NZ724003B2 - Mice that make binding proteins comprising vl domains - Google Patents
Mice that make binding proteins comprising vl domains Download PDFInfo
- Publication number
- NZ724003B2 NZ724003B2 NZ724003A NZ72400311A NZ724003B2 NZ 724003 B2 NZ724003 B2 NZ 724003B2 NZ 724003 A NZ724003 A NZ 724003A NZ 72400311 A NZ72400311 A NZ 72400311A NZ 724003 B2 NZ724003 B2 NZ 724003B2
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- NZ
- New Zealand
- Prior art keywords
- mouse
- human
- gene
- light chain
- heavy chain
- Prior art date
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Classifications
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- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A01K2267/01—Animal expressing industrially exogenous proteins
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
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- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
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- C—CHEMISTRY; METALLURGY
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
- C12N2015/8518—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic expressing industrially exogenous proteins, e.g. for pharmaceutical use, human insulin, blood factors, immunoglobulins, pseudoparticles
Abstract
Disclosed is a rat or mouse comprising in its germline an immunoglobulin hybrid chain locus comprising at least one unrearranged light chain variable region (VL) gene segment, at least one unrearranged light chain joining(JL) gene segment, and an immunoglobulin heavy chain constant region capable of associating with a light chain constant region, wherein each of the unrearranged VL and JL gene segments comprise recombination signal sequences that allow the unrearranged VL and JL gene segments to recombine such that the rat or mouse further comprises a rearranged immunoglobulin hybrid gene comprising light chain variable region (VL/JL) nucleotide sequence operably linked with the immunoglobulin heavy chain constant region, wherein the heavy chain constant region comprises an endogenous constant region gene selected from the group consisting of an endogenous IgM gene, an endogenous IgD gene, an endogenous IgG gene, an endogenous IgE gene, an endogenous IgA gene, and a combination thereof. associating with a light chain constant region, wherein each of the unrearranged VL and JL gene segments comprise recombination signal sequences that allow the unrearranged VL and JL gene segments to recombine such that the rat or mouse further comprises a rearranged immunoglobulin hybrid gene comprising light chain variable region (VL/JL) nucleotide sequence operably linked with the immunoglobulin heavy chain constant region, wherein the heavy chain constant region comprises an endogenous constant region gene selected from the group consisting of an endogenous IgM gene, an endogenous IgD gene, an endogenous IgG gene, an endogenous IgE gene, an endogenous IgA gene, and a combination thereof.
Description
MICE THAT MAKE BINDING PROTEINS COMPRISING VL DOMAINS
The present application is a divisional application from New Zealand patent application number
707327, which is in turn a divisional application from New Zealand patent application number
606824, the entire disclosures of which are incorporated herein by reference.
FIELD OF INVENTION
Immunoglobulin-like binding proteins comprising an immunoglobulin heavy chain
constant region fused with an immunoglobulin light chain variable domain are provided, as well
as binding proteins having an immunoglobulin light chain variable domain fused to a light chain
constant domain and an immunoglobulin light chain variable domain fused to a heavy chain
constant domain. Cells expressing such binding proteins, mice that make them, and related
methods and compositions are also provided.
BACKGROUND
Antibodies typically comprise a tetrameric structure having two identical heavy chains
that each comprise a heavy chain constant region (C ) fused with a heavy chain variable domain
(V ) associated with a light chain constant region (C ) fused with a light chain variable domain
(V ). For a typical human IgG, an antibody molecule is approximately about 150 kDa to about
170 kDa in size (e.g., for IgG3, which comprises a longer hinge region), depending on the
subclass of IgG (e.g., IgG1, IgG3, IgG4) and (varying) length of the variable region.
In a typical antibody, V and V domains associate to form a binding site that binds a
target antigen. Characteristics of the antibody with respect to affinity and specificity therefore
can depend in large part on characteristics of the V and V domains. In typical antibodies in
humans and in mice, V domains couple with either or V domains. It is also known,
however, that V domains can be made that specifically bind a target antigen in the absence of a
cognate V domain (e.g., a V domain that naturally expresses in the context of an antibody and
is associated with the particular V domain), and that V domains can be isolated that specifically
bind a target antigen in the absence of a cognate V domain. Thus, useful diversity in
immunoglobulin-based binding proteins is generally conferred by recombination leading to a
particular V or V (and somatic hypermutation, to the extent that it occurs), as well as by
combination of a cognate V /V pair. It would be useful to develop compositions and methods to
exploit other sources of diversity.
There is a need in the art for binding proteins based on immunoglobulin structures,
including immunoglobulin variable regions such as light chain variable regions, and including
binding proteins that exhibit enhanced diversity over traditional antibodies. There is also a need
for further methods and animals for making useful binding proteins, including binding proteins
that comprise diverse light chain immunoglobulin variable region sequences. Also in need are
useful formats for immunoglobulin-based binding proteins that provide an enhanced diversity of
binding proteins from which to choose, and enhanced
diversity of immunoglobulin variable domains, including compositions and methods for
generating somatically mutated and clonally selected immunoglobulin variable domains for
use, e.g., in making human therapeutics.
SUMMARY
In one aspect, binding proteins are described that comprise immunoglobulin
variable domains that are derived from light chain (i.e., kappa (κ) and/or lambda (λ))
immunoglobulin variable domains, but not from full-length heavy chain immunoglobulin
variable domains. Methods and compositions for making binding proteins, including
genetically modified mice, are also provided.
[0005a] In one aspect, the invention provides a rat or a mouse, comprising in its
germline an immunoglobulin hybrid chain locus comprising at least one unrearranged light
chain variable region (V ) gene segment, at least one unrearranged light chain joining (J )
gene segment, and an immunoglobulin heavy chain constant region capable of associating
with a light chain constant region, wherein each of the unrearranged V and J gene
segments comprise recombination signal sequences that allow the unrearranged V and J
gene segments to recombine such that the rat or mouse further comprises a rearranged
immunoglobulin hybrid gene comprising light chain variable region (V /J ) nucleotide
sequence operably linked with the immunoglobulin heavy chain constant region, and wherein
the heavy chain constant region comprises a gene selected from the group consisting of IgM,
IgD, IgG, IgE, IgA and a combination thereof.
[0005b] In another aspect, the invention provides a rat or mouse comprising a hybrid
immunoglobulin comprising a first polypeptide comprising a first human light chain variable
region sequence fused with an immunoglobulin heavy chain constant region, and a second
polypeptide comprising a second human light chain variable region fused with an
immunoglobulin light chain constant region.
[0005c] In another aspect, the invention provides a rat or mouse, comprising a
replacement in the germline of the rat or mouse at an endogenous immunoglobulin heavy
chain locus of all or substantially all functional endogenous heavy chain variable (V ) gene
segments with at least six or more unrearranged human light chain V gene segments and
one or more unrearranged human light chain J gene segments, wherein each of the
unrearranged human light chain V and J gene segments comprise recombination signal
sequences that allow the unrearranged V and J gene segments to recombine such that the
rat or mouse further comprises a rearranged immunoglobulin hybrid gene comprising light
chain variable region (V /J ) nucleotide sequence operably linked to an immunoglobulin
heavy chain constant region, wherein the heavy chain constant region comprises a gene
selected from the group consisting of IgM, IgD, IgG, IgE, IgA and a combination thereof,
wherein the rat or mouse is incapable of expressing an immunoglobulin heavy chain derived
from a heavy chain V gene segment, and wherein the rat or mouse comprises a splenic B cell
population (B220 /IgM ) that is at least about 75% the size of a splenic B cell population
(B220 /IgM ) of a wild-type rat or mouse.
[0005d] In another aspect, the invention provides a mouse, comprising in its germline
genome a modified endogenous mouse immunoglobulin heavy chain locus comprising a
replacement of all functional endogenous mouse immunoglobulin heavy chain variable V
gene segments, all functional endogenous mouse immunoglobulin heavy chain diversity D
gene segments and all functional endogenous mouse immunoglobulin heavy chain joining J
gene segments with a plurality of unrearranged human immunoglobulin light chain variable V
(hV ) gene segments and all five contiguous unrearranged functional human immunoglobulin
light chain joining J (hJ ) gene segments, wherein the plurality of unrearranged hV gene
κ κ κ
segments and all five contiguous unrearranged functional hJ are operably linked to an intact
endogenous mouse immunoglobulin heavy chain constant region at the endogenous mouse
immunoglobulin heavy chain locus, wherein the plurality of unrearranged hV gene segments
and the five unrearranged hJ gene segments rearrange in a B cell during B cell development
to form a rearranged human immunoglobulin light chain variable region V /J nucleotide
sequence operably linked to the endogenous mouse immunoglobulin heavy chain constant
region at the endogenous mouse immunoglobulin heavy chain locus, and wherein the mouse
comprises the B cell, which further comprises a polypeptide encoded by the rearranged
human immunoglobulin light chain variable region V /J nucleotide sequence operably linked
to the endogenous mouse immunoglobulin heavy chain constant region.
[0005e] In another aspect, the invention provides a mouse comprising in its germline a
first unrearranged human kappa light chain variable (V ) gene segment and an unrearranged
human kappa light chain J (J ) gene segment operably linked with the endogenous mouse
heavy chain constant region at the endogenous mouse heavy chain locus, wherein the first
unrearranged human V gene segment and the unrearranged human J gene segment
replace all functional endogenous mouse heavy chain variable (V ) gene segments, all
functional endogenous mouse D gene segments and all functional endogenous mouse
heavy chain J (J ) gene segments, wherein the first unrearranged human V gene segment
and unrearranged human J gene segment participate in rearrangement to form a rearranged
V / J sequence operably linked to the endogenous mouse heavy chain constant region in
the mouse, and wherein the mouse further comprises in its germline a second human light
chain variable (V ) gene segment and a human light chain J (J ) gene segment operably
linked to a mouse light chain constant gene.
[0005f] In another aspect, the invention provides an isolated rat or mouse cell,
comprising an immunoglobulin hybrid chain locus comprising at least one unrearranged light
chain variable region (V ) gene segment, at least one unrearranged light chain joining (J )
gene segment, and an immunoglobulin heavy chain constant region capable of associating
with a light chain constant region, wherein each of the unrearranged V and J gene
segments comprise recombination signal sequences that allow the unrearranged V and J
gene segments to recombine in a B cell during B cell rearrangement to form a rearranged
immunoglobulin hybrid gene comprising light chain variable region (V /J ) nucleotide
sequence operably linked with the immunoglobulin heavy chain constant region, and wherein
the heavy chain constant region comprises a gene selected from the group consisting of IgM,
IgD, IgG, IgE, IgA and a combination thereof.
[0005g] In another aspect, the invention provides an isolated mouse cell comprising a
first unrearranged human kappa light chain variable (V ) gene segment and an unrearranged
human kappa light chain J (J ) gene segment operably linked with the endogenous mouse
heavy chain constant region at the endogenous mouse heavy chain locus, wherein the first
unrearranged human V gene segment and the unrearranged human J gene segment
replace all functional endogenous mouse heavy chain variable (V ) gene segments, all
functional endogenous mouse D gene segments and all functional endogenous mouse
heavy chain J (J ) gene segments, wherein the first unrearranged human V gene segment
and unrearranged human J gene segment participate in rearrangement to form a rearranged
V / J sequence operably linked to the endogenous mouse heavy chain constant region, and
wherein the mouse cell further comprises a second human light chain variable (V ) gene
segment and a human light chain J (J ) gene segment operably linked to a mouse light chain
constant gene.
[0005h] In another aspect, the invention provides an isolated mouse cell comprising a
modified endogenous mouse immunoglobulin heavy chain locus comprising a replacement of
all functional endogenous mouse immunoglobulin heavy chain variable V gene segments, all
functional endogenous mouse immunoglobulin heavy chain diversity D gene segments and
all functional endogenous mouse immunoglobulin heavy chain joining J gene segments with
a plurality of unrearranged human immunoglobulin light chain variable V (hV ) gene
segments and all five unrearranged human immunoglobulin light chain joining Jk (hJ ) gene
segments, wherein the plurality of unrearranged hV gene segments and all five unrearranged
hJ are operably linked to an intact endogenous mouse immunoglobulin heavy chain constant
region at the endogenous mouse immunoglobulin heavy chain locus, wherein the plurality of
unrearranged hV gene segments and the five unrearranged hJ gene segments are capable
of rearranging in a B cell during B cell development to form a rearranged human
immunoglobulin light chain variable region V /J nucleotide sequence operably linked to the
endogenous mouse immunoglobulin heavy chain constant region at the endogenous mouse
immunoglobulin heavy chain locus.
In one aspect, nucleic acids constructs, cells, embryos, mice, and methods are
provided for making proteins that comprise one or more κ and/or λ light chain variable region
immunoglobulin sequences and an immunoglobulin heavy chain constant region sequence,
including proteins that comprise a human λ or κ light chain variable domain and a human or
mouse heavy chain constant region sequence.
In one aspect, a mouse is provided, comprising an immunoglobulin heavy
chain locus comprising a replacement of one or more immunoglobulin heavy chain variable
region (V ) gene segments, heavy chain diversity (D ) gene segments, and heavy chain
joining (J ) gene segments at an endogenous mouse immunoglobulin heavy chain locus with
one or more light chain variable region (V ) gene segments and one or more light chain
joining region (J ) gene segments.
In one aspect, a mouse is provided, comprising an immunoglobulin heavy
chain locus that comprises a replacement of all or substantially all V , D , and J gene
H H H
segments with one or more V gene segments and one or more J gene segments to form a
V gene segmen sequence at an endogenous heavy chain locus (VL locus), wherein the
L t H
VL locus is capable of recombining with an endogenous mouse C gene to form a
rearranged gene that is derived from a V gene segment, a J gene segment, and an
endogenous mouse C gene.
In one embodiment, the V segments are human V . In one embodiment, the
J segments are human J . In a specific embodiment, the V and J segments are human V
L L L L L
and human J segments.
In one embodiment, all or substantially all V , D , and J gene segments are
H H H
replaced with at least six human Vκ gene segments and at least one Jκ gene segment. In one
embodiment, all or substantially all V , D , and J gene segments are replaced with at least
H H H
16 human Vκ gene segments (human Vκ) and at least one Jκ gene segment. In one
embodiment, all or substantially all V , D , and J gene segments are replaced with at least
H H H
PCT/0S2011/046196
human VK gene segments and at least one JK gene segment. In one embodiment, all or
substantially all VH, DH, and JH gene segments are replaced with at least 40 human VK gene
segments and at least one JK gene segment. In one embodiment, the at least one JK gene
segment comprises two, three, four, or five human JK gene segments.
In one embodiment, the VL segments are human VK segments. In one
embodiment, the human VK segments comprise 4-1, 5-2, 7-3, 2-4, 1-5, and 1-6. In one
embodiment, the K VL comprise 3-7, 1-8, 1-9, 2-10, 3-11, 1-12, 1-13, 2-14, 3-15, 1-16. In
one embodiment, the human VK segments comprise 1-17, 2-18, 2-19, 3-20, 6-21, 1-22, 1-
23, 2-24, 3-25, 2-26, 1-27, 2-28, 2-29, and 2-30. In one embodiment, the human VK
segments comprise 3-31, 1-32, 1-33, 3-34, 1-35, 2-36, 1-37, 2-38, 1-39, and 2-40.
In one embodiment, the VL segments are human VK segments and comprise 4-1,
-2, 7-3, 2-4, 1-5, 1-6, 3-7, 1-8, 1-9, 2-10, 3-11, 1-12, 1-13, 2-14, 3-15, and 1-16. In one
embodiment, the VK segments further comprise 1-17, 2-18, 2-19, 3-20, 6-21, 1-22, 1-23, 2-
24, 3-25, 2-26, 1-27, 2-28, 2-29, and 2-30. In one embodiment, the VK segments further
comprise 3-31, 1-32, 1-33, 3-34, 1-35, 2-36, 1-37, 2-38, 1-39, and 2-40.
In one embodiment, the VL segments are human VA segments and comprise a
fragment of cluster A of the human A light chain locus. In a specific embodiment, the
fragment of cluster A of the human A light chain locus extends from hV113-27 through hV13-
In one embodiment, the VL segments comprise a fragment of cluster B of the
human A light chain locus. In a specific embodiment, the fragment of cluster B of the human
A light chain locus extends from hV15-52 through hVA 1-40.
In one embodiment, the VL segments comprise a human A light chain variable
region sequence that comprises a genomic fragment of cluster A and a genomic fragment of
cluster B. In a one embodiment, the human A light chain variable region sequence
comprises at least one gene segment of cluster A and at least one gene segment of cluster
In one embodiment, the VLsegments comprise at least one gene segment of
cluster B and at least one gene segment of cluster C.
In one embodiment, the VL segments comprise hVA 3-1, 4-3, 2-8, 3-9, 3-10, 2-11,
and 3-12. In a specific embodiment, the VL segments comprise a contiguous sequence of
the human A light chain locus that spans from VA3-12 to V13-1. In one embodiment, the
contiguous sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hVAs. In a specific
embodiment, the hV1s include 3-1, 4-3, 2-8, 3-9, 3-10, 2-11, and 3-12. In a specific
PCT/0S2011/046196
embodiment, the hVAs comprises a contiguous sequence of the human A locus that spans
from VA3-12 to VA3-1 .
In one embodiment, the hVAs comprises 13 to 28 or more hVAs. In a specific
embodiment, the hVAs include 2-14, 3-16, 2-18, 3-19, 3-21, 3-22, 2-23, 3-25, and 3-27. In a
specific embodiment, the hVAs comprise a contiguous sequence of the human A locus that
spans from VA3-27 to VA3-1.
In one embodiment, the VL segments comprise 29 to 40 hVAs. In a specific
embodiment, the VL segments comprise a contiguous sequence of the human A locus that
spans from VA3-29 to VA3-1, and a contiguous sequence of the human A locus that spans
from VA5-52 to VA 1-40. In a specific embodiment, all or substantially all sequence between
hVA 1-40 and hVA3-29 in the genetically modified mouse consists essentially of a human A
sequence of approximately 959 bp found in nature (e.g., in the human population)
downstream of the hVA 1-40 gene segment (downstream of the 3' untranslated portion), a
restriction enzyme site (e.g., Pl-Seel), followed by a human A sequence of approximately
3,431 bp upstream of the hVA3-29 gene segment found in nature.
In one embodiment, the JK is human and is selected from the group consisting of
JK1, JK2, JK3, JK4, JK5, and a combination thereof. In a specific embodiment, the JK
comprises JK1 through JK5.
In one embodiment, the VL segments are human VA segments, and the JK gene
segment comprises an RSS having a 12-mer spacer, wherein the RSS is juxtaposed at the
upstream end of the JK gene segment. In one embodiment, the VL gene segments are
human VA and the VL locus comprises two or more JK gene segments, each comprising an
RSS having a 12-mer spacer wherein the RSS is juxtaposed at the upstream end of each JK
gene segment.
In a specific embodiment, the VL segments comprise contiguous human K gene
segments spanning the human K locus from VK4-1 through VK2-40, and the J
segments
comprise contiguous gene segments spanning the human K locus from JK1 through JK5.
In one embodiment, where the VL segments are VA segments and no D
segment is present between the VL segments and J segments, the VL segments are flanked
downstream (i.e., juxtaposed on the downstream side) with 23-mer RSS, and JK segments if
present or JA segments if present are flanked upstream (i.e., juxtaposed on the upstream
side) with 12-mer RSS.
In one embodiment, where the V gene segments are VK gene segments and no
D gene segment is present between the V gene segments and J gene segments, the VK
gene segments are each juxtaposed on the downstream side with a 12-mer RSS, and JK
PCT/0S2011/046196
segments if present or JA segments if present are each juxtaposed on the upstream side
with a 23-mer RSS.
In one embodiment, the mouse comprises a rearranged gene that is derived from
a V gene segment, a JL gene segment, and an endogenous mouse C gene. In one
embodiment, the rearranged gene is somatically mutated. In one embodiment, the
rearranged gene comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N additions. In one
embodiment, the N additions and/or the somatic mutations observed in the rearranged gene
derived from the V segment and the J segment are 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-
fold, 4-fold, 4.5-fold, or at least 5-fold more than the number of N additions and/or somatic
mutations observed in a rearranged light chain variable domain (derived from the same V
gene segment and the same J gene segment) that is rearranged at an endogenous light
chain locus. In one embodiment, the rearranged gene is in a B cell that specifically binds an
antigen of interest, wherein the B cell binds the antigen of interest with a Ko in the low
nanomolar range or lower (e.g., a Ko of 10 nanomolar or lower). In a specific embodiment,
the V segment, the JL segment, or both, are human gene segments. In a specific
embodiment, the V and J segments are human K gene segments. In one embodiment, the
mouse C gene is selected from lgM, lgD, lgG, lgA and lgE. In a specific embodiment, the
mouse lgG is selected from lgG1, lgG2A, lgG2B, lgG2C and lgG3. In another specific
embodiment, the mouse lgG is lgG1.
In one embodiment, the mouse comprises a B cell, wherein the B cell makes
from a locus on a chromosome of the B cell a binding protein consisting essentially of four
polypeptide chains, wherein the four polypeptide chains consist essentially of (a) two
identical polypeptides that comprise an endogenous mouse C region fused with a V ; and,
(b) two identical polypeptides that comprise an endogenous mouse C region fused with a V
region that is cognate with respect to the V region that is fused with the mouse C region,
and, in one embodiment, is a human (e.g., a human K) V region. In one embodiment, the
V region fused to the endogenous mouse C region is a human V region. In a specific
L H L
embodiment, the human VL region fused with the mouse C region is a VK region. In a
specific embodiment, the human V region fused with the mouse C region is identical to a V
region encoded by a rearranged human germline light chain nucleotide sequence. In a
specific embodiment, the human V region fused to the mouse C region comprises two,
three, four, five, six, or more somatic hypermutations. In one embodiment, the human V
region fused to the mouse C region is encoded by a rearranged gene that comprises 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.
In one embodiment, at least 50% of all lgG molecules made by the mouse
comprise a polypeptide that comprises an lgG isotype CH region and a V region, wherein
PCT/0S2011/046196
the length of said polypeptide is no longer than 535, 530, 525, 520, or 515 amino acids. In
one embodiment, at least 75% of all lgG molecules comprise the polypeptide recited in this
paragraph. In one embodiment, at least 80%, 85%, 90%, or 95% of all lgG molecules
comprise the polypeptide recited in this paragraph. In a specific embodiment, all lgG
molecules made by the mouse comprise a polypeptide that is no longer than the length of
the polypeptide recited in this paragraph.
In one embodiment, the mouse makes a binding protein comprising a first
polypeptide that comprises an endogenous mouse C region fused with a variable domain
encoded by a rearranged human V gene segment and a J gene segment but not a D gene
segment, and a second polypeptide that comprises an endogenous mouse C region fused
with a V domain encoded by a rearranged human V gene segment and a J gene segment
but not a D gene segment, and the binding protein specifically binds an antigen with an
afinity in the micromolar, nanomolar, or picomolar range. In one embodiment, the J
segment is a human J segment (e.g., a human K gene segment). In one embodiment, the
human V segment is a human VK segment. In one embodiment, the variable domain that is
fused with the endogenous mouse C region comprises a greater number of somatic
hypermutations than the variable region that is fused with the endogenous mouse C
region;
in a specific embodiment, the variable region fused with the endogenous mouse C region
comprises about 1.5, 2-, 3-, 4-, or 5-fold or more somatic hypermutations than the V region
fused to the endogenous mouse C region; in a specific embodiment, the V region fused with
the mouse C region comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more somatic
hypermutations than the V region fused with the mouse C
region. In one embodiment, the
region is encoded by a rearranged gene that comprises 1,
V region fused with the mouse C
2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.
In one embodiment, the mouse expresses a binding protein comprising a first
1) fused with an immunoglobulin heavy chain constant region
light chain variable domain (V
sequence and a second light chain variable domain (VL2) fused with an immunoglobulin light
chain constant region, wherein V 1 comprises a number of somatic hypermutations that is
about 1.5- to about 5-fold higher or more than the number of somatic hypermutations
present in V 2, In one embodiment, the number of somatic hypermutations in V 1 is about
2- to about 4-fold higher than in V 2. In one embodiment, the number of somatic
hypermutations in V 1 is about 2- to about 3-fold higher than in V 2, In one embodiment,
V 1 is encoded by a sequence that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1 0 or more N
additions.
In one aspect, a genetically modified mouse is provided that expresses an
immunoglobulin that consists essentially of the following polypeptides: a first two identical
PCT/0S2011/046196
polypeptides that each consists essentially of a C region fused with a variable domain that
is derived from gene segments that consist essentially of a VL gene segment and a JL gene
segment, and a second two identical polypeptides that each consists essentially of a CL
region fused with a variable domain that is derived from gene segments that consist
essentially of a VL segment and a JL segment.
In a specific embodiment, the two identical polypeptides that have the C region
have a mouse C region.
In a specific embodiment, the two identical polypeptides that have the CL region
have a mouse CL region.
In one embodiment, the variable domain fused with the CL region is a variable
domain that is cognate with the variable domain fused to the C region.
[003 ] In one embodiment, the variable domain that is fused with the endogenous
mouse C region comprises a greater number of somatic hypermutations than the variable
domain that is fused with the endogenous mouse CL region; in a specific embodiment, the
variable domain fused with the endogenous mouse C region comprises about 1.5-fold, 2-
fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold or more somatic hypermutations than
the variable domain fused to the endogenous mouse CL region. In one embodiment, the
variable domain fused with the endogenous mouse CL region is encoded by a gene that
comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N additions.
In one embodiment, one or more of the V segments and the J segments are
human gene segments. In a specific embodiment, both the V segments and the J segments
are human K gene segments. In another specific embodiment, both of the V segments and
the J segments are human A gene segments. In one embodiment, the V segments and the
J segments are independently selected from human K and human A gene segments. In a
specific embodiment, the V segments are VK segments and the J segments are JA
segments. In another specific embodiment, the V segments are VA segments and the J
segments are JK segments.
In one embodiment, one or more of the variable domains fused with the CL region
and the variable domains fused with the C region are human variable domains. In a
specific embodiment, the human variable domains are human VK domains. In another
specific embodiment, the human variable domains are VA domains. In one embodiment, the
human domains are independently selected from human VK and human VA domains. In a
specific embodiment, the human variable domain fused with the CL region is a human VA
domain and the human variable domain fused with the C region is a human VK domain. In
another embodiment, the human variable domain fused with the CL region is a human VK
domain and the human variable domain fused with the C is a human VA domain.
PCT/0S2011/046196
In one embodiment, the V
gene segment of the first two identical polypeptides is
selected from a human VA segment and a human VK segment. In one embodiment, the V
segment of the second two identical polypeptides is selected from a human VA segment and
a human VK segment. In a specific embodiment, the V segment of the first two identical
polypeptides is a human VK segment and the V segment of the second two identical
polypeptides is selected from a human VK segment and a human VA segment. In a specific
embodiment, the V segment of the first two identical polypeptides is a human VA segment
and the V segment of the second two identical polypeptides is selected from a human VA
segment and a human VK segment. In a specific embodiment, the human V segment of the
first two identical polypeptides is a human VK segment, and the human V segment of the
second two identical polypeptides is a human VK segment.
In one embodiment, the lgG of the mouse comprises a binding protein made in
response to an antigen, wherein the binding protein comprises a polypeptide that consists
essentially of a variable domain and a CH region, wherein the variable domain is encoded by
a nucleotide sequence that consists essentially of a rearranged VL segment and a
rearranged J segment, and wherein the binding protein specifically binds an epitope of the
antigen with a Ko in the micromolar, nanomolar, or picomolar range.
In one aspect, a mouse is provided, wherein all or substantially all of the lgG
made by the mouse in response to an antigen comprises a heavy chain that comprises a
variable domain, wherein the variable domain is encoded by a rearranged gene derived from
gene segments that consist essentially of a V gene segment and a J gene segment. In one
embodiment, the rearranged gene comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N
additions.
In one embodiment, the V segment is a V segment of a light chain. In one
embodiment, the light chain is selected from a K light chain and a " light chain. In a specific
embodiment, the light chain is a K light chain. In a specific embodiment, the V segment is a
human V segment. In a specific embodiment, the V segment is a human VK segment and
the J segment is a human JK segment.
In one embodiment, the J segment is a J segment of a light chain. In one
embodiment, the light chain is selected from a K light chain and a " light chain. In a specific
embodiment, the light chain is a K light chain. In a specific embodiment, the J segment is a
human J segment. In another embodiment, the J segment is a J segment of a heavy chain
(i.e., a J )- In a specific embodiment, the heavy chain is of mouse origin. In another specific
embodiment, the heavy chain is of human origin.
In one embodiment, the variable domain of the heavy chain that is made from no
more than a V segment and a J segment is a somatically mutated variable domain.
PCT/0S2011/046196
[004 ] In one embodiment, the variable domain of the heavy chain that is made from no
more than a V segment and a J segment is fused to a mouse C region.
In a specific embodiment, all or substantially all of the lgG made by the mouse in
response to an antigen comprises a variable domain that is derived from no more than one
human V segment and no more than one human J segment, and the variable domain is
fused to a mouse lgG constant region, and the lgG further comprises a light chain that
comprises a human V domain fused with a mouse C region. In a specific embodiment, the
V domain fused with the mouse C region is derived from a human VK segment and a
human JK segment. In a specific embodiment, the V domain fused with the mouse C
region is derived from a human VA segment and a human JA segment.
In one aspect, a mouse is provided that makes an lgG comprising a first CDR3
on a polypeptide comprising a C region and a second CDR3 on a polypeptide comprising a
C region, wherein both the first CDR3 and the second CDR3 are each independently
derived from no more than two gene segments, wherein the two gene segments consist
essentially of a V gene segment and a J gene segment. In one embodiment, the CDR3 on
the polypeptide comprising the C region comprises a sequence that is derived from a CDR3
nucleotide sequence that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1 0 or more N additions.
[0046) In one embodiment, the V segment and the J segment are human gene
segments. In one embodiment, the V segment and the J segment are K gene segments.
In one embodiment, the V segment and the J segment are A gene segments.
In one aspect, a mouse is provided that makes an lgG comprising a first CDR3
on a first polypeptide comprising a C region and a second CDR3 on a second polypeptide
region, wherein both the first CDR3 and the second CDR3 each comprise a
comprising a C
sequence of amino acids wherein more than 75% of the amino acids are derived from a V
gene segment. In one embodiment, the CDR3 on the polypeptide comprising the C region
comprises a sequence that is derived from a CDR3 nucleotide sequence that comprises 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.
In one embodiment, more than 80%, more than 90%, or more than 95% of the
amino acids of the first CDR3, and more than 80%, more than 90%, or more than 95% of the
amino acids of the second CDR3, are derived from a light chain V segment.
In one embodiment, no more than two amino acids of the first CDR3 are derived
from a gene segment other than a light chain V segment. In one embodiment, no more than
two amino acids of the second CDR3 are derived from a gene segment other than a light
chain V segment. In a specific embodiment, no more than two amino acids of the first CDR3
and no more than two amino acids of the second CDR3 are derived from a gene segment
other than a light chain V segment. In one embodiment, no CDR3 of the lgG comprises an
PCT/0S2011/046196
amino acid sequence derived from a D gene segment. In one embodiment, the CDR3 of the
first polypeptide does not comprise a sequence derived from a D segment.
In one embodiment, the V segment is a human V gene segment. In a specific
embodiment, the V segment is a human VK gene segment.
In one embodiment, the first and/or the second CDR3 have at least one, two,
three, four, five, or six somatic hypermutations. In one embodiment, the first CDR3 is
encoded by a nucleic acid sequence that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N
additions.
In one embodiment, the first CDR3 consists essentially of amino acids derived
from a human light chain V gene segment and a human light chain J gene segment, and the
second CDR3 consists essentially of amino acids derived from a human light chain V gene
segment and a human light chain J gene segment. In one embodiment, the first CDR3 is
derived from a nucleic acid sequence that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N
additions. In one embodiment, the first CDR3 is derived from no more than two gene
segments, wherein the no more than two gene segments are a human VK gene segment
and a human JK gene segment; and the second CDR3 is derived from no more than two
gene segments, wherein the no more than two gene segments are a human VK gene
segment and a J gene segment selected from a human JK segment, a human JA segment,
and a human J segment. In one embodiment, the first CDR3 is derived from no more than
two gene segments, wherein the no more than two gene segments are a human VA
segment and a J segment selected from a huma'n JK segment, a human JA segment, and a
human JH segment.
In one aspect, a mouse is provided that makes an lgG that does not contain an
amino acid sequence derived from a DH gene segment, wherein the lgG comprises a first
polypeptide having a first V domain fused with a mouse C region and a second polypeptide
having a second V domain fused with a mouse C region, wherein the first V domain and
the second V domain are not identical. In one embodiment, the first and second V
domains are derived from different V segments. In another embodiment, the first and
second V domains are derived from different J segments. In one embodiment, the first and
second V domains are derived from identical V and J segments, wherein the second V
domain comprises a higher number of somatic hypermutations as compared to the first V
domain.
[005 ] In one embodiment, the first and the second VL domains are independently
selected from human and mouse V domains. In one embodiment, the first and second V
domains are independently selected from VK and VA domains. In a specific embodiment,
the first V domain is selected from a VK domain and a VA domain, and the second V
PCT/0S2011/046196
domain is a VK domain. In another specific embodiment, the VK domain is a human VK
domain.
In one aspect, a mouse is provided, wherein all or substantially all of the lgG
made by the mouse consists essentially of a light chain having a first human V domain
fused with a mouse C domain, and a heavy chain having a second human V domain fused
with a mouse C domain.
In one embodiment, the human V domain fused with the mouse C domain is a
human VK domain.
In one embodiment, the first and the second human V domains are not identical.
In one aspect, a mouse is provided, wherein at least 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, or about 100% of the immunoglobulin G made by the mouse
consists essentially of a dimer of (a) a first polypeptide that consists essentially of an
immunoglobulin V domain and an immunoglobulin C region; and, (b) a second polypeptide
of no more than 535 amino acids in length, wherein the second polypeptide consists
essentially of a C region and a V domain that lacks a sequence derived from a D gene
segment.
In one embodiment, the second polypeptide is about 435-535 amino acids in
length. In a specific embodiment, the second polypeptide is about 435-530 amino acids in
length. In a specific embodiment, the second polypeptide is about 435-525 amino acids in
length. In a specific embodiment, the second polypeptide is about 435-520 amino acids in
length. In a specific embodiment, the second polypeptide is about 435-515 amino acids in
length.
In one embodiment, in about 90% or more of the lgG made by the mouse the
second polypeptide is no more than about 535 amino acids in length.
In one embodiment, in about 50% or more of the lgG made by the mouse the
second polypeptide is no more than about 535 amino acids in length. In one embodiment, in
about 50% or more of the immunoglobulin G made by the mouse the second polypeptide is
no more than about 530, 525, 520, 515, 510, 505, 500, 495, 490, 485, 480, 475, 470, 465,
460, 455, or 450 amino acids in length. In one embodiment, about 60%, 70%, 80%, 90%, or
95 % or more of the lgG made by the mouse is of the recited length. In a specific
embodiment, all or substantially all of the lgG made by the mouse is of the recited length.
In one embodiment, the V domain of the second polypeptide is a V domain. In a
specific embodiment, the V domain of the second polypeptide is selected from a VK and a
VA domain. In a specific embodiment, the V domain of the second polypeptide is a human
VK or VA domain.
PCT/0S2011/046196
In one aspect, a mouse is provided that expresses from a nucleotide sequence in
its germline a polypeptide that comprises a light chain variable sequence (e.g., a V and/or J
sequence), a D sequence, and a heavy chain constant region.
In one embodiment, the mouse expresses the polypeptide from an endogenous
mouse heavy chain locus that comprises a replacement of all or substantially all functional
endogenous mouse heavy chain variable locus gene segments with a plurality of human
gene segments at the endogenous mouse heavy chain locus.
In one embodiment, the polypeptide comprises a VL sequence derived from a V'A
or a VK gene segment, the polypeptide comprises a CDR3 derived from a D gene segment,
or J1 or JK gene segment.
and the polypeptide comprises a sequence derived from a J
In one embodiment, the mouse comprises an endogenous mouse heavy chain
immunoglobulin locus comprising a replacement of all functional V gene segments with one
or more human light chain V1v gene segments wherein the one or more human V1v segments
each have juxtaposed on the downstream side a 23-mer spaced recombination signal
sequence (RSS), wherein the VA segments are operably linked to a human or mouse D
segment that has juxtaposed upstream and downstream a 12-mer spaced RSS; the D gene
segment is operably linked with a J segment juxtaposed upstream with a 23-mer spaced
RSS that is suitable for recombining with the 12-mer spaced RSS juxtaposing the D
gene
segment; wherein the V, D , and J segments are operably linked to a nucleic acid sequence
encoding a heavy chain constant region.
In one embodiment, the mouse comprises an endogenous mouse heavy chain
immunoglobulin locus comprising a replacement of all functional V gene segments with one
or more human VK gene segments each juxtaposed on the downstream side with a 12-mer
spaced recombination signal sequence (RSS), wherein the V segments are operably linked
segment that is juxtaposed both upstream and downstream with a
to a human or mouse D
23-mer spaced RSS; the D segment is operably linked with a J segment juxtaposed on the
upstream side with a 12-mer spaced RSS that is suitable for recombining with the 23-mer
segment; wherein the V, DH, and gene segments are
spaced RSS juxtaposing the D
operably linked to a nucleic acid sequence encoding a heavy chain constant region.
In one embodiment, the heavy chain constant region is an endogenous mouse
heavy chain constant region. In one embodiment, the nucleic acid sequence encodes a
sequence selected from a C 1, a hinge, a C 2, a C 3, and a combination thereof. In one
embodiment, one or more of the C 1, hinge, C 2, and C 3 are human.
H H H
In one embodiment, the mouse comprises an endogenous mouse heavy chain
immunoglobulin locus comprising a replacement of all functional V gene segments with a
plurality of human V1 or VK gene segments each juxtaposed downstream with 23-mer
PCT/0S2011/046196
spaced RSS, a plurality of human D segments juxtaposed both upstream and downstream
a 12-mer spaced RSS, a plurality of human J segments (J or JA or JK) juxtaposed both
upstream and downstream with a 23-mer spaced RSS, wherein the locus comprises an
endogenous mouse constant region sequence selected from C 1, hinge, C 2, C 3, and a
H H H
combination thereof. In a specific embodiment, the mouse comprises all or substantially all
functional human VA or VK segments, all or substantially all functional human D segments,
and all or substantially all JH or JA or JK segments.
In one embodiment, the mouse expresses an antigen-binding protein comprising
(a) a polypeptide that comprises a human light chain sequence linked to a heavy chain
constant sequence comprising a mouse sequence; and (b) a polypeptide that comprises a
human light chain variable region linked to a human or mouse light chain constant
sequence. In a specific embodiment, the light chain sequence is a human light chain
sequence, and upon exposure to a protease that is capable of cleaving an antibody into an
Fe and a Fab, a fully human Fab is formed that comprises at least four light chain CDRs,
wherein the at least four light chain CDRs are selected from A sequences, K sequences, and
a combination thereof. In one embodiment, the Fab comprises at least five light chain
CDRs. In one embodiment, the Fab comprises six light chain CDRs. In one embodiment, at
least one CDR of the Fab comprises a sequence derived from a VA segment or a VK
segment, and the at least one CDR further comprises a sequence derived from a D
segment. In one embodiment, the at least one CDR is a CDR3 and the CDR is derived from
a human VK segment, a human D segment, and a human JK segment.
In one embodiment, the polypeptide of comprises a variable region derived from
a human VA or VK gene segment, a human D gene segment, and a human J or JA or JK
gene segment. In a specific embodiment, the heavy chain constant sequence is derived
from a human C 1 and a mouse C 2 and a mouse C 3 sequence.
H H H
In one aspect, a mouse is provided that comprises in its germline an
unrearranged human VK or VA gene segment operably linked to a human J gene segment
and a heavy chain constant region sequence, wherein the mouse expresses a VL binding
protein that comprises a human VK domain fused with a heavy chain constant region, and
wherein the mice exhibit a population of splenic B cells that express VL binding proteins in
hi i
CD1 g B cells, including transitional B cells (CD19 IgM lgD nt), and mature B cells
i hi
(CD19 IgM ntIgD ).
In one aspect, a mouse is provided that comprises in its germline an
unrearranged human VK or VA gene segment operably linked to a human J gene segment
and a heavy chain constant region sequence, wherein the mouse expresses on a B cell an
immunoglobulin that comprises a light chain variable domain fused with a heavy chain
PCT/0S2011/046196
constant region, wherein the lymphocyte population in bone marrow of the mice exhibit a
pro/pre B cell population that is about the same in number as in a pro/pre B cell population
of a wild-type mouse (lymphocytes in bone marrow).
In one embodiment, the mice comprise at least 6 unrearranged hVK gene
segments and one or more unrearranged hJK gene segments, and the mice comprise a
lymphocyte-gated and lgM spleen cell population expressing a V binding protein, wherein
the population is at least 75% as large as a lymphocyte-gated and lgM spleen cell
population of a wild-type mouse.
In one embodiment, the mice exhibit a mature B cell-gated (CD19 ) splenocyte
population of lgD cells and lgM cells that total about 90%; in one embodiment, the mature
+ + +
B cell-gated (CD19 ) splenocyte population of lgD cells and lgM cells of the modified
mouse is about the same (e.g., within 10%, or within 5%) as the total of lgD cells and lgM
cells of a wild-type mouse that are mature B cell-gated (CD19 ) splenocytes.
In one aspect, a mouse is provided that expresses an immunoglobulin protein
from a modified endogenous heavy chain locus in its germline, wherein the modified
endogenous heavy chain locus lacks a functional mouse heavy chain V gene segment and
the locus comprises unrearranged light chain V gene segments and unrearranged J gene
segments, wherein the unrearranged light chain V gene segments and unrearranged J gene
segments are operably linked with a heavy chain constant region sequence; wherein the
immunoglobulin protein consists essentially of a first polypeptide and a second polypeptide,
wherein the first polypeptide comprises an immunoglobulin light chain sequence and an
immunoglobulin heavy chain constant sequence, and the second polypeptide comprises an
immunoglobulin light chain variable domain and a light chain constant region.
In one aspect, a mouse is provided that expresses an immunoglobulin protein,
wherein the immunoglobulin protein lacks a heavy chain immunoglobulin variable domain,
and the immunoglobulin protein comprises a first variable domain derived from a light chain
gene, and a second variable domain derived from a light chain gene, wherein the first
variable domain and the second variable domain are cognate with respect to one another,
wherein the first and the second light chain variable domains are not identical, and wherein
the first and the second light chain variable domains associate and when associated
specifically bind an antigen of interest.
In one aspect, a mouse is provided that makes from unrearranged gene
segments in its germline an immunoglobulin protein comprising variable regions that are
wholly derived from gene segments that consist essentially of unrearranged human gene
segments, wherein the immunoglobulin protein comprises an immunoglobulin light chain
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constant sequence and an immunoglobulin heavy chain constant sequence selected from
the group consisting of a CH1, a hinge, a CH2, a CH3, and a combination thereof.
In one aspect, a mouse is provided that makes from unrearranged gene
segments in its germline an immunoglobulin protein comprising variable regions, wherein all
CDR3s of all variable regions are generated entirely from light chain V and J gene
segments, and optionally one or more somatic hypermutations, e.g., one or more N
additions.
In one aspect, a mouse is provided that makes a somatically mutated
immunoglobulin protein derived from unrearranged human immunoglobulin light chain
variable region gene segments in the germ line of the mouse, wherein the immunoglobulin
protein lacks a CDR that comprises a sequence derived from a D gene segment, wherein
the immunoglobulin protein comprises a first CDR3 on a light chain variable domain fused
with a light chain constant region, comprises a second CDR3 on a light chain variable
domain fused with a heavy chain constant region, and wherein the second CDR3 is derived
from a rearranged light chain variable region sequence that comprises 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10 or more N additions.
In one aspect, a mouse as described herein is provided, wherein the mouse
comprises a functionally silenced light chain locus selected from a 1 locus, a K locus, and a
combination thereof. In one embodiment, the mouse comprises a deletion of a 1 and/or a K
locus, in whole or in part, such that the 1 and/or K locus is nonfunctional.
In one aspect, a mouse embryo is provided, comprising a cell that comprises a
modified immunoglobulin locus as described herein. In one embodiment, the mouse is a
chimera and at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the cells
of the embryo comprise a modified immunoglobulin locus as described herein. In one
embodiment, at least 96%. 97%, 98%, 99%, or 99.8% of the cells of the embryo comprise a
modified immunoglobulin locus as described herein. In one embodiment, the embryo
comprises a host cell and a cell derived from a donor ES cell, wherein the cell derived from
the donor ES cell comprises a modified immunoglobulin locus as described herein. In one
embodiment, the embryo is a 2-, 4-, 8, 16-, 32, or 64-cell stage host embryo, or a blastocyst,
and further comprises a donor ES cell comprising a modified immunoglobulin locus as
described herein.
In one aspect, a mouse or a cell made using a nucleic acid construct as
described herein is provided.
In one aspect, a mouse made using a cell as described herein is provided. In
one embodiment, the cell is a mouse ES cell.
PCT/0S2011/046196
(0085] In one aspect, use of a mouse as described herein to make a nucleic acid
sequence encoding a first human light chain immunoglobulin variable sequence (V 1) that is
cognate with a second human light chain immunoglobulin variable sequence (V 2), wherein
the V 1 fused with a human immunoglobulin light chain constant region (polypeptide 1)
expresses with V 2 fused with a human immunoglobulin heavy chain constant region
(polypeptide 2), as a dimer of polypeptide1/polypeptide 2, to form a V 1-V 2 antibody.
(0086] In one aspect, use of a mouse as described herein to make a nucleic acid
sequence encoding a human immunoglobulin light chain variable sequence that is fused
with a human immunoglobulin heavy chain sequence, wherein the nucleic acid sequence
encodes a human VL-CH polypeptide, wherein the human V -CH polypeptide expresses as a
dimer, and wherein the dimer expresses in the absence of an immunoglobulin light chain
(e.g., in the absence of a human A or human K light chain). In one embodiment, the V -CH
dimer specifically binds an antigen of interest in the absence of a A light chain and in the
absence of a K light chain.
(0087] In one aspect, use of a mouse as described herein to make a nucleic acid
sequence encoding all or a portion of an immunoglobulin variable domain. In one
embodiment, the immunoglobulin variable domain is a human VA or human VK domain.
(0088] In one aspect, use of a mouse as described herein to make a fully human Fab
(comprising a first hu11an V fused with a human light chain constant region, and a second
human V fused with a human heavy chain constant region sequence) or a fully human
F(ab)2 is provided.
(0089] In one aspect, use of a mouse as described herein to make an immortalized cell
line is provided. In one embodiment, the immortalized cell line comprises a nucleic acid
sequence encoding a human VA or VK domain operably linked to a nucleic acid sequence
that comprises a mouse constant region nucleic acid sequence.
(0090] In one aspect, use of a mouse as described herein to make a hybridoma or
quadroma is provided.
In one aspect, a cell is provided, comprising a modified immunoglobulin locus as
described herein. In one embodiment, the cell is selected from a totipotent cell, a pluripotent
cell, an induced pluripotent stem cell (iPS), and an ES cell. In a specific embodiment, the
cell is a mouse cell, e.g., a mouse ES cell. In one embodiment, the cell is homozygous for
the modified immunoglobulin locus.
(0092] In one aspect, a cell is provided, comprising a nucleic acid sequence encoding a
first polypeptide that comprises a first somatically mutated human VK or VA sequence fused
to a human heavy chain constant region sequence.
PCT/0S2011/046196
In one embodiment, the cell furher comprises a second polypeptide chain that
comprises a second somatically mutated human VK or VA sequence fused to a human light
chain constant region sequence.
[00 4] In one embodiment, the human VK or VA sequence of the first polypeptide is
cognate with the human VK or VA sequence of the second polypeptide.
[00 ] In one embodiment, the VK or VA of the first polypeptide and the human VK or VA
of the second polypeptide when associated specifically bind an antigen of interest. In a
specific embodiment, the first polypeptide comprises a variable domain consisting
essentially of a human VK, and the second polypeptide comprises a variable domain
consisting of a human VK that is cognate with the human VK of the first polypeptide, and the
human constant region sequence is an lgG sequence.
In one embodiment, the cell is selected from a CHO cell, a COS cell, a 293 cell, a
Hela cell, and a human retinal cell expressing a viral nucleic acid sequence (e.g., a
PERC.6 cell.
In one aspect, a somatic mouse cell is provided, comprising a chromosome that
comprises a genetic modification as described herein.
98] In one aspect, a mouse germ cell is provided, comprising a nucleic acid
sequence that comprises a genetic modification as described herein.
In one aspect, a pluripotent, induced pluripotent, or totipotent cell derived from a
mouse as described herein is provided. In a specific embodiment, the cell is a mouse
embryonic stem (ES) cell.
In one aspect, use of a cell as described herein for the manufacture of a mouse,
a cell, or a therapeutic protein (e.g., an antibody or other antigen-binding protein) is
provided.
In one aspect, a nucleic acid construct is provided that comprises a human DH
gene segment juxtaposed upstream and downstream with a 23-mer spaced RSS. In a
specific embodiment, the nucleic acid construct comprises a homology arm that is
homologous to a human genomic sequence comprising human VK gene segments. In one
embodiment, the targeting construct comprises all or substantially all human DH gene
segments each juxtaposed upstream and downstream with a 23-mer spaced RSS.
In one aspect, a nucleic acid construct is provided that comprises a human JK
gene segment juxtaposed upstream with a 12-mer spaced RSS. In a specific embodiment,
the nucleic acid construct comprises a first homology arm that contains homology to a
human genomic DH gene sequence that is juxtaposed upstream and downstream with a 23-
mer spaced RSS. In one embodiment, the nucleic acid construct comprises a second
homology arm that contains homology to a human genomic J gene sequence or that
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contains homology to a mouse heavy chain constant region sequence or that contains
homology to a J-C intergenic sequence upstream of a mouse constant region heavy chain
sequence.
In one aspect, a nucleic acid construct is provided that comprises a human VA
segment juxtaposed downstream with a 23-mer spaced RSS, a human D segment
juxtaposed upstream and downstream with a 12-mer spaced RSS, and a human J segment
selected from a JK segment juxtaposed upstream with a 23-mer spaced RSS, a human JA
segment juxtaposed upstream with a 23-mer spaced RSS, and a human J segment
juxtaposed upstream with a 23-mer spaced RSS. In one embodiment, the construct
comprises a homology arm that contains homology to a mouse constant region sequence, a
J-C intergenic mouse sequence, and/or a human VA sequence.
In one embodiment, the nucleic acid construct comprises a human " light chain
variable region sequence that comprises a fragment of cluster A of the human " light chain
locus. In a specific embodiment, the fragment of cluster A of the human " light chain locus
extends from hV"3-27 through hV"3-1.
In one embodiment, the nucleic acid construct comprises a human " light chain
variable region sequence that comprises a fragment of cluster B of the human " light chain
locus. In a specific embodiment, the fragment of cluster B of the human " light chain locus
extends from hV"S-52 through hV" 1-40.
In one embodiment, nucleic acid construct comprises a human " light chain
variable region sequence that comprises a genomic fragment of cluster A and a genomic
fragment of cluster B. In a one embodiment, the human " light chain variable region
sequence comprises at least one gene segment of cluster A and at least one gene segment
of cluster B.
In one embodiment, the human " light chain variable region sequence comprises
at least one gene segment of cluster B and at least one gene segment of cluster C.
In one aspect, a nucleic acid construct is provided, comprising a human D
segment juxtaposed upstream and downstream with a 23-mer spaced RSS normally found
in nature flanking either a JK, a J , a V", or a V segment. In one embodiment, the nucleic
acid construct comprises a first homology arm homologous to a human V-J intergenic region
or homologous to a human genomic sequence comprising a human V gene segment. In
one embodiment, the nucleic acid construct comprises a second homology arm homologous
to a human or mouse heavy chain constant region sequence. In a specific embodiment, the
human or mouse heavy chain constant region sequence is selected from a C
1, hinge, C 2,
C 3, and a combination thereof. In one embodiment, the nucleic acid construct comprises a
human J gene segment flanked upstream with a 12-mer RSS. In one embodiment, the
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nucleic acid construct comprises a second homology arm that contains homology to a J
gene segment flanked upstream with a 12-mer RSS. In one embodiment, the J gene
segment is selected from a human JK, a human JA, and a human JH,
In one aspect, a nucleic acid construct is provided that comprises a human DH
segment juxtaposed upstream and downstream with a 23-mer spaced RSS, and a site
specific recombinase recognition sequence, e.g., a sequence recognized by a site-specific
recombinase such as a Cre, a Flp, or a Dre protein.
[0011 0] In one aspect, a nucleic acid construct is provided that comprises a human VA or
a human VK segment, a DH segment juxtaposed upstream and downstream with a 12-mer or
a 23-mer spaced RSS, and a human J segment with a 12-mer or a 23-mer spaced RSS,
wherein the 12-mer or 23-mer spaced RSS is positioned immediately 5' to the human J
segment (i.e., with respect to the direction of transcription). In one embodiment, the
construct comprises a human VA juxtaposed with a 3' 23-mer spaced RSS, a human DH
segment juxtaposed upstream and downstream with a 12-mer spaced RSS, and a human JK
segment juxtaposed with a 5' 23-mer spaced RSS. In one embodiment, the construct
comprises a human VK juxtaposed with a 3' 12-mer spaced RSS, a human DH segment
juxtaposed upstream and downstream with a 23-mer spaced RSS, and a human JA segment
juxtaposed with a 5' 12-mer spaced RSS.
In one aspect, a targeting vector is provided, comprising (a) a first targeting arm
and a second targeting arm, wherein the first and second targeting arms are independently
selected from human and mouse targeting arms, wherein the targeting arms direct the
vector to an endogenous or modified immunoglobulin V region gene locus; and, (b) a
contiguous sequence of human VL gene segments or a contiguous sequence of human VL
gene segments and at least one human JK gene segment, wherein the contiguous sequence
is selected from the group consisting of (i) hVK4-1 through hVK1-6 and JK1, (ii) hVK4-1
through hVK1-6 and JK1 through JK2, (iii) hVK4-1 through hVK1-6 and JK1 through JK3, (iv)
hVK4-1 through hVK1-6 and JK1 through JK4, (v) hVK4-1 through hVK1-6 and JK1 through
JK5, (vi) hVK3-7 through hVK1-16, (vii) hVK1-17 through hVK2-30, (viii) hVK3-31 through
hVK2-40, and (ix) a combination thereof.
In one embodiment, the targeting arms that direct the vector to an endogenous or
modified immunoglobulin locus are identical or substantially identical to a sequence at the
endogenous or modified immunoglobulin locus.
In one aspect, use of a nucleic acid construct as described herein for the
manufacture of a mouse, a cell, or a therapeutic protein (e.g., an antibody or other antigen
binding protein) is provided.
PCT/0S2011/046196
In one aspect, use of a nucleic acid sequence from a mouse as described herein
to make a cell line for the manufacture of a human therapeutic is provided. In one
embodiment, the human therapeutic is a binding protein comprising a human light chain
variable sequence (e.g., derived from a human VA or human VK segment) fused with a
human heavy chain constant sequence. In one embodiment, the human therapeutic
comprises a first polypeptide that is a human " or K immunoglobulin light chain, and a
second polypeptide that comprises a human V" or human VK variable sequence fused with
a human heavy chain constant sequence.
In one aspect, an expression system is provided, comprising a mammalian cell
transfected with a DNA construct that encodes a polypeptide that comprises a somatically
mutated human V
domain fused with a human C domain.
In one embodiment, the expression system further comprises a nucleotide
sequence that encodes an immunoglobulin V domain fused with a human C domain,
wherein the V domain fused with the human C
domain is a cognate light chain with the V
L L L
domain fused with the human C domain.
In one embodiment, the mammalian cell is selected from a CHO cell, a COS cell,
a Vero cell, a 293 cell, and a retinal cell that expresses a viral gene (e.g., a PER.C6 cell).
In one aspect, a method for making a binding protein is provided, comprising
obtaining a nucleotide sequence encoding a V domain from a gene encoding a V region
fused to a C region from a cell of a mouse as described herein, and cloning the nucleotide
sequence encoding the V region sequence in frame with a gene encoding a human C
region to form a human binding protein sequence, expressing the human binding protein
sequence in a suitable cell.
In one embodiment, the mouse has been immunized with an antigen of interest,
and the V region fused to the C region specifically binds (e.g., with a Ko in the micromolar,
nanomolar, or picomolar range) an epitope of the antigen of interest. In one embodiment,
nucleotide sequence encoding the V region fused to the C region is somatically mutated in
the mouse.
In one embodiment, the suitable cell is selected from a B cell, a hybridoma,
quadroma, a CHO cell, a COS cell, a 293 cell, a Hela cell, and a human retinal cell
expressing a viral nucleic acid sequence (e.g., a PERC.6 cell).
In one embodiment, the CH region comprises a human lgG isotype. In a specific
embodiment, the human lgG is selected from an lgG1, lgG2, and lgG4. In another specific
embodiment, the human lgG is lgG1. In another specific embodiment, the human lgG is
lgG4. In another specific embodiment, the human lgG4 is a modified lgG4. In one
embodiment, the modified lgG4 comprises a substitution in the hinge region. In a specific
PCT/0S2011/046196
embodiment, the modified lgG4 comprises a substitution at amino acid residue 228 relative
to a wild-type human lgG4, numbered according to the EU numbering index of Kabat. In a
specific embodiment, the substitution at amino acid residue 228 is a S228P substitution,
numbered according to the EU numbering index of Kabat.
In one embodiment, the cell further comprises a nucleotide sequence encoding a
V domain from a light chain that is cognate to the V domain fused to the C region, and the
method further comprises expressing the nucleotide sequence encoding the cognate V
domain fused to a human CK or Ct domain.
In one aspect, a method for making a genetically modified mouse is provided,
comprising replacing at an endogenous mouse heavy chain locus one or more
immunoglobulin heavy chain gene segments of a mouse with one or more human
immunoglobulin light chain gene segments. In one embodiment, the replacement is of all or
substantially all functional mouse immunoglobulin heavy chain segments (i.e., V , D , and J
segments) with one or more functional human light chain segments (i.e., V and J
segments). In one embodiment, the replacement is of all or substantially all functional
mouse heavy chain VH, DH, and J segments with all or substantially all human VA or VK
segments and at least one JA or JK segment. In a specific embodiment, the replacement
includes all or substantially all functional human JA or JK segments.
In one aspect, a method is provided for making a mouse that expresses a
polypeptide that comprises a sequence derived from a human immunoglobulin VA or VK
and/or J, or JK segment fused with a mouse heavy chain constant region, comprising
replacing endogenous mouse heavy chain immunoglobulin variable segments (VH, D , and
J ) with at least one human VA or VK segment and at least one human JA or JK segment,
wherein the replacement is in a pluripotent, induced pluripotent, or totipotent mouse cell to
form a genetically modified mouse progenitor cell; the genetically modified mouse progenitor
cell is introduced into a mouse host; and, the mouse host comprising the genetically
modified progenitor cell is gestated to form a mouse comprising a genome derived from the
genetically modified mouse progenitor cell. In one embodiment, the host is an embryo. In a
specific embodiment, the host is selected from a mouse pre-morula (e.g., 8- or 4-cell stage),
a tetraploid embryo, an aggregate of embryonic cells, or a blastocyst.
In one aspect, a method is provided for making a genetically modified mouse as
described herein, comprising introducing by nuclear transfer a nucleic acid containing a
modification as described herein into a cell, and maintaining the cell under suitable
conditions (e.g., including culturing the cell and gestating an embryo comprising the cell in a
surrogate mother) to develop into a mouse as described herein.
PCT/0S2011/046196
In one aspect, a method for making a modified mouse is provided, comprising
modifying as described herein a mouse ES cell or pluripotent or totipotent or induced
pluripotent mouse cell to include one or more unrearranged immunoglobulin light chain
variable gene segments operably linked to an immunoglobulin heavy chain constant
sequence, culturing the ES cell, introducing the cultured ES cell into a host embryo to form a
chimeric embryo, and introducing the chimeric embryo into a suitable host mouse to develop
into a modified mouse. In one embodiment, the one or more unrearranged immunoglobulin
light chain variable region gene segments are human " or human K gene segments. In one
embodiment, the one or more unrearranged immunoglobulin light chain variable region gene
segments comprise human V" or human VK segments and one or more J", JK, or JH
segments. In one embodiment, the heavy chain constant gene sequence is a human
sequence selected from C 1, hinge, C
2, C 3, and a combination thereof. In one
H H H
embodiment, the one or more unrearranged immunoglobulin light chain variable gene
segments replace all or substantially all functional endogenous mouse heavy chain variable
region gene segments at the endogenous mouse heavy chain locus, and the heavy chain
constant sequence is a mouse sequence comprising a C 1, a hinge, a C 2, and a C 3,
In one aspect, an immunoglobulin variable region (VR) (e.g., comprising a human
VL sequence fused with a human JL, or J , or D and J , or D and JL) made in a mouse as
H H H
described herein is provided. In a specific embodiment, the immunoglobulin VR is derived
from a germline human gene segment selected from a VK segment and a V" segment,
wherein the VR is encoded by a rearranged sequence from the mouse wherein the
rearranged sequence is somatically hypermutated. In one embodiment, the rearranged
sequence comprises 1 to 5 somatic hypermutations. In one embodiment, the rearranged
sequence comprises at least 6, 7, 8, 9, or 1 O somatic hypermutations. In one embodiment,
the rearranged sequence comprises more than 10 somatic hypermutations. In one
embodiment, the rearranged sequence is fused with one or more human or mouse heavy
chain constant region sequences (e.g., selected from a human or mouse C 1, hinge, C
C 3, and a combination thereof).
In one aspect, an immunoglobulin variable domain amino acid sequence of a
binding protein made in a mouse as described herein is provided. In one embodiment, the
VR is fused with one or more human or mouse heavy chain constant region sequences
(e.g., selected from a human or mouse C 1, hinge, C 2, CH3, and a combination thereof).
In one aspect, a light chain variable domain encoded by a nucleic acid sequence
derived from a mouse as described herein is provided.
PCT/0S2011/046196
In one aspect, an antibody or antigen-binding fragment thereof (e.g., Fab, F(ab)2,
scFv) made in a mouse as described herein, or derived from a sequence made in a mouse
as described herein, is provided.
BRIEF DESCRIPTION OF THE FIGURES
illustrates a schematic (not to scale) of the mouse heavy chain locus.
The mouse heavy chain locus is about 3 Mb in length and contains approximately 200 heavy
chain variable (V ) gene segments, 13 heavy chain diversity (D ) gene segments and 4
heavy chain joining (J ) gene segments as well as enhancers (Enh) and heavy chain
constant (C ) regions.
B illustrates a schematic (not to scale) of the human K light chain locus.
The human K light chain locus is duplicated into distal and proximal contigs of opposite
polarity spanning about 440 kb and 600 kb, respectively. Between the two contigs is about
800 kb of DNA that is believed to be free of VK gene segments. The human K light chain
locus contains about 76 VK gene segments, 5 JK gene segments, an intronic enhancer
(Enh) and a single constant region (CK).
shows a targeting strategy for progressive insertion of 40 human VK and 5
human JK gene segments into the mouse heavy chain locus. Hygromycin (HYG) and
Neomycin (NEO) selection cassettes are shown with recombinase recognition sites (R1, R2,
etc.).
shows a modified mouse heavy chain locus comprising human VK and JK
gene segments operably linked to mouse C regions.
shows an exemplary targeting strategy for progressive insertion of
human VA and a single human JA gene segment into the mouse heavy chain locus.
Hygromycin (HYG) and Neomycin (NEO) selection cassettes are shown with recombinase
recognition sites (R1, R2, etc.).
shows an exemplary targeting strategy for progressive insertion of
human VA and four human JA gene segments into the mouse heavy chain locus.
Hygromycin (HYG) and Neomycin (NEO) selection cassettes are shown with recombinase
recognition sites (R1, R2, etc.).
shows an exemplary targeting strategy for progressive insertion of
human VA, human D and human JH gene segments into the mouse heavy chain locus.
Hygromycin (HYG) and Neomycin (NEO) selection cassettes are shown with recombinase
recognition sites (R1, R2, etc.).
shows an exemplary targeting strategy for progressive insertion of
human VA, human DH and human JK gene segments into the mouse heavy chain locus.
PCT/0S2011/046196
Hygromycin (HYG) and Neomycin (NEO) selection cassettes are shown with recombinase
recognition sites (R1, R2, etc.).
shows contour plots of splenocytes stained for surface expression of
B220 and lgM from a representative wild type (W) and a representative mouse
homozygous for six human VK and five human JK gene segments positioned at the
endogenous heavy chain locus (6hVK-5hJK HO).
shows contour plots of splenocytes gated on CD1 g B cells and stained
for immunoglobulin D (lgD) and immunoglobulin M (lgM) from a representative wild type
(W) and a representative mouse homozygous for six human VK and five human JK gene
segments positioned at the endogenous heavy chain locus (6hVK-5hJK HO).
shows the total number of CD1 g B cells, transitional B cells
hi i t int hi
(CD19 IgM lgD n ) and mature B cells (CD19 IgM lgD ) in harvested spleens from wild type
(W) and mice homozygous for six human VK and five human JK gene segments positioned
at the endogenous heavy chain locus (6hVK-5hJK HO).
A shows contour plots of bone marrow gated on singlets stained for
immunoglobulin M (lgM) and B220 from a wild type mouse (W) and a mouse homozygous
for six human VK and five human JK gene segments positioned at the endogenous heavy
chain locus (6hVK-5hJK HO). Immature, mature and pro/pre B cells are noted on each of
the dot plots.
shows the total number of pre/pro (B220 IgM), immature (B220 lgM )
and mature (B220 ilgM ) B cells in bone marrow isolated from the femurs of wild type mice
(W) and mice homozygous for six human VK and five human JK gene segments positioned
at the endogenous heavy chain locus (6hVK-5hJK HO).
shows contour plots of bone marrow gated on CD19 and stained for
ckit and CD43 from a wild type mouse (W) and a mouse homozygous for six human VK
and five human JK gene segments positioned at the endogenous heavy chain locus (6hVK-
5hJK HO). Pro and pre B cells are noted on each of the dot plots.
+ + +
shows the number of pro B (CD19 CD43 ckit} and pre B (CD19 CD43-
ckin cells in bone marrow harvested from the femurs of wild type mice (W) and mice
homozygous for six human VK and five human JK gene segments positioned at the
endogenous heavy chain locus (6hVK-5hJK HO).
shows contour plots of bone marrow gated on singlets stained for CD19
and CD43 from a wild type mouse (W) and a mouse homozygous for six human VK and
five human JK gene segments positioned at the endogenous heavy chain locus (6hVK-5hJK
HO). Immature, pre and pro B cells are noted on each of the dot plots.
PCT/0S2011/046196
+ int
shows histograms of bone marrow gated on pre B cells (CD19 co43 )
and expressing immunoglobulin M (lgM) from a wild type mouse (WT) and a mouse
homozygous for six human VK and five human JK gene segments positioned at the
endogenous heavy chain locus (6hVk-5hJk HO).
shows the number of lgM pre B cells (CD19
I9M co43 ) and immature
B cells (CD19 IgM CD43-) in bone marrow harvest from the femurs of wild type (WT) and
mice homozygous for six human VK and five human JK gene segments positioned at the
endogenous heavy chain locus (6hVK-5hJK HO).
FIG.SA shows contour plots of splenocytes gated on CD1 g and stained for 191
and lgK expression from a mouse containing a wild type heavy chain locus and a
replacement of the endogenous VK and JK gene segments with human VK and JK gene
segments (WT) and a mouse homozygous for thirty hVK and five JK gene segments at the
endogenous heavy chain locus and a replacement of the endogenous VK and JK gene
segments with human VK and JK gene segments (30hVK-5hJK HO).
int +
shows contour plots of bone marrow gated on immature (B220 lgM )
and mature (B220 lgM ) B cells stained for 1911 and lgK expression isolated from the femurs
of a mouse containing a wild type heavy chain locus and a replacement of the endogenous
VK and JK gene segments with human VK and JK gene segments (WT) and a mouse
homozygous for thirty hVK and five JK gene segments at the endogenous heavy chain locus
and a replacement of the endogenous VK and JK gene segments with human VK and JK
gene segments (30hVK-5hJK HO).
shows a nucleotide sequence alignment of the VK-JK-mlgG junction of
twelve independent RT-PCR clones amplified from splenocyte RNA of na've mice
homozygous for thirty hVK and five JK gene segments at the mouse heavy chain locus and a
replacement of the endogenous VK and JK gene segments with human VK and JK gene
segment. Lower case bases indicate non-germline bases resulting from either mutation
and/or N addition during recombination. Artificial spaces (periods) are included to properly
align the Framework 4 region and show alignment of the mouse heavy chain lgG nucleotide
sequence for lgG1, lgG2a/c, and lgG3 primed clones.
DETAILED DESCRIPTION
The phrase "bispecific binding protein" includes a binding protein capable of
selectively binding two or more epitopes. Bispecific binding proteins comprise two different
polypeptides that comprise a first light chain variable domain (V 1) fused with a first CH
region and a second light chain variable domain (V
2) fused with a second CH region. In
general, the first and the second CH regions are identical, or they differ by one or more
PCT/0S2011/046196
amino acid substitutions (e.g., as described herein). VL 1 and VL2 specifically binding
diferent epitopes-either on two different molecules (e.g., antigens) or on the same
molecule (e.g., on the same antigen). If a bispecific binding protein selectively binds two
different epitopes (a first epitope and a second epitope), the affinity of VL 1 for the first
epitope will generally be at least one to two or three or four orders of magnitude lower than
the affinity of VL 1 for the second epitope, and vice versa with respect to VL2. The epitopes
recognized by the bispecific binding protein can be on the same or a different target (e.g., on
the same or a different antigen). Bispecific binding proteins can be made, for example, by
combining a VL 1 and a VL2 that recognize diferent epitopes of the same antigen. For
example, nucleic acid sequences encoding VL sequences that recognize different epitopes
of the same antigen can be fused to nucleic acid sequences encoding different CH regions,
and such sequences can be expressed in a cell that expresses an immunoglobulin light
chain, or can be expressed in a cell that does not express an immunoglobulin light chain. A
typical bispecific binding protein has two heavy chains each having three light chain CDRs,
followed by (N-terminal to C-terminal) a CH1 domain, a hinge, a CH2 domain, and a CH3
domain, and an immunoglobulin light chain that either does not confer antigen-binding
specificity but that can associate with each heavy chain, or that can associate with each
heavy chain and that can bind one or more of the epitopes bound by VL 1 and/or VL2, or that
can associate with each heavy chain and enable binding or assist in binding of one or both
of the heavy chains to one or both epitopes.
Therefore, two general types of bispecific binding proteins are (1) VL 1-CH(dimer),
and (2) VL 1-CH:light chain + VL2-CH:light chain, wherein the light chain is the same or
different. In either case, the CH (i.e., the heavy chain constant region) can be differentially
modified (e.g., to differentially bind protein A, to increase serum half-life, etc.) as described
herein, or can be the same.
The term "cell," when used in connection with expressing a sequence, includes
any cell that is suitable for expressing a recombinant nucleic acid sequence. Cells include
those of prokaryotes and eukaryotes (single-cell or multiple-cell), bacterial cells (e.g., strains
of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria cells, fungal cells, yeast cells
(e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells
(e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal
cells, human cells, B cells, or cell fusions such as, for example, hybridomas or quadromas.
In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In
some embodiments, the cell is eukaryotic and is selected from the following cells: CHO
(e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CV1,
kidney (e.g., HEK293, 293 EBNA, MSR 293, MOCK, HaK, BHK), Hela, HepG2, Wl38, MRC
, Colo205, HB 8065, HL-60, (e.g., BHK21 ), Jurkat, Daudi, A431 (epidermal), CV-1, U937,
PCT/0S2011/046196
3T3, L cell, C127 cell, SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell,
myeloma cell, tumor cell, and a cell line derived from an aforementioned cell. In some
embodiments, the cell comprises one or more viral genes, e.g. a retinal cell that expresses a
viral gene (e.g., a PER.C6 cell).
The term "cognate," when used in the sense of "cognate with," e.g., a first V
domain that is "cognate with" a second V domain, is intended to include reference to the
relation between two V domains from a same binding protein made by a mouse in
accordance with the invention. For example, a mouse that is genetically modified in
accordance with an embodiment of the invention, e.g., a mouse having a heavy chain locus
in which V , D , and J regions are replaced with VL and J regions, makes antibody-like
H H H L
binding proteins that have two identical polypeptide chains made of the same mouse C
region (e.g., an lgG isotype) fused with a first human V domain, and two identical
polypeptide chains made of the same mouse C region fused with a second human V
domain. During clonal selection in the mouse, the first and the second human V domains
were selected by the clonal selection process to appear together in the context of a single
antibody-like binding protein. Thus, first and second V domains that appear together, as
the result of the clonal selection process, in a single antibody-like molecule are referred to as
being "cognate." In contrast, a V domain that appears in a first antibody-like molecule and a
V domain that appears in a second antibody-like molecule are not cognate, unless the first
and the second antibody-like molecules have identical heavy chains (i.e., unless the V
domain fused to the first human heavy chain region and the V domain fused to the second
human heavy chain region are identical).
The phrase "complementarity determining region," or the term "CDR," includes an
amino acid sequence encoded by a nucleic acid sequence of an organism's immunoglobulin
genes that normally (i.e., in a wild-type animal) appears between two framework regions in a
variable region of a light or a heavy chain of an immunoglobulin molecule (e.g., an antibody
or a T cell receptor). A CDR can be encoded by, for example, a germline sequence or a
rearranged or unrearranged sequence, and, for example, by a na"ve or a mature B cell or a
T cell. In some circumstances (e.g., for a CDR3), CDRs can be encoded by two or more
sequences (e.g., germline sequences) that are not contiguous (e.g., in an unrearranged
nucleic
acid sequence) but are contiguous in a B cell nucleic acid sequence, e.g., as the
result of splicing or connecting the sequences (e.g., V-D-J recombination to form a heavy
chain CDR3).
The phrase "gene segment," or "segment" includes reference to a V (light or
heavy) or D or J (light or heavy) immunoglobulin gene segment, which includes
unrearranged sequences at immunoglobulin loci (in e.g., humans and mice) that can
participate in a rearrangement (mediated by, e.g., endogenous recombinases) to form a
PCT/0S2011/046196
rearranged V/J or V/O/J sequence. Unless indicated otherwise, the V, D, and J segments
comprise recombination signal sequences (RSS) that allow for V/J recombination or V/O/J
recombination according to the 12/23 rule. Unless indicated otherwise, the segments further
comprise sequences with which they are associated in nature or functional equivalents
thereof (e.g., for V segments promoter(s) and leader(s)).
The phrase "heavy chain," or "immunoglobulin heavy chain" includes an
immunoglobulin heavy chain constant region sequence from any organism, and unless
otherwise specified includes a heavy chain variable domain (VH)- V domains include three
heavy chain CDRs and four framework (FR) regions, unless otherwise specified. Fragments
of heavy chains include CDRs, CDRs and FRs, and combinations thereof. A typical heavy
chain consists essentially of, following the variable domain (from N-terminal to C-terminal), a
1 domain, a hinge, a C 2 domain, a C 3 domain, and optionally a C 4 domain (e.g., in the
H H H H
of lgM or lgE) and a transmembrane (M) domain (e.g., in the case of membrane-bound
case
immunoglobulin on lymphocytes). A heavy chain constant region is a region of a heavy
chain that extends (from N-terminal side to C-terminal side) from outside FR4 to the C
terminal of the heavy chain. Heavy chain constant regions with minor deviations, e.g.,
truncations of one, two, three or several amino acids from the C-terminal, would be
encompassed by the phrase "heavy chain constant region," as well as heavy chain constant
regions with sequence modifications, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
substitutions. Amino acid substitutions can be made at one or more positions selected from,
e.g. (with reference to EU numbering of an immunoglobulin constant region, e.g., a human
lgG constant region), 228, 233, 234, 235, 236, 237, 238, 239, 241, 248, 249, 250, 252, 254,
255,256,258,265,267,268,269,270,272,276,278,280,283,285,286,289,290,292,
293,294,295,296,297,298,301,303,305,307,308,3 09,311,312,315,318,320,322,
324,326,327,328,329,330,331,332,333,334,335,337,338,33 9,340,342,344,356,
358,359,360,361,362,373,375,376,378,380,382,383,384,386,388,389,398,414,
416, 419,428,430,433,434,435,437,438, and 439.
For example, and not by way of limitation, a heavy chain constant region can be
modified to exhibit enhanced serum half-life (as compared with the same heavy chain
constant region without the recited modification(s)) and have a modification at position 250
(e.g., E or Q); 250 and 428 (e.g., Lor F); 252 (e.g., L/Y/F/W or T), 254 (e.g., Sor T), and
256 (e.g., S/R/Q/E/O or T); or a modification at 428 and/or 433 (e.g., L/R/S1/P/Q or K) and/or
434 (e.g., H/F or Y); or a modification at 250 and/or 428; or a modification at 307 or 308
(e.g., 308F, V308F), and 434. In another example, the modification can comprise a 428L
(e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591 (e.g., V259I), and a 308F
(e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252,
PCT/0S2011/046196
254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 2500 and 428L modification
(e.g., T250Q and M428L); a 307 and/or 308 modification (e.g., 308F or 308P).
The phrase "light chain" includes an immunoglobulin light chain constant (C )
region sequence from any organism, and unless otherwise specified includes human K and
" light chains. Light chain variable (V ) domains typically include three light chain CDRs and
four framework (FR) regions, unless otherwise specified. Generally, a full-length light chain
(V + C ) includes, from amino terminus to carboxyl terminus, a V domain that includes
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a CL region. Light chains (V + C ) that can be
used with this invention include those, e.g., that do not selectively bind either a first or
second (in the case of bispecific binding proteins) epitope selectively bound by the binding
protein (e.g., the epitope(s) selectively bound by the V domain fused with the C domain).
V domains that do not selectively bind the epitope(s) bound by the V that is fused with the
C domain include those that can be identified by screening for the most commonly
employed light chains in existing antibody libraries (wet libraries or in silica), wherein the
light chains do not substantially interfere with the affinity and/or selectivity of the epitope
binding domains of the binding proteins. Suitable light chains include those that can bind
(alone or in combination with its cognate V fused with the C region) an epitope that is
specifically bound by the V fused to the C region.
The phrase "micromolar range" is intended to mean 1-999 micromolar; the
phrase "nanomolar range" is intended to mean 1-999 nanomolar; the phrase "picomolar
range" is intended to mean 1-999 picomolar.
The term "non-human animals" is intended to include any vertebrate such as
cyclostomes, bony fish, cartilaginous fish such as sharks and rays, amphibians, reptiles,
mammals, and birds. Suitable non-human animals include mammals. Suitable mammals
include non-human primates, goats, sheep, pigs, dogs, cows, and rodents. Suitable non
human animals are selected from the rodent family including rat and mouse. In one
embodiment, the non-human animals are mice.
Mice, Nucleotide Sequences, and Binding Proteins
Binding proteins are provided that are encoded by elements of immunoglobulin
loci, wherein the binding proteins comprise immunoglobulin heavy chain constant regions
fused with immunoglobulin light chain variable domains. Further, multiple strategies are
provided to genetically modify an immunoglobulin heavy chain locus in a mouse to encode
binding proteins that contain elements encoded by immunoglobulin light chain loci. Such
genetically modified mice represent a source for generating unique populations of binding
PCT/0S2011/046196
proteins that have an immunoglobulin structure, yet exhibit an enhanced diversity over
traditional antibodies.
[00164) Binding protein aspects described herein include binding proteins that are
encoded by modified immunoglo_bulin loci, which are modified such that gene segments that
normally (i.e., in a wild-type animal) encode immunoglobulin light chain variable domains (or
portions thereof) are operably linked to nucleotide sequences that encode heavy chain
constant regions. Upon rearrangement of the light chain gene segments, a rearranged
nucleotide sequence is obtained that comprises a sequence encoding a light chain variable
domain fused with a sequence encoding a heavy chain constant region. This sequence
encodes a polypeptide that has an immunoglobulin light chain variable domain fused with a
heavy chain constant region. Thus, in one embodiment, the polypeptide consists essentially
of, from N-terminal to C-terminal, a VL domain, a CH1 region, a hinge, a CH2 region, a C
region, and optionally a CH4 region.
In modified mice described herein, such binding proteins are made that also
comprise a cognate light chain, wherein in one embodiment the cognate light chain pairs
with the polypeptide described above to make a binding protein that is antibody-like, but the
binding protein comprises a VL region-not a VH region-fused to a C region.
In various embodiments, the modified mice make binding proteins that comprise
a VL region fused with a CH region (a hybrid heavy chain), wherein the V region of the hybrid
heavy chain exhibits an enhanced degree of somatic hypermutation. In these embodiments,
the enhancement is over a V region that is fused with a CL region (a light chain). In some
embodiments, a VL region of a hybrid heavy chain exhibits about 1.5-fold, 2-fold, 2.5-fold, 3-
fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold or more somatic hypermutations than a V region
fused with a C region. In some embodiments, the modified mice in response to an antigen
exhibit a population of binding proteins that comprise a V region of a hybrid heavy chain,
wherein the population of binding proteins exhibits an average of about 1.5-fold, 2-fold, 2.5-
fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold or more somatic hypermutations in the V region
of the hybrid heavy chain than is observed in a wild-type mouse in response to the same
antigen. In one embodiment, the somatic hypermutations in the V region of the hybrid
heavy chain comprise one or more or two or more N additions in a CDR3.
In various embodiments, the binding proteins comprise variable domains
encoded by immunoglobulin light chain sequences that comprise a larger number of N
additions than observed in nature for light chains rearranged from an endogenous light chain
locus, e.g., a binding protein comprising a mouse heavy chain constant region fused with a
variable domain derived from human light chain V gene segments and human (light or
heavy) J gene segments, wherein the human V and human J segments rearrange to form a
rearranged gene that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.
PCT/0S2011/046196
(00168] In various embodiments, the mice of the invention make binding proteins that are
on average smaller than wild-type antibodies (i.e., antibodies that have a V domain), and
possess advantages associated with smaller size. Smaller size is realized at least in part
through the absence of an amino acid sequence encoded by a D region, normally present
in a V domain. Smaller size can also be realized in the formation of a CDR3 that is derived,
e.g., from a VK region and a JK region.
(00169] In another aspect, a mouse and a method is provided for providing a population
of binding proteins having somatically hypermutated V domains, e.g., somatically mutated
human VK domains, and, e.g., human VK domains encoded by rearranged K variable genes
that comprise 1-10 or more N additions. In one embodiment, in the absence of a V region
for generating antibody diversity, a mouse of the invention will generate binding proteins,
e.g., in response to challenge with an antigen, whose V domains are only or substantially V
domains. The clonal selection process of the mouse therefore is limited to selecting only or
substantially from binding proteins that have V domains, rather than V domains. Somatic
hypermutation of the V domains will be as frequent, or substantially more frequent (e.g., 2-
to 5-fold higher, or more), than in wild-type mice (which also mutate V domains with some
frequency). The clonal selection process in a mouse of the invention will generate high
affinity binding proteins from the modified immunoglobulin locus, including binding proteins
that specifically bind an epitope with an affinity in the nanomolar or picomolar range.
Sequences that encode such binding proteins can be used to make therapeutic binding
proteins containing human variable regions and human constant regions using an
appropriate expression system.
(00170] In other embodiments, a mouse according to the invention can be made wherein
the mouse heavy chain and/or light chain immunoglobulin loci are disabled, rendered non
functional, or knocked out, and fully human or chimeric human-mouse transgenes can be
placed in the mouse, wherein at least one of the transgenes contains a modified heavy chain
locus (e.g., having light chain gene segments operably linked to one or more heavy chain
gene sequences). Such a mouse may also make a binding protein as described herein.
(00171] In one aspect, a method is provided for increasing the diversity, including by
somatic hypermutation or by N additions in a V domain, comprising placing an
unrearranged light chain V gene segment and an unrearranged J gene segment in operable
linkage with a mouse C gene sequence, exposing the animal to an antigen of interest, and
isolating from the animal a rearranged and somatically hypermutated V(light)/J gene
sequence of the animal, wherein the rearranged V(light)/J gene sequence is fused with a
nucleotide sequence encoding an immunoglobulin C region.
PCT/0S2011/046196
In one embodiment, the immunoglobulin heavy chain fused with the
hypermutated V is an lgM; in another embodiment, an lgG; in another embodiment, an lgE;
in another embodiment, an lgA.
In one embodiment, the somatically hypermutated and class-switched VL domain
contains about 2- to 5-fold or more of the somatic hypermutations observed for a rearranged
and class-switched antibody having a V sequence that is operably linked to a CL sequence.
In one embodiment, the observed somatic hypermutations in the somatically hypermutated
V domain are about the same in number as observed in a VH domain expressed from a VH
gene fused to a CH region.
[0017 4] In one aspect, a method for making a high-affinity human VL domain is provided,
comprising exposing a mouse of the invention to an antigen of interest, allowing the mouse
to develop an immune response to the antigen of interest, and isolating a somatically
mutated, class-switched human V domain from the mouse that specifically binds the
antigen of interest with high affinity.
In one embodiment, the Ko of a binding protein comprising the somatically
mutated, class-switched human V domain is in the nanomolar or picomolar range.
In one embodiment, the binding protein consists essentially of a polypeptide
dimer, wherein the polypeptide consists essentially of the somatically mutated, class
switched binding protein comprising a human V domain fused with a human CH region.
In one embodiment, the binding protein consists essentially of a polypeptide
dimer and two light chains, wherein the polypeptide consists essentially of the somatically
mutated, class-switched binding protein having a human V domain fused with a human CH
region; and wherein each polypeptide of the dimer is associated with a cognate light chain
comprising a cognate light chain V domain and a human C region.
In one aspect, a method is provided for somatically hypermutating a human V
gene sequence, comprising placing a human VL gene segment and a human J gene
segment in operable linkage with an endogenous mouse CH gene at an endogenous mouse
heavy chain immunoglobulin locus, exposing the mouse to an antigen of interest, and
obtaining from the mouse a somatically hypermutated human V gene sequence that binds
the antigen of interest.
In one embodiment, the method further comprises obtaining from the mouse a V
gene sequence from a light chain that is cognate to the human somatically hypermutated
human V
gene sequence that binds the antigen of interest.
V Binding Proteins with DH Sequences
In various aspects, mice comprising an unrearranged immunoglobulin light chain
V gene segment and an unrearranged (e.g., light or heavy) J gene segment also comprise
PCT/0S2011/046196
an unrearranged DH gene segment that is capable of recombining with the J segment to
form a rearranged D/J sequence, which in turn is capable of rearranging with the light chain
V segment to form a rearranged variable sequence derived from (a) the light chain V
segment,
(b) the DH segment, and (c) the (e.g., light or heavy) J segment; wherein the
rearranged variable sequence is operably linked to a heavy chain constant sequence (e.g.,
selected from CH1, hinge, CH2, CH3, and a combination thereof; e.g., operably linked to a
mouse or human CH1, a hinge, a CH2, and a CH3).
In various aspects, mice comprising unrearranged human light chain V segments
and J segments that also comprise a human D segment are useful, e.g., as a source of
increased diversity of CDR3 sequences. Normally, CDR3 sequences arise in light chains
from V/J recombination, and in heavy chains from V/D/J recombination. Further diversity is
provided by nucleotide additions that occur during recombination (e.g., N additions), and
also as the result of somatic hypermutation. Binding characteristics conferred by CDR3
sequences are generally limited to those conferred by the light chain CDR3 sequence, the
heavy chain CDR3 sequence, and a combination of the light and the heavy chain CDR3
sequence, as the case may be. In mice as described herein, however, an added source of
diversity is available due to binding characteristics conferred as the result of a combination
of a first light chain CDR3 (on the heavy chain polypeptide) and a second light chain CDR3
(on the light chain polypeptide). Further diversity is possible where the first light chain CDR3
may contain a sequence derived from a D gene segment, as from a mouse as described
herein that comprises an unrearranged V segment from a light chain V region operably
linked to a D segment and operably linked to a J segment (light or heavy), employing the
RSS engineering as taught here.
Another source of diversity is the N and/or P additions that can occur in the
V(light)/J or V(light)/D/J recombinations that are possible in mice as described. Thus, mice
described herein not only provide a diferent source of diversity (light chain-light chain) but
also a further source of diversity due to the addition of, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1 O or
more N additions in a rearranged V(light)/J or a rearranged V(light)/D/J gene in a mouse as
described herein.
In various aspects the use of a D gene segment operably linked to a J gene
segment and a light chain V gene segment provides an enhanced diversity. Operable
linkage of a D segment in this instance will require that that D segment is capable of
recombining with the J segment with which it is recited. Thus, the D segment will require to
have juxtaposed a downstream RSS that is matched to the RSS juxtaposed upstream of the
J segment such that the D segment and the J segment may rearrange. Further, the D
segment will require an appropriate RSS juxtaposed upstream that is matched to the RSS
PCT/0S2011/046196
juxtaposed downstream of the V segment such that the rearranged D/J segment and the V
segment may rearrange to form a gene encoding a variable domain.
An RSS, or a recombination signal sequence, comprises a conserved nucleic
acid heptamer sequence separated, by 12 base pairs (bp) or 23 base pairs (bp) of
a conserved nucleic acid nonamer sequence. RSS's are used
unconserved sequence, from
by recombinases to achieve joining of immunoglobulin gene segments during the
rearrangement process following the 12/23 rule. According to the 12/23 rule, a gene
segment juxtaposed with an RSS having a 12 bp (unconserved) spacer rearranges with a
gene segment juxtaposed with an RSS having a 23 bp (unconserved) spacer; i.e.,
rearrangements between gene segments each having an RSS with a 12 bp spacer, or each
having an RSS with a 23 bp spacer, are generally not observed.
In the case of the A light chain locus, variable gene segments (VA gene
segments) are flanked downstream (with respect to the direction of transcription of the V
sequence) with an RSS having a 23-mer spacer, and joining gene segments (Jt. gene
segments) are flanked upstream (with respect to the direction of transcription of the J
sequence) with an RSS having a 12-mer spacer. Thus, Vt and Jt segments are flanked
with RSS's that are compatible under the 12/23 rule, and therefore are capable of recombine
during rearrangement.
At the K locus in a wild-type organism, however, each functional VK segment is
flanked downstream with an RSS having a 12-mer spacer. JK segments, therefore, have
23-mer spaces juxtaposed on the upstream side of the JK segment. At the heavy chain
locus, V gene segments are juxtaposed downstream with an RSS having a 23-mer spacer,
followed by D segment juxtaposed upstream and downstream with a 12-mer spacer, and J
segments each with a 23-mer segment juxtaposed on the upstream side of the J segment.
At the heavy chain locus, D/J recombination occurs first, mediated by the downstream
RSS with the 12-mer spacer and the upstream J RSS with the 23-mer spacer, to yield an
intermediate rearranged D-J sequence having an RSS juxtaposed on the upstream side that
has an RSS with a 12-mer spacer. The rearranged D-J segment having the RSS with the
12-mer juxtaposed on the upstream side then rearranges with the V
segment having the
RSS with the 23-mer juxtaposed on its downstream side to form a rearranged V/D/J
sequence.
In one embodiment, a Vt segment is employed at the heavy chain locus with a J
gene segment that is a Jt. segment, wherein the Vt. segment comprises an RSS juxtaposed
on the downstream side of the Vt sequence, and the RSS comprises a 23-mer spacer, and
the J segment is a Jt segment with an RSS juxtaposed on its upstream side having a 12-
mer spacer (e.g., as found in nature).
PCT/0S2011/046196
In one embodiment, a VA segment is employed at the heavy chain locus with a J
gene segment that is a JK or a J
gene segment, wherein the VA sequence has juxtaposed
on its downstream side an RSS comprising a 23-mer spacer, and the JK or J segment has
juxtaposed on its upstream side an RSS comprising a 12-mer spacer.
In one embodiment, a VA segment is employed at the heavy chain locus with a
D gene segment and a J gene segment. In one embodiment, the VA segment comprises
an RSS juxtaposed on the downstream side of the VA sequence with an RSS having a 23-
mer spacer; the D segment comprises an RSS juxtaposed on the upstream side and on the
downstream side of the DH sequence with an RSS having a 12-mer spacer; and a J segment
having an RSS juxtaposed on its upstream side having a 23-mer spacer, wherein the J
segment is selected from a JA, a JK, and a J ,
In one embodiment, a VK segment is employed at the heavy chain locus with a J
gene segment (with no intervening D segment), wherein the VK segment has an RSS
juxtaposed on the downstream side of the VK segment that comprises a 12-mer spaced
RSS, and the J segment has juxtaposed on its upstream side a 23-mer spaced RSS, and
the JK segment is selected from a JK segment, a JA segment, and a J segment. In one
embodiment, the V segment and/or the J segment are human.
In one embodiment, the VK segment is employed at the heavy chain locus with a
D segment and a J segment, wherein the VK segment has an RSS juxtaposed on the
downstream side of the VK segment that comprises a 12-mer spaced RSS, the D segment
has juxtaposed on its upstream and downstream side a 23-mer spaced RSS, and the J
segment has juxtaposed on its upstream side a 12-mer spaced RSS. In one embodiment,
the J segment is selected from a JK segment, a JA segment, and a J segment. In one
embodiment, the V segment and/or the J segment are human.
(00192] A JA segment with an RSS having a 23-mer spacer juxtaposed at its upstream
end, or a JK or J
segment with an RSS having a 12-mer spacer juxtaposed at its upstream
end, is made using any suitable method for making nucleic acid sequences that is known in
the art. A suitable method for making a J segment having an RSS juxtaposed upstream
wherein the RSS has a selected spacer (e.g., either 12-mer or 23-mer) is to chemically
synthesize a nucleic acid comprising the heptamer, the nonamer, and the selected spacer
and fuse it to a J segment sequence that is either chemically synthesized or cloned from a
suitable source (e.g., a human sequence source), and employ the fused J segment
sequence and RSS in a targeting vector to target the RSS-J to a suitable site.
A D segment with a 23-mer spaced RSS juxtaposed upstream and downstream
can be made by any method known in the art. One method comprises chemically
synthesizing the upstream 23-mer RSS and D segment sequence and the downstream 23-
PCT/0S2011/046196
mer RSS, and placing the RSS-flanked D segment in a suitable vector. The vector may be
directed to replace one or more mouse D segments with a human D segment with 12-mer
RSS sequences juxtaposed on the upstream and downstream sides, or directed to be
inserted into, e.g., a humanized locus at a position between a human V segment and a
human or mouse J segment.
Suitable nonamers and heptamers for RSS construction are known in the art
(e.g., see Janeway's lmmunobiology, 7th ed., Murphy et al., (2008, Garland Science, Taylor
& Francis Group, LLC) at page 148, Fig. 4.5, incorporated by reference). Suitable
nonconserved spacer sequences include, e.g., spacer sequences observed in RSS
sequences at human or mouse immunoglobulin loci.
Bispecific-Binding Proteins
The binding proteins described herein, and nucleotide sequences encoding them,
can be used to make multispecific binding proteins, e.g., bispecific binding proteins. In this
aspect, a first polypeptide consisting essentially of a first V domain fused with a C region
can associate with a second polypeptide consisting essentially of a second V domain fused
domain and the second V
with a C region. Where the first V domain specifically bind a
H L L
different epitope, a bispecific-binding molecule can be made using the two VL domains. The
C region can be the same or different. In one embodiment, e.g., one of the C
regions can
be modified so as to eliminate a protein A binding determinant, whereas the other heavy
chain constant region is not so modified. This particular arrangement simplifies isolation of
the bispecific binding protein from, e.g., a mixture of homodimers (e.g., homodimers of the
first or the second polypeptides).
In one aspect, the methods and compositions described herein are used to make
bispecific-binding proteins. In this aspect, a first VL that is fused to a C region and a second
V that is fused to a C region are each independently cloned in frame with a human lgG
sequence of the same isotype (e.g., a human lgG1, lgG2, lgG3, or lgG4). The first V
specifically binds a first epitope, and the second V specifically binds a second epitope. The
first and second epitopes may be on different antigens, or on the same antigen.
In one embodiment, the lgG isotype of the C region fused to the first VL and the
lgG isotype of the C region fused to the second V are the same isotype, but differ in that
one lgG isotype comprises at least one amino acid substitution. In one embodiment, the at
least one amino acid substitution renders the heavy chain bearing the substitution unable or
substantially unable to bind protein A as compared with the heavy chain that lacks the
substitution.
In one embodiment, the first C region comprises a first C 3 domain of a human
lgG selected from lgG1, lgG2, and lgG4; and the second C region comprises a second C 3
PCT/0S2011/046196
domain of a human lgG selected from lgG1, lgG2, and lgG4, wherein the second C 3
domain comprises a modification that reduces or eliminates binding of the second C 3
domain to protein A
In one embodiment, the second C 3 domain comprises a 435R modification,
numbered according to the EU index of Kabat. In another embodiment, the second C
domain further comprises a 436F modification, numbered according to the EU index of
Kabat.
In one embodiment, the second C 3 domain is that of a human lgG1 that
comprises a modification selected from the group consisting of D356E, L358M, N384S,
K392N, V397M, and V422I, numbered according to the EU index of Kabat.
In one embodiment, the second C 3 domain is that of a human lgG2 that
comprises a modification selected from the group consisting of N384S, K392N, and V422I,
numbered according to the EU index of Kabat.
In one embodiment, the second C 3 domain is that of a human lgG4 comprising a
modification selected from the group consisting of Q355R, N384S, K392N, V397M, R409K,
E419Q, and V422I, numbered according to the EU index of Kabat.
In one embodiment, the binding protein comprises C regions having one or more
modifications as recited herein, wherein the constant region of the binding protein is
nonimmunogenic or substantially nonimmunogenic in a human. In a specific embodiment,
the C regions comprise amino acid sequences that do not present an immunogenic epitope
in a human. In another specific embodiment, the binding protein comprises a C region that
is not found in a wild-type human heavy chain, and the C region does not comprise a
sequence that generates a T-cell epitope.
EXAMPLES
The following examples are provided so as to describe how to make and use
methods and compositions of the invention, and are not intended to limit the scope of what
the inventors regard as their invention. Unless indicated otherwise, temperature is indicated
in Celsius, and pressure is at or near atmospheric.
Example I
Introduction of Light Chain Gene Segments Into A Heavy Chain Locus
Various targeting constructs were made using VELOCIGENE® genetic
engineering technology (see, e.g., US Pat. No. 6,586,251 and Valenzuela, D.M., Murphy,
AJ., Frendewey, D., Gale, N.W., Economides, AN., Auerbach, W., Poueymirou, W.T.,
Adams, N.C., Rojas, J., Yasenchak, J., Chernomorsky, R., Boucher, M., Elsasser, AL.,
Esau, L., Zheng, J., Grifiths, J.A, Wang, X., Su, H., Xue, Y., Dominguez, M.G., Noguera, I.,
PCT/0S2011/046196
Torres, R., Macdonald, L.E., Stewart, AF., DeChiara, T.M., Yancopoulos, G.D. (2003). High
throughput engineering of the mouse genome coupled with high-resolution expression
analysis. Nat Biotechnol 21, 652-659) to modify mouse genomic Bacterial Artificial
Chromosome (BAC) libraries. Mouse BAC DNA was modified by homologous
recombination to inactivate the endogenous mouse heavy chain locus through targeted
deletion of V , D and J gene segments for the ensuing insertion of unrearranged human
H H H
germline K light chain gene sequences (top of FIG 2).
(00206] Briefly, the mouse heavy chain locus was deleted in two successive targeting
events using recombinase-mediated recombination. The first targeting event included a
targeting at the 5' end of the mouse heavy chain locus using a targeting vector comprising
from 5' to 3' a 5' mouse homology arm, a recombinase recognition site, a neomycin cassette
and a 3' homology arm. The 5' and 3' homology arms contained sequence 5' of the mouse
heavy chain locus. The second targeting event included a targeting at the 3' end of the
mouse heavy chain locus in the region of the J gene segments using a second targeting
vector that contained from 5' to 3' a 5' mouse homology arm, a 5' recombinase recognition
site, a second recombinase recognition site, a hygromycin cassette, a third recombinase
recognition site, and a 3' mouse homology arm. The 5' and 3' homology arms contained
sequence flanking the mouse J gene segments and 5' of the intronic enhancer and
constant regions. Positive ES cells containing a modified heavy chain locus targeted with
both targeting vectors (as described above) were confirmed by karyotyping. DNA was then
isolated from the double-targeted ES cells and subjected to treatment with a recombinase
thereby mediating the deletion of genomic DNA of the mouse heavy chain locus between the
' recombinase recognition site in the first targeting vector and the 5' recombinase
recognition site in the second targeting vector, leaving a single recombinase recognition site
and the hygromycin cassette flanked by two recombinase recognition sites (see top of . Thus a modified mouse heavy chain locus containing intact C genes was created for
progressively inserting human K germline gene segments in a precise manner using
targeting vectors described below.
Four separate targeting vectors were engineered to progressively insert 40 human
VK gene segments and five human JK gene segments into the inactivated mouse heavy
chain locus (described above) using standard molecular techniques recognized in the art
(. The human K gene segments used for engineering the four targeting constructs
are naturally found in proximal contig of the germ line human K light chain locus (B and
Table 1).
A ~110,499 bp human genomic fragment containing the first six human VK gene
segments and five human JK gene segments was engineered to contain a Pl-Seel site 431
PCT/0S2011/046196
bp downstream (3') of the human JK5 gene segment. Another Pl-Seel site was engineered
at the 5' end of a ~7,852 bp genomic fragment containing the mouse heavy chain intronic
enhancer, the lgM switch region (Sµ) and the lgM gene of the mouse heavy chain locus.
This mouse fragment was used as a 3' homology arm by ligation to the ~110.5 kb human
fragment, which created a 3' junction containing, from 5' to 3', ~110.5 kb of genomic
sequence of the human K light chain locus containing the first six consecutive VK gene
segments and five JK gene segments, a Pl-Seel site, ~7,852 bp of mouse heavy chain
sequence containing the mouse intronic enhancer, Sµ and the mouse lgM constant gene.
Upstream (5') from the human VK1-6 gene segment was an additional 3,710 bp of human K
sequence
before the start of the 5' mouse homology arm, which contained 19,752 bp of
mouse genomic DNA corresponding to sequence 5' of the mouse heavy chain locus.
Between the 5' homology arm and the beginning of the human K sequence was a neomycin
cassette flanked by three recombinase recognition sites (see Targeting Vector 1, .
The final targeting vector for the first insertion of human K sequence from 5' to 3' included a
' homology arm containing ~20 kb of mouse genomic sequence 5' of the heavy chain locus,
a first recombinase recognition site (R1 ), a neomycin cassette, a second recombinase
recognition site (R2), a third recombinase recognition site (R3), ~110.5 kb of human genomic
K sequence containing the first six consecutive human VK gene segments and five human
JK gene segments, a Pl-Seel site, and a 3' homology arm containing ~8 kb of mouse
genomic sequence including the intronic enhancer, Sµ and the mouse lgM constant gene
(see Targeting Vector 1 ). Homologous recombination with this targeting vector
created a modified mouse heavy chain locus containing six human VK gene segments and
five human JK gene segments operably linked to the endogenous mouse heavy chain
constant genes which, upon recombination, leads to the formation of a hybrid heavy chain
(i.e., a human VK domain and a mouse CH region).
PCT/0S2011/046196
Table 1
Human K Gene Segments Added
Size of
Targeting
Vector Human K Sequence
~110.5 kb 4-1, 5-2, 7-3, 2-4, 1-5, 1-6 1-5
3-7, 1-8, 1-9, 2-10, 3-11,
2 ~140 kb
1-12, 1-13, 2-14, 3-15, 1-16
1-17, 2-18, 2-19, 3-20, 6-21,
3 ~161 kb 1-22, 1-23, 2-24, 3-25, 2-26,
1-27, 2-28, 2-29, 2-30
3-31, 1-32, 1-33, 3-34, 1-35,
4 ~90 kb
2-36, 1-37, 2-38, 1-39, 2-40
Introduction of ten additional human V1 gene segments into a hybrid heavy
chain locus. A second targeting vector was engineered for introduction of 1 O additional
human VK gene segments to the modified mouse heavy chain locus described above (see
Targeting Vector 2). A 140,058 bp human genomic fragment containing 12
consecutive human VK gene segments from the human K light chain locus was engineered
with a 5' homology arm containing mouse genomic sequence 5' of the mouse heavy chain
locus and a 3' homology arm containing human genomic K sequence. Upstream (5') from
the human VK1-16 gene segment was an additional 10,170 bp of human K sequence before
the start of the 5' mouse homology arm, which was the same 5' homology arm used for
construction of Targeting Vector 1 (see . Between the 5' homology arm and the
beginning of the human K sequence was a hygromycin cassette flanked by recombinase
recognition sites. The 3' homology arm included a 31,165 bp overlap of human genomic K
sequence corresponding to the equivalent 5' end of the ~110.5 kb fragment of human
genomic K sequence of Targeting Vector 1 (. The final targeting vector for the
insertion of 10 additional human VK gene segments from 5' to 3' included a 5' homology arm
containing ~20 kb of mouse genomic sequence 5' of the heavy chain locus, a first
recombinase recognition site (R1 ), a hygromycin cassette, a second recombinase
recognition site (R2) and ~140 kb of human genomic K sequence containing 12 consecutive
human VA gene segments, ~31 kb of which overlaps with the 5' end of the human K
sequence of Targeting Vector 1 and serves as the 3' homology arm for this targeting
construct. Homologous recombination with this targeting vector created a modified mouse
heavy chain locus containing 16 human VK gene segments and five human JK gene
PCT/0S2011/046196
segments operably linked to the mouse heavy chain constant genes which, upon
recombination, leads to the formation of a hybrid heavy chain.
Introduction of fourteen additional human VK gene segments into a hybrid
heavy chain locus. A third targeting vector was engineered for introduction of 14 additional
human VK gene segments to the modified mouse heavy chain locus described above (see
Targeting Vector 3). A 160,579 bp human genomic fragment containing 15
consecutive human VK gene segments was engineered with a 5' homology arm containing
mouse genomic sequence 5' of the mouse heavy chain locus and a 3' homology arm
containing human genomic K sequence. Upstream (5') from the human VK2-30 gene
segment was an additional 14,687 bp of human K sequence before the start of the 5' mouse
homology arm, which was the same 5' homology used for the previous two targeting vectors
(described above, see also . Between the 5' homology arm and the beginning of the
human K sequence was a neomycin cassette flanked by recombinase recognition sites. The
3' homology arm included a 21,275 bp overlap of human genomic K sequence
corresponding to the equivalent 5' end of the ~140 kb fragment of human genomic K
sequence of Targeting Vector 2 (. The final targeting vector for the insertion of 14
additional human VK gene segments from 5' to 3' included a 5' homology arm containing
~20 kb of mouse genomic sequence 5' of the mouse heavy chain locus, a first recombinase
recognition site (R1 ), a neomycin cassette, a second recombinase recognition site (R2) and
~161 kb of human genomic K sequence containing 15 human VK gene segments, ~21 kb of
which overlaps with the 5' end of the human K sequence of Targeting Vector 2 and serves
as the 3' homology arm for this targeting construct. Homologous recombination with this
targeting vector created a modified mouse heavy chain locus containing 30 human VK gene
segments and five human JK gene segments operably linked to the mouse heavy chain
constant genes which, upon recombination, leads to the formation of a chimeric K heavy
chain.
Introduction of ten additional human VK gene segments into a hybrid heavy
chain locus. A fourth targeting vector was engineered for introduction of 10 additional
human VK gene segments to the modified mouse heavy chain locus described above (see
Targeting Vector 4). A 90,398 bp human genomic fragment containing 16
consecutive human VK gene segments was engineered with a 5' homology arm containing
mouse genomic sequence 5' of the mouse heavy chain locus and a 3' homology arm
containing human genomic K sequence. Upstream (5') from the human VK2-40 gene
segment was an additional 8,484 bp of human K sequence before the start of the 5' mouse
homology arm, which was the same 5' homology as the previous targeting vectors
PCT/0S2011/046196
(described above, see also . Between the 5' homology arm and the beginning of the
human K sequence was a hygromycin cassette flanked by recombinase recognition sites.
The 3' homology arm included a 61,615 bp overlap of human genomic K sequence
corresponding to the equivalent 5' end of the ~160 kb fragment of human genomic K
sequence of Targeting Vector 3 (. The final targeting vector for the insertion of 1 0
additional human VK gene segments from 5' to 3' included a 5' homology arm containing
~20 kb of mouse genomic sequence 5' of the mouse heavy chain locus, a first recombinase
recognition site (R 1 ), a hygromycin cassette, a second recombinase recognition site (R2)
and ~90 kb of human genomic K sequence containing 16 human VK gene segments, ~62 kb
of which overlaps with the 5' end of the human K sequence of Targeting Vector 3 and serves
as the 3' homology arm for this targeting construct. Homologous recombination with this
targeting vector created a modified mouse heavy chain locus containing 40 human VK gene
segments and five human JK gene segments operably linked to the mouse heavy chain
constant genes which, upon recombination, leads to the formation of a chimeric K heavy
chain (.
Using a similar approach as described above, other combinations of human light
chain variable domains in the context of mouse heavy chain constant regions are
constructed. Additional light chain variable domains may be derived from human VA and JA
gene segments ( and 4B).
The human A light chain locus extends over 1,000 kb and contains over 80 genes
that encode variable (V) or joining (J) segments. Among the 70 VA gene segments of the
human A light chain locus, anywhere from 30-38 appear to be functional gene segments
according to published reports. The 70 VA sequences are arranged in three clusters, all of
which contain different members of distinct V gene family groups (clusters A, B and C).
Within the human A light chain locus, over half of all observed VA domains are encoded by
the gene segments 1-40, 1-44, 2-8, 2-14, and 3-21. There are seven JA gene segments,
only four of which are regarded as generally functional JA gene segments-JA 1, JA2, JA3,
and JA7. In some alleles, a fifh JA-CA gene segment pair is reportedly a pseudo gene
(CA6). Incorporation of multiple human JA gene segments into a hybrid heavy chain locus,
as described herein, is constructed by de novo synthesis. In this way, a genomic fragment
containing multiple human JA gene segments in germline configuration is engineered with
multiple human VA gene segments and allow for normal V-J recombination in the context of
a heavy chain constant region.
Coupling light chain variable domains with heavy chain constant regions
represents a potentially rich source of diversity for generating unique VL binding proteins with
PCT/0S2011/046196
human V regions in non-human animals. Exploiting this diversity of the human " light chain
locus (or human K locus as described above) in mice results in the engineering of unique
hybrid heavy chains and gives rise to another dimension of binding proteins to the immune
repertoire of genetically modified animals and their subsequent use as a next generation
platform for the generation of therapeutics.
Additionally, human D and J (or JK) gene segments can be incorporated with
either human VK or VA gene segments to construct novel hybrid loci that will give rise, upon
recombination, to novel engineered variable domains ( and 5B). In this latter case,
engineering combinations of gene segments that are not normally contained in a single
locus would require specific attention to the recombination signal sequences (RSS) that are
associated with respective gene segments such that normal recombination can be achieved
when they are combined into a single locus. For example, V(D)J recombination is known to
be guided by conserved noncoding DNA sequences, known as heptamer and nonamer
sequences that are found adjacent to each gene segment at the precise location at which
recombination takes place. Between these noncoding DNA sequences are nonconserved
spacer regions that either 12 or 23 base pairs (bp) in length. Generally, recombination only
occurs at gene segments located on the same chromosome and those gene segments
flanked by a 12-bp spacer can be joined to a gene segment flanked by a 23-bp spacer, i.e.
the 12/23 rule, although joining two of D gene segments ( each flanked by 12-bp spacers)
has been observed in a small proportion of antibodies. To allow for recombination between
gene segments that do not normally have compatible spacers (e.g., VK and a D or D and
JA), unique, compatible spacers are synthesized in adjacent locations with the desired gene
segments for construction of unique hybrid heavy chains that allow for successful
recombination to form unique heavy chains containing light chain variable regions.
Thus, using the strategy outlined above for incorporation of human K light chain
gene segments into an endogenous heavy chain locus allows for the use of other
combinations of human " light chain gene segments as well as specific human heavy chain
gene segments (e.g., D and J ) and combinations thereof.
Example II
Identification of Targeted ES cells Bearing
Human Light Chain Gene Segments at an Endogenous Heavy Chain Locus
The targeted BAC DNA made in the foregoing Examples was used to
electroporate mouse ES cells to created modified ES cells for generating chimeric mice that
express V binding proteins (i.e., human K light chain gene segments operably linked to
mouse heavy chain constant regions). ES cells containing an insertion of unrearranged
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human K light chain gene segments were identified by the quantitative PCR assay,
TAQMAN (Lie and Petropoulos, 1998. Curr. Opin. Biotechnology 9:43-48). Specific primers
sets and probes were design for insertion of human K sequences and associated selection
cassettes, loss of mouse heavy chain sequences and retention of mouse sequences
flanking the endogenous heavy chain locus.
ES cells bearing the human K light chain gene segments can be transfected with a
construct that expresses a recombinase in order to remove any undesired selection cassette
introduced by the insertion of the targeting construct containing human K gene segments.
Optionally, the selection cassette may be removed by breeding to mice that express the
recombinase (e.g., US 6,774,279). Optionally, the selection cassette is retained in the mice.
Example Ill
Generation and Analysis of Mice Expressing VL Binding Proteins
Targeted ES cells described above were used as donor ES cells and introduced
into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., US Pat. No.
7,294,754 and Poueymirou, W.T., Auerbach, W., Frendewey, D., Hickey, J.F., Escaravage,
J.M., Esau, L., Dore, A.T., Stevens, S., Adams, N.C., Dominguez, M.G., Gale, N.W.,
Yancopoulos, G.D., DeChiara, T.M., Valenzuela, D.M. (2007). F0 generation mice fully
derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses.
Nat Biotechnol 25, 91-99). VELOCIMICE® (F0 mice fully derived from the donor ES cell)
independently bearing human K gene segments at the mouse heavy chain locus were
identified by genotyping using a modification of allele assay (Valenzuela et al., supra) that
detected the presence of the unique human K gene segments at the endogenous heavy
chain locus (supra). Pups are genotyped and a pup heterozygous for the hybrid heavy chain
gene locus is selected for characterizing expression of VL binding proteins.
Flow Cytometry. The introduction of human K light chain gene segments into the
mouse heavy chain locus was carried out in an F1 ES line (F1 H4; Valenzuela et al. 2007,
supra) derived from 129S6/SvEvTac and C57BL/6NTac heterozygous embryos that further
contained an in situ replacement of the mouse K light chain gene segments with human K
light chain gene segments (US 6,596,541). The human K light chain germ line variable gene
segments are targeted to the 129S6 allele, which carries the lgM
haplotype, whereas the
unmodified mouse C576BL/6N allele bears the lg M
haplotype. These allelic forms of lgM
can be distinguished by flow cytometry using antibodies specific to the polymorphisms found
in the lgM or lgM alleles. Heterozygous mice bearing human K light chain gene segments
at the endogenous heavy chain locus as described in Example I were evaluated for
expression of human VL binding proteins using flow cytometry.
PCT/0S2011/046196
Briefly, blood was drawn from groups of mice (n 6 per group) and grinded using
glass slides. C57BL/6 and Balb/c mice were used as control groups. Following lysis of red
blood cells (RBCs) with ACK lysis buffer (Lonza Walkersville), cells were resuspended in BD
Pharmingen FAGS staining buffer and blocked with anti-mouse CD16/32 (BD Pharmingen).
Lymphocytes were stained with anti-mouse lgM -FITC (BD Pharmingen), anti-mouse lgM
PE (BD Pharmingen), anti-mouse CD19 (Clone 1 D3; BD Biosciences), and anti-mouse CD3
(17A2; BIOLEGEND®) followed by fixation with BD CYTOFIX all according to the
manufacturer's instructions. Final cell pellets were resuspended in staining buffer and
and BD CELLQUEST PRO
analyzed using a BD FACSCALIBUR software. Table 2
sets forth the average percent values for B cells (CD19 ), T cells (CD3 ), hybrid heavy chain
+ + + +
(CD19 IgM ), and wild type heavy chain (CD19 IgM ) expression observed in groups of
animals bearing each genetic modification.
(00222] In a similar experiment, B cell contents of the spleen, blood and bone marrow
compartments from mice homozygous for six human VK and five human JK gene segments
operably linked to the mouse heavy chain constant region (described in Example I,
were analyzed for progression through B cell development using flow cytometry of various
cell surface markers.
(00223] Briefly, two groups (n 3 each, 8 weeks old females) of wild type and mice
homozygous for six human VK and five human JK gene segments operably linked to the
mouse heavy chain constant region were sacrificed and blood, spleens and bone marrow
were harvested. Blood was collected into microtainer tubes with EDTA (BD Biosciences).
Bone marrow was collected from femurs by flushing with complete RPMI medium (RPMI
medium supplemented with fetal calf serum, sodium pyruvate, Hepes, 2-mercaptoethanol,
non-essential amino acids, and gentamycin). RBCs from spleen and bone marrow
preparations were lysed with ACK lysis buffer (Lonza Walkersville), followed by washing with
complete RPMI medium.
(00224] Cells (1x10 ) were incubated with anti-mouse CD16/CD32 (2.4G2, BD) on ice for
ten minutes, followed by labeling with the following antibody cocktail for thirty minutes on ice:
anti-mouse FITC-CD43 (1 B11, BIOLEGEND®), PE-ckit (2B8, BIOLEGEND®), PeCy7-lgM
(11/41, EBIOSCIENCE®), PerCP-CyS.5-lgD (11-26c.2a, BIOLEGEND®), APC-eFluor 780-
B220 (RA3-6B2, EBIOSCIENCE®), APC-CD19 (MB19-1, EBIOSCIENCE®). Bone marrow:
in h + + +
ilgM ), pro B cells (CD19
immature B cells (B220 tlgM), mature B cells (B220 ckitCD43 ),
+ + in1 +1
pre B cells (CD19 ckit-CD43-), pre-B cells (CD19 co43 IgM -), immature B cells
+ +1 + + in1
(CD19 CD43-lgM l Blood and spleen: B cells (CD19 ), mature B cells (CD19 IgM I9D ),
+ hi int
lgD ).
transitional/immature B cells (CD19 IgM
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Following staining, cells were washed and fixed in 2% formaldehyde. Data
acquisition was performed on a LSRII flow cytometer and analyzed with FLOWJO
software (Tree Star, Inc.). , 68 and 6C show the results for the splenic
compartment. A - 7G show the results for the bone marrow compartment. The
results obtained for the blood compartment from each group of mice demonstrated similar
results as compared to the results from the splenic compartment from each group (data not
shown).
In a similar experiment, B cell contents of the spleen, blood and bone marrow
compartments from mice homozygous for thirty human VK and five human JK gene
segments operably linked to the mouse heavy chain constant region (described in Example
I, were analyzed for progression through B cell development using flow cytometry of
various cell surface markers.
Briefly, two groups (N=3 each, 6 week old females) of mice containing a wild-type
heavy chain locus and a replacement of the endogenous VK and JK gene segments with
human VK and JK gene segments (WT) and mice homozygous for thirty hVK and five JK
gene segments and a replacement of the endogenous VK and JK gene segments with
human VK and JK gene segments (30hVK-5hJK HO) were sacrificed and spleens and bone
marrow were harvested. Bone marrow and splenocytes were prepared for staining with
various cell surface markers (as described above).
Cells (1 x 10 ) were incubated with anti-mouse CD16/CD32 (2.4G2, BO
Biosciences) on ice for ten minutes, followed by labeling with bone marrow or splenocyte
panels for thirty minutes on ice. Bone marrow panel: anti-mouse FITC-CD43 (1811,
BIOLEGEND®), PE-ckit (288, BIOLEGEND®), PeCy7-lgM (11/41, EBIOSCIENCE®), APC
CD19 (MB19-1, EBIOSCIENCE®). Bone marrow and spleen panel: anti-mouse FITC-lgK
(187.1 BO Biosciences), PE-lgA (RML-42, BIOLEGEND®), PeCy7-lgM (11/41,
EBIOSCIENCE®), PerCP-Cy5.5-lgD (11-26c.2a, BIOLEGEND®), Pacific Blue-CD3 (17A2,
BIOLEGEND®), APC-8220 (RA3-682, EBIOSCIENCE®), APC-H7-CD19 (1D3, BO). Bone
int + hi +
marrow: immature B cells (B220 lgM ), mature B cells (B220 lgM ), pro B cells
+ + +
(CD19 ckitCD43 ), pre B cells (CD19+ ckit-CD43-), immature lgK B cells
+ + + + + +
int int
lgK lgA-), immature lgA B cells (B220 lgM lgK-lgA ), mature lgK B cells
(B220 lgM
+ + + +
+ + h
B cells (B220 ilgM lgK-lgA ). Spleen: B cells (CD19 ),
(B220 lgM lgK lgA-), mature lgA
hi int int hi
mature B cells (CD19 1gD lgM ), transitional/immature B cells (CD19 1gD lgM ). Bone
+ + + + +
marrow and spleen: lgK B cells (CD19 1gK lgA-), lgA B cells (CD19 lgK-lg1. ).
Following staining, cells were washed and fixed in 2% formaldehyde. Data
acquisition was performed on a LSRII flow cytometer and analyzed with FLOWJO
software (Tree Star, Inc.). The results demonstrated similar staining patterns and cell
PCT/0S2011/046196
populations for all three compartments as compared to mice homozygous for six human VK
and five human JK gene segments (described above). However, these mice demonstrated a
loss in endogenous " light chain expression in both the splenic and bone marrow
compartments ( and 88, respectively}, despite the endogenous " light chain locus
being intact in these mice. This may reflect an inability of rearranged human K light chain
domains, in the context of heavy chain constant regions, to pair or associate with murine "
light chain domains, leading to deletion of lg" cells.
lsotype Expression. Total and surface (i.e., membrane bound) immunoglobulin
M (lgM) and immunoglobulin G1 (lgG1) was determined for mice homozygous for human
heavy and K light chain variable gene loci (VELCOIMMUNE® Humanized Mice, see US
7,105,348) and mice homozygous for six human VK and 5 human JK gene segments
engineered into the endogenous heavy chain locus (6hVK-5hJK HO) by a quantitative PCR
assay using TAQMAN probes (as described above in Example II}.
Briefly, CD1 g B cells were purified from the spleens of groups of mice (n 3 to 4
mice per group) using mouse CD19 Microbeads (Miltenyi Biotec) according to
manufacturer's instructions. Total RNA was purified using the RNEASY Mini kit (Qiagen).
Genomic RNA was removed using an RNase-free DNase on-column treatment (Qiagen).
About 200 ng mRNA was reverse-transcribed into cDNA using the First Stand cDNA
Synthesis kit (lnvitrogen) and then amplified with the TAQMAN Universal PCR Master Mix
(Applied Biosystems) using the ABI 7900 Sequence Detection System (Applied Biosystems).
Unique primer/probe combinations were employed to specifically determine expression of
total, surface (i.e., transmembrane) and secreted forms of lgM and lgG1 isotypes (Table 3).
Relative expression was normalized to the mouse K constant region (mCK).
Table 2
Mouse Genotype % CD3 % CD19 % lgM % lgM
C57BL/6 22 63 0 100
Balb/c 11 60 100 0
43 30 7 85
6hVK-5hJK HET
16hVK-5hJK HET 41 81
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Table 3
Sequence (5' 3')
SEQ ID NOs·
sotype t
sense: GAGAGGACCG TGGACAAGTC 1
antisense: TGACGGTGGT GCTGTAGAAG 2
Surface lgM
probe: ATGCTGAGGA GGAAGGCTTT GAGAACCT 3
sense: GCTCGTGAGC AACTGAACCT 4
antisense: GCCACTGCAC ACTGATGTC 5
Total lgM
probe: AGTCAGCCAC AGTCACCTGC CTG 6
sense: GCCTGCACAA CCACCATAC 7
antisense: GAGCAGGAAG AGGCTGATGA AG 8
Surface lgG1
probe: AGAAGAGCCT CTCCCACTCT CCTGG 9
sense: CAGCCAGCGG AGAACTACAA G 10
antisense: GCCTCCCAGT TGCTCTTCTG 11
Total lgG1
probe: AACACTCAGC CCA TCA TGGA CACA 12
sense: TGAGCAGCAC CCTCACGTT 13
antisense: GTGGCCTCAC AGGT ATAGCT GTT 14
probe: ACCAAGGACG AGTA TGAA 15
The results from the quantitative TAQMAN PCR assay demonstrated a
decrease in total lgM and total lgG1. However, the ratio of secreted versus surface forms of
lgM and lgG1 appeared normal as compared to VElCOIMMUNE® humanized mice (data
not shown).
Human K gene segment usage and V1-J1 junction analysis. Na"ve mice
homozygous for thirty hVK and five JK gene segments and a replacement of the endogenous
VK and JK gene segments with human VK and JK gene segments (30hVK-5hJK HO) were
analyzed for unique human VK-JK rearrangements on mouse heavy chain (lgG) by reverse
transcription polymerase chain reaction (RT-PCR) using RNA isolated from splenocytes.
Briefly, spleens were harvested and perfused with 10 ml RPMl-1640 (Sigma)
with 5% HI-FBS in sterile disposable bags. Each bag containing a single spleen was then
placed into a STOMACHER (Seward) and homogenized at a medium setting for 30
seconds. Homogenized spleens were filtered using a 0.7µm cell strainer and then pelleted
with a centrifuge (1000 rpm for 10 minutes) and RBCs were lysed in BD PHARM l YSE
(BD Biosciences) for three minutes. Splenocytes were diluted with RPMl-1640 and
centrifuged again, followed by resuspension in 1 ml of PBS (Irvine Scientific). RNA was
isolated from pelleted splenocytes using standard techniques known in the art.
RT-PCR was performed on splenocyte RNA using primers specific for human
hVK gene segments and the mouse lgG. The mouse lgG primer was designed such that it
was capable of amplifying RNA derived from all mouse lgG isotypes. PCR products were
gel-purified and cloned into pCR2.1-TOPO TA vector (lnvitrogen) and sequenced with
PCT/0S2011/046196
primers M13 Forward (GTAAAACGAC GGCCAG; SEQ ID NO:16) and M13 Reverse
(CAGGAAACAG CTATGAC; SEQ ID NO:17) located within the vector at locations flanking
the cloning site. Human VK and JK gene segment usage among twelve selected clones are
shown in Table 4. sets forth the nucleotide sequence of the hVK-hJK-mlgG junction
for the twelve selected RT-PCR clones.
As shown in this Example, mice homozygous for six human VK and five human
JK gene segments or homozygous for thirty human VK and five human JK gene segments
operably linked to the mouse heavy chain constant region demonstrated expression human
light chain variable regions from a modified heavy chain locus containing light chain variable
gene segments in their germline configuration. Progression through the various stages of B
cell development was observed in these mice, indicating multiple productive recombination
events involving light chain variable gene segments from an endogenous heavy chain locus
and expression of such hybrid heavy chains (i.e., human light chain variable region linked to
a heavy chain constant region) as part of the antibody repertoire.
Table 4
Hybrid Heavy Chain
Clone -------- SEQ ID NO:
VK JK C
1E 1-5 4 lgG2AC 18
1G 1- 9 4 lgG2A/C 19
20
1A 1- 16 lgG3
2E 1- 12 2 lgG1 21
1C 1-27 4 lgG2A/C 22
2H 2-28 1 lgG1 23
3D 3-11 4 lgG1
3-20 4 lgG2A/C 25
4B 4-1 5 lgG2A/C 26
4C 2 lgG3
5A 5-2 2 lgG2A/C 28
5D 5-2 1 lgG1 29
Example IV
Propagation of Mice Expressing VL Binding Proteins
To create a new generation of VL binding proteins, mice bearing the unrearranged
human K gene segments can be bred to another mouse containing a deletion of the other
endogenous heavy chain allele. In this manner, the progeny obtained would express only
hybrid heavy chains as described in Example I. Breeding is performed by standard
techniques recognized in the art and, alternatively, by commercial companies, e.g., The
PCT/0S2011/046196
Jackson Laboratory. Mouse strains bearing a hybrid heavy chain locus are screened for
presence of the unique hybrid heavy chains and absence of traditional mouse heavy chains.
(00238] Alternatively, mice bearing the unrearranged human K gene segments at the
mouse heavy chain locus can be optimized by breeding to other mice containing one or
more deletions in the mouse light chain loci (K and 11). In this manner, the progeny obtained
would express unique human K heavy chain only antibodies as described in Example I.
Breeding is similarly performed by standard techniques recognized in the art and,
alternatively, by commercial companies, e.g., The Jackson Laboratory. Mouse strains
bearing a hybrid heavy chain locus and one or more deletions of the mouse light chain loci
are screened for presence of the unique hybrid heavy chains containing human K light chain
domains and mouse heavy chain constant domains and absence of endogenous mouse
light chains.
(00239] Mice bearing an unrearranged hybrid heavy chain locus are also bred with mice
that contain a replacement of the endogenous mouse K light chain variable gene locus with
the human K light chain variable gene locus (see US 6,596,541, Regeneron
Pharmaceuticals, The VELOCIMMUNE® Humanized Mouse Technology). The
VELOCIMMUNE® Humanized Mouse includes, in part, having a genome comprising human
K light chain variable regions operably linked to endogenous mouse K light chain variable
constant region loci such that the mouse produces antibodies comprising a human K light
chain variable domain and a mouse heavy chain constant domain in response to antigenic
stimulation. The DNA encoding the variable regions of the light chains of the antibodies can
be isolated and operably linked to DNA encoding the human light chain constant regions.
The DNA can then be expressed in a cell capable of expressing the fully human light chain
of the antibody. Upon a suitable breeding schedule, mice bearing a replacement of the
endogenous mouse K light chain with the human K light chain locus and an unrearranged
hybrid heavy chain locus is obtained. Unique VL binding proteins containing somatically
mutated human VK domains can be isolated upon immunization with an antigen of interest.
Example V
Generation of VL Binding Proteins
(00240] Afer breeding mice that contain the unrearranged hybrid heavy chain locus to
various desired strains containing modifications and deletions of other endogenous lg loci
(as described in Example IV), selected mice can be immunized with an antigen of interest.
(00241] Generally, a VELOCIMMUNE® humanized mouse containing at least one hybrid
heavy chain locus is challenged with an antigen, and cells (such as B-cells) are recovered
from the animal (e.g., from spleen or lymph nodes). The cells may be fused with a myeloma
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cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are
screened and selected to identify hybridoma cell lines that produce antibodies containing
hybrid heavy chains specific to the antigen used for immunization. DNA encoding the
human VK regions of the hybrid heavy chains may be isolated and linked to desirable
constant regions, e.g., heavy chain and/or light chain. Due to the presence of human VK
gene segments fused to the mouse heavy chain constant regions, a unique antibody-like
repertoire is produced and the diversity of the immunoglobulin repertoire is dramatically
increased as a result of the unique antibody format created. This confers an added level of
diversity to the antigen specific repertoire upon immunization. The resulting cloned antibody
sequences may be subsequently produced in a cell, such as a CHO cell. Alternatively, DNA
encoding the antigen-specific V binding proteins or the variable domains may be isolated
directly from antigen-specific lymphocytes (e.g., B cells).
Initially, high affinity V binding proteins are isolated having a human VK region and
a mouse constant region. As described above, the V binding proteins are characterized
and selected for desirable characteristics, including afinity, selectivity, epitope, etc. The
mouse constant regions are replaced with a desired human constant region to generate
unique fully human V binding proteins containing somatically mutated human VK domains
from an unrearranged hybrid heavy chain locus of the invention. Suitable human constant
regions include, for example wild type or modified lgG1 or lgG4 or, alternatively CK or CA.
Separate cohorts of mice containing a replacement of the endogenous mouse
heavy chain locus with six human VK and five human JK gene segments (as described in
Example I) and a replacement of the endogenous VK and JK gene segments with human VK
and JK gene segments were immunized with a human cell-surface receptor protein (Antigen
X). Antigen Xis administered directly onto the hind footpad of mice with six consecutive
injections every 3-4 days. Two to three micrograms of Antigen X are mixed with 10 µg of
CpG oligonucleotide (Cat # tlrl-modn - ODN1826 oligonucleotide; lnVivogen, San Diego,
CA) and 25 µg of Adju-Phos (Aluminum phosphate gel adjuvant, Cat# H250;
Brenntag Biosector, Frederikssund, Denmark) prior to injection. A total of six injections are
given prior to the final antigen recall, which is given 3-5 days prior to sacrifice. Bleeds afer
the 4th and 6th injection are collected and the antibody immune response is monitored by a
standard antigen-specific immunoassay.
When a desired immune response is achieved splenocytes are harvested and
fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines.
The hybridoma cell lines are screened and selected to identify cell lines that produce
Antigen X-specific V binding proteins. Using this technique several anti-Antigen X-specific
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V binding proteins (i.e., binding proteins possessing human VK domains in the context of
mouse heavy and light chain constant domains) are obtained.
Alternatively, anti-Antigen X V binding proteins are isolated directly from antigen
positive B cells without fusion to myeloma cells, as described in U.S. 2007/0280945A1,
herein specifically incorporated by reference in its entirety. Using this method, several fully
human anti-Antigen X V binding proteins (i.e., antibodies possessing human VK domains
and human constant domains) were obtained.
Human K Gene Segment Usage. To analyze the structure of the anti-Antigen X
V binding proteins produced, nucleic acids encoding the human VK domains (from both the
heavy and light chains of the V binding protein) were cloned and sequenced using methods
adapted from those described in US 200710280945A 1 (supra). From the nucleic acid
sequences and predicted amino acid sequences of the antibodies, gene usage was
binding proteins obtained
identified for the hybrid heavy chain variable region of selected V
from immunized mice (described above). Table 5 sets for the gene usage of human VK and
JK gene segments from selected anti-Antigen X V binding proteins, which demonstrates
that mice according to the invention generate antigen-specific V binding proteins from a
variety of human VK and JK gene segments, due to a variety of rearrangements at the
endogenous heavy chain and K light chain loci both containing unrearranged human VK and
JK gene segments. Human VK gene segments rearranged with a variety of human JK
segments to yield unique antigen-specific V binding proteins.
Table 5
Hybrid Heavy Chain Light Chain
Antibody --------
VK JK
A 4-1 3 3-20 1
B 4-1 3-20 1
C 4-1 3 3-20 1
D 4-1 3 3-20 1
E 4-1 3 3-20 1
F 1 3 3-20 1
G 3 3-20 1
H 4 1 3 3-20 1
I 4-1 3 3-20 1
J 1-33
1-5 3 3
K 4-1 3-20 1
L 4-1 1-9
M 4-1 1 1-33 4
N 4-1 1 1- 33 3
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1-5 1-9 2
p 1-5 3 1- 16 4
Q 4-1 3 3-20 1
R 4-1 3 3-20 1
1 1-9 2
s 1- 5
T 1 1- 9 2
u 5-2 2 3
2 1- 9 2
V 1- 5
4-1 1 1- 33 4
Enzyme-linked immunosorbent assay (ELISA). Human V binding proteins
raised against Antigen X were tested for their ability to block binding of Antigen X's natural
ligand (Ligand Y) in an ELISA assay.
Briefly, Ligand Y was coated onto 96-well plates at a concentration of 2 µg/mL
diluted in PBS and incubated overnight followed by washing four times in PBS with 0.05%
Tween-20. The plate was then blocked with PBS (Irvine Scientific, Santa Ana, CA)
containing 0.5% (w/v) BSA (Sigma-Aldrich Corp., St. Louis, MO) for one hour at room
temperature. In a separate plate, supernatants containing anti-Antigen X V binding proteins
were diluted 1: 1 O in buffer. A mock supernatant with the same components of the V binding
proteins was used as a negative control. The extracellular domain (ECO) of Antigen X was
conjugated to the Fe portion of mouse lgG2a (Antigen X-mFc). Antigen X-mFc was added
to a final concentration of 0.150 nM and incubated for one hour at room temperature. The
V binding protein/Antigen X-mFc mixture was then added to the plate containing Ligand Y
and incubated for one hour at room temperature. Detection of Antigen X-mFc bound to
Ligand Y was determined with Horse-Radish Peroxidase (HRP) conjugated to anti-Penta-His
antibody (Qiagen, Valencia, CA) and developed by standard colorimetric response using
tetramethylbenzidine (TMB) substrate (BD Biosciences, San Jose, CA) neutralized by
sulfuric acid. Absorbance was read at OD450 for 0.1 sec. Background absorbance of a
sample without Antigen X was subtracted from all samples. Percent blocking was calculated
for >250 (three 96 well plates) Antigen X-specific V binding proteins by division of the
background-subtracted MFI of each sample by the adjusted negative control value,
multiplying by 100 and subtracting the resulting value from 100.
The results showed that several V binding proteins isolated from mice
immunized with Antigen X specifically bound the extracellular domain of Antigen X fused to
the Fe portion of mouse lgG2a (data not shown).
Afinity Determination. Equilibrium dissociation constants (Ko) for selected
Antigen X-specific V binding protein supernatants were determined by SPR (Surface
Plasmon Resonance) using a BIACORE T100 instrument (GE Healthcare). All data were
1 -9
obtained using HBS-EP (10mM HEPES, 150mM NaCI, 0.3mM EDTA, 0.05% Surfactant P20, pH
7.4) as both the running and sample buffers, at 25°C.
Briefly, V binding proteins were captured from crude supernatant samples on a
CM5 sensor chip surface previously derivatized with a high density of anti-human Fc antibodies
using standard amine coupling chemistry. During the capture step, supernatants were injected
across the anti-human Fc surface at a flow rate of 3 µL/min, for a total of 3 minutes. The capture
step was followed by an injection of either running buffer or analyte at a concentration of 100 nM
for 2 minutes at a flow rate of 35 µL/min. Dissociation of antigen from the captured V binding
protein was monitored for 6 minutes. The captured V binding protein was removed by a brief
injection of 10 mM glycine, pH 1.5. All sensorgrams were double referenced by subtracting
sensorgrams from buffer injections from the analyte sensorgrams, thereby removing artifacts
caused by dissociation of the V binding protein from the capture surface. Binding data for each V
binding protein was fit to a 1:1 binding model with mass transport using BIAcore T100 Evaluation
software v2.1.
The binding affinities of thirty-four selected V binding proteins varied, with all
exhibiting a K in the nanomolar range (1.5 to 130 nM). Further, about 70% of the selected V
binding proteins (23 of 34) demonstrated single digit nanomolar affinity. T measurements for
these selected V binding proteins demonstrated a range of about 0.2 to 66 minutes. Of the thirty-
four V binding proteins, six showed greater than 3 nM affinity for Antigen X (1.53, 2.23, 2.58,
2.59, 2.79, and 2.84). The affinity data is consistent with the V binding proteins resulting from the
combinatorial association of rearranged human light chain variable domains linked to heavy and
light chain constant regions (described in Table 4) being high-affinity, clonally selected, and
somatically mutated. The V binding proteins generated by the mice described herein comprise a
collection of diverse, high-affinity unique binding proteins that exhibit specificity for one or more
epitopes on Antigen X.
In another experiment, selected human V binding proteins raised against Antigen
X were tested for their ability to block binding of Antigen X’s natural ligand (Ligand Y) to Antigen X
in a LUMINEX® bead-based assay (data not shown). The results demonstrated that in addition to
specifically binding the extracellular domain of Antigen X with affinities in the nanomolar range
(described above), selected V binding proteins were also capable of binding Antigen X from
cynomolgus monkey (Macaca fascicularis).
Throughout the description and claims of the specification, the word "comprise"
and variations of the word, such as "comprising" and "comprises", is not intended to exclude other
additives, components, integers or steps.
A reference herein to a patent document or other matter which is given as prior art
is not to be taken as an admission that that document or matter was known or that the information
it contains was part of the common general knowledge as at the priority date of any of the claims.
Claims (79)
1. A rat or a mouse, comprising in its germline an immunoglobulin hybrid chain locus comprising at least one unrearranged light chain variable region (V ) gene segment, at least one unrearranged light chain joining (J ) gene segment, and an immunoglobulin heavy chain constant region capable of associating with a light chain constant region, wherein each of the unrearranged V and J gene segments comprise recombination signal sequences that allow the unrearranged V and J gene segments to recombine such that the rat or mouse further comprises a rearranged immunoglobulin hybrid gene comprising light chain variable region (V /J ) nucleotide sequence operably linked with the immunoglobulin heavy chain constant region, and wherein the heavy chain constant region comprises an endogenous constant region gene selected from the group consisting of an endogenous IgM gene, an endogenous IgD gene, an endogenous IgG gene, an endogenous IgE gene, an endogenous IgA gene, and a combination thereof.
2. The rat or mouse of claim 1, wherein the at least one unrearranged light chain V gene segment is selected from an unrearranged human κ gene segment, an unrearranged human λ gene segment, and a combination thereof.
3. The rat or mouse of claim 1, wherein the at least one unrearranged light chain V gene segment and the at least one unrearranged JL gene segment respectively replace an endogenous heavy chain V gene segment and an endogenous heavy chain J gene segment at the endogenous heavy chain locus.
4. The rat or mouse of claim 3, wherein the at least one unrearranged light chain V gene segment replaces all or substantially all functional endogenous heavy chain V gene segments of the endogenous heavy chain locus.
5. The rat or mouse of claim 3, wherein the at least one unrearranged J gene segment replaces all or substantially all functional endogenous heavy chain J gene segments of the endogenous heavy chain locus.
6. The rat or mouse of claim 5, wherein the at least one unrearranged light chain V gene segment and the unrearranged light chain J gene segment are operably linked and the rat or mouse lacks a functional heavy chain D gene segment between the at least one unrearranged light chain V gene segment and the at least one unrearranged light chain J gene segment.
7. The rat or mouse of claim 6, wherein the at least one unrearranged light chain V gene segment is an unrearranged human κ gene segment and the unrearranged light chain J gene segment is an unrearranged human κ gene segment.
8. The rat or mouse of claim 1, comprising a B cell that comprises in its genome the rearranged immunoglobulin hybrid gene, and wherein the rearranged hybrid gene comprises a rearranged human κ variable region operably linked to the endogenous constant region gene selected from the group consisting of an endogenous IgM gene, an endogenous IgD gene, an endogenous IgG gene , an endogenous IgE gene, an endogenous IgA gene, and a combination thereof.
9. The rat or mouse of claim 1, wherein the rearranged immunoglobulin hybrid gene is at an endogenous immunoglobulin heavy chain locus.
10. The rat or mouse of claim 1, further comprising in its germline an unrearranged human light chain V gene segment and an unrearranged human light chain J gene segment operably linked to a light chain constant gene.
11. The rat or mouse of claim 10, wherein the light chain constant gene is a rat or mouse light chain constant gene.
12. The rat or mouse of claim 11, wherein the unrearranged human light chain V gene segment is an unrearranged human κ V gene segment.
13. The rat or mouse of claim 12, wherein the rat or mouse light chain constant gene is a rat or mouse κ light chain constant gene.
14. A rat or mouse comprising a hybrid immunoglobulin comprising a first polypeptide comprising a first human light chain variable region sequence fused with an endogenous immunoglobulin heavy chain constant region, and a second polypeptide comprising a second human light chain variable region fused with an immunoglobulin light chain constant region.
15. The rat or mouse of claim 14, wherein the first human light chain variable region sequence comprises a human κ variable region sequence, and the second human light chain variable region is selected from a human κ variable region and a human λ variable region.
16. The rat or mouse of claim 15, wherein the immunoglobulin heavy chain constant region comprises an IgG isotype.
17. The rat or mouse of claim 15, wherein the immunoglobulin light chain constant region is selected from a human light chain constant region, a rat heavy chain constant region, and a mouse light chain constant region.
18. The rat or mouse of claim 15, wherein the first polypeptide is expressed from a modified endogenous immunoglobulin heavy chain locus that lacks a functional endogenous heavy chain V gene segment.
19. The rat or mouse of claim 18, wherein the second polypeptide is expressed from a modified endogenous immunoglobulin light chain locus that lacks a functional endogenous light chain V gene segment.
20. A rat or mouse, comprising a replacement in the germline of the rat or mouse at an endogenous immunoglobulin heavy chain locus of all or substantially all functional endogenous heavy chain variable (V ) gene segments with at least six or more unrearranged human light chain V gene segments and one or more unrearranged human light chain J gene segments, wherein each of the unrearranged human light chain V and J gene segments comprise recombination signal sequences that allow the unrearranged V and J gene segments to recombine such that the rat or mouse further comprises a rearranged immunoglobulin hybrid gene comprising light chain variable region (V /J ) nucleotide sequence operably linked to an endogenous immunoglobulin heavy chain constant region, wherein the endogenous heavy chain constant region comprises a gene selected from the group consisting of an endogenous IgM gene, an endogenous IgD gene, an endogenous IgG gene, an endogenous IgE gene, an endogenous IgA gene, and a combination thereof, wherein the rat or mouse is incapable of expressing an immunoglobulin heavy chain derived from a heavy chain V gene segment, and wherein the rat or mouse comprises a splenic B cell population (B220+/IgM+) that is at least about 75% the size of a splenic B cell population (B220+/IgM+) of a wild-type rat or mouse.
21. The rat or mouse of any one of claims 1 to 20, which is a mouse.
22. A mouse, comprising in its germline genome a modified endogenous mouse immunoglobulin heavy chain locus comprising a replacement of all functional endogenous mouse immunoglobulin heavy chain variable V gene segments, all functional endogenous mouse immunoglobulin heavy chain diversity D gene segments and all functional endogenous mouse immunoglobulin heavy chain joining J gene segments with a plurality of unrearranged human immunoglobulin light chain variable V (hV ) gene segments and all five contiguous unrearranged functional human immunoglobulin light chain joining J (hJ ) gene segments, wherein the plurality of unrearranged hV gene segments and all five contiguous unrearranged functional hJ are operably linked to an intact endogenous mouse immunoglobulin heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus, wherein the plurality of unrearranged hV gene segments and the five unrearranged hJ gene segments rearrange in a B cell during B cell development to form a rearranged human immunoglobulin light chain variable region V /J nucleotide sequence operably linked to the endogenous mouse immunoglobulin heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus, and wherein the mouse comprises the B cell, which further comprises a polypeptide encoded by the rearranged human immunoglobulin light chain variable region V /J nucleotide sequence operably linked to the endogenous mouse immunoglobulin heavy chain constant region.
23. The mouse of claim 22, wherein the endogenous mouse immunoglobulin heavy chain constant region comprises an intact endogenous C region gene.
24. The mouse of claim 22, wherein all the functional endogenous mouse V , D , and J H H H gene segments are replaced with at least 6 human Vκ gene segments and the five human Jκ gene segments.
25. The mouse of claim 22, wherein all the functional endogenous mouse V , D , and J H H H gene segments are replaced with at least 16 human Vκ gene segments and the five human Jκ gene segments.
26. The mouse of claim 22, wherein all the functional endogenous mouse V , D , and J H H H gene segments are replaced with at least 30 human Vκ gene segments and the five Jκ gene segments.
27. The mouse of claim 22, wherein all the functional endogenous mouse V , D , and J H H H gene segments are replaced with at least 40 human Vκ gene segments and the five Jκ gene segments.
28. The mouse of claim 22, wherein the rearranged human immunoglobulin light chain variable region V /J gene sequence comprises at least one N addition.
29. The mouse of claim 22, wherein the mouse is homozygous or heterozygous for the modified endogenous immunoglobulin heavy chain locus.
30. The mouse of claim 22, wherein the B cell is a pre B cell, a pro B cell, an immature B cell, a transitional B cell, or a mature B cell.
31. A mouse comprising in its germline a first unrearranged human kappa light chain variable (V ) gene segment and an unrearranged human kappa light chain J (J ) gene segment operably linked with the endogenous mouse heavy chain constant region at the endogenous mouse heavy chain locus, wherein the first unrearranged human V gene segment and the unrearranged human J gene segment replace all functional endogenous mouse heavy chain variable (V ) gene segments, all functional endogenous mouse D gene segments and all functional endogenous mouse heavy chain J (J ) gene segments, wherein the first unrearranged human V gene segment and unrearranged human J gene segment participate in rearrangement to form a rearranged V /J sequence operably linked to the endogenous mouse heavy chain constant region in the mouse, and wherein the mouse further comprises in its germline a second human light chain variable (V ) gene segment and a human light chain J (J ) gene segment operably linked to a mouse light chain constant gene.
32. The mouse of claim 31, comprising a B cell that comprises in its genome a rearranged human immunoglobulin kappa light chain variable region nucleic acid sequence operably linked to the endogenous mouse heavy chain constant region at an endogenous mouse immunoglobulin heavy chain locus
33. The mouse of claim 31, wherein the second human V gene segment is a human V gene segment.
34. The mouse of claim 33, wherein the light chain constant gene is a κ light chain constant gene.
35. The mouse of claim 33, wherein the second human V gene segment is a human Vκ gene segment.
36. The mouse of claim 35, wherein the light chain constant gene is a κ light chain constant gene.
37. The mouse of claim 31 that expresses an antigen-binding protein, wherein the antigen-binding protein comprises a first polypeptide comprising a first human kappa light chain variable region fused with a mouse immunoglobulin heavy chain constant region, and a second polypeptide comprising a second human light chain variable region fused with a mouse immunoglobulin light chain constant region.
38. The mouse of claim 37, wherein the second human light chain variable region is selected from a human V variable region and a human Vλ variable region.
39. The mouse of claim 31, which is homozygous or heterozygous for said endogenous heavy chain locus comprising a first unrearranged human V gene segment and an unrearranged human J gene segment operably linked with the endogenous mouse heavy chain constant region at the endogenous mouse heavy chain locus, wherein the first unrearranged human V gene segment and the unrearranged human J gene segment replace all functional endogenous mouse V , D and J gene segments. H H H
40. Use of a rat or mouse according to any one of claims 1 to 39 to produce a hybrid immunoglobulin binding protein that comprises a human light chain variable domain operably linked to heavy chain constant region.
41. Use of a rat or mouse according to any one of claims 1-39 to produce an antigen binding protein comprising (i) a first polypeptide comprising a first light chain variable domain fused with a rat or mouse heavy chain constant region and (ii) a second polypeptide comprising a second light chain variable domain fused with a rat or mouse light chain constant region.
42. The use according to claim 41, wherein at least the first light chain variable domain is a kappa light chain variable domain.
43. The use according to claim 42, wherein the first light chain variable domain is a human kappa light chain variable domain and the second light chain variable domain is a human light chain variable domain.
44. The use according to claim 43, wherein the first human kappa light chain variable domain is fused with a mouse immunoglobulin heavy chain constant region, and wherein the second human light chain variable domain is fused with a mouse immunoglobulin light chain constant region.
45. Use of a mouse according to any of claims 31-39, to produce a hybridoma or quadroma for producing an antibody as defined in claim 44.
46. Use of a rat or mouse according to any one of claims 1-39 to produce a bispecific antibody.
47. A cell or tissue derived from a rat or mouse according to any one of claims 1-39.
48. A cell according to claim 47, wherein the cell is selected from an ES cell, a B cell, and a hybridoma.
49. A cell according to claim 47, wherein the cell is the B cell which further comprises the polypeptide encoded by the rearranged human immunoglobulin light chain variable region V /J nucleotide sequence operably linked to the endogenous immunoglobulin heavy chain constant region.
50. An isolated hybridoma comprising a myeloma cell line fused with the B cell of claim 49, wherein the hybridoma produces the polypeptide encoded by the rearranged human immunoglobulin light chain variable region V /J nucleotide sequence operably linked to the endogenous mouse immunoglobulin heavy chain constant region.
51. An isolated rat or mouse cell, comprising an immunoglobulin hybrid chain locus comprising at least one unrearranged light chain variable region (V ) gene segment, at least one unrearranged light chain joining (J ) gene segment, and an immunoglobulin heavy chain constant region capable of associating with a light chain constant region, wherein each of the unrearranged V and J gene segments comprise recombination signal sequences that allow the unrearranged V and J gene segments to recombine in a B cell during B cell rearrangement to form a rearranged immunoglobulin hybrid gene comprising light chain variable region (V /J ) nucleotide sequence operably linked with the immunoglobulin heavy chain constant region, and wherein the heavy chain constant region comprises a gene selected from the group consisting of IgM, IgD, IgG, IgE, IgA and a combination thereof.
52. An isolated mouse cell comprising a first unrearranged human kappa light chain variable (V ) gene segment and an unrearranged human kappa light chain J (J ) gene segment operably linked with the endogenous mouse heavy chain constant region at the endogenous mouse heavy chain locus, wherein the first unrearranged human V gene segment and the unrearranged human J gene segment replace all functional endogenous mouse heavy chain variable (V ) gene segments, all functional endogenous mouse D gene segments and all functional endogenous mouse heavy chain J (J ) gene segments, wherein the first unrearranged human V gene segment and unrearranged human J gene segment participate in rearrangement to form a rearranged V /J sequence operably linked to the endogenous mouse heavy chain constant region, and wherein the mouse cell further comprises a second human light chain variable (V ) gene segment and a human light chain J (JL) gene segment operably linked to a mouse light chain constant gene.
53. An isolated mouse cell comprising a modified endogenous mouse immunoglobulin heavy chain locus comprising a replacement of all functional endogenous mouse immunoglobulin heavy chain variable V gene segments, all functional endogenous mouse immunoglobulin heavy chain diversity D gene segments and all functional endogenous mouse immunoglobulin heavy chain joining J gene segments with a plurality of unrearranged human immunoglobulin light chain variable Vκ (hV ) gene segments and all five unrearranged human immunoglobulin light chain joining Jk (hJ ) gene segments, wherein the plurality of unrearranged hV gene segments and all five unrearranged hJ are operably linked to an intact endogenous mouse immunoglobulin heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus, wherein the plurality of unrearranged hV gene segments and the five unrearranged hJ gene segments are capable of rearranging in a B cell during B cell development to form a rearranged human immunoglobulin light chain variable region V -J nucleotide sequence operably linked to the endogenous mouse immunoglobulin heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus.
54. The isolated cell of any one of claims 51-53, wherein the cell is an embryonic stem cell.
55. A rat or mouse made with the embryonic stem cell of claim 54.
56. The tissue of claim 47, which comprises a B cell comprising a rearranged human immunoglobulin kappa light chain variable region nucleic acid sequence operably linked to an endogenous heavy chain constant region at the endogenous heavy chain locus and a rearranged human immunoglobulin light chain variable region nucleic acid sequence operably linked to an endogenous light chain constant region gene.
57. An antigen binding protein comprising an immunoglobulin hybrid chain comprising a light chain variable domain operably linked to a heavy chain constant region derived from a rat or mouse according to any one of claims 1 to 41 or made according to the use of any one of claims 42-48.
58. The antigen binding protein of claim 57, wherein the light chain variable domain is a human light chain variable domain.
59. The antigen binding protein of claim 58, wherein the human light chain variable domain is a human κ light chain variable domain.
60. The antigen binding protein of any one of claims 57 to 59, further comprising an immunoglobulin light chain comprising a light chain variable domain operably linked to a light chain constant region.
61. The antigen-binding protein of any one of claims 57-60 wherein the antigen binding protein comprises a human immunoglobulin light chain variable domain fused to a mouse light chain constant domain and a human immunoglobulin kappa light chain variable domain fused to a mouse heavy chain constant domain.
62. A method of making an antigen binding protein comprising exposing a rat or mouse according to any one of claims 1 to 39 to an antigen of interest and isolating from the rat or mouse a human V domain that specifically binds the antigen of interest with high affinity or a gene sequence encoding the V domain.
63. The method of claim 62, wherein the gene sequence is a rearranged human V /J gene sequence fused with a nucleotide sequence encoding an immunoglobulin C region.
64. The method of claim 62, further comprising expressing a nucleotide sequence comprising the rearranged human V /J gene sequence in a suitable cell.
65. The method of claim 62, further comprising cloning the rearranged human V /J gene sequence in frame with a gene encoding a human C region to form a human binding protein sequence.
66. The method of claim 65, further comprising expressing the human binding protein sequence in a suitable isolated cell.
67. A nucleotide comprising the gene sequence obtained from the method of any one of claims 62, 63 or 65.
68. An antigen binding protein made according to any one of methods 63 to 66.
69. A method for making a genetically modified rat or mouse according to any one of claims 1 to 39 comprising inserting at an endogenous heavy chain locus at least one unrearranged light chain variable region (V ) gene segment and at least one unrearranged light chain joining (J ) gene segment in operable linkage with an immunoglobulin heavy chain constant region capable of associating with a light chain constant region, wherein each of the unrearranged V and J gene segments comprise recombination signal sequences that allow the unrearranged V and J gene segments to recombine such that the rat or mouse further comprises a rearranged immunoglobulin hybrid gene comprising light chain variable region (V /J ) nucleotide sequence operably linked with the immunoglobulin heavy chain constant region.
70. A method of making a genetically modified mouse according to any one of claims 22- 30, comprising replacing all functional endogenous mouse immunoglobulin heavy chain variable V gene segments, all functional endogenous mouse immunoglobulin heavy chain diversity DH gene segments and all functional endogenous mouse immunoglobulin heavy chain joining J gene segments with a plurality of unrearranged human immunoglobulin light chain variable V (hV ) gene segments and all five unrearranged human immunoglobulin light chain joining J (hJ ) gene segments, wherein the plurality of unrearranged hV gene κ κ κ segments and all five unrearranged hJ are operably linked to an intact endogenous mouse immunoglobulin heavy chain constant region gene at the endogenous mouse immunoglobulin heavy chain locus.
71. A method for making a genetically modified mouse according to any one of claims 31 to 39, comprising replacing at an endogenous heavy chain locus of the mouse all functional V , D and J gene segments of the mouse with a first unrearranged human V gene H H H K segment and an unrearranged human J gene segment, to thereby operably link the first unrearranged human V gene segment and unrearranged human J gene segment to the endogenous heavy chain constant region, and also inserting into the germline of the mouse a second human V gene segment and a human J gene segment operably linked to a mouse light chain constant region.
72. The mouse according to any one of claims 1, 14, 20, 21 or 31, substantially as herein described and exemplified and/or described with reference to the accompanying figures.
73. The use according to any one of claims 40, 41, 45 or 46, substantially as herein described and exemplified and/or described with reference to the accompanying figures.
74. The antigen binding protein according to claim 57, substantially as herein described and exemplified and/or described with reference to the accompanying figures.
75. The method according to any one of claims 62, 69-71 substantially as herein described and exemplified and/or described with reference to the accompanying figures.
76. The nucleotide sequence according to claim 67, substantially as herein described and exemplified and/or described with reference to the accompanying figures.
77. The mouse cell according to any one of claims 47 and 51-53, substantially as herein described and exemplified and/or described with reference to the accompanying figures.
78. The tissue of claim 47, substantially as herein described and exemplified and/or described with reference to the accompanying figures.
79. The antigen-binding protein of claim 57, substantially as herein described and Exemplified and/or described with reference to the accompanying figures.
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