U.S. patent application number 13/140529 was filed with the patent office on 2011-12-22 for antibody production.
Invention is credited to Roger Kingdon Craig, Franklin Gerardus Grosveld, Richard Wilhelm Janssens, Mariuns Johannes Van Haperen.
Application Number | 20110314563 13/140529 |
Document ID | / |
Family ID | 41818841 |
Filed Date | 2011-12-22 |
United States Patent
Application |
20110314563 |
Kind Code |
A1 |
Craig; Roger Kingdon ; et
al. |
December 22, 2011 |
ANTIBODY PRODUCTION
Abstract
A non-human mammal containing an endogenous lambda light chain
gene locus, an endogenous kappa light chain gene locus and an
endogenous heavy chain gene locus, each of which can re-arrange so
that immunoglobulin heavy and light chain genes are formed and
expressed in B-cells following antigen challenge but said loci have
been mutated so that the ability to form functional immunoglobulin
tetramers comprising re-arranged heavy and light chains produced
from said mutated loci has been substantially reduced or
eliminated.
Inventors: |
Craig; Roger Kingdon;
(Cheshire, GB) ; Grosveld; Franklin Gerardus; (DR
Rotterdam, NL) ; Janssens; Richard Wilhelm; (DR
Rotterdam, NL) ; Van Haperen; Mariuns Johannes;
(Prinsenbeek, NL) |
Family ID: |
41818841 |
Appl. No.: |
13/140529 |
Filed: |
November 30, 2009 |
PCT Filed: |
November 30, 2009 |
PCT NO: |
PCT/GB2009/002781 |
371 Date: |
September 6, 2011 |
Current U.S.
Class: |
800/4 ; 800/14;
800/18; 800/21 |
Current CPC
Class: |
C07K 2317/515 20130101;
A61P 37/00 20180101; A01K 2217/072 20130101; A01K 2217/15 20130101;
C12N 15/8509 20130101; A01K 2207/15 20130101; C07K 2317/52
20130101; A01K 67/0278 20130101; C07K 2317/24 20130101; C07K
2317/56 20130101; A01K 2267/01 20130101; A01K 2217/206 20130101;
C07K 2317/51 20130101; C12P 21/005 20130101; C07K 16/00 20130101;
A01K 2227/105 20130101; A01K 67/0276 20130101 |
Class at
Publication: |
800/4 ; 800/14;
800/18; 800/21 |
International
Class: |
A01K 67/027 20060101
A01K067/027; A01K 67/02 20060101 A01K067/02; C12P 21/00 20060101
C12P021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 18, 2008 |
GB |
0823147.4 |
Apr 17, 2009 |
GB |
0906673.9 |
May 28, 2009 |
GB |
0909207.3 |
May 28, 2009 |
GB |
0909208.1 |
Claims
1. A non-human mammal containing an endogenous lambda light chain
gene locus, an endogenous kappa light chain gene locus and an
endogenous heavy chain gene locus, each of which can re-arrange so
that immunoglobulin heavy and light chain genes are formed and
expressed in B-cells following antigen challenge but said loci have
been mutated so that the ability to form functional immunoglobulin
tetramers comprising re-arranged heavy and light chains produced
from said mutated loci has been substantially reduced or
eliminated.
2. The non-human mammal of claim 1, wherein at least one of the
endogenous lambda light chain gene locus, the endogenous kappa
light chain gene locus and the endogenous heavy chain gene locus
has been mutated by the introduction of a frame shift mutation, a
polypeptide-encoding sequence and/or one or more stop codons into
the at least one endogenous locus.
3. The non-human mammal of claim 2, wherein the mutation is an
insertion of less than 50 nucleotides.
4. The non-human mammal of claim 1, wherein the expression of at
least one of the endogenous lambda light chain gene locus, the
endogenous kappa light chain gene locus and the endogenous heavy
chain gene locus has been substantially reduced by elimination of
part or all of the LCR in the at least one locus.
5. The non-human mammal of claim 2 or claim 3, in which the
introduction is in the endogenous kappa light chain gene locus.
6. The non-human mammal of claim 2 or claim 3, in which the
introduction is in the endogenous lambda light chain gene
locus.
7. The non-human mammal of claim 2 or claim 3, in which the
introduction is in the endogenous kappa light chain gene locus and
the endogenous lambda light chain gene locus.
8. The non-human mammal of claim 2 or claim 3, in which the
introduction is in the endogenous heavy chain gene locus.
9. The non-human mammal of claim 2 or claim 3, in which the
introduction is in the endogenous kappa light chain gene locus and
in the endogenous heavy chain gene locus.
10. The non-human mammal of claim 2 or claim 3, in which the
introduction is in the endogenous lambda light chain gene locus and
in the endogenous heavy chain gene locus.
11. The non-human mammal of claim 2 or claim 3, in which the
introduction is in the endogenous lambda light chain gene locus, in
the endogenous kappa light chain gene locus and in the endogenous
heavy chain gene locus.
12. The non-human mammal of claim 1, wherein there is an
introduction of a frame shift mutation, a polypeptide-encoding
sequence, and/or one or more stop codons in the at least one
endogenous kappa light chain gene locus, or an insertion of less
than 50 nucleotides in the at least one endogenous kappa light
chain gene locus and there is a partial or complete LCR deletion in
the endogenous lambda light chain gene locus.
13. The non-human mammal of claim 1, in which the endogenous heavy
chain gene locus is mutated such that heavy chain gene
rearrangement, mRNA transcription and protein synthesis occurs but
that B-cell activation is blocked.
14. The non-human mammal of claim 1 comprising a transgene
comprising one or more heterologous kappa light chain gene loci and
associated B-cell specific regulatory elements.
15. The non-human mammal of claim 1 comprising a transgene
comprising one or more heterologous lambda light chain gene loci
and associated B-cell specific regulatory elements.
16. The non-human mammal of claim 14 or claim 15, wherein the
transgene comprising a heterologous light chain gene locus
comprises a dominant selective marker gene.
17. The non-human mammal of claim 1 comprising a transgene
comprising one or more heterologous heavy chain gene loci and
associated B-cell specific regulatory elements.
18. The non-human mammal of claim 17 comprising two or more
transgenes comprising two or more different heterologous heavy
chain gene loci and associated B-cell specific regulatory
elements.
19. The non-human mammal of claim 17 or claim 18, wherein the one
or more transgene comprising a heterologous heavy chain gene locus
comprises a dominant selective marker gene.
20. The non-human mammal of claim 17 or claim 18, in which each
heterologous heavy chain gene locus comprises a CTCF binding
site.
21. The non-human mammal of claim 14 or claim 17 comprising a
transgene comprising a heterologous kappa light chain gene locus
and a transgene comprising one or more heterologous heavy chain
loci.
22. The non-human mammal of claim 15 or claim 17 comprising a
transgene comprising a heterologous lambda light chain gene locus
and a transgene comprising one or more heterologous heavy chain
loci.
23. The non-human mammal of any one of claims 14, 15, or 17
comprising a transgene comprising a heterologous kappa light chain
gene locus, a transgene comprising a lambda light chain gene locus
and a transgene comprising one or more heterologous heavy chain
gene loci.
24. The non-human mammal of any one of claims 14, 15, or 17,
wherein each heterologous locus incorporates a cognate LCR.
25. The non-human mammal of any one of claims 14, 15, or 17,
wherein each heterologous locus is a human locus.
26. The non-human mammal of any one of claims 14, 15, or 17,
wherein each heterologous locus is a hybrid locus comprising
variable regions and constant regions derived from more than one
species.
27. The non-human mammal of claim 26, wherein each heterologous
locus is a hybrid locus comprising human variable regions and rat
or murine constant regions.
28. The non-human mammal of any one of claims 14, 15, or 17,
comprising groups of transgenes comprising different groups of
different heterologous heavy chain gene loci, wherein each group of
transgenes comprises a different dominant selective marker
gene.
29. The non-human mammal of any one of claims 14, 15, or 17,
comprising transgenes comprising heterologous light chain loci and
transgenes comprising heterologous heavy chain loci, wherein
transgenes comprising heterologous light chain loci and transgenes
comprising heterologous heavy chain loci each comprise a different
dominant selective marker gene.
30. The non-human mammal of claim 1, which is a rodent.
31. The non-human mammal of claim 30, which is a mouse.
32. A method of producing an antigen-specific heterologous
monoclonal antibody comprising: (a) immunising a non-human
transgenic mammal of any claim 16 or claim 19, with an antigen; (b)
preparing hybridomas or immortalised B-cell lines each of which
produces a monoclonal antibody from the B-cells of the immunised
transgenic mammal; (c) optionally selecting at least one hybridoma
or immortalised B-cell line expressing the heterologous antibody by
use of the dominant selective marker genes present in the
transgenes comprising the heterologous immunoglobulin light chain
and heavy chain loci; and (d) selecting at least one hybridoma or
immortalised B-cell line which produces an antibody which binds
specifically to the antigen.
33. A method of deriving a mammalian, preferably human, antibody
from a hybrid antibody comprising: (a) carrying out the method of
claim 32, wherein the transgenes are hybrid; (b) selecting at least
one hybridoma or immortalised B-cell line which produces an
antibody which binds specifically to the antigen and comprises
V.sub.H and V.sub.L binding domains of the species of choice; (c)
cloning and sequencing the V.sub.H and V.sub.L domains; (d)
recloning selected sequences comprising the V.sub.H and V.sub.L
binding domain coding sequences with constant effectors domains of
choice from the same species; and (e) co-expressing the recloned
sequences encoding heavy and light chain polypeptides of the
desired species using an expression vector in a cell type of
choice.
34. A method for the production of the non-human mammal of claim 1
comprising mutating the endogenous heavy chain gene locus, the
endogenous kappa light chain gene locus, and, optionally, the
endogenous lambda light chain gene locus in a mammalian progenitor
cell and producing a mammal from said progenitor cell, wherein the
mutation is such that, in the mammal, each locus can re-arrange so
that immunoglobulin heavy and light chain genes are formed and
expressed in B-cells following antigen challenge but the ability to
form functional immunoglobulin tetramers comprising re-arranged
heavy and light chains produced from said mutated loci has been
substantially reduced or eliminated.
35. The method of claim 34, wherein the progenitor cell is a
non-human embryonic stem cell.
36. The method of claim 34 or claim 35, wherein the non-human
mammal is a rodent.
37. The method of claim 34 or claim 35, wherein the non-human
mammal is a mouse.
38. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to improved methods for the
derivation and selection using transgenic non-human mammals of a
diverse repertoire of functional, affinity-matured tetrameric
immunoglobulins comprising heavy and light chains in response to
antigen challenge and uses thereof.
[0002] In particular, the present invention relates to a non-human
mammal, preferably a mouse, engineered such that either its ability
to generate endogenous mouse kappa and/or lambda light chain
immunoglobulins is substantially reduced, or the ability of light
chains to complex with heavy chain is reduced, eliminated or
blocked. The non-human mammals of the invention also have a reduced
ability to generate functional endogenous mouse heavy chains. Thus,
their ability to form functional immunoglobulin tetramers
comprising re-arranged heavy and light chains produced from said
mutated loci has been substantially reduced or eliminated. Methods
of generating such mammals and methods of using such mammals to
generate human tetrameric antibodies and hybrid tetrameric
antibodies using immunoglobulin heavy and light chain transgenes
are also described.
[0003] In the following description, all amino acid residue
position numbers are given according to the numbering system
devised by Kabat et al. (1991) US Public Health Services
publication No 91-3242.
BACKGROUND TO THE INVENTION
Antibodies
[0004] The structure of antibodies is well known in the art. Most
natural antibodies are tetrameric, comprising two heavy chains and
two light chains. The heavy chains are joined to each other via
disulphide bonds between hinge domains located approximately half
way along each heavy chain. A light chain is associated with each
heavy chain on the N-terminal side of the hinge domain. Each light
chain is normally bound to its respective heavy chain by a
disulphide bond close to the hinge domain.
[0005] When an antibody molecule is correctly folded, each chain
folds into a number of distinct globular domains joined by more
linear polypeptide sequences.
[0006] For example, the light chain folds into a variable (V.sub.L)
and a constant (C.sub..kappa. or C.sub..lamda.) domain. Heavy
chains have a single variable domain V.sub.H, a first constant
domain (C.sub.H1), a hinge domain and two or three further constant
domains. The heavy chain constant domains and the hinge domain
together form what is generally known as the constant region of an
antibody heavy chain. Interaction of the heavy (V.sub.H) and light
(V.sub.L) chain variable domains results in the formation of an
antigen binding region (Fv). Interaction of the heavy and light
chains is facilitated by the C.sub.H1 domain of the heavy chain and
the C.sub..kappa. or C.lamda. domain of the light chain. Generally,
both V.sub.H and V.sub.L are required for antigen binding, although
heavy chain dimers and amino-terminal fragments have been shown to
retain activity in the absence of light chain (Jaton et al. (1968)
Biochemistry, 7, 4185-4195). Generally the proportion of
circulating .lamda. light chain is low, representing perhaps 2-5%
of the total light chain complexed as a tetrameric immunoglobulin
in plasma (Goldsby et al. (2003) Immunology, 5th edition, W.H.
Freeman & Co NY).
[0007] The in vitro manipulation of heavy chain immunoglobulin
genes to construct novel antibodies was first described in the
1980s. Much of the early antibody engineering work used a
rearranged mouse immunoglobulin .mu. gene (IgM) raised against a
well-characterised antigen. A feature of this antibody was that
antigen binding specificity was known to reside in the V.sub.H
domains, since assembly and secretion with an irrelevant light
chain showed retention of antigen binding (Neuberger and Williams
(1986) Phil. Trans. R. Soc. Lond., A317, 425-432). Using this
system, it was shown that a mouse antigen-specific V.sub.H binding
domain could be used to derive a novel antibody comprising a human
.epsilon. constant effector region fused to a mouse
antigen-specific V.sub.H domain. The resulting hybrid IgE retained
antigen specificity and showed effector activity expected of an IgE
(Neuberger et al. (1985) Nature, 314, 268-270). Other literature
examples of heavy chain engineering include: hybrid mouse-human
antibody genes encoding mouse V.sub.H human/IgA or IgG antibody
fusions which retain anti-phosphocholine activity (Morrison et al.
(1984) PNAS, 81, 6851-6855); an anti-carcinoma-associated antigen
17-1A antibody comprising mouse V.sub.H and human IgG (.gamma.3)
constant region (Sun et al. (1987) PNAS, 84, 214-218); and an
anti-human T-cell antibody (anti CD7) comprising human IgG
(.gamma.1) constant region and mouse V.sub.H domains (see Heinrich
et al. (1989) J. Immunol., 143, 3589-97).
Normal human B cells contain a single immunoglobulin heavy chain
locus on chromosome 14 from which the gene encoding a heavy chain
is produced by rearrangement. In the mouse, the heavy chain locus
is located on chromosome 12. A normal heavy chain locus comprises a
plurality of V gene segments, a number of D gene segments and a
number of J gene segments. Most of a V.sub.H domain is encoded by a
V gene segment, but the C terminal end of each V.sub.H domain is
encoded by a D gene segment and a J gene segment. VDJ rearrangement
in B-cells, followed by affinity maturation, provides each V.sub.H
domain with its antigen-binding specificity. Sequence analysis of
normal H.sub.2L.sub.2 tetramers derived from a heavy chain
immunoglobulin comprising a single V segment demonstrates that
diversity in response to antigen challenge results primarily from a
combination of VDJ rearrangement and somatic hypermutation (Xu and
Davies (2000) Immunity, 13, 37-45). There are over 50 human V gene
segments present in the human genome of which only 39 are
functional. In normal diploid antibody-producing B-cells, each cell
produces an antibody tetramer (H.sub.2L.sub.2) from a single set of
heavy and light chain antibody loci. The other set of loci are not
used productively as the result of a process called allelic
exclusion (Singh et al. (2003) J. Exp. Med., 197, 743-750 and
references therein).
[0008] Fully human antibodies (H.sub.2L.sub.2) can now be derived
from transgenic mice in response to antigen challenge. Such
transgenic mice generally comprise a single human heavy chain
immunoglobulin locus and a separate human light chain
immunoglobulin locus. The corresponding endogenous mouse heavy
chain, kappa light chain and, optionally, lambda light chain loci
coding sequences are deleted or partially deleted. Thus, only human
antibodies comprising a kappa light chain are produced in a low
background of mouse/human antibodies comprising a human heavy chain
and a mouse lambda light chain (WO90/04036; WO93/12227; WO98/24893;
U.S. Pat. No. 5,877,397, U.S. Pat. No. 5,814,318 and U.S. Pat. No.
6,162,963). The deletion of segments of all endogenous murine heavy
and light chain immunoglobulin genes to eliminate endogenous heavy
and light chain gene expression completely has been achieved but
remains technically demanding, particularly if the elimination of
all lambda light chain coding sequence is deemed necessary.
Elimination of the murine lambda light chain coding sequence has
been achieved through the complete deletion of all functional V and
J gene segments and the C1, C2 and C3 constant regions of the
lambda locus, resulting in a mouse with a silenced lambda light
chain locus (see EP1399559).
[0009] A different approach is to limit mouse B-cell development
and immunoglobulin secretion by disruption of membrane exons of the
gene encoding the murine heavy chain gene. Thus, whilst the
endogenous murine heavy chain gene is functional, in that it is
transcribed and undergoes VDJ rearrangement in response to antigen
challenge, since the IgM is never expressed on the cell surface of
pre-B cells, further development is arrested, resulting in a
non-productive response to antigen challenge (Kitamura et al.
(1991) Nature, 350, 423-426), even though both endogenous mouse
kappa and lambda light chain genes remain structurally intact and
functional (Tuaillon (2000) Molecular Immunology, 37, 221-231).
[0010] Where endogenous mouse heavy chain and light chain gene loci
remain functional, any additional introduced immunoglobulin heavy
chain transgene is also regulated by allelic exclusion, so that
some B-cells functionally express mouse heavy and light chain loci
only and others human heavy chain loci only and mouse light chain
loci (Nussenzweig et al. (1987) Science, 236, 816-819). In any
single non-human transgenic animal, there is a highly diverse
population of B-cells expressing antibodies derived from
potentially all immunoglobulin loci in response to disparate
antigen challenge. The subsequent selection of antigen-specific
antibodies using established hybridoma technology using HAT
selection (Davis et al. (1982) J. Immunol. Methods, 50, 161-171)
does not distinguish between hybridomas expressing one as opposed
to another heavy chain immunoglobulin locus.
[0011] Regulatory elements present in immunoglobulin heavy chain
transgenes comprise essential tissue-specific enhancer elements to
ensure B-cell specific expression in a copy number dependent
manner. The presence of a 5' intronic enhancer and the 3' LCR
ensures that transgenes are active at all stages of B-cell
maturation (Guglielmi et al. (2003) Biochim Biophys. Acta, 1642,
181-190). The inclusion of heavy and light chain specific LCRs in
the transgene loci ensures not only that expression is B-cell
specific, but that expression occurs irrespective of the site of
integration into the genome (WO90/10077, Mills et al. (1997) J.
Exp. Med., 186, 845-858 and Pettersson et al. (1997) Immunobiol.,
198, 236-248)). Thus, provided an LCR is present, every transgene
is functional irrespective of its position in the genome. In the
event that the LCR present on the transgene is partially deleted,
the chromatin surrounding the transgene is only partially open to
transcription at any point in time, leading to positional effect
mosaic expression, and so limited levels of expression of the
transgene across the target tissue (Festenstein et al. (1996)
Science, 23, 271 (5252):1123-5; Milot et al. (1996) Cell, 87(1),
105-14)
[0012] An alternative approach for the production of human
immunoglobulins in a mouse background is to replace murine
immunoglobulin gene segments with the homologous gene segments from
humans. Thus, if only the mouse V, D and J gene segments are
replaced by human homologues, a functional mouse/human hybrid
antibody comprising human V.sub.H and V.sub.L domains and mouse
constant (effector) regions will result following antigen challenge
(WO94/04667). If all murine gene segments are replaced by human
homologues, then an entirely human immunoglobulin will result
following antigen challenge (U.S. Pat. No. 6,596,541). One
perceived advantage of this approach is that, provided only coding
regions are exchanged, then the resultant transgene retains all
mouse regulatory elements, so ensuring maximal response to antigen
challenge. This approach provides high serum titres of high
affinity human antibodies or mouse/human hydrid antibodies
depending on the final configuration of the transgenes. In reality,
however, the replacement of all the individual V, D and J segments
in the mouse genome by homologous recombination is a long and
arduous task. Similarly, the construction of a heavy chain
transgene comprising all 39 functional human V, D and J segments
with constant (effector) regions is technically very demanding.
[0013] Therefore, there remains a need in the art for methods not
dependent on the deletion of large segments of genomic DNA, or
multiple deletions, which allow for (i) the simplified generation
of mice either with a substantially reduced ability to express
endogenous heavy and light chain immunoglobulin genes in a B-cell
specific manner in response to antigen challenge, or which express
endogenous immunoglobulin heavy and/or light chain genes in B-cells
following antigen challenge but encode immunoglobulin heavy and
light chain proteins which lack the ability to assemble as
functional immunoglobulin tetramers, resulting in a non-productive
response to antigen challenge; (ii) simplified and reproducible
methods for the construction and B-cell-specific expression, of
multiple heavy chain transgenic loci which may collectively
comprise all 39 human V gene segments, but individually comprise
preferred smaller groups of V gene segments, each in combination
with all D and J gene segments and some or all constant (effector)
regions, and whose functional expression is antigen dependent and
ultimately determined by allelic exclusion; and (iii) the ability
to select against hybridomas expressing residual endogenous mouse
immunoglobulins and to select for hybridomas expressing and
secreting assembled immunoglobulin tetramers comprising the full V
segment repertoire present on the heavy chain transgenic loci, or
alternatively to select for hybridomas which express a subset of V
gene segments present on one as opposed to another heavy chain
transgene locus.
THE INVENTION
[0014] According to a first aspect of the present invention, there
is provided a non-human mammal containing an endogenous lambda
light chain gene locus, an endogenous kappa light chain gene locus
and an endogenous heavy chain gene locus, each of which can
re-arrange so that immunoglobulin heavy and light chain genes are
formed and expressed in B-cells following antigen challenge but
said loci have been mutated so that the ability to form functional
immunoglobulin tetramers comprising re-arranged heavy and light
chains produced from said mutated loci has been substantially
reduced or eliminated.
[0015] In the non-human mammal, at least one of the endogenous
lambda light chain gene locus, the endogenous kappa light chain
gene locus and the endogenous heavy chain gene locus may have been
mutated by the introduction of a frame shift mutation, a
polypeptide-encoding sequence and/or one or more stop codons into
the or each endogenous locus.
[0016] The mutation is preferably an insertion of less than 50
nucleotides.
[0017] In the non-human mammal, the expression of at least one of
the endogenous lambda light chain gene locus, the endogenous kappa
light chain gene locus and the endogenous heavy chain gene locus
may have been substantially reduced by elimination of part or all
of the LCR in the or each locus.
[0018] Preferably, the introduction is in the endogenous kappa
light chain gene locus.
[0019] Alternatively, the introduction is in the endogenous lambda
light chain gene locus.
[0020] In another alternative, the introduction is in the
endogenous kappa light chain gene locus and the endogenous lambda
light chain gene locus.
[0021] The introduction may also be in the endogenous heavy chain
gene locus.
[0022] The introduction may in a further alternative be in the
endogenous kappa light chain gene locus and in the endogenous heavy
chain gene locus.
[0023] The introduction may in a yet further alternative be in the
endogenous lambda light chain gene locus and in the endogenous
heavy chain gene locus.
[0024] The introduction may in an even further alternative be in
the endogenous lambda light chain gene locus, in the endogenous
kappa light chain gene locus and in the endogenous heavy chain gene
locus.
[0025] Preferably, there is an introduction, as defined in above,
in the endogenous kappa light chain gene locus and there is a
partial or complete LCR deletion, as defined above, in the
endogenous lambda gene locus.
[0026] If desired, the endogenous heavy chain gene locus may be
mutated such that heavy chain gene rearrangement, mRNA
transcription and protein synthesis occurs but that B-cell
activation is blocked.
[0027] Preferably, the non-human mammal as defined above comprises
a transgene comprising one or more heterologous kappa light chain
gene loci and associated B-cell specific regulatory elements.
[0028] The non-human mammal may further comprises a transgene
comprising one or more heterologous lambda light chain gene loci
and associated B-cell specific regulatory elements.
[0029] In the non-human mammal as defined above, the transgene may
comprise a heterologous light chain gene locus comprises a dominant
selective marker gene.
[0030] The non-human mammal as defined above may also comprise a
transgene comprising one or more one or more heterologous heavy
chain gene loci and associated B-cell specific regulatory
elements.
[0031] If desired, the non-human mammal may comprise two or more
transgenes comprising two or more different heterologous heavy
chain gene loci and associated B-cell specific regulatory
elements.
[0032] In the non-human mammal the or each transgene may comprise a
heterologous heavy chain gene locus comprises a dominant selective
marker gene.
[0033] In the non-human mammal, each heterologous heavy chain gene
locus may comprise a CTCF binding sites.
[0034] Preferably, the non-human mammal comprises a transgene
comprising a heterologous kappa light chain gene locus and a
transgene comprising one or more heterologous heavy chain loci.
[0035] Alternatively, the non-human mammal may comprise a transgene
comprising a heterologous lambda light chain gene locus and a
transgene comprising one or more heterologous heavy chain loci.
[0036] In a further alternative, the non-human mammal may comprises
a transgene comprising a heterologous kappa light chain gene locus,
a transgene comprising a lambda light chain gene locus and a
transgene comprising one or more heterologous heavy chain gene
loci.
[0037] Preferably, each heterologous locus incorporates a cognate
LCR.
[0038] Each heterologous locus is preferably a human locus.
[0039] However, each heterologous locus may be a hybrid locus
comprising variable regions and constant regions derived from more
than one species, such as a hybrid locus comprising human variable
regions and rat or murine constant regions.
[0040] The non-human mammal may comprise groups of transgenes
comprising different groups of different heterologous heavy chain
gene loci, wherein each group of transgenes comprises a different
dominant selective marker gene.
[0041] Alternatively, the non-human mammal may comprise transgenes
comprising heterologous light chain loci and transgenes comprising
heterologous heavy chain loci, wherein transgenes comprising
heterologous light chain loci and transgenes comprising
heterologous heavy chain loci each comprise a different dominant
selective marker gene.
[0042] The non-human mammal is preferably a rodent, such as a
mouse.
[0043] According to a second aspect, the present invention provides
a method of producing an antigen-specific heterologous monoclonal
antibody comprising:
(a) immunising a non-human transgenic mammal of any of the
preceding claims with the antigen; (b) preparing hybridomas or
immortalised B-cell lines each of which produces a monoclonal
antibody from the B-cells of the immunised transgenic mammal; (c)
optionally selecting at least one hybridoma or immortalised B-cell
line expressing the heterologous antibody by use of the dominant
selective marker genes present in the transgenes comprising the
heterologous immunoglobulin light chain and heavy chain loci; and
(d) selecting at least one hybridoma or immortalised B-cell line
which produces an antibody which binds specifically to the
antigen.
[0044] According to a further aspect of the present invention,
there is provided a method of deriving a mammalian, preferably
human, antibody from a hybrid antibody comprising:
(a) carrying out the method as described above; (b) selecting at
least one hybridoma or immortalised B-cell line which produces an
antibody which binds specifically to the antigen and comprises
V.sub.H and V.sub.L binding domains of the species of choice; (c)
cloning and sequencing the V.sub.H and V.sub.L domains; (d)
recloning selected sequences comprising the V.sub.H and V.sub.L
binding domain coding sequences with constant effectors domains of
choice from the same species; and (e) co-expressing the recloned
sequences encoding heavy and light chain polypeptides of the
desired species using an expression vector in a cell type of
choice.
[0045] According to a yet further aspect of the invention, there is
provided a method for the production of the non-human mammal as
defined above comprising mutating the endogenous heavy chain gene
locus, the endogenous kappa light chain gene locus, and optionally
the endogenous lambda light chain gene locus in a mammalian
progenitor cell and producing a mammal from said progenitor cell,
wherein the mutation is such that, in the mammal, each locus can
re-arrange so that immunoglobulin heavy and light chain genes are
formed and expressed in B-cells following antigen challenge but the
ability to form functional immunoglobulin tetramers comprising
re-arranged heavy and light chains produced from said mutated loci
has been substantially reduced or eliminated.
[0046] Preferably, the progenitor cell is a non-human embryonic
stem cell.
[0047] The non-human mammal is preferably a rodent, such as a
mouse.
[0048] The present invention also provides use of antigen-specific,
heterologous, functional immunoglobulin tetramers, preferably
human, derived using a non-human mammal or the method as defined
above as medicaments, diagnostics or reagents.
[0049] The present inventors have surprisingly overcome the
limitations of the prior art, through the development of simplified
methods for the production of non-human mammals, particularly mice,
wherein the functional expression of endogenous kappa and/or lambda
light chain genes has been substantially reduced through either the
constant regions being rendered non-functional as a result of a
small insertional event preferably in the kappa constant region
and/or lambda constant region, leading to a frame-shift or
premature termination of mRNA translation, or elimination of part
or all of the cognate endogenous LCR This contrasts with
alternative strategies requiring the functional silencing of
endogenous immunoglobulin light chain genes by complete or partial
deletion of some or all light chain gene coding sequence.
[0050] The strategies described can be equally well applied to
immunoglobulin heavy chain gene loci. Thus, for instance, complete
or partial deletion of the LCR will result in a substantial
reduction of heavy chain gene expression. The introduction of
sequences leading to a frame-shift or premature termination of mRNA
translation preferably in the C.sub.H1 domain of the .mu.C region,
but alternatively in the C.sub.H1 region of all immunoglobulin
isotype constant regions, will substantially reduce, eliminate or
block the formation of immunoglobulin tetramers with light chains.
Similarly, as previously described, if IgM expression on the cell
surface of pre-B cells is blocked in vivo by premature termination
of mRNA translation, then further development is arrested,
resulting in a non-productive response to antigen challenge
(Kitamura et al. (1991) Nature, 350, 423-426). The introduction of
a heavy chain immunoglobulin transgene rescues B-cell expansion and
functional immunoglobulin tetramers comprising transgene-encoded
heavy chain and endogenous murine light chains circulate in the
plasma.
[0051] Thus, the ability of the endogenous light chain loci and/or
heavy chain loci to produce heavy and light chains that interact
and form functional immunoglobulin tetramers can be eliminated or
substantially reduced following the introduction of a frame shift
mutation, leading to the synthesis of irrelevent protein sequence,
generally accompanied by premature termination of protein synthesis
due to the presence of out-of-frame stop codons.
[0052] This can be achieved by the insertion of foreign DNA or a
small deletion of DNA in or upstream of heavy or light chain
polypeptide sequences responsible for the formation of functional
immunoglobulin tetramers. The preferred approach is to insert new
sequence. Effective insertional events designed either to cause a
frame shift in the amino acid coding sequence, resulting in the
premature termination of translation of the encoded mRNA, can be
limited to the introduction of a single nucleotide. Thus, the
insertion of one or more nucleocleotides within the coding region
may lead to a frameshift. Alternatively the insertion of in-frame
sequence encoding additional peptide sequences comprising a stop
codon will also result in the synthesis of a truncated protein
(U.S. Pat. No. 5,591,669). Targeted insertional events may also
include the introduction of selective marker genes and other
functionalities, provided that all the endogenous sequence is
retained and the resulting fusion protein disrupts the formation of
immunoglobulin tetramers, or the presence of one or more in-frame
stop codon(s) leads to premature termination of mRNA translation,
resulting in the synthesis of a truncated protein unable to form
functional immunoglobulin tetramers.
[0053] Preferably, insertional events are in immunoglobulin .kappa.
light chain constant regions. Optionally, insertional events are in
immunoglobulin .kappa. and/or .lamda. light chain constant regions.
In practice, insertions may comprise any recombinase recognition
site(s) such as a lox site. This alone may lead to a frame shift.
The inclusion of additional nucleotides to ensure a frameshift, or
codons for one or more stop codons, may also add to the
effectiveness of the inserted sequence in the disruption of heavy
and light chain dimerisation through the interaction of the heavy
chain C.sub.H1 domains and the light chain .kappa. and/or .lamda.
constant regions. An insertion can comprise a single nucleotide,
and is preferably less than 50 nucleotides. The insertion may
result in only a frameshift, but preferably is designed such that
one or more stop codons results in premature termination of mRNA
translation. The insertion is performed using homologous
recombination using arms which flank the site of insertion.
Preferably, a selective marker is included during the manipulation
process and then subsequently excised, leaving the recognition
sites alone plus any additional inserted sequence in situ in the
genome, e.g. lox sites.
[0054] It will be obvious to one skilled in the art that frame
shifts and the synthesis of truncated proteins can also be achieved
by the deletion of one or more nucleotides in the coding sequence
and the inclusion of minimal additional sequence comprising stop
codons. Preferably the insertion or deletion event occurs in the
constant regions of the heavy and light chain genes and not in the
multiple V, D and J regions of the endogenous loci. The preferred
choice is the kappa light chain constant region.
[0055] Thus, there is no dependency on large scale gene deletion
strategies for the elimination of endogenous immunoglobulin gene
rearrangement or mRNA transcription. Identical insertional
strategies can be used to inhibit the ability of endogenous heavy
chain immunoglobulin to form functional tetrameric complexes with
light chain through targeted insertional events within the C.sub.H1
regions of heavy chain isotypes. Preferably, the expression of
endogenous immunoglobulin heavy chain genes is blocked at the
pre-B-cell stage such that the endogenous heavy chains are not
expressed on the surface of B cells and productive expression
resulting from B-cell expansion is blocked, using strategies
similar to those described by Kitamura et al. (1991) Nature, 350,
423-426, whilst light chain association with the functional
C.sub.H1 of the endogenous IgM is inhibited by an insertional event
leading to the translation of kappa and/or lambda light chain mRNA
encoding light chain constant region(s) unable to functionally
interact with the immunoglobulin heavy chain, preventing the
formation of a functional endogenous immunoglobulin tetramer.
[0056] Provided the functional assembly of endogenous
immunoglobulin tetramers is functionally impaired, B-cell expansion
with associated affinity maturation of V.sub.H domains will be
limited to and be dependent on the presence and expression of
exogenous immunoglobulin heavy and light chain transgenes. The
immunoglobulin transgenes will participate in the allelic exclusion
process of the chosen non-human mammalian host in a B-cell specific
manner, resulting in a productive response to antigen challenge,
B-cell expansion and circulating, transgene-encoded
antigen-specific immunoglobulin tetramers.
[0057] There is also provided a non-human mammal in which
endogenous lambda light chain gene expression is substantially
reduced by elimination of part or all of the lambda light chain LCR
and endogenous kappa light chain gene expression is substantially
reduced by elimination of part or all of the kappa light chain LCR.
In one embodiment of the invention, only endogenous kappa light
chain expression, or only lambda light chain expression, is
substantially reduced by elimination of part or all of the relevant
LCR.
[0058] The endogenous light chain loci may retain functionality in
that they can rearrange and be transcribed into functional mRNA,
but that the levels of transcription are substantially reduced
through the elimination of some or all of the endogenous LCR
functionality (WO90/10077). LCR functionality is removed by gene
targeting nuclease hypersensitive sites in mouse ES cells or, in
the absence of ES cells, by cloning using either nuclear transfer
(Soulter (1998) Nature, 394, 315-316) or iPS cells (see Gottweiss.
and Minger (2008) Nature Biotechnology, 26, 271-272) derived from
other mammalian species. Alternatively, disruption of the heavy or
light chains could be achieved through targeted mutagenesis, such
as zinc finger nuclease technology and DNA repair (e.g
http://www.sigmaaldrich.com/life-science/functional-genoinics-and-rnai/zi-
nc-finger-nuclease-technology).
[0059] Endogenous kappa light chain gene expression may be
substantially reduced by elimination of part or all of the kappa
light chain LCR, and the lambda light chain gene may be
functionally silenced following deletion or insertional events.
[0060] Endogenous lambda light chain gene expression may be
substantially reduced by elimination of part or all of the lambda
light chain LCR, and the kappa light chain gene may be functionally
silenced following deletion or insertional events.
[0061] Endogenous kappa light chain gene may be functionally
silenced following LCR elimination or insertional events.
[0062] The invention also provides non-human mammals in which
either or both endogenous kappa light chain gene expression and
endogenous lambda light chain gene expression are substantially
reduced by elimination of part or all of the kappa light chain LCR
and/or elimination of part or all of the lambda light chain LCR.
The endogenous kappa gene may be functionally silenced and
endogenous lambda gene activity substantially reduced by
elimination of part or all of the lambda light chain LCR.
[0063] Kappa light chain gene expression may be substantially
reduced by elimination of part or all of the kappa light chain LCR
reduced and lambda gene expression functionally silenced.
[0064] Only endogenous kappa light chain gene expression may be
substantially reduced by elimination of the kappa chain LCR or
functionally silenced by deletion or insertional events.
[0065] The non-human mammals having functionally silenced
endogenous kappa and/or lambda light chain gene expression or
substantially reduced endogenous kappa and/or lambda light chain
gene expression as described above may also have reduced or
functionally silenced endogenous heavy chain gene expression.
According to one embodiment, endogenous heavy chain gene expression
is reduced following the deletion of some or all nuclease
hypersensitive sites which comprise the LCR or functionally
silenced following deletion or insertional events in the non-human
mammals of the invention. Preferably, the expression of endogenous
heavy chain genes is blocked at the pre-B-cell stage such that the
endogenous heavy chains are not expressed on the surface of B cells
and productive expression resulting from B-cell expansion is
blocked using strategies similar to those described by Kitamura et
al. (1991) Nature, 350, 423-426.
[0066] The non-human mammals described above may further comprise
one or more transgenes comprising heterologous heavy and light
chain loci and associated B-cell specific regulatory elements,
preferably comprising cognate LCRs.
[0067] In the context of the present invention, the term
`heterologous` means a nucleotide sequence or a locus as herein
described which is not endogenous to the mammal in which it is
located.
[0068] The non-human mammal may thus comprise a transgene
comprising a heterologous kappa light chain locus and associated
B-cell specific regulatory elements, preferably comprising an LCR
and/or a transgene comprising a heterologous lambda light chain
locus and associated B-cell specific regulatory elements,
preferably comprising an LCR.
[0069] The presence of cognate LCRs is not essential for B-cell
specific expression. Their inclusion within loci ensures that high
level transgene expression occurs at every site of integration and
is not dependent on random integration events, only some of which
fortuitously occur within chromatin regions actively transcribed in
B-cells.
[0070] The use of cognate LCRs significantly reduces the number of
transgenic animals required to be screened for antibody expression
and allows the insertion of more than one gene locus, with the
certainty that all loci inserted will be expressed at essentially
normal levels in a B-cell specific manner. Thus, the use of LCR
technology, combined with the surprising observation that allelic
exclusion mechanisms will discriminate between endogenous
immunoglobulin genes and multiple competing transgenes, opens the
way for the assembly of transgenic non-human mammals comprising one
or more immunoglobulin heavy or light chain gene loci, each locus
being of reduced V gene complexity relative to the endogenous genes
and comprising a relatively manageable piece of DNA (<300 Kb) to
assemble in vitro relative to the endogenous loci (1-2 Mb). For
example, the 39 functional human immunoglobulin heavy chain V gene
segments may be cloned into two or more immunoglobulin heavy chain
loci. Each will comprise different V gene segments, but have in
common D and J gene segments, and constant (effector) regions. The
inclusion of the LCR ensures that each is expressed in an identical
manner, irrespective of the site of integration within the genome.
Thus, the inclusion of two or more small loci in this manner
provides the same V gene complexity of a single, more complex gene
present in a single, large gene fragment which is technically
difficult to manipulate.
[0071] Each heterologous light chain locus may comprise V.sub.L
gene segments, J gene segments and a light chain constant region
segment. Preferably, the V.sub.L and J gene segments and light
chain constant region segment are derived from the same mammalian
source, for example rodent, pig, cow, goat or sheep. Preferably,
they are of human origin.
[0072] Alternatively, the heterologous light chain loci may be
hybrid loci comprising variable domains of mammalian origin,
preferably of human origin, and constant (effector) regions from a
different mammal, such as, but not limited to, mouse, rat, hamster,
rabbit, pig, goat and cow. Where the host mammal is a mouse,
preferably the constant regions are of rodent origin, more
preferably mouse or rat. Such heterologous light chain loci
comprise V.sub.L and J segments preferably from one species only
and a light chain constant region from another species.
[0073] Where hybrid kappa light chain transgenes are contemplated,
the V.sub.L and J gene segments are preferably from the same
species, contributing the heavy chain V, D and J gene segments, and
are preferably of human origin. The kappa light chain constant and
heavy chain constant regions are also preferably derived from the
same species but a species different from that contributing
variable domains and are preferably of rodent origin, and
preferably derived from rat or mouse.
[0074] A feature of all light chain transgenes contemplated is
that, following antigen challenge, the light chain rearranges in a
B-cell specific manner and that, following transcription and
translation, the resulting light chain is capable of complexing
with transgene-derived heavy chain immunoglobulin produced in the
same B-cell. The productive expression of immunoglobulin tetramers
gives rise to B-cell expansion and transgene-encoded,
antigen-specific tetravalent immunoglobulin complexes accumulate in
serum in the absence of significant levels of endogenous
immunoglobulin tetramers.
[0075] Where endogenous lambda light chain expression has not been
functionally suppressed, then low levels of host or hybrid antibody
comprising endogenous lambda light chains will be detectable. These
may be discarded following screening of hybridoma supernatants.
[0076] In humans, there are 36 functional kappa V.sub.L gene
segments, five J.sub.L gene segments and a single kappa light chain
constant region (http://imgt.cines.fr). Preferably, a heterologous
kappa light chain locus present in a transgene in the non-human
mammal of the invention comprises one or more human V.sub.L gene
segments, all human J.sub.L gene segments and a single human kappa
light chain constant region. Optionally, the human kappa light
chain constant region may be replaced by an alternate mammalian
kappa light chain constant region, preferably of rat or mouse
origin.
[0077] A heterologous lambda light chain locus present in a
transgene in the non-human mammal of the invention preferably
comprises a murine lambda LCR, and human lambda light chain V1 and
V2 gene segments, human lambda J1, J2, J3 and J4 gene segments, and
human lambda light chain C1, C2, C3 and C4 constant region segments
(WO90/10077 and WO2003/000737). Optionally, the human lambda light
chain C1, C2, C3 and C4 constant region segments may be replaced by
alternative lambda light chain constant regions, preferably of rat
or mouse origin.
[0078] A heterologous heavy chain locus present in a transgene in
the non-human mammal of the invention preferably comprises a heavy
chain immunoglobulin LCR, preferably of murine origin, one or more
human V gene segments, one or more J gene segments and one or more
D gene segments. Preferably, 10 or more human different V gene
segments and all human J and D gene segments are present.
[0079] The locus also may comprise one or more human constant
(effector) regions, preferably the .mu. and .gamma. constant
regions. Optionally, the human constant effector regions may be
replaced by effector regions from other non-human mammals. Where
the non-human mammalian host is a mouse or a rat, then preferably
constant (effector) regions are derived from rat or mouse. In
contrast with human, the transmembrane domains of the mouse and rat
B-cell receptor complex (BCR) are 100% conserved. Thus, mice
transgenic for antibody loci comprising rat constant (effector)
region genes should function as well as those comprising mouse
constant (effector) region genes following antigen challenge, and
may be superior to those comprising human constant (effector)
region genes (De Franco et al. (1995) Ann NY Acad. Sci., 766,
195-201). The transgenes may comprise heavy chain, kappa and lambda
light chain LCRs, preferably of mouse or human origin. LCRs which
function across all mammalian species are known and may be
substituted for human or mouse LCRs in the transgenes (Li et al.
(1999) Trends Genet., 10, 403-8).
[0080] Where the generation of fully human antibodies is
contemplated, cloned human antigen-specific V.sub.H and V.sub.L
binding domains derived from hybrid antibodies expressed by
hybridomas can be fused to human constant heavy and light chain
constant regions, so deriving fully human tetrameric antibodies of
any class.
[0081] As a further refinement, each immunoglobulin kappa and/or
lambda light chain locus may also comprise a dominant selective
marker gene.
[0082] The dominant selective marker genes incorporated in the loci
may have the same or different mechanisms of action. For the
purposes of the invention, any dominant selective marker gene can
be used, provided that expression of the gene confers a selective
benefit to hybridomas or transformed B-cells derived from the
non-human transgenic mammal in the presence of a selective or toxic
challenge. Typically, the dominant selective marker genes will be
of prokaryotic origin and will be selected from a group which
either confer resistance to toxic drugs, such as puromycin (Vara et
al. (1986) NAR, 14, 4617-4624), hygromycin (Santerre et al. (1984)
Gene, 30, 147-156) and G418 (Colbere-Garapin et al. (1981) 150,
1-14), or comprise genes which obviate certain nutritional
requirements such that their expression converts a toxic substance
into an essential amino acid, for example the conversion of indole
to tryptophan or the conversion of histidinol to histidine
(Hartmann and Mulligan, (1988) PNAS, 85, 8047-8051).
[0083] A necessary requirement of this aspect of the invention is
that the dominant selective marker is incorporated within the
immunoglobulin light chain transgenic locus and is co-expressed
with the desired immunoglobulin light chain allele, so ensuring
B-cell specific expression. Alternatively, the drug resistance gene
may be inserted into an endogenous or exogenous (transgenic)
immunoglobulin locus using homologous recombination in combination
with ES cells or nuclear transfer approaches (te Riele et al.
(1992), PNAS, 89, 11, 5128-5132).
[0084] The non-human mammal may also comprise a transgene or
transgenes comprising a heterologous heavy chain locus and
associated B-cell specific LCR and regulatory elements. More than
one different transgenic heavy chain gene locus may be present,
each comprising an LCR and regulatory elements.
[0085] The heavy chain gene loci, each comprising one or more V
gene segments, one or more D gene segments, one or more J gene
segments, and one or more constant (effector) regions are
introduced as transgenes, each locus comprising a cognate LCR.
[0086] Each locus comprises the 5' and 3' regulatory elements
necessary to drive B-cell specific expression. Each heavy or light
chain locus is expressed in an essentially identical manner to the
endogenous loci in response to antigen challenge, leading to the
circulation in mouse serum of transgene-encoded, antigen-specific
affinity-matured, tetrameric immunoglobulins.
[0087] Preferably, each heavy chain gene locus comprises one or
multiple V gene segments, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60 or more
V gene segments, which may be derived from any vertebrate species,
preferably a non-human mammal. Preferably, not more than 20 V gene
segments are present on any single heavy chain locus.
[0088] In one embodiment, each locus may comprise only one V gene
segment. In one alternative of this embodiment, a number of V gene
segments are present and each V gene segment is different from all
other V gene segments. In this embodiment, the V gene segments in
any one locus may all be derived from an organism of the same
species, e.g. all V gene segments may be of human origin.
[0089] Alternatively, the V gene segments in any one locus may be
derived from organisms of different species, e.g. some V gene
segments from human and others from sheep, cattle, rabbits,
camelids or even shark. In a second alternative, each V gene
segment is identical to all the other V gene segments. Irrespective
of the number and nature of the V gene segments present, the
remaining D and J gene segments in each locus may be the same as or
may be different from those in all the other loci.
[0090] It is thus envisaged that the non-human mammal may contain
multiple copies of a heavy chain gene locus. This has the advantage
of optimising the chances that a productive re-arrangement in a
B-cell will take place, thus allowing the optimal production of an
immunoglobulin heavy chain for antigen recognition.
In another embodiment, each locus comprises multiple V gene
segments.
[0091] Preferably, the V gene segments are of human origin.
[0092] The term `V gene segment` encompasses any naturally
occurring V gene segment derived from a vertebrate, including, but
not limited to, sharks, rodents, camelids and human. The V gene
segment must be capable of recombining with a D gene segment, a J
gene segment and a gene segment encoding a heavy chain constant
(effector) region to generate an immunoglobulin heavy chain
antibody capable of complexing with either a kappa or lambda
immunoglobulin light chain when the re-arranged nucleic acid is
expressed in B-cells.
[0093] A V gene segment also includes within its scope any gene
sequence encoding a natural or engineered homologue, derivative or
protein fragment which is capable of recombining with a D gene
segment, a J gene segment and a gene segment encoding a heavy chain
constant region to generate an immunoglobulin heavy chain antibody
capable of complexing with either a kappa or lambda immunoglobulin
light chain when the re-arranged nucleic acid is expressed in
B-cells. A V gene segment may, for example, be derived from a
T-cell receptor locus.
[0094] Preferably, the multiple heavy chain loci of the invention
comprise any number or combination of the 39 functional human V
gene segments and engineered variants thereof. These may be on any
number of loci, e.g. four loci comprising eight V gene segments
plus one locus comprising seven V gene segments; seven loci
comprising four V gene segments plus one locus comprising three V
gene segments; or thirty-nine loci comprising one V gene segment
each.
[0095] Human V genes are classified into seven families, V.sub.H1
to V.sub.H7, and the individual genes within each family numbered.
The frequency at which each gene is used is dependent on the
varying requirements of the particular immune response. For
example, the genes of family V.sub.H3 may be preferentially used in
comparison to those of family V.sub.H5 when responding to bacterial
antigens. Therefore, in a further preferred embodiment of the
invention, groups of V gene segments which have been shown to be
useful for generating an antibody response against specific
antigens are grouped into separate loci, each comprising a
different dominant selective marker gene. The V gene segments may
be grouped according to family or they may be grouped according to
individual function. For example, if the V genes of family V.sub.H3
are shown to be useful for generating an immune response against
bacterial antigens, then these may be used to generate a locus
which is particularly useful for generating heavy chain-only
antibodies against bacterial antigens. Alternatively, if it is
shown that several individual genes from families V.sub.H3 and
V.sub.H5 are useful for generating an immune response against
bacterial antigens, then these may be grouped together and used to
generate a locus which is particularly useful for generating
antibodies against bacterial antigens.
[0096] An "immunoglobulin heavy chain locus" in the context of the
present invention relates to a minimal micro-locus encoding a
V.sub.H domain comprising one or more V gene segments, one or more
D gene segments and one or more J gene segments, operationally
linked to one or more gene segments encoding heavy chain constant
(effector) regions. Preferably, the primary source of antibody
repertoire variability is the CDR3 region formed by the selection
of V, D and J gene segments and by the V-D and D-J junctions.
[0097] The advantage of the present invention is that antibody
repertoire and diversity obtained in the rearranged V, D and J gene
segments can be maximised through the use of multiple
immunoglobulin heavy chain gene loci in the same transgenic
non-human mammal by exploiting allelic exclusion. The process of
allelic exclusion, which randomly chooses one of the loci to start
recombination, followed by the next locus if the first
recombination was non-productive, etc., until a productive
recombination has been produced from one of the loci, would ensure
that actually all the V gene segments present in the combined loci
would be part of the overall recombination process.
[0098] To enhance the probability of all V.sub.H gene segments in
any given immunoglobulin heavy chain locus participating
productively in VDJ rearrangements, CTCF sites may be
interdispersed between groups of VH gene segments.
[0099] The immunoglobulin locus in its normal configuration appears
to have a three dimensional folded structure based on distance
measurements made in B cells and measuring in the direction of and
through the VH region (Jhunjhunwala et al. (2008) Cell, 133,
265-279). Such a folded or looped structure explains why different
V.sub.H region can be used equally efficiently even when they are
arranged at very different distances from the D, J and constant
region of the immunoglobulin heavy chain locus.
[0100] It has also recently become clear that a folded structure
formed by looping in a number of loci is mediated through a
particular chromatin binding protein called CTCF. CTCF appears to
be directly involved in the formation of chromatin looping as
demonstrated by mutagenesis experiments (Splinter et al. (2006)
Genes Dev., 20, 2349-2354). More recently it has been shown that
cohesin, the protein complex that holds sister chromatids together,
is present at CTCF binding sites (Wendt et al. (2008) Nature, 451,
796-801). The inclusion of a number of CTCF sites from the
immunoglobulin V.sub.H region (Kim et al. (2007) Cell, 128,
1231-1245; Denger, Wong, Jankevicius and Feeney (2009) J. Immunol.,
182, 44-48) increases the probability that the V.sub.H region of a
transgenic immunoglobulin heavy chain locus can be folded properly
and allow efficient usage of all the different V gene segments
present in that locus.
[0101] Each transgene comprising a heterologous heavy chain locus
may further comprise a dominant selective marker. Preferably, the
dominant selective marker is different from the dominant selective
marker introduced within the kappa or lambda light chain loci.
[0102] For the purpose of the invention, any dominant selective
marker gene can be used, provided that expression of the gene
confers a selective benefit to hybridomas or transformed B-cells
derived from the non-human transgenic mammal in the presence of a
selective or toxic challenge. Typically, the dominant selective
marker genes will be of prokaryotic origin and will be selected
from a group which either confer resistance to toxic drugs, such as
puromycin (Vara et al. (1986) NAR, 14, 4617-4624), hygromycin
(Santerre et al. (1984) Gene, 30, 147-156) and G418
(Colbere-Garapin et al. (1981) 150, 1-14), or comprise genes which
obviate certain nutritional requirements such that their expression
converts a toxic substance into an essential amino acid, for
example the conversion of indole to tryptophan or the conversion of
histidinol to histidine (see Hartmann and Mulligan (1988) PNAS, 85,
8047-8051).
[0103] A necessary requirement of the invention is that the
dominant selective marker(s), if used, reside within the
immunoglobulin heavy chain transgenic loci, so ensuring B-cell
specific co-expression. Alternatively, the drug resistance gene may
be inserted into an endogenous or exogenous (transgenic)
immunoglobulin locus using homologous recombination in combination
with ES cells or nuclear transfer approaches (e.g. to Riele,
Robanus Maandag and Berns (1992), PNAS, 89, 11, 5128-5132).
[0104] The same dominant selective marker gene may be incorporated
within all heavy chain loci. Alternatively, different heavy chain
loci or groups of heavy chain loci may comprise different dominant
selective marker genes.
[0105] Hybridomas or transformed B-cells, preferably transformed
long-lived plasma cells (Slifka et al (1998) Immunity, 8, 363-372),
derived from transgenic mice of the invention expressing tetrameric
antibodies may be selected for, free of cells expressing endogenous
immunoglobulin, due to the co-expression of a functional dominant
selective marker gene within the transgenic light chain loci.
Furthermore, hybridomas or transformed B-cell lines expressing
antibodies derived from specific groups of V segments present on
transgenic heavy chain loci may also be selected for due to the
presence and co-expression of different dominant selective markers
within heavy chain loci relative to the dominant selective markers
incorporated within the light chain loci. For example, the
inclusion of a puromycin resistance gene within the kappa light
chain transgenic locus would allow selection of all cells
expressing the kappa light chain transgene. Alternatively, the
inclusion of the G418 resistance gene within a heavy chain
transgenic locus comprising preferred V gene segments would allow
the selection of all cells expressing the preferred V gene
segments.
[0106] In particular, the invention provides a method of producing
an antigen-specific heterologous monoclonal antibody
comprising:
(a) immunising a non-human transgenic mammal as described above
with the antigen; (b) preparing hybridomas or immortalised B-cell
lines each of which produces a monoclonal antibody from the B-cells
of the immunised transgenic mammal; (c) selecting at least one
hybridoma or immortalised B-cell line expressing the heterologous
antibody by use of the dominant selective marker genes present in
the transgenes comprising the heterologous immunoglobulin light
chain and heavy chain loci; and (d) selecting at least one
hybridoma or immortalised B-cell line which produces an antibody
which binds specifically to the antigen.
[0107] The invention is now described, by way of example only, in
the following detailed description with reference to the following
Figures.
FIGURES
[0108] FIG. 1A: The 3' end of the mouse IgH locus.
[0109] The map is copied from the IMGT database
(http://imgt.cines.f). The scale is in kilobases (kb). Green
squares, functional V.sub.H segments; red and yellow squares,
non-functional V.sub.H segments; orange squares, J.sub.H segments;
blue squares, constant regions. The intronic IgH enhancer and the
LCR at the 3' end of the locus are not indicated.
[0110] FIG. 1B: Strategy to disable IgH
[0111] The top line shows the C.mu. region of the mouse with the
different exons including the two exons coding for the membrane
form of IgM. To the left are the J, D and V.sub.H region of the
locus, to the right the other constant regions starting with
C.delta.. The bottom lines show part of the amino acid sequence of
the normal M1 exon after recombination. The DNA sequence shows the
integration sequence. The stop codon is in red, the Spe I site in
red and blue.
[0112] FIG. 1C: Recombination in ES cells to disable C.mu.
[0113] This figure shows two of the recombination positive clones
of ES cells by PCR analysis covering the 3' side of the recombinant
insert. The larger fragment corresponds to the insertion of a neo
selectable marker into the M1 exon at the position indicated in
FIG. 5B.
[0114] FIG. 1D: FACS analysis of the C.mu. knockout mice
[0115] After the M1 exon has been interrupted by the stop codon and
neo gene, the ES cells are introduced into blastocysts to obtain
chimeras. These are bred to homozygosity and the blood is analyzed
for the presence of B cells. The top two panels show a FACS
analysis of a normal wild type mouse and a heterozygous interrupted
M1 exon mouse using the B cell markers B220 and CD19. The bottom
two panels show two homozygous mice, which show no B220.sup.+,
CD19.sup.+ cells, i.e. no functional B cells.
[0116] FIG. 1E: Deletion of neo after breeding to recombinant
mice
[0117] The mice of FIG. 5D are crossed with recombinase-expressing
mice to delete the neo gene. The two lanes on the right show a long
range PCR product over the neo gene in the parent animals, the next
two lanes to the left two heterozygous mice carrying a deletion of
neo and a wt allele. The next four lanes lane to the left are wild
type mice, while the lane furthest left show a mouse carrying a
homozygous deletion of the neo gene with an inactivated M1
exon.
[0118] FIG. 2: A map of the mouse Ig.sub..kappa. locus
[0119] The map is copied from the IMGT database
(http://imgt.cines.f). The scale is in kilobases (kb). Green
squares, V.sub..kappa. segments; orange squares, J.sub..kappa.
segments; blue square, constant region; black circle
.kappa.-enhancer and red circle .kappa.-LCR sequences.
[0120] FIG. 3: Mouse V.sub..kappa. knockdown.
[0121] Scheme to functionally inactivate the mouse Ig.sub..kappa.
locus by deletion of the LCR
[0122] A lox neomycin resistance gene cassette is inserted by
homologous recombination in ES cells replacing the
3'.sub..kappa.-LCR (bottom line). Treatment with cre recombinase
(+cre) will remove all sequences between the lox sites, leaving a
single lox site in the .kappa. locus (top line).
[0123] FIG. 4: A mouse C.sub..kappa. insertion to inactivate the
.kappa. locus.
[0124] The locus (top line) is the same as in FIG. 3. The bottom
shows the sequence at the 5' end of the C.sub..kappa. exon (blue in
top line) with the amino acid coding written above the bases. The
GG base pair at the start is immediately flanking the splice
acceptor site coding for the amino acid R after splicing. The
middle line shows the insertion of a 34 basepair lox site insertion
(blue and red inverted repeat sequence), which puts the codon usage
of the constant region out of frame and creating downstream stop
codons (e.g. TGA fat print underlined). Black circle
.kappa.-enhancer and red circle .kappa.-LCR sequences.
[0125] FIG. 5: A mouse C.sub..kappa. insertion
[0126] The locus (top line) is the same as in FIG. 3. The bottom
shows the sequence at the 5' end of the C.sub..kappa. exon (blue in
top line) with the amino acid coding written above the bases. The
GG base pair at the start is immediately flanking the splice
acceptor site coding for the amino acid R after splicing. The
middle line shows the insertion of a 46 basepair insertion
containing a lox sequence (blue and red inverted repeat sequence)
and 4 stop codons, which also puts the codon usage of the constant
region out of frame and creating downstream stop codons (e.g. TGA
fat print underlined). Black circle .kappa.-enhancer and red circle
.kappa.-LCR sequences.
[0127] FIG. 6A: A mouse C.sub..kappa. constant region stop codon
and frame shift insertion
[0128] The locus (top line) is the same as in FIG. 3. The bottom
shows part of the sequence of the C.sub..kappa. exon (black) with
the amino acid coding written above the bases. The middle line
shows part of the sequence of the C.sub..kappa. coding region. The
line above it shows the insertion of a 44 basepair insertion
containing a lox sequence (blue and red inverted repeat sequence),
3 stop codons, which also puts the codon usage of the constant
region out of frame and creating downstream stop codons (TAA).
Black circle .kappa.-enhancer and red circle .kappa.-LCR sequences,
the Hpa I site used for the insertion is shown in red.
[0129] FIG. 6B: Recombination in ES cells to disable
C.sub..kappa.
[0130] The gel shows the result of a PCR amplification over the
insertion site of a number of the clones of the C.sub..kappa.
recombination in ES cells illustrated in FIG. 13A. Clones 351 and
623 are positive and will be injected into blastocysts to generate
Ig.sub..kappa. negative mice.
[0131] FIG. 7: The human Ig.sub..kappa. locus.
[0132] The map is copied from the IMGT database
(http://imgt.cines.f). The scale is in kilobases (kb). Green
squares, functional V.kappa. segments; red and yellow squares,
non-functional V.sub..kappa. segments; orange squares,
J.sub..kappa. segments; blue squares, constant region; black circle
.kappa.-enhancer and red circle .kappa.-LCR sequences.
[0133] FIG. 8: Generation of a hybrid human/rat Ig.sub..kappa.
locus for transgenesis
[0134] The 3' end of the locus is obtained from the mouse (yellow)
containing the mouse .kappa. 3' enhancer (yellow). The mouse
constant coding sequences are replaced with those of the rat,
including its 5' enhancer obtained by long range PCR from rat
genomic DNA (red). The human segment downstream from
V.sub..kappa.4-1 through to the human J.sub..kappa. sequences are
obtained from the mouse (yellow) to maintain the proper spacing
between the V and J regions. The human J.sub..kappa. segments are
obtained from a PAC covering this part of the human locus (green).
The green squares are V.sub..kappa. segments added individually or
as a block (see text). The puromycin resistance gene present in the
PAC vector is in red, black circle .kappa.-enhancer and red circle
.kappa.-LCR sequences.
[0135] FIG. 9: A map of the V.sub.H4 heavy chain locus
[0136] This locus containing a neomycin selectable marker at the 5'
end is used as the starting material for the construction of the
human/mouse hybrid locus. This locus is built as described in
WO2008/035216. The scale is in kilobases. The locus contains four
V.sub.H regions (1-46, 3-53, 3-23, 3-11), all of the human D
segments, all of the human J segments and IgG constant regions and
the 3' human IgH LCR.
[0137] FIG. 10: Generation of a 4 V.sub.H human/rat constant region
IgH locus
[0138] A CeuI site present in the V.sub.H4 human locus (FIG. 6) is
used to generate a human/rat locus by adding the rat constant
coding and switch regions that have been amplified by PCR from rat
genomic DNA. Similarly the mouse LCR region is amplified from mouse
genomic DNA as several fragments which are first cloned together to
generate the complete mouse IgH LCR. The 5' end of the human
V.sub.H4 locus containing the 4 V.sub.H segments, all of the human
D and all the human J segments (FIG. 6). All human sequences are in
blue, all mouse sequences in light green, the rat sequences in red
and the neomycin resistance gene is shown in purple.
[0139] FIG. 11: Deletion of the Ig.lamda. enhancers comprising the
.lamda. LCR
[0140] The .lamda. locus enhancers are removed by homologous
recombination using standard replacement vectors using the
hygromycin resistance gene flanked by sequences homologous to the
regions flanking the enhancer 2-4 and the blasticin S resistance
gene flanked by segments homologous to the regions flanking the
4-10 enhancer. Replacement results in a .lamda. locus that has lost
the enhancers and shows decreased expression. Hygromycin resistance
gene is shown in red, the blasticin S resistance gene in orange.
The mouse V.sub..lamda. segments are in green, the J segments in
yellow and the constant regions in blue. The maps are copied from
the IGMT database.
[0141] FIG. 12: A human rat V.sub..kappa. locus for
transgenesis
[0142] The locus is the same as in FIG. 8, but additional human
V.sub..kappa. segments have been added to the locus. The resulting
locus contains all of the frequently and moderately frequently used
human V.sub..kappa. segments. Green circle and green line, murine
.kappa.-enhancer and intron; red square and circle, rat
.kappa.-constant region and enhancer; blue squares, human V.sub.H
segments; dark blue, puromycin selectable marker.
[0143] FIG. 13A: Generation of a human rat hybrid locus
[0144] The locus is generated by ligating V.sub.H regions together
to a concatemer of 17 consecutive human V.sub.H regions cloned
between SceI sites. A mouse spacer region is added to a human 40 kb
fragment containing all the human D.sub.H and J.sub.H segments to
keep the appropriate distance between the D.sub.H and V.sub.H
segments. This is followed by the addition of a V.sub.H6-1 segment
containing an SceI site. The concatemer is then added onto the
V.sub.H6-1. Finally, the various rat constant regions and the
murine LCR are added at the 3' side. The resulting locus contains
all of the frequently and moderately frequently used human V.sub.H
segments.
[0145] FIG. 13B: The starting construct of the human/rat IgH
locus
[0146] The gel shows the result of the first steps of the human rat
IgH locus construction after the addition of V.sub.H6-1. The lanes
on the right show a NotI digest of two of the cloned plasmids. The
lanes (NotI.times.MluI) show the plasmid in lane 1 to have the
correct orientation in the vector, whereas the plasmid in lane 2
has the wrong orientation. The lane 1 plasmid is used for the next
step in the generation of the locus.
[0147] FIG. 13C: Concatemer of V.sub.H segments
[0148] The gel shows an XhoI/SalI digest of a concatemer of 17
different V.sub.H regions. The marker lane contains a .lamda. phage
DNA digested with BstEII.
[0149] FIG. 14: Transgenic human rat heavy chain immunoglobulin
loci.
[0150] An example of two heavy chain gene loci introduced into the
same animal. Selection of one as opposed to another is through
allelic exclusion. Additional V.sub.H segments could be added to
each of the loci. Alternatively, additional V.sub.H segments could
be introduced using further heavy chain gene loci. Obviously, the
same strategy could be used to increase diversity with .kappa. or
.lamda. light chain loci.
[0151] FIG. 15: Human/rat .lamda. transgenic light chain locus
[0152] An example of a human/rat locus is shown. Its 3' end is
obtained from the mouse (yellow) containing the mouse .lamda. 3'
LCR (yellow). The mouse constant coding sequences are replaced with
those of the rat by long range PCR from rat genomic DNA (red). The
human segment downstream from V.sub..lamda.2-14 through to the
human J.sub..kappa. sequences are obtained from the mouse (yellow)
to maintain the proper spacing between the V and J regions. The
human J.sub..lamda. segments are obtained by long range PCR of a
human PAC covering this part of the human locus (blue). The blue
squares are V.sub..lamda. segments added individually or as a
block. The hygromycin resistance gene present in the PAC vector is
in purple.
EXAMPLES
[0153] In the following examples, transgenic mice are generated
that express hybrid human/rat heavy chain and light chain loci as
transgenes introduced by microinjection in fertilised eggs, a
routine transgenesis procedure. The egg-donating mice are modified
to have no or very low expression of the endogenous mouse heavy
chain genes and mouse light chain genes. There are two light chain
loci in mice, for .kappa. and .lamda. chains, of which .lamda. is
used only in approximately 2% of the mouse H2L2 antibodies.
[0154] The examples are therefore in either mice which have the IgH
locus and only the endogenous .kappa. locus inactivated or in mice
which have the IgH and .kappa. locus inactivated and the regulatory
sequences of the .lamda. locus removed to lower the expression of
the .lamda. locus even further.
[0155] Methodology used for the construction of heavy and light
chain loci, the generation and screening of transgenic mice
following antigen challenge are essentially as previously described
(Janssens et al. (2006) PNAS, 10, 103(41), 15130-5, WO2006/008548,
WO2007/096779, GB0805281.3 and the PCT application filed on 11 Jun.
2008 claiming priority from GB0805281.3) excepting that the
C.sub.H1 domain is retained in all heavy chain loci. General
methods for deriving vertebrates, including mammals, other than
mice, which express functional heterologous immunoglobulin loci
and/or have engineered endogenous loci are as described in
WO2006/047367. In the examples below, recombination in ES cells is
used and the modified ES cells are used to generate mice with the
desired properties. However, the same procedures could be carried
out in induced pluripotent stem cells (iPS cells) which are then
used to generate mice (e.g. Boland, Hazen, Nazor, Rodriguez,
Gifford, Martin, Kupriyanov and Baldwin (2009), 461, 7260, 91-4 and
references therein).
[0156] Alternatively, the modifications are carried out in somatic
cells or somatic stem cells which are subsequently reprogrammed
into iPS cells to generate modified mice. Also, modified
hematopoietic stem cells could be transplanted into recipient mice
lacking B cells to generate human or human hybrid antibodies.
Example 1
[0157] In this example, the IgH locus (FIG. 1A) is inactivated by a
strategy similar to that published by Kitamura and Rajewsky with
the difference that the stop codon is introduced into the
C.sub..mu. regions at a position one amino acid before that
described by Kitamura et al. (1991) Nature, 350, 423-426. ES (or
iPS) cells were transfected with a construct that changes the
second codon of the first membrane exon of the mouse IgM gene into
a stop codon. This involves routine procedure including a neo
selection for transfection. A SpeI site was included in the
recombination sequences to be able to monitor the successful
recombination (FIG. 1B). ES cells are subsequently screened by
Southern blots to confirm successful recombinant clones. This
resulted in 10 correct recombinants (e.g. FIG. 1C). Of these, 3
were injected into mouse blastocysts to obtain chimeras which were
subsequently bred to obtain mice that are homozygous for the IgH
mutation. FACS analysis (B220 versus CD19) of the B cells of such
mice shows the absence of B cells in peripheral blood (FIG. 1D).
The mice were subsequently crossed with recombinase-expressing mice
to remove the neo gene (FIG. 1E). Similarly, the mouse
Ig.sub..kappa. locus (FIG. 2) is inactivated or reduced by
recombination in ES cells (FIG. 2, 3, 4-6). The resulting .kappa.
inactivated mice are crossed to the heavy chain KO mice. A knock
down of the activity is achieved by replacing the 3' .kappa. gene
LCR with a neo resistance marker flanked by lox sites (FIG. 3). The
neo gene is optionally removed by treatment with recombinase.
[0158] Alternatively, the .kappa. alleles could be knocked out in
the IgH KO cells directly. Several different strategies can be used
to achieve the .kappa. inactivation. Blocking of the activity of
the .kappa. gene can be achieved by using homologous recombination
in ES cells or iPS cells to insert into the 5' end of the
C.sub..kappa. exon a neo gene flanked by lox sites (FIG. 4) or by
inserting a neo gene flanked by lox sites and an additional
sequence coding for stop codons (FIGS. 5 and 6A). Treatment of the
recombined ES cells with cre will leave the sequence out of frame
(FIG. 4) or additionally contain new stop codons (FIG. 5-6A). FIG.
6B shows a recombination result in ES cells after transfection of
the construct shown in FIG. 6A, resulting in two ES cells clones
that have one C.sub..kappa. allele inactivated (5 such clones were
obtained in total). The cells are treated with recombinase by a
standard transient transfection with an actin-driven recombinase
plasmid to remove the region between the two lox sites. The cells
are subsequently used to generate mice by routine methods and the
progeny bred to obtain homozygous mice. Such mice comprise B-cells
in which the assembly of immunoglobulin tetramers comprising kappa
light chains is substantially impaired or completely blocked.
[0159] Next, the most frequently used V.sub..kappa. genes of the
human Ig.sub..kappa. locus (assessed using the Ig database;
http://imgt.cines.fr/; see FIGS. 7, 8) are amplified by standard
PCR and subcloned between XhoI/SalI sites, as described previously
for human V.sub.H segments. This allows the multimerisation of the
V.sub..kappa. regions, keeping the multimer between XhoI and SalI
sites.
[0160] Also, the 3' end of the mouse .kappa. locus, including the
3' .kappa. enhancer, and the rat constant (C.sub..kappa.) region
plus the rat 5' enhancer are cloned together (FIG. 8). Next, the
human J.sub..kappa. region and the region (17 kb) from between
mouse V.sub..kappa. and J.sub..kappa. (FIG. 2) are cloned in to
maintain the normal spacing between V.sub..kappa. regions and
J.sub..kappa.. Finally, the human V.sub..kappa. are inserted into
the PAC (FIG. 8) containing a puromycin selectable marker by
routine procedures (e.g. Janssens et al. (2006), supra).
[0161] In the example shown, the most frequently used V.sub..kappa.
segments (4-1, 3-11, 3-15, 3-20 and 1-39) are multimerized and
ligated into the PAC vector containing the human J regions and the
mouse enhancers and rat C.sub..kappa. regions. This results in a
human-rat hybrid locus consisting of a puro resistance marker gene,
human V.sub..kappa. segments and a rat constant (C.sub..kappa.)
region (FIG. 8).
[0162] In parallel, a hybrid human/rat IgH locus is constructed.
Again, there are a number of possibilities in terms of starting
material. In this example, the starting material is a human PAC
containing 4 human V.sub.H regions, all of the human D.sub.H and
J.sub.H segments and two human constant regions and the human LCR
(FIG. 9 and UK patent application No 0905023.8).
[0163] The latter PAC has a unique CeuI meganuclease site in
between the J regions and the constant regions. To allow easy
construction of the hybrid locus, this CeuI site is used to remove
the human 3' end sequence and replace these with rat constant and
switch regions (C.mu., C.gamma.3, C.gamma.1 and C.gamma.2). These
have been amplified by standard long range PCR from rat genomic
DNA. Finally, the mouse heavy chain LCR is added. This regulatory
sequence is amplified from mouse genomic DNA in three parts,
subcloned together to restore the complete LCR and added to the 3'
side of the rat constant regions (FIG. 10). The resulting hybrid
IgH locus thus contains a neo selection marker, human V, D and J
regions and rat constant regions with mouse regulatory
sequences.
[0164] The hybrid loci inserts are subsequently isolated from the
PAC as large DNA fragments and injected into fertilized mouse eggs
derived from the IgH/Ig.sub..kappa. heterozygous or homozygous null
mice to generate mice that are transgenic for the human/rat hybrid
IgH and Ig.sub..kappa. loci. All of this is done by routine methods
(e.g. Janssens et al. (2006), supra).
[0165] The hybrid IgH and hybrid Ig.sub..kappa. transgenic mice are
subsequently bred to obtain mice that are homozygous null for the
endogenous mouse IgH and Ig.sub..kappa. expression and positive for
the human/rat hybrid IgH and Ig.sub..kappa. expression. These mice
are subsequently immunized to generate antigen-specific hybrid
human/rat H.sub.2L.sub.2 antibodies by routine procedures. When
generating monoclonal human/rat Ig.sub..kappa. through hybridomas,
double selection in puromycin and neomycin will ensure that only
myeloma fusions containing both an IgH and an Ig.sub..kappa. locus
will selected for.
[0166] The skilled person will appreciate that variations to this
procedure may be made to generate the hybrid transgenic mice, such
as the use of different vectors, different selection markers,
different recombination positions to inactivate the mouse genes or
variations in the actual (routine) cloning strategy of the hybrid
loci. The same procedure can be used to generate any normal or
hybrid locus using immunoglobulin DNA derived from any single
mammalian species, or hybrid loci derived using DNA from two or
more species.
Example 2
[0167] This example is in principle the same as Example 1 with the
exception that the high frequency of obtaining IgH/Ig.sub..kappa.
H.sub.2L.sub.2 antibodies is increased even further by lowering the
frequency of expression of the endogenous mouse Ig.sub..lamda.
locus. This can be achieved by replacing the regulatory regions of
both Ig.sub..lamda. with a selectable marker (FIG. 11), in this
case the hygromycin resistance gene and the TK-BSD gene.
[0168] The latter allows positive selection, resistance to
blasticidin S (Karreman, (1998) NAR, 26, (10), 2508-2510). This
combination of markers allows for positive selection in the two ES
cell recombinations when replacing the regulatory regions. The
recombination would be carried out in the ES cells generated in
Example 1 or alternatively in parallel in normal ES cells and bred
into the mice described above in Example 1.
[0169] The resulting transgenic mice would contain the hybrid human
rat IgH and Ig.sub..kappa. loci, be negative for endogenous mouse
IgH and Ig.sub..kappa. (or express C.sub..kappa. at very low
levels) and express Ig.sub..lamda. at very low levels. After
immunization and the generation of hybridomas by routine methods,
the hybridomas expressing only human rat hybrid H2L2 antibodies
would be selected for expression of the transgenic hybrid loci by
neo (FIG. 10) and puro (FIG. 8) selection.
Example 3
[0170] Example 3 is analogous to the Examples described above but
the hybrid Ig.sub..kappa. locus would be extended by the addition
of V.sub..kappa. segments that are used less frequently (FIG. 12;
V.sub..kappa. 1-9, 1-33, 2-30, 2-28, 1-27, 1-5). Alternatively,
mutated/modified V.sub..kappa. segments or V.sub..lamda. segments
could be added in addition. The addition of further segments would
be carried out by using the same XhoI/SalI cloning strategy
described above. Immunization of mice generated in this example
would allow a greater complexity in response to the immunization
with antigen. The number of V.sub.L regions could be varied further
by adding other V.sub..kappa. segments or the use of combinations
of all of the above V.sub.L segments.
Example 4
[0171] Example 4 is analogous to that described in the examples
described above, but here the hybrid human/rat IgH locus has been
generated by using 18 V.sub.H segments and 5 rat constant regions
(FIG. 13A; human V.sub.H 6-1, 1-2, 4-4, 2-5, 3-07, 1-8, 4-39, 3-15,
1-18, 3-23, 3-30, 3-33, 3-48, 4-34, 3-49, 3-53, 4-59, 1-69). First
a central 70 kb DJ region of the human locus is extended at the 5'
end with 8 kb from the mouse IgH intron to maintain the proper
distance between V.sub.H segments and the D region. Next the first
V.sub.H region (6-1) of 10 kb with an artificial SceI meganuclease
site is cloned at the 5' end of the mouse intron sequences (FIG.
10B). In a separate plasmid, all the remaining V.sub.H region are
cloned together by slotting in XhoI/SalI V.sub.H segments as
described above (FIG. 10C). One could also add to these loci more
V.sub.H segments or V.sub.H segments that have been
modified/mutated. One can also include CTCF sites. In the example
shown, three such sites have been used. They are obtained by long
range PCR of the V.sub.H region including the upstream CTCF site.
Furthermore, the rat C.sub.a has been added when compared to the
locus in FIG. 10. In this Example, immunization would allow an even
greater complexity in response to the immunization with antigen,
particularly in combination with Example 3. The V.sub.H multimer is
cloned into the VH6-1DJ plasmid, after which the rat constant
regions are added to complete the locus.
[0172] In all of these Examples, the complexity of the response
will be enhanced even further by adding V segments as part of
additional heavy or light chain transgenic loci present in the same
mouse. Since all the loci are subject to allelic exclusion (see
WO2007/096779), only the preferred rearrangement will be selected
in vivo following antigen challenge, resulting in B-cell expansion
and the accumulation of antibody in serum. FIG. 14 shows an example
of two heavy chain gene loci that can be introduced into the same
animal and each will be used through allelic exclusion. Obviously,
more V.sub.H segments could be added (including modified V.sub.H
segments) or even more loci could be introduced to increase the
complexity of the transgenic immune repertoire. The method could
also be applied to other species using V segments specific for
these species.
Example 5
[0173] In this example, the diversity of the human/rat hybrid
antibody is increased even further by the addition of a human/rat
Ig.lamda. locus through breeding to the mice that carry human/rat
IgH and/or Ig.kappa. loci described in the examples above. The
human/rat hybrid .lamda. locus is generated very much as described
for the human/rat Ig.kappa. locus described in the previous
examples. The difference is caused by the fact that J.sub..lamda.
and C.lamda. regions occur in pairs and hence 2 rat C.sub..lamda.
regions are cloned onto 2 human J.sub..lamda. regions (FIG. 15).
The spacing between the V.sub..lamda. and J.sub..lamda. segments is
maintained by cloning the normal mouse sequences that occur in that
position (see FIG. 11). In this example, 2 human J.sub..lamda. and
rat C.sub..lamda. segments are used together with four human
V.sub.x, segments. Together, these cover more than 80% of the human
Ig.lamda. response. The regulatory sequences (LCR, FIG. 15) are
derived from the mouse to ensure optimal expression, and a
selectable marker is added at the 5' end of the locus. As described
above, the locus is isolated as a restriction fragment and injected
into fertilised eggs to generate mice carrying the transgenic
.lamda. locus.
[0174] In all of these examples V.sub.H, V.sub.L, D, J and constant
regions from different species can be used to generate other single
species antibodies or hybrid species antibodies. It will also be
apparent to one skilled in the art that, once an antigen-specific
antibody has been identified, the V.sub.HDJ and V.sub.LJ regions
can be cloned onto alternative constant regions from the same
species or from different species by completely routine methods.
Sequence CWU 1
1
1316PRTMouse 1Glu Glu Gly Phe Glu Asn1 5237DNAMouse 2gaggaaggct
ttgagaacta gtcgagaagt tcctatt 37311PRTMouse 3Glu Glu Gly Phe Glu
Asn Leu Trp Ala Thr Ala1 5 10412PRTArtificial SequenceLox site
insertion 4Ile Thr Ser Tyr Cys Ile His Tyr Thr Leu Leu Cys1 5
10560DNAArtificial SequenceLox site insertion 5ggataacttc
gtatagcata cattatacga agttatgctg atgctgcacc aactgtatcc 6068PRTMouse
6Ala Asp Ala Ala Pro Thr Val Ser1 5726DNAMouse 7gggctgatgc
tgcaccaact gtatcc 26812PRTArtificial SequenceLox site insertion
8Arg Ile Thr Ser Tyr Cys Ile His Tyr Thr Leu Leu1 5
10972DNAArtificial SequenceLox site insertion 9ggataacttc
gtatagcata cattatacga agttatgatg auaauagagc tgatgctgca 60ccaactgtat
cc 721011PRTArtificial SequenceLox site insertion 10Ile Thr Ser Tyr
Ser Ile His Tyr Thr Lys Leu1 5 101163DNAArtificial SequenceLox site
insertion 11gagcagttat gataatagat aacttcgtat agcatacatt atacgaagtt
ataacatctg 60gag 631211PRTMouse 12Val Ser Ile Phe Pro Pro Ser Ser
Glu Gln Leu1 5 101335DNAMouse 13gtatccatct tcccaccatc cagtgagcag
ttaac 35
* * * * *
References