Transgenic mice
Inventors
TENG, Yumin • Young, Joyce • MCGUINNESS, Brian • ROMANOS, Mike • Brueggemann, Marianne
Assignees
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Publication Number
US-11547099-B2
Publication Date
2023-01-10
Expiration Date
Abstract
The invention relates to nucleic acid constructs for expression in mice for the production of heavy chain only antibodies and VH domains, transgenic mice, related methods and uses.
Core Innovation
The invention relates to transgenic mice that comprise in their genome a vector encoding chimeric immunoglobulin heavy chains and heavy-chain-only antibody (HCAb) expression. The vector includes at least 10 functional human heavy chain V genes in their natural configuration, together with at least one human heavy chain D gene and at least one human heavy chain J gene, and murine regulatory elements that drive expression in response to antigen challenge, including a murine μ enhancer, a murine switch μ element, and a murine 3′ enhancer region.
A key feature of the vector is a murine C gene that lacks the CH1 exon, wherein the C gene consists of a Cμ gene selected from Cμ1, Cμ2b and/or Cμ2a. The design specifies that Cμ is absent in the mouse, or Cμ is partially absent to the extent that it is non-functional, to support expression of a chimeric immunoglobulin heavy chain encoded by the vector.
The described approach uses a triple knockout (TKO) mouse lacking functional endogenous light and heavy chains, supporting in vivo V_H maturation without a partner light chain. The invention is directed to producing soluble, stable, high-yield V_H domains and to enabling characterization of V_H repertoire diversity, including CDR3 diversity, somatic hypermutation, and sequencing of V_H diversity.
Claims Coverage
The independent claim covers a transgenic mouse containing a single genomic vector with multiple human heavy-chain gene segments in natural configuration plus specified murine regulatory elements and a CH1-lacking murine C region, arranged so Cμ is absent or non-functional while the mouse can express a chimeric immunoglobulin heavy chain upon antigen challenge. The claim set further includes disruption of endogenous immunoglobulin loci and methods for generating in vitro display libraries and isolating HCAb-derived V_H domain sequences.
Transgenic mouse with multi-V gene human heavy-chain locus plus murine regulatory elements and CH1-lacking C gene
A transgenic mouse comprising in its genome a vector containing at least 10 functional human heavy chain V genes in their natural configuration, at least one human heavy chain D gene and at least one human heavy chain J gene, a murine μ enhancer, a murine switch μ element, a murine C gene lacking the CH1 exon selected from Cμ1, Cμ2b and/or Cμ2a, and a murine 3′ enhancer region, wherein Cμ is absent or non-functional and the mouse is capable of expressing a chimeric immunoglobulin heavy chain encoded by the vector in response to antigen challenge.
Non-functional endogenous immunoglobulin loci by disruption
The transgenic mouse wherein one or more endogenous immunoglobulin loci are disrupted so that those loci are non-functional.
Producing an in vitro display library using the transgenic mouse
A method for making an in vitro display library using the transgenic mouse.
Using a naive library
The method wherein the in vitro display library is a naive library.
Isolating HCAb-expressing samples and constructing a V_H transcript-based library
A method that isolates a heavy chain antibody-expressing cell or tissue, clones the V_H domain-encoding sequence from mRNA, constructs a library from cloned transcripts, and isolates the V_H domain.
Overall, the claims cover a transgenic mouse genome encoding a chimeric heavy-chain locus with multiple human heavy V genes in natural configuration and specified murine regulatory elements, implemented so that murine Cμ is absent or non-functional via a CH1-lacking murine C gene. Dependent claims require disruption of endogenous immunoglobulin loci and include methods for generating in vitro display libraries, including naive libraries, by isolating HCAb-expressing samples, cloning V_H sequences from mRNA, constructing transcript-based libraries, and isolating V_H domains.
Stated Advantages
Produces soluble, stable, high-yield V_H domains.
Enables V_H maturation in vivo without a partner light chain.
Documented Applications
Generating phage-display libraries, including a naive library and an immunized library.
Constructing a library by isolating heavy chain antibody-expressing cells or tissue, cloning V_H domain-encoding sequences from mRNA, constructing a library from cloned transcripts, and isolating the V_H domain.
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