Identification of polynucleotides associated with a sample
Inventors
Robinson, William H. • Tan, Yann Chong • Sokolove, Jeremy
Assignees
US Department of Veterans Affairs • Leland Stanford Junior University
Publication Number
US-11098302-B2
Publication Date
2021-08-24
Expiration Date
2032-04-27
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Abstract
Disclosed herein are compositions and methods for sequencing, analyzing, and utilizing samples such as single samples. Also disclosed herein are compositions and methods for matching together two or more sequences from a sample. Also disclosed herein are compositions and methods for expressing and screening molecules of interest.
Core Innovation
The invention provides compositions and methods for sequencing, analyzing, and utilizing samples such as single samples, specifically focusing on polynucleotides associated with B cells, including single activated B cells like plasmablasts. The invention also involves compositions and methods for matching two or more sequences from a single sample and for expressing and screening molecules of interest derived from such sequences. The core compositions include polynucleotides comprising a first region of expressed B cell variable immunoglobulin regions coupled to a second region comprising identification regions, such as sample and plate identification regions, facilitating unique labeling and tracking of sequences.
The problem being solved addresses the technical challenges in producing therapeutic monoclonal antibodies from human sources. Existing methods, including human hybridomas, transgenic mice with human immunoglobulins, and phage display libraries, each have limitations such as inefficient hybridoma generation, spontaneous gene loss, low affinity antibodies, and limited access to proprietary transgenic mice. The invention aims to overcome these issues by providing compositions, kits, and methods that can produce large numbers of affinity-matured human antibodies, avoiding laborious humanization or extensive screening, and is broadly applicable beyond human antibodies to match polynucleotides from a single sample with high fidelity.
Claims Coverage
The patent claims comprise twenty-six inventive features focused on polynucleotide libraries of compositions encoding paired immunoglobulin heavy and light chains derived from single cells, particularly plasmablasts, including unique sample identification and adapter regions for tracking and sequencing.
Polynucleotide library of paired immunoglobulin sequences from single plasmablasts
The invention provides a polynucleotide library comprising a plurality of compositions, where each composition includes cDNA molecules derived from a single plasmablast encoding cognate pairs of immunoglobulin heavy and light chain variable regions. These cDNA molecules are coupled to identical and unique sample identification regions that distinguish each composition in the library, providing an unbiased representation of the antibody repertoire.
Use of adapter regions attaching sample identification to cDNA
Each cDNA molecule is attached to its sample identification region via an adapter region, characterized by at least one G nucleotide at its 3′ end complementary to a C nucleotide at the 3′ end of the first strand cDNA, allowing stable attachment and facilitating downstream processing.
Inclusion of universal primer regions
The compositions include a universal primer region attached to the sample identification region, where the universal primer sequence is substantially identical across all polynucleotides in the library, supporting efficient amplification and sequencing.
Clonal family representation in the library
The library comprises cDNAs encoding immunoglobulin heavy and light chain variable regions from the same clonal family, derived from a single B cell, with sample identification adapted regions covalently attached to cDNA molecules, ensuring accurate pairing and unique identification of sequences from single cells.
Specificity for plasmablasts and cell markers
The single B cells in the compositions can be single plasmablasts characterized by markers such as CD19+CD20−CD27+CD38hi, ensuring that the libraries represent activated and affinity matured antibody repertoires.
Unlinked cDNA molecules within containers
Within each container, the cDNA molecules encoding heavy and light chain variable regions are not physically linked, maintaining separate polynucleotide identities tied only via the sample identification regions.
Presence of untranslated regions and sequence lengths
The cDNA molecules can include 5′ untranslated regions and contiguous sequences approximately 700 bp for heavy chains and 600 bp for light chains, consistent with full-length variable regions for antibody sequences.
Inclusion of constant regions
Each composition may further include sequences encoding immunoglobulin heavy chain constant regions (alpha, delta, gamma, epsilon, or mu) attached to the cDNA molecules, enabling expression of full-length immunoglobulin molecules.
Covalent and hybridization attachments
The sample identification-adapter regions are covalently attached or hybridized to the cDNA molecules by complementary binding between the adaptor region’s G nucleotides and the cDNA molecule’s C nucleotides at their respective 3' ends, stabilizing the constructs for sequencing and analysis.
Overall, the claims focus on uniquely barcoded polynucleotide libraries derived from single B cells, especially plasmablasts, encoding paired heavy and light chain immunoglobulin variable regions, coupled via specific adapter regions. The invention enables accurate pairing, high-throughput sequencing, analysis of clonal families, and expression of antibodies, providing improvements over previous methods in antibody discovery and repertoire characterization.
Stated Advantages
Provides a means to produce large numbers of affinity-matured human antibodies without laborious humanization or extensive screening.
Enables unambiguous identification and pairing of immunoglobulin heavy and light chain sequences derived from single cells.
Improves sequencing accuracy and error correction by using unique sample identification barcodes.
Facilitates matching of two or more sequences from a single sample, broadly applicable beyond human antibodies.
Allows for high-throughput sequencing and assembly of antibody repertoires with detailed clonal family analysis.
Documented Applications
Discovery and development of therapeutic human monoclonal antibodies with high affinity and specificity.
Discovery and development of diagnostics based on antibodies and T cell receptors representing immune responses.
Development of research tools comprising antibodies against specific antigens.
Monitoring and assessment of vaccine-induced immune responses by characterizing antibody and TCR repertoires.
Identification of antigens targeted by effective immune responses to guide vaccine component selection.
Generation of recombinant human monoclonal antibodies from single sorted B cells for binding and neutralization assays.
Development of human antibody libraries for expression, screening, cloning, and therapeutic evaluation.
Antibody-mediated detection, neutralization, and killing of pathogens including bacteria such as Staphylococcus aureus.
Identification and cloning of antibodies recognizing tumor antigens for cancer diagnostics and therapeutics.
Methods for linking and barcoding polynucleotide sequences to preserve cognate receptor pairs for sequencing and cloning.
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