U.S. patent application number 14/674206 was filed with the patent office on 2015-07-16 for mice that make heavy chain antibodies.
The applicant listed for this patent is Regeneron Pharmaceuticals, Inc.. Invention is credited to Lynn Macdonald, Andrew J. Murphy, Sean Stevens.
Application Number | 20150196015 14/674206 |
Document ID | / |
Family ID | 43534479 |
Filed Date | 2015-07-16 |
United States Patent
Application |
20150196015 |
Kind Code |
A1 |
Macdonald; Lynn ; et
al. |
July 16, 2015 |
MICE THAT MAKE HEAVY CHAIN ANTIBODIES
Abstract
Genetically modified non-human animals and methods and
compositions for making and using them are provided, wherein the
genetic modification comprises a deletion in an immunoglobulin
constant region CH1 gene (optionally a deletion in a hinge region)
of an IgG, IgA, IgD, and/or IgE, and wherein the mouse is capable
of expressing a functional IgM. Genetically modified mice are
described, including mice having a functional IgM gene and modified
to have a deletion of a CH1 domain and a hinge region in a heavy
chain constant domain that is not an IgM, e.g., in an IgG heavy
chain constant domain. Genetically modified mice that make human
variable/mouse constant chimeric heavy chain antibodies (antibodies
that lack a light chain), fully mouse heavy chain antibodies, or
fully human heavy chain antibodies are provided.
Inventors: |
Macdonald; Lynn; (White
Plains, NY) ; Stevens; Sean; (San Diego, CA) ;
Murphy; Andrew J.; (Croton-on-Hudson, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Regeneron Pharmaceuticals, Inc. |
Tarrytown |
NY |
US |
|
|
Family ID: |
43534479 |
Appl. No.: |
14/674206 |
Filed: |
March 31, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14267279 |
May 1, 2014 |
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14674206 |
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12965050 |
Dec 10, 2010 |
8754287 |
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14267279 |
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61285250 |
Dec 10, 2009 |
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Current U.S.
Class: |
800/18 ; 435/328;
536/23.53 |
Current CPC
Class: |
A01K 67/0276 20130101;
A01K 2267/01 20130101; C07K 2317/21 20130101; A01K 2217/072
20130101; C07K 2317/52 20130101; A01K 2217/075 20130101; C07K
2317/50 20130101; C07K 2317/14 20130101; A01K 67/0275 20130101;
C07K 2317/20 20130101; A01K 2227/105 20130101; C07K 16/462
20130101; C12N 15/8509 20130101; A01K 67/0278 20130101; C07K 16/00
20130101 |
International
Class: |
A01K 67/027 20060101
A01K067/027; C07K 16/46 20060101 C07K016/46 |
Claims
1.-20. (canceled)
21. A transgenic mouse comprising a germline modification, which
modification comprises: (a) a deletion of a nucleic acid sequence
encoding a CH1 domain of an endogenous IgG1 constant region gene;
(b) a deletion of an endogenous IgD constant region gene; (c) a
deletion of an endogenous IgG3 constant region gene; (d) a deletion
of an endogenous IgG2a constant region gene; (e) a deletion of an
endogenous IgG2b constant region gene; (f) a deletion of an
endogenous IgE constant region gene; (g) a deletion of an
endogenous IgA constant region gene; and (d) an inclusion of one or
more human heavy chain variable region gene segments, wherein the
one or more human heavy chain variable region gene segments is
operably linked to the endogenous IgG1 constant region of (a);
wherein the mouse comprises an intact IgM constant region gene.
22. The transgenic mouse of claim 21, characterized in that the
mouse expresses an IgG1 heavy chain antibody comprising a human
variable domain, lacking a CH1 domain, in whole or in part, and
lacking a cognate light chain, and secretes said IgG heavy chain
antibody into its serum.
23. The transgenic mouse of claim 22, wherein the IgG1 heavy chain
antibody lacks the CH1 domain in whole.
24. The transgenic mouse of claim 22, wherein the IgG1 heavy chain
antibody comprises the human variable domain, an IgG1 hinge, an
IgG1 CH2 domain, and an IgG1 CH3 domain.
25. The transgenic mouse of claim 21, wherein the mouse comprises a
functional immunoglobulin light chain gene locus.
26. The transgenic mouse of claim 25, wherein the immunoglobulin
light chain locus is a .kappa. light chain gene locus.
27. The transgenic mouse of claim 25, wherein the immunoglobulin
light chain gene locus is a .lamda. light chain gene locus.
28. The transgenic mouse of claim 21, wherein the mouse is selected
from the group consisting of a 129 strain, a C57BL/6 strain, and a
mixed 129.times.C57BL/6 strain.
29. The transgenic mouse of claim 28, wherein the mouse is 50% 129
and 50% C57BL/6.
30. A mouse cell comprising a germline modification, which
modification comprises: (a) a deletion of a nucleic acid sequence
encoding a CH1 domain of an endogenous IgG1 constant region gene;
(b) a deletion of an endogenous IgG2a constant region gene; (c) a
deletion of an endogenous IgG2b constant region gene; (d) a
deletion of an endogenous IgG3 constant region gene; (e) a deletion
of an endogenous IgE constant region gene; (f) a deletion of an
endogenous IgA constant region gene; (g) a deletion of an
endogenous IgD constant region gene; and (d) an inclusion of one or
more human heavy chain variable region gene segments, wherein the
one or more human heavy chain variable region gene segments is
operably linked to the endogenous IgG1 constant region of (a);
wherein the mouse cell comprises an intact IgM constant region
gene.
31. The mouse cell of claim 30, wherein the cell is a B cell.
32. The B cell of claim 31, characterized in that the B cell
expresses an IgG1 heavy chain antibody, which IgG1 heavy chain
antibody comprises a heavy chain variable domain, an IgG1 hinge, an
IgG1 CH2 domain, and an IgG1 CH3 domain.
33. A hybridoma made from the B cell of claim 31.
34. A hybridoma made from the B cell of claim 32.
35. A nucleic acid encoding a human heavy chain variable region
isolated from the hybridoma of claim 33.
36. A nucleic acid encoding a human heavy chain variable region
isolated from the hybridoma of claim 34.
37. A nucleic acid encoding a human heavy chain variable region
isolated from the B cell of claim 31.
38. A nucleic acid encoding a human heavy chain variable region
isolated from the B cell of claim 32.
39. The mouse cell of claim 30, wherein the cell is an embryonic
stem (ES) cell.
40. The mouse ES cell of claim 39, wherein the cell is from a
strain selected from the group consisting of a 129 strain, a
C57BL/6 strain, and a mixed 129.times.C57BL/6 strain.
41. The mouse ES cell of claim 40, wherein the cell is 50% 129 and
50% C57BL/6.
42. The mouse ES cell of claim 41, wherein the cell has a genome
comprising a functional immunoglobulin light chain gene locus.
43. A mouse embryo made from or comprising the ES cell of claim
39.
44. A mouse embryo made from or comprising the ES cell of claim
41.
45. A mouse embryo made from or comprising the ES cell of claim 42.
Description
[0001] This application claims the benefit of the filing date under
35 USC .sctn.119(e), and is a nonprovisional, of U.S. Provisional
Patent Application Ser. No. 61/285,250, filed 10 Dec. 2009, which
provisional application is hereby incorporated by reference.
FIELD OF INVENTION
[0002] The field of invention is genetically modified non-human
animals that make heavy chain antibodies, in particular genetically
modified animals that comprise a nucleotide sequence deletion in a
sequence encoding a CH1 domain (or CH1 domain and hinge region) of
an immunoglobulin gamma (IgG) gene but that are capable of
expressing an IgM that does not lack a functional CH1 domain, and
in particular mice that are capable of making wild-type IgM
molecules (i.e., with CH1 domains) but that make heavy chain IgG
antibodies devoid of a functional CH1 domain (or CH1 domain and
hinge region).
BACKGROUND
[0003] In most animals, normal immunoglobulin heavy chains are only
well-expressed when coupled with their cognate light chains. In
humans, lone heavy chains are found in heavy chain disease that is
manifested by dysfunctional heavy chains that lack sequences of the
variable heavy, the CH1, or the variable heavy and CH1 domains.
Heavy chains devoid of light chains are encountered in certain
species of fish and in camels. Such heavy chains lack a functional
CH1 domain and have non-human features in their heavy chain
variable domains. Attempts have been made to make camelized
antibodies by modifying mice to express camelized genes that mimic
VHH domains found in camels or certain species of fish, in part by
removal of IgM and IgG CH1 domains and conforming the heavy chain
variable regions to resemble those of camels and/or certain species
of fish. However, camelized antibodies would be expected to induce
immune responses in non-camel animals.
[0004] There is a need in the art for genetically modified
non-human animals that make heavy chain antibodies that have
non-camelid VH domains.
BRIEF DESCRIPTION OF THE FIGURES
[0005] FIG. 1 illustrates a wild-type IgG1 locus in a mouse (IgG1,
top), showing the JH region gene segment fusing to a CH1 gene
segment, followed by a hinge region, a CH2 gene segment, and a CH3
gene segment; an IgG1 locus targeted with a construct that deletes
a CH1 domain (IgG1.DELTA.CH1, middle); and an IgG1 locus targeted
with a construct that deletes both a CH1 domain and a hinge region
(IgG1.DELTA.CH1-.DELTA.hinge, bottom).
[0006] FIG. 2 illustrates targeting a mouse IgG1 gene to make a
genetically modified locus that expresses an IgG1 lacking a CH1
domain.
[0007] FIG. 3 illustrates targeting a mouse IgG1 gene to make a
genetically modified locus that expresses an IgG1 lacking a CH1
domain and lacking a hinge region.
[0008] FIG. 4 illustrates targeting a mouse heavy chain constant
region locus to make a genetically modified locus that expresses an
IgG1 lacking a CH1 domain, and does not express an IgG2b or an
IgG2a.
[0009] FIG. 5 illustrates a mouse heavy chain constant region
targeted with a construct that deletes a CH1 domain and deletes a
hinge region and that deletes an IgG2b gene and an IgG2a gene.
[0010] FIG. 6 illustrates a heavy chain constant region of a
genetically modified mouse having an IgG1 that lacks a CH1 domain
or lacks a CH1 domain and a hinge region (top), and a heavy chain
constant region of a genetically modified mouse having an IgG1 that
lacks a CH1 domain or lacks a CH1 domain and a hinge region, and
that lacks an IgG2a gene and lacks an IgG2b gene (bottom).
[0011] FIG. 7 shows Western blots of CHO cell supernatants from CHO
cells engineered to independently express control (cytokine
ectodomain fusion with a mouse Fc), chimeric (human VR)/(mouse Fc)
heavy chain antibody lacking a CH1 domain (hVR-mFc.DELTA.CH1),
camelized chimeric (human VR)/(mouse Fc) heavy chain antibody
lacking a CH1 domain (hVR*-mFc.DELTA.CH1), chimeric (human
VR)/(mouse Fc) heavy chain antibody (hVR-mFc), camelized chimeric
(human VR)/(mouse Fc) heavy chain antibody (hVR*-mFc), mFc with
(mFc) or without (mFc.DELTA.CH1) a CH1 domain.
[0012] FIG. 8 shows Western blot images from a reducing SDS-PAGE of
mouse sera from a wild-type mouse (left) and from a genetically
modified mouse whose IgG1 lacks a CH1 domain and lacks a hinge
region (heterozygous) (right), blotted with anti-mouse IgG;
schematics of the heavy chains are provided, as are molecular
weight marker positions.
[0013] FIG. 9 shows Western blots images from a non-reducing
SDS-PAGE of mouse sera from a wild-type mouse (WT) and four
genetically modified mice whose IgG1 lacks a CH1 domain and lacks a
hinge region (homozygous; noted as HO 1, HO 2, HO 3, HO 4,
respectively), blotted with anti-mouse IgG; each mouse (WT or HO)
is represented by two lanes indicated by brackets above the lanes
corresponding to 1:5 and 1:10 dilutions of serum for each animal
(consecutive lanes from left to right for each).
[0014] FIG. 10 provides a schematic diagram of a normal IgG1
antibody (left) and a heavy chain antibody that lacks a CH1 domain
and lacks a hinge region.
[0015] FIG. 11 shows separate IgG1 and IgG2b serum immunoglobulin
assays from wild type mice (WT) and genetically modified mice that
contain an IgG1 lacking a CH1 domain and lacking a hinge region
(HO; homozygous mouse that expresses a heavy chain antibody that
lacks a CH1 domain and lacks a hinge region). Control is pooled
human serum.
[0016] FIG. 12 shows the protein sequences of eleven independent
RT-PCR clones amplified from splenoctye RNA of mice bearing mouse
heavy chain gene sequences at a modified endogenous mouse heavy
chain locus devoid of IgG1 CH1 and hinge region sequences. B1=SEQ
ID NO:19; B2=SEQ ID NO:21; B3=SEQ ID NO:23; B5=SEQ ID NO:25; D2=SEQ
ID NO:27; D5=SEQ ID NO:29; D6=SEQ ID NO:31; E2=SEQ ID NO:33; E8=SEQ
ID NO:35; E10=SEQ ID NO:37; F6=SEQ ID NO:39. Lower case bases
indicate non-germline bases resulting from either mutation and/or N
addition during recombination. Dots represent artificial gaps in
the sequence for proper alignment of framework (FR) and
complementary determining regions (CDR), which are noted above the
sequences. The first nine amino acids from the CH2 region of the
endogenous IgG1 (CH2) constant region are shown for each clone.
[0017] FIG. 13 shows the protein sequences of seven independent
RT-PCR clones amplified from splenoctye RNA of mice bearing human
heavy chain gene sequences at a modified endogenous mouse heavy
chain locus devoid of an IgG1 CH1 region sequence. A8=SEQ ID NO:51;
C2=SEQ ID NO:53; D9=SEQ ID NO:55; C4=SEQ ID NO:57; H8=SEQ ID NO:59;
A5=SEQ ID NO:61; A2=SEQ ID NO:63. Lower case bases indicate
non-germline bases resulting from either mutation and/or N addition
during recombination. Dots represent artificial gaps in the
sequence for proper alignment of framework (FR) and complementary
determining regions (CDR), which are noted above the sequences. The
first seven amino acids of the 13 amino acid hinge region of the
endogenous IgG1 (HINGE) constant region are shown for each
clone.
SUMMARY
[0018] Genetically modified cells, non-human embryos, non-human
animals and methods and compositions for making and using them are
provided, wherein the animals are genetically modified to lack a
functional CH1 sequence in an immunoglobulin G (IgG), optionally
modified to lack a functional IgG hinge region on the modified IgG,
and wherein the cells, embryos, and animals comprise a functional
IgM CH1 sequence. In some aspects, the mice comprise a replacement
of one or more, or all, endogenous mouse immunoglobulin heavy chain
variable region gene segments with one or more human immunoglobulin
heavy chain variable region gene segments. In some aspects, all
endogenous mouse V, D, and J gene segments are replaced with one or
more human V, one or more human D, and one or more human J gene
segments.
[0019] In one aspect, a genetically modified mouse is provided,
wherein the genetic modification comprises a modification of a
nucleotide sequence encoding an IgG constant region, wherein the
modification results in a loss of function of the CH1 domain of the
IgG constant region. In one embodiment, the loss of function
modification is a deletion of a nucleotide sequence encoding the
CH1 domain, or a deletion within the nucleotide sequence encoding
the CH1 domain.
[0020] In one embodiment, the IgG is selected from IgG1, IgG2a,
IgG2b, and a combination thereof. In one embodiment, the IgG is an
IgG1. In one embodiment, the IgG is an IgG1, an IgG2a, and an
IgG2b.
[0021] In one embodiment, the modification further comprises a
deletion of a nucleotide sequence for a hinge region of the IgG
that comprises the CH1 modification.
[0022] In one embodiment, the genetically modified mouse is
selected from a 129 strain, a C57BL/6 strain, and a mix of
129.times.C57BL/6. In a specific embodiment, the mouse is 50% 129
and 50% C57BL/6.
[0023] In one embodiment, the genetically modified mouse is a 129
strain selected from the group consisting of a 129P1, 129P2, 129P3,
129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm), 129S2, 129S4, 129S5,
129S9/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2 (see,
e.g., Festing et al. (1999) Revised nomenclature for strain 129
mice, Mammalian Genome 10:836). 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,
C57BL/Ola. 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.
[0024] In one embodiment, the mouse comprises one or more
unrearranged endogenous mouse heavy chain immunoglobulin variable
region (mVR) gene segments operably linked to the modified IgG
constant region sequence. In one embodiment, the one or more mVR
gene segments are from a mouse VH gene family selected from VH1,
VH3, VH5, VH7, VH14, and a combination thereof. In one embodiment,
the one or more mVR gene segments are selected from a mVH 1-26,
1-42, 1-50, 1-58, 1-72, 3-6, 5-6, 7-1, 14-2, and a combination
thereof.
[0025] In one embodiment, the mouse comprises a rearranged gene
that encodes an FR1, FR2, and an FR3 in an IgG heavy chain that
lacks a functional CH1 region, wherein the FR1, FR2, and FR3 are
each independently at least 90%, 95%, 96%, 97%, 98%, or 99%
identical to an FR1, FR2, and FR3 derived from a mVH germline
sequence selected from a VH1, VH3, VH5, VH7, and VH14 gene family.
In one embodiment, the mVH germline sequence is selected from a
1-26, 1-42, 1-50, 1-58, 1-72, 3-6, 5-6, 7-1, and 14-2 sequence.
[0026] In one embodiment, the mouse comprises a CDR3 derived from a
DH gene segment selected from DH 1-1, 2-14, 3-1, 3-2, 3-3, 4-1, and
a combination thereof. In one embodiment, the mouse CDR3 comprises
a sequence encoded by a JH that is a JH1, JH2, JH3, or JH4.
[0027] In one embodiment, the mouse comprises a rearranged antibody
sequence that encodes a CDR3 that is derived from a rearrangement
of a DH 1-1, 2-14, 3-1, 3-2, 3-3, 4-1, and a JH1, JH2, JH3, or
JH4.
[0028] In one embodiment, the mouse comprises a rearranged gene
that encodes an FR4 in an IgG heavy chain that lacks a functional
CH1 region, wherein the FR4 is at least 90%, 95%, 96%, 97%, 98%, or
99% identical to an FR4 encoded by a rearrangement of a DH1-1,
2-14, 3-1, 3-2, 3-3, or 4-1 with a JH1, JH2, JH3, or JH4.
[0029] In one embodiment, the mouse comprises an unrearranged human
heavy chain immunoglobulin variable region (hVR) gene segment at an
endogenous mouse heavy chain variable region locus. In one
embodiment, the mouse comprises an unrearranged hVR gene segment
operably linked to the modified IgG constant region sequence at an
endogenous mouse heavy chain variable region locus. In one
embodiment, the hVR gene segments are from a human VH gene family
selected from VH1, VH3, VH4, and a combination thereof. In one
embodiment, the one or more hVR gene segments are selected from
1-2, 1-8, 1-18, 1-46, 1-69, 3-21, 3-72, and 4-59. In a specific
embodiment, the one or more hVR gene segments are selected from
1-8, 1-18, and 1-69.
[0030] In one embodiment, all or substantially all mouse heavy
chain V gene segments are replaced by one or more human heavy chain
V gene segments. In one embodiment, all mouse heavy chain V and D
gene segments are replaced by one or more human heavy chain V and D
gene segments. In one embodiment, all mouse heavy chain V, D, and J
gene segments are replaced with one or more human heavy cahin V,
one or more human heavy chain D, and and one or more human heavy
chain J gene segments. In these embodiments, the human heavy chain
V and/or D and/or J gene segments are at the mouse endogenous heavy
chain locus and are operably linked to the mouse constant region
gene(s) or modified mouse constant region gene(s).
[0031] In one embodiment, the mouse comprises a nucleotide sequence
that encodes a FR1, FR2, and FR3 sequence of an IgG heavy chain
that lacks a functional CH1 region, that is at least 80% identical
to an FR1, FR2, and FR3 from a human germline nucleotide sequence
of a 1-8, 1-18, or 1-69 human immunoglobulin heavy chain variable
region gene segment; wherein the FR1+FR2+FR3 sequence of the
modified mouse is optimally aligned with the recited human germline
sequence without regard to the sequence of the CDRs of the mouse
and human sequences (i.e., optimally aligning the FRs while not
considering the identities of amino acids of any CDRs in the
comparison). In specific embodiments, the FR1, FR2, and FR3 are
about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a human
germline nucleotide sequence of a FR1+FR2+FR3 of of a heavy chain
variable region gene segment that is a 1-8, 1-18, or 1-69 gene
segment.
[0032] In one embodiment, the mouse further comprises a FR4 that is
at least 80% identical to a FR4 formed by a human D6-19/J6
rearrangement, a D6-7/J4 rearrangement, a D4-4/J4 rearrangement, a
D6-6/J2 rearrangement, a D3-16/J6 rearrangement, a D6-6/J4
rearrangement, and a D1-7/J4 rearrangement. In specific
embodiments, the FR4 is about 85%, 90%, 95%, 96%, 97%, 98%, or 99%
identical to an FR4 formed by the aforementioned D/J
rearrangement.
[0033] In one embodiment, the mouse comprises a nucleotide sequence
encoding a FR1 whose amino acid sequence differs by no more than 1,
no more than 2, no more than 3, no more than 4, or no more than 5
amino acids from a FR1 encoded by a germline sequence of human
heavy chain variable region gene segment selected from V1-8, V1-18,
and V1-69. In a specific embodiment, the nucleotide sequence
encoding the FR1 is a rearranged sequence operably linked to a
sequence encoding an IgG constant region that lacks a functional
CH1 sequence.
[0034] In one embodiment, the mouse comprises a nucleotide sequence
encoding a FR2 whose amino acid sequence differs by no more than 1,
no more than 2, no more than 3, no more than 4, or no more than 5
amino acids from a FR2 encoded by a germline sequence of human
heavy chain variable region gene segment selected from V1-8, V1-18,
and V1-69. In a specific embodiment, the nucleotide sequence
encoding the FR2 is a rearranged sequence operably linked to a
sequence encoding an IgG constant region that lacks a functional
CH1 sequence.
[0035] In one embodiment, the mouse comprises a nucleotide sequence
encoding a FR3 whose amino acid sequence differs by no more than 1,
no more than 2, no more than 3, no more than 4, no more than 5, no
more than 6, no more than 7, no more than 8, no more than 9, no
more than 10, or no more than 11 amino acids from a FR3 encoded by
a germline sequence of human heavy chain variable region gene
segment selected from V1-8, V1-18, and V1-69. In a specific
embodiment, the nucleotide sequence encoding the FR3 is a
rearranged sequence operably linked to a sequence encoding an IgG
constant region that lacks a functional CH1 sequence.
[0036] In one embodiment, the mouse comprises a nucleotide sequence
encoding a FR4 whose amino acid sequence differs by no more than 1,
no more than 2, or no more than 3 amino acids from a FR4 amino acid
sequence encoded by a rearrangement of a human D6-19/J6, a D6-7/J4,
a D4-4/J4, a D6-6/J2, a D3-16/J6, a D6-6/J4, and a D1-7/J4. In a
specific embodiment, the nucleotide sequence encoding the FR4 is a
rearranged sequence operably linked to a sequence encoding an IgG
constant region that lacks a functional CH1 sequence.
[0037] In one embodiment, the mouse comprises a nucleotide sequence
encoding a heavy chain CDR3 derived from a human heavy chain D
region gene segment (hDH). In one embodiment, the hDH is selected
from D1-7, D3-16, D4-4, D6-6, D6-7, and D6-19.
[0038] In one embodiment, the mouse comprises a nucleotide sequence
encoding a heavy chain CDR3 derived from a human heavy chain
joining gene segment (JH). In a specific embodiment, the JH is
selected from J2, J4, and J6.
[0039] In one embodiment, the mouse comprises a heavy chain CDR3
encoded by a nucleotide sequence derived from a human DH and a
human JH rearrangement. In a specific embodiment, the CDR3 is
derived from a D1-7/J4, D3-16/J6, D4-4/J4, D6-6/J2, D6-6/J4,
D6-7/J4, or a D6-19/J6 rearrangement.
[0040] In one embodiment, the mouse comprises a replacement of an
endogenous mVR gene segment with an hVR gene segment. In a specific
embodiment, the replacement of the mVR gene segment with the hVR
gene segment is on the same allele as the modified heavy chain
constant region. In another specific embodiment, the replacement of
the mVR gene segment with the hVR gene segment is on a different
allele than the modified heavy chain constant region.
[0041] In one embodiment, 90-100% of mVR gene segments are replaced
with at least one hVR gene segment. In a specific embodiment, all
or substantially all of the endogenous mVR gene segments are
replaced with at least one hVR gene segment. In one embodiment, the
replacement is with at least 18, at least 39, or at least 80 or 81
hVR gene segments. In one embodiment, the replacement is with at
least 12 functional hVR gene segments, at least 25 functional hVR
gene segments, or at least 43 functional hVR gene segments.
[0042] In one embodiment, the genetically modified mouse comprises
a transgene that comprises at least one unrearranged hVR gene
segment, at least one unrearranged human D segment, at least one
unrearranged human J segment, and at least one human heavy chain
constant sequence. In one embodiment, the endogenous mouse heavy
chain variable region and kappa light chain variable region loci
are functionally silenced. In a specific embodiment, the mouse is
capable of trans-switching to produce a chimeric human/mouse
antibody comprising a human heavy chain variable domain contiguous
with a mouse IgG sequence that lacks a functional CH1 domain and,
optionally, lacks a hinge region of the IgG that lacks the
functional CH1 domain. In a specific embodiment, the transgene
further comprises an IgG sequence that lacks a CH1 domain, and
optionally comprises an IgM having a functional CH1 domain. In a
further specific embodiment, the IgG sequence lacks a hinge
region.
[0043] In one embodiment, the mouse comprises a first heavy chain
variable region allele and a second heavy chain variable region
allele, wherein the first allele and the second allele are both
from the same mouse strain. In one embodiment, the first allele is
from a first mouse strain and the second allele is from a second
mouse strain. In one embodiment, one allele of the first and the
second alleles comprises a replacement of an mVR with at least one
hVR. In another embodiment, both alleles comprise a replacement of
an mVR with at least on hVR.
[0044] In one aspect, a genetically modified mouse is provided,
wherein the mouse expresses an IgM that comprises a CH1 domain, and
the mouse expresses an IgG that lacks a functional CH1 domain or
that expresses an IgG that lacks both a functional CH1 domain and
that lacks a functional hinge region.
[0045] In one embodiment, the IgG is an IgG1.
[0046] In one embodiment, the mouse expresses four IgGs that are: a
modified IgG1 and a wild-type IgG3, IgG2a, and IgG2b. In another
embodiment, the mouse expresses no more than two IgGs that are: a
modified IgG1 and a wild-type IgG3. In a specific embodiment the
mouse expresses heavy chain isotypes that are: a wild-type IgM, a
wild-type IgD, a wild-type IgG3, a modified IgG1, a wild-type
IgG2a, a wild-type IgG2b, a wild-type IgA, and a wild-type IgE. In
another specific embodiment, the mouse expresses heavy chain
isotypes that are: a wild-type IgM, a wild-type IgD, a wild-type
IgG3, a modified IgG1, a wild-type IgA, and a wild-type IgE. In
various embodiments, the modification of the IgG1 comprises a
deletion of a CH1 domain and, optionally, a deletion of a hinge
region.
[0047] In one embodiment, the mouse is from a strain selected from
129, C56BL/6, a mixed 129.times.C57BL/6.
[0048] In one aspect, a mouse that expresses a heavy chain antibody
is provided, wherein the heavy chain antibody consists essentially
of a dimeric heavy chain, wherein the heavy chain lacks a
functional CH1 domain or lacks both a functional CH1 domain and a
functional hinge region, the heavy chain comprises a mammalian
heavy chain variable domain that comprises a sequence that is not
identical to a mammalian heavy chain variable domain encoded by a
germline variable region gene, and the heavy chain comprises a
human or mouse CH2 domain and a human or mouse CH3 domain, wherein
the mouse expresses a wild-type human or mouse IgM.
[0049] In one embodiment, the mouse comprises a functional
immunoglobulin light chain gene locus.
[0050] In one embodiment, wherein the mammalian heavy chain
variable domain is a human or mouse heavy chain variable
domain.
[0051] In one embodiment, the heavy chain antibody consists
essentially of a dimeric heavy chain lacking a functional CH1
domain and lacking a functional hinge region, wherein the heavy
chain comprises a human variable domain that comprises at least one
somatic mutation and comprises a CH2 domain and a CH3 domain. In a
specific embodiment, the CH2 domain and the CH3 domain are
independently selected from mouse and human domains. In a specific
embodiment, both the CH2 and the CH3 domain are human; in another
embodiment, both the CH2 and the CH3 domain are mouse.
[0052] In one aspect, a heavy chain antibody is provided, wherein
the heavy chain antibody comprises a heavy chain comprising a
non-camelid variable domain and a heavy chain constant region
lacking a CH1 domain.
[0053] In one embodiment, the heavy chain antibody further lacks a
hinge region.
[0054] In one embodiment, the heavy chain antibody comprises a
constant region that consists essentially of a hinge region, a CH2
domain, and a CH3 domain. In another embodiment, the heavy chain
antibody comprises a constant region that consists essentially of a
CH2 domain and a CH3 domain.
[0055] In one embodiment, the non-camelid variable domain is a
somatically mutated human or mouse heavy chain variable domain
obtained from an IgM- or an IgG-encoding nucleotide sequence of a B
cell from a mouse or a genetically modified mouse comprising a
human heavy chain variable region gene segment. In a specific
embodiment, the mouse comprises a humanized heavy chain variable
region gene segment. In another embodiment, the mouse comprises a
replacement of the endogenous mouse heavy chain variable region
gene segment locus with at least one human variable region gene
segment. In another embodiment, the mouse comprises a replacement
of the endogenous mouse heavy chain locus with at least one human
variable gene segment, at least one human D gene segment, and at
least one human J gene segment. In a specific embodiment, the
endogenous mouse immunoglobulin variable region locus is all or
substantially all replaced with a human immunoglobulin variable
region locus comprising a plurality of human V, D, and J gene
segments.
[0056] In one embodiment, the non-camelid variable domain is a
human or a mouse variable domain. In another embodiment, the
non-camelid variable domain is a human or a mouse variable domain
comprising one or more camelizing modifications. In a specific
embodiment, the camelizing modification is selected from L11S,
V37F, G44E, L45C, L45R, and W47G (Kabat numbering). In a specific
embodiment, the camelizing modification is selected from V37F,
G44E, and L45C. In a specific embodiment, the heavy chain variable
domain comprises a complementarity determining region 3 (CDR3) that
comprises two cysteines.
[0057] In one embodiment, the heavy chain antibody comprises a
dimer of a first heavy chain comprising a first heavy chain
variable domain and a second heavy chain comprising a second heavy
chain variable domain, wherein each of the first and the second
heavy chains lacks a CH1 domain (or lacks a CH1 domain and a hinge
region). In one embodiment, the human variable domain of the first
heavy chain of the dimer binds a first epitope, and the human
variable domain of the second heavy chain of the dimer binds a
second epitope, wherein the first and the second epitope are not
identical. In a specific embodiment, the heavy chain variable
domains of the first and the second heavy chains comprise human
heavy chain variable domains and/or human heavy chain FR regions as
described herein.
[0058] In one aspect, a genetically modified non-human cell is
provided, wherein the genetic modification comprises a deletion of
an IgG CH1 domain and the cell expresses a functional IgM. In a
specific embodiment, the cell comprises an IgM gene comprising a
sequence encoding a CH1 domain.
[0059] In one embodiment, the cell is selected from a non-human ES
cell, a pluripotent cell, and a totipotent cell. In a specific
embodiment, the non-human ES cell is selected from a mouse ES cell
and a rat ES cell.
[0060] In one aspect, a genetically modified non-human embryo is
provided, wherein the genetic modification comprises a modification
as described herein. In one embodiment, the genetic modification
comprises a deletion of an IgG CH1 domain and the non-human embryo
expresses a functional IgM. In a specific embodiment, the non-human
embryo comprises an IgM gene comprising a CH1 domain.
[0061] In one embodiment, the non-human embryo is a mouse embryo or
a rat embryo.
[0062] In one aspect, a non-human embryo comprising a donor cell is
provided, wherein the donor cell is genetically modified, and
wherein the genetic modification is a modification as described
herein. In one embodiment, the genetic modification comprises a
deletion of an IgG CH1 domain and the cell comprises an IgM gene
comprising a CH1 domain.
[0063] In one embodiment, the non-human embryo is a mouse embryo or
a rat embryo, and the donor cell is a mouse ES cell or a rat ES
cell, respectively.
[0064] In one aspect, a DNA construct is provided, wherein the DNA
construct comprises (a) a mouse homology arm homologous to a first
sequence 5' and immediately adjacent to the start of an IgG CH1
region; (b) a marker or drug selection cassette; and, (c) a
homology arm homologous to a second sequence 3' and immediately
adjacent to the end of an IgG CH1 region or, alternatively, a
homology arm homologous to a second sequence 3' and immediately
adjacent to the end of an IgG hinge region.
[0065] In one aspect, a method for making an antibody that lacks a
CH1 domain is provided, comprising: (a) immunizing a non-human
animal as described herein that lacks a functional CH1 domain in an
IgG or lacks a functional CH1 domain and lacks a functional hinge
region in the IgG, wherein the mouse expresses an IgM that
comprises a functional CH1 domain; (b) maintaining the non-human
animal under conditions sufficient for the non-human animal to make
an antibody; (c) identifying an antibody made by the mouse that
lacks a functional CH1 domain or that lacks a functional hinge
region; and, (d) isolating from the mouse the antibody, a cell that
makes the antibody, or a nucleotide sequence that encodes a
sequence of the antibody.
[0066] In one embodiment, the non-human animal comprises a
functional immunoglobulin light chain gene locus.
[0067] In one aspect, a method for humanizing a mouse heavy chain
antibody is provided, comprising immunizing a genetically modified
mouse that makes heavy chain antibodies with an antigen of
interest, allowing the mouse to mount an immune response,
identifying a mouse VH region of the mouse that is encoded in a B
cell of the mouse, wherein the B cell specifically binds the
antigen of interest, and humanizing the VH region.
[0068] In one embodiment, the genetically modified mouse that makes
heavy chain antibodies is a mouse as described herein. In one
embodiment, the mouse comprises at least one mVR gene segment
operably linked to a heavy chain constant locus that comprises an
intact IgM gene and that comprises an IgG gene that lacks a CH1
domain or that lacks a CH1 domain and lacks a hinge domain. In one
embodiment, the IgG gene is an IgG1 gene. In one embodiment, the
IgG gene is selected from IgG1, IgG2A, IgG2B, IgG3, and a
combination thereof.
[0069] In one embodiment, the method further comprises cloning a
nucleotide sequence encoding the humanized VH region onto a
nucleotide sequence of a human immunoglobulin constant region.
[0070] In one embodiment, the mouse mVR gene segment is from a
mouse VH gene family selected from VH1 and VH14, and the
humanization comprises replacing a mouse framework of VH1 or VH14
with a framework from a human VH1 gene. In one embodiment, the
human VH1 gene is selected from 1-2, 1-3, 1-8, 1-17, 1-18, 1-24,
1-45, 1-46, 1-58, and 1-69. In specific embodiments, the mVR gene
is a 1-58 gene and the human gene is a 1-18 gene; the mVR gene is a
1-26 gene and the human gene is a 1-2 gene; the mVR gene is a 1-50
gene and the human gene is a 1-46 gene; the mVR gene is a 1-17 gene
and the human gene is a 1-2 gene; the mVR gene is a 1-42 gene and
the human gene is a 1-2 gene; the mVR is a 14-1 gene and the human
gene is a 1-2 gene; or the mVR is a 14-2 gene and the human gene is
a 1-2 gene.
[0071] In one embodiment, the mVR gene segment is from a mouse VH
gene selected from a VH4, VH5, VH6, VH7, VH10, VH11, and VH13 gene,
and the humanization comprises replacing a mouse framework with a
framework from a human VH3 gene. In one embodiment, the human VH3
gene is selected from 3-7, 3-9, 3-11, 3-13, 3-15, 3-16, 3-20, 3-21,
3-23, 3-30, 3-33, 3-35, 3-38, 3-43, 3-48, 3-49, 3-53, 3-64, 3-66,
3-72, 3-73, and 3-74. In a specific embodiment, the mVR gene is a
7-1 gene and the human gene is a 3-72 gene; the mVR gene is a 3-6
gene and the human gene is a 4-59 gene; the mVR gene is a 5-6 gene
and the human gene is a 3-21 gene.
[0072] In one embodiment, the mVR gene segment is from a mouse VH
gene family selected from VH3 and VH12, and the humanization
comprises replacing a mouse framework with a framework from a human
VH4 gene. In one embodiment, the human VH4 gene is selected from
4-4, 4-28, 4-31, 4-34, 4-39, 4-59, and 4-61.
[0073] In one embodiment, the mVR gene segment is from a mouse VH4
gene family, and the humanization comprises replacing a mouse VH4
framework with a framework from a human VH6 gene. In one
embodiment, the human VH6 gene is 6-1.
[0074] In one embodiment, the mVR gene segment is from a mouse VH9
gene family, and the humanization comprises replacing a mouse VH9
framework with a framework from a human VH gene of the human VH7
family. In one embodiment, the human VH gene is selected from 7-4-1
and 7-81.
[0075] In one embodiment, the humanization further comprises making
one or more conservative or non-conservative substitutions, one or
more deletions, and/or one or more insertions in a mouse CDR such
that the mouse CDR corresponds more closely to a human CDR.
[0076] In one embodiment, the humanization further comprises making
one or more conservative or nonconservative substitutions, one or
more deletions, and/or one or more insertions in a human framework
such that the human framework corresponds more closely to the mouse
framework.
[0077] In one aspect, a genetically modified mouse is provided that
comprises a functional immunoglobulin light chain gene, wherein the
mouse expresses a heavy chain antibody that lacks a light chain and
that lacks a CH1 region or that lacks a CH1 region and a hinge
region.
[0078] In one embodiment, the mouse comprises an immunoglobulin
gene that lacks a sequence encoding a CH1 region, or lacks a
sequence encoding a hinge and a CH1 region. In one embodiment, the
immunoglobulin gene that lacks the sequence is one or more heavy
chain constant genes. In a specific embodiment, the immunoglobulin
gene that lacks the sequence is selected from an IgG1, IgG2a,
IgG2b, and IgG3 gene. In a specific embodiment, the mouse comprises
an IgM gene with a CH1 region, and/or a hinge region, and/or a CH1
region and hinge region.
[0079] In one embodiment, the antibody is expressed in response to
an antigen, and the antibody specifically binds the antigen.
[0080] In one embodiment, the antibody comprises a mouse VH domain.
In a specific embodiment, the mouse VH domain comprises a mouse VH
gene segment selected from 1-26, 1-42, 1-50, 1-58, 1-72, 3-6, 5-6,
7-1, 14-1, and 14-2.
[0081] In one embodiment, the antibody comprises a human VH domain.
In a specific embodiment, the human VH domain comprises a sequence
derived from a human VH gene segment selected from 1-2, 1-18, 1-46,
3-21, 3-72, and 4-59.
[0082] In one aspect, a genetically modified mouse is provided that
expresses a binding protein that consists essentially of two IgG1
heavy chains that each lack a CH1 domain, wherein the mouse
expresses an IgM that comprises a CH1 region, and wherein the mouse
is incapable of expressing from its genome an mRNA that comprises a
nucleotide sequence encoding a CH1 domain of an IgG1.
[0083] In one embodiment, the immunoglobulin heavy chains that each
lack a CH1 domain consist essentially of, from N-terminal to
C-terminal, a human or mouse heavy chain immunoglobulin variable
region, optionally a hinge region, a mouse CH2 region, and a mouse
CH3 region. In a specific embodiment, the heavy chain
immunoglobulin variable region is a human variable region, a hinge
region is present, and the mouse comprises a functional
immunoglobulin light chain gene locus.
[0084] In one aspect, a mouse is provided that expresses a heavy
chain antibody that lacks a light chain and that lacks a CH1 region
in whole or in part, wherein the mouse expresses a B cell receptor
on a B cell, wherein the B cell receptor on its surface displays a
binding molecule that comprises an immunoglobulin heavy chain
variable domain fused directly to an immunoglobulin hinge region or
fused directly to a CH2 region, wherein the binding molecule lacks
a CH1 region. In one embodiment, the binding molecule comprises an
IgG1 CH2 and CH3 region.
[0085] In one aspect, a method for making a heavy chain antibody is
provided, comprising immunizing a mouse with an antigen of
interest, wherein the mouse comprises an IgG gene that lacks a
sequence encoding a CH1 region, wherein the mouse comprises an
intact IgM constant region gene, allowing the mouse to mount an
immune response against the antigen of interest, and isolating from
the mouse a cell or protein that specifically recognizes the
antigen of interest, wherein the cell or protein comprises a heavy
chain antibody that lacks a CH1 domain and that lacks a cognate
light chain and that specifically binds the antigen of
interest.
[0086] In one embodiment, the mouse comprises a functional light
chain gene. In one embodiment, the mouse comprises a functional
light chain gene selected from lambda, kappa, and a combination
thereof.
[0087] In one embodiment, the mouse comprises a replacement of all
or substantially all mouse heavy chain V, D, J gene segments with
one or more human V, D, J gene segments.
[0088] In one embodiment, the IgG gene that lacks the sequence
encoding a CH1 is selected from an IgG1, IgG2a, IgG2b, IgG3, and a
combination thereof.
[0089] In one embodiment, the IgG gene that lacks the CH1 sequence
is IgG1, and the mouse lacks a gene encoding IgG2a, IgG2b, IgG3, or
a combination thereof. In one embodiment, the IgG gene that lacks
the CH1 sequence is IgG2a, and the mouse lacks a gene encoding
IgG1, IgG2b, IgG3, or a combination thereof. In one embodiment, the
IgG gene that lacks the CH1 sequence is IgG2b, and the mouse lacks
a gene encoding IgG1, IgG2a, IgG3, or a combination thereof. In one
embodiment, the IgG gene that lacks the CH1 sequence is IgG3, and
the mouse lacks a gene encoding IgG1, IgG2a, IgG2b, or a
combination thereof.
[0090] In one embodiment, the mouse comprises a B cell that bears
on its surface a B cell receptor, wherein the B cell receptor
comprises a rearranged heavy chain VDJ that binds the antigen of
interest, and wherein the B cell receptor comprises an IgM that
comprises a CH1 region, and wherein the IgM comprises a light
chain. In one embodiment, the light chain is VJ rearranged. In a
specific embodiment, the light chain is a kappa or a lambda light
chain that is cognate with the rearranged heavy chain VDJ that
binds the antigen of interest.
[0091] In one aspect, a mouse heavy chain antibody, human heavy
chain antibody, or chimeric human/mouse heavy chain antibody made
in a mouse according to the invention is provided.
[0092] In one aspect, a mouse heavy chain antibody, human heavy
chain antibody, chimeric human/mouse heavy chain antibody, or
humanized heavy chain antibody made using a heavy chain variable
region nucleotide sequence or fragment thereof made in a mouse
according to the invention is provided.
[0093] Other embodiments are described and will become apparent to
those skilled in the art from a review of the ensuing detailed
description.
DETAILED DESCRIPTION
[0094] The invention is not limited to particular methods, and
experimental conditions described, as such methods and conditions
may vary. 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 will be limited
only by the claims.
[0095] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by those
of ordinary skill in the art to which this invention belongs.
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 herein are incorporated
herein by reference in their entirety.
CH1 Domains and Antibody Production
[0096] Genetically modified non-human animals are provided that
make antibodies that lack a CH1 domain, including heavy chain
antibodies, i.e., antibodies that lack light chains. The
genetically modified non-human animals comprise a genetic
modification that includes a lack of a functional immunoglobulin
heavy chain domain (a CH1 domain), e.g., an IgG1 CH1 domain, and in
some embodiments a further modification comprising a deletion of a
hinge region in the immunoglobulin heavy chain that lacks the
functional CH1 domain, wherein the non-human animal expresses a
functional IgM. Other modifications include rendering isotypes
other than IgG1 and IgM to be nonfunctional, e.g., making deletions
in genes, or deletions of genes, for IgD, IgG3, IgG2a, IgG2b, IgA,
and IgE. Genetically modified non-human embryos, cells, and
targeting constructs for making the non-human animals, non-human
embryos, and cells are also provided.
[0097] Efforts at making genetically modified cells that can make
heavy chain antibodies (i.e., antibodies that lack a light chain)
have focused on mimicking heavy chain antibodies in other species,
e.g., in camelids and certain fish. This approach has been used to
genetically modify a mouse ES cell to delete CH1 domains in
immunoglobulin constant region genes of IgMs and IgGs, and also to
introduce heavy chain variable regions into the ES cell that are
camelid, or camelized VHH or VHH-like). The deletion of IgM and IgG
CH1 domains is undertaken presumably to prevent formation of
endogenous, natural antibodies to compete with camelized antibody
formation from a genetically modified locus. The addition of VHH
gene segments is undertaken presumably to mimic heavy chain
antibody formation in combination with the CH1 deletion. Heavy
chain antibodies from such animals will contain the VHH gene
segment. VHH gene segments are presumably thought to be necessary
for the proper expression of a heavy chain antibody, since in vitro
studies indicate that non-camelid VH domains do not satisfactorily
form expressible heavy chain antibodies when present in heavy
chains lacking a CH1 domain.
[0098] In camelids, however (and in some cartilaginous fish), genes
are present that include CH1 domains, or CH1-like domains.
VHH-containing antibodies that lack CH1 domains are believed to
result from RNA splicing or from rearrangement of DNA sequences
that can encode a CH1 region. Thus, even camelids have retained DNA
sequences encoding CH1 regions. Because humans (under some
circumstances) can make heavy chain antibodies lacking a CH1 region
in whole or in part (e.g., in human heavy chain disease), it might
be possible to compel non-camelids, such as mice, to form heavy
chains lacking a CH1 region under a given set of circumstances.
This approach relies upon not disturbing the germline structure of
a CH, but instead rendering the animal's light chain locus
nonfunctional. This approach assumes that with a nonfunctional
light chain locus those heavy chains that require a cognate light
chain for expression (e.g., full-length heavy chains having CH1
regions) are not made due to the lack of any kappa or lambda light
chain, such that only those heavy chains that can express and
secrete without a light chain (i.e., heavy chains lacking a CH1
region) will be expressed and secreted. The approach relies upon
the absence of functional kappa or lambda gene segments that can
rearrange to form a functional light chain gene, and on the absence
of any functional rearranged light chain gene, and thus requires a
genetic manipulation (e.g., a knockout) to destroy functionality of
both germline light chain loci. The approach relies upon "natural"
processes leading to non-use of the endogenous CH1 nucleotide
sequence, and that the "natural" process of CH1 silencing occurs in
class switching. There does not appear to be any possibility of
using such a process in any animal that contains a functional light
chain gene. Furthermore, it appears that the "natural" process
includes the synthesis of large amounts of normal RNA, i.e., RNA
that includes a region encoding a CH1 region.
[0099] Compositions and methods are provided for making a mouse
that makes an antibody that lacks an immunoglobulin CH1 domain (and
optionally a hinge region), including heavy chain antibodies, and
including antibodies that comprise VH domains (e.g., mouse or human
VH domains). The methods include selectively rendering an
endogenous non-IgM CH1 region to be nonfunctional (e.g., by a
deletion of a sequence of a CH1 domain), and employing either
unrearranged endogenous mouse variable region (mVR) gene segments
or unrearranged human variable region (hVR) gene segments at the
endogenous mouse variable region locus to make a chimeric
human/mouse antibody in a mouse. The deletion of the CH1 domain is
made in one or more IgG genes, but not in an IgM gene. The approach
selectively renders one or more IgG CH1 domains nonfunctional while
retaining a functional IgM. In addition to a deletion of the one or
more IgG CH1 domains, a further embodiment provides for deleting or
rendering nonfunctional the hinge region of the IgG(s) in which the
CH1 domain is deleted or rendered nonfunctional.
[0100] The IgG CH1 deletion approach employs a relatively
conservative disruption in natural B cell development in the
animal, because not all Ig isotypes of the genetically modified
non-human animal will exhibit a nonfunctional CH1 or a deletion of
the CH1 domain (and, optionally, hinge). Thus, the CH1 modification
does not occur in IgM molecules and thus does not affect those
steps in early B cell development that depend on an IgM having a
functional CH1. Because the IgM is not modified, animals bearing
one or more deletions of the CH1 domain of an IgG (and optionally a
hinge region of the IgG), but not an the CH1 domain of an IgM,
should be able to process a satisfactorily large repertoire of
variable regions in clonal selection steps prior to presentation of
the variable domain in the context of an IgG. Thus in various
embodiments, any deleterious affect of the genetic modification(s)
on the diversity of variable regions available for use in a heavy
chain antibody should not negatively impact the pool of variable
regions available for selection in an IgG context. Further, where
the CH1 sequence that is rendered nonfunctional (e.g., deleted) in
the germline is an IgG1, the mouse will lack the ability to make
any RNA that encodes a CH1 domain.
[0101] Genetically modifying a non-human animal to render a CH1
domain or a CH1 domain and a hinge region of one or more IgG
isotypes nonfunctional may result in a mouse that is able to
select, from a full or substantially full repertoire of VH regions,
a suitable VH region to express in a heavy chain antibody.
Selectively modifying IgG isotypes (but not IgM) avoids a potential
reduction in the number of VH regions that survive selection due to
a lack of a CH1 domain or a lack of a CH1 domain in IgM. Thus, a
fuller repertoire of VH regions is available for selection in the
context of an IgG (that lacks a CH1 domain or that lacks a CH1
domain and that lacks a hinge region). Thus, selection of a VH
domain in a genetically modified mouse in accordance with the
invention does not depend, e.g., on which VH domain might help
overcome early IgM-dependent B cell developmental hurdles that are
due to modified IgM structures. Instead, early IgM-dependent steps
should occur as normal, resulting in a large repertoire of heavy
chains available for selection as to their suitability to express
in the context of an IgG that lacks a CH1 domain or that lacks a
CH1 domain and lacks a hinge region.
[0102] Thus, in various embodiments, a genetically modified mouse
in accordance with the invention should maintain functional IgM
expression, which should provide an opportunity for a more natural
clonal selection process. For example, with a functional IgM (e.g.,
an IgM that does not lack a CH1 domain), both surrogate light chain
and the cognate light chain will be able to associate through the
IgM's CH1 domain and participate in selection processes in early B
cell development. In a genetically modified mouse in accordance
with the invention, it is believed that class switching to an IgG
isotype is the first selection step where any selection of heavy
chain variable domains that can be expressed in the context of a
constant domain lacking a functional CH1 domain or lacking a
functional CH1 domain and a functional hinge is encountered.
IgM in B Cell Development
[0103] Although observations in camelids, certain fish, and in
pathological conditions reveal that under some circumstances an
antibody that lacks a CH1 domain of its heavy chain constant region
can be expressed in the absence of a cognate light chain, normal
development of antibody-producing B cells generally requires the
presence of a CH1 domain. All heavy chain isotypes, including IgM,
comprise a CH1 domain. Both the surrogate light chain and a cognate
light chain are believed to interact with a given heavy chain
through the heavy chain's CH1 domain in the context of an IgM. To
the extent that development of heavy chain antibodies depends upon
structural integrity or functionality of an IgM isotype heavy
chain, disruption of the IgM's structural integrity or function
would be undesirable.
[0104] Normal development of antibodies requires that antibodies
survive throughout a multiplicity of complex selection schemes that
result in the survival and ultimate expression of functional and
useful antibodies. Disruptions in antibody structure can prove
deleterious to the survival and ultimate expression of an antibody
to the extent that the structural disruption results in the
inability of the antibody to effectively compete and evolve to the
satisfaction of one or more of nature's antibody selection
schemes.
[0105] Early in antibody development, antibody heavy chains undergo
a selection process wherein nature chooses, through a variety of
selection schemes, suitable heavy chains to undergo further
selection to eventually form functional and affinity-matured
antibodies. Antibody heavy chains expressed from recombined heavy
chain gene segments in progenitor B cells (or, pro-B cells) are
normally paired with a surrogate light chain for presentation on
the surface of the pro-B cell in an IgM isotype to form a structure
(which includes other co-receptors) referred to as a pre-B cell
receptor, or pre-BCR. Once the pre-BCR is presented on the cell
surface, the pre-BCR is believed to signal its appropriate
formation of the complex to the cell, effectively instructing the
cell that the heavy chain has passed this early selection step.
Thus the cell is informed that the heavy chain may undergo further
selection. If the heavy chain contains a defect that is deleterious
to the formation of a pre-BCR when presented in the context of an
IgM and a surrogate light chain, the cell will undergo apoptosis.
If the cell undergoes apoptosis, the usefulness, or contribution to
diversity, of the heavy chain variable region of the heavy chain
will be lost. Thus, a very early step in antibody selection
requires presentation of the heavy chain together with a surrogate
light chain in the context of an IgM isotype. The surrogate light
chain is believed to interact with IgM at least in part through
IgM's CH1 domain. A failure or disruption in antibody structure at
this early juncture (e.g., a nonfunctional CH1 domain) can result
in clonal selection failure, loss of the pro-B cell that expresses
the heavy chain, and loss of the possibility of employing the
particular heavy chain variable domain in a useful antibody.
[0106] Once the cell bearing the pre-BCR passes this selection
step, the next selection step requires that the heavy chain be
paired with a cognate light chain (e.g., either kappa or lambda in
mice and humans). The paired heavy chain/cognate light chain
structure is again presented on the surface of the cell, now a
naive pre-B cell, in the context of an IgM isotype through the
IgM's CH1 domain. This complex on the surface results in a
functional, membrane-bound, B cell receptor (BCR). This BCR is
believed to signal to the cell that the heavy chain is suitable for
further selection, and that the cell may now commit to expressing
this particular light chain and proceed to further B cell
maturation steps, including affinity maturation and class
switching. If the heavy chain contains a defect that is deleterious
to the formation of a BCR when presented in the context of an IgM
and its cognate light chain, the cell will undergo apoptosis. If
the cell undergoes apoptosis, the usefulness, or contribution to
diversity, of the heavy chain variable region of the heavy chain
will be lost. Thus, a very early step in antibody selection
requires presentation of the heavy chain together with a surrogate
light chain in the context of an IgM isotype. Again, a failure or
disruption in antibody structure (e.g., a non-functional CH1
domain) at this early juncture can result in clonal selection
failure and concomitant loss of the pre-B cell that expresses the
heavy chain.
[0107] Having survived selection thus far, the pre-B cell that
presents the heavy chain paired with its cognate light chain in the
IgM context then undergoes a maturation process that ultimately
results in class switching and further selection mechanisms in
which the heavy chain and cognate light chain are presented on the
B cell surface in the context of an IgG isotype. It would be at
this step that any selection of IgG heavy chains that lack a CH1
domain or that lack a CH1 domain and a hinge region would occur. In
animals according to the invention, it is believed that a normal
repertoire of heavy chain variable regions would be available for
selection based upon whether the variable domain would survive to
be expressed in an IgG heavy chain that lacks a CH1 domain or that
lacks a CH1 domain and a hinge region. In contrast, mice that have
impaired IgMs would likely not present a full repertoire of heavy
chain variable regions, since only those variable regions capable
of surviving selection in the context of an impaired IgM would be
available for class switching.
[0108] Thus, an animal lacking a functional IgM may experience a
marked reduction in the ability to make a B cell population
following rearrangement of otherwise suitable heavy chain variable
gene segments. In such a case, even where an ample supply of heavy
chain variable regions is available (i.e., the animal has a
suitable number of heavy chain variable region gene segments
capable of rearranging), a satisfactory population of B cells that
display a desirable degree of diversity may not form because of an
IgM impairment that mitigates against survival of a heavy chain
during the selection process.
Heavy Chain Antibody Production with a Functional IgM Gene
[0109] A suitable number of rearranged heavy chain variable regions
that can effectively survive selection when presented during B cell
development in the context of an IgM is desirable to be maintained
in order to generate sufficient diversity to make antibodies by
immunizing a non-human animal with an immunogen of interest. Thus,
a genetically modified non-human animal that comprises a
nonfunctional CH1 domain or a nonfunctional CH1 domain and a
nonfunctional hinge region in an immunoglobulin heavy chain should
not comprise a CH1 deletion in both IgM alleles.
[0110] In some embodiments, it is not desirable to delete CH1
domains of all Ig isotypes in order to make a heavy chain antibody
in a genetically modified animal. Thus, methods and compositions
are provided for making a heavy chain antibody in a genetically
modified non-human animal by disabling, deleting, or otherwise
rendering non-functional a nucleotide sequence encoding a CH1
domain or fragment thereof of an IgG (and in some embodiments also
disabling, deleting, or otherwise rendering nonfunctional a hinge
region of the IgG) while allowing other isotypes (e.g., IgM) to
retain functional CH1 domains. It is believed that functionality of
other isotype CH1 domains (other than one or more selected IgG CH1
domains) results in a B cell development process that does not
disrupt or substantially disrupt developmental steps in which the
heavy chain variable domain is presented in the context of a
non-IgG isotype, e.g., in an IgM isotype. Thus disruption of, e.g.,
IgM-dependent steps during B cell development is relatively
minimized. Without limitation as to the invention (which is
described by the claims) the inventors propose that minimalizing
disruption of early selection steps associated with presentation of
the heavy chain variable domain in an IgM context will result in
more cells that bear the heavy chain variable regions surviving to
undergo class-switching to an IgG isotype and selection in the
context of an IgG that lacks a functional CH1 domain or that lacks
a functional CH1 domain and lacks a functional hinge region.
[0111] Accordingly, a genetically modified non-human animal is
provided, along with methods and compositions for making the
animal, wherein the genetic modification results in lack of a
functional CH1 domain (in a further embodiment lack of a functional
hinge region) in an Ig domain that is not an IgM domain. In various
embodiments, a sequence encoding CH1 or the CH1 and the hinge
region (or a substantially functional portion thereof) are deleted
in the genome of the genetically modified animal. The genetically
modified non-human animal is useful in making heavy chain
antibodies (i.e., antibodies that lack a light chain), including
fully human antibodies (in a mouse genetically modified to include
human immunoglobulin genes) and chimeric human/mouse antibodies
(e.g., in a mouse genetically modified to include human variable
region gene segments, D regions, and J regions, or in a mouse
having a human transgene capable of trans-switching to a
genetically modified IgG isotype that lacks a functional CH1 domain
or that lacks a functional CH1 domain and lacks a functional hinge
region).
Heavy Chain Antibodies
[0112] Antibodies are useful as human therapeutics. Heavy chain
antibodies, i.e., antibodies that lack a light chain, are also
useful as human therapeutics. Because heavy chain antibodies lack a
light chain, they are smaller and thus expected to exhibit better
tissue penetration than antibodies that contain light chains, yet
have a similar or more favorable pharmacokinetic profile and yet
retain similar effector function as compared to a conventional
antibody. Because they are smaller, heavy chain antibodies are also
capable of administration at a higher dose in a given volume. A
frequent method of administering antibodies is by subcutaneous
injection, and a reduction in administration volume for a given
dosage of antibody can provide benefits to patients and avoid
complications and pain due to subcutaneous injections of large
volumes.
[0113] Another advantage of heavy chain antibodies is the ability
to make bispecific antibodies by heterodimerizing heavy chains with
specificity for two different epitopes in a single therapeutics.
Because heavy chain antibodies lack a light chain, they are
particularly suited for making bispecific antibodies since there is
no requirement to engineer a common light chain that would not
interfere with binding affinity or specificity of either heavy
chain but also enable suitable expression of the bispecific
antibody.
[0114] The genetically modified animals of the invention can be
used to make a wide variety of heavy chain antibodies. The genetic
modifications described herein can be made, e.g., in any suitable
mouse strain. The mouse strain can have any genetic background
suitable for making a heavy chain antibody of choice. Some genetic
backgrounds that encompass particular embodiments are provided
below.
[0115] The genetically modified animal can be a mouse comprising a
genetic modification in accordance with the invention and one or
more unrearranged human variable region gene segments, one or more
unrearranged D region gene segments, and one or more unrearranged J
region gene segments replacing an endogenous mouse heavy chain
variable region locus. In such a mouse, the humanized variable
region locus is capable of recombining to form a rearranged
variable region gene upstream of endogenous mouse constant domain
sequences (wherein one or more of the immunoglobulin constant
region genes is modified as described herein). The mouse would thus
be capable of making a chimeric human variable/mouse constant heavy
chain antibody. Upon exposure to an immunogen of interest, the
mouse would be capable of generating a heavy chain antibody in
accordance with the invention that is affinity matured and capable
of specifically binding an epitope of the immunogen of
interest.
[0116] The genetically modified animal can be a mouse comprising an
endogenous mouse variable region that includes unrearranged
endogenous mouse variable region gene segments, unrearranged
endogenous mouse D region gene segments, and unrearranged
endogenous mouse J region gene segments, wherein the mouse
comprises a genetic modification of a mouse heavy chain constant
region as described herein. The mouse would thus be capable of
making a mouse heavy chain antibody. Upon exposure to an immunogen
of interest, the mouse would be capable of generating a heavy chain
antibody in accordance with the invention that is affinity matured
and capable of specifically binding an epitope of the immunogen of
interest.
[0117] The genetically modified animal can be a mouse comprising a
human transgene that comprises unrearranged human variable region
gene segments, unrearranged human D gene segments, and unrearranged
human J gene segments, a mu gene, and a sequence that allows for
trans-switching. The mouse would further comprise a mouse heavy
chain constant region modification as described herein. The mouse
would be thus capable of making a fully human IgM antibody, and
through trans-switching a chimeric human variable/mouse constant
antibody, wherein the constant domain comprises a genetic
modification as described herein. Upon exposure to an immunogen of
interest, the mouse would be capable of generating a heavy chain
antibody in accordance with the invention that is affinity matured
and capable of specifically binding an epitope of the immunogen of
interest.
In Vitro Expression of Heavy Chain Antibodies
[0118] The inventors have established that a normal human or mouse
heavy chain variable region (hVR or mVR) can be expressed in an in
vitro system in the context of an IgG that lacks a functional CH1
domain. The inventors expressed an hVR from an unrearranged hVR
minilocus in a mouse with a wild-type mouse IgM. The expressed hVR
was cloned onto an IgG2b lacking a CH1 domain, and the resulting
hVR-IgG2b.DELTA.CH1 expressed and was secreted by a CHO cell
transiently transfected with the hVR-IgG2b.DELTA.CH1 construct,
effectively establishing that an hVR selected in a mouse having a
wild-type IgM can be expressed and secreted by a cell when switched
to an IgG lacking a functional CH1 domain, i.e., as a heavy chain
antibody.
[0119] The inventors constructed an in vitro system to express
heavy chains that lack CH1 domains and that have hVRs or human
camelized VRs (hVR*s) in CHO cells. The VRs were obtained from a
RAG mouse that contained a replacement of the endogenous mouse
heavy chain locus with a human heavy chain variable region
minilocus (having three human V region gene segments, 6-1, 1-2, and
1-3, all human DH gene segments, and all human JH gene segments).
The endogenous mouse immunoglobulin kappa and lambda light chain
loci were intact and functional.
[0120] Chimeric heavy chain (hVR-mFc) and camelized heavy chain
(hVR*-mFc) constructs were made, for expression in CHO cells, using
the VR sequences obtained from the mouse bearing the minilocus
described above. The chimeric heavy chains were the product of
normal V-D-J recombination during B cell development in the mouse
to form a functional antibody comprising a chimeric heavy chain
(hVR-mFc) and a mouse light chain. hVR-mFc and hVR*-mFc constructs
were made both having a CH1 domain and lacking a CH1 domain.
[0121] Transient transfection of hVR-mFc and hcVR-mFc constructs in
CHO cells showed that in the absence of a CH1 domain, heavy chains
having hVRs and hVR*s were expressed and remained soluble in the
supernatant. In the presence of a CH1 domain, heavy chains
containing either hVRs or hVR*s did not express in supernatant.
This observation suggested that such heavy chain antibodies could
be made without employing camelid VHH domains, e.g., with human or
mouse VH domains, in heavy chain antibodies that lacked a CH1
domain.
Humanized Heavy Chain Antibodies
[0122] To produce a humanized version of a heavy chain antibody of
the present invention, an animal homozygous for the modification is
immunized with an antigen and once a specific immune response of
the animal has been established, cells from the spleen of the
immunized animal are fused with a suitable immortal cell (e.g., a
myeloma cell) to produce hybridoma cells. Alternatively, the
antibodies can be obtained directly from B cells of the immunized
animal. Supernatants from the hybridoma cells (or, e.g., from
isolated B cells) are screened for the presence of antibody by
enzyme-linked immunosorbent assay (ELISA) and antibodies specific
for the antigen can be selected based on desired
characteristics.
[0123] Heavy chain variable region (VH) nucleic acids can be
isolated from hybridoma and/or B cells using standard molecular
biology techniques known in the art (Sambrook, et al. 1989.
Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring
Harbor, N.Y.; Ausubel, et al. 1995. Short Protocols in Molecular
Biology, 3rd ed., Wiley & Sons). Once the VH nucleic acid
sequence has been determined, the deduced amino acid sequence can
be obtained and compared to other human VH sequences to identify a
group of related VH sequences that have a similar sequence. Related
VH sequences can be obtained using antibody databases available to
those of skill in the art, e.g., The International ImMunoGeneTics
Information System.RTM. (IMGT.RTM.). This comparison may be
performed by alignment of the sequences accomplished either by eye
or, alternatively, electronically by employing an alignment program
(e.g., CLUSTAL). In this comparison, the complementary determining
regions (CDRs) and framework regions (FRs) are identified. CDR and
FR residues are determined according to a standard sequence
definition (e.g., Kabat et al. 1987, Sequences of Proteins of
Immunological Interest, National Institutes of Health, Bethesda
Md.; Chothia and Lesk, 1987. J. Mol Biol. 196:901-917). Those
skilled in the art will appreciate that there may occasionally
exist discrepancies in methods of numbering and determining the CDR
and FR regions of an immunoglobulin heavy chain sequence. In such
cases, the structural definition is preferred, however, the
residues identified by the sequence definition method are
considered important FR residues for determination of which
framework residues to substitute based on a comparison of heavy
chain sequences.
[0124] Once aligned, substitutable positions in the VH sequences
are identified. If the identity of an amino acid at a position in
the isolated VH sequence varies when compared to the other human VH
sequences, that position is evaluated for the suitability of a
substitution at that position of the isolated VH sequence.
Therefore, any position in the isolated VH sequence that varies
with the other related human VH sequence(s) to which it is being
compared can potentially serve as a position that could be
substituted with the amino acid at the corresponding position found
in one or any of the other related human VH sequences. Positions
that share identity with the other related human VH sequences,
i.e., those that do not demonstrate variability, are determined to
be nonsubstitutable positions. In various embodiments, the above
methods are employed to provide a consensus human heavy chain
antibody sequence.
[0125] A humanized heavy chain antibody for the purposes described
herein is an immunoglobulin heavy chain amino acid sequence variant
or fragment thereof that is capable of binding to a predetermined
antigen and that comprises a FR region having a substantially
similar or an identical amino acid sequence as compared with a
human FR amino acid sequence, and a CDR having a substantially
similar or an identical amino acid sequence to a non-human CDR
amino acid sequence. In general, a humanized heavy chain antibody
has one or more amino acid residues that are derived from a
non-human source. Such residues are typically derived from a heavy
chain variable domain. Further, these residues may have associated
characteristics such as, for example, affinity and/or specificity
as well as other desirable biological activity associated with
antibody function.
[0126] In various embodiments, the humanized heavy chain antibody
comprises substantially all of at least one, and in other
embodiments at least two, VH domains in which all or substantially
all of the CDR regions correspond to those of a non-human VH domain
and all or substantially all of the FR regions are those of a human
VH domain sequence. The humanized heavy chain antibody will
comprise a unique immunoglobulin constant region (Fc), that in one
embodiment lacks at least the CH1 domain, and in one embodiment
also lacks the hinge region of a human Fc. In one embodiment, the
heavy chain antibody will not comprise a light chain and will
comprise the CH2 and CH3 regions of an immunoglobulin G (IgG) heavy
chain constant region. In one embodiment, the constant region of
the heavy chain antibody will include the hinge, CH2 and CH3
regions of the IgG heavy chain Fc. In one embodiment, the constant
region of the heavy chain antibody will include a CH1 region of an
IgM.
[0127] The humanized heavy chain antibody will be selected from any
class of IgGs, including IgG1, IgG2, IgG3 and IgG4. In various
embodiments, the constant region may comprise sequences from more
than one class of IgG, and selecting particular constant regions to
optimize desired effector functions is within the ordinary skill in
the art.
[0128] In general, the heavy chain FR and heavy chain CDR regions
of the humanized heavy chain antibody need not correspond precisely
to the parental sequences, e.g., the non-human heavy chain CDR or
the human heavy chain FRs may be altered by substitution, insertion
or deletion of at least one residue so that the heavy chain CDR or
heavy chain FR residue at a given site does not correspond to
either the human heavy chain FR sequence or the non-human heavy
chain CDR sequence. Such mutations, however, will not be extensive.
In one embodiment, at least 75% of the humanized heavy chain
antibody residues will correspond to those of the parental heavy
chain FR and heavy chain CDR sequences, in another embodiment 90%,
and in another embodiment greater than 95%.
[0129] Humanized heavy chain antibodies as disclosed herein are, in
one embodiment, prepared by a process of analyzing parental
sequences and various conceptual humanized composite sequences in
silico, using computer programs available and known to those
skilled in the art. Sequence modifications to make humanized
versions and/or for changing characteristics such as
immunogenicity, affinity, etc. are made employing methods known in
the art (e.g., U.S. Pat. No. 5,565,332 Hoogenboom et al.; U.S. Pat.
No. 5,639,641 Pedersen et al.; U.S. Pat. No. 5,766,886 Studnicka et
al.; U.S. Pat. No. 5,859,205 Adair et al.; U.S. Pat. No. 6,054,297
Carter et al.; U.S. Pat. No. 6,407,213 Carter et al.; U.S. Pat. No.
6,639,055 Carter et al.; U.S. Pat. No. 6,849,425 Huse et al.; U.S.
Pat. No. 6,881,557 Foote; U.S. Pat. No. 7,098,006 Gorman et al.;
U.S. Pat. No. 7,175,996 Watkins et al.; U.S. Pat. No. 7,235,643
Nicolaides et al.; U.S. Pat. No. 7,393,648 Rother et al.; U.S. Pat.
No. 7,462,697 Couto et al.).
[0130] In various embodiments, desired substitutions to a parental
heavy chain antibody sequence to make a variant of a parental heavy
chain antibody are those that in one embodiment maintain, or in
another embodiment increase, the antigen binding activity of the
parental heavy chain antibody. In general, a heavy chain antibody
variant of a parental heavy chain antibody has an antigen binding
affinity that is at least 10%, at least 20%, at least 30%, at least
40%, at least 50%, at least 60%, at least 70%, at least 80%, at
least 90% or at least 100% (e.g., at least 150%, at least 200%, at
least 500%, at least 1000%, or up to at least 10,000%) of the
binding affinity of the parental heavy chain antibody to a
particular antigen. In some embodiments, a variant heavy chain
antibody will comprise a single substitution as compared to a
parental heavy chain antibody. However, in other embodiments,
several amino acids, e.g., up to about 5 or 10 or more, are
substituted as compared to the parental heavy chain antibody
sequence that are derived from other human heavy chain sequences
that share identity at a given position. Substitutions in one
embodiment are conservative (i.e., an amino acid sharing similar
properties to the residue to be replaced), and in another
embodiment non-conservative (i.e., an amino acid sharing different
properties to the residue to be replaced). In various embodiments,
the resultant variant heavy chain antibody is tested to confirm
that the desired binding affinity and/or specificity has not been
significantly decreased by the replacement residues. In some
embodiments, an improved variant heavy chain antibody is produced
by the substitution of amino acids from a different human heavy
chain sequence.
[0131] Naturally occurring heavy chain antibodies (e.g., found in
camelids) have been demonstrated to contain unique amino acid
residues at positions corresponding to the interface between heavy
and light chain variable regions in traditional antibody molecules
(i.e., two heavy chains and two light chains). These interface
residues are known to affect the proximity or orientation of the
two chains relative to one another in traditional antibodies.
Although these natural heavy chain antibodies are known to contain
replacement of residues that correlate with the absence of light
chain variable regions, they retain the residues at other positions
in the sequence as compared to traditional antibodies for
preserving the characteristic immunoglobulin fold. The
substitutions found in natural heavy chain antibodies are L11S,
V37F, G44E, L45R or L45C, W47G and additional cysteine residues
that contribute to a disulfide bond between the CDR1 and CDR3 of
the heavy chain variable region. In some embodiments, heavy chain
antibodies of the present invention may retain the residue of the
parental antibody at these positions. In other embodiments, the
parental antibody may display mutations at these positions that are
associated with the residues in natural heavy chain antibodies. In
some embodiments, it may be desirable to retain the same residue as
is found in the parental heavy chain antibody at at least one of
these positions or, in one embodiment, all of these positions when
making a humanized heavy chain antibody derived from an isolated VH
sequence from a genetically modified mouse as described herein. In
various embodiments, a person of skill in the art will understand
that these interface residues are not reasonably expected to be
involved in interchain interactions in heavy chain antibodies made
by the genetically modified mouse as described herein.
Making Genetically Modified Animals
[0132] Genetic modifications for making an animal that expresses a
heavy chain antibody are conveniently described by using the mouse
as an illustration. A genetically modified mouse according to the
invention can be made in a variety of ways, particular embodiments
of which are discussed below.
[0133] A schematic illustration (not to scale) of an IgG1 locus is
provided in FIG. 1 (top) to show CH domain arrangement at the IgG1
locus. As illustrated, domains CH1, CH2, and CH3 and the hinge
region are present in readily identifiable spans of nucleotide
downstream of a switch region.
[0134] A genetically modified mouse lacking a nucleotide sequence
encoding a CH1 domain of an IgG1 but containing a hinge region can
be made by any method known in the art. For example, a targeting
vector can be made that replaces the IgG1 gene with a truncated
IgG1 lacking a CH1 domain but containing the hinge. FIG. 2
illustrates a mouse genome (top) targeted by a targeting construct
having a 5' (with respect to the direction of transcription of the
genomic IgG1 gene) homology arm containing sequence upstream of the
endogenous CH1 domain, followed by nucleotide sequences that encode
an IgG1 hinge, an IgG1 CH2 domain, an IgG1 CH3 domain, a drug
selection cassette (e.g., a loxed resistance gene), and an IgG1
transmembrane domain, and a 3' homology arm containing sequence 3'
with respect to the transmembrane domain. Upon homologous
recombination at the locus and removal of the drug selection
cassette (e.g., by Cre treatment), the endogenous IgG1 is replaced
by an IgG1 that lacks a CH1 domain (bottom of FIG. 2; lox site not
shown). FIG. 1 (IgG1.DELTA.CH1, middle) shows the structure of the
resulting locus, which will express an IgG1 having a J region
sequence fused to the hinge sequence.
[0135] A genetically modified mouse lacking a nucleotide sequence
encoding a CH1 domain of an IgG1 and lacking a nucleotide sequence
encoding a hinge region can be made by any method known in the art.
For example, a targeting vector can be made that replaces the IgG1
gene with a truncated IgG1 lacking a sequence encoding a CH1 domain
and lacking a sequence encoding the hinge region. FIG. 3
illustrates a mouse genome (top) targeted by a targeting construct
having a 5' (with respect to the direction of transcription of the
genomic IgG1 gene) homology arm containing sequence upstream of the
endogenous CH1 domain, followed by nucleotide sequences that encode
an IgG1 CH2 domain, an IgG1 CH3 domain, a drug selection cassette
(e.g., a loxed resistance gene), and an IgG1 transmembrane domain,
and a 3' homology arm containing sequence 3' with respect to the
transmembrane domain. Upon homologous recombination at the locus
and removal of the drug selection cassette (e.g., by Cre
treatment), the endogenous IgG1 gene is replaced by an IgG1 gene
that lacks a sequence encoding a CH1 domain (bottom of FIG. 3; lox
site not shown). FIG. 1 (IgG1.DELTA.CH1-.DELTA.hinge, bottom) shows
the structure of the resulting locus, which will express an IgG1
having a J region sequence fused to the CH2 domain.
[0136] A genetically modified mouse lacking an IgG1 CH1 sequence
(IgG1.DELTA.CH1), or lacking an IgG1 CH1 sequence and lacking a
hinge (IgG1.DELTA.CH1-.DELTA.hinge), can be further modified to
favor usage of the modified IgG1 isotype by deleting one or more
other IgG isotypes, e.g., by deleting or functionally disabling
sequences encoding IgG2b and IgG2a. For example, a targeting
construct is made having a 5' homology arm containing sequence
upstream of the endogenous hinge region sequence (or upstream of
the endogenous CH1 domain sequence), sequences that encode the IgG1
CH2 and CH3 domains, a drug selection cassette followed by a
sequence encoding the IgG1 transmembrane domain, followed by
another drug selection cassette if desired. Upon homologous
recombination at the locus and removal of the drug selection
cassette(s) (e.g., by Cre treatment), the endogenous heavy chain
constant locus contains only two IgG genes: an endogenous IgG3 and
the IgG1.DELTA.CH1 (see FIG. 4, bottom; recombinase site(s) not
shown; see FIG. 6, bottom) or IgG1.DELTA.CH1-.DELTA.hinge (see FIG.
5, bottom; recombinase site(s) not shown; see FIG. 6, bottom).
[0137] An IgG1 expressed in a genetically modified mouse having an
IgG1.DELTA.CH1-.DELTA.hinge or an
IgG1.DELTA.CH1.DELTA.IgG2a.DELTA.IgG2b allele will have a structure
as shown on the right panel of FIG. 10, i.e., the VH domain will be
fused to the CH2 domain. The left panel of FIG. 10 provides, for
comparison, a wild-type IgG1 antibody, showing its CH1 domain
linked via a hinge region to the CH2 domain, and linked by
disulfide linkage to the light chain constant domain CL. In
contrast, the antibody made by the genetically modified mouse lacks
the hinge and CH1 domains and thus lacks any CL domain.
[0138] Genetically modified mice as described above, and others,
are made by introducing a suitable targeting construct into a
suitable mouse ES cell (in one or more independent targetings), and
positive clones comprising a marker or selection cassette of the
targeting construct are identified and grown. Clones are then
employed as donor ES cells in a host embryo under conditions
suitable for making a chimeric mouse or a fully ES cell-derived
mouse. The marker or selection cassette can be optionally removed,
either at the ES cell stage or in the chimeric or ES cell-derived
mouse, e.g., by employing a loxed cassette and breeding to a
Cre-containing strain, or by electroporating the ES cell with a Cre
expression vector.
[0139] A genetically modified mouse having an
IgG1.DELTA.CH1-.DELTA.hinge allele (heterozygous) was made in
accordance with an embodiment of the invention. Serum was isolated
from the mouse and blotted in a Western (reducing conditions) using
an anti-mouse IgG1 antibody to detect heavy chain. In contrast to a
wild-type mouse, which displayed a band corresponding in size to a
wild-type IgG1 heavy chain, the mouse genetically modified to
contain the IgG1.DELTA.CH1-.DELTA.hinge allele also expressed a
heavy chain that reacted with anti-mouse IgG1 antibody that had the
expected size of a heavy chain antibody consisting of the VH, CH2,
and CH3 domains (see FIG. 8).
EXAMPLES
Example 1
In Vitro Expression of Heavy Chain Antibodies
[0140] Chimeric heavy chain constructs were made using molecular
biology techniques (e.g., see Maniatis et al. 1982. Molecular
Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory) to
fuse human variable regions with a murine IgG2b (mIgG2b) constant
region. The human variable gene segment for each construct was a
full-length human variable gene segment containing both exons
(i.e., leader sequence plus mature sequence), identified from an
hVR of an IgM isolated from a naive RAG mouse that contained a
replacement of the endogenous mouse immunoglobulin heavy chain
locus with three hVR gene segments, all hDH gene segments, and all
hJH gene segments. The light chain of the IgM antibody was a mouse
light chain.
[0141] Two versions of the mIgG2b sequence were used; one with and
one without a CH1 domain. Several other constructs were also made
to serve as transfection and expression controls. A first control
construct was made using a cytokine receptor fused to the CH2 and
CH3 domains of mouse IgG2a (mIgG2a) constant region (Control I).
Two other controls were constructed by fusing a murine ROR signal
sequence to a murine IgG2a sequence with and without CH1 domains
(Control II and III, respectively).
[0142] Camelized versions of each human variable region were also
made using PCR site-directed mutagenesis techniques (e.g., see
Hutchinson et al. 1978. Mutagenesis at a specific position in a DNA
sequence. J. Biol. Chem. 253(18):6551-60). Two specific primer sets
were used for each variable region to create specific mutations
within the human variable region sequence resulting in a human
variable region sequence containing camel-like features. Primers L1
(SEQ ID NO:1) and HH1.2 mut BOT (SEQ ID NO:2) were used to amplify
one product comprising the 5' half of the variable region while
primers HH1.2 mut TOP (SEQ ID NO:3) and m18.3.1 (SEQ ID NO:4) were
used to amplify the 3' half of the variable region. These products
were purified and mixed together to serve as a template for a third
PCR reaction using primers L1 and m18.3.1. The resulting camelized
human variable region PCR product was cloned, purified and
confirmed by sequencing.
[0143] The full length heavy chain constructs (variable and
constant) were made by amplifying the human variable regions
(camelized and non-camelized) and constant regions with primers
containing restriction enzyme sites to allow for subsequent
ligation together via cohesive ends. All full length heavy chain
constructs were cloned into expression vectors, purified and
confirmed again by sequencing. Table 1 sets forth each heavy chain
construct, their SEQ ID NOs and a short description for each
construct.
TABLE-US-00001 TABLE 1 SEQ ID NO Construct Description
(DNA/Protein) hVR-mFc Non-camelized human variable region fused 5/6
to mouse IgG2b hVR*-mFc Camelized human variable region fused to
7/8 mouse IgG2b hVR- Non-camelized human variable region fused 9/10
mFc.DELTA.CH1 to mouse IgG2b lacking a CH1 domain hVR*- Camelized
human variable region fused to 11/12 mFc.DELTA.CH1 mouse IgG2b
lacking a CH1 domain
[0144] Chimeric heavy chain constructs were transiently transfected
into Chinese Hamster Ovary cells (CHO-K1) to analyze expression in
the absence of immunoglobulin light chain. Supernatants and cell
lysates were examined by Western blot to detect presence of heavy
chain using horseradish peroxidase (HRP) conjugated anti-mouse IgG
antibody (Promega) by chemilumescence. All the chimeric heavy chain
constructs were transiently transfected six (6) independent times.
A representative Western blot of the transfections is shown in FIG.
7.
[0145] All chimeric heavy chain constructs, with and without the
CH1 domain, as well as the control constructs, were detected in the
cell lysate. Only constructs lacking a CH1 domain were observed in
the supernatants (FIG. 7, left). Control I and Control III (mouse
Fc protein lacking a CH1 domain) were also detected (FIG. 7), but
mouse Fc protein containing a CH1 domain was not detected. Both
non-camelized and camelized heavy chain constructs containing a CH1
domain were not detected in the supernatant for any transfection
(FIG. 7, right). However, both non-camelized and camelized human
heavy chain constructs lacking a CH1 domain were detected in the
supernatant for all transfections. Together, the results establish
that hVRs (normal or camelized) that lack a CH1 domain can be
expressed and secreted from transiently transfected CHO cells in
the absence of immunoglobulin light chain, whereas hVRs (normal or
camelized) that contain a CH1 domain could not be secreted in the
absence of light chain.
Example 2
Modification of the Mouse Heavy Chain IgG1 Constant Region
A. Preparation of a Mouse IgG1-CH1-Hinge Targeting Vector (FIG.
3)
[0146] A targeting construct for introducing a deletion of the CH1
and hinge regions of the mouse IgG1 constant domain for the C57BL/6
allele from an ES cell of a VELOCIMMUNE.RTM. mouse (described
below) was constructed.
[0147] The targeting construct was made using VELOCIGENE.RTM.
technology (see, e.g., U.S. 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 the Bacterial Artificial Chromosome (BAC)
BMQ 70p08. BMQ 70p08 BAC DNA was modified to delete the CH1 and
hinge regions of the IgG1 constant domain while leaving the
remainder of the IgG1 gene intact (e.g., CH2, CH3 and transmembrane
exons).
[0148] Briefly, upstream and downstream homology arms were made
employing primers m102 (SEQ ID NO:13) and m104 (SEQ ID NO:14) and
m100 (SEQ ID NO:15) and m99 (SEQ ID NO:16), respectively. These
homology arms were used to make a cassette that deleted the CH1 and
hinge regions of the IgG1 constant domain while retaining the CH2,
CH3 and transmembrane regions of the IgG1 constant domain (see,
e.g., FIG. 3). The targeting construct included a loxed hygromycin
resistance gene positioned between the CH3 and transmembrane domain
exons of the IgG1 gene. Genes upstream of the CH1 and hinge exons
(e.g., IgG3, IgD, IgM) and downstream of the IgG1 transmembrane
exon (e.g., IgG2b, IgG21, IgE, IgA, etc.) were unmodified by the
targeting construct. Switch regions for all constant domains were
unmodified by the targeting construct. The nucleotide sequence
across the deletion included the following, which indicates a
splice acceptor sequence that is present at the deletion point:
TGACAGTGTA ATCACATATA CTTTTTCTTG T(AG)TCCCAGA AGTATCATC (SEQ ID
NO:17). The deletion sequence comprises a splice acceptor (the AG
contained within parentheses above) with pre-CH1 sequences 5' of
the splice acceptor and CH2 exon sequences 3' of the splice
acceptor.
B. Preparation of a Mouse IgG1-CH1 Targeting Vector (FIG. 2)
[0149] A second targeting construct for introducing a deletion of
the CH1 of the mouse IgG1 constant domain for the 129/SvEvTac
allele from an ES cell of a VELOCIMMUNE.RTM. mouse (described
below) was constructed in a similar fashion as described in section
A of this Example.
[0150] The targeting construct was made using VELOCIGENE.RTM.
technology (see, e.g., U.S. 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 the Bacterial Artificial Chromosome (BAC)
BMQ 70p08. BMQ 70p08 BAC DNA was modified to delete the CH1 region
of the IgG1 constant domain while leaving the remainder of the IgG1
gene intact (e.g., hinge, CH2, CH3 and transmembrane exons; see
FIG. 2).
[0151] The homology arms for the second targeting construct were
the same as those for the CH1-Hinge targeting vector (as described
above in section A of this Example). These homology arms were used
to make a cassette that deleted the CH1 region of the IgG1 constant
domain while retaining the hinge, CH2, CH3 and transmembrane
regions of the IgG1 constant domain (see, e.g., FIG. 2). The
targeting construct included a loxed hygromycin resistance gene
positioned between the CH3 and transmembrane domain exons of the
IgG1 gene. Genes upstream of the CH1 exon (e.g., IgG3, IgD, IgM)
and downstream of the IgG1 transmembrane exon (e.g., IgG2b, IgG21,
IgE, IgA, etc.) were unmodified by the targeting construct. Switch
regions for all constant domains were unmodified by the targeting
construct. The nucleotide sequence across the deletion included the
following, which indicates a splice acceptor sequence that is
present at the deletion point: TGACAGTGTA ATCACATATA CTTTTTCTTG
T(AG)TGCCCAG GGATTGTGGT TGTAAGCCTT GCATATGTAC AGGTAAGTCA GTAGGCCTTT
CACCCTGACC C (SEQ ID NO:64). The deletion sequence comprises a
splice acceptor (the AG contained within parentheses above) with
pre-CH1 sequences 5' of the splice acceptor and hinge exon
sequences 3' of the splice acceptor.
Example 3
Modification of the Mouse Heavy Chain Constant Region in ES
Cells
[0152] A. Targeting Mouse ES Cells with an IgG1-CH1-Hinge Targeting
Vector
[0153] A mouse ES cell was targeted with the targeting construct
described above (i.e., a targeting construct introducing a deletion
of the CH1 and hinge regions of the IgG1 gene). The ES cell was
from a VELOCIMMUNE.RTM. mouse that was a 50/50 mix of a 129 strain
and a C57BL/6 strain, bearing genetic modifications that comprise
replacement of mouse heavy and light chain variable region gene
segments with unrearranged human heavy and light chain variable
region gene segments. The 129 strain employed to cross with C57BL/6
is a strain that comprises a replacement of mouse heavy chain and
light chain variable region gene segments with human heavy chain
and light chain variable region gene segments.
[0154] The heterozygous VELOCIMMUNE.RTM. mice bear a single set of
endogenous mouse heavy chain constant region genes from the 129
strain at one allele and a single set of endogenous mouse heavy
chain constant region genes from the C57BL/6 strain at the other
allele. The 129 heavy chain allele is contiguous with a locus of
heavy chain variable region gene segments that are human heavy
chain variable region gene segments that have replaced the
endogenous mouse heavy chain variable region gene segments (i.e.,
at the endogenous mouse locus). The BL/6 heavy chain allele is
contiguous with wild-type mouse heavy chain variable region gene
segments. The VELOCIMMUNE.RTM. mice also bear wild-type endogenous
mouse light chain constant region genes. Thus, by targeting the 129
allele with a construct comprising an IgG, D, E, or A CH1 deletion
a chimeric human/mouse heavy chain antibody could be produced,
whereas by targeting the C57BL/6 allele with a similar
construction, a fully mouse heavy chain antibody lacking a CH1
domain and lacking a hinge could be produced.
[0155] ES cells from the VELOCIMMUNE.RTM. mice described above were
electroporated with linearized targeting vector of section A in
Example 2 and selected for the presence of the hygromycin
resistance gene.
B. Targeting Mouse ES Cells with an IgG1-CH1 Targeting Vector
[0156] In a similar fashion, a mouse ES cell was targeted with the
CH1 targeting construct described in section B of Example 2 (see
also FIG. 2). The ES cell was from a VELOCIMMUNE.RTM. mouse that
was a 50/50 mix of a 129/SvEvTac strain and a C57BL/6 strain,
bearing genetic modifications that comprise replacement of mouse
heavy and light chain variable region gene segments with
unrearranged human heavy and light chain variable region gene
segments. The 129/SvEvTac strain employed to cross with C57BL/6 is
a strain that comprises a replacement of mouse heavy chain and
light chain variable region gene segments with human heavy chain
and light chain variable region gene segments.
[0157] The heterozygous VELOCIMMUNE.RTM. mice bear a single set of
endogenous mouse heavy chain constant region genes from the
129/SvEvTac strain at one allele and a single set of endogenous
mouse heavy chain constant region genes from the C57BL/6 strain at
the other allele. The 129/SvEvTac heavy chain allele is contiguous
with a locus of heavy chain variable region gene segments that are
human heavy chain variable region gene segments that have replaced
the endogenous mouse heavy chain variable region gene segments
(i.e., at the endogenous mouse locus). The BL/6 heavy chain allele
is contiguous with wild-type mouse heavy chain variable region gene
segments. The VELOCIMMUNE.RTM. mice also bear wild-type endogenous
mouse light chain constant region genes. Thus, by targeting the
129/SvEvTac allele with a construct comprising an IgG, D, E, or A
CH1 deletion a chimeric human/mouse heavy chain antibody could be
produced, whereas by targeting the C57BL/6 allele with a similar
construction, a fully mouse heavy chain antibody lacking a CH1
domain and lacking a hinge could be produced.
[0158] ES cells from the VELOCIMMUNE.RTM. mice described above were
electroporated with linearized targeting vector, described in
section B in Example 2, and selected for the presence of the
hygromycin resistance gene.
Example 4
Generation of Mice Carrying a Modified IgG1 Constant Region
A. Mice Carrying an IgG1-CH1-Hinge Deletion
[0159] Targeted ES cells described above were used as donor ES
cells and introduced into an 8-cell stage mouse embryo by the
VELOCIMOUSE.RTM. method (see, e.g., U.S. Pat. No. 7,294,754 and
Poueymirou et al. (2007) F0 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.RTM. (F0 mice fully derived from the donor ES cell)
bearing targeted C57BL/6 IgG1 alleles were identified by genotyping
using a modification of allele assay (Valenzuela et al., supra)
that detected the presence of sequences positioned upstream and
downstream of the deleted hinge and CH1 regions.
[0160] Mice genotyped for the IgG1 CH1 and hinge deletion (in the
C57BL/6 allele, i.e., the mouse allele) were bred to a Cre deleter
mouse strain (see, e.g., International Patent Application
Publication No. WO 2009/114400) in order to remove the loxed hyg
cassette downstream of the IgG1 CH3 exon and upstream of the IgG1
transmembrane exon, introduced by the targeting construct (see,
e.g., FIG. 3). Pups were genotyped and a pup heterozygous for the
IgG1 CH1 and hinge deletion was selected to examine IgG1 heavy
chain expressed from the C57BL/6 allele in the pup's serum.
B. Mice Carrying an IgG1-CH1 Deletion
[0161] In a similar fashion, targeted ES cells carrying a deletion
of the IgG1 CH1 region were used as donor ES cells and introduced
into an 8-cell stage mouse embryo by the VELOCIMOUSE.RTM. method
(see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007) F0
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.RTM. (F0 mice fully derived
from the donor ES cell) bearing targeted 129SvEv/Tac alleles were
identified by genotyping using a modification of allele assay
(Valenzuela et al., supra) that detected the presence of sequences
positioned upstream and downstream of the deleted CH1 region.
[0162] Mice genotyped for the IgG1 CH1 deletion (in the 129/SvEvTac
allele, i.e., the human allele) were bred to a Cre deleter mouse
strain (see, e.g., International Patent Application Publication No.
WO 2009/114400) in order to remove the loxed hyg cassette
downstream of the IgG1 CH3 exon and upstream of the IgG1
transmembrane exon, introduced by the targeting construct (see,
e.g., FIG. 2). Pups were genotyped and a pup homozygous for the
IgG1 CH1 deletion was selected to examine modified IgG1 heavy chain
expression.
Example 5
Heavy Chain Antibodies from Mice Carrying a Modified IgG1 Gene
A. IgG1-.DELTA.CH1-.DELTA.Hinge Mice
[0163] A mouse pup identified above as containing the CH1 and hinge
deletion, and a wild-type pup, were bled and sera from the bled
mice were prepared for Western blotting to identify any expressed
IgG in the sera using an anti-mIgG1 antibody. Briefly, 10 .mu.L of
a 1:100 dilution of mouse sera was used in reducing SDS-PAGE, and
the gel was transferred to a PVDF membrane. The blot was blocked
overnight with 5% nonfat milk in Tris-Buffered Saline with 0.05%
Tween-20 (TBST; Sigma), washed 4 times for 5 minutes per wash with
TBST, and then exposed to primary antibody (goat anti-mIgG1
conjugated to HRP, Southern Biotech) diluted 1:1,000 in 1% nonfat
milk in TBST for two hours at room temperature. The blot was washed
6 times for 5 minutes per wash. The blot was developed for 5
minutes with SUPERSIGNAL.TM. West Pico Chemiluminescent Substrate
(Thermo Scientific) and then exposed to film for 1 minute.
[0164] Serum from the VELOCIMOUSE.RTM. (50% wild-type BL/6; 50%
.DELTA.CH1-.DELTA.hinge BL/6) derived from the targeted donor ES
cell revealed a mixture of bands: one band of about 57.5 kD, the
expected size for a wild-type IgG, and one band at about 45 kD, the
expected size for an IgG lacking a CH1 domain and a hinge (FIG. 8).
The results are consistent with the VELOCIMOUSE.RTM. expressing a
normal mouse heavy chain from the wild-type BL/6 allele and a
.DELTA.CH1/.DELTA.hinge mouse heavy chain from its
.DELTA.CH1-.DELTA.hinge BL/6 allele. This result establishes that
genetically modified mice bearing a functional IgM gene and an IgG
gene that lacks a CH1 domain and a hinge domain are capable of
expressing heavy chain antibodies in serum.
B. IgG1-.DELTA.CH1 Mice
[0165] In a similar fashion, mouse pups homozygous for the CH1
deletion, and wild-type pups, were bled. Plasma and serum (for five
homozygotes; two wild-type) from the bled mice were prepared for
Western blotting to identify any expressed IgG in the sera using an
anti-mIgG1 antibody (described above). Western blots of serum and
plasma from mice homozygous for the IgG1-.DELTA.CH1 deletion
revealed a mixture of bands: one band of about 45 kD, the expected
size for a single chain IgG1 lacking a CH1 domain, and one band at
about 75 kD, the expected size for a dimer IgG lacking a CH1 domain
(data not shown). The results are consistent with the homozygous
VELOCIMICE.RTM. expressing an IgG1-.DELTA.CH1 heavy chain from
either one or both heavy chain loci. This result establishes that
genetically modified mice bearing a functional IgM gene and an IgG
gene that lacks a CH1 domain are capable of expressing heavy chain
antibodies in the peripheral lymphocyte compartment of the animals'
immune system.
Example 6
Characterization of Mice Homozygous for IgG1-CH1-Hinge Deletion
[0166] VELOCIMICE.RTM. heterozygous for the CH1-hinge deletion were
bred together to obtain mice homozygous for the deletion. Four
mouse pups were identified as homozygous for IgG1
.DELTA.CH1-.DELTA.hinge. These four mice and a wild-type mouse were
bled and sera from the bled mice were prepared for Western blotting
to identify any expressed IgG in the sera using an anti-mIgG1
antibody (as described above). FIG. 9 shows the film developed from
the PVDF-membrane used in this experiment. Serum was diluted 1:5
and 1:10 and 10 .mu.L of each dilution was loaded onto the gel
side-by-side for each mouse. On the top portion of the gel images,
the lanes are labeled for each mouse as well as IgG1 (1) and IgG2a
(2a) controls.
[0167] Serum from the wild-type mouse showed an expected pattern
for a wild-type mouse that expresses normal antibodies comprising
two heavy chains and two light chains (approximately 150 kD). All
four mice (homozygous for IgG1 .DELTA.CH1-.DELTA.hinge) each showed
a mixture of bands: one band of about 150 kD, the expected size for
a wild-type IgG other than IgG1 (e.g., IgG2a, IgG2b or IgG3), and
one band at about 45 kD, the expected size for an IgG lacking a CH1
domain and a hinge (FIG. 9). These results are consistent with the
mice expressing an IgG1 heavy chain antibody lacking a CH1 domain
and a hinge region and lacking a light chain. This result further
establishes that genetically modified mice bearing a functional IgM
gene and an IgG gene that lacks a CH1 domain and a hinge region are
capable of expressing heavy chain antibodies in serum.
[0168] In another experiment, serum expression of IgG was
determined from mice homozygous for the IgG1
.DELTA.CH1-.DELTA.hinge using an ELISA assay. Briefly, antibodies
specific for either mIgG1 or mIgG2b (Pharmingen) were separately
diluted and 100 .mu.l/well was coated onto plates at 2 .mu.g/mL in
1.times.PBS (Irvine Scientific) and incubated at 4.degree. C.
overnight. The following day the plates were washed four times with
PBS with 0.05% Tween-20 (PBST; Sigma). After the fourth wash,
plates were blocked with 250 .mu.L/well of PBST with 5% BSA (Sigma)
and incubated at room temperature for one hour. Serum and standards
were serially diluted (dilution factor of 0.316) in PBST in 0.5%
BSA down the plate (from top to bottom) at a starting concentration
of 400 ng/mL (mIgG1) or 600 ng/mL (mIgG2b). After blocking, the
plates were washed again four times with PBST. Following the fourth
wash, 100 .mu.L of serum or standard was added to the plates and
incubated for one hour at room temperature. The plates were again
washed four times with PBST. Following the washes, 100 .mu.L of a
biotinylated detection antibody (10 ng/mL of rat anti-mIgG1 or 250
ng/mL of anti-mIgG2b; Pharmingen) was added to the plates and
incubated for one hour at room temperature. The plates were again
washed as described above. Following the wash, 100 .mu.L/well of a
1:20,000 dilution of horseradish peroxidase conjugated to
streptavidin (HRP-SA) in PBST was added to the plates and the
plates were incubated for 30 minutes at room temperature. The
plates were then washed six times with PBST, after which 100
.mu.L/well of a 1:1 dilution of Substrate A and B (BD OPTEIA.TM.;
BD Biosciences) was added and the plates were maintained in the
dark. The reaction was developed in the dark and stopped as desired
(approx. 15 minutes) with 1N phosphoric acid. Stopped reactions
were read on a Wallac 1420 Work Station VICTOR.TM. Plate Reader at
an absorption wavelength of 450 nm (1.0 sec/reading) and the
results plotted on graphs (FIG. 11).
[0169] Serum from wild-type mice showed normal levels of IgG1 and
IgG2b. Mice homozygous for IgG1 .DELTA.CH1-.DELTA.hinge were
capable of expressing an IgG1 lacking a CH1 domain and a hinge
region in the periphery (serum; left side of FIG. 11). Further,
serum levels of other IgG isotypes (e.g., IgG2b) were not
noticeably reduced from wild-type levels (right side of FIG. 11).
This result further establishes that genetically modified mice
bearing a functional IgM gene and an IgG gene that lacks a CH1
domain and a hinge region are capable of expressing a modified IgG1
isotype (i.e., lacking a CH1 domain and a hinge) that can be
detected in serum.
Example 7
Analysis of V-D-J Rearrangements in IgG1 Modified Mice
A. Mice Homozygous for an IgG1-CH1-Hinge Deletion
[0170] Mice homozygous for the IgG1 .DELTA.CH1-.DELTA.hinge
modification were analyzed for V-D-J recombination and heavy chain
gene usage by reverse-transcriptase polymerase chain reaction
(RT-PCR) using RNA isolated from splenocytes.
[0171] Briefly, spleens were harvested and perfused with 10 mL
RPMI-1640 (Sigma) with 5% HI-FBS in sterile disposable bags. Each
bag containing a single spleen was then placed in a STOMACHER.TM.
(Seward) and homogenized at a medium setting for 30 seconds.
Homogenized spleens were filtered using a 0.7 .mu.m cell strainer
and then pelleted with a centrifuge (1000 rpm for 10 minutes) and
red blood cells (RBCs) were lysed in BD PHARM LYSE.TM. (BD
Biosciences) for three minutes. Splenocytes were diluted with
RPMI-1640 and centrifuged again followed by resuspension in 1 mL of
PBS (Irvine Scientific). RNA was isolated from pelleted splenocytes
using standard techniques known in the art.
[0172] RT-PCR was performed on splenocyte RNA using a set of
degenerate primers specific for mouse heavy chain variable region
(VH) gene segments (Novagen) and a mouse IgG1 CH2 primer
(CGATGGGGGC AGGGAAAGCT GCAC; SEQ ID NO:40). PCR products were
gel-purified and cloned into pCR2.1-TOPO TA (Invitrogen) and
sequenced with M13 Forward (GTAAAACGAC GGCCAG; SEQ ID NO:41) and
M13 Reverse (CAGGAAACAG CTATGAC; SEQ ID NO:42) primers located
within the vector sequence at positions flanking the cloning site.
Nineteen clones were sequenced to determine heavy chain gene usage
and sequence of the junction of the rearranged VH and the CH2 of
the IgG1 constant region (Table 2).
TABLE-US-00002 TABLE 2 Heavy Chain Gene Usage Clone V.sub.H D.sub.H
J.sub.H B1 1-58 3-2 2 B2 1-26 4-1 1 B3 1-50 2-14 2 B4 1-58 3-2 2 B5
14-2 4-1 4 D2 3-6 1-1 4 D5 14-1 3-3 2 D6 14-2 4-1 3 D7 3-6 1-1 4 E2
7-1 3-1 4 E3 1-50 2-14 2 E4 1-50 2-14 2 E7 1-50 2-14 2 E8 1-72 1-1
4 E10 1-42 1-1 1 F6 5-6 1-1 1 F7 5-6 1-1 1 F8 5-6 1-1 1 F10 5-6 1-1
1
[0173] FIG. 12 shows the sequence alignment of the VH domains
rearranged to the CH2 of the IgG1 constant region for eleven of the
nineteen RT-PCR clones. The sequences shown in FIG. 12 illustrate
unique rearrangements involving different mouse heavy chain V, D
and J gene segments and mouse IgG1 devoid of CH1 and hinge regions.
Mice homozygous for a deletion of the CH1 and hinge regions of the
endogenous IgG1 constant region gene were able to produce heavy
chains containing mouse VH domains operably linked to a CH2-CH3
region from a mouse IgG1 constant region devoid of CH1 and hinge
regions and produce B cells that expressed mouse IgG1 heavy chains
devoid of CH1 and hinge regions and lacking a light chain (FIGS. 8
and 9). These rearrangements demonstrate that the modified loci
were able to independently rearrange mouse heavy chain gene
segments in multiple, independent B cells in these mice to produce
heavy chain antibodies that are similar to those normally found in
camels. Further, this Example demonstrates that the deletion of the
endogenous IgG1 CH1 and hinge regions did not render the locus
inoperable or prevent recombination involving the modified IgG1
constant region. These mice made functional heavy chain antibodies
containing an IgG1 devoid of CH1 and hinge regions as part of the
endogenous repertoire without any detectable defect in B cell
development.
B. Mice Homozygous for an IgG1-CH1 Deletion
[0174] In a similar fashion, mice homozygous for the IgG1
.DELTA.CH1 modification were analyzed for V-D-J recombination and
human heavy chain gene usage by reverse-transcriptase polymerase
chain reaction (RT-PCR) using RNA isolated from splenocytes.
[0175] Briefly, spleens were isolated from two homozygous
IgG1-.DELTA.CH1 mice as described above in section A of this
Example. CD19.sup.+ B cells were isolated using magnetic cell
sorting (MACS, Miltenyi Biotec) from pooled splenocytes. RNA was
extracted from the sorted CD19.sup.+ B cells using Qiagen
ALLPREP.TM. DNA/RNA mini kit (Qiagen). First-strand cDNA was
synthesized with SUPERSCRIPT.TM. III Reverse Transcriptase and
Oligo (dT)20 primers (Invitrogen). The cDNA was then used as a
template for PCR performed with a 3' mouse IgG1 hinge specific
primer and 5' degenerate primers designed to bind human heavy
variable leader sequences (Table 3). PCR products were cloned into
pCR2.1 TOPO.TM. TA vector (Invitrogen) and sequenced with M13
Forward and M13 Reverse primers (as described above in section A of
this Example).
TABLE-US-00003 TABLE 3 SEQ Primer Sequence (5'-3') ID NO: hVHL-1
TCACCATGGA CTGSACCTGG A 43 hVHL-2 CCATGGACAC ACTTTGYTCC AC 44
hVHL-3 TCACCATGGA GTTTGGGCTG AGC 45 hVHL-4 AGAACATGAA ACAYCTGTGG
TTCTT 46 hVHL-5 ATGGGGTCAA CCGCCATCCT 47 hVHL-6 ACAATGTCTG
TCTCCTTCCT CAT 48 3' mIgG1 GCAAGGCTTA CAACCACAAT C 49 Hinge
[0176] To determine heavy chain gene usage in mice homozygous for
IgG1 .DELTA.CH1, twenty-eight RT-PCR clones were sequenced. Within
these clones, seven unique rearrangements of human V, D and J gene
segments were observed (Table 4).
TABLE-US-00004 TABLE 4 Heavy Chain Gene Usage Clone V.sub.H D.sub.H
J.sub.H A2 1-69 6-19 6 A5 1-69 6-7 4 A8 1-8 4-4 4 C2 1-18 6-6 2 C4
1-18 3-16 6 D9 1-18 6-6 4 H8 1-18 1-7 4
[0177] FIG. 13 shows the sequence alignment of the VH domains
rearranged to the hinge-CH2-CH3 of the IgG1 constant region for the
seven rearrangements shown in Table 4. The sequences shown in FIG.
13 illustrate unique rearrangements involving different human heavy
chain V, D and J gene segments and mouse IgG1 devoid of the CH1
region. Mice homozygous for a deletion of the CH1 region of the
endogenous IgG1 constant region gene were able to produce heavy
chains containing human VH domains operably linked to a
hinge-CH2-CH3 region from a mouse IgG1 constant region devoid of
CH1 and produce B cells that expressed mouse IgG1 heavy chains
devoid of CH1 regions and lacking a light chain (data not shown).
These rearrangements demonstrate that either one or both modified
loci (IgG1 .DELTA.CH1-.DELTA.hinge and IgG1 .DELTA.CH1) were able
to independently rearrange heavy chain gene segments (mouse and
human) in multiple, independent B cells in these mice to produce
heavy chain antibodies that are similar to those normally found in
camels. Further, this Example demonstrates that the deletion of the
endogenous IgG1 CH1 did not render the locus inoperable or prevent
recombination involving human heavy chain V, D and J gene segments
and the modified mouse IgG1 constant region. These mice made
functional heavy chain antibodies containing human heavy chain V
domains and a mouse IgG1 devoid of CH1 as part of the endogenous
repertoire without any detectable defect in B cell development.
Sequence CWU 1
1
64121DNAArtificial Sequencesynthetic 1tcaccatgga ctggacctgg a
21240DNAArtificial Sequencesynthetic 2cccatcaact cacactcttg
tccaggggcc tgtcgaaacc 40340DNAArtificial Sequencesynthetic
3ctggtttcga caggcccctg gacaagagtg tgagttgatg 40433DNAArtificial
Sequencesynthetic 4acgttccgga tgaggagacg gtgaccaggg ttc
3351449DNAArtificial Sequencesynthetic 5atggactgga cctggaggat
ccttttcttg gtggcagcag ccacaggagc ccactcccag 60gtgcagctgg tgcagtctgg
ggctgaggtg aagaagcctg gggcctcagt gaaggtctcc 120tgcaaggctt
ctggatacac cttcaccggc tactatatgc actgggtgcg acaggcccct
180ggacaagggc ttgagtggat gggatggatc aaccctaaca gtggtggcac
aaactatgca 240cagaagtttc agggcagggt caccatgacc ggggacacgt
ccatcagcac agcctacatg 300gagctgagca ggctgagatc tgacgacacg
gccgtgtatt actgtgcgag aggctcctta 360tattgtacta atggtgtatg
ctttgactac tggggccagg gaaccctggt caccgtctcc 420tcaaagcttt
ccaaaacaac acccccatca gtctatccac tggcccctgg gtgtggagat
480acaactggtt cctccgtgac tctgggatgc ctggtcaagg gctacttccc
tgagtcagtg 540actgtgactt ggaactctgg atccctgtcc agcagtgtgc
acaccttccc agctctcctg 600cagtctggac tctacactat gagcagctca
gtgactgtcc cctccagcac ttggccaagt 660cagaccgtca cctgcagcgt
tgctcaccca gccagcagca ccacggtgga caaaaaactt 720gtccggagcg
agcccagcgg gcccatttca acaatcaacc cctgtcctcc atgcaaggag
780tgtcacaaat gcccagctcc taacctcgag ggtggaccat ccgtcttcat
cttccctcca 840aatatcaagg atgtactcat gatctccctg acacccaagg
tcacgtgtgt ggtggtggat 900gtgagcgagg atgacccaga cgtccagatc
agctggtttg tgaacaacgt ggaagtacac 960acagctcaga cacaaaccca
tagagaggat tacaacagta ctatccgggt ggtcagcacc 1020ctccccatcc
agcaccagga ctggatgagt ggcaaggagt tcaaatgcaa ggtcaacaac
1080aaagacctcc catcacccat cgagagaacc atctcaaaaa ttaaagggct
agtcagagct 1140ccacaagtat acatcttgcc gccaccagca gagcagttgt
ccaggaaaga tgtcagtctc 1200acttgcctgg tcgtgggctt caaccctgga
gacatcagtg tggagtggac cagcaatggg 1260catacagagg agaactacaa
ggacaccgca ccagtcctgg actctgacgg ttcttacttc 1320atatatagca
agctcaatat gaaaacaagc aagtgggaga aaacagattc cttctcatgc
1380aacgtgagac acgagggtct gaaaaattac tacctgaaga agaccatctc
ccggtctccg 1440ggtaaatga 14496482PRTArtificial Sequencesynthetic
6Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly1 5
10 15 Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe 35 40 45 Thr Gly Tyr Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Trp Ile Asn Pro Asn
Ser Gly Gly Thr Asn Tyr Ala65 70 75 80 Gln Lys Phe Gln Gly Arg Val
Thr Met Thr Gly Asp Thr Ser Ile Ser 85 90 95 Thr Ala Tyr Met Glu
Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys
Ala Arg Gly Ser Leu Tyr Cys Thr Asn Gly Val Cys Phe 115 120 125 Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Lys Leu Ser 130 135
140 Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly
Asp145 150 155 160 Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val
Lys Gly Tyr Phe 165 170 175 Pro Glu Ser Val Thr Val Thr Trp Asn Ser
Gly Ser Leu Ser Ser Ser 180 185 190 Val His Thr Phe Pro Ala Leu Leu
Gln Ser Gly Leu Tyr Thr Met Ser 195 200 205 Ser Ser Val Thr Val Pro
Ser Ser Thr Trp Pro Ser Gln Thr Val Thr 210 215 220 Cys Ser Val Ala
His Pro Ala Ser Ser Thr Thr Val Asp Lys Lys Leu225 230 235 240 Val
Arg Ser Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Cys Pro 245 250
255 Pro Cys Lys Glu Cys His Lys Cys Pro Ala Pro Asn Leu Glu Gly Gly
260 265 270 Pro Ser Val Phe Ile Phe Pro Pro Asn Ile Lys Asp Val Leu
Met Ile 275 280 285 Ser Leu Thr Pro Lys Val Thr Cys Val Val Val Asp
Val Ser Glu Asp 290 295 300 Asp Pro Asp Val Gln Ile Ser Trp Phe Val
Asn Asn Val Glu Val His305 310 315 320 Thr Ala Gln Thr Gln Thr His
Arg Glu Asp Tyr Asn Ser Thr Ile Arg 325 330 335 Val Val Ser Thr Leu
Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys 340 345 350 Glu Phe Lys
Cys Lys Val Asn Asn Lys Asp Leu Pro Ser Pro Ile Glu 355 360 365 Arg
Thr Ile Ser Lys Ile Lys Gly Leu Val Arg Ala Pro Gln Val Tyr 370 375
380 Ile Leu Pro Pro Pro Ala Glu Gln Leu Ser Arg Lys Asp Val Ser
Leu385 390 395 400 Thr Cys Leu Val Val Gly Phe Asn Pro Gly Asp Ile
Ser Val Glu Trp 405 410 415 Thr Ser Asn Gly His Thr Glu Glu Asn Tyr
Lys Asp Thr Ala Pro Val 420 425 430 Leu Asp Ser Asp Gly Ser Tyr Phe
Ile Tyr Ser Lys Leu Asn Met Lys 435 440 445 Thr Ser Lys Trp Glu Lys
Thr Asp Ser Phe Ser Cys Asn Val Arg His 450 455 460 Glu Gly Leu Lys
Asn Tyr Tyr Leu Lys Lys Thr Ile Ser Arg Ser Pro465 470 475 480 Gly
Lys71449DNAArtificial Sequencesynthetic 7atggactgga cctggaggat
ccttttcttg gtggcagcag ccacaggagc ccactcccag 60gtgcagctgg tgcagtctgg
ggctgaggtg aagaagcctg gggcctcagt gaaggtctcc 120tgcaaggctt
ctggatacac cttcaccggc tactatatgc actggtttcg acaggcccct
180ggacaagagt gtgagttgat gggatggatc aaccctaaca gtggtggcac
aaactatgca 240cagaagtttc agggcagggt caccatgacc ggggacacgt
ccatcagcac agcctacatg 300gagctgagca ggctgagatc tgacgacacg
gccgtgtatt actgtgcgag aggctcctta 360tattgtacta atggtgtatg
ctttgactac tggggccagg gaaccctggt caccgtctcc 420tcaaagcttt
ccaaaacaac acccccatca gtctatccac tggcccctgg gtgtggagat
480acaactggtt cctccgtgac tctgggatgc ctggtcaagg gctacttccc
tgagtcagtg 540actgtgactt ggaactctgg atccctgtcc agcagtgtgc
acaccttccc agctctcctg 600cagtctggac tctacactat gagcagctca
gtgactgtcc cctccagcac ttggccaagt 660cagaccgtca cctgcagcgt
tgctcaccca gccagcagca ccacggtgga caaaaaactt 720gtccggagcg
agcccagcgg gcccatttca acaatcaacc cctgtcctcc atgcaaggag
780tgtcacaaat gcccagctcc taacctcgag ggtggaccat ccgtcttcat
cttccctcca 840aatatcaagg atgtactcat gatctccctg acacccaagg
tcacgtgtgt ggtggtggat 900gtgagcgagg atgacccaga cgtccagatc
agctggtttg tgaacaacgt ggaagtacac 960acagctcaga cacaaaccca
tagagaggat tacaacagta ctatccgggt ggtcagcacc 1020ctccccatcc
agcaccagga ctggatgagt ggcaaggagt tcaaatgcaa ggtcaacaac
1080aaagacctcc catcacccat cgagagaacc atctcaaaaa ttaaagggct
agtcagagct 1140ccacaagtat acatcttgcc gccaccagca gagcagttgt
ccaggaaaga tgtcagtctc 1200acttgcctgg tcgtgggctt caaccctgga
gacatcagtg tggagtggac cagcaatggg 1260catacagagg agaactacaa
ggacaccgca ccagtcctgg actctgacgg ttcttacttc 1320atatatagca
agctcaatat gaaaacaagc aagtgggaga aaacagattc cttctcatgc
1380aacgtgagac acgagggtct gaaaaattac tacctgaaga agaccatctc
ccggtctccg 1440ggtaaatga 14498482PRTArtificial Sequencesynthetic
8Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly1 5
10 15 Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe 35 40 45 Thr Gly Tyr Tyr Met His Trp Phe Arg Gln Ala
Pro Gly Gln Glu Cys 50 55 60 Glu Leu Met Gly Trp Ile Asn Pro Asn
Ser Gly Gly Thr Asn Tyr Ala65 70 75 80 Gln Lys Phe Gln Gly Arg Val
Thr Met Thr Gly Asp Thr Ser Ile Ser 85 90 95 Thr Ala Tyr Met Glu
Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys
Ala Arg Gly Ser Leu Tyr Cys Thr Asn Gly Val Cys Phe 115 120 125 Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Lys Leu Ser 130 135
140 Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly
Asp145 150 155 160 Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val
Lys Gly Tyr Phe 165 170 175 Pro Glu Ser Val Thr Val Thr Trp Asn Ser
Gly Ser Leu Ser Ser Ser 180 185 190 Val His Thr Phe Pro Ala Leu Leu
Gln Ser Gly Leu Tyr Thr Met Ser 195 200 205 Ser Ser Val Thr Val Pro
Ser Ser Thr Trp Pro Ser Gln Thr Val Thr 210 215 220 Cys Ser Val Ala
His Pro Ala Ser Ser Thr Thr Val Asp Lys Lys Leu225 230 235 240 Val
Arg Ser Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Cys Pro 245 250
255 Pro Cys Lys Glu Cys His Lys Cys Pro Ala Pro Asn Leu Glu Gly Gly
260 265 270 Pro Ser Val Phe Ile Phe Pro Pro Asn Ile Lys Asp Val Leu
Met Ile 275 280 285 Ser Leu Thr Pro Lys Val Thr Cys Val Val Val Asp
Val Ser Glu Asp 290 295 300 Asp Pro Asp Val Gln Ile Ser Trp Phe Val
Asn Asn Val Glu Val His305 310 315 320 Thr Ala Gln Thr Gln Thr His
Arg Glu Asp Tyr Asn Ser Thr Ile Arg 325 330 335 Val Val Ser Thr Leu
Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys 340 345 350 Glu Phe Lys
Cys Lys Val Asn Asn Lys Asp Leu Pro Ser Pro Ile Glu 355 360 365 Arg
Thr Ile Ser Lys Ile Lys Gly Leu Val Arg Ala Pro Gln Val Tyr 370 375
380 Ile Leu Pro Pro Pro Ala Glu Gln Leu Ser Arg Lys Asp Val Ser
Leu385 390 395 400 Thr Cys Leu Val Val Gly Phe Asn Pro Gly Asp Ile
Ser Val Glu Trp 405 410 415 Thr Ser Asn Gly His Thr Glu Glu Asn Tyr
Lys Asp Thr Ala Pro Val 420 425 430 Leu Asp Ser Asp Gly Ser Tyr Phe
Ile Tyr Ser Lys Leu Asn Met Lys 435 440 445 Thr Ser Lys Trp Glu Lys
Thr Asp Ser Phe Ser Cys Asn Val Arg His 450 455 460 Glu Gly Leu Lys
Asn Tyr Tyr Leu Lys Lys Thr Ile Ser Arg Ser Pro465 470 475 480 Gly
Lys91149DNAArtificial Sequencesynthetic 9atggactgga cctggaggat
ccttttcttg gtggcagcag ccacaggagc ccactcccag 60gtgcagctgg tgcagtctgg
ggctgaggtg aagaagcctg gggcctcagt gaaggtctcc 120tgcaaggctt
ctggatacac cttcaccggc tactatatgc actgggtgcg acaggcccct
180ggacaagggc ttgagtggat gggatggatc aaccctaaca gtggtggcac
aaactatgca 240cagaagtttc agggcagggt caccatgacc ggggacacgt
ccatcagcac agcctacatg 300gagctgagca ggctgagatc tgacgacacg
gccgtgtatt actgtgcgag aggctcctta 360tattgtacta atggtgtatg
ctttgactac tggggccagg gaaccctggt caccgtctcc 420tcatccggag
agcccagcgg gcccatttca acaatcaacc cctgtcctcc atgcaaggag
480tgtcacaaat gcccagctcc taacctcgag ggtggaccat ccgtcttcat
cttccctcca 540aatatcaagg atgtactcat gatctccctg acacccaagg
tcacgtgtgt ggtggtggat 600gtgagcgagg atgacccaga cgtccagatc
agctggtttg tgaacaacgt ggaagtacac 660acagctcaga cacaaaccca
tagagaggat tacaacagta ctatccgggt ggtcagcacc 720ctccccatcc
agcaccagga ctggatgagt ggcaaggagt tcaaatgcaa ggtcaacaac
780aaagacctcc catcacccat cgagagaacc atctcaaaaa ttaaagggct
agtcagagct 840ccacaagtat acatcttgcc gccaccagca gagcagttgt
ccaggaaaga tgtcagtctc 900acttgcctgg tcgtgggctt caaccctgga
gacatcagtg tggagtggac cagcaatggg 960catacagagg agaactacaa
ggacaccgca ccagtcctgg actctgacgg ttcttacttc 1020atatatagca
agctcaatat gaaaacaagc aagtgggaga aaacagattc cttctcatgc
1080aacgtgagac acgagggtct gaaaaattac tacctgaaga agaccatctc
ccggtctccg 1140ggtaaatga 114910382PRTArtificial Sequencesynthetic
10Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly1
5 10 15 Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe 35 40 45 Thr Gly Tyr Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Trp Ile Asn Pro Asn
Ser Gly Gly Thr Asn Tyr Ala65 70 75 80 Gln Lys Phe Gln Gly Arg Val
Thr Met Thr Gly Asp Thr Ser Ile Ser 85 90 95 Thr Ala Tyr Met Glu
Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys
Ala Arg Gly Ser Leu Tyr Cys Thr Asn Gly Val Cys Phe 115 120 125 Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ser Gly Glu 130 135
140 Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Cys Pro Pro Cys Lys
Glu145 150 155 160 Cys His Lys Cys Pro Ala Pro Asn Leu Glu Gly Gly
Pro Ser Val Phe 165 170 175 Ile Phe Pro Pro Asn Ile Lys Asp Val Leu
Met Ile Ser Leu Thr Pro 180 185 190 Lys Val Thr Cys Val Val Val Asp
Val Ser Glu Asp Asp Pro Asp Val 195 200 205 Gln Ile Ser Trp Phe Val
Asn Asn Val Glu Val His Thr Ala Gln Thr 210 215 220 Gln Thr His Arg
Glu Asp Tyr Asn Ser Thr Ile Arg Val Val Ser Thr225 230 235 240 Leu
Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys 245 250
255 Lys Val Asn Asn Lys Asp Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser
260 265 270 Lys Ile Lys Gly Leu Val Arg Ala Pro Gln Val Tyr Ile Leu
Pro Pro 275 280 285 Pro Ala Glu Gln Leu Ser Arg Lys Asp Val Ser Leu
Thr Cys Leu Val 290 295 300 Val Gly Phe Asn Pro Gly Asp Ile Ser Val
Glu Trp Thr Ser Asn Gly305 310 315 320 His Thr Glu Glu Asn Tyr Lys
Asp Thr Ala Pro Val Leu Asp Ser Asp 325 330 335 Gly Ser Tyr Phe Ile
Tyr Ser Lys Leu Asn Met Lys Thr Ser Lys Trp 340 345 350 Glu Lys Thr
Asp Ser Phe Ser Cys Asn Val Arg His Glu Gly Leu Lys 355 360 365 Asn
Tyr Tyr Leu Lys Lys Thr Ile Ser Arg Ser Pro Gly Lys 370 375 380
111149DNAArtificial Sequencesynthetic 11atggactgga cctggaggat
ccttttcttg gtggcagcag ccacaggagc ccactcccag 60gtgcagctgg tgcagtctgg
ggctgaggtg aagaagcctg gggcctcagt gaaggtctcc 120tgcaaggctt
ctggatacac cttcaccggc tactatatgc actggtttcg acaggcccct
180ggacaagagt gtgagttgat gggatggatc aaccctaaca gtggtggcac
aaactatgca 240cagaagtttc agggcagggt caccatgacc ggggacacgt
ccatcagcac agcctacatg 300gagctgagca ggctgagatc tgacgacacg
gccgtgtatt actgtgcgag aggctcctta 360tattgtacta atggtgtatg
ctttgactac tggggccagg gaaccctggt caccgtctcc 420tcatccggag
agcccagcgg gcccatttca acaatcaacc cctgtcctcc atgcaaggag
480tgtcacaaat gcccagctcc taacctcgag ggtggaccat ccgtcttcat
cttccctcca 540aatatcaagg atgtactcat gatctccctg acacccaagg
tcacgtgtgt ggtggtggat 600gtgagcgagg atgacccaga cgtccagatc
agctggtttg tgaacaacgt ggaagtacac 660acagctcaga cacaaaccca
tagagaggat tacaacagta ctatccgggt ggtcagcacc 720ctccccatcc
agcaccagga ctggatgagt ggcaaggagt tcaaatgcaa ggtcaacaac
780aaagacctcc catcacccat cgagagaacc atctcaaaaa ttaaagggct
agtcagagct 840ccacaagtat acatcttgcc gccaccagca gagcagttgt
ccaggaaaga tgtcagtctc 900acttgcctgg tcgtgggctt caaccctgga
gacatcagtg tggagtggac cagcaatggg 960catacagagg agaactacaa
ggacaccgca ccagtcctgg actctgacgg ttcttacttc 1020atatatagca
agctcaatat gaaaacaagc aagtgggaga aaacagattc cttctcatgc
1080aacgtgagac acgagggtct gaaaaattac tacctgaaga agaccatctc
ccggtctccg 1140ggtaaatga 114912382PRTArtificial Sequencesynthetic
12Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly1
5 10 15 Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe 35 40
45 Thr Gly Tyr Tyr Met His Trp Phe Arg Gln Ala Pro Gly Gln Glu Cys
50 55 60 Glu Leu Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn
Tyr Ala65 70 75 80 Gln Lys Phe Gln Gly Arg Val Thr Met Thr Gly Asp
Thr Ser Ile Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg
Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Ser Leu
Tyr Cys Thr Asn Gly Val Cys Phe 115 120 125 Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Ser Gly Glu 130 135 140 Pro Ser Gly Pro
Ile Ser Thr Ile Asn Pro Cys Pro Pro Cys Lys Glu145 150 155 160 Cys
His Lys Cys Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser Val Phe 165 170
175 Ile Phe Pro Pro Asn Ile Lys Asp Val Leu Met Ile Ser Leu Thr Pro
180 185 190 Lys Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro
Asp Val 195 200 205 Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His
Thr Ala Gln Thr 210 215 220 Gln Thr His Arg Glu Asp Tyr Asn Ser Thr
Ile Arg Val Val Ser Thr225 230 235 240 Leu Pro Ile Gln His Gln Asp
Trp Met Ser Gly Lys Glu Phe Lys Cys 245 250 255 Lys Val Asn Asn Lys
Asp Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser 260 265 270 Lys Ile Lys
Gly Leu Val Arg Ala Pro Gln Val Tyr Ile Leu Pro Pro 275 280 285 Pro
Ala Glu Gln Leu Ser Arg Lys Asp Val Ser Leu Thr Cys Leu Val 290 295
300 Val Gly Phe Asn Pro Gly Asp Ile Ser Val Glu Trp Thr Ser Asn
Gly305 310 315 320 His Thr Glu Glu Asn Tyr Lys Asp Thr Ala Pro Val
Leu Asp Ser Asp 325 330 335 Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asn
Met Lys Thr Ser Lys Trp 340 345 350 Glu Lys Thr Asp Ser Phe Ser Cys
Asn Val Arg His Glu Gly Leu Lys 355 360 365 Asn Tyr Tyr Leu Lys Lys
Thr Ile Ser Arg Ser Pro Gly Lys 370 375 380 1319DNAArtificial
Sequencesynthetic 13caacacaagt gcgatgcac 191421DNAArtificial
Sequencesynthetic 14gattagcctc catgcctact c 211521DNAArtificial
Sequencesynthetic 15gatgatcatg tgggtagacc t 211620DNAArtificial
Sequencesynthetic 16tctatgctat ctcagtgcta 201749DNAArtificial
Sequencesynthetic 17tgacagtgta atcacatata ctttttcttg tagtcccaga
agtatcatc 4918384DNAArtificial Sequencesynthetic 18gaggtccagc
ttcagcagtc tggagctgag ctggtgaggc ctgggtcctc agtgaagatg 60tcctgcaaga
cttctggata tacattcaca agctacggta taaactgggt gaagcagagg
120cctggacagg gcctggaatg gattggatat atttatattg gaaatggtta
tactgagtac 180aatgagaagt tcaagggcaa ggccacactg acttcagaca
catcctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac
tctgcaatct atttccgtgc aagaggacgg 300gtcggcccgt actactttga
ctactggggc caaggcacca ctctcacagt ctcctcagtc 360ccagaagtat
catctgtctt catc 38419128PRTArtificial Sequencesynthetic 19Glu Val
Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser1 5 10 15
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Ser Tyr 20
25 30 Gly Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45 Gly Tyr Ile Tyr Ile Gly Asn Gly Tyr Thr Glu Tyr Asn
Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Thr Ser
Ser Ser Thr Ala Tyr65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu
Asp Ser Ala Ile Tyr Phe Arg 85 90 95 Ala Arg Gly Arg Val Gly Pro
Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val
Ser Ser Val Pro Glu Val Ser Ser Val Phe Ile 115 120 125
20381DNAArtificial Sequencesynthetic 20gaggtccagc tgcaacagtc
tggacgtgag ctggtcaagc ctggggcttc agtgatgata 60tcttgtacgg cttctggata
cacgttcatt gactacttca taaactggat gaagcggagc 120catggacaga
gccttgagtg gattggagat attaatccta acaatggtgg ttctaactac
180aaccagaagt tcaagggcaa ggccacattg actgtagaca agtcctccag
cacagcctac 240atggatctcc gcagcctgac atctgaagac tctgcagtct
attactgtgc aaaactggga 300cgggactggt acttcgatgt ctggggcaca
gggaccacgg tcaccgtctc ctcagtccca 360gaagtatcat ctgtcttcat c
38121127PRTArtificial Sequencesynthetic 21Glu Val Gln Leu Gln Gln
Ser Gly Arg Glu Leu Val Lys Pro Gly Ala1 5 10 15 Ser Val Met Ile
Ser Cys Thr Ala Ser Gly Tyr Thr Phe Ile Asp Tyr 20 25 30 Phe Ile
Asn Trp Met Lys Arg Ser His Gly Gln Ser Leu Glu Trp Ile 35 40 45
Gly Asp Ile Asn Pro Asn Asn Gly Gly Ser Asn Tyr Asn Gln Lys Phe 50
55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala
Tyr65 70 75 80 Met Asp Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95 Ala Lys Leu Gly Arg Asp Trp Tyr Phe Asp Val
Trp Gly Thr Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Val Pro Glu
Val Ser Ser Val Phe Ile 115 120 125 22375DNAArtificial
Sequencesynthetic 22caggtccaac tgcagcagcc tggggctgag cttgtgaagc
ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga
tgcagtgggt aaaacagagg 120cctggacagg gccttgagtg gatcggagag
attgatcctt ctgatagcta tactaactac 180aatcaaaagt tcaagggcaa
ggccacattg actgtagaca catcctccag cacagcctac 240atgcagctca
gcagcctgac acctgaggac tctgcggtct attactgtgc aagatgtagg
300tactactttg actactgggg ccaaggcacc actctcacag tctcctcagt
cccagaagta 360tcatctgtct tcatc 37523125PRTArtificial
Sequencesynthetic 23Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val
Lys Pro Gly Ala1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Gln Trp Val Lys Gln Arg
Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser
Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala
Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr65 70 75 80 Met Gln
Leu Ser Ser Leu Thr Pro Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Cys Arg Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100
105 110 Thr Val Ser Ser Val Pro Glu Val Ser Ser Val Phe Ile 115 120
125 24393DNAArtificial Sequencesynthetic 24gaggttcagt tgcagcagtc
tggggcagag attgtgaagt caggggcctc agtcaagttg 60tcctgcacag cttctggctt
caacatgaaa gactacttta tccactgggt gaagcagagg 120actgaacagg
gcctggagtg gattggaagg cttgatcctg aggatggtaa aactaaatat
180gccccgaaat tccagggcaa ggccactata acagcagaca catcctccaa
cacagcctac 240ctgcacctca gcagcctgac atctgaggac actgccgtct
attactgtgc tagaggggga 300ctgggacgtg aggaatacta tgctgtggac
tactggggtc aaggaacctc agtcaccgtc 360tcctcagtcc cagaagtatc
atctgtcttc atc 39325131PRTArtificial Sequencesynthetic 25Glu Val
Gln Leu Gln Gln Ser Gly Ala Glu Ile Val Lys Ser Gly Ala1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Met Lys Asp Tyr 20
25 30 Phe Ile His Trp Val Lys Gln Arg Thr Glu Gln Gly Leu Glu Trp
Ile 35 40 45 Gly Arg Leu Asp Pro Glu Asp Gly Lys Thr Lys Tyr Ala
Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser
Ser Asn Thr Ala Tyr65 70 75 80 Leu His Leu Ser Ser Leu Thr Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Gly Arg
Glu Glu Tyr Tyr Ala Val Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Ser
Val Thr Val Ser Ser Val Pro Glu Val Ser Ser 115 120 125 Val Phe Ile
130 26387DNAArtificial Sequencesynthetic 26gatgtacagc ttcaggagtc
aggacctggc ctcgtgaaac cttctcagtc tctgtctctc 60acctgctctg tcactggcta
ctccatcacc agtggttatt actggaactg gatccggcag 120tttccaggaa
acaaactgga atggatgggc tacataagct acgatggtag gaataactac
180aacccatctc tcaaaaatcg aatctccatc actcgtgaca catctaagaa
ccagtttttc 240ctgaagttga attctgtgac tactgaggac acagccacat
attactgtgc aatccatacg 300gtagtagggg actatgttat ggactactgg
ggtcaaggaa cctcagtcac cgtctcctca 360gtcccagaag tatcatctgt cttcatc
38727129PRTArtificial Sequencesynthetic 27Asp Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15 Ser Leu Ser Leu
Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Tyr
Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45
Met Gly Tyr Ile Ser Tyr Asp Gly Arg Asn Asn Tyr Asn Pro Ser Leu 50
55 60 Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe
Phe65 70 75 80 Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr
Tyr Tyr Cys 85 90 95 Ala Ile His Thr Val Val Gly Asp Tyr Val Met
Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser Val
Pro Glu Val Ser Ser Val Phe 115 120 125 Ile28378DNAArtificial
Sequencesynthetic 28gaggttcagc tgcagcagtc tggggcagag cttgtgaggc
caggggcctc agtcaagttg 60tcctgcacag cttctggctt caacattaaa gactactata
tacactgggt gaagaagagg 120cctgaacagg gcctggagtg gattggaagg
attgatcctg aggatggtga tactgagtat 180gccccgaagt tccagggcaa
ggccactatg actgcagaca catcctccaa cacagcctac 240cttcagctca
gcagcctgac atctgaggac attgccgtct attactgtac tacatctagg
300cctttttatt ttgactactg gggccaaggc accactctca cagtctcctc
agtcccagaa 360gtatcatctg tcttcatc 37829126PRTArtificial
Sequencesynthetic 29Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val
Arg Pro Gly Ala1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly
Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Ile His Trp Val Lys Lys Arg
Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Glu
Asp Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Lys Ala
Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75 80 Leu Gln
Leu Ser Ser Leu Thr Ser Glu Asp Ile Ala Val Tyr Tyr Cys 85 90 95
Thr Thr Ser Arg Pro Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100
105 110 Leu Thr Val Ser Ser Val Pro Glu Val Ser Ser Val Phe Ile 115
120 125 30369DNAArtificial Sequencesynthetic 30gaggttcagc
tgcagcagtc tgggacagaa cttgtgaagc caggggcctc agccaagttg 60tcctgcacag
cttctggctt caacgttaaa gactacttta tgcactgggt gaagcagaag
120actgaacagg gcctggagtg gattggaagg attgttcctg aggatggtga
aactaagtct 180gccccgaaat tccaggacag gaccactata agaacagaca
catcctccaa cacatctcac 240ctacaactca acagcctgac atctgaggac
actgccgtct attactgtgc tagacctaac 300cccccttact ggggccaagg
gactctggtc actgtctctg tagtcccaga agtatcatct 360gtcttcatc
36931123PRTArtificial Sequencesynthetic 31Glu Val Gln Leu Gln Gln
Ser Gly Thr Glu Leu Val Lys Pro Gly Ala1 5 10 15 Ser Ala Lys Leu
Ser Cys Thr Ala Ser Gly Phe Asn Val Lys Asp Tyr 20 25 30 Phe Met
His Trp Val Lys Gln Lys Thr Glu Gln Gly Leu Glu Trp Ile 35 40 45
Gly Arg Ile Val Pro Glu Asp Gly Glu Thr Lys Ser Ala Pro Lys Phe 50
55 60 Gln Asp Arg Thr Thr Ile Arg Thr Asp Thr Ser Ser Asn Thr Ser
His65 70 75 80 Leu Gln Leu Asn Ser Leu Thr Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Pro Asn Pro Pro Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val 100 105 110 Ser Val Val Pro Glu Val Ser Ser Val
Phe Ile 115 120 32399DNAArtificial Sequencesynthetic 32gaggtgaaac
tggtggaatc tggaggaggc ttggttcagt ctgggcgttc tctgagactc 60tcctgtgcaa
cttctgggtt caccttcagt gatttctaca tggagtgggt ccgccaagct
120ccagggaagg gactggagtg gattgctaca agtagaaaca aacttaatga
ttatacacca 180gaattcagtg catctgtgaa gggtcgattc atcgtctcca
gagacacttc ccaaaacatc 240ctctaccttc agatgaatgc cctgagacct
gaggacactg ccatttatta ctgtgcaaga 300gcctgtagtg actacgaccg
ttactatgct atggactatt ggggtcaagg aacctcagtc 360accgtctcct
cagtcccaga agtatcatct gtcttcatc 39933133PRTArtificial
Sequencesynthetic 33Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Ser Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly
Phe Thr Phe Ser Asp Phe 20 25 30 Tyr Met Glu Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ala Thr Ser Arg Asn Lys
Leu Asn Asp Tyr Thr Pro Glu Phe Ser Ala 50 55 60 Ser Val Lys Gly
Arg Phe Ile Val Ser Arg Asp Thr Ser Gln Asn Ile65 70 75 80 Leu Tyr
Leu Gln Met Asn Ala Leu Arg Pro Glu Asp Thr Ala Ile Tyr 85 90 95
Tyr Cys Ala Arg Ala Cys Ser Asp Tyr Asp Arg Tyr Tyr Ala Met Asp 100
105 110 Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Val Pro Glu
Val 115 120 125 Ser Ser Val Phe Ile 130 34399DNAArtificial
Sequencesynthetic 34caggtccaac tgcagcagcc tggggctgag cttgtgaagc
ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga
tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg
attgatccta atagtggtgg tactaagtac 180aatgagaagc tcaagaacaa
ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca
gcagcctgac atctgaggac tctgcggtct attattgtgc aagagaggag
300ataaattact acggtagtac ctacggtgct atggactact ggggtcaagg
aacctcagtc 360accgtctcct cagtcccaga agtatcatct gtcttcatc
39935133PRTArtificial Sequencesynthetic 35Gln Val Gln Leu Gln Gln
Pro Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15 Ser Val Lys Leu
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met
His Trp Val Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45
Gly Arg Ile Asp Pro Asn Ser Gly Gly Thr Lys Tyr Asn Glu Lys Leu 50
55 60 Lys Asn Lys Ala Thr Leu Thr Val Asp Lys Pro Ser Ser Thr Ala
Tyr65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Glu Glu Ile Asn Tyr Tyr Gly Ser Thr
Tyr Gly Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Ser Val Thr
Val Ser Ser Val Pro Glu Val 115 120 125 Ser Ser Val Phe Ile 130
36375DNAArtificial Sequencesynthetic 36gaggtccagc tgcagcagtc
tggacctgag ctggtgaagc ctggggcttc agtgaagata 60tcctgcaagg cttctggtta
ctcattcact ggctactaca tgaactgggt gaagcaaagt 120cctgaaaaga
gccttgagtg gattggagag attaatccta gcactggtgg tactacctac
180aaccagaagt tcaaggccaa ggccacattg actgtagaca aatcctccag
cacagcctac 240atgcagctca agagcctgac atctgaggac tctgcagtct
attactgtgc aggtggttac 300tggtacttcg atgtctgggg cacagggacc
acggtcaccg tctcctcagt cccagaagta 360tcatctgtct
tcatc 37537125PRTArtificial Sequencesynthetic 37Glu Val Gln Leu Gln
Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15 Ser Val Lys
Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Tyr
Met Asn Trp Val Lys Gln Ser Pro Glu Lys Ser Leu Glu Trp Ile 35 40
45 Gly Glu Ile Asn Pro Ser Thr Gly Gly Thr Thr Tyr Asn Gln Lys Phe
50 55 60 Lys Ala Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr
Ala Tyr65 70 75 80 Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 85 90 95 Ala Gly Gly Tyr Trp Tyr Phe Asp Val Trp
Gly Thr Gly Thr Thr Val 100 105 110 Thr Val Ser Ser Val Pro Glu Val
Ser Ser Val Phe Ile 115 120 125 38396DNAArtificial
Sequencesynthetic 38gaggtgcagc tggtggagtc tgggggagac ttagtggagc
ctggagggtc cctgaaactc 60tcctgtgcag cctctggatt cactttcagt agctatggca
tgtcttgggt tcgccagact 120ccagacaaga ggctggagtg ggtcgcaacc
attagtagtg gtggtagtta cacctactat 180ccagacagtg tgaaggggcg
attcaccatc tccagagaca atgccaagaa caccctgtac 240ctgcaaatga
gcagtctgaa gtctgaggac acagccatgt attactgtgc aagacatgat
300tactacggta gtagctacgg gtggtacttc gatgtctggg gcacagggac
cacggtcacc 360gtctcctcag tcccagaagt atcatctgtc ttcatc
39639132PRTArtificial Sequencesynthetic 39Glu Val Gln Leu Val Glu
Ser Gly Gly Asp Leu Val Glu Pro Gly Gly1 5 10 15 Ser Leu Lys Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met
Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45
Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met
Tyr Tyr Cys 85 90 95 Ala Arg His Asp Tyr Tyr Gly Ser Ser Tyr Gly
Trp Tyr Phe Asp Val 100 105 110 Trp Gly Thr Gly Thr Thr Val Thr Val
Ser Ser Val Pro Glu Val Ser 115 120 125 Ser Val Phe Ile 130
4024DNAArtificial Sequencesynthetic 40cgatgggggc agggaaagct gcac
244116DNAArtificial Sequencesynthetic 41gtaaaacgac ggccag
164217DNAArtificial Sequencesynthetic 42caggaaacag ctatgac
174321DNAArtificial Sequencevariation(14)...(14)s = c or g
43tcaccatgga ctgsacctgg a 214422DNAArtificial
Sequencevariation(17)...(17)y = c or t 44ccatggacac actttgytcc ac
224523DNAArtificial Sequencesynthetic 45tcaccatgga gtttgggctg agc
234625DNAArtificial Sequencevariation(14)...(14)y = c or t
46agaacatgaa acayctgtgg ttctt 254720DNAArtificial Sequencesynthetic
47atggggtcaa ccgccatcct 204823DNAArtificial Sequencesynthetic
48acaatgtctg tctccttcct cat 234921DNAArtificial Sequencesynthetic
49gcaaggctta caaccacaat c 2150381DNAArtificial Sequencesynthetic
50caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc
60tcctgcaagg cttctggata caccttcacc agttatgata tcaactgggt gcgacaggcc
120actggacaag ggcttgagtg gatgggatgg atgaacccta atagtggtaa
gacaggctat 180gcacagaagt tccagggcag agtcgccatg accaggaaaa
cctccataag cacagcctac 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc gagagaggac 300tacagtaact acggggactt
tgactactgg ggccagggaa ccctggtcac cgtctcctca 360gtgcccaggg
attgtggttg t 38151127PRTArtificial Sequencesynthetic 51Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25
30 Asp Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met
35 40 45 Gly Trp Met Asn Pro Asn Ser Gly Lys Thr Gly Tyr Ala Gln
Lys Phe 50 55 60 Gln Gly Arg Val Ala Met Thr Arg Lys Thr Ser Ile
Ser Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Asp Tyr Ser Asn Tyr
Gly Asp Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val
Ser Ser Val Pro Arg Asp Cys Gly Cys 115 120 125 52378DNAArtificial
Sequencesynthetic 52caggttcagc tggtgcagtc tggagctgag atgaagaagc
ctggggcctc agtgaaggtc 60tcctgcaagg cttctggtta cacctttacc agctatggta
tcagctgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggatgg
atcagcgctt acaatggtaa cacatactat 180gcacagaacc tccagggcag
agtcaccatg accacagaca catccacgag cgcagccttc 240atggacctga
ggagcctgag atctgacgac acggccgtgt attactgtgc gagagatggg
300tatagtacct cgtccttaga ctactggggc cagggaaccc tggtcaccgt
ctcctcagtg 360cccagggatt gtggttgt 37853126PRTArtificial
Sequencesynthetic 53Gln Val Gln Leu Val Gln Ser Gly Ala Glu Met Lys
Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr
Asn Gly Asn Thr Tyr Tyr Ala Gln Asn Leu 50 55 60 Gln Gly Arg Val
Thr Met Thr Thr Asp Thr Ser Thr Ser Ala Ala Phe65 70 75 80 Met Asp
Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Gly Tyr Ser Thr Ser Ser Leu Asp Tyr Trp Gly Gln Gly 100
105 110 Thr Leu Val Thr Val Ser Ser Val Pro Arg Asp Cys Gly Cys 115
120 125 54378DNAArtificial Sequencesynthetic 54caggttcagc
tggtgcagtc tggagctgag atgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg
cttctggtta cacctttacc agctatggta tcagctgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggatgg atcagcgctt acaatggtaa
cacatactat 180gcacagaacc tccagggcag agtcaccatg accacagaca
catccacgag cgcagccttc 240atggacctga ggagcctgag atctgacgac
acggccgtgt attactgtgc gagagatggg 300tatagtacct cgtccttaga
ctactggggc cagggaaccc tggtcaccgt ctcctcagtg 360cccagggatt gtggttgt
37855126PRTArtificial Sequencesynthetic 55Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Met Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Tyr Tyr Ala Gln Asn Leu 50
55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Ala Ala
Phe65 70 75 80 Met Asp Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Tyr Ser Thr Ser Ser Leu Asp
Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Val Pro
Arg Asp Cys Gly Cys 115 120 125 56408DNAArtificial
Sequencesynthetic 56caggttcagc tggtgcagtc tggagctgag gtgaagaagc
ctggggcctc agtgaaggtc 60tcctgcaagg cttctggtta cacctttacc agctatggta
tcagctgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggatgg
atcagcgctt acaatggtaa cacaaactat 180gcacagaagc tccagggcag
agtcaccatg accacagaca catccacgag cacagcctac 240atggagctga
ggagcctgag atctgacgac acggccgtgt attactgtgc gagagatgat
300atgattacgt ttgggggagt tatcgccaac tactactact acggtatgga
cgtctggggc 360caagggacca cggtcaccgt cacctcagtg cccagggatt gtggttgt
40857136PRTArtificial Sequencesynthetic 57Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50
55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala
Tyr65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Asp Asp Met Ile Thr Phe Gly Gly Val
Ile Ala Asn Tyr Tyr 100 105 110 Tyr Tyr Gly Met Asp Val Trp Gly Gln
Gly Thr Thr Val Thr Val Thr 115 120 125 Ser Val Pro Arg Asp Cys Gly
Cys 130 135 58372DNAArtificial Sequencesynthetic 58caggttcagt
tgctgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctacaagg
cttctgatta cacctttacc agctatggta tcagctgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggatgg atcagcgctt acaatggtaa
cacaaactat 180gcacagaacc tccagggcag agtcaccatg accacagaca
catccacgag cacagcctac 240atggaactga ggagcctgag atctgacgac
tcggccgtgt attactgtgc gagagaggag 300ctggaacttt ttgactactg
gggccaggga accctggtca ccgtctcctc agtgcccagg 360gattgtggtt gt
37259124PRTArtificial Sequencesynthetic 59Gln Val Gln Leu Leu Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val
Ser Tyr Lys Ala Ser Asp Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Asn Leu 50
55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala
Tyr65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Ser Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Glu Glu Leu Glu Leu Phe Asp Tyr Trp
Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Val Pro Arg Asp
Cys Gly Cys 115 120 60378DNAArtificial Sequencesynthetic
60caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggaggg atcatcccta tctttggtac
agcaaactac 180gcacagaagt tccagggcag agtcacgatt accacggacg
aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc gagagatggg 300tatagtacct cgtccttaga
ctactggggc cagggaaccc tggtcaccgt ctcctcagtg 360cccagggatt gtggttgt
37861126PRTArtificial Sequencesynthetic 61Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Ser Thr Ala
Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Tyr Ser Thr Ser Ser Leu Asp
Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Val Pro
Arg Asp Cys Gly Cys 115 120 125 62390DNAArtificial
Sequencesynthetic 62caggtccagc tggtgcagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60tcctgcaagg cttctggagg caccttcagc agctatgcta
tcagctgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggaggg
atcatcccta tctttggtac agcaaactac 180gcacagaagt tccagggcag
agtcacgatt accacggacg aatccacgag cacagcctac 240atggagctga
gcagcctgag atctgaggac acggccgtgt attactgtgc ggttatagca
300gtggctggta cctactacta ctacggtatg gacgtctggg gccaagggac
cacggtcacc 360gtctccttag tgcccaggga ttgtggttgt
39063130PRTArtificial Sequencesynthetic 63Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Ser Thr Ala
Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Val Ile Ala Val Ala Gly Thr Tyr Tyr Tyr
Tyr Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Leu Val Pro Arg Asp Cys 115 120 125 Gly Cys 130
64101DNAArtificial Sequencesynthetic 64tgacagtgta atcacatata
ctttttcttg tagtgcccag ggattgtggt tgtaagcctt 60gcatatgtac aggtaagtca
gtaggccttt caccctgacc c 101
* * * * *