Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR MAKING FULLY HUMAN BISPECIFIC ANTIBODIES
USING A COMMON LIGHT CHAIN
[0001] This application is being filed on 5 June 2013, as a PCT
International
patent application, and claims priority to U.S. Patent Application No.
13/488,628,
filed June 5, 2012, the disclosure of which is hereby incorporated by
reference
herein in its entirety.
FIELD
[0002] A genetically modified mouse is provided that expresses antibodies
having a common human variable/mouse constant light chain associated with
diverse human variable/mouse constant heavy chains. A method for making a
human bispecific antibody from human variable region gene sequences of B cells
of
the mouse is provided.
BACKGROUND
[0003] Antibodies typically comprise a homodimeric heavy chain component,
wherein each heavy chain monomer is associated with an identical light chain.
Antibodies having a heterodimeric heavy chain component (e.g., bispecific
antibodies) are desirable as therapeutic antibodies. But making bispecific
antibodies
having a suitable light chain component that can satisfactorily associate with
each of
the heavy chains of a bispecific antibody has proved problematic.
[0004] In one approach, a light chain might be selected by surveying
usage
statistics for all light chain variable domains, identifying the most
frequently
employed light chain in human antibodies, and pairing that light chain in
vitro with
the two heavy chains of differing specificity.
[0005] In another approach, a light chain might be selected by observing
light
chain sequences in a phage display library (e.g., a phage display library
comprising
human light chain variable region sequences, e.g., a human scFv library) and
selecting the most commonly used light chain variable region from the library.
The
light chain can then be tested on the two different heavy chains of interest.
[0006] In another approach, a light chain might be selected by assaying a
phage
display library of light chain variable sequences using the heavy chain
variable
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sequences of both heavy chains of interest as probes. A light chain that
associates
with both heavy chain variable sequences might be selected as a light chain
for the
heavy chains.
[0007] In another approach, a candidate light chain might be aligned with
the
heavy chains' cognate light chains, and modifications are made in the light
chain to
more closely match sequence characteristics common to the cognate light chains
of
both heavy chains. If the chances of immunogenicity need to be minimized, the
modifications preferably result in sequences that are present in known human
light
chain sequences, such that proteolytic processing is unlikely to generate a T
cell
epitope based on parameters and methods known in the art for assessing the
likelihood of immunogenicity (i.e., in silico as well as wet assays).
[0008] All of the above approaches rely on in vitro methods that subsume
a
number of a priori restraints, e.g., sequence identity, ability to associate
with specific
pre-selected heavy chains, etc. There is a need in the art for compositions
and
methods that do not rely on manipulating in vitro conditions, but that instead
employ
more biologically sensible approaches to making human epitope-binding proteins
that include a common light chain.
SUMMARY
[0009] Genetically modified mice that express human immunoglobulin heavy
and light chain variable domains, wherein the mice have a limited light chain
variable repertoire, are provided. A biological system for generating a human
light
chain variable domain that associates and expresses with a diverse repertoire
of
affinity-matured human heavy chain variable domains is provided. Methods for
making binding proteins comprising immunoglobulin variable domains are
provided, comprising immunizing mice that have a limited immunoglobulin light
chain repertoire with an antigen of interest, and employing an immunoglobulin
variable region gene sequence of the mouse in a binding protein that
specifically
binds the antigen of interest. Methods include methods for making human
immunoglobulin heavy chain variable domains suitable for use in making multi-
specific antigen-binding proteins.
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[00103 Genetically engineered mice are provided that select suitable
affinity-
matured human immunoglobulin heavy chain variable domains derived from a
repertoire of unrearranged human heavy chain variable region gene segments,
wherein the affinity-matured human heavy chain variable domains associate and
express with a single human light chain variable domain derived from one human
light chain variable region gene segment. Genetically engineered mice that
present a
choice of two human light chain variable region gene segments are also
provided. In
various aspects, the one or two gene segments include human Vx1-39 and human
Vic3-20.
[0011] Genetically engineered mice are provided that express a limited
repertoire of human light chain variable domains, or a single human light
chain
variable domain, from a limited repertoire of human light chain variable
region gene
segments. The mice are genetically engineered to include a single unrearranged
human light chain variable region gene segment (or two human light chain
variable
region gene segments) that rearranges to form a rearranged human light chain
variable region gene (or two rearranged light chain variable region genes)
that
express a single light chain (or that express either or both of two light
chains). The
rearranged human light chain variable domains are capable of pairing with a
plurality of affinity-matured human heavy chains selected by the mice, wherein
the
heavy chain variable regions specifically bind different epitopes.
[0012] Genetically engineered mice are provided that express a limited
repertoire of human light chain variable domains, or a single human light
chain
variable domain, from a limited repertoire of human light chain variable
region
sequences. The mice are genetically engineered to include a single V/J human
light
chain sequence (or two V/J sequences) that express a variable region of a
single light
chain (or that express either or both of two variable regions). A light chain
comprising the variable sequence is capable of pairing with a plurality of
affinity-
matured human heavy chains clonally selected by the mice, wherein the heavy
chain
variable regions specifically bind different epitopes.
[0013] In one aspect, a genetically modified mouse is provided that
comprises a
single human immunoglobulin light chain variable (VI) region gene segment that
is
capable of rearranging with a human J gene segment (selected from one or a
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plurality of JL segments) and encoding a human VL domain of an immunoglobulin
light chain. In another aspect, the mouse comprises no more than two human VL
gene segments, each of which is capable of rearranging with a human J gene
segment (selected from one or a plurality of JL segments) and encoding a human
VL
domain of an immunoglobulin light chain.
[0014] In one embodiment, the single human VL gene segment is operably
linked to a human JL gene segment selected from Jx1, JK2, JK3, Jic4, and Jic5,
wherein the single human VL gene segment is capable of rearranging to form a
sequence encoding a light chain variable region gene with any of the one or
more
human JL gene segments.
[0015] In one embodiment, the genetically modified mouse comprises an
immunoglobulin light chain locus that does not comprise an endogenous mouse VL
gene segment that is capable of rearranging to form an immunoglobulin light
chain
gene, wherein the VL locus comprises a single human VL gene segment that is
capable of rearranging to encode a VL region of a light chain gene. In a
specific
embodiment, the human VL gene segment is a human Vx.1-39k5 gene segment or a
human Vic3-20J-K1 gene segment. In one embodiment, the genetically modified
mouse comprises a VL locus that does not comprise an endogenous mouse VL gene
segment that is capable of rearranging to form an immunoglobulin light chain
gene,
wherein the VL locus comprises no more than two human VL gene segments that
are
capable of rearranging to encode a VL region of a light chain gene. In a
specific
embodiment, the no more than 2 human VL gene segments are a human Vic1-39Jx5
gene segment and a human Vx3-20JK1 gene segment.
[0016] In one aspect, a genetically modified mouse is provided that
comprises a
single rearranged (V/J) human immunoglobulin light chain variable (VI) region
(i.e.,
a VL/JL region) that encodes a human VL domain of an immunoglobulin light
chain.
In another aspect, the mouse comprises no more than two rearranged human VL
regions that are capable of encoding a human VL domain of an immunoglobulin
light chain.
[0017] In one embodiment, the VL region is a rearranged human Vx1-39/J
sequence or a rearranged human Vic3-204 sequence. In one embodiment, the
human JL segment of the rearranged VOL sequence is selected from JK1, Jx2,
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.1x4, and Jx5. In a specific embodiment, the VL region is a human Vk1-39JO
sequence or a human Vx3-20R1 sequence. In a specific embodiment, the mouse
has both a human W1-39JO sequence and a human W3-20.fie1 sequence.
[0018] In one embodiment, the human VL gene segment is operably linked to
a
human or mouse leader sequence. In one embodiment, the leader sequence is a
mouse leader sequence. In a specific embodiment, the mouse leader sequence is
a
mouse W3-7 leader sequence. In a specific embodiment, the leader sequence is
operably linked to an unrearranged human VL gene segment. In a specific
embodiment, the leader sequence is operably linked to a rearranged human VOL
sequence.
[0019] In one embodiment, the VL gene segment is operably linked to an
immunoglobulin promoter sequence. In one embodiment, the promoter sequence is
a human promoter sequence. In a specific embodiment, the human immunoglobulin
promoter is a human W3-15 promoter. In a specific embodiment, the promoter is
operably linked to an unrearranged human VL gene segment. In a specific
embodiment, the promoter is operably linked to a rearranged human VOL
sequence.
[0020] In one embodiment, the light chain locus comprises a leader
sequence
flanked 5' (with respect to transcriptional direction of a VL gene segment)
with a
human immunoglobulin promoter and flanked 3' with a human VL gene segment
that rearranges with a human J segment and encodes a VL domain of a reverse
chimeric light chain comprising an endogenous mouse light chain constant
region
(CO. In a specific embodiment, the VI, gene segment is at the mouse Vic locus,
and
the mouse CL is a mouse Cic.
[0021] In one embodiment, the light chain locus comprises a leader
sequence
flanked 5' (with respect to transcriptional direction of a VL gene segment)
with a
human immunoglobulin promoter and flanked 3' with a rearranged human VL region
(VOL sequence) and encodes a VL domain of a reverse chimeric light chain
comprising an endogenous mouse light chain constant region (CL). In a specific
embodiment, the rearranged human VOL sequence is at the mouse kappa (le)
locus,
and the mouse CL is a mouse Cic.
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[0022] In one embodiment, the VL locus of the modified mouse is a lc
light chain
locus, and the ic light chain locus comprises a mouse lc intronic enhancer, a
mouse lc
3' enhancer, or both an intronic enhancer and a 3' enhancer.
[0023] In one embodiment, the mouse comprises a nonfunctional
immunoglobulin lambda (20 light chain locus. In a specific embodiment, the X
light
chain locus comprises a deletion of one or more sequences of the locus,
wherein the
one or more deletions renders the X light chain locus incapable of rearranging
to
form a light chain gene. In another embodiment, all or substantially all of
the VL
gene segments of the X light chain locus are deleted.
[0024] In one embodiment, mouse makes a light chain that comprises a
somatically mutated VL domain derived from a human VL gene segment. In one
embodiment, the light chain comprises a somatically mutated VL domain derived
from a human VL gene segment, and a mouse CT( region. In one embodiment, the
mouse does not express a 2 light chain.
[0025] In one embodiment, the genetically modified mouse is capable of
somatically hypermutating the human VL region sequence. In a specific
embodiment, the mouse comprises a cell that comprises a rearranged
immunoglobulin light chain gene derived from a human VL gene segment that is
capable of rearranging and encoding a VI, domain, and the rearranged
immunoglobulin light chain gene comprises a somatically mutated VL domain.
[0026] In one embodiment, the mouse comprises a cell that expresses a
light
chain comprising a somatically mutated human VL domain linked to a mouse CK,
wherein the light chain associates with a heavy chain comprising a somatically
mutated VH domain derived from a human VH gene segment and wherein the heavy
chain comprises a mouse heavy chain constant region (CH). In a specific
embodiment, the heavy chain comprises a mouse CHI, a mouse hinge, a mouse
C112,
and a mouse CH3. In a specific embodiment, the heavy chain comprises a human
CH1, a hinge, a mouse CH2, and a mouse CH3.
[0027] In one embodiment, the mouse comprises a replacement of endogenous
mouse VII gene segments with one or more human VH gene segments, wherein the
human VH gene segments are operably linked to a mouse CH region gene, such
that
the mouse rearranges the human VH gene segments and expresses a reverse
chimeric
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immunoglobulin heavy chain that comprises a human VH domain and a mouse CH.
In one embodiment, 90-100% of unrearranged mouse VH gene segments are
replaced with at least one unrearranged human VH gene segment. In a specific
embodiment, all or substantially all of the endogenous mouse VH gene segments
are
replaced with at least one unrearranged human VH gene segment. In one
embodiment, the replacement is with at least 19, at least 39, or at least 80
or 81
unrearranged human VH gene segments. In one embodiment, the replacement is
with at least 12 functional unrearranged human VH gene segments, at least 25
functional unrearranged human VH gene segments, or at least 43 functional
unrearranged human VH gene segments. In one embodiment, the mouse comprises a
replacement of all mouse DH and JH segments with at least one unrearranged
human
DH segment and at least one unrearranged human JH segment. In one embodiment,
the at least one unrearranged human DH segment is selected from 1-1, D1-7, 1-
26, 2-
8, 2-15, 3-3, 3-10, 3-16, 3-22, 5-5, 5-12, 6-6, 6-13, 7-27, and a combination
thereof.
In one embodiment, the at least one unrearranged human JH segment is selected
from 1, 2, 3, 4, 5, 6, and a combination thereof. In a specific embodiment,
the one
or more human VH gene segment is selected from a 1-2, 1-8, 1-24, 1-69, 2-5, 3-
7, 3-
9, 3-11, 3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 3-53, 4-31, 4-39, 4-59, 5-
51, a 6-1
human VH gene segment, and a combination thereof.
[0028] In one embodiment, the mouse comprises a B cell that expresses a
binding protein that specifically binds an antigen of interest, wherein the
binding
protein comprises a light chain derived from a human Vic1-39/ix5 rearrangement
or
a human Vic3-20/.1x1 rearrangement, and wherein the cell comprises a
rearranged
immunoglobulin heavy chain gene derived from a rearrangement of human VH gene
segments selected from a 1-69, 2-5, 3-13, 3-23, 3-30, 3-33, 3-53, 4-39, 4-59,
and 5-
51 gene segment. In one embodiment, the one or more human VH gene segments
are rearranged with a human heavy chain Jli gene segment selected from 1, 2,
3, 4, 5,
and 6. In one embodiment, the one or more human VH and JH gene segments are
rearranged with a human DH gene segment selected from 1-1, 1-7, 1-26, 2-8, 2-
15,
3-3, 3-10, 3-16, 3-22, 5-5, 5-12, 6-6, 6-13, and 7-27. In a specific
embodiment, the
light chain gene has 1, 2, 3, 4, or 5 or more somatic hypen-nutations.
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[0029] In one embodiment, the mouse comprises a B cell that comprises a
rearranged immunoglobulin heavy chain variable region gene sequence comprising
a
VH/DH/JH region selected from 2-5/6-6/1, 2-5/3-22/1, 3-13/6-6/5, 3-23/2-8/4, 3-
23/3-
3/4, 3-23/3-10/4, 3-23/6-6/4, 3-23/7-27/4, 3-30/1-1/4, 3-30/1-7/4, 3-30/3-3/3,
3-
30/3-3/4, 3-30/3-22/5, 3-30/5-5/2, 3-30/5-12/4, 3-30/6-6/1, 3-30/6-6/3, 3-30/6-
6/4,
3-30/6-6/5, 3-30/6-13/4, 3-30/7-27/4, 3-30/7-27/5, 3-30/7-27/6, 3-33/1-7/4, 3-
33/2-
15/4, 4-39/1-26/3, 4-59/3-16/3, 4-59/3-16/4, 4-59/3-22/3, 5-51/3-16/6, 5-51/5-
5/3, 5-
51/6-13/5, 3-53/1-1/4, 1-69/6-6/5, and 1-69/6-13/4. In a specific embodiment,
the B
cell expresses a binding protein comprising a human immunoglobulin heavy chain
variable region fused with a mouse heavy chain constant region, and a human
immunoglobulin light chain variable region fused with a mouse light chain
constant
region.
[0030] In one embodiment, the rearranged human VL region is a human Vx1-
39Jic5 sequence, and the mouse expresses a reverse chimeric light chain
comprising
(i) a VL domain derived from the human VOL sequence and (ii) a mouse CL;
wherein the light chain is associated with a reverse chimeric heavy chain
comprising
(i) a mouse CH and (ii) a somatically mutated human VH domain derived from a
human VH gene segment selected from a 1-2, 1-8, 1-24, 1-69, 2-5, 3-7, 3-9, 3-
11, 3-
13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 3-53, 4-31, 4-39, 4-59, 5-51, a 6-1
human VH
gene segment, and a combination thereof. In one embodiment, the mouse
expresses
a light chain that is somatically mutated. In one embodiment the CL is a mouse
CK.
In a specific embodiment, the human VI; gene segment is selected from a 2-5, 3-
13,
3-23, 3-30, 4-59, 5-51, and 1-69 gene segment. In a specific embodiment, the
somatically mutated human VH domain comprises a sequence derived from a DH
segment selected from 1-1, 1-7, 2-8, 3-3, 3-10, 3-16, 3-22, 5-5, 5-12, 6-6, 6-
13, and
7-27. In a specific embodiment, the somatically mutated human VH domain
comprises a sequence derived from a JH segment selected from 1, 2, 3, 4, 5,
and 6.
In a specific embodiment, the somatically mutated human VH domain is encoded
by
a rearranged human Vii/DH/JH sequence selected from 2-5/6-6/1, 2-5/3-22/1, 3-
13/6-
6/5, 3-23/2-8/4, 3-23/3-3/4, 3-23/3-10/4, 3-23/6-6/4, 3-23/7-27/4, 3-30/1-1/4,
3-
30/1-7/4, 3-30/3-3/4, 3-30/3-22/5, 3-30/5-5/2, 3-30/5-12/4, 3-30/6-6/1, 3-30/6-
6/3,
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3-30/6-6/4, 3-30/6-6/5, 3-30/6-13/4, 3-30/7-27/4, 3-30/7-27/5, 3-30/7-27/6, 4-
59/3-
16/3, 4-59/3-16/4, 4-59/3-22/3, 5-51/5-5/3, 1-69/6-6/5, and 1-69/6-13/4.
[0031] In one embodiment, the rearranged human VL region is a human Vic3-
20.1x1 sequence, and the mouse expresses a reverse chimeric light chain
comprising
(i) a VL domain derived from the rearranged human VL/IL sequence, and (ii) a
mouse
CL; wherein the light chain is associated with a reverse chimeric heavy chain
comprising (i) a mouse CH, and (ii) a somatically mutated human VH derived
from a
human VH gene segment selected from a 1-2, 1-8, 1-24, 1-69, 2-5, 3-7, 3-9, 3-
11, 3-
13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 3-53, 4-31, 4-39, 4-59, 5-51, a 6-1
human VH
gene segment, and a combination thereof. In one embodiment, the mouse
expresses
a light chain that is somatically mutated. In one embodiment the CL is a mouse
Cic.
In a specific embodiment, the human VH gene segment is selected from a 3-30, 3-
33,
3-53, 4-39, and 5-51 gene segment. In a specific embodiment, the somatically
mutated human VH domain comprises a sequence derived from a DH segment
selected from 1-1, 1-7, 1-26, 2-15, 3-3, 3-16, and 6-13. In a specific
embodiment,
the somatically mutated human VH domain comprises a sequence derived from a JH
segment selected from 3, 4, 5, and 6. In a specific embodiment, the
somatically
mutated human VH domain is encoded by a rearranged human VH/DH/JH sequence
selected from 3-30/1-1/4, 3-30/3-3/3, 3-33/1-7/4, 3-33/2-15/4, 4-39/1-26/3, 5-
51/3-
16/6, 5-51/6-13/5, and 3-53/1-1/4.
[0032] In one embodiment, the mouse comprises both a rearranged human
Vic1-
39.1x5 sequence and a rearranged human Vic3-20Jic1 sequence, and the mouse
expresses a reverse chimeric light chain comprising (i) a VL domain derived
from
the human Vx1-39.1x5 sequence or the human Vic3-20.fic1 sequence, and (ii) a
mouse CL; wherein the light chain is associated with a reverse chimeric heavy
chain
comprising (i) a mouse CH, and (ii) a somatically mutated human VH derived
from a
human VH gene segment selected from a 1-2, 1-8, 1-24, 1-69, 2-5, 3-7, 3-9, 3-
11, 3-
13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 3-53, 4-31, 4-39, 4-59, 5-51, a 6-1
human VH
gene segment, and a combination thereof. In one embodiment, the mouse
expresses
a light chain that is somatically mutated. In one embodiment the CL is a mouse
ex..
[0033] In one embodiment, 90-100% of the endogenous unrearranged mouse VH
gene segments are replaced with at least one unrearranged human VH gene
segment.
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In a specific embodiment, all or substantially all of the endogenous
unrearranged
mouse VH gene segments are replaced with at least one unrearranged human VH
gene segment. In one embodiment, the replacement is with at least 18, at least
39, at
least 80, or 81 unrearranged human VH gene segments. In one embodiment, the
replacement is with at least 12 functional unrearranged human VII gene
segments, at
least 25 functional unrearranged human VH gene segments, or at least 43
unrearranged human VH gene segments.
[0034] In one embodiment, the genetically modified mouse is a C57BL
strain, in
a specific embodiment selected from C57BL/A, C57BL/An, C57BL/GrFa,
C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10,
C57BL/10ScSn, C57BL/10Cr, and C57BL/01a. In a specific embodiment, the
genetically modified mouse is a mix of an aforementioned 129 strain and an
aforementioned C57BL/6 strain. In another specific embodiment, the mouse is a
mix of aforementioned 129 strains, or a mix of aforementioned BL/6 strains. In
a
specific embodiment, the 129 strain of the mix is a 129S6 (129/SvEvTac)
strain.
[0035] In one embodiment, the mouse expresses a reverse chimeric antibody
comprising a light chain that comprises a mouse Cic and a somatically mutated
human VL domain derived from a rearranged human Vic1-39k5 sequence or a
rearranged human Vic3-20Jicl sequence, and a heavy chain that comprises a
mouse
CH and a somatically mutated human VH domain derived from a human VH gene
segment selected from a 1-2, 1-8, 1-24, 1-69, 2-5, 3-7, 3-9, 3-11, 3-13, 3-15,
3-20, 3-
23, 3-30, 3-33, 3-48, 3-53, 4-31, 4-39, 4-59, 5-51, and a 6-1 human VII gene
segment, wherein the mouse does not express a fully mouse antibody and does
not
express a fully human antibody. In one embodiment the mouse comprises a lc
light
chain locus that comprises a replacement of endogenous mouse x light chain
gene
segments with the rearranged human Vic.1-39Jx5 sequence or the rearranged
human
Vic3-20Jx1 sequence, and comprises a replacement of all or substantially all
endogenous mouse VH gene segments with a complete or substantially complete
repertoire of human VH gene segments.
[0036] In one aspect, a population of antigen-specific antibodies derived
from a
mouse as described herein is provided, wherein the antibodies comprise a light
chain
gene derived from a human Vx1-39/Jia rearrangement or a human Vic3-20/Jx1
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rearrangement, and wherein the antibodies comprise a rearranged immunoglobulin
heavy chain gene derived from a rearrangement of a human VH gene segment
selected from a 1-2, 1-3, 1-8, 1-18, 1-24, 1-46, 1-58, 1-69, 2-5, 2-26, 2-70,
3-7, 3-9,
3-11, 3-13, 3-15, 3-16, 3-20, 3-21, 3-23, 3-30, 3-33, 3-43, 3-48, 3-53, 3-64,
3-72, 3-
73, 4-31, 4-34, 4-39, 4-59, 5-51, and a 6-1 human VH gene segment. In one
embodiment, the one or more human VH gene segments are rearranged with a
human heavy chain JH gene segment selected from 1, 2, 3, 4, 5, and 6. In a
specific
embodiment, the light chain has 1, 2, 3, 4, or 5 or more somatic
hypermutations.
[0037] In one embodiment, the light chain has 1, 2, 3, or 4 somatic
hypermutations. In one embodiment, the light chain gene has 1 or 2 mutations.
In
various embodiments, the light chain gene is capable of incurring multiple
mutations
along its sequence.
[0038] In one embodiment, the light chain is derived from a human Vic1-
39/R5
rearrangement and the light chain has at least one or no more than four
somatic
hypermutations. In one embodiment, the light chain comprises at least two
somatic
hypermutations. In one embodiment, the light chain comprises at least three
somatic
hypermutations. In one embodiment, the light chain comprises at least four
somatic
hypermutations. In a specific embodiment, the mutations are present in one or
more
framework regions (FWs) of the light chain. In a specific embodiment, the
mutations are present in one or more complementarity determining regions
(CDRs)
of the light chain. In a specific embodiment, the mutations are present in one
or
more FWs and/or one or more CDRs of the light chain. In various embodiments,
the
framework regions are selected from framework 1 (FW1), framework 2 (FW2),
framework 3 (FW3), and/or a combination thereof. In various embodiments, the
CDRs are selected from CDR1, CDR2, CDR3, and/or a combination thereof.
[0039] In one embodiment, the heavy chain comprises at least one mutation
in
one or more FWs or one or more CDRs. In one embodiment, the heavy chain
comprises at least one mutation in one or more FWs and one or more CDRs. In
one
embodiment, the heavy chain comprises at least two mutations in one or more
FWs
and one or more CDRs. In one embodiment, the heavy chain comprises at least
three mutations in one or more FWs and one or more CDRs. In one embodiment,
the heavy chain comprises at least four mutations in one or more FWs and one
or
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more CDRs. In one embodiment, the heavy chain comprises at least five or more
than five mutations in one or more FWs and one or more CDRs; in a specific
embodiment, the heavy chain comprises at least five or more than five
mutations in
two FWs; in a specific embodiment, the heavy chain comprises at least five or
more
than five mutations in one FW and one CDR.
[0040] In one embodiment, the light chain is derived from a human Vx1-
39/Jic5
rearrangement and about 9% of the Vic1-39/.10-derived light chains have at
least
one mutation present in FW1; in one embodiment, at least 9% of the light
chains
comprise one mutation present in FW I . In one embodiment, the light chain is
derived from a human Vic1-39516 rearrangement and about 25% of the Vic I -
39/Jx5-derived light chains have at least one or no more than two mutations
present
in CDR1; in one embodiment, at least 19% of the light chains have one mutation
present in CDR1; in one embodiment, at least 5% of the light chains have two
mutations present in CDR1.
[0041] In one embodiment, the light chain is derived from a human Vic1-
39/Jx5
rearrangement and about 20% of the Vx1-39/Jx5-derived light chains have at
least
one or no more than three mutations present in FW2; in one embodiment, at
least
17% of the light chains have one mutation present in FW2; in one embodiment,
at
least 1% of the light chains have two mutations present in FW2; in one
embodiment,
at least 1% of the light chains have three mutations present in FW2.
[0042] In one embodiment, the light chain is derived from a human Vic I-
39/Jx5 rearrangement and about 10% of the Vic1-39/Jx5-derived light chains
have at
least one or no more than two mutations present in CDR2; in one embodiment, at
least 10% of the light chains have one mutation present in CDR2; in one
embodiment, at least 1% of the light chains have two mutations present in
CDR2.
[0043] In one embodiment, the light chain is derived from a human Vic1-
39/Jx5
rearrangement and about 29% of the Vic1-39/R5-derived light chains have at
least
one or no more than four mutations present in FW3; in one embodiment, at least
21% of the light chains have one mutation present in FW3; in one embodiment,
at
least 5% of the light chains have two mutations present in FW3; in one
embodiment,
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at least 2% of the light chains have three mutations present in FW3; in one
embodiment, at least 2% of the light chains have four mutations present in
FW3.
[0044] In one embodiment, the light chain is derived
from a human Vic1-39/.1x5
rearrangement and about 37% of the Vic1-39/Jic5-derived light chains have at
least
one or no more than four mutations present in CDR3; in one embodiment, at
least
27% of the light chains have one mutation present in CDR3; in one embodiment,
at
least 8% of the light chains have two mutations present in CDR3; in one
embodiment, at least 1% of the light chains have three mutations present in
CDR3;
in one embodiment, at least 1% of the light chains have four mutations present
in
CDR3.
[0045] In one embodiment, a population of antigen-
specific antibodies derived
from a mouse as described herein is provided, wherein the antibodies comprise
a
light chain derived from a human Vic1-39/Jx5 rearrangement and about 9% of the
Vx1-39/Jx5-derived light chains have one or more mutations present in FW I,
about
25% of the Vic1-395x5-derived light chains have one or more mutations present
in
CDR1, about 20% of the Vx1-39/Jx5-derived light chains have one or more
mutations present in FW2, about 10% of the Vx1-39/.1x5-derived light chains
have
one or more mutations present in CDR2, about 29% of the Vic1-39/Jx5-derived
light
chains have one or more mutations present in FW3, and about 37% of the Vxl-
39/JO-derived light chains have one or more mutations present in CDR3.
[0046] In one embodiment, the light chain is derived
from a human Vic1-39/Jx5
rearrangement and about 35% of the heavy chains have at least one mutation
present
=
in FW1; in one embodiment, at least 25% of the heavy chains have one mutation
present in FW1; in one embodiment, at least 9 % of the heavy chains have two
mutations present in FW1; in one embodiment, at least 1% of the heavy chains
have
three mutations present in FW1; in one embodiment, at least 1% of the heavy
chains
have more than five mutations present in FW1.
[0047] In one embodiment, the light chain is derived
from a human Yid -
39/Jx5 rearrangement and about 92% of the heavy chains have at least one or no
more than four mutations present in CDR 1; in one embodiment, at least 92% of
the
heavy chains have at least one, at least two, at least three, or at least four
mutations
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present in CDR1; in one embodiment, at least 26% of the heavy chains have one
mutation present in CDR1; in one embodiment, at least 44% of the heavy chains
have two mutations present in CDR1; in one embodiment, at least 19% of the
heavy
chains have three mutations present in CDR1; in one embodiment, at least 3% of
the
heavy chains have four mutations present in CDR 1.
[0048] In one embodiment, the light chain is derived from a human Vic1-
39/1K5
rearrangement and about 66% of the heavy chains have at least one or no more
than
three mutations present in FW2; in one embodiment, at least 66% of the heavy
chains have at least one, at least two, or at least three mutations present in
FW2; in
one embodiment, at least 35% of the heavy chains have one mutation present in
FW2; in one embodiment, at least 23% of the heavy chains have two mutations
present in FW2; in one embodiment, at least 8% of the heavy chains have three
mutations present in FW2.
[0049] In one embodiment, the light chain is derived from a human Vicl -
39/Jx5 rearrangement and about 70% of the heavy chains have at least one or no
more than four mutations present in CDR2; in one embodiment, at least 70% of
the
heavy chains have at least two, at least three, or at least four mutations
present in
CDR2; in one embodiment, at least 34% have one mutation present in CDR2; in
one
embodiment, at least 20% of the heavy chains have two mutations present in
CDR2;
in one embodiment, at least 12% of the heavy chains have three mutations
present in
CDR2; in one embodiment, at least 5% of the heavy chains have four mutations
present in CDR2.
[0050] In one embodiment, the light chain is derived from a human Vic1-
39/Jx5
rearrangement and about 91% of the heavy chains have at least one or up to
five or
more mutations present in FW3; in one embodiment, at least 91% of the heavy
chains have at least two, at least three, at least four, or at least five or
more
mutations present in FW3; in one embodiment, at least 19% of the heavy chains
have one mutation present in FW3; in one embodiment, at least 33% of the heavy
chains have two mutations present in FW3; in one embodiment, at least 22% of
the
heavy chains have three mutations present in FW3; in one embodiment, at least
11%
of the heavy chains have four mutations present in FW3; in one embodiment, at
least
7% of the heavy chains have five or more mutations present in FW3.
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[0051] In one embodiment, the light chain is derived from a human Vx1-
39/R5
rearrangement and about 63% of the heavy chains have at least one or no more
than
two mutations present in CDR3; in one embodiment, at least 63% of the heavy
chains have at one mutation present in CDR3; in one embodiment, at least 54%
of
the heavy chains have one mutation present in CDR3; in one embodiment, at
least
9% of the heavy chains have two mutations present in CDR3.
[0052] In one embodiment, a population of antigen-specific antibodies
derived
from a mouse as described herein is provided, wherein the antibodies comprise
a
light chain derived from a human Vx1-39/.1K5 rearrangement and at least 35% of
the
heavy chains have one or more mutations present in FW1, about 92% of the heavy
chains have one or more mutations present in CDR1, about 66% of the heavy
chains
have one or more mutations present in FW2, about 70% of the heavy chains have
one or more mutations present in CDR2, about 91% of the heavy chains have one
or
more mutations present in FW3, and about 63% of the heavy chains have one or
more mutations present in CDR3.
[0053] In one embodiment, the light chain is derived from a human Vx3-
20/R1
rearrangement and the light chain gene has at least one or no more than two
somatic
hypermutations; in one embodiment, the light chain gene has at least two, at
least
three, at least four or more somatic hypermutations. In a specific embodiment,
the
mutations are present in one or more framework regions of the light chain. In
a
specific embodiment, the mutations are present in one or more CDR regions of
the
light chain. In a specific embodiment, the mutations are present in one or
more
framework regions and/or one or more CDR regions of the light chain. In
various
embodiments, the framework regions are selected from framework 1 (FW1),
framework 2 (FW2), framework 3 (FW3), and/or a combination thereof.
[0054] In one embodiment, the light chain is derived from a human Vic3-
20/.1x1
rearrangement and about 10% of the Vx3-20/.1x1-derived light chains have at
least
one mutation present in FW1; in one embodiment, at least 10% of the light
chains
have one mutation in FW1.
[0055] In one embodiment, the light chain is derived from a human Vic3-
20/R1
rearrangement and about 53% of the Vx3-20/Jx1-derived light chains have at
least
one or no more than two mutations present in CDR1; in one embodiment, at least
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27% of the light chains have one or more mutations in CDR1; in one embodiment,
about 54% of the light chains have one or two mutations present in CDR1.
[0056] In one embodiment, the light chain is derived from a human Vic3-
20/Jic I
rearrangement and about 6% of the Vic3-20/Jicl-derived light chains have at
least
one or no more than two mutations present in FW2; in one embodiment, at least
6%
of light chains have at least one mutation present in FW2; in one embodiment,
at
least 3% of the light chains have one mutation present in FW2; in one
embodiment,
at least 3% of the light chains have two mutations present in FW2.
[0057] In one embodiment, the light chain is derived from a human Vx3-
20acl
rearrangement and at least about 3% of the Vtc3-20/JKI -derived light chains
have at
least one mutation present in CDR2; in one embodiment, at least 3% of the
light
chains have one mutation in CDR2.
[0058] In one embodiment, the light chain is derived from a human Vic3-
20/Jx1
rearrangement and about 17% or more of the Vic3-20/Jid-derived light chains
have
at least one or no more than two mutations present in FW3; in one embodiment,
at
least 20% of the light chain have one mutation present in FW3; in one
embodiment,
at least 17% of the light chains have two mutations present in FW3.
[0059] In one embodiment, the light chain is derived from a human Vic3-
20/Jx1
rearrangement and at least 43% of the V1c3-20/hcl-derived light chains have at
least
one mutation present in CDR3; in one embodiment, at least 43% of the light
chains
have one mutation in CDR3.
[0060] In one embodiment, a population of antigen-specific antibodies
derived
from a mouse as described herein is provided, wherein the antibodies comprise
a
light chain derived from a human V1(3-20/Jicl rearrangement and about 10% of
the
Vic3-20/R1-derived light chains have one or more mutations present in at
least,
about 53% of the Vx3-20/Jx1-derived light chains have one or more mutations
present in CDR I, about 6% of the Vx3-20/Jic1-derived light chains have one or
more mutations present in FW2, about 3% of the VO-20/Jx1-derived light chains
have one or more mutations present in CDR2, about 37% of the Vx3-20/Jicl-
derived
light chains have one or more mutations present in FW3, and about 43% of the
Vic3-
20/JO-derived light chains have one or more mutations present in CDR3.
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[0061] In one embodiment, the light chain is derived from a human V1c3-
20/Jx1
rearrangement and about 43% of the heavy chains have at least one or no more
than
two mutations present in FW1; in one embodiment, at least 41% of the heavy
chains
have at least one mutation present in FW1; in one embodiment, about 41% of the
heavy chains have one mutation present in FW1; in one embodiment, about 2% of
the heavy chains have two mutations present in FW1.
[0062] In one embodiment, the light chain is derived from a human Vic3-
20/.1x1
rearrangement and about 92% of the heavy chains have at least one or no more
than
four mutations present in CDR1; in one embodiment, at least 43% of heavy
chains
have at least one mutation present in CDR1; in one embodiment, at least 25% of
heavy chains have at least two mutations present in CDR1; in one embodiment,
at
least 15% of heavy chains have at least 3 mutations present in CDR1; in one
embodiment, at least 10% of heavy chains have 4 or more mutations present in
CDR1.
[0063] In one embodiment, the light chain is derived from a human V13-
20/J11
rearrangement and about 46% of the heavy chains have at least one or no more
than
three mutations present in FW2; in one embodiment, at least 34% of heavy
chains
have at least one mutation present in FW2; in one embodiment, at least 10% of
heavy chains have two or more mutations present in FW2; in one embodiment, at
least 2% of heavy chains have three or more mutations present in FW2.
[0064] In one embodiment, the light chain is derived from a human Vx.3-
20/1K1
rearrangement and about 84% of the heavy chains have at least one or up to
five or
more than five mutations present in CDR2; in one embodiment, at least 39% of
the
heavy chains have one or more mutations present in CDR2; in one embodiment, at
least 18% of the heavy chains have two or more mutations present in CDR2; in
one
embodiment, at least 21% of the heavy chains have three or more mutations
present
in CDR2; in one embodiment, at least 3% of the heavy chains have four or more
mutations present in CDR2; in one embodiment, at least 2% of the heavy chains
have five or more mutations present in CDR2.
[0065] In one embodiment, the light chain is derived from a human Vic3-
20/JK 1
rearrangement and about 92% of the heavy chains have at least one or up to
five or
more than five mutations present in FW3; in one embodiment, at least 21% of
the
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light chains have at least one mutation present in FW3; in one embodiment, at
least
20% of heavy chains have at least two mutations present in FW3; in one
embodiment, at least 13% of the heavy chains have at least three mutations
present
in FW3; in one embodiment, at least 20% of the heavy chains have at least four
mutations in FW3; in one embodiment, at least 18% of the heavy chains have at
lest
5 mutations in FW3.
[0066] In one embodiment, the light chain is derived from a human Vx3-
20/JK1
rearrangement and about 7% of the heavy chains have at least one mutation
present
in CDR3; in one embodiment, about 7% of the heavy chains have one mutation in
CDR3.
[0067] In one embodiment, a population of antigen-specific antibodies
derived
from a mouse as described herein is provided, wherein the antibodies comprise
a
light chain derived from a human Vx3-205x1 rearrangement and about 43% of the
heavy chains have one or more mutations present in FW1, about 92% of the heavy
chains have one or more mutations present in CDR1, about 46% of the heavy
chains
have one or more mutations present in FW2, about 84% of the heavy chains have
one or more mutations present in CDR2, about 92% of the heavy chains have one
or
more mutations present in FW3, and about 7% of the heavy chains have one or
more
mutations present in CDR3.
[0068] In one aspect, a mouse that expresses an immunoglobulin light chain
from a rearranged immunoglobulin light chain sequence is provided, wherein the
rearranged immunoglobulin light chain sequence is present in the germline of
the
mouse, wherein the immunoglobulin light chain comprises a human variable
sequence. In one embodiment, the germline of the mouse comprises a rearranged
immunoglobulin light chain sequence that is derived from the same V segment
and
the same J segment as all non-surrogate light chain sequences present in every
B cell
of the mouse that comprises a rearranged light chain sequence.
[0069] In one embodiment, the germline of the mouse lacks a functional
unrearranged immunoglobulin light chain V gene segment. In one embodiment, the
germline of the mouse lacks a functional unrearranged immunoglobulin light
chain J
gene segment.
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[0070] In one embodiment, the germline of the mouse comprises no more
than
one, no more than two, or no more than three rearranged (V/J) light chain
sequences.
[0071] In one embodiment, the rearranged V/J sequence comprises a ic
light
chain sequence. In a specific embodiment, the lc light chain sequence is a
human x
light chain sequence. In a specific embodiment, the lc light chain sequence is
selected from a human Vx1-39/J sequence, a human Vx3-20/J sequence, and a
combination thereof. In a specific embodiment, the lc light chain sequence is
a
human Vic1-39/Jx5 sequence. In a specific embodiment, the lc light chain
sequence
is a human Vid-20/Jx1 sequence.
[0072] In one embodiment, the mouse further comprises in its germline a
sequence selected from a mouse lc intronic enhancer 5' with respect to the
rearranged immunoglobulin light chain sequence, a mouse x 3' enhancer, and a
combination thereof.
[0073] In one embodiment, the mouse comprises an unrearranged human VH
gene segment, an unrearranged human DH gene segment, and an unrearranged
human JH gene segment, wherein said VH, DH, and JH gene segments are capable
of
rearranging to form an immunoglobulin heavy chain variable gene sequence
operably linked to a heavy chain constant gene sequence. In one embodiment,
the
mouse comprises a plurality of human VH, DH, and JH gene segments. In a
specific
embodiment, the human VH, DH, and JH gene segments replace endogenous mouse
VH, DH, and Ill gene segments at the endogenous mouse immunoglobulin heavy
chain locus. In a specific embodiment, the mouse comprises a replacement of
all or
substantially all functional mouse VH, DH, and JH gene segments with all or
substantially all functional human VH, DH, and JH gene segments.
[0074] In one embodiment, the mouse expresses an immunoglobulin light chain
that comprises a mouse constant sequence. In one embodiment, the mouse
expresses an immunoglobulin light chain that comprises a human constant
sequence.
[0075] In one embodiment, the mouse expresses an immunoglobulin heavy
chain that comprises a mouse sequence selected from a CHI sequence, a hinge
sequence, a CH2 sequence, a C113 sequence, and a combination thereof.
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[0076] In one embodiment, the mouse expresses an immunoglobulin heavy
chain that comprises a human sequence selected from a CHI sequence, a hinge
sequence, a CH2 sequence, a CH3 sequence, and a combination thereof.
[0077] In one embodiment, the rearranged immunoglobulin light chain
sequence
in the germline of the mouse is at an endogenous mouse immunoglobulin light
chain
locus. In a specific embodiment, the rearranged immunoglobulin light chain
sequence in the germline of the mouse replaces all or substantially all mouse
light
chain V and J sequences at the endogenous mouse immunoglobulin light chain
locus.
[0078] In one aspect, a mouse is provided that comprises a B cell
population
characterized by each B cell that comprises a non-surrogate light chain
sequence
comprises a rearranged light chain gene that is derived from a single human V
segment and a single human J segment, wherein the only light chain variable
sequence in the germline of the mouse is a rearranged sequence derived from
the
single human V segment and the single human J segment, and wherein each B ell
that comprises the rearranged light chain gene further comprises a gene
encoding a
cognate human heavy chain variable domain, and wherein the rearranged light
chain
gene comprises at least one, at least two, at least three, or at least four
somatic
hyperrnutations.
[0079] 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.
[0080] In one aspect, a tissue derived from a mouse as described herein
is
provided. In one embodiment, the tissue is derived from spleen, lymph node or
bone
marrow of a mouse as described herein.
[0081] In one aspect, a nucleus derived from a mouse as described herein
is
provided. In one embodiment, the nucleus is from a diploid cell that is not a
B cell.
[0082] In one aspect, a mouse cell is provided that is isolated from a
mouse as
described herein. In one embodiment, the cell is an ES cell. In one
embodiment, the
cell is a lymphocyte. In one embodiment, the lymphocyte is a B cell. In one
embodiment, the B cell expresses a chimeric heavy chain comprising a variable
domain derived from a human gene segment; and a light chain derived from a
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rearranged human Vic1-39/J sequence, rearranged human Vic3-20/I sequence, or a
combination thereof; wherein the heavy chain variable domain is fused to a
mouse
constant region and the light chain variable domain is fused to a mouse or a
human
constant region.
[0083] In one aspect, a hybridoma is provided, wherein the hybridoma is
made
with a B cell of a mouse as described herein. In a specific embodiment, the B
cell is
from a mouse as described herein that has been immunized with an immunogen
comprising an epitope of interest, and the B cell expresses a binding protein
that
binds the epitope of interest, the binding protein has a somatically mutated
human
Yu domain and a mouse CH, and has a human VL domain derived from a rearranged
human Vic1-39.ha or a rearranged human Vk3-20JK1 and a mouse CL.
[0084] In one aspect, a mouse embryo is provided, wherein the embryo
comprises a donor ES cell that is derived from a mouse as described herein.
[0085] In one aspect, a targeting vector is provided, comprising, from 5'
to 3' in
transcriptional direction with reference to the sequences of the 5' and 3'
mouse
homology arms of the vector, a 5' mouse homology arm, a human or mouse
immunoglobulin promoter, a human or mouse leader sequence, and a human VL
region selected from a rearranged human Vx1-39JK5 or a rearranged human Vic3-
20JK1, and a 3' mouse homology arm. In one embodiment, the 5' and 3' homology
arms target the vector to a sequence 5' with respect to an enhancer sequence
that is
present 5' and proximal to the mouse Cic gene. In one embodiment, the promoter
is
a human immunoglobulin variable region gene segment promoter. In a specific
embodiment, the promoter is a human W3-15 promoter. In one embodiment, the
leader sequence is a mouse leader sequence. In a specific embodiment, the
mouse
leader sequence is a mouse Vic3-7 leader sequence.
[0086] In one aspect, a targeting vector is provided as described above,
but in
place of the 5' mouse homology arm the human or mouse promoter is flanked 5'
with a site-specific recombinase recognition site (SRRS), and in place of the
3'
mouse homology arm the human VL region is flanked 3' with an SRRS.
[0087] In one aspect, a reverse chimeric antibody made by a mouse as
described
herein, wherein the reverse chimeric antibody comprises a light chain
comprising a
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human VL and a mouse CL, and a heavy chain comprising a human VH and a mouse
CH.
[0088] In one aspect, a method for making an antibody is provided,
comprising
expressing in a single cell (a) a first VH gene sequence of an immunized mouse
as
described herein fused with a human CH gene sequence; (b) a VL gene sequence
of
an immunized mouse as described herein fused with a human CL gene sequence;
and, (c) maintaining the cell under conditions sufficient to express a fully
human
antibody, and isolating the antibody. In one embodiment, the cell comprises a
second VH gene sequence of a second immunized mouse as described herein fused
with a human CH gene sequence, the first VH gene sequence encodes a VH domain
that recognizes a first epitope, and the second VH gene sequence encodes a VH
domain that recognizes a second epitope, wherein the first epitope and the
second
epitope are not identical.
[0089] In one aspect, a method for making an epitope-binding protein is
provided, comprising exposing a mouse as described herein with an immunogen
that
comprises an epitope of interest, maintaining the mouse under conditions
sufficient
for the mouse to generate an immunoglobulin molecule that specifically binds
the
epitope of interest, and isolating the immunoglobulin molecule that
specifically
binds the epitope of interest; wherein the epitope-binding protein comprises a
heavy
chain that comprises a somatically mutated human VH and a mouse CH, associated
with a light chain comprising a mouse CL and a human VL derived from a
rearranged
human Vx1-39.1x5 or a rearranged human Vic3-20Jic1.
[0090] In one aspect, a cell that expresses an epitope-binding protein is
provided, wherein the cell comprises: (a) a human nucleotide sequence encoding
a
human VL domain that is derived from a rearranged human Vic1-391K5 or a
rearranged human Vic3-20.11(1, wherein the human nucleotide sequence is fused
(directly or through a linker) to a human immunoglobulin light chain constant
domain cDNA sequence (e.g., a human lc constant domain DNA sequence); and, (b)
a first human V11 nucleotide sequence encoding a human VH domain derived from
a
first human VH nucleotide sequence, wherein the first human VH nucleotide
sequence is fused (directly or through a linker) to a human immunoglobulin
heavy
chain constant domain cDNA sequence; wherein the epitope-binding protein
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recognizes a first epitope. In one embodiment, the epitope-binding protein
binds the
first epitope with a dissociation constant of lower than le M, lower than 10-
8M,
lower than le M, lower than 10-1 M, lower than 1011 M, or lower than 10'12 M.
[0091] In one embodiment, the cell comprises a second human nucleotide
sequence encoding a second human VH domain, wherein the second human
sequence is fused (directly or through a linker) to a human immunoglobulin
heavy
chain constant domain cDNA sequence, and wherein the second human VH domain
does not specifically recognize the first epitope (e.g., displays a
dissociation constant
of, e.g., 10-6 M, 10 M, 10-4 M, or higher), and wherein the epitope-binding
protein
recognizes the first epitope and the second epitope, and wherein the first and
the
second immunoglobulin heavy chains each associate with an identical light
chain of
(a).
[0092] In one embodiment, the second VH domain binds the second epitope
with
a dissociation constant that is lower than 10'6 M, lower than 10-7M, lower
than 10-8
M, lower than 10-9M, lower than 10-1 M, lower than 10-11M, or lower than 10-
12 M.
[0093] In one embodiment, the epitope-binding protein comprises a first
immunoglobulin heavy chain and a second immunoglobulin heavy chain, each
associated with an identical light chain derived from a rearranged human VL
region
selected from a human Vic1-39J1c5 or a human Vx3-20Jx1, wherein the first
immunoglobulin heavy chain binds a first epitope with a dissociation constant
in the
nanomolar to picomolar range, the second immunoglobulin heavy chain binds a
second epitope with a dissociation constant in the nanomolar to picomolar
range, the
first epitope and the second epitope are not identical, the first
immunoglobulin heavy
chain does not bind the second epitope or binds the second epitope with a
dissociation constant weaker than the micromolar range (e.g., the millimolar
range),
the second immunoglobulin heavy chain does not bind the first epitope or binds
the
first epitope with a dissociation constant weaker than the micromolar range
(e.g., the
millimolar range), and one or more of the VL, the VH of the first
immunoglobulin
heavy chain, and the VH of the second immunoglobulin heavy chain, are
somatically
mutated.
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[0094] In one embodiment, the first immunoglobulin heavy chain comprises
a
protein A-binding residue, and the second immunoglobulin heavy chain lacks the
protein A-binding residue.
[0095] In one embodiment, the cell is selected from CHO, COS, 293, HeLa,
and
a retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6TM
cell).
[0096] In one aspect, a reverse chimeric antibody is provided, comprising
a
human VH and a mouse heavy chain constant domain, a human VL and a mouse light
chain constant domain, wherein the antibody is made by a process that
comprises
immunizing a mouse as described herein with an immunogen comprising an
epitope,
and the antibody specifically binds the epitope of the immunogen with which
the
mouse was immunized. In one embodiment, the VL domain is somatically mutated.
In one embodiment the VH domain is somatically mutated. In one embodiment,
both
the VL domain and the VH domain are somatically mutated. In one embodiment,
the
VL is linked to a mouse etc domain.
[0097] In one aspect, a mouse is provided, comprising human VH gene
segments
replacing all or substantially all mouse VH gene segments at the endogenous
mouse
heavy chain locus; no more than one or two rearranged human light chain VOL
sequences selected from a rearranged Vic1-39/J and a rearranged Vic3-20/.1 or
a
combination thereof, replacing all mouse light chain gene segments; wherein
the
human heavy chain variable gene segments are linked to a mouse constant gene,
and
the rearranged human light chain sequences are linked to a human or mouse
constant
gene.
[0098] In one aspect, a mouse ES cell comprising a replacement of all or
substantially all mouse heavy chain variable gene segments with human heavy
chain
variable gene segments, and no more than one or two rearranged human light
chain
VOL sequences, wherein the human heavy chain variable gene segments are linked
to a mouse immunoglobulin heavy chain constant gene, and the rearranged human
light chain VL/JL sequences are linked to a mouse or human immunoglobulin
light
chain constant gene. In a specific embodiment, the light chain constant gene
is a
mouse constant gene.
[0099] In one aspect, an antigen-binding protein made by a mouse as
described
herein is provided. In a specific embodiment, the antigen-binding protein
comprises
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a human immunoglobulin heavy chain variable region fused with a mouse constant
region, and a human immunoglobulin light chain variable region derived from a
Vx1-39 gene segment or a Vx3-20 gene segment, wherein the light chain constant
region is a mouse constant region.
[00100] In one aspect, a fully human antigen-binding protein made from an
immunoglobulin variable region gene sequence from a mouse as described herein
is
provided, wherein the antigen-binding protein comprises a fully human heavy
chain
comprising a human variable region derived from a sequence of a mouse as
described herein, and a fully human light chain comprising a Vic1-39 or a Vic3-
20.
In one embodiment, the light chain variable region comprises one to five
somatic
mutations. In one embodiment, the light chain variable region is a cognate
light
chain variable region that is paired in a B cell of the mouse with the heavy
chain
variable region.
[00101] In one embodiment, the fully human antigen-binding protein comprises a
first heavy chain and a second heavy chain, wherein the first heavy chain and
the
second heavy chain comprise non-identical variable regions independently
derived
from a mouse as described herein, and wherein each of the first and second
heavy
chains express from a host cell associated with a human light chain derived
from a
Vic1-39 gene segment or a Vx3-20 gene segment. In one embodiment, the first
heavy chain comprises a first heavy chain variable region that specifically
binds a
first epitope of a first antigen, and the second heavy chain comprises a
second heavy
chain variable region that specifically binds a second epitope of a second
antigen. In
a specific embodiment, the first antigen and the second antigen are different.
In a
specific embodiment, the first antigen and the second antigen are the same,
and the
first epitope and the second epitope are not identical; in a specific
embodiment,
binding of the first epitope by a first molecule of the binding protein does
not block
binding of the second epitope by a second molecule of the binding protein.
[00102] In one aspect, a fully human binding protein derived from a human
immunoglobulin sequence of a mouse as described herein comprises a first
immunoglobulin heavy chain and a second immunoglobulin heavy chain, wherein
the first immunoglobulin heavy chain comprises a first variable region that is
not
identical to a variable region of the second immunoglobulin heavy chain, and
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wherein the first immunoglobulin heavy chain comprises a wild type protein A
binding determinant, and the second heavy chain lacks a wild type protein A
binding
determinant. In one embodiment, the first immunoglobulin heavy chain binds
protein A under isolation conditions, and the second immunoglobulin heavy
chain
does not bind protein A or binds protein A at least 10-fold, a hundred-fold,
or a
thousand-fold weaker than the first immunoglobulin heavy chain binds protein A
under isolation conditions. In a specific embodiment, the first and the second
heavy
chains are IgG1 isotypes, wherein the second heavy chain comprises a
modification
selected from 95R (EU 435R), 96F (EU 436F), and a combination thereof, and
wherein the first heavy chain lacks such modification.
[00103] In one aspect, a method for making a bispecific antigen-binding
protein
is provided, comprising exposing a first mouse as described herein to a first
antigen
of interest that comprises a first epitope, exposing a second mouse as
described
herein to a second antigen of interest that comprises a second epitope,
allowing the
first and the second mouse to each mount immune responses to the antigens of
interest, identifying in the first mouse a first human heavy chain variable
region that
binds the first epitope of the first antigen of interest, identifying in the
second mouse
a second human heavy chain variable region that binds the second epitope of
the
second antigen of interest, making a first fully human heavy chain gene that
encodes
a first heavy chain that binds the first epitope of the first antigen of
interest, making
a second fully human heavy chain gene that encodes a second heavy chain that
binds
the second epitope of the second antigen of interest, expressing the first
heavy chain
and the second heavy chain in a cell that expresses a single fully human light
chain
derived from a human Vic1-39 or a human W3-20 gene segment to form a
bispecific antigen-binding protein, and isolating the bispecific antigen-
binding
protein.
[00104] In one embodiment, the first antigen and the second antigen are not
identical.
[00105] In one embodiment, the first antigen and the second antigen are
identical,
and the first epitope and the second epitope are not identical. In one
embodiment,
binding of the first heavy chain variable region to the first epitope does not
block
binding of the second heavy chain variable region to the second epitope.
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[00106] In one embodiment, the human light chain when paired with the first
heavy chain specifically binds the first epitope of the first antigen and when
paired
the second heavy chain specifically binds the second epitope of the second
antigen.
[00107] In one embodiment, the first antigen is selected from a soluble
antigen
and a cell surface antigen (e.g., a tumor antigen), and the second antigen
comprises a
cell surface receptor. In a specific embodiment, the cell surface receptor is
an
immunoglobulin receptor. In a specific embodiment, the immunoglobulin receptor
is an Fc receptor. In one embodiment, the first antigen and the second antigen
are
the same cell surface receptor, and binding of the first heavy chain to the
first
epitope does not block binding of the second heavy chain to the second
epitope.
[00108] In one embodiment, the light chain variable domain of the light chain
comprises 2 to 5 somatic mutations. In one embodiment, the light chain
variable
domain is a somatically mutated cognate light chain expressed in a B cell of
the first
or the second immunized mouse with either the first or the second heavy chain
variable domain. In one embodiment, the light chain of the cell comprises a
germline sequence.
[00109] In one embodiment, the first fully human heavy chain bears an amino
acid modification that reduces its affinity to protein A, and the second fully
human
heavy chain does not comprise a modification that reduces its affinity to
protein A.
[00110] In one aspect, a method of preparing a bispecific antibody that
specifically binds to a first and a second antigen is provided, wherein the
method
comprises (a) identifying a first nucleic acid sequence that encodes a first
human
heavy chain variable (VH) domain that is specific for the first antigen; (b)
identifying
a second nucleic acid sequence that encodes a second human heavy chain
variable
(VII) domain that is specific for the second antigen; (c) providing a third
nucleic acid
sequence that encodes a human light chain variable (VL) region which, when
paired
with the VH region of (a) specifically binds the first antigen, and when
paired with
the VH region of (b) specifically binds to the second antigen; (d) culturing a
host cell
comprising the first, second, and third nucleic acid sequences to allow
expression of
the first and second human VH regions and the human VL region to form the
bispecific antibody; and (d) recovering said bispecific antibody. In various
aspects,
the first and second antigens are different from one another. In various
aspects the
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first and second nucleic acid sequences are isolated from an immunized mouse
that
expresses a human immunoglobulin VL region from a rearranged immunoglobulin
light chain sequence, wherein the rearranged immunoglobulin sequence is in the
germline of the mouse.
[00111] In one embodiment, the human VL region is derived from a rearranged
human light chain sequence comprising a human Vx1-39 gene segment or a human
Vx3-20 gene segment. In a specific embodiment, the rearranged human light
chain
sequence is a germline sequence (i.e., does not comprise a somatic
hypermutation
within the V gene segment sequence).
[00112] In one embodiment, the third nucleic acid sequence is isolated from a
mouse that expresses a human immunoglobulin VL region from a rearranged
immunoglobulin light chain sequence in the germline of the mouse. In one
embodiment, the rearranged immunoglobulin light chain sequence comprises a
human Vx1-39 or human Vic3-20 gene segment. In a specific embodiment, the
rearranged immunoglobulin light chain sequence comprises a human VK-1-39 gene
segment. In one embodiment, the human immunoglobulin VL region is expressed
from a modified endogenous immunoglobulin light chain locus.
[00113] In one embodiment, the first and second antigens are present on one
molecule. In one embodiment, the first and second antigens are present on
different
molecules. In various embodiments, the first or second nucleic acid sequence
comprises a modification that reduces the affinity of the encoded heavy chain
to
protein A.
[00114] In one embodiment, the first or second nucleic acid sequences comprise
a
rearranged human heavy chain variable region sequence comprising a human heavy
chain gene segment selected from V111-2, VH1-3, VH1-8, VH1-18, VH1-24, VI11-
46,
V111-58, VH1-69, VH2-5, VH2-26, VH2-70, VH3-7, VH3-9, VH3-11, VH3-13, VH3-15,
V113-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-43, VH3-48, VH3-53, Vn3-64, V113-
72, VH3-73, VH4-31, VH4-34, VH4-39, VH4-59, VH5-51, and VH6-1. In a specific
embodiment, the heavy chain gene segment is VH2-5, V113-23 or V113-30.
[00115] In one aspect, a method of preparing a bispecific antibody that
specifically binds to a first and a second antigen is provided, wherein the
method
comprises (a) identifying a first nucleic acid sequence that encodes a first
human
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heavy chain variable (VH) domain that is specific for the first antigen; (b)
identifying
a second nucleic acid sequence that encodes a second human heavy chain
variable
(VH) domain that is specific for the second antigen; (c) providing a third
nucleic acid
sequence that encodes a human light chain variable (VL) region derived from a
human V-K1-39 or human VK3-20 gene segment which, when paired with the VH
region of (a) specifically binds the first antigen, and when paired with the
VH region
of (b) specifically binds to the second antigen; (d) culturing a host cell
comprising
the first, second, and third nucleic acid sequences to allow expression of the
first and
second human VH regions and the human VL region to form the bispecifie
antibody;
and (d) recovering said bispecific antibody. In various aspects, the first and
second
antigens are different from one another. In various aspects, the first and
second
nucleic acid sequences are isolated from an immunized mouse that expresses a
human immunoglobulin VL region from a rearranged immunoglobulin sequence that
is derived from a human W1-39 or human VK3-20 gene segment, wherein the
rearranged human VK1-39 or VK3-30 gene segment is in the germline of the
mouse.
[00116] In one embodiment, the third nucleic acid sequence is a germline
sequence (i e., does not comprise a somatic hypermutation within the V gene
segment sequence). In one embodiment, the third nucleic acid sequence is
isolated
from the mouse that expresses a human immunoglobulin VL region derived from a
human VK1-39 or human Vx3-20 gene segment from a rearranged immunoglobulin
light chain sequence in the germline of the mouse. In a specific embodiment,
the
third nucleic acid sequence comprises two to five somatic hypermutations in a
complementary determining region (CDR) and/or a framework region (FWR). In
one embodiment, the human immunoglobulin VL region is expressed from a
modified endogenous immunoglobulin light chain locus.
[00117] In one embodhnent, the first and second antigens are present on one
molecule. In one embodiment, the first and second antigens are present on
different
molecules. In one embodiment, the first or second nucleic acid sequence
comprises
a modification that reduces the affinity of the encoded heavy chain to protein
A.
[00118] In one embodiment, the first or second nucleic acid sequences comprise
a
rearranged human heavy chain variable region sequence comprising a human heavy
chain gene segment selected from VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-46,
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VH1-58, VH 1-69, VH2-5, VH2-26, VH2-70, VH3-7, VH3-9, VH3-11, VH3-13, VH3-15,
VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-43, VH3-48, VH3-53, VH3-64, V113-
72, V1.3-73, VH4-31, VH4-34, VH4-39, VH4-59, VH5-51, and VH6-I. In a specific
embodiment, the heavy chain gene segment is VH2-5, VH3-23 or VH3-30.
[00119] In one aspect, a method for making a bispecific antibody is provided,
comprising exposing a mouse as described herein to an antigen of interest,
allowing
the mouse to mount an immune response to the antigen of interest, identifying
a first
human heavy chain variable region that binds a first epitope of the antigen of
interest, identifying a second human heavy chain variable region that binds a
second
epitope of the antigen of interest, making a first fully human heavy chain
gene that
encodes the first heavy chain that binds the first epitope of the antigen of
interest,
making a second fully human heavy chain gene that encodes a second heavy chain
that binds the second epitope of the antigen of interest, expressing the first
heavy
chain and the second htavy chain in a cell that expresses a single fully human
light
chain derived from a human Vx1-39 or a human Vic3-20 gene segment to form a
bispecific antibody, and isolating the bispecific antigen-binding protein.
[00120] In one embodiment, the first epitope and the second epitope are not
identical. In one embodiment, binding of the first heavy chain variable region
to the
first epitope does not block binding of the second heavy chain variable region
to the
second epitope. In one embodiment, the first and second heavy chains are
capable
of binding the first and second epitopes simultaneously.
[00121] In one embodiment, the bispecific antibody binds the first and second
epitopes simultaneously. In one embodiment, the bispecific antibody binds the
first
epitope and second epitope independently.
[00122] In one embodiment, the binding response of the bispecific antibody to
the
antigen is about 2-fold higher than the binding response of the first heavy
chain
variable region to the antigen. In one embodiment, the binding response of the
bispecific antibody to the antigen is about 2-fold higher than the binding
response of
the second heavy chain variable region to the antigen. In one embodiment, the
binding response of the bispecific antibody to the antigen is about the same
as, or
about equal to, the binding response of the first heavy chain variable region
and or
the second heavy chain variable region to the antigen.
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[00123] In one embodiment, the antigen is selected from a soluble antigen, a
cell
surface antigen (e.g., a tumor antigen) and a cell surface receptor. In a
specific
embodiment, the cell surface receptor is an immunoglobulin receptor. In a
specific
embodiment, the immunoglobulin receptor is an Fc receptor.
[00124] In one embodiment, the light chain variable domain of the light chain
comprises 2 to 5 somatic mutations. In one embodiment, the light chain
variable
domain is a somatically mutated cognate light chain expressed in a B cell of
the
immunized mouse with either the first or the second heavy chain variable
domain.
[00126] In one embodiment, the first fully human heavy chain bears an amino
acid modification that reduces its affinity to protein A, and the second fully
human
heavy chain does not comprise a modification that reduces its affinity to
protein A.
[00126] In various embodiments, methods for making bispecific antibodies are
enhanced by employing a common light chain to pair with each heavy chain
variable
regions of the bispecific antibodies. In various embodiments, employing a
common
light chain as described herein reduces the number of inappropriate species of
immunoglobulins lacking bispecificity as compared to employing original
cognate
light chains. In various embodiments, the heavy chain variable regions of the
bispecific antibodies are identified from monospecific antibodies comprising a
common light chain. In various embodiments, the heavy chain variable regions
of
the bispecific antibodies comprise human heavy chain variable gene segments
that
are rearranged in vivo within mouse B cells that have been previously
engineered to
express a limited human light chain repertoire, or a single human light chain,
cognate with human heavy chains and, in response to exposure with an antigen
of
interest, generate a chimeric antibody repertoire containing a plurality of
human
heavy chain variable regions that are cognate with one or one of two possible
human
light chain variable regions, wherein the chimeric antibodies are specific for
the
antigen of interest.
[00127] In various aspects, a method of preparing a bispecific antibody is
provided, the bispecific antibody comprising 1) a first polypeptide and a
second
polypeptide, wherein the first and second polypeptides each include a
multimerization domain (e.g., an immunoglobulin Fc domain) allowing the first
and
second polypeptides to form a dimer, and the multimerization domains promote
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stable interaction between first and second polypeptides, and wherein one of
the
multimerization domains bears an amino acid modification that reduces its
affinity
to protein A and the other multimerization domain lacks the modification, 2) a
binding domain in each of the first and second polypeptide, each binding
domain
comprising a variable heavy chain and a variable light chain, wherein the
variable
light chain of the first polypeptide and the variable light chain of the
second
polypeptide have a common amino acid sequence, which common sequence has an
amino acid sequence identity to an original light chain of each of the
polypeptides of
at least 80%, of at least 85%, preferably at least 90%, more preferably at
least 95%
and most preferably 100V0 sequence identity. In various embodiments, the
variable
light chain is derived from a human Vid -39 or a human Vic3-20 gene segment.
In
various embodiments, the variable light chain is a rearranged human light
chain
sequence. In various embodiments, the variable light chain is isolated from a
mouse
as described herein.
[00128] In various embodiments, the method comprises the steps of (i)
culturing a
host cell comprising a nucleic acid encoding the first polypeptide, the second
polypeptide, and the common light chain, wherein the nucleic acid is
expressed; and
(ii) recovering the bispecific antibody from the host cell culture; in one
embodiment,
the nucleic acid encoding the first polypeptide or the nucleic acid encoding
the
second polypeptide, bears an amino acid modification that reduces its affinity
to
protein A. In one embodiment, the nucleic acid encoding the first polypeptide,
the
second polypeptide, and the common light chain is present in a single vector
or in
separate vectors. In one embodiment, the host cell is used to make a
bispecific
antibody according to the preceding paragraph.
[00129] In one aspect, a method of preparing a bispecifie antibody is
provided,
comprising (a) selecting a first nucleic acid encoding a first human heavy
chain
variable region isolated from a mouse as described herein; (b) selecting a
second
nucleic acid encoding a second human heavy chain variable region isolated from
the
same or separate mouse as described herein; (c) providing a third nucleic acid
encoding a human light chain variable region isolated from a mouse as
described
herein or derived from a rearranged human light chain variable region as
described
herein; (c) introducing into a host cell the first, second and third nucleic
acids and
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culturing the host cell so that expression of the first, second and third
nucleic acid
occurs; and (d) recovering the bispecific antibody formed from the cell
culture.
[00130] In one embodiment, the first and second human heavy chain variable
regions are somatically mutated. In a specific embodiment, the first and
second
human heavy chain variable regions are independently derived from a rearranged
human VH gene segment selected from 1-2, 1-3, 1-8, 1-18, 1-24, 1-46, 1-58, 1-
69, 2-
5, 2-26, 2-70, 3-7, 3-9, 3-11, 3-13, 3-15, 3-16, 3-20, 3-21, 3-23, 3-30, 3-33,
3-43, 3-
48, 3-53, 3-64, 3-72, 3-73, 4-31, 4-34, 4-39, 4-59, 5-51, and a 6-1 human VH
gene
segment. In one embodiment, the first and second human heavy chain variable
regions are independently derived from a rearranged human VH gene segment
selected from 2-5, 3-30 and 3-23. In one embodiment, the first human heavy
chain
variable region is derived from a human VH2-5 gene segment and the second
human
heavy chain variable region is derived from a human VH3-30 gene segment. In
one
embodiment, the first human heavy chain variable region is derived from a
human
VH3-30 gene segment and the second human heavy chain variable region is
derived
from a human VH3-23 gene segment. In one embodiment, the first human heavy
chain variable region is derived from a human VH3-23 gene segment and the
second
human heavy chain variable region is derived from a human VH3-30 gene segment.
[00131] In one embodiment, the first or second nucleic acid is modified prior
to
step (c), wherein the first or second nucleic acid is modified such that it
has a
reduced affinity to protein A.
[00132] In one embodiment, the third nucleic acid is isolated from a mouse as
described herein. In one embodiment, the third nucleic acid comprises 2 to 5
somatic mutations. In one embodiment, the third nucleic acid encodes a human
light
chain variable region derived from a human Vic1-39 gene segment. In one
embodiment, the third nucleic acid encodes a human light chain variable region
derived from a human Vx3-20 gene segment.
[00133] In one embodiment, the third nucleic acid is derived from a rearranged
human light chain variable region. In one embodiment, the rearranged human
light
chain variable region comprises a sequence derived from a human Vx1-39 gene
segment or a human Vic3-20 gene segment. In one embodiment, the rearranged
human light chain variable region comprises a gennline human VK1-39 sequence
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(i.e., does not comprise a somatic hypennutation within the V gene segment
sequence). In one embodiment, the rearranged human light chain variable region
comprises a germline human Vx3-20 sequence.
[00134] In various embodiments, a method of preparing a bispecific antibody
that
incorporates a first human heavy chain comprising a variable domain derived
from a
modified mouse that lacks a rearranged human light chain sequence in its
germline
is provided, wherein the first human heavy chain is paired with a cognate
human
light chain that comprises a rearranged human light chain variable region
derived
from a human Vx1-39 or a human W3-20 gene segment. In various embodiments,
a second human heavy chain with a different specificity from the first human
heavy
chain is identified from an immunized mouse as described herein. Nucleic acids
encoding the two heavy chains and the common light chain are introduced into a
host cell as described in the preceding paragraphs so that expression of all
three
chains occurs and the bispecific antibody is recovered from the cell culture.
[00135] In one embodiment, the mouse is immunized with the same antigen used
to generate the first human heavy chain variable domain. In one embodiment,
the
mouse is immunized with a different antigen used to generate the first human
heavy
chain variable domain.
[00136] In one aspect, a method of selecting human heavy chains that can pair
with a single human light chain to make a bispecific antibody is provided,
including
nucleic acids that encode the bispecific antibody and a host cell comprising
the
nucleic acids.
[00137] In one aspect, a method of increasing the amount of a desired
bispecific
antibody in a cell culture over undesired products such as monospecific
antibodies is
provided, wherein one of the heavy chains of the bispecific antibody is
modified to
reduce its affinity to protein A.
[00138] In one aspect, an isolated host cell is provided, wherein the host
cell
comprises (a) a first nucleic acid sequence encoding a first human heavy chain
variable region that binds a first antigen, wherein the first nucleic acid
sequence is
isolated from a mouse immunized with the first antigen that expresses a human
immunoglobulin VL region from a rearranged immunoglobulin light chain sequence
in the gennline of the mouse; (b) a second nucleic acid sequence encoding a
second
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human heavy chain variable region that binds a second antigen, wherein the
second
nucleic acid sequence is isolated from a mouse immunized with the second
antigen
that expresses a human immunoglobulin VL region from a rearranged
immunoglobulin light chain sequence in the germline of the mouse; (c) a third
nucleic acid sequence encoding a human light chain variable region which, when
paired with the heavy chain variable region of (a) specifically binds the
first antigen,
and when paired with the heavy chain variable region of (b) specifically binds
to the
second antigen.
[00139] In various aspects, the first and second antigens are different from
one
another. In various aspects, the expression of the first, second and third
nucleic acid
sequences leads to the formation of a bispecific antibody that specifically
binds to
the first and second antigens.
[00140] In one embodiment, the human VL region is derived from a rearranged
human light chain sequence comprising a human Vx1-39 gene segment or a human
Vx3-20 gene segment. In a specific embodiment, the rearranged human light
chain
sequence is a germline sequence (i.e., does not comprise a somatic
hypermutation
within the variable domain). In one embodiment, the third nucleic acid
sequence is
isolated from a mouse that expresses a human immunoglobulin VL region from a
rearranged immunoglobulin light chain sequence, wherein the rearranged human
light chain sequence is present in the germline of the mouse. In one
embodiment,
the rearranged immunoglobulin light chain sequence comprises a human V1c1-39
gene segment or a human Vx3-20 gene segment. In a specific embodiment, the
human Vx1-39 gene segment or human Vx3-20 gene segment comprises at least one
somatic hypermutation in a complementary determining region (CDR) or framework
region (FWR). In a specific embodiment, the first, second and third nucleic
acid
sequences are isolated from a mouse that expresses a human immunoglobulin VL
region derived from a human Vic1-39 or human Vx3-20 gene segment from a
rearranged immunoglobulin light chain sequence, wherein the rearranged
immunoglobulin light chain sequence is present in the germline of the mouse.
[00141] In various embodiments, the mouse does not contain an endogenous light
chain variable region gene segment that is capable of rearranging to form an
immunoglobulin light chain.
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[00142] In one embodiment, the human immunoglobulin VL region is expressed
from a modified endogenous immunoglobulin light chain locus. In one
embodiment, the first and second antigens are present on one molecule. In one
embodiment, the first and second antigens are present on different molecules.
In one
embodiment, the first or second nucleic acid sequence comprises a modification
that
reduces the affinity of the encoded heavy chain to protein A.
[00143] In one embodiment, the first or second nucleic acid sequences comprise
a
rearranged human heavy chain variable region sequence comprising a human heavy
chain gene segment selected from VH 1 -2, VH 1 -3 , VH I-8, VH 1 - 18, VH1-24,
VH1-46,
VH1-58, VH1-69, V112-5, VH2-26, VH2-70, VH3-7, V113-9, V113-11, VH3-13, VH3-
15,
VH3-20, VH3-21, VH3-23, V113-30, V113-33, VH3-43, VH3-48, V113-53, VH3-64,
V113-
72, V113-73, VH4-31, VH4-34, VH4-39, VH4-59, VHS-51, and V116-1. In a specific
embodiment, the heavy chain gene segment is VH2-5, V113-23 or VH3-30.
[00144] In one aspect, an antibody or a bispecific antibody comprising a human
heavy chain variable domain made in accordance with the invention is provided.
In
another aspect, use of a mouse as described herein to make a fully human
antibody
or a fully human bispecific antibody is provided.
[00145] In one aspect, a genetically modified mouse, embryo, or cell described
herein comprises a K light chain locus that retains endogenous regulatory or
control
elements, e.g., a mouse x intronic enhancer, a mouse lc 3' enhancer, or both
an
intronic enhancer and a 3' enhancer, wherein the regulatory or control
elements
facilitate somatic mutation and affinity maturation of an expressed sequence
of the lc
light chain locus.
[00146] In one aspect, a mouse is provided that comprises a B cell population
characterized by having immunoglobulin light chains derived from no more than
one, or no more than two, rearranged or unrearranged immunoglobulin light
chain V
and J gene segments, wherein the mouse exhibits a K:2. light chain ratio that
is about
the same as a mouse that comprises a wild type complement of immunoglobulin
light chain V and J gene segments.
[00147] In one embodiment, the immunoglobulin light chains are derived from no
more than one, or no more than two, rearranged immunoglobulin light chain V
and J
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gene segments. In a specific embodiment, the light chains are derived from no
more
than one rearranged immunoglobulin light chain V and J gene segments.
[00148] In one aspect, a mouse as described herein is provided that expresses
an
immunoglobulin light chain derived from no more than one, or no more than two,
human Vx/Ix sequences, wherein the mouse comprises a replacement of all or
substantially all endogenous mouse heavy chain variable region gene segments
with
one or more human heavy chain variable region gene segments, and the mouse
exhibits a ratio of (a) CD19 B cells that express an immunoglobulin having a
k
light chain, to (b) CD19 B cells that express an immunoglobulin having a ic
light
chain, of about 1 to about 20.
[00149] In one embodiment, the mouse expresses a single ic light chain derived
from a human Vx1-39.1x5 sequence, and the ratio of CD19f B cells that express
an
immunoglobulin having a light chain to CD19+ B cells that express an
immunoglobulin having a K light chain is about 1 to about 20; in one
embodiment,
the ratio is about 1 to at least about 66; in a specific embodiment, the ratio
is about 1
to 66.
[00150] In one embodiment, the mouse expresses a single x light chain derived
from a human Vx3-20Jx5 sequence, and the ratio of CD19+ B cells that express
an
immunoglobulin having a light chain to CD19+ B cells that express an
immunoglobulin having a lc light chain is about 1 to about 20; in one
embodiment,
the ratio is about 1 to about 21. In specific embodiments, the ratio is 1 to
20, or 1 to
21.
[00151] In one aspect, a genetically modified mouse is provided that expresses
a
single rearranged lc light chain, wherein the mouse comprises a functional 2
light
chain locus, and wherein the mouse expresses a B cell population that
comprises
Igie cells that express a ic light chain derived from the same single
rearranged lc
light chain. In one embodiment, the percent of Igtc+Ig%+ B cells in the mouse
is
about the same as in a wild type mouse. In a specific embodiment, the percent
of
Igx+IgX+ B cells in the mouse is about 2 to about 6 percent. In a specific
embodiment, the percent of Igx+Ig%+ B cells in a mouse wherein the single
rearranged K light chain is derived from a Vx1-39.1x5 sequence is about 2 to
about 3;
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in a specific embodiment, the percent is about 2.6. In a specific embodiment,
the
percent of Igx+Igk+ B cells in a mouse wherein the single rearranged lc light
chain is
derived from a Vx3-20Jx1 sequence is about 4 to about 8; in a specific
embodiment,
the percent is about 6.
[00152] In one aspect, a genetically modified mouse is provided, wherein the
mouse expresses a single rearranged lc light chain derived from a human Vic
and Ix
gene segment, wherein the mouse expresses a B cell population that comprises a
single lc light chain derived from the single rearranged lc light chain
sequence,
wherein the genetically modified mouse has not been rendered resistant to
somatic
hypermutations. In one embodiment, at least 90% of the lc light chains
expressed on
a B cell of the mouse exhibit from at least one to about five somatic
hypermutations.
[00153] In one aspect, a genetically modified mouse is provided that is
modified
to express a single x light chain derived from no more than one, or no more
than
two, rearranged lc light chain sequences, wherein the mouse exhibits a lc
light chain
usage that is about two-fold or more, at least about three-fold or more, or at
least
about four-fold or more greater than the ic light chain usage exhibited by a
wild type
mouse, or greater than the x light chain usage exhibited by a mouse of the
same
strain that comprises a wild type repertoire of lc light chain gene segments.
In a
specific embodiment, the mouse expresses the single lc light chain from no
more
than one rearranged lc light chain sequence. In a more specific embodiment,
the
rearranged ic light chain sequence is selected from a Vx1-39.1x5 and Vx3-20Jx1
sequence. In one embodiment, the rearranged x light chain sequence is a Yid -
39Jia sequence. In one embodiment, the rearranged lc light chain sequence is a
Vic3-20JK1 sequence.
[00154] In one aspect, a genetically modified mouse is provided that expresses
a
single lc light chain derived from no more than one, or no more than two,
rearranged
lc light chain sequences, wherein the mouse exhibits a lc light chain usage
that is
about 100-fold or more, at least about 200-fold or more, at least about 300-
fold or
more, at least about 400-fold or more, at least about 500-fold or more, at
least about
600-fold or more, at least about 700-fold or more, at least about 800-fold or
more, at
least about 900-fold or more, at least about 1000-fold or more greater than
the same
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K light chain usage exhibited by a mouse bearing a complete or substantially
complete human x light chain locus. In a specific embodiment, the mouse
bearing a
complete or substantially complete human lc light chain locus lacks a
functional
unrearranged mouse lc light chain sequence. In a specific embodiment, the
mouse
expresses the single lc light chain from no more than one rearranged lc light
chain
sequence. In one embodiment, the mouse comprises one copy of a rearranged lc
light chain sequence (e.g., a heterozygote). In one embodiment, the mouse
comprises two copies of a rearranged lc light chain sequence (e.g., a
homozygote).
In a more specific embodiment, the rearranged K light chain sequence is
selected
from a Vx1-39.1k5 and Vx3-20Jx1 sequence. In one embodiment, the rearranged lc
light chain sequence is a Vic1-39.1x5 sequence. In one embodiment, the
rearranged
x light chain sequence is a Vx3-20Jx1 sequence.
[00155] In one aspect, a genetically modified mouse is provided that expresses
a
single light chain derived from no more than one, or no more than two,
rearranged
light chain sequences, wherein the light chain in the genetically modified
mouse
exhibits a level of expression that is at least 10-fold to about 1,000-fold,
100-fold to
about 1,000-fold, 200-fold to about 1,000-fold, 300-fold to about l,000-fold,
400-
fold to about 1,000-fold, 500-fold to about 1,000-fold, 600-fold to about
1,000-fold,
700-fold to about 1,000-fold, 800-fold to about 1,000-fold, or 900-fold to
about
1,000-fold higher than expression of the same rearranged light chain exhibited
by a
mouse bearing a complete or substantially complete light chain locus. In one
embodiment, the light chain comprises a human sequence. In a specific
embodiment, the human sequence is a lc sequence. In one embodiment, the human
sequence is a 9 sequence. In one embodiment, the light chain is a fully human
light
chain.
[00156] In one embodiment, the level of expression is characterized by
quantitating mRNA of transcribed light chain sequence, and comparing it to
transcribed light chain sequence of a mouse bearing a complete or
substantially
complete light chain locus.
[00157] In one aspect, a genetically modified mouse is provided that expresses
a
single lc light chain derived from no more than one, or no more than two,
rearranged
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x light chain sequences, wherein the mouse, upon immunization with antigen,
exhibits a serum titer that is comparable to a wild type mouse immunized with
the
same antigen. In a specific embodiment, the mouse expresses a single ic light
chain
from no more than one rearranged ic light chain sequence. In one embodiment,
the
serum titer is characterized as total immunoglobulin. In a specific
embodiment, the
serum titer is characterized as IgM specific titer. In a specific embodiment,
the
serum titer is characterized as IgG specific titer. In a more specific
embodiment, the
rearranged x light chain sequence is selected from a Vx1-39J16 and Vic3-20.1x1
sequence. In one embodiment, the rearranged x light chain sequence is a Vx1 -
39.hc5 sequence. In one embodiment, the rearranged ic light chain sequence is
a
Vx3-20Jicl sequence.
[00158] In one aspect, a genetically modified mouse is provided that expresses
a
population of antigen-specific antibodies, wherein all of the immunoglobulin
light
chains of the population of antigen-specific antibodies comprise a human light
chain
variable (VL) region derived from the same single human VL gene segment and
the
immunoglobulin heavy chains comprise a human heavy chain variable (VH) region
derived from one of a plurality of human VH gene segments.
[00159] In various embodiments, the human VH gene segments are selected from
VH1-2, VH1-3, VH1-8, V111-18, VH1-24, VH1-46, VH1-58, VH1-69, V112-5, VH2-26,
V12-70, VH3-7, VH3-9, VH3-11, VH3-13, VH3-15, V113-20, V113-21, VH3-23, VH3-
30, VH3-33, VH3-43, VH3-48, V113-53, V113-64, VH3-72, V113-73, VH4-31, VH4-34,
V114-39, VH4-59, VH5-51, and V116-1.
[00160] In various embodiments, same single human VL gene segment is selected
from a human Vx1-39 gene segment and a human Vx3-20 gene segment. In various
embodiments, all of the immunoglobulin light chains comprise a human light
chain J
(JL) gene segment selected from a Jic and a A gene segment. In a specific
embodiment, the human JL gene segment is selected from a human .1x1 and a JO
gene segment. In various embodiments, the mouse lacks a sequence selected from
a
mouse immunoglobulin VL gene segment, a mouse immunoglobulin .11, gene
segment, and a combination thereof. In various embodiments, the human VL
region
is operably linked to a human, mouse, or rat immunoglobulin light chain
constant
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(CL) region. In a specific embodiment, the human VL region is operably linked
to a
mouse CI( region. In a specific embodiment, the human VL region is operably
linked to a rat CI( region.
[00161] In various embodiments, the human VL region is expressed from an
endogenous immunoglobulin light chain locus. In various embodiments, the human
VH region is operably linked to a human, mouse, or rat immunoglobulin heavy
chain
constant (CH) region. In various embodiments the (CH) region comprises a human
sequence selected from a CHI, a hinge, a C112, a CH3, a CH4, and/or a
combination
thereof. In various embodiments, the human VH region is expressed from an
endogenous immunoglobulin heavy chain locus.
[00162] In one aspect, a genetically modified mouse is provided that expresses
a
plurality of immunoglobulin heavy chains associated with a single light chain.
In
one embodiment, the heavy chain comprises a human sequence. In various
embodiments, the human sequence is selected from a variable sequence, a CH1, a
hinge, a CH2, a CH3, and a combination thereof. In one embodiment, the single
light
chain comprises a human sequence. In various embodiments, the human sequence
is
selected from a variable sequence, a constant sequence, and a combination
thereof.
In one embodiment, the mouse comprises a disabled endogenous immunoglobulin
locus and expresses the heavy chain and/or the light chain from a transgene or
extrachromosomal episome. In one embodiment, the mouse comprises a
replacement at an endogenous mouse locus of some or all endogenous mouse heavy
chain gene segments (i.e., V, D, J), and/or some or all endogenous mouse heavy
chain constant sequences (e.g., CH1, hinge, CH2, CH3, or a combination
thereof),
and/or some or all endogenous mouse light chain sequences (e.g., V, J,
constant, or a
combination thereof), with one or more human immunoglobulin sequences.
[00163] In one aspect, a mouse suitable for making antibodies that have the
same
light chain is provided, wherein all or substantially all antibodies made in
the mouse
are expressed with the same light chain. In one embodiment, the light chain is
expressed from an endogenous light chain locus.
[00164] In one aspect, a method for making a light chain for a human antibody
is
provided, comprising obtaining from a mouse as described herein a light chain
sequence and a heavy chain sequence, and employing the light chain sequence
and
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the heavy chain sequence in making a human antibody. In one embodiment, the
human antibody is a bispecific antibody.
[00165] In one aspect, a method for identifying a human heavy chain variable
domain that is capable of binding an antigen of interest with an engineered
light
chain as described herein is provided, wherein the method comprises providing
a
heavy chain variable domain derived from a first antibody that is capable of
binding
the antigen, repairing the heavy chain variable domain with a germline light
chain
sequence and transfecting a cell so that each are expressed to form a second
antibody, exposing the second antibody to the antigen, and measuring binding
of the
second antibody to the antigen.
[00166] In one embodiment, the light chain of the first antibody comprises a
human VK1-39 sequence. In one embodiment, the light chain of the first
antibody
comprises a human VK3-20 sequence. In one embodiment, the germline light chain
sequence comprises a human VK1-39 or Vic3-20 sequence. In various embodiments,
binding of the second antibody to the antigen is determined by comparison of
binding of the first antibody to the antigen.
(00167] In some embodiments, a method for making a bispecific antigen-binding
protein is provided, wherein the method comprises exposing a first mouse that
expresses a single human immunoglobulin light chain to a first antigen of
interest
that comprises a first epitope, exposing a second mouse that expresses a
single
human immunoglobulin light chain to a second antigen of interest that
comprises a
second epitope, allowing the first and the second mouse to each mount immune
responses to the antigens of interest, identifying in the first mouse a first
human
heavy chain variable region that binds the first epitope of the first antigen
of interest,
identifying in the second mouse a second human heavy chain variable region
that
binds the second epitope of the second antigen of interest, making a first
fully
human heavy chain gene that encodes a first heavy chain that binds the first
epitope
of the first antigen of interest, making a second fully human heavy chain gene
that
encodes a second heavy chain that binds the second epitope of the second
antigen of
interest, expressing the first heavy chain and the second heavy chain in a
cell that
expresses a single fully human light chain to form a bispecific antigen-
binding
protein, and isolating the bispecific antigen-binding protein.
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[00168] In some embodiments, a method for making a bispecific antigen-binding
protein is provided, wherein the method comprises exposing a first mouse that
expresses a single human immunoglobulin light chain to a first antigen of
interest
that comprises a first epitope, exposing a second mouse that expresses a
single
human immunoglobulin light chain to a second antigen of interest that
comprises a
second epitope, allowing the first and the second mouse to each mount immune
responses to the antigens of interest, identifying in the first mouse a first
human
heavy chain variable region that binds the first epitope of the first antigen
of interest,
identifying in the second mouse a second human heavy chain variable region
that
binds the second epitope of the second antigen of interest, making a first
fully
human heavy chain gene that encodes a first heavy chain that binds the first
epitope
of the first antigen of interest, making a second fully human heavy chain gene
that
encodes a second heavy chain that binds the second epitope of the second
antigen of
interest, expressing the first heavy chain and the second heavy chain in a
cell that
expresses a single fully human light chain to form a bispecific antigen-
binding
protein, and isolating the bispecific antigen-binding protein, and wherein the
single
fully human light chain is derived from a rearranged human Vic l-39 or a human
Vx3-20 gene segment.
[00169] In some embodiments, a method for making a bispecific antigen-binding
protein is provided, wherein the method comprises exposing a first mouse that
expresses a single human immunoglobulin light chain to a first antigen of
interest
that comprises a first epitope, exposing a second mouse that expresses a
single
human immunoglobulin light chain to a second antigen of interest that
comprises a
second epitope, allowing the first and the second mouse to each mount immune
responses to the antigens of interest, identifying in the first mouse a first
human
heavy chain variable region that binds the first epitope of the first antigen
of interest,
identifying in the second mouse a second human heavy chain variable region
that
binds the second epitope of the second antigen of interest, making a first
fully
human heavy chain gene that encodes a first heavy chain that binds the first
epitope
of the first antigen of interest, making a second fully human heavy chain gene
that
encodes a second heavy chain that binds the second epitope of the second
antigen of
interest, expressing the first heavy chain and the second heavy chain in a
cell that
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expresses a single fully human light chain to form a bispecific antigen-
binding
protein, and isolating the bispecific antigen-binding protein; and wherein (a)
the
first antigen and the second antigen are not identical; or (b) the first
antigen and the
second antigen are identical, and the first epitope and the second epitope are
not
identical.
[00170] In some embodiments, a method for making a bispecific antigen-binding
protein is provided, wherein the method comprises exposing a first mouse that
expresses a single human immunoglobulin light chain to a first antigen of
interest
that comprises a first epitope, exposing a second mouse that expresses a
single
human immunoglobulin light chain to a second antigen of interest that
comprises a
second epitope, allowing the first and the second mouse to each mount immune
responses to the antigens of interest, identifying in the first mouse a first
human
heavy chain variable region that binds the first epitope of the first antigen
of interest,
identifying in the second mouse a second human heavy chain variable region
that
binds the second epitope of the second antigen of interest, making a first
fully
human heavy chain gene that encodes a first heavy chain that binds the first
epitope
of the first antigen of interest, making a second fully human heavy chain gene
that
encodes a second heavy chain that binds the second epitope of the second
antigen of
interest, expressing the first heavy chain and the second heavy chain in a
cell that
expresses a single fully human light chain to form a bispecific antigen-
binding
protein, and isolating the bispecific antigen-binding protein; and wherein the
human
light chain when paired with the first heavy chain specifically binds the
first epitope
of the first antigen and when paired with the second heavy chain specifically
binds
the second epitope of the second antigen.
[00171] In some embodiments, a method for making a bispecific antigen-binding
protein is provided, wherein the method comprises exposing a first mouse that
expresses a single human immunoglobulin light chain to a first antigen of
interest
that comprises a first epitope, exposing a second mouse that expresses a
single
human immunoglobulin light chain to a second antigen of interest that
comprises a
second epitope, allowing the first and the second mouse to each mount immune
responses to the antigens of interest, identifying in the first mouse a first
human
heavy chain variable region that binds the first epitope of the first antigen
of interest,
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identifying in the second mouse a second human heavy chain variable region
that
binds the second epitope of the second antigen of interest, making a first
fully
human heavy chain gene that encodes a first heavy chain that binds the first
epitope
of the first antigen of interest, making a second fully human heavy chain gene
that
encodes a second heavy chain that binds the second epitope of the second
antigen of
interest, expressing the first heavy chain and the second heavy chain in a
cell that
expresses a single fully human light chain to form a bispecific antigen-
binding
protein, and isolating the bispecific antigen-binding protein; and wherein the
first
fully human heavy chain gene bears an amino acid modification that reduces its
affinity to protein A, and the second fully human heavy chain does not
comprise a
modification that reduces its affinity to protein A. In some embodiments, the
modification that reduces affinity to protein A is selected from a 95R (EUR
435R),
96F (EUR 436F), and a combination thereof.
[00172] In some embodiments, a method for making a bispecific antigen-binding
protein is provided, wherein the method comprises exposing a first mouse that
expresses a single human immunoglobulin light chain to a first antigen of
interest
that comprises a first epitope, exposing a second mouse that expresses a
single
human immunoglobulin light chain to a second antigen of interest that
comprises a
second epitope, allowing the first and the second mouse to each mount immune
responses to the antigens of interest, identifying in the first mouse a first
human
heavy chain variable region that binds the first epitope of the first antigen
of interest,
identifying in the second mouse a second human heavy chain variable region
that
binds the second epitope of the second antigen of interest, making a first
fully
human heavy chain gene that encodes a first heavy chain that binds the first
epitope
of the first antigen of interest, making a second fully human heavy chain gene
that
encodes a second heavy chain that binds the second epitope of the second
antigen of
interest, expressing the first heavy chain and the second heavy chain in a
cell that
expresses a single fully human light chain to form a bispecific antigen-
binding
protein, and isolating the bispecific antigen-binding protein; and wherein the
human
light chain of the cell comprises a germline sequence.
[00173] In some embodiments, a method for making a human bispecific antibody
is provided comprising a step of employing in the bispecific antibody two
human
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heavy chain variable region sequences of two different B cells a mouse that
expresses a single human light chain variable domain.
[00174] In some embodiments, a method for making a human bispecific antibody
is provided comprising a step of employing in the bispecific antibody two
human
heavy chain variable region sequences of two different B cells a mouse that
expresses a single human light chain variable domain, wherein the single human
light chain variable domain comprises a rearranged human Vx1-39 or human Vic3-
20 gene segment.
[00175] In some embodiments, a method for making a human bispecific antibody
is provided comprising a step of employing in the bispecific antibody two
human
heavy chain variable region sequences of two different B cells a mouse that
expresses a single human light chain variable domain, wherein in the single
human
light chain variable domain further comprises a rearranged human Jic5 or human
JO
gene segment.
[00176] In some embodiments, a method for making a human bispecific antibody
is provided comprising a step of employing in the bispecific antibody two
human
heavy chain variable region sequences of two different B cells a mouse that
expresses a single human light chain variable domain, wherein the mouse
further
comprises a humanized heavy chain locus containing one or more unrearranged
human VII gene segments, one or more unrearranged human Du gene segments, and
one or more unrearranged human Ju gene segments, operably linked to one or
more
non-human heavy chain constant region genes. In some embodiments, the
humanized heavy chain locus comprises 80 unrearranged human Vu gene segments,
27 unrearranged human Du gene segments and six unrearranged human Ju gene
segments operably linked to one or more mouse heavy chain constant region
genes.
In some embodiments, the method comprises, wherein the one or more human VH
gene segments comprises human VH1-2, V111-8, VH1-24, VH1-69, VH2-5, VH3-7,
VH3-9, VH3-11, VH3-13, VH3-15, VH3-20, VH3-23, VH3-30, VH3-33, V113-48, Vii3-
53, VH4-31, VH4-39, VH4-59, VH5-51, VH6-1, or a combination thereof.
[00177] In some embodiments, a method for making a human bispecific antibody
is provided comprising a step of employing in the bispecific antibody two
human
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heavy chain variable region sequences of two different B cells a mouse that
expresses a single human light chain variable domain, wherein the mouse does
not
contain an endogenous light chain variable gene segment that is capable of
rearranging to form an immunoglobulin light chain.
[00178] In some embodiments, a method of selecting two human
immunoglobulin heavy chain variable domains for use in a bispecific antibody
is
provided comprising immunizing a mouse with an antigen of interest, wherein
the
mouse expresses a single human light chain variable domain, and wherein the
two
human immunoglobulin heavy chain variable domains independently associate with
the single human light chain variable domain to bind the antigen of interest.
[00179] In some embodiments, a method for making a human bispecific antibody
is provided comprising a step of employing in the bispecific antibody two
human
heavy chain variable region sequences of two different B cells a mouse that
expresses a single human light chain variable domain, wherein the single human
light chain variable domain is derived from a rearranged human Vx1-39 gene
segment. In some embodiments, the single human light chain variable domain
further comprises a rearranged human JO gene segment. In some embodiments, the
single human light chain variable domain is derived from a rearranged human
Vic3-
gene segment. In some embodiments, the single human light chain variable
20 domain further comprises a rearranged human JO gene segment.
[01801 In some embodiments, a method of selecting two human
immunoglobulin heavy chain variable domains for use in a bispecific antibody
is
provided comprising immunizing a mouse with an antigen of interest, wherein
the
mouse expresses a single human light chain variable domain, and wherein the
two
human immunoglobulin heavy chain variable domains independently associate with
the single human light chain variable domain to bind the antigen of interest,
wherein
the mouse does not contain an endogenous light chain variable gene segment
that is
capable of rearranging to form an immunoglobulin light chain.
[00181] Any of the embodiments and aspects described herein can be used in
conjunction with one another, unless otherwise indicated or apparent from the
context. Other embodiments will become apparent to those skilled in the art
from a
review of the ensuing description.
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BRIEF DESCRIPTION OF FIGURES
[00182] FIG. 1 illustrates a targeting strategy for replacing endogenous mouse
immunoglobulin light chain variable region gene segments with a human Vicl-
39.10 gene region.
[00183] FIG. 2 illustrates a targeting strategy for replacing endogenous mouse
immunoglobulin light chain variable region gene segments with a human Vic3-
20JK1 gene region.
[00184] FIG. 3 illustrates a targeting strategy for replacing endogenous mouse
immunoglobulin light chain variable region gene segments with a human VpreB/A5
gene region.
[00185] FIG. 4 shows the percent of CD19+ B cells (y-axis) from peripheral
blood for wild type mice (WT), mice homozygous for an engineered human
rearranged Vx1-391K5 light chain region (Vic1-39JK5 HO) and mice homozygous
for an engineered human rearranged VK3-20JK1 light chain region (Vic3-20JK1
HO).
[00186] FIG. 5A shows the relative mRNA expression (y-axis) of a Vic1-39-
derived light chain in a quantitative PCR assay using probes specific for the
junction
of an engineered human rearranged Vic1-39JK5 light chain region (Vic1-39JK5
Junction Probe) and the human V-K1-39 gene segment (VK1-39 Probe) in a mouse
homozygous for a replacement of the endogenous Vic and JK gene segments with
human Vic and JK gene segments (HK), a wild type mouse (WT), and a mouse
heterozygous for an engineered human rearranged VK1-39J-K5 light chain region
(VK1-39JK5 HET). Signals are normalized to expression of mouse CK. N.D.: not
detected.
[00187] FIG. 5B shows the relative mRNA expression (y-axis) of a VK1-39-
derived light chain in a quantitative PCR assay using probes specific for the
junction
of an engineered human rearranged VK1-39JK5 light chain region (VK1-39.1K5
Junction Probe) and the human Vic1-39 gene segment (Vic1-39 Probe) in a mouse
homozygous for a replacement of the endogenous VIC and JK gene segments with
human VK and JK gene segments (HK), a wild type mouse (WT), and a mouse
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homozygous for an engineered human rearranged Vic1-39J1c5 light chain region
(Vic1-39.1x5 HO). Signals are normalized to expression of mouse Cic.
[00188] FIG. 5C shows the relative mRNA expression (y-axis) of a Vic3-20-
derived light chain in a quantitative PCR assay using probes specific for the
junction
of an engineered human rearranged Vic3-20Jic1 light chain region (Vx3-20JK1
Junction Probe) and the human Vx3-20 gene segment (Vic3-20 Probe) in a mouse
homozygous for a replacement of the endogenous Vic and Iic gene segments with
human Vic and Jic gene segments (Hx), a wild type mouse (WT), and a mouse
heterozygous (HET) and homozygous (HO) for an engineered human rearranged
Vic3-20.1k1 light chain region. Signals are normalized to expression of mouse
GK.
[00189] FIG. 6A shows IgM (left) and IgG (right) titer in wild type (WT; N-2)
and mice homozygous for an engineered human rearranged Vic1-39.1x5 light chain
region (Vic1-39Jx5 HO; N=2) immunized with 13 -galactosidase.
[00190] FIG. 6B shows total immunoglobulin (IgM, IgG, IgA) titer in wild type
(WT; N=5) and mice homozygous for an engineered human rearranged Vic3-20Jic1
light chain region (Vic3-20k1 HO; N=5) immunized with 13 -galactosidase.
[00191] FIG. 7A shows a schematic of monospecific antibodies (Parent-1 and
Parent-2) and a bispecific antibody (Bispecific) constructed from heavy chain
variable regions from each parent monospecific antibody. A common light chain
variable region (darkened) is indicated in the bispecific antibody.
[00192] FIG. 7B shows a schematic for the binding characteristics of two
parent
monoclonal antibodies (Parent-1 and Parent-2) for an antigen of interest, as
well as
the binding characteristic of a bispecific antibody constructed from pairing
the heavy
chain variable regions from each monospeeifie parent antibody with a common
light
chain. The capability of the bispecific antibody to bind to two distinct
epitopes of
the antigen of interest either separately (bottom left) or simultaneously
(bottom
right) is indicated.
[00193] FIG. 8 shows a bar graph of the binding of 300nM bispecific (darkened
bars) and monospecifie (striped and gray bars) antibodies to a captured
monomeric
Antigen E surface in BIACORETM units (RU). Monoclonal parent-1 antibody (P1
Ab), monoclonal parent-2 (P2 Ab) and bispecific antibodies (BsAb) are
indicated.
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DETAILED DESCRIPTION
[00194] This invention is not limited to particular methods, and experimental
conditions described, as such methods and conditions may vary. It is also to
be
understood that the terminology used herein is for the purpose of describing
particular embodiments only, and is not intended to be limiting, since the
scope of
the present invention is defined by the claims.
[00195] Unless defined otherwise, all terms and phrases used herein include
the
meanings that the terms and phrases have attained in the art, unless the
contrary is
clearly indicated or clearly apparent from the context in which the term or
phrase is
used. Although any methods and materials similar or equivalent to those
described
herein can be used in the practice or testing of the present invention,
particular
methods and materials are now described. All publications mentioned are hereby
incorporated by reference in their entirety.
[00196] The term "antibody", as used herein, includes immunoglobulin molecules
comprising four polypeptide chains, two heavy (H) chains and two light (L)
chains
inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain
variable (VH) region and a heavy chain constant region (CH). The heavy chain
constant region comprises three domains, CH1, CH2 and CH3. Each light chain
comprises a light chain variable (VL) region and a light chain constant region
(Ca
The VH and VL regions can be further subdivided into regions of
hypervariability,
termed complementarity determining regions (CDR), interspersed with regions
that
are more conserved, termed framework regions (FR). Each VH and VL comprises
three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in
the
following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs
may be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs may be
abbreviated as LCDR1, LCDR2 and LCDR3. The term "high affinity" antibody
refers to an antibody that has a KD with respect to its target epitope about
of 1e M
or lower (e.g., about 1 x 10-9M, 1 x 1040 M, 1 x M, or about 1 x
1042M). In
one embodiment, KD is measured by surface plasmon resonance, e.g., BIACORETM;
in another embodiment, KD is measured by ELISA.
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[00197] The phrase "bispecific antibody" includes an antibody capable of
selectively binding two or more epitopes. Bispecific antibodies include
fragments of
two different monoclonal antibodies (FIG. 7A) and generally comprise two
nonidentical heavy chains derived from the two different monoclonal
antibodies,
with each heavy chain specifically binding a different epitope¨either on two
different molecules (e.g., different epitopes on two different immunogens; see
FIG.
7B, bottom left) or on the same molecule (e.g., different epitopes on the same
immunogen; see FIG. 7B, bottom right). If a bispecific antibody is capable of
selectively binding two different epitopes (a first epitope and a second
epitope), the
affinity of the first heavy chain for the first epitope will generally be at
least one to
two or three or four or more orders of magnitude lower than the affinity of
the first
heavy chain for the second epitope, and vice versa. Epitopes specifically
bound by
the bispecific antibody can be on the same or a different target (e.g., on the
same or
a different protein; see FIG. 7B). Exemplary bispecific antibodies include
those
with a first heavy chain specific for a tumor antigen and a second heavy chain
specific for a cytotoxic marker, e.g., an Fc receptor (e.g., FeyRI, FeyRII,
FcyRIII,
etc.) or a T cell marker (e.g., CD3, CD28, etc.). Further, the second heavy
chain
variable region can be substituted with a heavy chain variable region having a
different desired specificity. For example, a bispecific antibody with a first
heavy
chain specific for a tumor antigen and a second heavy chain specific for a
toxin can
be paired so as to deliver a toxin (e.g., saporin, vinca alkaloid, etc.) to a
tumor cell.
Other exemplary bispecific antibodies include those with a first heavy chain
specific
for an activating receptor (e.g., B cell receptor, FcyRI, FcyRIIA, FcyRIIIA,
FcaRI, T
cell receptor, etc.) and a second heavy chain specific for an inhibitory
receptor (e.g.,
FcyRIIB, CD5, CD22, CD72, CD300a, etc.). Such bispecific antibodies can be
constructed for therapeutic conditions associated with cell activation (e.g.
allergy
and asthma). Bispecitic antibodies can be made, for example, by combining
heavy
chains that recognize different epitopes of the same or different immunogen
(FIG.
7B). For example, nucleic acid sequences encoding heavy chain variable
sequences
that recognize different epitopes of the same or different immunogen can be
fused to
nucleic acid sequences encoding the same or different heavy chain constant
regions,
and such sequences can be expressed in a cell that expresses an immunoglobulin
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light chain. A typical bispecific antibody has two heavy chains each having
three
heavy chain CDRs, followed by (N-terminal to C-terminal) a CH1 domain, a
hinge, a
CH2 domain, and a CH3 domain, and an immunoglobulin light chain that either
does
not confer epitope-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 the heavy chain epitope-binding regions, or that can
associate
with each heavy chain and enable binding or one or both of the heavy chains to
one
or both epitopes.
[00198] The term "cell" 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-infccted insect cells, Trichoplusia ni,
etc.), non-
human animal cells, human 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 Kl, 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, W138, MRC 5, Co1o205, HB 8065, HL-60,
(e.g., BIIK21), 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.C6TM cell).
[00199] 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. A CDR
can be
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somatically mutated (e.g., vary from a sequence encoded in an animal's
germline),
humanized, and/or modified with amino acid substitutions, additions, or
deletions.
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).
[00200] The term "conservative," when used to describe a conservative amino
acid substitution, includes substitution of an amino acid residue by another
amino
acid residue having a side chain R group with similar chemical properties
(e.g.,
charge or hydrophobicity). In general, a conservative amino acid substitution
will
not substantially change the functional properties of interest of a protein,
for
example, the ability of a variable region to specifically bind a target
epitope with a
desired affinity. Examples of groups of amino acids that have side chains with
similar chemical properties include aliphatic side chains such as glycine,
alanine,
valine, leucine, and isoleucine; aliphatic-hydroxyl side chains such as serine
and
threonine; amide-containing side chains such as asparagine and glutamine;
aromatic
side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains
such
as lysine, arginine, and histidine; acidic side chains such as aspartic acid
and
glutamic acid; and, sulfur-containing side chains such as cysteine and
methionine.
Conservative amino acids substitution groups include, for example,
valine/leucine/isoleucine, phenylalanine/tyrosine, lysine/arginine,
alanine/valine,
glutamate/aspartate, and asparagine/glutamine. In some embodiments, a
conservative amino acid substitution can be substitution of any native residue
in a
protein with alanine, as used in, for example, alanine scanning mutagenesis.
In
some embodiments, a conservative substitution is made that has a positive
value in
the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Exhaustive
Matching of the Entire Protein Sequence Database, Science 256:1443-45, hereby
incorporated by reference. In some embodiments, the substitution is a
moderately
conservative substitution wherein the substitution has a nonnegative value in
the
PAM250 log-likelihood matrix.
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[00201] In some embodiments, residue positions in an immunoglobulin light
chain or heavy chain differ by one or more conservative amino acid
substitutions. In
some embodiments, residue positions in an immunoglobulin light chain or
functional fragment thereof (e.g., a fragment that allows expression and
secretion
from, e.g., a B cell) are not identical to a light chain whose amino acid
sequence is
listed herein, but differs by one or more conservative amino acid
substitutions.
[00202] The phrase "epitope-binding protein" includes a protein having at
least
one CDR and that is capable of selectively recognizing an epitope, e.g., is
capable of
binding an epitope with a KD that is at about one micromolar or lower (e.g., a
KD
that is about 1 x 10-6M, 1 x 10-7M, 1 x 10-9 IA 1 x 10-9 m, 1 x 10-10 M, 1 x
10-11 M,
or about 1 x 10-12 M). Therapeutic epitope-binding proteins (e.g., therapeutic
antibodies) frequently require a KD that is in the nanomolar or the picomolar
range.
[00203] The phrase "functional fragrnent" includes fragments of epitope-
binding
proteins that can be expressed, secreted, and specifically bind to an epitope
with a
KD in the micromolar, nanomolar, or picomolar range. Specific recognition
includes
having a KD that is at least in the micromolar range, the nanomolar range, or
the
picomolar range.
[00204] The term "germline" includes reference to an immunoglobulin nucleic
acid sequence in a non-somatically mutated cell, e.g., a non-somatically
mutated B
cell or pre-B cell or hematopoietic cell.
[00205] The phrase "heavy chain," or "immunoglobulin heavy chain" includes an
immunoglobulin heavy chain constant region sequence from any organism. Heavy
chain variable domains include three heavy chain CDRs and four FR regions,
unless
otherwise specified. Fragments of heavy chains include CDRs, CDRs and FRs, and
combinations thereof. A typical heavy chain has, following the variable domain
(from N-terminal to C-terminal), a CHI domain, a hinge, a CH2 domain, and a
CH3
domain. A functional fragment of a heavy chain includes a fragment that is
capable
of specifically recognizing an epitope (e.g., recognizing the epitope with a
KD in the
micromolar, nanomolar, or picomolar range), that is capable of expressing and
secreting from a cell, and that comprises at least one CDR.
[00206] The term "identity" when used in connection with sequence includes
identity as determined by a number of different algorithms known in the art
that can
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be used to measure nucleotide ancUor amino acid sequence identity. In some
embodiments described herein, identities are determined using a ClustalW v.
1.83
(slow) alignment employing an open gap penalty of 10.0, an extend gap penalty
of
0.1, and using a Gonnet similarity matrix (MACVECTORTm 10Ø2, MacVector
Inc., 2008). The length of the sequences compared with respect to identity of
sequences will depend upon the particular sequences, but in the case of a
light chain
constant domain, the length should contain sequence of sufficient length to
fold into
a light chain constant domain that is capable of self-association to form a
canonical
light chain constant domain, e.g., capable of forming two beta sheets
comprising
beta strands and capable of interacting with at least one CHI domain of a
human or a
mouse. In the case of a CH1 domain, the length of sequence should contain
sequence of sufficient length to fold into a CH1 domain that is capable of
forming
two beta sheets comprising beta strands and capable of interacting with at
least one
light chain constant domain of a mouse or a human.
[002071 The phrase "immunoglobulin molecule" includes two immunoglobulin
heavy chains and two immunoglobulin light chains. The heavy chains may be
identical or different, and the light chains may be identical or different.
[002081 The phrase "light chain" includes an immunoglobulin light chain
sequence from any organism, and unless otherwise specified includes human lc
and
X. light chains and a VpreB, as well as surrogate light chains. Light chain
variable
(VI) domains typically include three light chain CDRs and four framework (FR)
regions, unless otherwise specified. Generally, a full-length light chain
includes,
from amino terminus to carboxyl terminus, a VL domain that includes FR I-CDRI-
FR2-CDR2-FR3-CDR3-FR4, and a light chain constant domain. Light chains
include those, e.g., that do not selectively bind either a first or a second
epitope
selectively bound by the epitope-binding protein in which they appear. Light
chains
also include those that bind and recognize, or assist the heavy chain with
binding
and recognizing, one or more epitopes selectively bound by the epitope-binding
protein in which they appear. Common light chains are those derived from a
rearranged human Vic1-391K5 sequence or a rearranged human Vic3-20.hcl
sequence, and include somatically mutated (e.g., affinity matured) versions.
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[00209] 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.
[00210] The phrase "somatically mutated" includes reference to a nucleic acid
sequence from a B cell that has undergone class-switching, wherein the nucleic
acid
sequence of an immunoglobulin variable region (e.g., a heavy chain variable
domain
or including a heavy chain CDR or FR sequence) in the class-switched B cell is
not
identical to the nucleic acid sequence in the B cell prior to class-switching,
such as,
for example, a difference in a CDR or framework nucleic acid sequence between
a B
cell that has not undergone class-switching and a B cell that has undergone
class-
switching. "Somatically mutated" includes reference to nucleic acid sequences
from
affinity-matured B cells that are not identical to corresponding
immunoglobulin
variable region sequences in B cells that are not affinity-matured (i.e.,
sequences in
the genome of germline cells). The phrase "somatically mutated" also includes
reference to an immunoglobulin variable region nucleic acid sequence from a B
cell
after exposure of the B cell to an epitope of interest, wherein the nucleic
acid
sequence differs from the corresponding nucleic acid sequence prior to
exposure of
the B cell to the epitope of interest. The phrase "somatically mutated" refers
to
sequences from antibodies that have been generated in an animal, e.g., a mouse
having human immunoglobulin variable region nucleic acid sequences, in
response
to an immunogen challenge, and that result from the selection processes
inherently
operative in such an animal.
No2113 The term "unrearranged," with reference to a nucleic acid sequence,
includes nucleic acid sequences that exist in the germline of an animal cell.
[00212] The phrase "variable domain" includes an amino acid sequence of an
immunoglobulin light or heavy chain (modified as desired) that comprises the
following amino acid regions, in sequence from N-terminal to C-terminal
(unless
otherwise indicated): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
Universal Light Chain
[00213] Prior efforts to make useful multispecifie epitope-binding proteins,
e.g.,
bispecific antibodies, have been hindered by variety of problems that
frequently
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share a common paradigm: in vitro selection or manipulation of sequences to
rationally engineer, or to engineer through trial-and-error, a suitable format
for
pairing a heterodimeric bispecific human immunoglobulin. Unfortunately, most
if
not all of the in vitro engineering approaches provide largely ad hoc fixes
that are
suitable, if at all, for individual molecules. On the other hand, in vivo
methods for
employing complex organisms to select appropriate pairings that are capable of
leading to human therapeutics have not been realized.
[00214] Generally, native mouse sequences are frequently not a good source for
human therapeutic sequences. For at least that reason, generating mouse heavy
chain immunoglobulin variable regions that pair with a common human light
chain
is of limited practical utility. More in vitro engineering efforts would be
expended
in a trial-and-error process to try to humanize the mouse heavy chain variable
sequences while hoping to retain epitope specificity and affinity while
maintaining
the ability to couple with the common human light chain, with uncertain
outcome.
At the end of such a process, the final product may maintain some of the
specificity
and affinity, and associate with the common light chain, but ultimately
immunogenicity in a human would likely remain a profound risk.
[00215] Therefore, a suitable mouse for making human therapeutics would
include a suitably large repertoire of human heavy chain variable region gene
segments in place of endogenous mouse heavy chain variable region gene
segments.
The human heavy chain variable region gene segments should be able to
rearrange
and recombine with an endogenous mouse heavy chain constant domain to form a
reverse chimeric heavy chain (i.e., a heavy chain comprising a human variable
domain and a mouse constant region). The heavy chain should be capable of
class
switching and somatic hypermutation so that a suitably large repertoire of
heavy
chain variable domains are available for the mouse to select one that can
associate
with the limited repertoire of human light chain variable regions.
[00216] A mouse that selects a common light chain for a plurality of heavy
chains
has a practical utility. In various embodiments, antibodies that express in a
mouse
that can only express a common light chain will have heavy chains that can
associate
and express with an identical or substantially identical light chain. This is
particularly useful in making bispecific antibodies. For example, such a mouse
can
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be immunized with a first immunogen to generate a B cell that expresses an
antibody that specifically binds a first epitope. The mouse (or a mouse
genetically
the same) can be immunized with a second immunogen to generate a B cell that
expresses an antibody that specifically binds the second epitope. Variable
heavy
regions can be cloned from the B cells and expresses with the same heavy chain
constant region, and the same light chain, and expressed in a cell to make a
bispecific antibody, wherein the light chain component of the bispecific
antibody
has been selected by a mouse to associate and express with the light chain
component.
[00217] The inventors have engineered a mouse for generating immunoglobulin
light chains that will suitably pair with a rather diverse family of heavy
chains,
including heavy chains whose variable regions depart from germline sequences,
e.g.,
affinity matured or somatically mutated variable regions. In various
embodiments,
the mouse is devised to pair human light chain variable domains with human
heavy
chain variable domains that comprise somatic mutations, thus enabling a route
to
high affinity binding proteins suitable for use as human therapeutics.
[00218] The genetically engineered mouse, through the long and complex process
of antibody selection within an organism, makes biologically appropriate
choices in
pairing a diverse collection of human heavy chain variable domains with a
limited
number of human light chain options. In order to achieve this, the mouse is
engineered to present a limited number of human light chain variable domain
options in conjunction with a wide diversity of human heavy chain variable
domain
options. Upon challenge with an immunogen, the mouse maximizes the number of
solutions in its repertoire to develop an antibody to the immunogen, limited
largely
or solely by the number or light chain options in its repertoire. In various
embodiments, this includes allowing the mouse to achieve suitable and
compatible
somatic mutations of the light chain variable domain that will nonetheless be
compatible with a relatively large variety of human heavy chain variable
domains,
including in particular somatically mutated human heavy chain variable
domains.
[00219] To achieve a limited repertoire of light chain options, the mouse is
engineered to render nonfunctional or substantially nonfunctional its ability
to make,
or rearrange, a native mouse light chain variable domain. This can be
achieved, e.g.,
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by deleting the mouse's light chain variable region gene segments. The
endogenous
mouse locus can then be modified by an exogenous suitable human light chain
variable region gene segment of choice, operably linked to the endogenous
mouse
light chain constant domain, in a manner such that the exogenous human
variable
region gene segments can combine with the endogenous mouse light chain
constant
region gene and form a rearranged reverse chimeric light chain gene (human
variable, mouse constant). In various embodiments, the light chain variable
region
is capable of being somatically mutated. In various embodiments, to maximize
ability of the light chain variable region to acquire somatic mutations, the
appropriate enhancer(s) is retained in the mouse. For example, in modifying a
mouse x light chain locus to replace endogenous mouse K light chain gene
segments
with human x light chain gene segments, the mouse lc intronic enhancer and
mouse
x 3' enhancer are functionally maintained, or undisrupted.
[00220] A genetically engineered mouse is provided that expresses a limited
repertoire of reverse chimeric (human variable, mouse constant) light chains
associated with a diversity of reverse chimeric (human variable, mouse
constant)
heavy chains. In various embodiments, the endogenous mouse x light chain gene
segments are deleted and replaced with a single (or two) rearranged human
light
chain region, operably linked to the endogenous mouse CK gene. In embodiments
for maximizing somatic hypermutation of the rearranged human light chain
region,
the mouse x intronic enhancer and the mouse lc 3' enhancer are maintained. In
various embodiments, the mouse also comprises a nonfunctional X light chain
locus,
or a deletion thereof or a deletion that renders the locus unable to make a X
light
chain.
[00221] A genetically engineered mouse is provided that, in various
embodiments, comprises a light chain variable region locus lacking endogenous
mouse light chain VL and .11, gene segments and comprising a rearranged human
light chain variable region, in one embodiment a rearranged human VOL
sequence,
operably linked to a mouse constant region, wherein the locus is capable of
undergoing somatic hypermutation, and wherein the locus expresses a light
chain
comprising the human VOL sequence linked to a mouse constant region. Thus, in
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various embodiments, the locus comprises a mouse K 3' enhancer, which is
correlated with a normal, or wild type, level of somatic hypermutation.
[00222] The genetically engineered mouse in various embodiments when
immunized with an antigen of interest generates B cells that exhibit a
diversity of
rearrangements of human immunoglobulin heavy chain variable regions that
express
and function with one or with two rearranged light chains, including
embodiments
where the one or two light chains comprise human light chain variable regions
that
comprise, e.g., 1 to 5 somatic mutations. In various embodiments, the human
light
chains so expressed are capable of associating and expressing with any human
immunoglobulin heavy chain variable region expressed in the mouse.
Epitope-binding Proteins Binding More Than One Epitope
[00223] The compositions and methods of described herein can be used to make
binding proteins that bind more than one epitope with high affinity, e.g.,
bispecific
antibodies. Advantages of the invention include the ability to select suitably
high
binding (e.g., affinity matured) heavy chain immunoglobulin chains each of
which
will associate with a single light chain.
[00224] Several techniques for making bispecific antibody fragments from
recombinant cell culture have been reported. However, synthesis and expression
of
bispeeific binding proteins has been problematic, in part due to issues
associated
with identifying a suitable light chain that can associate and express with
two
different heavy chains, and in part due to isolation issues. In various
embodiments,
compositions and methods described herein provide the advantage of full length
bispecific antibodies that do not require special modification(s) to maintain
traditional immunoglobulin structure by increasing stability/interaction of
the
components (FIG. 7A). In various embodiments, such modification(s) has proven
cumbersome and served as an obstacle to development of bispecific antibody
technology and their potential use in treating for human disease. Thus, in
various
embodiments, through providing a natural immunoglobulin structure (i.e., full
length) having the added property of multiple specificities, full length
bispecific
antibodies maintain their critical effector functions that previous bispecific
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fragments lack, and further provide therapeutics that demonstrate the
important
pharmacokinetic parameter of a longer half-life.
[00225] The methods and compositions described herein allow for a genetically
modified mouse to select, through otherwise natural processes, a suitable
light chain
.. that can associate and express with more than one heavy chain, including
heavy
chains that are somatically mutated (e.g., affinity matured). Human VL and VH
sequences from suitable B cells of immunized mice as described herein that
express
affinity matured antibodies having reverse chimeric heavy chains (i.e., human
variable and mouse constant) can be identified and cloned in frame in an
expression
.. vector with a suitable human constant region gene sequence (e.g., a human
IgG1).
Two such constructs can be prepared, wherein each construct encodes a human
heavy chain variable domain that binds a different epitope. One of the human
VLs
(e.g., human Vic1-39Jk5 or human Vx3-20Jx1), in germline sequence or from a B
cell wherein the sequence has been somatically mutated, can be fused in frame
to a
.. suitable human constant region gene (e.g., a human K constant gene). These
three
fully human heavy and light constructs can be placed in a suitable cell for
expression. The cell will express two major species: a homodimeric heavy chain
with the identical light chain, and a heterodimeric heavy chain with the
identical
light chain. To allow for a facile separation of these major species, one of
the heavy
.. chains is modified to omit a Protein A-binding determinant, resulting in a
differential affinity of a homodimeric binding protein from a heterodimeric
binding
protein. Compositions and methods that address this issue are described in
USSN
12/832,838, filed 25 June 2010, entitled "Readily Isolated Bispecific
Antibodies
with Native Immunoglobulin Format," published as US 2010/0331527A1, hereby
.. incorporated by reference.
[00226] In one aspect, an epitope-binding protein as described herein is
provided,
wherein human VL and VH sequences are derived from mice described herein that
have been immunized with an antigen comprising an epitope of interest.
[00227] In one embodiment, an epitope-binding protein is provided that
.. comprises a first and a second polypeptide, the first polypeptide
comprising, from
N-terminal to C-terminal, a first epitope-binding region that selectively
binds a first
epitope, followed by a constant region that comprises a first C113 region of a
human
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igG selected from IgGl, IgG2, IgG4, and a combination thereof; and, a second
polypeptide comprising, from N-terminal to C-terminal, a second epitope-
binding
region that selectively binds a second epitope, followed by a constant region
that
comprises a second CH3 region of a human IgG selected from IgGl, IgG2, IgG4,
and a combination thereof, wherein the second CH3 region comprises a
modification
that reduces or eliminates binding of the second CH3 domain to protein A.
[00228] In one embodiment, the second CH3 region comprises an H95R
modification (by IMGT exon numbering; H435R by EU numbering). In another
embodiment, the second CH3 region further comprises a Y96F modification (IMGT;
Y436F by EU).
[00229] In one embodiment, the second CH3 region is from a modified human
IgGl, and further comprises a modification selected from the group consisting
of
D16E, Ll8M, N44S, K52N, V571VI, and V82I (IMGT; D356E, L358M, N384S,
K392N, V397M, and V422I by EU).
[00230] In one embodiment, the second CH3 region is from a modified human
IgG2, and further comprises a modification selected from the group consisting
of
N44S, K52N, and V82I (IMGT; N384S, K392N, and V4221 by EU).
[00231] In one embodiment, the second CH3 region is from a modified human
IgG4, and further comprises a modification selected from the group consisting
of
Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT; Q355R, N384S,
K392N, V397M, R409K, E419Q, and V422I by EU).
[00232] One method for making an epitope-binding protein that binds more than
one epitope is to immunize a first mouse in accordance with the invention with
an
antigen that comprises a first epitope of interest, wherein the mouse
comprises an
endogenous immunoglobulin light chain variable region locus that does not
contain
an endogenous mouse VL that is capable of rearranging and forming a light
chain,
wherein at the endogenous mouse immunoglobulin light chain variable region
locus
is a single rearranged human VL region operably linked to the mouse endogenous
light chain constant region gene, and the rearranged human VL region is
selected
from a human VIC1-39.1K5 and a human Vx3-20Jicl, and the endogenous mouse VH
gene segments have been replaced in whole or in part with human VH gene
segments, such that immunoglobulin heavy chains made by the mouse are solely
or
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substantially heavy chains that comprise human variable domains and mouse
constant domains. When immunized, such a mouse will make a reverse chimeric
antibody, comprising only one of two human light chain variable domains (e.g.,
one
of human Vic1-39.bc5 or human Vx3-20,k1). Once a B cell is identified that
encodes a VH that binds the epitope of interest, the nucleotide sequence of
the VH
(and, optionally, the VI) can be retrieved (e.g., by PCR) and cloned into an
expression construct in frame with a suitable human immunoglobulin constant
domain. This process can be repeated to identify a second VH domain that binds
a
second epitope, and a second VH gene sequence can be retrieved and cloned into
an
expression vector in frame to a second suitable immunoglobulin constant
domain.
The first and the second immunoglobulin constant domains can the same or
different
isotype, and one of the immunoglobulin constant domains (but not the other)
can be
modified as described herein or in US 2010/0331527A1, and epitope-binding
protein can be expressed in a suitable cell and isolated based on its
differential
affinity for Protein A as compared to a homodimeric epitope-binding protein,
e.g., as
described in US 2010/0331527A1.
(002331 In one embodiment, a method for making a bispecific epitope-binding
protein is provided, comprising identifying a first affinity-matured (e.g.,
comprising
one or more somatic hypermutations) human VH nucleotide sequence (VH1) from a
mouse as described herein, identifying a second affinity-matured (e.g.,
comprising
one or more somatic hypermutations) human VH nucleotide sequence (VH2) from a
mouse as described herein, cloning VH1 in frame with a human heavy chain
lacking
a Protein A-determinant modification as described in US 2010/0331527A1 for
form
heavy chain 1 (HC1), cloning VH2 in frame with a human heavy chain comprising
a
Protein A-determinant as described in US 2010/0331527A1 to form heavy chain 2
(HC2), introducing an expression vector comprising HC1 and the same or a
different
expression vector comprising HC2 into a cell, wherein the cell also expresses
a
human immunoglobulin light chain that comprises a human Vx1-39/human Jic5 or a
human Vic3-20/human .1x1 fused to a human light chain constant domain,
allowing
the cell to express a bispecific epitope-binding protein comprising a VH
domain
encoded by VH1 and a VH domain encoded by VH2, and isolating the bispecific
epitope-binding protein based on its differential ability to bind Protein A as
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compared with a monospecific homodimeric epitope-binding protein. In a
specific
embodiment, HC1 is an IgGI, and HC2 is an IgG1 that comprises the modification
H95R (IMGT; H435R by EU) and further comprises the modification Y96F (IMGT;
Y436F by EU). In one embodiment, the VH domain encoded by Vill, the VH
domain encoded by VH2, or both, are somatically mutated.
Human Vif Genes That Express with a Common Human VL
[00234] A variety of human variable regions from affinity-matured antibodies
raised against four different antigens were expressed with either their
cognate light
chain, or at least one of a human light chain selected from human Vic1-39/Jx5,
human Vx3-20/kl, or human VpreB/JX5 (see Example 1). For antibodies to each
of the antigens, somatically mutated high affinity heavy chains from different
gene
families paired successfully with rearranged human germline V0-39.116 and Vic3-
20.1x1 regions and were secreted from cells expressing the heavy and light
chains.
For Vic1-39J1c5 and Vx3-20.11(1, VH domains derived from the following human
VH
gene families expressed favorably: 1-2, 1-8, 1-24, 2-5, 3-7, 3-9, 3-11, 3-13,
3-15, 3-
20, 3-23, 3-30, 3-33, 3-48, 4-31, 4-39, 4-59, 5-51, and 6-1. Thus, a mouse
that is
engineered to express a limited repertoire of human VL domains from one or
both of
Vx1-39.11c5 and Vx3-20.TK I will generate a diverse population of somatically
mutated human VH domains from a VH locus modified to replace mouse VH gene
segments with human VH gene segments.
[00235] Mice genetically engineered to express reverse chimeric (human
variable,
mouse constant) immunoglobulin heavy chains associated with a single
rearranged
light chain (e.g., a VK1-39/J or a Vx3-20/J), when immunized with an antigen
of
interest, generated B cells that comprised a diversity of human VH
rearrangements
and expressed a diversity of high-affinity antigen-specific antibodies with
diverse
properties with respect to their ability to block binding of the antigen to
its ligand,
and with respect to their ability to bind variants of the antigen (see
Examples 5
through 10).
[00236] Thus, the mice and methods described herein are useful in making and
selecting human immunoglobulin heavy chain variable domains, including
somatically mutated human heavy chain variable domains, that result from a
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diversity of rearrangements, that exhibit a wide variety of affinities
(including
exhibiting a KD of about a nanomolar or less), a wide variety of specificities
(including binding to different epitopes of the same antigen), and that
associate and
express with the same or substantially the same human immunoglobulin light
chain
variable region.
Fully Human Bispecifie Antibodies Having a Common Light Chain
[00237] As a first step in various embodiments, the first and second nucleic
acid
sequences that each encode human heavy chain variable domains (and any
additional nucleic acid sequences forming the bispecific antibody) are
selected from
parent monoclonal antibodies having desired characteristics such as, for
example,
capable of binding different epitopes (see FIGs. 7A and 7B), having different
affinities, etc. Normally, the nucleic acid sequences encoding the human heavy
chain variable domains are isolated from immunized mice, as described herein,
to
allow for fusing with human heavy chain constant regions to be suitable for
human
administration. Further modifications to the sequence(s) can be made by
introducing mutations that add additional functionality to the bispecific
antibody can
be achieved, which include, for example, increasing serum half-life (e.g., see
U.S.
7,217,797) and/or increasing antibody-dependent cell-mediated cytotoxicity
(e.g.,
see U.S. 6,737,056). Introducing mutations into the constant regions of
antibodies is
known in the art. Additionally, part of the bispecific antibody can be made
recombinantly in cell culture and other part(s) of the molecule can be made by
those
techniques mentioned above.
[00238] Several techniques for the producing antibodies have been described.
For example, in various embodiments chimeric antibodies are produced in mice
as
described herein. Antibodies can be isolated directly from B cells of an
immunized
mouse (e.g., see U.S. 2007/0280945AI) and/or the B cells of the immunized
mouse
can be used to make hybridomas (Kohler and Milstein, 1975, Nature 256:495-
497).
DNA encoding the antibodies (human heavy and/or light chains) from mice as
described herein is readily isolated and sequenced using conventional
techniques.
Hybridoma and/or B cells of derived from mice as described herein serve as a
preferred source of such DNA. Once isolated, the DNA may be placed into
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expression vectors, which are then transfected into host cells that do not
otherwise
produce immunoglobulin protein, to obtain the synthesis of monoclonal
antibodies
in the recombinant host cells. The DNA also may be modified, for example, by
substituting the coding sequence for human heavy and light chain constant
domains
in place of the murine sequences.
[00239] In various embodiments, following isolation of the DNA and selection
of
the first and second nucleic acid sequences that encode the first and second
human
heavy chain variable domains having the desired specificities/affinities, and
a third
nucleic acid sequence that encodes a human light chain domain (a germline
rearranged sequence or a light chain sequence isolated from a mouse as
described
herein), the three nucleic acids sequences encoding the molecules are
expressed to
form the bispecific antibody using recombinant techniques which are widely
available in the art. Often, the expression system of choice will involve a
mammalian cell expression vector and host so that the bispecific antibody is
appropriately glycosylated (e.g., in the case of bispecific antibodies
comprising
antibody domains which are glycosylated). However, the molecules can also be
produced in the prokaryotic expression systems. Normally, the host cell will
be
transformed with DNA encoding both the first human heavy chain variable
domain,
the second human heavy chain variable domain, the human light chain domain on
a
single vector or independent vectors. However, it is possible to express the
first
human heavy chain variable domain, second human heavy chain variable domain,
and human light chain domain (the bispecific antibody components) in
independent
expression systems and couple the expressed polypeptides in vitro. In various
embodiments, the human light chain domain comprises a germline sequence. In
various embodiments, the human light chain domain comprises no more than one,
no more than two, no more than three, no more than four, or no more than five
somatic hypermutations with the light chain variable sequence of the light
chain
domain.
[00240] In various embodiments, the nucleic acid(s) (e.g., cDNA or genomic
DNA) encoding the two heavy chains and single human light chain is inserted
into a
replicable vector for further cloning (amplification of the DNA) and/or for
expression. Many vectors are available, and generally include, but are not
limited
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to, one or more of the following: a signal sequence, an origin of replication,
one or
more marker genes, an enhancer element, a promoter, and a transcription
termination sequence. Each component may be selected individually or based on
a
host cell choice or other criteria determined experimentally. Several examples
of
each component are known in the art.
[00241] Expression and cloning vectors usually contain a promoter that is
recognized by the host organism and is operably linked to the nucleic acid
sequences
that encode each or all the components of the bispecific antibody. A large
number
of promoters recognized by a variety of potential host cells are well known.
These
promoters are operably linked to bispecific antibody-encoding DNA by removing
the promoter from the source DNA by restriction enzyme digestion and inserting
the
isolated promoter sequence into the vector.
[00242] Expression vectors used in eukaryotic host cells (yeast, fungi,
insect,
plant, animal, human, or nucleated cells from other multicellular organisms)
may
also contain sequences necessary for the termination of transcription and for
stabilizing the mRNA. Such sequences are commonly available from the 5' and,
occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs.
These
regions contain nucleotide segments transcribed as polyadenylated fragments in
the
untranslated portion of the mRNA encoding the bispecific antibody components.
Suitable expression vectors for various embodiments include those that provide
for
the transient expression in mammalian cells of DNA encoding the bispecific
antibody. In general, transient expression involves the use of an expression
vector
that is able to replicate efficiently in a host cell, such that the host cell
accumulates
many copies of the expression vector and, in turn, synthesizes high levels of
a
desired polypeptide encoded by the expression vector. Transient expression
systems, comprising a suitable expression vector and a host cell, allow for
the
convenient positive identification of polypeptides encoded by cloned DNAs, as
well
as for the rapid screening of bispecific antibodies having desired binding
specificities/affinities or the desired gel migration characteristics relative
to the
parental antibodies having homodimers of the first or second human heavy chain
variable domains.
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[00243] In various embodiments, once the DNA encoding the components of the
bispecific antibody are assembled into the desired vector(s) as described
above, they
are introduced into a suitable host cell for expression and recovery.
Transfecting
host cells can be accomplished using standard techniques known in the art
appropriate to the host cell selected (e.g., electroporation, nuclear
microinjection,
bacterial protoplast fusion with intact cells, or polycations, e.g.,
polybrene,
polyornithine, etc.).
[00244] A host cell is chosen, in various embodiments, that best suits the
expression vector containing the components and allows for the most efficient
and
favorable production of the bispecific antibody species. Exemplary host cells
for
expression 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, or cell fusions such as, for example, hybridomas or quadromas. In
various embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse
cell.
In various embodiments, the cell is eukaryotic cell selected from 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, Co1o205, HB 8065, HL-60, (e.g., BHX-21), 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 various embodiments, the cell
comprises
one or more viral genes, e.g. a retinal cell that expresses a viral gene
(e.g., a
PER.C6TM cell).
[00245] Mammalian host cells used to produce the bispecific antibody may be
cultured in a variety of media. Commercially available media such as Ham's F10
(Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and
Dulbeccols Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing
the host cells. Media may be supplemented as necessary with hormones and/or
other growth factors (such as insulin, transferrin, or epidermal growth
factor), salts
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(such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as
HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as
GENTAMYCINTm), trace elements (defined as inorganic compounds usually
present at final concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other supplements may also be included at
appropriate concentrations as known to those skilled in the art. The culture
conditions, such as temperature, pH, and the like, are, in various
embodiments, those
previously used with the host cell selected for expression, and will be
apparent to
those skilled in the art.
[00246] The bispecific antibody is in various embodiments recovered from the
culture medium as a secreted polypeptide, although it also may be recovered
from
host cell lysate when directly produced without a secretory signal. If the
bispecific
antibody is membrane-bound, it can be released from the membrane using a
suitable
detergent solution (e.g., Triton-X 100). Preferably, the bispecific antibodies
described herein involves the use of a first immunoglobulin CH3 domain and a
second immunoglobulin CH3 domain, wherein the first and second immunoglobulin
CH3 domains differ from one another by at least one amino acid, and wherein at
least one amino acid difference reduces binding of the bispecific antibody to
Protein
A as compared to a bi-specific antibody lacking the amino acid difference (see
U.S.
2010/0331527A1; herein incorporated by reference). In one embodiment, the
first
immunoglobulin CH3 domain binds Protein A and the second immunoglobulin CH3
domain contains a mutation that reduces or abolishes Protein A binding such as
an
H95R modification (by IMGT exon numbering; H435R by EU numbering). The
second CH3 may further comprise a Y96F modification (by IMGT; Y436F by EU).
Further modifications that may be found within the second CH3 include: D16E,
Ll 8M, N44S, K52N, V57M, and V82I (by IMGT; D356E, 1,358M, N384S, K392N,
V397M, and V422I by EU) in the case of IgG1 antibodies; N44S, K52N, and V82I
(IMGT; N384S, K392N, and V422I by EU) in the case of IgG2 antibodies; and
Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S,
K392N, V397M, R409K, E419Q, and V422I by EU) in the case of IgG4 antibodies.
Variations on the bi-specific antibody format described above are contemplated
within the scope of the present invention.
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[00247] Because of the dual nature of bispecific antibodies (i.e., may be
specific
for different epitopes of one polypeptide or may contain antigen-binding
domains
specific for more than one target polypeptide, see FIG. 7B; see also, e.g.,
Tun et al.,
1991, .1 Innnunol. 147:60-69; Kufer et aL, 2004, Trends Biotechnol. 22:238-
244),
they offer many useful advantages for therapeutic application. For example,
the
bispecific antibodies can be used for redirected cytotoxicity (e.g., to kill
tumor
cells), as a vaccine adjuvant, for delivering thrombolytic agents to clots,
for
converting enzyme activated prodrugs at a target site (e.g., a tumor), for
treating
infectious diseases, targeting immune complexes to cell surface receptors, or
for
delivering immunotoxins to tumor cells.
[00248] The bispecific antibodies described herein can also be used in several
therapeutic and non-therapeutic and/or diagnostic assay methods, such as,
enzyme
immunoassays, two-site immunoassays, in vitro or in vivo immunodiagnosis of
various diseases (e.g., cancer), competitive binding assays, direct and
indirect
sandwich assays, and immunoprecipitation assays. Other uses for the bispecific
antibodies will be apparent to those skilled in the art.
[00249] The following examples are provided so as to describe to those of
ordinary skill in the art 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. Efforts have been made to ensure accuracy with respect to
numbers
used (e.g., amounts, temperature, etc.) but some experimental errors and
deviations
should be accounted for. Unless indicated otherwise, parts are parts by
weight,
molecular weight is average molecular weight, temperature is indicated in
Celsius,
and pressure is at or near atmospheric.
EXAMPLES
[00250] The following examples are provided so as to describe to those of
ordinary skill in the art 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. Efforts have been made to ensure accuracy with respect to
numbers
used (e.g., amounts, temperature, etc.) but some experimental errors and
deviations
should be accounted for. Unless indicated otherwise, temperature is indicated
in
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14257
Celsius, pressure is at or near atmospheric, parts are by parts by weight, and
molecular weight is average molecular weight.
Example 1
Identification of Human VH Regions That Associate with Selected Human VL
Regions
[00251] An in vitro expression system was constructed to determine if a single
rearranged human germline light chain could be co-expressed with human heavy
chains from antigen specific human antibodies.
[00252] Methods for generating human antibodies in genetically modified mice
are known (see e.g., US 6,596,541, Regeneron Pharmaceuticals,
VELOCIMMUNE ). The VELOCIMMUNE technology involves generation of a
genetically modified mouse having a genome comprising human heavy and light
chain variable regions operably linked to endogenous mouse constant region
loci
such that the mouse produces an antibody comprising a human variable region
and a
mouse constant region in response to antigenic stimulation. The DNA encoding
the
variable regions of the heavy and light chains of the antibodies produced from
a
VELOCIMMUNE mouse are fully human. Initially, high affinity chimeric
antibodies are isolated having a human variable region and a mouse constant
region.
As described below, the antibodies 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 a fully
human
antibody containing a non-IgM isotype, for example, wild type or modified
IgGl,
IgG2, IgG3 or IgG4. While the constant region selected may vary according to
specific use, high affinity antigen-binding and target specificity
characteristics reside
in the variable region.
[00253] A VELOC EC mouse was immunized with a growth factor that
promotes angiogenesis (Antigen C) and antigen-specific human antibodies were
isolated and sequenced for V gene usage using standard techniques recognized
in the
art. Selected antibodies were cloned onto human heavy and light chain constant
regions and 69 heavy chains were selected for pairing with one of three human
light
chains: (1) the cognate i< light chain linked to a human lc constant region,
(2) a
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rearranged human germline Vic1-39.1x5 linked to a human K constant region, or
(3) a
rearranged human gerrnline Vic3-20Jic1 linked to a human x constant region.
Each
heavy chain and light chain pair were co-transfected in CHO-Kl cells using
standard
techniques. Presence of antibody in the supernatant was detected by anti-human
lgG
in an ELBA assay. Antibody titer (ng/ml) was determined for each heavy
chain/light chain pair and titers with the different rearranged germline light
chains
were compared to the titers obtained with the parental antibody molecule (i.
e., heavy
chain paired with cognate light chain) and percent of native titer was
calculated
(Table 1). VH: Heavy chain variable gene. ND: no expression detected under
current experimental conditions.
Table 1
Antibody Titer (ng/mL) Percent of Native Titer
VH _____________________________________
Cognate LC VK I -39.10 Vic3-20Jic1 Vic1-39,1K5 Vx3-20JK1
3-15 63 23 11 36.2 17.5 _
1-2 103 53 ND 51.1 -
-
3-23 83 60 23 72.0 27.5
_
3-33 15 77 ND 499.4 -
_
4-31 22 69 17 _ 309.4 76.7
3-7 53 35 28_ 65.2 53.1
- 22 32 19 148.8 89.3
,_
1-24 3 13 ND 455.2 -
_
3-33 1 47 ND- 5266.7 -
3-33 58 37 ND 63.1 -
- 110 67 18 60.6 16.5
3-23 127 123 21 _ 96.5 16.3
3-33 28 16 2 57.7 7.1
3-23 32 50 38 157.1 119.4 ,
_
- 18 45 18 254.3 101.7
3-9 1 30 23 2508.3 1900.0
_
3-11 12 26 6 225.9 48.3
1-8 16 ND 13 - 81.8
3-33 54 81 10 150.7 19.1
- 34 9 ND 25.9 -
3-20 7 14 54 203.0 809.0
_
3-33 19 38 ND- 200.5
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, 3-11 48 ND 203 - 423.6
- 11 23 8 , 212.7 74.5
3-33 168 138 182 82.0 108.2
_
3-20 117 67 100 57.5 86.1
3-23 86 ., 61 132 70.7 154.1
3-33 20 12 33 60.9 165.3
4-31 69 92 52 133.8 75.0
_
3-23 87 78 62 89.5 71.2
_
1-2 31 82 51 263.0 164.6
3-23 ' 53 93, 151 175.4 285.4
- 11 8 . 17 75.7 151.4
t
3-33 114 36 27 31.6 23.4
_
3-15 73 _ 39 44 53.7 59.6
3-33 1 34 16 5600.0 2683.3
3-9 58 112 57 192.9 97.6
3-33 67 20 105 30.1 157.0
3-33 34 , 21 24 62.7 70.4 _
_
3-20 10 , 49 91 478.4 888.2
3-33 66 32 25 48.6 38.2
3-23 17 59 56 342.7 329.8 _
,
- 58 108 19 184.4 32.9
- 68 54 20 79.4 29.9
, 3-33 42 35 32 83.3 75.4
7
- 29 19 13 67.1 43.9
3-9 24 34 29 137.3 118.4 _
3-30/33 17 33 7 195.2 43.1 ,
_
3-7 25 70 74 284.6 301.6
-q
3-33 87 127 ND 145.1 -
6-1 28 56 ND 201.8 -
3-33 56 39 20 69.9 36.1
3-33 10 53 1 520.6 6.9
3-33 20 67 10 337.2 52.3
_
3-33 11 36 18 316.8 158.4 _
3-23 12 42 32 356.8 272.9
-
3-33 66 95 15 143.6 22.5
3-15 55 68 ND 123.1 -
_ -
- 32 68 3 210.9 10.6
1-8 28 48 ND 170.9 -
3-33 124 192 21 154.3 17.0
3-33 0 113 ND 56550.0 -
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r-
3-33 10 157 1 1505.8 12.5
3-33 6 86 15 1385.5 243.5
3-23 70 115 22 163.5 31.0
3-7 71 117 21 164.6 29.6
3-33 82 100 47 122.7 57.1
3-7 124 161 41 130.0 33.5
[00254] In a similar experiment, VELOCIMMUNE mice were immunized with
several different antigens and selected heavy chains of antigen specific human
antibodies were tested for their ability to pair with different rearranged
human
germline light chains (as described above). The antigens used in this
experiment
included an enzyme involved in cholesterol homeostasis (Antigen A), a serum
hormone involved in regulating glucose homeostasis (Antigen B), a growth
factor
that promotes angiogenesis (Antigen C) and a cell-surface receptor (Antigen
D).
Antigen specific antibodies were isolated from mice of each immunization group
and the heavy chain and light chain variable regions were cloned and
sequenced.
From the sequence of the heavy and light chains, V gene usage was determined
and
selected heavy chains were paired with either their cognate light chain or a
rearranged human germline Vx1-39JK5 region. Each heavy/light chain pair was co-
transfected in CHO-K1 cells and the presence of antibody in the supernatant
was
detected by anti-human IgG in an ELISA assay. Antibody titer (ttg/m1) was
determined for each heavy chain/light chain pairing and titers with the
different
rearranged human germline light chains were compared to the titers obtained
with
the parental antibody molecule (i.e., heavy chain paired with cognate light
chain)
and percent of native titer was calculated (Table 2). VII: Heavy chain
variable gene.
Vic: light chain variable gene. ND: no expression detected under current
experimental conditions.
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Table 2
Titer (p,g/m1)
Antigen Antibody VH VIC VT4 + Percent of
VH Alone VH + VK - Native Titer
Vic1-39Jic5
320 1-18 2-30 0.3 3.1 2.0 66
321 2-5 2-28 0.4 0.4 1.9 448
334 2-5 2-28 0.4 2.7 2.0 73
A 313 3-13 3-15 0.5 0.7 4.5 670
316 3-23 4-1 0.3 0.2 4.1 2174 '
315 3-30 4-1 0.3 0.2 3.2 1327
318 4-59 1-17 0.3 4.6 4.0 86
257 3-13 1-5 0.4 3.1 3.2 104
283 3-13 1-5 0.4 5.4 3.7 69
637 3-13 1-5 _ 0.4 4.3 3.0 70
638 , 3-13 1-5 0.4 4.1 3.3 82
B 624 3-23 1-17 0.3 5.0 3.9 79
284 3-30 1-17 0.3 4.6 3.4 75
653 3-33 1-17 0.3 4.3 0.3 7
268 4-34 1-27 0.3 5.5 3.8 69
633 4-34 1-27 0.6 6.9 3.0 44
730 3-7 1-5 0.3 1.1 2.8 249
728 3-7 1-5 0.3 2.0 3.2 157
691 3-9 3-20 0.3 2.8 3.1 109
749 3-33 3-15 0.3 3.8 2.3 62
750 3-33 1-16 0.3 3.0 2.8 92
724 3-33 1-17 0.3 2.3 3.4 151
706 3-33 1-16 0.3 3.6 3.0 84 ..
C 744 1-18 1-12 0.4 5.1 3.0 59
696 3-11 1-16 0.4 3.0 2.9 97
685 3-13 3-20 0.3 0.5 3.4 734
732 1 3-15 1-17 0.3 4.5 3.2 72
694 3-15 1-5 0.4 5.2 2.9 55
743 3-23 1-12 0.3 3.2 0.3 10
742 3-23 2-28 0.4 _ 4.2 3.1 74
693 3-23 1-12 0.5 4.2 4.0 94
136 3-23 2-28 0.4 5.0 2.7 55
D 155 3-30 1-16 0.4 1.0 2.2 221
163 3-30 1-16 0.3 , 0.6 3.0 506
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171 3-30 1-16 0.3 1.0 2.8 295
145 3-43 1-5 0.4 4.4 2.9 65
49 3-48 3-11 0.3 1.7 2.6 155
51 3-48 1-39 0.1 1.9 0.1 4
159 3-7 6-21 0.4 3.9 3.6 92
169 3-7 6-21 0.3 1.3 3.1 235
134 3-9 1-5 0.4 5.0 2.9 58
141 4-31 1-33 2.4 4.2 2.6 63
142 4-31 1-33 0.4 4.2 2.8 67
[00255] The results obtained from these experiments demonstrate that
somatically
mutated, high affinity heavy chains from different gene families are able to
pair with
rearranged human germline Vx1-39Jx5 and Vi(3-20.1x1 regions and be secreted
from the cell as a normal antibody molecule. As shown in Table 1, antibody
titer
was increased for about 61% (42 of 69) heavy chains when paired with the
rearranged human Vic1-39.ho light chain and about 29% (20 of 69) heavy chains
when paired with the rearranged human Vx3-20k1 light chain as compared to the
cognate light chain of the parental antibody. For about 20% (14 of 69) of the
heavy
chains, both rearranged human germline light chains conferred an increase in
expression as compared to the cognate light chain of the parental antibody. As
shown in Table 2, the rearranged human germline Vic1-39.10 region conferred an
increase in expression of several heavy chains specific for a range of
different
classes of antigens as compared to the cognate light chain for the parental
antibodies. Antibody titer was increased by more than two-fold for about 35%
(15/43) of the heavy chains as compared to the cognate light chain of the
parental
antibodies. For two heavy chains (315 and 316), the increase was greater than
ten-
fold as compared to the parental antibody. Within all the heavy chains that
showed
increase expression relative to the cognate light chain of the parental
antibody,
family three (VH3) heavy chains are over represented in comparison to other
heavy
chain variable region gene families. This demonstrates a favorable
relationship of
human VH3 heavy chains to pair with rearranged human germline Vx1-39R5 and
Vic3-20.1x1 light chains.
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Example 2
Generation of a Rearranged Human Germline Light Chain Locus
[00256] Various rearranged human germline light chain targeting vectors were
made using VELOCIGENE technology (see, e.g., US Pat. No. 6,586,251 and
Valenzuela et al. (2003) High-throughput engineering of the mouse genome
coupled
with high-resolution expression analysis, Nature Biotech. 21(6): 652-659) to
modify
mouse genomic Bacterial Artificial Chromosome (BAC) clones 302g12 and 254m04
(Invitrogen). Using these two BAC clones, genomic constructs were engineered
to
contain a single rearranged human germline light chain region and inserted
into an
endogenous x light chain locus that was previously modified to delete the
endogenous lc variable and joining gene segments.
[00257] Construction of Rearranged Human Germline Light Chain
Targeting Vectors. Three different rearranged human germline light chain
regions
were made using standard molecular biology techniques recognized in the art.
The
human variable gene segments used for constructing these three regions
included
rearranged human Vicl -39Jx5 sequence, a rearranged human Vic3-20Jx1 sequence
and a rearranged human VpreBA5 sequence.
[00258] A DNA segment containing exon 1 (encoding the leader peptide) and
intron 1 of the mouse Vx3-7 gene was made by de novo DNA synthesis (Integrated
DNA Technologies). Part of the 5' untranslated region up to a naturally
occurring
BlpI restriction enzyme site was included. Exons of human Vic1-39 and Vic3-20
genes were PCR amplified from human genomic BAC libraries. The forward
primers had a 5' extension containing the splice acceptor site of intron 1 of
the
mouse Vic3-7 gene. The reverse primer used for PCR of the human W1-39
sequence included an extension encoding human .1x5, whereas the reverse primer
used for PCR of the human W3-20 sequence included an extension encoding
human Jx1. The human VpreBA5 sequence was made by de novo DNA synthesis
(Integrated DNA Technologies). A portion of the human Iic-CK intron including
the
splice donor site was PCR amplified from plasmid pBS-296-HA18-PISceI. The
forward PCR primer included an extension encoding part of either a human JK5,
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Jxl, or A5 sequence. The reverse primer included a PI-SceI site, which was
previously engineered into the intron.
[00259] The mouse Vx3-7 exonl/intron I, human variable light chain exons, and
human Jx-Cx intron fragments were sewn together by overlap extension PCR,
digested with BlpI and PI-SceI, and ligated into plasmid pBS-296-HA18-PISceI,
which contained the promoter from the human Vic3-15 variable gene segment. A
foxed hygromycin cassette within plasmid pBS-296-HA18-PISceI was replaced with
a FRTed hygromycin cassette flanked by NotI and AscI sites. The NotI/PI-Scel
fragment of this plasmid was ligated into modified mouse BAC 254m04, which
contained part of the mouse Jic-Cic intron, the mouse CI( exon, and about 75
kb of
genomic sequence downstream of the mouse x locus, which provided a 3' homology
arm for homologous recombination in mouse ES cells. The NotI/Ascl fragment of
this BAC was then ligatal into modified mouse BAC 302g12, which contained a
FRTed neomycin cassette and about 23 kb of genomic sequence upstream of the
endogenous lc locus for homologous recombination in mouse ES cells.
[00260] Rearranged Human Germline Vic1-39,k5 Targeting Vector (FIG. 1).
Restriction enzyme sites were introduced at the 5' and 3' ends of an
engineered light
chain insert for cloning into a targeting vector: an AscI site at the 5' end
and a PI-
Seel site at the 3' end. Within the 5' AscI site and the 3' PI-SceI site the
targeting
construct from 5' to 3' included a 5' homology arm containing sequence 5' to
the
endogenous mouse ic light chain locus obtained from mouse BAC clone 302g12, a
FRTed neomycin resistance gene, an genomic sequence including the human Vic3-
15 promoter, a leader sequence of the mouse Vx3-7 variable gene segment, a
intron
sequence of the mouse Vx3-7 variable gene segment, an open reading frame of a
rearranged human germline Vic1-39.10 region, a genomic sequence containing a
portion of the human Jx-Cfc intron, and a 3' homology arm containing sequence
3'
of the endogenous mouse ha gene segment obtained from mouse BAC clone
254m04 (Figure 1, middle). Genes and/or sequences upstream of the endogenous
mouse lc light chain locus and downstream of the most 3' Jic gene segment
(e.g., the
endogenous 3' enhancer) were unmodified by the targeting construct (see Figure
1).
The sequence of the engineered human Vic1-39.1x5 locus is shown in SEQ ID NO:
1.
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[00261] Targeted insertion of the rearranged human germline Vic1-39JK5 region
into BAC DNA was confirmed by polymerase chain reaction (PCR) using primers
located at sequences within the rearranged human germline light chain region.
Briefly, the intron sequence 3' to the mouse Vic3-7 leader sequence was
confirmed
with primers ULC-m1F (AGGTGAGGGT ACAGATAAGT GTTATGAG; SEQ ID
NO: 2) and ULC-m1R (TGACAAATGC CCTAATTATA GTGATCA; SEQ ID
NO: 3). The open reading frame of the rearranged human germline Vx1-39Jx5
region was confirmed with primers 1633-h2F (GGGCAAGTCA GAGCATTAGC
A; SEQ ID NO: 4) and 1633-h2R (TGCAAACTGG ATGCAGCATA G; SEQ ID
NO: 5). The neomycin cassette was confirmed with primers neoF
(GGTGGAGAGG CTA n CGGC; SEQ ID NO: 6) and neoR (GAACACGGCG
GCATCAG; SEQ ID NO: 7). Targeted BAC DNA was then used to electroporate
mouse ES cells to created modified ES cells for generating chimeric mice that
express a rearranged human germline Vic1-39k5 region.
[00262] Positive ES cell clones were confirmed by TAQMANTm screening and
karyotyping using probes specific for the engineered Vic.1-39k5 light chain
region
inserted into the endogenous locus. Briefly, probe neoP (TGGGCACAAC
AGACAATCGG CTG; SEQ ID NO:8) which binds within the neomycin marker
gene, probe ULC-m1P (CCATTATGAT GCTCCATGCC TCTCTGTTC; SEQ ID
NO: 9) which binds within the intron sequence 3' to the mouse Vx3-7 leader
sequence, and probe 1633h2P (ATCAGCAGAA ACCAGGGAAA GCCCCT; SEQ
ID NO: 10) which binds within the rearranged human germline Vx1-39k5 open
reading frame. Positive ES cell clones were then used to implant female mice
to
give rise to a litter of pups expressing the germline Vic1-39.10 light chain
region.
[00263] Alternatively, ES cells bearing the rearranged human germ line W1-
39.1x5 light chain region are transfected with a construct that expresses FLP
in order
to remove the FRTed neomycin cassette introduced by the targeting construct.
Optionally, the neomycin cassette is removed by breeding to mice that express
FLP
recombinase (e.g., US 6,774,279). Optionally, the neomycin cassette is
retained in
the mice.
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[00264] Rearranged Human Germline Vic3-20.fic1 Targeting Vector (FIG. 2).
In a similar fashion, an engineered light chain locus expressing a rearranged
human
germline W3-20J-K1 region was made using a targeting construct including, from
5'
to 3', a 5' homology arm containing sequence 5' to the endogenous mouse K
light
chain locus obtained from mouse BAC clone 302g12, a FRTed neomycin resistance
gene, a genomic sequence including the human VK.3-15 promoter, a leader
sequence
of the mouse VK3-7 variable gene segment, an intron sequence of the mouse VK3-
7
variable gene segment, an open reading frame of a rearranged human gerrnline
VK3-
20JKI region, a genomic sequence containing a portion of the human JK-CK
intron,
and a 3' homology arm containing sequence 3' of the endogenous mouse JK5 gene
segment obtained from mouse BAC clone 254m04 (Figure 2, middle). The
sequence of the engineered human V1c3-20JK1 locus is shown in SEQ ID NO: 11.
[00265] Targeted insertion of the rearranged human germline VK3-20JK1 region
into BAC DNA was confirmed by polymerase chain reaction (PCR) using primers
located at sequences within the rearranged human germline VK3-20JK1 light
chain
region. Briefly, the intron sequence 3' to the mouse Vx.3-7 leader sequence
was
confirmed with primers ULC-m1F (SEQ ID NO: 2) and ULC-m IR (SEQ ID NO: 3).
The open reading frame of the rearranged human germline VK3-20JK1 region was
confirmed with primers 1635-h2F (TCCAGGCACC CTGTCIT1G; SEQ ID NO:
12) and 1635-h2R (AAGTAGCTGC TGCTAACACT CTGACT; SEQ ID NO: 13).
The neomycin cassette was confirmed with primers neoF (SEQ ID NO: 6) and neoR
(SEQ ID NO: 7). Targeted BAC DNA was then used to electroporate mouse ES
cells to created modified ES cells for generating chimeric mice that express
the
rearranged human germline Vx3-20JK1 light chain.
[00266] Positive ES cell clones were confirmed by TAQMANTm screening and
karyotyping using probes specific for the engineered VK3-20JK1 light chain
region
inserted into the endogenous K light chain locus. Briefly, probe neoP (SEQ ID
NO:8) which binds within the neomycin marker gene, probe ULC-m1P (SEQ ID
NO: 9) which binds within the mouse W3-7 leader sequence, and probe 1635h2P
(AAAGAGCCAC CCTCTCCTGC AGGG; SEQ ID NO: 14) which binds within the
human VK3-20JK1 open reading frame. Positive ES cell clones were then used to
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implant female mice. A litter of pups expressing the human germline Vic3-
20.1k1
light chain region.
[00267] Alternatively, ES cells bearing human germline Vic3-20JK1 light chain
region can be transfected with a construct that expresses FLP in order to
remove the
FRTed neomycin cassette introduced by the targeting construct. Optionally, the
neomycin cassette may be removed by breeding to mice that express FLP
recombinase (e.g., US 6,774,279). Optionally, the neomycin cassette is
retained in
the mice.
[00268] Rearranged Human Germline VpreBn5 Targeting Vector (FIG. 3).
In a similar fashion, an engineered light chain locus expressing a rearranged
human
germline VpreaR5 region was made using a targeting construct including, from
5'
to 3', a 5' homology arm containing sequence 5' to the endogenous mouse K
light
chain locus obtained from mouse BAC clone 302g12, a FRTed neomycin resistance
gene, an genomic sequence including the human Vx3-15 promoter, a leader
sequence of the mouse Vic3-7 variable gene segment, an intron sequence of the
mouse Vic3-7 variable gene segment, an open reading frame of a rearranged
human
germline Vprella5 region, a genomic sequence containing a portion of the human
intron, and a 3' homology arm containing sequence 3' of the endogenous
mouse Jx5 gene segment obtained from mouse BAC clone 254m04 (Figure 3,
middle). The sequence of the engineered human VpreBJX,5 locus is shown in SEQ
ID NO: 15.
[00269] Targeted insertion of the rearranged human germline VpreBA5 region
into BAC DNA was confirmed by polymerase chain reaction (PCR) using primers
located at sequences within the rearranged human germline VpreBA5 region light
chain region. Briefly, the intron sequence 3' to the mouse Vic3-7 leader
sequence
was confirmed with primers ULC-m1F (SEQ ID NO: 2) and ULC-m1R (SEQ ID
NO: 3). The open reading frame of the rearranged human germline VpreBA5
region was confirmed with primers 1616-h1F (TGTCCTCGGC CCTTGGA; SEQ
ID NO: 16) and 1616-h1R (CCGATGTCAT GGTCGTTCCT; SEQ ID NO: 17).
The neomycin cassette was confirmed with primers neoF (SEQ ID NO: 6) and neoR
(SEQ ID NO: 7). Targeted BAC DNA was then used to electroporate mouse ES
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cells to created modified ES cells for generating chimeric mice that express
the
rearranged human germline VpreBA5 light chain.
[00270] Positive ES cell clones are confirmed by TAQMANTm screening and
karyotyping using probes specific for the engineered Vprella5 light chain
region
inserted into the endogenous lc light chain locus. Briefly, probe neoP (SEQ ID
NO:8), which binds within the neomycin marker gene, probe ULC-m1P (SEQ ID
NO: 9), which binds within the mouse IgV1c3-7 leader sequence, and probe 1616h
1P
(ACAATCCGCC TCACCTGCAC CCT; SEQ ID NO: 18) which binds within the
human VpreB.11.5 open reading frame. Positive ES cell clones are then used to
implant female mice to give rise to a litter of pups expressing a germline
light chain
region.
[00271] Alternatively, ES cells bearing the rearranged human germline VpreBJX5
light chain region are transfected with a construct that expresses FLP in
order to
remove the FRTed neomycin cassette introduced by the targeting construct.
Optionally, the neomycin cassette is removed by breeding to mice that express
FLP
recombinase (e.g., US 6,774,279). Optionally, the neomycin cassette is
retained in
the mice.
Example 3
Generation of Mice expressing a single rearranged human light chain
[00272] Targeted ES cells described above were used as donor ES cells and
introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE method
(see, e.g., US Pat. No. 7,294,754 and Poueymirou et al. (2007) FO generation
mice
that are essentially fully derived from the donor gene-targeted ES cells
allowing
immediate phenotypic analyses Nature Biotech. 25(1): 91-99. VELOCIMICE
independently bearing an engineered human germline Vx1-391K5 light chain
region,
a VIc3-20J-K1 light chain region or a Vprel3J2L5 light chain region are
identified by
genotyping using a modification of allele assay (Valenzuela et al., supra)
that
detects the presence of the unique rearranged human germline light chain
region.
[00273] Pups are genotyped and a pup heterozygous or homozygous for the
unique rearranged human germline light chain region are selected for
characterizing
expression of the rearranged human germline light chain region.
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[00274) Flow Cytametry. Expression of the rearranged human light chain region
in the normal antibody repertoire of common light chain mice was validated by
analysis of immunoglobulin lc and X expression in splenocytes and peripheral
blood
of common light chain mice. Cell suspensions from harvested spleens and
peripheral blood of wild type (n=5), Vx1-39.Ix5 common light chain
heterozygote
(n=3), Vx1-39.1x5 common light chain homozygote (n=3), Vx3-20.1x1 common
light chain heterozygote (n=2), and Vx3-20JK1 common light chain homozygote
(n=2) mice were made using standard methods and stained with CD19+, IgX+ and
Tgx+ using fluoreseently labeled antibodies (BD Pharmigen).
(002751 Briefly, 1x106 cells were incubated with anti-mouse CD16/CD32 (clone
2.4G2, BD Pharmigen) on ice for 10 minutes, followed by labeling with the
following antibody cocktail for 30 minutes on ice: APC conjugated anti-mouse
CD19 (clone 1D3, BD Pharmigen), PerCP-Cy5.5 conjugated anti-mouse CD3 (clone
17A2, BioLegend), FITC conjugated anti-mouse Ig x (clone 187.1, BD Pharmigen),
PE conjugated anti-mouse Igk (clone RML-42, BioLegend). Following staining,
cells were washed and fixed in 2% formaldehyde. Data acquisition was performed
on an LSRII flow cytometer and analyzed with FlowJo. Gating: total B cells
(CD19+CD.3"), Igx+ B cells (Igic +IgX-CD19+CD3-), IgX+ B cells (Igx-
Igk+CD19+CD3-). Data gathered from blood and splenocyte samples demonstrated
similar results. Table 3 sets forth the percent positive CD19+ B cells from
peripheral
blood of one representative mouse from each group that are IgX+, Ig le, or
Igk+Igx+.
Percent of CD19+ B cells in peripheral blood from wild type (WT) and mice
homozygous for either the Vx1-39k5 or Vx3-20.1x1 common light chain are shown
in FIG. 4.
Table 3
CD19 B cells
Mouse
Igk+ Igx+ Igk+Igx'
Wild type 4.8 93 0.53
Vx1-39.1x5 1.4 93 2.6
Vx3-20J1c1 4.2 88 6
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[00276] Common Light Chain Expression. Expression of each common light
chain (Vx1-39Jx5 and Vx3-20Jx1) was analyzed in heterozygous and homozygous
mice using a quantitative PCR assay (e.g. TAQMANTm).
[00277] Briefly, CD19 B cells were purified from the spleens of wild type,
mice
homozygous for a replacement of the mouse heavy chain and x light chain
variable
region loci with corresponding human heavy chain and lc light chain variable
region
loci (Hx), as well as mice homozygous and heterozygous for each rearranged
human
light chain region (Vx1-39Jx5 or Vx3-20Jx1) using mouse CD19 Microbeads
(Miltenyi Biotec) according to manufacturer's specifications. Total RNA was
purified from CD19+ B cells using RNeasy Mini kit (Qiagen) according to
manufacturer's specifications and genomic RNA was removed using an RNase-free
DNase on-column treatment (Qiagen). 200 ng mRNA was reverse-transcribed into
cDNA using the First Stand cDNA Synthesis kit (Invitrogen) and the resulting
cDNA was amplified with the Taqman Universal PCR Master Mix (Applied
Biosystems). All reactions were performed using the ABI 7900 Sequence
Detection
System (Applied Biosystems) using primers and Taqman MGB probes spanning (1)
the Vx-Jx. junction for both common light chains, (2) the Vic gene alone (i.e.
Vx1-39
and Vic3-20), and (3) the mouse CI( region. Table 4 sets forth the sequences
of the
primers and probes employed for this assay. Relative expression was normalized
to
expression of the mouse CK region. Results are shown in FIG. 5A, 5B and 5C.
Table 4
Region Primer/Probe Description (5'-3') SEQ ID
NOs:
(Sense) AGCAGTCTGC AACCTGAAGA ITT
19
Vx1-39k5 (Anti-sense) G1T1AATCTC CAGTCGTGTC
Junction CCTT
21
(Probe) CCTCCGATCA CCU C
(Sense) AAACCAGGGA AAGCCCCTAA 22
Vx1-39 (Anti-sense) ATGGGACCCC ACTTTGCA 23
(Probe) CTCCTGATCT ATGCTGCAT 24
(Sense) CAGCAGACTG GAGCCTGAAG A 25
Vx3-20k1
(Anti-sense) TGAIII CCAC C'TTGGTCCCT T 26
Junction
(Probe) TAGCTCACCT TGGACGTT 27
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(Sense) CTCCTCATCT ATGGTGCATC CA 28
Vx3-20 (Anti-sense) GACCCACTGC CACTGAACCT 29
(Probe) CCACTGGCAT CCC 30
(Sense) TGAGCAGCAC CCTCACGTT'
31
Mouse
(Anti-sense) GTGGCCTCAC AGGTATAGCT
Cic
GTT 32
33
(Probe) ACCAAGGACG AGTATGAA
[00278] Antigen Specific Common Light Chain Antibodies. Common light
chain mice bearing either a WI-39116 or Vic3-20J1c1 common light chain at the
endogenous mouse K light chain locus were immunized with P-galactosidase and
antibody titer was measured.
[00279] Briefly, fl-galactosidase (Sigma) was emulsified in titennax adjuvant
(Sigma), as per manufacturers directions. Wild type (n=7), Vic1-39J0 common
light chain homozygotes (n=2) and Vic3-203K1 common light chain homozygotes
(n=5) were immunized by subcutaneous injection with 100 ug 13-
galactosidase/Titermax. Mice were boosted by subcutaneous injection two times,
3
weeks apart, with 50 lig J3-galactosidase/Titermax. After the second boost,
blood
was collected from anaesthetized mice using a retro-orbital bleed into serum
separator tubes (BD Biosciences) as per manufacturer's directions. To measure
anti-
13-galactosidase IgM or IgG antibodies, EL1SA plates (Nunc) were coated with 1
pg/mL P-galactosidase overnight at 4 C. Excess antigen was washed off before
blocking with PBS with 1% BSA for one hour at room temperature. Serial
dilutions
of serum were added to the plates and incubated for one hour at room
temperature
before washing. Plates were then incubated with HRP conjugated anti-IgM
(Southern Biotech) or anti-IgG (Southern Biotech) for one hour at room
temperature. Following another wash, plates were developed with TMB substrate
(BD Biosciences). Reactions were stopped with 1N sulfuric acid and 0D450 was
read using a Victor X5 Plate Reader (Perkin Elmer). Data was analyzed with
GraphPad Prism and signal was calculated as the dilution of serum that is two
times
above background. Results are shown in FIG. 6A and 6B.
[00280] As shown in this Example, the ratio of KA, B cells in both the splenic
and
peripheral compartments of Vicl -39.10 and Vx3-20.1X1 common light chain mice
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demonstrated a near wild type pattern (Table 3 and FIG. 4). VpreB.125 common
light chain mice, however, demonstrated fewer peripheral B cells, of which
about 1-
2% express the engineered human light chain region (data not shown). The
expression levels of the Vic1-39R5 and Vic3-20Jx1 rearranged human light chain
regions from the endogenous lc light chain locus were elevated in comparison
to an
endogenous x light chain locus containing a complete replacement of mouse Vic
and
Ix gene segments with human Vic and Jic gene segments (FIG. 5A, 5B and 5C).
The
expression levels of the VpreBR.5 rearranged human light chain region
demonstrated similar high expression from the endogenous lc light chain locus
in
both heterozygous and homozygous mice (data not shown). This demonstrates that
in direct competition with the mouse k, ic, or both endogenous light chain
loci, a
single rearranged human VOL sequence can yield better than wild type level
expression from the endogenous ic light chain locus and give rise to normal
splenic
and blood B cell frequency. Further, the presence of an engineered ic light
chain
locus having either a human Vic1-39.1x5 or human Vx3-20Jx1 sequence was well
tolerated by the mice and appear to function in wild type fashion by
representing a
substantial portion of the light chain repertoire in the humoral component of
the
immune response (FIG 6A and 6B).
Example 4
Breeding of mice expressing a single rearranged human germline light chain
[00281] This Example describes several other genetically modified mouse
strains
that can be bred to any one of the common light chain mice described herein to
create multiple genetically modified mouse strains harboring multiple
genetically
modified immunoglobulin loci.
[00282] Endogenous IgX, Knockout (KO). To optimize the usage of the
engineered light chain locus, mice bearing one of the rearranged human
germline
light chain regions are bred to another mouse containing a deletion in the
endogenous k light chain locus. In this manner, the progeny obtained will
express,
as their only light chain, the rearranged human germline light chain region as
described in Example 2. Breeding is performed by standard techniques
recognized
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in the art and, alternatively, by a commercial breeder (e.g., The Jackson
Laboratory).
Mouse strains bearing an engineered light chain locus and a deletion of the
endogenous light chain locus are screened for presence of the unique light
chain
region and absence of endogenous mouse k light chains.
[00283] Humanized Endogenous Heavy Chain Locus. Mice bearing an
engineered human germline light chain locus are bred with mice that contain a
replacement of the endogenous mouse heavy chain variable gene locus with the
human heavy chain variable gene locus (see US 6,596,541; the VELOCIMMUNE
mouse, Regeneron Pharmaceuticals, Inc.). The VELOCIMMUNE mouse
comprises a genome comprising human heavy chain variable regions operably
linked to endogenous mouse constant region loci such that the mouse produces
antibodies comprising a human heavy chain variable region and a mouse heavy
chain constant region in response to antigenic stimulation. The DNA encoding
the
variable regions of the heavy chains of the antibodies is isolated and
operably linked
to DNA encoding the human heavy chain constant regions. The DNA is then
expressed in a cell capable of expressing the fully human heavy chain of the
antibody.
[00284] Mice bearing a replacement of the endogenous mouse VH locus with the
human VH locus and a single rearranged human germline VL region at the
endogenous lc light chain locus are obtained. Reverse chimeric antibodies
containing somatically mutated heavy chains (human VH and mouse CH) with a
single human light chain (human VL and mouse CL) are obtained upon
immunization
with an antigen of interest. VH and VL nucleotide sequences of B cells
expressing
the antibodies are identified and fully human antibodies are made by fusion
the VH
and VL nucleotide sequences to human CH and CL nucleotide sequences in a
suitable
expression system.
Example 5
Generation of Antibodies from Mice Expressing Human Heavy Chains and a
Rearranged Human Germline Light Chain Region
[00285] After breeding mice that contain the engineered human light chain
region
to various desired strains containing modifications and deletions of other
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endogenous Ig loci (as described in Example 4), selected mice can be immunized
with an antigen of interest.
[00286] Generally, a VELOCIMMUNE mouse containing one of the single
rearranged human gennline light chain regions is challenged with an antigen,
and
lymphatic cells (such as B-cells) are recovered from serum of the animals. The
lymphatic cells are 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 human heavy chain
variables and a rearranged human germline light chains which are specific to
the
antigen used for immunization. DNA encoding the variable regions of the heavy
chains and the light chain are isolated and linked to desirable isotypic
constant
regions of the heavy chain and light chain. Due to the presence of the
endogenous
mouse sequences and any additional cis-acting elements present in the
endogenous
locus, the single light chain of each antibody may be somatically mutated.
This adds
additional diversity to the antigen-specific repertoire comprising a single
light chain
and diverse heavy chain sequences. The resulting cloned antibody sequences are
subsequently expressed in a cell, such as a CHO cell. Alternatively, DNA
encoding
the antigen-specific chimeric antibodies or the variable domains of the light
and
heavy chains are identified directly from antigen-specific lymphocytes.
[00287] Initially, high affinity chimeric antibodies are isolated having a
human
variable region and a mouse constant region. As described above, the
antibodies 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 the fully human antibody containing a
somatically mutated human heavy chain and a single light chain derived from a
rearranged human gennline light chain region of the invention. Suitable human
constant regions include, for example wild type or modified IgG I or IgG4.
[00288] Separate cohorts of VELOCIMMUNE mice containing a replacement
of the endogenous mouse heavy chain locus with human VH, DR, and JH gene
segments and a replacement of the endogenous mouse K light chain locus with
either
the engineered germline Vx.1-393k5 human light chain region or the engineered
germline Vic3-20.ficl human light chain region (described above) were
immunized
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with a human cell-surface receptor protein (Antigen E). Antigen E is
administered
directly onto the hind footpad of mice with six consecutive injections every 3-
4
days. Two to three micrograms of Antigen E are mixed with 10 lig of CpG
oligonucleotide (Cat # tlrl-modn - 0DN1826 oligonucleotide; InVivogen, San
Diego, CA) and 25 ug 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.
[00289] 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 E-specific common light chain antibodies. Using this
technique several anti-Antigen E-specific common light chain antibodies (i.e.,
antibodies possessing human heavy chain variable domains, the same human light
chain variable domain, and mouse constant domains) are obtained.
[00290] Alternatively, anti-Antigen E common light chain antibodies 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 E common light
chain
antibodies (i.e., antibodies possessing human heavy chain variable domains,
either
an engineered human Vk1-39R5 light chain or an engineered human Vic3-20Jic1
light chain region, and human constant domains) were obtained.
[00291] The biological properties of the exemplary anti-Antigen E common light
chain antibodies generated in accordance with the methods of this Example are
described in detail in the sections set forth below.
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Example 6
Heavy Chain Gene Segment Usage in Antigen-Specific Common Light Chain
Antibodies
[00292] To analyze the structure of the human anti-Antigen E common light
chain antibodies produced, nucleic acids encoding heavy chain antibody
variable
regions were cloned and sequenced. From the nucleic acid sequences and
predicted
amino acid sequences of the antibodies, gene usage was identified for the
heavy
chain variable region (HCVR) of selected common light chain antibodies
obtained
from immunized VELOCIMMUNE mice containing either the engineered human
Vic1-39.1K5 light chain or engineered human Vx3-20JK1 light chain region.
Results
are shown in Tables 5 and 6, which demonstrate that mice according to the
invention
generate antigen-specific common light chain antibodies from a variety of
human
heavy chain gene segments, due to a variety of rearrangements, when employing
either a mouse that expresses a light chain from only a human Vx1-39- or a
human
Vx3-20-derived light chain. Human VH gene segments of the 2, 3, 4, and 5
families
rearranged with a variety of human DH segments and human JH segments to yield
antigen-specific antibodies.
Table 5
Vx1-39.1x5
Common Light Chain Antibodies
HCVRHCVR
Antibody Antibody ____________
VH DH JH VH DH JH
2952 2-5 6-6 1 6030 3-30 6-6 5
5978 2-5 6-6 1 6032 3-30 6-6 5
5981 2-5 3-22 1 2985 3-30 6-13 4
6027 3-13 6-6 5 2997 3-30 6-13 4
3022 3-23 3-10 4 3011 3-30 6-13 4
3028 3-23 3-3 4 3047 3-30 6-13 4
5999 3-23 6-6 4 5982 3-30 6-13 4
6009 3-23 2-8 4 6002 3-30 6-13 4
6011 3-23 7-27 4 6003 3-30 6-13 4
5980 3-30 1-1 4 6012 3-30 6-13 4
3014 3-30 1-7 4 6013 3-30 6-13 4
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3015 3-30 1-7 4 6014 3-30 6-13 4
3023 3-30 1-7 4 6015 3-30 6-13 4
3024 3-30 1-7 4 6016 3-30 6-13 4
3032 3-30 1-7 4 6017 3-30 6-13 4
6024 3-30 1-7 4 6020 3-30 6-13 4
6025 3-30 1-7 4 6034 3-30 6-13 4
6031 3-30 1-7 4 2948 3-30 7-27 4
6007 3-30 3-3 4 2987 3-30 7-27 4
2982 3-30 3-22 5 2996 3-30 7-27 4
6001 3-30 3-22 5 3005 3-30 7-27 4
6005 3-30 3-22 5 3012 3-30 7-27 4
6035 3-30 5-5 2 3020 3-30 7-27 4
3013 3-30 5-12 4 3021 3-30 7-27 4
3042 3-30 5-12 4 3025 3-30 7-27 4 ,
2955 3-30 6-6 1 3030 3-30 7-27 4
3043 3-30 6-6 3 3036 3-30 7-27 4
3018 3-30 6-6 4 5997 3-30 7-27 4
2949 3-30 6-6 5 6033 3-30 7-27 4
2950 3-30 6-6 5 3004 3-30 7-27 5
2954 3-30 6-6 5 6028 3-30 7-27 6
_
2978 3-30 6-6 5 3010 4-59 3-16 3
3016 3-30 6-6 5 3019 4-59 3-16 3
3017 3-30 6-6 5 6018 4-59 3-16 3
3033 3-30 6-6 5 6026 4-59 3-16 3
3041 3-30 6-6 5 6029 4-59 3-16 3
5979 3-30 6-6 5 6036 4-59 3-16 3
5998 3-30 , 6-6 5 6037 4-59 3-16 3
6004 3-30 6-6 5 2964 4-59 3-22 3
6010 3-30 6-6 5 3027 4-59 3-16 4
6019 3-30 6-6 5 3046 5-51 5-5 3
6021 3-30 6-6 5 6000 1-69 6-13 4
6022 3-30 6-6 5 6006 , 1-69 6-6 5
6023 3-30 6-6 5 6008 1-69 6-13 4
Table 6
VO-20.1K1
Common Light Chain Antibodies
HCVR HCVR
Antibody _________________________ Antibody _______
VH DH JH VH DH JH
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5989 3-30 3-3 3 5992 4-39 1-26 3
5994 3-33 1-7 4 2975 5-51 6-13 5
5985 3-33 2-15 4 2972 5-51 3-16 6
5987 3-33 2-15 4 5986 5-51 3-16 6
5995 3-33 2-15 4 5993 5-51 3-16 6
2968 4-39 1-26 3 5996 5-51 3-16 6
5988 4-39 1-26 3 5984 3-53 1-1 4
5990 4-39 1-26 3
Example 7
Determination of Blocking Ability of Antigen-Specific Common Light Chain
Antibodies by LUMINEXTm Assay
[00293] Ninety-eight human common light chain antibodies raised against
Antigen E were tested for their ability to block binding of Antigen E's
natural ligand
(Ligand Y) to Antigen E in a bead-based assay.
[00294] The extracellular domain (ECD) of Antigen E was conjugated to two
myc epitope tags and a 6X histidine tag (Antigen E-rnmH) and amine-coupled to
carboxylated microspheres at a concentration of 20 ug,/mL in MES buffer. The
mixture was incubated for two hours at room temperature followed by bead
deactivation with 1M Tris pH 8.0 followed by washing in PI3S with 0.05% (v/v)
Tween-20. The beads were then blocked with PBS (Irvine Scientific, Santa Ana,
CA) containing 2% (w/v) BSA (Sigma-Aldrich Corp., St. Louis, MO). In a 96-well
filter plate, supernatants containing Antigen E-specific common light chain
antibodies were diluted 1:15 in buffer. A negative control containing a mock
supernatant with the same media components as for the antibody supernatant was
prepared. Antigen E-labeled beads were added to the supernatants and incubated
overnight at 4 C. Biotinylated-Ligand Y protein was added to a final
concentration
of 0.06 nM and incubated for two hours at room temperature. Detection of
biotinylated-Ligand Y bound to Antigen E-myc-myc-6His labeled beads was
determined with R-Phycoerythrin conjugated to Streptavidin (Moss Inc,
Pasadena,
MD) followed by measurement in a LUMINEXTm flow cytometry-based analyzer.
Background Mean Fluorescence Intensity (MFI) of a sample without Ligand Y was
subtracted from all samples. Percent blocking was calculated by division of
the
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background-subtracted MFI of each sample by the adjusted negative control
value,
multiplying by 100 and subtracting the resulting value from 100.
[00295] In a similar experiment, the same 98 human common light chain
antibodies raised against Antigen E were tested for their ability to block
binding of
Antigen E to Ligand Y-labeled beads.
[00296] Briefly, Ligand Y was amine-coupled to carboxylated microspheres at a
concentration of 20 tig/mL diluted in MES buffer. The mixture and incubated
two
hours at room temperature followed by deactivation of beads with 1M Tris pH 8
then washing in PBS with 0.05% (v/v) Tween-20. The beads were then blocked
with PBS (Irvine Scientific, Santa Ana, CA) containing 2% (w/v) BSA (Sigma-
Aldrich Corp., St. Louis, MO). In a 96-well filter plate, supernatants
containing
Antigen E-specific common light chain antibodies were diluted 1:15 in buffer.
A
negative control containing a mock supernatant with the same media components
as
for the antibody supernatant was prepared. A biotinylated-Antigen E-mmH was
added to a final concentration of 0.42 nM and incubated overnight at 4 C.
Ligand
Y-labeled beads were then added to the antibody/Antigen E mixture and
incubated
for two hours at room temperature. Detection of biotinylated-Antigen E-mmH
bound to Ligand Y-beads was determined with R-Phycoerythrin conjugated to
Streptavidin (Moss Inc, Pasadena, MD) followed by measurement in a
LUMINEXTm flow cytometry-based analyzer. Background Mean Fluorescence
Intensity (MFI) of a sample without Antigen E was subtracted from all samples.
Percent blocking was calculated 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.
[00297] Tables 7 and 8 show the percent blocking for all 98 anti-Antigen E
common light chain antibodies tested in both LUMINEXTm assays. ND: not
determined under current experimental conditions.
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Table 7
V1c1-393K5
Common Light Chain Antibodies
Antibody % Blocking of % Blocking of
Antigen E-Labeled Beads Antigen E In Solution
2948 81.1 47.8
2948G 38.6 ND
2949 97.6 78.8
2949G 97.1 73.7
2950 96.2 81.9
29500 89.8 31.4
2952 96.1 74.3
2952G 93.5 39.9
2954 93.7 70.1
2954G 91.7 30.1
2955 75.8 30.0
2955G 71.8 ND
2964 92.1 31.4
2964G 94.6 43.0
2978 98.0 95.1
2978G 13.9 94.1
2982 92.8 78.5
2982G 41.9 52.4
2985 39.5 31.2
2985G 2.0 5.0
2987 81.7 67.8
2987G 26.6 29.3
2996 87.3 55.3
2996G 95.9 38.4
2997 93.4 70.6
2997G 9.7 7.5
3004 79.0 48.4
3004G 60.3 40.7
3005 97.4 93.5
3005G 77.5 75.6
3010 98.0 82.6
30100 97.9 81.0
3011 87.4 42.8
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3011G 83.5 41.7
3012 91.0 60.8
3012G 52.4 16.8
3013 80.3 65.8
3013G 17.5 15.4
3014 63.4 20.7
3014G 74.4 28.5
3015 89.1 55.7
3015G 58.8 17.3
3016 97.1 81.6
3016G 93.1 66.4
3017 94.8 70.2
3017G 87.9 40.8
3018 85.4 54.0
3018G 26.1 12.7
3019 99.3 92.4
3019G 99.3 88.1
3020 96.7 90.3
3020G 85.2 41.5
3021 74.5 26.1
3021G 81.1 27.4
3022 65.2 17.6
3022G 67.2 9.1
3023 71.4 28.5
3023G 73.8 29.7
3024 73.9 32.6
3024G 89.0 10.0
3025 70.7 15.6
3025G 76.7 24.3
3027 96.2 61.6
3027G 98.6 75.3
3028 92.4 29.0
3028G 87.3 28.8
3030 6.0 10.6
3030G 41.3 14.2
3032 76.5 31.4
3032G 17.7 11.0
3033 98.2 86.1
30330 93.6 64.0
3036 74.7 32.7
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3036G 90.1
51.2
3041 95.3 75.9
3041G 92.4 51.6
3042 88.1 73.3
3042G 60.9 25.2
3043 90.8 65.8
3043G 92.8 60.3
Table 8
Vic3-20.1x1
Common Light Chain Antibodies
Antibody
Antigen / o EBlocking-abe
l of edBeads Anti/g eBnlEocIkninSgolouftion
2968 97.1 73.3
2968G 67.1 14.6
2969 51.7 20.3
2969G 37.2 16.5
2970 92.2 =34.2
2970G 92.7 27.2
2971 23.4 11.6
297IG 18.8 18.9
2972 67.1 38.8
2972G 64.5 39.2
2973 77.7 27.0
2973G 51.1 20.7
2974 57.8 12.4
2974G 69.9 17.6
2975 49.4 18.2
2975G 32.0 19.5
2976 1.0 1.0
29760 50.4 20.4
[002981 In the first LUMINEXTm experiment described above, 80 common light
chain antibodies containing the Vx1-39JK5 engineered light chain were tested
for
their ability to block Ligand Y binding to Antigen E-labeled beads. Of these
80
common light chain antibodies, 68 demonstrated >50% blocking, while 12
demonstrated <50% blocking (6 at 25-50% blocking and 6 at (25% blocking). For
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the 18 common light chain antibodies containing the Vic3-20.1x1 engineered
light
chain, 12 demonstrated >50% blocking, while 6 demonstrated <50% blocking (3 at
25-50% blocking and 3 at <25% blocking) of Ligand Y binding to Antigen E-
labeled beads.
[00299] In the second LUMINEXTm experiment described above, the same 80
common light chain antibodies containing the Vic1-39.1k5 engineered light
chain
were tested for their ability to block binding of Antigen E to Ligand Y-
labeled
beads. Of these 80 common light chain antibodies, 36 demonstrated >50%
blocking, while 44 demonstrated <50% blocking (27 at 25-50% blocking and 17 at
<25% blocking). For the 18 common light chain antibodies containing the Vk3-
20.1k1 engineered light chain, 1 demonstrated >50% blocking, while 17
demonstrated <50% blocking (5 at 25-50% blocking and 12 at <25% blocking) of
Antigen E binding to Ligand Y-labeled beads.
[00300] The data of Tables 7 and 8 establish that the rearrangements described
in
Tables 5 and 6 generated anti-Antigen E-specific common light chain antibodies
that
blocked binding of Ligand Y to its cognate receptor Antigen E with varying
degrees
of efficacy, which is consistent with the anti-Antigen E common light chain
antibodies of Tables 5 and 6 comprising antibodies with overlapping and non-
overlapping epitope specificity with respect to Antigen E.
Example 8
Determination of Blocking Ability of Antigen-Specific Common Light Chain
Antibodies by ELISA
[00301] Human common light chain antibodies raised against Antigen E were
tested for their ability to block Antigen E binding to a Ligand Y-coated
surface in an
ELISA assay.
[00302] Ligand Y was coated onto 96-well plates at a concentration of 2 ug/mL
diluted in PBS and incubated overnight followed by washing four times in PBS
with
0.05% Tween-20. The plate was then blocked with PBS (Irvine Scientific, Santa
Ana, CA) containing 0.5% (w/v) BSA (Sigma-Aldrich Corp., St. Louis, MO) for
one
hour at room temperature. In a separate plate, supernatants containing anti-
Antigen
E common light chain antibodies were diluted 1:10 in buffer. A mock
supernatant
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with the same components of the antibodies was used as a negative control.
Antigen
E-mmH (described above) was added to a final concentration of 0.150 nM and
incubated for one hour at room temperature. The antibody/Antigen E-mmH mixture
was then added to the plate containing Ligand Y and incubated for one hour at
room
temperature. Detection of Antigen E-mmH bound to Ligand Y was determined with
Horse-Radish Peroxidase (HRP) conjugated to anti-Penta-His antibody (Qiagen,
Valencia, CA) and developed by standard colorimetric response using
tetramethylbenzidine (TMB) substrate (BD Biosciences, San Jose, CA)
neutralized
by sulfuric acid. Absorbance was read at 0D450 for 0.1 sec. Background
absorbance of a sample without Antigen E was subtracted from all samples.
Percent
blocking was calculated 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.
[00303] Tables 9 and 10 show the percent blocking for all 98 anti-Antigen E
common light chain antibodies tested in the ELISA assay. ND: not determined
under current experimental conditions.
Table 9
Vic1-391x5
Common Light Chain Antibodies
% Blocking of% Blocking of
Antibody Antibody
Antigen E In Solution Antigen E In Solution
2948 21.8 3015 23.7
2948G 22.9 3015G 10.2
2949 79.5 3016 78.1
2949G 71.5 3016G 37.4
2950 80.4 3017 61.6
2950G 30.9 3017G 25.2
2952 66.9 3018 40.6
29520 47.3 3018G 14.5
2954 55.9 3019 94.6
29540 44.7 3019G 92.3
2955 12.1 3020 80.8
29550 25.6 30200 ND
2964 34.8 3021 7.6
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2964G 47.7 ' 3021G 20.7
2978 90.0 3022 2.4
2978G 90.2 3022G 15.0
2982 59.0 3023 9.1
2982G 20.4 3023G 19.2
2985 10.5 3024 7.5
2985G ND 3024G 15.2
2987 31.4 3025 ND
2987G ND 3025G 13.9
2996 29.3 3027 61.4
2996G ND 3027G 82.7
2997 48.7 3028 40.3
2997G ND 3028G 12.3
3004 16.7 3030 ND
3004G 3.5 3030G 9.5
3005 87.2 3032 ND
3005G 54.3 3032G 13.1
3010 74.5 3033 77.1
3010G 84.6 3033G 32.9
3011 19.4 3036 17.6
3011G ND 3036G 24.6
3012 45.0 3041 59.3
3012G 12.6 3041G 30.7
3013 39.0 3042 39.9
3013G 9.6 3042G 16.1
3014 5.2 3043 57.4
]
3014G 17.1 3043G 46.1
Table 10
VK3-20JK1
Common Light Chain Antibodies
% Blocking of% Blocking of
Antibody
Antigen E In Solution Antibody
Antigen E In Solution
2968 68.9 2972G 35.7 ,
2968G 15.2 2973 20.7 .
2969 10.1 2973G 23.1
2969G 23.6 2974 ND
2970 34.3 2974G 22.0
2970G 41.3 2975 8.7
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2971 6.3 2975G 19.2
29710 27.1 2976 4.6
2972 _ 9.6 2976G 26.7
[00304] As described in this Example, of the 80 common light chain antibodies
containing the Vicl -39.10 engineered light chain tested for their ability to
block
Antigen E binding to a Ligand Y-coated surface, 22 demonstrated >50% blocking,
while 58 demonstrated <50% blocking (20 at 25-50% blocking and 38 at <25%
blocking). For the 18 common light chain antibodies containing the Vic3-20.1x1
engineered light chain, one demonstrated >50% blocking, while 17 demonstrated
<50% blocking (5 at 25-50% blocking and 12 at <25% blocking) of Antigen E
binding to a Ligand Y-coated surface.
[00305] These results are also consistent with the Antigen E-specific common
light chain antibody pool comprising antibodies with overlapping and non-
overlapping epitope specificity with respect to Antigen E.
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Example 9
BIACORETM Affinity Determination for Antigen-Specific Common Light
Chain Antibodies
[00306] Equilibrium dissociation constants (1(D) for selected antibody
supernatants were determined by SPR (Surface Plasmon Resonance) using a
BIACORETM T100 instrument (GE Healthcare). All data was obtained using HBS-
EP (10mM Hepes, 150mM NaC1, 0.3mM EDTA, 0.05% Surfactant P20, pH 7.4) as
both the running and sample buffers, at 25 C. Antibodies 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 ullmin, 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 uL/min. Dissociation of antigen from the captured
antibody was monitored for 6 minutes. The captured antibody 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 antibody from the
capture
surface. Binding data for each antibody was fit to a 1:1 binding model with
mass
transport using BIAeore T100 Evaluation software v2.1. Results are shown in
Tables 11 and 12.
Table 11
Vx1-391K5
Common Light Chain Antibodies
100 nM Antigen E100 nM Antigen E
Antibody _________________________ Antibody __________
KD (nM) T112 (min) KD (nM) T112 (min)
2948 8.83 28 3015 29.1 11
2948G 95.0 1 3015G 65.9
2949 3.57 18 3016 4.99 17
2949G 6.37 9 3016G 18.9 4
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2950 4.91 ' 17 3017 9.83 8
2950G 13.6 5 . 3017G 55.4 2
2952 6.25 7 3018 11.3 36
2952G 7.16 4 3018G 32.5 3
2954 2.37 24 3019 1.54 59
2954G 5.30 9 3019G 2.29 42
2955 14.4 6 3020 5.41 39
2955G 12.0 4 3020G 41.9 6
_
2964 14.8 6 3021 50.1 6
2964G 13.0 9 3021G 26.8 4
2978 1.91 r 49 3022 25.7 17
2978G 1.80 58 3022G 20.8 12
2982 6.41 19 3023 263 9
2982G 16.3 9 3023G 103 5
2985 64.4 9 3024 58.8 7
2985G 2.44 , 8 3024G 7.09 10
2987 21.0 11 =3025 35.2 6
2987G 37.6 4 3025G 42.5 8
2996 10.8 9 3027 7.15 6
_
2996G 24.0 2 3027G 4.24 18
,
2997 7.75 19 3028 6.89 37
2997G 151 1 =3028G 7.23 22
3004 46.5 14 3030 46.2 7
3004G 1.93 91 3030G 128 3
3005 2.35 108 3032 53.2 9
3005G 6.96 27 3032G 13.0 1
3010 4.13 26 3033 4.61 17
3010G 2.10 49 3033G 12.0 5
_=
3011 59.1 5 3036 284 12
3011G 41.7 5 3036G 18.210
3012 9.71 20 3041 6.90 ' 12
3012G 89.9 2 3041G 22.9 2
3013 20.2 20 3042 9.46 34
3013G 13.2 4 3042G 85.5 3
3014 213 4 3043 9.26 29
3014G 36.8 3 3043G 13.1 22
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Table 12
Vic3-20.rx1
Common Light Chain Antibodies
100 nM Antigen E 100 nM Antigen E
Antibody _________________________ Antibody __________
KD (nM) T1r2 (min) KD (nM) T112 (min)
2968 5.50 8 2973 5.35 39
2968G 305 0 2973G 11.0 44
2969 34.9 2 2974 256 0
2969G 181 1 2974G 138 0
2970G - 12.3 3 2975 38.0 2
2971G - 32.8 22 2975G 134 1
2972 6.02 13 2976 6.73 10
2972G 74.6 26 2976G 656 8
[00307] The binding affinities shown in Tables 11 and 12 of common light chain
antibodies comprising the rearrangements shown in Tables 5 and 6 vary, with
nearly
all exhibiting a KD in the nanomolar range. The affinity data is consistent
with the
common light chain antibodies resulting from the combinatorial association of
rearranged variable domains described in Tables 5 and 6 being high-affinity,
clonally selected, and somatically mutated. Coupled with data previously
shown,
the common light chain antibodies described in Tables 5 and 6 comprise a
collection
of diverse, high-affinity antibodies that exhibit specificity for one or more
epitopes
on Antigen E.
Example 10
Determination of Binding Specificities of Antigen-Specific Common Light
Chain Antibodies by LUMINEXTm Assay
[00308] Selected anti-Antigen E common light chain antibodies were tested for
their ability to bind to the ECD of Antigen E and Antigen E ECD variants,
including
the cynomolgous monkey ortholog (Mf Antigen E), which differs from the human
protein in approximately 10% of its amino acid residues; a deletion mutant of
Antigen E lacking the last 10 amino acids from the C-terminal end of the ECD
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(Antigen E-ACT); and two mutants containing an alanine substitution at
suspected
locations of interaction with Ligand Y (Antigen E-Alal and AntigenE-A1a2). The
Antigen E proteins were produced in CHO cells and each contained a myc-myc-His
C-terminal tag.
[00309] For the binding studies, Antigen E ECD protein or variant protein
(described above) from 1 mL of culture medium was captured by incubation for 2
hr
at room temperature with l x 106microsphere (LUMINEXTm) beads covalently
coated with an anti-mye monoclonal antibody (MAb 9E10, hybridoma cell line
CRL1729TM; ATCC, Manassas, VA). The beads were then washed with PBS
before use. Supernatants containing anti-Antigen E common light chain
antibodies
were diluted 1:4 in buffer and added to 96-well filter plates. A mock
supernatant
with no antibody was used as negative control. The beads containing the
captured
Antigen E proteins were then added to the antibody samples (3000 beads per
well)
and incubated overnight at 4 C. The following day, the sample beads were
washed
and the bound common light chain antibody was detected with a R-phycoerythrin-
conjugated anti-human IgG antibody. The fluorescence intensity of the beads
(approximately 100 beads counted for each antibody sample binding to each
Antigen
E protein) was measured with a LUMINEXThl flow cytometry-based analyzer, and
the median fluorescence intensity (MFI) for at least 100 counted beads per
bead/antibody interaction was recorded. Results are shown in Tables 13 and 14.
Table 13
Vic1-39J0 Common Light Chain Antibodies
Mean Fluorescence Intensity (MEI)
Antibody Antigen E- Antigen E- Antigen E- Antigen E-
MfAntigen E
ECD ACT Alal A1a2
2948 1503 2746 4953 3579 1648
2948G 537 662 2581 2150 863
2949 3706 4345 8169 5678 5142
29496 3403 3318 7918 5826 5514
2950 3296 4292 7756 5171 4749
2950G 2521 2408 7532 5079 3455
2952 3384 1619 1269 168 911
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2952G 3358 1001 108 55 244
2954 2808 3815 7114 5039 3396
2954G 2643 2711 7620 5406 3499
2955 1310 2472 4738 3765 1637
2955G 1324 1802 4910 3755 1623
2964 5108 1125 4185 346 44
2964G 4999 729 4646 534 91
2978 6986 2800 14542 10674 8049
2978G 5464 3295 11652 8026 6452
2982 4955 2388 13200 9490 6772
2982G 3222 2013 8672 6509 4949
2985 1358 832 4986 3892 1669
2985G 43 43 128 244 116
2987 3117 1674 7646 5944 2546
2987G 3068 1537 9202 6004 4744
2996 4666 1917 12875 9046 6459
2996G 2752 1736 8742 6150 4873
2997 5164 2159 12167 8361 5922
2997G 658 356 3392 2325 1020
3004 2794 1397 8542 6268 3083
3004G 2753 1508 8267 5808 4345
3005 5683 2221 12900 9864 5868
3005G 4344 2732 10669 7125 5880
_
3010 4829 1617 2642 3887 44 _
3010G 3685 1097 2540 3022 51
3011 2859 2015 7855 5513 3863
3011G 2005 1072 6194 4041 3181
3012 3233 2221 8543 5637 3307
30120 968 378 3115 2261 1198 _
3013 2343 1791 6715 4810 2528
3013G 327 144 1333 1225 370
3014 1225 1089 5436 3621 1718
3014G 1585 851 5178 3705 2411
3015 3202 2068 8262 5554 3796
3015G 1243 531 4246 2643 1611
3016 4220 2543 8920 5999 5666
3016G 2519 1277 6344 4288 4091
3017 3545 2553 8700 5547 5098
3017G 1972 1081 5763 3825 3038
3018 2339 1971 6140 4515 2293
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3018G 254 118 978 1020 345 1
3019 5235 1882 7108 4249 54
3019G 4090 1270 4769 3474 214
3020 _ 3883 3107 8591 6602 4420 ,
3020G 2165 1209 6489 4295 2912
3021 1961 1472 6872 4641 2742
3021G 2091 1005 6430 3988 2935
3022 2418 793 7523 2679 , 36
3022G 2189 831 6182 3051 132
3023 1692 1411 5788 3898 2054
3023G 1770 825 = 5702 3677 _ 2648
3024 1819 1467 6179 4557 2450
3024G 100 87 268 433 131
3025 1853 1233 6413 4337 2581
3025G 1782 791 5773 3871 2717
3027 4131 1018 582 2510 22
3027G 3492 . 814 1933 2596 42
3028 4361 2545 9884 5639 975
3028G 2835 1398 7124 3885 597
3030 463 277 1266 1130 391
3030G , 943 302 3420 2570 1186
3032 2083 1496 6594 4402 2405
_
3032G 295 106 814 902 292
3033 4409 2774 8971 6331 5825
3033G 2499 1234 6745 4174 4210
3036 1755 1362 6137 4041 1987
3036G 2313 1073 6387 4243 3173
3041 3674 , 2655 8629 5837 4082
3041G 2519 1265 6468 4274 3320
3042 2653 2137 7277 5124 3325
3042G 1117 463 4205 2762 1519
3043 3036 2128 7607 5532 3366
3043G 2293 1319 6573 4403 3228
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Table 14
Vx3-20R1 Common Light Chain Antibodies
Mean Fluorescence Intensity (MFI)
Antibody Antigen E- Antigen E- Antigen E- Antigen E-
ECD ACT Alai A1a2 Mf Antigen E
2968 1 6559 i 3454 14662 3388 29
2968G 2149 375 9109 129 22
_ 2969 2014 1857 7509 5671 3021
_ 2969G 1347 610 6133 4942 2513
2970 5518 1324 14214 607 32
2970G 4683 599 12321 506 31
-
2971 501 490 2506 2017 754
2971G 578 265 2457 2062 724
_
2972 2164 2158 8408 6409 3166
2972G 1730 992 6364 4602 2146
_
2973 3527 1148 3967 44 84
-
2973G 1294 276 1603 28 44
_
2974 1766 722 8821 241 19
_
2974G 2036 228 8172 135 26
2975 1990 1476 8669 6134 2468
_
2975G 890 315 4194 3987 1376
2976 147 140 996 1079 181
29760 1365 460 6024 3929 1625
_
[00310] The anti-Antigen E common light chain antibody supernatants exhibited
high specific binding to the beads linked to Antigen E-ECD. For these beads,
the
negative control mock supernatant resulted in negligible signal (<10 MFI) when
combined with the Antigen E-ECD bead sample, whereas the supernatants
containing anti-Antigen E common light chain antibodies exhibited strong
binding
signal (average MFI of 2627 for 98 antibody supernatants; MFI > 500 for 91/98
antibody samples).
[00311] As a measure of the ability of the selected anti-Antigen E common
light
chain antibodies to identify different epitopes on the ECD of Antigen E, the
relative
binding of the antibodies to the variants were determined. All four Antigen E
variants were captured to the anti-myc EUMINEXTm beads as described above for
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the native Antigen E-ECD binding studies, and the relative binding ratios
(MFIvanant8AFIAntigen E-Eco) were determined. For 98 tested common light chain
antibody supernatants shown in Tables 12 and 13, the average ratios
(MnanantiMPIAntigen E-ECD) differed for each variant, likely reflecting
different
capture amounts of proteins on the beads (average ratios of 0.61, 2.9, 2.0,
and 1.0 for
Antigen E-ACT, Antigen E-Alal, Antigen E-A1a2, and MfAntigen E, respectively).
For each protein variant, the binding for a subset of the 98 tested common
light
chain antibodies showed greatly reduced binding, indicating sensitivity to the
mutation that characterized a given variant. For example, 19 of the common
light
chain antibody samples bound to the Mf Antigen E with MFIvanant/MFIAntigen E-
ECD of
<8%. Since many in this group include high or moderately high affinity
antibodies
(5 with KD < 5nM, 15 with KD < 50 nM), it is likely that the lower signal for
this
group results from sensitivity to the sequence (epitope) differences between
native
Antigen E-ECD and a given variant rather than from lower affinities.
[00312] These data establish that the common light chain antibodies described
in
Tables 5 and 6 represent a diverse group of Antigen-E-specifie common light
chain
antibodies that specifically recognize more than one epitope on Antigen E.
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Example 11
Light Chain Shuffling in Common Light Chain Antibodies
[00313] Heavy chains of selected antigen-specific common light chain
antibodies
were tested for binding to Antigen E after repairing the heavy chains with
either a
germline VK 1-39JK5 or a germline VK3-20JKI engineered light chain (as
described
in Example 1).
[00314] Briefly, 247 heavy chains of Antigen E-specific common light chain
antibodies (VKI-39JK5 and VK3-20TK1) were transfected with either a germline
VK1-39 or a germline W3-20 engineered light chain and rescreened for binding
to
Antigen E by a LUMINEXTm assay (as described in Example 7 and Example 10).
Binding to Antigen E was confirmed by BIACORETM (as described in Example 9).
The results are shown in Table 15.
[00315] As shown in this Example, twenty-eight common light chain antibodies
specific for Antigen E were capable of binding to Antigen E when repaired with
a
germline form of the light chain.
Table 15
Original Light Chain Repaired Light Chain No. Tested No. Confirmed Binders
1-39 1-39 198 23
3-20 3-20 49 5
Example 12
Heavy Chain Gene Usage and Somatic Hypermutation Frequency in Common
Light Chain Antibodies
[00316] Heavy and light chain sequences (>6000) of antibodies raised in
VELCOIMMUNE mice (e.g., US 6,596,541 and US 7,105,348) were compiled
with heavy and light chain sequences (>600) of common light chain antibodies
obtained by a multi-antigen immunization scheme employing the engineered light
chain mice (described above) to compare heavy chain gene segment usage and
somatic hypermutation frequencies of the antibody chains.
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[00317] Heavy Chain Gene Usage. Heavy and light chain sequences obtained
from VELOCIMMUNE mice containing a replacement of the endogenous mouse
heavy chain locus with human VH, DH, and JH gene segments and a replacement of
the endogenous mouse K light chain locus with either the engineered germline
VKl-
39.11a human light chain region or the engineered germline Vx3-20k1 human
light
chain region (as described in Example 2) immunized with a human cell-surface
receptor (Antigen E), a heterodimer of two human cell-surface glycoproteins
(Antigen F), a human cytokine receptor (Antigen G) and a human tumor
differentiation antigen (Antigen H) were analyzed for heavy chain gene segment
usage and VH and JH gene segments were recorded. Results are shown in Tables
16
¨ 18. Percentages in Tables 16 ¨ 18 represent rounded values and in some cases
may not equal 100% when added together.
[00318] Table 16 sets forth the percent heavy chain family usage for
antibodies
from VELCOIMMUNE mice (VI), antibodies from VELCOIMMUNE mice
having a cognate Vic1-39 light chain (VI ¨ Vx1-39), antibodies from Vic1-39
engineered light chain mice (Vx1-39), antibodies from 'VELCOIMMUNE mice
having a cognate Vx3-20 light chain (VI ¨ Vx3-20), and antibodies from Vx3-20
engineered light chain mice (Vic3-20). Table 17 sets forth the percent VH and
J11
gene usage for antibodies from VELCOIMMUNE mice (VI), antibodies from
VELCOIMMUNE mice having a cognate Vx1-39 light chain (VI ¨ Vic1-39),
antibodies from Vic1-39 engineered light chain mice (Vx1-39), antibodies from
VELCOIMMUNE mice having a cognate Vx3-20 light chain (VI ¨ Vic3-20), and
antibodies from Vx3-20 engineered light chain mice (Vx3-20). Table 18 sets
forth
the percent VH gene usage for antibodies from Vic1-39 engineered light chain
mice
(Vx1-39 Mice) from each immunization group (Antigens E, F, G and H) and the
percent VH gene usage for antibodies from V1(3-20 engineered light chain mice
(Vx3-20 Mice) from selected immunization groups (Antigens E and G).
[00319] As shown in this Example, heavy chain gene usage for antigens tested
in
Vic1-39R5-engineered light chain mice was characterized by a preponderance of
V11
family III subgroups (VH3-7, VH3-9, VH3-11, V113-13, VH3-20, VH3-23, VH3-30,
V113-33 and VH3-48). Notable usage of other VH family subgroups was
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characterized by usage of VH 1-18, VH 1 -69, VH2-5, VH4-59 and V116-1. For
antigens
tested in Vx3-20k1 engineered light chain mice, heavy chain gene usage was
characterized by a preponderance of VH family III, VH family IV and VH family
V
subgroups (VH3-11, VH3-30, VH3-33, VH4-39, VH4-59 and V115-51). Notable usage
of other VH family subgroups was characterized by usage of VH1-18, VH1-69,
V112-
70 and V116-1.
[00320] Somatic Hypermutation Frequency. Heavy and light chains from
antibodies raised in VELCOIMMUNE mice and the engineered light chain mice
(described above) were aligned to germline sequences according to the heavy
and
light chain gene usage demonstrated for each heavy and/or light chain. Amino
acid
changes for each framework region (FW) and complementarity determining region
(CDR) for both heavy and light chain of each sequence were calculated. Results
are
shown in Tables 19 ¨ 22. Percentages in Tables 21 ¨ 24 represent rounded
values
and in some cases may not equal 100% when added together.
[00321] Table 19 sets forth the number of amino acid (AA) changes observed in
each FW and CDR region of heavy chains of antibodies from VELCOIMMUNE
mice, heavy chains of antibodies from Vi(1-39 engineered light chain mice
(Vic1-39
Mice) and heavy chains of antibodies from Vk3-20 engineered light chain mice
(W3-20 Mice). Table 20 sets forth the number of amino acid (AA) changes
observed in each FW and CDR region of light chains of antibodies from
VELCOIMMUNE mice, the light chain of antibodies from W1-39 engineered
mice (W1-39 Mice) and the light chain of antibodies from Vx3-20 engineered
mice
(V1c3-20 Mice). Table 21 sets forth the number of amino acid (AA) changes
observed in each FW and CDR region of heavy chains of antibodies from Vx1-39
engineered light chain mice (Vx1-39 Mice) for selected immunization groups
(Antigens E, F and H). Table 22 sets forth the number of amino acid (AA)
changes
observed in each FW and CDR region of heavy chains of antibodies from Vx3-20
engineered light chain mice (Vx3-20 Mice) for selected immunization groups
(Antigens E and G).
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Table 16
VH Family VI VI - VK1-39 VK1-39 VI - VK3-20 VK3-20
1 9.0 14.8 3.3 7.1 4.9
2 2.2 1.8 4.6 0 1.6
3 77.8 69.8 77.3 _ 61.4 29.5
4 8.4 8.3 11.2 27.1 39.3
5 0.9 0 0.7 4.3 23.0
6 1.7 5.3 3.0 0 1.6
Table 17
VII Gene VI VI - VK1-39 VK1-39 VI - VK3-20 VK3-20
1-2 3.9 8.3 0 2.9 0
_
1-3 0 0 0 0 0
1-8 1.3 0.6 0 1.4 0
1-18 3.0 0.6 1.3 2.1 1.6
1-24 0.4 3.6 0 0.7 0
-
1-46 0.1 0 0 0 0
1-58 0 0 0 0 0
1-69 0.3 1.8 2.0 0 3.3
2-5 1.9 0 _ 4.6 0 0
2-26 0.2 1.8 0.0 0 ,_ 0
2-70 0.1 0 0 0 1.6
3-7 3.0 14.8 0 1.4 0
3-9 8.5 3.6 29.6 16.4 0
3-11 5.4 10.7 _ 0 7.1 1.6
3-13 3.2 1.8 0.7 2.1 0
3-15 4.0 4.7 0.3 0.7 0
3-20 1.0 0.6 0.3 5.0 0
3-21 0.8 0.6- 0 2.1 0
3-23 20.4 8.9 3.3 8.6 0
3-30 17.6 4.1 35.2 12.9 1.6
3-33 12.6 14.8 0 5.0 26.2
3-43 0.2 0.6 0 0 0
3-48 0.8 1.2 7.2 0 0
3-53 0.3 3.6 0.3 0 0
3-64 0 0 0.3 0 0
3-72 0 0 0 0 0
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3-73 0 0 0 0 0
4-31 2.7 0 0.7 8.6 0
4-34 1.8 0.6 0.3 14.3 0
4-39 1.6 0.6 3.0 2.1 14.8
4-59 2.3 7.1 7.2 2.1 24.6
5-51 0.9 0 0.7 4.3 23.0
6-1 1.7 5.3 3.0 0 1.6
JH Gene VI VI - Vx1-39 Vx1-39 VI - Vx3-20 Vx3-20
1 1.5 1.2 7.1 0 0
2 4.5 2.4 0.7 5.0 26.9
3 10.5 16.6 13.1 13.6 26.9
4 44.0 34.3 32.3 50.7 9.6
9.6 10.1 16.8 7.9 1.9
6 29.7 35.5 30.0 22.9 34.6
Table 18
V-K1-39 Mice Vic3-20 Mice
VH _______________________________________
Gene Antigen Antigen Antigen Antigen Antigen Antigen
E F G H E G
1-2 0 0 0 0 0 = 0
1-3 0 0 0 0 0 0
1-8 0 0 0 0 0 0
1-18 0 0 0 8.3 0 3.1
1-24 0 0 0 0 0 0
1-46 0 0 0 0 0 0
1-58 0 0 0 0 0 0
1-69 2.9 0 25.0 0 0 6.3
2-5 8.2 0 0 0 0 0
2-26 0 0 0 0 0 0
2-70 0 0 0 0 0 3.1
3-7 0 0 0 0 0 0
3-9 1.2 98.8 0 14.6 0 0
3-11 0 0 0 0 0 3.1
3-13 0.6 0 25.0 0 0 0
3-15 0 1.2 0 0 0 0
3-20 0 0 25.0 0 0 0
3-21 0 0 0 0 0 0
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3-23 4.1 0 25.0 4.2 0 0
3-30 62.9 0 0 0 3.4 0
3-33 0 0 0 0 13.8 37.5
3-43 0 0 0 0 0 0
3-48 0.6 0 0 43.8 0 0
3-53 1.6 0 0 0 0 0
3-64 1.6 0 0 0 0 0
3-72 0 0 0 0 0 0
3-73 0 0 0 0 0 0
,-
4-31 0 0 0 4.2 0 0
4-34 0 0 0 2.1 0 0
4-39 5.3 0 0 0 31.0 0
4-59 11.8 0 0 4.2 3.4 43.8
5-51 1.2 0 0 0 48.3 0
6-1 0 0 0 18.8 0 3.1
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Table 19
Heavy Chains of Antibodies from VELCOIMMUNE Mice
# AA Changes
FW1 CDR1 FW2 CDR2 F W3 CDR3
0 63 32 36 26 12 82
1 23 32 41 31 22 17
,
2 9 25 17 23 27 1
3 4 10 5 16 13 0
4 0 1 1 3 12 0
>5 1 0 0 1 14 0
Heavy Chains of Antibodies from VK1-39 Mice
# AA Changes
FW1 CDRI FW2 CDR2 FW3 CDR3
0 65 ' 8 34 30 i 9 37
1 25 26 35 34 19 54
1
2 9 44 23 , 20 33 9
3 1 ' 19 8 12 ' 22 0
r
4 0 3 0 , 5 11 0
>5 1 0 0 0 7 0 ,
Heavy Chains of Antibodies from VK3-20 Mice
# AA Changes
FW1 CDR1 FW2 CDR2 F W3 CDR3
0 57 1 8 1 54 1 16 8 93
1 41 43 34 39 21 7
2 2 25 10 18 20_ 0
3 0 15 2 21 13 0
4 0 10 0 3 20 0
>5 0 0 0 2 18 0
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Table 20
Light Chains of Antibodies from VELCOIMMUNE Mice
# AA Changes
FW1 CDR I FW2 CDR2 FW3 CDR3
_
0 65 24 49 60 33 23
_
1 24 20 34 31 27 38
2 9 27 16 9 18 28
3 1 , 20 1 0 14 7
_
4 0 7 0 0 4 3
>5 1 1 0 0 3 0
Light Chains of Antibodies from Vx1-39 Mice
# AA Changes
FW1 CDR1 FW2 CDR2 FW3 CDR3
0 91 75 80 90 71 63
1 9 19 17 10 21 27
2 0 5 1 1 5 8
3 0 0 1 0 2 1
4 0 0 0 0 2 1
>5 0 0 0 0 0 0
Light Chains of Antibodies from VK3-20 Mice
# AA Changes
FW1 CDR1 FW2 CDR2 FW3 CDR3
_
0 90 47 93 97 63 57
_
1 10 27 3 3 20 43
2 0 27 3 0 17 0 1
_
3 0 0 0 0 0 0
4 0 0 0 0 0 0 _
>5 0 0 0 0 0 0
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Table 21
Heavy Chains of Anti-Antigen E Antibodies from Vic1-39 Mice
# AA Changes
FW1 CDR1 FW2 CDR2 FW3 CDR3
0 75 8 49 41 14 36
1 21 25 33 35 25 52
2 4 43 14 18 28 12
3 0 20 4 5 16 0
4 0 5 0 1 12 0
>5 1 0 0 0 5 0
Heavy Chains of Anti-Antigen F Antibodies from Vx1-39 Mice
# AA Changes
FW1 CDRI FW2 CDR2 FW3 CDR3
0 52 0 6 6 2 15 I
1 35 24 32 35 15 78
2 11 59 46 22 49 7
3 0 17 16 24 29 0
4 0 0 0 12 4 0
>5 1 0 0 0 1 , 0
Heavy Chains of Anti-Antigen H Antibodies from Vx1-39 Mice
# AA Changes
FW1 CDR1 FW2 CDR2 FW3
CDR3 ,
0 54 21 29 33 4 77
1 17 35 50 27 6 23
2 23 21 15 21 25 0
3 6 21 4 15 27 0
4 0 2 2 2 15 0
>5 0 0 0 2 23 0
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Table 22
Heavy Chains of Anti-Antigen E Antibodies from Vx3-20 Mice
# AA Changes
FW1 CDR I FW2 CDR2 FW3 CDR3
0 79 17 62 24 17 90
1 21 28 34 55 31 10
2 0 28 3 21 24 0
3 0 14 0 0 10 0
4 0 14 0 0 3 0
>5 0 0 0 0 14 0
Heavy Chains of Anti-Antigen G Antibodies from Vx3-20 Mice
# AA Changes
FW1 CDR1 FW2 CDR2 FW3 CDR3
0 38 0 47 9 0 97
1 59 56 34 25 13 3
2 3 22 16 16 16 0
3 0 16 3 41 16 0
_
4 0 6 0 6 34 0
>5 0 0 0 3 22 0
Example 13
_
Binding Affinity of Bispecific Antibodies Having Universal Light Chains
[00322] Fully human bispecific antibodies were constructed from cloned human
heavy chain variable regions of selected monospecific anti-Antigen E common
light
chain antibodies (described in Example 5) using standard recombinant DNA
techniques known in the art. Table 23 sets forth the pairing of human heavy
chains
(HC-1 and HC-2) from selected parental monospecific antibodies, each pair
employed with a germline rearranged human Vic1-39/Jic5 light chain for
construction of each bispecific antibody.
[00323] Binding of bispecific or parental monospecific anti-Antigen E
antibodies
to the extracellular domain (ECD) of Antigen E was determined using a real-
time
surface plasmon resonance biosensor assay on a BIACORETM 2000 instrument (GE
Healthcare). A CM5 BIACORETM sensor surface derivatized with anti-c-myc-
specific monoclonal antibody (Clone# 9E10) using EDC-NHS chemistry was used
to capture the C-terminal myc-myc-hexahistidine tagged ECD of Antigen E
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(AntigenE-mmh). Around 190 RUs of AntigenE-mmh was captured on the
BIACORETM sensor surface, followed by the injection of 300nM and 50nM
concentrations of different bispecific or parental monospecific anti-Antigen E
antibodies at a flow rate of 50 1/min. The experiment was performed at 25 C
in
HBST running buffer (0.01M HEPES pH 7.4, 0.15M NaC1, 3mM EDTA, 0.05% v/v
Surfactant P20). The amount of antibody binding to AntigenE-mmh surface at
300nM concentration was recorded three seconds before the end of antibody
injection and plotted.
[00324] Table 24 and FIG. 8 set forth the binding responses (BIACORETM units;
RU) observed for each bispecific antibody (BsAb) and monospecific parental
antibody (PAb-1, PAb-2). Since each antibody was injected under saturating
conditions over an identical AntigenE-mmh surface, the binding response
reflects
the binding stoichiometry for each antibody binding to the antigen capture
surface.
[00325] As shown in this Example, the observed binding response for each
bispecific antibody was approximately 2-fold greater than the binding response
for
each parental monospecific antibody (Table 24 and FIG. 8), demonstrating
functional construction of bispecific antibodies using heavy chains of antigen-
specific monoclonal antibodies and a common light chain where each Fab arm in
the
bispecific antibody molecule binds simultaneously to distinct epitopes on the
extracelluar domain of a cell surface receptor (Antigen E; see FIG. 7B, bottom
left).
Table 23
Bispecific Antibody Parent HC-1 Parent HC-2
3108 2952 2978
3109 2978 3022
3111 2952 3005
3112 3622 3005
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Table 24
Binding Response (RU)
Bispecific Antibody
PAb-1 PAb-2 BsAb
3108 236 229 485
3109 236 197 408
3111 202 229 435
3112 202 197 345
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