U.S. patent application number 14/135518 was filed with the patent office on 2014-05-08 for mice that make vl binding proteins.
This patent application is currently assigned to Regeneron Pharmaceuticals, Inc.. The applicant listed for this patent is Regeneron Pharmaceuticals, Inc.. Invention is credited to Cagan Gurer, Karolina A. Hosiawa, Lynn Macdonald, Andrew J. Murphy, Sean Stevens.
Application Number | 20140130194 14/135518 |
Document ID | / |
Family ID | 44543797 |
Filed Date | 2014-05-08 |
United States Patent
Application |
20140130194 |
Kind Code |
A1 |
Macdonald; Lynn ; et
al. |
May 8, 2014 |
MICE THAT MAKE VL BINDING PROTEINS
Abstract
Genetically modified mice and methods for making an using them
are provided, wherein the mice comprise a replacement of all or
substantially all immunoglobulin heavy chain V gene segments, D
gene segments, and J gene segments with at least one light chain V
gene segment and at least one light chain J gene segment. Mice that
make binding proteins that comprise a light chain variable domain
operably linked to a heavy chain constant region are provided.
Binding proteins that contain an immunoglobulin light chain
variable domain, including a somatically hypermutated light chain
variable domain, fused with a heavy chain constant region, are
provided. Modified cells, embryos, and mice that encode sequences
for making the binding proteins are provided.
Inventors: |
Macdonald; Lynn; (White
Plains, NY) ; Stevens; Sean; (San Francisco, CA)
; Gurer; Cagan; (Valhalla, NY) ; Hosiawa; Karolina
A.; (Tarrytown, NY) ; Murphy; Andrew J.;
(Croton-on-Hudson, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Regeneron Pharmaceuticals, Inc. |
Tarrytown |
NY |
US |
|
|
Assignee: |
Regeneron Pharmaceuticals,
Inc.
Tarrytown
NY
|
Family ID: |
44543797 |
Appl. No.: |
14/135518 |
Filed: |
December 19, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13195951 |
Aug 2, 2011 |
|
|
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14135518 |
|
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|
|
61369909 |
Aug 2, 2010 |
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Current U.S.
Class: |
800/18 ;
435/328 |
Current CPC
Class: |
C07K 2317/14 20130101;
C07K 16/082 20130101; A01K 67/0278 20130101; A01K 67/0275 20130101;
C07K 16/461 20130101; C07K 16/00 20130101; C07K 2317/31 20130101;
C12N 2015/8518 20130101; C07K 2317/21 20130101; C07K 16/468
20130101; C07K 2317/92 20130101; C12N 15/8509 20130101; A01K
2267/01 20130101; C07K 2317/56 20130101; A01K 2217/072 20130101;
A01K 2227/105 20130101 |
Class at
Publication: |
800/18 ;
435/328 |
International
Class: |
A01K 67/027 20060101
A01K067/027; C07K 16/08 20060101 C07K016/08 |
Claims
1.-21. (canceled)
22. A mouse whose genome comprises a sequence that includes an
unrearranged human light chain variable (V.sub.L) gene segment and
an unrearranged human light chain joining (J.sub.L) gene segment
operably linked with one another to allow for recombination to form
a rearranged human V.sub.L/J.sub.L region, and further operably
linked with an intact heavy chain constant (C.sub.H) region gene,
which sequence lacks a functional D.sub.H gene segment between the
unrearranged V.sub.L gene segment and the unrearranged J.sub.L gene
segment, so that when immunized with an antigen, the mouse
generates an antibody in which at least one chain is comprised of
an immunoglobulin light chain variable region amino acid sequence
that lacks a D amino acid sequence, fused to an immunoglobulin
heavy chain constant region amino acid sequence.
23. The mouse of claim 22, wherein the unrearranged V.sub.L and
J.sub.L gene segments are V.kappa. and J.kappa. gene segments.
24. The mouse of claim 23, wherein the sequence replaces all of the
endogenous V.sub.H, D.sub.H and J.sub.H segments at the endogenous
heavy chain locus.
25. The mouse of claim 24, wherein: the sequence includes a
plurality of unrearranged human V.sub.L gene segments and a
plurality of unrearranged human J.sub.L gene segments; and wherein
the intact C.sub.H region is in an endogenous C.sub.H region.
26. The mouse of claim 25, wherein the endogenous C.sub.H region is
one of a plurality of endogenous C.sub.H regions.
27. The mouse of claim 26, wherein the sequence that includes an
unrearranged human light chain variable (V.sub.L) gene segment and
an unrearranged human light chain joining (J.sub.L) gene segment is
operably linked with each of the plurality of endogenous C.sub.H
regions.
28. The mouse of claim 25, wherein the sequence includes (a) six
human V.kappa. gene segments and five human J.kappa. gene segments;
(b) 16 human V.kappa. gene segments and five human J.kappa. gene
segments; (c) 30 human V.kappa. gene segments and five human
J.kappa. gene segments; or (d) 40 human V.kappa. gene segments and
five human J.kappa. gene segments.
29. The mouse of claim 22, wherein the unrearranged V.sub.L and
J.sub.L gene segments are V.lamda. and J.lamda. gene segments.
30. The mouse of claim 29, wherein the sequence replaces all of the
endogenous V.sub.H, D.sub.H and J.sub.H segments at the endogenous
heavy chain locus.
31. The mouse of claim 30, wherein the sequence comprises a
plurality of unrearranged human V.sub.L gene segments and a
plurality of unrearranged human J.sub.L gene segments; and wherein
the intact C.sub.H region is an endogenous C.sub.H region.
32. The mouse of claim 31, wherein the sequence includes (a) 12
human V.lamda. gene segments and one human J.lamda. gene segment;
(b) 28 human V.lamda. gene segments and one human J.lamda. gene
segment; or (c) 40 human V.lamda. gene segments and one human
J.lamda. gene segment.
33. The mouse of claim 32, wherein the one human J.lamda. gene
segment is human J.lamda.1.
34. The mouse of claim 31, wherein the sequence includes (a) 12
human V.lamda. gene segments and four human J.lamda. gene segments;
(b) 28 human V.lamda. gene segments and four human J.lamda. gene
segments; or (c) 40 human V.lamda. gene segments and four human
J.lamda. gene segments.
35. The mouse of claim 34, wherein the four human J.lamda. gene
segments are J.lamda.1, J.lamda.2, J.lamda.3 and J.lamda.7.
36. The mouse of claim 22, which mouse contains B cells that
comprise a rearranged V.sub.L/J.sub.L region operably linked to a
mouse C.sub.H region.
37. The mouse of claim 36, wherein the rearranged V.sub.L/J.sub.L
region is a rearranged V.kappa./J.kappa. region.
38. The mouse of claim 22, which mouse, when immunized with an
antigen, generates antibodies comprised of four chains: two of
which include a light chain variable region amino acid sequence
fused to a light chain constant region amino acid sequence, and two
of which include an immunoglobulin light chain variable region
amino acid sequence that lacks a D.sub.H amino acid sequence, fused
to an immunoglobulin heavy chain constant region amino acid
sequence.
39. An isolated mouse cell whose genome comprises a sequence that
includes an unrearranged human light chain variable (V.sub.L) gene
segment and an unrearranged human light chain joining (J.sub.L)
gene segment operably linked with one another to allow for
recombination to form a rearranged human V.sub.L/J.sub.L region,
and further operably linked with an intact heavy chain constant
(C.sub.H) region gene, which sequence lacks a functional D.sub.H
gene segment between the unrearranged V.sub.L gene segment and the
unrearranged J.sub.L gene segment.
40. The isolated mouse cell of claim 39, wherein the cell is an
embryonic stem (ES) cell.
41. The isolated mouse cell of claim 39, wherein the unrearranged
V.sub.L and J.sub.L gene segments are V.kappa. and J.kappa. gene
segments.
42. The isolated mouse cell of claim 39, wherein the unrearranged
V.sub.L and J.sub.L gene segments are V.lamda. and J.lamda. gene
segments.
43. The isolated mouse cell of claim 39, wherein the intact C.sub.H
region gene is a mouse C.sub.H region gene.
44. The isolated mouse cell of claim 39, wherein the unrearranged
human V.sub.L and J.sub.L gene segments replace all of the
endogenous V.sub.H, D.sub.H and J.sub.H segments at the endogenous
heavy chain locus.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 USC
.sctn.119(e) of U.S. Provisional Application Ser. No. 61/369,909,
filed 2 Aug. 2010, which application is hereby incorporated by
reference.
FIELD OF INVENTION
[0002] 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
[0003] Antibodies typically comprise a tetrameric structure having
two identical heavy chains that each comprise a heavy chain
constant region (C.sub.H) fused with a heavy chain variable domain
(V.sub.H) associated with a light chain constant region (C.sub.L)
fused with a light chain variable domain (V.sub.L). 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.
[0004] In a typical antibody, V.sub.H and V.sub.L 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.sub.H and
V.sub.L domains. In typical antibodies in humans and in mice,
V.sub.H domains couple with either .lamda. or .kappa. V.sub.L
domains. It is also known, however, that V.sub.L domains can be
made that specifically bind a target antigen in the absence of a
cognate V.sub.H domain (e.g., a V.sub.H domain that naturally
expresses in the context of an antibody and is associated with the
particular V.sub.L domain), and that V.sub.H domains can be
isolated that specifically bind a target antigen in the absence of
a cognate V.sub.L domain. Thus, useful diversity in
immunoglobulin-based binding proteins is generally conferred by
recombination leading to a particular V.sub.H or V.sub.L (and
somatic hypermutation, to the extent that it occurs), as well as by
combination of a cognate V.sub.H/V.sub.L pair. It would be useful
to develop compositions and methods to exploit other sources of
diversity.
[0005] 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
[0006] In one aspect, binding proteins are described that comprise
immunoglobulin variable domains that are derived from light chain
(i.e., kappa (.kappa.) and/or lambda (.lamda.)) 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.
[0007] In one aspect, nucleic acids constructs, cells, embryos,
mice, and methods are provided for making proteins that comprise
one or more .kappa. and/or .lamda. light chain variable region
immunoglobulin sequences and an immunoglobulin heavy chain constant
region sequence, including proteins that comprise a human .lamda.
or .kappa. light chain variable domain and a human or mouse heavy
chain constant region sequence.
[0008] 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.sub.H) gene
segments, heavy chain diversity (D.sub.H) gene segments, and heavy
chain joining (J.sub.H) gene segments at an endogenous mouse
immunoglobulin heavy chain locus with one or more light chain
variable region (V.sub.L) gene segments and one or more light chain
joining region (J.sub.L) gene segments.
[0009] In one aspect, a mouse is provided, comprising an
immunoglobulin heavy chain locus that comprises a replacement of
all or substantially all V.sub.H, D.sub.H, and J.sub.H gene
segments with one or more V.sub.L gene segments and one or more
J.sub.L gene segments to form a V.sub.L gene segment sequence at an
endogenous heavy chain locus (VL.sub.H locus), wherein the VL.sub.H
locus is capable of recombining with an endogenous mouse C.sub.H
gene to form a rearranged gene that is derived from a V.sub.L gene
segment, a J.sub.L gene segment, and an endogenous mouse C.sub.H
gene.
[0010] In one embodiment, the V.sub.L segments are human V.sub.L.
In one embodiment, the segments are human J.sub.L. In a specific
embodiment, the V.sub.L and J.sub.L segments are human V.sub.L and
human J.sub.L segments.
[0011] In one embodiment, all or substantially all V.sub.H,
D.sub.H, and J.sub.H gene segments are replaced with at least six
human V.kappa. gene segments and at least one J.kappa. gene
segment. In one embodiment, all or substantially all V.sub.H,
D.sub.H, and J.sub.H gene segments are replaced with at least 16
human V.kappa. gene segments (human V.sub..kappa.) and at least one
J.kappa. gene segment. In one embodiment, all or substantially all
V.sub.H, D.sub.H, and J.sub.H gene segments are replaced with at
least 30 human V.kappa. gene segments and at least one J.kappa.
gene segment. In one embodiment, all or substantially all V.sub.H,
D.sub.H, and J.sub.H gene segments are replaced with at least 40
human V.kappa. gene segments and at least one J.kappa. gene
segment. In one embodiment, the at least one J.kappa. gene segment
comprises two, three, four, or five human J.kappa. gene
segments.
[0012] In one embodiment, the V.sub.L segments are human V.kappa.
segments. In one embodiment, the human V.kappa. segments comprise
4-1, 5-2, 7-3, 2-4, 1-5, and 1-6. In one embodiment, the .kappa.
V.sub.L 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 V.kappa. 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 V.kappa. segments
comprise 3-31, 1-32, 1-33, 3-34, 1-35, 2-36, 1-37, 2-38, 1-39, and
2-40.
[0013] In one embodiment, the V.sub.L segments are human V.kappa.
segments and comprise 4-1, 5-2, 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 V.kappa. 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 V.kappa. segments further comprise 3-31, 1-32,
1-33, 3-34, 1-35, 2-36, 1-37, 2-38, 1-39, and 2-40.
[0014] In one embodiment, the V.sub.L segments are human V.lamda.
segments and comprise a fragment of cluster A of the human light
chain locus. In a specific embodiment, the fragment of cluster A of
the human .lamda. light chain locus extends from hV.lamda.3-27
through hV.lamda.3-1.
[0015] In one embodiment, the V.sub.L segments comprise a fragment
of cluster B of the human .lamda. light chain locus. In a specific
embodiment, the fragment of cluster B of the human .lamda. light
chain locus extends from hV.lamda.5-52 through hV.lamda.1-40.
[0016] In one embodiment, the V.sub.L segments comprise a human
.lamda. 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 .lamda. light chain variable region
sequence comprises at least one gene segment of cluster A and at
least one gene segment of cluster B.
[0017] In one embodiment, the V.sub.L segments comprise at least
one gene segment of cluster B and at least one gene segment of
cluster C.
[0018] In one embodiment, the V.sub.L segments comprise hV.lamda.
3-1, 4-3, 2-8, 3-9, 3-10, 2-11, and 3-12. In a specific embodiment,
the V.sub.L segments comprise a contiguous sequence of the human
.lamda. light chain locus that spans from V.lamda.3-12 to
V.lamda.3-1. In one embodiment, the contiguous sequence comprises
at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hV.lamda.s. In a
specific embodiment, the hV.lamda.s include 3-1, 4-3, 2-8, 3-9,
3-10, 2-11, and 3-12. In a specific embodiment, the hV.lamda.s
comprises a contiguous sequence of the human .lamda. locus that
spans from V.lamda.3-12 to V.lamda.3-1.
[0019] In one embodiment, the hV.lamda.s comprises 13 to 28 or more
hV.lamda.s. In a specific embodiment, the hV.lamda.s 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 hV.lamda.s comprise a contiguous sequence of the
human .lamda. locus that spans from V.lamda.3-27 to
V.lamda.3-1.
[0020] In one embodiment, the V.sub.L segments comprise 29 to 40
hV.lamda.s. In a specific embodiment, the V.sub.L segments comprise
a contiguous sequence of the human .lamda. locus that spans from
V.lamda.3-29 to V.lamda.3-1, and a contiguous sequence of the human
.lamda. locus that spans from V.lamda.5-52 to V.lamda.1-40. In a
specific embodiment, all or substantially all sequence between
hV.lamda.1-40 and hV.lamda.3-29 in the genetically modified mouse
consists essentially of a human .lamda. sequence of approximately
959 bp found in nature (e.g., in the human population) downstream
of the hV.lamda.1-40 gene segment (downstream of the 3'
untranslated portion), a restriction enzyme site (e.g., PI-Scel),
followed by a human .lamda. sequence of approximately 3,431 bp
upstream of the hV.lamda.3-29 gene segment found in nature.
[0021] In one embodiment, the J.kappa. is human and is selected
from the group consisting of J.kappa.1, J.kappa.2, J.kappa.3,
J.kappa.4, J.kappa.5, and a combination thereof. In a specific
embodiment, the J.kappa. comprises J.kappa.1 through J.kappa.5.
[0022] In one embodiment, the V.sub.L segments are human V.lamda.
segments, and the J.kappa. gene segment comprises an RSS having a
12-mer spacer, wherein the RSS is juxtaposed at the upstream end of
the J.kappa. gene segment. In one embodiment, the V.sub.L gene
segments are human V.lamda. and the VL.sub.H locus comprises two or
more J.kappa. gene segments, each comprising an RSS having a 12-mer
spacer wherein the RSS is juxtaposed at the upstream end of each
J.kappa. gene segment.
[0023] In a specific embodiment, the V.sub.L segments comprise
contiguous human .kappa. gene segments spanning the human .kappa.
locus from V.kappa.4-1 through V.kappa.2-40, and the J.sub.L
segments comprise contiguous gene segments spanning the human
.kappa. locus from J.kappa.1 through J.kappa.5.
[0024] In one embodiment, where the V.sub.L segments are V.lamda.
segments and no D.sub.H segment is present between the V.sub.L
segments and J segments, the V.sub.L segments are flanked
downstream (i.e., juxtaposed on the downstream side) with 23-mer
RSS, and J.kappa. segments if present or J.lamda. segments if
present are flanked upstream (i.e., juxtaposed on the upstream
side) with 12-mer RSS.
[0025] In one embodiment, where the V gene segments are V.kappa.
gene segments and no D.sub.H gene segment is present between the V
gene segments and J gene segments, the V.kappa. gene segments are
each juxtaposed on the downstream side with a 12-mer RSS, and
J.kappa. segments if present or J.lamda. segments if present are
each juxtaposed on the upstream side with a 23-mer RSS.
[0026] In one embodiment, the mouse comprises a rearranged gene
that is derived from a V.sub.L gene segment, a J.sub.L gene
segment, and an endogenous mouse C.sub.H 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.sub.L
segment and the J.sub.L 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.sub.L gene segment and the same J.sub.L 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 K.sub.D in the low nanomolar range or lower (e.g.,
a K.sub.D of 10 nanomolar or lower). In a specific embodiment, the
V.sub.L segment, the J.sub.L segment, or both, are human gene
segments. In a specific embodiment, the V.sub.L and J.sub.L
segments are human .kappa. gene segments. In one embodiment, the
mouse C.sub.H gene is selected from IgM, IgD, IgG, IgA and IgE. In
a specific embodiment, the mouse IgG is selected from IgG1, IgG2A,
IgG2B, IgG2C and IgG3. In another specific embodiment, the mouse
IgG is IgG1.
[0027] 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.sub.H
region fused with a V.sub.L; and, (b) two identical polypeptides
that comprise an endogenous mouse C.sub.L region fused with a
V.sub.L region that is cognate with respect to the V.sub.L region
that is fused with the mouse C.sub.H region, and, in one
embodiment, is a human (e.g., a human .kappa.) V.sub.L region. In
one embodiment, the V.sub.L region fused to the endogenous mouse
C.sub.H region is a human V.sub.L region. In a specific embodiment,
the human V.sub.L region fused with the mouse C.sub.H region is a
V.kappa. region. In a specific embodiment, the human V.sub.L region
fused with the mouse C.sub.H region is identical to a V region
encoded by a rearranged human germline light chain nucleotide
sequence. In a specific embodiment, the human V.sub.L region fused
to the mouse C.sub.H region comprises two, three, four, five, six,
or more somatic hypermutations. In one embodiment, the human
V.sub.L region fused to the mouse C.sub.H region is encoded by a
rearranged gene that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or
more N additions.
[0028] In one embodiment, at least 50% of all IgG molecules made by
the mouse comprise a polypeptide that comprises an IgG isotype
C.sub.H region and a V.sub.L region, wherein 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 IgG molecules
comprise the polypeptide recited in this paragraph. In one
embodiment, at least 80%, 85%, 90%, or 95% of all IgG molecules
comprise the polypeptide recited in this paragraph. In a specific
embodiment, all IgG molecules made by the mouse comprise a
polypeptide that is no longer than the length of the polypeptide
recited in this paragraph.
[0029] In one embodiment, the mouse makes a binding protein
comprising a first polypeptide that comprises an endogenous mouse
C.sub.H region fused with a variable domain encoded by a rearranged
human V gene segment and a J gene segment but not a D.sub.H gene
segment, and a second polypeptide that comprises an endogenous
mouse C.sub.L region fused with a V domain encoded by a rearranged
human V gene segment and a J gene segment but not a D.sub.H gene
segment, and the binding protein specifically binds an antigen with
an affinity in the micromolar, nanomolar, or picomolar range. In
one embodiment, the J segment is a human J segment (e.g., a human
.kappa. gene segment). In one embodiment, the human V segment is a
human V.kappa. segment. In one embodiment, the variable domain that
is fused with the endogenous mouse C.sub.H region comprises a
greater number of somatic hypermutations than the variable region
that is fused with the endogenous mouse C.sub.L region; in a
specific embodiment, the variable region fused with the endogenous
mouse C.sub.H 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.sub.L region; in a specific embodiment, the V
region fused with the mouse C.sub.H 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.sub.L region. In one
embodiment, the V region fused with the mouse C.sub.H region is
encoded by a rearranged gene that comprises 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10 or more N additions.
[0030] In one embodiment, the mouse expresses a binding protein
comprising a first light chain variable domain (V.sub.L1) fused
with an immunoglobulin heavy chain constant region sequence and a
second light chain variable domain (V.sub.L2) fused with an
immunoglobulin light chain constant region, wherein V.sub.L1
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.sub.L2. In one embodiment, the number
of somatic hypermutations in V.sub.L1 is about 2- to about 4-fold
higher than in V.sub.L2. In one embodiment, the number of somatic
hypermutations in V.sub.L1 is about 2- to about 3-fold higher than
in V.sub.L2. In one embodiment, V.sub.L1 is encoded by a sequence
that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N
additions.
[0031] In one aspect, a genetically modified mouse is provided that
expresses an immunoglobulin that consists essentially of the
following polypeptides: a first two identical polypeptides that
each consists essentially of a C.sub.H region fused with a variable
domain that is derived from gene segments that consist essentially
of a V.sub.L gene segment and a J.sub.L gene segment, and a second
two identical polypeptides that each consists essentially of a
C.sub.L region fused with a variable domain that is derived from
gene segments that consist essentially of a V.sub.L segment and a
J.sub.L segment.
[0032] In a specific embodiment, the two identical polypeptides
that have the C.sub.H region have a mouse C.sub.H region.
[0033] In a specific embodiment, the two identical polypeptides
that have the C.sub.L region have a mouse C.sub.L region.
[0034] In one embodiment, the variable domain fused with the
C.sub.L region is a variable domain that is cognate with the
variable domain fused to the C.sub.H region.
[0035] In one embodiment, the variable domain that is fused with
the endogenous mouse C.sub.H region comprises a greater number of
somatic hypermutations than the variable domain that is fused with
the endogenous mouse C.sub.L region; in a specific embodiment, the
variable domain fused with the endogenous mouse C.sub.H 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 C.sub.L region. In
one embodiment, the variable domain fused with the endogenous mouse
C.sub.L region is encoded by a gene that comprises 1, 2, 3, 4, 5,
6, 7, 8, 9, 10 or more N additions.
[0036] 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 .kappa. gene segments.
In another specific embodiment, both of the V segments and the J
segments are human .lamda. gene segments. In one embodiment, the V
segments and the J segments are independently selected from human
.kappa. and human .lamda. gene segments. In a specific embodiment,
the V segments are V.kappa. segments and the J segments are
J.lamda. segments. In another specific embodiment, the V segments
are V.lamda. segments and the J segments are J.kappa. segments.
[0037] In one embodiment, one or more of the variable domains fused
with the C.sub.L region and the variable domains fused with the
C.sub.H region are human variable domains. In a specific
embodiment, the human variable domains are human V.kappa. domains.
In another specific embodiment, the human variable domains are
V.lamda. domains. In one embodiment, the human domains are
independently selected from human V.kappa. and human V.lamda.
domains. In a specific embodiment, the human variable domain fused
with the C.sub.L region is a human V.lamda. domain and the human
variable domain fused with the C.sub.H region is a human V.kappa.
domain. In another embodiment, the human variable domain fused with
the C.sub.L region is a human V.kappa. domain and the human
variable domain fused with the C.sub.H is a human V.lamda.
domain.
[0038] In one embodiment, the V.sub.L gene segment of the first two
identical polypeptides is selected from a human V.lamda. segment
and a human V.kappa. segment. In one embodiment, the V.sub.L
segment of the second two identical polypeptides is selected from a
human V.lamda. segment and a human V.kappa. segment. In a specific
embodiment, the V.sub.L segment of the first two identical
polypeptides is a human V.kappa. segment and the V.sub.L segment of
the second two identical polypeptides is selected from a human
V.kappa. segment and a human V.lamda. segment. In a specific
embodiment, the V.sub.L segment of the first two identical
polypeptides is a human V.lamda. segment and the V.sub.L segment of
the second two identical polypeptides is selected from a human
V.lamda. segment and a human V.kappa. segment. In a specific
embodiment, the human V.sub.L segment of the first two identical
polypeptides is a human V.kappa. segment, and the human V.sub.L
segment of the second two identical polypeptides is a human
V.kappa. segment.
[0039] In one embodiment, the IgG 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 C.sub.H region, wherein the variable domain is encoded
by a nucleotide sequence that consists essentially of a rearranged
V.sub.L segment and a rearranged J segment, and wherein the binding
protein specifically binds an epitope of the antigen with a K.sub.D
in the micromolar, nanomolar, or picomolar range.
[0040] In one aspect, a mouse is provided, wherein all or
substantially all of the IgG 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.
[0041] In one embodiment, the V segment is a V segment of a light
chain. In one embodiment, the light chain is selected from a
.kappa. light chain and a .lamda. light chain. In a specific
embodiment, the light chain is a .kappa. light chain. In a specific
embodiment, the V segment is a human V segment. In a specific
embodiment, the V segment is a human V.kappa. segment and the J
segment is a human J.kappa. segment.
[0042] In one embodiment, the J segment is a J segment of a light
chain. In one embodiment, the light chain is selected from a
.kappa. light chain and a .lamda. light chain. In a specific
embodiment, the light chain is a .kappa. 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.sub.H). In a specific embodiment, the heavy chain is of mouse
origin. In another specific embodiment, the heavy chain is of human
origin.
[0043] 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.
[0044] 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.sub.H region.
[0045] In a specific embodiment, all or substantially all of the
IgG 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 IgG constant region, and the IgG further
comprises a light chain that comprises a human V.sub.L domain fused
with a mouse C.sub.L region. In a specific embodiment, the V.sub.L
domain fused with the mouse C.sub.L region is derived from a human
V.kappa. segment and a human J.kappa. segment. In a specific
embodiment, the V.sub.L domain fused with the mouse C.sub.L region
is derived from a human V.lamda. segment and a human J.lamda.
segment.
[0046] In one aspect, a mouse is provided that makes an IgG
comprising a first CDR3 on a polypeptide comprising a C.sub.H
region and a second CDR3 on a polypeptide comprising a C.sub.L
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.sub.L gene segment
and a J.sub.L gene segment. In one embodiment, the CDR3 on the
polypeptide comprising the C.sub.H 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.
[0047] In one embodiment, the V.sub.L segment and the J.sub.L
segment are human gene segments. In one embodiment, the V.sub.L
segment and the J.sub.L segment are .kappa. gene segments. In one
embodiment, the V.sub.L segment and the J.sub.L segment are .lamda.
gene segments.
[0048] In one aspect, a mouse is provided that makes an IgG
comprising a first CDR3 on a first polypeptide comprising a C.sub.H
region and a second CDR3 on a second polypeptide comprising a
C.sub.L region, wherein both the first CDR3 and the second CDR3
each comprise a 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.sub.H
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.
[0049] 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.
[0050] 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 IgG comprises an
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.
[0051] In one embodiment, the V segment is a human V gene segment.
In a specific embodiment, the V segment is a human V.kappa. gene
segment.
[0052] 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.
[0053] 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 V.kappa. gene segment and a human J.kappa. 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 V.kappa.
gene segment and a J gene segment selected from a human J.kappa.
segment, a human J.lamda. segment, and a human J.sub.H 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 V.lamda. segment and a J segment selected from a human
J.kappa. segment, a human J.lamda. segment, and a human J.sub.H
segment.
[0054] In one aspect, a mouse is provided that makes an IgG that
does not contain an amino acid sequence derived from a D.sub.H gene
segment, wherein the IgG comprises a first polypeptide having a
first V.sub.L domain fused with a mouse C.sub.L region and a second
polypeptide having a second V.sub.L domain fused with a mouse
C.sub.H region, wherein the first V.sub.L domain and the second
V.sub.L domain are not identical. In one embodiment, the first and
second V.sub.L domains are derived from different V segments. In
another embodiment, the first and second V.sub.L domains are
derived from different J segments. In one embodiment, the first and
second V.sub.L domains are derived from identical V and J segments,
wherein the second V.sub.L domain comprises a higher number of
somatic hypermutations as compared to the first V.sub.L domain.
[0055] In one embodiment, the first and the second V.sub.L domains
are independently selected from human and mouse V.sub.L domains. In
one embodiment, the first and second V.sub.L domains are
independently selected from V.kappa. and V.lamda. domains. In a
specific embodiment, the first V.sub.L domain is selected from a
V.kappa. domain and a V.lamda. domain, and the second V.sub.L
domain is a V.kappa. domain. In another specific embodiment, the
V.kappa. domain is a human V.kappa. domain.
[0056] In one aspect, a mouse is provided, wherein all or
substantially all of the IgG made by the mouse consists essentially
of a light chain having a first human V.sub.L domain fused with a
mouse C.sub.L domain, and a heavy chain having a second human
V.sub.L domain fused with a mouse C.sub.H domain.
[0057] In one embodiment, the human V.sub.L domain fused with the
mouse C.sub.H domain is a human V.kappa. domain.
[0058] In one embodiment, the first and the second human V.sub.L
domains are not identical.
[0059] 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.sub.L domain and an immunoglobulin C.sub.L region;
and, (b) a second polypeptide of no more than 535 amino acids in
length, wherein the second polypeptide consists essentially of a
C.sub.H region and a V domain that lacks a sequence derived from a
D.sub.H gene segment.
[0060] 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.
[0061] In one embodiment, in about 90% or more of the IgG made by
the mouse the second polypeptide is no more than about 535 amino
acids in length.
[0062] In one embodiment, in about 50% or more of the IgG 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 IgG
made by the mouse is of the recited length. In a specific
embodiment, all or substantially all of the IgG made by the mouse
is of the recited length.
[0063] In one embodiment, the V domain of the second polypeptide is
a V.sub.L domain. In a specific embodiment, the V domain of the
second polypeptide is selected from a V.kappa. and a V.lamda.
domain. In a specific embodiment, the V domain of the second
polypeptide is a human V.kappa. or V.lamda. domain.
[0064] 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.sub.H sequence, and a heavy chain constant region.
[0065] 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.
[0066] In one embodiment, the polypeptide comprises a V.sub.L
sequence derived from a V.lamda. or a V.kappa. gene segment, the
polypeptide comprises a CDR3 derived from a D.sub.H gene segment,
and the polypeptide comprises a sequence derived from a J.sub.H or
J.lamda. or J.kappa. gene segment.
[0067] In one embodiment, the mouse comprises an endogenous mouse
heavy chain immunoglobulin locus comprising a replacement of all
functional V.sub.H gene segments with one or more human light chain
V.lamda. gene segments wherein the one or more human V.lamda.
segments each have juxtaposed on the downstream side a 23-mer
spaced recombination signal sequence (RSS), wherein the V.lamda.
segments are operably linked to a human or mouse D.sub.H segment
that has juxtaposed upstream and downstream a 12-mer spaced RSS;
the D.sub.H 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.sub.H gene
segment; wherein the V, D.sub.H, and J segments are operably linked
to a nucleic acid sequence encoding a heavy chain constant
region.
[0068] In one embodiment, the mouse comprises an endogenous mouse
heavy chain immunoglobulin locus comprising a replacement of all
functional V.sub.H gene segments with one or more human V.kappa.
gene segments each juxtaposed on the downstream side with a 12-mer
spaced recombination signal sequence (RSS), wherein the V segments
are operably linked to a human or mouse D.sub.H segment that is
juxtaposed both upstream and downstream with a 23-mer spaced RSS;
the D.sub.H 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 spaced RSS juxtaposing the D.sub.H
segment; wherein the V, D.sub.H, and gene segments are operably
linked to a nucleic acid sequence encoding a heavy chain constant
region.
[0069] 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.sub.H1, a hinge, a C.sub.H2, a C.sub.H3, and a combination
thereof. In one embodiment, one or more of the C.sub.H1, hinge,
C.sub.H2, and C.sub.H3 are human.
[0070] In one embodiment, the mouse comprises an endogenous mouse
heavy chain immunoglobulin locus comprising a replacement of all
functional V.sub.H gene segments with a plurality of human V.lamda.
or V.kappa. gene segments each juxtaposed downstream with 23-mer
spaced RSS, a plurality of human D.sub.H segments juxtaposed both
upstream and downstream by a 12-mer spaced RSS, a plurality of
human J segments (J.sub.H or J.lamda. or J.kappa.) juxtaposed both
upstream and downstream with a 23-mer spaced RSS, wherein the locus
comprises an endogenous mouse constant region sequence selected
from C.sub.H1, hinge, C.sub.H2, C.sub.H3, and a combination
thereof. In a specific embodiment, the mouse comprises all or
substantially all functional human V.lamda. or V.kappa. segments,
all or substantially all functional human D.sub.H segments, and all
or substantially all J.sub.H or J.lamda. or J.kappa. segments.
[0071] 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 Fc 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 .lamda. sequences, .kappa. 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 V.lamda. segment or a V.kappa. 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 V.kappa. segment, a human D segment,
and a human J.kappa. segment.
[0072] In one embodiment, the polypeptide of comprises a variable
region derived from a human V.lamda. or V.kappa. gene segment, a
human D.sub.H gene segment, and a human J.sub.H or J.lamda. or
J.kappa. gene segment. In a specific embodiment, the heavy chain
constant sequence is derived from a human C.sub.H1 and a mouse
C.sub.H2 and a mouse C.sub.H3 sequence.
[0073] In one aspect, a mouse is provided that comprises in its
germline an unrearranged human V.kappa. or V.lamda. gene segment
operably linked to a human J gene segment and a heavy chain
constant region sequence, wherein the mouse expresses a V.sub.L
binding protein that comprises a human V.kappa. domain fused with a
heavy chain constant region, and wherein the mice exhibit a
population of splenic B cells that express V.sub.L binding proteins
in CD19.sup.+ B cells, including transitional B cells
(CD19.sup.+IgM.sup.hiIgD.sup.int), and mature B cells
(CD19.sup.+IgM.sup.intIgD.sup.hi).
[0074] In one aspect, a mouse is provided that comprises in its
germline an unrearranged human V.kappa. or V.lamda. 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 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).
[0075] In one embodiment, the mice comprise at least 6 unrearranged
hV.kappa. gene segments and one or more unrearranged hJ.kappa. gene
segments, and the mice comprise a lymphocyte-gated and IgM.sup.+
spleen cell population expressing a V.sub.L binding protein,
wherein the population is at least 75% as large as a
lymphocyte-gated and IgM.sup.+ spleen cell population of a
wild-type mouse.
[0076] In one embodiment, the mice exhibit a mature B cell-gated
(CD19.sup.+) splenocyte population of IgD.sup.+ cells and IgM.sup.+
cells that total about 90%; in one embodiment, the mature B
cell-gated (CD19.sup.+) splenocyte population of IgD.sup.+ cells
and IgM.sup.+ cells of the modified mouse is about the same (e.g.,
within 10%, or within 5%) as the total of IgD.sup.+ cells and
IgM.sup.+ cells of a wild-type mouse that are mature B cell-gated
(CD19.sup.+) splenocytes.
[0077] 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.
[0078] 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.
[0079] 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 constant sequence and an immunoglobulin
heavy chain constant sequence selected from the group consisting of
a C.sub.H1, a hinge, a C.sub.H2, a C.sub.H3, and a combination
thereof.
[0080] 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.
[0081] 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
germline 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.
[0082] In one aspect, a mouse as described herein is provided,
wherein the mouse comprises a functionally silenced light chain
locus selected from a .lamda. locus, a .kappa. locus, and a
combination thereof. In one embodiment, the mouse comprises a
deletion of a .lamda. and/or a .kappa. locus, in whole or in part,
such that the .lamda. and/or .kappa. locus is nonfunctional.
[0083] 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.
[0084] In one aspect, a mouse or a cell made using a nucleic acid
construct as described herein is provided.
[0085] In one aspect, a mouse made using a cell as described herein
is provided. In one embodiment, the cell is a mouse ES cell.
[0086] 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.sub.L1) that is cognate with a
second human light chain immunoglobulin variable sequence
(V.sub.L2), wherein the V.sub.L1 fused with a human immunoglobulin
light chain constant region (polypeptide 1) expresses with V.sub.L2
fused with a human immunoglobulin heavy chain constant region
(polypeptide 2), as a dimer of polypeptide1/polypeptide 2, to form
a V.sub.L1-V.sub.L2 antibody.
[0087] 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
V.sub.L-C.sub.H polypeptide, wherein the human V.sub.L-C.sub.H
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 .lamda. or human .kappa. light chain). In one
embodiment, the V.sub.L-C.sub.H dimer specifically binds an antigen
of interest in the absence of a .lamda. light chain and in the
absence of a .kappa. light chain.
[0088] 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 V.lamda. or human
V.kappa. domain.
[0089] In one aspect, use of a mouse as described herein to make a
fully human Fab (comprising a first human V.sub.L fused with a
human light chain constant region, and a second human V.sub.L fused
with a human heavy chain constant region sequence) or a fully human
F(ab).sub.2 is provided.
[0090] 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 V.lamda. or V.kappa. domain operably linked to a nucleic acid
sequence that comprises a mouse constant region nucleic acid
sequence.
[0091] In one aspect, use of a mouse as described herein to make a
hybridoma or quadroma is provided.
[0092] 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.
[0093] In one aspect, a cell is provided, comprising a nucleic acid
sequence encoding a first polypeptide that comprises a first
somatically mutated human V.kappa. or V.lamda. sequence fused to a
human heavy chain constant region sequence.
[0094] In one embodiment, the cell further comprises a second
polypeptide chain that comprises a second somatically mutated human
V.kappa. or V.lamda. sequence fused to a human light chain constant
region sequence.
[0095] In one embodiment, the human V.kappa. or V.lamda. sequence
of the first polypeptide is cognate with the human V.kappa. or
V.lamda. sequence of the second polypeptide.
[0096] In one embodiment, the V.kappa. or V.lamda. of the first
polypeptide and the human V.kappa. or V.lamda. 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 V.kappa., and
the second polypeptide comprises a variable domain consisting of a
human V.kappa. that is cognate with the human V.kappa. of the first
polypeptide, and the human constant region sequence is an IgG
sequence.
[0097] 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.TM.
cell.
[0098] In one aspect, a somatic mouse cell is provided, comprising
a chromosome that comprises a genetic modification as described
herein.
[0099] In one aspect, a mouse germ cell is provided, comprising a
nucleic acid sequence that comprises a genetic modification as
described herein.
[0100] 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.
[0101] 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.
[0102] In one aspect, a nucleic acid construct is provided that
comprises a human D.sub.H 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 V.kappa. gene
segments. In one embodiment, the targeting construct comprises all
or substantially all human D.sub.H gene segments each juxtaposed
upstream and downstream with a 23-mer spaced RSS.
[0103] In one aspect, a nucleic acid construct is provided that
comprises a human J.kappa. 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 D.sub.H 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
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.
[0104] In one aspect, a nucleic acid construct is provided that
comprises a human V.lamda. segment juxtaposed downstream with a
23-mer spaced RSS, a human D.sub.H segment juxtaposed upstream and
downstream with a 12-mer spaced RSS, and a human J segment selected
from a J.kappa. segment juxtaposed upstream with a 23-mer spaced
RSS, a human J.lamda. segment juxtaposed upstream with a 23-mer
spaced RSS, and a human J.sub.H 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 V.lamda.
sequence.
[0105] In one embodiment, the nucleic acid construct comprises a
human .lamda. light chain variable region sequence that comprises a
fragment of cluster A of the human .lamda. light chain locus. In a
specific embodiment, the fragment of cluster A of the human .lamda.
light chain locus extends from hV.lamda.3-27 through
hV.lamda.3-1.
[0106] In one embodiment, the nucleic acid construct comprises a
human .lamda. light chain variable region sequence that comprises a
fragment of cluster B of the human .lamda. light chain locus. In a
specific embodiment, the fragment of cluster B of the human .lamda.
light chain locus extends from hV.lamda.5-52 through
hV.lamda.1-40.
[0107] In one embodiment, nucleic acid construct comprises a human
.lamda. 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 .lamda. light chain variable region
sequence comprises at least one gene segment of cluster A and at
least one gene segment of cluster B.
[0108] In one embodiment, the human .lamda. light chain variable
region sequence comprises at least one gene segment of cluster B
and at least one gene segment of cluster C.
[0109] In one aspect, a nucleic acid construct is provided,
comprising a human D.sub.H segment juxtaposed upstream and
downstream with a 23-mer spaced RSS normally found in nature
flanking either a J.kappa., a J.sub.H, a V.lamda., or a V.sub.H
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.sub.H1, hinge, C.sub.H2, C.sub.H3, 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
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 J.kappa., a human J.lamda., and a human J.sub.H.
[0110] In one aspect, a nucleic acid construct is provided that
comprises a human D.sub.H 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.
[0111] In one aspect, a nucleic acid construct is provided that
comprises a human V.lamda. or a human V.kappa. segment, a D.sub.H
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 V.lamda. juxtaposed with a 3' 23-mer spaced RSS,
a human D.sub.H segment juxtaposed upstream and downstream with a
12-mer spaced RSS, and a human J.kappa. segment juxtaposed with a
5' 23-mer spaced RSS. In one embodiment, the construct comprises a
human V.kappa. juxtaposed with a 3' 12-mer spaced RSS, a human
D.sub.H segment juxtaposed upstream and downstream with a 23-mer
spaced RSS, and a human J.lamda. segment juxtaposed with a 5'
12-mer spaced RSS.
[0112] 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 V.sub.L gene
segments or a contiguous sequence of human V.sub.L gene segments
and at least one human J.kappa. gene segment, wherein the
contiguous sequence is selected from the group consisting of (i)
hV.kappa.4-1 through hV.kappa.1-6 and J.kappa.1, (ii) hV.kappa.4-1
through hV.kappa.1-6 and J.kappa.1 through J.kappa.2, (iii)
hV.kappa.4-1 through hV.kappa.1-6 and J.kappa.1 through J.kappa.3,
(iv) hV.kappa.4-1 through hV.kappa. 1-6 and J.kappa.1 through
J.kappa.4, (v) hV.kappa.4-1 through hV.kappa.1-6 and J.kappa.1
through J.kappa.5, (vi) hV.kappa.3-7 through hV.kappa.1-16, (vii)
hV.kappa.1-17 through hV.kappa.2-30, (viii) hV.kappa.3-31 through
hV.kappa. 2-40, and (ix) a combination thereof.
[0113] 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.
[0114] 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.
[0115] 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 V.lamda. or human
V.kappa. segment) fused with a human heavy chain constant sequence.
In one embodiment, the human therapeutic comprises a first
polypeptide that is a human .lamda. or .kappa. immunoglobulin light
chain, and a second polypeptide that comprises a human V.lamda. or
human V.kappa. variable sequence fused with a human heavy chain
constant sequence.
[0116] 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.sub.L
domain fused with a human C.sub.H domain.
[0117] In one embodiment, the expression system further comprises a
nucleotide sequence that encodes an immunoglobulin V.sub.L domain
fused with a human C.sub.L domain, wherein the V.sub.L domain fused
with the human C.sub.L domain is a cognate light chain with the
V.sub.L domain fused with the human C.sub.H domain.
[0118] 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.TM. cell).
[0119] In one aspect, a method for making a binding protein is
provided, comprising obtaining a nucleotide sequence encoding a
V.sub.L domain from a gene encoding a V.sub.L region fused to a
C.sub.H region from a cell of a mouse as described herein, and
cloning the nucleotide sequence encoding the V.sub.L region
sequence in frame with a gene encoding a human C.sub.H region to
form a human binding protein sequence, expressing the human binding
protein sequence in a suitable cell.
[0120] In one embodiment, the mouse has been immunized with an
antigen of interest, and the V.sub.L region fused to the C.sub.H
region specifically binds (e.g., with a K.sub.D in the micromolar,
nanomolar, or picomolar range) an epitope of the antigen of
interest. In one embodiment, nucleotide sequence encoding the
V.sub.L region fused to the C.sub.H region is somatically mutated
in the mouse.
[0121] In one embodiment, the suitable cell is selected from a B
cell, a hybridoma, a 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.TM. cell).
[0122] In one embodiment, the C.sub.H region comprises a human IgG
isotype. In a specific embodiment, the human IgG is selected from
an IgG1, IgG2, and IgG4. In another specific embodiment, the human
IgG is IgG1. In another specific embodiment, the human IgG is IgG4.
In another specific embodiment, the human IgG4 is a modified IgG4.
In one embodiment, the modified IgG4 comprises a substitution in
the hinge region. In a specific embodiment, the modified IgG4
comprises a substitution at amino acid residue 228 relative to a
wild-type human IgG4, 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.
[0123] In one embodiment, the cell further comprises a nucleotide
sequence encoding a V.sub.L domain from a light chain that is
cognate to the V.sub.L domain fused to the C.sub.H region, and the
method further comprises expressing the nucleotide sequence
encoding the cognate V.sub.L domain fused to a human C.kappa. or
C.lamda. domain.
[0124] 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.sub.H, D.sub.H, and J.sub.H segments) with one or
more functional human light chain segments (i.e., V.sub.L and
J.sub.L segments). In one embodiment, the replacement is of all or
substantially all functional mouse heavy chain V.sub.H, D.sub.H,
and J.sub.H segments with all or substantially all human V.lamda.
or V.kappa. segments and at least one J.lamda. or J.kappa. segment.
In a specific embodiment, the replacement includes all or
substantially all functional human J.lamda. or J.kappa.
segments.
[0125] In one aspect, a method is provided for making a mouse that
expresses a polypeptide that comprises a sequence derived from a
human immunoglobulin V.lamda. or V.kappa. and/or J.lamda. or
J.kappa. segment fused with a mouse heavy chain constant region,
comprising replacing endogenous mouse heavy chain immunoglobulin
variable segments (V.sub.H, D.sub.H, and J.sub.H) with at least one
human V.lamda. or V.kappa. segment and at least one human J.lamda.
or J.kappa. 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.
[0126] 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.
[0127] 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 .lamda. or human .kappa. gene segments. In one
embodiment, the one or more unrearranged immunoglobulin light chain
variable region gene segments comprise human V.lamda. or human
V.kappa. segments and one or more J.lamda., J.kappa., or J.sub.H
segments. In one embodiment, the heavy chain constant gene sequence
is a human sequence selected from C.sub.H1, hinge, C.sub.H2,
C.sub.H3, and a combination thereof. In one 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.sub.H1, a hinge, a C.sub.H2, and a
C.sub.H3.
[0128] In one aspect, an immunoglobulin variable region (VR) (e.g.,
comprising a human V.sub.L sequence fused with a human J.sub.L, or
J.sub.H, or D.sub.H and J.sub.H, or D.sub.H and J.sub.L) made in a
mouse as described herein is provided. In a specific embodiment,
the immunoglobulin VR is derived from a germline human gene segment
selected from a V.kappa. segment and a V.lamda. 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 10 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.sub.H1, hinge,
C.sub.H2, C.sub.H3, and a combination thereof).
[0129] 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.sub.H1, hinge, C.sub.H2, C.sub.H3,
and a combination thereof).
[0130] In one aspect, a light chain variable domain encoded by a
nucleic acid sequence derived from a mouse as described herein is
provided.
[0131] In one aspect, an antibody or antigen-binding fragment
thereof (e.g., Fab, F(ab).sub.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
[0132] FIG. 1A 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.sub.H) gene segments, 13 heavy chain diversity (D.sub.H) gene
segments and 4 heavy chain joining (J.sub.H) gene segments as well
as enhancers (Enh) and heavy chain constant (C.sub.H) regions.
[0133] FIG. 1B illustrates a schematic (not to scale) of the human
.kappa. light chain locus. The human .kappa. 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
V.kappa. gene segments. The human .kappa. light chain locus
contains about 76 V.kappa. gene segments, 5 J.kappa. gene segments,
an intronic enhancer (Enh) and a single constant region
(C.kappa.).
[0134] FIG. 2 shows a targeting strategy for progressive insertion
of 40 human V.kappa. and 5 human J.kappa. gene segments into the
mouse heavy chain locus. Hygromycin (HYG) and Neomycin (NEO)
selection cassettes are shown with recombinase recognition sites
(R1, R2, etc.).
[0135] FIG. 3 shows a modified mouse heavy chain locus comprising
human V.kappa. and J.kappa. gene segments operably linked to mouse
C.sub.H regions.
[0136] FIG. 4A shows an exemplary targeting strategy for
progressive insertion of human V.lamda. and a single human J.kappa.
gene segment into the mouse heavy chain locus. Hygromycin (HYG) and
Neomycin (NEO) selection cassettes are shown with recombinase
recognition sites (R1, R2, etc.).
[0137] FIG. 4B shows an exemplary targeting strategy for
progressive insertion of human V.lamda. and four human J.kappa.
gene segments into the mouse heavy chain locus. Hygromycin (HYG)
and Neomycin (NEO) selection cassettes are shown with recombinase
recognition sites (R1, R2, etc.).
[0138] FIG. 5A shows an exemplary targeting strategy for
progressive insertion of human V.lamda. human D.sub.H and human
J.sub.H gene segments into the mouse heavy chain locus. Hygromycin
(HYG) and Neomycin (NEO) selection cassettes are shown with
recombinase recognition sites (R1, R2, etc.).
[0139] FIG. 5B shows an exemplary targeting strategy for
progressive insertion of human V.lamda. human D.sub.H and human
J.kappa. gene segments into the mouse heavy chain locus. Hygromycin
(HYG) and Neomycin (NEO) selection cassettes are shown with
recombinase recognition sites (R1, R2, etc.).
[0140] FIG. 6A shows contour plots of splenocytes stained for
surface expression of B220 and IgM from a representative wild type
(WT) and a representative mouse homozygous for six human V.kappa.
and five human J.kappa. gene segments positioned at the endogenous
heavy chain locus (6hV.kappa.-5hJ.kappa. HO).
[0141] FIG. 6B shows contour plots of splenocytes gated on
CD19.sup.+ B cells and stained for immunoglobulin D (IgD) and
immunoglobulin M (IgM) from a representative wild type (WT) and a
representative mouse homozygous for six human V.kappa. and five
human J.kappa. gene segments positioned at the endogenous heavy
chain locus (6hV.kappa.-5hJ.kappa. HO).
[0142] FIG. 6C shows the total number of CD19.sup.+ B cells,
transitional B cells (CD19.sup.+IgM.sup.hiIgD.sup.int) and mature B
cells (CD19.sup.+IgM.sup.intIgD.sup.hi) in harvested spleens from
wild type (WT) and mice homozygous for six human V.kappa. and five
human J.kappa. gene segments positioned at the endogenous heavy
chain locus (6hV.kappa.-5hJ.kappa. HO).
[0143] FIG. 7A shows contour plots of bone marrow gated on singlets
stained for immunoglobulin M (IgM) and B220 from a wild type mouse
(WT) and a mouse homozygous for six human V.kappa. and five human
J.kappa. gene segments positioned at the endogenous heavy chain
locus (6hV.kappa.-5hJ.kappa. HO). Immature, mature and pro/pre B
cells are noted on each of the dot plots.
[0144] FIG. 7B shows the total number of pre/pro
(B220.sup.+IgM.sup.-), immature (B220.sup.intIgM.sup.+) and mature
(B220.sup.hiIgM.sup.+) B cells in bone marrow isolated from the
femurs of wild type mice (WT) and mice homozygous for six human
V.kappa. and five human J.kappa. gene segments positioned at the
endogenous heavy chain locus (6hV.kappa.-5hJ.kappa. HO).
[0145] FIG. 7C shows contour plots of bone marrow gated on
CD19.sup.+ and stained for ckit.sup.+ and CD43.sup.+ from a wild
type mouse (WT) and a mouse homozygous for six human V.kappa. and
five human J.kappa. gene segments positioned at the endogenous
heavy chain locus (6hV.kappa.-5hJ.kappa. HO). Pro and pre B cells
are noted on each of the dot plots.
[0146] FIG. 7D shows the number of pro B
(CD19.sup.+CD43.sup.+ckit.sup.+) and pre B
(CD19.sup.+CD43.sup.-ckit.sup.-) cells in bone marrow harvested
from the femurs of wild type mice (WT) and mice homozygous for six
human V.kappa. and five human J.kappa. gene segments positioned at
the endogenous heavy chain locus (6hV.kappa.-5hJ.kappa. HO).
[0147] FIG. 7E shows contour plots of bone marrow gated on singlets
stained for CD19 and CD43 from a wild type mouse (WT) and a mouse
homozygous for six human V.kappa. and five human J.kappa. gene
segments positioned at the endogenous heavy chain locus
(6hV.kappa.-5hJ.kappa. HO). Immature, pre and pro B cells are noted
on each of the dot plots.
[0148] FIG. 7F shows histograms of bone marrow gated on pre B cells
(CD19.sup.+CD43.sup.int) and expressing immunoglobulin M (IgM) from
a wild type mouse (WT) and a mouse homozygous for six human
V.kappa. and five human J.kappa. gene segments positioned at the
endogenous heavy chain locus (6hVk-5hJk HO).
[0149] FIG. 7G shows the number of IgM.sup.+ pre B cells
(CD19.sup.+IgM.sup.+CD43.sup.int) and immature B cells
(CD19.sup.+IgM.sup.+CD43.sup.-) in bone marrow harvest from the
femurs of wild type (WT) and mice homozygous for six human V.kappa.
and five human J.kappa. gene segments positioned at the endogenous
heavy chain locus (6hV.kappa.-5hJ.kappa. HO).
[0150] FIG. 8A shows contour plots of splenocytes gated on
CD19.sup.+ and stained for Ig.lamda..sup.+ and Ig.kappa..sup.+
expression from a mouse containing a wild type heavy chain locus
and a replacement of the endogenous V.kappa. and J.kappa. gene
segments with human V.kappa. and J.kappa. gene segments (WT) and a
mouse homozygous for thirty hV.lamda. and five J.kappa. gene
segments at the endogenous heavy chain locus and a replacement of
the endogenous V.kappa. and J.kappa. gene segments with human
V.kappa. and J.kappa. gene segments (30hV.kappa.-5hJ.kappa.
HO).
[0151] FIG. 8B shows contour plots of bone marrow gated on immature
(B220.sup.intIgM.sup.+) and mature (B220.sup.hiIgM.sup.+) B cells
stained for Ig.lamda. and Ig.kappa. expression isolated from the
femurs of a mouse containing a wild type heavy chain locus and a
replacement of the endogenous V.kappa. and J.kappa. gene segments
with human V.kappa. and J.kappa. gene segments (WT) and a mouse
homozygous for thirty hV.kappa. and five J.kappa. gene segments at
the endogenous heavy chain locus and a replacement of the
endogenous V.kappa. and J.kappa. gene segments with human V.kappa.
and J.kappa. gene segments (30hV.kappa.-5hJ.kappa. HO).
[0152] FIG. 9 shows a nucleotide sequence alignment of the
V.kappa.-J.kappa.-mIgG junction of twelve independent RT-PCR clones
amplified from splenocyte RNA of naive mice homozygous for thirty
hV.kappa. and five J.kappa. gene segments at the mouse heavy chain
locus and a replacement of the endogenous V.kappa. and J.kappa.
gene segments with human V.kappa. and J.kappa. 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 IgG nucleotide sequence for
IgG1, IgG2a/c, and IgG3 primed clones.
DETAILED DESCRIPTION
[0153] 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.sub.L1) fused
with a first C.sub.H region and a second light chain variable
domain (V.sub.L2) fused with a second C.sub.H region. In general,
the first and the second C.sub.H regions are identical, or they
differ by one or more amino acid substitutions (e.g., as described
herein). V.sub.L1 and V.sub.L2 specifically binding different
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 V.sub.L1 for the
first epitope will generally be at least one to two or three or
four orders of magnitude lower than the affinity of V.sub.L1 for
the second epitope, and vice versa with respect to V.sub.L2. 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 V.sub.L1 and a V.sub.L2 that recognize different
epitopes of the same antigen. For example, nucleic acid sequences
encoding V.sub.L sequences that recognize different epitopes of the
same antigen can be fused to nucleic acid sequences encoding
different C.sub.H 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 C.sub.H1 domain, a hinge, a C.sub.H2 domain, and a
C.sub.H3 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 V.sub.L1 and/or
V.sub.L2, 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.
[0154] Therefore, two general types of bispecific binding proteins
are (1) V.sub.L1-C.sub.H(dimer), and (2) V.sub.L1-C.sub.H:light
chain+V.sub.L2-C.sub.H:light chain, wherein the light chain is the
same or different. In either case, the C.sub.H (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.
[0155] 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, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205,
HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal),
CV-1, U937, 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.TM.
cell).
[0156] The term "cognate," when used in the sense of "cognate
with," e.g., a first V.sub.L domain that is "cognate with" a second
V.sub.L domain, is intended to include reference to the relation
between two V.sub.L 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.sub.H, D.sub.H, and J.sub.H regions are replaced with V.sub.L and
J.sub.L regions, makes antibody-like binding proteins that have two
identical polypeptide chains made of the same mouse C.sub.H region
(e.g., an IgG isotype) fused with a first human V.sub.L domain, and
two identical polypeptide chains made of the same mouse C.sub.L
region fused with a second human V.sub.L domain. During clonal
selection in the mouse, the first and the second human V.sub.L
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.sub.L 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.sub.L
domain that appears in a first antibody-like molecule and a V.sub.L
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.sub.L domain fused
to the first human heavy chain region and the V.sub.L domain fused
to the second human heavy chain region are identical).
[0157] 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 naive 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).
[0158] 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 rearranged V/J or V/D/J sequence. Unless indicated
otherwise, the V, D, and J segments comprise recombination signal
sequences (RSS) that allow for V/J recombination or V/D/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)).
[0159] 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 (V.sub.H). V.sub.H 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 C.sub.H1 domain, a hinge, a C.sub.H2 domain, a
C.sub.H3 domain, and optionally a C.sub.H4 domain (e.g., in the
case of IgM or IgE) and a transmembrane (M) domain (e.g., in the
case of membrane-bound 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
IgG 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, 309, 311, 312, 315, 318,
320, 322, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335,
337, 338, 339, 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.
[0160] 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., L or F); 252 (e.g., L/Y/F/W or
T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a
modification at 428 and/or 433 (e.g., L/R/SUP/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, 259I (e.g., V259I), and a
308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434
(e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T,
and 256E) modification; a 250Q and 428L modification (e.g., T250Q
and M428L); a 307 and/or 308 modification (e.g., 308F or 308P).
[0161] The phrase "light chain" includes an immunoglobulin light
chain constant (C.sub.L) region sequence from any organism, and
unless otherwise specified includes human .kappa. and .lamda. light
chains. Light chain variable (V.sub.L) domains typically include
three light chain CDRs and four framework (FR) regions, unless
otherwise specified. Generally, a full-length light chain
(V.sub.L+C.sub.L) includes, from amino terminus to carboxyl
terminus, a V.sub.L domain that includes
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a C.sub.L region. Light chains
(V.sub.L+C.sub.L) 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.sub.L domain fused with the C.sub.H domain). V.sub.L
domains that do not selectively bind the epitope(s) bound by the
V.sub.L that is fused with the C.sub.H domain include those that
can be identified by screening for the most commonly employed light
chains in existing antibody libraries (wet libraries or in silico),
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.sub.L fused with the
C.sub.H region) an epitope that is specifically bound by the
V.sub.L fused to the C.sub.H region.
[0162] 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.
[0163] 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
[0164] 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 proteins that have an immunoglobulin
structure, yet exhibit an enhanced diversity over traditional
antibodies.
[0165] Binding protein aspects described herein include binding
proteins that are encoded by modified immunoglobulin 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 V.sub.L domain, a C.sub.H1 region, a hinge, a C.sub.H2 region, a
C.sub.H3 region, and optionally a C.sub.H4 region.
[0166] 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 V.sub.L region--not a V.sub.H
region--fused to a C.sub.H region.
[0167] In various embodiments, the modified mice make binding
proteins that comprise a V.sub.L region fused with a C.sub.H region
(a hybrid heavy chain), wherein the V.sub.L region of the hybrid
heavy chain exhibits an enhanced degree of somatic hypermutation.
In these embodiments, the enhancement is over a V.sub.L region that
is fused with a C.sub.L region (a light chain). In some
embodiments, a V.sub.L 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.sub.L
region fused with a C.sub.L region. In some embodiments, the
modified mice in response to an antigen exhibit a population of
binding proteins that comprise a V.sub.L 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.sub.L 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.sub.L region of the hybrid
heavy chain comprise one or more or two or more N additions in a
CDR3.
[0168] 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.
[0169] 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.sub.H 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.sub.H region, normally present in a V.sub.H
domain. Smaller size can also be realized in the formation of a
CDR3 that is derived, e.g., from a V.kappa. region and a J.kappa.
region.
[0170] In another aspect, a mouse and a method is provided for
providing a population of binding proteins having somatically
hypermutated V.sub.L domains, e.g., somatically mutated human
V.kappa. domains, and, e.g., human V.kappa. domains encoded by
rearranged .kappa. variable genes that comprise 1-10 or more N
additions. In one embodiment, in the absence of a V.sub.H 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.sub.L domains.
The clonal selection process of the mouse therefore is limited to
selecting only or substantially from binding proteins that have
V.sub.L domains, rather than V.sub.H domains. Somatic hypermutation
of the V.sub.L 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.sub.L 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.
[0171] 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.
[0172] In one aspect, a method is provided for increasing the
diversity, including by somatic hypermutation or by N additions in
a V.sub.L domain, comprising placing an unrearranged light chain V
gene segment and an unrearranged J gene segment in operable linkage
with a mouse C.sub.H 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.sub.H region.
[0173] In one embodiment, the immunoglobulin heavy chain fused with
the hypermutated V.sub.L is an IgM; in another embodiment, an IgG;
in another embodiment, an IgE; in another embodiment, an IgA.
[0174] In one embodiment, the somatically hypermutated and
class-switched V.sub.L domain contains about 2- to 5-fold or more
of the somatic hypermutations observed for a rearranged and
class-switched antibody having a V.sub.L sequence that is operably
linked to a C.sub.L sequence. In one embodiment, the observed
somatic hypermutations in the somatically hypermutated V.sub.L
domain are about the same in number as observed in a V.sub.H domain
expressed from a V.sub.H gene fused to a C.sub.H region.
[0175] In one aspect, a method for making a high-affinity human
V.sub.L 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.sub.L domain from the
mouse that specifically binds the antigen of interest with high
affinity.
[0176] In one embodiment, the K.sub.D of a binding protein
comprising the somatically mutated, class-switched human V.sub.L
domain is in the nanomolar or picomolar range.
[0177] 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.sub.L domain fused with a human
C.sub.H region.
[0178] 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.sub.L domain fused
with a human C.sub.H region; and wherein each polypeptide of the
dimer is associated with a cognate light chain comprising a cognate
light chain V.sub.L domain and a human C.sub.L region.
[0179] In one aspect, a method is provided for somatically
hypermutating a human V.sub.L gene sequence, comprising placing a
human V.sub.L gene segment and a human J.sub.L gene segment in
operable linkage with an endogenous mouse C.sub.H 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.sub.L gene sequence that binds the
antigen of interest.
[0180] In one embodiment, the method further comprises obtaining
from the mouse a V.sub.L gene sequence from a light chain that is
cognate to the human somatically hypermutated human V.sub.L gene
sequence that binds the antigen of interest.
V.sub.L Binding Proteins with D.sub.H Sequences
[0181] 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 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).
[0182] 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.
[0183] 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 different 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 10 or more N additions in a
rearranged V(light)/J or a rearranged V(light)/D/J gene in a mouse
as described herein.
[0184] 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 DH 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 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.
[0185] 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 unconserved sequence, from a
conserved nucleic acid nonamer sequence. RSS's are used 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.
[0186] In the case of the .lamda. light chain locus, variable gene
segments (V.lamda. 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 (J.lamda.
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, V.lamda. and J.lamda. segments are flanked with RSS's
that are compatible under the 12/23 rule, and therefore are capable
of recombine during rearrangement.
[0187] At the .kappa. locus in a wild-type organism, however, each
functional V.kappa. segment is flanked downstream with an RSS
having a 12-mer spacer. J.kappa. segments, therefore, have 23-mer
spaces juxtaposed on the upstream side of the J.kappa. segment. At
the heavy chain locus, V.sub.H gene segments are juxtaposed
downstream with an RSS having a 23-mer spacer, followed by D.sub.H
segment juxtaposed upstream and downstream with a 12-mer spacer,
and J.sub.H segments each with a 23-mer segment juxtaposed on the
upstream side of the J.sub.H segment. At the heavy chain locus, D/J
recombination occurs first, mediated by the downstream D.sub.H RSS
with the 12-mer spacer and the upstream J.sub.H 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.sub.H
segment having the RSS with the 23-mer juxtaposed on its downstream
side to form a rearranged V/D/J sequence.
[0188] In one embodiment, a V.lamda. segment is employed at the
heavy chain locus with a J gene segment that is a J.lamda. segment,
wherein the V.lamda. segment comprises an RSS juxtaposed on the
downstream side of the V.lamda. sequence, and the RSS comprises a
23-mer spacer, and the J segment is a J.lamda. segment with an RSS
juxtaposed on its upstream side having a 12-mer spacer (e.g., as
found in nature).
[0189] In one embodiment, a V.lamda. segment is employed at the
heavy chain locus with a J gene segment that is a J.kappa. or a
J.sub.H gene segment, wherein the V.lamda. sequence has juxtaposed
on its downstream side an RSS comprising a 23-mer spacer, and the
J.kappa. or J.sub.H segment has juxtaposed on its upstream side an
RSS comprising a 12-mer spacer.
[0190] In one embodiment, a V.lamda. segment is employed at the
heavy chain locus with a D.sub.H gene segment and a J gene segment.
In one embodiment, the V.lamda. segment comprises an RSS juxtaposed
on the downstream side of the V.lamda. sequence with an RSS having
a 23-mer spacer; the D.sub.H segment comprises an RSS juxtaposed on
the upstream side and on the downstream side of the D.sub.H
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 J.lamda. a J.kappa., and a
J.sub.H.
[0191] In one embodiment, a V.kappa. segment is employed at the
heavy chain locus with a J gene segment (with no intervening D
segment), wherein the V.kappa. segment has an RSS juxtaposed on the
downstream side of the V.kappa. 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 J.kappa. segment is selected from a
J.kappa. segment, a J.lamda. segment, and a J.sub.H segment. In one
embodiment, the V segment and/or the J segment are human.
[0192] In one embodiment, the V.kappa. segment is employed at the
heavy chain locus with a D segment and a J segment, wherein the
V.kappa. segment has an RSS juxtaposed on the downstream side of
the V.kappa. 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 J.kappa. segment, a J.lamda. segment, and a J.sub.H segment.
In one embodiment, the V segment and/or the J segment are
human.
[0193] A J.lamda. segment with an RSS having a 23-mer spacer
juxtaposed at its upstream end, or a J.kappa. or J.sub.H 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.
[0194] 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-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.
[0195] Suitable nonamers and heptamers for RSS construction are
known in the art (e.g., see Janeway's Immunobiology, 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
[0196] 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.sub.L domain
fused with a C.sub.H region can associate with a second polypeptide
consisting essentially of a second V.sub.L domain fused with a
C.sub.H region. Where the first V.sub.L domain and the second
V.sub.L domain specifically bind a different epitope, a
bispecific-binding molecule can be made using the two V.sub.L
domains. The C.sub.H region can be the same or different. In one
embodiment, e.g., one of the C.sub.H 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).
[0197] In one aspect, the methods and compositions described herein
are used to make bispecific-binding proteins. In this aspect, a
first V.sub.L that is fused to a C.sub.H region and a second
V.sub.L that is fused to a C.sub.H region are each independently
cloned in frame with a human IgG sequence of the same isotype
(e.g., a human IgG1, IgG2, IgG3, or IgG4). The first V.sub.L
specifically binds a first epitope, and the second V.sub.L
specifically binds a second epitope. The first and second epitopes
may be on different antigens, or on the same antigen.
[0198] In one embodiment, the IgG isotype of the C.sub.H region
fused to the first V.sub.L and the IgG isotype of the C.sub.H
region fused to the second V.sub.L are the same isotype, but differ
in that one IgG 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.
[0199] In one embodiment, the first C.sub.H region comprises a
first C.sub.H3 domain of a human IgG selected from IgG1, IgG2, and
IgG4; and the second C.sub.H region comprises a second C.sub.H3
domain of a human IgG selected from IgG1, IgG2, and IgG4, wherein
the second C.sub.H3 domain comprises a modification that reduces or
eliminates binding of the second C.sub.H3 domain to protein A.
[0200] In one embodiment, the second C.sub.H3 domain comprises a
435R modification, numbered according to the EU index of Kabat. In
another embodiment, the second C.sub.H3 domain further comprises a
436F modification, numbered according to the EU index of Kabat.
[0201] In one embodiment, the second C.sub.H3 domain is that of a
human IgG1 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.
[0202] In one embodiment, the second C.sub.H3 domain is that of a
human IgG2 that comprises a modification selected from the group
consisting of N384S, K392N, and V422I, numbered according to the EU
index of Kabat.
[0203] In one embodiment, the second C.sub.H3 domain is that of a
human IgG4 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.
[0204] In one embodiment, the binding protein comprises C.sub.H
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.sub.H 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.sub.H region that is
not found in a wild-type human heavy chain, and the C.sub.H region
does not comprise a sequence that generates a T-cell epitope.
EXAMPLES
[0205] 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
[0206] Various targeting constructs were made using VELOCIGENE.RTM.
genetic engineering technology (see, e.g., U.S. Pat. No. 6,586,251
and Valenzuela, D. M., Murphy, A. J., Frendewey, D., Gale, N. W.,
Economides, A. N., Auerbach, W., Poueymirou, W. T., Adams, N. C.,
Rojas, J., Yasenchak, J., Chernomorsky, R., Boucher, M., Elsasser,
A. L., Esau, L., Zheng, J., Griffiths, J. A., Wang, .lamda., Su,
H., Xue, Y., Dominguez, M. G., Noguera, I., Torres, R., Macdonald,
L. E., Stewart, A. F., 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.sub.H, D.sub.H and J.sub.H gene segments for
the ensuing insertion of unrearranged human germline .kappa. light
chain gene sequences (top of FIG. 2).
[0207] 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.sub.H
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.sub.H 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 5' 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 FIG. 2). Thus a modified
mouse heavy chain locus containing intact C.sub.H genes was created
for progressively inserting human .kappa. germline gene segments in
a precise manner using targeting vectors described below.
[0208] Four separate targeting vectors were engineered to
progressively insert 40 human V.kappa. gene segments and five human
J.kappa. gene segments into the inactivated mouse heavy chain locus
(described above) using standard molecular techniques recognized in
the art (FIG. 2). The human .kappa. gene segments used for
engineering the four targeting constructs are naturally found in
proximal contig of the germline human .kappa. light chain locus
(FIG. 1B and Table 1).
[0209] A .about.110,499 bp human genomic fragment containing the
first six human V.kappa. gene segments and five human J.kappa. gene
segments was engineered to contain a PI-Scel site 431 bp downstream
(3') of the human J.kappa.5 gene segment. Another PI-Scel site was
engineered at the 5' end of a .about.7,852 bp genomic fragment
containing the mouse heavy chain intronic enhancer, the IgM switch
region (S.mu.) and the IgM gene of the mouse heavy chain locus.
This mouse fragment was used as a 3' homology arm by ligation to
the .about.110.5 kb human fragment, which created a 3' junction
containing, from 5' to 3', .about.110.5 kb of genomic sequence of
the human .kappa. light chain locus containing the first six
consecutive V.kappa. gene segments and five J.kappa. gene segments,
a PI-Scel site, .about.7,852 bp of mouse heavy chain sequence
containing the mouse intronic enhancer, S.mu. and the mouse IgM
constant gene. Upstream (5') from the human V.kappa.1-6 gene
segment was an additional 3,710 bp of human .kappa. 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 .kappa. sequence was a neomycin cassette flanked by three
recombinase recognition sites (see Targeting Vector 1, FIG. 2). The
final targeting vector for the first insertion of human .kappa.
sequence from 5' to 3' included a 5' homology arm containing
.about.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), .about.110.5 kb of human genomic .kappa.
sequence containing the first six consecutive human V.kappa. gene
segments and five human J.kappa. gene segments, a PI-Scel site, and
a 3' homology arm containing .about.8 kb of mouse genomic sequence
including the intronic enhancer, S.mu. and the mouse IgM constant
gene (see FIG. 2, Targeting Vector 1). Homologous recombination
with this targeting vector created a modified mouse heavy chain
locus containing six human V.kappa. gene segments and five human
J.kappa. 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 V.kappa. domain
and a mouse C.sub.H region).
TABLE-US-00001 TABLE 1 Targeting Size of Human .kappa. Gene
Segments Added Vector Human .kappa. Sequence V.kappa. J.kappa. 1
~110.5 kb 4-1, 5-2, 7-3, 2-4, 1-5, 1-6 1-5 2 ~140 kb 3-7, 1-8, 1-9,
2-10, 3-11, -- 1-12, 1-13, 2-14, 3-15, 1-16 3 ~161 kb 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, 2-30 4 ~90 kb 3-31, 1-32, 1-33, 3-34, 1-35, -- 2-36, 1-37,
2-38, 1-39, 2-40
[0210] Introduction of Ten Additional Human V.kappa. Gene Segments
into a Hybrid Heavy Chain Locus.
[0211] A second targeting vector was engineered for introduction of
10 additional human V.kappa. gene segments to the modified mouse
heavy chain locus described above (see FIG. 2, Targeting Vector 2).
A 140,058 bp human genomic fragment containing 12 consecutive human
V.kappa. gene segments from the human .kappa. 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 .kappa. sequence. Upstream (5') from the human
V.kappa.1-16 gene segment was an additional 10,170 bp of human
.kappa. 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 FIG. 2). Between the 5' homology arm and
the beginning of the human .kappa. sequence was a hygromycin
cassette flanked by recombinase recognition sites. The 3' homology
arm included a 31,165 bp overlap of human genomic .kappa. sequence
corresponding to the equivalent 5' end of the .about.110.5 kb
fragment of human genomic .kappa. sequence of Targeting Vector 1
(FIG. 2). The final targeting vector for the insertion of 10
additional human V.kappa. gene segments from 5' to 3' included a 5'
homology arm containing .about.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 .about.140 kb of human genomic .kappa. sequence containing
12 consecutive human V.lamda. gene segments, .about.31 kb of which
overlaps with the 5' end of the human .kappa. 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
V.kappa. gene segments and five human J.kappa. gene segments
operably linked to the mouse heavy chain constant genes which, upon
recombination, leads to the formation of a hybrid heavy chain.
[0212] Introduction of Fourteen Additional Human V.kappa. Gene
Segments into a Hybrid Heavy Chain Locus.
[0213] A third targeting vector was engineered for introduction of
14 additional human V.kappa. gene segments to the modified mouse
heavy chain locus described above (see FIG. 2, Targeting Vector 3).
A 160,579 bp human genomic fragment containing 15 consecutive human
V.kappa. 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 V.kappa.2-30 gene segment was an additional
14,687 bp of human .kappa. 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 FIG. 2).
Between the 5' homology arm and the beginning of the human .kappa.
sequence was a neomycin cassette flanked by recombinase recognition
sites. The 3' homology arm included a 21,275 bp overlap of human
genomic .kappa. sequence corresponding to the equivalent 5' end of
the .about.140 kb fragment of human genomic .kappa. sequence of
Targeting Vector 2 (FIG. 2). The final targeting vector for the
insertion of 14 additional human V.kappa. gene segments from 5' to
3' included a 5' homology arm containing .about.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 .about.161 kb of human
genomic .kappa. sequence containing 15 human V.kappa. gene
segments, .about.21 kb of which overlaps with the 5' end of the
human .kappa. 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 V.kappa. gene segments and five human
J.kappa. gene segments operably linked to the mouse heavy chain
constant genes which, upon recombination, leads to the formation of
a chimeric .kappa. heavy chain.
[0214] Introduction of Ten Additional Human V.kappa. Gene Segments
into a Hybrid Heavy Chain Locus.
[0215] A fourth targeting vector was engineered for introduction of
10 additional human V.kappa. gene segments to the modified mouse
heavy chain locus described above (see FIG. 2, Targeting Vector 4).
A 90,398 bp human genomic fragment containing 16 consecutive human
V.kappa. 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 .kappa. sequence.
Upstream (5') from the human V.kappa.2-40 gene segment was an
additional 8,484 bp of human .kappa. sequence before the start of
the 5' mouse homology arm, which was the same 5' homology as the
previous targeting vectors (described above, see also FIG. 2).
Between the 5' homology arm and the beginning of the human .kappa.
sequence was a hygromycin cassette flanked by recombinase
recognition sites. The 3' homology arm included a 61,615 bp overlap
of human genomic .kappa. sequence corresponding to the equivalent
5' end of the .about.160 kb fragment of human genomic .kappa.
sequence of Targeting Vector 3 (FIG. 2). The final targeting vector
for the insertion of 10 additional human V.kappa. gene segments
from 5' to 3' included a 5' homology arm containing .about.20 kb of
mouse genomic sequence 5' of the mouse heavy chain locus, a first
recombinase recognition site (R1), a hygromycin cassette, a second
recombinase recognition site (R2) and .about.90 kb of human genomic
.kappa. sequence containing 16 human V.kappa. gene segments,
.about.62 kb of which overlaps with the 5' end of the human .kappa.
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 V.kappa. gene segments and five human J.kappa.
gene segments operably linked to the mouse heavy chain constant
genes which, upon recombination, leads to the formation of a
chimeric .kappa. heavy chain (FIG. 3).
[0216] 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 V.lamda. and
J.lamda. gene segments (FIGS. 4A and 4B).
[0217] The human .lamda. light chain locus extends over 1,000 kb
and contains over 80 genes that encode variable (V) or joining (J)
segments. Among the 70 V.lamda. gene segments of the human .lamda.
light chain locus, anywhere from 30-38 appear to be functional gene
segments according to published reports. The 70 V.lamda. 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 .lamda. light chain locus, over half of all
observed V.lamda. domains are encoded by the gene segments 1-40,
1-44, 2-8, 2-14, and 3-21. There are seven J.lamda. gene segments,
only four of which are regarded as generally functional J.lamda.
gene segments--J.lamda.1, J.lamda.2, J.lamda.3, and J.lamda.7. In
some alleles, a fifth J.lamda.-C.lamda. gene segment pair is
reportedly a pseudo gene (C.lamda.6). Incorporation of multiple
human J.lamda. 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 J.lamda. gene segments
in germline configuration is engineered with multiple human
V.lamda. gene segments and allow for normal V-J recombination in
the context of a heavy chain constant region.
[0218] Coupling light chain variable domains with heavy chain
constant regions represents a potentially rich source of diversity
for generating unique V.sub.L binding proteins with human V.sub.L
regions in non-human animals. Exploiting this diversity of the
human .lamda. light chain locus (or human .kappa. 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.
[0219] Additionally, human D.sub.H and J.sub.H (or J.kappa.) gene
segments can be incorporated with either human V.kappa. or V.lamda.
gene segments to construct novel hybrid loci that will give rise,
upon recombination, to novel engineered variable domains (FIGS. 5A
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.sub.H 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., V.kappa. and a
D.sub.H or D.sub.H and J.lamda.), 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.
[0220] Thus, using the strategy outlined above for incorporation of
human .kappa. light chain gene segments into an endogenous heavy
chain locus allows for the use of other combinations of human
.lamda. light chain gene segments as well as specific human heavy
chain gene segments (e.g., D.sub.H and J.sub.H) and combinations
thereof.
Example II
Identification of Targeted ES Cells Bearing Human Light Chain Gene
Segments at an Endogenous Heavy Chain Locus
[0221] 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.sub.L binding proteins
(i.e., human .kappa. light chain gene segments operably linked to
mouse heavy chain constant regions). ES cells containing an
insertion of unrearranged human .kappa. light chain gene segments
were identified by the quantitative PCR assay, TAQMAN.RTM. (Lie and
Petropoulos, 1998. Curr. Opin. Biotechnology 9:43-48). Specific
primers sets and probes were design for insertion of human .kappa.
sequences and associated selection cassettes, loss of mouse heavy
chain sequences and retention of mouse sequences flanking the
endogenous heavy chain locus.
[0222] ES cells bearing the human .kappa. 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 .kappa. gene
segments. Optionally, the selection cassette may be removed by
breeding to mice that express the recombinase (e.g., U.S. Pat. No.
6,774,279). Optionally, the selection cassette is retained in the
mice.
Example III
Generation and Analysis of Mice Expressing V.sub.L Binding
Proteins
[0223] Targeted ES cells described above were used as donor ES
cells and introduced into an 8-cell stage mouse embryo by the
VELOCIMOUSE.RTM. method (see, e.g., U.S. 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.RTM. (F0 mice fully
derived from the donor ES cell) independently bearing human .kappa.
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 .kappa. 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 V.sub.L binding
proteins.
[0224] Flow Cytometry.
[0225] The introduction of human .kappa. light chain gene segments
into the mouse heavy chain locus was carried out in an F1 ES line
(F1H4; Valenzuela et al. 2007, supra) derived from 129S6/SvEvTac
and C57BL/6NTac heterozygous embryos that further contained an in
situ replacement of the mouse .kappa. light chain gene segments
with human .kappa. light chain gene segments (U.S. Pat. No.
6,596,541). The human .kappa. light chain germline variable gene
segments are targeted to the 129S6 allele, which carries the
IgM.sup.a haplotype, whereas the unmodified mouse C576BL/6N allele
bears the IgM.sup.b haplotype. These allelic forms of IgM can be
distinguished by flow cytometry using antibodies specific to the
polymorphisms found in the IgM.sup.a or IgM.sup.b alleles.
Heterozygous mice bearing human .kappa. light chain gene segments
at the endogenous heavy chain locus as described in Example I were
evaluated for expression of human V.sub.L binding proteins using
flow cytometry.
[0226] 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 FACS staining buffer and blocked with anti-mouse CD16/32
(BD Pharmingen). Lymphocytes were stained with anti-mouse
IgM.sup.b-FITC (BD Pharmingen), anti-mouse IgM.sup.a-PE (BD
Pharmingen), anti-mouse CD19 (Clone 1D3; BD Biosciences), and
anti-mouse CD3 (17A2; BIOLEGEND.RTM.) followed by fixation with BD
CYTOFIX.TM. all according to the manufacturer's instructions. Final
cell pellets were resuspended in staining buffer and analyzed using
a BD FACSCALIBUR.TM. and BD CELLQUEST PRO.TM. software. Table 2
sets forth the average percent values for B cells (CD19.sup.+), T
cells (CD3.sup.+), hybrid heavy chain (CD19.sup.+IgM.sup.a+), and
wild type heavy chain (CD19.sup.+IgM.sup.b+) expression observed in
groups of animals bearing each genetic modification.
[0227] In a similar experiment, B cell contents of the spleen,
blood and bone marrow compartments from mice homozygous for six
human V.kappa. and five human J.kappa. gene segments operably
linked to the mouse heavy chain constant region (described in
Example I, FIG. 2) were analyzed for progression through B cell
development using flow cytometry of various cell surface
markers.
[0228] Briefly, two groups (n=3 each, 8 weeks old females) of wild
type and mice homozygous for six human V.kappa. and five human
J.kappa. 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.
[0229] Cells (1.times.10.sup.6) 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 (1B11, BIOLEGEND.RTM.), PE-ckit (2B8,
BIOLEGEND.RTM.), PeCy7-IgM (11/41, EBIOSCIENCE.RTM.),
PerCP-Cy5.5-IgD (11-26c.2a, BIOLEGEND.RTM.), APC-eFluor 780-B220
(RA3-6B2, EBIOSCIENCE.RTM.), APC-CD19 (MB19-1, EBIOSCIENCE.RTM.).
Bone marrow: immature B cells (B220.sup.intIgM.sup.+), mature B
cells (B220.sup.hiIgM.sup.+), pro B cells
(CD19.sup.+ckit.sup.+CD43.sup.+), pre B cells
(CD19.sup.+ckit.sup.-CD43.sup.-), pre-B cells
(CD19.sup.+CD43.sup.intIgM.sup.+/-), immature B cells
(CD19.sup.+CD43.sup.-IgM.sup.+/-). Blood and spleen: B cells
(CD19.sup.+), mature B cells (CD19.sup.+IgM.sup.intIgD.sup.hi)
transitional/immature B cells
(CD19.sup.+IgM.sup.hiIgD.sup.int).
[0230] Following staining, cells were washed and fixed in 2%
formaldehyde. Data acquisition was performed on a LSRII flow
cytometer and analyzed with FLOWJO.TM. software (Tree Star, Inc.).
FIGS. 6A, 6B and 6C show the results for the splenic compartment.
FIG. 7A-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).
[0231] In a similar experiment, B cell contents of the spleen,
blood and bone marrow compartments from mice homozygous for thirty
human V.kappa. and five human J.kappa. gene segments operably
linked to the mouse heavy chain constant region (described in
Example I, FIG. 2) were analyzed for progression through B cell
development using flow cytometry of various cell surface
markers.
[0232] 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 V.kappa. and J.kappa. gene segments with human V.kappa.
and J.kappa. gene segments (WT) and mice homozygous for thirty
hV.kappa. and five J.kappa. gene segments and a replacement of the
endogenous V.kappa. and J.kappa. gene segments with human V.kappa.
and J.kappa. gene segments (30hV.kappa.-5hJ.kappa. 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).
[0233] Cells (1.times.10.sup.6) were incubated with anti-mouse
CD16/CD32 (2.4G2, BD 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 (1B11,
BIOLEGEND.RTM.), PE-ckit (2B8, BIOLEGEND.RTM.), PeCy7-IgM (II/41,
EBIOSCIENCE.RTM.), APC-CD19 (MB19-1, EBIOSCIENCE.RTM.). Bone marrow
and spleen panel: anti-mouse FITC-Ig.kappa. (187.1 BD Biosciences),
PE-Ig.lamda. (RML-42, BIOLEGEND.RTM.), PeCy7-IgM (II/41,
EBIOSCIENCE.RTM.), PerCP-Cy5.5-IgD (11-26c.2a, BIOLEGEND.RTM.),
Pacific Blue-CD3 (17A2, BIOLEGEND.RTM.), APC-B220 (RA3-6B2,
EBIOSCIENCE.RTM.), APC-H7-CD19 (1D3, BD). Bone marrow: immature B
cells (B220.sup.intIgM.sup.+), mature B cells
(B220.sup.hiIgM.sup.+), pro B cells (CD19.sup.30
ckit.sup.+CD43.sup.+), pre B cells (CD19+ckit-CD43-), immature
Ig.kappa..sup.+ B cells
(B220.sup.intIgM.sup.+Ig.kappa..sup.+Ig.lamda..sup.-), immature
Ig.lamda..sup.+ B cells
(B220.sup.intIgM.sup.+Ig.kappa..sup.-Ig.lamda..sup.+), mature
Ig.kappa..sup.+ B cells
(B220.sup.hiIgM.sup.+Ig.kappa..sup.+Ig.lamda..sup.-), mature
Ig.lamda..sup.+ B cells
(B220.sup.hiIgM.sup.+Ig.kappa..sup.-Ig.lamda..sup.+). Spleen: B
cells (CD19.sup.+), mature B cells
(CD19.sup.+IgD.sup.hiIgM.sup.int), transitional/immature B cells
(CD19.sup.+IgD.sup.intIgM.sup.hi). Bone marrow and spleen:
Ig.kappa..sup.+ B cells (CD19.sup.+Ig.kappa..sup.+Ig.lamda..sup.-),
Ig.lamda..sup.+ B cells (CD19.sup.+
Ig.kappa..sup.-Ig.lamda..sup.+).
[0234] Following staining, cells were washed and fixed in 2%
formaldehyde. Data acquisition was performed on a LSRII flow
cytometer and analyzed with FLOWJO.TM. software (Tree Star, Inc.).
The results demonstrated similar staining patterns and cell
populations for all three compartments as compared to mice
homozygous for six human V.kappa. and five human J.kappa. gene
segments (described above). However, these mice demonstrated a loss
in endogenous .lamda. light chain expression in both the splenic
and bone marrow compartments (FIGS. 8A and 8B, respectively),
despite the endogenous .lamda. light chain locus being intact in
these mice. This may reflect an inability of rearranged human
.kappa. light chain domains, in the context of heavy chain constant
regions, to pair or associate with murine .lamda. light chain
domains, leading to deletion of Ig.lamda..sup.+ cells.
[0235] Isotype Expression.
[0236] Total and surface (i.e., membrane bound) immunoglobulin M
(IgM) and immunoglobulin G1 (IgG1) was determined for mice
homozygous for human heavy and .kappa. light chain variable gene
loci (VELCOIMMUNE.RTM. Humanized Mice, see U.S. Pat. No. 7,105,348)
and mice homozygous for six human V.kappa. and 5 human J.kappa.
gene segments engineered into the endogenous heavy chain locus
(6hV.kappa.-5hJ.kappa. HO) by a quantitative PCR assay using
TAQMAN.RTM. probes (as described above in Example II).
[0237] Briefly, CD19.sup.+ 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.TM. 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 (Invitrogen) and then amplified with the TAQMAN.RTM. 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 IgM and
IgG1 isotypes (Table 3). Relative expression was normalized to the
mouse .kappa. constant region (mC.kappa.).
TABLE-US-00002 TABLE 2 Mouse Genotype % CD3 % CD19 % IgM.sup.a %
IgM.sup.b C57BL/6 22 63 0 100 Balb/c 11 60 100 0
6hV.kappa.-5hJ.kappa. HET 43 30 7 85 16hV.kappa.-5hJ.kappa. HET 33
41 7 81
TABLE-US-00003 TABLE 3 SEQ ID Isotype Sequence (5'-3') NOs: Surface
sense: GAGAGGACCG TGGACAAGTC 1 IgM antisense: TGACGGTGGT GCTGTAGAAG
2 probe: ATGCTGAGGA GGAAGGCTTT GAGAACCT 3 Total sense: GCTCGTGAGC
AACTGAACCT 4 IgM antisense: GCCACTGCAC ACTGATGTC 5 probe:
AGTCAGCCAC AGTCACCTGC CTG 6 Surface sense: GCCTGCACAA CCACCATAC 7
IgG1 antisense: GAGCAGGAAG AGGCTGATGA AG 8 probe: AGAAGAGCCT
CTCCCACTCT CCTGG 9 Total sense: CAGCCAGCGG AGAACTACAA G 10 IgG1
antisense: GCCTCCCAGT TGCTCTTCTG 11 probe: AACACTCAGC CCATCATGGA
CACA 12 C.kappa. sense: TGAGCAGCAC CCTCACGTT 13 antisense:
GTGGCCTCAC AGGTATAGCT GTT 14 probe: ACCAAGGACG AGTATGAA 15
[0238] The results from the quantitative TAQMAN.RTM. PCR assay
demonstrated a decrease in total IgM and total IgG1. However, the
ratio of secreted versus surface forms of IgM and IgG1 appeared
normal as compared to VELCOIMMUNE.RTM. humanized mice (data not
shown).
[0239] Human .kappa. Gene Segment Usage and V.kappa.-J.kappa.
Junction Analysis.
[0240] Naive mice homozygous for thirty hV.kappa. and five J.kappa.
gene segments and a replacement of the endogenous V.kappa. and
J.kappa. gene segments with human V.kappa. and J.kappa. gene
segments (30hV.kappa.-5h.kappa. HO) were analyzed for unique human
V.kappa.-J.kappa. rearrangements on mouse heavy chain (IgG) by
reverse transcription polymerase chain reaction (RT-PCR) using RNA
isolated from splenocytes.
[0241] Briefly, spleens were harvested and perfused with 10 mL
RPMI-1640 (Sigma) with 5% HI-FBS in sterile disposable bags. Each
bag containing a single spleen was then placed into a STOMACHER.TM.
(Seward) and homogenized at a medium setting for 30 seconds.
Homogenized spleens were filtered using a 0.7 .mu.m cell strainer
and then pelleted with a centrifuge (1000 rpm for 10 minutes) and
RBCs were lysed in BD PHARM LYSE.TM. (BD Biosciences) for three
minutes. Splenocytes were diluted with RPMI-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.
[0242] RT-PCR was performed on splenocyte RNA using primers
specific for human hV.kappa. gene segments and the mouse IgG. The
mouse IgG primer was designed such that it was capable of
amplifying RNA derived from all mouse IgG isotypes. PCR products
were gel-purified and cloned into pCR2.1-TOPO TA vector
(Invitrogen) and sequenced with 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 V.kappa. and J.kappa. gene segment usage among twelve
selected clones are shown in Table 4. FIG. 9 sets forth the
nucleotide sequence of the hV.kappa.-hJ.kappa.-mIgG junction for
the twelve selected RT-PCR clones.
[0243] As shown in this Example, mice homozygous for six human
V.kappa. and five human J.kappa. gene segments or homozygous for
thirty human V.kappa. and five human J.kappa. 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-US-00004 TABLE 4 Hybrid Heavy Chain Clone V.kappa. J.kappa.
C.sub.H SEQ ID NO: 1E 1-5 4 IgG2A/C 18 1G 1-9 4 IgG2A/C 19 1A 1-16
5 IgG3 20 2E 1-12 2 IgG1 21 1C 1-27 4 IgG2A/C 22 2H 2-28 1 IgG1 23
3D 3-11 4 IgG1 24 3A 3-20 4 IgG2A/C 25 4B 4-1 5 IgG2A/C 26 4C 4-1 2
IgG3 27 5A 5-2 2 IgG2A/C 28 5D 5-2 1 IgG1 29
Example IV
Propagation of Mice Expressing V.sub.L Binding Proteins
[0244] To create a new generation of V.sub.L binding proteins, mice
bearing the unrearranged human .kappa. 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 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.
[0245] Alternatively, mice bearing the unrearranged human .kappa.
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 (.kappa. and .lamda.). In this manner, the
progeny obtained would express unique human .kappa. 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
.kappa. light chain domains and mouse heavy chain constant domains
and absence of endogenous mouse light chains.
[0246] Mice bearing an unrearranged hybrid heavy chain locus are
also bred with mice that contain a replacement of the endogenous
mouse .kappa. light chain variable gene locus with the human
.kappa. light chain variable gene locus (see U.S. Pat. No.
6,596,541, Regeneron Pharmaceuticals, The VELOCIMMUNE.RTM.
Humanized Mouse Technology). The VELOCIMMUNE.RTM. Humanized Mouse
includes, in part, having a genome comprising human .kappa. light
chain variable regions operably linked to endogenous mouse .kappa.
light chain variable constant region loci such that the mouse
produces antibodies comprising a human .kappa. 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 .kappa. light
chain with the human .kappa. light chain locus and an unrearranged
hybrid heavy chain locus is obtained. Unique V.sub.L binding
proteins containing somatically mutated human V.kappa. domains can
be isolated upon immunization with an antigen of interest.
Example V
Generation of V.sub.L Binding Proteins
[0247] After breeding mice that contain the unrearranged hybrid
heavy chain locus to various desired strains containing
modifications and deletions of other endogenous Ig loci (as
described in Example IV), selected mice can be immunized with an
antigen of interest.
[0248] Generally, a VELOCIMMUNE.RTM. 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
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 V.kappa. 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 V.kappa.
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.sub.L binding proteins or the variable
domains may be isolated directly from antigen-specific lymphocytes
(e.g., B cells).
[0249] Initially, high affinity V.sub.L binding proteins are
isolated having a human V.kappa. region and a mouse constant
region. As described above, the V.sub.L binding proteins are
characterized and selected for desirable characteristics, including
affinity, selectivity, epitope, etc. The mouse constant regions are
replaced with a desired human constant region to generate unique
fully human V.sub.L binding proteins containing somatically mutated
human V.kappa. domains from an unrearranged hybrid heavy chain
locus of the invention. Suitable human constant regions include,
for example wild type or modified IgG1 or IgG4 or, alternatively
C.kappa. or C.lamda..
[0250] Separate cohorts of mice containing a replacement of the
endogenous mouse heavy chain locus with six human V.kappa. and five
human J.kappa. gene segments (as described in Example I) and a
replacement of the endogenous V.kappa. and J.kappa. gene segments
with human V.kappa. and J.kappa. gene segments were immunized with
a human cell-surface receptor protein (Antigen X). Antigen X is
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 .mu.g of CpG oligonucleotide (Cat #
tlrl-modn-ODN1826 oligonucleotide; InVivogen, San Diego, Calif.)
and 25 .mu.g of Adju-Phos (Aluminum phosphate gel adjuvant, Cat#
H-71639-250; 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
after the 4th and 6th injection are collected and the antibody
immune response is monitored by a standard antigen-specific
immunoassay.
[0251] 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.sub.L binding proteins. Using this technique
several anti-Antigen X-specific V.sub.L binding proteins (i.e.,
binding proteins possessing human V.kappa. domains in the context
of mouse heavy and light chain constant domains) are obtained.
[0252] Alternatively, anti-Antigen X V.sub.L 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.sub.L binding proteins
(i.e., antibodies possessing human V.kappa. domains and human
constant domains) were obtained.
[0253] Human .kappa. Gene Segment Usage.
[0254] To analyze the structure of the anti-Antigen X V.sub.L
binding proteins produced, nucleic acids encoding the human
V.kappa. domains (from both the heavy and light chains of the
V.sub.L binding protein) were cloned and sequenced using methods
adapted from those described in US 2007/0280945A1 (supra). From the
nucleic acid sequences and predicted amino acid sequences of the
antibodies, gene usage was identified for the hybrid heavy chain
variable region of selected V.sub.L binding proteins obtained from
immunized mice (described above). Table 5 sets for the gene usage
of human V.kappa. and J.kappa. gene segments from selected
anti-Antigen X V.sub.L binding proteins, which demonstrates that
mice according to the invention generate antigen-specific V.sub.L
binding proteins from a variety of human V.kappa. and J.kappa. gene
segments, due to a variety of rearrangements at the endogenous
heavy chain and .kappa. light chain loci both containing
unrearranged human V.kappa. and J.kappa. gene segments. Human
V.kappa. gene segments rearranged with a variety of human J.kappa.
segments to yield unique antigen-specific V.sub.L binding
proteins.
TABLE-US-00005 TABLE 5 Hybrid Heavy Chain Light Chain Antibody
V.kappa. J.kappa. V.kappa. J.kappa. A 4-1 3 3-20 1 B 4-1 3 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 4-1 3 3-20 1 G 4-1 3
3-20 1 H 4-1 3 3-20 1 I 4-1 3 3-20 1 J 1-5 3 1-33 3 K 4-1 3 3-20 1
L 4-1 3 1-9 3 M 4-1 1 1-33 4 N 4-1 1 1-33 3 O 1-5 1 1-9 2 P 1-5 3
1-16 4 Q 4-1 3 3-20 1 R 4-1 3 3-20 1 S 1-5 1 1-9 2 T 1-5 1 1-9 2 U
5-2 2 1-9 3 V 1-5 2 1-9 2 W 4-1 1 1-33 4
[0255] Enzyme-Linked Immunosorbent Assay (ELISA).
[0256] Human V.sub.L 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.
[0257] Briefly, Ligand Y was coated onto 96-well plates at a
concentration of 2 .mu.g/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,
Calif.) 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.sub.L binding proteins
were diluted 1:10 in buffer. A mock supernatant with the same
components of the V.sub.L binding proteins was used as a negative
control. The extracellular domain (ECD) of Antigen X was conjugated
to the Fc portion of mouse IgG2a (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.sub.L 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,
Calif.) and developed by standard colorimetric response using
tetramethylbenzidine (TMB) substrate (BD Biosciences, San Jose,
Calif.) 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.sub.L
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.
[0258] The results showed that several V.sub.L binding proteins
isolated from mice immunized with Antigen X specifically bound the
extracellular domain of Antigen X fused to the Fc portion of mouse
IgG2a (data not shown).
[0259] Affinity Determination.
[0260] Equilibrium dissociation constants (K.sub.D) for selected
Antigen X-specific V.sub.L binding protein supernatants were
determined by SPR (Surface Plasmon Resonance) using a BIACORE.TM.
T100 instrument (GE Healthcare). All data were obtained using
HBS-EP (10 mM HEPES, 150 mM NaCl, 0.3 mM EDTA, 0.05% Surfactant
P20, pH 7.4) as both the running and sample buffers, at 25.degree.
C.
[0261] Briefly, V.sub.L 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 .mu.L/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 .mu.L/min. Dissociation of antigen from the captured V.sub.L
binding protein was monitored for 6 minutes. The captured V.sub.L
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.sub.L
binding protein from the capture surface. Binding data for each
V.sub.L binding protein was fit to a 1:1 binding model with mass
transport using BIAcore T100 Evaluation software v2.1.
[0262] The binding affinities of thirty-four selected V.sub.L
binding proteins varied, with all exhibiting a K.sub.D in the
nanomolar range (1.5 to 130 nM). Further, about 70% of the selected
V.sub.L binding proteins (23 of 34) demonstrated single digit
nanomolar affinity. T.sup.1/2 measurements for these selected
V.sub.L binding proteins demonstrated a range of about 0.2 to 66
minutes. Of the thirty-four V.sub.L 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.sub.L
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.sub.L 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.
[0263] In another experiment, selected human V.sub.L 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.RTM. 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.sub.L binding proteins were
also capable of binding Antigen X from cynomolgus monkey (Macaca
fascicularis).
Sequence CWU 1
1
29120DNAArtificial Sequencesynthetic 1gagaggaccg tggacaagtc
20220DNAArtificial Sequencesynthetic 2tgacggtggt gctgtagaag
20328DNAArtificial Sequencesynthetic 3atgctgagga ggaaggcttt
gagaacct 28420DNAArtificial Sequencesynthetic 4gctcgtgagc
aactgaacct 20519DNAArtificial Sequencesynthetic 5gccactgcac
actgatgtc 19623DNAArtificial Sequencesynthetic 6agtcagccac
agtcacctgc ctg 23719DNAArtificial Sequencesynthetic 7gcctgcacaa
ccaccatac 19822DNAArtificial Sequencesynthetic 8gagcaggaag
aggctgatga ag 22925DNAArtificial Sequencesynthetic 9agaagagcct
ctcccactct cctgg 251021DNAArtificial Sequencesynthetic 10cagccagcgg
agaactacaa g 211120DNAArtificial Sequencesynthetic 11gcctcccagt
tgctcttctg 201224DNAArtificial Sequencesynthetic 12aacactcagc
ccatcatgga caca 241319DNAArtificial Sequencesynthetic 13tgagcagcac
cctcacgtt 191423DNAArtificial Sequencesynthetic 14gtggcctcac
aggtatagct gtt 231518DNAArtificial Sequencesynthetic 15accaaggacg
agtatgaa 181616DNAArtificial Sequencesynthetic 16gtaaaacgac ggccag
161717DNAArtificial Sequencesynthetic 17caggaaacag ctatgac
1718145DNAArtificial Sequencesynthetic 18tctgggacag aattcactct
caccatcagc agcctgcagc ctgatgattt tgcaacttat 60tactgccaac agtataatac
cctcactttc ggcggaggga ccaaggtgga gatcaaaccc 120aaaacaacag
ccccatcggt ctatc 14519151DNAArtificial Sequencesynthetic
19tctgggacag aatccactct cacaatcagc agcctgcagc ctgaagattt tgcaacttat
60tactgtcaac agcttaatag ttaccctttc actttcggcg gagggaccaa ggtggagatc
120aaacccaaaa caacagcccc atcggtctat c 15120151DNAArtificial
Sequencesynthetic 20tctgggacag atttcactct caccatcagc agcctgcagc
ctgaagattt tgcaacttat 60tactgccaac agtataatag ttaccctccc accttcggcc
aagggacacg actggagatt 120aaacctacaa caacagcccc atctgtctat c
15121153DNAArtificial Sequencesynthetic 21tctgggacag atttcactct
caccatcagc agcctgcagc ctgaagattt tgcaacttac 60tattgtcaac aggctaacag
tttcccgtac acttttggcc aggggaccaa gctggagatc 120aaacccaaaa
cgacaccccc atctgtctat cca 15322151DNAArtificial Sequencesynthetic
22tctgggacag atttcactct caccatcagc agcctgcagc ctgaagatgt tgcaacttat
60tactgtcaaa agtataacag tgcccctcac actttcggcg gagggaccaa ggtggagatc
120aaacccaaaa caacagcccc atcggtctat c 15123153DNAArtificial
Sequencesynthetic 23tcaggcacag attttacact gaaaatcagc agagtggagg
ctgaggatgt tggggtttat 60tactgcatgc aagctctaca aatttcgtgg acgttcggcc
aagggaccaa ggtggaaatc 120aaacccaaaa cgacaccccc atctgtctat cca
15324150DNAArtificial Sequencesynthetic 24tctgggacag acttcactct
caccatcagc agcctagagc ctgaagattt tgcagtttat 60tactgtcagc agcgtagccc
ccgtttcact ttcggcggag ggaccaaggt ggagatcaaa 120cccaaaacga
cacccccatc tgtctatcca 15025148DNAArtificial Sequencesynthetic
25tctgggacag acttcactct caccatcagc agactggagc ctgaagattt tgccgtgtat
60tactgtcagc agtatggtag ctcactcact ttcggcggag ggaccaaggt ggagatcaaa
120cccaaaacaa cagccccatc ggtctatc 14826151DNAArtificial
Sequencesynthetic 26tctgggacag atttcactct caccatcagc agcctgcagg
ctgaagatgt ggcagtttat 60tactgtcagc aatattatag tactccgatc accttcggcc
aagggacacg actggagatt 120aaacccaaaa caacagcccc atcggtctat c
15127151DNAArtificial Sequencesynthetic 27tctgggacag atttcactct
caccatcagc agcctgcagg ctgaagatgt ggcagtttat 60tactgtcagc aatattatag
tactgggccc acttttggcc aggggaccaa gctggagatc 120aaacctacaa
caacagcccc atctgtctat c 15128151DNAArtificial Sequencesynthetic
28tatggaacag attttaccct cacaattaat aacattgaat gtgaggatgc tgcatattac
60ttctgtctac aacatgataa tttcccgtac acttttggcc aggggaccaa gctggagatc
120aaacccaaaa caacagcccc atcggtctat c 15129147DNAArtificial
Sequencesynthetic 29tatggaacag attttaccct cacaattaat aacatagaat
ctgaggatgc tgcatattac 60ttctgtctac aacatgataa ttggacgttc ggccaaggga
ccaaggtgga aatcaaaccc 120aaaacgacac ccccatctgt ctatcca 147
* * * * *