Binding polypeptides and methods of making the same
Inventors
Shriver, Zachary • Babcock, Gregory • Robinson, Luke
Assignees
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Publication Number
US-11820979-B2
Publication Date
2023-11-21
Expiration Date
Abstract
Polypeptides, such as antibody molecules and TCR molecules, and methods of making the same, are disclosed. The polypeptides can be used to treat, prevent, and/or diagnose disorders.
Core Innovation
The invention relates to a method of making a nucleic acid sequence encoding a matched antibody heavy chain variable region (HCVR) and a matched antibody light chain variable region (LCVR) by producing and then covalently linking heavy-chain and light-chain elements derived from a single cell. The method acquires an isolated production reaction site comprising an HC strand and an LC strand derived from the cell, where the HC strand is a strand of HC double-stranded cDNA and the LC strand is a strand of LC double-stranded cDNA. Within the isolated production reaction site, the HC strand and the LC strand are single stranded.
The method covalently links the single stranded HC strand to the single stranded LC strand to produce a single stranded nucleic acid sequence. The isolated production reaction site does not comprise a nucleic acid encoding an HCVR or an LCVR from a cell other than the cell, thereby making a nucleic acid sequence with matched HCVR and LCVR derived from the same cell. Compartmentalized workflows include isolated production reaction sites and isolated linkage reaction sites.
Additional embodiments include linkage in an isolated linkage reaction site that includes a ligase, and linkage using a splint oligonucleotide that hybridizes at the junction to form a duplexed region at the site of linkage. The approach is extended to generating binding polypeptides such as antibody molecules and T-cell receptor molecules, and it supports constructing display libraries, including phage, yeast, and mammalian display libraries. Further embodiments construct libraries of unique matched members with defined diversity and uniqueness constraints and extend matched pairing concepts to T cell receptor chains.
Claims Coverage
The provided independent claim set includes two core inventive features. It covers acquiring an isolated production reaction site with single-stranded HC and LC strands derived from the same cell, and covalently linking those strands to produce a single-stranded nucleic acid sequence encoding matched HCVR and LCVR elements.
Matched HCVR/LCVR nucleic acid production from a single cell
A method of making a nucleic acid sequence comprising a sequence that encodes a heavy chain element of an antibody heavy chain variable region (HCVR) and a light chain element of an antibody light chain variable region (LCVR), and wherein the HCVR and LCVR are matched, comprising acquiring an isolated production reaction site with an HC strand from HC ds cDNA and an LC strand from LC ds cDNA, wherein the HC strand is single stranded and the LC strand is single stranded, and wherein the isolated production reaction site does not comprise a nucleic acid encoding an HCVR or an LCVR from a cell other than the cell.
Covalent linking of single-stranded HC and LC strands
The method further comprises covalently linking the single stranded HC strand to the single stranded LC strand to produce a single stranded nucleic acid sequence.
The claimed coverage centers on generating matched antibody nucleic acid sequences by using an isolated production reaction site that provides single-stranded HC and LC strands derived from the same cell, followed by covalent linkage to yield a single stranded nucleic acid encoding matched HCVR and LCVR elements. The provided material further mentions isolated linkage sites with ligase or a splint oligonucleotide, as well as library constraints for unique matched members.
Stated Advantages
Produces a single stranded nucleic acid sequence encoding a matched antibody HCVR and LCVR by covalently linking HC and LC strands derived from the same cell within an isolated production reaction site.
Preserves matched heavy/light chain pairing by using an isolated production reaction site that does not comprise nucleic acid encoding HCVR or LCVR from a cell other than the cell.
Supports construction of display libraries, including phage display library, yeast display library, and mammalian display library.
Enables making a nucleic acid sequence that encodes matched HCVR and LCVR for generating binding polypeptides.
Documented Applications
Diagnostic method involving contacting a biological sample (in vitro or in vivo) with a disclosed antibody/polypeptide, optionally comparing to a control, and detecting complex formation or change in complex formation via labeled polypeptide or standard immunoassays.
Combination therapy use cases including administering polypeptides in combination with surgery, radiation therapy, cell therapy, stem cell therapy, or organ/tissue transplantation.
Creating antibody/TCR molecules and binding polypeptides, including contexts described as display libraries.
Use in generating display libraries including phage display, yeast display, and mammalian display.
Construction of display-library formats including phage display library, yeast display library, and mammalian display library for antibody molecules and matched functional antigen-binding molecules.
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