Note: Descriptions are shown in the official language in which they were submitted.
WO 93/12227 PCT/US92/10983
2124967
Transgenic non-human animals capable of producing heterologous antibodies
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 inactivating or suppressing
expression.of endogenousimmunoglobulin loci, methods and
transgenes for producing heterologous antibodies wherein a
variable regicn sequence comprises somatic mutation as
compared to germline rearranged variable region sequences, and
transgenic nonhuman animals which produce antibodies having a
human primary sequence and which bind to human antigens.
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
immunocompetent-human patients are administere'd therapeut'ic
doses of rodent monoclonal antibodies, the patients produce
antibodies against the rodent immunoglobulin sequences; these
human anti-mouse antibodies (HAMA) neutralize the therapeutic 35 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
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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 sourceof 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 Oevelopmentpathway,. Such maturation requires the
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
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
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required for B cell maturation to a subsequent-isotype that
has superior therapeutic utility.
P. 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., 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
jBuchini, 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.
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 Fc or Fc=y, 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
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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
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
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.
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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.
5 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
admissionthatthe inventors are not entitled to antedate such
disclosure by virtue of prior invention.
SUMMARY OF THE INVENTION
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
isotypes, including: IgGi, IgG2, IgG3, IgG4, IgM, IgAl, IgA2,
IgABeC, IgD, of IgE. Inlorder 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
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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 s
(secreted ) and M (membrane-bound g) forms, and the and b'
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 nonhumar. :nimals harboring such
transgenes, wherein the transgenic animal is capable of
producing heterologous antibodies of multiple isotypes by
undergoing isotype switching. Classical isotype switching
occursby 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 (d-
associated deletion). Alternative non-classical switching
mechanisms, such as intertransgene and/or interchromosomal
recombination, among others, may occur and effectuate isotype
switchir.3. 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
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
encoded byhuman 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
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immortalized to provide a source of a monoclonal antibodv
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 o= 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.
This invention further provides methods that
facilitate isotype, switching of the transgene, so that
switching between particular isotypes may occur at much'high'er
or lower frequencies or in different temporal orders than
typically-occurs in germline immunoglobulin loci. Switch
regions may be grafted from various C. 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
~j. . . .,.... . . . ,
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associated switch regions. Alternatively, or in combination
with switch sequences, 6-associated deletion sequences may be
linked to various C. 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 methocs for producing immunoglobulin transgene
constructs, some of wt:.ch contain a subset of germline
immunoglobulin loci sequences (which may include deletions).
The invention includes a s;-ecific method for facilitated
cloning and constri.iction of immunoglobulin transgenes,
involving a vectozthat employs unique XhoI and SalI
restriction sites flanked by two unique NotI sites. This
method exploits the complementa-y termini of XhoI and SalI
restrictions sites and is useful for creating large constructs
by ordered concatemerization of restriction fragments in a
vector.
The transgenes of the invention incl;ude a heavy
chain transgene comprising DNA encoding at least one variable
gene segment, one diversity.gene segment, one joining gene
seament and one constant region gene segment. The
immunoqlobulin light-chain transgene comprises DNA encoding at
least one variable gene segment, one joining gene segment and
one constant region gene segment. The gene segments encoding
the light and heavy chin' gene segments are heterologous !to
the transgenic non-human animal in t'-3t they are derived f'rom,
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
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unrearranged transgenes permit recombination ofthe 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 gen.e segments may be combined in a transgene
with arodent 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-ce11.
Also included in the invention are vectors'and
methods to disrupt the endogenous immunoglobulin loci in the
non-human aniinal'to'b'e used''i'n,the invention. Such vectors ~
and methods utilize a transgene, preferably positive-negative
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
segments. In this aspect of the invention, the
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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
5 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 chrotnosomal DNA. Such immune deficient non-human animals
10 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 immunc. tobulin locus. Such methods are useful for
suppressing expre,-aion of one or more endogenous
immunoglobulin chains while permitting the expression of one
ormore 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 Iggene 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
antisense RNA that specifically hybridizes to an endogenous Ig
chain gene sequence, (2) antisense oligonucleatides that
specif ically . hybridiie to an , endogenous Ig ch&in gene
sequence, and (3) immunoglobulins that bind specifically to a.,
endogenous Ig chain polypeptide.
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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Various embodiments of this invention pr vide an immunoglobulin (Ig)
light chain transgene construct comprising DNA sequen es that encode human
variable
(V), joining (J) and constant (C) regions of a human Ig p otein, which
sequences are
operably linked to transcription regulatory sequences and capable of
undergoing gene
rearrangement in vivo, when integrated in a transgenic aftimal, to produce a
rearranged gene encoding a human light chain polypepti4e, wherein the
construct
comprises a human 3' kappa (K) enhancer and at least one of each of the
following: a
functional variable VK gene segment, a functional joining JK region gene
segment and, a
functional constant CK region gene segment.
Various embodiments of this invention provide a method for generating a
plurality of B cells expressing heterologous antibody sequences, the method
comprising:
providing a transgenic mouse comprising the aforementioned
immunoglobulin light chain transgene and immunizing the transgenic non-human
mammal to generate B cells producing a population of heterologous antibodies.
The
transgene may be expressed in B cells of the transgenic non-human animal
containing at
least one integrated copy of a polynucleotide comprising a DNA sequence of the
formula:
(VH)x-(D)y-(JH)z-(SD)m-(Cl)n-((T)-(SA)p-(C2)]q, wherein x, y, z, m, n, p, and
q are
integers, and x is 2-100, n is 1-10, y is 2-50, p is 1-10, z is 1-50, q is 1-
50, and m is 0-10.
Various embodiments of this invention provide a method for generating
hybridomas, said method comprising the aforementioned method for generating a
plurality of B cells expressing human antibody sequences, and further
comprising fusing
the B cells producing a population of heterologous antibodies with
immortalized:cells to
form hybridomas.
Various embodiments of this invention provide a method of generating
antigen-specific hybridomas secreting heterologous antibody, the method
comprising:
immunizing a transgenic mouse comprising the aforemehtioned
immunoglobulin light chain transgene with a predetermined antigen; fusing
lymphocytes
from the transgenic mouse with immortalized cells to fonn hybridoma
cells; and determining binding of the heterologous antibody produced by the
hybridoma
cells to the predetermined antigen.
Various embodiments of this invention provide a method for generating a
human sequence antibody that binds to a predetermined antigen, the method
comprising
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10b
the following steps: immunizing a transgenic mouse com~rising the
aforementioned immunoglobulin light chain transgene with a predetermined
antigen;
generating hybridomas using the aforementioned method for generating
hybridomas and
screening hybridoma cells formed for the presence of antigen reactive
antibodies.
Various embodiments of this invention provide a method for producing
rearranged immunoglobulin light chain sequences comprising: providing a
transgenic
mouse comprising the aforementioned immunoglobulin 4ht chain
transgene, and obtaining rearranged immunoglobulin light chain sequences from
the
transgenic mouse.
Various embodiments of this invention provide isolated nucleic acids
encoding light chain variable region sequences produced according to this
invention,
including vectors comprising such nucleic acids and host cells comprising such
nucleic
acids or vectors.
Various embodiments of this invention provide a method for producing
rearranged immunoglobulin sequences, comprising: culturing the host cell of
this
invention under conditions such that the nucleic acid is expressed; and
recovering the
nucleic acid from the cultured host cell or its cultured medium.
Various embodiments of this invention provide a hybridoma comprising a
transgenic mouse B cell fused with a second cell capable of immortalizing said
B
cell, wherein said hybridoma produces a monoclonal antibody having heavy and
light
chains heterologous to said mouse B cell.
Various embodiments of this invention provide a transgenic mouse B
cell comprising an Ig light chain transgene construct according to this
invention.
r~ , . . , . . . .. ._ . .. :;,._ , ..;
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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 yl constant regions followed by a 700 bp fragment
containing the rat chain 3' enhancer sequence.
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 pGPl.
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 C71 fragments which are
in'serted 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. 15depictsthefragments covering the human'Jrc=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.
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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. 20 a through e depict the structure of a kappa
light chain targeting vector.
Figs. 21 a through f 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,
pHCI and pHC2.
Fig. 26 depicts the structure of p7e2.
Fig. 27 depicts tl~e structure of pVGE1.
Fig. 28 depicts tne assay results of human Ig
expression in a pHCi transgenic mouse.
Fig,. 29 depicts the structure of pJCK1.
Fig. 30 depicts the construction of a synthetic
heavy chain variable region.
Fig: 31 isa schematic representation of the-heavy
chain minilocus constructs pIGMl, pHCl, and pHC2.
Fig. 32 is a schematic representation of the heavy
chain minilocus construct pIGGl 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
.pBCEl.
Fig. 37 depicts 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 chains; each panel shows reciprocal
PCT/US92/10983
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13
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 reciproc&l
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
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 th conventional single-letter
notation.
Fig, 41 shows the data from Fig. 40 in histogram
format; deduced amino acid residue position is shown as the
ordinate (left is the amino-terminal direction, right is in
the direction towards the carboxy-terminus) and frequency of
sequence variation is shown as the abscissa.
Fig.,42 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. 43. show ithe,,nucleotide sequence of a human DNA
fragment, designated vk65.5, containing a V. gene segment; the
deduced amino acid sequences of the Vx 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 vk65.8, containing a V. gene segment; the
deduced amino acid sequences of the V. coding regions are also
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14
shown; splicing and recombination signal sequences
(beptamer?nonamer) are shown boxed.
Fig. 45 show the nucleotide sequence of a human DNA
fragment, designated vk65.15, 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. 46 shows formation of a light chain minilocus
by homologous recombination between two overlapping fragments
which were co-inject, .
Table 1 depicts the sequence of vector pGPe.
Table 2 depicts the sequence of gene VH49.8.
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 dep. ts tne distribution of J segments
incorpo ed into pf?C1 transgene encoded transcripts to J
segment: aund in a:..lt human peripheral blood lymphocytes
(PBL).
Table 6 depicts the distr:.bution of D segments
incorporated into pHCl 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 pHCl transgenic
mouse and ir, uman PBL.
Table 8 depicts the predicted amino acid sequences
of the VDJregions from 30 clones analyzed from a pHC1
transgenic.
Table 9 shows transgenic mice of line 112 that were
used in the indicated e'xpeiiments; (+)'indicates the presence
of the respective transgene, (++) indicates that the animal is
homozygous for the JHD knockout transgene.
. ,.
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
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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
5 presented antigen. In order for an animal to make an iinmune
response, the antigen presented must be foreign and the animal
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
10 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).
15 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-
ceils for a hybridoma, such autoimmunization would be
considered unethical by contemporary standards.
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
PCT/US91/06185 filed August 28, 1991) which comprise essential
functional elements of the human heavy and light chain loci,
are employed' to prodtice a transgenic nonhuman,animal. Such a,
trarisgenic nonhuman animal will have the capacity to produce
immunoglobulin chains that are encoded by human immunoglobulin
genes, and additionally 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
WO 93/12227 PCT/US92/10983
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16
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.
pefinitions
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 anti;body"'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.
As used herein, a "heterohybrid antibody" refers to
an antibody having a light and heavy chains of different
WO 93/12227 PCT/US92/10983
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organismal origins. For example, an antibody having a human
heavy chain associated with a murine light chain is a
heterohybrid antibody.
As used herein, "isotype" refers to the antibody
class (e.g., IgM or IgGl) 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 CH gene encoding the
nonswitched isotype is typically the first CH 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 switch region, will be
5" (i.e., upstream) of the construct region tobe 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 sequencewill typically not be predictable from the
construct.
As usedherein, "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
producedby the specie~ ot'the nonhumantransgenic 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 CHgenes of the transgene were derived.
As used herein, "specific binding" refers to the
property of the antibody: (1) to bind to a predetermined
.. . . . ,. ..
WO 93/12227 PCT/US92/10983
18
antigen with an affinity of at least 1 x 10' M-1, and (2) to
preferentially bind to the predetermined antigen with an
affinity that is at least two-fold greater than its affinity
for binding t_- a r.on-specific antigen (e.g., BSA, casein)
other than the predetermined 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 bycomparison 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.
Transgenic Nonhuman Animals Capable
ofProducinc Heterologous Antibodies
The design of a transgenic non-human animal that
responds to foreign antigen stimulation with a heterologous
antibody.:repertoir##; 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
CA 02124967 2003-04-28
19
response to a].lelic exclusion, (4) expression of a sufficient
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: foregoi.ni3 criteria need be met. For example, in
those embodimE:nts wherein the endogenous immunoglobulin loci
of the transgE:nic animal are functionally disrupted, the
transgene neeci not activate allelic exclusion. Further, in
those embodimE:nts wherein the transgene comprises a
functionally rearranged heavy andJor light chain
immunoglobuliii gene, the second criteria of functional gene
rearrangement is unnecessary, at least for that transgene
which is aiready rearranged. For background on molecular
immunology, see, Fundamental ImmunoloQV, 2nd edition (1989),
Paul William 1::., 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
immunoglobuliin heavy and light chain transgenes in the
germline of the transgenic animal. Each of the heavy chain
transgenes comprises at least one CH 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 CH 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 C. genes, or such switch sequences may be derived
from those which occur in the species that is to receive the
transgene construct (the transgeneic 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 iri the mouse heavy chain locus, as
presumably the mouse switch sequences are optimized to
function with the mouse switch recombinase enzyme system,
CA 02124967 2003-04-28
whereas the human switch sequences are not. Switch sequences
made be isolated and cloned by conventional cloning methods,
or may be synthesized de novo from overlapping syntr.etic
oligonucleotides designed on the basis of published sequence
5 information relating to immunoglobulin switch region sequences
(Mills et al., Nucl. Acids Res. 18:7305-7316 (1991);
and Sideras et: al., Intl . Immunol. 1:631 : 642 (1.989) ).
For each of the foregoing transgenic animals,
10 functionally rearranged heterologous heavy and light chain
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
15 gene segment, one diversity gene segment, one joining gene
segment and at: least one constant region gene segment. The
immunoglobulir.i 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
20 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
immunoglobuli.n 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 chairi. Such unrearranged transgenes support
recombination of the V, D, and J gene segments (functional
rearrangement) and preferably support incorporation of all or
a portion of a D region gene segment in the resultant
rearranged immunoglobulin heavy chain within the transgenic
non-human aninaal 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,
:~.. ~.
WO 93/12227 PCT/US92/10983
19.1249 6 7
21
enhancers, class switch regions, splice-donor and splice-
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 maybe 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.
The invention also includes transgenic animals
containing germ line cells having a heavy and light transgene
wherein oneof 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 regi.on. 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
constant region gene (CH) segments. The use of a particular
heavy chain gene segment defines the class of immunoglobulin.
For example, in humans, the constant region gene segments
WO 93/1222" PCT/US92/10983
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22
define the IgM class of antibody whereas the use of a=y, y2,
y3 or 74 constant region gene segment defines the IgG class of
antibodies as well as the IgG subclasses IgGl through IgG4.
Similarly, the use of a al or a2 cons::.ant 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 d,1ersity (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
include recognizable D region sequences, more preferably at
least about 30 percent, and most preferably more than 50
percent include recognizable D region sequences.
WO 93/12227 PCr/US92/10983
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23
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 is recognizable as containing a D region sequence,
specifically a DHQ52 sequence.
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
side by an amino acid sequence encoded by a transgene J gene
sequence is recognizable as a D region sequence.
Therefore, because sofiatic 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)
WO 93/12227 PCT/US92/10983
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?1 24 9 6
24
and on the other side by a J gene sequence (nuc,leotide
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 isat 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
can result from a single nucleotide substitution; a first
amino acid is replaced by a second amino acid where a codon
for the first aminoacidand a codon for the.second amino acid
can differ by a single nucleotide substitution. Thus, for
example, the.sequence -Lys-Glu-Arg-Val- is suDstantially
similar to the sequence -Asn-Asp-Ser-Val-, since the codon
sequence -AAA-GAA-AGA-GUU- can be mutated to -AAC-GAC-AGC-GW-
by introducing only 3 substitution mutations, single
nucleotide substitutions in three of the four original codons.
The reference sequence may be a subset of a larger sequence,
WO 93/12227 PCT/US92/10983
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.
5
The Primary Repertoire
The process for generating DNA encoding the heavy
and light chain immunoglobulin genes occurs primarily in
10 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
15 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-celldifferentiation, one of each of the appropriate family
20 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
25 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 th3s initial form of a functional variable region in
a light and/or heavy chain is referred to as "functionally
rearranged DNA" or "rearranged DNA". In the case of the heavy
chain, such DNA is referred'toas "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
WO 93/12227 pCT/[JS92/10983
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26
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 preced2.:lg the JL, D or Jx
segments with which they recombine. The spacers betwecn the
heptameric and nonameric sequences are either 12 base pairs
long or between 22 and 24 base pairs long.
In additi-on 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 lightchain typically occurs within the last
codon of the V gene segment and the first codon of the J
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 andD 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
"'0 93/12227 pC'T/L;S92/10983
21,2 4 967
27
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 m.RNA 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
ailelic exclusion.
The Seeondary Repertoire
B-cell clones expressing immunoglobulins from within
the set of sequences comprising the primary repertoire are
immediately~available~to respond to foreign antigens. Because
of the limited diversity gerierated 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., Ce 1 59:1049-1059 (1989)).
The first type of B-cell matures into IgM-secreting plasma
CA 02124967 2003-04-28
28
cells in response to certain antigens. The other B-cells
respond to initial exposure to antigen by entering a T-cell
dependent maturation pathway.
Duririg the T-cell dependent maturation of antigen
stimula.ted B-ce:ll 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 thrqughout 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.
Seauences 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 refei-red to as framework regions designated FR1, FR2,
FR3 and FR4. See Figõ 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, giviing rise to affinity maturation of the expressed
antibody. Clonal selection typically results in expression of
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.
40
NN'O 93/12227 PCT/US92/10983
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29
Transgenic Non-Human Animals Capable
of Producing Heterologous 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.
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
segments, contain immunoglobulin DNA sequences which are also
WO 93/12227 PCT/L'S92/109$3
21?49fi7
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
5 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.
10 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
15 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.
Unrearranged Transgenes
20 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
25 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
30 at least,.one variable gene $egment, 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 =
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
WO 93/12227 PCT/US92/10983
20 12 ~9 6 7
31
an unrearranged form. Thus, interposed between the V, D and J
segments in the heavy chain transgene and between the V and J
seginents 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 andC-yi 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 presenceof 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., Intlj Immunol.
2,s621-627:(1990); Reid et al., Proc. Natl. Acad. Sci. USA
":840-844 ,~198=9) ;;;, ~Stavnezer, ~t al. Pxoc. Natl. Acad. Sci.
-85:7704-7708 (1988) ; and Mills et al.,Nucl. Acids Res.
JI:7305-7316 (1991), each of which is incorporated herein by
reference. These sequences typically include about at least
50 bp immediately upstream of a switch region, preferably
about at least 200 bpupstream 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, Sy21 Sy3, Sy4, Sa1, Sa2, and SE switch
WO 93/1222 i PCT/US92/10983
9 6
32
regions, although the sequences immediately upstream of the
human SY1, and S,,3 switch regions are preferable. In
particular, 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 ap ig-mediatedlmmune response to a specific antigen
administe'red 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.
WO 93/12227 PCT/US92/10983
21249,67
33
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 teachinqs 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 CH 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 withthe 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.
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
35. 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
, ,. ,.... r .
:. , õ _. .. ..
_.. : . _, ,.
WO 93/12227 pCT/US92/10983
MIJ4967
34
concatenated to form the synthetic immunoglc'-~lin V segment
repertoire. Such synthetic variable region ht.3vy chain
transgenes shall have at least two CH genes and the associated
sequer.ces required for isotype switc,ing.
Isotype Switching
In the development of a B lymphocyte, the cell
initially produces IgM with a binding specificity determined
by the productively rearranged VH 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 theisotype of the H chain.
The use of or 6 constant regions is largely
,determined by alternate splicing, permitting IgM and IgD to be
iG coexpressed in a single cell. The other heavy chain isotvpes
(y, a, and E) are only expressed natively after a gene
rizarrangement event deletes the C and Gd exons. This gene
rearrangement process, termed isotype switching, typically
occurs by recombination between so called switch segments
located immediately upstream of each heavychain gene (except
d). The individual switch segments are between 2 and 10 kb in
length, and consist primarily of short repeatedsequences.
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
(GAGCT)n(GGGGT), where-n is usuaily 2 to 5 but can.range as
high as 17. (Ieg T. Nikaido et al.Naturg M: 845-848 (1981) )
Similar internally repetitive switch sequences
'spanning several kilobases have beenfound 5' of tne other CH
qenes. The Sa region has been sequenced and found to consist
of tandemly repeated 80-bp homology units, whereas Sy2a, SY2b,
and Sy3 all contain repeated 49-bp homology units very similar
to each other. (See, P. Szurek et al., J. Immunol 135:620-626
CA 02124967 2003-04-28
(1985) and T. Nikaido et al., J. Biol. Chem. 257:7322-7329
(1982)). A11 the
sequenced S regions include numerous occurrences of the
pentamers GAGCT and GGGGT that are the basic repeated elements
5 of the SN gene: (T. Nikaido et al., J. Siol. Chem. 257:7322-
7329 (1982)); in the
other S regioris these pentamers are not precisely tandemly
repeated as in S., but instead are embedded in larger repeat
units. The S,,1 region has an additional higher-order
10 structure: two direct repeat sequences flank each of two
clusters of 49-bp tandem repeats. (See M. R. Mowatt et al.,
J. Immunol. -I:36:2674-2 683 (1986) ) .
Swii""ch regions of human H chain genes have been
15, found to be very similar to their mouse homologs. Indeed,
similarity between pairs of human and mouse clones 5' to the
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 and a genes
2(i produces a composite S~-Sa sequence. Typically, there is no
specific site, either in SP or in any other S region, where
the recombination always occurs.
Generally, unlike the enzymatic machinery of V-J
recombination, the switch machinery can apparently accommodate
25 different alignments of the repeated homologous regions of
germline S precursors and then join the sequences at different
positions within the alignment. (See, T. H. Rabbits et al.,
Nucleic .cids Res. 9:4509-4524 (1981) and J. Ravetch et al.,
Proc. Natl. Acad. Sci. USA 77:6734-6738 (1980))
3 ~0
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
35 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.
CA 02124967 2003-04-28
36
The T-cell-derived lymphokines IL-4 and IFNY have
been shown to specifically promote the expression of certain
isotypes: IL-4 decreases IgM, IgG2a, IgG2b, and IgG3
expression and increases IgE and IgGl expression; while IFNY
selectively stimulates IgG2a expression and antagonizes the
IL-4-induced increase in IgE and IgGl expression (Coffman et
al., J. Immunol. 136:949-954 (1986) and Snapper et al.,
Science 236:944-947 i(,..987) ) .
A combin..:iti.cn of IL-4 and IL-5 promotes IgA
expression (Coffman et al., J. Immunol. 139:3685-3690 (1987)
Most of the experiments implicating T-cell effects
on switchi,ng have not:.ruled out the possibility that the
observed increase in cells with particular switch
recombinatior.is might reflect selection of preswitched or
precommitted cells; but the most likely explanation is that
the lymphokirkes 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. Nat1.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. Im;u o. 2:621 (1990)).
For example, the observed induction of the 71
sterile transcript by IL-4 and inhibition by IFN-ry correlates
with the observation that IL-4 promotes class switching to -yi
in B-cells in culture, while IFN-7 inhibits 71 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
transcription of a particular sterile transcript typically can
be expected 'to enhance the frequency of isotype switch
recombination involving adjacent switch sequences.
:15 For these reasons, it is preferable that transgenes
incorporate transcriptional regulatory sequences withia about
1-2 kb upstream of each switch region that is to be utilized
for isotype switching., These transcriptional regulatory
WO 93/1222; PCT/US92J10983
.2i7
37
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
aerml;.ne 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., the 5' flanking sequence from
the human SYl switch can be grafted immediately upstream of the
Sal switch), 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.
The Transgenic Primary Repertoire
A. The Human Immunogiobulin Loci
An important requirement for transgene function is
20' 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
.=egion genes encode the constant portion of a different class
of immunoglobulins. During H-cell development, V region
proximal constant regions are deleted leading to the
expression of new heavy chain classes. For each heavy chain
class, alt6rnative patterns of RNA splicing give rise to both
transmembrane and secreted immunoglobulins.
The human heavy chain locus consists of
approximately 200 V qene segments spanning 2 Mb, approximately
30 D gene segments spanning about 40 kb, six J segments
clustered-within a 3 kbspan, 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.
CA 02124967 2003-04-28
38
B. Gene Fragment Transaenes
1. Heavy Chain Transgene
In a preferred embodiment, immunoglobulin heavy and
light chain t.ransgenes comprise unrearranged genomic DNA from
humans. In the case of the heavy chain, a preferred transgene
comprises a NotI fragment having a length between 670 to 830
kb. The length of this fragment is ambiguous because the 3'
restriction site has not been accurately mapped. It is known,
however, to reside between the al and ~a gene segments. This
110 fragment contains members of all six of the known VH famil-xes,
the D and J clene segments, as well as the u, 6, y3, 71 and a1
constant regions (Berman et al., EMBO J. 7.727-738i(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. Lialht 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.
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
:30 example, the 670-830 kb NotI fragment from the human
immunoglobul.in heavy chain locus may be formed in vivo in the
non-human ariimal during transgenesis. Such in vivo transgene
constructiori is produced by introducing two or more
overlapping DNA fragments into an embryonic nucleus of the
non-human ariimal. 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
WO 93/12227 PCT/US92/10983
19W124967
39
homologously recombined in proper orientation to form the
670-830 kb NotI heavy chain fragment.
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
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
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
Examples.
In the preferredembodiments 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
end portion of a second DNA fragment. Such overlapping
portions of the DNA fragments preferably comprise about 500 bp
to about 2000 bp, mostpreferably 1.0 kb to 2.0 kb.
Homologous recombination of overlapping DNA fragments to form
transgenes in vivo is further described in commonly assigned
PCT Publication No. WO 92/03917 entitled "Homologous
Recombination in Mammalian Cells" published March 19, 1992.
D. Mini'locus TrarYsQenes
As used herein, the term "imnnunoglobulin 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
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
WO 93/12227 PGT/1JS92/.10983
2; 1.94967
that said DNA sequence contains at least one substantial
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
5 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
1.0 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
15 minilocus comprising at least two CH genes and the requisite
switching sequences is typically capable of undergoing isotype
.switching, so that functional antibody molecules of differer:t
immunoglobulin classes will be generated. Such isotype
switching may occurin vivo in B-cells residing within the
20 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,
25 D, and JH 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
CH segments for the heavy chain transgene, it is preferred
that the transgene contain at least one gene segment and at
30 least one 'other con'stant region gene s'egment, laore preferably,
a7 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.
35 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
CA 02124967 2003-04-28
41
genes will be different from the naturally-occurring spatial
order found in the ger.mline of the species serving as the
donor of the i-H 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 switc:hing; the resultant 'transgenic nonhuman animal
may then, in some embodiments, make antibodies of various
1CI classes including all of the allotypes represented in the
species from which the transgene C. genes were obtained.
Still further=, those skilled in the art can select
CH genes fr.om 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
217 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
2.5 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 wit.tt the gene and the 5' portion of the 6 gene on a
single 23 kb SFiI/SpeI fragment (Sado et al., Biochem.
30 BioDhvs. Res., Comm. 1 4:264271 (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 and 6 genes, it is
likely to contain all of the 3' cis-linked regulatory elements
35 required for. 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
CA 02124967 2003-04-28
42
(1986) ) . It also
contains the transcription start sites which trigger VDJ
joining to form primary repertoire B-cells (Yancopoulos and
Alt, Cell 40:271-281 (1985)).
AlternatiNrely, a 36 kb BssHII/Spell fragment,
which includes part on the D region, may be used in place of
the 23 }:.b SfiI/SpeI1 fragment. The use of such a fragment
increases the. amount of 51 flanking sequence to facilitate
efficient D-t.o-J joining.
The human D region cctisists of 4 or 5 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 usijzg 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., 7,
4141-4150). Twelve D segments should be sufficient for a
primary repertoire. }iowever, given the dispersed nature of
the D region, an alternative strategy is to ligate together
several non-contiguous D-seqment 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 :solated as described PCT
Publication No. WO.92/03918, publisned March 19, 1992,
entitled "Transgenic Non-Human Animals Capable of Producing
Heterologous Antibodies."
WO 93/12227 PC't'/US92/10983
i4~
'~1- 2 4 9 6A' 43
Rearranged or unrearranged V segments, D segments, J
segments, and C genes, with or without flanking sequences, can
be isolated as described in PCT Publication No. WO 92/03918,
published March 19, 1992.
A minilocus light chain transgene may be sirailarly
constructed from the human X or x 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 sequerices
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.
E. Transgere Constructs Capable of Isotype Switchinq
Ideally, transgene constructs that are intended to
undergo class switching should include all of the cis-acting
sequencesnecessary 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 lqast;,500 to; .1,000 basep,,a,irs or more of sequence
'immediately upstream of a switch region, preferably a huiaan.ryl
switch region, should be operably linked to a switch sequence,
preferably a human 71 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
yl
region. For example, but not for limitation, a human
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.
WO 93/12227 PCr/US92/10983
24 9 6'7
44
An alternative method for obtaining non-classical
isotype switching (e.g., S-associated deletion) in transgenic
mice involves the inclusion of the 400 bp direct repeat
sequences (aA and E ) that flank the human gene (Yasui et
al., Eur. J. Immunol. 19:1399 (1989)). Homologous
recombination between these two sequences deletes the A gene
in IgD-only B-cells. Heavy chain transgenes can be
represented by the following formulaic description:
(VH)X-(D)y-(Jg)Z-(SD)m-(C1)n-I (Z') '(SA)p-(C2) I q
where:
VH is a heavy chain variable region gene segment,
D is a heavy chain D (diversity) region gene segment,
JH 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.,
or 8),
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. ,'yl, 72, y31 "Ya, a1,
Gt2 , E ) .
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 93/12227 9124967 PC.'T/[,'S92/10983
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,
5 but are preferably selected from V. segments that occur
naturally in the human germline, such as UH251= 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.
10 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
15 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, an.d further preferred embodiments include
six human JH segments and no nonhuman JH segments.
20 S. segments are donor regions capable of
participating in recombinatlon events with the SA segment of
the transgene. For .classicalisotype switching, SD and SA are
switch regions such as S SY1, Sy2, Sy3 f S-y4 f Scr , Sa2 , and SE .
Preferably the switch regions are murine or human, more
25 preferably SD is a human or murine S and SA is a human or
murine Sy1. For nonclassical isotype switching (d'-associated
deletion), SD and SA are preferably the 400 basepair direct
repeat sequences that flank the human gene.
Cl segments are typically or d genes, preferably a
30 gene, andi, mor'e p'referably '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
nucleotides in length, preferably about at least 200
35 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 93/12227 PCl'/US92/10983
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46
evident to those of skill in the art that T segments may
comprise cis-acting transcriptio~al 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 yl, 72, 73, y4, al, a2,
or E CH gene, preferably a human CH gene of these isotypes, and
more preferably a human -yl or ~y3 gene. Murine 72a 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 .:g locus. Thus, for example, deletion of
unnecessary regionT )r substitutions with corresponding
regions from other ~pecies will be present.
F. Methods for Determining Functional
Isotyne 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 6 and an adjacentsequence homologous to a transgene VH-
DH-JH rearranged gene; such detection may be by Northern
hybridization, Sl nuclease protection assays, PCR
ampfification; cDNAlcloning-; 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 -if the transgenic
animal or in genomic DNA from hybridom:~ .;-ells, of DNA
WO 93/12227 919~(C~~61 1/y PCT/US92/10983
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47
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.
G. Functional Disruption of
Endo.genous Immunocrlobu linLoci
The expression of successfully rearranged
immmunoglobu3in h'ea'ty andlight 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
i.mmundglobulin 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.
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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
(reviewedby Capecchi (1989), Science, 244, 1288-1292).
Because *he mouse X locus contributes to only 5% of
the immunoglobuli:.s, inactivation of the heavy chain and/or
x-ligr.t 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 introduc,:ion of a stop codon. The
last option is the most straight_=orward, in terms of DNA
construct design. E:.imination of the 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 re genes, targeting
vectors are used based,on the.design employed by Jaenisch and
co-workers (Zijlstra, et al.-(1989), Nature, 342, 435-438) for
the successful disruption of the mouse 02-microglobulin gene.
The neomycin resistance gene (neo), from the plasmid pMCIneo
is inserted into the coding region of the target gene. The
35, pMCIneo 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
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49
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
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 pMCIneo. The HSV tk gene
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
sequences 5' of the KpnI site in the D region.
Alternatively, the heavy-chain locus is knocked out
by disrupting the codingregion 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
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.
Theseare detected from pools of selected clones by PCR
amplification. One of the PCR primers is derived from neo
sequences and the other from mouse sequences outside of the
targetix-g vector. The functional disruption of the mouse
immunoglobulin loci is presented in the Examples.
G. Suppressing Expression of
EndogenQus 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
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.
CA 02124967 2003-04-28
Antisense Polynucleotides
Aritisense 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,
5 J. (1990) Biochimi.ca 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).
"wAntisense polynucleotides" are polynucleotides
10 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
complemerLta:cy target sequence, such as a chromosomal gene
locus-or a Ig mRNA. Such complementary antisense
15 polynucleot:ides 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
20 oligonucleo-tides 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. 8~:10006-10010 (1989); Broder et al., Ann. Int. Med.
25 =:604-618 (1990); Loreau et al., FEBS Letters 274:53-56
(1990); Holcenberg et al., W091/11535; W091/09865; W091/04753;
W090/13641; and EP 386563).
An antisense sequence is a
polynuclaot.ide sequence that is complementary to at least one
30 immunogloba.lin genE equence 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, in some embodiments, antisense sequences may
have substitutions, additions, or deletions as compared to the
35 complementary immunoglobulin gene sequence, so long as
specific hybridization is retained as a property of the
antisense polynucleotide. Generally, an antisense sequence is
complementILry to an endogenous immunoglobulin gene sequence
WO 93/12227 PCT/US92/10983
2124967
51
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. (1995) Science ~5 :;; 149.7) . For general methods
relating to antisense polynucleotides, see Antisense RNA and
(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
CA 02124967 2003-04-28
52
provide a therapeutic function by inhibiting the formation of
one or more endogenows ig chains in vivo.
Whether as soluble antiser.se oligonucleotides or as
antisense RN)- transcribed from ari antisense transgene, the
'5 antisense polynucleotides of this invention are selected so as
to hybridize preferentially to endogenous Ig sequences at
physiologica], 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).
Antiserum Suppression
Pa:=tia1 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. Nat7:.. Acad. Sci.. (U.S.A.) 81 211.
Antisera are selected so
as to react specifically with one or more endogenous 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. og=cit. will suppress
endogenous Ig chain expression but permits expression of
heterologous Ig chain(s) encoded by a transgene of the present
invention.
Nucleic Acidis
The nucleic acids, the term "substantial homology"
indicates*that two nucleic acids, or designated sequences
thereof, whE:n 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 segment:; will hybridize under selective hybridization
conditions, to the complement of the strand. The nucleic
4=.). .. . .. . . . . . . . . . . . : .. . .. . : :". . . . . .
WO 93/12227 PCT/US92/10983
53
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
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. F'or;~switch~sequences;; operably linked indicates that
. , ;
the sequences are capable of effecting switch recombination.
Sgegif ic Preferred Embodiments
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,
WO 93/12227 PCT/US92/10983
4 9 6 7 54
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 hemizyc7ous 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 J. 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 ef the single copy of the rearranged
human K light chain gene wiil allelically and isotypicaily
exclude the rearrangement of the endogenous mouse K and A
chain genes in a signif icant fraction of B-cells.
Toe transgenic mwouse= of the, preferred embodiment
'wi1l exhibit immunoglobulin production with a significant=
repertoire, ideally substantially similar to that of a native
mouse. Thus, for example, in embodiments where the endogenous
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/mi.
When a transgene capable of effecting a switch to IgG from IgM
has been introduced into the transgenic mouse, the adult mouse
WO 93112227
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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
5 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
10 104 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
15 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
20 107M-1, preferably 108M-1 to 109M-1 or greater.
Thus, prior to rearrangement of a transgene
containingvarious 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
25 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.
30 Fourcategories of transgenic animal may be defined:
I. Transgenic animal's containing an unrearranged heavy
and rearranged light immunoglobulin transgene.
II. Transgenic animals containing an unrearranged heavy
and unrearranged light immunoglobulin transgene
35 III. Transgenic animal containing rearranged heavy and an
unrearranged light immunoglobulin transgene, and
IV. Transgenic animals containing rearranged heavy and
rearranged light immunoglobulin transgenes.
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56
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 I+IATERIALS
Transgenic mice are derived according to Hogan, et
al., "Manipu.lating the Mouse Embryo: A Laboratory Manual",
Cold Spring :Harbor Laboratory.
Jl5 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 Schwartz,bera et al.. Science 246:799-803 (1989)).
DNA cloning procedures are carried out according to
J. Sambrook,, et al. in Molecular Cloning: A Laboratory
Manual, 2d i.d., 1989, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.
Oligonucleotides are synthesized on an Applied Bio
Systems oligonucleotide synthesizer according to
specifications provided by the manufacturer.
Hybridoma cells and antibodies are manipulated
according to "Antibodies: A Laboratory Manual", Ed Harlow and
David Lane, Cold Spring Harbor Laboratory (1988).
EXAMPLE 1
Genomic Heavy Chain Human Ig Transaene
This Example describes the cloning and
microinjection of a human genomic heavy chain immunoglobulin
transgene iahich is microinjected into a murine zygote.
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5/
Nuclei are isolated from fresh human 'placental
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
EDTA and proteinase K to expose high molecular weight DNA,
which is then digested in the agarose with the restriction
enzyme NotI as described by M. Finney in Current Protocols in
3_0 Molecular Biology (F. Ausubel, et al., eds. John Wiley & Sons,
Supp. 4, 1988, Section 2.5.1).
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.
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
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 51 - AAT TGC GGC CGC - 3'.
YAC clones containing the heavy chain NotI fragment
are isolated as described by Brownstein et al., Science
3D ZAA:1348-1351 (1989), and Green et al., Proc. Natl. Acad. Sci.
.PU.87:1213-1217 (1990).
The cloned Notl 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.
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EXAMPLE 2
Genomic x Light Chain Human Ig Transgene
Formed by In Vivo Homologous RecomAination
A map of the human K light chain has been described
in Lorenz et al., Nuci. Acids Res. 15:9667-9677 (1987),
A 450 kb XhoI to NotI fragment that includes all of
Cx, the 3' enhancer, all J segments, and at least five
different V segments is isolated and microinjected 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 M1uI 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 Notl fragment plus the
approximately 400 kb M1uI to BssHII fragment have sequence
overlap defined by the BssHII and XhoI restriction sites.
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 Xhol/NotI fragment (Example 2).
EXAMPLE 4
Construction of Heavy Chain Mini-Locus
A. Construction of vGP1 and pGP2
pBR322. is digested with EcoRI and StyI 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.
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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 - 31
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 qan be
isolated from vector sequencesfor 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, pGPl contains a strong transcription
terminator sequence derived from trpA (Christie et al., oc.
Nat1: A;pad. Sci. USA 78:4180 (1981)) inserted between the
ampicillin resistance gene and the polylinker. This further
reduces the toxicity associated with certain inserts by
preventing readthrough transcription coming from the
ampicillin promoters.
Plasmid pGP2 is derived from pGP1 to introduce an
additional restriction site (Sfil) in the polylinker. pGPl is
digested with MluI 'anct Sp"eI o 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'
51 CTA GTG GCC ATT GCG GCC A 3'
,. ;_._. -.- .,.... ..
... . , .. ,
.... . , . .. , . ,::: - ._.
CA 02124967 2003-04-28
pGP2 is identical to pGPl except that it contains an
additional Sfi I site located between the M1uI and Spel sites.
This allows inserts to be completely e-cised with Sfil as well
as with NotI.
5
B. Construction of nRE3 (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 bv
10 Petterson et al., Natur 344:165-168 (1990)
is isolated and cloned. The
rat IGH 3' enhancer sequence is PCR amplified by using the
following.oligonucleotides:
15 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
20 cut pGP2 to yield pRE3 (rat enhancer 3').
C. Cloning of Human J-u Recion
A substantial portion of this region is cloned by
combining two or more fragments isolated from phage lambda
25 inserts. See Fig. 9<,
A 6.3 kb BamHI/HindIZl fragment that includes all
human J segments (Matsuda et al., EMBO J., 7:1047-1051 (1988);
Ravetech et al.m CelY, 27:583-591 (1981)
is isolated from human
30 genomic DNA library using the oligonucleotide GGA CTG TGT CCC
TGT GTG ATG CTT TTG ATG TCT GGG GCC AAG.
An adjacent 10 kb HindIII/BamiI fragment that
contains enhancer, switch and constant region coding exons
(Yasui et al., Eur. J. Immunol. 19:1399-1403 (1989)) is
35 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
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EcoRI/HindIII 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.
pGP1 is digested with BamHI and BglII 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 HindilI digest. The resultant
plasmid pHIG1 (Fig. 10) contains an 18 kb insert encoding J
and C segments.
D. Cloning of Cu Region
pGP1 is digested with BamHI and HindIII 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/HindIIl cut pGPl. Proper
orientation of fragment (c) is checked by HindIIl digestion to
form pCONi containing a 12 kb insert encoding the CA region.
Whereas pHIGi contains J segments, switch and
sequences in its 18 kb insert with an Sf iI 3' site and a Spel
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
rearranged VDJ segments will be described hereinafter.
E. Cloning of 7-1 Constanp Region (pREG2)
The cloning of the human 7-1 region is depicted in
Fig. 16.
Yamamura et al., Proc. Natl. Acad. Sci. USA
&2:2152-2156 (1986) reported the expression of inembrane bound
human -y-1 from a transgene construct that had been partially
deleted on integration. Their results indicate that the 3'
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BamHI site delineates a sequence that includes the
transmembrane rearranged and switched copy of the gamma gene
with a V-C intron of le~s than 5kb. Therefore, in the
unrearranged, unswitchea gene, the entire switch region is
included in a sequence beginning less than 5 kb from the 5'
end of the first y-1 constant exon. Therefore it is included
in the 5' 5.3 kb HindIII fragment (Ellison et al., Nucleic
Acids Res. Jg:4071-4079 (1982)).
Takahashi et al., Cell 29: 671-679 (1982),
11D also reports that
this fragment contains the switch sequence, and this fragment
together with the 7.7 kb HindIII 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 specif-.ed gene.
Phage clones containing the 7-1 region are
identified and isolated using the following oligonucleotide
which is specific for the third exon of y-Z (CH3).
5' TGA GCC ACG AAG ACC CTG AGG
TCA AGT TCA ACT GGT ACG TGG 3'
A 7.7 kb HindIII to BglII fragment (fragment (a) in
25, Fig. 11) is cloned into HindIII/BglII cut pRE3 to form pREG1.
The upstream 5.3 kb HindIII fragment (fragment (b) in Fig. 11)
is cloned into Hindii:C digested pREG1 to form pREG2. Correct
orientation is confirmed by BamHI/SpeI digestion.
F. Combininv CY and Cu
- The previously described plasmid pHIG1 contains
human J segments and the C constant region exons. To provide
a transgene containing the C constant region gene segments,
pHIGl was digested with Sfii (Fig. 10). The plasmid pi.EG2 was
also digested with Sfi.i to produce a 13.5 kb insert containing
human C-y 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
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region, the human C-yl constant region and the rat 3' enhancer
contained on a 31.5 kb insert.
A second plasmid encoding human C and human C71
without J segments is constructed by digesting pCON1 with Sfii
and combining that with the SfiI fragment containing the human
Cry region and the rat 3' enhancer by digesting pREG2 with
Sfil. The resultant plasmid, pCON (Fig. 12) contains a 26 kb
Noti/Spel insert containing human C , human =yl 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., IIKBO J. 7:4141-4150 (1988)). The following
oligonucleotides are used:
DXP1: 5' s 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
GCA CCT GAC ACC - 3'
A 5.2 kb XhoI fragment (fragment (b) in Fig. 13)
containing DLR1, DXP1, DXP'l, and DA1 is isolated from a phage
clone identified w.ith 9ligo,pXP1.
A'3.2 kb XbaI fragment= (fragment (cj 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 Xbal/XhoI site of pGPl 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
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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 ir.sert in the proper orientation as
determined by BamHI digestion. The 3.2 kb Xbal 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' SpeI site. The entire
polylinker is flanked by NotI sites.
H. Construction of Heavv Chain Minilocus
The follow:ing 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. 11:1511-1518 (1988),
is isolated using the
following oligonucleotide:
5' - GAT CCT GGT TTA GTT AAA GAG GAT TTT
ATT CAC CCC TGT GTC - 3'
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 SfiI 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
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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
5 cyl 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 pGP1 to produce pHIG which contains
insert encoding a V segment, nine D segments, six functional J
10 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
15 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
20 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 C7l, human J
25 segments, human D segments and a multiplicity of human V
segments.
A 6.3 kb BamHI/HindliI 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 pHIG5'O
(overlap). The insert contained in this plasmid contains
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human V, D and J segments. When the single V segment from
pVHl 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 , 71õ,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)
DNA encoding a synthetic V segment repertoire is cloned into
pHIG2 at the SfiI site to generate FiIG5' 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
sec :nts and J segments which overlap with the J segments
conzained 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
Dsegments, :he C region, the Gyl region (all from human) and
the rat 3' :lhancer sequence.
EXAMPLE 5
Construction of Light Chain Minilocus
A. Construction of pEul
The construotio4,of,pE 1 is depicted in Fig. 16.
The mouse heavy chain enhancer is isolated on the XbaI to
EcoRI 678 bp fragment (Banerji et al., Ce 21:729-740 (1983))
from phage clones using oligo:
5' GAA TGG GAG TGA GGC TCT CTC ATA CCC
TAT TCA GAA CTG ACT 3'
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This Ep fragment is cloned into EcoRV/XbaI digested
pGP1 by blunt end filling in EcoRI site. The resultant plasmid is
designated pEmul.
B. Construction of K Light chain Minilocus
The K construct contains at least one human Vx segment,
all five human JK segments, the human J-Cx enhancer, human K
constant region exon, and, ideally, the human 3' K enhancer (Meyer
et al., EMBO J. 8:1959-1964 (1989)). The K enhancer in mouse is 9
kb downstream from C,, and the human enhancer can be found on a 4 kb
BamHI fragment (Judde and Max, Mol. Cell. Biol. 12:5206-5215
(1992)). In addition, the construct contains a copy of the mouse
heavy chain J-Cp enhancers.
The minilocus is constructed from four component
fragments:
(a) A 16 kb SmaI fragment that contains the human
C. exon and the 3' human enhancer by analogy with the mouse-
locus;
(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
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 rc enhancer); and
(d) A fragment containing one or more V segments.
The preparation of this construct is as follows.
Human placental DNA is digested with SmaI and fractionated on
aqarose gel by electrophoresis. Similarly, human placental
DNA is digested with BamHI and fractionated.by
electrophoresis. The 16 kb fraction is isolated from the SmaI
diqested gel and the 11 kb region is similarly isolated from
the gel containing DNA digested with BamHI.
The 16 kb SmaI fraction is cloned into Lambda FIX II
(Stratagene, La Jolla, California) which has been digested
with Xhol, treated with klenow fragment DNA polymerase to fill
in the XhoI restriction diqest product. Ligation of the 16 kb
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SmaI 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 CCC CAT CTG 3'
A 16 kb XhoI insert that was subcloned into the XhoI
cut pE l so that Cu is adjacent.to the Smai site. The
resultant plasmid was designated pKapl.
The above Cx specific oligonucleotide is used to
probe the X EMBL3/BamHI library to identify an 11 kb clone. A
5 kb SmaI.fragment (fragment (b) in Fig. 20) is subcloned and
subsequently inserted into pKapl digested with Smai. Those
piasmids containing the correct orientation of J segments, CK
and the Eg enhancer are designatedpKap2.
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 Cre segments and the human E enhancer. This insert is
excised by digesting pKapH with NotI and purified by agarose
gel electrophoresis. The thus purified insert is
microinjectedinto the pronucleus of a mouse zygote as
previously described.
G. Construction of K Light Chain Minilocus by
in Vivo Hoinoloaous Recombination
The 11 kb BamHI fragment, is cloned into BamHI'
digested pGP1 such that the 31 end is towardthe Sfii site.
The resultant plasmid is designated pKAPint. One or more VK segments is
inserted into the polylinker between the BamHI and
SpeI 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
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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.
EXAMPLE 6
Isolation of Genomic Clones
Corresponding to Rearranged and Expressed
Couies of Immunoqlobulin 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 theimznunoglobulin 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. DouLale Stranded cDNA
Cells from human hybridoma, or lymphoma, or other
cell line that synthesizes either cell surface or secreted or
both forms of IqM with a K light chain are used for the
isolation of polyA+ RNA. The RNA is then used for the
synthesis of,o.ligo;dT,primed cDNA using the enzyme reverse
transcriptase. The single stranded cDNA is 'then isolated and
G residues are added to the 31 end using the enzyme
polynucleotide terminal transferase. The Gtailed
.single-stranded cDNA is then purified and used as template for
seeond=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 - 31
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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
immunoglobulin molecule. Genomic clones of these exptessed
5 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
10 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
GGGAAG 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'
5' - GTA CGC 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 C rAC ,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
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including SmaI, 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 thex}õ cut
with SmaI 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 usingthe unique probe o-kappa. DNA is isolated from
positive clones and subcloned into the polylinker of pKapi.
The resulting clone is calledpRKL.
F,XAMPLE 7
Isolation of Genomic Clones
Corresponding to Rearranged Expressed Copies
of Immunoalobulin Heavy Chain u Genes
This example describes the cloning of immunoglobulin
heavy chain genes from cultured cells of expressed and
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 i a'templ'ate 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
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that can be used as a primer for the PCF 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 immunoglobulinmessage 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 ceil line and digested individually and in pairwise
combinations with several different restriction.endonucleases
including MluI (MluI is a rare cutting enzyme that cleaves
between the J segmentand mu CHI), is then probed with the
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 MluI and second enzyme. MluI or SpeI adapter linkers are
3-0 then ligiited onto the endw: and' qut ta,convert; the upstream
site to MluI or Spel. The DNA is then size fractionated by
aqarose 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, andthe insert is
subcloned intoMluI or MluI/SpeI cut plasmid pCON2. The.
resulting plasmid is called pRMGH.
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EXAMPLE 8
Construction of Human K Miniloci Transcrenes
Light Chain Minilocus
A human genomic DNA phage library was screened with
kappa light chain specific oligonucleotide probes and isolated
clones spanning the JK-C region. A 5.7 kb ClaI/XhoI fragment
containing JK 1 together with a 13 kb XhoI fragment containing
JK2-5 and CK into pGPid was cloned and used to create the
plasmid pKcor. This plasmid contains Jxi-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' SalI site for
inserting additional cis-acting regulatory sequences.
V kaAVa c,ienes
A human genomic DNA phage library was screened with
Vx light chain specific oligonucleotide probes and isolated
clones containing human V. segments. Functional V segments
were identified by DNA sequence analysis. These clones
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. Three of the clones were mapped and sequenced.
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.
pxcl
The kappa light chain minilocus transgene pKCl (Fig.
32) was generated by inserting a 7.5 kb XhoI/SalI fragment
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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
+-ransferred the surviving embryos into pseudopregnant females
described by Hogan et al. (in Methods of Manipulating the
"ise Embryo, 1986, Cold Spring Harbor Laboratory, New York).
e that developed from injected embryos were analyzed for
a 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 t-e presence of transgene encoded
human Ig kappa protein by ELISA as described by Harlow and
Lane (,in Antibodies: A Laboratory Manual, .988, 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 IgGi
(clone JL512, #02$0, 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-
=eactivity 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
eig2 'h mouse is 'derived from -a mouse generated by
microinjection of the pHC1 transgene (desctibed previously).
Two of the seven mice from KC1 injected embryos (I.D.Ps 666 =}
aand 664) did not contain the transgene insert as assayed by
DAN Southern blot analysis, and five of the mice (I.D.Ps 676,
674, 673, 670, and 665) contained the transgene. All but one
of the KCl transgene positive animals express detectable
levels of human Ig kappa protein, and the single non-
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expressing animal appears to be a genetic mosaic on the basis
of DNA Southern blot analysis. The pHC1 positive transgenic
mouse expresses human IgM and IgGi but not Ig kappa,
demonstrating the specificity of the reagents used in the
5 assay.
8KC2
The kappa light chain minilocus transgene pKC2 was
generated by inserting an 8 kb XhoI/SalI fragment containing
10 VK 65.5 into the 5" XhoI site of pKC1. The resulting
transgene insert, which contains two V. 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-m
intronic enhancer (Banerji et al., Cell 33:729-740 (1983))
together with a 1.4 kb M1uI Hindlil fragment containing the
human heavy chain J-m intronic enhancer (Hayday et al., a u e
3f3,7:334-340 (1984)) inserted downstream. This construct tests
the feasibility of initiating early rearrangement of the light
chain minilocus to effect allelic and isotypic exclusion.
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)).
Rearranaed Light Chain Transaenes
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 pKSnx that includes 3.7 kb of 5'
~ ._ .
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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, o-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 XhoI
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
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 chaiwminilocus ,trarlsgenics to ~produce a strain og mice
that express a spectrum of'fully human a:=,ibodies 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
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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, pHCl, pIGG1, pKC1,
and pKC2 were isolated away from vector sequences by agarose
gel electrophoresis. The purified inserts were microinjected
into the pronuclei of fertilized (CS7BL/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,Maninulating the Mouse Embryo, Cold Spring
Harbor Laboratory, New York (1986)).
EXAMPLE 9
Inactivation of the Mouse Kaooa Light Chain Gene by Homoloarous
Recombination
This example describes the inactivation of the mouse
endogenous kappalocus 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).
The strategy is to delete J. and C. by homologous
recombination with a vector containing DNA sequences
homologous to the mouse kappa locus in which a 4.5 kb segment
ofthe locus, spanning the Jx gene and C. segments, is deleted
and,replaced by the selectable marker neo.
Constr-uction of the kagga targgt}ng 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., Gene 60:65-74 (1987)) in the cloning vector pGEM-
7Zf(+)'. The plasmid also includes a heterologous
CA 02124967 2003-04-28
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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. rl he 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 CK locus:
5'- GGC TGA TGC TGC ACC AAC TGT ATC CAT CTT Cc:C ACC ATC CAG
-3 '
and for the JK5 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 Ca 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 JR
region was also isolated from a positive phage clone. An
SphI/Xbal/Bg1II/EcoR.I 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).
:30 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 33 :348-352 (1988).
The HSV TK cassette was
:35 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
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modified to a BamHI site and the TK cassette was then excised
as a BamHI/HindIII fragment and subcloned into pGP1b to
generate pGPlb-TK. This plasmid was linearized at the XhoI
site and the Xhol fragment from pNEO-K5'3', containinS 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 K1 is
shown in Fig. 20e.
Generation and analysis of ES cells with targeted inactivation
of a kapPa allele
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 a1. (1985) J. Embrvol.
E2p. Morvh. 87: 27-45), and the CCE line (Robertson et al.
(1986) Nature 323_t 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
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 qnly, 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
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majority of these chimeras can transmit the ES genome to
progany.
The kappa chain inactivation vector J/C Ki was
digested with NotI and electroporated into AB-1 cellsby the
5 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
10 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.
15 DNA analysis was carried out by Southern blot
hybridization. DNA was isolated from the clones as described
(Laird et al., Nucl. Acids Res. I9:4293 (1991)) digested with
Xbal and probed with the 8'00 bp EcoRI/XbaI fragment indicated
in Fig. 20e as probe A. This probe detects a 3.7 kb XbaI
20 fragment in the wild type locus, and a diagnostic 1.8 kb band'
in a locus which has homologously recombined with the
targetingvector (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
25 recombination into one of the kappa genes. These 7 clones
were further digested with the enzymes BglII, Sacl, and PstI
to verify that the vector integrated homologously into one of
tb,ekappa genes. When probed with the diagnostic 800 bp
EcoRI/XbaI fragment (probe A), Bg1II, SacI, and PstI digests
30 of wild. type; DNA prqdu9e fragments --of 4.1, 5.4, and 7 kb,
respectively, whereas the presence-of a targeted kappa ailele
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,
35 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
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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 M
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 anda majority oE 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
indicative of germline transmission of the ES genome.
Chimeras from two of theseclones 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 a-f, inheztiting, 'the; mutated locus. . Screenizig for
the targetedgene 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.
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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 hetero~ygote
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 J. 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 MouseHeavy Chain Gene by Homologous
Recombination
This examp..=.., 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
sequences from which the JH region has been deleted and
replaced by the gene f.or the selectable marker neo.
Construction of a heavy chain tarcreting vector
Mouse heayy qhaip,sequencesõ containing the JH 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. 12:9065-9089 (1986)) using a JH4 specific oligonucleotide probe:
5'-.ACT ATG CTA TGG ACT ACT GGG GTC AAG GAA CCT CAG TCA CCG
_3i
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A 3.5 kb genomic Sacl/StuI fragment, spanning the JH
region, was isolated from a positive phage clone and subclonad
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) Ptoc. 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
HindIIl site.in the prepared pGEM7 (KJ1) was converted to a
SaIT 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 polI, and the neo fragment was
subcioned 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. pGPlb was
digested with the restriction enzyme NotI'and ligated with the
following oligonucleotide as an adaptor:
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 imcluded 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
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84
digestion with EcoRI and HindIII. The TK DNA fragment was
subcloned between the EcoRI and HindIII 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
J. This fragment was blunte:. by Klenow polymerase and
subcloned into the similarly blunted XhoI site of
pGMT-TK-JH5'. The resulting construct, JHKO1 (Fig. 21e),
contains6.9 kb of genomic sequences flanking the JH locus,
with a 2.3 kbdeletion 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.
EXAMPLE 11
Generation and analysis of taraeted ES cells
AB-1 ES cells (McMahon and Bradley, Cell
~1:1073-1085 (1990)) were grown on mitotically inactive
S147,76/7 cell feeder;layers essentially as described
(i-.,bertson; E.J. (1987) Teratocarcinomas and'Embryonic Stem
Q.1.1s: A Practical Aptiroach. 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.
WO 93/12227 PCT/US92/10983
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.The heavy chain inactivation vector JHKOl 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
5 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
10 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
15 (Laird et al. (1991) Nucleic Acids Res. 19: 4293), digested
with StuI and probed with the 500 bp EcoRI/StuI 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
20 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
25 locus (as shown in Fig. 21f) was confirmed by further
digestion with HindliI, SpeI and HpaI. Hybridization of probe
A (see Fig. 21f) to Southern blots of HindIIl, SpeI, and HpaI
digested DNA produces bands of 2.3 kb, >10 kb, and >10kb,
respectively, for the wild-type locus (see Fig. 21a), whereas
30 bands of ~ 5.3 kb, 3: 8, kb, ~andi 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 HindIII, SpeI, and HpaI bands diagnostic of a
targeted JH gene. In addition, Southern blot analysis of a
35 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.
~,. ~... ...._ _ . . . _
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Generation of mice carrying the J. deletion
Three of the targeted ES clones described in the
previous section were thawed and injected :.nto 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
aells. 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
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 s.howed a hybri,dizing Stul bapd
of approximately 3 kb in addition tb 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
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locus, heterozygotes caring one targeted copy of the gene and
one normal copy, and mice homozygous for the J. 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 J. (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 Alasmid vectors for clonincx larcxe DNA
sequences
1. gGPTa
The plasmid pBR322 was digested with EcoRI and StyI
and ligated with the following oligonucleotides:
oligo-42 5'- caa gag ccc gcc taa tga gcg ggc ttt ttt ttg cat
act gcg gcc gct -3'
oligo-43 5'- aat tag cgg ccg cag tat gca aaa aaa agc ccg ctc
att agg cgg gct -31
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
resistance gene, AmpR) of a strong transcription termination
signal derived from the trpA gene (Christie et al., Proc.
Hati. 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.
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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 Clal BamHI HindIil NotI
GCggccgcctcgagatcactatcgattaattaaggatccagcagtaagcttgcGGCCGC
..i
pGIlc
NotI SmaI XhoI SalI HindIII BamHI Sacil NotI
C ?gccc-.=3tcccgggtctcgaggtcgacaagctttcgaggu:ccgcGGCCGC
pGPld
No-: aall HindIli Clal ?amHI Xhc NotI
GCggcc.~ -jtcgacaagcttatcgat;, 3atcctcga:. gcGG 'CGC
pGPlf
NotI Sall Hindlli EcoRI C1aI KpnI BamHI XhoI NotI
GCggccgctqtcgacaagcttcgaattcagatcgatgtggtacctggatcctcgagtgcGGCCGC
Each of these plasmids canbe 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.' pGPlb
. r' .
pGPla was digested with -I and ligated with the
following oligonucleotides:
oligo-47 5'- ggc cgc aag ctt act gct 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 gct tgc -3'
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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 gct
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.
Theamplified product was digested with BamHI and
SphT and cloned into BamHI/SphI digested pNN03 (pNN03 is a pUC
-derived plasmid that contains a polylinker with the following
restriction sites, listed in order: NotI, BamHI, NcoI, C1aI,
EcoRV, XbaI,, SacI, XhoI, SphI, PstI, BglII, EcoRI, Smal, Kpnl,
HindIII, 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),
contains several unique restriction sites into which sequences
can be cloned and subsequently excised together with the 3'
enhancer by Notl digestion.
f .""f. :::. ......._. .. i'...,.
.. ... . . ..-...... - ,...;...... ....+sn+.. .-rr_. ........
. . . . . .,.. .-~.:. r. . . . . .
WO 93/12227 PCT/US92/10983
~~2~967 90
7ABLE-:
AAT:Wr~.ggc=cctc.-
,agatcactat:..:aLLadLLa3QgaLCC3QaLdLCdGL'Cc:7:~:.3.Z3CaQggCr.- rC_:'3ac?.
: _.-~~ _...:tgtetcrct~cr~~~~..:..gLCCCr-gtctcrcctcrgLCtct~~~~~~~~~w~-===
_ -=-=RRr': C3C3CdC3C3C3CaC3CaCaG3CaCaCaCCLCG?aQCg3RC3RCr'".gCaQC]CfLL'"gC==
=Cgqggc3CaLgcaiaLQCJatgtttrttC:
atQCdQiiaa~CatgLtt~caLLCt=r?aQCc"~a"taQCatcaatCa
==cccccaccczqcaqctgcagqttcaccc:zccr:~qccaqqctgaccaccrttgqgqatqqggccggqqctc:aLCa
c
=cR
aacggzgaaattgaatt:aqL'tt=cccatt:atcqacaezqccgqaatctqacccr.aggaqgqaatgac,aaqaa.
aLaqqcaaqqtccaaAcaccc:aqqqaaat gcczqzgctccaqqLC~.:..gcatqctgcaqa
,:ZQaaLLCG accaAg CL~gGGGCCwOILGT O
~ . ~Aabv.. aa www = ww = i = = w
~wI~~ "Z'~TGn
... . .. ~..Vi~aAir~= Vw.iV~r~= w w . i= ~W =r
. .~ ~a r ~ ~.. ~ =bi= e.~. i+ aA
_ '~'.""~.i'~=V~ ' . =w.v
wwi+
. .= = ==A = . r
a = a1~
= r=
= = = =
~ . . ..~~AV~ .. = ~ =~= ~ ==~= '.= =~r =
~:-...= =~ .... i' = ==Ja~ ~~=~ <_~ ~ ~ ~ . \rW-\s/M/Wr~t\'
. .. ,_. .. ,,,..~ . .... www~.w - .~ , : ~~~~w =
~.. . ~ J= . ~.~aVaN- :., n"QCCCA TIN.4V\ti=iIMM~.~
. ~ ~.:. . : ~ . .. . . . ~'
.~~ . = ~' ~ = y
" . . ~ : = . ~ . . . = . = . . , .
. .. . . .... ~~.: , .. .. . w
~ ., , ..., . i
.... . =~ , := ~ .
~ ~ . ~ =. .
. , ._. . . . ~ ===
.. . .. .... .. =
Seqoeace of vectoc pGPe.
WO 93/12227 PCT/US92/10983
?1.24967
91
B. Construction of IgM expressing minilocus transgene.pIGM1
1. Isolation of J-o constant region clones and construction
of pJMi
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- intronic
enhancer, was isolated. The same library was screened with
the human 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 switch region and all of the
constant region exons, was isolated from this clone. These
two fragments were ligated together with KpnI/XhoI digested
pNNO3 to obtain the plasmid pJMl.
2. i2JM2
A 4 kb XhoI fragment was isolated from phage clone
A2.1 that contains sequences immediately downstream of the
sequences in pJMi, including the so called E element involved
isn d-associ'ated deleteon of the in certain IgD expreseing,
B-cells (Yasui et al., Eur. J. Immunol. 19:1399 (1989), which
is incorporated herein by reference). This fragment was
treated with the Klenow fragment of DNA polymerase I and
ligated to XhoI cut, Klenow treated, pJM1. 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- intronic enhancer, the -switch region and all
WO 93/12227 PCT/US92/10983
.~-.
/.,4 9 6 7 92
of the constant region exons, as well as the two 0.4 kb
direct repeats, a 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 Xhol fragment, that includes the D elements Dx11 DN1-
and DIõ2 (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 DLR1t DXp2, DXp.l, and
DAl, was isolated from phage clone X4.3. Each of these D
region XhoI fragments were cloned into the Sa1I site of the
plasmid vectorpSP72 (Promega, Madison, WI) so as to destroy
the XhoI site linking the two sequences. The upstream
fragment was then excised with XhoI and SmaI, and the
downstream fragment with EcoRV and XhoI. The resulting
isolated fragments were ligated together with Sall digested
pSP72.to give the plasmid pDHl-. 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. j~CORI
The plasmid pJM2 was digested with Asp718 (an
.iso$chizomer of KpnI) and 'the overhang filled, in with the
Klenow fragment of DNA polymerase 2. The resulting DNA was
then digested with ClaI and the insert isolated. This insert
was ligated to the_XhoI/EcoRV insert of pDH1 and XhoI/ClaI
digested pGPe to generate pCORl (Fig. 24).
5. pVH25~
A 10.3 kb genomic HindIii fragment containing the
two human heavy chain variable region segments Va251 and VH105
CA 02124967 2003-04-28
93
(Humphries et al., lature 331:446 (1988))
incorporated herein by reference) was subcloned into pSP72 to
give the plasmid pVH251.
6. Ip GMl
The plasmid pCORl was partially digested with XhoI
and the isolated Xhol/SalI insert of pVH251 cloned into the
upstream XhoI site to generate the plasmid pIGM1 (Fig. 25).
pIGM1 contains 2 furictional human variable region segments, at
least 8 human D segments all 6 human JH segments, the human
J- enhancer, the human a element, the human switch region,
all of the human c:oding exons, and the human E 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 IgG expressing minilocus
transgene. cHCl
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 HindiII fragment of
phage clone X29.4, containing a-y switch region, was used to
probe a human placentagenomic DNA library cloned into the
phage vector lambda FIX'm 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'
WO 93/12227 PCT/US92/10983
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94
Phage clones X29.5 and XSy1.13 were'both determined
to be of the 71 subclass.
2. pwe1.
A 7.8 kb HindiII fragment of phage clone X29..F.
containing the yl coding region was cloned into pUC18. T;:e
resulting plasmid, pLT1, was digested with XhoI, Klenow
treated, and reli-Tated to destroy the internal XhoI site. The
resulting clone, pLTlxk, was digested with HindIIl 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 71 constant region coding exons. This
7.6 kb X',-~oI/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 plasanid
clone pyel. p7e1 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= g3'e~i
A 5.3 kb HindIIl fragment containing the yl 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 ASry1.l3 and cloned into pSP72 with
the polylinker XhoI siteadjacent to the 5' end of the insert,
to generate the plasznid clone pS7ls. The XhoI/SalI insert of
pSy1s 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 XhoI site and the 3' rat
enhancer can be excised from vector sequences by digestion
with NotI.
WO 93/12227 PC,'T/US92/10983
2 12 49 67
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 pHCl (Fig. 25). pHCI contains 2 M
5 functional human variable region segments, at least 8 human D
segments all 6 human JH segments, the human J- enhancer, the
human v element, the human switch region, all of the human
coding exons, the human E element, and the human 71
constant region, including the associated switch region and
10 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 IctM and IgG expressinc minilocus
transgene, nHC2
1. Isolation of human heavy chain V region qene VH49.8
The human placental genomic DNA library lambda, FIX"
.20 II, Stratagene, La Jolla, CA) was screened with the following
human VH1 family specific oligonucleotide:
oliga.-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
pNNO3 (such that the polylinker Clal 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
thatthe gene is functional (Table 2).
. . . . :_ ...., .,..=_~=r-',..,rr,..:-. ...._. ..,z,..:r.:-; , ,....: i
_ .. _, . -,..,.... ,. . ...: . :..,.... ,
.. .._ ......_ .. ..::. . .t.r,:: ...._... . . .
WO 93/12227 PC"T/US92/ 10983
9
,,124967 96
TABLE 4
~..r...+v ~ ~.-~ _.,, .......: ~ -.. ~..-,._ _ ~ . ~'*..c- x.c u~ .-s-
.ty...~a.r=,+,.~ =1
....r
=
,_..41
~~~Z'!LY iL ~ -r+ T T-T nv~~~ ~n~r.~a ~ ~~
-"~'AAA~'rs'.~A C'~e-:~ "'~"~ _'.~r~C'r. '',~."r~~r-s~~i LvU
L"SlY.7!"AG '~~i+..+i+q 1.7L.~rw-'il~y_~ Ar a""'q =,VTI ~~
300
:,,--T---mAraE)h eireu?heVai. 'IalAlaAlaA IaTtir
act~,.aaac ~cc caLCc~c~ =t~:.~ua. Qca.t-=~~~
?50
:acc:rrgcg ~c~ rrrc.~c ag : DCY~.r..r~G 400
G1yValGln Ser.GlnVa1G a.nLeuVa.1M
GIC'."7,7.3= "'';PM A~JCy'f,~~ 450
nSerGlyAla GluValLysL vsPrvGlyySe rSezVal.Lys Va1SezCysL
11.CI'Ccs AGM Af~C~C-jATG uxAICP,G'~ ~7. ,MG 50Q
ysAlaSerGl yG1.yThrPhe SerSe='IyrA 1alleSet'TM_ JValArgGln
C ~ C x . x . ' ~;,C , - . GT 3 3 TC333G;, AGGATCA= C _ 5m
AlaProGl,yG 1.*iGlyLewGl - y ArgIleTleP roIleleuGl
TACAGTTC=;= CMGAGI'C'ADG ATI'AO~' 600
y ZyrALaGlnL ysPhe+GLnGl: yAtgValnr T1eZ~irAlaP,
-nuG:CT GPZATCTMG 650
spLv~Se~Th rSerl'hrAla TyrMetGluL euSerSerLe vAtqSezGlu
A~:~TC 70Q
-WMA V alWrTtrtQi sALaArg
'G~aAAG a'~"L1~IC"' CJO-,~~-..~~ '750 ,
, s G~TAZ'Z'P~C~G ?"7C~NAGM'C's T1'II~CAAA= 3331'rATATA 800
8i2
=lw a M
~egu~e o~.ht~man vHI f~ad~y ~ne VH49.8
WO 93/12227 PCT/US92/10983
~_2496'~~
97
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 Sma.I and
HindIII, and treated with the Klenow fragment of polymerase I.
The blunt ended fragment was then cloned into C1aI 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 Sa1I site at the 3' end of
the insert and a unique XhoI site at the 5' end.
3. pS3~l-5'
A 0.7 kb XbaI/HindIII fragment (representing
sequences immediately upstream of, and adjacent to, the 5.3 kb
71 switch region containing fragment in the plasmid p7e2)
together with the neighboring upstream 3.1 kb Xbal fragment
were isolated from the phage clone XSg1.13 and cloned into
HindIII/XbaI digested pUC18 vector. The resulting plasmid,
pS71-5', 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. 2,: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.rdi
The pS71-5' insert was . excised with SmaI and
HindIII, treated with Klenow enzyme, and ligated with the
following oligonucleotide linker:
5'- ccg gtc gac cgg -3'
The ligation product was digested with SalI and ligated to
Sall digested pV2. The resulting plasmid, pVP, contains 3.8
WO 93/12227 PC'T/US92/10983
?,!,4 -967 98
kb of -yi 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 SaII
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 p7e2 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 Sall site between the
variable and constant regions can be used to clone in D, J,
and gene segments. The rat heavy chain 3' enhancer is
linked to the 3' end of the ryl gene and the entire insert is
flanked by NotI sites.
5. pHC2
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 JK
segments, the human J-m enhancer, the human o element, the
human switch region, all of the human coding exons, the
human E element,and the human -yl constant region, including
the associated switch region and sterile transcript associated
exons, together with 4 kf 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
trensgeni,c animals., .,, A; unique Xhol site at the 5' end of the
Ansert can be used to clone in additional human variable gene
segments to further expand the recombinational diversity of
this heavy chain minilocus.
E. Transqenic mice
The NotI inserts of plasmids pIGMl and pHCl were
isolated from vector sequences by agarose gel electrophoresis.
The purified inserts were microinjected into the pronuclei of
WO 93/12227 PCT/US92/10983
~ -t 2 4 ~.'7~ 67
99
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
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 IgGl 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 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 (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 IgG1 in the
serum of two animals that developed from embryos injected with
the transgene insert of plasmid pHCl. All of the control non-
transgenic mice tested negative for expression of human IgM
and IgGl by this assay. Mice from two lines containing the
pIGM1 NotI insert (lines #6 and 15) express human IgM but not
human IqGl. We tested mice from 6 lines that contain the pHCi
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 pHCi transgenic mice
that did not express human immunoglobulins were so-called G.
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
WO 93/12227 PCT/US92/10983
;: .
2124967 100
detection of human IgM in the serum of pIGM1 transgenics, and
human IgM and IgGi in pHCl 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 buman IgM or
IgGl. The second animal (#38) contained appraximately 5
copies of the transgene, as assayed by Southern blotting, and
showed detectable levels of both human IgM and IgGl. 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 # transaene conies per cell human IgM human IaGl
6 pIGMl 1 + + -
7 pIGM1 0 - -
9 pIGMl 0 - -
10 pIGMl 0 - -
12 pIGMl 0 , - -
15 pIGMl 10. + -}' -
; õ. ,
18 pHCl 0 - =
19 pHcl 1 - - -
21 pHCi <1 - -
26 pHCl 2 ++
38 pHCl 5
WO 93/12227 PCT/US92/10983
2 124 9 67
101
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),
10. 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
pIGMl transgenics, and human IgM and IgGl in pHCl 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 pHCl 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
transgeneencoded heavy chains, focusing on D and J segment
usage, N region addition, CDR3 lengthdistribution, and the
frequency of joints,resulting in functional mRNA molecules was
examined. Transcripts encoding IgM and IgG incorporating
VH105 and VFI251 were examined.
Polyadenylated RNA was isolated from an eleven week
old male,second geneiration''linei~57pHCl 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 gct cga gtc caa gga gtc tgt gcc gag gtg cag ctg
(g,a,t,c); VH105 specific o-150, gtt gct cga gtg'aaa ggt gtc
cag tqtgag gtg cag ctg (g,a,t,c); human gammal specific
oligo-151, ggc gct cga gtt cca cga cac cgt cac cgg ttc; and
WO 93/12227 PCT/US92/10983
2124967 102
human mu specific oligo-152, cct gct 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 o-
152 to amplify VH251-mu transcripts, reaction 3 used o-150 and
o-151 to amplify VH105-gammal transcripts, and reaction 4 used
o-150 and o-152 to amplify VH105-mu transcripts. The
resulting 0.5 kb PCR products were isolated from an agarose
gel; the transcript products were more abundant than the 7
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 DNAsamplesturned 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 5-cells is dominant. The fact that no
two sequences are alike is also an indication of the large
diversity of immunoglobulins thatcan be expressed from a
compact minilocus containing only 2 V segments, 10 D segments,
and6 J segments. Both of the V segments, all six of the J
segments,! and -7 o,f,.t~e 10. 9 segments that are included in the
transgene-are used in VDJ joints=. In addition, both'constant
region genes (muand gammal) are ir~orporated into
transcripts. The VH105 primer turned out not to be specific for VH105 in
thereactions performed. Therefore many of the
clones from reactions3 and 4 contained VH251 transcripts.
Additionally, clones isolated from ligated reaction 3 PCR
product turned out to encode IqM rather than IgG; however this
may reflect contamination with PCR product from reaction 4 as
CA 02124967 2003-04-28
103
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 pHCl
transgenic mouse.
WO 93/1222 i 104 PCT/U592/10983
7ABLEa
. . v == ,J ,J v u y V y u = v v v v u v. = u :J u u u u u .
< < < <
< < < c - - =- < < < . V r r- r ,
4c Ic
~ u v v u < .c ., 'u u a u < . < < < <
~ 8 8
_ < K x
t < < ! ' t t < < < < t u' ~ ~
J Li
V U :7 U U V VV u U U V V -J~ L U U U V V J~~ ' G L G G~ G~ C~ G G G G C v G r
G~~ G G~
~ u u u u v u u u u u u u v v ~ u u
G G C C G C C G G C G C C G C C t t G C r~ C G G G
u G G G G G G G G G G G G G G G G G G G G G a G n G~ G G G
u u u ~ u u u u v u v u v u u u u u u u u u u u v
dc~~
it u ~- ~< C G G C't c G G'~i C G< C.c C r~ t~
'.y vG u u u u u v ~ u u u u u c y u u .~ u
~ u u v u :+ u u u u u u < s ~ u u u ~ ~
~ . ~ ..
I V V
G ,
t;
13
C
dg
< y~
~ do 42
C
> ~ ~ ~
11111111, 3G
; L S L 1C . G s s s s s s a a R a S t A a S S a a a a a a a a
. . = ~ = =. : ~ q:. A ~ ' ..= =. w~r = = A = r M = ro ~ = = = I~1 w~ = = =
=~ ~ y "Y 1 9.. ~l . ,=~ ' 9 ~ : ~! _ ' y ~l
_ ~ h - ~ ~ ~ .~ N _ N N
~ Pa _
.~ . ~ i i ' .~ ..
~ ~~ o'a ~ a.' ~ o o H o 0 0 0
~ ~ ~r ~ w~> ~ ~ ~ ~~ ~ .~I- ~ ~ r r . ~ Y~ A s 11~ ~ A
A N M: ~ AA A I~ J~ I~ A A M A O_ M A w ~A N .~ A O_ = A ~ A O h
}~ ~S'i S S s~ S S S S 5~ S~~~ S S 5~ S S 5 S S s S S S
.. .. .. = ~ ~ ~ ~ ~ ~i :~. w : w .' i w w tii ~i ~
~ == == w ~ r+ = w 2
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G. J seqment choice
Table 5 compared the distribution of.J segments
incorporated into pHCl 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-t
H. D sectment choice
49% (40 of 82) of the clones analyzed by Yamada et
al.incorporated D segments that are included in the pHCl
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 froman inversion of the DIR2 segments which is
included in the pHCi construct. This mechanism, which was
predicted by Ichiharaet al. (EMBO J. 7:4141 (1988)) and
observed by Sanz (J.,Immunol. 14,7:1720-1729 (1991)), was not
considered by Yamada et al. (,I;EXp. Med. 173:395-407 (1991) ).
Table 5 is :a compalrisan 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 thepHCi 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'l and DN1, are also found with high frequency in
the transgenic mouse. The most dramatic dissimilarity between
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=
106
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 theMheavy
chain transcripts contained DHQ52 sequences. If D segments
not found in pHCi 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 pHCi transgenic
mouse.
TABLE 6 D Segment Choice
Percent Usage ( 3%)
D. Sectment HC1 transgenic Human PBL
DLRl <1 <1
DXP1 3 6
DXP'l 25 19
DAl <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 ioints
Table 7 shows the predicted amino acid sequences of
the VDJ regions from 30 clones that were analyzed from the
pHCl transgenic. The translated sequences indicate that 23 of
the 30 VDJ joints (77%) are in-frame with respect to the
variable and J segments.
, ; i, ; ~
9 12 4 9 67
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T,ARj.E? Functionality of V-D-J Joirits
FR3 CDR3 FR4 t YHZS: l:so52 J3 .t Y~tA :5.."GVOiIFDZ ~GiiGI:lV'NSS~S:X
1 V'riZSl J4 !t ':CiA :MIAAAGM AGQG:==VMSA+'TK
3 NF;2!1 Z7 Jo ~t ':CAit ="715S+1SMA
vHZ:: Yt1A !?Yflz;.. . . . RStslp~t!
VH2~1 ~~"1 3. Yt YCAA '4GOC.TZVTVSSASTlC
e YI~Z.SI t:' J3 7o Y'C~IA eiC'~Y'SOMZ
VRZSl D}=SZ TJ u YM A1WTDZ
6 VR2,:I :t1Q'S2 Jo u YM
9 VHZ~2 - :I u ':G1A :~'0!i WOQGZ'LYT'VSSGSAS
:3 '1R2S1 CZ~2 33 tl '!'t71A FtVJNSIClC ~100~.'1'ZVlV~Si'.SAS
VSZSI pQr' : J4 r{ YCAR Q1T!lNM.'VlTVY i~pG'rL'JTY~Si~S
:2 V11251 D? Jl w YM Q!!~ N=TLVTVSStSLS
:~ v1QSl 0=52 .: i r YG~ '~~YCiOK
vfQSl D7Q' 1 Ji r YM
19 vlZ31 D~ = 2 Ji tt YGG1t .
: o W10S 0~ =:.?S 1t YCVR
f :. Vl2Sl DOQ' I J4 tt YG~
I ' .
Gl1R~Y MfipGiZy~1'VSS~S='!C
li J1~2'JI D8=Z Ji r YM
VSZ91 D=l Ji Y! '!Wt
n V112SI D9053 A YM QZGIDMW IIDo!TLYPVXSGS74S.
YC7Nt'
n VMI M .T2 11
22 V=ZSl =2 Ji i ! zvrvsgam
23: .:' VMI ' AW; J4 owsrmm,
24. VQOS 07 Ji M xC.11!
23 VitOS mQl J( Y'CVft Oz:.TG=M
26 YM2 Ol JZ w YGR ~2
27 VQOS DM J3 YM stwicau
2r v~'iS1 m A n ~ ZNNUM&M
29 VQM al A M
80 Vn1 DsM J,* M YliZ C~
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J. CDR3 length 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 skeweQ 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) )-.o -
TABLE 8 CDR3 Length Distribution
A Percent Occurrence ( 3%)
#am%no acids in CDR3 HC1 transgenic Human PBL
3-g_ 26 14
9-12 48 41
13=18 26 37
19-23 <1 7
>23 <1 1
100% 100%
EXAMPLE 13
Rearranged Heavy Chain Transc3enes
A. Isolat'.on of Rearranged Human Heavy Chain VDJ secxments.
Two human leukocyte genomic DNA libraries cloned
into the phage vector XEMSL3/SP6/T7 (Clonetech Laboratories,
Inc., Palo Alto, CA) are screened with a 1 kb Paci/HindIII
fragment of X1.3 containing the human heavy chain J- intronic
enhancer. Pbsitive 'clones-' a're 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-31
oligo-8 51-tcc ctg aga ctc tcc tgt gca gcc tct gga ttc4acc
ttc agt-3'
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Clones that hybridized with both V and J- 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 Xhol site
is adjacent to the 5' end of the insert sequence. A subclone
containing a functional VDJ segment is digested with XhoI and
PacI (PacI, 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- intronic enhancer, the
switch element, the constant region coding exons, and the yl
constant region, including the sterile transcript associated
sequences, the yi 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
Lj,ght Chain Transgenes
A. Const uction of Plasm,ig vectors
1. Plasmid vector pGPlc
Plasmidvector pGPla is digested with NotI and the
followingoligonucleotides 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 gct tgt cga cct cga gac
ccg gga tgc-3'
The.resulting plasmid, pGP1c, contains a polylinker with XmaI,
XhoI, Sall, HindIII, and BamHI restriction sites flanked by
Noti sites.
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2. Plasmid vector pGPld
Plasmid vector pGPla is digested with NotI and the
following oligonucleotides ligated in:
oligo-87 5'-ggc cgc tgt cga caa gct tat cga tgg atc ctc gag
tgc -3'
oligo-88 5'-ggc cgc act cga gga tcc atc gat aag ctt gtc gac
agc -3'
The resulting plasmid, pGPld, contains a polylinker with SalI;
HindiII, Clal; BamHI, and XhoI.restriction sites flanked by
NotI sites.
B. Isolation of Jx and CK clones
A human placental genomic DNA library cloned into
the phage vector XEMBL3/SF6/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 -31
and the phage clones 136.2 and 136.5 isolated. A 7.4 kb Xhol
fracgment that includes the Jrcl segment was isolated from
136.2 and subcloned into the plasmid pNN03 to generate the
plasmid clone p36.2. A neighboring 13 kb XhoI fragment that
includes Jk segments 2 through 5 together with the Crc gene
segment was isolated from phage clone 136.5 and,subcloned into
th'eplasmid,' pNN03'"to gerieralte' the plasmid clqne p3,6. 5. .- ,
Togetherthese two clones span the region beginning 7.2 kb'
upstream ot Jx1 and ending 9 kb downstream of Cx.
C. C,ons.~ruction ofrearranced liaht chain tran enes
1. pCKi, a Ck vector for expressing rearranged variable
segments
The 13 kb XhoI insert of plasmid clone p36.5
ccntaining the Cx gene, together with 9 kb of downstream
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1~1
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 VJK segments into the unique
51 Xhol site. The transgene can then be excised with NotI and
purified from vector sequences by gel electrophoresis. The
resulting transgene construct will cantain the human J-Crc
intronic enhancer and may contain the human 3' K enhancer.
2. pCK2, a Cx 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- 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 HindliI
and a 1.4 kb M1uI/HindIiI fragment of phage clone X1.3
(previous example), containing the human heavy chain J-
intronic enhancer (Hayday et al., Nature 307:334-340 (1984)),
to it. The resulting plasmid, pMHEl, consists of the mouse
and human heavy chain J- intronic enhancers ligated together
into pUC18 such that they are excised on a single
BamHI/Hindill fragment. This 2.3 kb fragment is isolated and
cloned into pGPlc to generate pMHE2. pMHE2 is digested with
Salz 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- 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,
$:1959-1964 (1989); Petterson et al., Nature, 344:165-168
(1990)).
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3. 'Isolation of rearranged kappa light chain variable
segments
Two human leukocyte genomic DNA libraries cloned
into the phage vector XEMBL3/SP6/T7 (Clcnetech Laboratgries,
= ! 5 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 51-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 frameand splice signals) are subcloned into the
unique XhoI sites of vectors pCK1 and pCK2 to generate
rearranged kappalightchain transgenes. The transgene
constructs are isolated from vector sequences by digestion
with NotI. Agarose gel purified insert is microinjected into
mouse embryopronuclei 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, og,; different ,heavx . chain VDJ com'-;inations, several
different light chain transgene=construc:-- are generated, each
using a different rearranged VJk clone, &.; 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
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Diego, CA) and analyzed by flow cytometry using a FACScan
analyzer (Becton Dickinson, San Jose, CA). Rearranged light
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.
D. Construction of unrearranged 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 HindIIl digested,
Klenow-treated, plasmid pGPld. A plasmid clone is selected
such that the 5' end of the insert'is adjacent to the vector
derived C1aI site. The resulting plasmid, p36.5-1d, is
digested with C1aI and Klenow-treated. The Jui containing 7.4
kb XhoI insert of p36.2 is then Klenow-treated and cloned into
the C1aI, Kienow-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, pJCK1 (Fig. 34), contains the entire
human Jrc.region and Cx, together with 7.2 kb of upstream
sequences and 9 kb of downstream sequences. The insert also
contains the human J-Cx 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 Xhol site i$,.located at the 5' end of the insert for
the purpose of cloning in unrearranged VK gene segments. The
unique Sall and XhoI sites are in turn flanked by NotI sites
that are used to isolate the completed transgene construct
away from vector sequences.
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2. Isolation of unrearranged Vx 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 DNAlibrary
cloned into the phage vector 1EMBL3/SP6/T7 (Clonetech
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 :~nstructs. The resulting clones are
digested wi}h NotI and the inserts isolated and injected into
mouse embr,: 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 Ig 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),
Transcriution and Translation: A Practical Approach, B.D.
Hammes and $.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 1mg/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 500C, and then washing extensively with TE. The
DNA is then digested in the agarose with the restriction
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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 byMpulsed
field gel electrophoresis as described by Anand et al., Nuc.
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., Technicrue 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. NLttl. Acad.
Sci. USA, 8,6:5898-5902 (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., EMO J. 10:1629-1634 (1991)),
or the YAC is introduced into.ES cells by spheroplast fusion.
EXAMPLE 16
Discontinuous Ggnomic Heavy Chain Ia T$ansqene
An 85.kb Spel fragment of human genomic DNA,
containing VH6, D segments, J segments, the constant region
and part of the 7 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
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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.- '
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 SpeI fragment.
DNP, 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 rearranc,ed variable reaion
secuences in transcrenic B cells
An antigen of interest is used to immunize (see
Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor, New;,York (1988)) armouse with;.the following: genetic
traits: homozyqosity at the endogenous having chain locus for
a deletion of JH (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.
CA 02124967 2003-04-28
117
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
uniquesix 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 p7el (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 Xhol site. The resulting clones
are used together to transfect SPa 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.
Technolocv, 7:934-938 (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 Comolex 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
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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.
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 (JD) and
harboring a human heavy chain minigene construct (HC1); these
mice are designated as line,112. Blood was washed and
resuspended in 50 m1s Hanks' and diluted to 1x108 cells/ml 0.2
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 theproduction 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 - ++
5 234' 112 F - ++
2348 112 F + ++
7 2349 112 F - +
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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)
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 cytometrv
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 andthen 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 celis 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
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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.4NS) 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
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 Endocrenous Mouse Immunoglobulin Expression
by Antisense RNA
A. Vector for Expressiori,of Antisense Ig Sequences
1. Construction of the cloning vector pGPlh
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'
5'- tcg acg aat tca caa gat ctg gta cct ggt ctc gag -3'
to generate the plasmid pGPlh. This plasmid contains a
polylinker that includes the following restriction sites:
NotI, EcoRI, BglII, Asp718, XhoI, BamHI, HindIII, NotI.
Construction of pBCE1.
A 0.8 kb XbaI/BglII fragment of pVH251 (referred to
in a previous.examp,le), that ;include,s, the promoter, leader
sequence exon, first intron, and part of the second exonof
the human VH-V family immunoglobulin variable gene segment,
was inserted into XbaI/Bg1II 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) PM 1:239-249) is cloned into BglII/EcoRI
digested pGHlh. The resulting plasmid is digested with BamHI
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and the BamHI/BglII of pVH251N is inserted in the same
orientation as the rGH gene to generate the plasmid pVhgh.
A 0.9 kb kbal fragment of mouse genomic DNA
containing the mouse heavy chain J- 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
enzyme. The klenow treated DNA was then digested with HindIil
and a 1.4 kb MluI(klenow)/HindIII fragment of phage clone X1.3
(previous example), containing the human heavy chain J-
intronic enhancer (Hayday et al., (1984) Nature 307:334-340),
to it. . The resulting plasmid, pMHE1, consists of the mouse
and human heavy chainJ- intron enhancers ligated together
into pUC18 such that they can be excised on a single
BamHI/HindIII fragment:
The BamHi/HindiII fragment of pMHEl is cloned into
BamHI/HindIIl cut pVhgh to generate the B-cell expression
vector pBCEl. 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
polyadenylationsequences in addition to intron sequences that
promote the expression of transgene constructs. Antisense
transgene constructs generated from pBCE1 can be separated
from vector sequences bydigestion with NotI.
B. An IgM antisense transgene construct.
The following two oligonucleotides:
~ 30
5'- cgc ggt acc gag aqt cag tcc'ttc cca aat g'tc -3'
51- cgc ctc gag aca gct 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 pBCEl to generate the antisense transgene
;~. , .
.._a..
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construct pMAS1. The purified Notl 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 constr4ct
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
human heavy chain transgene minilocus such as pHCl (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 h=.iman 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 lev,.~ls 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 gct gat gct gca cca act gta tcc -3'
5'- cgc ctc gag cta aca ctc att cct gtt gaa gct -3'
are used as primers for the amplification of mouse IgKappa
constant,region sequence,s,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
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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 tran'sgenic mouse of the
present invention.
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 g dimethyldioctadecyl Ammonium Bromide in 100 L 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 370C 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')-
ho=seradish peroxidase or anti-human IgM Fc-horseradish
peroxidase in the same diluent. After 1 hour at room
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temperature enzyme activity was assessed by addition of ABTS
substrate (Sigma, St. Louis, Misscuri) and read after 30
minutes at 415-490 nm.
Human Heavv Chain Participation in Immune Response in
Transgenic Mice
Figure 37 illustrates the response of three mouse
littermates to immunization with KLH-DNP. Mouse number 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
antiqens 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 resDonses comgrising humgn
immun2glokUlin heavy chains aaainst a human alvcoDrotein
aI]ti3=
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 (suora) resulting in the absence of
functional endogenous (murine) heavy chain production. The
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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 -yl 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
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 chain-containing immunoglobulin
and human 7 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 7 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 ry chain Ig continued to
show an.incr:ease over time during the course of the
experiment. The observed human 'chain Ig response, followed
by a plateau, combined with a later geveloping 7 chain
response which continues to rise is characteristic of the
pattern seen with affinity maturation. Analysis of Day 21
.35 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.
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These data indicat- that animals transgenic for
human unrearranged immunoglr .lin gene loci: (1) can respond
to a human antigen (e.g., the human glycoprotein, CEA), (2)
can undergo isotype switching ("class switching) as
exemplified by the observed to 7 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
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
transceng minilocus.
Line HC1-57 transgenic mice, containing multiple
copies of the HCi transgene, were bred with immunoglobulin
heavy chain deletion mice to obtain mice that contain the HCi
transgeneand contain disruptions at both alleles of the
endogenous mouse heavy chain (supra). These mice express
human mu and gammai 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
ficom the-spleen, inquinal ;,and ;mesen;teric lymph nodes, and
peyer:, 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 51-cta gct cga gtc caa.gga gtc tgt gcc gag gtg cag ctg
(g/a/t/c) -3 +
151 51-ggc gct cga gtt cca cga cac cgt cac cgg ttc-3'
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These primers specifically amplify VH251/gammal cDNA
sequences. The amplified sequences were digested with Xhol
and cloned into the vector pNNO3. DNA sequence from the
inserts of 23 random clones is shown in Fig. 40; sequence
variations from germl.ine sequence are indicated, dots indicate
sequence is identical to germline. Comparison of the cDNA
sequences with the c,;ermline sequence of the VH251 transgene
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. Exy. Med. 176: 1073).
The overall frequency of somatic mutations is
approximately 1%; however, the frequency goes up to about 51
within CDR1, indicating selection for amino acid changes that
affect antigen binding. This demonstrates antigen driven
affinity maturation of the human heavy chain sequences.
.20
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 transaene
by co-injection of two overlappingDNA fraqments
1. Isolation of unrearranaed functional Vx gene segments
vk65.3. vk65.5. vk65.8 and vk65.15
The V. specific oligonucleotide, oligo-65 (5'-agg
ttc aqt 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 XEMHL3/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
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selected. Criteria for judging functionalitv include: open
reading frames, intact splice acceptor and donor sequences,
and intact recombination sequence. DNA sequences of 4
functional V. ge:e 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.
(1 a) p65.3f
A 3 kb Xba fragment of phage clone X65.3 was
subcloned into pUC19 so that the vector derived SalI 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 Xhol site is
proximal to the 5' end of the insert and the vector derived
Sall site 3'. The resulting plasmid is designated p65.5g1.
(l c) p65.8
A 6.5 kb HindiiI 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 piasmid p65.15.3. The Vx
gene segment within the plasmid insert was mapped to a 4.6 kb
EcoRI/Hindlli subfr~g3aentR , 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. gM
The XhoI/SalI insert of p65.8 was cloned into the
XhoI site of p65.15f to generate the plasmid pKV2. The
Xhol/Sall insert of p65.5g1 was cloned into the XhoI site of
pIiV2 to generate pKV3. The XhoI/Sall insert of pKV3 was
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cloned into the XhoI site of p65.3f to generate the plasmid
pKV4. This plasmid contains a single 21 kb Xhol/SalI insert
that includes 4 functional V. gene segments. The entire
insert can also be excised with NotI.
3. pKC1B
(3 a) pKcor
Two XhoI fragments derived from human genomic DNA
phage X clones were subcloned into plasmid vectors. The
first, a 13 kb Jx2-Jx5/Cx containing fragment, was treated with
Klenow enzyme and cloned into HindiII digested, Klenow
treated, plasmid pGPid. A plasmid clone (pK-31) was selected
such that the 5' end of the insert is adjacent to the vector
derived ClaI site. The second Xhol fragment, a 7.4 kb piece
of DNA containing Jx1 was cloned into XhoI/SalI-digested
pSP72, such that the 3' insert Xhol site was destroyed by
ligation to the vector SalI site. The resulting clone,
p36.2s, includes an insert derived C1aI site 4.5 kb upstream
of Jxl and a polylinker derived C1aI site downstream in place
of the naturally occurring XhoI site between Jxl and Jx2. This
clone was digested with Clal to release a 4.7 kb fragment
which was cloned into C1aI digested pK-31 in the correct 5' to
3' orientation to generate a plasmid containing all 5 human J.
segments, the human intronic enhancer human Cx, 4.5 kb of 5'
flanking sequence, and 9 kb of 3' flanking sequence. This
plasmid, pKcor, includes unique flanking Xhol and SalI 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.
12: 5206) was cloned into
pGPif such that the 5' end is proximal to the vector XhoI
site. The resulting plasmid, p24Bf, was cut with XhoI and the
:35 17.7 kb XhoI/Sa1I fragment of pKcor cloned into it in the same
orientation as the enhancer fragment. The resulting plasmid,
pKcorB, includes unique XhoI and SalI sites at the 5' and 3'
ends of the insert respectively.
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(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 V. segment, all 5 human J. segments, the human
intronic enhancer, human Cx, and the hunan 3' kappa enhancer.
The entire 25 kb insert can be isolated by NotI digestion.
4. Co4
The two NotI inserts from plasmids pKV4 and pICC1B
were mixed at a concentration of 2.5 Ag/mi each in
microin,jection buffer, and co-injected into the pronuclei of
half day mouse embryos as descri.bed, 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 pKCiB 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.
The foregoinq description of the preferred
embodiments of the present invention has been presented for
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 and variations
are possible in light of the above teaching.
Although the present invention has been described in
some detail by way of illustration for purposes of clarity of
understanding, it will be apparent that certain changes and
modifications may be practiced within the scope of the claims.
