Immune repertoire sequence amplification methods and applications
Inventors
Emig, Christopher J. • Mena, Marco
Assignees
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Publication Number
US-10858649-B2
Publication Date
2020-12-08
Expiration Date
Abstract
The present invention relates generally to the field of immune binding proteins and method for obtaining immune binding proteins from genomic or other sources. The present invention also relates to nucleic acids encoding the immune binding proteins in which the natural multimeric association of chains is maintained in the nucleic acids and the immune binding proteins made therefrom. For example nucleic acids encoding antibodies that are amplified from a B-cell using the methods of the invention maintain the natural pairing of heavy and light chains from the B-cell. This maintenance of pairing (or multimerization) produces libraries and/or repertoires of immune binding proteins that are enriched for useful binding molecules.
Core Innovation
The disclosed subject matter provides a method for making a linked nucleic acid encoding an antibody light chain and an antibody heavy chain. Host cells are obtained from a subject that has a protective immune response, where the host cells comprise nucleic acid encoding an antibody heavy chain and nucleic acid encoding an antibody light chain. A host cell is isolated from the plurality of host cells, lysed, and then used with a linked primer and a primer set to amplify variable regions for the antibody light chain and the antibody heavy chain.
The method uses a linked primer that comprises a primer for the antibody heavy chain and a primer for the antibody light chain, where the 5′ end of the primer for the antibody heavy chain is linked to the 5′ end of the primer for the antibody light chain. The linked primer and the second primer form a primer set for amplifying a variable region of the antibody light chain, and the linked primer and the third primer form a primer set for amplifying a variable region of the antibody heavy chain. Variable-region nucleic acids are produced by amplification in the presence of dNTPs and a polymerase.
After amplification, a circular joined nucleic acid is made by joining the 5′ end of the first nucleic acid with the 3′ end of the second nucleic acid and joining the 3′ end of the first nucleic acid with the 5′ end of the second nucleic acid. A nucleic acid encoding the antibody heavy chain and the antibody light chain is then made by amplifying the circular joined nucleic acid. In the broader disclosure, this approach is framed as immune repertoire amplification intended to preserve natural heavy–light chain pairing and multimeric associations.
Claims Coverage
The partial content provides one independent claim and several dependent claims. The independent claim contains four inventive features for producing a linked nucleic acid encoding both antibody light and heavy chains by linked-primer amplification and formation of a circular joined nucleic acid.
Linked primer for paired heavy- and light-chain variable region amplification
Using a linked primer comprising a primer for the antibody heavy chain and a primer for the antibody light chain, where the 5′ end of the primer for the antibody heavy chain is linked to the 5′ end of the primer for the antibody light chain, and using the linked primer together with second and third primers to form primer sets that amplify variable regions of the antibody light chain and the antibody heavy chain.
Circular joined nucleic acid joining of amplified light and heavy variable-region nucleic acids
Making a circular joined nucleic acid by joining the 5′ end of the first nucleic acid with the 3′ end of the second nucleic acid and joining the 3′ end of the first nucleic acid with the 5′ end of the second nucleic acid.
Amplification of the circular joined nucleic acid to encode linked antibody heavy and light chains
Making a nucleic acid encoding the antibody heavy chain and the antibody light chain by amplifying the circular joined nucleic acid.
Use of host cells obtained from a subject with a protective immune response
Providing a plurality of host cells obtained from a subject that has a protective immune response, wherein the host cells comprise nucleic acid encoding an antibody heavy chain and nucleic acid encoding an antibody light chain, and isolating a host cell from the plurality of host cells.
The claims center on generating linked heavy–light chain information from host-cell nucleic acids using a linked primer for variable-region amplification, converting the amplified products into a circular joined nucleic acid, and amplifying the circular joined nucleic acid to produce a nucleic acid encoding both antibody heavy and light chains.
Stated Advantages
Preserving natural heavy–light chain pairing/multimeric associations during immune repertoire amplification.
Documented Applications
Antibody applications across infectious diseases including influenza, HIV, Ebola, Zika, RSV, and CMV.
Cancer-related applications.
Allergy-related applications.
Autoimmune disease-related applications.
Neurodegenerative context applications.
Antigen barcoding and antibody–antigen coupling using sequencing.
Multiplex antigen staining and multiplexed antigen library sequencing workflows described as example embodiments.
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