Branford, CT, United States
Branford, CT, United States

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The identification of binding moieties capable of selectively interacting with one or more target antigens is of scientific, medical, and commercial value. Disclosed herein are methods and compositions for the identification, labeling, and/or retrieval of cognate binding moieties.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 216.00K | Year: 2015

DESCRIPTION provided by applicant Current methods for converting an antibody fragment for example scFv Fab Fabandapos etc involves subcloning from an M phagemid or yeast display vector by either restriction enzyme digestion or PCR followed by ligation into or more vectors to produce the heavy and light chains transformation into E coli and subsequent DNA sequencing validation The proposed scFv andgt IgG method using recombineering changes this paradigm for subcloning antibody fragments to produce IgG molecules into a simple transduction of a properly modified phagemid or yeast display vector into a genetically modified strain of E coli harboring a specially modified IgG expression shuttle plasmid The costs and labor are reduced from approximately $ $ for traditional subcloning and DNA sequencing analysis to under $ per clone conversion Additionally automation equipment is unnecessary and the low error rate of recombineering is expected to obviate the need for DNA sequence validation AxioMx is developing a pipeline for the rapid discovery less than two weeks of recombinant Abs Completion of the objectives of this proposal will allow researchers to develop and quickly assemble IgG molecules which could be useful for high throughput proteome analysis diagnostics and immunotherapeutics The ability to clone immuno pools from phage yeast and other display technologies while keeping the heavy and light chains linked is a significant advantage to the proposed method We expect this method to increase the efficient production of better antibodies with implications for both diagnostics and therapeutics PUBLIC HEALTH RELEVANCE The market for antibodies in research is approximately $ B with the market for custom antibodies andgt $ M and expected to grow at least until C Bird lead analyst Frost and Sullivan report Strategic Analysis of the Research Antibody Market March Most recombinant antibodies are screened in display as antibody fragments Current methods for converting an antibody fragment for example scFv Fab Fabandapos etc involves subcloning from an M phagemid or yeast display vector by either restriction enzyme digestion or PCR followed by ligation into or more vectors to produce the heavy and light chains transformation into E coli and subsequent DNA sequencing validation The proposed method will reduce the costs and labor from $ $ for traditional subcloning including DNA sequencing analysis to under $ per clone conversion Additionally the need for automation equipment will be limited even in high throughput mode And the low error rate of recombineering is expected to obviate the need for DNA sequence validation Finally unlike almost all other methods the recombineering method links the heavy and light chains together So that transformation of the novel strain of E coli with a pool of display clones will keep together within the bacterium the heavy and light chains that bind to the target Clones will be able to be tested in mammalian cells faster and cheaper


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 222.50K | Year: 2014

DESCRIPTION (provided by applicant): The overarching goal of Phase I is to produce a method that will generate, identify and clone a catalog of protein-protein interactions in a single rapid experiment. In this proposal we will model the technology using antibody-antigen interactions. Recombinant antibody libraries against a proteome could be produced prior to any need, and then desired affinity reagents systematically cloned as needed. Libraries of recombinational biopanning integration product against a proteome could also be distributed along with primer sets against a set of barcodes for facile cloning. In future endeavors, the method will be used to clone tumor-cell or other diseased tissue cDNAs to use as an epitope library to quickly identify potential biomarkers. Successful completion of this phase will allow th commercialization of antibodies that can be used for diagnostic and therapeutic applications at a speed and comprehensiveness that is not easily achieved by current methods. Kits composed


Grant
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 218.69K | Year: 2014

DESCRIPTION (provided by applicant): Site-specific binding of proteins to DNA plays an important role in cell development, cell signaling, the cell cycle, and diseases such as cancer. There are two overarching goals to this proposal: (1) to develop a meansfor the identification of proteins bound to specific DNA sequences, and (2) to develop a better method for the identification of immunoprecipitation (IP) functional affinity reagents against DNA-bound proteins found on a complex antigen source. We proposeto apply the proof-of-principle using the interaction of transcription factors (TFs) and their specific DNA binding sites as a model. TFs are one major example of such DNA-binding proteins. Once bound to their binding site(s), TF proteins can regulate thetranscription of genes, thereby making individual genes either more or less active. Although this is a proposal to develop a better means toward obtaining functional affinity reagents against proteins when they are bound to DNA, the method itself can


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 334.66K | Year: 2013

DESCRIPTION: We intend to refine the affinity-reagent discovery process so that it is not only cheaper and faster than currently possible, but also so that we produce reagents that are more versatile and useful than current monoclonal antibodies. We propose to use in vitro combinatorial recombination of pre-defined synthetic complementarity determining regions (CDRs) of single chain variable fragment (scFv) antibodies to synthesize a novel type of highly diverse synthetic display library. We have termed this new library-type as a 'pre-defined CDR' (PDC) library. This novel library system will have properties that will also enable it to greatly facilitate both the downstream affinity-reagent selection and the maturation processes. Methods for testing this library using microfluidics and emulsion screening are discussed. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: We intend to refine the affinity-reagent discovery process so that it is not only cheaper and faster than currently possible, but also so that we produce reagents that are more versatile and useful than current monoclonal antibodies. A novel library system is proposed that will have properties that will enable it to greatly facilitate both the downstream affinity-reagent selection and the maturation processes. Methods for testing this library using microfluidics and emulsion screening are discussed. This library and the method proposed recapitulates in vitro the way antibodies in vivo are enriched as a response to an antigen assault. Butwe will be able to achieve a comparable analysis on the much larger recombinant libraries (for example 109-1012) we can generate in vitro. Completion of this method has the potential to save time and money and result in a better product over current methods for recombinant affinity-reagent generation.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: STTR | Phase: Phase I | Award Amount: 220.32K | Year: 2015

DESCRIPTION provided by applicant Although tyrosine phosphorylation is an important controlling element in cell signaling no tRNA suppressor for phosphotyrosine pTyr incorporation has yet been made We propose to use directed molecular evolution of several aminoacyl tRNA synthetases aaRSs to identify mutations that enable binding of pTyr to the aaRS Specifically ATP molecules will be attached to beads which will then be incubated with free pTyr and a phage display library of a mutated aaRS If a mutant aaRS can catalyze the formation of pTyr AMP which is the intermediate for the generation of charged tRNA it will bind to the beads and can be enriched The mutated aaRSs will be used in in vitro translation to incorporate the pTyr into the protein structure of an assayable gene for example galactosidase Mass Spectrometry and a set of already existing anti pTyr specific antibodies will be used to validate incorporation of the pTyr in the assayable protein Phase I is focused on in vitro incorporation PUBLIC HEALTH RELEVANCE The ability to generate tyrosyl phosphorylated proteins will have significant utility in studying the role of phosphoproteins that are involved in cell signalin inflammation cancer and other diseases Conventional approaches toward making phosphorylated proteins require kinases which are promiscuous and often lead to phosphorylation at multiple undesirable residues Successful completion of this proposal will be the first method to allow site specific incorporation of phosphotyrosine in a protein


Disclosed are methods of identifying binding moieties that recognize antigens displayed on cells, such as membrane proteins or recombinant proteins that display eptiopes on the surface of cells. Binding moieties capable of binding membrane proteins can be difficult to obtain because these proteins can depend on their native environments for structural integrity. In some methods scFv phage display libraries are panned against whole cells expressing a membrane protein in an emulsion. Certain methods further permit discrimination of binding moieties according to their affinity or avidity for a target. This approach allows rapid identification of cell surface epitope specific antibodies for research, diagnostics, and immunotherapeutics.


The present invention provides methods and compositions for converting a first polypeptide into a chimeric polypeptide. The invention includes two vectors: a first vector including the sequence of the first polypeptide and a second vector including a second polypeptide. The vectors include complementary site-specific recombination motifs such that site-specific recombination between the two vectors results in the generation of a chimeric polypeptide including at least a portion of the first polypeptide and at least a portion of the second polypeptide. A site-specific recombination motif may be positioned within an intron or within a coding sequence on the first or second vector.


Disclosed herein is an efficient method of generating a library of variants of a sequence of interest, such as may be used in directed evolution. In one embodiment, the method includes an amplification reaction, e.g., error-prone PCR, to generate double-stranded DNA (dsDNA) variants of a sequence of interest, after which one strand of the dsDNA variants may be selectively degraded to produce single-stranded DNA (ssDNA) variants. The ssDNA variants may be hybridized to ssDNA intermediaries, e.g., uracilated circular ssDNA intermediaries, to form heteroduplex DNA, which may be transformed into cells, such as E. coli cells, yielding a library of variants. This method eliminates the inefficient sub-cloning steps and the need for costly primer sets required by many prior methods.


Disclosed herein is an efficient method of generating a library of variants of a sequence of interest, such as may be used in directed evolution, in one embodiment, the method includes an amplification reaction, e.g. error-prone PCR, to generate double-stranded DNA (dsDNA) variants of a sequence of interest, after which one strand of the dsDNA variants may be selectively degraded to produce single-stranded DNA (ssDNA) variants. The ssDNA variants may be hybridized to ssDNA intermediaries, e.g., uracilated circular ssDNA intermediaries, to form heteroduplex DNA, which may be transformed into cells, such as E. coli cells, yielding a library of variants. This method eliminates the inefficient sub-cloning steps and the need for costly primer sets required by many prior methods.

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