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Publication Number
US-11396717-B2
Publication Date
2022-07-26
Expiration Date
Abstract
The present invention relates to methods for efficiently generating recombinant monoclonal antibodies derived from B cells of a non-human host which has been immunochallenged with one or more target antigens. The methods comprise the steps of identifying and isolating B cell that bind to the antigen by FACS, and recombining and enriching for thousands of cells to create a B cell library. Related products and methods, such as methods of producing expression libraries, are also disclosed.
Core Innovation
The invention provides a method of generating a recombinant antibody that binds at least one target antigen using B cells from a non-human host selected from rabbit, mouse, rat, hamster, guinea pig, and goat. cDNA is amplified from mRNA of antigen-capable B cells to prepare an immunoglobulin library comprising cDNA encoding VH and VL domains, and antibodies are generated from the VH and VL domains so that the antibodies comprise light and heavy chain combinations.
A subset of antibodies capable of binding to the at least one target antigen is identified by screening the antibodies with the at least one target antigen. The method further includes humanizing at least one antibody of the subset to generate the recombinant antibody, and the outcome is a recombinant antibody that binds at least one target antigen.
The approach also supports generating antibody diversity from B cells, including maintaining native VH/VL pairing when amplifying VH and VL domains, and increasing VH/VL combinations by recombining or pooling. Antibody libraries are enriched and screened for antigen binders, and at least one selected antibody can be humanized and further evolved, including approaches described as CDR grafting and framework reshaping and evolution approaches described as CPE and CPS.
The document describes characterization of antigen-binding antibodies using binding and kinetic/functional characterization assays, and includes discussion of ELISA and kinetic assays in connection with binding affinity, sequence diversity, and large combinatorial libraries. The methods are positioned for efficient generation of recombinant monoclonal antibodies from B cells of non-human hosts, optionally immunized, and from human donors, including display-based library screening with full-length IgG display.
Claims Coverage
The independent claim covers the core workflow: amplifying immunoglobulin variable-region cDNA from antigen-capable B cells of specified non-human hosts, generating light/heavy chain combinations from VH and VL domains, screening for antigen binders, and humanizing a selected antibody subset to produce a recombinant antibody binding the target antigen. Dependent claims refine this core with additional constraints and specific implementation options.
Antigen-capable B-cell immunoglobulin library amplification and VH/VL domain preparation
Amplifying cDNA obtained from mRNA of B cells from a non-human host selected from a rabbit, a mouse, a rat, a hamster, a guinea pig, and a goat, which are capable of binding to the at least one target antigen to prepare an immunoglobulin library comprising cDNA encoding VH and VL domains.
Light and heavy chain combination antibody generation from VH and VL domains
Generating antibodies from the VH and VL domains whereby the antibodies comprise light and heavy chain combinations.
Screening antibody panel against the target antigen to identify a binding subset
Screening the antibodies with the at least one target antigen to identify a subset of antibodies capable of binding to the at least one target antigen.
Humanizing a selected antibody from the binding subset to generate the recombinant antibody
Humanizing at least one antibody of the subset of antibodies to generate the recombinant antibody, whereby a recombinant antibody that binds at least one target antigen is generated.
Combination scale exceeding B-cell library size
Providing a B cell library such that the number of combinations generated in step b is greater than the number of B cells in the B cell library, and the B cell library contains at least 10^3 B cells.
Screening using fluorescence-activated cell sorting (FACS)
Screening the cell population using fluorescence-activated cell sorting (FACS).
Binding affinity threshold greater than 10 μM
Binding between a binding agent and at least one target antigen with a binding affinity greater than 10 μM.
Humanization by CDR grafting
Specifying that the humanizing step is performed by CDR grafting.
Comprehensive property characterization of selected antibodies
Characterizing the selected properties from a listed group of biochemical/biophysical and functional attributes, including isoelectric point, thermal stability, sedimentation rate, folding rate, neutralization of antigen activity, antagonistic/agonistic activity, expression level, inhibition of enzymatic activity, and stability measures, among others.
Overall, the claim set centers on preparing an immunoglobulin library from mRNA of antigen-capable B cells of specified non-human hosts, generating antibody light/heavy chain combinations from VH and VL domains, screening for target-antigen binders, and humanizing an antibody from the binding subset to generate a recombinant antibody binding the target antigen. Dependent claims further constrain library/combination scale, specify screening via FACS, introduce an affinity threshold, limit humanization to CDR grafting, and expand characterization into a broad list of biochemical/biophysical and functional properties.
Stated Advantages
Documented Applications
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