Allen J.E.,Pennsylvania State University |
Allen J.E.,University of Pennsylvania |
Ferrini R.,Anaphore Inc. |
Dicker D.T.,Pennsylvania State University |
And 8 more authors.
Molecular Cancer Therapeutics | Year: 2012
TRAIL is a trimeric protein that potently induces apoptosis in cancer cells by binding to the trimeric death receptors (DR4 or DR5). Death receptors are attractive therapeutic targets through both the recombinant TRAIL ligand as well as receptor agonist monoclonal antibodies. Although efficacy of the ligand is hampered by its short half-life, agonistic antibodies have a much longer half-life and have shown some clinical efficacy as antitumor agents. However, the efficacy of these antibodies may be limited by their bivalent nature that does not optimally mimic the trimeric ligand. To overcome limitations of currently used death receptor-targeting agents, we engineered trimeric proteins called Atrimer complexes that selectively bind DR4 and potently induce apoptosis in a variety of cancer cells. Atrimer complexes are based on human tetranectin, a trimeric plasma protein of approximately 60 kDa. Loop regions within the tetranectin C-type lectin domains (CTLD) were randomized to create a large phage display library that was used to select DR4-binding complexes. A panel of unique and potent agonist DR4 Atrimer complexes with subnanomolar affinity to DR4 and no detectable binding to DR5 or the decoy receptors was identified. Mechanism of action studies with a selected Atrimer complex, 1G2, showed that Atrimer complexes induce caspase-dependent and DR4-specific apoptosis in cancer cells while sparing normal human fibroblasts and, importantly, hepatocytes. This proof-of-principle study supports the use of alternative proteins engineered to overcome limitations of therapeutically desirable molecules such as TRAIL. ©2012 AACR.
Anaphore Inc. and Hoffmann-La Roche | Date: 2011-04-13
A method for the preparation of a polypeptide of interest in authentic form by enzymatic cleavage of fusion proteins using Granzyme B protease (EC 188.8.131.52). There is also provided fusion proteins comprising a polypeptide of interest an a fusion partner, wherein the junction region between the polypeptide of interest and the fusion partner comprises a Grazyme B protease cleavage site adjacent to the polypeptide of interest, and a human Granzyme B protease variant wherein the Cystein residue no. 228 (chymotrupsinogen numbering) is mutated to Phenylalanine.
Sajish M.,Scripps Research Institute |
Zhou Q.,Scripps Research Institute |
Zhou Q.,Soochow University of China |
Kishi S.,Scripps Research Institute |
And 12 more authors.
Nature Chemical Biology | Year: 2012
Interferon-γ (IFN-γ) engenders strong antiproliferative responses, in part through activation of p53. However, the long-known IFN-γ-dependent upregulation of human Trp-tRNA synthetase (TrpRS), a cytoplasmic enzyme that activates tryptophan to form Trp-AMP in the first step of protein synthesis, is unexplained. Here we report a nuclear complex of TrpRS with the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) and with poly(ADP-ribose) polymerase 1 (PARP-1), the major PARP in human cells. The IFN-γ-dependent poly(ADP-ribosyl)ation of DNA-PKcs (which activates its kinase function) and concomitant activation of the tumor suppressor p53 were specifically prevented by Trp-SA, an analog of Trp-AMP that disrupted the TrpRS-DNA-PKcs-PARP-1 complex. The connection of TrpRS to p53 signaling in vivo was confirmed in a vertebrate system. These and further results suggest an unexpected evolutionary expansion of the protein synthesis apparatus to a nuclear role that links major signaling pathways. © 2012 Nature America, Inc. All rights reserved.
Anaphore Inc. | Date: 2011-04-20
The present invention pertains to the provision of trimeric binding units which bind to trimeric cytokines. In particular there is provided a trimeric polypeptide comprising a trimerising domain and three monomers with binding members capable of binding a trimeric cytokine. Preferably, the trimeric binding units bind in a manner such that upon binding, all receptor binding sites of the trimeric cytokine are substantially blocked, and hence the potential biological activity of the trimeric cytokine is suppressed. In one aspect the invention relates to trimeric binders capable of binding to trimeric cytokines of the Tumor necrosis factor ligand superfamily, such as TNF, TRAIL, RANKL, TWEAK, APRIL and BAFF.
Anaphore Inc. | Date: 2011-07-26
Agonists for TRAIL death receptors including polypeptides that bind to TRAIL death receptor TRAIL-R1 (DR4) and/or TRAIL-R2 (DR5) and optionally having a multimerizing, e.g. trimerizing domain. Agonists are described that do not bind to TRAIL decoy receptors. The multimerizing domain may be derived from human tetranectin. The agonists can induce apoptosis in pathogenic cells expressing a TRAIL death receptor. Pharmaceutical compositions are described for treating diseases associated with cells expressing DR4 and DR5, such as tumor cells. Methods for selecting polypeptides and preparing multimeric complexes.
Anaphore Inc. | Date: 2011-02-16
A novel family of protein libraries comprising CTLDs (C-type Lectin-Like Domains) in which internal polypeptide loopregions lining the ligand binding sites in CTLDs have been replaced with ensembles of completely or partially randomized polypeptide segments. Tetranectin CTLDs were chosen as framework for the preferred embodiment of the invention; and versatile phagemid vectors useful in the generation and manipulation of human and murine tetranectin CTLD libraries are disclosed as part of this invention. Tetranectin CTLDs in monomeric as well as in trimeric form are efficiently displayed as gene III fusions in fully functional form by the recombinant fd phage display vector. CTLD derivatives with affinity for new ligands may readily be isolated from libraries of vectors displaying CTLDs, in which loop-regions have been randomised, using one or more rounds of enrichment by screening or selection followed by amplification of the enriched subpopulation in each round. The efficiency with which protein products containing CTLDs with new binding properties can be produced, e.g. by bacterial expression and in vitro refolding, in mono-, tri-, or multimeric formats provides important advantages in terms of simplicity, cost and efficiency of generation, production and diagnostic or therapeutic applications in comparison to recombinant antibody derivatives.
Anaphore Inc. | Date: 2011-06-08
Novel polypeptides having the scaffold structure of a C-type lectin-like domain (CTLD) and a randomized loop region for specifically binding a variety of target compounds and also provides nucleic acids encoding the polypeptides. Combinatorial CTLD libraries, methods for constructing the libraries, and methods for screening the libraries to identify and isolate the novel CTLD polypeptides. Libraries of nucleic acids encoding polypeptides having a scaffold CTLD with a randomized loop region, as well as nucleic acid sequences, vectors, and methods for preparing and expressing the libraries. Exemplary nucleic acids useful in the combinatorial libraries are derived from tetranectin and other proteins having a CTLD.
Anaphore Inc. | Date: 2012-01-26
Agonists for TRAIL death receptors including polypeptides having a multimerizing, e.g. trimerizing, domain and a polypeptide sequence that binds to at least one of TRAIL death receptors TRAIL-R1 and TRAIL-R2. Agonists are described that do not bind to TRAIL decoy receptors. The multimerizing domain may be derived from human tetranectin. The agonists can induce apoptosis in pathogenic cells expressing a TRAIL death receptor. Pharmaceutical compositions are described for treating diseases associated with cells expressing DR4 and DR5, such as tumor cells. Methods for selecting polypeptides and preparing multimeric complexes.
Anaphore Inc. | Date: 2010-10-19
Pharmaceutical compositions and preparations for use in therapeutics for human, animal and livestock diseases, namely, use for cardiovascular disease, metabolic disease, autoimmune disease, diabetes, obesity, inflammatory disease, vascular disease, dermatological disease, ocular disease, hematological disease, neurological disease, gastrointestinal disease, respiratory disease, viral and infectious disease, cancer, allergies, allograft rejection, genetic disorders, diseases of the musculoskeletal and nervous system, analgesics and wound healing; pharmaceutical compositions and preparations for the diagnosis, prophylaxis and treatment of human, animal and livestock diseases, namely, cardiovascular disease, metabolic disease, autoimmune disease, diabetes, obesity, inflammatory disease, vascular disease, dermatological disease, ocular disease, hematological disease, neurological disease, gastrointestinal disease, respiratory disease, viral and infectious disease, cancer, allergies, allograft rejection, genetic disorders, diseases of the musculoskeletal and nervous system, analgesics and wound healing; vaccines; pharmaceutical compositions and preparations in the nature of contrast media for use with medical imaging equipment; biological reagents for use in clinical medical and non-clinical medical tests for the diagnosis and measurement of disease progression in human, animal and livestock diseases; biological reagents for use in clinical medical and non-clinical medical imaging.
Anaphore Inc. | Date: 2010-10-19
Biological reagents for laboratory, clinical medical and non-clinical medical research applications.