NON-HUMAN ANIMALS WITH MODIFIED IMMUNOGLOBULIN HEAVY CHAIN SEQUENCES
Non-human animals, e.g., mammals, e.g., mice or rats, are provided comprising an immunoglobulin heavy chain locus that comprises a rearranged human immunoglobulin heavy chain variable region nucleotide sequence. The rearranged human immunoglobulin heavy chain variable region nucleotide sequence may be operably linked to a heavy or light chain constant region nucleic acid sequence. Also described are genetically modified non-human animals comprising an immunoglobulin light chain locus comprising one or more but less than the wild type number of human immunoglobulin light chain variable region gene segments, which may be operably linked to a light chain constant region nucleic acid sequence. Also provided are methods for obtaining nucleic acid sequences that encode immunoglobulin light chain variable domains capable of binding an antigen in the absence of a heavy chain.
1 . A non-human animal comprising in its germline genome an immunoglobulin heavy chain locus that comprises a rearranged human immunoglobulin heavy chain variable region nucleotide sequence.
2 . The non-human animal of claim 1 , wherein the non-human animal is a mammal.
3 . The non-human animal of claim 2 , wherein the mammal is a rodent.
4 . The non-human animal of claim 3 , wherein the rodent is selected from the group consisting of a mouse, a rat, and a hamster.
5 . The non-human animal of claim 1 , wherein the non-human animal is homozygous for the rearranged human immunoglobulin heavy chain variable region nucleotide sequence.
6 . The non-human animal of claim 1 , wherein the rearranged human immunoglobulin heavy chain variable region nucleotide sequence is operably linked to a non-human heavy chain constant region gene sequence.
7 . The non-human animal of claim 6 , wherein the non-human heavy chain constant region gene sequence encodes an Fc.
8 . The non-human animal of claim 7 , wherein the non-human heavy chain constant region gene sequence is a mouse or a rat heavy chain constant region gene sequence.
9 . The non-human animal of claim 1 , wherein the non-human animal is a rodent, and the rearranged human immunoglobulin heavy chain variable region nucleotide sequence is operably linked to a human heavy chain constant region gene sequence.
10 . The non-human animal of claim 6 , wherein the heavy chain constant region gene sequence is selected from a C H 1, a hinge, a C H 2, a C H 3, and a combination thereof.
11 . The non-human animal of claim 1 , wherein the rearranged human immunoglobulin heavy chain variable region nucleotide sequence is derived from a human heavy chain V H gene segment, a human heavy chain D gene segment, and a human heavy chain J H gene segment.
12 . The non-human animal of claim 11 , wherein the rearranged human immunoglobulin heavy chain variable region nucleotide sequence is derived from a human germline heavy chain V H segment, a human germline heavy chain D segment, and a human germline heavy chain J H segment.
13 . The non-human animal of claim 1 , wherein the rearranged human immunoglobulin heavy chain variable region nucleotide sequence encodes the sequence of human V H 3-23/GY/J H 4-4.
14 . The non-human animal of claim 1 , wherein substantially all endogenous functional V H , D, and J H gene segments are deleted from the immunoglobulin heavy chain locus of the non-human animal or rendered non-functional.
15 . The non-human animal of claim 1 , wherein the non-human animal comprises a modification that deletes or renders non-functional endogenous functional V H , D, and J H gene segments; and the non-human animal comprises the rearranged human immunoglobulin heavy chain variable region nucleotide sequence, wherein the rearranged human immunoglobulin heavy chain variable region nucleotide sequence is present ectopically.
16 . The non-human animal of claim 1 , wherein an immunoglobulin heavy chain variable domain encoded by the rearranged heavy chain variable region nucleotide sequence is not immunogenic to the non-human animal.
17 . The non-human animal of claim 1 , wherein the non-human animal comprises an Adam6a gene, an Adam6b gene, or both.
18 . The non-human animal of claim 1 , wherein the non-human animal further comprises a nucleotide sequence encoding an unrearranged human immunoglobulin light chain V gene segment (V L ) and an unrearranged human immunoglobulin light chain J gene segment (J L ).
19 . The non-human animal of claim 18 , wherein the nucleotide sequence encoding the unrearranged light chain V gene segment (V L ) and the unrearranged light chain (J L ) gene segment is operably linked to an immunoglobulin light chain constant region gene sequence.
20 . The non-human animal of claim 19 , wherein the light chain constant region gene sequence is selected from a rodent and a human constant region gene sequence.
21 . The non-human animal of claim 20 , wherein the rodent is selected from a mouse, a rat, and a hamster.
22 . The non-human animal of claim 18 , wherein the unrearranged human immunoglobulin light chain (V L ) gene segment and the unrearranged human immunoglobulin (J L ) gene segment are operably linked, at an endogenous rodent locus, to a rodent immunoglobulin constant region gene sequence.
23 . The non-human animal of claim 1 , wherein the immunoglobulin heavy chain locus comprises a plurality of copies of the rearranged human immunoglobulin heavy chain variable region nucleotide sequence.
24 . A non-human immunoglobulin heavy chain locus in a genome of a non-human germ cell comprising a rearranged human immunoglobulin heavy chain variable region nucleotide sequence operably linked to a heavy chain constant region gene sequence, wherein the constant region gene sequence comprises a non-human sequence, a human sequence, or a combination thereof.
25 . The immunoglobulin heavy chain locus of claim 24 , wherein the rearranged human immunoglobulin heavy chain variable region nucleotide sequence is operably linked to an endogenous non-human immunoglobulin constant region gene sequence.
26 . The immunoglobulin heavy chain locus of claim 25 , wherein the endogenous non-human immunoglobulin constant region gene sequence is a mouse or a rat heavy chain constant region gene sequence.
27 . A method of making a non-human animal, the method comprising:
(a) modifying a genome of a non-human animal to delete or render non-functional endogenous functional immunoglobulin heavy chain V H , D, and J H gene segments; and
(b) placing in the genome a rearranged human immunoglobulin heavy chain variable region nucleotide sequence.
28 . The method of claim 27 , wherein the rearranged human immunoglobulin heavy chain variable region nucleotide sequence is operably linked to a non-human immunoglobulin heavy chain constant region gene sequence.
29 . The method of claim 28 , wherein the non-human immunoglobulin heavy chain constant region gene sequence is a mouse or rat immunoglobulin heavy chain constant region gene sequence.
30 . The method of claim 27 , wherein the rearranged human immunoglobulin heavy chain variable region nucleotide sequence is placed at an endogenous immunoglobulin heavy chain locus in the genome.
31 . The method of claim 27 , wherein the rearranged human immunoglobulin heavy chain variable region nucleotide sequence is present in a germline genome of the non-human animal.
32 . The method of claim 27 , wherein the rearranged human immunoglobulin heavy chain variable region nucleotide sequence is present at an ectopic locus in the genome.
33 . The method of claim 27 , wherein the non-human animal comprises a plurality of copies of the rearranged human immunoglobulin heavy chain variable region nucleotide sequence.
34 . The method of claim 27 , wherein the non-human animal comprises an Adam6a gene, an Adam6b gene or both.
35 . The method of claim 27 , wherein the non-human animal is a rodent selected from the group consisting of a mouse, a rat, or a hamster.
36 . A non-human animal that is heterozygous for the immunoglobulin heavy chain locus of claim 24 , wherein the non-human animal expresses the rearranged human immunoglobulin heavy chain variable region nucleotide sequence predominantly from the immunoglobulin heavy chain locus.
37 . A non-human animal comprising a genetically modified immunoglobulin locus comprising:
(a) a rearranged human immunoglobulin heavy chain variable region nucleotide sequence that is operably linked to a light chain constant region gene sequence; and
(b) an unrearranged human immunoglobulin light chain variable region nucleotide sequence that is operably linked to a heavy chain constant region gene sequence.
38 - 60 . (canceled)
61 . An immunoglobulin locus in a germline genome of a non-human animal comprising:
(1) a rearranged human immunoglobulin heavy chain variable region nucleotide sequence that is operably linked to a light chain constant region gene sequence, and
(2) an unrearranged human immunoglobulin light chain variable region nucleotide sequence that is operably linked to a heavy chain constant region gene sequence.
62 - 64 . (canceled)
65 . A method of making a non-human animal that comprises a modified immunoglobulin locus, the method comprising:
(a) modifying a genome of a non-human animal to delete or render non-functional:
(i) endogenous functional immunoglobulin heavy chain V, D, and J gene segments, and
(ii) endogenous functional immunoglobulin light chain V and J gene segments; and
(b) placing in the genome:
(i) a rearranged human immunoglobulin heavy chain variable region nucleotide sequence that is operably linked to a light chain constant region gene sequence, and
(ii) an unrearranged human immunoglobulin light chain variable region nucleotide sequence that is operably linked to a heavy chain constant region gene sequence.
66 - 73 . (canceled)
74 . A non-human animal comprising a modified immunoglobulin heavy chain locus that comprises a rearranged human immunoglobulin heavy chain variable region nucleotide sequence comprising a heavy chain V segment (V H ) sequence that is operably linked, via a spacer, to a heavy chain J segment (J H ) sequence, wherein the spacer comprises at least one amino acid residue.
75 - 93 . (canceled)
94 . An immunoglobulin heavy chain locus in a germline genome of a non-human animal, comprising a rearranged human immunoglobulin heavy chain variable region nucleotide sequence comprising a heavy chain variable gene segment (V H ) that is operably linked, via a spacer, to a heavy chain J gene segment (J H ), wherein the spacer encodes at least one amino acid residue.
95 - 97 . (canceled)
98 . A method of making a non-human animal comprising a modified immunoglobulin heavy chain locus, the method comprising:
(a) modifying a genome of a non-human animal to delete or render non-functional endogenous functional immunoglobulin heavy chain V H , D, and J H gene segments; and
(b) placing in the genome a rearranged human immunoglobulin heavy chain variable region nucleotide sequence comprising a heavy chain variable gene segment (V H ) that is operably linked, via a spacer, to a heavy chain J gene segment (J H ), wherein the spacer comprises at least one amino acid residue.
99 - 106 . (canceled)