U.S. patent application number 10/753554 was filed with the patent office on 2004-11-18 for murine expression of human iga lambda locus.
This patent application is currently assigned to The Babraham Institute. Invention is credited to Bruggemann, Marianne.
Application Number | 20040231012 10/753554 |
Document ID | / |
Family ID | 10841679 |
Filed Date | 2004-11-18 |
United States Patent
Application |
20040231012 |
Kind Code |
A1 |
Bruggemann, Marianne |
November 18, 2004 |
Murine expression of human IgA Lambda locus
Abstract
In humans, approximately 60% of expressed immunoglobulin light
chains are of the Kappa type and 40% of the Lambda type. In mice,
there is almost no expression from the Lambda locus and over 95% of
light chains are of Kappa type. The present invention discloses,
among other things, transgenic mice carrying most of the human Ig
Lambda light chain locus in their genome. The resulting mice
express light chains with Kappa/Lambda ratio similar to the human
ratio. Breeding of HuIg Lamda mice to Kappa-deficient mice also is
described, as well as the generation of human monoclonal antibodies
from transgenic mice with human Ig Lambda locus.
Inventors: |
Bruggemann, Marianne;
(Cambridge, GB) |
Correspondence
Address: |
HELLER EHRMAN WHITE & MCAULIFFE LLP
1666 K STREET,NW
SUITE 300
WASHINGTON
DC
20006
US
|
Assignee: |
The Babraham Institute
Babraham Hall
Cambridge
GB
CB2 4AT
|
Family ID: |
10841679 |
Appl. No.: |
10/753554 |
Filed: |
January 9, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10753554 |
Jan 9, 2004 |
|
|
|
09734613 |
Dec 13, 2000 |
|
|
|
Current U.S.
Class: |
800/18 |
Current CPC
Class: |
A01K 2217/00 20130101;
A01K 2227/105 20130101; C07K 16/00 20130101; C12N 15/8509 20130101;
A01K 2207/15 20130101; A01K 2217/072 20130101; A01K 2217/05
20130101; A01K 2267/01 20130101; C07K 2317/24 20130101; A01K
2267/0381 20130101; A01K 67/0278 20130101; A01K 2217/075
20130101 |
Class at
Publication: |
800/018 |
International
Class: |
A01K 067/027 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 3, 1999 |
WO |
PCT/GB99/03632 |
Nov 3, 1998 |
GB |
GB9823930.4 |
Claims
1-34 (Canceled)
35. A transgenic mouse comprising as a translocus a yeast
artificial chromosome (YAC) of about 410 Kb, wherein the YAC
contains at least a majority of the human V.lambda. genes of
cluster A and all the human J.lambda.-C.lambda. segments in
germline configuration, wherein the translocus shows high
expression, and is able to compete equally with the endogenous
mouse .kappa. locus.
36. A transgenic mouse comprising as a translocus a yeast
artificial chromosome (YAC) of about 410 Kb, wherein the YAC
contains at least a majority of the human V.lambda. genes of
cluster A and all the human J.lambda.-C.lambda. segments in
germline configuration, wherein the mouse has one or both
endogenous Ig.kappa. alleles disrupted, and wherein the translocus
shows high expression.
37. A transgenic mouse comprising a 380 Kb region of the human
immunoglobulin (Ig) .lambda. light (L) chain locus in germline
configuration, wherein the 380 Kb region resides on a yeast
artificial chromosome (YAC) that accommodates the most proximal V
(variable gene) .lambda. cluster, wherein the 380 Kb region has 15
V.lambda. genes and all J.lambda.-C.lambda. segments with the 3'
region, wherein the 3' region includes a downstream enhancer.
38. The transgenic mouse according to claim 35, wherein the mouse
includes a HuIg.lambda. YAC that accommodates a 380 Kb region of
the human .lambda. light chain locus in authentic configuration
with all V.lambda. genes of cluster A, the J.lambda.-C.lambda.
segments and the 3' enhancer.
Description
[0001] This application claims priority to PCT/GB99/03632, filed
Nov. 3, 1999, the entirety of which is hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] The light chain component of the Ig protein is encoded by 2
separate loci, Ig.kappa. and Ig.lambda.. The proportion of
antibodies containing .kappa. or .lambda. light chains varies
considerably between different species (1-3), e.g., in mice the
.kappa.:.lambda. ratio is 95:5, compared to 60:40 in humans. Two
models have evolved to account for this apparent bias in the
expression of .kappa. in the mouse. First, from the observation
that murine Ig.lambda.-producing myelomas have rearranged .kappa.
light chain genes, and that Ig.lambda.-producing cells have the
.lambda. light chain locus in germline configuration, it was
proposed that .kappa. rearrangement must occur before .lambda.
rearrangement can commence (4, 5). In the human situation, however,
while almost all .lambda. producing cells have both .kappa. alleles
rearranged, the proportion of .kappa. and .lambda. producing cells
are similar (4). The second proposal is that .kappa. and .lambda.
loci are both available for rearrangement at the same time, but the
mouse .kappa. locus is more efficient at engaging the rearrangement
process (6). The occasional finding of cells with rearranged
.lambda. and the .kappa. locus in germline configuration may
support this (5, 7, 8). The influence of antigen selection on the
biased .kappa.:.lambda. ratio is discounted by the finding that the
ratio is similar in fetal liver and in cells that have not
encountered antigen (9-13).
[0003] Light chain V-J rearrangement occurs at the transition from
pre B-II to immature B cells, where the surrogate light chain
associated with membrane Ig.mu. is replaced by .kappa. or .lambda.
light chain (14). Although the timing of light chain rearrangement
is essentially defined, the processes which activate light chain
locus rearrangement are not fully understood. From locus silencing
experiments, it became clear that .kappa. rearrangement is not a
prerequisite for .lambda. recombination (15). Indeed, .kappa. and
.lambda. rearrangements are independent events (16), the activation
of which may be affected by differences in the strength of the
respective enhancers. A region believed to be important in the
regulation of the accessibility of the human .lambda. locus has
been identified about 10 Kb downstream of C.lambda.7 (17, 18).
Functional comparisons in reporter gene assays identified a core
enhancer region that is flanked by elements which can drastically
reduce enhancer activity in pre-B cells (17). Although transfection
studies showed that the .kappa. and .lambda.3' enhancer regions
appear to be functionally equivalent, other (functional) sequences
flanking the core enhancer motifs are remarkably dissimilar.
Targeted deletion of the .kappa.3' enhancer in transgenic mice
showed that this region is not essential for .kappa. locus
rearrangement and expression but is required to establish the
.kappa.:.lambda. ratio (19).
[0004] The human Ig.lambda. locus on chromosome 22q11.2 is 1.1 Mb
in size and typically contains 70 V.lambda. genes and 7
J.lambda.-C.lambda. gene segments (20, 21 and references therein).
About half of the V.lambda. genes are regarded as functional and
J.lambda.-C.lambda.1, 2, 3 and 7 are active. The V.lambda. genes
are organized in 3 clusters which contain distinct V gene family
groups. There are 10 V.lambda. gene families, with the largest
V.lambda.III being represented by 23 members. In human peripheral
blood lymphocytes, the most J-C proximal V gene segments in cluster
A, from families I, II and III, are preferentially rearranged, with
the contribution of the 2a2 V.lambda. segment (2-14 in the new
nomenclature (22) being unusually high (23). All .lambda. gene
segments have the same polarity which allows deletional
rearrangement (24). Sequence diversity of the Ig.lambda. repertoire
is provided mainly by V.lambda.-J.lambda. combination. Additional
CDR3 diversity due to N (nonencoded)- or P (palindromic)-nucleotide
additions at the V to J junction, although not as extensive as seen
in IgH rearrangement, seems to be much more frequently used in
humans than in mice (25, 26, 27, 28), where the TdT (terminal
deoxyribonucleotide transferase) activity is down-regulated at the
time of light chain rearrangement.
[0005] It has been shown that human Ig can be produced in
transgenic mice carrying human Ig genes on miniloci or yeast
artificial chromosomes (YACs) (58, 59, 60, 61, 62) and that
silencing of the endogenous mouse heavy and .kappa. loci
enhances-human antibody production in such transgenic animals.
However, in all such mice reported to date, only the human .kappa.
light chain genes have been incorporated and there have been no
reports of the human .lambda. light chain locus being integrated
into transgenic mice. Therefore, until the present invention, no
.lambda.-containing human antibodies have been made from transgenic
mice, nor has there been any information on the expressibility of
human .lambda. genes in such animals or on the relative
contributions of human .kappa. and .lambda. in mice carrying both
transgenic human loci. Thus it was not known whether
.lambda.-transgenic mice would be suitable for the production of
human antibodies.
SUMMARY OF THE INVENTION
[0006] It is an object of the present invention to provide
transgenic animals, such as a mouse, that can express human
.lambda. sequences. In accomplishing this and other objects, there
is provided, in accordance with one aspect of the invention,
transgenic mice comprising as a translocus a YAC of about 410 Kb,
wherein the YAC contains most of the human V.lambda. genes of
cluster A and all the human J.lambda.-C.lambda. segments in
germline configuration, wherein the translocus shows high
expression, and is able to compete equally with the endogenous
mouse .kappa. locus.
[0007] There also is provided, in accordance with another aspect of
the invention, transgenic mice comprising as a translocus a YAC of
about 410 Kb, wherein the YAC contains most of the human V.lambda.
genes of cluster A and all the human J.lambda.-C.lambda. segments
in germline configuration, wherein the mouse has one or both
endogenous Ig.kappa. alleles disrupted, and wherein the translocus
shows high expression.
[0008] In accordance with yet another aspect of the invention,
there are provided a transgenic mouse carrying a 380 Kb region of
the human immunoglobulin (Ig) .lambda. light (L) chain locus in
germline configuration, wherein the introduced translocus resides
on a yeast artificial chromosome (YAC) that accommodates the most
proximal V (variable gene) .lambda. cluster--with 15 V.lambda.
genes that contribute to over 60% of .lambda. light chains in
man--and all J.lambda.-C.lambda. segments with the 3' region
including the downstream enhancer.
[0009] In accordance with still another aspect of the invention,
there are provided transgenic mice comprising human Ig lambda genes
in which the proportion of the .kappa. and .lambda. light chains
expressed by said human lambda genes resembles that found in
humans, and exhibits relative proportions of .ltoreq.60% .kappa.
light chains and .gtoreq.40% .lambda. light chains.
[0010] The transgenic mice according to the invention can include a
HuIg.lambda. YAC that accommodates a 380 Kb region of the human
.lambda. light chain locus in authentic configuration with all
V.lambda. genes of cluster A, the J.lambda.-C.lambda. segments and
the 3' enhancer, such as the HuIg.lambda. YAC shown in FIG. 1.
[0011] In accordance with a further aspect of the invention, there
are provided methods for producing transgenic mice, comprising:
[0012] (a) introducing a HuIg.lambda. YAC into murine embryonic
stems cells; and
[0013] (b) deriving a transgenic mouse from the cells of step (a).
The HuIg.kappa. YAC can be about 410 Kb and accommodate a 380 Kb
region (V.lambda.-JC.lambda.) of the human .lambda. light chain
locus with V, J and C genes in germline configuration when it is
introduced into said stem cells. Additionally, selectable markers,
such as two copies of the neomycin resistance gene (NEO.sup.r) can
be site-specifically integrated into the ampicillin gene on the
left (centromeric) YAC arm in order to permit selection. The
methods can further comprise steps where YAC-containing yeast cells
are fused with HM-1 embryonic stem (ES) cells and G418 resistance
colonies are picked and analyzed 2-3 weeks after protoplast fusion.
The ES cells can contain a complete HuIg.lambda. YAC copy, and can
be used for blastocyte injection to produce a transgenic animal.
The breeding of a transgenic animal with a Balb/c mouse, for
example, results in germline transmission. Breeding partners
include .kappa..sup.-/- mice to establish lines of transgenic
mice.
[0014] In accordance with another aspect of the invention, there
are provided hybridomas obtainable from HuIg.lambda.
YAC/.kappa..sup.+/- mice (preferably one that is 3 months old), for
example, by fusion of splenocytes with NSO myeloma cells, and
subsequent selection of single clones. Antibodies obtainable from
these hybridomas also are provided.
[0015] In accordance with another aspect of the invention, there is
provided transgenic mmice comprising as a translocus a yeast
artificial chromosome (YAC) of greater than 100 Kb which contains a
proportion of the human V.lambda. genes proximal to the
J.lambda.-C.lambda. cluster in germline configuration. The YAC can
include a 380 Kb region of the human Ig.lambda. locus in authentic
configuration with most V.lambda. genes of cluster A,
J.lambda.-C.lambda. segments and the 3' enhancer.
[0016] In accordance with yet another aspect of the invention,
there are provided transgenic mice comprising variable, joining and
constant genes of the human .lambda. light chain locus as a
transgenic locus on a YAC, wherein B cells of said mice rearrange
said .lambda. light chain genes and the mice express serum
immunoglobulins containing human .lambda. light chains. the
.lambda. translocus is rearranged with similar efficiency as
endogenous mouse .kappa. and at the same time as or before the
endogenous .kappa. locus. Additionally, the endogenous .kappa.
locus can be silenced, and the mouse expresses serum
immunoglobulins containing human .lambda. light chains. The
transgenic mice can further comprise human heavy chain genes as a
second transgenic locus integrated on a separate YAC, wherein the
mice express serum immunoglobulin molecules containing combinations
of human heavy and .lambda. light chains. Moreover, the second
transgenic locus can carry a diversity of human heavy chain
constant region genes, including .mu., .delta. and .gamma. genes.
For example, the heavy chain transgenic locus can carry a diversity
of human heavy chain constant region genes, including .mu., .delta.
and .gamma. genes, in authentic germline configuration. Also
permissible are transgenic mice carrying human .lambda. light chain
genes, wherein the mice comprise human .kappa. light chain genes as
a second transgenic light chain locus integrated on a separate YAC,
wherein the mice express serum immunoglobulin molecules containing
human .kappa. and .lambda. light chains. Additionally, there are
provided transgenic mice carrying human .lambda. light chain genes
comprising human heavy chain genes as a second transgenic locus and
human .kappa. light chain genes as a third transgenic locus,
wherein the mice express serum immunoglobulin molecules containing
human heavy chains in combination with human .kappa. or .lambda.
light chains. Expression of the endogenous mouse heavy and/or light
chain loci in the transgenic mice of the invention can be
prevented, if desired, through gene targeting or other means and
which expresses serum immunoglobulin containing human heavy and/or
light chains and which are deficient in production of mouse
immunoglobulin.
[0017] In accordance with still a further aspect of the invention,
there are provided transgenic mice carrying human .lambda. light
chain genes in which expression of the human .lambda. locus is
equal to or greater than that of the endogenous or transgenic human
.kappa. locus. The .lambda. translocus can be bred to homozygosity.
Additionally, the there can be rearranged variable genes in the
.kappa. translocus are subject to somatic hypermutation.
[0018] In accordance with yet a further aspect of the invention,
there are provided methods for production of human antibodies
comprising stimulating with antigen transgenic mice incorporating
human .lambda. light chain genes into their genome and collecting
the human antibodies which bind to the antigen. Hybridomas for the
production of antibodies can be created through fusion to an
appropriate mouse myeloma cell line.
[0019] In accordance with still a further aspect of the invention,
there, are provided human monoclonal antibodies comprising human
heavy and light chains of diverse isotypes and chain combinations
produced from transgenic mice carrying the human .lambda.
translocus. The variable regions of the human .lambda. light chains
of such antibodies can undergo somatic mutation. The antibodies
preferably have an affinity for antigen of greater than 10.sup.-8
M.
[0020] These and other aspects of the invention will become
apparent to the skilled person upon a review of the specification,
including the examples, figures and sequences.
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIG. 1 shows that the HuIg.lambda.YAC accommodates a 380 Kb
region of the human .lambda. light chain locus in authentic
configuration with all V.lambda. genes of cluster A (21, 40, 54),
the J.lambda.-C.lambda. segments and the 3' enhancer (17). Black
boxes represent functional V.lambda. genes (3-27, 3-25, 2-23, 3-22,
3-21, 3-19, 2-18, 3-16, 2-14, 2-11, 3-10, 3-9,2-8, 4-3,3-1) and
white boxes show V.lambda. genes with open reading frames (2-33,
3-32, 3-12) which have not been identified in productive
rearrangements of human lymphocytes (40). Pseudogenes are not
shown. Black triangles indicate rearranged V genes found by RT-PCR
in spleen and sorted Peyer's patch cells from HuIg.lambda. mice.
The unique NotI restriction site is indicated. Probes to assess the
integrity of the HuIg.lambda.YAC, LA (left arm) and C.lambda.2+3
are indicated.
[0022] FIG. 2 depicts a Southern blot analysis of HuIg.lambda.YAC
Integration. (Left) NotI digested testis DNA resolved on PFGE and
hybridized with the C.lambda.2+3 probe. The same size band was
obtained with the left arm probe (not shown). The majority of the
hybridization signal remains in the compression band (CB)
presumably due to protection of the NotI site by methylation.
(Right) EcORI/HindIII digests hybridized with the C.lambda.2+3
probe. Lane 1: HuIg.lambda.YAC ES cell DNA from a protoplast fusion
clone; lane 2: normal ES cell DNA; lane 3: human genomic DNA, (XZ);
lane 4: human KB carcinoma (55) DNA; lane 5 and 6: tail DNA from 2
HuIg.lambda.YAC germline transmission mice. Note that the human DNA
shows an additional 5.2 Kb band which represents an allelic
variation (56).
[0023] FIG. 3 shows human Ig.lambda., mouse Ig.kappa. and mouse
Ig.lambda. serum titers for Hu.lambda.YAC/Mo.kappa..sup.+/- and
Hu.lambda.YAC/Mo.kappa..sup.-/- mice (5-6 mice per group kept in
pathogermfree conditions and 5 human sera). Antibody levels
presented were obtained from 2-3 months old animals but the serum
titers from older mice were similar. From the 5
Hu.lambda.YAC/Mo.kappa..sup.+/- mice tested 3 animals had somewhat
higher mouse Ig.kappa. titers than human Ig.lambda. while 2 animals
showed higher human Ig.lambda. levels.
[0024] The controls show light chain distribution in human and
normal mouse serum. Total Ig levels are in good agreement with the
sum of individual titers (not shown).
[0025] FIG. 4 depicts a flow cytometric analysis of light chain
expression in the developing B-cell. (A) .kappa. and .lambda. light
chain distribution of CD 19.sup.+ human peripheral lymphocytes and
B220.sup.+ mouse spleen cells from Hu.lambda.YAC/Mo.kappa..sup.+/-
and Hu.lambda.YAC/Mo.kappa..sup.-/- mice. (B) Mouse Ig.lambda. and
human Ig.lambda. light chain distribution in gated populations of
CD19.sup.+/c-kit.sup.+ and CD19.sup.+/CD25.sup.+ bone marrow
cells.
[0026] FIG. 5 shows human V.lambda. sequences from sorted
B220.sup.+ and PNA.sup.+ Peyer's patches B-cells from
HuIg.lambda..sup.+YAC/.kappa..sup.- +/- mice.
[0027] FIG. 6 illustrates the occurrence of somatic hypermutation
in the H, .kappa., and .lambda. transloci of 5-feature mice after
immunization. The number of mutations in individual sequenced
chains are indicated in the pie chart which shows the frequency of
their occurrence. `Total analyzed` refers to the number of
individual chains sequenced.
[0028] FIG. 7 depicts serum antibody titers in 5-feature transgenic
mice following immunization with 4 antigens. The responses to human
fodrin, human placental alkaline phosphatase (PLAP), the B subunit
of cholera toxin and human carcinoembryonic antigen (CES) are
shown, as measured by ELISA. In all cases the uppermost (bold) line
is the response after 2 or 3 immunizations (background subtracted).
ELISAs were developed with anti-human IgM antibodies linked to
horseradish peroxidase.
[0029] FIG. 8 shows the properties of a human %-containing
monoclonal antibody (7783.26) against human placental alkaline
phosphatase (PLAP), produced from an immunized 5-feature mouse. (A)
Titration of anti-PLAP from supernatant of an individual hybridoma
clone of antibody 7783.26 against immobilized PLAP antigen in an
ELISA assay, developed with anti-human X antibodies linked to
horseradish peroxidase. (B) Inhibition of binding of human
anti-PLAP antibody 7783.26 by free PLAP. (C) Affinity determination
of human anti-PLAP antibody 7783/26 by Scatchard plot after the
method of Friguet et al (63). From the plot, the affinity (Ka) of
this antibody was estimated to be 2.times.10.sup.9 M.sup.-1
DETAILED DESCRIPTION OF ASPECTS OF THE INVENTION
[0030] The present invention provides transgenic mice (`lambda
mice`, or `.lambda. mice`) into which a YAC of about 410 Kb has
been introduced as a transgenic locus (translocus) containing most
of the human V.lambda. genes of cluster A and all the
J.lambda.-C.lambda. segments in germline configuration. As the
skilled person will recognize, sizes of polynucleotides provided
herein are approximate, and can be readily changed in view of the
teachings contained herein without departing from the
invention.
[0031] The translocus leads to high expression of human .lambda.
light chains in plasma and on B cells and is able to compete
equally with the endogenous mouse .kappa. locus. A number of
different transgenic mice are further described in which the human
.lambda. light chain is present in different combinations with YACs
encoding genes of the human heavy chain locus (IgH) and genes of
the human .kappa. light chain locus (Ig.kappa.), and in which the
endogenous mouse alleles for heavy chain or .kappa. light chain may
have been disrupted. Mice with these features are suitable for the
production of fully human antibodies carrying the .lambda. light
chain. After immunization with antigens, such mice produce fully
human antibodies containing the .lambda. light chain with at least
as high a frequency as they do .kappa.-containing antibodies, and
often with an excess of .lambda.-containing antibodies over
.kappa.. Moreover, the mice according to the invention can be used
to produce antigen specific monoclonal human % containing
antibodies of high affinity. Isolation of human V.lambda. genes
from the transgenic mice by RT-PCR cloning showed that many
V.lambda. genes are rearranged and exhibit somatic hypermutation.
Such DNA products can be used to construct human
.lambda.-containing antibodies for expression in prokaryotic or
eukaryotic cells. Thus human .lambda.-expressing transgenic mice
provide an improved method of producing fully human antibodies,
either from hybridomas or by in vitro recovery and manipulation of
V.lambda. genes.
[0032] The present invention provides the first transgenic mice
carrying unrearranged human Ig.lambda. genes on a YAC as a
translocus. They demonstrate that the human .lambda. genes are
well-expressed in the translocus mouse similar to or better than
their expression in man relative to .kappa.. The
.lambda.-containing antibodies made by such translocus mice may be
of value as therapeutic reagents.
[0033] According to the invention, transgenic mice were created
carrying a 380 Kb region of the human immunoglobulin (Ig) .lambda.
light (L) chain locus in germline configuration. The introduced
translocus on a yeast artificial chromosome (YAC) accommodates the
most proximal V (variable gene) .lambda. cluster--with 15 V.lambda.
genes that contribute to over 60% of .lambda. light chains in
man--and all J.lambda.-C.lambda. segments with the 3' region
including the downstream enhancer. The HuIg.lambda. YAC mice were
bred with animals in which mouse .kappa. L chain production was
silenced by gene targeting. Human Ig .lambda. expression in mouse
.kappa..sup.-/- animals was dominant with up to 84% of B220.sup.+
B-cells expressing surface human L chain. In serum human Ig.lambda.
was up to 1.9 mg/ml, while mouse L chain levels were reduced to 0.2
mg/ml. However, a striking result was that in heterozygous
.kappa..sup.+/-- and normal .kappa..sup.+/+ translocus mice both
human .lambda. and mouse .kappa. were expressed at similar high
levels (38% and 45% of cells, respectively). Interestingly, in
HuIg.lambda.YAC/Mo.kappa. mice human .lambda. is predominantly
expressed at the pre B-cell stage with subsequent upregulation of
cells expressing mouse L chain at the immature B-cell stage. The
human V.lambda. genes hypermutate readily but show restricted P or
N sequence variability at the V-J junction. The finding that human
.lambda. genes can be utilized with similar efficiency in mouse and
man implies that L chain expression is dependent on the
configuration of the locus. Thus, the transfer of large transloci
may circumvent many expression problems encountered with small gene
constructs introduced into cells and animals, with the advantage
that some silencing approaches such as exploiting human antibody
production may prove unnecessary.
[0034] Furthermore, the .lambda.-expressing transgenic mice with
the .kappa..sup.-/- background were mated with those in which the
human heavy (H) chain genes were incorporated as a translocus (65)
and in which the endogenous mouse H chain locus had been silenced
by the .mu.MV.sup.-/- modification (57), producing so-called
'4-feature'.lambda.-mice (human H and .lambda. transloci on a
endogenous H and .kappa. knockout background). These mice produced
human IgM,.lambda. immunoglobulin in their plasma and responded to
immunization by production of human IgM,.lambda. antibodies. The
mice were further crossed with those having, in addition to the
other characteristics, the human .kappa. genes as a YAC translocus
(65) to produce mice which express both human IgM,.lambda. and
IgM,.kappa. antibodies, so called `5-feature` mice. In these
animals, the B lymphocyte population shows preferential (3:1)
expression of human .lambda. over human .kappa.. Human IgM is found
in the serum at between 50 and 400 .mu.g per ml. The 5-feature mice
were immunized with several different antigens, including human
antigens, leading to production of specific human antibodies in
their serum. Hybridomas secreting fully human monoclonal antibodies
were prepared from the spleen cells of such mice. Among such
hybridomas, the ratio of .lambda.:.kappa. is often in favor of
.lambda., in some cases by as much as 8:1. This is remarkable in
view of the .kappa. bias (60.kappa.:40.lambda.) seen in normal
human plasma and the extreme .kappa. bias (95.kappa.:5.lambda.) in
plasma of normal mice. Thus, transgenic mice have been produced in
which the proportion of .lambda. to .kappa. light chains resembles
or exceeds that normally found in humans. In general, transgenic
loci are not highly expressed in authentic fashion or as well as
endogenous genes. Moreover, in normal mice the endogenous .lambda.
genes are not efficiently expressed and it was therefore assumed
that other .lambda. genes would also be expressed at low frequency.
Thus, the equally high expression in man and mouse of human
.lambda. is very unexpected and could not have been predicted.
[0035] The 4 and 5 feature .lambda. mice develop a highly effective
repertoire of .lambda.-containing antibodies which can be used to
make hybridomas and monoclonal antibodies of high affinity. The
.lambda. translocus undergoes somatic hypermutation and could
therefore contribute to increased antibody affinity. Also described
herein is a human monoclonal antibody, anti-human placental
alkaline phosphatase (PLAP), with a .lambda. light chain from a
5-feature mouse, with an affinity of greater than 10.sup.8
M.sup.-1. Thus, according to the invention there have produced mice
suitable for immunization with human antigens and for the isolation
of high affinity human antibodies containing .lambda. light chains
which are suitable for therapeutic applications.
[0036] The present invention is further illustrated by the
following examples, which do not limit the invention in any manner
or way.
EXAMPLE I
Production Methodologies
[0037] The HuIg.lambda.YAC, Introduction into ES Cells and
Derivation of Transgenic Mice
[0038] The 410 Kb HuIg.lambda.YAC, accommodating a 380 Kb region
(V.lambda.-J.lambda.) of the human .lambda. light chain locus with
V, J and C genes in germline configuration, was constructed as
described (29). To allow selection, 2 copies of the neomycin
resistance gene (NEO') were site-specifically integrated into the
ampicillin gene on the left (centromeric) YAC arm. YAC-containing
yeast cells were fused with HM-1 ES cells, a kind gift from D.
Melton, as described (30) and G418 resistant colonies were picked
and analyzed 2-3 weeks after protoplast fusion. ES cells containing
a complete HuIg.lambda.YAC copy, confirmed by Southern
hybridization, were used for blastocyst injection to produce
chimeric animals (31). Breeding of chimeric animals with Balb/c
mice resulted in germline transmission. Further breeding with
.kappa..sup.-/- mice (32) established the lines for analysis.
[0039] Southern Blot Analysis.
[0040] Either conventional DNA was obtained (33) or high molecular
weight DNA was prepared in agarose blocks (34). For the preparation
of testis DNA, tissues were homogenized and passed through 70 .mu.M
nylon mesh. PFGE conditions to separate in the 50-900 Kb range were
1% agarose, 180V, 70 s switch time and 30 hours running time at
3.5.degree. C. Hybridization probes were C.lambda.2+3 and the left
YAC arm probe (LA) comprising LYS2 (29).
[0041] Production of 4 and 5 Feature Mice
[0042] The 4 and 5 feature mice were produced by crossing the
transgenic .lambda. mice with transgenic mice described previously
carrying the IgH YAC and the Ig.kappa. YAC as transloci, and in
which the endogenous loci for H and .kappa. were disrupted
(.mu.MT.sup.-/-, Mo.kappa..sup.-/- knockouts) (65 and references
therein). The transgenic status of the offspring was confirmed by
Southern hybridization of genomic DNA with appropriate-probes. The
strains were bred to homozygosity to carry 2 alleles of each of the
transloci and for each of the knockout features. Test breeding
showed that the 3 transloci and 2 knockouts were not linked.
[0043] Immunization of Mice, Hybridoma Production and ELISA
Assay
[0044] Four and 5 feature mice were initially immunized with 50
.mu.g of antigen in complete Freund's adjuvant and boosted at 4 and
8 weeks with 50 .mu.g in IFA. A final boost was given at 12 weeks
and 3 days later hybridomas were prepared by fusion of splenocytes
with NS/0 myeloma cells using polyethylene glycol. Fusion
supernatants were screened for reactivity with the immunogen by
ELISA and selected clones expanded for further analysis and cloned.
Human IgM expression levels and light chain isotype were determined
by ELISA. Specificity of hybridomas was confirmed by testing for
cross-reactivity to unrelated antigens.
[0045] Affinity determination was performed by the method of
Friguet et al. (63), i.e. a fixed concentration of antibody was
incubated with varying amounts of PLAP to equilibrium in tubes and
the free antibody determined by quantitative ELISA on an
PLAP-coated microwell plate. The free and bound antibody
concentrations were calculated and the Scatchard plot of B/F
antigen versus B antigen was plotted. The affinity was given by the
slope of the graph.
[0046] For the detection of human or mouse Ig.lambda., coating
reagents were a 1:500 dilution of anti-human .lambda. light chain
monoclonal antibody (mAB) HP-6054 (L 6522, Sigma, St. Louis, Mo.)
or a 1:500 dilution of the 2.3 mg/ml rat anti-mouse .lambda. mAB (L
2280, Sigma), respectively. Respective binding was detected with
biotinylated antibodies: polyclonal anti-human X (B 0900, Sigma), a
1:1000 dilution of polyclonal anti-mouse .lambda. (RPN 1178,
Amersham Intl., Amersham, UK) or rat anti-mouse Ig.lambda. (#
021172D, Pharmingen, San Diego, USA) followed by
streptavadin-conjugated horseradish peroxidase (Amersham). Mouse
IgG2a.lambda. myeloma protein from HOPC1 (M 6034, Sigma) and human
serum IgG.lambda. (I 4014, Sigma) were used to standardize the
assays. To determine mouse .kappa. light chain levels, plates were
coated with a 1:1000 dilution of rat anti-mouse .kappa., clone
EM34.1 (K 2132, Sigma), and bound Ig was detected using
biotinylated rat mAB anti-mouse Ig.kappa. (Cat. no. 04-6640, Zymed,
San Francisco). Mouse myeloma proteins IgG2a.kappa. and IgG1.kappa.
(UPC10 and MOPC21, Sigma) were used as standards. For detection of
mouse IgM, plates were coated with polyclonal anti-mouse .mu. (The
Binding Site, Birmingham, UK) and bound Ig was detected with
biotinylated goat anti-mouse .mu. (RPN1176, Amersham) followed by
streptavadin-conjugated horseradish peroxidase. Mouse plasmacytoma
TEPC183, IgM.kappa., (Sigma) was used as a standard.
[0047] Flow Cytometry Analysis.
[0048] Cell suspensions were obtained from bone marrow (BM), spleen
and Peyer's patches (PPs). Multicolor staining was then carried out
with the following reagents in combinations illustrated in FIG. 4:
FITC-conjugated anti-human .lambda. (F5266, Sigma), PE-conjugated
anti-mouse c-kit (CD117) receptor (clone ACK45, cat. no. 09995B,
Pharmingen, San Diego, USA), PE-conjugated anti-mouse CD25 (IL-2
receptor) (Sigma, clone 3C7, P 3317), biotin-conjugated anti-human
.kappa. (clone G20-193, cat. no. 08172D, Pharmingen),
biotin-conjugated anti-mouse CD19 (clone 1D3, cat. no. 09654D,
Pharmingen), followed by Streptavadin-Quantum Red (S2899, Sigma) or
Streptavadin-PerCP (cat. no. 340130, Becton-Dickinson) and rat
monoclonal anti-mouse .kappa. light chain (clone MRC-OX-20, cat.
MCA152, Serotec, Oxford, UK) coupled according to the
manufacturer's recommendations with allophycocyanin (APC) (PJ25C,
ProZyme, San Leandro, USA). Data were collected from
1.times.10.sup.6 stained cells on a FACScalibur flow cytometer
(Becton Dickinson Immunocytometry Systems, Mountain View, Calif.,
USA) as described (32). Cells were first gated on forward and side
scatter to exclude dead cells. To obtain accurate percentage
distribution for comparison, cells from normal mice were stained in
parallel. In addition, human peripheral blood lymphocytes were
purified on Ficoll gradients (1.077 g/ml) and stained with
PE-conjugated anti-human CD19 antibody (P7437, clone SJ25-C1,
Sigma), biotinylated anti-human .kappa. followed by
Streptavadin-Quantum Red and FITC-conjugated anti-human .lambda.
antibodies as above.
[0049] For RT-PCR cloning of V, genes PPs cells were stained with
FITC-conjugated peanut agglutinin (PNA) (L 7381, Sigma) and
PE-conjugated anti-mouse B220 antibodies (Sigma P 3567).
Double-positive cells were sorted on the FACStar.sup.Plus flow
cytometer (Becton Dickinson Immunocytometry Systems, Mountain View,
Calif.) as described (32) and 5.times.10.sup.3 cells were lysed in
denaturing solution (37). 5'RACE was carried out as described below
with 1 modification--2 .mu.g carrier RNA was added to the cell
lysates before RNA extraction and precipitation.
[0050] Cloning and Sequencing of 5'RACE Products.
[0051] Spleen RNA was prepared as described (37) and for cDNA
preparation 2-3 .mu.g of RNA was ethanol precipitated and
air-dried. For rapid amplification of 5' cDNA ends (5'RACE) (38)
first strand cDNA was primed with oligo(dT)22 and 0.100 units of
Super Script II reverse transcriptase (Gibco BRL, Gaithersburg,
Md.) was used at 46.degree. C. according to manufacturer's
instructions with 20 units of RNAse placental inhibitor (Promega,
Madison, Wis.). The DNA/RNA duplex was passed through 1 ml G-50
equilibrated with TE (10 mM Tris-HCl pH 7.8, 1 mM EDTA) in a
hypodermic syringe to remove excess oligo(dT). For G-tailing 20
units of TdT (Cambio, Cambridge, UK) were used according to
standard protocols (39). Double stranded (ds) cDNA was obtained
from G-tailed ss cDNA by addition of oligonucleotide Pr1 (see
below), 100 .mu.M dNTP and 2.5 units of Klenow fragment (Cambio)
and incubation for 10 min at 40.degree. C. After heating the
reaction for 1 min at 94.degree. C. and extraction with
phenol-chloroform the ds cDNA was passed through G-50 to remove
primer Pr1. PCR amplifications, 35 cycles, were carried out in the
RoboCycler Gradient 96 Thermal Cycler (Stratagene, LaJolla, Calif.,
USA) using oligonucleotides Pr2 and Pr3. For PCR of PPs cDNA 50
cycles were used: 40 cycles in the first amplification and 10
cycles in-additional amplifications. Pfu Thermostable Polymerase
(Stratagene, LaJolla, Calif., USA) was used instead of Taq
polymerase to reduce-PCR error rates. The amplification products
were purified using a GENECLEAN II kit (BIO 101, Vista, Calif.,
USA) and re-amplified for 5 cycles with primers Pr2 and Pr4 to
allow cloning into Eco RI sites. Oligonucleotide for 5'RACE of
V.lambda. genes were:
1 Pr1 5'-AATTCTAAAACTACAAACTG CCCCCCCCA/T/G-3' Pr2
5'-AATTCTAAAACTACAAACTGC-3' (sense) Pr3 -5'-CTCCCGGGTAGAAGTCAC-3'
(reverse) Pr4 5'-AATTCGTGTGGCCTTGTTGGCT-3'. (reverse nested)
[0052] A PCR protocol (A. Sudarikov) was used to clone V.lambda.
PCR products. PCR products of about 500 bp were cut out from
agarose gels and purified on GENECLEAN II. The DNA was incubated in
50 mM Tris-HCl, pH 7.4, 10 mM MgCl.sub.2, with 100 .mu.M dGTP/dCTP
and 1 unit of Klenow fragment for 10 min at RT. Under these
conditions the Klenow fragment removes the 3' ends of the PCR
products (AATT) leaving ligatable Eco RI overhangs. DNA was ligated
with Eco RI restricted pUC19, transformed into competent E. coli
XL1Blue and colonies were selected on X-Gal/IPTG/amp plates.
Plasmid DNA prepared from white colonies was used for sequencing.
Sequencing of both strands was done on the ABI 373 automated
sequencer in the Babraham Institute Microchemical Facility.
EXAMPLE 2
Characterization of the Transgenic Mice, Production Methodologies
and Produced Antibodies
[0053] The transgenic human Ig.lambda. locus. The human Ig.lambda.
translocus (FIG. 1) was assembled on a YAC by recombining 1 YAC
containing about half of all V.lambda. gene segments with 3
overlapping cosmids containing V.lambda. and J.lambda.-C.lambda.
gene segments and the 3' enhancer (29). This resulted in a 410 Kb
YAC accommodating a 380 Kb region of the human .lambda. light chain
locus with 15 V.lambda. genes regarded as functional, 3 V.lambda.s
with open reading frames but not found to be expressed and 13
V.lambda. pseudogenes (40). This HuIg.lambda.YAC was introduced
into ES cells by protoplast-fusion (30) and chimeric mice were
produced by blastocyst injection (31). The ES cell clone used for
this showed a 450 Kb NotI fragment corresponding to
HuIg.lambda.YAC, as identified by PFGE and Southern hybridization
with the 3' probe, C.lambda.2+3, and the 5' probe, LA comprising
LYS2, present in the left centromeric YAC arm (not shown). Germline
transmission was obtained, and PFGE analysis of testis DNA from 1
animal is illustrated in FIG. 2. A NotI fragment larger than 380 Kb
is necessary to accommodate this region of the HuIg.lambda.YAC and
the 450 Kb band obtained indicates random integration involving the
single NotI site 3' of J.lambda.-C.lambda. and a NotI site in the
mouse chromosome. Digests with EcORI/HindIII and hybridization with
the C.lambda.2+3 probe further confirmed the integrity of the
transferred HuIg.lambda.YAC (FIG. 2). The results indicated that
one complete copy of the HuIg.lambda.YAC was integrated in the
mouse genome.
[0054] Human Ig.lambda. Expression is Dominant in Mouse
.kappa..sup.-/- Animals.
[0055] To assess the human .lambda. light chain repertoire for the
production of authentic human antibodies the HuIg.lambda.YAC mice
were bred with mice in which endogenous Ig.kappa. production was
silenced by gene targeting (32). In these .kappa..sup.+/+ mice, the
mouse Ig.lambda. titers are elevated compared to .kappa..sup.+/+
strains (32, 41). Serum titrations (FIG. 3) showed that human
Ig.lambda. antibody titers in HuIg.lambda.YAC/.kappa..sup.-/- mice
are between 1 and 2 mg/ml which in some cases is up to 10-fold
higher than the mouse Ig.lambda..sup.+ levels. Interestingly, the
mouse Ig.lambda. levels remained low in the
HuIg.lambda.YAC/.kappa..sup.-/- mice, similar to the levels found
in normal mice. High levels of human Ig.lambda..sup.+ cells were
also identified in flow cytometric analysis of splenic B-cells from
HuIg.lambda.YAC/.kappa..sup.-/- mice (FIG. 4A) with human .lambda.
expressed on the surface of >80% of the B-cells while the number
of mouse Ig.lambda..sup.+ cells was always below 5% (data not
shown).
[0056] Human Ig.lambda. Expression Equals Mouse Ig.kappa.
Production.
[0057] Assessment of human Ig.lambda. production in heterozygous
HuIg.lambda.YAC.sup.+/.kappa..sup.+/- mice allowed a detailed
comparison of expression and activation of endogenous versus
transgenic light chain loci present at equal functional numbers.
Serum analysis (FIG. 3) of mice capable of expressing both human
.lambda. and mouse .kappa. showed similar titers for human and
mouse light chains. Human Ig.lambda. levels in
HuIg.lambda.YAC/.kappa..sup.+/+ transgenic mice were very similar
to those in HuIg.lambda.YAC/.kappa..sup.+/- mice. Total Ig levels
in HuIg.lambda.YAC.sup.+/.kappa..sup.+/- mice were 1-2 mg/ml, with
a typical contribution of about 51% mouse Ig.kappa., 43% human
Ig.lambda. and 6% mouse Ig.lambda.. However, a comparison of
endogenous .kappa. and human .lambda. expression in individual sera
from HuIg.lambda.YAC mice, and similarly from human volunteers,
showed that .lambda./.kappa. ratios can vary. For example, 3 of the
HuIg.lambda.YAC/.kappa..sup.+/- mice produced somewhat higher
.kappa. levels while in 2 mice the human .lambda. levels were
higher than the Ig.kappa. titers. In
HuIg.lambda.YAC/.kappa..sup.+/- mice, similar high translocus
expression was also found in B220.sup.+ B-cells from different
tissues, for example 38% of spleen cells expressed human .lambda.
and 45% mouse .kappa. (FIG. 4A). These values resemble very much
the levels in human volunteers as illustrated in FIG. 4A with 34%
Ig.lambda..sup.+ versus 51% Ig.kappa..sup.+ in CD19.sup.+
peripheral blood lymphocytes.
[0058] To assess whether the high contribution of the human
.lambda. translocus to the mature B cell repertoire is the result
of selection at the mature B-cell stage, or alternatively from
early translocus rearrangement, light chain expression in bone
marrow precursor B-cells was examined. For this, early B-cell
markers, c-kit or CD25, were used in 4-color stainings in
combination with the B-cell lineage marker CD19 and human .lambda.
and mouse .kappa. specific antibodies. FIG. 4B shows that human
.lambda. expression in HuIg.lambda.YAC/.kappa..sup.+/- mice occurs
at an earlier stage of development than mouse .kappa. light chain
expression. Human .lambda. expression can be detected at the
unusually early CD19.sup.+/c-kit.sup.+ pre B-I stage and is
maintained in CD19.sup.+/CD25.sup.+ pre B-II cells. However, at the
later immature to mature B-cell stage
(CD19.sup.+/c-kit.sup.-/CD25.sup.-) the proportion of mouse
Ig.kappa..sup.+ cells is significantly increased. This suggests
that human .lambda. light chains can rearrange at an earlier stage
than mouse Ig.kappa. but that upregulation at the mature B-cell
stage balances any disadvantages in the timing of
rearrangement.
[0059] DNA Rearrangement and Diversification of a Highly Active
Human .lambda. Translocus.
[0060] In order to assess whether the translocus expression levels
were a direct result of its rearrangement capacity, individual
hybridoma clones were analyzed. Results from 2 fusions suggest that
human .lambda. and mouse .kappa. light chain producing cells were
present in the spleen of HuIg.lambda.YAC/.kappa..sup.-/+ mice at
similar frequencies. Furthermore, the antibody expression rates of
human .lambda. (2-20 .mu.g/ml) or mouse .kappa. (4-25 .mu.g/ml)
producing hybridomas were similar. In order to assess if human
Ig.lambda. rearrangement must precede mouse Ig.kappa. rearrangement
or vice versa, endogenous and transgene rearrangements were
analyzed. Southern blot hybridization of randomly picked human
Ig.lambda. or mouse Ig.kappa. expressing hybridoma clones showed
the following: from 11 human Ig.lambda. expressers, 7 had the mouse
.kappa. locus in germline configuration and only 1 clone had mouse
Ig.kappa. rearranged, and from 19 mouse Ig.kappa. expressers, 17
had the human Ig.lambda. locus in germline configuration. The
analysis of 8 more Ig.lambda. producers showed that in 2 the human
Ig.lambda. locus was rearranged (data not shown). This result
suggests that there is no locus activation bias and further
emphasizes that the human .lambda. translocus performs with similar
efficiency than the endogenous locus.
[0061] Hence the human .lambda. locus is particularly well
expressed in transgenic mice, even on a normal .kappa..sup.+/+ or
heterozygous .kappa..sup.+/- background, a result which was
unexpected given the dominance of mouse .kappa. over human .kappa.
in HuIg.kappa. transgenic mice (64). FIG. 4 and the hybridoma
results show that this has a developmental basis, with human
.lambda. often rearranging before mouse .kappa., which is also
unexpected given the normal progression from .kappa. to .lambda.
rearrangement for the endogenous mouse loci. The ability of the
human 3' .lambda. enhancer to function in the mouse background may
be the reason that human .lambda. and mouse .kappa., levels are
similar in HuIg.lambda.YAC.sup.+/- mice and that .lambda. and
.kappa. light chain 3' enhancers compete at the pre B-cell stage to
initiate light chain rearrangement.
[0062] The capacity of the human .lambda. locus to produce an
antibody repertoire is further documented in the V gene usage. V-J
rearrangement was determined from spleen cells and Peyer's patch
cells by PCR reactions, not biased by specific V gene primers. The
results show that a substantial proportion of the V.lambda. genes
on the translocus are being used with V.lambda.3-1 and
V.lambda.3-10 being most frequently expressed. In DNA
rearrangement, J.lambda.2 and J.lambda.3 were preferentially used
and J.lambda.1 rarely, and as expected J.lambda.4, 5 and 6 were not
utilized as they are adjacent to .psi.Cs. Sequences obtained from
RT-PCR products from FACS-sorted germinal centre
PNA.sup.+/B220.sup.+ Peyer's patches revealed that somatic
hypermutation is operative in HuIg.lambda.YAC mice (with somewhat
more extensive changes in CDRs than in framework regions).
Extensive variability due to N- or P-sequence additions, which is
found in human but not mouse light chain sequences (25, 27, 28),
was not observed.
[0063] Hybridomas and Human Monoclonal Antibodies from 5-Feature
.lambda. Mice
[0064] Mice carrying the human .lambda. translocus in the 5-feature
genotype, i.e. together with human heavy and .kappa. chain
transloci and with endogenous heavy and .kappa. chains silenced,
were immunized with several human proteins, including fodrin,
placental alkaline phosphatase (PLAP), carcinoembryonic antigen
(CEA), the Fc fragment of human IgE, the steroid progesterone
coupled to bovine serum albumin, and the bacterial protein cholera
toxin subunit B. Periodic bleeds post-immunization showed good
responses of IgM containing human .lambda. and .kappa.; mouse
.lambda.-containing Ig was barely detectable and was considerably
lower than in 4-feature mice lacking human .lambda.. The human
antibody (IgM) responses of 5-feature mice to fodrin, PLAP, cholera
toxin and CEA are shown in FIG. 7.
[0065] Hybridomas were produced from spleen cells of the immunized
5 feature mice and Ig producing clones were screened for human
light chain production in order to determine the proportions of
.kappa. and .lambda.. The number expressed in hybridomas is a good
reflection of the expression of the light chains among B cells and
in immune sera. The results summarized in Table 1 below show that
in 7 fusions, there was a majority of human .lambda.-producing
hybridomas in 6, while in only one fusion was there a small
preponderance of .kappa.. The proportion of human .lambda. ranged
from a minimum of 75% of the human .kappa. level to a maximum of 8
times greater than the .kappa. level. In most cases (5/7) the
number of human .lambda.-producing hybridomas exceeded those making
human .kappa. by a factor of 4 fold or greater. This demonstrates
the unexpectedly high expression of human .lambda. in transgenic
mice.
2TABLE 1 Antigen Hybridomas .lambda. % .kappa. % .lambda.:.kappa.
Progest 73 51 49 1.04 Progest 16 43 57 0.75 IGF 82 87 13 6.7 IGF 42
81 19 4.3 IgE 45 89 11 8.1 IgE 21 81 19 4.3 IgE 23 62 38 1.63
Frequency of occurrence of human .lambda. and .kappa. light chains
among monoclonal immunoglobulins produced by hybridomas from
immunized 5-feature translocus mice. The mice were immunized with
the antigens shown in the far left column (Progest =
progesterone-bovine serum albumin; IGF = insulin related growth
factor; IgE = Fc fragment of human immunoglobulin E). Hybridomas
were prepared and the number # expressing .lambda. or .kappa. light
chains were determined. The ratio of .lambda.:.kappa. is shown in
the far right column.
[0066] Diversity of Rearrangements of the .lambda. Light Chain
Genes
[0067] The utilization of individual V.lambda. genes is indicated
by the triangles in FIG. 1, and shows that a substantial proportion
of the V.lambda. genes on the translocus are being used in
productive rearrangements, with V.lambda. 3-1 and V.lambda. 3-10
being most frequently expressed. In V.lambda.-J.lambda.
rearrangements, J.lambda. 2 was preferentially used and J.lambda. 3
and J .lambda.1 less frequently, and, as expected J.lambda. 4, 5
and 6 were not utilized as they are adjacent to .psi.Cs. Extensive
variability due to N-- or P-- sequence additions, which is found in
human but not mouse L chain sequences, was not observed. Sequences
obtained by RT-PCR from FACS-sorted PP germinal centre B cells
(B220+/PNA+) revealed that somatic hypermutation is operative in
HuIg.lambda. YAC mice (FIG. 5). Provided herein are unique 11V
.lambda.-J.lambda. rearrangements with 2 or more changes in the V
region, excluding CDR3, which may be affected by
V.lambda.-J.lambda. recombination. The majority of mutations lead
to amino acid replacements, but there was no preferential
distribution in CDR1 and CDR2. Extensive somatic hypermutation of
many rearranged human Ig.lambda. sequences were found, indicating
that they were able to participate in normal immune responses.
[0068] Somatic Hypermutation in Human Ig.lambda. Rearrangements in
5-Feature .lambda. Mice
[0069] The occurrence of somatic mutations was determined by
sequencing of rearrangements from B cells or hybridomas
and-comparison with germline sequences. The results shown in FIG. 6
show that the .lambda. locus undergoes mutation with up to 10 point
mutations being observed, with a comparable frequency to the
.kappa. locus and a considerably higher frequency than that seen in
the IgH translocus. The 6 Ig.lambda. rearrangements were obtained
by RT-PCR from a single 5 feature animal, and show a limited use of
the V gene segments, with V.lambda.3-19 used in 5 sequences (FIG.
6). Given the high contribution to the B cell repertoire seen in
FACS and serum analysis, it is likely that the rearrangement of the
locus in the 5 feature mice is similar to what is seen in mice
where the HuIgLambda YAC is in the presence of a functional mouse
Ig.kappa. locus. Little or no `N` insertion is found in the
translocus-derived L chains, either in the 4 and 5 feature mice, or
in mice with the HuIgKappa or HuIgLambda YAC in the presence of a
functional mouse H chain locus. This would suggest that the L chain
translocus rearranges at the same developmental stage as the
endogenous L chains, at which time terminal deoxynucleotide
transferase activity is reduced.
[0070] High Affinity Monoclonal Human Antibody from a 5-Feature
.lambda. Mouse.
[0071] The occurrence of somatic hypermutation suggested that
5-feature mice would be capable of producing high affinity human
antibodies, including those against human antigens of clinical
importance. This was demonstrated for the IgM antibody 7783.26
against human placental alkaline phosphatase PLAP (FIG. 8). After
cloning, the monoclonal antibody bound strongly to PLAP in ELISA,
was sensitively inhibited by free PLAP (50% inhibition at about 2
nM) and from a Scatchard plot had an affinity of 2.times.10.sup.9
M.sup.-1. Hence, the mice are capable of giving rise to human
antibodies with a high affinity which would be suitable for
therapeutic purposes.
[0072] Efficient DNA rearrangement and high antibody expression
levels are rarely achieved in transgenic mice carrying
immunoglobulin regions in germline configuration on minigene
constructs. Competition with the endogenous locus can be eliminated
in Ig knock-out strains, where transgene expression is usually good
(42). Poor transloci expression levels could be a result of the
failure of human sequences in the mouse background, or
alternatively the lack of locus specific control regions which are
more likely to be included on larger transgenic regions (43, 44,
45). The latter is supported by the finding that HuIg.lambda.YAC
mice express human Ig.lambda. and mouse Ig.kappa. at similar
levels. The 410 Kb HuIg.lambda.YAC translocus accommodates V-gene
region cluster A containing at least 15 functional V.lambda. genes
(see FIG. 1). In man, cluster A is the main contributor to the
.lambda. antibody repertoire, with V.lambda. 2-14 (2a2) expressed
most frequently at 27% in blood lymphocytes (23). Expression of
V.lambda. 2-14 in the transgenic mice was found, but the main
contributors to .lambda. light chain usage were 3-1, the V.lambda.
gene most proximal to the C-J region, and 3-10, both of which are
expressed at about 3% in man. Although the validity to draw
conclusions about gene contribution is dependent on the numbers
compared, from the 31 sequences obtained 11 showed were
V.lambda.3-1 and 8 were V.lambda.3-10 which suggests that
rearrangement or selection preferences are different in mouse and
man. Sequence analysis revealed that there was very little further
diversification by insertion of N or P nucleotides. In contrast,
somatic hypermutation of some rearranged human Ig.lambda. sequences
was found, indicating that they are able to participate in normal
immune responses. Indeed mutation levels in B220.sup.+/PNA.sup.+
PPs from HuIg.lambda.YAC translocus mice were similar to what has
been reported for mouse light chains (46). In the mouse, unlike in
humans, untemplated light chain diversification is essentially
absent and it was believed that this is because deoxynucleotidyl
transferase is no longer expressed at the stage of light chain
rearrangement (28, 47). This concept has been challenged by the
discovery that mouse light chain rearrangement can occur at the
same time as V.sub.H to DJ.sub.H rearrangements (48). Indeed, these
results also show light chain rearrangement at the pre B-I stage,
with a substantial percentage of CD19.sup.+ cells expressing human
X (see FIG. 4). Although the human .lambda. translocus appears to
be earlier activated than the .kappa. locus in the mouse,
rearranged human .lambda. light chains did not accumulated much N
region diversity as found in human peripheral B-cells (27).
[0073] In the different species, the ratio of .lambda. and .kappa.
light chain expression varies considerably (1-3, 49, 50) and in the
mouse the low .lambda. light chain levels are believed to be a
result of inefficient activation of the mouse .lambda. locus during
B-cell differentiation (reviewed in 6). The Ig.lambda. (.sup.-40%)
and Ig.kappa. (.sup.-60%) ratio in humans is more balanced and
suggests that both .lambda. and .kappa. play an equally important
role in immune responses. This notion is supported by the finding
that the mouse V.lambda. genes are most similar to the less
frequently used human V.lambda. gene families, while no genes
comparable to the major contributors to the human V.lambda.
repertoire are present in mice (40). With the HuIg.lambda.YAC,
these V.lambda. genes are available, and are able to make a
significant contribution to the antibody repertoire, and the bias
towards V.kappa. gene utilization is removed.
[0074] Comparison of size and complexity of light chain loci
between different species suggests that larger loci with many more
V genes may contribute much more efficiently to the antibody
repertoire (6, 51). Recently, this question was addressed in
transgenic mice by the introduction of different size human .kappa.
light chain loci (45). The result showed that the size of the V
gene cluster and the V gene numbers present are not relevant to
achieving high translocus expression levels. It is possible,
however, that a presently undefined region with cis-controlled
regulatory sequences may be crucial in determining expressibility
and subsequently light chain choice. That the
HuIg.lambda.YAC.sup.+/.kappa..sup.+/- mice do not exhibit a bias in
the selection of light chain locus for expression is shown by the
absence of rearrangement of the non-expressed locus in hybridoma
cells. This supports the model that .lambda. and .kappa.
rearrangements are indeed independent (52) and that poor Ig.lambda.
expression levels in mice may be the result of an inefficient
recombination signal (53). A possible signal that initiates light
chain recombination has been identified in gene targeting
experiments where the 3'.kappa. enhancer has been deleted (19). The
.kappa.:.lambda. ratio was essentially equal in mice where the
3'E.kappa. had been deleted or replaced by neo (down to 1:1 and not
20:1 as in normal mice). In addition, the .kappa. locus was largely
in germline configuration in .lambda. expressing cells, a result
also seen in the HuIg.lambda.YAC.sup.+/- mice. Taken together, the
results suggest that the ability of the human 3' .lambda. enhancer
to function in the mouse background may be the reason that human
.lambda. and mouse .kappa. levels are similar in
HuIg.lambda.YAC.sup.+/.kappa..sup.+/- mice and that .lambda. and
.kappa. light chain 3' enhancers compete at the pre B-cell stage to
initiate light chain rearrangement.
REFERENCES
[0075] The following are hereby incorporated by reference.
[0076] 1. Hood, L., Gray, W. R., Sanders, B. G. and Dreyer, W. Y.
(1967) Cold Spring Harbor Symp. Quant. Biol. 32:13346.
[0077] 2. McIntire, K. R. and Rouse, A. M. (1970) Fed. Proc. 19:
704.
[0078] 3. Arun, S. S., Breuer, W. and Herrmanns, W. (1996)
Zentralbl. Veterinarmed. A. 43: 573-76.
[0079] 4. Hieter, P. A., Korsmeyer, S. J., Waldmann, T. A and
Leder, P. (1981) Nature 290: 368-72.
[0080] 5. Coleclough, C., Perry, R. P., Karjalainen, K. and
Weigert, M. (1981) Nature 290: 372-78.
[0081] 6. Selsing, E. and Daitch, L. E. (1995) Immunoglobulin
.lambda. genes. In Immunoglobulin Genes, 2nd Ed., eds. T. Honjo and
F. W. Alt, Rabbitts. Academic Press: 193-203.
[0082] 7. Berg, J., McDowell, M., Jack, H. M. and Wabl, M. (1990)
Dev. Immunol. 1, 53-57.
[0083] 8. Abken, H. and Buitzler, C (1991) Immunol. 74:
709-713.
[0084] 9. Takemori, T. and Rajewsky, K. (p1981) Eur. J. Immunol.
11: 618-25.
[0085] 10. McGuire, K. L. and Vitetta, E. S. (1981) J. Immunol.
127: 1670-73.
[0086] 11 Kessler, S., Kim, K. J. and Scher, I. (1981) J. Immunol.
127: 1674-78.
[0087] 12. Lejeune, J. M., Briles, D. E., Lawton, A. R. and
Kearney, J. F. (1982) J. Immunol. 129: 673-677.
[0088] 13. Rolink, A., Streb, M. and Melchers, F. (1991) Eur. J.
Immunol. 21, 2895-98.
[0089] 14. Osmond, D. J., Rolink, A. and Melchers, F (1998)
Immunol. Today 19, 65-68.
[0090] 15. Zou, Y. R., Takeda, S. and Rajewsky, K. (1993) EMBO J.
12: 811-20.
[0091] 16. Arakawa, H., Shimizu, T. and Takeda, S. (1996). Int.
Immunol. 8: 91-99.
[0092] 17. Glozak, M. and Blomberg, B. B. (1996) Mol. Immunol., 33:
427-38.
[0093] 18. Asenbauer, H and Klobeck, H. G. (1996) Eur. J. Immunol.
26: 142-50.
[0094] 19. Gorman, J. R., van der Stoep, N., Monroe, R., Cogne, M.,
Davidson, L. and Alt, F. W. (1996) Immunity 5, 241-52.
[0095] 20. Frippiat, J. -P., Williams, S. C., Tomlinson, I. M.,
Cook, G. P., Cherif, D., Le Paslier, D., Collins, J. E., Dunham,
I., Winter, G. and Lefranc, M. -P (1995) Hum. Mol. Genet. 4:
983-91.
[0096] 21. Kawasaki, K., Minoshima, S., Nakato, E., Shibuya, K.,
Shintani, A., Schmeits, J. L., Wang, J. and Shimizu, N. (1997)
Genome Res. 7: 260-61.
[0097] 22. Giudicelli, V., Chaume, D., Bodmer, J., Muller, W.,
Busin, C., Marsh, S., Bontrop, R., Marc, L., Malik, A. and Lefranc,
M. -P. (1997) Nucl. Acids Res., 25: 206-11.
[0098] 23. Ignatovich, O., Tomlinson, I. M., Jones, P. T. and
Winter, G., (1997) J. Mol. Biol. 268: 69-77.
[0099] 24. Combriato, G. and Klobeck, H. -G. (1991) Eur. J.
Immunol., 21: 1513-22.
[0100] 25. Foster, S. J., Brezinschek, H. -P., Brezinschek, R. I
and Lipsky, P. E. (1997) Clin. Invest., 99, 1614-27.
[0101] 26. Ignatovich, O. The creation of diversity in the human
immunoglobulin V.lambda. repertoire. PhD thesis, University of
Cambridge, 1998.
[0102] 27. Bridges, S. L., Lee, S. K., Johnson, M. L., Lavelle, J.
C., Fowler, P. G., Koopman, W. J. and Schroeder, (1995) J. Clin.
Invest., 96, 831A41.
[0103] 28. Victor, K. D., Vu, K. anf Feeney, A. J. (1994) J.
Immunol., 152: 3467-75.
[0104] 29. Popov, A V., Butzler, C., Frippiat, J -P., Lefranc, M
-P., Bruggemann, M. (1996). Gene 177: 195-201.
[0105] 30. Davies, N. P., Popov, A. V., Zou, X. and Bruggemann, M.
(1996). Human antibody repertoires in transgenic mice: Manipulation
and transfer of YACs. Antibody Engineering: A Practical Approach,
eds. J. McCafferty, H. R. Hoogenboom and D. J. Chiswell, IRL,
Oxford: 59-76.
[0106] 31. Hogan, B., Beddington, R., Costantini, F. and Lacy E.
(1994) Manipulating the Mouse Embryo: A Laboratory Manual. Cold
Spring Harbor Laboratory Press.
[0107] 32. Zou, X., Xian, J., Popov, A. V., Rosewell, I. R, Muller,
M and Bruggemann, M (1995) Eur J. Immunol. 25: 2154-62.
[0108] 33. Wurst, W. and Joyner, A. L. Production of targeted
embryonic stem cell DNA. In: Gene targeting, ed. A. L. Joyner. IRL
Press, Oxford, 1993: 33-61.
[0109] 34. Herrmann B. G., Barlow D. P., and Lehrach, H., (1987)
Cell 48: 813-25.
[0110] 35. Galfr, G. and Milstein, C. (1981) Methods Enzymol, 73
:346.
[0111] 36. Tijssen, P. Practice and theory of enzyme immunoassays.
Laboratory techniques in biochemistry and molecular biology. Vol.
15. Burdon, R. H. and Knippenberg, P. H. (eds.) Elsevier, 1985.
[0112] 37. Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem.
162: 156-159.
[0113] 38. Frohman, M. A., Dush, M. K. and Martin, G. R. (1988)
Proc. Natl. Acad. Sci, USA 85: 8998-9002.
[0114] 39. Current protocols in molecular biology (1995) eds.
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman,
J. G., Struhl, K., Smith, J. A. Massachusetts General Hospital,
Boston, Mass.: Harvard Medical School, Boston, Mass.; University of
Alabama, Birmingham, Ala.; Wiley & Sons, USA.
[0115] 40. Williams, S. C., Frippiat, J. -P., Tomlinson, I. M.,
Ignatovich, O., Lefranc, M. -P. and Winter, G. (1996) J. Mol. Biol.
264: 220-32.
[0116] 41. Chen, J., Trounstine, M., Kurahara, C., Young, F., Kuo,
C. -C., Xu, Y., Loring, J. F., Alt, F. W. and Huszar, (1997) D.
EMBO J. 12: 821-30.
[0117] 42. Bruggemann, M. and Neuberger, M. S. (1996) Immunol.
Today, 17: 391-97.
[0118] 43. Green, L. L. and Jakobovits, A. (1998) J. Exptl. Med.
188: 483-95.
[0119] 44. Zou, X., Xian, J., Davies, N. P., Popov, A. V. and
Bruggemann, M. (1996) FASEB J., 10: 1227-32.
[0120] 45. Xian, J., Zou, X., Popov, A. V., Mundt, C. A., Miller,
N., Williams, G. T., Davies, S. L., Neuberger, M. S. and
Bruggemann, M. (1998) Transgenics 2: 333-43.
[0121] 46. Gonzalez-Fernandez, A., Gupta, S. K., Pannell, R.,
Neuberger, M. S. and Milstein, C. (1994) Proc. Natl. Acad. Sci. USA
91: 12614-18.
[0122] 47. Li, Y -S., Hayakawa, K. and Hardy, R. R. (1993) J. Exp.
Med. 178: 951-60.
[0123] 48. Hardy, R. R., Carmack, C. E., Shinton, S. A., Kemp, J.
D. and Hayakawa, K. (1991) J. Exp. Med., 173, 1213.
[0124] 49. Saitta, M., Iavarone A., Cappello, N., Bergami, M R.,
Fiorucci, G. C. and Aguzzi, F. (1992) Clin Chem. 38: 2454-57.
[0125] 50. Hood, L., Eichmann, H., Lackland, H., Krause, R. M and
Ohms, J. J. (1970) Nature 228: 1040.
[0126] 51. Lansford, R., Okada, A., Chen, J., Oltz, E. M.,
Blackwell, T. K., Alt, F. W. and Rathburn, G. (1996) Mechanisms and
control of immunoglobulin gene rearrangement. In Molecular
Immunology, B. D. Hames and D. M. Glover, eds. (New York: IRL
Press): 1-100.
[0127] 52. Nadel, B., Cazenave, P. -A. and Sanchez, P. (1990) EMBO
J., 9: 435-40.
[0128] 53. Arakawa, H., Shimizu, T. and Takeda, S. (1996). Int.
Immunol. 8: 91-99.
[0129] 54. Giudicelli, V., Chaume, D., Bodmer, J., Muller, W.,
Busin, C., Marsh, S., Bontrop, R., Marc, L., Malik, A. and Lefranc,
M. -P. (1997) Nucl. Acids Res. 25: 206-11.
[0130] 55. Eagle, H. (1955) Proc. Soc. Exptl. Biol. Med. 89:
362-64.
[0131] 56. Taub, R. A., Hollis, G. F., Hieter, P. A., Korsmeyer,
S., Waldmann, T. A. and Leder, P. (1983) Nature (London) 304:
172-74.
[0132] 57. Kitamura, D., Roes, J., Kahn, R., and Rajewsky, K,
(1991) Nature 350: 423.
[0133] 58. PCT/GB89/01207.
[0134] 59. Bruggemann and Neuberger (1996) Immunology Today 17:
391-97.
[0135] 60. Bruggemann and Taussig (1997) Curr. Opinion Biotech. 8:
455-58.
[0136] 61. Mendez et al. (1997) Nat. Genet. 15: 146-56.
[0137] 62. Fishwild et al. (1996) Nat. Biotechnol. 14: 845-51.
[0138] 63. Friguet et al., (1985) J. Immunol. Methods 77:
305-19.
[0139] 64. Xian et al. (1998). Transgenics 2: 33344.
[0140] 65. Nicholson, I. et al. (1999) J. Immunology 163:
6898-6906.
[0141] This application claims priority to GB 9823930.4, filed Nov.
3, 1998, the entirety of which is hereby incorporated by
reference.
[0142] It is to be understood that the description, specific
examples and data, while indicating exemplary embodiments, are
given by way of illustration and are not intended to limit the
present invention. Various changes and modifications within the
present invention will become apparent to the skilled artisan from
the discussion, disclosure and data contained herein, and thus are
considered part of the invention.
Sequence CWU 1
1
27 1 29 DNA Homo sapiens 1 aattctaaaa ctacaaactg cccccccca 29 2 21
DNA Homo sapiens 2 aattctaaaa ctacaaactg c 21 3 18 DNA Homo sapiens
3 ctcccgggta gaagtcac 18 4 22 DNA Homo sapiens 4 aattcgtgtg
gccttgttgg ct 22 5 234 DNA Homo sapiens 5 gccagcatca cctgctctgg
agataaattg ggggataaat atgcttgctg gtatcagcag 60 aagccaggcc
agtcccctgt gctggtcatc tatcaagata gcaagcggcc ctcagggatc 120
cctgagcgat tctctggctc caactctggg aacacagcca ctctgaccat cagcgggacc
180 caggctatgg atgaggctga ctattactgt caggcgtggg acagcagcac tgca 234
6 231 DNA Homo sapiens 6 gccaacatca cctgttctgg agataaattg
ggggataaat atgcttgctg gtatcagcag 60 aagccaggcc agtcccctat
tctgatcatc tatcaagata acaggcggcc ctcagggatc 120 cctgagcgat
tctctggctc caactctggg aacacagcca ctctgaccat cagcgggacc 180
caggctatgg atgaggctga ctattattgt caggcgtggg accgcagcac t 231 7 37
DNA Homo sapiens 7 ttgggtgttc ggcggaggga ccaagctgac cgtccta 37 8 36
DNA Homo sapiens 8 tgggtattcg gcggagggac ctacctgacc gtcctg 36 9 232
DNA Homo sapiens 9 gccagcatca cctgctcgag agataaattg ggggaaacat
atgtttcctg gtatcggcag 60 aagccaggcc agtcccctgt gctgctcatc
tatcaagata ccaagcgacc ctcagggatc 120 cctgagcgat tctctggctc
caactctggg aacacagccg ctctgaccat caccgggacc 180 caggctttgg
atgaggctga ctattactgt caggcgtggg acagcgccac tg 232 10 37 DNA Homo
sapiens 10 tgtggtattc ggcggaggga ccaagctgac cgtccta 37 11 35 DNA
Homo sapiens 11 tggttttcgg cggagggacc aaactgacca tccta 35 12 239
DNA Homo sapiens 12 gccaggatca cctgctctgg agatgcattg ccaaaaaaat
atgcttattg gtaccagcag 60 aagtcaggcc aggcccctgt gctggtcatc
tatgaggaca gcaaacgacc ctccgggatc 120 cctgagagat tctctggctc
cagctcaggg acaatggcca ccttgactat cagtggggcc 180 caggtggagg
atgaagctga ctactactgt tactcaacag acagcagtgg taatcatag 239 13 239
DNA Homo sapiens 13 gccaggatca cctgctctgg agatgcattg ccaaaaaaat
atgcttattg gtaccagcag 60 aagtcaggcc aggcccctgt gctggtcatc
tctgaggaca gcaaacgacc ctccgggatc 120 cctgagagaa tctctggctc
cagctcaggg acaatggcca ccttgactat cagtggggcc 180 caggtggaag
atgaagctga ctactactgt tactcaacag acagcagtag tactcatag 239 14 34 DNA
Homo sapiens 14 ggtgttcggc ggagggacca agctgaccgt ccta 34 15 246 DNA
Homo sapiens 15 atcaccatct cctgcactgg aaccagcagt gacgttggtg
gttataacta tgtctcctgg 60 taccaacagc acccaggcaa agcccccaaa
ctcatgattt atgaggtcag taatcggccc 120 tcaggggttt ctaatcgctt
ctctggctcc aagtctggca acacggcctc cctgaccatc 180 tctgggctcc
aggctgagga cgaggctgat tattactgca gctcatatac aagcagcagc 240 actctc
246 16 243 DNA Homo sapiens 16 atcaccatct cctgcactgg aaccagcagt
gacgttggtg gttctaactt tgtctcctgg 60 taccaacaac acccaggcaa
agcccccaaa ctcatgattt atgatgtcag ttatcggccc 120 tcaggggttt
ctaatcgctt ctctggctcc aagtctggca acacggcctc cctgaccatc 180
tctgggctcc aggctgagga cgaggctgat tattactgcg gctcatatac aagcagcagc
240 act 243 17 36 DNA Homo sapiens 17 tgggtgttcg gcggagggac
caagctgacc gtccta 36 18 239 DNA Homo sapiens 18 gtcaggatca
catgccaagg agacagcctc agaagctatt atgcaagctg gtaccagcag 60
aagccaggac aggcccctgt acttgtcatc tatggtaaaa acaaccggcc ctcagggatc
120 ccagaccgat tctctggctc cagctcagga aacacagctt ccttgaccat
cactggggct 180 caggcggaag atgaggctga ctattactgt aactcccggg
acagcagtgg taaccatct 239 19 237 DNA Homo sapiens 19 gtcaggatca
catgccaagg agacagcctc agaagctatt atgcaagctg gttccagcag 60
aagccaggac aggcccctgt acttgtcatc tatgctaaaa acaagcggcc ctcagggatc
120 ccagaccgat tctctggctc cagctcagga aacacagctt ccttgaccat
cactgggact 180 caggcggaag atgaggctga ctattactgt aactcccggg
acagcagtgg tgaacat 237 20 36 DNA Homo sapiens 20 gtggtattcg
gcggagggac caagctgacc gtccta 36 21 246 DNA Homo sapiens 21
atcaccatct cctgcactgg aaccagcagt gatgttggga gttataacct tgtctcctgg
60 taccaacagc acccaggcaa agcccccaaa ctcatgattt atgaggtcag
taagcggccc 120 tcaggggttt ctaatcgctt ctctggctcc aagtctggca
acacggcctc cctgacaatc 180 tctgggctcc aggctgagga cgaggctgat
tattactgct gctcatatgc aggtagtagc 240 actttc 246 22 241 DNA Homo
sapiens 22 atcaccatct cctgcactgg aaccagcggt gatgttggga gttataactt
tgtctcctgg 60 taccaactac acccaggcaa agtccccaaa ctcatgattt
atgaagacat taagcggccc 120 tcaggggttt ctaatcgctt ttctgcctcc
aagtctggca acacggcctc cctgacaatc 180 tctgggctcc aggctgagga
cgaggctgat tattactgct gctcatatgc aagtcgtgac 240 a 241 23 38 DNA
Homo sapiens 23 ggtgggtgtt cggcggaggg accaacctga ccgtccta 38 24 31
DNA Artificial Sequence Primer 24 aattctaaaa ctacaaactg ccccccccat
g 31 25 21 DNA Artificial Sequence Primer 25 aattctaaaa ctacaaactg
c 21 26 18 DNA Artificial Sequence Primer 26 ctcccgggta gaagtcac 18
27 22 DNA Artificial Sequence Primer 27 aattcgtgtg gccttgttgg ct
22
* * * * *