Biomadison, Inc.

Neoclone Biotechnology International, LLC

Ward C Tucker

Francis Mark Dunning

Timothy M Piazza

Dhammika Atapattu

Tim Piazza

Füsûn N Zeytin

Rachel H Kravitz

Mark W Jackson

Compositions and methods for characterization binding proteins are described that include use of a reporting construct that incorporates a bait region that interacts with the binding protein and a reporter that is susceptible to degradation in the reporter's local environment. Complex formation between the bait region and the binding protein provides protection of the susceptible reporter from degradation. Such a reporting construct can be utilized to identify cells expressing a binding protein when the susceptible reporter is susceptible to degradation in cytosol.

Compositions and methods for cell-based assays

Recombinant nucleic acids encoding reporting constructs for characterizingneurotoxin protease activity and cells that incorporate such recombinant nucleic acids and that are suitable for use in cell-based assays for the neurotoxin are provided. The encoded reporting constructs are a pair of recombinant hybrid proteins that act in concert, and can include an N-terminal palmitoylation sequence. The reporting constructs are a pair of recombinant hybrid proteins that act in concert, and that include aneurotoxin protease recognition and cleavage sequence positioned to release a fluorophore upon cleavage.

Peptides for characterization ofneurotoxins

Compositions and methods for improved cell-based methods of characterizing botulinum neurotoxins are provided. Cells utilized in these methods include a reporting construct that is cleaved following uptake and processing of botulinum neurotoxin by the cell, resulting in proteolysis of the portion of the reporting construct that is released following cleavage. The released portion includes a fluorophore and amino acid substitutions or sequences that enhance the rate of proteolysis. A pair of reporting constructs can be utilized in which one member of the pair is modified to resist cleavage by the botulinum neurotoxin while co-localizing with the remaining member of the pair.

Reporting construct with synaptobrevin based moiety

Cells are described that are useful in rapid, sensitive, and accurate cell-based assays for enzyme activity, particularly for enzyme activities associated with botulinum toxins. Such cell expresses a construct that includes an anchor region, a cleavage site, and a reporting region having two or more identical reporter peptides. Enzymatic activity at the cleavage site releases the reporter region into the cytosol of the cell, where multiple degradation events occur. The observed change in the signal is proportional to the enzymatic activity.

Cell for use in enzyme assays

Systems are described in which a primary detergent or surfactant in an aqueous solution is removed by the addition of a secondary detergent or surfactant in concentrations that exceed the critical micellar concentration (CMC) of the secondary detergent or surfactant using a size separation device. These systems are particularly applicable to protein-containing solutions. Typical primary detergents/surfactants include polysorbate 20, polysorbate 80, and Triton X-100. Suitable secondary detergents or surfactants can be ionic, nonionic, or zwitterionic. Typical secondary detergents/surfactants include, but are not limited to, galactoside detergents (e.g. octyl-β-galactoside), glucamide detergents (e.g. MEGA 8, MEGA 9, MEGA 10), cholamide detergents (e.g. CHAPS, CHAPSO, BIGCHAPS), and sulfobetaine detergents (such as sulfobetaine 3-10).

Systems for removal of detergents from aqueous solutions

Recombinant nucleic acids encoding reporting constructs for characterizingneurotoxin protease activity and cells that incorporate such recombinant nucleic acids and that are suitable for use in cell-based assays for the neurotoxin are provided. The encoded reporting constructs are a pair of recombinant hybrid proteins that act in concert. The reporting constructs are a pair of recombinant hybrid proteins that act in concert, and that include aneurotoxin protease recognition and cleavage sequence positioned to release a fluorophore upon cleavage.

Cells for characterization of botulinum neurotoxins

Assay with synaptobrevin based moiety

Compositions and methods are disclosed that provide a rapid, sensitive, and accurate cell-based assay for enzyme activity, particularly for enzyme activities associated withtoxins. A cell is provided that expresses a construct that includes an anchor region, a cleavage site, and a reporting region having two or more identical reporter peptides. Enzymatic activity at the cleavage site releases the reporter region into the cytosol, where multiple degradation events occur. The observed change in the signal is proportional to the enzymatic activity.

Enzyme assay with duplicate fluorophores

Vesicles that incorporate reporting constructs for characterizing Botulinum neurotoxin protease activity and suitable for use in an assay are provided. The reporting constructs are a pair of recombinant hybrid proteins that act in concert. The reporting constructs are a pair of recombinant hybrid proteins that act in concert, and that include a Botulinum neurotoxin protease recognition and cleavage sequence positioned to release a fluorophore upon cleavage.

Vesicles for characterization of botulinum neurotoxins

Compositions and methods are described in which a primary detergent or surfactant in an aqueous solution is removed by the addition of a secondary detergent or surfactant in concentrations that exceed the critical micellar concentration (CMC) of the secondary detergent or surfactant. These compositions and methods are particularly applicable to protein-containing solutions. Typical primary detergents/surfactants include polysorbate 20, polysorbate 80, and Triton X-100. Suitable secondary detergents or surfactants can be ionic, nonionic, or zwitterionic. Typical secondary detergents/surfactants include, but are not limited to, galactoside detergents (e.g. octyl-&#x3b2;-galactoside), glucamide detergents (e.g. MEGA 8, MEGA 9, MEGA 10), cholamide detergents (e.g. CHAPS, CHAPSO, BIGCHAPS), and sulfobetaine detergents (such as sulfobetaine 3-10).

Compositions and methods for removal of detergents from aqueous solutions

Compositions and methods for determining the efficacy and/or potency of a vaccine preparation are described herein. Splenocytes from immunized animals are isolated and frozen. Upon thawing aliquots these cells are activated by exposure to a series of dilutions of q vaccine preparation being tested and a series of dilutions of a reference vaccine with known characteristics. Cells secreting immunogen-specific antibody and cells secreting nonspecific antibody are enumerated, as is the amount of immunogen-specific and nonspecific antibody produced. Comparison between the results from the vaccine preparations provides a measure of relative vaccine efficacy and/or potency.

Methods for determining vaccine potency

A protease directed to a non-neuronal SNARE protein is described. The protease is produced by selective mutation of aneurotoxin light chain, and is characterized utilizing a reporting construct that includes all or part of the non-neuronal SNARE protein. Such a protease has utility in the treatment of diseases associated with hypersecretion, where the hypersecretion is mediated by a non-neuronal SNARE protein.

Botulinum neurotoxins with modified light chain specificity and methods for producing same

Methods for increasing specific uptake of aneurotoxin are provided. Specific neurotoxin uptake by cells capable of being intoxicated byneurotoxin is enhanced by increasing temperature from about 37&#xb0; C. to up to about 41&#xb0; C., as indicated by a decrease in the ECfound for cells so treated. The effect requires the presence of both heavy and light chains of theneurotoxin, and is serotype selective.

Methods for improving uptake ofneurotoxin

Compositions for characterization oftoxin (BoNT) are described that include a genetically modified cell that is transfected with an artificial construct comprising a nucleic acid sequence that encodes for a hybrid protein having (a) a reporter-containing portion chemically coupled to (b) a cleavage site and (c) a control fluorophore. The cleavage site interacts with a BoNT in a manner that cleaves the reporter-containing portion from remainder of the construct. The cleaved portion is destroyed or otherwise degraded by the local environment, and presence of BoNT is evidenced by reduction in signal from the reporter. The cleavage sequence is all or part of a SNARE protein, the cleavable reporter-containing portion is preferably Yellow Fluorescent Protein (YFP), Citrine, Venus, or a YPet protein and the control fluorophore is preferably CFP, mStrawberry, or a mCherry protein.

Compositions and methods for stability testing of botulinum toxin

Compositions and methods are provided that reduce the time required for detection of botulinum neurotoxins in cell-based assays. In one aspect an isoquinolynyl compound can be used for this purpose. In such cell-based assays the cell can include an enzyme that facilitates degradation of the reporter significantly faster after the cleavage than before the cleavage, and presence of the Botulinum toxin correlates with reduction of the signal from a baseline signal. The cell can advantageously express both the construct that includes the reporter, and an enzyme that facilitates the degradation.

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Monona, WI, United States of America

Ward C Tucker