Sequence diversity generation in immunoglobulins
AssigneeInnovative Targeting Solutions Inc.
InventorsMichael Gallo, Jaspal Singh Kang, Craig Robin Pigott
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Abstract
Compositions and methods are disclosed for generating immunoglobulin structural diversity in vitro, and in particular, for reducing biases in V region and J segment gene utilization, and for generating immunoglobulin V-D-J recombination events in a manner that does not require D-J recombination to precede V-DJ recombination. Selection of advantageous combinations of immunoglobulin gene elements, including introduction of artificial diversity (D) segment genes and optimization of recombination signal sequence (RSS) efficiency, are disclosed.
Claims22
An isolated host cell that is capable of in vitro immunoglobulin gene rearrangement and which comprises a first nucleic acid composition for generating immunoglobulin structural diversity comprising a tripartite recombination substrate, wherein the tripartite recombination substrate comprises:
- (a) a first nucleic acid that comprises one or a plurality of mammalian immunoglobulin heavy chain variable (VH) region genes each operably linked to an expression control sequence, each VH region gene consisting essentially of (i) an isolated immunoglobulin VH region encoding polynucleotide sequence, and (ii) a non-endogenous VH region recombination signal sequence that is located 3′ to the isolated immunoglobulin VH region encoding polynucleotide sequence;
- (b) a second nucleic acid that comprises one or a plurality of mammalian immunoglobulin heavy chain diversity (D) segment genes, each of said immunoglobulin D segment genes consisting essentially of (i) an isolated D segment encoding polynucleotide sequence, (ii) a D segment upstream recombination signal sequence that is located 5′ to each of said D segment encoding polynucleotide sequences and that is capable of functional recombination with the non-endogenous VH region recombination signal sequence, and (iii) a D segment downstream recombination signal sequence that is located 3′ to each of said isolated D segment encoding polynucleotide sequences;
- and
- (c) a third nucleic acid that comprises one or a plurality of mammalian immunoglobulin heavy chain joining (JH) segment genes, each of said immunoglobulin JH segment genes consisting essentially of (i) an isolated JH segment encoding polynucleotide sequence in operable linkage to an immunoglobulin heavy chain constant (CH) region gene, and (ii) a nonendogenous JH segment recombination signal sequence that is located 5′ to the JH segment encoding polynucleotide sequence and that is capable of functional recombination with the D segment downstream recombination signal sequence of (b)
- wherein the first nucleic acid composition is maintained extrachromosomally in the isolated host cell and wherein the tripartite recombination substrate can undergo at least two or more recombination events in any order in the isolated host cell to form a recombined polynucleotide that encodes an immunoglobulin heavy chain polypeptide, and wherein the isolated host cell expresses said immunoglobulin heavy chain polypeptide.
Dependencies are inferred from the claim text.