Note: Descriptions are shown in the official language in which they were submitted.
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1
TRANSGENIC NON-HUMAN ANIMALS CAPABLE OF PRODUCING
HETEROLOGOUS ANTIBODIES
10
TECHNICAL FIELD
The invention relates to transgenic non-human
animals capable of producing heterologous antibodies,
transgenes used to produce such transgenic animals,
transgenes capable of functionally rearranging a heterologous
D gene in V-D-J recombination, immortalized B-cells capable of
producing heterologous antibodies, methods and transgenes for
producing heterologous antibodies of multiple isotypes,
methods and transgenes for producing heterologous antibodies
wherein a variable region sequence comprises somatic mutation
as compared to germline rearranged variable region sequences,
transgenic nonhuman animals which produce antibodies having a
human primary sequence and which bind to human antigens,
hybridomas made from B cells of such transgenic animals, and
monclonal antibodies expressed by such hybridomas.
BACKGROUND OF THE INVENTION
One of the major impediments facing the development
of in vivo therapeutic and diagnostic applications for
monoclonal antibodies in humans is the intrinsic
immunogenicity of non-human immunoglobulins. For example, when
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2161351 2
immunocompetent human patients are administered therapeutic
doses of rodent monoclonal antibodies, the patients produce
antibodies against the rodent immunoglobulin sequences; these
human anti-mouse antibodies (HAMA) neutralize the therapeutic
antibodies and can cause acute toxicity. Hence, it is
desirable to produce human immunoglobulins that are reactive
with specific human antigens that are promising therapeutic
and/or diagnostic targets. However, producing human
immunoglobulins that bind specifically with human antigens is
problematic.
The present technology for generating monoclonal
antibodies involves pre-exposing, or priming, an animal
(usually a rat or mouse) with antigen, harvesting B-cells from
that animal, and generating a library of hybridoma clones. By
screening a hybridoma population for antigen binding
specificity (idiotype) and also screening for immunoglobulin
class (isotype), it is possible to select hybridoma clones
that secrete the desired antibody.
However, when present methods for generating
monoclonal antibodies are applied for the purpose of
generating human antibodies that have binding specificities
for human antigens, obtaining B-lymphocytes which produce
human immunoglobulins a serious obstacle, since humans will
typically not make immune responses against self-antigens.
Hence, present methods of generating human
monoclonal antibodies that are specifically reactive with
human antigens are clearly insufficient. It is evident that
the same limitations on generating monoclonal antibodies to
authentic self antigens apply where non-human species are used
as the source of B-cells for making the hybridoma.
The construction of transgenic animals harboring a
functional heterologous immunoglobulin transgene are a method
by which antibodies reactive with self antigens may be
produced. However, in order to obtain expression of
therapeutically useful antibodies, or hybridoma clones
producing such antibodies, the transgenic animal must produce
transgenic B cells that are capable of maturing through the B
lymphocyte development pathway. Such maturation requires the
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presence of surface IgM on the transgenic B cells, however
isotypes other than IgM are desired for therapeutic uses.
Thus, there is a need for transgenes and animals harboring
such transgenes that are able to undergo functional V-D-J
y 5 rearrangement to generate recombinational diversity and
junctional diversity. Further, such transgenes and transgenic
animals preferably include cis-acting sequences that
facilitate isotype switching from a first isotype that is
required for B cell maturation to a subsequent isotype that
has superior therapeutic utility.
A number of experiments have reported the use of
transfected cell lines to determine the specific DNA sequences
required for Ig gene rearrangement (reviewed by Lewis and
Gellert (1989), Cell, 59, 585-588). Such reports have
identified putative sequences and concluded that the
accessibility of these sequences to the recombinase enzymes
used for rearrangement is modulated by transcription
(Yancopoulos and Alt (1985), Cell, 40, 271-281). The
sequences for V(D)J joining are reportedly a highly conserved,
near-palindromic heptamer and a less well conserved AT-rich
nanomer separated by a spacer of either 12 or 23 bp (Tonegawa
(1983), Nature, 302, 575-581; Hesse, et al. (1989), Genes in
Dev., 3, 1053-1061). Efficient recombination reportedly
occurs only between sites containing recombination signal
sequences with different length spacer regions.
Ig gene rearrangement, though studied in tissue
culture cells, has not been extensively examined in transgenic
mice. Only a handful of reports have been published
describing rearrangement test constructs introduced into mice
[Buchini, et al. (1987), Nature, 326, 409-411 (unrearranged
chicken X transgene); Goodhart, et al. (1987) , Proc. Natl.
Acad. Sci. USA, 84, 4229-4233) (unrearranged rabbit K gene);
and Bruggemann, et al. (1989), Proc. Natl. Acad. Sci. USA, 86,
6709-6713 (hybrid mouse-human heavy chain)]. The results of
such experiments, however, have been variable, in some cases,
producing incomplete or minimal rearrangement of the
transgene.
WO 94/25585 PCT/US94/04580
k Further, a variety of biological functions of
antibody molecules are exerted by the Fc portion of molecules,
such as the interaction with mast cells or basophils through
Fcc, and binding of complement by Fcg or Fcy, it further is
desirable to generate a functional diversity of antibodies of
a given specificity by variation of isotype.
Although transgenic animals have been generated that
incorporate transgenes encoding one or more chains of a
heterologous antibody, there have been no reports of
heterologous transgenes that undergo successful isotype
switching. Transgenic animals that cannot switch isotypes are
limited to producing heterologous antibodies of a single
isotype, and more specifically are limited to producing an
isotype that is essential for B cell maturation, such as IgM
and possibly IgD, which may be of limited therapeutic utility.
Thus, there is a need for heterologous immunoglobulin
transgenes and transgenic animals that are capable of
switching from an isotype needed for B cell development to an
isotype that has a desired characteristic for therapeutic use.
Based on the foregoing, it is clear that a need
exists for methods of efficiently producing heterologous
antibodies, e.g. antibodies encoded by genetic sequences of a
first species that are produced in a second species. More
particularly, there is a need in the art for heterologous
immunoglobulin transgenes and transgenic animals that are
capable of undergoing functional V-D-J gene rearrangement that
incorporates all or a portion of a D gene segment which
contributes to recombinational diversity. Further, there is a
need in the art for transgenes and transgenic animals that can
support V-D-J recombination and isotype switching so that (1)
functional B cell development may occur, and (2)
therapeutically useful heterologous antibodies may be
produced. There is also a need for a source of B cells which
can be used to make hybridomas that produce monoclonal
antibodies for therapeutic or diagnostic use in the particular
species for which they are designed. A heterologous
immunoglobulin transgene capable of functional V-D-J
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recombination and/or capable of isotype switching could
fulfill these needs.
In accordance with the foregoing object transgenic
nonhuman animals are provided which are capable of producing a
5 heterologous antibody, such as a human antibody.
= Further, it is an object to provide B-cells from
such transgenic animals which are capable of expressing
heterologous antibodies wherein such B-cells are immortalized
to provide a source of a monoclonal antibody specific for a
particular antigen.
In accordance with this foregoing object, it is a
further object of the invention to provide hybridoma cells
that are capable of producing such heterologous monoclonal
antibodies.
Still further, it is an object herein to provide
heterologous unrearranged and rearranged immunoglobulin-heavy
and light chain transgenes useful for producing the
aforementioned non-human transgenic animals.
Still further, it is an object herein to provide
methods to disrupt endogenous immunoglobulin loci in the
transgenic animals.
Still further, it is an object herein to provide
methods to induce heterologous antibody production in the
aforementioned transgenic non-human animal.
A further object of the invention is to provide
methods to generate an immunoglobulin variable region gene
segment repertoire that is used to construct one or more
transgenes of the invention.
The references discussed herein are provided solely
for their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an
admission that the inventors are not entitled to antedate such
disclosure by virtue of prior invention.
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5a
SUMMARY OF THE INVENTION
Various embodiments of this invention provide a method
of making a transgenic non-human animal comprising: (a)
functionally disrupting both endogenous heavy chain alleles;
(b) functionally disrupting both endogenous light chain
alleles; (c) introducing into the genome of the non-human
animal at least one copy of a heterologous immunoglobulin
heavy chain transgene; and (d) introducing into the genome
of the non-human animal at least one copy of a heterologous
immunoglobulin kappa light chain transgene; wherein said
transgenic non-human animal makes a heterologous antibody
response following immunization with an antigen.
Various other embodiments of this invention provide a
non-human B-cell derived from a transgenic non-human animal
made according to this invention, and immunized with a
predetermined antigen.
Various other embodiments of this invention provide
a hybridoma prepared from the non-human B-cell of this
invention, wherein the hybridoma produces a monoclonal
antibody comprising a heterologous immunoglobulin heavy chain
and a heterologous immunoglobulin light chain.
Various other embodiments of this invention provide the
use of a hybridoma of this invention or produced by a method
of this invention, to produce a monoclonal antibody. Some of
these embodiments provide a human monoclonal antibody
produced by a hybridoma of this invention.
Various other embodiments of this invention provide a
method of producing a heterologous immunoglobulin from a
transgenic non-human animal, the method comprising immunizing
a transgenic non-human animal made according to the method of
this invention, with an antigen to induce the production of
said heterologous immunoglobulin. These embodiments may
further comprise a step of immortalizing at least one of the
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5b
B-cells producing the immunoglobulin. Such immortalizing may be
by fusion of the B-cell with a myeloma cell line to form a
hybridoma.
Various embodiments of this invention provide a method of
making a transgenic mouse comprising: (a) functionally
disrupting both endogenous heavy chain alleles; (b) functionally
disrupting both endogenous light chain alleles; (c) introducing
into the genome of the mouse at least one copy of a heterologous
immunoglobulin heavy chain transgene; and (d) introducing into
the genome of the mouse at least one copy of a heterologous
immunoglobulin kappa light chain transgene; wherein said
transgenic mouse makes a heterologous antibody response
following immunization with an antigen. The invention also
provides a B lymphocyte derived from such a transgenic mouse
immunized with a predetermined antigen as well as a hybridoma
prepared from the lymphocyte, wherein the hybridoma produces a
monoclonal antibody comprising a heterologous immunoglobulin
heavy chain and a heterologous immunoglobulin light chain. Also
included are human monoclonal antibodies produced by such a
hybridoma. Also provided is a method of producing a
heterologous immunoglobulin from a transgenic mouse, the method
comprising immunizing such a transgenic mouse with an antigen to
induce the production of said heterologous immunoglobulin. Also
provided is the use of the aforementioned hybridoma to produce a
monoclonal antibody.
Various embodiments of this invention provide a mouse B
lymphocyte, comprising: (a) both endogenous heavy chain alleles
functionally disrupted; (b) both endogenous light chain alleles
functionally disrupted; (c) a genome with at least one copy of a
heterologous immunoglobulin heavy chain transgene; and (d) a
genome with at least one copy of a heterologous immunoglobulin
kappa light chain transgene; said mouse B lymphocyte capable of
making a heterologous antibody to a predetermined antigen.
Various embodiments of this invention provide a method of
making a transgenic mouse, comprising: introducing into the
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genome of the mouse an unrearranged human heavy chain
immunoglobulin variable region, wherein the variable region
comprises a plurality of human VH gene segments, a plurality of
human D gene segments, and a plurality of human JH gene segments,
and wherein B lymphocytes of the transgenic mouse express a
chimeric antibody comprising a human immunoglobulin heavy chain
variable region and mouse immunoglobulin heavy chain constant
region.
Various embodiments of this invention provide a method of
making a transgenic mouse, comprising: introducing into the
genome of the mouse an unrearranged human light chain
immunoglobulin variable region, wherein the variable region
comprises a plurality of human VL gene segments and a plurality
of human JL gene segments, wherein B lymphocytes of the
transgenic mouse express a chimeric antibody comprising a human
immunoglobulin light chain variable region and mouse
immunoglobulin light chain constant region.
Also included are hybridomas comprising a B lymphocyte of a
transgenic mouse as described above, fused to a second cell
capable of immortalizing the B lymphocyte, wherein the hybridoma
produces a chimeric monoclonal antibody that specifically binds
to a pre-determined antigen. Also included are mouse B
lymphocytes derived from a transgenic mouse as described above,
immunized with a predetermined antigen.
Various embodiments of this invention provide a method for
obtaining a chimeric antibody that binds to a predetermined
antigen, comprising: (a) immunizing a transgenic mouse as
described above with the predetermined antigen; and (b)
obtaining from the transgenic mouse a chimeric antibody that
binds to the predetermined antigen.
various embodiments of this invention provide a method of
making a transgenic mouse, comprising: introducing into the
genome of the mouse an unrearranged human heavy chain
immunoglobulin variable region, wherein the variable region
comprises multiple human VH gene segments, multiple human D gene
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5d
segments, multiple human JH gene segments, and wherein B
lymphocytes of the transgenic mouse express an antibody
comprising a human immunoglobulin heavy chain variable region.
Various embodiments of this invention provide a method of
making a transgenic mouse comprising, introducing into the
genome of the mouse an unrearranged human light chain
immunoglobulin variable region, wherein the variable region
comprises multiple human VL gene segments, multiple human JL gene
segments, and wherein B lymphocytes of the transgenic mouse
express an antibody comprising a human immunoglobulin light
chain variable region.
Various embodiments of this invention provide a method for
preparing a human antibody, which method comprises culturing a
host cell comprising: (i) a human immunoglobulin heavy chain
recombinant construct comprising a nucleic acid molecule that
encodes a human heavy chain variable region from a mouse B
lymphocyte operably linked to a human heavy chain constant
region, and (ii) a human immunoglobulin light chain recombinant
construct comprising a nucleic acid molecule that encodes a
human light chain variable region from a mouse B lymphocyte
operably linked to a human light chain constant region, wherein
the mouse B lymphocyte is from a transgenic mouse having an
unrearranged human heavy chain immunoglobulin variable region
comprising a plurality of human VH gene segments, a plurality of
human D gene segments and a plurality of human JH gene segments,
and having an unrearranged human light chain immunoglobulin
variable region comprising a plurality of human VL gene segments
and a plurality of human JL gene segments; under conditions that
permit expression of the human heavy chain and human light chain
to form an antibody.
Various embodiments of this invention provide a method for
constructing a human heavy chain, which method comprises
operably linking a nucleic acid molecule encoding a human heavy
chain variable region to a nucleic acid molecule encoding a
human heavy chain constant region, wherein the human heavy chain
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variable region is isolated from a mouse B lymphocyte, wherein
the mouse B lymphocyte is from a transgenic mouse having an
unrearranged human heavy chain immunoglobulin variable region
comprising a plurality of human VH gene segments, a plurality of
human D gene segments, and a plurality of human JH gene segments.
Various embodiments of this invention provide a method for
constructing a human light chain, which method comprises
operably linking a nucleic acid molecule encoding a human light
chain variable region to a nucleic acid molecule encoding a
human light chain constant region, wherein the human light chain
variable region is isolated from a mouse B lymphocyte, wherein
the mouse comprises an unrearranged human light chain
immunoglobulin variable region comprising a plurality of human VL
gene segments and a plurality of human JL gene segments. Also
included is a method for preparing a human antibody, which
method comprises expressing a human heavy chain construct
prepared according to the preceding method with a human light
chain construct as described above, in a host cell under
conditions that permit expression of the human heavy chain and
the human light chain to form a human antibody.
Transgenic nonhuman animals are provided which are capable
of producing a heterologous antibody, such as a human antibody.
Such heterologous antibodies may be of various
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isotypes, including: IgG1, IgG2, IgG3, IgG4, IgM, IgAl, IgA2,
IgAsec, IgD, of IgE. In order for such transgenic nonhuman
animals to make an immune response, it is necessary for the
transgenic B cells and pre-B cells to produce surface-bound
immunoglobulin, particularly of the IgM (or possibly IgD)
isotype, in order to effectuate B cell development and
antigen-stimulated maturation. Such expression of an IgM (or
IgD) surface-bound immunoglobulin is only required during the
antigen-stimulated maturation phase of B cell development, and
mature B cells may produce other isotypes, although only a
single switched isotype may be produced at a time.
Typically, a cell of the B-cell lineage will produce
only a single isotype at a time, although cis or trans
alternative RNA splicing, such as occurs naturally with the As
(secreted ) and M (membrane-bound A) forms, and the and S
immunoglobulin chains, may lead to'the contemporaneous
expression of multiple isotypes by a single cell. Therefore,
in order to produce heterologous antibodies of multiple
isotypes, specifically the therapeutically useful IgG, IgA,
and IgE isotypes, it is necessary that isotype switching
occur. Such isotype switching may be classical class-
switching or may result from one or more non-classical isotype
switching mechanisms.
The invention provides heterologous immunoglobulin
transgenes and transgenic nonhuman animals harboring such
transgenes, wherein the transgenic animal is capable of
producing heterologous antibodies of multiple isotypes by
undergoing isotype switching. Classical isotype switching
occurs by recombination events which involve at least one
switch sequence region in the transgene. Non-classical
isotype switching may occur by, for example, homologous
recombination between human aA and human E sequences (6-
associated deletion). Alternative non-classical switching
mechanisms, such as intertransgene and/or interchromosomal
recombination, among others, may occur and effectuate isotype
switching. Such transgenes and transgenic nonhuman animals
produce a first immunoglobulin isotype that is necessary for
antigen-stimulated B cell maturation and can switch to encode
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and produce one or more subsequent heterologous isotypes that
have therapeutic and/or diagnostic utility. Transgenic
nonhuman animals of the invention are thus able to produce, in
one embodiment, IgG, IgA, and/or IgE antibodies that are
5 encoded by human immunoglobulin genetic sequences and which
also bind specific human antigens with high affinity.
= The invention also encompasses B-cells from such
transgenic animals that are capable of expressing heterologous
antibodies of various isotypes, wherein such B-cells are
10 immortalized to provide a source of a monoclonal antibody
specific for a particular antigen. Hybridoma cells that are
derived from such B-cells can serve as one source of such
heterologous monoclonal antibodies.
The invention provides heterologous unrearranged and
rearranged immunoglobulin heavy and light chain transgenes
capable of undergoing isotype switching in vivo in the
aforementioned non-human transgenic animals or in explanted
lymphocytes of the B-cell lineage from such transgenic
animals. Such isotype switching may occur spontaneously or be
induced by treatment of the transgenic animal or explanted B-
lineage lymphocytes with agents that promote isotype
switching, such as T-cell-derived lymphokines (e.g., IL-4 and
IFNY).
Still further, the invention includes methods to
induce heterologous antibody production in the aforementioned
transgenic non-human animal, wherein such antibodies may be of
various isotypes. These methods include producing an antigen-
stimulated immune response in a transgenic nonhuman animal for
the generation of heterologous antibodies, particularly
heterologous antibodies of a switched isotype (i.e., IgG, IgA,
and IgE).
This invention provides methods whereby the
transgene contains sequences that effectuate isotype
switching, so that the heterologous immunoglobulins produced
in the transgenic animal and monoclonal antibody clones
derived from the B-cells of said animal may be of various
isotypes.
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L N. This invention further provides methods that
facilitate isotype switching of the transgene, so that
switching between particular isotypes may occur at much higher
or lower frequencies or in different temporal orders than
typically occurs in germline immunoglobulin loci. Switch
regions may be grafted from various CH genes and ligated to
other CH genes in a transgene construct; such grafted switch
sequences will typically function independently of the
associated CH gene so that switching in the transgene
construct will typically be a function of the origin of the
associated switch regions. Alternatively, or in combination
with switch sequences, 6-associated deletion sequences may be
linked to various CH genes to effect non-classical switching
by deletion of sequences between two 6-associated deletion
sequences. Thus, a transgene may be constructed so that a
particular CH gene is linked to a different switch sequence
and thereby is switched to more frequently than occurs when
the naturally associated switch region is used.
This invention also provides methods to determine
whether isotype switching of transgene sequences has occurred
in a transgenic animal containing an immunoglobulin transgene.
The invention provides immunoglobulin transgene
constructs and methods for producing immunoglobulin transgene
constructs, some of which contain a subset of germline
immunoglobulin loci sequences (which may include deletions).
The invention includes a specific method for facilitated
cloning and construction of immunoglobulin transgenes,
involving a vector that employs unique XhoI and Sall
restriction sites flanked by two unique NotI sites. This
method exploits the complementary termini of XhoI and Sall
restrictions sites and is useful for creating large constructs
by ordered concatemerization of restriction fragments in a
vector.
The transgenes of the invention include a heavy
chain transgene comprising DNA encoding at least one variable
gene segment, one diversity gene segment, one joining gene
segment and one constant region gene segment. The
immunoglobulin light chain transgene comprises DNA encoding at
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least one variable gene segment, one joining gene segment and
one constant region gene segment. The gene segments encoding
the light and heavy chain gene segments are heterologous to
the transgenic non-human animal in that they are derived from,
or correspond to, DNA encoding immunoglobulin heavy and light
chain gene segments from a species not consisting of the
transgenic non-human animal. In one aspect of the invention,
the transgene is constructed such that the individual gene
segments are unrearranged, i.e., not rearranged so as to
encode a functional immunoglobulin light or heavy chain. Such
unrearranged transgenes permit recombination of the gene
segments (functional rearrangement) and expression of the
resultant rearranged immunoglobulin heavy and/or light chains
within the transgenic non-human animal when said animal is
exposed to antigen.
In one aspect of the invention, heterologous heavy
and light immunoglobulin transgenes comprise relatively large
fragments of unrearranged heterologous DNA. Such fragments
typically comprise a substantial portion of the C, J (and in
the case of heavy chain, D) segments from a heterologous
immunoglobulin locus. In addition, such fragments also
comprise a substantial portion of the variable gene segments.
In one embodiment, such transgene constructs
comprise regulatory sequences, e.g. promoters, enhancers,
class switch regions, recombination signals and the like,
corresponding to sequences derived from the heterologous DNA.
Alternatively, such regulatory sequences may be incorporated
into the transgene from the same or a related species of the
non-human animal used in the invention. For example, human
immunoglobulin gene segments may be combined in a transgene
with a rodent immunoglobulin enhancer sequence for use in a
transgenic mouse.
In a method of the invention, a transgenic non-human
animal containing germline unrearranged light and heavy
immunoglobulin transgenes - that undergo VDJ joining during
D-cell differentiation - is contacted with an antigen to
induce production of a heterologous antibody in a secondary
repertoire B-cell.
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Also included in the invention are vectors and
methods to disrupt the endogenous immunoglobulin loci in the
non-human animal to be used in the invention. Such vectors
and methods utilize a transgene, preferably positive-negative
5 selection vector, which is constructed such that it targets
the functional disruption of a class of gene segments encoding
a heavy and/or light immunoglobulin chain endogenous to the
non-human animal used in the invention. Such endogenous gene
segments include diversity, joining and constant region gene
10 segments. In this aspect of the invention, the
positive-negative selection vector is contacted with at least
one embryonic stem cell of a non-human animal after which
cells are selected wherein the positive-negative selection
vector has integrated into the genome of the non-human animal
by way of homologous recombination. After transplantation,
the resultant transgenic non-human animal is substantially
incapable of mounting an immunoglobulin-mediated immune
response as a result of homologous integration of the vector
into chromosomal DNA. Such immune deficient non-human animals
may thereafter be used for study of immune deficiencies or
used as the recipient of heterologous immunoglobulin heavy and
light chain transgenes.
The invention also provides vectors, methods, and
compositions useful for suppressing the expression of one or
more species of immunoglobulin chain(s), without disrupting an
endogenous immunoglobulin locus. Such methods are useful for
suppressing expression of one or more endogenous
immunoglobulin chains while permitting the expression of one
or more transgene-encoded immunoglobulin chains. Unlike
genetic disruption of an endogenous immunoglobulin chain
locus, suppression of immunoglobulin chain expression does not
require the time-consuming breeding that is needed to
establish transgenic animals homozygous for a disrupted
endogenous Ig locus. An additional advantage of suppression
as compared to engognous Ig gene disruption is that, in
certain embodiments, chain suppression is reversible within an
individual animal. For example, Ig chain suppression may be
accomplished with: (1) transgenes encoding and expressing
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antisense RNA that specifically hybridizes to an endogenous Ig
chain gene sequence, (2) antisense oligonucleotides that
specifically hybridize to an endogenous Ig chain gene
sequence, and (3) immunoglobulins that bind specifically to an
endogenous Ig chain polypeptide.
The invention provides transgenic non-human animals
comprising: a homozygous pair of functionally disrupted
endogenous heavy chain alleles, a homozygous pair of
functionally disrupted endogenous light chain alleles, at
least one copy of a heterologous immunoglobulin heavy chain
transgene, and at least one copy of a heterologous
immunoglobulin heavy chain transgene, wherein said animal
makes an antibody response following immunization with an
antigen, such as a human antigen (e.g., CD4). The invention
also provides such a transgenic non-human animal wherein said
functionally disrupted endogenous heavy chain allele is a JH
region homologous recombination knockout, said functionally
disrupted endogenous light chain allele is a J. region
homologous recombination knockout, said heterologous
immunoglobulin heavy chain transgene is the HC1 or HC2 human
minigene transgene, said heterologous light chain transgene
is the KC2 or KCle human K transgene, and wherein said antigen
is a human antigen.
The invention also provides various embodiments for
suppressing, ablating, and/or functionally disrupting the
endogenous nonhuman immunoglobulin loci.
The invention also provides transgenic mice
expressing both human sequence heavy chains and chimeric heavy
chains comprising a human sequence heavy chain variable region
and a murine sequence heavy chain constant region. Such
chimeric heavy chains are generally produced by trans-
switching between a functionally rearranged human transgene
and an endogenous murine heavy chain constant region (e.g.,
71, 72a, 72b, y3). Antibodies comprising such chimeric heavy
chains, typically in combination with a transgene-encoded
human sequence light chain or endogenous murine light chain,
are formed in response to immunization with a predetermined
antigen. The transgenic mice of these embodiments can
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comprise B cells which produce (express) a human sequence
heavy chain at a first timepoint and trans-switch to produce
(express) a chimeric heavy chain composed of a human variable
region and a murine constant region (e.g., yl, y2a, y2b, y3)
at a second (subsequent) timepoint; such human sequence and
chimeric heavy chains are incorporated into functional
antibodies with light chains; such antibodies are present in
the serum of such transgenic mice. This, to restate: the
transgenic mice of these embodiments can comprise B cells
which express a human sequence heavy chain and subsequently
switch (via trans-switching or cis-switching) to express a
chimeric or isotype-switched heavy chain composed of a human
variable region and an alternative constant region (e.g.,
murine yl, y2a, y2b, y3; human y, (x, E) ; such human sequence
and chimeric or isotype-switched heavy chains are
incorporated into functional antibodies with light chains
(human or mouse); such antibodies are present in the serum of
such transgenic mice.
In one aspect, the invention provides a method for
making a transgenic mouse that produces chimeric IgG
comprising: (a) inactivating the endogenous murine heavy
chain variable gene regions; (b) introducing into the mouse
genome, an unrearranged human heavy chain immunoglobulin
variable region operably linked to a mu constant region gene
segment wherein the unrearranged human heavy chain variable
region comprises multiple human VH gene segments, multiple
human D gene segments, and multiple human JH gene segments,
and wherein the mu constant region is selected from the group
consisting of (i) a human S-mu sequence and a human or mouse
mu coding sequence and (ii) a murine S-mu sequence and a
human or mouse mu coding sequence, and (c) identifying a
chimeric IgG antibody produced by the mouse comprising a
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12a
human immunoglobulin heavy chain variable region and mouse
immunoglobulin heavy chain constant region.
In another aspect, the invention provides a mouse
B lymphocyte derived from a transgenic mouse made by the
method as described above, and immunized with a predetermined
antigen.
In another aspect, the invention provides a
hybridoma, comprising a B lymphocyte of a transgenic mouse
made by the method as described above, fused to a second cell
capable of immortalizing the B lymphocyte, wherein the
hybridoma produces a chimeric monoclonal antibody that
specifically binds to the predetermined antigen.
In another aspect, the invention provides a method
for obtaining a chimeric antibody that binds to a
predetermined antigen, comprising: (a) immunizing a
transgenic mouse prepared according to the method as
described above with the predetermined antigen; and (b)
obtaining from the transgenic mouse a chimeric antibody that
binds to the predetermined antigen.
In another aspect, the invention provides a
B lymphocyte from a mouse prepared according to the method as
described above, or a cell derived therefrom, wherein the
B lymphocyte or cell derived therefrom expresses an antibody
comprising a heavy chain consisting of a human immunoglobulin
heavy chain variable region and a mouse immunoglobulin heavy
chain y constant region.
In another aspect, the invention provides a method
for preparing a human antibody, which method comprises
culturing a host cell comprising: (i) a human immunoglobulin
heavy chain recombinant construct comprising a nucleic acid
molecule that encodes a human heavy chain variable region
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from a mouse B lymphocyte, and (ii) a human immunoglobulin
light chain recombinant construct comprising a nucleic acid
molecule that encodes a human light chain variable region
from a mouse B lymphocyte, wherein the mouse B lymphocyte is
from a transgenic mouse prepared by (a) inactivating the
endogenous murine heavy chain variable gene regions;
(b) introducing into the mouse genome, an unrearranged human
heavy chain immunoglobulin variable region operably linked
to a mu constant region gene segment, wherein the
unrearranged human heavy chain variable region comprises
multiple human VH gene segments, multiple human D gene
segments, and multiple human JH gene segments, and wherein
the mu constant region is selected from the group consisting
of a human S-mu sequence and a human or mouse mu coding
sequence and a murine S-mu sequence and a human or mouse mu
coding sequence, and (c) identifying a chimeric IgG antibody
produced by the mouse comprising a human immunoglobulin
heavy chain variable region and mouse immunoglobulin heavy
chain constant region.
In another aspect, the invention provides a method
for constructing a human heavy chain, which method comprises
operably linking a nucleic acid molecule encoding a human
heavy chain variable region to a nucleic acid molecule
encoding a human heavy chain constant region, wherein the
human heavy chain variable region is isolated from a mouse
B lymphocyte, wherein the mouse B lymphocyte is from a
transgenic mouse prepared by (a) inactivating the endogenous
murine heavy chain variable gene regions; (b) introducing
into the mouse genome, an unrearranged human heavy chain
immunoglobulin variable region operably linked to a
mu constant region gene segment, wherein the unrearranged
human heavy chain variable region comprises multiple human VH
gene segments, multiple human D gene segments, and multiple
CA 02161351 2009-03-20
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12c
human JH gene segments, and wherein the mu constant region is
selected from the group consisting of a human S-mu sequence
and a human or mouse mu coding sequence and a murine
S-mu sequence and a human or mouse mu coding sequence, and
(c) identifying a chimeric IgG antibody produced by the mouse
comprising a human immunoglobulin heavy chain variable region
and mouse immunoglobulin heavy chain constant region.
In another aspect, the invention provides a method
for obtaining a somatically mutated antibody that binds to a
predetermined antigen, which method comprises: (a) immunizing
a transgenic mouse with a predetermined antigen, wherein the
transgenic mouse has a genome comprising inactivated
endogenous murine heavy chain variable gene regions, and
further comprising an unrearranged human heavy chain
immunoglobulin variable region operably linked to a
mu constant region gene segment, wherein the unrearranged
human heavy chain variable region comprises multiple human VH
gene segments, multiple human D gene segments, and multiple
human JH gene segments, and wherein the mu constant region is
selected from the group consisting of (i) a human S-mu
sequence and a human or mouse mu coding sequence and (ii) a
murine S-mu sequence and a human or mouse mu coding sequence,
(b) removing B lymphocytes from the transgenic mouse,
(c) identifying mouse IgG isotype immunoglobulin molecules
that bind to the predetermined antigen expressed by the
B lymphocytes; and (d) isolating the IgG isotype
immunoglobulin molecules that bind to the predetermined
antigen, whereby a somatically mutated antibody that binds to
the predetermined antigen is obtained from the B lymphocytes
of step (c).
CA 02161351 2009-03-20
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12d
The references discussed herein are provided solely
for their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an
admission that the inventors are not entitled to antedate such
disclosure by virtue of prior invention.
BRIEF DESCRIPTION OF. THE FIGURES
Fig. 1 depicts the complementarity determining
regions CDR1, CDR2 and CDR3 and framework regions FR1, FR2,
FR3 and FR4 in unrearranged genomic DNA and mRNA expressed
from a rearranged immunoglobulin heavy chain gene,
Fig. 2 depicts the human X. chain locus,
Fig. 3 depicts the human K chain locus,
Fig. 4 depicts the human heavy chain locus,
Fig."5 depicts a transgene construct containing a
rearranged IgM gene ligated to a 25 kb fragment that contains
human 73 and 71 constant regions followed by a 700 bp fragment
containing the rat chain 3' enhancer sequence.
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Fig. 6 is a restriction map of the human K chain
locus depicting the fragments to be used to form a light chain
transgene by way of in vivo homologous recombination.
Fig. 7 depicts the construction of pGP1.
= 5 Fig. 8 depicts the construction of the polylinker
contained in pGP1.
Fig. 9 depicts the fragments used to construct a
human heavy chain transgene of the invention.
Fig. 10 depicts the construction of pHIG1 and pCON1.
Fig. 11 depicts the human Cyl fragments which are
inserted into pRE3 (rat enhancer 3') to form pREG2.
Fig. 12 depicts the construction of pHIG3' and PCON.
Fig. 13 depicts the fragment containing human D
region segments used in construction of the transgenes of the
invention.
Fig. 14 depicts the construction of pHIG2 (D segment
containing plasmid).
Fig. 15 depicts the fragments covering the human JK
and human CK gene segments used in constructing a transgene of
the invention.
Fig. 16 depicts the structure of pE .
Fig. 17 depicts the construction of pKapH.
Figs. 18A through 18D depict the construction of a
positive-negative selection vector for functionally disrupting
the endogenous heavy chain immunoglobulin locus of mouse.
Figs. 19A through 19C depict the construction of a
positive-negative selection vector for functionally disrupting
the endogenous immunoglobulin light chain loci in mouse.
Figs. 20A through 20E depict the structure of a
kappa light chain targeting vector.
Figs. 21A through 21F depict the structure of a
mouse heavy chain targeting vector.
Fig. 22 depicts the map of vector pGPe.
Fig. 23 depicts the structure of vector pJM2.
Fig. 24 depicts the structure of vector pCOR1.
Fig. 25 depicts the transgene constructs for pIGM1,
pHC1 and pHC2.
Fig. 26 depicts the structure of pye2.
WO 94.2585 } 1 3 l PCT/US94/04580
14 to
Fig. 27 depicts the structure of pVGE1.
Fig. 28 depicts the assay results of human Ig
expression in a pHC1 transgenic mouse.
Fig. 29 depicts the structure of pJCK1.
Fig. 30 depicts the construction of a synthetic
heavy chain variable region.
Fig. 31 is a schematic representation of the heavy
chain minilocus constructs pIGM1, pHC1, and pHC2.
Fig. 32 is a schematic representation of the heavy
chain minilocus construct pIGG1 and the K light chain
minilocus construct pKC1, pKVel, and pKC2.
Fig. 33 depicts a scheme to reconstruct functionally
rearranged light chain genes.
Fig. 34 depicts serum ELISA results
Fig. 35 depicts the results of an ELISA assay of
serum from 8 transgenic mice.
Fig. 36 is a schematic representation of plasmid
pBCE1.
Figs. 37A-37C depict the immune response of
transgenic mice of the present invention against KLH-DNP, by
measuring IgG and IgM levels specific for KLH-DNP (37A), KLH
(37B) and BSA-DNP (37C).
Fig. 38 shows ELISA data demonstrating the presence
of antibodies that bind human carcinoembryonic antigen (CEA)
and comprise human g chains; each panel shows reciprocal
serial dilutions from pooled serum samples obtained from mice
on the indicated day following immunization.
Fig. 39 shows ELISA data demonstrating the presence
of antibodies that bind human carcinoembryonic antigen (CEA)
and comprise human y chains; each panel shows reciprocal
serial dilutions from pooled serum samples obtained from mice
on the indicated day following immunization.
Fig. 40 shows aligned variable region sequences of
23 randomly-chosen cDNAs generated from mRNA obtained from
lymphoid tissue of HC1 transgenic mice immunized with human
carcinoembryonic antigen (CEA) as compared to the germline
transgene sequence (top line); on each line nucleotide changes
relative to germline sequence are shown above the alteration
WO 94/25585 PCT/US94/04580
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in deduced amino acid sequence (if any); the regions
corresponding to heavy chain CDR1, CDR2, and CDR3 are
indicated. Non-germline encoded nucleotides are shown in
capital letters. Germline VH251 and JH are shown in lower case
letters. Deduced amino acid changes are given beneath
nucleotide sequences using the conventional single-letter
notation.
Fig. 41 show the nucleotide sequence of a human DNA
fragment, designated vk65.3, containing a V. gene segment; the
deduced amino acid sequences of the V. coding regions are also
shown; splicing and recombination signal sequences
(heptamer/nonamer) are shown boxed.
Fig. 42 show the nucleotide sequence of a human DNA
fragment, designated vk65.5, containing a V. gene segment; the
deduced amino acid sequences of the V. coding regions are also
shown; splicing and recombination signal sequences
(heptamer/nonamer) are shown boxed.
Fig. 43 show the nucleotide sequence of a human DNA
fragment, designated vk65.8, containing a V. gene segment; the
deduced amino acid sequences of the V. coding regions are also
shown; splicing and recombination signal sequences
(heptamer/nonamer) are shown boxed.
Fig. 44 show the nucleotide sequence of a human DNA
fragment, designated vk65.15, containing a VK gene segment;
the deduced amino acid sequences of the V. coding regions are
also shown; splicing and recombination signal sequences
(heptamer/nonamer) are shown boxed.
Fig. 45 shows formation of a light chain minilocus
by homologous recombination between two overlapping fragments
which were co-injected.
Fig. 46 shows ELISA results for monoclonal
antibodies reactive with CEA and non-CEA antigens showing the
specificity of antigen binding.
Fig. 47 shows the DNA sequences of 10 cDNAs
amplified by PCR to amplify transcripts having a human VDJ and
a murine constant region sequence.
Fig. 48 shows ELISA results for various dilutions of
serum obtained from mice bearing both a human heavy chain
WO 94/25585 PCT/US94/04580
16
minilocus transgene and a human K minilocus transgene; the
mouse was immunized with human CD4 and the data shown
represents antibodies reactive with human CD4 and possessing
human K, human g, or human y epitopes, respectively.
Fig. 49 shows relative distribution of lymphocytes
staining for human or mouse g as determined by FACS for
three mouse genotypes.
Fig. 50 shows relative distribution of lymphocytes
staining for human K or mouse K as determined by FACS for
three mouse genotypes.
Fig. 51 shows relative distribution of lymphocytes
staining for mouse X as determined by FACS for three mouse
genotypes.
Fig. 52 shows relative distribution of lymphocytes
staining for mouse X or human K as determined by FACS for four
mouse genotypes.
Fig. 53 shows the amounts of human , human y, human
K, mouse , mouse y, mouse K, and mouse X chains in the serum
of unimmunized 0011 mice.
Fig. 54 shows a scatter plot showing the amounts of
human , human y, human K, mouse A, mouse y, mouse K, and
mouse X chains in the serum of unimmunized 0011 mice of
various genotypes.
Fig. 55 shows the titres of'antibodies comprising
human , human y, or human K chains in anti-CD4 antibodies in
the serum taken at three weeks or seven weeks post-
immunization following immunization of a 0011 mouse with human
CD4.
Fig. 56 shows a schematic representation of the
human heavy chain minilocus transgenes pHC1 and pHC2, and the
light chain minilocus transgenes pKC1, pKCle, and the light
chain minilocus transgene created by homologous recombination
between pKC2 and Co4 at the site indicated.
Fig. 57 shows a linkage map of the murine lambda
light chain locus as taken from Storb et al. (1989) op.cit.;
the stippled boxes represent a pseudogene.
WO 94/25585 PCT/US94/04580
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Fig. 58 shows a schematic representation of
inactivation of the murine X locus by homologous gene
targeting.
Fig. 59 schematically shows the structure of a
homologous recombination targeting transgene for deleting
genes, such as heavy chain constant region genes.
Fig. 60 shows a map of the BALB/c murine heavy chain
locus as taken from Immunoglobulin Genes, Honjo, T, Alt, FW,
and Rabbits TH (eds.) Academic Press, NY (1989) p. 129.
Structural genes are shown by closed boxes in the top line;
second and third lines show restriction sites with symbols
indicated.
Fig. 61 shows a nucleotide sequence of mouse heavy
chain locus a constant. region gene.
Fig. 62 shows the construction of a frameshift
vector (plasmid B) for introducing a two bp frameshift into
the murine heavy chain locus J4 gene.
Fig. 63 shows isotype specific response of
transgenic animals during hyperimmunization. The relative
levels of reactive human and 71 are indicated by a
colorimetric ELISA assay (y-axis). We immunized three 7-10
week old male HC1 line 57 transgenic animals (#1991, #2356,
#2357), in a homozygous JHD background, by intraperitoneal
injections of CEA in Freund's adjuvant. The figure depicts
binding of 250 fold dilutions of pooled serum (collected prior
to each injection) to CEA coated microtiter wells.
Fig. 64A and 64B show expression of transgene
encoded yl isotype mediated by class switch recombination.
The genomic structure of integrated transgenes in two
different human 71 expressing hybridomas is consistent with
recombination between the and 71 switch regions. Fig. 64A
shows a Southern blot of Pacl/Sfii digested DNA isolated from
three transgene expressing hybridomas. From left to right:
clone 92-09A-5H1-5, human y1+/ -; clone 92-90A-4G2-2, human
71+//f; clone 92-09A-4F7-A5-2, human y1-, +. All three
hybridomas are derived from a 7 month old male mouse
hemizygous for the HC1-57 integration, and homozygous for the
JHD disruption (mouse #1991). The blot is hybridized with a
WO 94/25585 PCT/US94/04580
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probe derived from a 2.3 kb BglII/SfiI DNA fragment spanning
the 3' half of the human yl switch region. No switch product
is found in the expressing hybridoma, while the two 71
expressing hybridomas, 92-09A-5H1-5 and 92-09A-4G2-2, contain
switch products resulting in Pacl/SfiI fragments of 5.1 and
5.3 kb respectively, Fig. 64B is a diagram of two possible
deletional mechanisms by which a class switch from g to 71 can
occur. The human gene is flanked by 400 bp direct repeats
(ai and E ) which can recombine to delete A. Class switching
by this mechanism will always generate a 6.4 kb PacI/SfiI
fragment, while class switching by recombination between the
and the 71 switch regions will generate a Pacl/SfiI fragment
between 4 and 7 kb, with size variation between individual
switch events. The two yl expressing hybridomas examined in
Fig. 64A appear to have undergone recombination between the
and yi switch regions.
Fig. 65 shows chimeric human/mouse immunoglobulin
heavy chains generated by trans-switching. cDNA clones of
trans-switch products were generated by reverse transcription
and PCR amplification of a mixture of spleen and lymph node
RNA isolated from a hyperimmunized HC1 transgenic-JHD mouse
(#2357; see legend to Fig. 63 for description of animal and
immunization schedule). The partial nucleotide sequence of 10
randomly picked clones is shown. Lower case letters indicate
germline encoded, capital letters indicate nucleotides that
cannot be assigned to known germline sequences; these may be
somatic mutations, N nucleotides, or truncated D segments.
Both face type indicates mouse y sequences.
Figs. 66A and 66B show that the rearranged VH251
transgene undergoes somatic mutation in a hyperimmunized. The
partial nucleotide sequence of IgG heavy chain variable region
cDNA clones from CH1 line 26 mice exhibiting Fig. 66A primary
and Fig. 66B secondary responses to antigen. Germline
sequence is shown at the top; nucleotide changes from germline
are given for each clone. A period indicates identity with
germline sequence, capital letters indicate no identified
germline origin. The sequences are grouped according to J
segment usage. The germline sequence of each of the J
WO 94/25585 PCT/US94/04580
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segments if shown. Lower case letters within CDR3 sequences
indicate identity to known D segment included in the HC1
transgene. The assigned D segments are indicated at the end
of each sequence. Unassigned sequences could be derived from
N region addition or somatic mutation; or in some cases they
are simply too short to distinguish random N nucleotides from
known D segments. Fig. 66A primary response: 13 randomly
picked VH251-71 cDNA clones. A 4 week old female HC1 line
26-JHD mouse (#2599) was given a single injection of KLH and
complete Freunds adjuvant; spleen cell RNA was isolated 5 days
later. The overall frequency of somatic mutations within the
V segment is 0.06% (2/3,198 bp). Fig. 66B secondary response:
13 randomly picked VH251-y1 cDNA clones. A 2 month old female
HC1 line 26-JHD mouse (#3204) was given 3 injections of HEL
and Freunds adjuvant over one month (a primary injection with
complete adjuvant and boosts with incomplete at one week and 3
weeks); spleen and lymph node RNA was isolated 4 months later.
The overall frequency of somatic mutations within the V
segment is 1.6% (52/3,198 bp).
Figs. 67A and 67B show that extensive somatic
mutation is confined to 71 sequences: somatic mutation and
class switching occur within the same population of B cells.
Partial nucleotide sequence of VH251 cDNA clones isolated from
spleen and lymph node cells of HC1 line 57 transgenic-JHD
mouse (#2357) hyperimmunized against CEA (see Fig. 63 for
immunization schedule). Fig. 67A: IgM: 23 randomly picked
VH251-g cDNA clones. Nucleotide sequence of 156 bp segment
including CDRs 1 and 2 surrounding residues. The overall
level of somatic mutation is 0.1% (5/3,744 bp). Fig 67B: IgG:
23 randomly picked VH251-71 cDNA clones. Nucleotide sequence
of segment including CDRs 1 through 3 and surrounding
residues. The overall frequency of somatic mutation within
the V segment is 1.1% (65/5,658 bp). For comparison with the
p sequences in Fig. 67A: the mutation frequency for first
156 nucleotides is 1.1% (41/3,588 bp). See legend to
Figs. 66A and 66B for explanation of symbols.
Fig. 68 indicates that VH51P1 and VH56P1 show
extensive somatic mutation of in an unimmunized mouse. The
WO 94/25585 PCT/US94/04580
partial nucleotide sequence of IgG heavy chain variable region
cDNA clones from a 9 week old, unimmunized female HC2 line
2550 transgenic-JHD mouse (#5250). The overall frequency of
somatic mutation with the 19 VH56p1 segments is 2.2%
5 (101/4,674 bp). The overall frequency of somatic mutation
within the single VH51p1 segment is 2.0% (5/246 bp). See
legend to Figs. 66A and 66B for explanation of symbols.
Fig. 69. Double transgenic mice with disrupted
endogenous Ig loci contain human IgMrc positive B cells. FACS
10 of cells isolated from spleens of 4 mice with different
genotypes. Left column: control mouse (#9944, 6 wk old
female JH+/-, JCrc+/-; heterozygous wild-type mouse heavy and
K-light chain loci, non-transgenic). Second column: human
heavy chain transgenic.(#9877, 6 wk old female JH-/-, JCK-/-,
15 HC2 line 2550 +; homozygous for disrupted mouse heavy and rc-
light chain loci, hemizygous for HC2 transgene). Third
column: human K-light chain transgenic (#9878, 6 wk old
female JH-/-, JCrc-/-, KCo4 line 4437 +; homozygous for
disrupted mouse heavy and K-light chain loci, hemizygous for
20 KCo4 transgene). Right column: double transgenic (#9879, 6
wk old female JH-/-m JCK-/-, HC2 line 2550 +, KCo4 line 4437
+; homozygous for disrupted mouse heavy and ak-light chain
loci, hemizygous for HC2 and KCo4 transgenes). Top row:
spleen cells stained for expression of mouse X light chain (x-
axis) and human K light chain (y-axis). Second row: spleen
cells stained for expression of human A heavy chain (x-axis)
and human K light chain (y-axis). Third row: spleen cells
stained for expression of mouse A heavy chain (x-axis) and
mouse K light chain (y-axis). Bottom row: histogram of
spleen cells stained for expression of mouse B220 antigen (log
fluorescence: x-axis; cell number: y-axis). For each of the
two color panels, the relative number of cells in each of the
displayed quadrants is given as percent of a e-parameter gate
based on propidium iodide staining and light scatter. The
fraction of B220+ cells in each of the samples displayed in
the bottom row is given as a percent of the lymphocyte light
scatter gate.
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Fig. 70. Secreted immunoglobulin levels in the
serum of double transgenic mice. Human g, y, and K, and mouse
y and X from 18 individual HC2/KCo4 double transgenic mice
homozygous for endogenous heavy and K-light chain locus
disruption. Mice: (+) HC2 line 2550 ('5 copies of HC2 per
integration), KCo4 line 4436 (1-2 copies of KCo4 per
integration); (0) HC2 line 2550, KCo4 line 4437 ("10 copies of
KCo4 per integration); (x) HC2 line 2550, KCo4 line 4583 (-5
copies of KCo4 per integration); (0) HC2 line 2572 (30-50
copies of HC2 per integration, KCo4 line 4437; (n) HC2 line
5467 (20-30 copies of HC2 per integration, KCo4 line 4437.
Figs. 71A and 71B show human antibody responses to
human antigens. Fig. 71A: Primary response to recombinant
human soluble CD4. Levels of human IgM and human K light
chain are reported for prebleed (0) and post-immunization (=)
serum from four double transgenic mice. Fig. 71B: Switching to
human IgG occurs in vivo. Human IgG (circles) was detected
with peroxidase conjugated polyclonal anti-human IgG used in
the presence of 1.5 /ml excess IgE, K and 1% normal mouse
serum to inhibit non-specific cross-reactivity. Human K light
chain (squares) was detected using a peroxidase conjugated
polyclonal anti-human K reagent in the presence of 1% normal
mouse serum. A representative result from one mouse (#9344;
HC2 line 2550, KCo4 line 4436) is shown. Each point
represents an average of duplicate wells minus background
absorbance.
Fig. 72 shows FACS analysis of human PBL with a
hybridoma supernatant that discriminates human CD4+
lymphocytes from human CD8+ lymphocytes.
Fig. 73 shows human a-CD4 IgM anf IgG in transgenic
mouse serum.
Fig. 74 shows competition binding experiments
comparing a transgenic mouse a-human CD4 hybridoma monoclonal,
2C11-8, to the RPA-TA and Leu-3A monoclonals.
Fig. 75 shows production data for Ig expression of
cultured 2C11-8 hybridoma.
Table 1 depicts the sequence of vector pGPe.
Table 2 depicts the sequence of gene VH49.8.
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7
Table 3 depicts the detection of human IgM and IgG
in the serum of transgenic mice of this invention.
Table 4 depicts sequences of VDJ joints.
Table 5 depicts the distribution of J segments
incorporated into pHC1 transgene encoded transcripts to J
segments found in adult human peripheral blood lymphocytes
(PBL).
Table 6 depicts the distribution of D segments
incorporated into pHC1 transgene encoded transcripts to D
segments found in adult human peripheral blood lymphocytes
(PBL).
Table 7 depicts the length of the CDR3 peptides from
transcripts with in-frame VDJ joints in the pHC1 transgenic
mouse and in human PBL.
Table 8 depicts the predicted amino acid sequences
of the VDJ regions from 30 clones analyzed from a pHC1
transgenic.
Table 9 shows transgenic mice of line 112 that were
used in the indicated experiments; (+) indicates the presence
of the respective transgene, (++) indicates that the animal is
homozygous for the JHD knockout transgene.
Table 10 shows the genotypes of several 0011 mice.
Table 11 shows transgene V and J segment usage.
Table 12 shows the occurrence of somatic mutation in
the HC2 heavy chain transgene in transgenic mice.
DETAILED DESCRIPTION
As has been discussed supra, it is desirable to
produce human immunoglobulins that are reactive with specific
human antigens that are promising therapeutic and/or
diagnostic targets. However, producing human immunoglobulins
that bind specifically with human antigens is problematic.
First, the immunized animal that serves as the
source of B cells must make an immune response against the
presented antigen. In order for an animal to make an immune
response, the antigen presented must be foreign and the animal
CA 02161351 2003-10-23
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must not be tolerant to the antigen. Thus, for example, if it
is desired to produce a human monoclonal antibody with an
idiotype that binds to a human protein, self-tolerance will
prevent an immunized human from making a substantial immune
response to the human protein, since the only epitopes of the
antigen that may be immunogenic will be those that result from
polymorphism of the protein within the human population
(allogeneic epitopes).
Second, if the animal that serves as the source of
B-cells for forming a hybridoma (a human in the illustrative
given example) does make an immune response against an
authentic self antigen, a severe autoimmune disease may result
in the animal. Where humans would be used as a source of B-
cells for a hybridoma, such autoimmunization would be
considered unethical by contemporary standards. Thus,
developing hybridomas secreting human immunoglobulin
chainsspecifically reactive with predetermined human antigens
is problematic, since a reliable source of human antibody-
secreting B cells that can evoke an antibody response against
predetermined human antigens is needed.
One methodology that can be used to obtain human
antibodies that are specifically reactive with human antigens
is the production of a transgenic mouse harboring the human
immunoglobulin transgene constructs of this invention.
Briefly, transgenes containing all or portions of the human
immunoglobulin heavy and light chain loci, or transgenes
containing synthetic "miniloci" (described infra, and in
WO 92/03918 and WO 93/12227)
which comprise essential functional elements of the human
heavy and light chain loci, are employed to produce a
transgenic nonhuman animal. Such a transgenic nonhuman animal
will have the capacity to produce immunoglobulin chains that
are encoded by human immunoglobulin genes, and additionally
WO 94/25585 PCT/US94/04580
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will be capable of making an immune response against human
antigens. Thus, such transgenic animals can serve as a source
of immune sera reactive with specified human antigens, and B-
cells from such transgenic animals can be fused with myeloma
cells to produce hybridomas that secrete monoclonal antibodies
that are encoded by human immunoglobulin genes and which are
specifically reactive with human antigens.
The production of transgenic mice containing various
forms of immunoglobulin genes has been reported previously.
Rearranged mouse immunoglobulin heavy or light chain genes
have been used to produce transgenic mice. In addition,
functionally rearranged human Ig genes including the or yl
constant region have been expressed in transgenic mice.
However, experiments in which the transgene comprises
unrearranged (V-D-J or V-J not rearranged) immunoglobulin
genes have been variable, in some cases, producing incomplete
or minimal rearrangement of the transgene. However, there are
no published examples of either rearranged or unrearranged
immunoglobulin transgenes which undergo successful isotype
switching between CH genes within a transgene.
The invention also provides a method for identifying
candidate hybridomas which secrete a monoclonal antibody
comprising a human immunoglobulin chain consisting essentially
of a human VDJ sequence in polypeptide linkage to a human
constant region sequence. Such candidate hybridomas are
identified from a pool of hybridoma clones comprising: (1)
hybridoma clones that express immunoglobulin chains consisting
essentially of a human VDJ region and a human constant region,
and (2) trans-switched hybridomas that express heterohybrid
immunoglobulin chains consisting essentially of a human VDJ
region and a murine constant region. The supernatant(s) of
individual or pooled hybridoma clones is contacted with a
predetermined antigen, typically an antigen which is
immobilized by adsoption onto a solid substrate (e.g., a
microtitre well), under binding conditions to select
antibodies having the predetermined antigen binding
specificity. An antibody that specifically binds to human
constant regions is also contacted with the hybridoma
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supernatant and predetermined antigen under binding conditions
so that the antibody selectively binds to at least one human
constant region epitope but substantially does not bind to
murine constant region epitopes; thus forming complexes
consisting essentially of hybridoma supernatant (transgenic
monoclonal antibody) bound to a predetermined antigen and to
= an antibody that specifically binds human constant regions
(and which may be labeled with a detectable label or
reporter). Detection of the formation of such complexes
indicates hybridoma clones or pools which express a human
immunoglobulin chain.
Definitions
As used herein, the term "antibody" refers to a
glycoprotein comprising at least two light polypeptide chains
and two heavy polypeptide chains. Each of the heavy and light
polypeptide chains contains a variable region (generally the
amino terminal portion of the polypeptide chain) which
contains a binding domain which interacts with antigen. Each
of the heavy and light polypeptide chains also comprises a
constant region of the polypeptide chains (generally the
carboxyl terminal portion) which may mediate the binding of
the immunoglobulin to host tissues or factors including
various cells of the immune system, some phagocytic cells and
the first component (Clq) of the classical complement system.
As used herein, a "heterologous antibody" is defined
in relation to the transgenic non-human organism producing
such an antibody. It is defined as an antibody having an
amino acid sequence or an encoding DNA sequence corresponding
to that found in an organism not consisting of the transgenic
non-human animal, and generally from a species other than that
of the transgenic non-human animal.
As used herein, a "heterohybrid antibody" refers to
an antibody having a light and heavy chains of different
organismal origins. For example, an antibody having a human
heavy chain associated with a murine light chain is a
heterohybrid antibody.
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As used herein, "isotype" refers to the antibody
class (e.g., IgM or IgG1) that is encoded by heavy chain
constant region genes.
As used herein, "isotype switching" refers to the
phenomenon by which the class, or isotype, of an antibody
changes from one Ig class to one of the other Ig classes.
As used herein, "nonswitched isotype" refers to the
isotypic class of heavy chain that is produced when no isotype
switching has taken place; the C. gene encoding the
nonswitched isotype is typically the first C. gene immediately
downstream from the functionally rearranged VDJ gene.
As used herein, the term "switch sequence" refers to
those DNA sequences responsible for switch recombination. A
"switch donor" sequence, typically a a switch region, will be
51 (i.e., upstream) of the construct region to be deleted
during the switch recombination. The "switch acceptor" region
will be between the construct region to be deleted and the
replacement constant region (e.g., y, e, etc.). As there is
no specific site where recombination always occurs, the final
gene sequence will typically not be predictable from the
construct.
As used herein, "glycosylation pattern" is defined
as the pattern of carbohydrate units that are covalently
attached to a protein, more specifically to an immunoglobulin
protein. A glycosylation pattern of a heterologous antibody
can be characterized as being substantially similar to
glycosylation patterns which occur naturally on antibodies
produced by the species of the nonhuman transgenic animal,
when one of ordinary skill in the art would recognize the
glycosylation pattern of the heterologous antibody as being
more similar to said pattern of glycosylation in the species
of the nonhuman transgenic animal than to the species from
which the CH genes of the transgene were derived.
As used herein, "specific binding" refers to the
property of the antibody: (1) to bind to a predetermined
antigen with an affinity of at least 1 x 107 M-1, and (2) to
preferentially bind to the predetermined antigen with an
affinity that is at least two-fold greater than its affinity
WO 94/25585 PCT/US94/04580
! 27 2161351
for binding to a non-specific antigen (e.g., BSA, casein)
other than the predetermined antigen or a closely-related
antigen.
The term "naturally-occurring" as used herein as
applied to an object refers to the fact that an object can be
found in nature. For example, a polypeptide or polynucleotide
sequence that is present in an organism (including viruses)
that can be isolated from a source in nature and which has not
been intentionally modified by man in the laboratory is
naturally-occurring.
The term "rearranged" as used herein refers to a
configuration of a heavy chain or light chain immunoglobulin
locus wherein a V segment is positioned immediately adjacent
to a D-J or J segment in a conformation encoding essentially a
complete VH or VL domain, respectively. A rearranged
immunoglobulin gene locus can be identified by comparison to
germline DNA; a rearranged locus will have at least one
recombined heptamer/nonamer homology element.
The term "unrearranged" or "germline configuration"
as used herein in reference to a V segment refers to the
configuration wherein the V segment is not recombined so as to
be immediately adjacent to a D or J segment.
For nucleic acids, the term "substantial homology"
indicates that two nucleic acids, or designated sequences
thereof, when optimally aligned and compared, are identical,
with appropriate nucleotide insertions or deletions, in at
least about 80% of the nucleotides, usually at least about 90%
to 95%, and more preferably at least about 98 to 99.5% of the
nucleotides. Alternatively, substantial homology exists when
the segments will hybridize under selective hybridization
conditions, to the complement of the strand. The nucleic
acids may be present in whole cells, in a cell lysate, or in a
partially purified or substantially pure form. A nucleic acid
is "isolated" or "rendered substantially pure" when purified
away from other cellular components or other contaminants,
e.g., other cellular nucleic acids or proteins, by standard
techniques, including alkaline/SDS treatment, CsCl banding,
column chromatography, agarose gel electrophoresis and others
WO 94/25585 PCT/US94/04580
28
well known in the art. See, F. Ausubel, et al., ed. Current
Protocols in Molecular Biology, Greene Publishing and Wiley-
Interscience, New.York (1987).
The nucleic acid compositions of the present
invention, while often in a native sequence (except for
modified restriction sites and the like), from either cDNA,
genomic or mixtures may be mutated, thereof in accordance with
standard techniques to provide gene sequences. For coding
sequences, these mutations, may affect amino acid sequence as
desired. In particular, DNA sequences substantially
homologous to or derived from native V, D, J, constant,
switches and other such sequences described herein are
contemplated (where "derived" indicates that a sequence is
identical or modified from another sequence).
A nucleic acid is "operably linked" when it is
placed into a functional relationship with another nucleic
acid,sequence. For instance, a promoter or enhancer is
operably linked to a coding sequence if it affects the
transcription of the sequence. With respect to transcription
regulatory sequences, operably linked means that the DNA
sequences being linked are contiguous and, where necessary to
join two protein coding regions, contiguous and in reading
frame. For switch sequences, operably linked indicates that
the sequences are capable of effecting switch recombination.
Transgenic Nonhuman Animals Capable
of Producing Heterologous Antibodies
The design of a transgenic non-human animal that
responds to foreign antigen stimulation with a heterologous
antibody repertoire, requires that the heterologous
immunoglobulin transgenes contained within the transgenic
animal function correctly throughout the pathway of B-cell
development. In a preferred embodiment, correct function of a
heterologous heavy chain transgene includes isotype switching.
Accordingly, the transgenes of the invention are constructed
so as to produce isotype switching and one or more of the
following: (1) high level and cell-type specific expression,
(2) functional gene rearrangement, (3) activation of and
response to allelic exclusion, (4) expression of a sufficient
CA 02161351 2003-10-23
29
primary repertoire, (5) signal transduction, (6) somatic
hypermutation, and (7) domination of the transgene antibody
locus during the immune response.
As will be apparent from the following disclosure,
not all of the foregoing criteria need be met. For example, in
those embodiments wherein the endogenous immunoglobulin loci
of the transgenic animal are functionally disrupted, the
transgene need.not activate allelic exclusion. Further, in
those embodiments wherein the transgene comprises a
functionally rearranged heavy and/or light chain
immunoglobulin gene, the second criteria of functional gene
rearrangement is unnecessary, at least for that transgene
which is already rearranged. For background on molecular
immunology, see, Fundamental Immunology, 2nd edition (1989),
Paul William E., ed. Raven Press, N.Y..
In one aspect of the invention, transgenic non-human
animals are provided that contain rearranged, unrearranged or
a combination of rearranged and unrearranged heterologous
immunoglobulin heavy and light chain transgenes in the
germline of the transgenic animal. Each of the heavy chain
transgenes comprises at least one C. gene. In addition, the
heavy chain transgene may contain functional isotype switch
sequences, which are capable of supporting isotype switching
of a heterologous transgene encoding multiple C. genes in B-
cells of the transgenic animal. Such switch sequences may be
those which occur naturally in the germline immunoglobulin
locus from the species that serves as the source of the
transgene CH genes, or such switch sequences may be derived
from those which occur in the species that is to receive the
transgene construct (the transgenic animal). For example, a
human transgene construct that is used to produce a transgenic
mouse may produce a higher frequency of isotype switching
events if it incorporates switch sequences similar to those
that occur naturally in the mouse heavy chain locus, as
presumably the mouse switch sequences are optimized to
function with the mouse switch recombinase enzyme system,
whereas the human switch sequences are not. Switch sequences
CA 02161351 2003-10-23
made be isolated and cloned by conventional cloning methods,
or may be synthesized de novo from overlapping synthetic
oligonucleotides designed on the basis of published sequence
information relating to immunoglobulin switch region sequences
5 (Mills et al., Nucl. Acids Res. 18:7305-7316 (1991);
Sideras'et al., Intl. Immunol. 1:631-642 (1989)).
For each of the foregoing transgenic animals,
functionally rearranged heterologous heavy and light chain
10 immunoglobulin transgenes are found in a significant fraction
of the B-cells of the transgenic animal (at least 10 percent).
The transgenes of the invention include a heavy
chain transgene comprising DNA encoding at least one variable
gene segment, one diversity gene segment, one joining gene
15 segment and at least one constant region gene segment. The
immunoglobulin light chain transgene comprises DNA encoding at
least one variable gene segment, one joining gene segment and
at least one constant region gene segment. The gene segments
encoding the light and heavy chain gene segments are
20 heterologous to the transgenic non-human animal in that they
are derived from, or correspond to, DNA encoding
immunoglobulin heavy and light chain gene segments from a
species not consisting of the transgenic non-human animal. In
one aspect of the invention, the transgene is constructed such
25 that the individual gene segments are unrearranged, i.e., not
rearranged so as to encode a functional immunoglobulin light
or heavy chain. Such unrearranged transgenes support
recombination of the V, D, and J gene segments (functional
rearrangement) and preferably support incorporation of all or
30 a portion of a D region gene segment in the resultant
rearranged immunoglobulin heavy chain within the transgenic
non-human animal when exposed to antigen.
In an alternate embodiment, the transgenes comprise
an unrearranged "mini-locus". Such transgenes typically
comprise a substantial portion of the C, D, and J segments as
well as a subset of the V gene segments. In such transgene
constructs, the various regulatory sequences, e.g. promoters,
enhancers, class switch regions, splice-donor and splice-
WO 94/25585 PCT/US94/04580
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acceptor sequences for RNA processing, recombination signals
and the like, comprise corresponding sequences derived from
the heterologous DNA. Such regulatory sequences may be
incorporated into the transgene from the same or a related
species of the non-human animal used in the invention. For
example, human immunoglobulin gene segments may be combined in
a transgene with a rodent immunoglobulin enhancer sequence for
use in a transgenic mouse. Alternatively, synthetic regulatory
sequences may be incorporated into the transgene, wherein such
synthetic regulatory sequences are not homologous to a
functional DNA sequence that is known to occur naturally in
the genomes of mammals. Synthetic regulatory sequences are
designed according to consensus rules, such as, for example,
those specifying the permissible sequences of a splice-
acceptor site or a promoter/enhancer motif. For example, a
minilocus comprises a portion of the genomic immunoglobulin
locus having at least one internal (i.e., not at a terminus of
the portion) deletion of a non-essential DNA portion (e.g.,
intervening sequence; intron or portion thereof) as compared
to the naturally-occurring germline Ig locus.
The invention also includes transgenic animals
containing germ line cells having a heavy and light transgene
wherein one of the said transgenes contains rearranged gene
segments with the other containing unrearranged gene segments.
In the preferred embodiments, the rearranged transgene is a
light chain immunoglobulin transgene and the unrearranged
transgene is a heavy chain immunoglobulin transgene.
The Structure and Generation of Antibodies
The basic structure of all immunoglobulins is based
upon a unit consisting of two light polypeptide chains and two
heavy polypeptide chains. Each light chain comprises two
regions known as the variable light chain region and the
constant light chain region. Similarly, the immunoglobulin
heavy chain comprises two regions designated the variable
heavy chain region and the constant heavy chain region.
The constant region for the heavy or light chain is
encoded by genomic sequences referred to as heavy or light
WO 94/25585 PCT/US94/04580
~16A_151 32
constant region gene (CH) segments. The use of a particular
heavy chain gene segment defines the class of immunoglobulin.
For example, in humans, the g constant region gene segments
define the IgM class of antibody whereas the use of a y, y2,
73 or 74 constant region gene segment defines the IgG class of
antibodies as well as the IgG subclasses IgGi through IgG4.
Similarly, the use of a al or a2 constant region gene segment
defines the IgA class of antibodies as well as the subclasses
IgAl and IgA2. The S and e constant region gene segments
define the IgD and IgE antibody classes, respectively.
The variable regions of the heavy and light
immunoglobulin chains together contain the antigen binding
domain of the antibody. Because of the need for diversity in
this region of the antibody to permit binding to a wide range
of antigens, the DNA encoding the initial or primary
repertoire variable region comprises a number of different DNA
segments derived from families of specific variable region
gene segments. In the case of the light chain variable
region, such families comprise variable (V) gene segments and
joining (J) gene segments. Thus, the initial variable region
of the light chain is encoded by one V gene segment and one J
gene segment each selected from the family of V and J gene
segments contained in the genomic DNA of the organism. In the
case of the heavy chain variable region, the DNA encoding the
initial or primary repertoire variable region of the heavy
chain comprises one heavy chain V gene segment, one heavy
chain diversity (D) gene segment and one J gene segment, each
selected from the appropriate V, D and J families of
immunoglobulin gene segments in genomic DNA.
In order to increase the diversity of sequences that
contribute to forming antibody binding sites, it is preferable
that a heavy chain transgene include cis-acting sequences that
support functional V-D-J rearrangement that can incorporate
all or part of a D region gene sequence in a rearranged V-D-J
gene sequence. Typically, at least about 1 percent of
expressed transgene-encoded heavy chains (or mRNAs) include
recognizable D region sequences in the V region. Preferably,
at least about 10 percent of transgene-encoded V regions
WO 94/25585 PCT/US94/04580
Is 33 i t351
include recognizable D region sequences, more preferably at
least about 30 percent, and most preferably more than 50
percent include recognizable D region sequences.
A recognizable D region sequence is generally at
least about eight consecutive nucleotides corresponding to a
sequence present in a D region gene segment of a heavy chain
transgene and/or the amino acid sequence encoded by such D
region nucleotide sequence. For example, if a transgene
includes the D region gene DHQ52, a transgene-encoded mRNA
containing the sequence 5'-TAACTGGG-3' located in the V region
between a V gene segment sequence and a J gene segment
sequence is recognizable as containing a D region sequence,
specifically a DHQ52 sequence. Similarly, for example, if a
transgene includes the D region gene DHQ52, a transgene-
encoded heavy chain polypeptide containing the amino acid
sequence -DAF- located in the V region between a V gene
segment amino acid sequence and a J gene segment amino acid
sequence may be recognizable as containing a D region
sequence, specifically a DHQ52 sequence. However, since D
region segments may be incorporated in VDJ joining to various
extents and in various reading frames, a comparison of the D
region area of a heavy chain variable region to the D region
segments present in the transgene is necessary to determine
the incorporation of particular D segments. Moreover,
potential exonuclease digestion during recombination may lead
to imprecise V-D and D-J joints during V-D-J recombination.
However, because of somatic mutation and N-region
addition, some D region sequences may be recognizable but may
not correspond identically to a consecutive D region sequence
in the transgene. For example, a nucleotide sequence 5'-
CTAAXTGGGG-3', where X is A, T, or G, and which is located in
a heavy chain V region and flanked by a V region gene sequence
and a J region gene sequence, can be recognized as
corresponding to. the DHQ52 sequence 5'-CTAACTGGG-3'.
Similarly, for example, the polypeptide sequences -DAFDI-,
-DYFDY-, or -GAFDI- located in a V region and flanked on the
amino-terminal side by an amino acid sequence encoded by a
transgene V gene sequence and flanked on the carboxyterminal
WO 94/25585 PCT/US94/04580
34
side by an amino acid sequence encoded by a transgene J gene
sequence is recognizable as a D region sequence.
Therefore, because somatic mutation and N-region
addition can produce mutations in sequences derived from a
transgene D region, the following definition is provided as a
guide for determining the presence of a recognizable D region
sequence. An amino acid sequence or nucleotide sequence is
recognizable as a D region sequence if: (1) the sequence is
located in a V region and is flanked on one side by a V gene
sequence (nucleotide sequence or deduced amino acid sequence)
and on the other side by a J gene sequence (nucleotide
sequence or deduced amino acid sequence) and (2) the sequence
is substantially identical or substantially similar to a known
D gene sequence (nucleotide sequence or encoded amino acid
sequence).
The term "substantial identity" as used herein
denotes a characteristic of a polypeptide sequence or nucleic
acid sequence, wherein the polypeptide sequence has at least
50 percent sequence identity compared to a reference sequence,
and the nucleic acid sequence has at least 70 percent sequence
identity compared to a reference sequence. The percentage of
sequence identity is calculated excluding small deletions or
additions which total less than 35 percent of the reference
sequence. The reference sequence may be a subset of a larger
sequence, such as an entire D gene; however, the reference
sequence is at least 8 nucleotides long in the case of
polynucleotides, and at least 3 amino residues long in the
case of a polypeptide. Typically, the reference sequence is
at least 8 to 12 nucleotides or at least 3 to 4 amino acids,
and preferably the reference sequence is 12 to 15 nucleotides
or'more, or at least 5 amino acids.
The term "substantial similarity" denotes a
characteristic of an polypeptide sequence, wherein the
polypeptide sequence has at least 80 percent similarity to a
reference sequence. The percentage of sequence similarity is
calculated by scoring identical amino acids or positional
conservative amino acid substitutions as similar. A
positional conservative amino acid substitution is one that
WO 94/25585 PCT/US94/04580
2161351
can result from a single nucleotide substitution; a first
amino acid is replaced by a second amino acid where a codon
for the first amino acid and a codon for the second amino acid
can differ by a single nucleotide substitution. Thus, for
5 example, the sequence -Lys-Glu-Arg-Val- is substantially
similar to the sequence -Asn-Asp-Ser-Val-, since the codon
sequence -AAA-GAA-AGA-GUU- can be mutated to -AAC-GAC-AGC-GUU-
by introducing only 3 substitution mutations, single
nucleotide substitutions in three of the four original codons.
10 The reference sequence may be a subset of a larger sequence,
such as an entire D gene; however, the reference sequence is
at least 4 amino residues long. Typically, the reference
sequence is at least 5 amino acids, and preferably the
reference sequence is 6 amino acids or more.
The Primary Repertoire
The process for generating DNA encoding the heavy
and light chain immunoglobulin genes occurs primarily in
developing B-cells. Prior to the joining of various
immunoglobulin gene segments, the V, D, J and constant (C)
gene segments are found, for the most part, in clusters of V,
D, J and C gene segments in the precursors of primary
repertoire B-cells. Generally, all of the gene segments for a
heavy or light chain are located in relatively close proximity
on a single chromosome. Such genomic DNA prior to
recombination of the various immunoglobulin gene segments is
referred to herein as "unrearranged" genomic DNA. During
B-cell differentiation, one of each of the appropriate family
members of the V, D, J (or only V and J in the case of light
chain genes) gene segments are recombined to form functionally
rearranged heavy and light immunoglobulin genes. Such
functional rearrangement is of the variable region segments to
form DNA encoding a functional variable region. This gene
segment rearrangement process appears to be sequential.
First, heavy chain D-to-J joints are made, followed by heavy
chain V-to-DJ joints and light chain V-to-J joints. The DNA
encoding this initial form of a functional variable region in
a light and/or heavy chain is referred to as "functionally
WO 94125585 PCT/US94/04580
36
rearranged DNA" or "rearranged DNA". In the case of the heavy
chain, such DNA is referred to as "rearranged heavy chain DNA"
and in the case of the light chain, such DNA is referred to as
"rearranged light chain DNA". Similar language is used to
describe the functional rearrangement of the transgenes of the
invention.
The recombination of variable region gene segments
to form functional heavy and light chain variable regions is
mediated by recombination signal sequences (RSS's) that flank
recombinationally competent V, D and J segments. RSS's
necessary and sufficient to direct recombination, comprise a
dyad-symmetric heptamer, an AT-rich nonamer and an intervening
spacer region of either 12 or 23 base pairs. These signals
are conserved among the different loci and species that carry
out D-J (or V-J) recombination and are functionally
interchangeable. See Oettinger, et al. (1990), Science, 248,
1517-1523 and references cited therein. The heptamer
comprises the sequence CACAGTG or its analogue followed by a
spacer of unconserved sequence and then a nonamer having the
sequence ACAAAAACC or its analogue. These sequences are found
on the J, or downstream side, of each V and D gene segment.
Immediately preceding the germline D and J segments are again
two recombination signal sequences, first the nonamer and then
the heptamer again separated by an unconserved sequence. The
heptameric and nonameric sequences following a VL, VH or D
segment are complementary to those preceding the JL, D or J.
segments with which they recombine. The spacers between the
heptameric and nonameric sequences are either 12 base pairs
long or between 22 and 24 base pairs long.
In addition to the rearrangement of V, D and J
segments, further diversity is generated in the primary
repertoire of immunoglobulin heavy and light chain by way of
variable recombination between the V and J segments in the
light chain and between the D and J segments of the heavy
chain. Such variable recombination is generated by variation
in the exact place at which such segments are joined. Such
variation in the light chain typically occurs within the last
codon of the V gene segment and the first codon of the J
WO 94/25585 PCT/US94/04580
= 37 2161351
segment. Similar imprecision in joining occurs on the heavy
chain chromosome between the D and JH segments and may extend
over as many as 10 nucleotides. Furthermore, several
nucleotides may be inserted between the D and JH and between
the VH and D gene segments which are not encoded by genomic
DNA. The addition of these nucleotides is known as N-region
diversity.
After VJ and/or VDJ rearrangement, transcription of
the rearranged variable region and one or more constant region
gene segments located downstream from the rearranged variable
region produces a primary RNA transcript which upon
appropriate RNA splicing results in an mRNA which encodes a
full length heavy or light immunoglobulin chain. Such heavy
and light chains include a leader signal sequence to effect
secretion through and/or insertion of the immunoglobulin into
the transmembrane region of the B-cell. The DNA encoding this
signal sequence is contained within the first exon of the V
segment used to form the variable region of the heavy or light
immunoglobulin chain. Appropriate regulatory sequences are
also present in the mRNA to control translation of the mRNA to
produce the encoded heavy and light immunoglobulin
polypeptides which upon proper association with each other
form an antibody molecule.
The net effect of such rearrangements in the
variable region gene segments and the variable recombination
which may occur during such joining, is the production of a
primary antibody repertoire. Generally, each B-cell which has
differentiated to this stage, produces a single primary
repertoire antibody. During this differentiation process,
cellular events occur which suppress the functional
rearrangement of gene segments other than those contained
within the functionally rearranged Ig gene. The process by
which diploid B-cells maintain such mono-specificity is termed
allelic-exclusion.
The Secondary Repertoire
B-cell clones expressing immunoglobulins from within
the set of sequences comprising the primary repertoire are
CA 02161351 2003-10-23
38
immediately available to respond to foreign antigens. Because
of the limited diversity generated by simple VJ and VDJ
joining, the antibodies produced by the so-called primary
response are of relatively low affinity. Two different types
of B-cells make up this initial response: precursors of
primary-antibody-forming cells and precursors of secondary
repertoire B-cells (Linton et al., Cell 52:1049-1059 (1989)).
The first type of B-cell matures into IgM-secreting plasma
cells in response to certain antigens. The other B-cells
respond to initial exposure to antigen by entering a T-cell
dependent maturation pathway.
During the T-cell dependent maturation of antigen
stimulated B-cell clones, the structure of the antibody
molecule on the cell surface changes in two ways: the constant
region switches to a non-IgM subtype and the sequence of the
variable region can be modified by multiple single amino acid
substitutions to produce a higher affinity antibody molecule.
As previously indicated, each variable region of a
heavy or light Ig chain contains an antigen binding domain.
It has been determined by amino acid and nucleic acid
sequencing that somatic mutation during the secondary response
occurs throughout the V region including the three
complementary determining regions (CDR1, CDR2 and CDR3) also
referred to as hypervariable regions 1, 2 and 3 (Kabat et al.
Sequences of Proteins of Immunological Interest (1991) U.S.
Department of Health and Human Services, Washington, DC.
The CDR1 and CDR2 are
located within the variable gene segment whereas the CDR3 is
largely the result of recombination between V and J gene
segments or V, D and J gene segments. Those portions of the
variable region which do not consist of CDR1, 2 or 3 are
commonly referred to as framework regions designated FR1, FR2,
FR3 and FR4. See Fig. 1. During hypermutation, the
rearranged DNA is mutated to give rise to new clones with
altered Ig molecules. Those clones with higher affinities for
the foreign antigen are selectively expanded by helper
T-cells, giving rise to affinity maturation of the expressed
antibody. Clonal selection typically results in expression of
WO 94/25585 PCT/US94/04580
3 216t351
9
clones containing new mutation within the CDR1, 2 and/or 3
regions. However, mutations outside these regions also occur
which influence the specificity and affinity of the antigen
binding domain.
Transgenic Non-Human Animals Capable
of Producing Heteroloaous Antibody
Transgenic non-human animals in one aspect of the
invention are produced by introducing at least one of the
immunoglobulin transgenes of the invention (discussed
hereinafter) into a zygote or early embryo of a non-human
animal. The non-human animals which are used in the invention
generally comprise any mammal which is capable of rearranging
immunoglobulin gene segments to produce a primary antibody
response. Such nonhuman transgenic animals may include, for
example, transgenic pigs, transgenic rats, transgenic rabbits,
transgenic cattle, and other transgenic animal species,
particularly mammalian species, known in the art. A
particularly preferred non-human animal is the mouse or other
members of the rodent family.
However, the invention is not limited to the use of
mice. Rather, any non-human mammal which is capable of
mounting a primary and secondary antibody response may be
used. Such animals include non-human primates, such as
chimpanzee, bovine, ovine, and porcine species, other members
of the rodent family, e.g. rat, as well as rabbit and guinea
pig. Particular preferred animals are mouse, rat, rabbit and
guinea pig, most preferably mouse.
In one embodiment of the invention, various gene
segments from the human genome are used in heavy and light
chain transgenes in an unrearranged form. In this embodiment,
such transgenes are introduced into mice. The unrearranged
gene segments of the light and/or heavy chain transgene have
DNA sequences unique to the human species which are
distinguishable from the endogenous immunoglobulin gene
segments in the mouse genome. They may be readily detected in
unrearranged form in the germ line and somatic cells not
consisting of B-cells and in rearranged form in B-cells.
WO 94/25585 PCT/US94/04580
In an alternate embodiment of the invention, the
transgenes comprise rearranged heavy and/or light
immunoglobulin transgenes. Specific segments of such
transgenes corresponding to functionally rearranged VDJ or VJ
5 segments, contain immunoglobulin DNA sequences which are also
clearly distinguishable from the endogenous immunoglobulin
gene segments in the mouse.
Such differences in DNA sequence are also reflected
in the amino acid sequence encoded by such human
10 immunoglobulin transgenes as compared to those encoded by
mouse B-cells. Thus, human immunoglobulin amino acid
sequences may be detected in the transgenic non-human animals
of the invention with antibodies specific for immunoglobulin
epitopes encoded by human immunoglobulin gene segments.
15 Transgenic B-cells containing unrearranged
transgenes=from human or other species functionally recombine
the appropriate gene segments to form functionally rearranged
light and heavy chain variable regions. It will be readily
apparent that the antibody encoded by such rearranged
20 transgenes has a DNA and/or amino acid sequence which is
heterologous to that normally encountered in the nonhuman
animal used to practice the invention.
Unrearranaed Transaenes
25 As used herein, an "unrearranged immunoglobulin
heavy chain transgene" comprises DNA encoding at least one
variable gene segment, one diversity gene segment, one joining
gene segment and one constant region gene segment. Each of
the gene segments of said heavy chain transgene are derived
30 from, or has a sequence corresponding to, DNA encoding
immunoglobulin heavy chain gene segments from a species not
consisting of the non-human animal into which said transgene
is introduced. Similarly, as used herein, an "unrearranged
immunoglobulin light chain transgene" comprises DNA encoding
35 at least one variable gene segment, one joining gene segment
and at least one constant region gene segment wherein each
gene segment of said light chain transgene is derived from, or
has a sequence corresponding to, DNA encoding immunoglobulin
CA 02161351 2003-10-23
41
light chain gene segments from a species not consisting of the
non-human animal into which said light chain transgene is
introduced.
Such heavy and light chain transgenes in this aspect
of the invention contain the above-identified gene segments in
an unrearranged form. Thus, interposed between the V, D and J
segments in the heavy chain transgene and between the V and J
segments on the light chain transgene are appropriate
recombination signal sequences (RSS's). In addition, such
transgenes also include appropriate RNA splicing signals to
join a constant region gene segment with the VJ or VDJ
rearranged variable region.
In order to facilitate isotype switching within a
heavy chain transgene containing more than one C region gene
segment, e.g. C and Cyl from the human genome, as explained
below "switch regions" are incorporated upstream from each of
the constant region gene segments and downstream from the
variable region gene segments to permit recombination between
such constant regions to allow for immunoglobulin class
switching, e.g. from IgM to IgG. Such heavy and light
immunoglobulin transgenes also contain transcription control
sequences including promoter regions situated upstream from
the variable region gene segments which typically contain TATA
motifs. A promoter region can be defined approximately as a
DNA sequence that, when operably linked to a downstream
sequence, can produce transcription of the downstream
sequence. Promoters may require the presence of additional
linked cis-acting sequences in order to produce efficient
transcription. In addition, other sequences that participate
in the transcription of sterile transcripts are preferably
included. Examples of sequences that participate in
expression of sterile transcripts can be found in the
published literature, including Rothman et al., Intl. Immunol.
2:621-627 (1990); Reid et al., Proc. Natl. Acad. Sci. USA
86:840-844 (1989); Stavnezer et al., Proc. Natl. Acad. Sci.
USA 85:7704-7708 (1988); and Mills et al., Nucl. Acids Res.
18:7305-7316 (1991).
These sequences typically include about at least
WO 9,4/j1,550' a ) ~Q i j5 + PCTIUS94/04580
42
50 bp immediately upstream of a switch region, preferably
about at least 200 bp upstream of a switch region; and more
preferably about at least 200-1000 bp or more upstream of a
switch region. Suitable sequences occur immediately upstream
of the human Syl, Sy2, Sry3, Sy4, Sal, S 2, and SE switch
regions; the sequences immediately upstream of the human S,yl,
and Sy3 switch regions can be used to advantage, with Syl
generally preferred. Alternatively, or in combination, murine
Ig switch sequences may be used; it may frequently be
advantageous to employ Ig switch sequences of the same species
as the transgenic non-human animal. Furthermore, interferon
(IFN) inducible transcriptional regulatory elements, such as
IFN-inducible enhancers, are preferably included immediately
upstream of transgene switch sequences.
In addition to promoters, other regulatory sequences
which function primarily in B-lineage cells are used. Thus,
for example, a light chain enhancer sequence situated
preferably between the J and constant region gene segments on
the light chain transgene is used to enhance transgene
expression, thereby facilitating allelic exclusion. In the
case of the heavy chain transgene, regulatory enhancers and
also employed. Such regulatory sequences are used to maximize
the transcription and translation of the transgene so as to
induce allelic exclusion and to provide relatively high levels
of transgene expression.
Although the foregoing promoter and enhancer
regulatory control sequences have been generically described,
such regulatory sequences may be heterologous to the nonhuman
animal being derived from the genomic DNA from which the
heterologous transgene immunoglobulin gene segments are
obtained. Alternately, such regulatory gene segments are
derived from the corresponding regulatory sequences in the
genome of the non-human animal, or closely related species,
which contains the heavy and light transgene.
In the preferred embodiments, gene segments are
derived from human beings. The transgenic non-human animals
harboring such heavy and light transgenes are capable of
mounting an Ig-mediated immune response to a specific antigen
WO 94/25585 216135 1 PCT/US94/04580
0 43
administered to such an animal. B-cells are produced within
such an animal which are capable of producing heterologous
human antibody. After immortalization, and the selection for
an appropriate monoclonal antibody (Mab), e.g. a hybridoma, a
source of therapeutic human monoclonal antibody is provided.
Such human Mabs have significantly reduced immunogenicity when
therapeutically administered to humans.
Although the preferred embodiments disclose the
construction of heavy and light transgenes containing human
gene segments, the invention is not so limited. In this
regard, it is to be understood that the teachings described
herein may be readily adapted to utilize immunoglobulin gene
segments from a species other than human beings. For example,
in addition to the therapeutic treatment of humans with the
antibodies of the invention, therapeutic antibodies encoded by
appropriate gene segments may be utilized to generate
monoclonal antibodies for use in the veterinary sciences.
Rearranged Transgenes
In an alternative embodiment, transgenic nonhuman
animals contain functionally at least one rearranged
heterologous heavy chain immunoglobulin transgene in the
germline of the transgenic animal. Such animals contain
primary repertoire B-cells that express such rearranged heavy
transgenes. Such B-cells preferably are capable of undergoing
somatic mutation when contacted with an antigen to form a
heterologous antibody having high affinity and specificity for
the antigen. Said rearranged transgenes will contain at least
two C. genes and the associated sequences required for isotype
switching.
The invention also includes transgenic animals
containing germ line cells having heavy and light transgenes
wherein one of the said transgenes contains rearranged gene
segments with the other containing unrearranged gene segments.
In such animals, the heavy chain transgenes shall have at
least two CH genes and the associated sequences required for
isotype switching.
WO 94/25585 PCT/US94/04580
44
The invention further includes methods for
generating a synthetic variable region gene segment repertoire
to be used in the.transgenes of the invention. The method
comprises generating a population of immunoglobulin V segment
DNAs wherein each of the V segment DNAs encodes an
immunoglobulin V segment and contains at each end a cleavage
recognition site of a restriction endonuclease. The
population of immunoglobulin V segment DNAs is thereafter
concatenated to form the synthetic immunoglobulin V segment
repertoire. Such synthetic variable region heavy chain
transgenes shall have at least two C. genes and the associated
sequences required for isotype switching.
Isotype Switching
In the development of a B lymphocyte, the cell
initially produces IgM with a binding specificity determined
by the productively rearranged V. and VL regions.
Subsequently, each B cell and its progeny cells synthesize
antibodies with the same L and H chain V regions, but they may
switch the isotype of the H chain.
The use of or d constant regions is largely
determined by alternate splicing, permitting IgM and IgD to be
coexpressed in a single cell. The other heavy chain isotypes
(y, a, and e) are only expressed natively after a gene
rearrangement event deletes the C and CS exons. This gene
rearrangement process, termed isotype switching, typically
occurs by recombination between so called switch segments
located immediately upstream of each heavy chain gene (except
6). The individual switch segments are between 2 and 10 kb in
length, and consist primarily of short repeated sequences.
The exact point of recombination differs for individual class
switching events. Investigations which have used solution
hybridization kinetics or Southern blotting with cDNA-derived
CH probes have confirmed that switching can be associated with
loss of CH sequences from the cell.
The switch (S) region of the gene, S., is located
about 1 to 2 kb 5' to the coding sequence and is composed of
numerous tandem repeats of sequences of the form
CA 02161351 2003-10-23
(GAGCT)n(GGGGT), where n is usually 2 to 5 but can range as
high as 17. (See T. Nikaido et al. Nature 292:845-848 (1981))
Similar internally repetitive switch sequences
spanning several kilobases have been found 5' of the other C.
5 genes. The S. region has been sequenced and found to consist
of tandemly repeated 80-bp homology units, whereas murine SY2a,
SY2b, and S.,3 all contain repeated 49-bp homology units very
similar to each other. (See, P. Szurek et al., J. Immunol
135:620-626 (1985) and T. Nikaido et al., J. Biol. Chem.
10 257:7322-7329 (1982)).
All the sequenced S regions include numerous
occurrences of the pentamers GAGCT and GGGGT that are the
basic repeated elements of the S. gene (T. Nikaido et al.,
Biol. Chem. 257:7322-7329 (1982);
15 in the other S regions these pentamers are not
precisely tandemly repeated as in S., but instead are embedded
in larger repeat units. The SY1 region has an additional
higher-order structure: two direct repeat sequences flank
each of two clusters of 49-bp tandem repeats. (See M. R.
20 Mowatt et al., J. Immunol. 136:2674-2683 (1986)).
Switch regions of human H chain genes have been
found to be very similar to their mouse homology. Indeed,
similarity between pairs of human and mouse clones 5' to the
25 CH genes has been found to be confined to the S regions, a fact
that confirms the biological significance of these regions.
A switch recombination between g and a genes
produces a composite Sp-Sa sequence. Typically, there is no
specific site, either in SF, or in any other S region, where
30 the recombination always occurs.
Generally, unlike the enzymatic machinery of V-J
recombination, the switch machinery can apparently accommodate
different alignments of the repeated homologous regions of
germline S precursors and then join the sequences at different
35 positions within the alignment. (See, T. H. Rabbits et al.,
Nucleic Acids Res. 9:4509-4524 (1981) and J. Ravetch et al.,
Proc. Natl. Acad. Sci. USA 77:6734-6738 (1980)).
CA 02161351 2003-10-23
46
The exact details of the mechanism(s) of selective
activation of switching to a particular isotype are unknown.
Although exogenous influences such as lymphokines and
cytokines might upregulate isotype-specific recombinases, it
is also possible that the same enzymatic machinery catalyzes
switches to all isotypes and that specificity lies in
targeting this machinery to specific. switch regions.
The T-cell-derived lymphokines IL-4 and IFNY have
been shown to specifically promote the expression of certain
isotypes: in the mouse, IL-4 decreases IgM, IgG2a, IgG2b, and
IgG3 expression and increases IgE and IgG1 expression; while
IFNY selectively stimulates IgG2a expression and antagonizes
the IL-4-induced increase in IgE and IgGl expression (Coffman
et al., J. Immunol. 136: 949 (1986) and Snapper et al.,
Science 236: 944 (1987)).
A combination of.IL-4 and IL-5 promotes IgA
expression (Coffman et al., J. Immunol. 139: 3685 (1987)).
Most of the experiments implicating T-cell effects
on switching have not ruled out the possibility that the
observed increase in cells with particular switch
recombinations might reflect selection of preswitched or
precommitted cells; but the most likely explanation is that
the lymphokines actually promote switch recombination.
Induction of class switching appears to be
associated with sterile transcripts that initiate upstream of
the switch segments (Lutzker et al., Mol. Cell. Biol. 8:1849
(1988); Stavnezer et al., Proc. Natl. Acad. Sci. USA 85:7704
(1988); Esser and Radbruch, EMBO J. 8:483 (1989); Berton et
al., Proc. Natl. Acad. Sci. USA 86:2829 (1989); Rothman et
al., Int. Immunol. 2:621 (1990)).
For example, the observed induction of the -yl
sterile transcript by IL-4 and inhibition by IFN-'y correlates
with the observation that IL-4 promotes class switching to yl
in B-cells in culture, while IFN-y inhibits 'yl expression.
Therefore, the inclusion of regulatory sequences that affect
the transcription of sterile transcripts may also affect the
rate of isotype switching. For example, increasing the
WO 94/25585 PCT/US94/04580
0 47 21 1351
transcription of a particular sterile transcript typically can
be expected to enhance the frequency of isotype switch
recombination involving adjacent switch sequences.
For these reasons, it is preferable that transgenes
incorporate transcriptional regulatory sequences within about
1-2 kb upstream of each switch region that is to be utilized
for isotype switching. These transcriptional regulatory
sequences preferably include a promoter and an enhancer
element, and more preferably include the 5' flanking (i.e.,
upstream) region that is naturally associated (i.e., occurs in
germline configuration) with a switch region. This 5'
flanking region is typically about at least 50 nucleotides in
length, preferably about at least 200 nucleotides in length,
and more preferably at least 500-1000 nucleotides.
Although a 5' flanking sequence from one switch
region can be operably linked to a different switch region for
transgene construction (e.g., a 5' flanking sequence from the
human Syl switch can be grafted immediately upstream of the Sal
switch; a murine S,1 flanking region can be grafted adjacent to
a human yl switch sequence; or the murine Syl switch can be
grafted onto the human yl coding region), in some embodiments
it is preferred that each switch region incorporated in the
transgene construct have the 5' flanking region that occurs
immediately upstream in the naturally occurring germline
configuration.
Monoclonal Antibodies
Monoclonal antibodies can be obtained by various
techniques familiar to those skilled in the art. Briefly,
spleen cells from an animal immunized with a desired antigen
are immortalized, commonly by fusion with a myeloma cell (see,
Kohler and Milstein, Eur. J. Immunol., 6:511-519 (1976)).
Alternative methods of immortalization include transformation
with Epstein Barr Virus, oncogenes, or retroviruses, or other
methods well known in the art. Colonies arising from single
immortalized cells are screened for production of antibodies
of the desired specificity and affinity for the antigen, and
yield of the monoclonal antibodies produced by such cells may
WO 94/25585 PCT/US94/04580
48
be enhanced by various techniques, including injection into
the peritoneal cavity of a vertebrate host. Various
techniques useful in these arts are discussed, for example, in
Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor, New York (1988) including: immunization of animals to
produce immunoglobulins; production of monoclonal antibodies;
labeling immunoglobulins for use as probes; immunoaffinity
purification; and immunoassays.
The Transgenic Primary Repertoire
A. The Human Immunoglobulin Loci
An important requirement for transgene function is
the generation of a primary antibody repertoire that is
diverse enough to trigger a secondary immune response for a
wide range of antigens. The rearranged heavy chain gene
consists of a signal peptide exon, a variable region exon and
a tandem array of multi-domain constant region regions, each
of which is encoded by several exons. Each of the constant
region genes encode the constant portion of a different class
of immunoglobulins. During B-cell development, V region
proximal constant regions are deleted leading to the
expression of new heavy chain classes. For each heavy chain
class, alternative patterns of RNA splicing give rise to both
transmembrane and secreted immunoglobulins.
The human heavy chain locus is estimated to consist
of approximately 200 V gene segments (current data supports
the existence of about 50-100 V gene segments) spanning 2 Mb,
approximately 30 D gene segments spanning about 40 kb, six J
segments clustered within a 3 kb span, and nine constant
region gene segments spread out over approximately 300 kb.
The entire locus spans approximately 2.5 Mb of the distal
portion of the long arm of chromosome 14.
B. Gene Fragment Transgenes
1. Heavy Chain Transgene
In a preferred embodiment, immunoglobulin heavy and
light chain transgenes comprise unrearranged genomic DNA from
humans. In the case of the heavy chain, a preferred transgene
CA 02161351 2003-10-23
49
comprises a Notl fragment having a length between 670 to 830
kb. The length of this fragment is ambiguous because the 31
restriction site has not been accurately mapped. It is known,
however, to reside between the al and to gene segments. This
fragment contains members of all six of the known VH families,
the D and J gene segments, as well as the , 6, 73, 71 and al
constant regions (Berman et al., EMBO J. 7:727-738 (1988)).
A transgenic
mouse line containing this transgene correctly expresses a
heavy chain class required for B-cell development (IgM) and at
least one switched heavy chain class (IgGl), in conjunction
with a sufficiently large repertoire of variable regions to
trigger a secondary response for most antigens.
2. Light Chain Transgene
A genomic fragment containing all of the necessary
gene segments and regulatory sequences from a human light
chain locus may be similarly constructed. Such transgenes are
constructed as described in the Examples and in
WO 92/03918 entitled "Transgenic Non-Human Animals Capable
of Producing Heterologous Antibodies".
C. Transgenes Generated Intracellularly
by in Vivo Recombination
It is not necessary to isolate the all or part of
the heavy chain locus on a single DNA fragment. Thus, for
example, the 670-830 kb NotI fragment from the human
immunoglobulin heavy chain locus may be formed in vivo in the
non-human animal during transgenesis. Such in vivo transgene
construction is produced by introducing two or more
overlapping DNA fragments into an embryonic nucleus of the
non-human animal. The overlapping portions of the DNA
fragments have DNA sequences which are substantially
homologous. Upon exposure to the recombinases contained
within the embryonic nucleus, the overlapping DNA fragments
homologously recombined in proper orientation to form the
670-830 kb NotI heavy chain fragment.
WO 94/25585 PCT/US94/04580
In vivo transgene construction can be used to form
any number of immunoglobulin transgenes which because of their
size are otherwise difficult, or impossible, to make or
manipulate by present technology. Thus, in vivo transgene
5 construction is useful to generate immunoglobulin transgenes
which are larger than DNA fragments which may be manipulated
by YAC vectors (Murray and Szostak, Nature 305:189-193
(1983)). Such in vivo transgene construction may be used to
introduce into a non-human animal substantially the entire
10 immunoglobulin loci from a species not consisting of the
transgenic non-human animal.
In addition to forming genomic immunoglobulin
transgenes, in vivo homologous recombination may also be
utilized to form "mini-locus" transgenes as described in the
15 Examples.
In the preferred embodiments utilizing in vivo
transgene construction, each overlapping DNA fragment
preferably has an overlapping substantially homologous DNA
sequence between the end portion of one DNA fragment and the
20 end portion of a second DNA fragment. Such overlapping
portions of the DNA fragments preferably comprise about 500 bp
to about 2000 bp, most preferably 1.0 kb to 2.0 kb. Homologous
recombination of overlapping DNA fragments to form transgenes
in vivo is further described in commonly assigned U.S. Patent
25 Application entitled "Intracellular Generation of DNA by
Homologous Recombination of DNA Fragments" filed August 29,
1990, under U.S.S.N. 07/574,747.
D. Minilocus Transgenes
30 As used herein, the term "immunoglobulin minilocus"
refers to a DNA sequence (which may be within a longer
sequence), usually of less than about 150 kb, typically
between about 25 and 100 kb, containing at least one each of
the following: a functional variable (V) gene segment, a
35 functional joining (J) region segment, at least one functional
constant (C) region gene segment, and--if it is a heavy chain
minilocus--a functional diversity (D) region segment, such
that said DNA sequence contains at least one substantial
WO 94/25585 - PCT/US94/04580
2161351
discontinuity (e.g., a deletion, usually of at least about 2
to 5 kb, preferably 10-25 kb or more, relative to the
homologous genomic DNA sequence). A light chain minilocus
transgene will be at least 25 kb in length, typically 50 to 60
kb. A heavy chain transgene will typically be about 70 to 80
kb in length, preferably at least about 60 kb with two
constant regions operably linked to switch regions.
Furthermore, the individual elements of the minilocus are
preferably in the germline configuration and capable of
undergoing gene rearrangement in the pre-B cell of a
transgenic animal so as to express functional antibody
molecules with diverse antigen specificities encoded entirely
by the elements of the minilocus. Further, a heavy chain
minilocus comprising at least two C. genes and the requisite
switching sequences is typically capable of undergoing isotype
switching, so that functional antibody molecules of different
immunoglobulin classes will be generated. Such isotype
switching may occur in vivo in B-cells residing within the
transgenic nonhuman animal, or may occur in cultured cells of
the B-cell lineage which have been explanted from the
transgenic nonhuman animal.
In an alternate preferred embodiment, immunoglobulin
heavy chain transgenes comprise one or more of each of the VH,
D, and J. gene segments and two or more of the CH genes. At
least one of each appropriate type gene segment is
incorporated into the minilocus transgene. With regard to the
C. segments for the heavy chain transgene, it is preferred
that the transgene contain at least one gene segment and at
least one other constant region gene segment, more preferably
a y gene segment, and most preferably 73 or 71. This
preference is to allow for class switching between IgM and IgG
forms of the encoded immunoglobulin and the production of a
secretable form of high affinity non-IgM immunoglobulin.
Other constant region gene segments may also be used such as
those which encode for the production of IgD, IgA and IgE.
Those skilled in the art will also construct
transgenes wherein the order of occurrence of heavy chain CH
genes will be different from the naturally-occurring spatial
CA 02161351 2003-10-23
52
order found in the germline of the species serving as the
donor of the CH genes.
Additionally, those skilled in the art can select CH
genes from more than one individual of a species (e.g.,
allogeneic CH genes) and incorporate said genes in the
transgene as supernumerary CH genes capable of undergoing
isotype switching; the resultant transgenic nonhuman animal
may then, in some embodiments, make antibodies of various
classes including all of the allotypes represented in the
species from which the transgene CH genes were obtained.
Still further, those skilled in the art can select
CH genes from different species to incorporate into the
transgene. Functional switch sequences are included with .each
CH gene, although the switch sequences used are not
necessarily those which occur naturally adjacent to the CH
gene. Interspecies CH gene combinations will produce a
transgenic nonhuman animal which may produce antibodies of
various classes corresponding to CH genes from various
species. Transgenic nonhuman animals containing interspecies
CH transgenes may serve as the source of B-cells for
constructing hybridomas to produce monoclonals for veterinary
uses.
The heavy chain J region segments in the human
comprise six functional J segments and three pseudo genes
clustered in a 3 kb stretch of DNA. Given its relatively
compact size and the ability to isolate these segments
together with the gene and the 5' portion of the 6 gene on a
single 23 kb SFiI/SpeI fragment (Sado et al., Biochem.
Biophys. Res. Comm. 154:264271 (1988)),
it is preferred that all of the J region
gene segments be used in the mini-locus construct. Since this
fragment spans the region between the g and 6 genes, it is
likely to contain all of the 3' cis-linked regulatory elements
required for p expression. Furthermore, because this fragment
includes the entire J region, it contains the heavy chain
enhancer and the switch region (Mills et al., Nature 306:809
(1983); Yancopoulos and Alt, Ann. Rev. Immunol. 4:339-368
(1986)). It also
CA 02161351 2003-10-23
53
contains the transcription start sites which trigger VDJ
joining to form primary repertoire B-cells (Yancopoulos and
Alt, Cell 40:271-281 (1985)).
Alternatively, a 36 kb BssHII/Spell fragment,
which includes part on the D region, may be used in place of
the 23 kb SfiI/Spell fragment. The use of such a fragment
increases the amount of 5' flanking sequence to facilitate
efficient D-to-J joining.
The human D region consists of 4 homologous 9 kb
subregions, linked in tandem (Siebenlist, et al. (1981),
Nature, 294, 631-635). Each subregion contains up to 10
individual D segments. Some of these segments have been
mapped and are shown in Fig. 4.' Two different strategies are
,used to generate a mini-locus D region. The first strategy
involves using only those D segments located in a short
contiguous stretch of DNA that includes one or two of the
repeated D subregions. A candidate is a single 15 kb fragment
that contains 12 individual D segments. This piece of DNA
consists of 2 contiguous EcoRI fragments and has been
completely sequenced (Ichihara, et al. (1988), EMBO J.,
4141-4150). Twelve D segments should be sufficient for a
primary repertoire. However, given the dispersed nature of
the D region, an alternative strategy is to ligate together
several non-contiguous D-segment containing fragments, to
produce a smaller piece of DNA with a greater number of
segments. Additional D-segment genes can be identified, for
example, by the presence of characteristic flanking nonamer
and heptamer sequences, supra, and by reference to the
literature.
At least one, and preferably more than one V gene
segment is used to construct the heavy chain minilocus
transgene. Rearranged or unrearranged V segments with or
without flanking sequences can be isolated as described in
WO 92/03918 and WO 93/12227.
CA 02161351 2003-10-23
54
Rearranged or unrearranged V segments, D segments, J
segments, and C genes, with or without flanking sequences, can
be isolated as described in WO 92/03918.
A minilocus light chain transgene may be similarly
constructed from the human X or K immunoglobulin locus.
Thus, for example, an immunoglobulin heavy chain minilocus
transgene construct, e.g., of about 75 kb, encoding V, D, J
and constant region sequences can be formed from a plurality
of DNA fragments, with each sequence being substantially
homologous to human gene sequences. Preferably, the sequences
are operably linked to transcription regulatory sequences and
are capable of undergoing rearrangement. With two or more
appropriately placed constant region sequences (e.g., and y)
and switch regions, switch recombination also occurs. An
exemplary light chain transgene construct can be formed
similarly from a plurality of DNA fragments, substantially
homologous to human DNA and capable of undergoing
rearrangement, as described in WO 92/01918.
E. Transgene Constructs Capable of Isotype Switching
Ideally, transgene constructs that are intended to
undergo class switching should include all of the cis-acting
sequences necessary to regulate sterile transcripts.
Naturally occurring switch regions and upstream promoters and
regulatory sequences (e.g., IFN-inducible elements) are
preferred cis-acting sequences that are included in transgene
constructs capable of isotype switching. About at least 50
basepairs, preferably about at least 200 basepairs, and more
preferably at least 500 to 1000 basepairs or more of sequence
immediately upstream of a switch region, preferably a human -yl
switch region, should be operably linked to a switch sequence,
preferably a human yi switch sequence. Further, switch
regions can be linked upstream of (and adjacent to) CH genes
that do not naturally occur next to the particular switch
region. For example, but not for limitation, a human yl
WO 94/25585 PCT/US94104580
46 55
switch region may be linked upstream from a human a2 CH gene,
or a murine 71 switch may be linked to a human CH gene.
An alternative method for obtaining non-classical
isotype switching (e.g., 6-associated deletion) in transgenic
mice involves the inclusion of the 400 bp direct repeat
sequences (ag and e ) that flank the human p gene (Yasui et
al., Eur. J. Immunol. L9:1399 (1989)). Homologous
recombination between these two sequences deletes the g gene
in IgD-only B-cells. Heavy chain transgenes can be
represented by the following formulaic description:
I (D) (T) - (SA) p- (C2) where:
VH is a heavy chain variable region gene segment,
D is a heavy chain D (diversity) region gene segment,
J. is a heavy chain J (joining) region gene segment,
SD is a donor region segment capable of participating in
a recombination event with the Sa acceptor region
segments such that isotype switching occurs,
Cl is a heavy chain constant region gene segment encoding
an isotype utilized in for B cell development (e.g.,
g or 6),
T is a cis-acting transcriptional regulatory region
segment containing at least a promoter,
SA is an acceptor region segment capable of participating
in a recombination event with selected SD donor
region segments, such that isotype switching occurs,
C2 is a heavy chain constant region gene segment encoding
an isotype other than (e.g., 71, 72' 73, 74, all
a2 l 0-
x, y, z, m, n, p, and q are integers. x is 1-100, n is
0-10, y is 1-50, p is 1-10, z is 1-50, q is 0-50, m
is 0-10. Typically, when the transgene is capable
of isotype switching, q must be at least 1, m is at
least 1, n is at least 1, and m is greater than or
equal to n.
WO 94/25585 160) 351 PCT/US94/04580
56
_.VH, D, JH' SD, C1, T, SA, and CZ segments may be
selected from various species, preferably mammalian species,
and more preferably from human and murine germline DNA.
VH segments may be selected from various species,
but are preferably selected from VH segments that occur
naturally in the human germline, such as VH251= Typically
about 2 VH gene segments are included, preferably about 4 VH
segments are included, and most preferably at least about 10
VH segments are included.
At least one D segment is typically included,
although at least 10 D segments are preferably included, and
some embodiments include more than ten D segments. Some
preferred embodiments include human D segments.
Typically at least one JH segment is incorporated in
the transgene, although it is preferable to include about six
JH segments, and some preferred embodiments include more than
about six JH segments. Some preferred embodiments include
human JH segments, and further preferred embodiments include
six human JH segments and no nonhuman JH segments.
SD segments are donor regions capable of
participating in recombination events with the SA segment of
the transgene. For classical isotype switching, SD and SA are
switch regions such as S , 5x,1, 5,y2, 5,y3, S,y4, Sot, Sot2,, and Sc.
Preferably the switch regions are murine or human, more
preferably SD is a human or murine S and SA is a human or
murine S. . For nonclassical isotype switching (6-associated
deletion), SD and SA are preferably the 400 basepair direct
repeat sequences that flank the human gene.
C1 segments are typically a or 6 genes, preferably a
gene, and more preferably a human or murine gene.
T segments typically include S' flanking sequences
that are adjacent to naturally occurring (i.e., germline)
switch regions. T segments typically at least about at least
50 nucleotides in length, preferably about at least 200
nucleotides in length, and more preferably at least 500-1000
nucleotides in length. Preferably T segments are 5' flanking
sequences that occur immediately upstream of human or murine
switch regions in a germline configuration. It is also
WO 94/25585 PCT/US94/04580
57 2191351
evident to those of skill in the art that T segments may
comprise cis-acting transcriptional regulatory sequences that
do not occur naturally in an animal germline (e.g., viral
enhancers and promoters such as those found in SV40,
adenovirus, and other viruses that infect eukaryotic cells).
C2 segments are typically a 7,, 72, 731' 741 Cell C'211
or E CH gene, preferably a human CH gene of these isotypes, and
more preferably a human 71 or 73 gene. Murine yea and 72b may
also be used, as may downstream (i.e., switched) isotype genes
form various species. Where the heavy chain transgene
contains an immunoglobulin heavy chain minilocus, the total
length of the transgene will be typically 150 kilo basepairs
or less.
In general, the transgene will be other than a
native heavy chain Ig locus. Thus, for example, deletion of
unnecessary regions or substitutions with corresponding
regions from other species will be present.
F. Methods for Determining Functional
Isotype Switching in Ig Transgenes
The occurrence of isotype switching in a transgenic
nonhuman animal may be identified by any method known to those
in the art. Preferred embodiments include the following,
employed either singly or in combination:
1. detection of mRNA transcripts that contain a sequence
homologous to at least one transgene downstream CH gene other
than d and an adjacent sequence homologous to a transgene VH-
DH-JH rearranged gene; such detection may be by Northern
hybridization, S1 nuclease protection assays, PCR
amplification, cDNA cloning, or other methods;
2. detection in the serum of the transgenic animal, or in
supernatants of cultures of hybridoma cells made from B-cells
of the transgenic animal, of immunoglobulin proteins encoded
by downstream CH genes, where such proteins can also be shown
by immunochemical methods to comprise a functional variable
region;
3. detection, in DNA from B-cells of the transgenic
animal or in genomic DNA from hybridoma cells, of DNA
WO 94/25585 PCT/US94/04580
ffil 58
rearrangements consistent with the occurrence of isotype
switching in the transgene, such detection may be accomplished
by Southern blot hybridization, PCR amplification, genomic
cloning, or other method; or
4. identification of other indicia of isotype switching,
such as production of sterile transcripts, production of
characteristic enzymes involved in switching (e.g., "switch
recombinase"), or other manifestations that may be detected,
measured, or observed by contemporary techniques.
Because each transgenic line may represent a
different site of integration of the transgene, and a
potentially different tandem array of transgene inserts, and
because each different configuration of transgene and flanking
DNA sequences can affect gene expression, it is preferable to
identify and use lines of mice that express high levels of
human immunoglobulins, particularly of the IgG isotype, and
contain the least number of copies of the transgene. Single
copy transgenics minimize the potential problem of incomplete
allelic expression. Transgenes are typically integrated into
host chromosomal DNA, most usually into germline DNA and
propagated by subsequent breeding of germline transgenic
breeding stock animals. However, other vectors and transgenic
methods known in the present art or subsequently developed may
be substituted as appropriate and as desired by a
practitioner.
Trans-switching to endogenous nonhuman heavy chain
constant region genes can occur and produce chimeric heavy
chains and antibodies comprising such chimeric human/mouse
heavy chains. Such chimeric antibodies may be desired for
certain uses described herein or may be undesirable.
G. Functional Disruption of
Endogenous Immunoglobulin Loci
The expression of successfully rearranged
immunoglobulin heavy and light transgenes is expected to have
a dominant effect by suppressing the rearrangement of the
endogenous immunoglobulin genes in the transgenic nonhuman
animal. However, another way to generate a nonhuman that is
devoid of endogenous antibodies is by mutating the endogenous
WO 94/25585 PCTIUS94/04580
j161351
immunoglobulin loci. Using embryonic stem cell technology and
homologous recombination, the endogenous immunoglobulin
repertoire can be readily eliminated. The following describes
the functional description of the mouse immunoglobulin loci.
The vectors and methods disclosed, however, can be readily
adapted for use in other non-human animals.
Briefly, this technology involves the inactivation
of a gene, by homologous recombination, in a pluripotent cell
line that is capable of differentiating into germ cell tissue.
A DNA construct that contains an altered, copy of a mouse
immunoglobulin gene is introduced into the nuclei of embryonic
stem cells. In a portion of the cells, the introduced DNA
recombines with the endogenous copy of the mouse gene,
replacing it with the altered copy. Cells containing the
newly engineered genetic lesion are injected into a host mouse
embryo, which is reimplanted into a recipient female. Some of
these embryos develop into chimeric mice that possess germ
cells entirely derived from the mutant cell line. Therefore,
by breeding the chimeric mice it is possible to obtain a new
line of mice containing the introduced genetic lesion
(reviewed by Capecchi (1989), Science, 244, 1288-1292).
Because the mouse X locus contributes to only 5% of
the immunoglobulins, inactivation of the heavy chain and/or
K-light chain loci is sufficient. There are three ways to
disrupt each of these loci, deletion of the J region, deletion
of the J-C intron enhancer, and disruption of constant region
coding sequences by the introduction of a stop codon. The
last option is the most straightforward, in terms of DNA
construct design. Elimination of the g gene disrupts B-cell
maturation thereby preventing class switching to any of the
functional heavy chain segments. The strategy for knocking
out these loci is outlined below.
To disrupt the mouse and !c genes, targeting
vectors are used based on the design employed by Jaenisch and
co-workers (Zijistra, et al. (1989), Nature, 342, 435-438) for
the successful disruption of the mouse (32-microglobulin gene.
The neomycin resistance gene (neo), from the plasmid pMClneo
is inserted into the coding region of the target gene. The
WO ~ PCTNS94/04580
pMClneo insert uses a hybrid viral promoter/enhancer sequence
to drive neo expression. This promoter is active in embryonic
stem cells. Therefore, neo can be used as a selectable marker
for integration of the knock-out construct. The HSV thymidine
5 kinase (tk) gene is added to the end of the construct as a
negative selection marker against random insertion events
(Zijlstra, et al., supra.).
A preferred strategy for disrupting the heavy chain
locus is the elimination of the J region. This region is
10 fairly compact in the mouse, spanning only 1.3 kb. To
construct a gene targeting vector, a 15 kb KpnI fragment
containing all of the secreted A constant region exons from
mouse genomic library is isolated. The 1.3 kb J region is
replaced with the 1.1 kb insert from pMClneo. The HSV tk gene
15 is then added to the 5' end of the KpnI fragment. Correct
integration of this construct, via homologous recombination,
will result in the replacement of the mouse JH region with the
neo gene. Recombinants are screened by PCR, using a primer
based on the neo gene and a primer homologous to mouse
20 sequences 5' of the KpnI site in the D region.
Alternatively, the heavy-chain locus is knocked out
by disrupting the coding region of the gene. This approach
involves the same 15 kb KpnI fragment used in the previous
approach. The 1.1 kb insert from pMClneo is inserted at a
25 unique BamHI site in exon II, and the HSV tk gene added to the
3' KpnI end. Double crossover events on either side of the
neo insert, that eliminate the tk gene, are then selected for.
These are detected from pools of selected clones by PCR
amplification. One of the PCR primers is derived from neo
30 sequences and the other from mouse sequences outside of the
targeting vector. The functional disruption of the mouse
immunoglobulin loci is presented in the Examples.
35 G. Suppressing Expression of
Endogenous Immunoglobulin Loci
In addition to functional disruption of endogenous
Ig loci, an alternative method for preventing the expression
of an endogenous Ig locus is suppression. Suppression of
CA 02161351 2003-10-23
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endogenous Ig genes may be accomplished with antisense RNA
produced from one or more integrated transgenes, by antisense
oligonucleotides, and/or by administration of antisera
specific for one or more endogenous Ig chains.
Antisense Polynucleotides
Antisense RNA transgenes can be employed to
partially or totally knock-out expression of specific genes
(Pepin et al. (1991) Nature 355: 725; Helene., C. and Toulme,
J. (1990) Biochimica Biophys. Acta 1049: 99; Stout, J. and
Caskey, T. (1990) Somat. Cell Mol. Genet. 16: 369; Munir et
al. (1990) Somat. Cell Mol. Genet. 16: 383).
"Antisense polynucleotides" are polynucleotides
that: (1) are complementary to all or part of a reference
sequence, such as a sequence of an endogenous Ig CH or,CL
region, and (2) which specifically hybridize to a
complementary target sequence, such as a chromosomal gene
locus or a Ig mRNA. Such complementary antisense
polynucleotides may include nucleotide substitutions,
additions, deletions, or transpositions, so long as specific
hybridization to the relevant target sequence is retained as a
functional property of the polynucleotide. Complementary
antisense polynucleotides include soluble antisense RNA or DNA
oligonucleotides which can hybridize specifically to
individual mRNA species and prevent transcription and/or RNA
processing of the mRNA species and/or translation of the
encoded polypeptide (Ching et al., Proc. Natl. Acad. Sci.
U.S.A. 86:10006-10010 (1989); Broder et al., Ann. Int. Med.
113:604-618 (1990); Loreau et al., FEBS Letters 274:53-56
(1990); Holcenberg et al., W091/11535; U.S. 5,256,643
("New human CRIPTO gene"); W091/09865; W091/04753; W090/13641;
and EP 386563).
An antisense sequence is a polynucleotide
sequence that is complementary to at least one immunoglobulin
gene sequence of at least about 15 contiguous nucleotides in
length, typically at least 20 to 30 nucleotides in length, and
preferably more than about 30 nucleotides in length. However,
WO 94125585 PCT/US94/04580
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in some embodiments, antisense sequences may have
substitutions, additions, or deletions as compared to the
complementary immunoglobulin gene sequence, so long as
specific hybridization is retained as a property of the
antisense polynucleotide. Generally, an antisense sequence is
complementary to an endogenous immunoglobulin gene sequence
that encodes, or has the potential to encode after DNA
rearrangement, an immunoglobulin chain. In some cases, sense
sequences corresponding to an immunoglobulin gene sequence may
function to suppress expression, particularly by interfering
with transcription.
The antisense polynucleotides therefore inhibit
production of the encoded polypeptide(s). In this regard,
antisense polynucleotides that inhibit transcription and/or
translation of one or more endogenous Ig loci can alter the
capacity and/or specificity of a non-human animal to produce
immunoglobulin chains encoded by endogenous Ig loci.
Antisense polynucleotides may be produced from a
heterologous expression cassette in a transfectant cell or
transgenic cell, such as a transgenic pluripotent
hematopoietic stem cell used to reconstitute all or part of
the hematopoietic stem cell population of an individual, or a
transgenic nonhuman animal. Alternatively, the antisense
polynucleotides may comprise soluble oligonucleotides that are
administered to the external milieu, either in culture medium
in vitro or in the circulatory system or interstitial fluid in
vivo. Soluble antisense polynucleotides present in the
external milieu have been shown to gain access to the
cytoplasm and inhibit translation of specific mRNA species. In
some embodiments the antisense polynucleotides comprise
methylphosphonate moieties, alternatively phosphorothiolates
or O-methylribonucleotides may be used, and chimeric
oligonucleotides may also be used (Dagle et al. (1990) Nucleic
Acids Res. 18: 4751). For some applications, antisense
oligonucleotides may comprise polyamide nucleic acids (Nielsen
et al. (1991) Science 254: 1497). For general methods
relating to antisense polynucleotides, see Antisense RNA and
CA 02161351 2003-10-23
63
DNA, (1988), D.A. Melton, Ed., Cold Spring Harbor Laboratory,
Cold Spring Harbor, NY).
Antisense polynucleotides complementary to one or
more sequences are employed to inhibit transcription, RNA
processing, and/or translation of the cognate mRNA species and
thereby effect a reduction in the amount of the respective
encoded polypeptide. Such antisense polynucleotides can
provide a therapeutic function by inhibiting the formation of
one or more endogenous Ig chains in vivo.
Whether as soluble antisense oligonucleotides or as
antisense RNA transcribed from an antisense transgene, the
antisense polynucleotides of this invention are selected so as
to hybridize preferentially to endogenous Ig sequences at
physiological conditions in vivo. Most typically, the
selected antisense polynucleotides will not appreciably
hybridize to heterologous Ig sequences encoded by a heavy or
light chain transgene of the invention (i.e., the antisense
oligonucleotides will not inhibit transgene Ig expression by
more than about 25 to 35 percent).
20,
Antiserum Suppression
Partial or complete suppression of endogenous Ig
chain expression can be produced by injecting mice with
antisera against one or more endogenous Ig chains (Weiss et
al. (1984) Proc. Natl. Acad. Sci. (U.S.A.) 81 211).
Antisera are selected so
as to react specifically with one or more endogenous (e.g.,
murine) Ig chains but to have minimal or no cross-reactivity
with heterologous Ig chains encoded by an Ig transgene of the
invention. Thus, administration of selected antisera
according to a schedule as typified by that of Weiss et al.
op.cit. will suppress endogenous Ig chain expression but
permits expression of heterologous Ig chain(s) encoded by a
transgene of the present invention. Suitable antibody sources
for antibody comprise:
(1) monoclonal antibodies, such as a monoclonal
antibody that specifically binds to a murine A, y, K, or X
WO 94/25585 PCT/US94/04580
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chains but does not react with the human immunoglobulin
chain(s) encoded by a human Ig transgene of the invention;
(2) mixtures of such monoclonal antibodies, so that
the mixture binds with multiple epitopes on a single species
of endogenous Ig chain, with multiple endogenous Ig chains
(e.g., murine and murine y, or with multiple epitopes and
multiple chains or endogenous immunoglobulins;
(3) polyclonal antiserum or mixtures thereof,
typically such antiserum/antisera is monospecific for binding
to a single species of endogenous Ig chain (e.g., murine ,
murine y, murine K, murine X) or to multiple species of
endogenous Ig chain, and most preferably such antisera
possesses negligible binding to human immunoglobulin chains
encoded by a transgene of the invention; and/or
(4) a mixture of polyclonal antiserum and monoclonal
antibodies binding to a single or multiple species of
endogenous Ig chain, and most preferably possessing negligible
binding to human immunoglobulin chains encoded by a transgene
of the invention. Generally, polyclonal antibodies are
preferred, and such substantially monospecific polyclonal
antibodies can be advantageously produced from an antiserum
raised against human immunoglobulin(s) by pre-adsorption with
antibodies derived from the nonhuman animal species (e.g.,
murine) and/or, for example, by affinity chromatography of the
antiserum or purified fraction thereof on an affinity resin
containing immobilized human Ig (wherein the bound fraction is
enriched for the desired anti-human Ig in the antiserum; the
bound fraction is typically eluted with conditions of low pH
or a chaotropic salt solution).
Cell separation and/or complement fixation can be
employed to provide the enhancement of antibody-directed cell
depletion of lymphocytes expressing endogenous (e.g., murine)
immunoglobulin chains. In one embodiment, for example,
antibodies are employed for ex vivo depletion of murine Ig-
expressing explanted hematopoietic cells and/or B-lineage
lymphocytes obtained from a transgenic mouse harboring a human
Ig transgene. Thus, hematopoietic cells and/or B-lineage
lymphocytes are explanted from a transgenic nonhuman animal
WO 94/25585 PCTIUS94/04580
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harboring a human Ig transgene (preferably harboring both a
human heavy chain transgene and a human light chain transgene)
and the explanted cells are incubated with an antibody (or
antibodies) which (1) binds to an endogenous immunoglobulin
(e.g., murine and/or K) and (2) lacks substantial binding to
human immunoglobulin chains encoded by the transgene(s). Such
antibodies are referred to as "suppression antibodies" for
clarity. The explanted cell population is selectively
depleted of cells which bind to the suppression antibody(ies);
such depletion can be accomplished by various methods, such as
(1) physical separation to remove suppression antibody-bound
cells from unbound cells (e.g., the suppression antibodies may
be bound to a solid support or magnetic bead to immobilize and
remove cells binding to the suppression antibody), (2)
antibody-dependent cell killing of cells bound by the
suppression antibody (e.g., by ADCC, by complement fixation,
or by a toxin linked to the suppression antibody), and (3)
clonal anergy induced by the suppression antibody, and the
like.
Frequently, antibodies used for antibody suppression
of endogenous Ig chain production will be capable of fixing
complement. It is frequently preferable that such antibodies
may be selected so as to react well with a convenient
complement source for ex vivo/in vitro depletion, such as
rabbit or guinea pig complement. For in vivo depletion, it is
generally preferred that the suppressor antibodies possess
effector functions in the nonhuman transgenic animal species;
thus, a suppression antibody comprising murine effector
functions (e.g., ADCC and complement fixation) generally would
be preferred for use in transgenic mice.
In one variation, a suppression antibody that
= specifically binds to a predetermined endogenous
immunoglobulin chain is used for ex vivo/in vitro depletion
of lymphocytes expressing an endogenous immunoglobulin. A
cellular explant (e.g., lymphocyte sample) from a transgenic
nonhuman animal harboring a human immunoglobulin transgene is
contacted with a suppression antibody and cells specifically
binding to the suppression antibody are depleted (e.g., by
WO 94/25585 PCT/US94/04580
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immobilization, complement fixation, and the like), thus
generating a cell subpopulation depleted in cells expressing
endogenous (nonhuman) immunoglobulins (e.g., lymphocytes
expressing murine Ig). The resultant depleted lymphocyte
population (T cells, human Ig-positive B-cells, etc.) can be
transferred into a immunocompatible (i.e., MHC-compatible)
nonhuman animal of the same species and which is substantially
incapable of producing endogenous antibody (e.g., SCID mice,
RAG-1 or RAG-2 knockout mice). The reconstituted animal
(mouse) can then be immunized with an antigen (or reimmunized
with an antigen used to immunize the donor animal from which
the explant was obtained) to obtain high-affinity (affinity
matured) antibodies and B-cells producing such antibodies.
Such B-cells may be used to generate hybridomas by
conventional cell fusion and screened. Antibody suppression
can be used in combination with other endogenous Ig
inactivation/suppression methods (e.g., J. knockout, C.
knockout, D-region ablation, antisense suppression,
compensated frameshift inactivation).
Complete Endogenous Ig Locus Inactivation
In certain embodiments, it is desirable to effect
complete inactivation of the endogenous Ig loci so that hybrid
immunoglobulin chains comprising a human variable region and a
non-human (e.g., murine) constant region cannot be formed
(e.g., by trans-switching between the transgene and endogenous
Ig sequences). Knockout mice bearing endogenous heavy chain
alleles with are functionally disrupted in the J. region only
frequently exhibit trans-switching, typically wherein a
rearranged human variable region (VDJ) encoded by a transgene
is expressed as a fusion protein linked to an endogenous
murine constant region, although other trans-switched
junctions are possible. To overcome this potential problem,
it is generally desirable to completely inactivate the
endogenous heavy chain locus by any of various methods,
including but not limited to the following: (1) functionally
disrupting and/or deleting by homologous recombination at
least one and preferably all of the endogenous heavy chain
WO 94/25585 PCT/US94/04580
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constant region genes, (2) mutating at least one and
preferably all of the endogenous heavy chain constant region
genes to encode a termination codon (or frameshift) to produce
a truncated or frameshifted product (if trans-switched), and
other methods and strategies apparent to those of skill in the
art. Deletion of a substantial portion or all of the heavy
chain,constant region genes and/or D-region genes may be
accomplished by various methods, including sequential deletion
by homologous recombination targeting vectors, especially of
the "hit-and-run" type and the like. Similarly, functional
disruption and/or deletion of at least one endogenous light
chain locus (e.g., K) to ablate endogenous light chain
constant region genes is often preferable.
Frequently, it is desirable to employ a frameshifted
transgene wherein the heterologous transgene comprises a
frameshift in the J segment(s) and'a compensating frameshift
(i.e., to regenerate the original reading frame) in the
initial region (i.e., amino-terminal coding portion) of one or
more (preferably all) of the transgene constant region genes.
Trans-switching to an endogenous IgH locus constant gene
(which does not comprise a compensating frameshift) will
result in a truncated or missense product that results in the
trans-switched B cell being deleted or non-selected, thus
suppressing the trans-switched phenotype.
Antisense suppression and antibody suppression may
also be used to effect a substantially complete functional
inactivation of endogenous Ig gene product expression (e.g.,
murine heavy and light chain sequences) and/or trans-switched
antibodies (e.g., human variable/murine constant chimeric
antibodies).
Various combinations of the inactivation and
suppression strategies may be used to effect essentially total
suppression of endogenous (e.g., murine) Ig chain expression.
Trans-Switching
In some variations, it may be desirable to produce a
trans-switched immunoglobulin. For example, such trans-
switched heavy chains can be chimeric (i.e., a non-murine
WO 94/25585 c I l 3 5 1 PCT/US94/04580
68
(h'uman) variable region and a murine constant region).
Antibodies comprising such chimeric trans-switched
immunoglobulins can be used for a variety of applications
where it is desirable to have a non-human (e.g., murine)
constant region (e.g., for retention of effector functions in
the host, for the presence of murine immunological
determinants such as for binding of a secondary antibody which
does not bind human constant regions). For one example, a
human variable region repertoire may possess advantages as
compared to the murine variable region repertoire with respect
to certain antigens. Presumably the human VH, D, JH, VL, and
JL genes have been selected for during evolution for their
ability to encode immunoglobulins that bind certain
evolutionarily important antigens; antigens which provided
evolutionary selective pressure for the murine repertoire can
be distinct from those antigens which provided evolutionary
pressure to shape the human repertoire. Other repertoire
adavantages may exist, making the human variable region
repertoire advantageous when combined with a murine constant
region (e.g., a trans-switched murine) isotype. The presence
of a murine constant region can afford advantages over a human
constant region. For example, a murine 'y constant region
linked to a human variable region by trans-switching may
provide an antibody which possesses murine effector functions
(e.g., ADCC, murine complement fixation) so that such a
chimeric antibody (preferably monoclonal) which is reactive
with a predetermined antigen (e.g., human IL-2 receptor) may
be tested in a mouse disease model, such as a mouse model of
graft-versus-host disease wherein the T lymphocytes in the
mouse express a functional human IL-2 receptor. Subsequently,
the human variable region encoding sequence may be isolated
(e.g., by PCR amplification or cDNA cloning from the source
(hybridoma clone)) and spliced to a sequence encoding a
desired human constant region to encode a human sequence
antibody more suitable for human therapeutic uses where
immunogenicity is preferably minimized. The polynucleotide(s)
having the resultant fully human encoding sequence(s) can be
expressed in a host cell (e.g., from an expression vector in a
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mammalian cell) and purified for pharmaceutical formulation.
For some applications, the chimeric antibodies may be used
directly without replacing the murine constant region with a
human constant region. Other variations and uses of trans-
switched chimeric antibodies will be evident to those of skill
in the art.
The present invention provides transgenic nonhuman
animals containing B lymphocytes which express chimeric
antibodies, generally resulting from trans-switching between a
human heavy chain transgene and an endogenous murine heavy
chain constant region gene. Such chimeric antibodies comprise
a human sequence variable region and a murine constant region,
generally a murine switched (i.e., non- , non-6) isotype. The
transgenic nonhuman animals capable of making chimeric
antibodies to a predetermined antigen are usually also
competent to make fully human sequence antibodies if both
human heavy chain and human light chain transgenes encoding
human variable and human constant region genes are integrated.
Most typically, the animal is homozygous for a functionally
disrupted heavy chain locus and/or light chain locus but
retains one or more endogenous heavy chain constant region
gene(s) capable of trans-switching (e.g., y,a, e) and
frequently retains a cis-linked enhancer. Such a mouse is
immunized with a predetermined antigen, usually in combination
with an adjuvant, and an immune response comprising a
detectable amount of chimeric antibodies comprising heavy
chains composed of human sequence variable regions linked to
murine constant region sequences is produced. Typically, the
serum of such an immunized animal can comprise such chimeric
antibodies at concentrations of about at least 1 g/ml, often
about at least 10 g/ml, frequently at least 30 g/m1, and up
to 50 to 100 g/ml or more. The antiserum containing
antibodies comprising chimeric human variable/mouse constant
region heavy chains typically also comprises antibodies which
comprise human variable/human constant region (complete human
sequence) heavy chains. Chimeric trans-switched antibodies
usually comprise (1) a chimeric heavy chain composed of a
human variable region and a murine constant region (typically
WO 94/25585 PCTIUS94/04580
t.-'` 70
a murine gamma) and (2) a human transgene-encoded light chain
(typically kappa) or a murine light chain (typically lambda in
a kappa knockout background). Such chimeric trans-switched
antibodies generally bind to a predetermined antigen (e.g.,
the immunogen) with an affinity of about at least 1 x 107 M-1,
preferably with an affinity of about at least 5 x 107 M-1, more
preferably with an affinity of at least 1 x 108 M-1 to 1 x 109
M'1 or more. Frequently, the predetermined antigen is a human
protein, such as for example a human cell surface antigen
(e.g., CD4, CDB, IL-2 receptor, EGF receptor, PDGF receptor),
other human biological macromolecule (e.g., thrombomodulin,
protein C, carbohydrate antigen, sialyl Lewis antigen, L-
selectin), or nonhuman disease associated macromolecule (e.g.,
bacterial LPS, virion capsid protein or envelope glycoprotein)
and the like.
The invention provides transgenic nonhuman animals
comprising a genome comprising: (1) a homozygous functionally
disrupted endogenous heavy chain locus comprising at least one
murine constant region gene capable of trans-switching (e.g.,
in cis linkage to a functional switch recombination sequence
and typically to a functional enhancer), (2) a human heavy
chain transgene capable of rearranging to encode end express a
functional human heavy chain variable region and capable of
trans-switching (e.g., having a cis-linked RSS); optionally
further comprising (3) a human light chain (e.g., kappa)
transgene capable of rearranging to encode a functional human
light chain variable region and expressing a human sequence
light chain; optionally further comprising (4) a homozygous
functionally disrupted endogenous light chain locus (K,
preferably K and X); and optionally further comprising (5) a
serum comprising an antibody comprising a chimeric heavy chain
composed of a human sequence variable region encoded by a
human transgene and a murine constant region sequence encoded
by an endogenous murine heavy chain constant region gene =
(e.g., 71, 72a, 72b, y3).
Such transgenic mice may further comprise a serum
comprising chimeric antibodies which bind a predetermined
human antigen (e.g., CD4, CD8, CEA) with an affinity of about
WO 94/25585 PCT/US94/04580
71 2161351
at least 1 x 104 M-1, preferably with an affinity of about at
least 5 x 104 M-1, more preferably with an affinity of at least
1 x 107 M-1 to 1 x 109 M-1 or more. Frequently, hybridomas can
be made wherein the monoclonal antibodies produced thereby
have an affinity of at least 8 X107 M-1. Chimeric antibodies
comprising a heavy chain composed of a murine constant region
and a human variable region, often capable of binding to a
nonhuman antigen, may also be present in the serum or as an
antibody secreted from a hybridoma.
Generally, such chimeric antibodies can be generated
by trans-switching, wherein a human transgene encoding a human
variable region (encoded by productive V-D-J rearrangement in
vivo) and a human constant region, typically human ,
undergoes switch recombination with a non-transgene
immunoglobulin constant gene switch sequence (RSS) thereby
operably linking the transgene-encoded human variable region
with a heavy chain constant region which is not encoded by
said transgene, typically an endogenous murine immunoglobulin
heavy chain constant region or a heterologous (e.g., human)
heavy chain constant region encoded on a second transgene.
Whereas cis-switching refers to isotype-switching by
recombination of RSS elements within a transgene, trans-
switching involves recombination between a transgene RSS and
an RSS element outside the transgene, often on a different
chromosome than the chromosome which harbors the transgene.
Trans-switching generally occurs between an RSS of
an expressed transgene heavy chain constant region gene and
either an RSS of an endogenous murine constant region gene (of
a non-g isotype, typically y) or an RSS of a human constant
region gene contained on a second transgene, often integrated
on a separate chromosome.
When trans-switching occurs between an RSS of a
first, expressed transgene heavy chain constant region gene
= (e.g., g) and an RSS of a human heavy chain constant region
gene contained on a second transgene, a non-chimeric antibody
having a substantially fully human sequence is produced. For
example and not limitation, a polynucleotide encoding a human
heavy chain constant region (e.g., 71) and an operably linked
WO 94/25585 a I L 35 , PCT/US94/04580
72 .10
RSS (e.g., a y1 RSS) can be introduced (e.g., transfected)
into a population of hybridoma cells generated from a
transgenic mouse B-cell (or B cell population) expressing an
antibody comprising a transgene-encoded human chain. The
resultant hybridoma cells can be selected for the presence of
the introduced polynucleotide and/or for the expression of
trans-switched antibody comprising a heavy chain having the
variable region (idiotype/antigen reactivity) of the human
chain and having the constant region encoded by the introduced
polynucleotide sequence (human yi). Trans-switch
recombination between the RSS of the transgene-encoded human
chain and the RSS of the introduced polynucleotide encoding a
downstream isotype (e.g., 71) thereby can generate a trans-
switched antibody.
The invention also provides a method for producing
such chimeric trans-switched antibodies comprising the step of
immunizing with a predetermined antigen a transgenic mouse
comprising a genome comprising: (1) a homozygous functionally
disrupted endogenous heavy chain locus comprising at least one
murine constant region gene capable of trans-switching (e.g.,
72a, 72b, yi, y3), (2) a human heavy chain transgene capable
of rearranging to encode a functional human heavy chain
variable region and expressing a human sequence heavy chain
and capable of undergoing isotype switching (and/or trans-
switching), and optionally further comprising (3) a human
light chain (e.g., kappa) transgene capable of rearranging to
encode a functional human light (e.g., kappa) chain variable
region and expressing a human sequence light chain, and
optionally further comprising (4) a homozygous functionally
disrupted endogenous light chain locus (typically K,
preferably both K and X), and optionally further comprising
(5) a serum comprising an antibody comprising a chimeric heavy
chain composed of a human sequence variable region encoded by
a human transgene and a murine constant region sequence
encoded by an endogenous murine heavy chain constant region
gene (e.g., 71, 72a, 72b, 73).
Affinity Tagging: Selecting for Switched Isotopes
WO 94/25585 PCT/US94/04580
= 73 2 i 6 135 1
Advantageously, trans-switching (and cis-switching)
is associated with the process of somatic mutation. Somatic
mutation expands the range of antibody affinities encoded by
clonal progeny of a B-cell. For example, antibodies produced
5. by hybridoma cells which have undergone switching (trans- or
cis-) represent a broader range of antigen-binding affinities
than is present in hybridoma cells which have not undergone
switching. Thus, a hybridoma cell population (typically
clonal) which expresses a first antibody comprising a heavy
chain comprising a first human heavy chain variable region in
polypeptide linkage to a first human heavy chain constant
region (e.g., p) can be screened for hybridoma cell clonal
variants which express an antibody comprising a heavy chain
containing said first human heavy chain variable region in
polypeptide linkage to a second heavy chain constant region
(e.g., a human y, a, or e constant region). Such clonal
variants can be produced by natural clonal variation producing
cis-switching in vitro, by induction of class switching
(trans- or cis-) as through the administration of agents that
promote isotype switching, such as T-cell-derived lymphokines
(e.g., IL-4 and IFNY) , by introduction of a polynucleotide
comprising a functional RSS and a heterologous (e.g. human)
heavy chain constant region gene to serve as a substrate for
trans-switching, or by a combination of the above, and the
like.
Class switching and affinity maturation take place
within the same population of B cells derived from transgenic
animals of the present invention. Therefore, identification
of class-switched B cells (or hybridomas derived therefrom)
can be used as a screening step for obtaining high affinity
monoclonal antibodies. A variety of approaches can be
employed to facilitate class switching events such as cis-
switching (intratransgene switching), trans-switching, or
both. For example, a single continuous human genomic fragment
comprising both and y constant region genes with the
associated RSS elements and switch regulatory elements (e.g.,
sterile transcript promoter) can be used as a transgene.
However, some portions of the desired single contiguous human
WO 94/25585 PCT/US94/04580
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gerlo is fragment can be difficult to clone efficiently, such
as due to instability problems when replicated in a cloning
host or the like; in particular, the region between 6 and y3
can prove difficult to clone efficiently, especially as a
contiguous fragment comprising the gene, 73 gene, a V gene,
D gene segments, and J gene segments.
Also for example, a discontinuous human transgene
(minigene) composed of a human gene, human 73 gene, a human
V gene(s), human D gene segments, and human J gene segments,
with one or more deletions of an intervening (intronic) or
otherwise nonessential sequence (e.g., one or more V, D,
and/or J segment and/or one or more non- constant region
gene(s)). Such minigenes have several advantages as compared
to isolating a single contiguous segment of genomic DNA
spanning all of the essential elements for efficient
immunoglobulin expression and switching. For example, such a
minigene avoids the necessity of isolating large pieces of DNA
which may contain sequences which are difficult to clone
(e.g., unstable sequences, poison sequences, and the like).
Moreover, miniloci comprising elements necessary for isotype
switching (e.g., human y sterile transcript promoter) for
producing cis- or trans-switching, can advantageously undergo
somatic mutation and class switching in vivo. As many
eukaryotic DNA sequences can prove difficult to clone,
omitting non-essential sequences can prove advantageous.
In a variation, hybridoma clones producing
antibodies having high binding affinity (e.g., at least 1 x
107 M-1, preferably at least 1 x 108 M-1, more preferably at
least 1 x 109 M-1 or greater) are obtained by selecting, from a
pool of hybridoma cells derived from B cells of transgenic
mice harboring a human heavy chain transgene capable of
isotype switching (see, supra) and substantially lacking
endogenous murine heavy chain loci capable of undergoing
productive (in-frame) V-D-J rearrangement, hybridomas which
express an antibody comprising a heavy chain comprising a
human sequence heavy chain variable region in polypeptide
linkage to a human (or mouse) non- heavy chain constant
region; said antibodys are termed "switched antibodies" as
WO 94/25585 PCT/US94/04580
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they comprise a "switched heavy chain" which is produced as a
consequence of cis-switching and/or trans-switching in vivo or
in cell culture. Hybridomas producing switched antibodies
generally have undergone the process of somatic mutation, and
a pool of said hybridomas will generally have a broader range
of antigen binding affinities from which hybridoma clones
secreting high affinity antibodies can be selected.
Typically, hybridomas secreting a human sequence antibody
having substantial binding affinity (greater than 1 x 107 M-1
to i x 108 M-1) for a predetermined antigen and wherein said
human sequence antibody comprises human immunoglobulin
variable region(s) can be selected by a method comprising a
two-step process. One step is to identify and isolate
hybridoma cells which secrete immunoglobulins which comprise a
switched heavy chain (e.g., by binding hybridoma cells to an
immobilized immunoglobulin which specifically binds a switched
heavy chain and does not substantially bind to an unswitched
isotype, e.g., ). The other step is to identify hybridoma
cells which bind to the predetermined antigen with substantial
binding affinity (e.g., by ELISA of hybridoma clone
supernatants, FACS analysis using labeled antigen, and the
like). Typically, selection of hybridomas which secrete
switched antibodies is performed prior to identifying
hybridoma cells which bind predetermined antigen. Hybridoma
cells which express switched antibodies that have substantial
binding affinity for the predetermined antigen are isolated
and cultured under suitable growth conditions known in the
art, typically as individual selected clones. Optionally, the
method comprises the step of culturing said selected clones
under conditions suitable for expression of monocloanl
antibodies; said monoclonal antibodies are collected and can
be administered for therapeutic, prophylactic, and/or
diagnostic purposes.
Often, the selected hybridoma clones can serve as a
source of DNA or RNA for isolating immunoglobulin sequences
which encode immunoglobulins (e.g. a variable region) that
bind to (or confer binding to) the predetermined antigen.
Subsequently, the human variable region encoding sequence may
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2161351 76 .40
be isolated (e.g., by PCR amplification or cDNA cloning from
the source (hybridoma clone)) and spliced to a sequence
encoding a desired human constant region to encode a human
sequence antibody more suitable for human therapeutic uses
where immunogenicity is preferably minimized. The
polynucleotide(s) having the resultant fully human encoding
sequence(s) can be expressed in a host cell (e.g., from an
expression vector in a mammalian cell) and purified for
pharmaceutical formulation.
Xenoenhancers
A heterologous transgene capable of encoding a human
immunoglobulin (e.g., a heavy chain) advantageously comprises
a cis-linked enhancer which is not derived from the mouse
genome, and/or which is not naturally associated in cis with
the exons of the heterologous transgene. For example, a human
K transgene (e.g., a K minilocus) can advantageously comprise
a human VK gene, a human JK gene, a human CK gene, and a
xenoenhancer, typically said xenoenhancer comprises a human
heavy chain intronic enhancer and/or a murine heavy chain
intronic enhancer, typically located between a JK gene and the
CK gene, or located downstream of the CK gene. For example,
the mouse heavy chain J- intronic enhancer (Banerji et al.
(1983) Cell 33: 729) can be isolated on a 0.9 kb XbaI fragment
of the plasmid pKVe2 (see, infra). The human heavy chain J-g
intronic enhancer (Hayday et al. (1984) Nature 307: 334)
can be isolated as a 1.4 kb MluI/HindIII fragment (see,
infra). Addition of a transcriptionally active xenoenhancer
to a transgene, such as a combined xenoenhancer consisting
essentially of a human J-g intronic enhancer linked in cis to
a mouse J- intronic enhancer, can confer high levels of
expression of the transgene, especially where said transgene
encodes a light chain, such as human K. Similarly, a rat 3'
enhancer can be advantageously included in a minilocus
construct capable of encoding a human heavy chain.
Specific Preferred Embodiments
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A preferred embodiment of the invention is an animal
containing at least one, typically 2-10, and sometimes 25-50
or more copies of the transgene described in Example 12 (e.g.,
pHC1 or pHC2) bred with an animal containing a single copy of
a light chain transgene described in Examples 5, 6, 8, or 14,
and the offspring bred with the JH deleted animal described in
Example 10. Animals are bred to homozygosity for each of
these three traits. Such animals have the following genotype:
a single copy (per haploid set of chromosomes) of a human
heavy chain unrearranged mini-locus (described in Example 12),
a single copy (per haploid set of chromosomes) of a rearranged
human K light chain construct (described in Example 14), and a
deletion at each endogenous mouse heavy chain locus that
removes all of the functional JH segments (described in
Example 10). Such animals are bred with mice that are
homozygous for the deletion of the'JH segments (Examples 10)
to produce offspring that are homozygous for the JH deletion
and hemizygous for the human heavy and light chain constructs.
The resultant animals are injected with antigens and used for
production of human monoclonal antibodies against these
antigens.
B cells isolated from such an animal are
monospecific with regard to the human heavy and light chains
because they contain only a single copy of each gene.
Furthermore, they will be monospecific with regards to human
or mouse heavy chains because both endogenous mouse heavy
chain gene copies are nonfunctional by virtue of the deletion
spanning the JH region introduced as described in Example 9
and 12. Furthermore, a substantial fraction of the B cells
will be monospecific with regards to the human or mouse light
chains because expression of the single copy of the rearranged
human K light chain gene will allelically and isotypically
exclude the rearrangement of the endogenous mouse K and X
chain genes in a significant fraction of B-cells.
The transgenic mouse of the preferred embodiment
will exhibit immunoglobulin production with a significant
repertoire, ideally substantially similar to that of a native
mouse. Thus, for example, in embodiments where the endogenous
WO 94/251815 PCT/US94/04580
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Ig genes have been inactivated, the total immunoglobulin
levels will range from about 0.1 to 10 mg/ml of serum,
preferably 0.5 to 5 mg/ml, ideally at least about 1.0 mg/ml.
When a transgene capable of effecting a switch to IgG from IgM
has been introduced into the transgenic mouse, the adult mouse
ratio of serum IgG to IgM is preferably about 10:1. Of
course, the IgG to IgM ratio will be much lower in the
immature mouse. In general, greater than about 10%,
preferably 40 to 80% of the spleen and lymph node B cells
express exclusively human IgG protein.
The repertoire will ideally approximate that shown
in a non-transgenic mouse, usually at least about 10% as high,
preferably 25 to 50% or more. Generally, at least about a
thousand different immunoglobulins (ideally IgG), preferably
104 to 106 or more, will be produced, depending primarily on
the number-of different V, J and D regions introduced into the
mouse genome. These immunoglobulins will typically recognize
about one-half or more of highly antigenic proteins,
including, but not limited to: pigeon cytochrome C, chicken
lysozyme, pokeweed mitogen, bovine serum albumin, keyhole
limpit hemocyanin, influenza hemagglutinin, staphylococcus
protein A, sperm whale myoglobin, influenza neuraminidase, and
lambda repressor protein. Some of the immunoglobulins will
exhibit an affinity for preselected antigens of at least about
107M-1, preferably 108M-1 to 109M-1 or greater.
In some embodiments, it may be preferable to
generate mice with predetermined repertoires to limit the
selection of V genes represented in the antibody response to a
predetermined antigen type. A heavy chain transgene having a
predetermined repertoire may comprise, for example, human VH
genes which are preferentially used in antibody responses to
the predetermined antigen type in humans. Alternatively, some
VH genes may be excluded from a defined repertoire for various
reasons. (e.g., have a low likelihood of encoding high affinity
V regions for the predetermined antigen; have a low propensity
to undergo somatic mutation and affinity sharpening; or are
immunogenic to certain humans).
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Thus, prior to rearrangement of a transgene
containing various heavy or light chain gene segments, such
gene segments may be readily identified, e.g. by hybridization
or DNA sequencing, as being from a species of organism other
than the transgenic animal.
Although the foregoing describes a preferred
embodiment of the transgenic animal of the invention, other
embodiments are defined by the disclosure herein and more
particularly by the transgenes described in the Examples.
Four categories of transgenic animal may be defined:
I. Transgenic animals containing an unrearranged heavy
and rearranged light immunoglobulin transgene.
II. Transgenic animals containing an unrearranged heavy
and unrearranged light immunoglobulin transgene
III. Transgenic animal containing rearranged heavy and an
unrearranged light immunoglobulin transgene,,and
IV. Transgenic animals containing rearranged heavy and
rearranged light immunoglobulin transgenes.
Of these categories of transgenic animal, the
preferred order of preference is as follows II > I > III > IV
where the endogenous light chain genes (or at least the K
gene) have been knocked out by homologous recombination (or
other method) and.I > II > III >IV where the endogenous light
chain genes have not been knocked out and must be dominated by
allelic exclusion.
EXPERIMENTAL EXAMPLES
METHODS AND MATERIALS
Transgenic mice are derived according to Hogan, et
al., "Manipulating the Mouse Embryo: A Laboratory Manual",
Cold Spring Harbor Laboratory, which is incorporated herein by
reference.
Embryonic stem cells are manipulated according to
published procedures (Teratocarcinomas and embryonic stem
cells: a practical approach, E.J. Robertson, ed., IRL Press,
Washington, D.C., 1987; Zjilstra et al., Nature 342:435-438
(1989); and Schwartzberg et al., Science 246:799-803 (1989)).
CA 02161351 2003-10-23
DNA cloning procedures are carried out according to
J. Sambrook, et al. in Molecular Cloning: A Laboratory
Manual, 2d ed., 1989, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.
5
Oligonucleotides are synthesized on an Applied Bio
Systems oligonucleotide synthesizer according to
specifications provided by the manufacturer.
Hybridoma cells and antibodies are manipulated
10 according to "Antibodies: A Laboratory Manual", Ed Harlow and
David Lane, Cold Spring Harbor Laboratory (1988).
EXAMPLE 1
15 Genomic Heavy Chain Human Ig Transgene
This Example describes the cloning and
microinjection of a human genomic heavy chain immunoglobulin
transgene which is microinjected into a murine zygote.
Nuclei are isolated from fresh human placental
20 tissue as described by Marzluff et al., "Transcription and
Translation: A Practical Approach", B.D. Hammes and
S.J. Higgins, eds., pp. 89-129, IRL Press, Oxford (1985)).
The isolated nuclei (or PBS washed human spermatocytes) are
embedded in a low melting point agarose matrix and lysed with
25 EDTA and proteinase K to expose high molecular weight DNA,
which is then digested in the agarosq with the restriction
enzyme NotI as described by M. Finney in Current Protocols in
Molecular Biology (F. Ausubel, et al., eds. John Wiley & Sons,
Supp. 4, 1988, Section 2.5.1).
30 The NotI digested DNA is then fractionated by pulsed
field gel electrophoresis as described by Anand et al.,
Nucl. Acids Res. 17:3425-3433 (1989). Fractions enriched for
the NotI fragment are assayed by Southern hybridization to
detect one or more of the sequences encoded by this fragment.
35 Such sequences include the heavy chain D segments, J segments,
and y1 constant regions together with representatives of all
6 VH families (although this fragment is identified as 670 kb
fragment from HeLa cells by Berman et al. (1988), supra., we
CA 02161351 2003-10-23
81
have found it to be as 830 kb fragment from human placental an
sperm DNA). Those fractions containing this NotI fragment.
(see Fig. 4) are pooled and cloned into the NotI site of the
vector pYACNN in Yeast cells. Plasmid pYACNN is prepared by
digestion of pYAC-4 Neo (Cook et.al., Nucleic Acids Res. 16:
11817 (1988)) with EcoRI and ligation in the presence of the
oligonucleotide 5' - AAT TGC GGC CGC - 3'.
YAC clones containing the heavy chain NotI fragment
are isolated as described by Brownstein et al., Science
244:1348-1351 (1989), and Green et al., Proc. Natl. Acad. Sci.
USA 87:1213-1217 (1990).
The cloned NotI insert is isolated from high
molecular weight yeast DNA by pulse field gel electrophoresis
as described by M. Finney, op cit. The DNA is condensed by
the addition of 1 mM spermine and microinjected directly into
the nucleus of single cell embryos previously described.
EXAMPLE 2
Genomic K Light Chain Human Ig Transgene
Formed by In Vivo Homologous Recombination
A map of the human K light chain has been described
in Lorenz et al., Nucl. Acids Res. 15:9667-9677 (1987).
A 450 kb XhoI to NotI fragment that includes all of
C., the 3' enhancer, all J segments, and at least five
different V segments is isolated and0microinjected into the
nucleus of single cell embryos as described in Example 1.
EXAMPLE 3
Genomic K Light Chain Human Ig Transgene
Formed by In Vivo Homologous Recombination
A 750 kb MluI to NotI fragment that includes all of
the above plus at least 20 more V segments is isolated as
described in Example 1 and digested with BssHII to produce a
fragment of about 400 kb.
The 450 kb XhoI to NotI fragment plus the
approximately 400 kb Mlul to BssHII fragment have sequence
overlap defined by the BssHII and XhoI restriction sites.
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2161351 82 1
Homologous recombination of these two fragments upon
microinjection of a mouse zygote results in a transgene
containing at least an additional 15-20 V segments over that
found in the 450 kb XhoI/NotI fragment (Example 2).
EXAMPLE 4
Construction of Heavy Chain Mini-Locus
A. Construction of PGPl and pGP2
pBR322 is digested with EcoRI and Styl and ligated
with the following oligonucleotides to generate pGP1 which
contains a 147 base pair insert containing the restriction
sites shown in Fig. 8. The general overlapping of these
oligos is also shown in Fig. 9.
The oligonucleotides are:
oligo-1 5' - CTT GAG CCC GCC TAA TGA GCG GGC TTT
TTT TTG CAT ACT GCG GCC - 3'
oligo-2 5' - GCA ATG GCC TGG ATC CAT GGC GCG CTA
GCA TCG ATA TCT AGA GCT CGA GCA -3'
oligo-3 5' - TGC AGA TCT GAA TTC CCG GGT ACC AAG
CTT ACG CGT ACT AGT GCG GCC GCT -3'
oligo-4 5' - AAT TAG CGG CCG CAC TAG TAC GCG TAA
GCT TGG TAC CCG GGA ATT - 3'
oligo-5 5' - CAG ATC TGC ATG CTC GAG CTC TAG ATA
TCG ATG CTA GCG CGC CAT GGA TCC - 3'
oligo-6 5' - AGG CCA TTG CGG CCG CAG TAT GCA AAA
AAA AGC CCG CTC ATT AGG CGG GCT - 3'
This plasmid contains a large polylinker flanked by
rare cutting NotI sites for building large inserts that can be
isolated from vector sequences for microinjection. The
plasmid is based on pBR322 which is relatively low copy
compared to the pUC based plasmids (pGP1 retains the pBR322
copy number control region near the origin of replication).
Low copy number reduces the potential toxicity of insert
sequences. In addition, pGP1 contains a strong transcription
terminator sequence derived from trpA (Christie et al., oc.
Natl. Acad. Sci. USA 78:4180 (1981)) inserted between the
ampicillin resistance gene and the polylinker. This further
reduces the toxicity associated with certain inserts by
CA 02161351 2003-10-23
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preventing readthrough transcription coming from the
ampicillin promoters.
Plasmid pGP2 is derived from pGP1 to introduce an
additional restriction site (Sfil) in the polylinker. pGP1 is
digested with Mlul and Spel to cut the recognition sequences
in the polylinker portion of the plasmid.
The following adapter oligonucleotides are ligated
to the thus digested pGP1 to form pGP2.
5' CGC GTG GCC GCA ATG GCC A 3'
5' CTA GTG GCC ATT GCG GCC A 3'
pGP2 is identical to pGPl except that it contains an
additional Sfi I site located between the Miul and Spel sites.
This allows inserts to be completely excised with SfiI as well
as with NotI.
B. Construction of pRE3 (rat enhancer 3')
An enhancer sequence located downstream of the rat
constant region is included in the heavy chain constructs.
The heavy chain region 3' enhancer described by
Petterson et al., Nature 344:165-168 (1990)
is isolated and cloned. The
rat IGH 3' enhancer sequence is PCR amplified by using the
following oligonucleotides:
5' CAG GAT CCA GAT ATC AGT ACC TGA AAC AGG GCT TGC 3'
5' GAG CAT GCA CAG GAC CTG GAG CAC ACA CAG CCT TCC 3'
The thus formed double stranded DNA encoding the 3'
enhancer is cut with BamHI and SphI and clone into BamHI/SphI
cut pGP2 to yield pRE3 (rat enhancer 3').
C. Cloning of Human J-u Region
A substantial portion of this region is cloned by
combining two or more fragments isolated from phage lambda
inserts. See Fig. 9.
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A 6.3 kb BamHI/HindIII fragment that includes all
human J segments (Matsuda et al., EMBO J., 7:1047-1051 (1988);
Ravetech et al.m Cell, 27:583-591 (1981))
is isolated from human
genomic DNA library using the oligonucleotide GGA CTG TGT CCC
TGT GTG'ATG CTT TTG ATG TCT GGG GCC AAG.
An adjacent 10 kb HindIIl/Bamil fragment that
contains enhancer, switch and constant region coding exons
(Yasui et al., Eur. J. Immunol. 19:1399-1403 (1989)) is
similarly isolated using the oligonucleotide:
CAC CAA GTT GAC CTG CCT GGT CAC AGA CCT GAC CAC CTA TGA
An adjacent 3' 1.5 kb BamHI fragment is similarly
isolated using clone pMUM insert as probe (pMUM is 4 kb
EcoRI/HindIIl fragment.isolated from human genomic DNA library
with oligonucleotide:
CCT GTG GAC CAC CGC CTC CAC CTT CAT
CGT CCT CTT CCT CCT
mu membrane exon 1) and cloned into pUC19.
pGPl is digested with BamHI and Bg1II followed by
treatment with calf intestinal alkaline phosphatase.
Fragments (a) and (b) from Fig. 9 are cloned in the
digested pGPl. A clone is then isolated which is oriented
such that 5' BamHI site is destroyed by BamHI/Bgl fusion. It
is identified as pMU (see Fig. 10). pMU is digested with
BamHI and fragment (c) from Fig. 9 is inserted. The
orientation is checked with HindIIl digest. The resultant
plasmid pHIGi (Fig. 10) contains an 18 kb insert encoding J
and Cg segments.
D. Cloning of Cu Region
pGP1 is digested with BamHI and HindIIl is followed
by treatment with calf intestinal alkaline phosphatase (Fig.
14). The so treated fragment (b) of Fig. 14 and fragment (c)
of Fig. 14 are cloned into the BamHI/HindIII cut pGP1. Proper
orientation of fragment (c) is checked by HindIIl digestion to
form pCON1 containing a 12 kb insert encoding the Cg region.
Whereas pHIG1 contains J segments, switch and p
sequences in its 18 kb insert with an Sfil 3' site and a Spel
CA 02161351 2003-10-23
5' site in a polylinker flanked by NotI sites, will be used
for rearranged VDJ segments. pCON1 is identical except that
it lacks the J region and contains only a 12 kb insert. The
use of pCON1 in the construction of fragment containing
5 rearranged VDJ segments will be described hereinafter.
E. Cloning of 7-1 Constant Region (pREG2)
The cloning of the human 7-1 region is depicted in
Fig. 16.
10 Yamamura et al., Proc. Natl. Acad. Sci. USA
83:2152-2156 (1986) reported the expression of membrane bound
human 7-1 from a transgene construct that had been partially
deleted on integration. Their results indicate that the 3'
BamHI site delineates a sequence that includes the
15 transmembrane rearranged and switched copy of the gamma gene
with a V-C intron of less than 5kb. Therefore, in the
unrearranged, unswitched gene, the entire switch region is
included in a sequence beginning less than 5 kb from the 5'
end of the first 7-1 constant exon. Therefore it is included
20 in the 5' 5.3 kb Hindlll fragment (Ellison et al., Nucleic
Acids Res. 10:4071-4079 (1982)).
Takahashi et al., Cell 29: 671-679 (1982),
also reports that
this fragment contains the switch sequence, and this fragment
25 together with the 7.7 kb Hindlll to BamHI fragment must
include all of the sequences we need,for the transgene
construct. An intronic sequence is a nucleotide sequence of
at least 15 contiguous nucleotides that occurs in an intron of
a specified gene.
30 Phage clones containing the 7-1 region are
identified and isolated using the following oligonucleotide
which is specific for the third exon of 7-I (CH3).
5' TGA GCC ACG AAG ACC CTG AGG
35 TCA AGT TCA ACT GGT ACG TGG 3'
A 7.7 kb Hindlil to BglII fragment (fragment (a) in
Fig. 11) is cloned into HindIII/BglII cut pRE3 to form pREG1.
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The upstream 5.3 kb Hindlil fragment (fragment (b) in Fig. 11)
is cloned into Hindill digested pREG1 to form pREG2. Correct
orientation is confirmed by BamHI/SpeI digestion.
F. Combining Cy and Cu
The previously described plasmid pHIG] contains
human J segments and the C constant region exons. To provide
a transgene containing the C constant region gene segments,
pHIG1 was digested with Sfii (Fig. 10). The plasmid pREG2 was
also digested with Sfii to produce a 13.5 kb insert containing
human Cy exons and the rat 3' enhancer sequence. These
sequences were combined to produce the plasmid pHIG3' (Fig.
12) containing the human J segments, the human C constant
region, the human Cyl constant region and the rat 3' enhancer
contained on a 31.5 kb insert.
A second plasmid encoding human C and human Cyl
without J segments is constructed by digesting pCON1 with Sfii
and combining that with the Sfii fragment containing the human
Cy region and the rat 3' enhancer by digesting pREG2 with
Sfii. The resultant plasmid, pCON (Fig. 12) contains a 26 kb
Notl/Spel insert containing human C , human 71 and the rat 3'
enhancer sequence.
G. Cloning of D Segment
The strategy for cloning the human D segments is
depicted in Fig. 13. Phage clones from the human genomic
library containing D segments are identified and isolated
using probes specific for diversity region sequences (Ichihara
et al., EMBO J. 7:4141-4150 (1988)). The following
oligonucleotides are used:
DXP1: 5' - TGG TAT TAC TAT GGT TCG GGG AGT TAT TAT
AAC CAC AGT GTC - 3'
DXP4: 5' - GCC TGA AAT GGA GCC TCA GGG CAC AGT GGG
CAC GGA CAC TGT - 3'
DN4: 5' - GCA GGG AGG ACA TGT TTA GGA TCT GAG GCC
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GCA CCT GAC ACC - 3'
A 5.2 kb XhoI fragment (fragment (b) in Fig. 13)
containing DLR1, DXP1, DXP'1, and DA1 is isolated from a phage
clone identified with oligo DXP1.
A 3.2 kb XbaI fragment (fragment (c) in Fig. 13)
containing DXP4, DA4 and DK4 is isolated from a phage clone
identified with oligo DXP4.
Fragments (b), (c) and (d) from Fig. 13 are combined
and cloned into the XbaI/XhoI site of pGP1 to.form pHIG2 which
contains a 10.6 kb insert.
This cloning is performed sequentially. First, the
5.2 kb fragment (b) in Fig. 13 and the 2.2 kb fragment (d) of-
Fig. 13 are treated with calf intestinal alkaline phosphatase
and cloned into pGP1 digested with XhoI and XbaI. The
resultant clones are screened with the 5.2 and 2.2 kb insert.
Half of those clones testing positive with the 5.2 and 2.2 kb
inserts have the 5.2 kb insert in the proper orientation as
determined by BamHI digestion. The 3.2 kb XbaI fragment from
Fig. 13 is then cloned into this intermediate plasmid
containing fragments (b) and (d) to form pHIG2. This plasmid
contains diversity segments cloned into the polylinker with a
unique 5' SfiI site and unique 3' Spel site. The entire
polylinker is flanked by NotI sites.
H. Construction of Heavy Chain Min locus
The following describes the construction of a human
heavy chain mini-locus which contain one or more V segments.
An unrearranged V segment corresponding to that
identified as the V segment contained in the hybridoma
of Newkirk et al., J. Clin. Invest. 81:1511-1518 (1988),
is isolated using the
following oligonucleotide:
5' - GAT CCT GGT TTA GTT AAA GAG GAT TTT
ATT CAC CCC TGT GTC - 3'
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88 40
A restriction map of the unrearranged V segment is
determined to identify unique restriction sites which provide
upon digestion a DNA fragment having a length approximately 2
kb containing the unrearranged V segment together with 5' and
3' flanking sequences. The 5' prime sequences will include
promoter and other regulatory sequences whereas the 3'
flanking sequence provides recombination sequences necessary
for V-DJ joining. This approximately 3.0 kb V segment insert
is cloned into the polylinker of pGB2 to form pVH1.
pVH1 is digested with Sf11 and the resultant
fragment is cloned into the SfiI site of pHIG2 to form a
pHIG5'. Since pHIG2 contains D segments only, the resultant
pHIG5' plasmid contains a single V segment together with D
segments. The size of the insert contained in pHIG5 is 10.6
kb plus the size of the V segment insert.
The insert from pHIG5 is excised by digestion with
NotI and Spel and isolated. pHIG3' which contains J, C and
c71 segments is digested with Spel and NotI and the 3' kb
fragment containing such sequences and the rat 3' enhancer
sequence is isolated. These two fragments are combined and
ligated into NotI digested pGPl to produce pHIG which contains
insert encoding a V segment, nine D segments, six functional J
segments, C , Cy and the rat 3' enhancer. The size of this
insert is approximately 43 kb plus the size of the V segment
insert.
I. Construction of Heavy Chain Minilocus
by Homologous Recombination
As indicated in the previous section, the insert of
pHIG is approximately 43 to 45 kb when a single V segment is
employed. This insert size is at or near the limit of that
which may be readily cloned into plasmid vectors. In order to
provide for the use of a greater number of V segments, the
following describes in vivo homologous recombination of
overlapping DNA fragments which upon homologous recombination
within a zygote or ES cell form a transgene containing the rat
3' enhancer sequence, the human C , the human C71, human J
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segments, human D segments and a multiplicity of human V
segments.
A 6.3 kb BamHI/HindIII fragment containing human J
segments (see fragment (a) in Fig. 9) is cloned into MluI/SpeI
digested pHIG5' using the following adapters:
5' GAT CCA AGC AGT 3'
5' CTA GAC TGC TTG 3'
5' CGC GTC GAA CTA 3'
5' AGC TTA GTT CGA 3'
The resultant is plasmid designated pHIGS'O
(overlap). The insert contained in this plasmid contains
human V, D and J segments. When the single V segment from
pVH1 is used, the size of this insert is approximately 17 kb
plus 2 kb. This insert is isolated and combined with the
insert from pHIG3' which contains the human J, C , 71 and rat
3' enhancer sequences. Both inserts contain human J segments
which provide for approximately 6.3 kb of overlap between the
two DNA fragments. When coinjected into the mouse zygote, in
vivo homologous recombination occurs generating a transgene
equivalent to the insert contained in pHIG.
This approach provides for the addition of a
multiplicity of V segments into the transgene formed in vivo.
For example, instead of incorporating a single V segment into
pHIG5', a multiplicity of V segments contained on (1) isolated
genomic DNA, (2) ligated DNA derived from genomic DNA, or (3)
'30 DNA encoding a synthetic V segment repertoire is cloned into
pHIG2 at the Sfii site to generate pHIG5' VN. The J segments
fragment (a) of Fig. 9 is then cloned into pHIG5' VN and the
insert isolated. This insert now contains a multiplicity of V
segments and J segments which overlap with the J segments
contained on the insert isolated from pHIG3'. When
cointroduced into the nucleus of a mouse zygote, homologous
recombination occurs to generate in vivo the transgene
encoding multiple V segments and multiple J segments, multiple
D segments, the CA region, the Cyl region (all from human) and
the rat 3' enhancer sequence.
WO 94/25585 PCTIUS94/04580
%16j351 EXAMPLE 5
Construction of Licht Chain Minilocus
A. Construction of PEal
The construction of pEgl is depicted in Fig. 16.
5 The mouse heavy chain enhancer is isolated on the XbaI to
EcoRI 678 bp fragment (Banerji et al., Cell 33:729-740 (1983))
from.phage clones using oligo:
5' GAA TGG GAG TGA GGC TCT CTC ATA CCC
10 TAT TCA GAA CTG ACT 3'
This E fragment is cloned into EcoRV/XbaI digested
pGP1 by blunt end filling in EcoRI site. The resultant
15 plasmid is designated pEmul.
B. Construction Of K Light chain Minilocus
The Ic construct contains at least one human V.
segment, all five human JK segments, the human J-CK enhancer,
20 human !c constant region exon, and, ideally, the human 3' K
enhancer (Meyer et al., EMBO J. 8:1959-1964 (1989)). The !c
enhancer in mouse is 9 kb downstream from CK. However, it is
as yet unidentified in the human. In addition, the construct
contains a copy of the mouse heavy chain J-C enhancers.
25 The minilocus is constructed from four component
fragments:
(a) A 16 kb Smal fragment that contains the human
C. exon and the 3' human enhancer by analogy with the mouse
locus;
30 (b) A 5' adjacent 5 kb SmaI fragment, which
contains all five J segments;
(c) The mouse heavy chain intronic enhancer
isolated from pE l (this sequence is included to induce
expression of the light chain construct as early as possible
35 in B-cell development. Because the heavy chain genes are
transcribed earlier than the light chain genes, this heavy
chain enhancer is presumably active at an earlier stage than
the intronic K enhancer); and
(d) A fragment containing one or more V segments.
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The preparation of this construct is as follows.
Human placental DNA is digested with SmaI and fractionated on
agarose gel by electrophoresis. Similarly, human placental
DNA is digested with BamHi and fractionated by
electrophoresis. The 16 kb fraction is isolated from the Smal
digested gel and the 11 kb region is similarly isolated from
the gel containing DNA digested with BamHI.
The 16 kb Smal fraction is cloned into Lambda FIX II
(Stratagene, La Jolla, California) which has been digested
with XhoI, treated with klenow fragment DNA polymerase to fill
in the XhoI restriction digest product. Ligation of the 16 kb
Smal fraction destroys the SmaI sites and lases XhoI sites
intact.
The 11 kb BamHI fraction is cloned into X EMBL3
(Strategene, La Jolla, California) which is digested with
BamHI prior to cloning.
Clones from each library were probed with the CK
specific oligo:
5' GAA CTG TGG CTG CAC CAT CTG TCT
TCA TCT TCC CGC CAT CTG 3'
A 16 kb XhoI insert that was subcloned into the XhoI
cut pEil so that CK is adjacent to the SmaI site. The
resultant plasmid was designated pKapi.
The above CK specific oligonucleotide is used to
probe the X EMBL3/BamHI library to identify an 11 kb clone. A
5 kb Smal fragment (fragment (b) in Fig. 20) is subcloned and
subsequently inserted into pKapl digested with SmaI. Those
plasmids containing the correct orientation of J segments, CK
and the E enhancer are designated pKap2.
One or more VK segments are thereafter subcloned
into the M1uI site of pKap2 to yield the plasmid pKapH which
encodes the human VK segments, the human JK segments, the
human CK segments and the human Eg enhancer. This insert is
excised by digesting pKapH with NotI and purified by agarose
gel electrophoresis. The thus purified insert is
microinjected into the pronucleus of a mouse zygote as
previously described.
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61361 C. Construction of K Light Chain Minilocus by
In Vivo Homologous Recombination
The 11 kb BamHI fragment is cloned into BamHI
digested pGP1 such that the 3' end is toward the Sfii site.
The resultant plasmid is designated pKAPint. One or more VK
segments is inserted into the polylinker between the BamHI and
Spel sites in pKAPint to form pKapHV. The insert of pKapHV is
excised by digestion with NotI and purified. The insert from
pKap2 is excised by digestion with NotI and purified. Each of
these fragments contain regions of homology in that the
fragment from pKapHV contains a 5 kb sequence of DNA that
include the J. segments which is substantially homologous to
the 5 kb SmaI fragment contained in the insert obtained from
pKap2. As such, these inserts are capable of homologously
recombining when microinjected into a mouse zygote to form a
transgene encoding VK, J. and C.
EXAMPLE6
Isolation of Genomic Clones
Corresponding to Rearranged and Expressed
Copies of Immunoglobulin K Light Chain Genes
This example describes the cloning of immunoglobulin
K light chain genes from cultured cells that express an
immunoglobulin of interest. Such cells may contain multiple
alleles of a given immunoglobulin gene. For example, a
hybridoma might contain four copies of the K light chain gene,
two copies from the fusion partner cell line and two copies
from the original B-cell expressing the immunoglobulin of
interest. Of these four copies, only one encodes the
immunoglobulin of interest, despite the fact that several of
them may be rearranged. The procedure described in this
example allows for the selective cloning of the expressed copy
of the K light chain.
A. Double Stranded cDNA
Cells from human hybridoma, or lymphoma, or other
cell line that synthesizes either cell surface or secreted or
both forms of IgM with a K light chain are used for the
isolation of polyA+ RNA. The RNA is then used for the
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synthesis of oligo dT primed cDNA using the enzyme reverse
transcriptase (for general methods see, Goodspeed et al.
(1989) Gene 76: 1; Dunn et al. (1989) J. Biol. Chem. 264:
13057). The single stranded cDNA is then isolated and G
residues are added to the 3' end using the enzyme
polynucleotide terminal transferase. The G-tailed
single-stranded cDNA is then purified and used as template for
second strand synthesis (catalyzed by the enzyme DNA
polymerase) using the following oligonucleotide as a primer:
5' - GAG GTA CAC TGA CAT ACT GGC ATG CCC
CCC CCC CCC - 3'
The double stranded cDNA is isolated and used for
determining the nucleotide sequence of the 5' end of the mRNAs
encoding the heavy and light chains of the expressed
immunoglobiilin molecule. Genomic clones of these expressed
genes are then isolated. The procedure for cloning the
expressed light chain gene is outlined in part B below.
B. Light Chain
The double stranded cDNA described in part A is
denatured and used as a template for a third round of DNA
synthesis using the following oligonucleotide primer:
5' - GTA CGC CAT ATC AGC TGG ATG AAG TCA TCA GAT
GGC GGG AAG ATG AAG ACA GAT GGT GCA - 3'
This primer contains sequences specific for the
constant portion of the K light chain message (TCA TCA GAT GGC
GGG AAG ATG AAG ACA GAT GGT GCA) as well as unique sequences
that can be used as a primer for the PCR amplification of the
newly synthesized DNA strand (GTA CGC CAT ATC AGC TGG ATG
AAG). The sequence is amplified by PCR using the following
two oligonucleotide primers:
5' - GAG GTA CAC TGA CAT ACT GGC ATG -3'
=40 5' - GTA CGC CAT ATC AGC TGG ATG AAG -3'
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el
The PCR amplified sequence is then purified by g
electrophoresis and used as template for dideoxy sequencing
reactions using the following oligonucleotide as a primer:
5' - GAG GTA CAC TGA CAT ACT GGC ATG -3'
The first 42 nucleotides of sequence will then be
used to synthesize a unique probe for isolating the gene from
which immunoglobulin message was transcribed. This synthetic
42 nucleotide segment of DNA will be referred to below as
o-kappa.
A Southern blot of DNA, isolated from the Ig
expressing cell line and digested individually and in pairwise
combinations with several different restriction endonucleases
including Smal, is then probed with the 32-P labelled unique
oligonucleotide o-kappa. A unique restriction endonuclease
site is identified upstream of the rearranged V segment.
DNA from the Ig expressing cell line is then cut
with Smal and second enzyme-(or BamHI or KpnI if there is SmaI
site inside V segment). Any resulting non-blunted ends are
treated with the enzyme T4 DNA polymerase to give blunt ended
DNA molecules. Then add restriction site encoding linkers
(BamHI, EcoRI or XhoI depending on what site does not exist in
fragment) and cut with the corresponding linker enzyme to give
DNA fragments with BamHI, EcoRI or XhoI ends. The DNA is then
size fractionated by agarose gel electrophoresis, and the
fraction including the DNA fragment covering the expressed V
segment is cloned into lambda EMBL3 or Lambda FIX (Stratagene,
La Jolla, California). V segment containing clones are
isolated using the unique probe o-kappa. DNA is isolated from
positive clones and subcloned into the polylinker of pKapl.
The resulting clone is called pRKL.
EXAMPLE 7
Isolation of Genomic Clones
Corresponding to Rearranged Expressed Copies
of Immunoglobulin Heavy Chain u Genes
This example describes the cloning of immunoglobulin
heavy chain g genes from cultured cells of expressed and
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immunoglobulin of interest. The procedure described in this
example allows for the selective cloning of the expressed copy
of a heavy chain gene.
Double-stranded cDNA is prepared and isolated as
described herein before. The double-stranded cDNA is
denatured and used as a template for a third round of DNA
synthesis using the following oligonucleotide primer:
5' - GTA CGC CAT ATC AGC TGG ATG AAG ACA GGA GAC
GAG GGG GAA AAG GGT TGG GGC GGA TGC - 3'
This primer contains sequences specific for the
constant portion of the heavy chain message (ACA GGA GAC GAG
GGG GAA AAG GGT TGG GGC GGA TGC) as well as unique sequences
that can be used as a primer for the PCR amplification of the
newly synthesized DNA strand (GTA CGC CAT ATC AGC TGG ATG
AAG). The sequence is amplified by PCR using the following
two oligonucleotide primers:
5' - GAG GTA CAC TGA CAT ACT GGC ATG - 3'
5' - GTA CTC CAT ATC AGC TGG ATG AAG - 3'
The PCR amplified sequence is then purified by gel
electrophoresis and used as template for dideoxy sequencing
reactions using the following oligonucleotide as a primer:
5' - GAG GTA CAC TGA CAT ACT GGC ATG - 3'
The first 42 nucleotides of sequence are then used
to synthesize a unique probe for isolating the gene from
which immunoglobulin message was transcribed. This synthetic
42 nucleotide segment of DNA will be referred to below as
o-mu.
A Southern blot of DNA, isolated from the Ig
expressing cell line and digested individually and in pairwise
combinations with several different restriction endonucleases
including M1uI (MluI is a rare cutting enzyme that cleaves
between the J segment and mu CH1), is then probed with the
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32-P labelled unique oligonucleotide o-mu. A unique
restriction endonuclease site is identified upstream of the
rearranged V segment.
DNA from the Ig expressing cell line is then cut
with Mlul and second enzyme. Mlul or Spel adapter linkers are
then ligated onto the ends and cut to convert the upstream
site to Mlul or Spel. The DNA is then size fractionated by
agarose gel electrophoresis, and the fraction including the
DNA fragment covering the expressed V segment is cloned
directly into the plasmid pGPI. V segment containing clones
are isolated using the unique probe o-mu, and the insert is
subcloned into Mlul or Mlul/Spel cut plasmid pCON2. The
resulting plasmid is called pRMGH.
EXAMPLE 8
Construction of Human K Miniloci Transaenes
Light Chain Minilocus
A human genomic DNA phage library was screened with
kappa light chain specific oligonucleotide probes and isolated
clones spanning the J. -C region. A 5.7 kb ClaI/XhoI fragment
containing J K 1 together with a 13 kb XhoI fragment containing
JK2-5 and CK into pGP1d was cloned and used to create the
plasmid pKcor. This plasmid contains Jx1-5, the kappa
intronic enhancer and CK together with 4.5 kb of 5' and 9 kb
of 3' flanking sequences. It also has a unique 5' XhoI site
for cloning VK segments and a unique 3' Sall site for
inserting additional cis-acting regulatory sequences.
V kappa genes
A human genomic DNA phage library was screened with
VK light chain specific oligonucleotide probes and isolated
clones containing human VK segments. Functional V segments
were identified by DNA sequence analysis. These clones
contain TATA boxes, open reading frames encoding leader and
.35 variable peptides (including 2 cysteine residues), splice
sequences, and recombination heptamer-12 bp spacer-nonamer
sequences. Three of the clones were mapped and sequenced.
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Two of the clones, 65.5 and 65.8 appear to be functional, they
contain TATA boxes, open reading frames encoding leader and
variable peptides (including 2 cysteine residues), splice
sequences, and recombination heptamer-12 bp spacer-nonamer
sequences. The third clone, 65.4, appears to encode a VKI
pseudogene as it contains a non-canonical recombination
heptamer.
One of the functional clones, Vk 65-8, which encodes
a VkIII family gene, was used to build a light chain minilocus
construct.
pKC]
The kappa light chain minilocus transgene pKC1 (Fig.
32) was generated by inserting a 7.5 kb XhoI/SalI fragment
containing VK 65.8 into the 5' XhoI site of pKcor. The
transgene insert was isolated by digestion with NotI prior to
injection.
The purified insert was microinjected into the
pronuclei of fertilized (C57BL/6 x CBA)F2 mouse embryos and
transferred the surviving embryos into pseudopregnant females
as described by Hogan et al. (in Methods of Manipulating the
Mouse Embryo, 1986, Cold Spring Harbor Laboratory, New York).
Mice that developed from injected embryos were analyzed for
the presence of transgene sequences by Southern blot analysis
of tail DNA. Transgene copy number was estimated by band
intensity relative to control standards containing known
quantities of cloned DNA. Serum was isolated from these
animals and assayed for the presence of transgene encoded
human Ig kappa protein by ELISA as described by Harlow and
Lane (in Antibodies: A Laboratory Manual, 1988, Cold Spring
Harbor Laboratory, New York). Microtiter plate wells were
coated with mouse monoclonal antibodies specific for human Ig
kappa (clone 6E1, #0173, AMAC, Inc., Westbrook, ME), human IgM
(Clone AF6, #0285, AMAC, Inc., Westbrook, ME) and human IgGl
(clone JL512, #0280, AMAC, Inc., Westbrook, ME). Serum
samples were serially diluted into the wells and the presence
of specific immunoglobulins detected with affinity isolated
alkaline phosphatase conjugated goat anti-human Ig
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(polyvalent) that had been pre-adsorbed to minimize cross-
reactivity with mouse immunoglobulins.
Fig. 35 shows the results of an ELISA assay of serum
from 8 mice (I.D. #676, 674, 673, 670, 666, 665, 664, and
496). The first seven of these mice developed from embryos
that were injected with the pKC1 transgene insert and the
eighth mouse is derived from a mouse generated by
microinjection of the pHCl transgene (described previously).
Two of the seven mice from KC1 injected embryos (I.D.#'s 666
and 664) did not contain the transgene insert as assayed by
DAN Southern blot analysis, and five of the mice (I.D.#'s 676,
674, 673, 670, and 665) contained the transgene. All but one
of the KC1 transgene positive animals express detectable
levels of human Ig kappa protein, and the single non-
expressing animal appears to be a genetic mosaic on the basis
of DNA Southern blot analysis. The pHCl positive transgenic
mouse expresses human IgM and IgG] but not Ig kappa,
demonstrating the specificity of the reagents used in the
assay.
pKC2
The kappa light chain minilocus transgene pKC2 was
generated by inserting an 8 kb XhoI/SalI fragment containing
VK 65.5 into the 5' XhoI site of pKC1. The resulting
transgene insert, which contains two VK segments, was isolated
prior to microinjection by digestion with NotI.
PKVe2
This construct is identical to pKC1 except that it
includes 1.2 kb of additional sequence 5' of JK and is missing
4.5 kb of sequence 3' of VK 65.8. In additional it contains a
0.9 kb XbaI fragment containing the mouse heavy chain J- intronic enhancer
(Banerji et al., Cell 33:729-740 (1983))
together with a 1.4 kb Mlul/HindIll fragment containing the
human heavy chain J- intronic enhancer (Hayday et al., Nature
307:334-340 (1984)) inserted downstream. This construct tests
the feasibility of initiating early rearrangement of the light
chain minilocus to effect allelic and isotypic exclusion.
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Analogous constructs can be generated with different
enhancers, i.e., the mouse or rat 3' kappa or heavy chain
enhancer (Meyer and Neuberger, EMBO J. $:1959-1964 (1989);
Petterson et al. Nature 344:165-168 (1990)).
Rearranged Light Chain Transgenes
A kappa light chain expression cassette was designed
to reconstruct functionally rearranged light chain genes that
have been amplified by PCR from human B-cell DNA. The scheme
is outlined in Fig. 33. PCR amplified light chain genes are
cloned into the vector pK5nx that includes 3.7 kb of 5'
flanking sequences isolated from the kappa light chain gene
65.5. The VJ segment.fused to the 5' transcriptional
sequences are then cloned into the unique XhoI site of the
vector pK31s that includes JK2-4, the JK intronic enhancer, CK,
and 9 kb of downstream sequences. The resulting plasmid
contains a reconstructed functionally rearranged kappa light
chain transgene that can be excised with NotI for
microinjection into embryos. The plasmids also contain unique
SalI sites at the 3' end for the insertion of additional cis-
acting regulatory sequences.
Two synthetic oligonucleotides (0-130, 0-131) were
used to amplify rearranged kappa light chain genes from human
spleen genomic DNA. Oligonucleotide o-131 (gga ccc aga
(g,c)gg aac cat gga a(g,a)(g,a,t,c)), is complementary to the
5' region of VKIII family light chain genes and overlaps the
first ATC of the leader sequence. Oligonucleotide o-130 (gtg
caa tca att ctc gag ttt gac tac aga c) is complementary to a
sequence approximately 150 bp 3' of J K 1 and includes an Xhol
site. These two oligonucleotides amplify a 0.7 kb DNA
fragment from human spleen DNA corresponding to rearranged
VKIII genes joined to J K 1 segments. The PCR amplified DNA was
digested with NcoI and XhoI and cloned individual PCR products
into the plasmid pNN03. The DNA sequence of 5 clones was
determined and identified two with functional VJ joints (open
reading frames). Additional functionally rearranged light
chain clones are collected. The functionally rearranged clones
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can be individually cloned into light chain expression
cassette described above (Fig. 33). Transgenic mice generated
with the rearranged light chain constructs can be bred with
heavy chain minilocus transgenics to produce a strain of mice
that express a spectrum of fully human antibodies in which all
of the diversity of the primary repertoire is contributed by
the heavy chain. One source of light chain diversity can be
from somatic mutation. Because not all light chains will be
equivalent with respect to their ability to combine with a
variety of different heavy chains, different strains of mice,
each containing different light chain constructs can be
generated and tested. The advantage of this scheme, as
opposed to the use of unrearranged light chain miniloci, is
the increased light chain allelic and isotypic exclusion that
comes from having the light chain ready to pair with a heavy
chain as soon as heavy chain VDJ joining occurs. This
combination can result in an increased frequency of B-cells
expressing fully human antibodies, and thus it can facilitate
the isolation of human Ig expressing hybridomas.
NotI inserts of plasmids pIGM1, pHC1, pIGG1, pKC1,
and pKC2 were isolated away from vector sequences by agarose
gel electrophoresis. The purified inserts were microinjected
into the pronuclei of fertilized (C57BL/6 x CBA)F2 mouse
embryos and transferred the surviving embryos into
pseudopregnant females as described by Hogan et al. (Hogan et
al., Methods of Manipulating the Mouse Embryo, Cold Spring
Harbor Laboratory, New York (1986)).
EXAMPLE 9
Inactivation of the Mouse Kappa Light Chain Gene by Homologous
Recombination
This example describes the inactivation of the mouse
endogenous kappa locus by homologous recombination in
embryonic stem (ES) cells followed by introduction of the
mutated gene into the mouse germ line by injection of targeted
ES cells bearing an inactivated kappa allele into early mouse
embryos (blastocysts).
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The strategy is to delete JK and CK by homologous
recombination with a vector containing DNA sequences
homologous to the mouse kappa locus in which a 4.5 kb segment
of the locus, spanning the JK gene and CK segments, is deleted
and replaced by the selectable marker neo.
Construction of the kappa targeting vector
The plasmid pGEM7 (KJ1) contains the neomycin
resistance gene (neo), used for drug selection of transfected
ES cells, under the transcriptional control of the mouse
phosphoglycerate kinase (pgk) promoter (XbaI/TaqI fragment;
Adra et al. (1987) Gene 60: 65) in the cloning vector pGEM-
7Zf(+). The plasmid also includes a heterologous
polyadenylation site for the neo gene, derived from the 3'
region of the mouse pgk gene (PvuII/HindIII fragment; Boer et
al., Biochemical Genetics, 28:299-308 (1990)). This plasmid
was used as the starting point for construction of the kappa
targeting vector. The first step was to insert sequences
homologous to the kappa locus 3' of the neo expression
cassette.
Mouse kappa chain sequences (Fig. 20a) were isolated
from a genomic phage library derived from liver DNA using
oligonucleotide probes specific for the C!c locus:
5'- GGC TGA TGC TGC ACC AAC TGT ATC CAT CTT CCC ACC ATC CAG
-3'
and for the Jrc 5 gene segment :
5'- CTC ACG TTC GGT GCT GGG ACC AAG CTG GAG CTG AAA CGT AAG -
3'.
An 8 kb BglII/SacI fragment extending 3' of the
mouse CK segment was isolated from a positive phage clone in
two pieces, as a 1.2 kb BglII/SacI fragment and a 6.8 kb SacI
fragment, and subcloned into BglII/SacI digested pGEM7 (KJ1)
to generate the plasmid pNEO-K3' (Fig. 20b).
A 1.2 kb EcoRI/SphI fragment extending 5' of the JK
region was also isolated from a positive phage clone. An
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SphI/XbaI/BglII/EcoRI adaptor was ligated to the SphI site of
this fragment, and the resulting EcoRI fragment was ligated
into EcoRI digested pNEO-K3', in the same 5' to 3' orientation
as the neo gene and the downstream 3' kappa sequences, to
generate pNEO-K5'3' (Fig. 20c).
The Herpes Simplex Virus (HSV) thymidine kinase (TK)
gene was then included in the construct in order to allow for
enrichment of ES clones bearing homologous recombinants, as
described by Mansour et al., Nature 336:348-352 (1988).
The HSV TK cassette was
obtained from the plasmid pGEM7 (TK), which contains the
structural sequences for the HSV TK gene bracketed by the
mouse pgk promoter and polyadenylation sequences as described
above for pGEM7 (KJ1). The EcoRI site of pGEM7 (TK) was
modified to a BamHI site and the TK cassette was then excised
as a BamHI/HindIII fragment and subcloned into pGPlb to
generate pGPlb-TK. This plasmid was linearized at the XhoI
site and the XhoI fragment from pNEO-K5'3', containing the neo
gene flanked by genomic sequences from 5' of Jx and 3' of CK,
was inserted into pGPlb-TK to generate the targeting vector
J/C KI (Fig. 20d). The putative structure of the genomic
kappa locus following homologous recombination with J/C Ki is
shown in Fig. 20e.
Generation and analysis of ES cells with targeted inactivation
of a kappa allele 4
The ES cells used were the AB-1 line grown on
mitotically inactive SNL76/7 cell feeder layers (McMahon and
Bradley, Cell 62:1073-1085 (1990)) essentially as described
(Robertson, E.J. (1987) in Teratocarcinomas and Embryonic Stem
Cells: A Practical Approach. E.J. Robertson, ed. (Oxford: IRL
Press), p. 71-112). Other suitable ES lines include, but are
not limited to, the E14 line (Hooper et al. (1987) Nature 326:
292-295), the D3 line (Doetschman et al. (1985) J. Embryol.
Exp. Morph. 87: 27-45), and the CCE line (Robertson et al.
(1986) Nature 323: 445-448). The success of generating a
mouse line from ES cells bearing a specific targeted mutation
depends on the pluripotence of the ES cells (i.e., their
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ability, once injected into a host blastocyst, to participate
in embryogenesis and contribute to the germ cells of the
resulting animal).
The pluripotence of any given ES cell line can vary
with time in culture and the care with which it has been
handled. The only definitive assay for pluripotence is to
determine whether the specific population of ES cells to be
used for targeting can give rise to chimeras capable of
germline transmission of the ES genome. For this reason,
prior to gene targeting, a portion of the parental population
of AB-1 cells is injected into C57B1/6J blastocysts to
ascertain whether the cells are capable of generating chimeric
mice with extensive ES cell contribution and whether the
majority of these chimeras can transmit the ES genome to
progeny.
The kappa chain inactivation vector J/C Ki was
digested with NotI and electroporated into AB-1 cells by the
methods described (Hasty et al., Nature, 350:243-246 (1991)).
Electroporated cells were plated onto 100 mm dishes at a
density of 1-2 x 106 cells/dish. After 24 hours, G418
(200 g/ml of active component) and FIAU (0.5 M) were added to
the medium, and drug-resistant clones were allowed to develop
over 10-11 days. Clones were picked, trypsinized, divided
into two portions, and further expanded. Half of the cells
derived from each clone were then frozen and the other half
analyzed for homologous recombination between vector and
target sequences.
DNA analysis was carried out by Southern blot
hybridization. DNA was isolated from the clones as described
(Laird et al., Nuci. Acids Res. 19:4293 (1991)) digested with
XbaI and probed with the 800 bp EcoRI/XbaI fragment indicated
in Fig. 20e as probe A. This probe detects a 3.7 kb XbaI
fragment in the wild type locus, and a diagnostic 1.8 kb band
in a locus which has homologously recombined with the
targeting vector (see Fig. 20a and e). Of 901 G418 and FIAU
resistant clones screened by Southern blot analysis, 7
displayed the 1.8 kb XbaI band indicative of a homologous
recombination into one of the kappa genes. These 7 clones
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were further digested with the enzymes BglII, Sacl, and PstI
to verify that the vector integrated homologously into one of
the kappa genes. When probed with the diagnostic 800 bp
EcoRI/XbaI fragment (probe A), BglII, Sacl, and PstI digests
of wild type DNA produce fragments of 4.1, 5.4, and 7 kb,
respectively, whereas the presence of a targeted kappa allele
would be indicated by fragments of 2.4, 7.5, and 5.7 kb,
respectively (see Fig. 20a and e). All 7 positive clones
detected by the XbaI digest showed the expected BglII, Sacl,
and PstI restriction fragments diagnostic of a homologous
recombination at the kappa light chain. In addition, Southern
blot analysis of an NsiI digest of the targeted clones using a
neo specific probe (probe B, Fig. 20e) generated only the
predicted fragment of 4.2 kb, demonstrating that the clones
each contained only a single copy of the targeting vector.
Generation of mice bearing the inactivated kappa chain
Five of the targeted ES clones described in the
previous section were thawed and injected into C57B1/6J
blastocysts as described (Bradley, A. (1987) in
Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach. E.J. Robertson, ed. (Oxford: IRL Press), p. 113-151)
and transferred into the uteri of pseudopregnant females to
generate chimeric mice resulting from a mixture of cells
derived from the input ES cells and the host blastocyst. The
extent of ES cell contribution to the chimeras can be visually
estimated by the amount of agouti coat coloration, derived
from the ES cell line, on the black C57B1/6J background.
Approximately half of the offspring resulting from blastocyst
injection of the targeted clones were chimeric (i.e., showed
agouti as well as black pigmentation) and of these, the
majority showed extensive (70 percent or greater) ES cell
contribution to coat pigmentation. The AB1 ES cells are an XY
cell line and a majority of these high percentage chimeras
were male due to sex conversion of female embryos colonized by
male ES cells. Male chimeras derived from 4 of the 5 targeted
clones were bred with C57BL/6J females and the offspring
monitored for the presence of the dominant agouti coat color
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indicative of germline transmission of the ES genome.
Chimeras from two of these clones consistently generated
agouti offspring. Since only one copy of the kappa locus was
targeted in the injected ES clones, each agouti pup had a 50
percent chance of inheriting the mutated locus. Screening for
the targeted gene was carried out by Southern blot analysis of
Bgl II-digested DNA from tail biopsies, using the probe
utilized in identifying targeted ES clones (probe A, Fig.
20e). As expected, approximately 50 percent of the agouti
offspring showed a hybridizing Bgl II band of 2.4 kb in
addition to the wild-type band of 4.1 kb, demonstrating the
germline transmission of the targeted kappa locus.
In order to generate mice homozygous for the
mutation, heterozygotes were bred together and the kappa
genotype of the offspring determined as described above. As
expected, three genotypes were derived from the heterozygote
matings: wild-type mice bearing two copies of a normal kappa
locus, heterozygotes carrying one targeted copy of the kappa
gene and one NT kappa gene, and mice homozygous for the kappa
mutation. The deletion of kappa sequences from these latter
mice was verified by hybridization of the Southern blots with
a probe specific for JK (probe C, Fig. 20a). Whereas
hybridization of the JK probe was observed to DNA samples from
heterozygous and wild-type siblings, no hybridizing signal was
present in the homozygotes, attesting to the generation of a
novel mouse strain in which both copies of the kappa locus
have been inactivated by deletion as a result of targeted
mutation.
EXAMPLE 10
Inactivation of the Mouse Heavy Chain Gene by Homologous
Recombination
This example describes the inactivation of the
endogenous murine immunoglobulin heavy chain locus by
homologous recombination in embryonic stem (ES) cells. The
strategy is to delete the endogenous heavy chain J segments by
homologous recombination with a vector containing heavy chain
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sequences from which the J. region has been deleted and
replaced by the gene for the selectable marker neo.
Construction of a heavy chain targeting vector
Mouse heavy chain sequences containing the J. region
(Fig. 21a) were isolated from a genomic phage library derived
from the D3 ES cell line (Gossler et al., Proc. Natl. Acad.
Sci. U.S.A. 83:9065-9069 (1986)) using a JH4 specific
oligonucleotide probe:
5'- ACT ATG CTA TGG ACT ACT GGG GTC AAG GAA CCT CAG TCA CCG
-3'
A 3.5 kb genomic Sacl/Stul fragment, spanning the J.
region, was isolated from a positive phage clone and subcloned
into Sacl/Smal digested pUC18. The resulting plasmid was
designated=pUC18 JH. The neomycin resistance gene (neo), used
for drug selection of transfected ES cells, was derived from a
repaired version of the plasmid pGEM7 (KJ1). A report in the
literature (Yenofsky et al. (1990) Proc. Natl. Acad. Sci.
(U.S.A.) 87: 3435-3439) documents a point mutation the neo
coding sequences of several commonly used expression vectors,
including the construct pMClneo (Thomas and Cappechi (1987)
Cell 51: 503-512) which served as the source of the neo gene
used in pGEM7 (KJ1). This mutation reduces the activity of
the neo gene product and was repaired by replacing a
restriction fragment encompassing the mutation with the
corresponding sequence from a wild-type neo clone. The
Hindlll site in the prepared pGEM7 (KJ1) was converted to a
SalI site by addition of a synthetic adaptor, and the neo
expression cassette excised by digestion with XbaI/SalI. The
ends of the neo fragment were then blunted by treatment with
the Klenow form of DNA poll, and the neo fragment was
subcloned into the NaeI site of pUC18 JH, generating the
plasmid=pUC18 JH-neo (Fig. 21b).
Further construction of the targeting vector was
carried out in a derivative of the plasmid pGPlb. pGP1b was
digested with the restriction enzyme NotI and ligated with the
following oligonucleotide as an adaptor:
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5'-.GGC CGC TCG ACG ATA GCC TCG AGG CTA TAA ATC TAG AAG AAT
TCC AGC AAA GCT TTG GC -3'
The resulting plasmid, called pGMT, was used to
build the mouse immunoglobulin heavy chain targeting
construct.
The Herpes Simplex Virus (HSV) thymidine kinase (TK)
gene was included in the construct in order to allow for
enrichment of ES clones bearing homologous recombinants, as
described by Mansour et al.=(Nature 336, 348-352 (1988)). The
HSV TK gene was obtained from the plasmid pGEM7 (TK) by
digestion with EcoRI and Hindlll. The TK DNA fragment was
subcloned between the EcoRI and Hindlil sites of pGMT,
creating the plasmid pGMT-TK (Fig. 21c).
To provide an extensive region of homology to the
target sequence, a 5.9 kb genomic XbaI/XhoI fragment, situated
5' of the JH region, was derived from a positive genomic phage
clone by limit digestion of the DNA with XhoI, and partial
digestion with XbaI. As noted in Fig. 21a, this XbaI site is
not present in genomic DNA, but is rather derived from phage
sequences immediately flanking the cloned genomic heavy chain
insert in the positive phage clone. The fragment was
subcloned into XbaI/XhoI digested pGMT-TK, to generate the
plasmid pGMT-TK-JH5' (Fig. 21d).
The final step in the construction involved the
excision from pUC18 JH-neo of the 2.8 kb EcoRI fragment which
contained the neo gene and flanking genomic sequences 3' of
JH. This fragment was blunted by Klenow polymerase and
subcloned into the similarly blunted XhoI site of
pGMT-TK-J.51. The resulting construct, JHKO1 (Fig. 21e),
contains 6.9 kb of genomic sequences flanking the JH locus,
with a 2.3 kb deletion spanning the JH region into which has
been inserted the neo gene. Fig. 21f shows the structure of
an endogenous heavy chain gene after homologous recombination
with the targeting construct.
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EXAMPLE 11
Generation and analysis of targeted ES cells
AB-1 ES cells (McMahon and Bradley, Cell
62:1073-1085 (1990)) were grown on mitotically inactive
SNL76/7 cell feeder layers essentially as described
(Robertson, E.J. (1987) Teratocarcinomas and Embryonic Stem
Cells.: A Practical Approach. E.J. Robertson, ed. (Oxford: IRL
Press), pp. 71-112). As described in the previous example,
prior to electroporation of ES cells with the targeting
construct JHKO1, the pluripotency of the ES cells was
determined by generation of AB-1 derived chimeras which were
shown capable of germline transmission of the ES genome.
The heavy chain inactivation vector JHKO1 was
digested with NotI and electroporated into AB-1 cells by the
methods described (Hasty et al., Nature 350:243-246 (1991)).
Electroporated cells were plated into 100 mm dishes at a
density of 1-2 x 106 cells/dish. After 24 hours, G418
(200mg/ml of active component) and FIAU (0.5mM) were added to
the medium, and drug-resistant clones were allowed to develop
over 8-10 days. Clones were picked, trypsinized, divided into
two portions, and further expanded. Half of the cells derived
from each clone were then frozen and the other half analyzed
for homologous recombination between vector and target
sequences.
DNA analysis was carried out by Southern blot
hybridization. DNA was isolated from the clones as described
(Laird et al. (1991) Nucleic Acids Res. 19: 4293), digested
with Stul and probed with the 500 bp EcoRI/Stul fragment
designated as probe A in Fig. 21f. This probe detects a Stul
fragment of 4.7 kb in the wild-type locus, whereas a 3 kb band
is diagnostic of homologous recombination of endogenous
sequences with the targeting vector (see Fig. 21a and f). Of
525 G418 and FIAU doubly-resistant clones screened by Southern
blot hybridization, 12 were found to contain the 3 kb fragment
diagnostic of recombination with the targeting vector. That
these clones represent the expected targeted events at the JH
locus (as shown in Fig. 21f) was confirmed by further
digestion with Hindlil, Spel and HpaI. Hybridization of probe
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A (see Fig. 21f) to Southern blots of Hindlil, Spel, and HpaI
digested DNA produces bands of 2.3 kb, >10 kb, and >10kb,
respectively, for the wild-type locus (see Fig. 21a), whereas
bands of 5.3 kb, 3.8 kb, and 1.9 kb, respectively, are
expected for the targeted heavy chain locus (see Fig 21f).
All 12 positive clones detected by the Stul digest showed the
predicted Hindlll, Spel, and HpaI bands diagnostic of a
targeted J. gene. In addition, Southern blot analysis of a
Stul digest of all 12 clones using a neo-specific probe (probe
B, Fig. 21f) generated only the predicted fragment of 3 kb,
demonstrating that the clones each contained only a single
copy of the targeting vector.
Generation of mice carrying theJH deletion
Three of the targeted ES clones described in the
previous section were thawed and injected into C57BL/6J
blastocysts as described (Bradley, A. (1987) in
Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, E.J. Robertson, ed. (Oxford: IRL Press), p.113-151)
and transferred into the uteri of pseudopregnant females. The
extent of ES cell contribution to the chimera was visually
estimated from the amount of agouti coat coloration, derived
from the ES cell line, on the black C57BL/6J background. Half
of the offspring resulting from blastocyst injection of two of
the targeted clones were chimeric (i.e., showed agouti as well
as black pigmentation); the third targeted clone did not
generate any chimeric animals. The majority of the chimeras
showed significant (approximately 50 percent or greater) ES
cell contribution to coat pigmentation. Since the AB-1 ES
cells are an XY cell line, most of the chimeras were male, due
to sex conversion of female embryos colonized by male ES
cells. Males chimeras were bred with C57BL/6J females and the
offspring monitored for the presence of the dominant agouti
coat color indicative of germline transmission of the ES
genome. Chimeras from both of the clones consistently
generated agouti offspring. Since only one copy of the heavy
chain locus was targeted in the injected ES clones, each
agouti pup had a 50 percent chance of inheriting the mutated
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locus. Screening for the targeted gene was carried out by
Southern blot analysis of Stul-digested DNA from tail
biopsies, using the probe utilized in identifying targeted ES
clones (probe A, Fig. 21f). As expected, approximately 50
percent of the agouti offspring showed a hybridizing Stul band
of approximately 3 kb in addition to the wild-type band of 4.7
kb, demonstrating germline transmission of the targeted JH
gene segment.
In order to generate mice homozygous for the
mutation, heterozygotes were bred together and the heavy chain
genotype of the offspring determined as described above. As
expected, three genotypes were derived from the heterozygote
matings: wild-type mice bearing two copies of the normal JH
locus, heterozygotes carrying one targeted copy of the gene
and one normal copy, and mice homozygous for the JH mutation.
The absence of JH sequences from these latter mice was
verified by hybridization of the Southern blots of Stul-
digested DNA with a probe specific for JH (probe C, Fig. 21a).
Whereas hybridization of the JH probe to a 4.7 kb fragment in
DNA samples from heterozygous and wild-type siblings was
observed, no signal was present in samples from the JH-mutant
homozygotes, attesting to the generation of a novel mouse
strain in which both copies of the heavy chain gene have been
mutated by deletion of the JH sequences.
EXAMPLE 12
Heavy Chain Minilocus Transgene
A. Construction of plasmid vectors for cloning large DNA
sequences
1. Gp Pla
The plasmid pBR322 was digested with EcoRI and Styl
and ligated with the following oligonucleotides:
oligo-42 5'- caa gag ccc gcc taa tga gcg ggc ttt ttt ttg cat
act gcg gcc get -3'
oligo-43 5'- aat tag cgg ccg cag tat gca aaa aaa agc ccg ctc
att agg cgg get -3'
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The resulting plasmid, pGPla, is designed for
cloning very large DNA constructs that can be excised by the
rare cutting restriction enzyme NotI. It contains a NotI
restriction site downstream (relative to the ampicillin
= 5 resistance gene, AmpR) of a strong transcription termination
signal derived from the trpA gene (Christie et al., Proc.
Natl. Acad. Sci. USA 78:4180 (1981)). This termination signal
reduces the potential toxicity of coding sequences inserted
into the NotI site by eliminating readthrough transcription
from the AmpR gene. In addition, this plasmid is low copy
relative to the pUC plasmids because it retains the pBR322
copy number control region. The low copy number further
reduces the potential toxicity of insert sequences and reduces
the selection against large inserts due to DNA replication.
The vectors pGPlb, pGPlc, pGPld, and pGPlf are derived from
pGPla and contain different polylinker cloning sites. The
polylinker sequences are given below
pGPla
NotI
GCGGCCGC
pGPlb
NotI XhoI ClaI BamHI Hindill NotI
GCggccgcctcgagatcactatcgattaattaaggatccagcagtaagcttgcGGCCGC
pGIlc
NotI Smal XhoI Sall Hindill BamHi Sacil NotI
GCggccgcatcccgggtctcgaggtcgacaagctttcgaggatccgcGGCCGC
pGPld
NotI Sall Hindill Clal BamHI XhoI NotI
GCggccgctgtcgacaagcttatcgatggatcctcgagtgcGGCCGC
pGPlf
=4 5
NotI Sall Hindlll EcoRI Clal KpnI BamHI XhoI NotI
GCggccgctgtcgacaagcttcgaattcagatcgatgtggtacctggatcctcgagtgcGGCCGC
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Each of these plasmids can be used for the construction of
large transgene inserts that are excisable with NotI so that
the transgene DNA can be purified away from vector sequences
prior to microinjection.
2. G~ Plb
pGPla was digested with NotI and ligated with the
following oligonucleotides:
oligo-47 5'- ggc cgc aag ctt act get gga tcc tta att aat cga
tag tga tct cga ggc -3'
oligo-48 5'- ggc cgc ctc gag atc act atc gat taa tta agg atc
cag cag taa get tgc -3'
The resulting plasmid, pGPlb, contains a short
polylinker region flanked by NotI sites. This facilitates the
construction of large inserts that can be excised by NotI
digestion.
3. pGPe
The following oligonucleotides:
oligo-44 5'- ctc cag gat cca gat atc agt acc tga aac agg get
tgc -3'
oligo-45 5'- ctc gag cat gca cag gac ctg gag cac aca cag cct
tcc -3'
were used to amplify the immunoglobulin heavy chain 3'
enhancer (S. Petterson, et al., Nature 344:165-168 (1990))
from rat liver DNA by the polymerase chain reaction technique.
The amplified product was digested with BamHI and
SphI and cloned into BamHI/SphI digested pNNO3 (pNNO3 is a pUC
derived plasmid that contains a polylinker with the following
restriction sites, listed in order: NotI, BamHI, NcoI, Clai,
EcoRV, XbaI, Sacl, XhoI, SphI, PstI, BglII, EcoRI, Smal, KpnI, 40 HindIll, and
NotI). The resulting plasmid, pRE3, was digested
with BamHI and Hindill, and the insert containing the rat Ig
heavy chain 3' enhancer cloned into BamHI/HindIII digested
pGPlb. The resulting plasmid, pGPe (Fig. 22 and Table 1),
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contains several unique restriction sites into which sequences
can be cloned and subsequently excised together with the 3'
enhancer by NotI digestion.
L
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TABLE I
Sequence of vector pOPe.
AATTAGCggccgcctcgagatcactatcgatta&itaaggatccagaiatcagtacctgaaacagggcltgetcacaac
a
tctcIctctctgtctctctgtctctgtgtgtgtgtctctctctgtctctatetctctctgtctctctgtctcigtgtgt
g
tcL ct ci ct gt ci ctct ct cLgt cl ct ct gt ct at ct gL CL pt at ci at c L cL
at c l ci gi ci ci ci ct c l ci ci cl ctc
tctctctctctctctctcacacacacacacacacacacacacacacctgccgagtgactcactctgtgcagggttggcc
c
tcggggcacatgcaaatggatgtttgttccatgcagasaaacatgtttctcattctctgagccaaaaategcatcaatg
a
ttcccccaccetgcagctgcaggttcaceccacctggecaggttgaccagctttggggatggggctgggggitccatga
d
ccctaacggtgacattgaattcagtgttttcccatttatcgacactgctggaatctgaccctaggagggaatgacagga
g
ataggcaaggtccaaacaccccagggaagtgggagagacaggaaggctgtglgtgctccaggtcclglgcaIgetgcag
a
tctgaaticccgggtaccaagcttgcGGCCOCAGTATGCAAAAAAAAGCCCGCTCATTAGGCGGGCTCTTGGCAGAACA
T
ATCCATCGCGTCCGCCATCTCCAGCAGCCGCACGCGGCGCATCTCGGGCAGCGTTGGGTCCTGGCCACGGGCGCGCATG
A
TCGTGCTCCTOTCOCCGAGGACCCGGCTAGGCTOGCGGGGTTOCCTTACTGOTTAGCAGAATQAATCACCOACACGCGA
G
CGAACGTOAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGT
A
AAGTCTGOAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGOATGCTOCTGOCTACCCTGT
O
GAACACCTACATCTOTATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGCATCCATACCGC
C
AGTTGTTTACCCTCACAACGTTCCAGTAACCGGOCATGTTCATCATCAGTAACCCGTATCGTGAGCATCCTCTCTCGTT
T
CATCGGTATCATTACCCCCATGAACAGAAATTCCCCCTTACACGGAGOCATCAAGTGACCAAACAGOAAAAAACCGCCC
T
TAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTCTGOAGAAACTCAACGAGCTGGACGCGGATGAACAGOCA
G
ACATCTGTGAATCGCTTCACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAA
C
CTCTOACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATOCCGGGAGCAGACAAGCCCGTCAGGGCG
C
GTCAGCGGGT0TTGGCGGGTGTCGGGOCGCAGCCATGACCCAGTCACGTAGCGATAOCGGAGTGTATACTGGCTTAACT
A
TGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCOGTGTOAAATACCGCACAGATGCGTAAGGAGAAAATAC
C
GCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCA
C
TCAAAGGCGGTAATACGOTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGACCAAAAGGCCAGCAAAAGG
C
CAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACOC
T
CAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAOATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCC
T
GTTCCOACCCTGCCGCTTACCGGATACCTGTCCOCCTTTCTCCCTTCOGGAAGCOTGGCOCTTTCTCATAGCTCACGCT
G
TAGGTATCTCAGTTCGGTGTAQGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGC
G
CCTTATCCGGTAACTATCOTCTTGAGTCCAACCCOGTAAGACACOACTTATCOCCACTGOCAGCAGCCACTGGTAACAG
G
ATTAOCAGAOCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAO
T
ATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACC
G
CTOOTAGCGGTOGTTTTTTTGTTTGCAAGCAGCAGATTACGCOCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTT
T
TCTACGGGQTCTGACGCPCAOTGGAACOAAAACTCACOTTAAGGGATTTTOOTCATOAGATTATCAAAAAGGATCTTCA
C
CTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAA
T
GCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCOTTCATCCATAGTTGCCTGACTCCCCGTCGTOTAGAT
A
ACTACGATACGGGAGGGCTTACCATCTGOCCCCAOTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATT
T
ATCAGCAATAAACCAGCCAGCCOGAAGOGCCOAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATT
A
ATTGTTGCCGGGAAQCTAGAGTAAGTAGTTCGCCAOTTAATAGTTTGCOCAACGTTGTTGCCATTGCTGCAGGCATCGT
G
GTQTCACGCTCCfTCGTTTGGTATGQCTTCATTCAGCTCCCOGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCAT
GTT
GTGCAAAAAAGCGGTTAGCTCCTTCGOTCCTCCGATCGTTOTCAOAAGTAAGTTGGCCGCAGTQTTATCACTCATGGTT
A
TGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTOTGACTGOTGAGTACTCAACCAAGTC
A
TTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGOATAATACCGCGCCACATAGCAOAA
C
TTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCOAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCG
A
TGTAACCCACCCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAG
G
CAAAATGCCGCAAAAAAGOGAATAAGGGCGACACGGAAATOTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAA
G
CATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGCcC>CTTCCGC
GCA
CATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCAC
G
AGGCCCTTTCGTCTTCAAQ
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B. Construction of IgM expressing minilocus transgene, pIGM1
1. Isolation of J-u constant region clones and construction
of PJM1
A human placental genomic DNA library cloned into
the phage vector XEMBL3/SP6/T7 (Clonetech Laboratories, Inc.,
Palo Alto, CA) was screened with the human heavy chain J
region specific oligonucleotide:
oligo-1 5'- gga ctg tgt ccc tgt gtg atg ctt ttg atg tct ggg
gcc aag -3'
and the phage clone X1.3 isolated. A 6 kb HindIII/KpnI
fragment from this clone, containing all six J segments as
well as D segment DHQ52 and the heavy chain J-p intronic
enhancer, was isolated. The same library was screened with
the human p specific oligonucleotide:
oligo-2 5'- cac caa gtt gac ctg cct ggt cac aga cct gac cac
cta tga -3'
and the phage clone X2.1 isolated. A 10.5 kb HindIII/XhoI
fragment, containing the p switch region and all of the p
constant region exons, was isolated from this clone. These
two fragments were ligated together with Kpnl/XhoI digested
pNN03 to obtain the plasmid pJM1.
2. pJM2
A 4 kb XhoI fragment was isolated from phage clone
X2.1 that contains sequences immediately downstream of the
sequences in pJM1, including the so called EA element involved
in d-associated deleteon of the p in certain IgD expressing
B-cells (Yasui et al., Eur. J. Immunol. 19:1399 (1989)).
This fragment was
treated with the Klenow fragment of DNA polymerase I and
ligated to XhoI cut, Klenow treated, p3M1. The resulting
plasmid, pJM2 (Fig. 23), had lost the internal XhoI site but
retained the 3' XhoI site due to incomplete reaction by the
Klenow enzyme. pJM2 contains the entire human J region, the
heavy chain J-p intronic enhancer, the p switch region and all
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of the constant region exons, as well as the two 0.4 kb
direct repeats, ag and E , involved in 6-associated deletion
of the gene.
3. Isolation of D region clones and construction of pDH1
The following human D region specific
oligonucleotide:
oligo-4 5'- tgg tat tac tat ggt tcg ggg agt tat tat aac cac
agt gtc -3'
was used to screen the human placenta genomic library for D
region clones. Phage clones X4.1 and X4.3 were isolated. A
5.5 kb XhoI fragment, that includes the D elements DK1, DM1,
and DM2 (Ichihara et al., EMBO J. 7:4141 (1988)), was isolated
from phage clone X4.1. An adjacent upstream 5.2 kb XhoI
fragment, that includes the D elements DLR1, Dxpi, DXp.l, and
DA1, was isolated from phage clone X4.3. Each of these D
region XhoI fragments were cloned into the Sall site of the
plasmid vector pSP72 (Promega, Madison, WI) so as to destroy
the XhoI site linking the two sequences. The upstream
fragment was then excised with XhoI and Smal, and the
downstream fragment with EcoRV and XhoI. The resulting
isolated fragments were ligated together with Sall digested
pSP72 to give the plasmid pDH1. pDH1 contains a 10.6 kb
insert that includes at least 7 D segments and can be excised
with XhoI (5') and EcoRV (3').
4. Cp OR1
The plasmid pJM2 was digested with Asp718 (an
isoschizomer of KpnI) and the overhang filled in with the
Klenow fragment of DNA polymerase I. The resulting DNA was
then digested with Clal and the insert isolated. This insert
was ligated to the XhoI/EcoRV insert of pDHl and XhoI/ClaI
digested pGPe to generate pCORl (Fig. 24).
5. DVH251
A 10.3 kb genomic Hindlll fragment containing the
two human heavy chain variable region segments VH251 and VH105
CA 02161351 2003-10-23
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(Humphries et al., Nature 331:446 (1988))
was subcloned into pSP72 to
give the plasmid pVH251.
6. Ip GM1
The plasmid pCOR1 was partially digested with XhoI
and the isolated XhoI/SalI insert of pVH251 cloned into the
upstream XhoI site to generate the plasmid pIGM1 (Fig. 25).
pIGM1 contains 2 functional human variable region segments, at
least 8 human D segments all 6 human J. segments, the human
J-p enhancer, the human a element, the human A switch region,
all of the human A coding exons, and the human EA element,
together with the rat heavy chain 3' enhancer, such that all
of these sequence elements can be isolated on a single
fragment, away from vector sequences, by digestion with NotI
and microinjected into mouse embryo pronuclei to generate
transgenic animals.
C. Construction of IqM and IqG expressing minilocus
transgene, PHC]
1. Isolation of y constant region clones
The following oligonucleotide, specific for human Ig
g constant region genes:
oligo-29 5'- cag cag gtg cac acc caa tgc cca tga gcc cag aca
ctg gac -3'
was used to screen the human genomic library. Phage clones
129.4 and X29.5 were isolated. A 4 kb Hindlil fragment of
phage clone X29.4, containing a y switch region, was used to
probe a human placenta genomic DNA library cloned into the
phage vector lambda FIX"' II (Stratagene, La Jolla, CA). Phage
clone XSg1.13 was isolated. To determine the subclass of the
different y clones, dideoxy sequencing reactions were carried
out using subclones of each of the three phage clones as
templates and the following oligonucleotide as a primer:
oligo-67 5'- tga gcc cag aca ctg gac -3'
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Phage clones X29.5 and XSy1.13 were both determined
to be of the yi subclass.
2. pyel
A 7.8 kb Hindill fragment of phage clone X29.5,
containing the yl coding region was cloned into pUC18. The
resulting plasmid, pLT1, was digested with XhoI, Klenow
treated, and religated to destroy the internal XhoI site. The
resulting clone, pLTlxk, was digested with Hindlil and the
insert isolated and cloned into pSP72 to generate the plasmid
clone pLTlxks. Digestion of pLTlxks at a polylinker XhoI site
and a human sequence derived BamHI site generates a 7.6 kb
fragment containing the yl constant region coding exons. This
7.6 kb XhoI/BamHI fragment was cloned together with an
adjacent downstream 4.5 kb BamHI fragment from phage clone
X29.5 into XhoI/BamHI digested pGPe to generate the plasmid
clone pyel. pyel contains all of the yl constant region
coding exons, together with 5 kb of downstream sequences,
linked to the rat heavy chain 3' enhancer.
3. pye2
A 5.3 kb Hindill fragment containing the yi switch
region and the first exon of the pre-switch sterile transcript
(P. Sideras et al. (1989) International Immunol. 1, 631) was
isolated from phage clone XS71.13 and cloned into pSP72 with
the polylinker XhoI site adjacent to the 5' end of the insert,
to generate the plasmid clone pSyls. The XhoI/SalI insert of
pSyls was cloned into XhoI digested pyel to generate the
plasmid clone p7e2 (Fig. 26). p7e2 contains all of the 71
constant region coding exons, and the upstream switch region
and sterile transcript exons, together with 5 kb of downstream
sequences, linked to the rat heavy chain 3' enhancer. This
clone contains a unique XhoI site at the 5' end of the insert.
The entire insert, together with the Xhol site and the 3' rat
enhancer can be excised from vector sequences by digestion
with NotI.
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4. pHC1
The plasmid pIGM1 was digested with XhoI and the 43
kb insert isolated and cloned into XhoI digested pge2 to
generate the plasmid pHC1 (Fig. 25). pHC1 contains 2
functional human variable region segments, at least 8 human D
segments all 6 human JH segments, the human J-g enhancer, the
human aj element, the human switch region, all of the human
p coding exons, the human E element, and the human yl
constant region, including the associated switch region and
sterile transcript associated exons, together with the rat
heavy chain 3' enhancer, such that all of these sequence
elements can be isolated on a single fragment, away from
vector sequences, by digestion with NotI and microinjected
into mouse embryo pronuclei to generate transgenic animals.
D. Construction of IgM and IgG expressing minilocus
transgene, pHC2
1. Isolation of human heavy chain V region gene VH49.8
The human placental genomic DNA library lambda, FIXm
II, Stratagene, La Jolla, CA) was screened with the following
human VH1 family specific oligonucleotide:
oligo-49 5'- gtt aaa gag gat ttt att cac ccc tgt gtc ctc tcc
aca ggt gtc -3'
Phage clone X49.8 was isolated and a 6.1 kb XbaI
fragment containing the variable segment VH49.8 subcloned into
pNN03 (such that the polylinker ClaI site is downstream of
VH49.8 and the polylinker XhoI site is upstream) to generate
the plasmid pVH49.8. An 800 bp region of this insert was
sequenced, and VH49.8 found to have an open reading frame and
intact splicing and recombination signals, thus indicating
that the gene is functional (Table 2).
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TABLE 2
Sequence of human V]HI family gene VH 49.8
TTCCTCAGGC AGGATTTAGG GCTTGGTCTC TCAGCATCCC ACACTTGTAC 50
AGCTGATGTG GCATCTGTGT TTTCTTTCTC ATCCTAGATC AAGCTTTGAG 100
CTGTGAAATA CCCTGCCTCA TGAATATGCA AATAATCTGA GGTCTTCTGA 150
GATAAATATA GATATATTGG TGCCCTGAGA GCATCACATA ACAACCAGAT 200
TCCTCCTCTA AAGAAGCCCC TGGGAGCACA GCTCATCACC ATGGACTGGA 250
Met AspTrpT
CCTGGAGGTT CCTCTTTGTG GTGGCAGCAG CTACAGg t a a g g g g c t t c c t 300
hrTrpArgPh eLeuPheVal VaIAlaA1aA 1aThr
agtcctaagg ctgaggaagg gatcctggtt tagttaaaga ggattttatt 350
c a c c c c t g t g t c c t c t c c a c a g GTGTCCAG TCCCAGGTCC AGCTGGTGCA 400
GI yVal GI n SerG1 nVal G I nLeuVal G1
GTCTGGGGCT GAGGTGAAGA AGCCTGGGTC CTCGGTGAAG GTCTCCTGCA 450
n S e r Gl yAl a GI u Va 1 LysL y s P r oGl y S e r Se r Va 1 Ly s Val Se r
Cys L
AGGCTTCTGG AGGCACCTTC AGCAGCTATG CTATCAGCTG GGTGCGACAO 500
ysAlaSerGl yGlyThrPhe SerSerTyrAIal IeSerTr pVaIArgGI n
GCCCCTGGAC AAGGGCTTGA GTGGATGGGA AGGATCATCC CTATCCTTGG 550
A]aPro01yG 1n01yLeuGl uTrpMetGI y ArgllelleP roIIeLeuG1
TATAGCAAAC TACGCACAGA AGTTCCAGGG CAGAGTCACG ATTACCGCGG 600
yIIeAla Asn Tyr Al aGI aL ysPheGlnG1 yArgValThr IleThrAlaA
ACAAATCCAC GAGCACAGCC TACATGGAGC TGAGCAGCCT GAGGTCTGAG 650
spLysSerTh rSerThrAl a Tyr Met GI uL euSerSerLe uArgSerGlu
GACACGGCCG TCTATTACTG TGCGAGAG C ACAGTG TGAA AACCCACATC 700
As p Th r Al a V a I Ty r Ty r Cy s Al a Ar g
CTGAGAGTGIT CAGAAACC CT GAGGGAGAAG GCAGCTGTGC CGGGCTGAGG 750
AGATGACAGG GTTTATTAGG TTTAAGGCTC TTTACAAAAT GGGTTATATA 800
TTTGAGAAAA AA 812
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2. pV2
A 4 kb XbaI genomic fragment containing the human
VHIV family gene VH4-21 (Sanz= et al., EMBO J., 8:3741 (1989)),
subcloned into the plasmid pUC12, was excised with Smal and
HindIll, and treated with the Klenow fragment of polymerase I.
The blunt ended fragment was then cloned into Clai digested,
Klenow treated, pVH49.8. The resulting plasmid, pV2, contains
the human heavy chain gene VH49.8 linked upstream of VH4-21 in
the same orientation, with a unique Sall site at the 3' end of
the insert and a unique XhoI site at the 5' end.
3. pSyl-5'
A 0.7 kb XbaI/HindIII fragment (representing
sequences immediately upstream of, and adjacent to, the 5.3 kb
yl switch region containing fragment in the plasmid p7e2)
together with the neighboring upstream 3.1 kb XbaI fragment
were isolated from the phage clone XSg1.13 and cloned into
HindIII/XbaI digested pUCl8 vector. The resulting plasmid,
pS7l-5', contains a 3.8 kb insert representing sequences
upstream-of the initiation site of the sterile transcript
found in B-cells prior to switching to the yl isotype (P.
Sideras et al., International Immunol. 1:631 (1989)). Because
the transcript is implicated in the initiation of isotype
switching, and upstream cis-acting sequences-are often
important for transcription regulation, these sequences are
included in transgene constructs to promote correct expression
of the sterile transcript and the associated switch
recombination.
4. Vp GE1
The pSyl-5' insert was excised with Sinai and
Hindlll, treated with Klenow enzyme, and ligated with the
following oligonucleotide linker:
5'- ccg gtc gac cgg -3'
The ligation product was digested with Sall and ligated to
Sall digested pV2. The resulting plasmid, pVP, contains 3.8
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kb of y1 switch 5' flanking sequences linked downstream of the
two human variable gene segments VH49.8 and VH4-21 (see Table
2). The pVP insert is isolated by partial digestion with Sall
and complete digestion with XhoI, followed by purification of
the 15 kb fragment on an agarose gel. The insert is then
cloned into the XhoI site of pye2 to generate the plasmid
clone pVGE1 (Fig. 27). pVGE1 contains two human heavy chain
variable gene segments upstream of the human 71 constant gene
and associated switch region. A unique SalI site between the
variable and constant regions can be used to clone in D, J,
and g gene segments. The rat heavy chain 3' enhancer is
linked to the 3' end of the yl gene and the entire insert is
flanked by NotI sites.
5. nHC2
The plasmid clone pVGE1 is digested with Sall and
the XhoI insert of pIGM1 is cloned into it. The resulting
clone, pHC2 (Fig. 25), contains 4 functional human variable
region segments, at least 8 human D segments all 6 human JH
segments, the human J-m enhancer, the human ag element, the
human switch region, all of the human coding exons, the
human Eg element, and the human 71 constant region, including
the associated switch region and sterile transcript associated
exons, together with 4 kb flanking sequences upstream of the
sterile transcript initiation site. These human sequences are
linked to the rat heavy chain 3' enhancer, such that all of
the sequence elements can be isolated on a single fragment,
away from vector sequences, by digestion with NotI and
microinjected into mouse embryo pronuclei to generate
transgenic animals. A unique XhoI site at the 5' end of the
insert can be used to clone in additional human variable gene
segments to further expand the recombinational diversity of
this heavy chain minilocus.
E. Transgenic mice
The NotI inserts of plasmids pIGM1 and pHC1 were
isolated from vector sequences by agarose gel electrophoresis.
The purified inserts were microinjected into the pronuclei of
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fertilized (C57BL/6 x CBA)F2 mouse embryos and transferred the
surviving embryos into pseudopregnant females as described by
Hogan et al. (B. Hogan, F. Costantini, and E. Lacy, Methods of
Manipulating the Mouse Embryo, 1986, Cold Spring Harbor
= 5 Laboratory, New York). Mice that developed from injected
embryos were analyzed for the presence of transgene sequences
by Southern blot analysis of tail DNA. Transgene copy number
was estimated by band intensity relative to control standards
containing known quantities of cloned DNA. At 3 to 8 weeks of
age, serum was isolated from these animals and assayed for the
presence of transgene encoded human IgM and IgGi by ELISA as
described by Harlow and Lane (E. Harlow and D. Lane.
Antibodies: A Laboratory Manual, 1988, Cold Spring Harbor
Laboratory, New York). Microtiter plate wells were coated
with mouse monoclonal antibodies specific for human IgM (clone
AF6, #0285, AMAC, Inc. Westbrook,'ME) and human IgGi (clone
JL512, #0280, AMAC, Inc. Westbrook, ME). Serum samples were
serially diluted into the wells and the presence of specific
immunoglobulins detected with affinity isolated alkaline
phosphatase conjugated goat anti-human Ig (polyvalent) that
had been pre-adsorbed to minimize cross-reactivity with mouse
immunoglobulins. Table 3 and Fig. 28 show the results of an
ELISA assay for the presence of.human IgM and IgGi in the
serum of two animals that developed from embryos injected with
the transgene insert of plasmid pHC1. All of the control non-
transgenic mice tested negative for expression of human IgM
and IgGi by this assay. Mice from two lines containing the
pIGM1 NotI insert (lines #6 and 15) express human IgM but not
human IgGi. We tested mice from 6 lines that contain the pHC1
insert and found that 4 of the lines (lines #26, 38, 57 and
122) express both human IgM and human IgGi, while mice from
two of the lines (lines #19 and 21) do not express detectable
levels of human immunoglobulins. The pHC1 transgenic mice
that did not express human immunoglobulins were so-called Go
mice that developed directly from microinjected embryos and
may have been mosaic for the presence of the transgene.
Southern blot analysis indicates that many of these mice
contain one or fewer copies of the transgene per cell. The
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detection of human IgM in the serum of pIGM1 transgenics, and
human IgM and IgG1 in pHC1 transgenics, provides evidence that
the transgene sequences function correctly in directing VDJ
joining, transcription, and isotype switching. One of the
animals (#18) was negative for the transgene by Southern blot
analysis, and showed no detectable levels of human IgM or
IgGi. The second animal (#38) contained approximately 5 4
copies of the transgene, as assayed by Southern blotting, and
showed detectable levels of both human IgM and IgGi. The
results of ELISA assays for 11 animals that developed from
transgene injected embryos is summarized in the table below
(Table 3).
TABLE 3
Detection of human IgM and IgGl in the serum of transgenic
animals by ELISA assay
approximate
injected transgene
animal # transgene copies per cell human IgM human IgG1
6 pIGM1 1 + + -
7 pIGM1 0 - -
9 pIGM1 0 - -
10 pIGM1 0 - -
12 pIGM1 0 - -
15 pIGM1 10 + + -
18 pHC1 0 - -
19 pHC1 1
21 pHC1 <1 - - w
26 pHC 1 2 + + +
38 pHC1 5 + + +
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Table 3 shows a correlation between the presence of
integrated transgene DNA and the presence of transgene encoded
immunoglobulins in the serum. Two of the animals that were
found to contain the pHC1 transgene did not express detectable
levels of human immunoglobulins. These were both low copy
animals and may not have contained complete copies of the
transgenes, or the animals may have been genetic mosaics
(indicated by the <1 copy per cell estimated for animal #21),
and the transgene containing cells may not have populated the
hematopoietic lineage. Alternatively, the transgenes may have
integrated into genomic locations that are not conducive to
their expression. The detection of human IgM in the serum of
pIGM1 transgenics, and human IgM and IgGl in pHC] transgenics,
indicates that the transgene sequences function correctly in
directing VDJ joining, transcription, and isotype switching.
F. cDNA clones
To assess the functionality of the pHCI transgene in
VDJ joining and class switching, as well the participation of
the transgene encoded human B-cell receptor in B-cell
development and allelic exclusion, the structure of
immunoglobulin cDNA clones derived from transgenic mouse
spleen mRNA were examined. The overall diversity of the
transgene encoded heavy chains, focusing on D and J segment
usage, N region addition, CDR3 length distribution, and the
frequency of joints resulting in functional mRNA molecules was
examined. Transcripts encoding IgM and IgG incorporating
VH105 and VH251 were examined.
Polyadenylated RNA was isolated from an eleven week
old male second generation line-57 pHC1 transgenic mouse.
This RNA was used to synthesize oligo-dT primed single
stranded cDNA. The resulting cDNA was then used as template
for four individual PCR amplifications using the following
four synthetic oligonucleotides as primers: VH251 specific
oligo-149, cta get cga gtc caa gga gtc tgt gcc gag gtg cag ctg
(g,a,t,c); VH105 specific o-150, gtt get cga gtg aaa ggt gtc
cag tgt gag gtg cag ctg (g,a,t,c); human gametal specific
oligo-151, ggc get cga gtt cca cga cac cgt cac cgg ttc; and
WO 94/25585 PCT/US94/04580
tj~4j` . 126
human mu specific oligo-152, cct get cga ggc agc caa cgg cca
cgc tgc tcg. Reaction 1 used primers 0-149 and o-151 to
amplify VH251-gammal transcripts, reaction 2 used o-149 and 0-
152 to amplify VH251-mu transcripts, reaction 3 used o-150 and
0-151 to amplify VH105-gammal transcripts, and reaction 4 used
0-150 and o-152 to amplify VH105-mu transcripts. The
resulting 0.5 kb PCR products were isolated from an agarose
gel; the a transcript products were more abundant than the y
transcript products, consistent with the corresponding ELISA
data (Fig. 34). The PCR products were digested with XhoI and
cloned into the plasmid pNN03. Double-stranded plasmid DNA
was isolated from minipreps of nine clones from each of the
four PCR amplifications and dideoxy sequencing reactions were
performed. Two of the clones turned out to be deletions
containing no D or J segments. These could not have been
derived from normal RNA splicing products and are likely to
have originated from deletions introduced during PCR
amplification. One of the DNA samples turned out to be a
mixture of two individual clones, and three additional clones
did not produce readable DNA sequence (presumably because the
DNA samples were not clean enough). The DNA sequences of the
VDJ joints from the remaining 30 clones are compiled in Table
4. Each of the sequences are unique, indicating that no
single pathway of'gene rearrangement, or single clone of
transgene expressing B-cells is dominant. The fact that no
two sequences are alike is also an indication of the large
diversity of immunoglobulins that can be expressed from a
compact minilocus containing only 2 V segments, 10 D segments,
and 6 J segments. Both of the V segments, all six of the J
segments, and 7 of the 10 D segments that are included in the
transgene are used in VDJ joints. In addition, both constant
region genes (mu and gammal) are incorporated into
transcripts. The VH105 primer turned out not to be specific
for VH105 in the reactions performed. Therefore many of the
clones from reactions 3 and 4 contained VH251 transcripts.
Additionally, clones isolated from ligated reaction 3 PCR
product turned out to encode IgM rather than IgG; however this
may reflect contamination with PCR product from reaction 4 as
CA 02161351 2003-10-23
127
the DNA was isolated on the same gel. An analogous
experiment, in which immunoglobulin heavy chain sequences were
amplified from adult human peripheral blood lymphocytes (PBL),
and the DNA sequence of the VDJ joints determined, was
recently reported by Yamada et al. (J. Exp. Med. 173:395-407
(1991)).. We
compared the data from human PBL with our data from the pHC3
transgenic mouse.
WO 94/25585 PCT/US94/04580
128 0
r - u u u
.. is 2 .c s = = X < < S c t c < < < < = a
< < i 2 i s Z ~2 Z t d s s L t u Y
u u u u u u u ~ v v u u u u u u ~
G G G G t G r
G :. ~ t t G ~ G G L ~ L ~ C t t t G C C t
~ u G G G u n~~ C u` :: u G G G G G G G
U V U U U U V .U U U V V U ~U+ V V U U U V U
u 61
.3 Is"
C_ G t t G t C L C
c c c < < i < i c c c t Y
<
4 < i < < < < < < < < V
a < < t a a < '~ 4 < < i < Z < s i < tt < i S K -C W.
duC
d1l
2 2
u s ~
13
t i M S i i iJ 6i 44 t s j 3
iS u r w t7 t3
r G 6 6 G G 6 G G Cs G G G 6 G G ~, G G G 6 5 G 6 G
r r r r r r r r r. r r e. r r r r
r. r r
IL L a : a a a a a a L a L L
= + r S ^ i a a i i i i a a
w a - w - - . ti w wt a-
~ ~ ~ Q Q ~ ~ Q L f
3 ~ a o o `a II 8 v cn II o a o o II ~ a ~ c o a` a ~ ~ ~I ~ II
w h ^ h N A A A IA A A M N A N - _ N A A A A = O = N N _
w w = N
- s s s s s s "s s s s s s s s s s s s s s s s s s;
w =. .~ . = s . . . w w w w w wry a- w
WO 94125585 PCT/US94/04580
129 216131
G. J segment choice
Table 5 compared the distribution of J segments
incorporated into pHC1 transgene encoded transcripts to J
segments found in adult human PBL immunoglobulin transcripts.
The distribution profiles are very similar, J4 is the dominant
segment in both systems, followed by J6. J2 is the least
common segment in human PBL and the transgenic animal.
TABLE 5 J. Segment Choice
Percent Usage ( 3%)
J. Segment HC1 transgenic Human PBL
J1 7 1
J2 3 <1
J3 17 9
J4 44 53
J5 3 15
J6 26 22
100% 100%
H. P segment choice
49% (40 of 82) of the clones analyzed by Yamada et
al. incorporated D segments that are included in the pHC1
transgene. An additional 11 clones. contained sequences that
were not assigned by the authors to any of the known D
segments. Two of these 11 unassigned clones appear to be
derived from an inversion of the DIR2 segments which is
included in the pHC1 construct. This mechanism, which was
predicted by Ichihara et al. (EMBO J. 7:4141 (1988)) and
observed by Sanz (J. Immunol. 147:1720-1729 (1991)), was not
considered by Yamada et al. (J. Exp. Med. 173:395-407 (1991)).
Table 5 is a comparison of the D segment distribution for the
pHC1 transgenic mouse and that observed for human PBL
transcripts by Yamada et al. The data of Yamada et al. was
recompiled to include DIR2 use, and to exclude D segments that
are not in the pHC1 transgene. Table 6 demonstrates that the
distribution of D segment incorporation is very similar in the
transgenic mouse and in human PBL. The two dominant human D
segments, DXP'1 and DN1, are also found with high frequency in
the transgenic mouse. The most dramatic dissimilarity between
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130 0
the two distributions is the high frequency of DHQ52 in the
transgenic mouse as compared to the human. The high frequency
of DHQ52 is reminiscent of the D segment distribution in the
human fetal liver. Sanz has observed that 14% of the heavy
chain transcripts contained DHQ52 sequences. If D segments
not found in pHC1 are excluded from the analysis, 31% of the
fetal transcripts analyzed by Sanz contain DHQ52. This is
comparable to the 27% that we observe in the pHC1 transgenic
mouse.
TABLE 6 D Segment Choice
Percent Usage ( 3%)
D. Segment HC1 transgenic Human PBL
DLR1 <1 <1
DXP1 3 6
DXP'1 25 19
DA1 <1 12
DK1 7 12
DN1 12 22
DIR2 7 4
DM2 <1 2
DLR2 3 4
DHQ52 26 2
? 17 17
100% 100%
I. Functionality of VDJ joints
Table 7 shows the predicted amino acid sequences of
the VDJ regions from 30 clones that were analyzed from the
pHC1 transgenic. The translated sequences indicate that 23 of
the 30 VDJ joints (77%) are in-frame with respect to the
variable and J segments.
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131
TABLE 7
Functionality of V-D-J Joints
FR3 CDR3 FR4
1 VH251 DHQ52 J3 71 YCAR RLTGVDAFDI WGQGTMVTVSSASTK
2 VH251 DN1 J4 y1 YCAR HRIAAAGFDY WGQGTLVTVSSASTK
3 VH251 D? J6 y1 YCAR YYYYYGMDV WGQGTTVTVSSASTK
4 VH251 DXP'l J6 yl YCAR HYDILTGP'TI'ITVWTSGAKGPRSPSPQPPP
VH251 DXP'1 J4 yl YCAR RRYYGSGSYYNVTFDY WGQGTLVTVSSASTK
6 VH251 D? J3 yl YCAR RGVSDAFDI WGQGTMVTVSSASTK
7 VH251 DHQ52 J3 At YCAR ATGAFDI WGQGTMVTVSSGSAS
8 VH251 DHQ52 J6 q YCAR SANWGSYYYYGMDV WGQG'ITVFVSSGSAS
9 VH251 - J1 u YCAR YFQH WGQGTLVTVSSGSAS
VH251 DLR2 J4 u YCAR HVANSFDY WGQGTLVTVSSGSAS
11 VH251 DXP'l J4 # YCAR QITMVRGVPFDY WGQGTLVTVSSGSAS
12 VH251 D? J1 u YCAR QYFQH WGQGTLVTVSSGSAS
13 VH251 DHQ52 J6 u YCAR QTGDYYYYGMDV WGQGTTV FVSSGSAS
14 VH251 DXP'1 J6 p YCAR HYYGSGSYDYYYYGMDV WGQG'ITVI'VSSGSAS
VH251 DXP'l J4 yl YCVR QGVGPGNPGHRLLSLHQ
16 VH105 DXP'1 J5 ,u YCAR FWETGSTPGAREPWSPSPQGVH
17 VI-I251 DXP'l J4 y1 YCAR RRYYGSGYYNVFDY WGQGTLVIVSSASTK
18 VH251 DHQ52 J4 yl YCAR QTWGGDY WGQGTLVTVSSASTIC
19 VH251 DPQ J6 yl YCAR GYSGYDNYYYGIHV WGQGTTVTVSSASTK
VH251 DHQ52 J4 u YCAR QTGEDYFDY WGQGTLVTVSSGSAS
21 VH251 DK1 J2 At YCAR YSGYDYLLVLRSLGPWHPGHCLLSLHR
22 VH251 DIR2 36 yl YCAR ASLPSFDYYGMDV WGQGTTVTVSSASTK
23 VH251 DIR2 J4 u YCAR RGGGLTTGAREPWSPSPQGVH
24 VH105 D? J6 u YCVP VETLLLLLRYGRLGPRDHGHRLLRECI
VH105 DXPI J4 u YCVR DILTGXPDY WGQGTLVTVSSGSAS
26 VH251 DNI J3 ~u YCAR HGIAAAGTAFDI WGQGTMVTVSSGSAS
27 VH10S DHQ52 33 u YCVR STGVDAFDI WGQGTMVTVSSGSAS
28 VH251 DN1 J4 a YCAE IAAAALLXLLGPGNPGHRLLRECI
29 VH105 DN1 J4 Ai YCVC IAAAGKGNGY WGQGTLVTVSSGSAS
VH251 DHQS2 14 At YCAR QNWGDY WGQGTLVTVSSGSAS
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J. CDR3 lenctth distribution
Table 8 compared the length of the CDR3 peptides
from transcripts with in-frame VDJ joints in the pHC1
transgenic mouse to those in human PBL. Again the human PBL
data comes from Yamada et al. The profiles are similar with
the transgenic profile skewed slightly toward smaller CDR3
peptides than observed from human PBL. The average length of
CDR3 in the transgenic mouse is 10.3 amino acids. This is
substantially the same as the average size reported for
authentic human CDR3 peptides by Sanz (J. Immunol. 147:1720-
1729 (1991)).
TABLE 8 CDR3 Length Distribution
Percent occurrence ( 3%)
#amino acids in CDR3 HC1 transgenic Human PBL
3-8 26 14
9-12 48 41
13-18 26 37
19-23 <1 7
>23 <1 .
100% 100%
EXAMPLE 13
Rearranged Heavy Chain Transgenes
A. Isolation of Rearranged Human Heavy Chain VDJ segments.
Two human leukocyte genomic DNA libraries cloned
into the phage vector XEMBL3/SP6/T7 (Clonetech Laboratories,
Inc., Palo Alto, CA) are screened with a 1 kb Pacl/Hindlll
fragment of X1.3 containing the human heavy chain J-A intronic
enhancer. Positive clones are tested for hybridization with a
mixture of the following VH specific oligonucleotides:
oligo-7 5'-tca gtg aag gtt tcc tgc aag gca tct gga tac acc
ttc acc-3'
oligo-8 5'-tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc
ttc agt-3'
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Clones that hybridized with both V and J-g probes
are isolated and the DNA sequence of the rearranged VDJ
segment determined.
B. Construction of rearranged human heavy chain transgenes
Fragments containing functional VJ segments (open
reading frame and splice signals) are subcloned into the
plasmid vector pSP72 such that the plasmid derived XhoI site
is adjacent to the 5' end of the insert sequence. A subclone
containing a functional VDJ segment is digested with XhoI and
Pacl (Pacl, a rare-cutting enzyme, recognizes a site near the
J-m intronic enhancer), and the insert cloned into XhoI/PacI
digested pHC2 to generate a transgene construct with a
functional VDJ segment, the J-p intronic enhancer, the A
switch element, the A constant region coding exons, and the yi
constant region, including the sterile transcript associated
sequences, the 71 switch, and the coding exons. This
transgene construct is excised with NotI and microinjected
into the pronuclei of mouse embryos to generate transgenic
animals as described above.
EXAMPLE 14
Light Chain Transgenes
A. Construction of Plasmid vectors
1. Plasmid vector pGPlc
Plasmid vector pGPla is digested with NotI and the
following oligonucleotides ligated in:
oligo-81 5'-ggc cgc atc ccg ggt ctc gag gtc gac aag ctt tcg
agg atc cgc-3'
oligo-82 5'-ggc cgc gga tcc tcg aaa get tgt cga cct cga gac
ccg gga tgc-3'
The resulting plasmid, pGPlc, contains a polylinker with XmaI,
XhoI, Sall, Hindlll, and BamHI restriction sites flanked by
NotI sites.
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Ma'smid 134
2. vector pGPid
Plasmid vector pGPla is digested with NotI and the
following oligonucleotides ligated in:
oligo-87 5'-ggc cgc tgt cga caa get tat cga tgg atc ctc gag
tgc -3'
ti
oligo-88 5'-ggc cgc act cga gga tcc atc gat aag ctt gtc gac
agc -3'
The resulting plasmid, pGPid, contains a polylinker with Sall,
Hindlil, Clal, BamHI, and XhoI restriction sites flanked by
NotI sites.
B. Isolation of JK and CK clones
A human placental genomic DNA library cloned into
the phage vector XEMBL3/SP6/T7 (Clonetech Laboratories, Inc.,
Palo Alto, CA) was screened with the human kappa light chain J
region specific oligonucleotide:
oligo-36 5'- cac ctt cgg cca agg gac acg act gga gat taa acg
taa gca -3'
and the phage clones 136.2 and 136.5 isolated. A 7.4 kb XhoI
fragment that includes the JK1 segment was isolated from
136.2 and subcloned into the plasmid pNNO3 to generate the
plasmid clone p36.2. A neighboring 13 kb XhoI fragment that
includes Jk segments 2 through 5 together with the CK gene
segment was isolated from phage clone 136.5 and subcloned into
the plasmid pNNO3 to generate the plasmid clone p36.5.
Together these two clones span the region beginning 7.2 kb
upstream of Jxl and ending 9 kb downstream of Cx.
C. Construction of rearranged light chain transgenes
1. pCKl, a Cx vector for expressing rearranged variable
segments
The 13 kb XhoI insert of plasmid clone p36.5
containing the CK gene, together with 9 kb of downstream
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sequences, is cloned into the Sall site of plasmid vector
pGPlc with the 5' end of the insert adjacent to the plasmid
XhoI site. The resulting clone, pCK1 can accept cloned
fragments containing rearranged VJ segments into the unique
5' XhoI site. The transgene can then be excised with NotI and
purified from vector sequences by gel electrophoresis. The
s resulting transgene construct will contain the human J-Cx
intronic enhancer and may contain the human 3' K enhancer.
2. pCK2, a CK vector with heavy chain enhancers for
expressing rearranged variable segments
A 0.9 kb XbaI fragment of mouse genomic DNA
containing the mouse heavy chain J-g intronic enhancer (J.
Banerji et al., Cell 33:729-740 (1983)) was subcloned into
pUC18 to generate the plasmid pJH22.1. This plasmid was
linearized with SphI and the ends filled in with Klenow
enzyme. The Klenow treated DNA was then digested with Hindlil
and a 1.4 kb MluI/HindIII fragment of phage clone X1.3
(previous example), containing the human heavy chain J-g
intronic enhancer (Hayday et al., Nature 307:334-340 (1984)),
to it. The resulting plasmid, pMHE1, consists of the mouse
and human heavy chain J- intronic enhancers ligated together
into pUC18 such that they are excised on a single
BamHi/Hindlll fragment. This 2.3 kb fragment is isolated and
cloned into pGPlc to generate pMHE2. pMHE2 is digested with
Sall and the 13 kb XhoI insert of p36.5 cloned in. The
resulting plasmid, pCK2, is identical to pCK1, except that the
mouse and human heavy chain J-g intronic enhancers are fused
to the 3' end of the transgene insert. To modulate expression
of the final transgene, analogous constructs can be generated
with different enhancers, i.e. the mouse or rat 3' kappa or
heavy chain enhancer (Meyer and Neuberger, EMBO J.,
8:1959-1964 (1989); Petterson et al., Nature, 344:165-168
(1990)).
3. Isolation of rearranged kappa light chain variable
segments
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Two human leukocyte genomic DNA libraries cloned
into the phage vector XEMBL3/SP6/T7 (Clonetech Laboratories,
Inc., Palo Alto, CA) were screened with the human kappa light
chain J region containing 3.5 kb XhoI/SmaI fragment of p36.5.
Positive clones were tested for hybridization with the
following VK specific oligonucleotide:
oligo-65 5'-agg ttc agt ggc agt ggg tct ggg aca gac ttc act
ctc acc atc agc-3'
Clones that hybridized with both V and J probes are isolated
and the DNA sequence of the rearranged VJK segment determined.
4. Generation of transgenic mice containing rearranged human
light chain constructs.
Fragments containing functional VJ segments (open
reading frame and splice signals) are subcloned into the
unique XhoI sites of vectors pCK1 and pCK2 to generate
rearranged kappa light chain transgenes. The transgene
constructs are isolated from vector sequences by digestion
with NotI. Agarose gel purified insert is microinjected into
mouse embryo pronuclei to generate transgenic animals.
Animals expressing human kappa chain are bred with heavy chain
minilocus containing transgenic animals to generate mice
expressing fully human antibodies.
Because not all VJK combinations may be capable of
forming stable heavy-light chain complexes with a broad
spectrum of different heavy chain VDJ combinations, several
different light chain transgene constructs are generated, each
using a different rearranged VJk clone, and transgenic mice
that result from these constructs are bred with heavy chain
minilocus transgene expressing mice. Peripheral blood,
spleen, and lymph node lymphocytes are isolated from double
transgenic (both heavy and light chain constructs) animals,
stained with fluorescent antibodies specific for human and
mouse heavy and light chain immunoglobulins (Pharmingen, San
Diego, CA) and analyzed by flow cytometry using a FACScan
analyzer (Becton Dickinson, San Jose, CA). Rearranged light
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chain transgenes constructs that result in the highest level
of human heavy/light chain complexes on the surface of the
highest number of B cells, and do not adversely affect the
immune cell compartment (as assayed by flow cytometric
analysis with B and T cell subset specific antibodies), are
selected for the generation of human monoclonal antibodies.
Y
D. Construction of unrearranced light chain minilocus
transgenes
1. pJCK1, a JK, CK containing vector for constructing
minilocus transgenes
The 13 kb CK containing XhoI insert of p36.5 is
treated with Klenow enzyme and cloned into Hindlil digested,
Klenow-treated, plasmid pGP1d. A plasmid clone is selected
such that the 5' end of the insert is adjacent to the vector
derived Clal site. The resulting plasmid, p36.5-1d, is
digested with Clai and Klenow-treated. The JK1 containing 7.4
kb XhoI insert of p36.2 is then Klenow-treated and cloned into
the ClaI, Klenow-treated p36.5-1d. A clone is selected in
which the p36.2 insert is in the same orientation as the p36.5
insert. This clone, pJCKl (Fig. 34), contains the entire
human JK region and CK, together with 7.2 kb of upstream
sequences and 9 kb of downstream sequences. The insert also
contains the human J-CK intronic enhancer and may contain a
human 3' K enhancer. The insert is flanked by a unique 3'
Sall site for the purpose of cloning additional 3' flanking
sequences such as heavy chain or light chain enhancers. A
unique XhoI site is located at the 5' end of the insert for
the purpose of cloning in unrearranged VK gene segments. The
unique SalI and XhoI sites are in turn flanked by NotI sites
that are used to isolate the completed transgene construct
away from vector sequences.
=b
2. Isolation of unrearranged VK gene segments and generation
of transgenic animals expressing human Ig light chain protein
The VK specific oligonucleotide, oligo-65 (discussed
above), is used to probe a human' placental genomic DNA library
cloned into the phage vector 1EMBL3/SP6/T7 (Clonetech
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J ' s 138
.0
Laboratories, Inc., Palo Alto, CA). Variable gene segments
from the resulting clones are sequenced, and clones that
appear functional-are selected. Criteria for judging
functionality include: open reading frames, intact splice
acceptor and donor sequences, and intact recombination
sequence. DNA fragments containing selected variable gene
segments are cloned into the unique XhoI site of plasmid pJCK1
to generate minilocus constructs. The resulting clones are
digested with NotI and the inserts isolated and injected into
mouse embryo pronuclei to generate transgenic animals. The
transgenes of these animals will undergo V to J joining in
developing B-cells. Animals expressing human kappa chain are
bred with heavy chain minilocus containing transgenic animals
to generate mice expressing fully human antibodies.
EXAMPLE 15
Genomic Heavy Chain Human Iq Transgene
This Example describes the cloning of a human
genomic heavy chain immunoglobulin transgene which is then
introduced into the murine germline via microinjection into
zygotes or integration in ES cells.
Nuclei are isolated from fresh human placental
tissue as described by Marzluff, W.F., et al. (1985),
Transcription and Translation: A Practical Approach, B.D.
Hammes and S.J. Higgins, eds., pp. 89-129, IRL Press, Oxford).
The isolated nuclei (or PBS washed human spermatocytes) are
embedded in 0.5% low melting point agarose blocks and lysed
with 1 mg/ml proteinase K in 500mM EDTA, 1% SDS for nuclei, or
with lmg/ml proteinase K in 500mM EDTA, 1% SDS, 10mM DTT for
spermatocytes at 50 C for 18 hours. The proteinase K is
inactivated by incubating the blocks in 40 g/ml PMSF in TE for
30 minutes at 50 C, and then washing extensively with TE. The
a=
DNA is then digested in the agarose with the restriction
enzyme NotI as described by M. Finney in Current Protocols in
Molecular Biology (F. Ausubel et al., eds. John Wiley & Sons,
Supp. 4, 1988, e.g., Section 2.5.1).
The NotI digested DNA is then fractionated by pulsed
field gel electrophoresis as described by Anand et al., Nuc.
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Acids Res. 17:3425-3433 (1989). Fractions enriched for the
NotI fragment are assayed by Southern hybridization to detect
one or more of the sequences encoded by this fragment. Such
sequences include the heavy chain D segments, J segments, and
yl constant regions together with representatives of all 6 VH
families (although this fragment is identified as 670 kb
fragment from HeLa cells by Berman et al. (1988), supra., we
have found it to be an 830 kb fragment from human placental
and sperm DNA). Those fractions containing this NotI
fragment are ligated into the NotI cloning site of the vector
pYACNN as described (McCormick et al., Technique 2:65-71
(1990)). Plasmid pYACNN is prepared by digestion of pYACneo
(Clontech) with EcoRI and ligation in the presence of the
oligonucleotide 5' - AAT TGC GGC CGC - 3'.
YAC clones containing the heavy chain NotI fragment
are isolated as described by Traver et al., Proc. Natl. Acad.
Sci. USA, 86:5898-5902 (1989). The cloned NotI insert is
isolated from high molecular weight yeast DNA by pulse field
gel electrophoresis as described by M. Finney, op. cit. The
DNA is condensed by the addition of 1 mM spermine and
microinjected directly into the nucleus of single cell embryos
previously described. Alternatively, the DNA is isolated by
pulsed field gel electrophoresis and introduced into ES cells
by lipofection (Gnirke et al., EMBO J. 10:1629-1634 (1991)),
or the YAC is introduced into ES cells by spheroplast fusion.
EXAMPLE 16
Discontinuous Genomic Heavy Chain Ig Transctene
An 85 kb Spel fragment of human genomic DNA,
containing VH6, D segments, J segments, the A constant region
and part of the y constant region, has been isolated by YAC
cloning essentially as described in Example 1. A YAC carrying
a fragment from the germline variable region, such as a 570 kb
NotI fragment upstream of the 670-830 kb NotI fragment
described above containing multiple copies of V1 through V5, is
isolated as described. (Berman et al. (1988), supra. detected
two 570 kb NotI fragments, each containing multiple V
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segments.), The two fragments are coinjected into the nucleus
of a mouse single cell embryo as described in Example 1.
Typically, coinjection of two different DNA
fragments result in the integration of both fragments at the
same insertion site within the chromosome. Therefore,
approximately 50% of the resulting transgenic animals that
contain at least one copy of each of the two fragments will
have the V segment fragment inserted upstream of the constant
region containing fragment. Of these animals, about 50% will
carry out V to DJ joining by DNA inversion and about 50% by
deletion, depending on the orientation of the 570 kb NotI
fragment relative to the position of the 85 kb Spel fragment.
DNA is isolated from resultant transgenic animals and those
animals found to be containing both transgenes by Southern
blot hybridization (specifically, those animals containing
both multiple human V segments and human constant region
genes) are tested for their ability to express human
immunoglobulin molecules in accordance with standard
techniques.
EXAMPLE 17
Identification of functionally rearranged variable region
sequences in transgenic B cells
An antigen of interest is used to immunize (see
Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor, New York (1988)) a mouse with the following genetic
traits: homozygosity at the endogenous having chain locus for
a deletion of J. (Examples 10); hemizygous for a single copy
of unrearranged human heavy chain minilocus transgene
(examples 5 and 14); and hemizygous for a single copy of a
rearranged human kappa light chain transgene (Examples 6 and
14).
Following the schedule of immunization, the spleen
is removed, and spleen cells used to generate hybridomas.
Cells from an individual hybridoma clone that secretes
antibodies reactive with the antigen of interest are used to
prepare genomic DNA. A sample of the genomic DNA is digested
with several different restriction enzymes that recognize
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141
unique six base pair sequences, and fractionated on an agarose
gel. Southern blot hybridization is used to identify two DNA
fragments in the 2-10 kb range, one of which contains the
single copy of the rearranged human heavy chain VDJ sequences
and one of which contains the single copy of the rearranged
human light chain VJ sequence. These two fragments are size
fractionated on agarose gel and cloned directly into pUC18.
The cloned inserts are then subcloned respectively into heavy
and light chain expression cassettes that contain constant
region sequences.
The plasmid clone pyel (Example 12) is used as a
heavy chain expression cassette and rearranged VDJ sequences
are cloned into the XhoI site. The plasmid clone pCK1 is used
as a light chain expression cassette and rearranged VJ
sequences are cloned into the XhoI site. The resulting clones
are used together to transfect SPO'cells to produce antibodies
that react with the antigen of interest (Co. et al., Proc.
Natl. Acad. Sci. USA 88:2869 (1991)).
Alternatively, mRNA is isolated from the cloned
hybridoma cells described above, and used to synthesize cDNA.
The expressed human heavy and light chain VDJ and VJ sequence
are then amplified by PCR and cloned (Larrick et al., Biol.
Technology, 2:934-938 (1989)). After the nucleotide sequence
of these clones has been determined, oligonucleotides are
synthesized that encode the same polypeptides, and synthetic
expression vectors generated as described by Queen et al.,
Proc. Natl. Acad. Sci. USA., 84:5454-5458 (1989).
Immunization of Transgenic Animals with Complex Antigens
The following experiment demonstrates that
transgenic animals can be successfully immunized with complex
antigens such as those on human red blood cells and respond
with kinetics that are similar to the response kinetics
observed in normal mice.
Blood cells generally are suitable immunogens and
comprise many different types of antigens on the surface of
red and white blood cells.
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Immunization with human blood
Tubes of human blood from a single donor were
collected and used to immunize transgenic mice having
functionally disrupted endogenous heavy chain loci (JHD) and
harboring a human heavy chain minigene construct (HC1); these
mice are designated as line 112. Blood was washed and
resuspended in 50 mis Hanks' and diluted to 1x108 cells/ml 0.2
ti
mis (2x107 cells) were then injected interperitoneally using a
28 gauge needle and 1 cc syringe. This immunization protocol
was repeated approximately weekly for 6 weeks. Serum titers
were monitored by taking blood from retro-orbital bleeds and
collecting serum and later testing for specific antibody. A
pre-immune bleed was also taken as a control. On the very
last immunization, three days before these animals were
sacrificed for serum and for hybridomas, a single immunization
of 1 x 107 cells was given intravenously through the tail to
enhance the production of hybridomas.
Table 9
Animals
Mouse ID Line Sex HC1-112 JHD
1 2343 112 M + ++
2 2344 112 M - +
3 2345 112 F - +
4 2346 112 F - ++
5 2347 112 F - ++
6 2348 112 F + ++
7 2349 112 F - +
Mice # 2343 and 2348 have a desired phenotype: human heavy
chain mini-gene transgenic on heavy chain knock-out
background.
Generation of Hybridomas
Hybridomas were generated by fusing mouse spleen
cells of approximately 16 week-old transgenic mice (Table 9)
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143
that had been immunized as described (supra) to a fusion
partner consisting of the non-secreting HAT-sensitive myeloma
cell line, X63 Ag8.653. Hybridoma clones were cultivated and
hybridoma supernatants containing immunoglobulins having
specific binding affinity for blood cell antigens were
identified, for example, by flow cytometry.
Flow cytometry
Serum and hybridoma supernatants were tested using
flow cytometry. Red blood cells from the donor were washed 4X
in Hanks' balanced salt solution and 50,000 cells were placed
in 1.1 ml polypropylene microtubes. Cells were incubated with
antisera or supernatant from the hybridomas for 30 minutes on
ice in staining media (lx RPMI 1640 media without phenol red
or biotin (Irvine Scientific) 3% newborn calf serum, 0.1% Na
azide). Controls consisted of littermate mice with other
genotypes. Cells were then washed by centrifugation at 4 C in
Sorvall RT600B for 5-10 minutes at 1000 rpm. Cells were
washed two times and then antibody detected on the cell
surface with a fluorescent developing reagent. Two monoclonal
reagents were used to test. One was a FITC-labeled mouse
anti-human heavy chain antibody (Pharmagen, San Diego, CA)
and the other was a PE-labeled rat anti-mouse kappa light
chain (Becton-Dickenson, San Jose, CA). Both of these
reagents gave similar results. Whole blood (red blood cells
and white blood cells) and white blood cells alone were used
as target cells. Both sets gave positive results.
Serum of transgenic mice and littermate controls was
incubated with either red blood cells from the donor, or white
blood cells from another individual, washed and then developed
with anti-human IgM FITC labeled antibody and analyzed in a
flow cytometer. Results showed that serum from mice that are
transgenic for the human mini-gene locus (mice 2343 and 2348)
show human IgM reactivity whereas all littermate animals
(2344, 2345, 2346, 2347) do not. Normal mouse serum (NS) and
phosphate buffer saline (PBS) were used as negative controls.
Red blood cells were ungated and white blood cells were gated
to include only lymphocytes. Lines are drawn on the x and y
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j~ jc3sl 144 0
axis to provide a reference. Flow cytometry was performed on
100 supernatants from fusion 2348. Four supernatants showed
positive reactivity for blood cell antigens.
EXAMPLE 18
Reduction of Endogenous Mouse Immunoalobulin Expression
by Antisense RNA
A. Vector for Expression of Antisense Ig Sequences
1. Construction of the cloning vector pGP1h
The vector pGPlb (referred to in a previous example)
is digested with XhoI and BamHI and ligated with the following
oligonucleotides:
5'- gat cct cga gac cag gta cca gat ctt gtg aat tcg -3'
51- tcg acg aat tca caa gat ctg gta cct ggt ctc gag -3'
to generate the plasmid pGPih. This plasmid contains a
polylinker that includes the following restriction sites:
NotI, EcoRI, BglII, Asp718, XhoI, BamHI, Hindill, NotI.
Construction of pBCE1.
A 0.8 kb XbaI/BglII fragment of pVH251 (referred to
in a previous example), that includes the promoter leader
sequence exon, first intron, and part of the second exon of
the human VH-V family immunoglobulin variable gene segment,
was inserted into XbaI/BglII digested vector pNN03 to generate
the plasmid pVH251.
The 2.2 kb BamHI/EcoRI DNA fragment that includes
the coding exons of the human growth hormone gene (hGH;
Seeburg, (1982) DNA 1:239-249) is cloned into Bg1II/EcoRI
digested pGHlh. The resulting plasmid is digested with BamHI
and the BamHI/BglII of pVH251N is inserted in the same
orientation as the hGH gene to generate the plasmid pVhgh.
A 0.9 kb XbaI fragment of mouse genomic DNA
containing the mouse heavy chain J-g intronic enhancer
(Banerji et al., (1983) Cell 33:729-740) was subcloned into
pUC18 to generate the plasmid pJH22.1. This plasmid was
linearized with SphI and the ends filled in with klenow
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enzyme. The klenow treated DNA was then digested with Hindlil
and a 1.4 kb MluI(klenow)/HindIII fragment of phage clone X1.3
(previous example), containing the human heavy chain J-p
intronic enhancer (Hayday et al., (1984) Nature 307:334-340),
to it. The resulting plasmid, pMHE1, consists of the mouse
and human heavy chain J- intron enhancers ligated together
+ into pUC18 such that they can be excised on a single
BamHI/HindIII fragment.
The BamHI/HindIII fragment of pMHE1 is cloned into
BamHI/HindIII cut pVhgh to generate the B-cell expression
vector pBCE1. This vector, depicted in Fig. 36, contains
unique XhoI and Asp718 cloning sites into which antisense DNA
fragments can be cloned. The expression of these antisense
sequences is driven by the upstream heavy chain promoter-
enhancer combination the downstream hGH gene sequences provide
polyadenylation sequences in addition to intron sequences that
promote the expression of transgene constructs. Antisense
transgene constructs generated from pBCE1 can be separated
from vector sequences by digestion with NotI.
B. An IgM antisense transgene construct.
The following two oligonucleotides:
5'- cgc ggt acc gag agt cag tcc ttc cca aat gtc -3'
5'- cgc ctc gag aca get gga atg ggc aca tgc aga -3'
are used as primers for the amplification of mouse IgM
constant region sequences by polymerase chain reaction (PCR)
using mouse spleen cDNA as a substrate. The resulting 0.3 kb
PCR product is digested with Asp718 and XhoI and cloned into
Asp718/XhoI digested pBCE1 to generate the antisense transgene
=, construct pMAS1. The purified NotI insert of pMAS1 is
microinjected into the pronuclei of half day mouse embryos--
alone or in combination with one or more other transgene
constructs--to generate transgenic mice. This construct
expresses an RNA transcript in B-cells that hybridizes with
mouse IgM mRNA, thus down-regulating the expression of mouse
IgM protein. Double transgenic mice containing pMAS1 and a
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2,%Js~ 146 4
human heavy chain transgene minilocus such as pHCI (generated
either by coinjection of both constructs or by breeding of
singly transgenic mice) will express the human transgene
encoded Ig receptor on a higher percentage of B-cell than mice
transgenic for the human heavy chain minilocus alone. The
ratio of human to mouse Ig receptor expressing cells is due in
part to competition between the two populations for factors
and cells that promoter B-cell differentiation and expansion.
Because the Ig receptor plays a key role in B-cell
development, mouse Ig receptor expressing B-cells that express
reduced levels of IgM on their surface (due to mouse Ig
specific antisense down-regulation) during B-cell development
will not compete as well as cells that express the human
receptor.
C. An IgKappa antisense transgene construct.
The following two oligonucleotides:
5'- cgc ggt acc get gat get gca cca act gta tcc -3'
5'- cgc ctc gag cta aca ctc att cct gtt gaa get -3'
are used as primers for the amplification of mouse IgKappa
constant region sequences by polymerase chain reaction (PCR)
using mouse spleen cDNA as a substrate. The resulting 0.3 kb
PCR product is digested with Asp718 and XhoI and cloned into
Asp718/XhoI digested pBCE1 to generate the antisense transgene
construct pKAS1. The purified NotI insert of pKAS1 is
microinjected into the pronuclei of half day mouse embryos--
alone or in combination with one or more other transgene
constructs--to generate transgenic mice. This construct
expresses an RNA transcript in B-cells that hybridizes with
mouse IgK mRNA, thus down-regulating the expression of mouse
IgK protein as described above for pMAS1.
EXAMPLE 19
This example demonstrates the successful
immunization and immune response in a transgenic mouse of the
present invention.
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Immunization of Mice
Keyhole limpet hemocyanin conjugated with greater
than 400 dinitrophenyl groups per molecule (Calbiochem, La
Jolla, California) (KLH-DNP) was alum precipitated according
to a previously published method (Practical Immunology, L.
Hudson and F.C. Hay, Blackwell Scientific (Pubs.), p. 9,
* 1980). Four hundred g of alum precipitated KLH-DNP along
with 100 gg dimethyldioctadecyl Ammonium Bromide in 100 gL of
phosphate buffered saline (PBS) was injected intraperitoneally
into each mouse. Serum samples were collected six days later
by retro-orbital sinus bleeding.
Analysis of Human Antibody Reactivity in Serum
Antibody reactivity and specificity were assessed
using an indirect enzyme-linked immunosorbent assay (ELISA).
Several target antigens were tested to analyze antibody
induction by the immunogen. Keyhole limpet hemocyanin
(Calbiochem) was used to identify reactivity against the
protein component, bovine serum albumin-DNP for reactivity
against the hapten and/or modified amino groups, and KLH-DNP
for reactivity against the total immunogen. Human antibody
binding to antigen was detected by enzyme conjugates specific
for IgM and IgG sub-classes with no cross reactivity to mouse
immunoglobulin. Briefly, PVC microtiter plates were coated
with antigen drying overnight at 37 C of 5 g/mL protein in
PBS. Serum samples diluted in PBS, 5% chicken serum, 0.5%
Tween-20 were incubated in the wells for 1 hour at room
temperature, followed by anti-human IgG Fc and IgG F(ab')-
horseradish peroxidase or anti-human IgM Fc-horseradish
peroxidase in the same diluent. After 1 hour at room
temperature enzyme activity was assessed by addition of ABTS
substrate (Sigma, St. Louis, Missouri) and read after 30
minutes at 415-490 nm.
Human Heavy Chain Participation in Immune Response in
Transuenic Mice
Figures 37A-37D illustrate the response of three
mouse littermates to immunization with KLH-DNP. Mouse number
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1296 carried the human IgM and IgG unrearranged transgene and
was homozygous for mouse Ig heavy chain knockout. Mouse
number 1299 carried the transgene on a non-knockout
background, while mouse 1301 inherited neither of these sets
of genes. Mouse 1297, another littermate, carried the human
transgene and was hemizygous with respect to mouse heavy chain
knockout. It was included as a non-immunized control.
The results demonstrate that both human IgG and IgM
responses were developed to the hapten in the context of
conjugation to protein. Human IgM also developed to the KLH
molecule, but no significant levels of human IgG were present
at this time point. In pre-immunization serum samples from
the same mice, titers of human antibodies to the same target
antigens were insignificant.
EXAMPLE 20
This example demonstrates the successful
immunization with a human antigen and immune response in a
transgenic mouse of the present invention, and provides data
demonstrating that nonrandom somatic mutation occurs in the
variable region sequences of the human transgene.
Demonstration of antibody responses comprising human
immunoglobulin heavy chains against a human glycoprotein
antigen
Transgenic mice used for the experiment were
homozygous for functionally disrupted murine immunoglobulin
heavy chain loci produced by introduction of a transgene at
the joining (J) region (supra) resulting in the absence of
functional endogenous (murine) heavy chain production. The
transgenic mice also harbored at least one complete
unrearranged human heavy chain mini-locus transgene, (HC1,
supra), which included a single functional VH gene (VH251),
human constant region gene, and human 71 constant region
gene. Transgenic mice shown to express human immunoglobulin
transgene products (supra) were selected for immunization with
a human antigen to demonstrate the capacity of the transgenic
mice to make an immune response against a human antigen
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immunization. Three mice of the HC1-26 line and three mice of
the HC1-57 line (supra) were injected with human antigen.
One hundred g of purified human carcinoembryonic
antigen (CEA) insolubilized on alum was injected in complete
Freund's adjuvant on Day 0, followed by further weekly
injections of alum-precipitated CEA in incomplete Freund's
adjuvant on Days 7, 14, 21, and 28. Serum samples were
collected by retro-orbital bleeding on each day prior to
injection of CEA. Equal volumes of serum were pooled from
each of the three mice in each group for analysis.
Titres of human g chain-containing immunoglobulin
and human y chain-containing immunoglobulin which bound to
human CEA immobilized on microtitre wells were determined by
ELISA assay. Results of the ELISA assays for human chain-
containing immunoglobulins and human y chain-containing
immmunoglbulins are shown in Figs. 38 and 39, respectively.
Significant human chain Ig titres were detected for both
lines by Day 7 and were observed to rise until about Day 21.
For human y chain Ig, significant titres were delayed, being
evident first for line HC1-57 at Day 14, and later for line
HC1-26 at Day 21. Titres for human y chain Ig continued to
show an increase over time during the course of the
experiment. The observed human chain Ig response, followed
by a plateau, combined with a later geveloping y chain
response which continues to rise is characteristic of the
pattern seen with affinity maturation. Analysis of Day 21
samples showed lack of reactivity to an unrelated antigen,
keyhole limpet hemocyanin (KLC), indicating that the antibody
response was directed against CEA in a specific manner.
These data indicate that animals transgenic for
human unrearranged immunoglobulin gene loci: (1) can respond
=k to a human antigen (e.g., the human glycoprotein, CEA), (2)
can undergo isotype switching ("class switching) as
exemplified by the observed g to y class switch, and (3)
exhibit characteristics of affinity maturation in their
humoral immune responses. In general, these data indicate:
(1) the human Ig transgenic mice have the ability to induce
heterologous antibody production in response to a defined
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antigen, (2) the capacity of a single transgene heavy chain
variable region to respond to a defined antigen, (3) response
kinetics over a time period typical of primary and secondary
response development, (4) class switching of a transgene-
encoded humoral immune response from IgM to IgG, and (5) the
capacity of transgenic animal to produce human-sequence
antibodies against a human antigen.
Demonstration of somatic mutation in a human heavy chain
transgene minilocus.
Line HC1-57 transgenic mice, containing multiple
copies of the HC1 transgene, were bred with immunoglobulin
heavy chain deletion mice to obtain mice that contain the HC1
transgene and contain disruptions at both alleles of the
endogenous mouse heavy chain (supra). These mice express
human mu and gammal heavy chains together with mouse kappa and
lambda light chains (supra). One of these mice was
hyperimmunized against human carcinoembryonic antigen by
repeated intraperitoneal injections over the course of 1.5
months. This mouse was sacrificed and lymphoid cells isolated
from the spleen, inguinal and mesenteric lymph nodes, and
peyers patches. The cells were combined and total RNA
isolated. First strand cDNA was synthesized from the RNA and
used as a template for PCR amplification with the following 2
oligonucleotide primers:
149 5'-cta get cga gtc caa gga gtc tgt gcc gag gtg cag ctg
(g/a/t/c)-3'
151 5'-ggc get cga gtt cca cga cac cgt cac cgg ttc-3'
These primers specifically amplify VH251/gammal cDNA
sequences. The amplified sequences were digested with XhoI
and cloned into the vector pNN03. DNA sequence from the
inserts of 23 random clones is shown in Fig. 40; sequence
variations from germline sequence are indicated, dots indicate
sequence is identical to germline. Comparison of the cDNA
sequences with the germline sequence of the VH251 transgene
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reveals that 3 of the clones are completely unmutated, while
the other 20 clones contain somatic mutations. One of the 3
non-mutated sequences is derived from an out-of-frame VDJ
joint. Observed somatic mutations at specific positions of
occur at similar frequencies and in similar distribution
patterns to those observed in human lymphocytes (Cai et al.
(1992) J. Exp. Med. 176: 1073).
The overall frequency of somatic mutations is
approximately 1%; however, the frequency goes up to about 5%
within CDR1, indicating selection for amino acid changes that
affect antigen binding. This demonstrates antigen driven
affinity maturation of the human heavy chain sequences.
EXAMPLE 21
This example demonstrates the successful formation
of a transgene by co-introduction of two separate
polynucleotides which recombine to form a complete human light
chain minilocus transgene.
Generation of an unrearranged light chain minilocus transgene
by co-infection of two overlapping DNA fragments
1. Isolation of unrearranged functional V. gene segments
vk65 3 vk65.5, vk65.8 and vk65.15
The V. specific oligonucleotide, oligo-65 (5'-agg
ttc agt ggc agt ggg tct ggg aca gac ttc act ctc acc atc agc-
3'), was used to probe a human placental genomic DNA library
cloned into the phage vector XEMBL3/SP6/T7 (Clonetech
Laboratories, Inc., Palo Alto, CA). DNA fragments containing
V. segments from positive phage clones were subcloned into
plasmid vectors. Variable gene segments from the resulting
clones are sequenced, and clones that appear functional were
selected. Criteria for judging functionality include: open
reading frames, intact splice acceptor and donor sequences,
and intact recombination sequence. DNA sequences of 4
functional V. gene segments (vk65.3, vk65.5, vk65.8, and
vk65.15) from 4 different plasmid clones isolated by this
procedure are shown in Figs. 41-44. The four plasmid clones,
p65.3f, p65.5g1, p65.8, and p65.15f, are described below.
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(1 a) p65.3f
A 3 kb Xba fragment of phage clone X65.3 was
subcloned into pUC19 so that the vector derived Sall site was
proximal to the 3' end of the insert and the vector derived
BamHI site 5'. The 3 kb BamHI/SalI insert of this clone was
subcloned into pGP1f to generate p65.3f.
(1 b) p65.5g1
A 6.8 kb EcoRI fragment of phage clone X65.5 was
subcloned into pGPlf so that the vector derived XhoI site is
proximal to the 5' end of the insert and the vector derived
Sall site 3'. The resulting plasmid is designated p65.5g1.
(1 c) p65.8
A 6.5 kb Hindlil fragment of phage clone X65.8 was
cloned into pSP72 to generate p65.8.
(1 d) p65.15f
A 10 kb EcoRI fragment of phage clone X65.16 was
subcloned into pUC18 to generate the plasmid p65.15.3. The V.
gene segment within the plasmid insert was mapped to a 4.6 kb
EcoRI/Hindlll subfragment, which was cloned into pGPlf. The
resulting clone, p65.15f, has unique XhoI and Sall sites
located at the respective 5' and 3' ends of the insert.
2. pKV4
The XhoI/SalI insert of p65.8 was cloned into the
XhoI site of p65.15f to generate the plasmid pKV2. The
XhoI/SalI insert of p65.5g1 was cloned into the XhoI site of
pKV2 to generate pKV3. The XhoI/SalI insert of pKV3 was
cloned into the XhoI site of p65.3f to generate the plasmid
pKV4.. This plasmid contains a single 21 kb XhoI/SalI insert
that includes 4 functional V. gene segments. The entire
insert can also be excised with NotI.
3. pKC1B
(3 a) pKcor
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Two XhoI fragments derived from human genomic DNA
phage X clones were subcloned into plasmid vectors. The
first, a 13 kb JK2-JK5/CK containing fragment, was treated with
Klenow enzyme and cloned into Hindill digested, Klenow
treated, plasmid pGPld. A plasmid clone (pK-31) was selected
such that the 5' end of the insert is adjacent to the vector
derived Clal site. The second XhoI fragment, a 7.4 kb piece
of DNA containing JK1 was cloned into XhoI/SalI-digested
pSP72, such that the 3' insert XhoI site was destroyed by
ligation to the vector SalI site. The resulting clone,
p36.2s, includes an insert derived Clal site 4.5 kb upstream
of JK1 and a polylinker derived Clal site downstream in place
of the naturally occurring XhoI site between JK1 and JK2. This
clone was digested with Clal to release a 4.7 kb fragment
which was cloned into Clal digested pK-31 in the correct 5' to
3' orientation to generate a plasmid containing all 5 human JK
segments, the human intronic enhancer human CK, 4.5 kb of 5'
flanking sequence, and 9 kb of 3' flanking sequence. This
plasmid, pKcor, includes unique flanking XhoI and Sall sites
on the respective 5' and 3' sides of the insert.
(3 b) pKcorB
A 4 kb BamHI fragment containing the human 3' kappa
enhancer (Judde, J.-G. and Max, E.E. (1992) Mol. Cell. Biol.
122: 5206, incorporated herein by reference)=was cloned into
pGPlf such that the 5' end is proximal to the vector XhoI
site. The resulting plasmid, p24Bf, was cut with XhoI and the
17.7 kb XhoI/SalI fragment of pKcor cloned into it in the same
orientation as the enhancer fragment. The resulting plasmid,
pKcorB, includes unique XhoI and Sall sites at the 5' and 3'
ends of the insert respectively.
(3 c) pKC1B
The XhoI/SalI insert of pKcorB was cloned into the
SalI site of p65.3f to generate the light-chain minilocus-
transgene plasmid pKC1B. This plasmid includes a single
functional human VK segment, all 5 human JK segments, the human
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intronic enhancer, human C,, and the human 3' kappa enhancer.
The entire 25 kb insert can be isolated by NotI digestion.
4. Co4
The two NotI inserts from plasmids pKV4 and pKC1B
were mixed at a concentration of 2.5 g/ml each in
microinjection buffer, and co-injected into the pronuclei of
half day mouse embryos as described in previous examples.
Resulting transgenic animals contain transgene inserts
(designated Co4, product of the recombination shown in Fig.
45) in which the two fragments co-integrated. The 3' 3 kb of
the pKV4 insert and the 5'3 kb of the pKC1B insert are
identical. Some of the integration events will represent
homologous recombinations between the two fragments over the 3
kb of shared sequence. The Co4 locus will direct the
expression of a repertoire of human sequence light chains in a
transgenic mouse.
EXAMPLE 22
This example demonstrates the successful production
of a murine hybridoma clone secreting a monoclonal antibody
reactive with a specific immunogen, wherein the monoclonal
antibody comprises a human immunoglobulin chain encoded by a
human Ig transgene.
Generation of Monoclonal Antibodies Incorporating Human Heavy
Chain Transgene Product
1. Immunization of Mouse Harboring Human Heavy Chain
Transgene
A mouse containing a human heavy chain encoding
transgene and homozygous for knockout (i.e., functional
disruption) of the endogenous heavy chain locus (see, EXAMPLE
20, supra) was immunized with purified human CEA, and spleen
cells were subsequently harvested after a suitable immune
response period. The murine spleen cells were fused with
mouse myeloma cells to generate hybridomas using conventional
techniques (see, Kohler and Milstein, Eur. J. Immunol., 6:511-
519 (1976); Harlow and Lane, Antibodies: A Laboratory Manual,
Cold Spring Harbor, New York (1988)). The mouse used for
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immunization contained a human unrearranged heavy chain
minilocus transgene which comprised a single functional VH
gene (VH251), human D and J segments, human constant region,
and human yi constant region genes. The transgenic line from
which it originated was designated HC1-57 (supra).
One hundred g of purified human carcinoembryonic
antigen (CEA) (Cyrstal Chem, Chicago, IL or Scripps Labs, San
Diego, CA) insolubilized on alum was injected in complete
Freund's adjuvant on Day 0, followed by further weekly
injections of alum-precipitated CEA in incomplete Freund's
adjuvant on Days 7, 14, 21, and 28. An additional 20 E.tg of
soluble CEA was administered intravenously on Day 83, followed
by 50 g alum-precipitated CEA in incomplete Freund's adjuvant
on Day 92. Human heavy chain responses to CEA were confirmed
in serum samples prior to fusion of spleen cells with myleoma
cells. The animal was sacrificed on Day 95, the spleen
removed and fused with P3X63-Ag8.653 mouse myeloma cells (ATCC
CRL 1580, American Type Culture Collection, Rockville, MD)
using polyethylene glycol. Two weeks later, supernates from
fusion wells were screened for the presence of antibodies
specifically reactive with CEA, and which contained human
heavy chain or y constant region epitopes by ELISA.
Briefly, purified human CEA was coated onto PVC microtitre
plates at 2.5 g/ml, and incubate with culture supernate
diluted 1:4 or 1:5 in PBS, 0.5% Tween-20, 5% chicken serum.
Plates were washed, followed by addition of horseradish
peroxidase-conjugated goat antiserum specific for human IgG Fc
or rabbit antiserum specific for human IgM Fc5Mu (Jackson
ImmunoResearch, West Grove, PA). Presence of conjugate bound
to captured antibody was determined, after further washing, by
the addition of ABTS substrate. Two independent fusion wells
were found to contain antibody with substantial binding to
CEA. After cloning, both hybridomas were found to be positive
for the presence of human chain and murine K chain by ELISA.
No mouse IgG or IgM were detected using similar assays.
Subcloning of the two independent parent hybridomas
resulted in two clones, designated 92-09A-4F7-A5-2 and 92-09A-
1D7-1-7-1. Both lines were deposited with the ATCC Patent
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Culture Depository under the Budapest Treaty and were assigned
ATCC Designation HB 11307 and HB 11308, respectively. Culture
supernatants from these cell lines were assessed for
specificity by testing for reactivity to several purified
target proteins using ELISA. As shown in Fig. 46, ELISA
assays for determining the reactivity of the monoclonal
antibodies to various antigens demonstrate that only CEA and
the CEA-related antigen NCA-2 show significant reactivity,
indicating the development of a restricted reactivity for the
variable regions of the heterohybrid immunoglobulin molecules.
EXAMPLE 23
This example demonstrates that a rearranged human
VDJ gene encoded by a human Ig minilocus transgene may be
transcribed as a transcript which includes an endogenous Ig
constant region gene, for example by the mechanism of trans-
switching, to encode a chimeric human/mouse Ig chain.
Identification of Trans-Switch Transcripts Encoding Chimeric
Human-Mouse Heavy Chains
RNA was isolated from a hyperimmunized HC1 line 57
transgenic mouse homozygous for the endogenous heavy chain J
segment deletion (supra). cDNA was synthesized according to
Taylor et al. (1993) Nucleic Acids Res. 20: 6287,
and amplified by PCR using the following
two primers:
0-149 (human VH251)
5'-CTA GCT CGA GTC CAA GGA GTC TGT GCC GAG GTG CAG CTG (G,A,T,C)-3'
o-249 (mouse gamma):
5'-GGC GCT CGA GCT GGA CAG GG(A/C) TCC A(G/T)A GTT CCA-3'
Oligonucleotide o-149 is specific for the HC1-
encoded variable gene segment VH251+ while o-249 hybridizes to
both mouse and human gamma sequences with the following order
of specificities:
mouse -yl = mouse 72b = mouse 73 > mouse 72a >> human yi.
DNA sequences from 10 randomly chosen clones generated from
the PCR products was determined and is shown in Fig. 47. Two
clones comprised human VDJ and mouse yl; four clones comprised
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human VDJ and mouse y2b; and four clones comprised human VDJ
and mouse y3. These results indicate that in a fraction of
the transgenic B cells, the transgene-encoded human VDJ
recombined into the endogenous murine heavy chain locus by
class switching or an analogous recombination.
EXAMPLE 24
This example describes a method for screening a pool
of hybridomas to discriminate clones which encode chimeric
human/mouse Ig chains from clones which encode and express a
human Ig chain. For example, in a pool of hybridoma clones
made from a transgenic mouse comprising a human Ig heavy chain
transgene and homozygous for a J region-disrupted endogenous
heavy chain locus, hybridoma clones encoding trans-switched
human VDJ-murine constant region heavy chains may be
identified and separated from hybridoma clones expressing
human VDJ-human constant region heavy chains.
Sceening Hybridomas to Eliminate Chimeric Ia Chains
The screening process involves two stages, which may
be conducted singly or optionally in combination: (1) a
preliminary ELISA-based screen, and (2) a secondary molecular
characterization of candidate hybridomas. Preferably, a
preliminary ELISA-based screen is used for initial
identification of candidate hybridomas which express a human
VDJ region and a human constant region.
Hybridomas that show positive reactivity with the
antigen (e.g., the immunogen used to elicit the antibody
response in the transgenic mouse) are tested using a panel of
monoclonal antibodies that specifically react with mouse g, y,
K, and X, and human g, y, and K. Only hybridomas that are
positive for human heavy and light chains, as well as negative
for mouse chains, are identified as candidate hybridomas that
express human immunoglobulin chains. Thus, candidate
hybridomas are shown to have reactivity with specific antigen
and to possess epitopes characteristic of a human constant
region.
RNA is isolated from candidate hybridomas and used
to synthesize first strand cDNA. The first strand cDNA is
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then ligated to a unique single-stranded oligonucleotide of
predetermined sequence (oligo-X) using RNA ligase (which
ligates single-stranded DNA). The ligated cDNA is then
amplified in two reactions by PCR using two sets of
oligonucleotide primers. Set H (heavy chain) includes an
oligo that specifically anneals to either human or human 71
(depending on the results of the ELISA) and an oligo that
anneals to the oligo-X sequence. This prevents bias against
detection of particular V segments, including mouse V segments
that may have trans-rearranged into the human minilocus. A
second set of primers, Set L (light chain), includes an oligo
that specifically anneals to human K and an oligo that anneals
specifically to oligo-X. The PCR products are molecularly
cloned and the DNA sequence of several are determined to
ascertain whether the hybridoma is producing a unique human
antibody on the basis of sequence comparison to human and
murine Ig sequences.
EXAMPLE 25
This example demonstrates production of a transgenic
mouse harboring a human light chain (K) minilocus.
Human K Minilocus transaenic mice
CC1
A 13 kb XhoI JK2-KK containing fragment from a phage
clone (isolated from a human genomic DNA phage library by
hybridization to a K specific oligonucleotide, e.g., supra)
was treated with Klenow enzyme and cloned into the Klenow
treated Hindlil site of pGPld to produce pK-31. This
destroyed the insert XhoI sites and positioned the unique
polylinker derived XhoI site at the 5' end next to JK2. A
unique polylinker derived Clal site is located between this
XhoI site and the inset sequences, while a unique polylinker
derived Sall site is located at the 3' end of the insert. A
7.5 kb XhoI fragment, containing JK1 and upstream sequences,
was also isolated from a human genomic DNA phage clone
(isolated from a human genomic DNA phage library by
hybridization to a K specific oligonucleotide, e.g. supra).
This 7.5 kb XhoI fragment was cloned into the Sall site of
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pSP72 (Promega, Madison, Wisconsin), thus destroying both XhoI
sites and positioning a polylinker Clal site 3' of Jlcl.
Digestion of the resulting clone with Clal released a 4.7 kb
fragment containing Jkl and 4.5 kb of upstream sequences.
This 4.7 kb fragment was cloned into the Clal site of pK-31 to
create pKcor. The remaining unique 5' XhoI site is derived
from polylinker sequences. A 6.5 kb XhoI/SalI DNA fragment
containing the unrearranged human VKIII gene segment 65.8
(plasmid p65.8, EXAMPLE 21) was cloned into the XhoI site of
pKcor to generate the plasmid pKC1. The Notl insert of pKC1
was microinjected into 1/2 day mouse embryos to generate
transgenic mice. Two independent pKC1 derived transgenic
lines were established and used to breed mice containing both
heavy and light chain miniloci. These lines, KC1-673 and KC1-
674, were estimated by Southern blot hybridization to contain
integrations of approximately 1 and 10-20 copies of the
transgenes respectively.
KCle
The plasmid pMHE1 (EXAMPLES 13 and 18) was digested
with BamHI and Hindill to excise the 2.3 kb insert containing
both the mouse and human heavy chain J- intronic enhancers.
This fragment was. Klenow treated, ligated to Sall linkers (New
England Biolabs, Beverly, Massachusetts), and cloned into the
unique 3' Sall site of pKC1 to generate the plasmid pKC1e.
The NotI insert of pKCle was microinjected into 1/2 day mouse
embryos to generate transgenic mice. Four independent pKCle
derived transgenic lines were established and used to breed
mice containing both heavy and light chain miniloci. These
lines, KCle-1399, KCle-1403, KCle-1527, and KCle-1536, were
estimated by Southern blot hybridization to contain
integrations of approximately.20-50, 5-10, 1-5, and 3-5 copies
of the transgene, respectively.
pKC2
A 6.8 kb XhoI/SalI DNA fragment containing the
unrearranged human VKIII gene segment 65.5 (plasmid p65.5g1,
EXAMPLE 21) was cloned into the unique 5' XhoI site of pKC1 to
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generate the plasmid pKC2. This minilocus transgene contains
two different functional VIII gene segments. The NotI insert
of pKC2 was microinjected into 1/2 day mouse embryos to
generate transgenic mice. Five independent pKC2 derived
transgenic lines were established and used to breed mice
containing both heavy and light chain miniloci. These lines,
KC2-1573, KC2-1579, KC2-1588, KC2-1608, and KC2-1610, were
estimated by Southern blot hybridization to contain
integrations of approximately 1-5, 10-50, 1-5, 50-100, and 5-
20 copies of the transgene, respectively.
EXAMPLE 26
This example shows that transgenic mice bearing the
human K transgene can make an antigen-induced antibody
response forming antibodies comprising a functional human K
chain.
Antibody Responses Associated with Human Ig K Light Chain
A transgenic mouse containing the HC1-57 human heavy
chain and KCle human K transgenes was immunized with purified
human soluble CD4 (a human glycoprotein antigen). Twenty g
of purified human CD4 (NEN Research products, Westwood, MA)
insolublized by conjugation to polystyrene latex particles
(Polysciences, Warrington, PA) was injected intraperitoneally
in saline with dimethyldioctadecyl ammonium bromide
(Calbiochem, San Diego, CA) on Day 0, followed by further
injections on Day 20 and Day 34.
Retro-orbital bleeds were taken on Days 25 and 40,
and screened for the presence of antibodies to CD4, containing
human IgM or human IgG heavy chain by ELISA. Briefly,
purified human CD4 was coated onto PVC microtitre plates at
2.5 Ag/ml and incubated with culture supernate diluted 1:4/1:5
in PBS, 0.5% Tween-20, 5% chicken serum. Plates were washed,
followed by addition of horseradish peroxidase-conjugated goat
antiserum specific for human IgG Fc or rabbit antiserum
specific for human IgM Fc5Mu (Jackson ImmunoResearch, Westr
Grove, PA). Presence of conjugate bound to captured antibody
was determined after further washing by addition of ABTS
substrate. Human A reactive with antigen was detected in both
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bleeds, while there was essentially undetectable 7 reactivity.
The Day 40 sample was also tested for antigen-reactive human K
chain using the same assay with goat anti-human K peroxidase
conjugate (Sigma, St. Louis, MO). CD4-binding K reactivity
was detected at this time point. The assay results are shown
in Fig. 48.
EXAMPLE 27
This example shows the successful generation of mice
which are homozygous for functionally disrupted murine heavy
and light chain loci (heavy chain and K chain loci) and which
concomitantly harbor a human heavy chain transgene and a human
light chain transgene capable of productively rearranging to
encode functional human heavy chains and functional human
light chains. Such mice are termed "0011" mice, indicating by
the two 0's in the first two digits that the mice lack
functional heavy and light chain loci and indicating by the
1's in the second two digits that the mice are hemizygous for
a human heavy chain transgene and a human light chain
transgene. This example shows that such 0011 mice are capable
of making a specific antibody response to a predetermined
antigen, and that such an antibody response can involve
isotype switching.
0011/0012 Mice: Endogenous Ia Knockout + Human Ig Transgenes
Mice which were homozygous for a functionally
disrupted endogenous heavy chain locus lacking a functional J.
region (designated JHD++ or JHO++) and also harboring the
human HC1 transgene, such as the HC1-26 transgenic mouse line
described supra, were interbred with mice homozygous for a
functionally disrupted endogenous kappa chain locus lacking a
functional J. region (designated here as JKD++ or JKD++; see
Example 9) to produce mice homozygous for functionally
disrupted heavy chain and kappa chain loci (heavy chain/kappa
chain knockouts), designated as JHD++/JKD++ and containing a
HC1 transgene. Such mice were produced by interbreeding and
selected on the basis of genotype as evaluated by Southern
blot of genomic DNA. These mice, designated HC1-
26+/JKD++/JHD++ mice, were interbred with mice harboring a
human kappa chain transgene (lines KC2-1610, KCle-1399, and
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KCle-1527; see Example 25), and Southern blot analysis of
genomic DNA was used to identify offspring mice homozygous for
functionally disrupted heavy and light chain loci and also
hemizygous for the HC1 transgene and the KC2 or KCle
transgene. Such mice are designated by numbers and were
identified as to their genotype, with the following
abbreviations: HC1-26+ indicates hemizygosity for the HC1-26 x
line human heavy chain minilocus transgene integration; JHD++
indicates homozygosity for JH knockout; JKD++ indicates
homozygosity for JK knockout; KC2-1610+ indicates hemizygosity
for a KC2 human K transgene integrated as in line KC2-1610;
KCle-1527+ indicates hemizygosity for a KCle human K transgene
integrated as in line KCle-1527; KCle-1399+ indicates
hemizygosity for a KCle human K transgene integrated as in
line KCle-1399.
The resultant individual offspring were each given a
numerical designation (e.g., 6295, 6907, etc.) and each was
evaluated for the presence of J. knockout alleles, J. knockout
alleles, HC1-26 transgene, and K transgene (KC2 or KC1e) and
determined to be either hemizygous (+) or homozygous (++) at
each locus. Table 10 shows the number designation, sex, and
genotypes of several of the offspring mice.
Table 10
No. Sex Ia Code Genotype
6295 M 0011 HC1-26+;JHD++;JKD++;KC2-1610+
6907 M 0011 HC1-26+;JHD++;JKD++;KCle-1527+
7086 F 0011 HC1-26+;JHD++;JKD++;KCIe-1399+
7088 F 0011 HC1-26+;JHD++;JKD++;KCle-1399+
7397 F 0011 HC1-26+;JHD++;JKD++;KCle-1527+
7494 F 0012 HC1-26+;JHD++;JKD++;KC2-1610++
7497 M 0011 HC1-26+;JHD++;JKD++;KCle-1399+
7648 F 0011 HC1-26+;JHD++;JKD++;KC2-1610+
7649 F 0012 HC1-26+;JHD++;JKD++;KC2-1610++
7654 F 0011 HC1-26+;JHD++;JKD++;KC2-1610+
7655 F 0011 HC1-26+;JHD++;JKD++;KC2-1610+
7839 F 0011 HC1-26+;JHD++;JKD++;KCle-1399+
7656 F 0001 HC1-26-;JHD++;JKD++;KC2-1610+
7777 F 1100 Col-2141-;JHD+;JKD+
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We removed spleens from three 6 week old female
mice. Mouse # 7655 was determined by Southern blot
hybridization to be hemizygous for the HC1 (line 26) and KC2
(line 1610) transgene integrations, and homozygous for the JHn
and Jun targeted deletions of the mouse g and KJ regions.
Mouse #7656 was determined by Southern blot hybridization to
be hemizygous for the KC2 (line 1610) transgene integration
and homozygous for the JHn and Jun targeted deletions of the
mouse g and uJ regions. Mouse # 7777 was determined by
Souther blot hybridization to be hemizygous for the JHn and
Jun targeted deletions of the mouse and uJ regions. Because
of the recessive nature of these deletions, this mouse should
be phenotypically wild-type.
Expression of Endogenous Ia Chains in 0011 Mice
FACS analysis using a panel of antibodies reactive
with either human , mouse , hman u, mouse K, or mouse X was
used to sort lymphocytes explanted from (1) a wildtype mouse
(7777), (2) a 0001 mouse homozygous for heavy chain and kappa
knockout alleles and harboring a human light chain transgene
(7656), and (3) a 0011 mouse homozygous for heavy chain and
kappa knockout alleles and harboring a human light chain
transgene and a human heavy chain transgene (7655).
We prepared single cell suspensions from spleen and
lysed the red cells with NH4C1, as described by Mishell and
Shiigi (Mishell, B.B. & Shiigi, S.M. (eds) Selected Methods in
Cellular Immunology. W.H. Freeman & Co., New York, 1980).
The lymphocytes are stained with the following reagents:
propidium iodide (Molecular Probes, Eugene, OR), FITC
conjugated anti-human IgM (clone G20-127; Pharmingen, San
Diego, CA), FITC conjugated anti-mouse IgM (clone R6-60.2;
Pharmingen, San Diego, CA), phycoerythrin conjugated anti-
human IgK (clone HP6062; CalTag, South San Francisco, CA),
FITC conjugated.anti-mouse IgX (clone R26-46; Pharmingen, San
Diego, CA) FITC conjugated anti-mouse B220 (clone RA3-6B2;
Pharmingen, San Diego, CA), and Cy-Chrome conjugated anti-
mouse B220 (clone RA3-6B2; Pharmingen, San Diego, CA). We
analyzed the stained cells using a FACScan flow cytometer and
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LYSIS II software (Becton Dickinson, San Jose, CA).
Macrophages and residual red cells are excluded by gating on
forward and side scatter. Dead cells are excluded by gating
out propidium iodide positive cells. The flow cytometric data
in Figs. 49 and 50 confirms the Southern blot hybridization
data and demonstrates that mouse #7655 expresses both human
and human K and relatively little if any mouse or mouse K.
Nevertheless a significant fraction of the B cells (about 70-
80%) appear to express hybrid Ig receptors consisting of human
heavy and mouse X light chains.
Fig. 49 shows the relative distribution of B cells
expressing human or mouse on the cell surface; 0011 mouse
(7655) lymphocytes are positive for human but relatively
lack mouse ; 0001 mouse (7656) lymphocytes do not express
much human g or mouse ; wildtype mouse (7777) lymphocytes
express mouse but lack human .
Fig. 50 shows the relative distribution of B cells
expressing human K or mouse K on the cell surface; 0011 mouse
(7655) lymphocytes are positive for human K but relatively
lack mouse K; 0001 mouse (7656) lymphocytes do not express
much human K or mouse K; wildtype mouse (7777) lymphocytes
express mouse K but lack human K. .
Fig. 51 shows the relative distribution of B cells
expressing mouse X on the cell surface; 0011 mouse (7655)
lymphocytes are positive for mouse X; 0001 mouse (7656)
lymphocytes do not express significant mouse X; wildtype mouse
(7777) lymphocytes express mouse X but at a relatively lower
level than the 0011 mouse (7655).
Fig. 52 shows the relative distribution of B cells
positive for endogenous mouse X as compared to human K
(transgene-encoded). The upper left panel shows the results
of cells from a wildtype mouse possessing functional
endogenous heavy and light chain alleles and lacking human
transgene(s); the cells are positive for mouse lambda. The
upper right panel shows cells from a mouse (#5822) having a K
knockout background (JKD++) and harboring the human K
transgene intergration of the KCle-1399 line; the cells are
positive for human K or mouse X in roughly proportional
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amounts. The lower left panel shows cells from a mouse
(#7132) having a K knockout background (JKD++) and harboring
the human K transgene intergration of the KC2-1610 line; more
cells are positive for mouse X than for human K, possibly
indicating that the KC2-1610 transgene integration is less
efficient than the KCle-1399 transgene integration. The lower
right panel shows cells from a mouse harboring a human K
minilocus transgene (KCo4) and lacking a functional endogenous
murine K allele. The data presented in Fig. 52 also
demonstrates the variability of phenotypic expression between
transgenes. Such variability indicates the desirability of
selecting for individual transgenes and/or transgenic lines
which express one or more desired phenotypic features
resulting from the integrated transgene (e.g., isotype
switching, high level expression, low murine Ig background).
Generally, single or multiple transgene species (e.g.,'pKC1e,
pKC2, KCo4) are employed separately to form multiple
individual transgenic lines differing by: (1) transgene, (2)
site(s) of transgene integration, and/or (3) genetic
background. Individual transgenic lines are examined for
desired parameters, such as: (1) capability to mount an immune
respone to a predetermined antigen, (2) frequency of isotype
switching within transgene-encoded constant regions and/or
frequency of trans-switching to endogenous (e.g., murine) Ig
constant region genes, (3) expression level of transgene-
encoded immmunoglobulin chains and antibodies, (4) expression
level of endogenous (e.g., murine) immunoglobulin
immunoglobulin sequences, and (5) frequency of productive VDJ
and VJ rearrangement. Typically, the transgenic lines which
produce the largest concentrations of transgene-encoded (e.g.,
human) immunoglobulin chains are selected; preferably, the
selected lines produce about at least 40 g/ml of transgene-
encoded heavy chain (e.g., human g or human y) in the serum of
the transgenic animal and/or about at least 100 gg/ml of
transgene-encoded light chain (e.g., human K).
Mice were examined for their expression of human and
murine immmunoglobulin chains in their unimmunized serum and
in their serum following immunization with a specific antigen,
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human CD4. Fig. 53 shows the relative expression of human g,
human y, murine g, murine y, human K, murine K, and murine X
chains present in the serum of four separate unimmunized 0011
mice of various genotypes (nt = not tested); human K
predominates as the most abundant light chain, and human and
murine y (putatively a product of trans-switching) are the
most abundant heavy chains, with variability between lines
present, indicating the utility of a selection step to
identify advantageous genotypic combinations that minimize
expression of murine chains while allowing expression of human
chains. Mice #6907 and 7088 show isotype switching (cis-
switching within the transgene) from human to human y.
Fig. 54 shows serum immunoglobulin chain levels for
human (hu ), human y (huy), human K (huK), murine (ms ),
murine y (msy), murine K (msK), and murine X (msX) in mice of
the various 0011 genotypes.
Specific Antibody Response in 0011 Mice
An 0011 mouse (#6295) was immunized with an
immunogenic dose of human CD4 according to the following
immunization schedule: Day 0, intraperitoneal injection of 100
gl of CD4 mouse immune serum; Day 1, inject 20 g of human CD4
(American Bio-Tech) on latex beads with DDA in 100 Al; Day 15
inject 20 g of human CD4 (American Bio-Tech) on latex beads
with DDA in 100 Al; Day 29 inject 20 g of human CD4 (American
Bio-Tech) on latex beads with DDA in 100 Al,*, Day 43 inject 20
g of human CD4 (American Bio-Tech) on latex beads with DDA in
100 Al.
Fig. 55 shows the relative antibody response to CD4
immunization at 3 weeks and 7 weeks demonstrating the presence
of human , human K, and human y chains in the anti-CD4
response. Human y chains are present at significantly
increased abundance in the 7 week serum, indicating that cis-
switching within the heavy chain transgene (isotype switching)
is occurring in a temporal relationship similar to that of
isotype switching in a wildtype animal.
Fig. 56 shows a schematic compilation of various
human heavy chain and light chain transgenes.
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EXAMPLE 28
This example provides for the targeted knockout of
the murine X light chain locus.
Targeted Inactivation of the Murine Lambda Light Chain Locus
Unlike the Ig heavy and kappa light chain loci, the
murine VXJX and CA gene segments are not grouped into 3
families arranged in a 5' to 3' array, but instead are
interspersed. The most 5' portion consists of two V segments
(VX2 and VAX) which are followed, proceeding in a 3'
direction, by two constant region exons, each associated with
its own J segment (JX2CX2 and the pseudogene JX4CX4). Next is
the most extensively used V segment (VA1) which is followed by
the second cluster of constant region exons (JX3CX3 and
JA1CA1,). Overall the locus spans approximate 200 kb, with
intervals of -20-90 kb between the two clusters.
Expression of the lambda locus involves
rearrangement of VX2 or VAX predominantly to JX2 and only
rarely further 3' to JX3 or Al. VA1 can recombine with both
JX3 and JX1. Thus the lambda locus can be mutated in order to
fully eliminate recombination and expression of the locus.
The distance between the two lambda gene clusters
makes it difficult to inactivate expression of the locus via
the generation of a single compact targeted deletion, as was
used in inactivating the murine Ig heavy and kappa light chain
loci. Instead, a small single deletion which would eliminate
expression lambda light chains spans approximately 120 kb,
extending from JX2CX2 to JX1CX1 (Fig. 57). This removes all
of the lambda constant region exons as well as the VX1 gene
segment, ensuring inactivation of the locus.
Replacement type targeting vectors (Thomas and
Capecchi (1987) op.cit) are constructed in which the deleted
=~ 120 kb is replaced with the selectable marker gene, neo, in a
PGK expression cassette. The marker is embedded within
genomic lambda sequences flanking the deletion to provide
homology to the lambda locus and can also contain the HSV-tk
gene, at the end of one of the regions of homology, to allow
for enrichment for cells which have homologously integrated
the vectors. Lambda locus genomic clone sequences are
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obtained by screening of a strain 129/Sv genomic phage library
isogenic to the ES line being targeted, since the use of
targeting vectors isogenic to the chromosomal DNA being
targeted has been reported to enhance the efficiency of
homologous recombination. Targeting vectors are constructed
which differ in their lengths of homology to the lambda locus.
The first vector (vector 1 in Fig. 58) contains the marker
gene flanked by total of approximately 8-12 kb of lambda locus
sequences. For targeting events in which replacement vectors
mediate addition or detection of a few kb of DNA this has been
demonstrated to be a more than sufficient extent of homology
(Hasty et al. (1991) op.cit; Thomas et al.(1992) on.cit.).
Vectors with an additional approximately 40-60 kb of flanking
lambda sequence are also constructed (vector 2 in Fig. 58).
Human Ig miniloci of at least 80 kb are routinely cloned and
propagated in the plasmid vector pGP1 (Taylor et al. (1993)
op.cit).
An alternative approach for inactivation of the
lambda locus employs two independent mutations, for example
mutations of the two constant region clusters or of the two V
region loci, in the same ES cell. Since both constant regions
are each contained within -6 kb of DNA, whereas one of the V
loci spans -19 kb, targeting vectors are constructed to
independently delete the JX2CX2/JX4CX4 and the JX3CX3/JX1CX1
loci. As shown in Fig. 58, each vector consists of a
selectable marker (e.g., neo or pac) in a PGK expression
cassette, surrounded by a total of -8-12 kb of lambda locus
genomic DNA blanking each deletion. The HSV-tk gene can be
added to the targeting vectors to enrich for homologous
recombination events by positive-negative selection. ES cells
are targeted sequentially with the two vectors, such that
clones are generated which carry a deletion of one of the
constant region loci; these clones are then targeted
sequentially with the two vectors, such that clones will be
generated which carry a deletion of one of the constant region
loci, and these clones are then targeted to generate a
deletion of the remaining functional constant region cluster.
Since both targeting events are thus being directed to the
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169
same cell, it is preferable to use a different selectable'
marker for the two targetings. In the schematic example shown
in Fig. 58, one of the vectors contains the neo gene and the
other the pac (puromycin N-acetyl transferase) gene. A third
potential dominant selectable marker is the hyg (hygromycin
phosphotransferase) gene. Both the pac and hyg genes can be
been inserted into the PGK expression construct successfully
used for targeting the neo gene into the Ig heavy and kappa
light chain loci. Since the two lambda constant region
clusters are tightly linked, it is important that the two
mutations reside on the same chromosome. There preferably is
a 50% probability of mutating the same allele by two
independent targeting events, and linkage of the mutations is
established by their co-segregation during breeding of
chimeras derived from the doubly targeted ES cells.
EXAMPLE 28
This example provides for the targeted knockout of
the murine heavy chain locus.
Targeted Inactivation of the Murine Heavy Chain Locus
A homologous recombination gene targeting transgene
having the structure shown in Fig. 59 is used to delete at
least one and preferably substantially all of the murine heavy
chain locus constant region genes by gene targeting in ES
cells. Fig. 59 shows a general schematic diagram of a
targeting transgene. Segment (a) is a cloned genomic DNA
sequence located upstream of the constant region gene(s) to be
deleted (i.e, proximal to the JH genes); segment (b) comprises
a positive selection marker, such as pgk-neo; segment (c) is a
cloned genomic DNA sequence located downstream of the constant
region gene(s) to be deleted (i.e, distal to the constan
region gene(s) and and JH genes); and segment (d), which is
optional, comprises a negative selection marker gene (e.g.,
HSV-tk). Fig. 60 shows a map of the murine heavy chain locus
as taken from Immunoglobulin Genes, Honjo, T, Alt, FW, and
Rabbits TH (eds.) Academic Press, NY (1989) p. 129.
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=
A targeting transgene having a structure according
to Fig. 59, wherein: (1) the (a) segment is the 11.5 kb insert
of clone JH8.1 (Chen et al. (1993) Int. Immunol. 5: 647) or an
equivalent portion comprising about at least 1-4 kb of
sequence located upstream of the murine C gene, (2) the (b)
segment is pgk-neo as described supra, (3) the (c) segment
comprises the 1674 bp sequence shown in Fig. 61 or a 4-6 kb
insert isolated from a phage clone of the mouse Ca gene
isolated by screening a mouse genomic clone library with the
end-labeled oligonucleotide having the sequence:
5'-gtg ttg cgt gta tca get gaa acc tgg aaa cag ggt gac cag-3'
and (4) the (d) segment comprises the HSV-tk expression
cassette described supra.
Alternatively, a stepwise deletion of one or more
heavy chain constant region genes is performed wherein a first
targeting transgene comprises homology regions, i.e., segments
(a) and (c), homologous to sequences flanking a constant
region gene or genes, a first species of positive selection
marker gene (pgk-neo), and an HSV-tk negative selection
marker. Thus, the (a) segment can comprise a sequence of at
least about 1-4 kb and homologous to a region located upstream
of C73 and the (c) segment can comprise a sequence of at least
about 1-4 kb and homologous to a region located upstream of
Cy2a. This targeting transgene deletes the C73, C71, Cy2b,
and Cy2a genes. This first targeting transgene is introduced
into ES cells and correctly targeted recombinants are selected
(e.g., with G418), producing a correctly targeted C region
deletion. Negative selection for loss of the HSV-tk cassette
is then performed (e.g., with ganciclovir or FIAU). The
resultant correctly targeted first round C deletion
recombinants have a heavy chain locus lacking the C73, Cyl,
C72b, and Cy2a genes.
A second targeting transgene comprises homology
regions, i.e., segments (a) and (c), homologous to sequences
flanking a constant region gene or genes, a second species of
positive selection marker gene different that the first
species (e.g., gpt or pac), and an HSV-tk negative selection
marker. Thus, the (a) segment can comprise a sequence of at
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least about 1-4 kb and homologous to a region located upstream
of Cc and the (c) segment can comprise a sequence of at least
about 1-4 kb and homologous to a region located upstream of
Ca. This targeting transgene deletes the Cc and Ca genes.
This second targeting transgene is introduced into
the correctly targeted C-region recombinant ES cells obtained
from the first targeting event. Cells which are correctly
targeted for the second knockout event (i.e., by homologous
recombination with the second targeting transgene) are
selected for with a selection drug that is specific for the
second species of positive selection marker gene (e.g.,
mycophenolic acid to select for gpt; puromycin to select for
pac). Negative selection for loss of the HSV-tk cassette is
then performed (e.g., with ganciclovir or FIAU). These
resultant correctly targeted second round C region
recombinants have a heavy chain locus lacking the C73, Cy1,
C72b, C72a, Ce, and Ca genes.
Correctly targeted first-round or second-round
recombinant ES cells lacking one or more C region genes are
used for blastocyst injections as described (supra) and
chimeric mice are produced. Germline transmission of the
targeted heavy chain alleles is established, and breeding of
the resultant founder mice is performed to generate mice
homozygous for C-region knockouts. Such C-region knockout
mice have several advantages as compared to,JH knockout mice;
for one example, C-region knockout mice have diminished
ability (or completely lack,the ability) to undergo trans-
switching between a human heavy chain transgene and an
endogenous heavy chain locus constant region, thus reducing
the frequency of chimeric human/mouse heavy chains in the
transgenic mouse. Knockout of the murine gamma genes is
preferred, although g and delta are frequently also deleted by
homologous targeting. C-region knockout can be done in
conjunction with other targeted lesions int he endogenous
murine heavy chain locus; a C-region deletion can be combined
with a J. knockout to preclude productive VDJ rearrangement of
the murine heavy chain locus and to preclude or reduce trans-
switching between a human heavy chain transgene and the murine
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heavy chain locus, among others. For some embodiments, it may
be desirable to produce mice which specifically lack one or
more C-region genes of the endogenous heavy chain locus, but
which retain certain other C-region genes; for example, it may
be preferable to retain the murine Ca gene to allow to
production of chimeric human/mouse IgA by trans-switching, if
such IgA confers an advantageous phenotype and does not
substantially interfere with the desired utility of the mice.
EXAMPLE 29
This example demonstrates ex vivo depletion of
lymphocytes expressing an endogenous (murine) immunoglobulin
from a lymphocyte sample obtained from a transgenic mouse
harboring a human transgene. The lymphocytes expressing
murine Ig are selectively depleted by specific binding to an
anti-murine immunoglobulin antibody that lacks substantial
binding to human immunoglobulins encoded by the transgene(s).
Ex Vivo Depletion of Murine Ia-Expressing B-cells
A mouse homozygous for a human heavy chain minilocus
transgene (HC2) and a human light chain minilocus transgene
(KCo4) is bred with a C57BL/6 (B6) inbred mouse to obtain 2211
mice (i.e., mice which: are homozygous for a functional
endogenous murine heavy chain locus, are homozygous for a
functional endogenous murine light chain locus, and which
possess one copy of a human heavy chain transgene and one copy
of a human light chain transgene). Such 2211 mice also
express B6 major and minor histocompatibility antigens. These
mice are primed with an immunogenic dose of an antigen, and
after approximately one week spleen cells are isolated. B
cells positive for murine Ig are removed by solid phase-
coupled antibody-dependent cell separation according to
standard methods (Wysocki et al. (1978) Proc. Natl. Acad. Sci.
(U.S.A.) 75: 2844; MACS magnetic cell sorting, Miltenyi Biotec
Inc., Sunnyvale, CA), followed by antibody-dependent
complement-mediated cell lysis (Selected Methods in Cellular
Immunology, Mishell BB and Shiigi SM (eds.), W.H. Freeman and
Company, New York, 1980, pp.211-212) to substantially remove
residual cells positive for murine Ig. The remaining cells in
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the depleted sample (e.g., T cells, B cells positive for human
Ig) are injected i.v., preferably together with additional
anti-murine Ig antibody to deplete arising B cells, into a
SCID/B6 or RAG/B6 mouse. The reconstitutued mouse is then
further immunized for the antigen to obtain antibody and
affinity matured cells for producing hybridoma clones.
EXAMPLE 30
Production of Fully Human Antibodies in Somatic Chimeras
A method is described for producing fully human
antibodies in somatic chimeric mice. These mice are generated
by introduction of embryonic stem (ES) cells, carrying human
immunoglobulin (Ig) heavy and light chain transgenes and
lacking functional murine Ig heavy and kappa light chain
genes, into blastocysts from RAG-i or RAG-2 deficient mice.
RAG-i and RAG-2 deficient mice (Mombaerts et al.
(1992) Cell 68: 869; Shinkai et al. (1992) Cell 68: 855) lack
murine B and T cells due to an inability to initiate VDJ
rearrangement and to assemble the gene segments encoding Igs
and T cell receptors (TCR). This defect in B and T cell
production can be complemented by injection of wild-type ES
cells into blastocysts derived from RAG-2 deficient animals.
The resulting chimeric mice produce mature B and T cells
derived entirely from the injected ES cells (Chen et al.
(1993) Proc. Natl. Acad. Sci. USA 90: 4528).
Genetic manipulation of the injected ES cells is
used for introducing defined mutations and/or exogenous DNA
constructs into all of the B and/or T cells of the chimeras.
Chen et al. (1993), Proc. Natl. Acad. Sci. USA 90:4528-4532)
generated ES cells carrying a homozygous inactivation of the
Ig heavy chain locus, which, when injected into RAG
blastocysts, produced chimeras which made T cells in the
absence of B cells. Transfection of a rearranged murine heavy
chain into the mutant ES cells results in the rescue of B cell
development and the production of both B and T cells in the
chimeras.
Chimeric mice which express fully human antibodies
in the absence of murine Ig heavy chain or kappa light chain
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synthesis can be generated. Human Ig heavy and light chain
constructs are introduced into ES cells homozygous for
inactivation of both the murine Ig heavy and kappa light chain
genes. The ES cells are then injected into blastocysts
ti
derived from RAG2 deficient mice. The resulting chimeras
contain B cells derived exclusively from the injected ES cells
which-are incapable of expressing murine Ig heavy and kappa
light chain genes but do express human Ig genes.
Generation of ES cells Homozygous for Inactivation of the
Immunoglobulin Heavy and Kappa Licrht Chain Genes
Mice bearing inactivated Ig heavy and kappa light
chain loci were generated by targeted deletion, in ES cells,
of Ig JH and JK/CK sequences, respectively according to known
procedures (Chen et al. (1993) EMBO J. 12: 821; and Chen et
al. (1993) Int. Immunol. op.cit). The two mutant strains of
mice were bred together to generate a strain homozygous for
inactivation of both Ig loci. This double mutant strain was
used for derivation of ES cells. The protocol used was
essentially that described by Robertson (1987, in
Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, p. 71-112, edited by E.J. Robertson, IRL Press).
Briefly, blastocysts were generated by natural matings of
homozygous double mutant mice. Pregnant females were
ovariectomized on day 2.5 of gestation and the "delayed"
blastocysts were flushed from the uterus on day 7 of gestation
and cultured on feeder cells, to help maintain their
undifferentiated state. Stem cells from the inner cell mass
of the blastocysts, identifiable by their morphology, were
picked, dissociated, and passaged on feeder cells. Cells with
a normal karyotype were identified, and male cell lines will
be tested for their ability to generate chimeras and
contribute to the germ cells of the mouse. Male ES cells are
preferable to female lines since a male chimera can produce
significantly more offspring.
Introduction of Human Ig Genes into Mouse Ia Heavy and Kappa
Light Chain Deficient ES cells
Human immunoglobulin heavy and light chain genes are
introduced into the mutant ES cells as either minilocus
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constructs, such as HC2 and KC-C04, or as YAC clones, such as
J1.3P. Transfection of ES cells with human Ig DNAs is carried
out by techniques such as electroporation or lipofection with
a cationic lipid. In order to allow for selection of ES cells
which have incorporated the human DNA, a selectable marker
either is ligated to the constructs or is co-transfected with
the constructs into ES cells. Since the mutant ES cells
contain the neomycin phosphotransferse (neo) gene as a result
of the gene targeting events which generated the Ig gene
inactivations, different selectable markers, such as
hygromycin phosphotransferase (hyg) or puromycin N-acetyl
transferase (pac), are used to introduce the human Ig genes
into the ES cells.
The human Ig.heavy and light chain genes can be
introduced simultaneously or sequentially, using different
selectable markers, into the mutant ES cells. Following
transfection, cells are selected with the appropriate
selectable marker and drug-resistant colonies are expanded for
freezing and for DNA analysis to verify and analyze the
integration of the human gene sequences.
Generation of Chimeras
ES clones containing human Ig heavy and light chain
genes are injected into RAG-2 blastocysts as described
(Bradley, A. (1987), in Teratocarcinomas and Embryonic Stem
Cells: A Practical Approach, p. 113-151, edited by E.J.
Robertson, IRL Press) and transferred into the uteri of
pseudopregnant females. Offspring are screened for the
presence of human antibodies by ELISA assay of serum samples.
Positive animals are used for immunization and the production
of human monoclonal antibodies.
EXAMPLE 31
This example describes the introduction, via
homologous recombination in ES cells, of a targeted frameshift
mutation into the murine heavy chain locus leading to a
deletion of B cells which undergo switch recombination. The
frameshifted mice are suitable hosts for harboring non-murine
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(e.g., human) transgenes encoding human sequence
immunoglobulins.
The novel frameshifted mice can be used for
expressing non-murine (e.g., human) sequence immunoglobulins
encoded by heavy chain transgene(s) and/or light chain
transgene(s), and for the isolation of hybridomas expressing
class-switched, affinity matured, human sequence antibodies
from introduced transgenes, among other uses. A frameshift is
introduced into one of the four mouse JH gene segments and
into the first exon of the mouse gene. The two introduced
frameshift mutations compensate for each other thus allowing
for the expression of fully functional murine heavy chain
when a B cell uses the frameshifted JH for a functional VDJ
joint. None of the other three JH segments can be used for
functional VDJ joining because of the frameshift in , which
is not compensated in the remaining JH genes. Alternatively,
compensating frameshifts can be engineered into multiple
murine JH genes.
A mouse homozygous for a compensated, frameshifted
immunoglobulin heavy chain allele has an approximately
physiological level of peripheral B cells, and an
approximately physiological level of serum IgM comprising both
murine and human A. However, B cells recruited into germinal
centers frequently undergo a class switch to a non- isotype.
Such a class switch in B cells expressing the endogenous
murine chain leads to the expression of a non-compensated
frameshift mRNA, since the remaining non- CH genes do not
possess a compensating frameshift. The resulting B cells do
not express a B cell receptor and are deleted. Hence, B cells
expressing a murine heavy chain are deleted once they reach
the stage of differentiation where isotype switching occurs.
However, B cells expressing heavy chains encoded by a non-
murine (e.g., human) transgene capable of isotype switching
and which does not contain such isotype-restrictive
frameshifts are capable of further development, including
isotype switching and/or affinity maturation, and the like.
Therefore, the frameshifted mouse has an impaired
secondary response with regard to murine heavy chain ( ) but a
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significant secondary response with regard to transgene-
encoded heavy chains. If a heavy chain transgene that is
capable of undergoing class switching is introduced into this
mutant background, the non-IgM secondary response is dominated
by transgene expressing B cells. It is thus possible to
isolate affinity matured human sequence immunoglobulin
expressing hybridomas from these frameshifted mice. Moreover,
the frameshifted mice generally possess immunoprotective
levels of murine IgM, which may be advantageous where the
human heavy chain transgene can encode only a limited
repertoire of variable regions.
For making hybridomas secreting human sequence
monoclonal antibodies, transgenic mutant mice are immunized;
their spleens fused with a myeloma cell line; and the
resulting hybridomas screened for expression of the transgene
encoded human non- isotype. Further, the frameshifted mouse
may be advantageous over a JH deleted mouse because it will
contain a functional g switch sequence adjacent to a
transcribed VDJ which serves as an active substrate for cis-
switching (Gu et al. (1993) Cell 73: 1155); thus reducing the
level of trans-switched B cells that express chimeric
human/mouse antibodies.
Construction of Frameshift Vectors
Two separate frameshift vectors are built. One of
the vectors is used to introduce 2 nucleotides at the 3' end
of the mouse J4 gene segment, and one of the vectors is used
to delete those same two nucleotides from the 5' end of exon 1
of the mouse g gene.
1. JH vector.
A 3.4 kb XhoI/EcoRI fragment covering the mouse
heavy chain J region and the g intronic enhancer is subcloned
into a plasmid vector that contains a neomycin resistance gene
as well-as a herpes thymidine kinase gene under the control of
a phosphoglycerate kinase promoter (tk/neo cassette; Hasty et
al., (1991) Nature 350: 243). This clone is then used as a
substrate for generating 2 different PCR fragments using the
following oligonucleotide primers:
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o-Al 5'- cca cac tct gca tgc tgc aga agc ttt tct gta -3'
o-A2 5'- ggt gac tga ggt acc ttg acc cca gta gtc cag -3'
o-A3 5'- ggt tac ctc agt cac cgt ctc ctc aga ggt aag aat
ggc ctc -3'
o-A4 5'- agg ctc cac cag acc tct cta gac agc aac tac -3'
Oligonucleotides o-Al and o-A2 are used to amplify a
1.2 kb fragment which is digested with SphI and KpnI.
Oligonucleotides o-A3 and o-A4 are used to amplify a 0.6 kb
fragment which is digested with KpnI and XbaI. These two
digested fragments are then cloned into SphI/XbaI digested
plasmid A to produce plasmid B.
Plasmid B contains the 2 nucleotide insertion at the
end of the J4 and, in addition, contains a new KpnI site
upstream of the insertion. The KpnI site is used as a
diagnostic marker for the insertion.
Additional flanking sequences may be cloned into the
5' Xhol site and the 3' EcoRI site of plasmid B to increase
its homologous recombination efficiency. The resulting
plasmid is then digested with SphI, or another restriction
enzyme with a single site within the insert, and
electroporated into embryonic stem cells which are then
selected with G418 as described by Hasty et al. (1991) on.cit.
Homologous recombinants are identified by Southern blot
hybridization and then selected with FIAU as described by
Hasty et al. to obtain deleted subclones which contain only
the 2 base pair insertion and the new KpnI site in JH4. These
are identified by Southern blot hybridization of KpnI digested
DNA and confirmed by DNA sequence analysis of PCR amplified
JH4 DNA.
The resulting mouse contains a JH4 segment that has
been converted from the unmutated sequence:
... TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGgtaagaatggcctctcc...
TrpGlyGlnGlyThrSerValThrVAlSerSerGlu
to the mutant sequence:
... TGGGGTCAAGGTACCTCAGTCACCGTCTCCTCAGAGgtaagaatggcctctcc...
TrpGlyGlnGlyThrSerValThrVAlSerSerGlu
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u Exon 1 Vector
Using similar in vitro mutagenesis methodology
described above to engineer a two base pair insertion into the
JH4 gene segment, PCR products and genomic subclones are
assembled to create a vector containing a two base pair
deletion at the 5' end of the first exon. In addition, to
mark the mutation, a new XmnI site is also introduced
downstream by changing an A to a G.
The sequence of the unmutated gene is:
... ctggtcctcagAGAGTCAGTCCTTCCCAAATGTCTTCCCCCTCGTC...
GluSerGlnSerPheProAsnValPheProLeuVal
The sequence of the mutated gene is:
XmnI
... ctggtcctcagAGTCAGTCCTTCCCGAATGTCTTCCCCCTCGTC...
SerGlnSerPheProAsnValPheProLeuVa1
The homologous recombination vector containing the mutant
sequence is linearized and electroporated into an ES cell line
containing the JH4 insertion. Homologous recombinants are
identified from neomycin-resistant clones. Those homologous
recombinants that contain the frameshift insertion on the same
chromosome as the JH4 insertion are identified by Southern
blot hybridization of KpnI/BamHI digested DNA. The JH4
insertion is associated with a new KpnI site that reduces the
size of the J-g intron containing Kpnl/BamHI fragment from the
wild type 11.3 kb to a mutant 9 kb. The resulting clones are
then selected for deletion of the inserted tk/neo cassette
using FIAU. Clones containing the mutant exon are
identified by Southern blot hybridization of XmnI digested
DNA. The mutation is confirmed by DNA sequence analysis of
PCR amplified g exonl DNA.
Generation of Frameshifted Mice
The ES cell line containing both the two base pair
insertion in JH4, and the two base pair deletion in A exon 1,
is then introduced into blastocyst stage embryos which are
inserted into pseudopregnant females to generate chimeras.
Chimeric animals are bred to obtain germline transmission, and
the resulting animals are bred to homozygosity to obtain
mutant animals homozygous for compensated frameshifted heavy
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chain loci and having impaired secondary humoral immune
responses in B cells expressing murine heavy chains.
A human heavy chain transgene, such as for example
pHC1 or pHC2 and the like, may be bred into the murine heavy
chain frameshift background by crossbreeding mice harboring
such a human transgene into mice having the frameshifted
murine IgH locus. Via interbreeding and backcrossing, mice
homozygous at the murine IgH locus for -compensated
frameshifted murine IgH alleles (i.e., capable of compensated
in-frame expression of only murine and not murine non-
chains) and harboring at least one integrated copy of a
functional human heavy chain transgene (e.g., pHC1 or pHC2)
are produced. Such mice may optionally contain knockout of
endogenous murine K and/or X loci as described supra, and may
optionally comprise a human or other non-murine light chain
transgene '(e.g., pKCle, pKC2, and the like).
Alternatively, the human transgene(s) (heavy and/or
light) may comprise compensating frameshifts, so that the
-transgene J gene(s) contain a frameshift that is compensated
by a frameshift in the transgene constant region gene(s).
Trans-switching to the endogenous constant region genes is
uncompensated and produces a truncated or nonsense product; B
cells expressing such uncompensated trans-switched
immunoglobulins are selected against and depleted.
EXAMPLE 32
Endogenous Heavy Chain Inactivation by D Region Ablation
This example describes a positive-negative selection
homologous recombination vector for replacing the mouse
germline immunoglobulin heavy chain D region with a
nonfunctional rearranged VDJ segment. The resulting allele
functions within a B cell as a normal non-productive allele,
with the allele undergoing intra-allele heavy chain class
switching, thereby reducing the level of trans-switching to an
active transgene locus.
D Region Targeting Construct
An 8-15 kb DNA fragment located upstream of the
murine D region is isolated and subcloned from a mouse strain
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129 phage library using an oligonucleotide probe comprising
approximately 50 consecutive nucleotides of the published
sequence for the DFL16.1 segment listed in GenBank. DFL16.1
is the upstream D segment (i.e., proximal to the V region gene
cluster and distal to the constant region gene cluster).
Similarly, a 9.5 kb BamHI fragment containing JH3,
JH4, E , S , and the first two coding exons of the constant
region is isolated and subcloned from a mouse strain 129
genomic phage library.
A 5-10 kb rearranged VDJ is then isolated from a
mouse hybridoma (any strain) and a synthetic linker containing
a stop codon is inserted into the J segment. The stop linker
within the J is preferable to an out-of-frame VDJ junction
because of the possibility of V replacement rearrangements.
These three fragments are assembled together with a
PGKneo positive selection cassette and a PGKHSVtk negative
selection cassette to form a positive-negative selection
vector for eliminating the mouse D region in 129-derived ES
cells (e.g., AB1) by homologous recombination. The targeting
vector is formed by ligating the 8-15 kb DNA fragment to the
positive selection cassette (e.g., PGKneo), which is itself
ligated to the rearranged 5-10 kb rearranged VDJ, which is
itself ligated to the 9.5 kb BamHI fragment; the negative
selection cassette (e.g., PGKHSVtk) is then ligated at either
end of the targeting construct. The construction of such a D
region targeting vector is shown schematically in Fig. 63.
The D region targeting construct is transferred into
AB1 ES cells, positive and negative selection is performed as
described above, and correctly targeted ES cells are cloned.
The correctly targeted ES cell clones are used for blastocyst
injections and chimeric mice are produced. The chimeric mice
are bred to produce founder mice harboring a D-region
inactivated heavy chain allele. Interbreeding of offspring is
performed to produce homozygotes lacking a functional
endogenous heavy chain locus. Such homozygotes are used to
crossbreed to mice harboring human Ig transgenes (e.g., pHC1,
pHC2, pKC2, pKC1e, KCo4) to yield (by further backcrossing to
the homozygotes lacking a functional D-region) mice lacking a
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functional endogenous heavy chain locus and harboring a human
heavy transgene (and preferably also a human light chain
transgene). In embodiments where some functional endogenous
light chain loci remain (e.g., X loci), it is generally
preferred that transgenes contain transcriptional control
sequences that direct high level expression of human light
chain (e.g., K) polypeptides, and thus allow the transgene
locus to compete effectively with the remaining endogenous
light chain (e.g., X) loci. For example, the Co4 kappa light
chain transgene is generally preferred as compared to pKC1
with regard to the ability to compete effeectively with the
endogenous X loci in the transgenic animal.
EXAMPLE 33
This example describes expansion of the human light
chain transgene V gene repertoire by co-injection of a human K
light chain minilocus and a yeast artificial chromosome
comprising a portion of the human VK locus.
Introduction of Functional Human Light Chain V Segments by Co-
Iniection of VK-Containing YAC DNA and a K Minilocus
An approximately 450 kb YAC clone containing part of
the human VK locus was obtained as a non-amplified YAC DNA
from clone 4x17E1 of the publicly available ICRF YAC library
(Larin et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 4123;
Genome Analysis Laboratory, Imperial Cancer Research Fund,
London, UK). The 450 kb YAC clone was isolated without prior
amplification by standard pulsed-field gel electrophoresis as
per the manufacturer's specifications (CHEF DR-II
electrophoresis cell, Bio-Rad Laboratories, Richmond, CA).
Six individual pulse field gels were stained with ethidium
bromide and the gel material containing the YAC clone DNA was
excised from the gel and then embedded in a new (low melting
point agarose in standard gel buffer) gel cast in a triangular
gel tray. The resulting triangular gel (containing the six
excised YAC-containing gel blocks) was extended at the apex
with a narrow agarose gel with 2 M NaOAc in addition to the
standard electrophoresis buffer. The gel was then placed in
an electrophoresis chamber immersed in standard gel buffer.
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The Y-shaped gel former rises above the surface of the buffer
so that current can only flow to the narrow high salt gel
portion. A plexiglas block was placed over the high salt gel
slice to prevent diffusion of the NaOAc into the buffer. The
YAC DNA was then electrophoresed out of the original excised
gel sliced (embedded) and into the narrow high salt gel
portion. At the point of transition from the low salt gel to
the high salt gel, there is a resistance drop that effectively
halts the migration of the DNA at the apex of the triangular
gel.
Following electrophoresis and staining with ethidium
bromide, the concentrated YAC DNA was cut away from the rest
of the gel and the agarose was digested with GELase (EpiCentre
Technologies, Madison, Wisconsin). Cesium chloride was then
added to the resultant YAC-containing liquid to obtain a
density of 1.68 g/ml. This solution was centrifuged at 37,000
rpm for 36 hours to separate the YAC DNA from any
contaminating material. 0.5 ml fractions of the resulting
density gradient were isolated and the peak DNA fraction was
dialyzed against 5 mM Tris (pH 7.4), 5 mM NaCl, 0.1 M EDTA.
Following dialysis, the concentration of the resulting 0.65 ml
solution of YAC DNA was found to contain 2 g/ml of DNA. This
YAC DNA was mixed with purified DNA insert from plasmids pKC1B
and pKV4 at a ratio of 20:1:1 (micrograms YAC4x17E1:KCIB:KV4).
The resulting 2 Ag/ml solution was injected into the pronuclei
of half-day B6CBF2 embryos, and 95 surviving microinjected
embryos were transferred into the oviducts of pseudopregnant
females. Twelve mice which developed from the microinjected
embryos were born.
EXAMPLE 34
This example describes class-switching, somatic
mutation, and B cell development in immunized transgenic mice
homozygous for an inactivated endogenous immunoglobulin locus
and containing the HC1 or HC2 heavy chain transgene(s).
To demonstrate that a human sequence germline
configuration minilocus can functionally replace the authentic
locus, we bred a mouse strain lacking endogenous IgH with
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strains containing human germline-configuration IgH
transgenes. The two transgene miniloci, HC1 and HC2, include
one and four functional variable (V) segments respectively 10
and 16 diversity (D) segments respectively, all six joining
(JH) segments, and both the and yl constant region segments.
The miniloci include human cis-acting regulatory sequences--
such as the JH- intronic enhancer and the and yi switch
sequences--that are closely linked to the coding segments.
They also include an additional enhancer element derived from
the 3' end of the rat IgH locus. We crossed HC1 and HC2
transgenic mice with stem-cell derived mutant mice that lack
JH segments (JHD mice) as described (supra) and cannot
therefore undergo functional heavy chain rearrangements. The
resulting transgenic-JHD mice contain B cells that are
dependent on the introduced heavy chain sequences.
Immunizations and hvbridomas.
We immunized mice by intraperitoneal injections of
50-100 g of antigen. Antigens included human carcinoembryonic
antigen (CEA; Crystal Chem, chicago, IL), hen eggwhite
lysozyme (HEL; Pierce, Rockford, IL), and keyhole limpet
hemocyanin (KLH; Pierce, Rockford, IL). For primary
injections we mixed the antigen with complete Freund's
adjuvant, for subsequent injections we used incomplete
Freund's adjuvant (Gibco BRL, Gaithersburg, MD). We fused
spleen cells with the non-secreting mouse myeloma P3X63-
Ag8.653 (ATCC, CRL1580). We assayed serum samples and
hybridoma supernatants for the presence of specific and non-
specific antibody comprising human heavy chain sequences by
ELISA. For detection of non-specific antibodies we coated
microtiter wells with human heavy chain isotype specific
antibody (mouse MAb a human IgGl, clone HP6069, Calbiochem, La
Jolla, CA; mouse MAb a human IgM, clone CH6, The Binding Site,
Birmingham, UK) and developed with peroxidase conjugated
antisera (horseradish peroxidase conjugated affinity purified
fab fragment from polyclonal goat a human IgG(fc), cat # 109-
036-098; affinity purified horseradish peroxidase conjugated
polyclonal rabbit a human IgM(fc), cat # 309-035-095. Jackson
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Immuno Research, West Grove, PA). For detection of antigen-
specific antibodies we coated microtiter wells with antigen
and developed with peroxidase-conjugated human heavy chain
isotype specific antisera. We detected bound peroxidase by
incubation with hydrogen peroxide and
2,2'-Azino-bis-(3-Ethylbenzthiazoline-6-Sulfonic Acid, Sigma
Chem. Co., St. Louis, MO). The reaction product is measured
by absorption at 415 nm, and corrected for absorption at
490 nm.
Flow cytometry.
We prepared single cell suspensions from spleen,
bone marrow, and peritoneal cavity, and lysed red cells with
NH4C1, as described by-Mishell and Shiigi. The lymphocytes
are stained with the following reagents: Phycoerythrin
conjugated anti-mouse IgK (clone X36; Becton Dickinson, San
Jose, CA), FITC conjugated anti-mouse IgD (clone SBA 1,
Southern Biotech, AL), FITC conjugated anti-mouse CD5 (clone
53-7.3; Becton Dickinson, San Jose, CA), FITC conjugated anti-
mouse IgX (clone R26-46; Pharmingen, San Diego, CA), and Cy-
Chrome conjugated anti-mouse B220 (clone RA3-6B2; Pharmingen,
San Diego, CA). We analyzed the stained cells using a FACScan
flow cytometer and LYSIS II software (Becton Dickinson, San
Jose, CA). Most macrophages, neutrophils, and residual red
cells are excluded by gating on forward and side scatter.
Rescue of B cell compartment
In the peritoneal cavity of HC1 transgenic-JHD
animals we find normal levels of CD5+ B cells and
approximately one-quarter the normal level of conventional
CD5- B cells. The transgenic peritoneal CD5+ B cells are
similar to the so-called B-i cells described in normal
animals: they are larger than conventional B and T
lymphocytes, they express lower levels of B220 than the
conventional B cells found in the spleen, and they include a
higher proportion of X light chain expressing cells. Over 90%
of the splenic B cells express K, while up to 50% of the
peritoneal B cells express X. Thus, while the level of
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conventional B cells is uniformly reduced in all tissues, the
level of B-1, which are reported to have a much greater
capacity for self-renewal, appears to be normal in the HC1
transgenic-JHD animals.
Class switching.
In transgenic-JHD mice, repeated exposure to antigen
results in the production of human 71 antibodies as well as
antibodies. We injected human CEA into transgenic-JHD mice at
weekly intervals and monitored the serum levels of antigen-
specific IgM and IgGl over a period of four weeks (Fig. 63).
At one week there is a detectable IgM response but no IgG1
response. However, the IgGi response is greater than the IgM
response after two weeks, and it continues to increase while
the IgM response remains relatively constant. This pattern--
an initial IgM reaction followed by an IgG reaction--is
typical of a secondary immune response; and it suggests that
cis-acting sequences included in the transgene may be
responding to cytokines that direct class switching. We have
considered three possible mechanisms for expression of non-
isotypes, each of which have been discussed in the literature.
These mechanisms are: alternative splicing, which does not
involve deletion of the gene; "5-type" switching, which
involved deletion of the gene via homologous recombination
between flanking repeat sequences; and non-homologous
recombination between switch regions. The results of our
experiments, described below, are indicative of a switch
region recombination model.
Two types of non-deletional alternative splicing
mechanisms can be invoked to explain an isotype shift. First,
it is possible that a single transcript covering both and yl
is expressed from the transgene; this transcript could be
alternatively spliced in response to cytokines induced by
exposure to antigen. Alternative, a cytokine induced sterile
transcript initiating upstream of 71 could be trans-spliced to
the transcript. If either of these mechanisms were
responsible for the expression of human yi sequences, then we
would expect to be able to isolate hybridomas that express
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both and y1. However, although we have screened several
hundred hybridomas expressing either human or human yl, we
have not found any such double producer ( +, y1+) hybridomas.
This indicates that expression of yl is accompanied by
deletion of the gene.
Deletion of the gene can be mediated by non-
homologous recombination between the and yl switch regions,
or by homologous recombination between the two flanking 400 bp
direct repeats (a and E ) that are included in the HC1 and
HC2 transgenes. Deletional recombination between aA and E
has been reported to be responsible for the IgD+, IgM-
phenotype of some human B cells. While the first mechanism,
non-homologous switch recombination, should generate switch
products of varying lengths, the second mechanism, v /E
recombination, should always generate the same product. We
performed a Southern blot analysis of genomic DNA isolated
from three hybridomas (Fig. 64A), one expressing and two
expressing yl. We find genomic rearrangements upstream of the
transgene yl only in the two the 71 switch regions (Fig. 64B).
Furthermore, neither of the observed structures is compatible
with homologous recombination between a and E. Our results
are therefore consistent with a model for 71 isotype
expression mediated by deletional non-homologous recombination
between the transgene encoded and y1 switch regions.
Trans-switching.
In addition to human 71, we find mouse y in the
serum of HC1 and HC2 transgenic-JHD mice. We have also
obtained mouse y expressing hybridomas from these animals.
Because the non-transgenic homozygous JHD animals do not
express detectable levels of mouse immunoglobulins, we
attribute the expression of mouse y in the HC1 and HC2
transgenic-JHD animals to the phenomenon of trans-switching.
All of the transgenic hybridomas that we have analyzed express
either mouse or human constant region sequences, but not both.
It is therefore unlikely that a trans-splicing mechanism is
involved. We used PCR amplification to isolate cDNA clones of
trans-switch products, and determined the nucleotide sequence
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of 10 of the resulting clones (Fig. 65). The 5'
oligonucleotide in the PCR amplification is specific for the
transgene encoded VH251, and the 3' oligonucleotide is
specific for mouse 71, 72b, and 73 sequences. We find
examples of trans-switch products incorporating all three of =
these mouse constant regions.
Somatic mutation.
Approximately 1% of the nucleotides within the
variable regions of the trans-switch products shown in Fig. 7
are not germline encoded. This is presumably due to somatic
mutation. Because the mutated sequence has been translocated
to the endogenous locus, the cis-acting sequences directing
these mutations could be located anywhere 3' of the mouse y
switch. However, as we discuss below, we also observe somatic
mutation in VDJ segments that have not undergone such
translocations; and this result indicates that sequences
required by heavy chain somatic mutation are included in the
transgene.
To determine if the HC1 and HC2 constructs include
sufficient cis-acting sequences for somatic mutation to occur
in the transgenic-JHD mice, we isolated and partially
sequenced cDNA clones derived from two independent HC1
transgenic lines and one HC2 line. We find that some of the
71 transcripts from transgenic-JHD mice contain V regions with
extensive somatic mutations. The frequency of these mutated
transcripts appears to increase with repeated immunizations.
Figs. 66A and 66B show two sets of cDNA sequences: one set is
derived form an HC1 (line 26) transgenic-JHD mouse that we
immunized with a single injection of antigen 5 days before we
isolated RNA; the second set is derived from an HC1 (line 26)
transgenic-JHD mouse that we hyperimmunized by injecting
antigen on three different days beginning 5 months before we
isolated RNA; the second set is derived from an HC1 (line 26)
transgenic-JHD mouse that we hyperimmunized by injecting
antigen on three different days beginning 5 months before we
isolated RNA. Only 2 of the 13 V regions from the 5 day post-
exposure mouse contain any non-germline encoded nucleotides.
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Each of these V's contains only a single nucleotide change,
giving an overall somatic mutation frequency of less than 0.1%
for this sample. In contrast, none of the 13 V sequences from
the hyperimmunized animal are completely germline, and the
overall somatic mutation frequency is 1.6%.
Comparison of and 71 transcripts isolated from a
single tissue sample, shows that the frequency of somatic
mutations is higher in transgene copies that have undergone a
class switch. We isolated and partially sequenced 47
independent and 71 cDNA clones from a hyperimmunized CH1
line 57 transgenic-JHD mouse (Fig. 67A and 67B). Most of the
g cDNA clones are unmodified relative to the germline
sequence, while over half of the 71 clones contain multiple
non-germline encoded nucleotides. The 71 expressing cells are
distinct from the /h expressing cells and, while the two
processes are not necessarily linked, class switching and
somatic mutation are taking place in the same sub-population
of B cells.
Although we do not find extensive somatic mutation
of the VH251 gene in non-hyperimmunized CH1 transgenic mice,
we have found considerable somatic mutation in VH56p1 and
VH51p1 genes in a naive HC2 transgenic mouse. We isolated
spleen and lymph node RNA from an unimmunized 9 week old
female HC2 transgenic animal. We individually amplified 71
transcripts that incorporate each of the four V regions in the
HC2 transgene using V and 71 specific primers. The relative
yields of each of the specific PCR products were
VH56p1>>VH51p1>VH4.21>VH251. Although this technique is not
strictly quantitative, it may indicate a bias in V segment
usage in the HC2 mouse. Fig. 68 shows 23 randomly picked 71
cDNA sequences derived from PCR amplifications using an
ti equimolar mix of all four V specific primers. Again we
observe a bias toward VH56p1 (19/23 clones). In addition, the
VH56pl sequences show considerable somatic mutation, with an
overall frequency of 2.1% within the V gene segment.
Inspection of the CDR3 sequences reveals that although 17 of
the 19 individual VH56p1 clones are unique, they are derived
from only 7 different VDJ recombination events. It thus
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appears that the VH56p1 expressing B cells are selected,
perhaps by an endogenous pathogen or self antigen, in the
naive animal. It may be relevant that this same gene is over-
represented in the human fetal repertoire.
Summary
Upstream cis-acting sequences define the
functionality of the individual switch regions, and are
necessary for class switching. Our observation--that class
switching within the HC1 transgene is largely confined to
cells involved in secondary response, and does not occur
randomly across the entire B cell population--suggests that
the minimal sequences contained with the transgene are
sufficient. Because the y sequences included in this
construct begin only 116 nucleotides upstream of the start
site of the 71 sterile transcript, the switch regulatory
region is compact.
Our results demonstrate that these important cis-
acting regulatory elements are either closely linked to
individual y genes, or associated with the 3' heavy chain
enhancer included in the HC1 and HC2 transgenes. Because the
HC1 and HC2 inserts undergo transgene-autonomous class
switching--which can serve as a marker for sequences that are
likely to have been somatically mutated--we were able to
easily find hypermutated transcripts that did not originate
from translocations to the endogenous locus. We found
somatically mutated y transcripts in three independent
transgenic lines (two HC1 lines and one HC2 line). It is
therefore unlikely that sequences flanking the integration
sites of the transgene affect this process; instead, the
transgene sequences are sufficient to direct somatic mutation.
EXAMPLE 35
This example describes the generation of hybridomas
from mice homozygous for an inactivated endogenous
immunoglobulin locus and containing transgene sequences
encoding a human sequence heavy chain and human sequence light
chain. The hybridomas described secrete monoclonal antibodies
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comprising a human sequence heavy chain and a human seqeunce
light chain and bind to a predetermined antigen expressed on T
lymphocytes. The example also demonstrates the capacity of
the mice to make a human sequence antibody in response to a
= 5 human-derived immunogen, human CD4, and the suitability of
such mice as a source for making hybridomas secreting human
% sequence monoclonal antibodies reactive with human antigens.
A. Generation of Human Ig Monoclonal Antibodies Derived from
HC1 Transgenic Mice Immunized with a Human CD4 Antigen
A transgenic mouse homozygous for a functionally
disrupted JH locus and harboring a transgene capable of
rearranging to encode a human sequence heavy chain and a
transgene capable of rearranging to encode a human sequence
light chain was immunized. The genotype of the mouse was HC1-
26' KCle-1536+ JHD+/+ JKD-, indicating homozygosity for murine
heavy chain inactivation and the presence of germline copies
of the HC1 human sequence heavy chain transgene and the KCle
human sequence light chain transgene.
The mouse was immunized with a variant of the EL4
cell line (ATCC) expressing a mouse-human hybrid CD4 molecule
encoded by a stably transfected polynucleotide. The expressed
CD4 molecule comprises a substantially human-like CD4
sequence. Approximately 5 x 106 cells in 100 Al of PBS
accompanied by 100 Al of Complete Freund's Adjuvant (CFA) were
introduced into the mouse via intraperitoneal injection on Day
0. The inoculation was repeated on Days 7, 14, 21, 28, 60,
= and 77, with test bleeds on Days 18, 35, and 67. The spleen
was removed on Day 81 and approximately 7.2 x 107 spleen cells
were fused to approximately 1.2 x 107 fusion partner cells
(P3x63Ag8.653 cell line; ATCC) by standard methods (PEG
fusion) and cultured in RPMI 1640 15 % FCS, 4 mM glutamine, 1
mM sodium pyruvate plus HAT and PSN medium. Multiple fusions
were performed. .
Hybridomas were grown up and supernatants were
tested with ELISA for binding to a commercial source of
purified recombinant soluble human sequence CD4 expressed in
CHO cells (American Bio-Technologies, Inc. (ABT), Cambridge,
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MA) and/or CD4 obtained from NEN-DuPont. The ABT sample
contained a purified 55 kD human CD4 molecule comprised the V1
through V3 domains of human CD4. The recombinant human
sequence CD4 (produced in CHO-K1 cells) was adsorbed to the
assay plate and used to capture antibody from hybridoma
supernatants, the captured antibodies were then evaluated for
binding to a panel of antibodies which bind either human g,
= r
human K, human y, murine g, or murine K.
One hybridoma was subcloned from its culture plate
well, designated 1F2. The 1F2 antibody bound to the ABT CD4
preparation, was positive for human g and human K, and was
negative for human y, mouse y, and mouse K.
B. Generation of Human Ig Monclonal Antibodies Derived from
HC2 Transgenic Mice Immunized with Human CD4 and Human IgE.
The heavy chain transgene, HC2, is shown in Fig. 56
and has been described supra (see, Example 34).
The human light chain transgene, KCo4, depicted in
Fig. 56 is generated by the cointegration of two individually
cloned DNA fragments at a single site in the mouse genome.
The fragments comprise 4 functional VK segments, 5J segments,
the CK exon, and both the intronic and downstream enhancer
elements (see Example 21) (Meyer and Neuberger (1989), EMBO J.
8:1959-1964; Judde and Max (1992), Mol. Cell Biol. 12:5206-
5216). Because the two fragments share a common 3 kb sequence
(see Fig. 56), they can potentially integrate into genomic DNA
as a contiguous 43 kb transgene, following homologous
recombination between the overlapping sequences. It has been
demonstrated that such recombination events frequently occur
upon microinjection of overlapping DNA fragments (Pieper et
al. (1992), Nucleic Acids Res. 20:1259-1264). Co-injected
DNA's also tend to co-integrate in the zygote, and the
sequences contained within the individually cloned fragments
would subsequently be jointed by DNA rearrangement during B
cell development. Table 11 shows that transgene inserts from
at least 2 of the transgenic lines are functional. Examples
of VJ junctions incorporating each of the 4 transgene encoded
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V segments, and each of the 5J segments, are represented in
this set of 36 clones.
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Table 11
line VK65.5 Vx65.& VK65.15 VK65.3 Jxl JK2 JK3 Jx4 Jx5
#4436 0 11 4 3 14 1 0 2 1
#4437 1 3 7 7 5 2 1 7 3
Table 11. Human light chain V and J segment usage in KCo4 transgenic
= mice. The table shows the number of PCR clones, amplified from cDNA
derived from two transgenic lines, which contain the indicated human kappa
sequences. cDNA was synthesized using spleen RNA isolated from w
individual KCo4 transgenic mice (mouse #8490, 3 mo., male, KCo4 line
4437; mouse #8867, 2.5 mo., female, KCo4 line 4436). The cDNA was
amplified by PCR using a CK specific oligonucleotide. 5'TAG AAG GAA
TTC AGC AGG CAC ACA ACA GAG GCA GTT CCA 3', AND A 1:3
mixture of the following 2 VK specific oligonucleotides: 5' AGC TTC TCG
AGC TCC TGC TGC TCT GTT TCC CAG GTG CC 3' and 5' CAG CTT
CTC GAG CTC CTG CTA CTC TGG CTC (C,A)CA GAT ACC 3' . The
PCR product was digested with Xhol and EcoRI, and cloned into a plasmid
vector. Partial nucleotide sequences were determined by the dideoxy chain
termination method for 18 randomly picked clones from each animal. The
sequences of each clone were compared to the germline sequence of the
unrearranged transgene.
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Twenty-three light chain minilocus positive and 18
heavy chain positive mice developed from the injected embryos.
These mice, and their progeny, were bred with mice containing
targeted mutations in the endogenous mouse heavy (strain JHD)
and K light chain loci (strain JCKD) to obtain mice containing
human heavy and K light chain in the absence of functional
mouse heavy and K light chain loci. These mice contain only
XB cells.
Table 12 shows that somatic mutation occurs in the
variable regions of the transgene-encoded human heavy chain
transcripts of the transgenic mice. Twenty-three cDNA clones
from a HC2 transgenic mouse were partially sequenced to
determine the frequency of non-germline encoded nucleotides
within the variable region. The data include only the
sequence of V segment codons 17-94 from each clone, and does
not include N regions. RNA was isolated from the spleen and
lymph node of mouse 5250 (HC2 line 2550 hemizygouc, JHD
homozygous). Single-stranded cDNA was synthesized and y
transcripts amplified by PCR as described [references]. The
amplified cDNA was cloned into plasmid vectors, and 23
randomly picked clones were partially sequenced by the dideoxy
chain-termination method. The frequency of PCR-introduced
nucleotide changes is estimated from constant region sequence
as <0.2%.
TABLE 12: The Variable Regions of Human y Transcripts in HC2
Transgenic Mice Contain Non-Germline-Encoded Nucleotides
Number of non- Frequency of non-
VH Number of germline encoded germline-encoded
Segment clones nucleotides nucleotides (%)
VH251 0 --
VH56P1 10 100 2.1
VH51P1 1 5 2.0
VH4.21 3 0 0.0
Flow cytometry
We analyzed the stained cells using a FACScan
flow cytometer and LYSIS II software (Becton Dickinson, San
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Jose, CA). Spleen cells were stained with the following
reagents: propidium iodide (Molecular Probes, Eugene, OR),
phycoerythrin conjugated a-human IgK (clone HP6062; Caltag, S.
San Francisco, CA), phycoerythrin conjugated a-mouse Igrc (clone
X36; Becton Dickinson, San Jose, CA), FITC conjugated a-mouse
IgX (clone R26-46; Pharmingen, San diego, CA), FITC conjugated
a-mouse Ig (clone R6-60.2; Pharmingen, San Diego, CA), FITC
conjugated a-human Ig (clone G20-127; Pharmingen, San Diego,
CA), and Cy-Chrome conjugated a-mouse B220 (clone RA3-6B2;
Pharmingen, San Diego, CA).
Expression of human Ig transgenes
Figure 69 shows a flow cytometric analysis of spleen cells from
KCo4 and HC2 mice that are homozygous for both the JHD and JCKD
mutations. The human sequence HC2 transgene rescued B cell
development in the JHD mutant background, restoring the
relative number of B220+ cells in the spleen to approximately
half that of a wild type animal. These B cells expressed cell
surface immunoglobulin receptors that used transgene encoded
heavy chain. The human KCo4 transgene was also functional, and
competed successfully with the intact endogenous X light chain
locus. Nearly 95% of the splenic B cells in JHD/JCKD
homozygous mutant mice that contain both heavy and light chain
human transgenes (double transgenic) expressed completely human
cell surface IgMi.
Serum Ig levels were determined by ELISA done as
follows: human : microtiter wells coated with mouse Mab a
human IgM (clone CH6, The Binding Site, Birmingham, UK) and
developed with peroxidase conjugated rabbit a human IgM(fc)
(cat # 309-035-095, Jackson Immuno Research, West Grove, PA).
Human y: microtiter wells coated with mouse MAb a human IgG1
(clone HP6069, Calbiochem, La Jolla, CA) and developed with
peroxidase conjugated goat a human IgG(fc) (cat # 109-036-098,
Jackson Immuno Research, West Grove, PA). Human rc: microtiter
wells coated with mouse Mab a human Igic (cat # 0173, AMAC, Inc.
Igrc (cat #A7164, Sigma Chem. Co., St. Louis, MO). Mouse y:
microtiter wells coated with goat a mouse IgG (cat #115-006-
071, Jackson Immuno Research, West Grove, PA). Mouse X:
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microtiter wells coated with rat MAb a mouse IgX (cat # 02171D,
Pharmingen, San Diego, CA) and developed with peroxidase
conjugated rabbit a mouse IgM(fc) (cat # 309-035-095, Jackson
Immuno Research, West Grove, PA). Bound peroxidase is detected
by incubation with hydrogen peroxide and 2,2'-Azino-bis-)3-
Ethylbenzthiazoline-6-Sulfonic Acid, Sigma Chem. Co., St.
Louis, MO). The reaction product is measured by absorption at
415 nm.
The double transgenic mice also express fully
human antibodies in the serum. Figure 70 shows measured serum
levels of immunoglobulin proteins for 18 individual double
transgenic mice, homozygous for endogenous heavy and kappa
light chain inactivations, derived from several different
transgenic founder animals. We found detectable levels of
human p, yl, and K. We have shown supra that the expressed
human yl results from authentic class switching by genomic
recombination between the transgene and yl switch regions.
Furthermore, we have found that intra-transgene class switching
was accompanied by somatic mutation of the heavy chain variable
regions. In addition to human immunoglobulins, we also found
mouse y and X in the serum. The present of mouse X protein is
expected because the endogenous locus is completely intact. We
have shown elsewhere that the mouse y expression is a
consequence of trans-switch recombination of transgene VDJ
segments into the endogenous heavy chain locus. This trans-
switching phenomenon, which was originally demonstrated for
wild-type heavy chain alleles and rearranged VDJ transgenes
(Durdik et al. (1989), Proc. Natl. Acad. Sci. USA 86:2346-2350;
Gerstein et al. (1990), Cell 63:537-548), occurs in the mutant
JHD background because the downstream heavy chain constant
regions and their respective switch elements are still intact.
ti The serum concentration of human IgMi in the
double transgenic mice was approximately 0.1 mg/ml, with very
little deviation between animals or between lines. However,
human yl, mouse -y, and mouse X levels range from 0.1 to
10 micrograms/ml. The observed variation in y levels between
individual animals may be a consequence of the fact that y is
an inducible constant region. Expression presumably depends on
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factors such as the health of the animal, exposure to antigens,
and possibly MHC type. The mouse X serum levels are the only
parameter that appears to correlate with individual transgenic
lines. KCo4 line 4436 mice which have the fewest number of
copies of the transgene per integration (approximately 1-2
copies) have the highest endogenous X levels, while KCo4 line
4437 mice ("10 copies per integration) have the lowest A
levels. This is consistent with a model in which endogenous X
rearranges subsequent to the K transgene, and in which the
serum X level is not selected for, but is instead a reflection
of the relative size of the precursor B cell pool. Transgene
loci containing multiple light chain inserts may have the
opportunity to undergo more than one V to J recombination
event, with an increased probability that one of them will be
functional. Thus high copy lines will have a smaller pool of
potential X cells.
Immunizations with human.CD4 and IgE
To test the ability of the transgenic B cells to
participate in an immune response, we immunized double
transgenic mice with human protein antigens, and measured serum
levels of antigen specific immunoglobulins by ELISA. Mice were
immunized with 50 g recombinant sCD4 (cat. # 013101, American
Bio-Technologies Inc., Cambridge, MA) covalently linked to
polystyrene beads (cat # 08226, Polysciences Inc., Warrington,
PA) in complete Freund's adjuvant by intraperitoneal injection.
Each of the mice are homozygous for disruptions of the
endogenous and K loci, and hemizygous for the human heavy
chain transgene HC2 line 2500 and human K light chain transgene
KCo4 line 4437.
Methods
Serum samples were diluted into microtiter wells
coated with recombinant sCD4. Human antibodies were detected
with peroxidase conjugated rabbit a human IgM(fc) (Jackson
Immuno Research, West Grove, PA) or peroxidase conjugated goat
anti-human IgK (Sigma, St. Louis, MO).
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Figure 71A shows the primary response of
transgenic mice immunized with recombinant human soluble CD4.
All four of the immunized animals show an antigen-specific
human IgM response at one week. The CD4-specific serum
antibodies comprise both human g heavy chain and human K light
chain.
To evaluate the ability of the HC2 transgene to
participate in a secondary response, we hyperimmunized the
transgenic mice by repeated injection with antigen, and
monitored the heavy chain isotype of the induced antibodies.
Mice homozygous for the human heavy chain transgene HC2 and
human K light chain transgene KCo4 were immunized with 25 Ag of
human IgEK (The Binding Site, Birmingham, UK) in complete
Freund's adjuvant on day = 0. Thereafter, animals were
injected with IgEK in incomplete Freund's adjuvant at
approximately weekly intervals. Serum samples were diluted
1:10, and antigen-specific ELISAs were performed on human IgE,
coated plates.
Figure 71B shows a typical time course of the
immune response from these animals: we injected double
transgenic mice with human IgE in complete Freund's adjuvant,
followed by weekly boosts of IgE in incomplete Freund's
adjuvant. The initial human antibody response was IgMK,
followed by the appearance of antigen specific human IgGK. The
induced serum antibodies in these mice showed no cross-
reactivity to human IgM or BSA. The development, over time, of
a human IgG
We have also tested the ability of the heavy
chain transgene to undergo class switching in vitro: splenic B
cells purified form animals hemizygous for the same heavy chain
construct (HC2, line 2550) switch from human IgM to human IgGl
in the presence of LPS and recombinant mouse IL-4. However, in
vitro switching did not take place in the presence of LPS and
recombinant mouse IL-2, or LPS alone.
In a transgenic mouse immunized with human CD4,
human IgG reactivity to the CD4 antigen was detectable at serum
concentrations ranging from 2 x 10-2 to 1.6 x 10-4.
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Identification of Anti-Human CD4 Hybridomas
A transgenic mouse homozygous for the human heavy
chain transgene HC2 and human K light chain transgene KCo4 were
immunized with 20 Ag of recombinant human CD4 in complete
Freund's adjuvant on day 0. Thereafter, animals were injected
with CD4 in incomplete Freund's adjuvant at approximately
weekly intervals. Fig. 73 shows human antibody response to
human CD4 in serum of the transgenic mouse. Serum samples were
diluted 1:50, and antigen-specific ELISAs were performed on
human CD4 coated plates. Each line represents individual
sample determinations. Solid circles represent IgM, open
squares represent IgG.
A mouse of line #7494 (0012;HC1-
26+;JHD++;JKD++;KC2-1610++) was immunized on days 0, 13, 20,
28, 33, and 47 with human CD4, and produced anti-human CD4
antibodies comprised of human K and human or y.
By day 28, human and human K were found present
in the serum. By day 47, the serum response against human CD4
comprised both human A and human y, as well as human K. On day
50, splenocytes were fused with P3X63-Ag8.653 mouse myeloma
cells and cultured. Forty-four out of 700 wells (6.3%)
contained human y and/or K anti-human CD4 monoclonal
antibodies. Three of these wells were confirmed to contain
human y anti-CD4 monoclonal antibodies, but lacked human K
chains (presumably expressing mouse X). Nine of the primary
wells contained fully human IgMK anti-CD4 monoclonal
antibodies, and were selected for further characterization.
One such hybridoma expressing fully human IgMK anti-CD4
monoclonal antibodies was designated 2C11-8.
Primary hybridomas were cloned by limiting
dilution and assessed for secretion of human A and K monoclonal
antibodies reactive against CD4. Five of the nine hybridomas
remained positive in the CD4 ELISA. The specificity of these
human IgMK monoclonal antibodies for human CD4 was demonstrated
by their lack of reactivity with other antigens including
ovalbumin, bovine serum albumin, human serum albumin, keyhole
limpet hemacyanin, and carcinoembryonic antigen. To determine
whether these monoclonal antibodies could recognize CD4 on the
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surface of cells (i.e., native CD4), supernatants from these
five clones were also tested for reactivity with a CD4+ T cell
line, Sup T1. Four of the five human IgMK monoclonal
antibodies reacted with these CD4+ cells. To further confirm
the specificity of these IgMK monoclonal antibodies, freshly
isolated human peripheral blood lymphocytes (PBL) were stained
with these antibodies. Supernatants from clones derived from
four of the five primary hybrids bound only to CD4+ lymphocytes
and not to CD8+ lymphocytes (Figure 72).
Fig. 72 shows reactivity of IgMK anti-CD4
monoclonal antibody with human PBL. Human PBL were incubated
with supernatant from each clone or with an isotype matched
negative control monoclonal antibody, followed by either a
mouse anti-human CD4 monoclonal antibody conjugated to PE (top
row) or a mouse anti-human CD8 Ab conjugated to FITC (bottom
row). Any bound human IgMK was detected with a mouse anti-
human conjugated to FITC or to PE, respectively.
Representative results for one of the clones, 2C11-8 (right
side) and for the control IgMK (left side) are shown. As
expected, the negative control IgMK did not react with T cells
and the goat anti-human g reacted with approximately 10% of
PBL, which were presumably human B cells.
Good growth and high levels of IgMK anti-CD4
monoclonal antibody production are important factors in
choosing a clonal hybridoma cell line for development. Data
from one of the hybridomas, 2C11-8, shows that up to 5
pg/cell/d can be produced (Figure 74). Similar results were
seen with a second clone. As is commonly observed, production
increases dramatically as cells enter stationary phase growth.
Fig. 74 shows cell growth and human IgMK anti-CD4 monoclonal
antibody secretion in small scale cultures. Replicate cultures
were seeded at 2x105 cells/ml in a total volume of 2 ml. Every
twenty-four hours thereafter for four days, cultures were
harvested. Cell growth was determined by counting viable cells
and IgMK production was quantitated by an ELISA for total human
(top panel). The production per cell per day was calculated
by dividing the amount of IgMK by the cell number (bottom
panel).
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Fig. 75 shows epitope mapping of a human IgMK
anti-CD4 monoclonal antibody. Competition binding flow
cytometric experiment's were used to localize the epitope
recognized by the IgMK anti-CD4 monoclonal antibody, 2011-8.
For these studies, the mouse anti-CD4 monoclonal antibodies,
Leu3a and RPA-T4, which bind to unique, nonoverlapping epitopes
on CD4 were used. PE fluorescence of CD4+ cells preincubated
with decreasing concentrations of either RPA-TA or Leu-3a
followed by staining with 2C11-8 detected with PE-conjugated
goat anti-human IgM. 1There was concentration-dependent
competition for the binding of the human IgMK anti-CD4
monoclonal antibody 2C11-8 by Leu3a but not by RPA-T4 (Figure
75). Thus, the epitope recognized by 2C11-8 was similar to or
identical with that recognized by monoclonal antibody Leu3a,
but distinct from that recognized by RPA-T4.
In summary, we have produced several hybridoma
clones that secrete human IgMK monoclonal antibodies that
specifically react with native human CD4 and can be used to
discriminate human PBLs into CD4+ and CD4- subpopulations. At
least one of these antibodies binds at or near the epitope
defined by monoclonal antibody Leu3a. Monoclonal antibodies
directed to this epitope have been shown to inhibit a mixed
leukocyte response (Engleman et al., J. Exp. Med. (1981)
153:193). A chimeric version of monoclonal antibody Leu3a has
shown some clinical efficacy in patients with mycosis fungoides
(Knox et al. (1991) Blood 77:20).
The association and dissociation rates of the
immunizing human CD4 antigen for the monoclonal antibodies
secreted by two of the hybridomas, 4E4.2 and 2C5.1, were
determined. The experimentally-derived binding constants (Ka)
were approximately 9 x 107 M-1 and 8 x 107 M-1 for antibodies
4E4.2 and 2C5.1, respectively. These Ka values fall within the
range of murine IgG anti-human CD4 antibodies that have been
used in clinical trials by others (Chen et al. (1993) Int.
Immunol. 6. 647).
The foregoing description of the preferred
embodiments of the present invention has been presented for
CA 02161351 2003-10-23
203
purposes of illustration and description. They are not
intended to be exhaustive or to limit the invention to the
precise form disclosed, and many modifications ancirvariations
are possible in light of the above teaching. It will be
apparent that certain changes and modifications may be
practiced within the scope of the claims.
15
