U.S. patent number RE47,131 [Application Number 15/019,852] was granted by the patent office on 2018-11-20 for humanized immunoglobulin loci.
This patent grant is currently assigned to Therapeutic Human Polyclonals, Inc.. The grantee listed for this patent is Therapeutic Human Polyclonals, Inc. Invention is credited to Roland Buelow, Josef Platzer, Wim van Schooten.
United States Patent |
RE47,131 |
Platzer , et al. |
November 20, 2018 |
Humanized immunoglobulin loci
Abstract
The present invention concerns methods and means to produce
humanized antibodies from transgenic non-human animals. The
invention specifically relates to novel immunoglobulin heavy and
light chain constructs, recombination and transgenic vectors useful
in making transgenic non-human animals expressing humanized
antibodies, transgenic animals, and humanized immunoglobulin
preparations.
Inventors: |
Platzer; Josef (Geretsried,
DE), Buelow; Roland (Palo Alto, CA), van Schooten;
Wim (Sunnyvale, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Therapeutic Human Polyclonals, Inc |
Mountain View |
CA |
US |
|
|
Assignee: |
Therapeutic Human Polyclonals,
Inc. (Mountain View, CA)
|
Family
ID: |
34079388 |
Appl.
No.: |
15/019,852 |
Filed: |
February 9, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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12511188 |
Apr 2, 2013 |
8410333 |
|
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10893483 |
Sep 8, 2009 |
7585668 |
|
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60487733 |
Jul 15, 2003 |
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Reissue of: |
13779585 |
Feb 27, 2013 |
8652842 |
Feb 18, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
15/67 (20130101); C12N 15/67 (20130101); C07K
16/00 (20130101); C07K 16/461 (20130101); A61P
35/00 (20180101); A61P 31/04 (20180101); C07K
16/00 (20130101); A61P 31/00 (20180101); A61P
37/02 (20180101); A61P 37/06 (20180101); C07K
16/461 (20130101); C12N 15/8509 (20130101); C12N
15/8509 (20130101); A01K 67/0278 (20130101); A01K
67/0278 (20130101); A01K 2227/105 (20130101); A01K
2227/107 (20130101); A01K 2217/00 (20130101); A01K
2267/01 (20130101); C07K 2317/24 (20130101); A01K
2207/15 (20130101); A01K 2207/15 (20130101); A01K
2267/01 (20130101); A01K 2227/105 (20130101); A01K
2217/00 (20130101); C07K 2317/24 (20130101); A01K
2227/107 (20130101) |
Current International
Class: |
C12N
5/10 (20060101); C07K 16/00 (20060101); C12N
15/67 (20060101); C07K 16/46 (20060101); C12N
15/85 (20060101); A01K 67/027 (20060101) |
Field of
Search: |
;435/326,328
;800/14 |
References Cited
[Referenced By]
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0491057 |
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EP |
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WO |
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WO |
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WO |
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WO |
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WO |
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WO |
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WO 2002/012437 |
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WO |
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WO 2004/003157 |
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Jan 2004 |
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WO |
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WO 2004/003157 |
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Aug 2004 |
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WO |
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|
Primary Examiner: Ponnaluri; Padmashri
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a divisional application of U.S. application
Ser. No. 12/511,188 filed Jul. 29, 2009, which is a divisional
application of U.S. application Ser. No. 10/893,483 filed Jul. 15,
2004 (now U.S. Pat. No. 7,585,668) which claims priority under 35
U.S.C. Section 119(e) and the benefit of U.S. Provisional
Application Ser. No. 60/487,733 filed Jul. 15, 2003, the entire
disclosures of which are incorporated herein by reference in their
entireties.
Claims
What is claimed is:
1. A B cell from a transgenic rabbit, wherein said transgenic
rabbit comprises a humanized immunoglobulin (Ig) locus comprising
multiple Ig gene segments, .[.present in a transgenic vector.].
wherein: (a) at least one of said gene segments is a human Ig gene
segment comprising two or more identical or different units
consisting of, from 5' to 3' direction, a 5' nucleotide sequence, a
human immunoglobulin heavy or light chain V gene segment, and a 3'
nucleotide sequence, wherein said 5' nucleotide sequence and said
3' nucleotide sequence have the nucleotide sequence of SEQ ID NO:
35; (b) said gene segments are juxtaposed in an unrearranged,
partially rearranged or fully rearranged configuration, and (c)
said humanized Ig locus is capable of undergoing gene
rearrangement, if necessary, and gene conversion and/or
hypermutation, and producing a repertoire of humanized
immunoglobulins in said transgenic rabbit.
2. The B cell of claim 1, wherein said transgenic rabbit preserves
an essentially intact endogenous regulatory and antibody production
machinery.
3. The B cell of claim 1 or claim 2, wherein said human Ig heavy
chain V gene segment is a member of the VH3, VH1, VH5, or VH4
family.
Description
In accordance with 37 CFR 1.821(e), we hereby expressly incorporate
herein by reference, in its entirety, the last-filed (filed Apr. 4,
2005) computer readable Sequence Listing, saved as "39691-0007A
saved Apr. 4, 2005.txt" date of creation Apr. 4, 2005, size 1,489
KB, submitted in U.S. application Ser. No. 10/893,483, filed Jul.
15, 2004.
FIELD OF THE INVENTION
The present invention concerns methods and means to produce
humanized antibodies from transgenic non-human animals. The
invention specifically relates to novel immunoglobulin heavy and
light chain constructs, recombination and transgenic vectors useful
in making transgenic non-human animals expressing humanized
antibodies, transgenic animals, and humanized immunoglobulin
preparations. The transgenic vectors contain humanized
immunoglobulin loci, which are capable of undergoing gene
rearrangement, gene conversion and hypermutation in transgenic
non-human animals to produce diversified humanized antibodies,
while leaving the endogenous regulatory and antibody production
machinery of the non-human animals essentially intact. The
humanized antibodies obtained have minimal immunogenicity to humans
and are appropriate for use in the therapeutic treatment of human
subjects.
BACKGROUND OF THE INVENTION
Antibodies are an important class of pharmaceutical products that
have been successfully used in the treatment of various human
diseases and conditions, such as cancer, allergic diseases,
prevention of transplant rejection and host-versus-graft
disease.
A major problem of antibody preparations obtained from animals is
the intrinsic immunogenicity of non-human immunoglobulins in human
patients. In order to reduce the immunogenicity of non-human
antibodies, it has been shown that by fusing animal variable (V)
region exons with human constant (C) region exons, a chimeric
antibody gene can be obtained.
Humanized monoclonal antibodies have also been developed and are in
clinical use. Humanized monoclonal antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in non-human
animal, e.g. rodent, antibodies. Humanization can be essentially
performed following the method of Winter and co-workers (Jones et
al., Nature, 321: 522 (1986); Riechmann et al., Nature, 332: 323
(1988); Verhoeyen et al., Science, 239: 1534 (1988)), by
substituting non-human animal, e.g. rodent, CDRs or CDR sequences
for the corresponding sequences of a human monoclonal antibody.
It has been described that the homozygous deletion of the antibody
heavy-chain joining region (JH) gene in chimeric and germ-line
mutant mice results in complete inhibition of endogenous antibody
production. Transfer of the human germ-line immunoglobulin gene
array in such germ-line mutant mice will result in the production
of human antibodies upon antigen challenge. See, e.g., Jakobovits
et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et
al., Nature, 362: 255 (1993); Bruggemann et al., Year in Immunol.,
7: 33 (1993). While this genetic engineering approach resulted in
the expression of human immunoglobulin polypeptides in genetically
engineered mice, the level of human immunoglobulin expression is
low. This may be due to species-specific regulatory elements in the
immunoglobulin loci that are necessary for efficient expression of
immunoglobulins. As demonstrated in transfected cell lines,
regulatory elements present in human immunoglobulin genes may not
function properly in non-human animals.
Indeed, several regulatory elements in immunoglobulin genes have
been described. Of particular importance are enhancers downstream
(3') of heavy chain constant regions and intronic enhancers in
light chain genes. In addition, other, yet to be identified,
control elements may be present in immunoglobulin genes. Studies in
mice have shown that the membrane and cytoplasmic tail of the
membrane form of immunoglobulin molecules play an important role in
expression levels of human-mouse chimeric antibodies in the serum
of mice homozygous for the human C.gamma.1 gene. Therefore, for the
expression of heterologous immunoglobulin genes in animals it is
desirable to replace sequences that contain enhancer elements and
exons encoding transmembrane (M1 exon) and cytoplasmic tail (M2
exon) with sequences that are normally found in the animal in
similar positions.
In addition to the issues raised by the potential immunogenicity of
the non-human antibodies, the use of monoclonal antibodies in
general, whether chimeric, humanized or human, is further limited
by the fact that devastating diseases, such as cancer and
infections with virulent pathogens, are difficult to treat by
attacking one target, due to their complexity, multifactorial
etiology and adaptivity. Monoclonal antibodies directed against
singularly defined targets fail when those targets change, evolve
and mutate. Thus, malignancies may gain resistance to standard
monoclonal antibody therapies. A solution to this problem is the
use of polyclonal antibodies, which have the ability to target and
attack a plurality of evolving targets linked with complex
diseases. Polyclonal antibodies also have the ability to neutralize
bacterial and viral toxins, and direct immune responses to kill and
eliminate pathogens.
Accordingly, there is a great clinical need for a new approach
suitable for the large-scale production of high-titer,
high-affinity, humanized poly- and monoclonal antibodies.
The introduction of human immunoglobulin genes into the genome of
mice resulted in expression of a diversified human antibody
repertoire in genetically engineered mice. In both mice and humans,
primary antibody diversity is generated by gene rearrangement. This
process results in the generation of many different recombined
V(D)J segments encoding a large number of antibody molecules with
different antigen binding sites. However, in other animals, like
rabbits, pigs, cows and birds, primary antibody diversity is
generated by substantially different mechanisms, namely templated
mutations or gene conversion and non-templated mutations or
hypermutation. For example, it is well established that in rabbit
and chicken, VDJ rearrangement is very limited (almost 90% of
immunoglobulin is generated with the 3' proximal VH1 element) and
antibody diversity is generated by gene conversion and
hypermutation. In contrast, mouse and human gene conversion occurs
very rarely, if at all. Therefore, it is expected that in animals
that diversify their primary antibody repertoire by gene conversion
and hypermutation a genetic engineering approach based on gene
rearrangement will result in animals with low antibody titers and
limited antibody diversity. Thus, the genetic engineering of large
animals for the production of non-immunogenic antibody preparations
for human therapy requires alternative genetic engineering
strategies.
The production of humanized antibodies in transgenic non-human
animals is described in PCT Publication No. WO 02/12437, published
on Feb. 14, 2002, the disclosure of which is hereby expressly
incorporated by reference in its entirety. WO 02/12437 describes
genetically engineered non-human animals containing one or more
humanized immunoglobulin loci which are capable of undergoing gene
rearrangement and gene conversion in transgenic non-human animals,
including animals in which antibody diversity is primarily
generated by gene conversion to produce diversified humanized
antibodies. The humanized antibodies obtained have minimal
immunogenicity to humans and are appropriate for use in the
therapeutic treatment of human subjects. It further describes novel
nucleotide sequences from the 5' and 3' flanking regions of
immunoglobulin heavy chain constant region segments of various
non-human mammalians, such as chickens, cows, sheep, and rabbits.
Recombinant vectors in which human immunoglobulin heavy chain gene
segments are flanked by sequences homologous to such 5' and 3'
sequences are shown to be useful for replacing an immunoglobulin
heavy chain gene segment of a non-human animal with the
corresponding human immunoglobulin heavy chain gene segment.
SUMMARY OF THE INVENTION
In one aspect, the present invention concerns an isolated nucleic
acid molecule comprising a human immunoglobulin gene segment,
flanked by nucleotide sequences, wherein the flanking sequences are
identical or different, and comprise at least about 20 contiguous
nucleotides of a spacer sequence from an immunoglobulin heavy or
light chain gene of an animal generating antibody diversity
primarily by gene conversion and/or hypermutation, or from a
consensus sequence of two or more of the spacer sequences.
In another aspect, the invention concerns an isolated nucleic acid
molecule comprising a human immunoglobulin heavy or light chain
constant region (C) gene segment, flanked by nucleotide sequences,
wherein the flanking sequences are identical or different, and
comprise at least about 20 contiguous nucleotides of a spacer
sequence from an immunoglobulin heavy or light chain gene of a
non-primate animal, or from a consensus sequence of two or more of
the spacer sequences.
In a further aspect, the invention concerns an isolated nucleic
acid molecule comprising a human immunoglobulin heavy or light
chain gene segment, flanked by nucleotide sequences, wherein the
flanking sequences are identical or different, and comprise at
least about 20 contiguous nucleotides of a spacer sequence selected
from the group consisting of SEQ ID NOS: 1 to 185 (Table 1), or
from a consensus sequence of two or more of the spacer
sequences.
In one embodiment, the flanking sequences comprise at least about
50 contiguous nucleotides of a spacer sequence.
In another embodiment, the human immunoglobulin gene segment is a
heavy chain V, D, or J segment, where the V fragment may, for
example be a member of the VH3, VH1, VH5, or VH4 family.
In a further embodiment, the human immunoglobulin gene segment is a
light chain V or J segment, where the V segment may, for example be
a .kappa. light chain gene segment, such as V.kappa.1, V.kappa.3,
or V.kappa.4, or a .lamda. light chain segment, e.g. V.lamda.1,
V.lamda.2 or V.lamda.3.
In a further embodiment, the non-primate animal which generates
antibody diversity primarily by gene conversion and/or somatic
hypermutation is, for example, rabbit, pig, bird, e.g. chicken,
turkey, duck, or goose, sheep, goat, cow, horse or donkey, however,
other non-primate animals, e.g. rodents are also specifically
included within the scope of the invention.
In another aspect, the invention concerns a recombination vector
comprising any of the foregoing nucleic acid molecules.
In yet another aspect, the invention concerns a transgenic vector
comprising a humanized immunoglobulin (Ig) locus, wherein the
humanized Ig locus is derived from an Ig locus or a portion of an
Ig locus of a non-human animal, comprising multiple Ig segments
wherein
(a) at least one of the gene segments is a human Ig gene segment
flanked by nucleotide sequences comprising at least about 20
contiguous nucleotides from a spacer sequence, or from a consensus
sequence or two or more of such spacer sequences;
(b) the gene segments are juxtaposed in an unrearranged, partially
rearranged or fully rearranged configuration, and
(c) the humanized Ig locus is capable of undergoing gene
rearrangement, if necessary, as well as gene conversion and/or
hypermutation, if the non-human animal is a gene converting animal,
and producing a repertoire of humanized immunoglobulins in the
non-human animal.
In a further embodiment, the humanized Ig heavy chain locus present
in the transgenic vector comprises about 5 to 100 V gene segments,
with at least one human V gene segment. In a specific embodiment,
the humanized Ig heavy chain locus comprises more than one human V
gene segments.
In another embodiment, the humanized Ig heavy chain locus present
in the transgenic vector comprises about 5 to 25 D gene segments,
In a specific embodiment, the humanized Ig heavy chain locus
comprises one or several human D gene segments.
In yet another embodiment, the humanized Ig heavy chain locus
present in the transgenic vector comprises about 1 to 10 J gene
segments, with at least one human J gene segment. In a specific
embodiment, the humanized Ig heavy chain locus comprises more than
one human J gene segments.
In another embodiment, the humanized Ig heavy chain locus present
in the transgenic vector comprises about 1-25 C region segments,
with at least one human C region segment. In a specific embodiment,
the humanized Ig heavy chain locus present in the transgenic vector
comprises more than one human C gene segment.
In a still further embodiment, the humanized Ig locus present in
the transgenic vector is a light chain locus of a non-human animal,
and it comprises at least one V gene segment, at least one J gene
segment and at least one constant (C) region gene segment, where at
least one gene segment is selected from the group of human light
chain V and J segments and human light chain C region segments. In
a specific embodiment, the constant region gene segment is a human
light chain constant region gene segment, which can, for example,
be a C.lamda. or C.kappa. gene segment. In another embodiment, the
humanized Ig light chain locus comprises two or more segments
selected from human V and J segments and human C region segments.
In a further embodiment, the humanized Ig light chain locus
comprises at least one human V segment, at least one human J
segment, and at least one human C region segment.
In a further embodiment, the humanized Ig light chain locus present
in the transgenic vector comprises about 5-100 V gene segments,
with at least one human V gene segment, wherein the human V gene
segment is placed downstream to the 5-100 V gene segments of the
non-human animal. In a specific embodiment, the human V gene
segment is placed immediately 5' to a J gene segment in a
rearranged configuration. In another embodiment, the humanized Ig
light chain locus present in the transgenic vector comprises more
than one human V gene segment.
In a still further embodiment, the humanized Ig light chain locus
present in the transgenic vector comprises about 1-10 J gene
segments, with at least one human J gene segment. In a specific
embodiment, the humanized Ig light chain locus present in the
transgenic vector comprises more than one human J gene segment.
In another embodiment, the humanized Ig light chain locus present
in the transgenic vector comprises about 1-25 C region segments,
with at least one human C region segment. In a specific embodiment,
the humanized Ig light chain locus present in the transgenic vector
comprises more than one human C gene segment.
In a still further embodiment, the humanized Ig locus present in
the transgenic vector is a light chain locus of a non-human animal,
and it comprises at least one V gene segment, at least one J gene
segment and at least one constant (C) region gene segment, where at
least one gene segment is selected from the group of human light
chain V and J segments and human light chain C region segments. In
a specific embodiment, the constant region gene segment is a human
light chain constant region gene segment, which can, for example,
be a C.lamda. or C.kappa. gene segment. In another embodiment, the
humanized Ig light chain locus comprises two or more segments
selected from human V and J segments and human C region segments.
In a further embodiment, the humanized Ig light chain locus
comprises at least one human V segment, at least one human J
segment, and at least one human C region segment.
In a different aspect, the invention concerns a nucleic acid
molecule comprising two or more units consisting of, from 5' to 3'
direction, a 5' nucleotide sequence, a human immunoglobulin
sequence, and a 3' nucleotide sequence, wherein the 5' and 3'
nucleotide sequences are identical or different, and comprise at
least about 20 contiguous nucleotides from a spacer sequence
separating the coding regions in a non-primate animal
immunoglobulin heavy or light chain gene, or from a consensus
sequence of two or more of the spacer sequences. In a specific
embodiment, the spacer sequences are selected from within SEQ ID
NOS: 1 to 185 (Table 1). In another particular embodiment, the 5'
and/or 3' nucleotide sequences in all repetitive units of the
nucleic acid molecule are identical. In another particular
embodiment, the repetitive units of the nucleic acid molecule
comprise at least two different 5' and/or 3' sequences. In a
further embodiment, the 5' and 3' nucleotide sequence are different
from each other, but all 5' and all 3' nucleotide sequences are
identical.
In a further aspect, the invention concerns a method for making a
transgenic vector comprising a humanized immunoglobulin (Ig) locus
capable of producing a functional repertoire of humanized
antibodies in a non-human animal, comprising:
(a) obtaining a DNA fragment comprising an Ig locus or a portion
thereof from the non-human animal which comprises at least one V
gene segment, at least one J gene segment and at least one constant
region gene segment; and
(b) integrating at least one human Ig gene segment into the DNA
fragment of step (a) to produce a humanized Ig locus, wherein the
human Ig gene segment flanked by nucleotide sequences comprising at
least about 20 contiguous nucleotides from a spacer sequence
separating the coding regions in a non-primate animal
immunoglobulin heavy or light chain gene, or from a consensus
sequence or two or more of such spacer sequences; wherein (i) the
gene segments are juxtaposed in an unrearranged, partially
rearranged or fully rearranged configuration, and (ii) the
humanized Ig locus is capable of undergoing gene rearrangement, if
necessary, and producing a repertoire of humanized immunoglobulins
in the non-human animal.
The humanized Ig locus can be a humanized Ig heavy chain or light
chain locus. In the case of a humanized Ig heavy chain locus the
DNA fragment obtained in step (a) additionally comprises at least
one D gene segment.
In another aspect, the invention concerns a transgenic animal
comprising a humanized immunoglobulin locus described above, and
methods for making such transgenic animals. In one embodiment, the
transgenic animal comprises both a humanized immunoglobulin heavy
chain locus and a humanized immunoglobulin light chain locus. In
another embodiment, only one of the heavy and light chain loci
present in the transgenic animal is humanized. In another
embodiment, all of the V, D, J and C regions of at least one of the
animal's immunoglobulin loci are humanized. In yet another
embodiment, all of the V, D, J, and C region of the transgenic
animals endogenous immunoglobulin loci are humanized.
In a further aspect, the invention concerns a B cell from the
transgenic animals produced in accordance with the present
invention.
In a still further aspect, the invention concerns a humanized
immunoglobulin produced using a transgenic animal of the present
invention, and an antibody preparation or a pharmaceutical
composition comprising the humanized immunoglobulin.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic depiction of the rabbit immunoglobulin gene
heavy chain locus.
FIGS. 2-4 show a comparison of rabbit heavy chain spacer
sequences.
FIG. 5 is a schematic depiction of the rabbit immunoglobulin light
chain locus.
FIG. 6 illustrates the building of an immunoglobulin gene V locus
using human V.sub.H and rabbit spacer elements.
FIGS. 7 (a) and (b): Insertion of two cassettes by homologous
recombination using the red .beta..gamma.-system. FIG. 7(a) shows
that the upper cassette contains two restriction sites (FseI and
AscI) flanking a gentamycin cassette. FIG. 7(b) shows that the
lower cassette contains an inverted (i) and mutated (71) loxP-site,
a FRT-site and a MluI-restriction site. After modification the BAC
is digested with FseI and AscI.
FIG. 8 shows a humanized rabbit light chain locus (rLC3-B) based on
the rabbit K1 light chain locus. Rabbit C.kappa.1 was replaced with
human C.kappa.. A human rearranged human V.kappa.J.kappa. was
inserted. The synthetic human V.kappa.J.kappa. shares more than 80%
sequence homology with rabbit V.kappa. elements
FIG. 9a shows the sequence of a BAC clone comprising a chicken
light chain genomic locus whose nucleotide sequence is shown in
FIG. 9b (SEQ ID NO: 186).
FIGS. 10a and 10b illustrate an outline showing the construction of
a humanized immunoglobulin locus using chicken immunoglobulin
spacer sequences and human V elements.
FIGS. 11a and 11b illustrate an outline showing the construction of
a humanized immunoglobulin locus using mouse or rabbit
immunoglobulin spacer sequences and human V elements.
FIGS. 12a to 12c show a humanized light chain locus. A synthetic
sequence (FIG. 12a, Unit 1, 12,235 bp, SEQ ID NO: 187) containing
17 human V pseudogenes and 18 chicken spacer sequences is shown in
(a). A second synthetic sequence (FIG. 12b, Unit 2, 13,283 bp, SEQ
ID NO: 188) containing a functional rearranged human VkJk gene
fragment, 11 human V pseudogenes, 12 chicken spacer sequences and 2
introns is shown in (b). Units 1 and 2 were combined with a
fragment derived from BAC 179L1 containing human Ck and rabbit
intron and spacer sequences (FIG. 12c).
FIG. 13a-e show a humanized heavy chain locus. Four synthetic DNA
fragments (Unit 1-4, FIG. 13a-d, SEQ ID NOS: 189, 190, 191, 192)
consisting of human VH3 gene fragments and rabbit spacer and intron
sequences were combined with parts of BAC 219D23, 27N5 and Fos15B
as shown in (FIG. 13e).
DETAILED DESCRIPTION OF THE INVENTION
A. Definitions
"Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins
having the same structural characteristics. While antibodies
exhibit binding specificity to a specific antigen, immunoglobulins
include both antibodies and other antibody-like molecules which
lack antigen specificity. Polypeptides of the latter kind are, for
example, produced at low levels by the lymph system and at
increased levels by myelomas.
"Native antibodies and immunoglobulins" are usually
heterotetrameric glycoproteins of about 150,000 daltons, composed
of two identical light (L) chains and two identical heavy (H)
chains. Each light chain is linked to a heavy chain by covalent
disulfide bond(s), while the number of disulfide linkages varies
between the heavy chains of different immunoglobulin isotypes. Each
heavy and light chain also has regularly spaced intrachain
disulfide bridges. Each heavy chain has at one end a variable
domain (VH) followed by a number of constant domains. Each light
chain has a variable domain at one end (VL) and a constant domain
at its other end; the constant domain of the light chain is aligned
with the first constant domain of the heavy chain, and the light
chain variable domain is aligned with the variable domain of the
heavy chain. Particular amino acid residues are believed to form an
interface between the light- and heavy-chain variable domains
(Clothia et al., J. Mol. Biol. 186:651 (1985); Novotny and Haber,
Proc. Natl. Acad. Sci. U.S.A. 82:4592 (1985)).
The term "variable" refers to the fact that certain portions of the
variable domains differ extensively in sequence among antibodies
and are used in the binding and specificity of each particular
antibody for its particular antigen. However, the variability is
not evenly distributed throughout the variable domains of
antibodies. It is concentrated in three segments called
complementarity-determining regions (CDRs) or hypervariable regions
both in the light-chain and the heavy-chain variable domains. The
more highly conserved portions of variable domains are called the
framework (FR). The variable domains of native heavy and light
chains each comprise four FR regions, connected by three CDRs. The
CDRs in each chain are held together in close proximity by the FR
regions and, with the CDRs from the other chain, contribute to the
formation of the antigen-binding site of antibodies (see Kabat et
al., Sequences of Proteins of Immunological Interest, Fifth
Edition, National Institute of Health, Bethesda, Md. (1991)). The
constant domains are not involved directly in binding an antibody
to an antigen, but exhibit various effector functions, such as
participation of the antibody in antibody-dependent cellular
toxicity.
The term "monoclonal antibody" is used to refer to an antibody
molecule synthesized by a single clone of B cells.
The term "polyclonal antibody" is used to refer to a population of
antibody molecules synthesized by many clones of B cells. In a
specific embodiment, polyclonal antibodies recognize several
epitopes.
The terms "a humanized antibody" and "a humanized immunoglobulin",
as used herein, mean an immunoglobulin molecule comprising at least
a portion of a human immunoglobulin polypeptide sequence (or a
polypeptide sequence encoded by a human immunoglobulin gene
segment). The humanized immunoglobulin molecules of the present
invention can be isolated from a transgenic non-human animal
engineered to produce humanized immunoglobulin molecules. Such
humanized immunoglobulin molecules are less immunogenic to
primates, especially humans, relative to non-humanized
immunoglobulin molecules prepared from the animal or prepared from
cells derived from the animal.
The term "non-human animal" as used herein includes, but is not
limited to mammals, and includes, for example, non-human primates,
rabbits, pigs, birds (e.g., chickens, turkeys, ducks, geese and the
like), sheep, goats, cows, horses, and rodents (e.g. mice and
rats). Preferred non-human animals are those animals which create
antibody diversity substantially by gene conversion and/or somatic
hypermutation, e.g., rabbit, pigs, birds (e.g., chicken, turkey,
duck, goose and the like), sheep, goat, and cow. Particularly
preferred non-human animals are rabbit and chicken.
The term "non-primate animal" as used herein includes, but is not
limited to, mammals other than primates, including those listed
above.
The phrase "animals which create antibody diversity substantially
by gene conversion and/or hypermutation" is used to refer to
animals in which the predominant mechanism of antibody
diversification is gene conversion and/or hypermutation as opposed
to gene rearrangement.
The term "Ig gene segment" as used herein refers to segments of DNA
encoding various portions of an Ig molecule, which are present in
the germline of animals and humans, and which are brought together
in B cells to form rearranged Ig genes. Thus, Ig gene segments as
used herein include V gene segments, D gene segments, J gene
segments and C region gene segments.
The term "human Ig gene segment" as used herein includes both
naturally occurring sequences of a human Ig gene segment,
degenerate forms of naturally occurring sequences of a human Ig
gene segment, as well as synthetic sequences that encode a
polypeptide sequence substantially identical to the polypeptide
encoded by a naturally occurring sequence of a human Ig gene
segment. By "substantially" is meant that the degree of amino acid
sequence identity is at least about 85%-95%. In a particular
embodiment, the human Ig gene segment renders the immunoglobulin
molecule non-immunogenic in humans.
A specific humanized immunoglobulin molecule of the present
invention contains at least a portion of a human heavy or light
chain variable region polypeptide sequence. Another specific
immunoglobulin molecule contains at least a portion of a human
heavy or light chain variable region polypeptide sequence, and at
least a portion of a human constant domain polypeptide
sequence.
By "a preparation of humanized antibodies" or "a humanized antibody
preparation" is meant an isolated antibody product or a purified
antibody product prepared from a transgenic non-human animal (e.g.,
serum, milk, or egg yolk of the animal) or from cells derived from
a transgenic non-human animal (e.g., a B-cell or a hybridoma
cell).
A humanized antibody preparation can be a preparation of polyclonal
antibodies, which includes a repertoire of humanized immunoglobulin
molecules. A humanized antibody preparation can also be a
preparation of a monoclonal antibody.
The terms "antibody diversity" and "antibody repertoire" are used
interchangeably, and refer to the total of all antibody
specificities that an organism is capable of expressing.
The term "spacer sequence" is used herein to refer to any
non-coding nucleotide sequence present in an immunoglobulin heavy
or light chain gene. Thus, the term specifically includes intron
sequences and any other non-coding sequences separating the coding
regions within the V, D, J segments and C region segments in an
immunoglobulin heavy chain gene, intron sequences and any other
non-coding sequences separating the coding regions within the V and
J segments an C region segments in an immunoglobulin light chain
gene, as well as non-coding sequence flanking regulatory elements,
such as enhancers, in an immunoglobulin heavy or light chain gene.
In addition, non-coding sequences between exons encoding parts of a
membrane-spanning helix and heavy and light chain enhancers are
specifically included.
An Ig locus having the capacity to undergo gene rearrangement and
gene conversion is also referred to herein as a "functional" Ig
locus, and the antibodies with a diversity generated by a
functional Ig locus are also referred to herein as "functional"
antibodies or a "functional" repertoire of antibodies.
B. Relevant Literature
Regulatory elements in immunoglobulin genes have been described by
Bradley et al. (1999), Transcriptional enhancers and the evolution
of the IgH locus; Lauster, R. et al., Embo J 12: 4615-23 (1993);
Volgina et al., J Immunol 165:6400 (2000); Hole et al., J Immunol
146:4377 (1991).
Antibody diversification by gene conversion in chicken and rabbit
has been described by Bucchini et al., Nature 326: 409-11 (1987);
Knight et al., Advances in Immunology 56: 179-218 (1994); Langman
et al., Res Immunol 144: 422-46 (1993). The generation of mice
expressing human-mouse chimeric antibodies has been described by
Pluschke et al., Journal of Immunological Methods 215: 27-37
(1998). The generation of mice expressing human-mouse chimeric
antibodies with mouse derived membrane and cytoplamic tails has
been described by Zou et al., Science 262: 1271-1274 (1993); Zou et
al. Curr Biol 4: 1099-1103. The generation of mice expressing human
immunoglobulin polypeptides has been described by Bruggemann et al.
Curr Opin Biotechnol 8(4): 455-8 (1997); Lonberg et al. Int Rev
Immunol 13(1):65-93 (1995); Neuberger et al., Nature 338: 350-2
(1989). Generation of transgenic mice using a BAC clone has been
described by Yang et al., Nat Biotechnol 15: 859-65 (1997). The
generation of cows expressing human antibodies has been described
by Kuroiwa et al., Nature Biotech 20(9): 889-894 (2002).
The generation of transgenic rabbits has been described by Fan, J.
et al., Pathol Int 49: 583-94 (1999); Brem et al., Mol Reprod Dev
44: 56-62 (1996). Rabbits with impaired immunoglobulin expression
have been described by McCartney-Francis et al., Mol Immunol 24:
357-64 (1987); Allegrucci, et al., Eur J Immunol 21: 411-7
(1991).
The production of transgenic chicken has been described by Sherman
et al., Nature Biotech 16:1050-1053 (1998); Etches et al., Methods
in Molecular Biology 62: 433-450; Pain et al., Cells Tissues Organs
165(3-4): 212-9 (1999); Sang, H., "Transgenic chickens--methods and
potential applications", Trends Biotechnol 12:415 (1994); and in
WO2004003157, "Gene regulation in transgenic animals using a
transposon based vector"; and in WO 200075300, "Introducing a
nucleic acid into an avian genome, useful for transfecting avian
blastodermal cells for producing transgenic avian animals with the
desired genes, by directly introducing the nucleic acid into the
germinal disc of the egg".
A gammaglobulinemic chicken have been described by Frommel et al.,
J Immunol 105(1): 1-6 (1970); Benedict et al., Adv Exp Med Biol
1977; 88(2): 197-205.
The cloning of animals from cells has been described by T. Wakayama
et al., Nature 1998; 394:369-374; J. B. Cibelli et al., Science
280:1256-1258 (1998); J. B. Cibelli et al., Nature Biotechnology
1998; 16:642-646; A. E. Schnieke et al., Science 278: 2130-2133
(1997); K. H. Campbell et al., Nature 380: 64-66 (1996), Kuroiwa et
al., Nature Genetics 2004, Jun. 6. Nuclear transfer cloning of
rabbits has been described by Stice et al., Biology of Reproduction
39: 657-664 (1988), and Challah-Jacques et al., Cloning and Stem
Cells 8(4):295-299 (2003).
Production of antibodies from transgenic animals is described in
U.S. Pat. No. 5,814,318, No. 5,545,807 and No. 5,570,429.
Homologous recombination for chimeric mammalian hosts is
exemplified in U.S. Pat. No. 5,416,260. A method for introducing
DNA into an embryo is described in U.S. Pat. No. 5,567,607.
Maintenance and expansion of embryonic stem cells is described in
U.S. Pat. No. 5,453,357.
The mechanisms involved in the diversification of the antibody
repertoire in pigs, sheep and cows are reviewed in Butler, J. E.
(1998), "Immunoglobulin diversity, B-cell and antibody repertoire
development in large farm animals", Rev Sci Tech 17:43. Antibody
diversification in sheep is described in Reynaud, C. A., C. Garcia,
W. R. Hein, and J. C. Weill (1995), "Hypermutation generating the
sheep immunoglobulin repertoire is an antigen-independent process",
Cell 80:115; and Dufour, V., S. Malinge, and F. Nau. (1996), "The
sheep Ig variable region repertoire consists of a single VH
family," J Immunol 156:2163.
C. Detailed Description
Immunoglobulin heavy and light chain genes comprise several
segments encoded by individual genes and separated by intron
sequences. Thus genes for the human immunoglobulin heavy chain are
found on chromosome 14. The variable region of the heavy chain (VH)
comprises three gene segments: V, D and J segments, followed by
multiple genes coding for the C region. The V region is separated
from the C region by a large spacer, and the individual genes
encoding the V D and J segments are also separated by spacers.
There are two types of immunoglobulin light chains: .kappa. and
.lamda.. Genes for the human .kappa. light chain are found on
chromosome 2 and genes for the human .lamda. light chain are found
on chromosome 22. The variable region of antibody light chains
includes a V segment and a J segment, encoded by separate gene
segments. In the germline configuration of the .kappa. light chain
gene, there are approximately 100-200 V region genes in linear
arrangement, each gene having its own leader sequence, followed by
approximately 5 J gene segments, and C region gene segment. All V
regions are separated by introns, and there are introns separating
the V, J and C region gene segments as well.
The immune system's capacity to protect against infection rests in
a genetic machinery specialized to create a diverse repertoire of
antibodies. Antibody-coding genes in B cells are assembled in a
manner that allows to countless combinations of binding sites in
the variable (V) region. It is estimated that more than 10.sup.12
possible binding structures arise from such mechanisms. In all
animals, including humans, the antibody-making process begins by
recombining variable (V), diversity (D) and joining (J) segments of
the immunoglobulin (Ig) locus. Following this step, depending on
the animal species, two general mechanisms are used to produce the
diverse binding structures of antibodies.
In some animals, such as human and mouse, there are multiple copies
of V, D and J gene segments on the immunoglobulin heavy chain
locus, and multiple copies of V and J gene segments on the
immunoglobulin light chain locus. Antibody diversity in these
animals is generated primarily by gene rearrangement, i.e.,
different combinations of gene segments to form rearranged heavy
chain variable region and light chain variable region. In other
animals (e.g., rabbit, birds, e.g., chicken, goose, and duck,
sheep, goat, and cow), however, gene rearrangement plays a smaller
role in the generation of antibody diversity. For example, in
rabbit, only a very limited number of the V gene segments, most
often the V gene segments at the 3' end of the V-region, is used in
gene rearrangement to form a contiguous VDJ segment. In chicken,
only one V gene segment (the one adjacent to the D region, or "the
3' proximal V gene segment"), one D segment and one J segment are
used in the heavy chain rearrangement; and only one V gene segment
(the 3' proximal V segment) and one J segment are used in the light
chain rearrangement. Thus, in these animals, there is little
diversity among initially rearranged variable region sequences
resulting from junctional diversification. Further diversification
of the rearranged Ig genes is achieved by gene conversion, a
process in which short sequences derived from the upstream V gene
segments replace short sequences within the V gene segment in the
rearranged Ig gene. Additional diversification of antibody
sequences may be generated by hypermutation.
Immunoglobulins (antibodies) belong into five classes (IgG, IgM,
IgA, IgE, and IgD, each with different biological roles in immune
defense. The most abundant in the blood and potent in response to
infection is the IgG class. Within the human IgG class, there are
four sub-classes (IgG1, IgG2, IgG3 and IgG4 isotypes) determined by
the structure of the heavy chain constant regions that comprise the
Fc domain. The F(ab) domains of antibodies bind to specific
sequences (epitopes) on antigens, while the Fc domain of antibodies
recruits and activates other components of the immune system in
order to eliminate the antigens.
Antibodies have been used successfully as therapeutics since the
1890s when it was found that polyclonal antiserum taken from
animals could treat life-threatening infections in humans. A
significant advance in antibody research occurred with the
development of methods for the recombinant production of
antibodies, followed by the development of antibody humanization
techniques and method for making fully human monoclonal antibodies
in non-human animals.
As a result, chimeric, humanized and human monoclonal antibodies
have recently emerged as an important class of pharmaceutical
products. While monoclonal antibody-based drugs are very effective
in treating diseases when blocking a particular target (e.g.
receptor or ligand) certain devastating diseases, such as cancer
and infections with virulent pathogens, may be difficult to treat
due to their complexity, multifactoral etiology and adaptivity.
Monoclonal antibodies address singularly defined targets that
change, evolve and mutate during the spread of diseases throughout
a population or within an individual. Such adaptive evolution is
the bane of mono-specific drugs (e.g. monoclonal antibodies), which
are quickly circumvented by resistant strains. Examples abound of
bacterial and viral resistance to high-potency antibiotics, and
malignant cancers that develop resistance to standard anticancer
drugs, such as monoclonal antibody therapies.
In contrast, polyclonal antibodies have the ability to bind and
eliminate a plurality of evolving targets linked with complex
diseases. By binding multiple antigens, polyclonal antibodies
saturate the target and retain activity even in the event of
antigen mutation. Following this, through a cascade of signals,
polyclonal antibodies induce a potent immune response to eliminate
the target antigen, pathogen or cell. These properties make
polyclonal antibodies ideal for treating infectious diseases and
cancer.
So far, the use of polyclonal antibodies has been severely limited
by either supply problems or unwanted reactions to non-human
proteins.
The present invention provides a new humanization approach, based
on selective humanization the immunoglobulin-coding elements of the
immunoglobulin (Ig) translocus. The creation of such human-animal
translocus allows for the creation of transgenic animals that
express diversified, high-affinity humanized (polyclonal)
antibodies in high yields.
As a first step, the genomic loci for non-human, including
non-primate, immunoglobulin heavy and light chains are identified
and sequenced. For example, as part of the present invention,
genomic sequences for rabbit and chicken immunoglobulin heavy and
light chains were determined, and are shown in FIGS. 1, 5, and
9.
Analysis of the rabbit Ig heavy chain genomic locus has shown that
the immunoglobulin heavy chain variable region (Vh) contains
numerous genes, including functional genes and non-functional
pseudogenes. Alignment of 18 Vh genes has revealed a high degree
(80-90%) sequence identity among rabbit heavy chain variable region
gene sequences (Vh1-Vh18). The rabbit heavy chain variable region
genes have been found to share highest homology with the Vh3 group
of the human heavy chain variable region genes. Specifically,
sequence comparison of the rabbit Vh1-a2 gene with the human Vh3-23
sequences has revealed 72.8% sequence identity.
In addition, the non-coding (e.g. intron) sequences separating the
rabbit heavy chain variable region gene sequences were analyzed.
FIGS. 2-4 show a comparison of rabbit heavy chain intron sequences.
It has been found that such intron sequences fall into two groups,
and are highly conserved. Especially members of the Group 1 introns
show a surprisingly high (80-90%) sequence identity.
Similar findings were made by analysis of rabbit immunoglobulin
light chain variable region genomic sequences. In particular,
analysis of the rabbit immunoglobulin light chain locus has shown
that the light chain variable region (V1) region contains numerous
gene segments, which show a high degree (80-90% sequence identity).
It has further been found that the rabbit light chain variable
region (V.kappa.) exhibits high homology with the V.kappa.1 group
of the human light chain variable region gene sequences. Most
V.kappa. sequences have been found to be functional and highly
conserved. Unlike in the rabbit heavy chain variable region genes,
in the rabbit light chain variable region genes the intron
sequences have been found to be heterogeneous.
Similar studies with chicken immunoglobulin heavy and light chain
genomic sequences provide analogous results.
In one aspect, the present invention provides spacer sequences,
which separate the coding regions in a non-primate animal heavy or
light chain gene. In one embodiment, the present invention provides
spacer sequences from the heavy and light chain genes of animals
which create antibody diversity substantially by gene conversion,
including, for example, rabbit and chicken. Such spacer sequences
are then used to flank human immunoglobulin heavy or light chain
gene segments used in the process of creating a humanized
immunoglobulin locus.
The spacer sequences typically comprise at least about 20
nucleotides, or at least about 30 nucleotides, or at least about 40
nucleotides, or at least about 50 nucleotides, and typically are
between about 20 and about 10000 nucleotides in length. The spacer
sequences may contain a contiguous stretch of nucleotides of
appropriate length from a naturally occurring intron sequence in a
non-human (e.g. non-primate) animal, or may include an artificial
sequence, which may, for example, be a consensus sequence of two or
more naturally occurring intron sequences.
The spacer sequences may comprise at least about 20 (30, 40, 50,
etc. up to 1000 in 10-nucleotide increments) contiguous nucleotides
from a sequence selected from the group consisting of SEQ ID NOS 1
to 185 (Table 1), or from a consensus sequence of two or more of
such sequences. It is possible, but not necessary, to separate
human heavy or light chain sequences (e.g. V, D, J, C region
sequences) used for humanization by spacer sequences that separate
the corresponding regions within the genomic sequence of the
non-human (non-primate) animal the immunoglobulin of which is
humanized.
In general, the humanization of an immunoglobulin (Ig) locus in a
non-human animal involves the integration of one or more human Ig
gene segments into the animal's genome to create humanized
immunoglobulin loci. Thus, creation of a humanized Ig heavy chain
locus involves the integration of one or more V and/or D and/or J
segments, and/or C region segments into the animal's genome.
Similarly, the creation of a humanized Ig light chain locus
involves the integration of one or more V and/or J segments, and/or
C region segments into the animal's genome.
Depending upon the approach used, the human Ig gene segment(s) can
be integrated at the chromosomal location where the endogenous Ig
locus of the animal ordinarily resides, or at a different locus of
the animal. Regardless of the chromosomal location, the humanized
Ig locus of the present invention has the capacity to undergo gene
rearrangement and gene conversion and hypermutation in the
non-human animal, thereby producing a diversified repertoire of
humanized Ig molecules. An Ig locus having the capacity to undergo
gene rearrangement and gene conversion is also referred to as a
"functional" Ig locus and the antibodies with a diversity generated
by a functional Ig locus are also referred to as "functional"
antibodies or a "functional" repertoire of antibody molecules.
In a further aspect, the invention provides nucleic acid molecules
comprising a human Ig gene segment, flanked by nucleotide sequences
which comprise at least bout 20 contiguous nucleotides from a
spacer sequence separating the coding regions in a non-primate
animal Ig heavy or light chain gene, or from a consensus sequence
of two or more of such spacer sequences. The flanking sequences
just as the spacer sequence-derived sections within the flanking
sequences can be identical or different. The contiguous nucleotides
derived from a spacer sequence or from a consensus sequence of two
or more spacer sequences can be fused directly to the human Ig gene
segment. Alternatively, there might be an intervening sequence
between the human Ig gene segment and at least one of the
spacer-originating nucleotide sequences. Thus, for example, a
flanking sequence at the 5' end of a human V gene segment may
include a promoter region, which is linked directly to the human V
gene segment, and separates it from the spacer-sequence derived
nucleotide stretch of at least 20 nucleotides.
In yet another aspect, the invention concerns a humanized Ig heavy
chain locus in which human heavy chain V, D and/or J gene segments
and/or C region segments are present in the same configuration as
in the original non-human animal immunoglobulin gene, and separated
by sequences including at least about 20 contiguous nucleotides
from an intron sequence separating the coding regions in a
non-primate animal Ig heavy or light chain gene. In another
embodiment, the present invention provides a humanized light chain
locus in which human light chain C region segments and/or J gene
segments and/or V region segments are separated by non-human animal
(e.g. non-primate) intron sequences in the same configuration as in
the original non-human animal immunoglobulin gene. In a particular
embodiment, the spacer sequences are designed based on non-coding,
e.g. intron sequences of the non-human (non-primate) animal. In one
embodiment, the spacers may retain the appropriate non-coding
sequences from the non-human (non-primate) animal. Alternatively,
in order to simplify the construct, a consensus sequence, designed
based upon the highly homologous non-coding (intron) sequences may
be designed, and used as a uniform spacer sequence for the
preparation of multiple human heavy or light chain gene
segments.
The invention specifically provides isolated nucleic acid sequences
and vectors useful in the construction of humanized immunoglobulin
loci.
In one embodiment, DNA fragments containing an Ig locus to be
humanized are isolated from animals which generate antibody
diversity by gene conversion, e.g., rabbit and chicken. Such large
DNA fragments can be isolated by screening a library of plasmids,
cosmids, yeast artificial chromosomes (YACs) or bacterial
artificial chromosomes (BACs), and the like, prepared from the
genomic DNA of the non-human, e.g. non-primate animal. An entire
animal C-region can be contained in one plasmid or cosmid clone
which is subsequently subjected to humanization. YAC clones can
carry DNA fragments of up to 2 megabases, thus an entire animal
heavy chain locus or a large portion thereof can be isolated in one
YAC clone, or reconstructed to be contained in one YAC clone. BAC
clones are capable of carrying DNA fragments of smaller sizes
(about 150-450 kb). However, multiple BAC clones containing
overlapping fragments of an Ig locus can be separately humanized
and subsequently injected together into an animal recipient cell,
wherein the overlapping fragments recombine in the recipient animal
cell to generate a continuous Ig locus.
Human Ig gene segments can be integrated into the Ig locus on a
vector (e.g., a BAC clone) by a variety of methods, including
ligation of DNA fragments, or insertion of DNA fragments by
homologous recombination. Integration of the human Ig gene segments
is done in such a way that the human Ig gene segment is operably
linked to the host animal sequence in the transgene to produce a
functional humanized Ig locus, i.e., an Ig locus capable of gene
rearrangement and gene conversion and hypermutation which lead to
the production of a diversified repertoire of humanized
antibodies.
In one embodiment, human Ig gene segments can be integrated into
the Ig locus by homologous recombination. Homologous recombination
can be performed in bacteria, yeast and other cells with a high
frequency of homologous recombination events. For example, a yeast
cell is transformed with a YAC containing an animal's Ig locus or a
large portion thereof. Subsequently, such yeast cell is further
transformed with a recombination vector as described hereinabove,
which carries a human Ig gene segment linked to a 5' flanking
sequence and a 3' flanking sequence. The 5' and the 3' flanking
sequences in the recombination vector are homologous to those
flanking sequences of the animal Ig gene segment on the YAC. As a
result of a homologous recombination, the animal Ig gene segment on
the YAC is replaced with the human Ig gene segment. Alternatively,
a bacterial cell such as E. coli is transformed with a BAC
containing an animal's Ig locus or a large portion thereof. Such
bacterial cell is further transformed with a recombination vector
which carries a human Ig gene segment linked to a 5' flanking
sequence and a 3' flanking sequence. The 5' and the 3' flanking
sequences in the recombination vector mediate homologous
recombination and exchange between the human Ig gene segment on the
recombination vector and the animal Ig gene segment on the BAC.
Humanized YACs and BACs can be readily isolated from the cells and
used in making transgenic animals.
In a further aspect of the present invention, methods of making
transgenic animals capable of producing humanized immunoglobulins
are provided.
According to the present invention, a transgenic animal capable of
making humanized immunoglobulins are made by introducing into a
recipient cell or cells of an animal one or more of the transgenic
vectors described herein above which carry a humanized Ig locus,
and deriving an animal from the genetically modified recipient cell
or cells.
The recipient cells may, for example, be from non-human animals
which generate antibody diversity by gene conversion and/or
hypermutation, e.g., bird (such as chicken), rabbit, cows and the
like. In such animals, the 3'proximal V gene segment is
preferentially used for the production of immunoglobulins.
Integration of a human V gene segment into the Ig locus on the
transgene vector, either by replacing the 3'proximal V gene segment
of the animal or by being placed in close proximity of the
3'proximal V gene segment, results in expression of human V region
polypeptide sequences in the majority of immunoglobulins.
Alternatively, a rearranged human V(D)J segment may be inserted
into the J locus of the immunoglobulin locus on the transgene
vector.
The transgenic vectors containing a humanized Ig locus is
introduced into the recipient cell or cells and then integrated
into the genome of the recipient cell or cells by random
integration or by targeted integration.
For random integration, a transgenic vector containing a humanized
Ig locus can be introduced into an animal recipient cell by
standard transgenic technology. For example, a transgenic vector
can be directly injected into the pronucleus of a fertilized
oocyte. A transgenic vector can also be introduced by co-incubation
of sperm with the transgenic vector before fertilization of the
oocyte. Transgenic animals can be developed from fertilized
oocytes. Another way to introduce a transgenic vector is by
transfecting embryonic stem cells and subsequently injecting the
genetically modified embryonic stem cells into developing embryos.
Alternatively, a transgenic vector (naked or in combination with
facilitating reagents) can be directly injected into a developing
embryo. Ultimately, chimeric transgenic animals are produced from
the embryos which contain the humanized Ig transgene integrated in
the genome of at least some somatic cells of the transgenic
animal.
In a particular embodiment, a transgene containing a humanized Ig
locus is randomly integrated into the genome of recipient cells
(such as fertilized oocyte or developing embryos) derived from
animal strains with an impaired expression of endogenous
immunoglobulin genes. The use of such animal strains permits
preferential expression of immunoglobulin molecules from the
humanized transgenic Ig locus. Examples for such animals include
the Alicia and Basilea rabbit strains, as well as Agammaglobinemic
chicken strain, as well as immunoglobulin knock-out mice.
Alternatively, transgenic animals with humanized immunoglobulin
transgenes or loci can be mated with animal strains with impaired
expression of endogenous immunoglobulins. Offspring homozygous for
an impaired endogenous Ig locus and a humanized transgenic Ig locus
can be obtained.
For targeted integration, a transgenic vector can be introduced
into appropriate animal recipient cells such as embryonic stem
cells or already differentiated somatic cells. Afterwards, cells in
which the transgene has integrated into the animal genome and has
replaced the corresponding endogenous Ig locus by homologous
recombination can be selected by standard methods See for example,
Kuroiwa et al, Nature Genetics 2004, Jun. 6. The selected cells may
then be fused with enucleated nuclear transfer unit cells, e.g.
oocytes or embryonic stem cells, cells which are totipotent and
capable of forming a functional neonate. Fusion is performed in
accordance with conventional techniques which are well established.
Enucleation of oocytes and nuclear transfer can also be performed
by microsurgery using injection pipettes. (See, for example,
Wakayama et al., Nature (1998) 394:369). The resulting egg cells
are then cultivated in an appropriate medium, and transferred into
synchronized recipients for generating transgenic animals.
Alternatively, the selected genetically modified cells can be
injected into developing embryos which are subsequently developed
into chimeric animals.
Further, according to the present invention, a transgenic animal
capable of producing humanized immunoglobulins can also be made by
introducing into a recipient cell or cells, one or more of the
recombination vectors described herein above, which carry a human
Ig gene segment, linked to 5' and 3' flanking sequences that are
homologous to the flanking sequences of the endogenous Ig gene
segment, selecting cells in which the endogenous Ig gene segment is
replaced by the human Ig gene segment by homologous recombination,
and deriving an animal from the selected genetically modified
recipient cell or cells.
Similar to the target insertion of a transgenic vector, cells
appropriate for use as recipient cells in this approach include
embryonic stem cells or already differentiated somatic cells. A
recombination vector carrying a human Ig gene segment can be
introduced into such recipient cells by any feasible means, e.g.,
transfection. Afterwards, cells in which the human Ig gene segment
has replaced the corresponding endogenous Ig gene segment by
homologous recombination, can be selected by standard methods.
These genetically modified cells can serve as nuclei donor cells in
a nuclear transfer procedure for cloning a transgenic animal.
Alternatively, the selected genetically modified embryonic stem
cells can be injected into developing embryos which can be
subsequently developed into chimeric animals.
Transgenic animals produced by any of the foregoing methods form
another embodiment of the present invention. The transgenic animals
have at least one, i.e., one or more, humanized Ig loci in the
genome, from which a functional repertoire of humanized antibodies
is produced.
In a specific embodiment, the present invention provides transgenic
rabbits having one or more humanized Ig loci in the genome. The
transgenic rabbits of the present invention are capable of
rearranging and gene converting the humanized Ig loci, and
expressing a functional repertoire of humanized antibodies.
In another specific embodiment, the present invention provides
transgenic chickens having one or more humanized Ig loci in the
genome. The transgenic chickens of the present invention are
capable of rearranging and gene converting the humanized Ig loci,
and expressing a functional repertoire of humanized antibodies In
another specific embodiment, the present invention provides
transgenic mice with one or more humanized V regions in the genome.
The humanized V region comprises at least two human V gene segments
flanked by non-human spacer sequences. The transgenic mice are
capable of rearranging the human V elements and expressing a
functional repertoire of antibodies.
Once a transgenic non-human animal capable of producing diversified
humanized immunoglobulin molecules is made, humanized
immunoglobulins and humanized antibody preparations against an
antigen can be readily obtained by immunizing the animal with the
antigen. A variety of antigens can be used to immunize a transgenic
host animal. Such antigens include, without limitation,
microorganisms, e.g. viruses and unicellular organisms (such as
bacteria and fungi), alive, attenuated or dead, fragments of the
microorganisms, or antigenic molecules isolated from the
microorganisms.
Exemplary bacterial antigens for use in immunizing an animal
include purified antigens from Staphylococcus aureus such as
capsular polysaccharides type 5 and 8, recombinant versions of
virulence factors such as alpha-toxin, adhesin binding proteins,
collagen binding proteins, and fibronectin binding proteins.
Exemplary bacterial antigens also include an attenuated version of
S. aureus, Pseudomonas aeruginosa, enterococcus, enterobacter, and
Klebsiella pneumoniae, or culture supernatant from these bacteria
cells. Other bacterial antigens which can be used in immunization
include purified lipopolysaccharide (LPS), capsular antigens,
capsular polysaccharides and/or recombinant versions of the outer
membrane proteins, fibronectin binding proteins, endotoxin, and
exotoxin from Pseudomonas aeruginosa, enterococcus, enterobacter,
and Klebsiella pneumoniae.
Exemplary antigens for the generation of antibodies against fungi
include attenuated version of fungi or outer membrane proteins
thereof, which fungi include, but are not limited to, Candida
albicans, Candida parapsilosis, Candida tropicalis, and
Cryptococcus neoformans.
Exemplary antigens for use in immunization in order to generate
antibodies against viruses include the envelop proteins and
attenuated versions of viruses which include, but are not limited
to respiratory synctial virus (RSV) (particularly the F-Protein),
Hepatitis C virus (HCV), Hepatits B virus (HBV), cytomegalovirus
(CMV), EBV, and HSV.
Therapeutic antibodies can be generated for the treatment of cancer
by immunizing transgenic animals with isolated tumor cells or tumor
cell lines; tumor-associated antigens which include, but are not
limited to, Her-2-neu antigen (antibodies against which are useful
for the treatment of breast cancer); CD19, CD20, CD22 and CD53
antigens (antibodies against which are useful for the treatment of
B cell lymphomas), (3) prostate specific membrane antigen (PMSA)
(antibodies against which are useful for the treatment of prostate
cancer), and 17-1A molecule (antibodies against which are useful
for the treatment of colon cancer).
The antigens can be administered to a transgenic host animal in any
convenient manner, with or without an adjuvant, and can be
administered in accordance with a predetermined schedule.
After immunization, serum or milk from the immunized transgenic
animals can be fractionated for the purification of pharmaceutical
grade polyclonal antibodies specific for the antigen. In the case
of transgenic birds, antibodies can also be made by fractionating
egg yolks. A concentrated, purified immunoglobulin fraction may be
obtained by chromatography (affinity, ionic exchange, gel
filtration, etc.), selective precipitation with salts such as
ammonium sulfate, organic solvents such as ethanol, or polymers
such as polyethyleneglycol.
For making a monoclonal antibody, spleen cells are isolated from
the immunized transgenic animal and used either in cell fusion with
transformed cell lines for the production of hybridomas, or cDNAs
encoding antibodies are cloned by standard molecular biology
techniques and expressed in transfected cells. The procedures for
making monoclonal antibodies are well established in the art. See,
e.g., European Patent Application 0 583 980 A1 ("Method For
Generating Monoclonal Antibodies From Rabbits"), U.S. Pat. No.
4,977,081 ("Stable Rabbit-Mouse Hybridomas And Secretion Products
Thereof"), WO 97/16537 ("Stable Chicken B-cell Line And Method of
Use Thereof"), and EP 0 491 057 B1 ("Hybridoma Which Produces Avian
Specific Immunoglobulin G"), the disclosures of which are
incorporated herein by reference. In vitro production of monoclonal
antibodies from cloned cDNA molecules has been described by
Andris-Widhopf et al., "Methods for the generation of chicken
monoclonal antibody fragments by phage display", J Immunol Methods
242:159 (2000), and by Burton, D. R., "Phage display",
Immunotechnology 1:87 (1995), the disclosures of which are
incorporated herein by reference.
Cells derived from the transgenic animals of the present invention,
such as B cells or cell lines established from a transgenic animal
immunized against an antigen, are also part of the present
invention.
In a further aspect of the present invention, methods are provided
for treating a disease in a primate, in particular, a human
subject, by administering a purified humanized antibody
composition, preferably, a humanized polyclonal antibody
composition, desirable for treating such disease.
In another aspect of the present invention, purified monoclonal or
polyclonal antibodies are admixed with an appropriate
pharmaceutical carrier suitable for administration in primates
especially humans, to provide pharmaceutical compositions.
Pharmaceutically acceptable carriers which can be employed in the
present pharmaceutical compositions can be any and all solvents,
dispersion media, isotonic agents and the like. Except insofar as
any conventional media, agent, diluent or carrier is detrimental to
the recipient or to the therapeutic effectiveness of the antibodies
contained therein, its use in the pharmaceutical compositions of
the present invention is appropriate. The carrier can be liquid,
semi-solid, e.g. pastes, or solid carriers. Examples of carriers
include oils, water, saline solutions, alcohol, sugar, gel, lipids,
liposomes, resins, porous matrices, binders, fillers, coatings,
preservatives and the like, or combinations thereof.
The humanized polyclonal antibody compositions used for
administration are generally characterized by containing a
polyclonal antibody population, having immunoglobulin
concentrations from 0.1 to 100 mg/ml, more usually from 1 to 10
mg/ml. The antibody composition may contain immunoglobulins of
various isotypes. Alternatively, the antibody composition may
contain antibodies of only one isotype, or a number of selected
isotypes.
In most instances the antibody composition consists of unmodified
immunoglobulins, i.e., humanized antibodies prepared from the
animal without additional modification, e.g., by chemicals or
enzymes. Alternatively, the immunoglobulin fraction may be subject
to treatment such as enzymatic digestion (e.g. with pepsin, papain,
plasmin, glycosidases, nucleases, etc.), heating, etc, and/or
further fractionated.
The antibody compositions generally are administered into the
vascular system, conveniently intravenously by injection or
infusion via a catheter implanted into an appropriate vein. The
antibody composition is administered at an appropriate rate,
generally ranging from about 10 minutes to about 24 hours, more
commonly from about 30 minutes to about 6 hours, in accordance with
the rate at which the liquid can be accepted by the patient.
Administration of the effective dosage may occur in a single
infusion or in a series of infusions. Repeated infusions may be
administered once a day, once a week once a month, or once every
three months, depending on the half-life of the antibody
preparation and the clinical indication. For applications on
epithelial surfaces the antibody compositions are applied to the
surface in need of treatment in an amount sufficient to provide the
intended end result, and can be repeated as needed. In addition,
antibodies can, for example, be administered as an intramuscular
bolus injection, which may, but does not have to, be followed by
continuous administration, e.g. by infusion.
The antibody compositions can be used to bind and neutralize
antigenic entities in human body tissues that cause disease or that
elicit undesired or abnormal immune responses. An "antigenic
entity" is herein defined to encompass any soluble or cell-surface
bound molecules including proteins, as well as cells or infectious
disease-causing organisms or agents that are at least capable of
binding to an antibody and preferably are also capable of
stimulating an immune response.
Administration of an antibody composition against an infectious
agent as a monotherapy or in combination with chemotherapy results
in elimination of infectious particles. A single administration of
antibodies decreases the number of infectious particles generally
10 to 100 fold, more commonly more than 1000-fold. Similarly,
antibody therapy in patients with a malignant disease employed as a
monotherapy or in combination with chemotherapy reduces the number
of malignant cells generally 10 to 100 fold, or more than
1000-fold. Therapy may be repeated over an extended amount of time
to assure the complete elimination of infectious particles,
malignant cells, etc. In some instances, therapy with antibody
preparations will be continued for extended periods of time in the
absence of detectable amounts of infectious particles or
undesirable cells. Similarly, the use of antibody therapy for the
modulation of immune responses may consist of single or multiple
administrations of therapeutic antibodies. Therapy may be continued
for extended periods of time in the absence of any disease
symptoms.
The subject treatment may be employed in conjunction with
chemotherapy at dosages sufficient to inhibit infectious disease or
malignancies. In autoimmune disease patients or transplant
recipients, antibody therapy may be employed in conjunction with
immunosuppressive therapy at dosages sufficient to inhibit immune
reactions. The invention is further illustrated, but by no means
limited, by the following examples.
EXAMPLE 1
Isolation and Sequencing of BAC Clones Containing Rabbit
Immunoglobulin Loci
High molecular weight DNA was isolated from a2b5 male rabbits. The
rabbits were euthanized, spleen and kidneys were removed and rinsed
in ice-cold PBS. Fat and connecting tissues were removed and
processed separately. The organs were cut into pieces and
homogenized in a pre-cooled Dounce homogenizer. The supernatant was
transferred into cooled 50 ml falcon tubes, mixed with cold PBS and
large tissue debris was allowed to sink to the bottom for 2
minutes. Cells in the supernatant were pelleted at 200 g for 10 min
at 4.degree. C., washed once with PBS, resuspended in 1 ml PBS and
counted. Sets of 5.times.10.sup.6, 5.times.10.sup.7 and
5.times.10.sup.8 cells were embedded in agarose plugs using the
CHEF Mammalian Genomic DNA Plug Kit (BIORAD) To optimize conditions
for partial digestion with HindIII, plugs were cut into 5 equal
pieces and digested with 1 to 10 units of HindIII for various times
and temperatures. Best results were obtained with 2 units HindIII
at 4.degree. C. for 3 hrs or 37.degree. C. for 25 min. Digested DNA
was double size fractioned on a Pulse Field Gel Electrophoresis
(PFGE) apparatus using the following parameters: 6 hr backwards, 15
s switch times; 6 hr forwards, 15 s switch times; 20 hr forwards,
90 s switch times; 200V 14.degree. C. The area of the gel with the
desired size of partial digested DNA was cut and DNA was isolated
using gelase. 11 ng of insert was ligated with 1 ng of HindIII
digested pBELOBAC 11 and electroporated into DH10B cells. 1% of the
resulting colonies was sized using NotI and revealed an average
insert size of 124 kb. 1.times.10.sup.5 clones were spotted on
Nylon filters and screened by hybridization with specific
probes.
Probes for screening were amplified by PCR using genomic DNA from
rabbits, cloned into pBlueScript, and verified by sequencing.
Primer pairs (SEQ ID NO: 193-208, Table 2) were designed according
to published sequences. Several BACs representing rabbit heavy and
light chain immunoglobulin loci were isolated and mapped (FIGS. 1
and 5). BACs 219D23 219D23 (GenBank Acc. No. AY386695), 225P18
(GenBank Acc. No. AY386697), 27N5 (GenBank Acc. No. AY386696), 38A2
(GenBankAcc. No. AY386694), 179L1 (GenBank Acc. No. AY495827),
215M22 (GenBank Acc. No. AY495826), 19 (GenBankAcc. No. AY495828)
and Fosmid Fos15B (GenBank Acc. No. AY3866968) were sequenced.
Shotgun libraries for sequencing were constructed in pCR-Blunt with
an insert size of 1.5-2 kb. For sequence analysis the STADEN
package (Roger Staden, Cambridge, UK) was used. The software
modules pregap and gap4 were used for assembly and gap closure. For
the quality clipping of sequences PHRED (Washington University) and
the STADEN package was coupled.
EXAMPLE 2
Construction of a Humanized Rabbit Immunoglobulin Heavy Chain
Locus
BAC and fosmid clones containing rabbit immunoglobulin heavy chain
locus sequences were isolated from genomic DNA libraries using
probes specific for the constant, variable, and joining gene
segments or the 3' enhancer region. Isolated BACs (FIG. 1) 27N5
(GenBank Acc. No. AY386696), 219D23 (GenBankAcc. No. AY386695),
225P18 (GenBank Acc. No. AY386697), 38A2 (GenBank Acc. No.
AY386694) and fosmid Fos15B (GenBank Acc. No. AY3866968) were
sequenced (Ros et al., Gene 330, 49-59).
Selected immunoglobulin coding sequences were exchanged with
corresponding human counterparts by homologous recombination in E.
Coli by ET cloning (E-Chiang Lee et al., Genomics 73, 56-65 (2001);
Daiguan Yu et al., PNAS 97, 5978-5983 (2000); Muyrers et al.,
Nucleic Acids Research 27, 1555-1557 (1999); Zhang et al., Nature
Biotechnology 18, 1314-1317 (2000)).
Alternatively, DNA fragments were recombined by ligation in vitro
and subsequent transformation of E. coli. BACs and/or Fos15B or
parts thereof were combined by in vitro ligation and
transformation, ET cloning, or by Cre recombinase mediated
integration.
For ET cloning, vectors containing target sequence were transformed
into a streptomycin resistant E. coli strain containing the
inducible lambda phage recombination enzymes Red.alpha., Red.beta.
and .gamma.. These recombination proteins were expressed either
from a co-transfected plasmid (DH10B E. coli cells with plasmid
pSC101) or from a genomic integrated lambda prophage (DY380 E. coli
strain). The ET cloning procedure encompassed two homologous
recombination steps.
In a first step the target locus was replaced by a
selection-counter selection cassette (e.g. neo-rpsL which confers
resistance to neomycin (neo) and sensitivity to streptomycin
(rpsL)). After isolation of neo-resistant colonies, insertion of
the selection cassette by homologous recombination was confirmed by
restriction enzyme analysis and partial sequencing.
In a second step, the rpsL-neo selection cassette was exchanged
with a new sequence. Streptomycin resistant clones were analyzed by
restriction analysis and sequencing. Fragments used for the ET
cloning procedure had flanking sequences of 20 to 50 bp length,
which were identical to target sequences. Sequences used for
ligation had appropriate restriction enzyme sites at their 3' and
5' ends. These sites were either naturally occurring sites or they
were introduced by PCR using primers containing appropriate
sites.
Alternatively, sequences were generated synthetically.
A humanized heavy chain was constructed by replacement of rabbit
J.sub.H, C.mu. in BAC 219D23 and C.gamma. in BAC 27N5 with their
corresponding human counterparts by ET cloning. Human sequences
used for the ET cloning procedures were amplified by PCR from human
genomic DNA.
Human C.mu., C.gamma. and J.sub.H gene segments was amplified using
primers (SEQ ID Nos: 209-214, Table 2) with 50 bp homologies to
rabbit target sequences.
After ligation of BAC clone 225P18 with clone 219D23 and BAC 27N5
with Fosmid 15B, the ligated constructs were transformation into E.
coli and connected by Cre recombinase mediated insertion. This
resulted in a functional locus consisting of 18 rabbit variable
genes, rabbit D region, human J region, human C.mu., human
C.gamma., rabbit C , rabbit C.alpha.4 and the 3'enhancer
element.
For the generation of transgenic animals the humanized BAC clones
were coinjected either separately as three overlapping BACs (225P18
and 219D23 and BAC 27N5) or two overlapping combined BACs
(225P18-219D23 and BAC 27N5-Fosmid 15B) or as one BAC
(225P18-219D23-27N5-Fosmid 15B). Founder animals with transgenes
were identified by PCR.
EXAMPLE 3
Construction of a Humanized Immunoglobulin Heavy Chain Locus Using
Synthetic Fragments
Four fragments denoted Unit1, Unit2, Unit3, and Unit 4 (FIG. 13,
SEQ ID Nos: 189-192) with human V sequences and rabbit spacers were
chemically synthesized. Each fragment was flanked 5' by an AscI
restriction endonuclease recognition sequence, 3' by a lox71 Cre
recombinase recognition sequence followed by Fse I and MluI
restriction enzyme recognition sequences. Unit 1 consisted of human
V.sub.H3-49, V.sub.H3-11, V.sub.H3-7 and V.sub.H3-15 variable genes
separated by rabbit spacers I29-30, I3-4, I2-3 and the 3' half of
I1-2 (I1-2B). Unit 2 consisted of human V.sub.H3-48, V.sub.H3-43
and V.sub.H3-64 separated by rabbit spacers I1-2A (5' half of
I1-2), I7-8, I6-7 and the 3' half of I4-5 (I4-5B). Unit 3 consisted
of human V.sub.H3-74, V.sub.H3-30, and V.sub.H3-9 separated by the
rabbit spacer sequences I4-5B, I26-27, I11-12 and I17-18.
Unit 3 had in addition to the afore mentioned upstream flanks an
Flp recombinase recognition target (FRT) sequence, followed by a
Sglf I restriction endonuclease recognition sequence preceding the
already mentioned Asc I site.
Unit 4 had the human V.sub.H3-23 gene 5' flanked by the rabbit
spacer I1-2, a lox66 Cre recombinase target sequence and an AscI
endonuclease recognition sequence, and 3' flanked by IV-C (5' half)
rabbit spacer sequence followed by a MluI endonuclease recognition
sequence.
A gentamycin selection cassette was PCR-amplified, using primers
SEQ ID NOs 215 and 216 (Table 2) containing AscI and FseI sites and
ligated into a pGEM vector with a modified cloning site including
AscI, FseI, and MluI endonuclease recognition sites (pGEM.Genta
modified by PCR using SEQ ID NOs 217 and 218, Table 2).
Units 1, 2 and 3 were cloned into pGEM.Genta (Promega) vectors.
Unit 4 was sub-cloned into a customized pBELOBAC11 (NEB) vector
linearized with Hind III, and PCR-amplified. The forward primer
(SEQ ID NO: 219, Table 2) had restriction sites for HindIII, PacI
and AatII, and the reverse primer (SEQ ID NO: 220) sites for Bam
HI, MluI and AscI. The primers were designed in such a way that the
pBELOBAC11 Chloramphenicol selection cassette was deleted.
Furthermore, a Neomycin selection cassette was PCR-amplified with
primers SEQ ID NOs 221 and 222 (Table 2) carrying Bam HI and Hind
III restriction sites, and ligated to the modified pBELOBAC11
vector (pBB11.1).
Units 1-4 were assembled by cre-mediated recombination as described
(Mejia et al, Genomics 70(2) 165-70 (2000)). First Unit 1 was
cloned into the customized pgem.Genta vector, digested with Fse I
and subsequently recircularized by ligation. This vectorless
construct was transformed into E. coli containing pBB11.1.Unit4 and
p706-Cre plasmid. Following recombination of Unit 1 with PBB11.1
unit 4, positive clones (Unit4/1) were selected on kanamycin and
gentamycin containing media. Clones were characterized by
restriction analyses using various enzymes.
For recombination of Unit 2, the Unit4/1 insert was excised by
double digestion with AscI and PacI, and cloned into pBELOBAC11
with a modified linker (pBB11.2: modified by PCR using primers SEQ
ID NOs 223 and 224, Table 2).
pBELOBAC11 was linearized with HindIII and PCR-amplified with a
forward primer encoding PacI and AatII endonuclease recognition
sites and a reverse primer encoding MluI and NotI endonuclease
recognition sites and a lox66 Cre recombinase target site. For
ligation with Unit1/4 the pBB11.2 vector was opened with MluI and
PacI. pGEM.Genta.Unit2 was converted into a circular vectorless
construct as described for pGEM.Genta.Unit1 and connected with
pBB11.2.Unit4/1 by in vivo Cre mediated recombination.
Subsequently, the resulting construct pBB11.2.Unit4/1/2 is prepared
for Cre mediated recombination with Unit 3 by replacing the wild
type loxp site with a lox66 target site by ET-cloning (Muyrers et
al., Nucleic Acids Research 27, 1555-1557 (1999); Muyrers et at
Trends Biochem. Sci. 26(5):325-31 (2001)). A chloramphenicol
selection cassette was amplified by PCR with primers (SEQ ID NOs
225 and 226, Table 2) containing 50 bp sequences homologous to the
BAC target sequence. The reverse primer included a lox66 site. The
gel-purified PCR product was transformed into cells carrying the
target BAC as well as the pSC101 plasmid, required for homologous
recombination. Positive clones were selected with chloramphenicol
and confirmed by restriction analysis and sequencing.
pGEM.Genta.Unit 3 was prepared for in vivo recombination as
described above for Unit1 and 2 and transformed into cells carrying
the receptor BAC, as well as the p706-Cre plasmid. Positive clones
pBB11.2.Unit4/1/2/3 were selected with gentamycin and confirmed by
restriction analysis. pBB11.2.Unit4/1/2/3 was further modified by
ET-cloning to generate a lox 71 target site. Subsequently,
pBB11.2.Unit4/1/2/3 was connected to fragments from BACs 219D23,
27N5 and Fos15B.
EXAMPLE 4
Construction of a Humanized Immunoglobulin Heavy Chain Locus Using
PCR Amplified Fragments
Human V.sub.H elements were amplified using genomic DNA (ClonTech)
and primers SEQ ID NOs 227-248 (Table 2). PCR products were
analyzed by gel-electrophoresis and gel purified using the
GENECLEAN kit (Q-Biogen). Subsequently, amplification products were
sub-cloned into Zero-Blunt TOPO.TM. (Invitrogen), according to the
manufacturer's instructions. The sequences of all amplified V
elements were confirmed. For the construction of the humanized V
region, V elements were amplified using plasmid DNA as template and
primers SEQ ID NOs 249-270 (Table 2). Forward primers contained an
AscI site, followed by a rabbit splice site. Reverse primers
contained a rabbit recombination signal sequence (RSS) and a MluI
restriction site. PCR products were gel purified using the
GENECLEAN kit.
Human V.sub.Hs, V3-33, V3-74, V3-49, V3-21, V3-48, V3-73, V3-7, and
V3-D could not be isolated by PCR and were synthesized chemically
(BlueHeron, Bothel, Wash.). Restriction sites and rabbit regulatory
sequences were added during synthesis.
Rabbit spacer sequences were amplified using BACs 38A2 and 225P18
as templates and primers SEQ ID NOs 271-288 (Table 2). BAC 225P18
was double digested with NheI and a 41 kb fragment was gel
purified. This fragment served as a template for the amplification
of spacers V1-2, V2-3, V3-4, V4-5, and V5-6.
BAC 225P18 was digested with BstBI and the template for spacers
V6-7 and V7-8 was gel-purified. A double digestion of BAC 38A2 with
PacI and RsrlI allowed gel purification of the template for spacers
V21-22, and V22-23.
Amplified spacer sequences were gel-purified, and subcloned into
XL-PCR-TOPO.TM. (Invitrogen) according to the manufacturer's
instructions.
V.sub.H elements and rabbit spacer sequences were sub-cloned into
modified pGEM (Promega) and pBS (Strategene) vectors. The pGEM
vector was cut with NotI and Hind III and ligated with chemically
synthesized oligonucleotide sequences containing FseI, AscI and
MluI sites (Oligo1: SEQ ID NO: 289; Oligo2: SEQ ID NO: 290; Table
2). Vector pBS was cut with SacI and KpnI and ligated with a
chemically synthesized oligonucleotide sequence containing the
restriction sites FseI, AscI and MluI (Oligo1: (SEQ ID NO: 291;
Oligo2: SEQ ID NO: 292, Table 2).
Gentamycin and neomycin selection cassettes were amplified using
primers (SEQ ID NOs: 293-296, Table 2) with Fse I or AscI sites and
ligated into the modified pGEM and pBS-vectors.
The final construct is built in a modified pBeloBAC II vector. The
pBeloBAC II vector was opened with BamHI and HindIII and the
cloning sites were modified to contain FseI, AscI, MluI sites using
a chemically synthesized oligonucleotide sequence (Oligo1: SEQ ID
NO: 297; Oligo2: SEQ ID NO: 298, Table 2).
BAC 219D23 was modified by introduction of restriction sites using
ET-cloning (Muyrers et al., Nucleic Acids Research 27, 1555-1557
(1999); Muyers et at Trends Biochem. Sci. 26(5):325-31 (2001)). The
Neomycin selection cassette was amplified with primers SEQ ID NO:
299 and SEQ ID NO: 300 (Table 2). The forward primer contained an
FseI site, the reverse primer an AscI site.
The purified PCR product was transformed into E. coli cells
carrying BAC 219D23 and plasmid pSC101 necessary for homologous
recombination. After homologous recombination of the cassette and
the target sites in the BAC, introduced restriction sites were
confirmed by restriction analysis. Subsequently, the modified BAC
219D23 was digested with FseI and MluI and the resulting 17 kb
fragment (containing the FseI-Neo-AscI cassette) was separated by
PFGE and purified by electro-elution. This purified fragment was
ligated with the modified pBeloBAC II vector opened with FseI and
MluI.
A purified DNA fragment encoding a human V.sub.H element is ligated
with the modified pGEM.neo vector opened with AscI and MluI.
Similarly a spacer sequence is sub-cloned into the modified
pGEM.genta vector. Subsequently, the pGEM.genta vector carrying the
spacer sequence is cut with FseI and MluI and the insert is ligated
with pGEM.neo.V.sub.H vector opened with FseI and AscI. This step
is repeated several times to build a fragment consisting of several
spacer and V.sub.H segments. Such fragments are excised with FseI
and MluI and ligated with the modified pBeloBAC II vector
linearized with FseI and AscI. These processes are repeated to
build a large immunoglobulin V region (FIG. 6). The humanized heavy
chain locus is used for the generation of transgenic animals.
EXAMPLE 5
Construction of a Humanized Rabbit Light Chain Locus Containing
Humanized Ck and Humanized Rearranged VJ
Screening of a rabbit genomic BAC libraries resulted in the
identification of three BACs (215M22, 179L1 and 196O2; Gene Bank
Accession Nos: AY495826, AY495827, and AY495828, respectively)
containing rabbit light chain K1 gene segments. Rabbit
C.kappa..quadrature. was exchanged with human C.kappa..quadrature.
allotype Km3 by ET cloning as described (E-Chiang Lee et al.,
Genomics 73, 56-65 (2001); Daiguan Yu et al., PNAS 97, 5978-5983
(2000); Muyrers et al., Nucleic Acids Research 27, 1555-1557
(1999); Zhang et al., Nature Biotechnology 18, 1314-1317 (2000)).
Human C.kappa. (allotype Km3) was amplified by PCR with primers
(SEQ ID Nos 301 and 302, Table 2) containing 50 bp sequences
homologous to target sequences. Homology arms were designed based
on the published sequence of rabbit germline kappa (b5; GenBank
Accession No. K01363) and matched the intron-exon boundary of
C.kappa.. The exchange of rabbit C.kappa.against the human C.kappa.
in BAC 179L1 was verified by sequencing.
BAC 179L1-huCk was modified by two ET cloning. A neomycin selection
cassette was amplified with primers (SEQ ID NOs 303 and 304, Table
2) containing 50 bp sequences homologous to BAC 179L1. The forward
primer additionally had an i-CeuI meganuclease site. The PCR
product was used for ET cloning. Positive clones were selected with
neomycin and checked for correctness by restriction enzyme digests
and sequencing. A zeocin selection cassette was amplified with
primers (SEQ ID NOs 305 and 306, Table 2) containing 50 bp
sequences homologous to BAC 179L1. The forward primer additionally
had an i-SceI meganuclease site. The PCR product was used for ET
cloning. Positive clones were selected with zeozin and checked for
correctness by restriction enzyme digests and sequencing.
BAC 215M22 was modified by one ET cloning. A gentamycin resistance
gene was amplified with primers (SEQ ID NOs 307 and 308, Table 2)
containing 50 bp sequences homologous to BAC215M22. The forward
primer additionally had an i-CeuI Meganuclease site and the reverse
primer an i-SceI meganuclease site. The PCR product was used for ET
cloning. Resulting clones were selected with gentamycin and
analyzed by restriction enzyme digests and sequencing.
Modified BAC179L1 and 225M22 were cut with i-CeuI and i-SceI.
Fragments of 98 kb and 132 kb were purified and ligated. Resulting
clones were selected with kanamycin and chloramphenicol and checked
for correctness by restriction enzyme digests, PCR of the regions
containing i-SceI and i-CeuI restriction sites, and sequencing. The
resulting BAC was termed 179-215-huCk.
Rabbit J.kappa.1 and J.kappa.2 of BAC 179-215-huCk were replaced by
ET cloning with a synthetic human rearranged V.kappa.J.kappa. gene.
A DNA fragment with rabbit promoter, rabbit leader, rabbit intron
and human V.kappa.J.kappa. gene was synthesized chemically. The
codon usage of the synthetic human VJ was optimised to achieve
highest DNA sequence homology to rabbit V kappa genes.
The synthetic human VJ was PCR amplified with a forward primer (SEQ
ID NO 309, Table 2) containing 50 bp sequences homologous to BAC
179L1 and a reverse primer (SEQ ID NO 310, Table 2) containing a
sequence homologous to the gentamycin resistance gene and a FRT
site. A gentamycin resistance gene was amplified with a forward
primer (SEQ ID NO 311, Table 2) containing a FRT site and a reverse
primer (SEQ ID NO 312, Table 2) with 50 bp homology to BAC 179L1
and a FRT site. The human synthetic human VJ and the gentamycin
resistance gene were combined by overlap extension PCR using the
forward primer for the synthetic human VJ gene and the reverse
primer for the gentamycin resistance gene. The resulting fragment
was used for ET cloning. Positive clones were selected with
gentamycin and checked for correctness by restriction enzyme
digests and sequencing.
The gentamycin resistance gene was removed by site specific
recombination via expression of Flp recombinase. After
recombination one FRT was left. The FRT site was deleted by ET
cloning. A 232 bp fragment from the synthetic human VJ was
amplified by PCR (using primers SEQ ID NOs 313 and 314, Table 2)
and used for ET cloning. Resulting colonies were screened by PCR
(using primers SEQ ID NOs 315 and 316, Table 2) for loss of the FRT
site and confirmed by sequencing.
The neomycin resistance gene of BAC179-215-huCk was replaced by ET
cloning. A gentamycin resistance (pRepGenta; Genebridges) gene was
amplified by PCR with primers (SEQ ID NOs 317 and 318, Table 2)
containing 50 bp sequences homologous to BAC 179-215-huCk. The
forward primer additionally had a loxP site, an attB site and a
PvuI restriction site. Resulting clones were selected with
gentamycin and checked for correctness by restriction enzyme
digests and sequencing.
The resulting BAC (rLC3-B; FIG. 8) was used for the generation of
transgenic animals.
EXAMPLE 6
Construction of a Humanized Rabbit Light Chain Locus Containing
Multipe Human Vk Elements, Chicken Spacer Elements and a Rearranged
Human VJ
Screening of a rabbit genomic BAC libraries resulted in the
identification of three BACs (215M22, 179L1 and 196O2; Gene Bank
Accession Nos: AY495826, AY495827, and AY495828, respectively)
containing rabbit light chain K1 gene segments. Rabbit
C.kappa..quadrature.0 was exchanged with human C.kappa..quadrature.
allotype Km3 by ET cloning as described (E-Chiang Lee et al.,
Genomics 73, 56-65 (2001); Daiguan Yu et al., PNAS 97, 5978-5983
(2000); Muyrers et al., Nucleic Acids Research 27, 1555-1557
(1999); Zhang et al., Nature Biotechnology 18, 1314-1317 (2000)).
Human C.kappa. allotype Km3) was amplified by PCR with primers (SEQ
ID Nos 301 and 302, Table 2) containing 50 bp sequences homologous
to target sequences. Homology arms were designed based on the
published sequence of rabbit germline kappa (b5; GenBank Accession
No. K01363) and matched the intron-exon boundary of C.kappa.. The
exchange of rabbit C.kappa. against the human C.kappa. in BAC 179L1
was verified by sequencing.
Two DNA fragments, Unit1 (12,235 bp, FIG. 12a, SEQ ID NO 187),
containing 17 human V pseudogenes and 18 chicken spacer sequences
and Unit 2 (13,283 bp, FIG. 12b, SEQ ID NO 188) containing one
functional rearranged human kappa VJ gene with leader, 11 human V
pseudogenes, 12 chicken spacer sequences and intron 1 and parts of
intron 2 were synthesized chemically and cloned into vector
pBR322.
Units 1 and 2 were digested with the restriction enzyme NgoMIV and
AsiSI or NgoMIV and AscI respectively and ligated into pBELOBAC11
with a modified linker by three fragment ligation. The modified
linker contained a BsiWI restriction site, a FRT5-site, a
rpsL-Neo-cassette, a AscI site and a AsiSI-site. The linker
fragment was amplified with High fidelity polymerase (Roche),
primers CE_1_001_012904 (SEQ ID NO 319, Table 2) and
CE_1_on005_013004 (SEQ ID NO 320, Table 2) and plasmid pRpsL-Neo
(Genebridges) as template. Subsequently, the amplified product was
ligated into BamHI and HindIII sites of pBELOBAC11. For ligation
with Unit 1 and 2 the modified pBELOBAC11 was opened with AsiSI and
AscI. Positive clones (pBELOBAC11 Unit1/2) were checked by
restriction enzyme digests.
BAC 179L1 (GENBANK Acc. No. AY495827) was modified by insertion of
two modified selection cassettes by ET cloning. Cassette 1 was a
gentamycin resistance gene amplified with primers (SEQ ID Nos 321
and 322, Table 2) containing 50 bp sequences homologous to BAC
179L1 and an AscI site in the reverse primer. Cassette 2 was a
rpsl-Neo selection cassette amplified with primers (SEQ ID Nos 323
and 324, Table 2) containing 50 bp sequences homologous to BAC
179L1 and an attB site, a FRT5 site and a BsiWI site in the forward
primer.
The purified PCR products were transformed into E. coli cells
carrying the BAC and plasmid pSC101 necessary for homologous
recombination. After homologous recombination successful
modification of the BAC was confirmed by restriction digest
analyses, Southern Blot and sequencing.
Modified BAC 179L1 was cut with the restriction enzymes AscI and
BsiWI. The fragment containing the human C.kappa. was purified and
ligated with pBELOBAC11 Unit1/2 opened with the same restriction
enzymes. Positive clones were checked by restriction enzyme
digests. The final construct (FIG. 12c) is used for the generation
of transgenic animals
EXAMPLE 7
Construction of a Humanized Rabbit Light Chain Locus Containing
Multiple Human V.kappa. Elements, Chicken Spacers and an
Unrearranged Human J Kappa Locus
The construct described in example 6 was modified by ET cloning as
follows: an. The rearranged functional VJ sequence was exchanged
with a functional V1 flanked by a functional recombination signal
sequence (RSS). The RSS was PCR amplified from BAC179L1 with a
forward primer (SEQ ID NO 325, Table 2) containing a 50 bp sequence
homologous to V1 of pBELOBAC11 Unit1/2 and a reverse primer (SEQ ID
NO 326, Table 2) containing an AscI restriction enzyme site and
homology to the gentamycin resistance gene. A gentamycin resistance
gene was amplified with a forward primer (SEQ ID NO 327, Table 2)
containing a sequence homologous to the reverse primer used for RSS
amplification and a reverse primer (SEQ ID NO 328, Table 2)
containing a 50 bp sequence homologous to pBELOBAC11 Unit1/2 and a
BsiWI restriction enzyme site.
The RSS and the gentamycin resistance gene were combined by overlap
extension PCR using the forward primer for RSS amplification and
the reverse primer for Gentamycin resistance gene amplification.
The resulting fragment was used to modify pBELOBAC11 Unit1/2 by ET
cloning. Positive clones were selected with gentamycin and analyzed
by restriction enzyme digests and sequencing.
BAC 179L1 with human C.kappa. was further modified by ET cloning. A
kanamycin selection cassette was amplified with a primers (SEQ ID
NO 329 and 330, Table 2) containing 50 bp sequences homologous to
BAC 179L1. The reverse primer contained also an AscI restriction
enzyme site and a FRT site. The PCR product was used for ET
cloning. An ampicillin selection cassette was amplified with
primers (SEQ ID Nos 331 and 332, Table 2) containing 50 bp
sequences homologous to BAC 179L1. The forward primer contained
also an attB site, an AsiSI restriction enzyme site and a FRT5 site
The reverse primer contained a BsiWI restriction enzyme site and a
FRT site. The PCR product was used for ET cloning. The human J
region was amplified from human genomic DNA with primers (SEQ ID
Nos 333 and 334, Table 2) containing 50 bp sequences homologous to
BAC 179L1. The PCR product was used for ET cloning. The resulting
clones were analyzed by restriction enzyme digest and
sequencing.
A positive clone was cut with AscI and BsiWI. The resulting
fragment was purified and ligated into the modified pBELOBAC11
Unit1/2 cut with the same enzymes. Positive clones were selected
with ampicillin and analyzed by restriction enzyme digests and
sequencing. Correct clones are used to generate transgenic
animals.
EXAMPLE 8
Construction of a Humanized Rabbit Light Chain Locus Containing
Multiple Human Vk Elements
Screening of a rabbit genomic BAC libraries resulted in the
identification of three BACs (215M22, 179L1 and 196O2; Gene Bank
Accession Nos: AY495826, AY495827, and AY495828, respectively)
containing rabbit light chain K1 gene segments. Rabbit C.kappa.1
was exchanged with human C.kappa..quadrature. allotype Km3 by ET
cloning as described (E-Chiang Lee et al., Genomics 73, 56-65
(2001); Daiguan Yu et al., PNAS 97, 5978-5983 (2000); Muyrers et
al., Nucleic Acids Research 27, 1555-1557 (1999); Zhang et al.,
Nature Biotechnology 18, 1314-1317 (2000)). Human C.kappa. allotype
Km3) was amplified by PCR with primers (SEQ ID NOs 301 and 302,
Table 2) containing 50 bp sequences homologous to target sequences.
Homology arms were designed based on the published sequence of
rabbit germline kappa (b5; GenBank Accession No. K01363) and
matched the intron-exon boundary of C.kappa.. The exchange of
rabbit C.kappa. against the human C.kappa. in BAC 179L1 was
verified by sequencing.
Human V.kappa. elements of the V.kappa.1 family (O2, L8, L4, A30,
L11, L1, L5, L15, O8, L19, L12, A20, O4, L14, L23, L9, A4, L24, O6,
L22, A9, A25, A15, O9) were amplified by PCR using primers (SEQ ID
NOs 335-382, Table 2) and human genomic DNA as a template.
Amplification products were analysed by gel-electrophoresis, gel
purified using GENECLEAN (Q-Biogen), subcloned into the Zero-Blunt
TOPO.TM. vectors (Invitrogen) and sequenced. A rearranged human
V.kappa. (O2) J.kappa. (J4) element was produced by PCR
amplification, subcloned and sequenced. To combine human V.kappa.
elements with rabbit spacers, human V.kappa. elements were
amplified by PCR with primers (SEQ ID Nos 383-430, Table 2) using
plasmid DNA as a template. Primers contained AscI or MluI
sites.
Rabbit spacer sequences are amplified by PCR using primers SEQ ID
NOs 431-450 (Table 2). BACs 179L1 and 215M22 are digested with
SpeI, NheI, AclI, SfoI, MluI, and SalI/XhoI. Fragments are gel
purified and used as amplification templates.
The spacer sequence located at the 5-end is amplified by an
upstream oligonucleotide containing a FRT and an attB site. PCR
products are gel purified using the GENECLEAN kit and subcloned
into XL-PCR-TOPO.TM. (Invitrogen) according to the manufacturer's
instructions.
Human Vk elements and rabbit spacer sequences were cloned into pGem
(Promega) modified as described in Example 4.
Human V kappa and the modified pGEM.genta vector are digested with
AscI and MluI and ligated. Similarly, rabbit spacer sequences are
cloned into pGEM.neo. Subsequently, pGem.neo. V.kappa. is cut with
FseI and AscI and ligated with a purified insert of
pGem.genta.spacer excised with FseI and MluI. Ligation of AscI and
MluI complementary ends destroys the restriction enzyme site and
allows repeated use of AscI and MluI for the construction of a
V.kappa. locus comprising several V.kappa. and spacer elements. The
final construct, consisting of fragments of a humanized BAC 179L1
and 215M22 and a humanized V.kappa. region is built in pBeloBAC.
BAC 179L1 and 215M22 were modified and combined. Subsequently, BAC
179L1-215M22-huCk was further modified by ET cloning. Two cassettes
containing restriction enzyme site, selection markers, and
additional functional sites were inserted into the vector by
ET-cloning as shown in FIG. 7. Primers (SEQ ID NOs 451-454) used
for the amplification of the cassettes are listed in Table 2.
To built the final construct, units consisting of human V elements,
rabbit spacer elements and a resistance marker are excised out of
pGEM with FseI and MluI and ligated with BAC 179L1-215M22 digested
with FseI and AscI. Subsequently, the resistance marker is replaced
with a new insert consisting of human V elements, rabbit spacer
elements and another resistance marker. After several repeats the
final construct will consist of many Vk segments (L8, L4, A30, L11,
L1, L5, L15, O8, L19, L12, A20, O4, L14, L23, L9, A4, L24, O6, L22,
A9, A25, A15, O9) separated by rabbit spacer sequences. The
humanized light chain locus is used for the generation of
transgenic animals.
EXAMPLE 9
Construction of a Humanized Heavy Chain Locus with Chicken Heavy
Chain Locus Spacer Sequences
A synthetic humanized heavy chain locus containing a rabbit D
region, a human J region, human C.mu., human C.gamma., rabbit
C.alpha.4, the rabbit 3'.alpha. enhancer and human VH elements
(including promotor and nonamer/heptamer sequences) separated by
chicken spacer sequences is constructed.
Modified rabbit BAC 27N5 (see "Example 2) was further modified by
ET cloning. The construct contained a humanized C.mu. and C.gamma.
and two unique restriction sites, BsiWI and AsiSI downstream of the
.alpha.-4 membrane exon. DNA is amplified with oligonucleotides SEQ
ID Nos 455 and 456 (Table 2).
Fosmid Fos15B is digested with NheI and the resulting 13 kb
fragment containing the 3'.alpha. enhancer is subcloned into a
cloning vector in such a way that the insert is flanked by BsiWI
and AsiSI sites. Subsequently, the insert is excised with BsiWI and
AsiSI and ligated with the modified BAC 27N5 to form BAC
27N5Fos.
The rabbit J region in BAC219D23 was exchanged with the
corresponding human J region by ET cloning. The human J region was
amplified by PCR using primers SEQ ID NOs 457 and 458 (Table
2).
Unique restriction enzyme sites are inserted in BAC219D23 upstream
of the D region (A) and upstream of C.mu.(B).quadrature.. In BAC
27N5Fos restriction site A is inserted upstream of the linker
region and B is inserted in sequences homologous to BAC219D23.
Following digestion with enzymes A and B, the fragment containing
human J and rabbit D regions is isolated and ligated with BAC
27N5Fos to create BAC 219D23/27N5Fos.
Chicken heavy chain spacer sequences are amplified from chicken
genomic DNA by PCR using primers (SEQ ID NOS 459 and 460, Table 2)
specific for chicken heavy chain V pseudogenes (Mansikka et al., J
Immunol 145(11), 3601-3609 (1990), Reynaud et al., Cell 59(1),
171-183 (1989)). Alternatively, spacer sequences are synthesized
chemically.
The PCR products are gel purified, cloned into pTOPO (Invitrogen)
and sequenced.
Human heavy chain variable elements are amplified by PCR using
primers designed according to published sequences in GENBANK (eg
Acc. No. NG_001019) or synthesized chemically. The human V elements
contain the human promoter region, the human leader sequence, the
human intron between leader and V-coding region, the human V-coding
region and the human recombination signal sequence. The amplified
or synthesized fragments are flanked by specific restriction
endonuclease recognition. Chicken spacer sequences and human V
elements are combined in one or several large DNA fragment
comprising a humanized immunoglobulin locus. The construct is used
to generate transgenic animals.
EXAMPLE 10
Construction of a Humanized Immunoglobulin Locus Containing Human V
Elements and Non-Human Spacer Sequences (without Promoter Region
and RSS)
A BAC library generated with non-human genomic DNA is screened with
probes specific for immunoglobulin and BAC clones containing heavy
and light chain immunoglobulin C, J and D regions are identified.
The BAC clones are modified to contain restriction enzyme sites.
Human heavy and light chain variable elements are amplified by PCR
using primers designed according to published sequences in GenBank
(eg., Acc. No. NG_001019). Sequences are amplified from genomic DNA
or synthesized chemically. The human V elements contain the human
promoter region, the human leader sequence, the human intron
between leader and V-coding region, the human V-coding region and
the human recombination signal sequence (RSS). The amplified or
synthesized fragments have specific restriction endonuclease
recognition sites at the ends. The non-human spacer sequences are
amplified by PCR or synthesized chemically. Non-human spacer
sequences and human V elements are combined in one or several large
DNA fragment comprising a humanized immunoglobulin locus. The
construct is used to generate transgenic animals. An example for
the construction of a humanized V region using chicken spacer
sequences is shown in FIG. 10.
EXAMPLE 11
Construction of a Humanized Immunoglobulin Locus Containing Human V
Elements and Non-Human Spacer Sequences
A BAC library generated with non-human genomic DNA is screened with
probes specific for immunoglobulin and BAC clones containing heavy
and light chain immunoglobulin C, J and D regions are identified.
The BAC clones are modified to contain restriction enzyme sites.
Human heavy and light chain variable elements are amplified by PCR
using primers designed according to published sequences in GenBank
(eg., Acc No. NG_001019). Sequences are amplified from genomic DNA
or synthesized chemically. The human V elements contain the human V
coding region. Non-human spacer sequences are amplified by PCR or
synthesized chemically and contain a recombination signal sequence,
a spacer sequence, a promoter region, a leader sequence and the
intron between leader and V coding region. Such non-human spacer
sequences are combined with human V elements in one or several
large DNA fragments and used for the generation of transgenic
animals. An example for the construction of a humanized V region
using mouse or rabbit spacer sequences is shown in FIG. 11.
EXAMPLE 12
Transgenic Rabbits Expressing Humanized Immunoglobulins
Transgenic rabbits were generated as described by Fan et al.
(Pathol. Int. 49: 583-594, 1999). Briefly, female rabbits are
superovulated using standard methods and mated with male rabbits.
Pronuclear-stage zygotes are collected from oviduct and placed in
an appropriate medium such as Dulbecco's phosphate buffered saline
supplemented with 20% fetal bovine serum. BAC containing humanized
immunoglobulin loci were microinjected into the male pronucleus
with the aid of a pair of manipulators. Morphological surviving
zygotes were transferred to the oviducts of pseudopregnant rabbits.
Pseudopregnancy was induced by the injection of human chorionic
gonadotrophin (hCG). Following injection of a humanized light chain
construct into 4645 pronuclei of fertilized oocytes, 4043 oocytes
were transferred into 132 recipients. In total, 253 live offspring
were born, 11 of which were transgenic. Expression of human kappa
light chain was detected by ELISA using human-kappa light chain
specific reagents (for example, mouse anti-human Kappa, Southern
Biotech, 9220-01; goat anti-human Kappa, Southern Biotech
2063-08).
A humanized heavy chain construct was injected into 4083 pronuclei
of fertilized oocytes. 3485 oocytes were transferred into 119
recipients which delivered 433 offspring. Analysis by PCR and FISH
revealed that 20 of these animals were transgenic. Humanized heavy
chain in the blood of founder animals was detected by ELISA using
antibodies specific for human IgM/IgG (for example, rabbit
anti-human IgM, Rockland 609-4131; rabbit anti-human IgM, Rockland
609-4631; rabbit anti-human IgG, Pierce 31142, rabbit anti-human
IgG, Southern Biotech 6145-08; rabbit anti-human IgG, Pierce
31784).
Sandwich-type ELISAs detecting humanized .kappa., .mu. and .gamma.
chains were performed using standard procedures. Briefly,
microtiter plates were coated with capture antibody and incubated
with diluted serum samples. Bound human immunoglobulin was detected
using a secondary labeled antibody and
peroxidase-streptavidin-conjugate (Sigma, S2438).
Double transgenic animals expressing both humanized heavy and light
immunoglobulin chains were generated by breeding of founder
animals.
EXAMPLE 13
Transgenic Mice Expressing Humanized Immunoglobulins
Transgenic mice were generated as described by Nagy et al.
(Manipulating the Mouse Embryo: A Laboratory Manual; Cold Spring
Harbor Laboratory Press, New York, 2003). Briefly, female mice were
superovulated using standard methods and mated with male mice.
Pronuclear-stage zygotes were collected from oviduct and placed in
a suitable medium such as M2 medium. BAC containing humanized
immunoglobulin loci were microinjected into the male pronucleus
with the aid of a pair of manipulators. Morphologically surviving
zygotes were transferred to the oviducts of pseudopregnant female
mice. Pseudopregnancy was induced by mating with sterile males.
Following injection of a humanized light chain construct into 1325
pronuclei of fertilized oocytes, 787 oocytes were transferred into
29 recipients. In total, 55 live offspring were born, 11 of which
were transgenic.
A humanized heavy chain construct was injected into 1050 pronuclei
of fertilized oocytes. 650 oocytes were transferred into 25
recipients which delivered 64 live offspring. Analysis by PCR
revealed that 19 of these animals were transgenic.
Double transgenic animals expressing both humanized heavy and light
immunoglobulin chains are generated by breeding of founder animals.
Expression of humanized .kappa., .mu. and .gamma. chains was
detected by ELISAs using standard procedures. Briefly, microtiter
plates were coated with capture antibody and incubated with diluted
serum samples. Bound human immunoglobulin was detected using a
secondary labeled antibody and peroxidase-streptavidin-conjugate
(Sigma, S2438).
All references cited throughout the specification are hereby
expressly incorporated by reference. While the invention is
illustrated by reference to certain embodiments, it is not so
limited. One skilled in the art will recognize that various
modifications and variations are possible without diverting from
the essence of the invention. All such modifications and variations
are specifically included within the scope herein.
TABLE-US-00001 TABLE 1 ID BAC Accession# Start Finish Description 1
38A2 AY386694 1 2137 Spacer 5' start-V34 2 38A2 AY386694 2433 9504
Spacer V34-V33 3 38A2 AY386694 9798 19384 Spacer V33-V32 4 38A2
AY386694 19690 35164 Spacer V32-V31 5 38A2 AY386694 35447 47669
Spacer V31-V30 6 38A2 AY386694 47973 52521 Spacer V30-V29 7 38A2
AY386694 52819 61798 Spacer V29-V28 8 38A2 AY386694 62100 74264
Spacer V28-V27 9 38A2 AY386694 74566 79145 Spacer V27-V26 10 38A2
AY386694 79449 84800 Spacer V26-V25 11 38A2 AY386694 85103 95717
Spacer V25-V24 12 38A2 AY386694 96009 102226 Spacer V24-V26 13 38A2
AY386694 102504 105307 Spacer V23-V22 14 38A2 AY386694 105603
107583 Spacer V22-V24 15 38A2 AY386694 107769 118033 Spacer V24-V20
16 38A2 AY386694 118334 125546 Spacer V20-V19 17 38A2 AY386694
125849 128059 Spacer V19-3' end 18 225P18 AY386697 1 4333 Spacer 5'
start-V18 19 225P18 AY386697 4629 9255 Spacer V18-V17 20 225P18
AY386697 9561 15841 Spacer V17-V16 21 225P18 AY386697 16135 22502
Spacer V16-V15 22 225P18 AY386697 22794 32821 Spacer V15-V14 23
225P18 AY386697 33118 37738 Spacer V14-V13 24 225P18 AY386697 38044
44571 Spacer V13-V12 25 225P18 AY386697 44865 49447 Spacer V12-V11
26 225P18 AY386697 49745 56909 Spacer V11-V10 27 225P18 AY386697
57205 63678 Spacer V10-V9 28 225P18 AY386697 63977 71204 Spacer
V9-V8 29 225P18 AY386697 71507 76261 Spacer V8-V7 30 225P18
AY386697 76560 79012 Spacer V7-V6 31 225P18 AY386697 79308 83467
Spacer V6-V5 32 225P18 AY386697 83768 88013 Spacer V5-V4 33 225P18
AY386697 88314 91233 Spacer V4-V3 34 225P18 AY386697 91531 95929
Spacer V3-V2 35 225P18 AY386697 96233 100963 Spacer V2-V1 36 225P18
AY386697 101262 133721 Spacer V1-D3 37 225P18 AY386697 133752
135212 Spacer D3-D1a 38 225P18 AY386697 135237 136922 Spacer D1a-D4
39 225P18 AY386697 136947 139446 Spacer D4-3' end 40 219D23
AY386695 8151 40612 Spacer V1-D3 41 219D23 AY386695 40643 42102
Spacer D3-D1a 42 219D23 AY386695 42127 43812 Spacer D4-D1b 43
219D23 AY386695 43837 48553 Spacer D1b-D6 44 219D23 AY386695 48577
48753 Spacer D6-D8 45 219D23 AY386695 48769 51181 Spacer D8-D2x 46
219D23 AY386695 51213 55826 Spacer D1c-Df 47 219D23 AY386695 55852
61112 Spacer Df-D1d 48 219D23 AY386695 61237 62445 Spacer D1d-D5 49
219D23 AY386695 62471 97024 Spacer D5-DQ52 50 219D23 AY386695 97036
97831 Spacer DQ52-J1 51 219D23 AY386695 97871 98090 Spacer J1-J2 52
219D23 AY386695 98140 98430 Spacer J2-J3 53 219D23 AY386695 98483
98642 Spacer J3-J4 54 219D23 AY386695 98690 98981 Spacer J4-J5 55
219D23 AY386695 99032 99550 Spacer J5-J6 56 219D23 AY386695 99604
106867 1501Spacer J6- IgM exon1 57 219D23 AY386695 107185 107290
Spacer IgM exon1- exon2 58 219D23 AY386695 107635 107863 Spacer IgM
exon2- exon3 59 219D23 AY386695 108181 108268 Spacer IgM exon3-
exon4 60 219D23 AY386695 108664 110499 Spacer IgM exon4- exonM1 61
219D23 AY386695 110615 110741 Spacer exonM1- exonM2 62 219D23
AY386695 108664 137302 Spacer IgM exon4- 3' end 63 27N5 AY386696
5099 55582 Spacer IgM exon4- IgG exon1 64 27N5 AY386696 55867 56071
Spacer IgG exon1- exon2 65 27N5 AY386696 56104 56201 Spacer IgG
exon2- exon3 66 27N5 AY386696 56531 56623 Spacer IgG exon3- exon4
67 27N5 AY386696 56946 58984 Spacer IgG exon4- exonM1 68 27N5
AY386696 56946 59205 Spacer IgG exon4- exonM2 69 27N5 AY386696
56946 69246 Spacer IgG exon4- IgE exon1 70 27N5 AY386696 69546
69719 Spacer IgE exon1- exon2 71 27N5 AY386696 70037 70132 Spacer
IgE exon2- exon3 72 27N5 AY386696 70453 70532 Spacer IgE exon3-
exon4 73 27N5 AY386696 70873 73061 Spacer IgE exon4- exonM1 74 27N5
AY386696 70873 73292 Spacer IgE exon4- exonM2 75 27N5 AY386696
70873 86059 Spacer IgE exon4- IgA4 exon1 76 27N5 AY386696 86362
86498 Spacer IgA4 exon1- exon2 77 27N5 AY386696 86876 87059 Spacer
IgA4 exon2- exon3 78 27N5 AY386696 87450 89577 Spacer IgA4 exon3-
exonM 79 27N5 AY386696 87450 103798 Spacer IgA4 exon3- IgA5b exon1
80 27N5 AY386696 104101 104231 Spacer IgA5b exon1- exon2 81 27N5
AY386696 104609 104787 Spacer IgA5b exon2- exon3 82 27N5 AY386696
105182 107227 Spacer IgA5b exon3- exonM 83 27N5 AY386696 105182
112077 Spacer IgA5b exon3- exonM 84 27N5 AY386696 105182 119223
Spacer IgA5b exon3- IgA1 exon1 85 27N5 AY386696 119511 119644
Spacer IgA1 exon1- exon2 86 27N5 AY386696 119984 120162 Spacer IgA1
exon2- exon3 87 27N5 AY386696 120557 122823 Spacer IgA1 exon3-
exonM 88 27N5 AY386696 120557 127750 Spacer IgA1 exon3- exonM* 89
27N5 AY386696 120557 135838 Spacer IgA1 exon3- IgA2 exon1 90 27N5
AY386696 136138 136274 Spacer IgA2 exon1- exon2 91 27N5 AY386696
136652 136831 Spacer IgA2 exon2- exon3 92 27N5 AY386696 137229
139433 Spacer IgA2 exon3- exonM 93 27N5 AY386696 137229 146676
Spacer IgA2 exon3- 3' end 94 Fos15B AY386698 1 828 Spacer 5'
start-IgA exonM 95 Fos15B AY386698 1043 3596 Spacer IgA exonM- end
contig1 96 Fos15B AY386698 1 3596 Spacer 5' start-end contig1 97
Fos15B AY386698 7404 7541 Spacer IgA exon1- exon2 98 Fos15B
AY386698 7908 8086 Spacer IgA exon2- exon3 99 Fos15B AY386698 8481
10538 Spacer IgA exon3- exonM 100 Fos15B AY386698 8481 13140 Spacer
IgA exon3- end contig2 101 Fos15B AY386698 8481 15871 Spacer IgA
exon3- 1,2 hs 3' enh 102 Fos15B AY386698 8481 21447 Spacer IgA
exon3- 4hs enh 103 Fos15B AY386698 21484 33297 Spacer 4hs enh-end
contig4 104 Fos15B AY386698 16633 33297 Spacer 1,2hs 3' enh- end
contig4 105 179L1 AY495827 1 124285 Spacer 5' end-enh 106 179L1
AY495827 125411 131350 Enhancer-C.quadrature. 107 179L1 AY495827
131664 134637 Spacer C.quadrature.-J5 108 179L1 AY495827 134684
134915 Spacer J5-J4 109 179L1 AY495827 134952 135196 Spacer J4-J3
110 179L1 AY495827 135241 135485 Spacer J3-J2 111 179L1 AY495827
135525 135863 Spacer J2-J1 112 179L1 AY495827 135897 155257 Spacer
J1-V1 113 179L1 AY495827 155572 170621 Spacer V1-V2 114 179L1
AY495827 170936 173443 Spacer V2-V3 115 179L1 AY495827 173752
177227 Spacer V3-V4 116 179L1 AY495827 177536 185356 Spacer V4-V5
117 179L1 AY495827 185664 200758 Spacer V5-V6 118 179L1 AY495827
201064 203580 Spacer V6-V7 119 179L1 AY495827 203886 205144 Spacer
V7-3' end 120 215M22 AY495826 1 12829 Spacer 5' end-V6 121 215M22
AY495826 13136 15653 Spacer V6-V7 122 215M22 AY495826 15957 22241
Spacer V7-V8 123 215M22 AY495826 22551 32876 Spacer V8-V9 124
215M22 AY495826 33188 38276 Spacer V9-V10 125 215M22 AY495826 38582
41476 Spacer V10-V11 126 215M22 AY495826 41780 47827 Spacer V11-V12
127 215M22 AY495826 48133 48547 Spacer V12-V13 128 215M22 AY495826
48841 51408 Spacer V13-V14 129 215M22 AY495826 51638 55438 Spacer
V14-V15 130 215M22 AY495826 55745 67437 Spacer V15-V16 131 215M22
AY495826 67743 77805 Spacer V16-V17 132 215M22 AY495826 78120 80628
Spacer V17-V18 133 215M22 AY495826 80937 84009 Spacer V18-V19 134
215M22 AY495826 84315 87339 Spacer V19-V20 135 215M22 AY495826
87648 89399 Spacer V20-V21 136 215M22 AY495826 89711 95414 Spacer
V21-V22 137 215M22 AY495826 95720 106650 Spacer V22-V23 138 215M22
AY495826 106956 110940 Spacer V23-V24 139 215M22 AY495826 111246
117877 Spacer V24-V25 140 215M22 AY495826 118183 122396 Spacer
V25-V26 141 215M22 AY495826 122706 126496 Spacer V26-V27 142 215M22
AY495826 126802 133358 Spacer V27-V28 143 196O2 AY495828 37134
48826 Spacer V15-V16 144 196O2 AY495828 49032 59195 Spacer V16-V17
145 196O2 AY495828 115057 125885 Spacer V28-V29 146 196O2 AY495828
126195 130012 Spacer V29-V30 147 196O2 AY495828 130318 136966
Spacer V30-V31 148 196O2 AY495828 137272 144512 Spacer V31-V32 149
196O2 AY495828 144819 148617 Spacer V32-V33 150 196O2 AY495828
148923 155402 Spacer V33-V34 151 196O2 AY495828 155714 171415
Spacer V34-V35 152 196O2 AY495828 171572 177676 Spacer V35-V36 153
196O2 AY495828 177979 178083 Spacer V36-3' end 154 CLC* NA 1 443
Spacer 5' end- pV28** 155 CLC* NA 486 1203 Spacer pV28- Pv27** 156
CLC* NA 1528 1635 Spacer pV27- pV26** 157 CLC* NA 1818 2242 Spacer
pV26- pV25** 158 CLC* NA 2585 2676 Spacer pV25- pV24** 159 CLC* NA
2781 3327 Spacer pV24- pV23** 160 CLC* NA 3464 3659 Spacer pV23-
pV22** 161 CLC* NA 3985 4241 Spacer pV22- pV21** 162 CLC* NA 4578
4994 Spacer pV21- pV20** 163 CLC* NA 5366 5425 Spacer pV20- pV19**
164 CLC* NA 5749 5842 Spacer pV19- pV18** 165 CLC* NA 6034 7043
Spacer pV18- pV17** 166 CLC* NA 7266 7493 Spacer pV17- pV16** 167
CLC* NA 7625 7625 Spacer pV16- pV15** 168 CLC* NA 7988 8758 Spacer
pV15- pV14** 169 CLC* NA 9100 9410 Spacer pV14- pV13** 170 CLC* NA
9787 10057 Spacer pV13- pV12** 171 CLC* NA 10441 11022 Spacer pV12-
pV11** 172 CLC* NA 11380 11911 Spacer pV11- pV10** 173 CLC* NA
12162 12349 Spacer pV10-pV9** 174 CLC* NA 12691 13357 Spacer
pV9-pV8** 175 CLC* NA 13708 13882 Spacer pV8-pV7** 176 CLC* NA
14229 14406 Spacer pV7-pV6** 177 CLC* NA 14599 15338 Spacer
pV6-pV5**
178 CLC* NA 15613 16578 Spacer pV5-pV4** 179 CLC* NA 16916 18219
Spacer pV4-pV3** 180 CLC* NA 18439 18879 Spacer pV3-pV2** 181 CLC*
NA 19248 19343 Spacer pV2-pV1** 182 CLC* NA 19609 22208 Spacer
pV1-V** 183 CLC* NA 22506 24313 Spacer V-J 184 CLC* NA 24350 26088
Spacer J-C.quadrature. 185 CLC* NA 26402 36259 Spacer C-3' end
*CLC--Chicken light chain locus SEQ ID 184, FIG. 9 **pV--pseudo V
gene (not functional) Comments: BAC sequences submitted to GenBank
were modified by deletion of vector sequences at the 5' and 3' end
as follows: BAC Accession# Removed from 5' end Removed from 3' end
38A2 AY386694 1-125 1281285-128225 219D23 AY386695 1-54
137357-137389 Fos15B* AY3866968 1-97 33395-33427 179L1 AY495827 0
205145-205968 196O2 AY495828 1-32 178117-178171 *In addition
contigs in GenBank are separated by 50 nt. In the Fos15B sequence
submitted with the provisional application contigs were separated
by 10 nt.
TABLE-US-00002 TABLE 2 ID Region Sequence 193 V.sub.H1
5'CGCGGATCCGAGACTGGGCTGCGCTG3' 194 V.sub.H1
5'CGCAAGCTTGAAATAGGTGGCCGTGTC3' 195 J.sub.H
5'CGCGGATCCAGGCACCCTGGTCACCG3' 196 J.sub.H
5'CGCAAGCTTGTGACCAGGGTGCCCTG3' 197 C.gamma.
5'CGCGGATCCCTGGAGCCGAAGGTCTAC3' 198 C.gamma.
5'CGCAAGCTTGAGATGGACTTCTGCGTG3' 199 3'Enh
5'CGCGGATCCCAGAGTGGGTCTGTGACA3' 200 3'Enh
5'CGCAAGCTTACAGGCGCATGCAAATGC3' 201 V.kappa.
5'CGCGGATCCGAGGCACAGTCACCATC3' 202 V.kappa.
5'CGCAAGCTTACAGTAGTAAGTGGCAGC3' 203 J.kappa.
5'CGCGGATCCGGAGGGACCGAGGTGGT3' 204 J.kappa.
5'CGCAAGCTTACCATGGTCCCTGAGCC3' 205 C.quadrature.
5'CGCGGATCCCCTCAGGTGATCCAGTTG3' 206 C.quadrature.
5'CGCAAGCTTCTATTGAAGCTCTGGACG3' 207 K 3'Enh
5'CGCGGATCCGTGACTGGCCCAAGAAG3' 208 K 3'Enh
5'CGCAAGCTTATACAACCTTGGCCAGG3' 209 C.quadrature.
5'AAACAGCTTTTCACACCTCCCCTTTCTCTCTTTGCTCCCC
TGGGCCCTCAGGGAGTGCATCCGCCCCAACCCTTTTCC3' 210 C.quadrature.
5'CAGGGTTAGTTTGCATGCACACACACACAGCGCCTGGTC
ACCCAGAGGGGTCAGTAGCAGGTGCCAGCTGTGTCGGACATG3' 211 C.quadrature.
5'GGTCAGGGGTCCTCCAGGGCAGGGGTCACATTGTGCCCC
TTCTCTTGCAGCCTCCACCAAGGGCCCATCGGTC3' 212 C.gamma.
5'CACAGCTGCGGCGTGGGGGGGAGGGAGAGGGCAGCTCG
CCGGCACAGCGCTCATTTACCCGGAGACAGGGAGAGGCTCTTC3' 213 J.sub.H
5'GTGTTATAAAGGGAGACTGAGGGGGCAGAGGCTGTGCTA
CTGGTACCTGGCTGAATACTTCCAGCACTGGGGCCAGG3' 214 J.sub.H
5'GGCCACAGAAAAGAGGAGAGAATGAAGGCCCCGGAGAG
GCCGTTCCTACCTGAGGAGACGGTGACCGTGGTCCCT TG-3' 215 Genta
5'CCAGGCCGGCCTGGAGTTGTAGATCCTCTACG3' 216 Genta
5'CCAGGCGCGCCAAGATGCGTGATCTGATCC3' 217 Linker
5'GGCCGCGGCCGGCCATCGATGGCGCGCCTTCGAAACGCGTA3' 218 Linker
5'AGCTTACGCGTTTCGAAGGCGCGCCATCGATGGCCGGCCGC3' 219 pBB11.1
5'ATTCCCAAGCTTTTAATTAAGACGTCAGCTTCCTTAGCTCCTG3' 220 pBB11.1
5'ATTCGCGGATCCACGCGTTTCGTTCCCAAAGGCGCGCCTAGCG ATGAGCTCGGAC3' 221
Neo 5'GCAGGCATGCAAAGCTTATTACACCAGTGTCAGTAAGCG3' 222 Neo
5'GGTACCCGGGGATCCTCAGAAGAACTCGTCAAGAAGGCG3' 223 pBB11.2
5'AAATTCCCTTAATTAAGACGTCAGCTTCCTTAGCTCCTG3' 224 pBB11.2
5'GAAACCGGGGACGCGTTACCGTTCGTATAATGTATGCTATACGAA
GTTATGCGGCCGCTAGCGATGAGCTCGGAC3' 225 CA
5'TTCTCTGTTTTTGTCCGTGGAATGAACAATGGAAGTCCGAGCTCA
TCGCTAAGGGCACCAATAACTGC3' 226 CA
5'CACAGGAGAGAAACAGGACCTAGAGGATGAGGAAGTCCCTGTAG
GCTTCCTACCGTTCGTATAATGTATGCTATACGAAGTTATTACCTGT GACGGAAGATC-3' 227
V.sub.H3-9 5'ATAGAGAGATTGAGTGTG3' 228 V.sub.H3-9
5'TCCTGTCTTCCTGCAG3' 229 V.sub.H3-11 5'AGAGACATTGAGTGGAC3' 230
V.sub.H3-11 5'AGGGAGGTTTGTGTC3' 231 V.sub.H3-13
5'ACTAGAGATATTGAGTGTG3' 232 V.sub.H3-13 5'AGGCATTCTGCAGGG3' 233
V.sub.H3-15 5'ACTAGAGAGATTAAGTGTG3' 234 V.sub.H3-15
5'TCACACTGACCTCCC3' 235 V.sub.H3-20 5'TCATGGATCAATAGAGATG3' 236
V.sub.H3-20 5'TGCAGGGACGTTTGTG3' 237 V.sub.H3-23
5'AGAAAAATTGAGTGTGAA3' 238 V.sub.H3-23 5'GTGTCTGGGCTCACAA3' 239
V.sub.H3-30 5'AGAGAGACTGAGTGTG3' 240 V.sub.H3-30
5'TGCAGGGAGGTTTGTG3' 241 V.sub.H3-43 5'TGAGTGTGAGTGAACATG3' 242
V.sub.H3-43 5'ACCAGCTCTTAACCTTC3' 243 V.sub.H3-64
5'TGAGTGTGAGTGGAC3' 244 V.sub.H3-64 5'TGACGCTGATCAGTG3' 245
V.sub.H3-66 5'TCTGACCAATGTCTCTG3' 246 V.sub.H3-66
5'AGGTTTGTGTCTGGGC3' 247 V.sub.H3-72 5'ACAAGGTGATTTATGGAG3' 248
V.sub.H3-72 5'AGGTTTGTGTCCGGG3' 249 V.sub.H3-9 5'TTGGCGCGCC
TGTCGTCTGTGTTTGCAG GTGTCC3' 250 V.sub.H3-9
5'TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTCA
GCCTGAGGGCCCCTCACTGTGTCATCTTTTGCAC3' 251 V.sub.H3-11 5'TTGGCGCGCC
TGTCGTCTGTGTTTGCAG GTGTCC3 252 V.sub.H3-11
5'TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTCA
GCCTGAGGGCCCCTCACTGTGTCTCTCG3' 253 V.sub.H3-13
5'TTGGCGCGCCTGTCGTCTGTGTTTGCAGGTGTCC3' 254 V.sub.H3-13
5'TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTCA
GCCTGAGGGCCCCTCACTGTGTCTCTTG3' 255 V.sub.H3-15
5'TTGGCGCGCCTGTCGTCTGTGTTTGCAGGTGTCC3' 256 V.sub.H3-15
5'TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTCA
GCCTGAGGGCCCCTCACTGTGTCTGTGG3' 257 V.sub.H3-20 5'TTGGCGCGCC
TGTCGTCTGTGTTTGCAGGTGTC3' 258 V.sub.H3-20
5'TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTC AGCCTGAGGGCCCCTCACTGTGTCTCTC3'
259 V.sub.H3-23 5'TTGGCGCGCCTGTCGTCTGTGTTTGCAG GTGTCCAGTGTG3' 260
V.sub.H3-23 5'TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTCAGCCTGA
GGGCCCCTCACTGTGTCTTTC3' 261 V.sub.H3-30
5'TTGGCGCGCCTGTCGTCTGTGTTTGCAGGTGTCCAGTGTC3' 262 V.sub.H3-30
5'TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTCAGCCTG
AGGGCCCCTCACTGTGTCTTTCG3' 263 V.sub.H3-43
5'TTGGCGCGCCTGTCGTCTGTGTTTGCAGGTGTCC3' 264 V.sub.H3-43
5'TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTCA
GCCTGAGGGCCCCTCACTGTGTCTCTTTTGCAC3' 265 V.sub.H3-64
5'TTGGCGCGCCTGTCGTCTGTGTTTGCAGGTGTCC3' 266 V.sub.H3-64
5'TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTCA
GCCTGAGGGCCCCTCACTGTGTCTCTCGCAC3' 267 V.sub.H3-66
5'TTGGCGCGCCTGTCGTCTGTGTTTGCAGGTGTCC3' 268 V.sub.H3-66
5'TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTCA GCCTGAGGGCCCCTCACTGTGTCTCCG3'
269 V.sub.H3-72 5'TTGGCGCGCCTGTCGTCTGTGTTTGCAG GTTTCC3' 270
V.sub.H3-72 5'TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTCA
GCCTGAGGGCCCCTCACTGTGTCTCTAGCAC3' 271 V1-2
5'TTGGCGCGCCAGGGGAGTGCGGCTCCAC3' 272 V1-2 5'TTGCACGCGT
TGGTCAGGACACTGTCACTCAC3' 273 V2-3 5'TTGGCGCGCCAGGGGCGCGCGGCTCCAC3'
274 V2-3 5'TTGCACGCGTTGATCACGAAACTGTCACTCACACTCTC3' 275 V3-4
5'TTGGCGCGCCAGGGGCGCGCGGCTCCAC3' 276 V3-4
5'TTGCACGCGTTCTGTTGGTCTCTTCTTCTCTTGCTATAAC3' 277 V4-5
5'TTGGCGCGCCAGGGGAGTGCGGCTCCAC3' 278 V4-5
5'TTGCACGCGTTGGTCAAGACACTGTCACTCAC3 279 V5-6
5'TTGGCGCGCCAGGGACGCACGGCTCCAC3' 280 V5-6
5'TTGCACGCGTTGGTCAGGAAGCTGTCACTCAC3' 281 V6-7
5'TTGGCGCGCCAGGGATGCGCGGCTCCAG3' 282 V6-7
5'TTGCACGCGTTGGTCAGGACACTGTCACTGACAC3' 283 V7-8 5'TT
GGCGCGCCAGGGGAGTGCGGCTCCAC3' 284 V7-8
5'TTGCACGCGTTGGTCAGGAAGCTGTCACTCACTCTC3' 285 V21-22
5'TTGGCGCGCCGGGGCCCGCGGCTCCAC3' 286 V21-22
5'TTGCACGCGTTGGTCAGGAAGCTGTCAC3' 287 V22-23
5'TTGGCGCGCCAGGGACGTGAGGCTCTAC3' 288 V22-23
5'TTGCACGCGTTGGTCAGGGCACTGTCAC3' 289 Linker
5'GGCCGCGGCCGGCCATCGATGGCGCGCC TTCGAAACGCGTA3' 290 Linker
3'CGCCGGCCGGTAGCTACCGCGCGGAAGCTT TGCGCATTCGA5' 291 Linker 5'CGG CCG
GCC ATC GAT GGC GCG CCT TCG AAA CGC GTG GTA C3' 292 Linker 3'TCG
AGC CGG CCG GTA GCT ACC GCG CGG AAG CTT TGC GCA C5' 293 Genta
5'CCAGGCCGGCCTGGAGTTGTAGATCCTCTACG3' 294 Genta
5'CCAGGCGCGCCAAGATGCGTGATCTGATCC-3' 295 Neo
5'CCAGGCCGGCCATTACACCAGTGTCAGTAAGCG3' 296 Neo
5'CCAGGCGCGCCTCAGAAGAACTCGTCAAGAAGGCG3' 297 Linker 5'GAT CCG GCC
GGC CAT CGA TGG CGC GCC TTC GAA ACG CGT TAG GGA TAA CAG GGT AAT A3'
298 Linker 3'GCC GGC CGG TAG CTA CCG CGC GGA AGC TTT GCG CAA TCC
CTA TTG TC CCA TTA TCGA5' 299 Neo
5'ATCTGCACTCAGTGCGTCTTGAGCGCCCCCTGGTAGAGCCG CGCGACCCT GGCGCGCC
ATTACACCAGTGTCAGTAAGCG3' 300 Neo
5'AAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTC TGTGCGAGA GGCCGGCC
TCAGAAGAACTCGTCAAGAAGGCG3' 301 C.kappa. Km3
5'GATGTCCACTGGTACCTAAGCCTCGCCCTCTGTGCTTCTTCCCTC
CTCAGGAACTGTGGCTGCACCATCTGTCTTC3'
302 C.kappa. Km3 5'GAGGCTGGGCCTCAGGGTCGCTGGCGGTGCCCTGGCAGGCGTC
TCGCTCTAACACTCTCCCCTGTTGAAGCTCTTTGTG3 303 Neo
5'CTTTCTCTGTCCTTCCTGTGCGACGGTTACGCCGCTCCATGAGCTT
ATCGTAACTATAACGGTCCTAAGGTAGCGATGGACAGCAAGCGAA CCGGA3' 304 Neo
5'GGACCAGTTTACAATCCCACCTGCCATCTAAGAAAGCTGGTCTCA
TCGTGTCAGAAGAACTCGTCAAGAAG3' 305 Zeo
5'CCCCCCCCGCCACTTCTCTTCTGTTTCGTTTAAGTTCTACACTGAC
ATACTAGGGATAACAGGGTAATAACGTTTACAATTTCGCCTGATG3 306 Zeo
5'AGTGGGTAGGCCTGGCGGCCGCCTGGCCGTCGACATTTAGGTGA
CACTATAGAAGGATCCTAGCACGTGTCAGTCCTGCT3' 307 Genta
5'TTACGCCAAGCTATTTAGGTGACACTATAGAATACTCAAGCTTTG
ATTGCTAACTATAACGGTCCTAAGGTAGCGATGAAGGCACGAACCC AGTTG3' 308 Genta
5'GCGGAATTCTATGTCTAGTGGAGGGTGAAGCTGGTGATTATAGA
GTGAAAATTACCCTGTTATCCCTATCGGCTTGAACGAATTGTTAG3' 309 VJ
5'CATAAATATACTGTCTTCCAGGATCTTAGAGCTCACCTAAGGAAA
CAAGAGTTCATTTGAAGTTTTTAAAGTG3' 310 VJ
5'ACTCCAGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAAT
AGGAACTTCCTTTGATCTCCACCTTGGTC3' 311 Genta
5'GAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAAC
TTCTGGAGTTGTAGATCCTCTACG3' 312 Genta
5'AAAACAAACCAATCAGGCAGAAACGGTGAGGAATCAGTGAAAC
GGCCACTTACGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGG
AATAGGAACTTCAAGATGCGTGATCTGATCC3' 313 FRT 5'TTATGCTGCATCCAGTTTGC3'
314 FRT 5'AAAACAAACCAATCAGGCAG3' 315 FRT 5'TGTGACATCCAGATGAC3' 316
FRT 5'AAAACAAACCAATCAGGCAG3' 317 Genta
5'GGACCAGTTTACAATCCCACCTGCCATCTAAGAAAGCTGGTCTCA
TCGTGGTGCCAGGGCGTGCCCTTGGGCTGGGGGCGCGATAACTTCG
TATAGCATACATTATACGAAGTTATCGATCGTGGAGTTGTAGATCC TCTACG3' 318 Genta
5'TTACGCCAAGCTATTTAGGTGACACTATAGAATACTCAAGCTTTG
ATTGCAAGATGCGTGATCTGATCCT3' 319 Linker
5'CGGGATCCGCGCGTACGGAAGTTCCTATACCTTTTGAAGAATAGG
AACTTCGGAATAGGAACTTCATTACACCAGTGTCAGTAAGCG3' 320 Linker
5'GGGAAGCTTCGCGCGATCGCCGCTTTCGCAAAGGCGCGCCTCAG
AAGAACTCGTCAAGAAGGCG3' 321 Genta
5'GGCGGCCGCCTGGCCGTCGACATTTAGGTGACACTATAGAAGGA
TCCGCGTGGAGTTGTAGATCCTCTACG3' 322 Genta
5'AACTCAGTAAGGAAAAGGACTGGGAAAGTGCACTTACATTTGAT
CTCCAGGCGCGCCAAGATGCGTGATCTGATCC3' 323 Neo
5'GGACCAGTTTACAATCCCACCTGCCATCTAAGAAAGCTGGTCTCA
TCGTGGTGCCAGGGCGTGCCCTTGGGCTGGGGGCGCGGAAGTTCCT
ATTCCGAAGTTCCTATTCTTCAAAAGGTATAGGAACTTCCGTACGA
TTACACCAGTGTCAGTAAGCG3' 324 Neo
5'GGACTGATGGGAAAATAGAGGAGAAAATTGACCAGAGGAAGTG
CAGATGGTCAGAAGAACTCGTCAAGAAGGCG3' 325 RSS
5'AACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTA
CCCCTTCCACAGTGATACAAGCCC3' 326 RSS
5'TGCCGGCCACGATGCGTCCGGCGTAGAGGATCTACAACTCCAGG
CGCGCCTGGTCATGTCAGTGCTGCTGC3' 327 Genta
5'CTCCTTTCCTCCTCCTTGGTGGCAGCAGCACTGACATGACCAGGC GCGCC
TGGAGTTGTAGATCCTCTACG3' 328 Genta
5'TGTAATACGACTCACTATAGGGCGAATTCGAGCTCGGTACCCGGG
GATCCCGTACGAAGATGCGTGATCTGATCC3' 329 Kana
5'GGCGGCCGCCTGGCCGTCGACATTTAGGTGACACTATAGAAGGA
TCCGCGACCCTGTTATCCCTAGATTTAAATGATATCGG3' 330 Kana
5'AACTTTCTCCTACAGATCCCAGATAACCATGAATTTATTACACCA
TCTTGGGCGCGCCGAAGTTCCTATACTTTCTAGAGAATAGGAACTT
CGGAATAGGAACTTCAGTTGGTGATTTTGAACTTTTGCTTTGCC3' 331 Amp
5'GGACCAGTTTACAATCCCACCTGCCATCTAAGAAAGCTGGTCTCA
TCGTGGTGCCAGGGCGTGCCCTTGGGCTGGGGGCGCGGCGATCGCG
AAGTTCCTATTCCGAAGTTCCTATTCTTCAAAAGGTATAGGAACTTC
TACGGGGTCTGACGCTCAG3' 332 Amp
5'GAATTCAGAGCTCAATGAGTTGCCTTGTTCAGAGCTCTATTTTCA
CTTGACGTACGACAGACAAGCTGTGACCGTC3' 333 J Region
5'GAGTTAGGCCTCAGAGCTGAGGCAGGGCTCGGTTCCCCTTGGGTG
AGAAGGGTTTCTGTTCAGCAAGAC3' 334 J Region
5'TGGCCAATTAGAGCAAAATTTCAGACAGTAATAGGAAAAAGGTA
CTTACGTTTAATCTCCAGTCGTGTC3' 335 O2 5'TCAGTACTGACTGGAAC3' 336 O2
5'CCAATGACTTTCAAAACC3' 337 L8 5'CCGTACAGCCTGGCTC3' 338 L8
5'AACACCATCAGAGTGTGC 339 L4 5'ATGATTAATTGTGTGGACC3' 340 L4
5'AGGTGATCTCATATCCTC3' 341 A30 5'CTCAGTACTGCTTTACTG3' 342 A30
5'TGACTTCATGTCCCCTTC3' 343 L11 5'ACATGATTAATTGTGTGGACC3' 344 L11
5'GGTGCAGAGGTGACTTCG3' 345 L1 5'CTCAGTACTGCTTTACTATTC3' 346 L1
5'GAGGAACACTCTCAGCTG3' 347 L5 5'CAGGGAACTTCTCTTACAG3' 348 L5
5'GAATTAGGGTGCAGAGGC3' 349 L15 5'TACTATTCAGGGAAATTC3' 350 L15
5'TGTCTGTGAAGTTGGTG3' 351 O8 5'TGGCTCTTGATGGAAGC3' 352 O8
5'ACTTCAAAGTGTGACTGC3' 353 L19 5'AGGGAACTTCTCTTACAGC3' 354 L19
5'AATTAGGGTGCAGAGGCG3' 355 L12 5'GAAGTCTTCCTATAATATGATC3' 356 L12
5'TGGCTGCATCTGAGGACC3' 357 A20 5'GCCACTAATGCCTGGCAC3' 358 A20
5'CTGCTGTCAGCAGAGGGC3' 359 O4 5'CTTCTTATAACATGATGG3' 360 O4
5'AAACGCTCTGAGCAGC3' 361 L14 5'CTCAGTACTGCTTTACTG3' 362 L14
5'GAGGAACAATCTCAGCCG3' 363 L23 5'AGCCAGGCTGTACGGAAC3' 364 L23
5CCCAGCCTCACACATCTC3' 365 L9 5'TGGCCCTTCAGGGAAG3' 366 L9
5'ACCATCAGAGTGTGGTTG3' 367 A4 5'CCAGTGTAGCCATTAATG3' 368 A4
5'TACCAAAACTTCCCAGGG3' 369 L24 5'GGGAAATTCTCTTACTAC3 370 L24
5'CCCCCTCTACCAATAC3' 371 O6 5'CCATTCAGGGAAGTCTTC3' 372 O6
5'TGAGTCTGAGAAGTGTTG3' 373 L22 5'GGAATTTTCTTAGCCCAC3' 374 L22
5'ATGTTCAGGCTTGTAACC3' 375 A9 5'TCATCTTACAAATAGTTG3' 376 A9
5'TCTGACCATTCCTGC3' 377 A25 5'GGGAAATCATCTTATAAATAG3' 378 A25
5'TGCAGATGAGACTTCTGG3' 379 A15 5'ATTCAGGAAAGTCCTCTC3' 380 A15
5'CAGTGACCTTCAGAGTG3' 381 O9 5'ATTCAGGAAAGTCCTCTC3' 382 O9
5'CAGTGACCTTCAGAGTG3' 383 O2
5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGACATC 384 O2
5'CGCACGCGTGTTTGATCTCCACCTTGGTCCCTCCGCCGAAAGTGA
GAGGGGTACTGTAACTCTGTTG3' 385 L8
5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGACATC 386 L8
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTAACTATTAAG3'
387 L4 5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGCCATC 388 L4
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTAACTATTAAAC3'
389 A30 5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGGTGTGACATC3' 7 390 A30
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTAACTATTATGC3'
391 L11 5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGCCATC3' 392 L11
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTAATTGTAATC3'
393 L1 5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGACATC3' 394 L1
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTAACTATTATAC3'
395 L5 5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTTCCAGATGCGACATC3' 396 L5
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGAAACTGTTAG3'
397 L15 5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGACATC 398 L15
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTAACTATTATAC3'
399 O8 5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGACATC 400 O8
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGAGATTATCATAC3'
401 L19 5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTTCCAGATGCGACATC3' 402 L19
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG
GAGGGAAACTGTTAG3' 403 L12
5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAAATGTGACATC3' 404 L12
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGAATAACTATTATAC3'
405 A20 5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGATACCAGATGTGACATCC3' 406 A20
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGGCACTGTTATAC3'
407 O4 5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGACATC3' 408 O4
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGGCATTGTAAG3'
409 L14 5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTAACATCC3' 410 L14
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTAACTATTATGC3'
411 L23 5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGCCATC3' 412 L23
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGGTACTATAATAC3'
413 L9 5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGCCATC3' 414 L9
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTAACTATAATAC3'
415 A4 5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGATACCAGATGTGACATCC3' 416 A4
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGGCACTGTTATAC3'
417 L24 5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGTCATC3' 418 L24
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGAAACTATAATAC3'
419 O6 5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGGACCAGAAGTGACATC3' 420 O6
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTAATTTTTATAC3'
421 L22 5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGTCAGATTTGACATCC3' 422 L22
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTAACTGAAGTC3'
423 A9 5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGAGTCAGATGTGATTTCC3' 424 A9
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GATGGCTGCTGTAAG3'
425 A25 5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGAGTCAGATGTGATTTC C3' 426 A25
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GATGGCTGCTGTAAG3'
427 A15 5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATATGACATGC3' 428 A15
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTCACTTTTATAC3'
429 O9 5'TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATATGACATGC3' 430 O9
5'CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTCACTTTTATAC3'
431 V7-8 5'TTGGCGCGCC GGAGGAAACAGAAACACAG3' 432 V7-8 5'CGCACGCGT
CAGCTGCTCGTCCTGGG3' 433 V11-10 5'TTGGCGCGCC GGAGGGAAACAGAAACAC3'
434 V11-10 5'CGCACGCGTAGCTGCTCCTCCTGGG3' 435 V15-14 5'TTGGCGCGCC
GAAGGGAAACAGAAACACAG3' 436 V15-14 5'CGCACGCGTAGCTGCTCCTCCTGGG3' 437
V18-17 5'TTGGCGCGCC GAGGGAAACAGAAACAC3' 438 V18-17
5'CGCACGCGTCAGCTGCTGCTCCTGGG3' 439 V19-18 5'TTGGCGCGCC
GAAGGGAAACAGAAACACAG3' 440 V19-18 5'CGCACGCGT AGCTGCTCCTCCTGGG3'
441 V20-19 5'TTGGCGCGCC GAGGAGGGAAACAGAAACAC3' 442 V20-19
5'CGCACGCGTCAGCTGCCCCTCCTGGG3' 443 V21-20 5'TTGGCGCGCC
GGAGGAAACAGAAACACAG3' 444 V21-20 5'CGCACGCGTCCCTAGCTGCTCCTGGG3' 445
V24-23 5'TTGGCGCGCC GGAGGGAAACAGACACAC3' 446 V24-23
5'CGCACGCGTCAGCTGCTCCTCCTGGC3' 447 V26-25 5'TTGGCGCGCC
GAAGGGAAAGAGAAACACAG3' 448 V26-25 5'CGCACGCGTAGCTGCTCCTCCTGGG3' 449
V27-26 5'TTGGCGCGCC GGAGGGAAACAGAAACAC3' 450 V27-26
5'CGCACGCGTCCCAGCTGCTCCTGGG3' 451 Genta
5'AGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGC
ATGCAAGCTTGGCCGGCCTGGAGTTGTAGATCCTCTACG3' 452 Genta
5'AAAACAAACCAATCAGGCAGAAACGGTGAGGAATCAGT
GAAACGGCCACTTACGGCGCGCCAAGATGCGTGATCTGATCC3' 453 Hygro
5'CGTTGGACCAGTTTACAATCCCACCTGCCATCTAAGAAAGC
TGGTCTCATATAACTTCGTATAATGTATGCTATACGAACGGTA
ACGCGTGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGT
ATAGGAACTTCTCAGAGCAGATTGTACTG3' 454 Hygro
5'GGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCT
GGTAAGGTTAAGATGCGTGATCTGATCC3' 455
5'ACTGCACCTCAGCGTCCCCCTGCCCATGTCAGGGCCGATGAA
GGGCACAGCGTACGATTACACCAGTGTCAGTAAGCG3' 456
5'TGTAATACGACTCACTATAGGGCGAATTGAGCTCGGTACCCG
GGGATCCTGCGATCGCTCAGAAGAACTCGTCAAGAAGGCG3' 457 J.sub.H
5'GTGTTATAAAGGGAGACTGAGGGAGGCAGAGGCTGTGCTA
CTGGTACCTGGCTGAATACTTCCAGCACTGGGGCCAGG3' 458 J.sub.H
5'GGCCACAGAAAAGAGGAGAGAATGAAGGCCCCGGAGAGG
CCGTTCCTACCTGAGGAGACGGTGACCGTGGTCCCTTG3' 459 Spacer
5'AACAACCTCAGGGCTGAGGACACC3' 460 Spacer
5'CTGCCCGTTGTCCCTCGAGATGGTGGCACGGCC3'
SEQUENCE LISTINGS
0 SQTB SEQUENCE LISTING The patent contains a lengthy "Sequence
Listing" section. A copy of the "Sequence Listing" is available in
electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=USRE047131E1)-
. An electronic copy of the "Sequence Listing" will also be
available from the USPTO upon request and payment of the fee set
forth in 37 CFR 1.19(b)(3).
* * * * *
References