoxins/immunology , Immunotoxins/pharmacology , Lectins/immunology , Animals , Antibodies , Cell Death , Diphtheria Toxin/immunology , Escherichia coli/genetics , Escherichia coli/immunology , Female , Leukemia, B-Cell , Lymphoma, B-Cell , Mice , Mice, Nude , Mice, SCID , Molecular Conformation , Sialic Acid Binding Ig-like Lectin 2 , Survival Analysis , Transplantation, Heterologous , Tumor Cells, Cultured
7.
Leuk Res ; 29(3): 331-41, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15661270

ABSTRACT

A novel bivalent single chain fusion protein, Bic3, was assembled consisting of the catalytic and translocation domains of diphtheria toxin (DT(390)) fused to two repeating sFv molecules recognizing human CD3 epsilon of the human T-cell receptor. Historically, problems with these constructs include low yield, toxicity, and reduced efficacy. Instead of using conventional Gly(4)Ser linkers to connect heavy/light chains, aggregation reducing linkers (ARL) were used which when combined with a new SLS-based refolding method reduced aggregation and enhanced the yield of final product. Toxicity was reduced at least 25-fold by repeating the two sFv molecules and adding a portion of the hinge-CH2-CH3 human constant regions. The resulting Bic3 was just as cytotoxic to HPB-MLT.UM T leukemia cells in vitro (IC(50)=4 pmol) as a monovalent construct made with the same DT and sFv. In vivo, Bic3 was effective in a new and aggressive therapy model in which it significantly prolonged survival of scid mice with established human T-cell leukemia (p<0.0001 compared to controls). Importantly, no toxicity measured by weight loss, enzyme function, or histology was observed at the highest dose of Bic3 tested (2000 ug/kg). Bic3 warrants investigation as a new drug for treating T-cell malignancy and other T-cell related disorders.


Subject(s)
Immunotoxins/adverse effects , Immunotoxins/chemistry , Leukemia, T-Cell/drug therapy , Recombinant Fusion Proteins/adverse effects , Recombinant Fusion Proteins/chemical synthesis , Animals , Antibodies, Monoclonal , CD3 Complex/immunology , Disease Models, Animal , Humans , Immunoglobulin G , Kidney/pathology , Mice , Mice, SCID , Receptors, Antigen, T-Cell , Recombinant Fusion Proteins/chemistry , T-Lymphocytes/drug effects
8.
Protein Eng Des Sel ; 17(2): 157-64, 2004 Feb.
Article in English | MEDLINE | ID: mdl-15047912

ABSTRACT

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.


Subject(s)
Immunotoxins/immunology , Immunotoxins/pharmacology , Receptors, Cell Surface/immunology , Receptors, Interleukin/metabolism , Xenograft Model Antitumor Assays , Animals , Antibodies, Bispecific , Antineoplastic Agents/adverse effects , Antineoplastic Agents/pharmacology , Binding Sites , Cell Division/drug effects , Cells, Cultured , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Female , Glioblastoma/drug therapy , Humans , Immunotoxins/genetics , Interleukin-13/genetics , Interleukin-13/metabolism , Interleukin-13 Receptor alpha1 Subunit , Kidney/drug effects , Mice , Mice, Nude , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Peptide Fragments , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Receptors, Interleukin-13 , Receptors, Urokinase Plasminogen Activator , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/pharmacology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Toxicity Tests
9.
Hum Gene Ther ; 14(18): 1787-98, 2003 Dec 10.
Article in English | MEDLINE | ID: mdl-14670129

ABSTRACT

In past studies, we showed that T cells transduced with retroviral diphtheria immunotoxin (IT) target genes could serve as vehicles for delivering IT to tumors in vivo. We took advantage of the observation that antigen-specific T cells are able to penetrate tumors to design an approach delivering combined cellular and humoral therapy directly to the tumor site. To improve tumor specificity, we selected interleukin (IL)-3 as a ligand because its receptor is selectively overexpressed on myeloid leukemia progenitors. Because Bcl-2 family proteins show structural similarity to diphtheria toxin (DT), we constructed a unique retroviral IT using Bax, a proapoptotic member of the Bcl-2 family, in place of DT. Bax was chosen because several studies showed that its transduction induces lethal apoptosis in different cancers. The retroviral construct for gene therapy included IL-3 positioned downstream of its 80 amino acid leader, and permitted cotranslational protein synthesis of hybrid IL-3/human Bax fusion protein. Other vectors were constructed with IL-3 fused to DT or Pseudomonas exotoxin. Retroviral vectors were used to transiently transduce C8, a CD4(+) T cell clone that specifically recognized FBL-3, a lethal myeloid leukemia. Supernatants collected from transduced cells showed proapoptotic activity and selectively inhibited FBL-3 cells in vitro. Intraperitoneal injection of transduced but not nontransduced C8 into mice with subcutaneous tumors or systemic cancer significantly inhibited tumor growth. These results indicate that retroviral IT made with IL-3 and various toxic proteins may be useful in patients with acute myelogenous leukemia (AML). Furthermore, the Bax construct may be particularly useful as a nonimmunogenic substitute for bacterial toxins in retIT.


Subject(s)
Genetic Therapy/methods , Immunotoxins , Interleukin-3/genetics , Leukemia/genetics , Leukemia/therapy , Proto-Oncogene Proteins c-bcl-2 , Proto-Oncogene Proteins/genetics , Retroviridae/genetics , Animals , CD4-Positive T-Lymphocytes , Genetic Vectors , Injections, Intraperitoneal , Mice , T-Lymphocytes , Transduction, Genetic , bcl-2-Associated X Protein
SELECTION OF CITATIONS
SEARCH DETAIL
...