NZ709608B2 - Polynucleotides encoding rodent antibodies with human idiotypes and animals comprising same - Google Patents
Polynucleotides encoding rodent antibodies with human idiotypes and animals comprising same Download PDFInfo
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- NZ709608B2 NZ709608B2 NZ709608A NZ70960813A NZ709608B2 NZ 709608 B2 NZ709608 B2 NZ 709608B2 NZ 709608 A NZ709608 A NZ 709608A NZ 70960813 A NZ70960813 A NZ 70960813A NZ 709608 B2 NZ709608 B2 NZ 709608B2
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- 239000002502 liposome Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 1
- 108010056929 lyticase Proteins 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
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- 239000012474 protein marker Substances 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- JFINOWIINSTUNY-UHFFFAOYSA-N pyrrolidin-3-ylmethanesulfonamide Chemical compound NS(=O)(=O)CC1CCNC1 JFINOWIINSTUNY-UHFFFAOYSA-N 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
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- 238000012216 screening Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000013605 shuttle vector Substances 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000037439 somatic mutation Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
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- 229940063673 spermidine Drugs 0.000 description 1
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- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/01—Animal expressing industrially exogenous proteins
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Abstract
The invention relates to polynucleotides, particularly chimeric polynucleotides useful for optimal production of functional immunoglobulins with human idiotypes in rodents. The invention further relates to rodents comprising such polynucleotides.
Description
POLYNUCLEOTIDES ENCODING RODENT ANTIBODIES WITH HUMAN
IDIOTYPES AND ANIMALS COMPRISING SAME
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority to U.S. provisional patent application U.S.S.N.
61/737,371 filed 14 December 2012. The application is expressly incorporated herein in its
entirety by reference.
FIELD OF INVENTION
The invention relates to polynucleotides, particularly chimeric polynucleotides useful
for the production of immunoglobulins with human idiotypes in rodents. The invention further
relates to rodent cells comprising such polynucleotides.
REFERENCE TO A SEQUENCE LISTING
A Sequence Listing is provided in this patent document as a txt file entitled “189314-
US_ST25.txt” and created December 12, 2012 (size 3MB). The contents of this file is
incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
Human monoclonal antibodies have proven to be invaluable in therapeutic
1, 2
applications, either as IgG of conventional size, single chains or domain modules . Despite the
successes there are still major shortcomings in their production, which relies either on specificity
selection of available human material and subsequent modification of individual products, or the
immunization of the limited availability of transgenic animals, mainly mice . Target antigen
restrictions are widely in place for the use of transgenic mice, as well as large transgenic animals
such as cattle, and the development of new specificities is company controlled .
DNA rearrangement and expression of human immunoglobulin (Ig) genes in
transgenic mice was pioneered over 20 years ago by stably inserting heavy-chain genes in
germline configuration . Although human antibody repertoires were obtained in these early
animals, major improvements, resulting in higher expression levels and exclusive production of
human Ig, combined two new strategies: gene knock-out in embryonic stem (ES) cells and locus
extension on artificial chromosomes .
Silencing of the endogenous Ig genes by gene targeting in ES cells produced several
inactive mouse lines without the ability to rearrange their IgH and Igκ locus or without
producing fully functional IgH, Igκ or Igλ products (summarized in ). More recently zinc finger
nucleases (ZFNs) were designed to generate site-specific double-strand breaks in Ig genes, which
allowed gene disruption by deletion and non-homologous DNA repair. Injection of ZFN
plasmids into fertilized eggs produced Ig silenced rats and rabbits with IgH and IgL
11-13
disruptions .
Efficient expression of antibodies requires functional regulatory elements in various
locations in immunoglobulin loci. Enhancer sequences have been identified near many active
genes by nuclease digest and hypersensitivity to degradation. Hypersensitive sites may precede
promoter sequences and the strength of their activity was correlated with the DNA sequence.
Linkage to reporter genes showed elevated transcription if enhancer function was present (Mundt
et al., J. Immunol., , 166, 3315[2001]. In the IgH locus two important transcription or expression
regulators have been identified, Eµ and the 3’E at the end of the locus (Pettersson et al., Nature,
344, 165 [1990]). In the mouse the removal of the entire 3’ regulatory region (containing hs3a,
hs1,2, hs3b and hs4) allows normal early B-cell development but abrogates class-switch
recombination (Vincent-Fabert et al., Blood, 116, 1895 [2010]) and possibly prevents the
optimization of somatic hypermutation (Pruzina et al., Protein Engineering, Design and
Selection, 1, [2011]).
The regulatory function to achieve optimal isotype expression is particularly
desirable when transgenic human IgH genes are being used. However, in a number of
laboratories, transgene constructs with an incomplete 3’E region, typically providing only the
hs1,2 element, led to disappointing expression levels in transgenic mice even when the
endogenous IgH locus was knocked-out. This may be one reason why the generation of antigen-
specific fully human IgGs from genetically engineered mice has been inefficient thus far.
(Lonberg et al., Nature 368, 856 [1994]; Nicholson et al., J. Immunol., 163, 6898 [1999]; Davis
et al., Cancer Metastasis Rev. 18, 421 [1999]; Pruzina et al., Protein Engineering, Design and
Selection, 1,[2011].
In the rat, the 3’E region has only been poorly analyzed. A comparison of mouse and
rat sequences does not allow identification of hs4, the crucial 4th E element with additional
important regulatory sequences further downstream (Chatterjee et al., J. Biol. Chem., 286,29303
). This could mean the region is not present in the rat, and perhaps not as important as in
the mouse, or it could be absent in the analyzed rat genome sequences.
Still needed are materials for the optimal production of immunoglobulins or
antibodies having human idiotypes using transgenic animals, which are useful for treating
humans in a broad range of disease areas; and/or which at least to provide the public with a
useful choice.
SUMMARY OF INVENTION
[0010a] In a first aspect, the invention relates to a chimeric polynucleotide comprising, in 5’ to 3’
order, a human immunoglobulin (Ig) variable (V) region gene, a human Ig diversity (D) region
gene, at least one human immunoglobulin (Ig) joining (J) region gene, an Ig constant region
gene, and a rat 3' enhancer comprising the sequence set forth as SEQ ID NO:1.
[0010b] In a second aspect, the invention relates to a chimeric polynucleotide according to the
first aspect, wherein said V-D-J regions are rearranged and form a complete exon encoding a
heavy chain variable domain.
[0010c] In a third aspect, the invention relates to an isolated rodent cell comprising the chimeric
polynucleotide according to the first or second aspect.
BRIEF DESCRIPTION
Disclosed herein are novel polynucleotides comprising nucleic acid sequences
encoding chimeric immunoglobulin chains, particularly chimeric heavy chains for use in creating
transgenic animals. The polynucleotides of the present description advantageously provide
optimal expression due, at least in part, to the inclusion of a 3’ enhancer since transloci lacking
this 3’ enhancer result in impaired isotype switching and low IgG expression. Accordingly, in
preferred embodiments the description includes chimeric polynucleotides comprising a rat 3’
enhancer sequence, an Ig constant region gene and at least one human immunoglubulin (Ig)
joining (J) region gene. In a preferred embodiment, the rat 3’ enhancer sequence comprises the
sequence set forth as SEQ ID NO:1, or a portion thereof.
The chimeric polynucleotides set forth herein may further comprise at least one
human variable (V) gene, at least one a diversity (D) gene, or a combination thereof. In one
embodiment, the constant region gene of the chimeric polynucleotide is selected from the group
consisting of a human constant region gene and a rat constant region gene. In a preferred
embodiment, the constant region gene is a rat constant region gene. In another preferred
embodiment, the constant region gene is selected from the group consisting of C μ and C γ.
In one embodiment, the chimeric polynucleotide comprises a nucleic acid sequence
substantially homologous to the bacterial artificial chromosome (BAC) Annabel disclosed herein
(e.g., SEQ ID NO:10), or a portion thereof, and may optionally further comprise at least one
human variable Ig gene isolatable from BAC6-V 3-11 and BAC3. In a preferred embodiment,
the chimeric polynucleotides contemplated herein comprise nucleic acid sequences (a) and (b) in
’ to 3’ order: (a) a human Ig variable region comprising human V genes in natural configuration
isolatable from BAC6-V 3-11 and/or BAC3, and (b) a human Ig joining region comprising
human J genes in natural configuration isolatable from the BAC Annabel. In another
embodiment, each of the human Ig variable region, human Ig diversity region, human Ig joining
region, the Ig constant region and the rat 3’ enhancer region of a chimeric polynucleotide as
disclosed herein are in the relative positions as shown in . In another embodiment, a
chimeric polynucleotide as disclosed has a sequence comprising or substantially homologous to
the sequence set forth as SEQ ID NO:2 or a portion thereof. In another embodiment, a chimeric
polynucleotide as disclosed has a sequence comprising or substantially homologous to the
sequence set forth as SEQ ID NO:11, or a portion thereof. In a further embodiment, a chimeric
polynucletoide as disclosed herein comprises a rearranged V-D-J regions, wherein said
rearranged V-D-J regions encode a heavy chain variable domain exon.
Also disclosed herein are polynucleotides encoding human kappa light chain genes.
In one embodiment, a polynucleotide as disclosed herein has a nucleic acid sequence comprising
or substantially homologous to a nucleic acid sequence selected from the group consisting of
RP11-1156D9 (set forth as SEQ ID NO:3) and RP11-1134E24 (set forth as SEQ ID NO:4). In
another embodiment, the isolated polynucleotide comprises nucleic acid sequences (a) and (b) in
’ to 3’ order: (a) a human Ig variable region comprising human V genes in natural configuration
isolatable from bacterial artificial chromosomes (BAC) RP11-156D9 and/or RP11-1134E24; (b)
a human Ig joining region comprising human J genes in natural configuration isolatable from the
bacterial artificial chromosomes (BAC) RP11-1134E24 and/or RP11-344F17 (set forth as SEQ
ID NO:5). In a preferred embodiment, each of the human Ig variable region, the human Ig
joining region, and the human Ig constant region are in relative position as shown in . In
another embodiment, a chimeric polynucleotide as disclosed has a sequence comprising or
substantially homologous to the sequence set forth as SEQ ID NO:6 or a portion thereof.
Also described herein is a rodent cell comprising one or more polynucleotides of the
description. For example, described herein is a rodent cell comprising a polynucleotide as
disclosed herein, preferably comprising a nucleic acid sequence encoding for a chimeric heavy
chain, e.g., a nucleic acid sequence encoding a rat 3’ enhancer sequence, an Ig constant region
gene and at least one human J region gene, and optionally, comprising a nucleic acid sequence
substantially homologous to the nucleic acid sequence selected from the group consisting of
RP11-1156D9, RP11-1134E24 and portions thereof. The rodent cell contemplated herein may
further comprise a polynucleotide encoding a functional light chain, e.g., having a nucleic acid
sequence comprising or substantially homologous to a nucleic acid sequence selected from the
group consisting of the sequence shown in (set forth as SEQ ID NO:6), the sequence
shown in (set forth as SEQ ID NO:7), and portions thereof. In one embodiment, one or
more of the polynucleotides are integrated into the rodent cell genome.
[0015a] Certain statements that appear herein are broader than what appears in the statements of
the invention. These statements are provided in the interests of providing the reader with a better
understanding of the invention and its practice. The reader is directed to the accompanying claim
set which defines the scope of the invention.
[0015b] In this specification where reference has been made to patent specifications, other
external documents, or other sources of information, this is generally for the purpose of
providing a context for discussing the features of the invention. Unless specifically stated
otherwise, reference to such external documents is not to be construed as an admission that such
documents, or such sources of information, in any jurisdiction, are prior art, or form part of the
common general knowledge in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
Integrated human Ig loci. (a) The chimeric human-rat IgH region contains 3
overlapping BACs with 22 different and potentially functional human V segments. BAC6-3 has
been extended with V 3-11 to provide a 10.6 kb overlap to BAC3, which overlaps 11.3 kb via
V 6-1 with the C region BAC Hu-Rat Annabel. The latter is chimeric and contains all human D
and J segments followed by the rat C region with full enhancer sequences. (b) The human Igk
BACs with 12 Vks and all Jks provide a ~14 kb overlap in the Vk region and ~40 kb in Ck to
include the KDE. (c) The human Igl region with 17 Vls and all J-Cls, including the 3’ enhancer,
is from a YAC .
-d: Flow cytometry analysis of lymphocyte-gated bone marrow and spleen
cells from 3 months old rats. Surface staining for IgM and CD45R (B220) revealed a similar
number of immature and mature B-cells in bone marrow and spleen of HC14 and wt rats, while
JKO/JKO animals showed no B-cell development. Plotting forward (FSC) against site (SSC)
scatter showed comparable numbers of lymphocyte (gated) populations, concerning size and
shape. Surface staining of spleen cells with anti-IgG (G1, G2a, G2b, G2c isotype) plotted against
cell count (x 102) revealed near normal numbers of IgG+ expressers in HC14 rats compared to
IgM ) and B refers to immature B-cells
wt. In , A refers to pro/pre B-cells (CD45R
+ + + -
(CD45R IgM ). In , A refers to transitional B cells (CD45R IgM ), B to follicular B cells
+ + low +
(CD45R IgM ) and C to marginal zone B cells (CD45R IgM ).
Mutational changes in IgH and IgL transcripts from PBLs. Unique
(VHDJH)s and VLs were from amplifications with V group specific primers: IGHV1, 2, 3, 4
and 6 in combination with the universal γCH2 reverse primer, IGLV2, 3 and 4 with reverse C λ
primer; and IGKV1, 3, 4 and 5 in with reverse C κ primer (Supplementary Table 1). Mutated
trans-switch products were identified for humanVH-rat C γ2a (4) and human VH-rat C γ2c (2).
Purification of rat Ig with human idiotypes and comparison to human and
normal rat Ig levels. OmniRat serum and human or rat wt control serum, 100 µl each, was used
for IgM/G purification. (a) IgM was captured with anti-IgM matrix, which identified 14 µg in wt
rat, and 30 µg and 10 µg in OmniRats [HC14(a) and HC14(c)]. (b) IgG was purified on protein
A and protein G columns, with a yield of up to ~3 mg/ml for OmniRats (Protein A: HC14(a)
1000 µg/ml; HC14(b) 350 µg/ml; wt rat 350 µg/ml; Protein G: HC14(a) 2970 µg/ml; HC14(b)
280 µg/ml; wt rat 1010 µg/ml). (c) Human Igκ and (d) human Igλ was purified on anti-Igκ and
anti-Igλ matrix, respectively. No purification product was obtained using wt rat serum (not
shown). Purified Ig, ~3 µg (concentration determined by nano drop), was separated on 4-15%
SDS-PAGE under reducing conditions. Comparison by ELISA titration of (e) human Igκ and (f)
human Igλ levels in individual OmniRats (8531, 8322, 8199, 8486, 8055), human and wt rat
serum. Serum dilution (1:10, 1:100, 1:1,000, 1:10,000) was plotted against binding measured by
adsorption at 492 nm. Matching name/numbers refer to samples from the same rat.
DETAILED DESCRIPTION
Described herein are chimeric polynucleotides encoding a recombinant or artificial
immunoglobulin chain or loci. As described above, the chimeric polynucleotides disclosed
herein are useful for the transformation of rodents to include human Ig genes and for the
production of immunoglobulins or antibodies having human idiotypes using such rodents.
[0020a] The term “comprising” as used in this specification and claims means “consisting at
least in part of”. When interpreting statements in this specification, and claims which include the
term “comprising”, it is to be understood that other features that are additional to the features
prefaced by this term in each statement or claim may also be present. Related terms such as
“comprise” and “comprised” are to be interpreted in similar manner.
Polynucleotides
Immunoglobulin refers to a protein consisting of one or more polypeptides
substantially encoded by immunoglobulin genes. The recognized human immunoglobulin genes
include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta,
epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region
genes. Full-length immunoglobulin "light chains" (about 25 Kd, or 214 amino acids) generally
comprise a variable domain encoded by an exon comprising one or more variable region gene(s)
and one or more joining region gene(s) at the NH -terminus (about 110 amino acids) and
constant domain encoded by a kappa or lambda constant region gene at the COOH-terminus.
Full-length immunoglobulin "heavy chains" (about 50 Kd, or 446 amino acids), similarly
comprise (1) a variable domain (about 116 amino acids) encoded by an exon comprising one or
more variable region genes, one or more diversity region genes and one or more joining region
genes, and (2) one of the aforementioned constant domains comprising one or more constant
region genes, e.g., alpha, gamma, delta, epsilon or mu (encoding about 330 amino acids). The
immunoglobulin heavy chain constant region genes encode for the antibody class, i.e., isotype
(e.g., IgM or IgG1).
As used herein, the term "antibody" refers to a protein comprising at least one, and
preferably two, heavy (H) chain variable domains (abbreviated herein as VH), and at least one
and preferably two light (L) chain variable domains (abbreviated herein as VL). An ordinarily
skilled artisan will recognize that the variable domain of an immunological chain is encoded in
gene segments that must first undergo somatic recombination to form a complete exon encoding
the variable domain. There are three types of regions or gene segments that undergo
rearrangement to form the variable domain: the variable region comprising variable genes, the
diversity region comprising diversity genes (in the case of an immunoglobulin heavy chain), and
the joining region comprising joining genes. The VH and VL domains can be further subdivided
into regions of hypervariability, termed "complementarity determining regions" ("CDRs")
interspersed with regions that are more conserved, termed "framework regions" ("FRs"). The
extent of the FRs and CDRs has been precisely defined (see, Kabat et al. (1991) Sequences of
Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242; and Chothia et al. (1987) J. Mol. Biol. 196:901-17,
which are hereby incorporated by reference). Each VH and VL domain is generally composed of
three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following
order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The antigen binding fragment of an antibody
(or simply "antibody portion," or "fragment"), as used herein, refers to one or more fragments of
a full-length antibody that retain the ability to specifically bind to an antigen (e.g., CD3).
Examples of binding fragments encompassed within the term "antigen binding
fragment" of an antibody include (i) an Fab fragment, a monovalent fragment consisting of the
VL, VH, CL and CH1 domains; (ii) an F(ab') fragment, a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the
VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm
of an antibody, (v) a dAb fragment (Ward et al. (1989) Nature 341:544-46), which consists of a
VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore,
although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they
may be joined, using recombinant methods, by a synthetic linker that enables them to be made as
a single protein chain in which the VL and VH regions pair to form monovalent molecules
(known as single chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-26; and Huston et
al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-83). Such single chain antibodies are also
intended to be encompassed within the term "antigen binding fragment" of an antibody. These
antibody fragments are obtained using conventional techniques known to those skilled in the art,
and the fragments are screened for utility in the same manner as are intact antibodies.
An antibody may further include a heavy and/or light chain constant domain to
thereby form a heavy and light immunoglobulin chain, respectively. In one embodiment, the
antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin
chains, wherein the heavy and light immunoglobulin chains are interconnected, e.g., by disulfide
bonds. The heavy chain constant domain is comprised of three gene segments, CH1, CH2 and
CH3. The light chain constant domain is comprised of one gene, CL. The variable domains of
the heavy and/or light chains contain a binding domain that interacts with an antigen. The
constant domains of the antibodies typically mediate the binding of the antibody to host tissues
or factors, including various cells of the immune system (e.g., effector cells) and the first
component (C1q) of the classical complement system.
By polynucleotide encoding an artificial immunoglobulin locus or artificial
immunoglobulin chain is meant an recombinant polynucleotide comprising multiple
immunoglobulin regions, e.g., a variable (V) region or gene segment comprising V genes, a
joining (J) gene region or gene segment comprising J genes, a diversity (D) region or gene
segment comprising D genes in the case of a heavy chain locus and/or at least one constant (C)
region comprising at least one C gene. Preferably, each region of the variable domain, e.g., V,
D, or J region, comprises or spans at least two genes of the same type. For example a variable
region as used herein comprises at least two variable genes, a joining region comprises at least
two joining genes and a diversity region comprises two diversity genes. A constant region may
comprise only one constant gene, e.g. a κ gene or λ gene, or multiple genes, e.g., CH1, CH2, and
CH3.
”Enhancer sequences” or “enhancer” as used herein refers to sequences that have
been identified near many active genes by nuclease digest and hypersensitivity to degradation.
Hypersensitive sites may precede promoter sequences and the strength of their activity was
correlated with the DNA sequence. Linkage to reporter genes showed elevated transcription if
enhancer function was present (Mundt et al., J. Immunol., 166, 3315[2001]). In the IgH locus
two important transcription or expression regulators have been identified, Eµ and the 3’E at the
end of the locus (Pettersson et al., Nature, 344, 165 [1990]). In the mouse the removal of the
whole 3’ regulatory region (containing hs3a, hs1,2, hs3b and hs4) allows normal early B-cell
development but abrogates class-switch recombination (Vincent-Fabert et al., Blood, 116, 1895
) and possibly prevents the optimization of somatic hypermutation (Pruzina et al., Protein
Engineering, Design and Selection, 1, [2011]). The regulatory function to achieve optimal
isotype expression is particularly desirable when transgenic human IgH genes are being used.
Transgene constructs with incomplete 3’E region, usually only providing the hs1,2 element, led
to disappointing expression levels in transgenic mice even when the endogenous IgH locus was
knocked-out. As a consequence, only few antigen-specific fully human IgGs have been isolated
from constructs produced in the last 20 years (Lonberg et al., Nature 368, 856 [1994]; Nicholson
et al., J. Immunol., 163, 6898 [1999]; Davis et al., Cancer Metastasis Rev. 18, 421 [1999];
Pruzina et al., Protein Engineering, Design and Selection, 1, [2011]). In the rat IgH locus, the
3’E region has only been poorly analyzed. A comparison of mouse and rat sequences did not
allow identification of hs4, the crucial 4 element with additional important regulatory sequences
further downstream (Chatterjee et al., J. Biol. Chem., 286,29303 [2011]). The polynucleotides
of the present description advantageously provide optimal expression due, at least in part, to the
inclusion of a rat 3’ enhancer since chimeric polynucleotides lacking this 3’ enhancer result in
impaired isotype switching and low IgG expression. In one embodiment, the rat 3’ enhancer has
a sequence comprising or substantially homologous to the sequence set forth as SEQ ID NO:1 or
a portion thereof.
As used herein, a polynucleotide having a sequence comprising or substantially
homologous to a portion, e.g., less than the entirety, of second sequence (e.g., SEQ ID NO:1,
SEQ ID NO:2, etc.) preferably retains the biological activity of the second sequence (e.g.,
retains the biological activity of a 3’ enhancer to provide optimal expression and/or isotype
switching of immunoglobulins, is capable of rearrangement to provide a humanized chimeric
heavy chain, etc.) . In one embodiment, a nucleic acid comprising a sequence comprising or
substantially homologous to a portion of SEQ ID NO:1 comprise at least 8 kB, preferably at least
kB of continuous nucleic acids that are substantially homologous to SEQ ID NO:1.
“Artificial Ig locus” as used herein may refer to polynucleotides that (e.g., a
sequence comprising V-,D-, and/or J regions in the case of heavy chain, or V- and/or J regions in
the case of light chain, and optionally a constant region for either or both a heavy and light
chgin) that are unrearranged, partially rearranged, or rearranged. Artificial Ig loci include
artificial Ig light chain loci and artificial Ig heavy chain loci. In one embodiment, an artificial
immunoglobulin locus of the description is functional and capable of rearrangement and
producing a repertoire of immunoglobulin chains. In a preferred embodiment, the variable
domain or portion thereof of a polynucleotide disclosed herein comprises genes in natural
configuration, i.e., naturally occurring sequences of an human Ig gene segment, degenerate
forms of naturally occurring sequences of a human Ig gene segment, as well as synthetic
sequences that encode a polypeptide sequence substantially identical to the polypeptide encoded
by a naturally occurring sequence of a human Ig gene segment. In another preferred
embodiment, the polynucleotide comprises a variable domain or portion thereof in a natural
configuration found in humans. For example, a polynucleotide encoding an artificial Ig heavy
chain as disclosed herein may comprise in natural configuration at least two human V genes, at
least two D genes, at least two J genes or a combination thereof.
In a preferred embodiment, an artificial Ig locus comprises a non-human C region
gene and is capable of producing a repertoire of immunoglobulins including chimeric
immunoglobulins having a non-human C region. In one embodiment, an artificial Ig locus
comprises a human C region gene and is capable of producing a repertoire of immunoglobulins
including immunoglobulins having a human C region. In one embodiment, an artificial Ig locus
comprises an ”artificial constant region gene”, by which is meant a constant region gene
comprising nucleotide sequences derived from human and non-human constant regions genes.
For example, an exemplary artificial C constant region gene is a constant region gene encoding a
human IgG CH1 domain and rat IgG CH2 and CH3 domain.
In a preferred embodiment, an artificial Ig locus comprises 3’ enhancer sequences,
including hs1,2, hs3a, hs3b and sequences (500-2500 nt) downstream of hs3b. In transgenic
animals, artificial loci comprising the full ~30 kb 3’E region from Calpha to 3’ hs3b result in
high level IgG expression, extensive hypermutation and large numbers of antigen-specific
hybridomas of high (pM) affinity. However, shorter enhancer sequences reduce Ig expression.
In a preferred embodiment, an artificial Ig locus comprises the 3’ enhancer sequence
shown in Fig 1a. This sequence is derived from the rat Ig heavy chain locus and contains about
30kb of the 3’ region from Calapha to 3’ hs3b. The sequence of the rat 3’ enhancer sequence is
set forth as SEQ ID NO:1. In another embodiment, the artificial Ig locus comprises a sequence
comprising or substantially homologous to the sequence set forth as SEQ ID NO:1, or a portion
thereof.
In some embodiments, an artificial Ig heavy chain locus lacks CH1, or an equivalent
sequence that allows the resultant immunoglobulin to circumvent the typical immunoglobulin:
chaperone association. Such artificial loci provide for the production of heavy chain-only
antibodies in transgenic animals which lack a functional Ig light chain locus and hence do not
express functional Ig light chain. Such artificial Ig heavy chain loci are used in methods
contemplated herein to produce transgenic animals lacking a functional Ig light chain locus, and
comprising an artificial Ig heavy chain locus, which animals are capable of producing heavy
chain-only antibodies. Alternatively, an artificial Ig locus may be manipulated in situ to disrupt
CH1 or an equivalent region and generate an artificial Ig heavy chain locus that provides for the
production of heavy chain-only antibodies. Regarding the production of heavy chain-only
antibodies in light chain-deficient mice, see for example Zou et al., JEM, 204:3271-3283, 2007.
By “human idiotype” is meant a polypeptide sequence present on a human
antibody encoded by an immunoglobulin V-gene segment. The term “human idiotype” as used
herein includes both naturally occurring sequences of a human antibody, as well as synthetic
sequences substantially identical to the polypeptide found in naturally occurring human
antibodies. By “substantially” is meant that the degree of amino acid sequence identity is at least
about 85%-95%. Preferably, the degree of amino acid sequence identity is greater than 90%,
more preferably greater than 95%.
By a “chimeric antibody” or a “chimeric immunoglobulin” is meant an
immunoglobulin molecule comprising a portion of a human immunoglobulin polypeptide
sequence (or a polypeptide sequence encoded by a human Ig gene segment) and a portion of a
non-human immunoglobulin polypeptide sequence. The chimeric immunoglobulin molecules of
the present description are immunoglobulins with non-human Fc-regions or artificial Fc-regions,
and human idiotypes. Such immunoglobulins can be isolated from animals of the description
that have been engineered to produce chimeric immunoglobulin molecules.
By “artificial Fc-region” is meant an Fc-region encoded by an artificial constant
region gene.
The term “Ig gene segment” as used herein refers to regions of DNA encoding
various portions of an Ig molecule, which are present in the germline of non-human animals and
humans, and which are brought together in B cells to form rearranged Ig genes. Thus, Ig gene
segments as used herein include V gene segments, D gene segments, J gene segments and C gene
segments.
The term “human Ig gene segment” as used herein includes both naturally occurring
sequences of a human Ig gene segment, degenerate forms of naturally occurring sequences of a
human Ig gene segment, as well as synthetic sequences that encode a polypeptide sequence
substantially identical to the polypeptide encoded by a naturally occurring sequence of a human
Ig gene segment. By “substantially” is meant that the degree of amino acid sequence identity is
at least about 85%-95%. Preferably, the degree of amino acid sequence identity is greater than
90%, more preferably greater than 95%
Polynucleotides related to the present description may comprise DNA or RNA and
may be wholly or partially synthetic. Reference to a nucleotide sequence as set out herein
encompasses a DNA molecule with the specified sequence, and encompasses an RNA molecule
with the specified sequence in which U is substituted for T, unless context requires otherwise.
Calculations of "homology" or "sequence identity" between two sequences (the terms
are used interchangeably herein) are performed as follows. The sequences are aligned for optimal
comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino
acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be
disregarded for comparison purposes). In a preferred embodiment, the length of a reference
sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more
preferably at least 50%, even more preferably at least 60%, and even more preferably at least
70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or
nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
When a position in the first sequence is occupied by the same amino acid residue or nucleotide
as the corresponding position in the second sequence, then the molecules are identical at that
position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or
nucleic acid "homology"). The percent identity between the two sequences is a function of the
number of identical positions shared by the sequences, taking into account the number of gaps,
and the length of each gap, which need to be introduced for optimal alignment of the two
sequences.
The comparison of sequences and determination of percent sequence identity between
two sequences may be accomplished using a mathematical algorithm. In a preferred
embodiment, the percent identity between two amino acid sequences is determined using the
Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-53) algorithm, which has been
incorporated into the GAP program in the GCG software package (available online at gcg.com),
using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6,
or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent
identity between two nucleotide sequences is determined using the GAP program in the GCG
software package (available at www.gcg.com), using a NWSgapdna.CMP matrix and a gap
weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred
set of parameters (and the one that should be used if the practitioner is uncertain about what
parameters should be applied to determine if a molecule is within a sequence identity or
homology limitation of the description) is a Blossum 62 scoring matrix with a gap penalty of 12,
a gap extend penalty of 4, and a frameshift gap penalty of 5. The percent identity between two
amino acid or nucleotide sequences can also be determined using the algorithm of Meyers and
Miller ((1989) CABIOS 4:11-17), which has been incorporated into the ALIGN program
(version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty
of 4.
Artificial Ig Loci
The present description is further directed to artificial Ig loci and their use in making
transgenic animals capable of producing immunoglobulins having a human idiotype. Each
artificial Ig locus comprises multiple immunoglobulin gene segments, which include at least one
V region gene segment, one or more J gene segments, one or more D gene segments in the case
of a heavy chain locus, and one or more constant region genes. In the present description, at
least one of the V gene segments encodes a germline or hypermutated human V-region amino
acid sequence. Accordingly, such transgenic animals have the capacity to produce a diversified
repertoire of immunoglobulin molecules, which include antibodies having a human idiotype. In
heavy chain loci human or non-human-derived D-gene segments may be included in the artificial
Ig loci. The gene segments in such loci are juxtaposed with respect to each other in an
unrearranged configuration (or “the germline configuration”), or in a partially or fully rearranged
configuration. The artificial Ig loci have the capacity to undergo gene rearrangement (if the gene
segments are not fully rearranged) in the subject animal thereby producing a diversified
repertoire of immunoglobulins having human idiotypes.
Regulatory elements like promoters, enhancers, switch regions, recombination
signals, and the like may be of human or non-human origin. What is required is that the
elements be operable in the animal species concerned, in order to render the artificial loci
functional. Preferred regulatory elements are described in more detail herein.
In one embodiment, the description includes transgenic constructs containing an
artificial heavy chain locus capable of undergoing gene rearrangement in the host animal thereby
producing a diversified repertoire of heavy chains having human idiotypes. An artificial heavy
chain locus of the transgene contains a V-region with at least one human V gene segment.
Preferably, the V-region includes at least about 5-100 human heavy chain V (or “VH”) gene
segments. As described above, a human VH segment encompasses naturally occurring
sequences of a human VH gene segment, degenerate forms of naturally occurring sequences of a
human VH gene segment, as well as synthetic sequences that encode a polypeptide sequence
substantially (i.e., at least about 85%-95%) identical to a human heavy chain V domain
polypeptide.
In a preferred embodiment, the artificial heavy chain locus contains at least one or
several rat constant region genes, e.g., C δ, C μ and C γ (including any of the C γ subclasses).
In another preferred embodiment, the artificial heavy chain locus contains artificial
constant region genes. In a preferred embodiment, such artificial constant region genes encode a
human CH1 domain and rat CH2 CH3 domains, or a human CH1 and rat CH2, CH3 and CH4
domains. A hybrid heavy chain with a human CH1 domain pairs effectively with a fully human
light chain.
In a preferred embodiment, an artificial Ig locus comprises 3’ enhancer sequences,
including hs1,2, hs3a, hs3b and sequences between rat Calpha and 3’hs3b.
In another preferred embodiment, the artificial heavy chain locus contains artificial
constant region genes lacking CH1 domains In a preferred embodiment, such artificial constant
region genes encode truncated IgM and/or IgG lacking the CH1 domain but comprising CH2,
and CH3, or CH1, CH2, CH3 and CH4 domains. Heavy chains lacking CH1 domains cannot pair
effectively with Ig light chains and form heavy chain only antibodies.
In another embodiment, the description includes transgenic constructs containing an
artificial light chain locus capable of undergoing gene rearrangement in the host animal thereby
producing a diversified repertoire of light chains having human idiotypes. An artificial light
chain locus of the transgene contains a V-region with at least one human V gene segment, e.g., a
V-region having at least one human VL gene and/or at least one rearranged human VJ segment.
Preferably, the V-region includes at least about 5-100 human light chain V (or “VL”) gene
segments. Consistently, a human VL segment encompasses naturally occurring sequences of a
human VL gene segment, degenerate forms of naturally occurring sequences of a human VL
gene segment, as well as synthetic sequences that encode a polypeptide sequence substantially
(i.e., at least about 85%-95%) identical to a human light chain V domain polypeptide. In one
embodiment, the artificial light chain Ig locus has a C-region having at least one rat C gene (e.g.,
rat C λ or C κ).
Another embodiment of the present description is directed to methods of making a
transgenic vector containing an artificial Ig locus. Such methods involve isolating Ig loci or
fragments thereof, and combining the same, with one or several DNA fragments comprising
sequences encoding human V region elements. The Ig gene segment(s) are inserted into the
artificial Ig locus or a portion thereof by ligation or homologous recombination in such a way as
to retain the capacity of the locus to undergo effective gene rearrangement in the subject animal.
Preferably, a non-human Ig locus is isolated by screening a library of plasmids,
cosmids, YACs or BACs, and the like, prepared from the genomic DNA of the same. YAC
clones can carry DNA fragments of up to 2 megabases, thus an entire animal heavy chain locus
or a large portion thereof can be isolated in one YAC clone, or reconstructed to be contained in
one YAC clone. BAC clones are capable of carrying DNA fragments of smaller sizes (about 50-
500 kb). However, multiple BAC clones containing overlapping fragments of an Ig locus can be
separately altered and subsequently injected together into an animal recipient cell, wherein the
overlapping fragments recombine in the recipient animal cell to generate a continuous Ig locus.
Human Ig gene segments can be integrated into the Ig locus on a vector (e.g., a BAC
clone) by a variety of methods, including ligation of DNA fragments, or insertion of DNA
fragments by homologous recombination. Integration of the human Ig gene segments is done in
such a way that the human Ig gene segment is operably linked to the host animal sequence in the
transgene to produce a functional humanized Ig locus, i.e., an Ig locus capable of gene
rearrangement which lead to the production of a diversified repertoire of antibodies with human
idiotypes. Homologous recombination can be performed in bacteria, yeast and other cells with a
high frequency of homologous recombination events. Engineered YACs and BACs can be
readily isolated from the cells and used in making transgenic animals
Rodent oocytes and transgenic animals comprising artificial Ig loci and capable of producing
antibodies having human idiotypes
In one embodiment, the description includes transgenic animals capable of producing
immunoglobulins having human idiotypes, as well as methods of making the same.
The transgenic animals used are selected from rodents (e.g., rats, hamsters, mice and
guinea pigs).
The transgenic animals used for humanized antibody production in the description
carry germline mutations in endogenous Ig loci. In a preferred embodiment, the transgenic
animals are homozygous for mutated endogenous Ig heavy chain and/or endogenous Ig light
chain genes. Further, these animals carry at least one artificial Ig locus that is functional and
capable of producing a repertoire of immunoglobulin molecules in the transgenic animal. The
artificial Ig loci used in the description include at least one human V gene segment.
In a preferred embodiment, the transgenic animals carry at least one artificial Ig
heavy chain locus and at least one artificial Ig light chain locus that are each functional and
capable of producing a repertoire of immunoglobulin molecules in the transgenic animal, which
repertoire of immunoglobulin molecules includes antibodies having a human idiotype. In one
embodiment, artificial loci including at least one non-human C gene are used, and animals
capable of producing chimeric antibodies having a human idiotype and non-human constant
region are provided. In one embodiment, artificial loci including at least one human C gene are
used, and animals capable of producing antibodies having a human idiotype and human constant
region are provided.
In another preferred embodiment, the transgenic animals carry at least one artificial Ig
heavy chain locus, and lack a functional Ig light chain locus. Such animals find use in the
production of heavy chain–only antibodies.
Production of such transgenic animals involves the integration of one or more
artificial heavy chain Ig loci and one or more artificial light chain Ig loci into the genome of a
transgenic animal having at least one endogenous Ig locus that has been or will be inactivated by
the action of one or more meganucleases. Preferably, the transgenic animals are nullizygous for
endogenous Ig heavy chain and/or endogenous Ig light chain and, accordingly, incapable of
producing endogenous immunoglobulins. Regardless of the chromosomal location, an artificial
Ig locus of the present description has the capacity to undergo gene rearrangement and thereby
produce a diversified repertoire of immunoglobulin molecules. An Ig locus having the capacity
to undergo gene rearrangement is also referred to herein as a “functional” Ig locus, and the
antibodies with a diversity generated by a functional Ig locus are also referred to herein as
“functional” antibodies or a “functional” repertoire of antibodies.
The artificial loci used to generate such transgenic animals each include multiple
immunoglobulin gene segments, which include at least one V region gene segment, one or more
J gene segments, one or more D gene segments in the case of a heavy chain locus, and one or
more constant region genes. In the present description, at least one of the V gene segments
encodes a germline or hypermutated human V-region amino acid sequence. Accordingly, such
transgenic animals have the capacity to produce a diversified repertoire of immunoglobulin
molecules, which include antibodies having a human idiotype.
In one embodiment, the artificial loci used comprise at least one non-human C region
gene segment. Accordingly, such transgenic animals have the capacity to produce a diversified
repertoire of immunoglobulin molecules, which include chimeric antibodies having a human
idiotype.
In one embodiment, the artificial loci used comprise at least one human C region gene
segment. Accordingly, such transgenic animals have the capacity to produce a diversified
repertoire of immunoglobulin molecules, which include antibodies having a human idiotype and
a human constant region.
In one embodiment, the artificial loci used comprise at least one artificial constant
region gene. For example, an exemplary artificial C constant region gene is a constant region
gene encoding a human IgG CH1 domain and rat IgG CH2 and CH3 domain. Accordingly, such
transgenic animals have the capacity to produce a diversified repertoire of immunoglobulin
molecules, which include antibodies having a human idiotype and an artificial constant region
comprising both human and non-human components.
The transgenic vector containing an artificial Ig locus is introduced into the recipient
cell or cells and then integrated into the genome of the recipient cell or cells by random
integration or by targeted integration.
For random integration, a transgenic vector containing an artificial Ig locus can be
introduced into a recipient cell by standard transgenic technology. For example, a transgenic
vector can be directly injected into the pronucleus of a fertilized oocyte. A transgenic vector can
also be introduced by co-incubation of sperm with the transgenic vector before fertilization of the
oocyte. Transgenic animals can be developed from fertilized oocytes. Another way to introduce
a transgenic vector is by transfecting embryonic stem cells or other pluripotent cells (for example
primordial germ cells) and subsequently injecting the genetically modified cells into developing
embryos. Alternatively, a transgenic vector (naked or in combination with facilitating reagents)
can be directly injected into a developing embryo. Ultimately, chimeric transgenic animals are
produced from the embryos which contain the artificial Ig transgene integrated in the genome of
at least some somatic cells of the transgenic animal. In another embodiment, the transgenic
vector is introduced into the genome of a cell and an animal is derived from the transfected cell
by nuclear transfer cloning.
In a preferred embodiment, a transgene containing an artificial Ig locus is randomly
integrated into the genome of recipient cells (such as fertilized oocyte or developing embryos).
In a preferred embodiment, offspring that are nullizygous for endogenous Ig heavy chain and/or
Ig light chain and, accordingly, incapable of producing endogenous immunoglobulins and
capable of producing transgenic immunoglobulins are obtained.
For targeted integration, a transgenic vector can be introduced into appropriate
recipient cells such as embryonic stem cells, other pluripotent cells or already differentiated
somatic cells. Afterwards, cells in which the transgene has integrated into the animal genome
can be selected by standard methods. The selected cells may then be fused with enucleated
nuclear transfer unit cells, e.g. oocytes or embryonic stem cells, cells which are totipotent and
capable of forming a functional neonate. Fusion is performed in accordance with conventional
techniques which are well established. See, for example, Cibelli et al., Science (1998) 280:1256;
Zhou et al. Science (2003) 301: 1179. Enucleation of oocytes and nuclear transfer can also be
performed by microsurgery using injection pipettes. (See, for example, Wakayama et al., Nature
(1998) 394:369.) The resulting cells are then cultivated in an appropriate medium, and
transferred into synchronized recipients for generating transgenic animals. Alternatively, the
selected genetically modified cells can be injected into developing embryos which are
subsequently developed into chimeric animals.
In one embodiment, a meganuclease is used to increase the frequency of homologous
recombination at a target site through double-strand DNA cleavage. For integration into a
specific site, a site specific meganuclease may be used. In one embodiment, a meganuclease
targeting an endogenous Ig locus is used to increase the frequency of homologous recombination
and replacement of an endogenous Ig locus, or parts thereof with an artificial Ig locus, or parts
thereof. In one embodiment, the transgenic animal lacks a functional Ig light chain locus and
comprises an artificial Ig heavy chain locus.
Immunoglobulins having a human idiotype
Once a transgenic animal capable of producing immunoglobulins having a human
idiotype is made, immunoglobulins and antibody preparations against an antigen can be readily
obtained by immunizing the animal with the antigen. “Polyclonal antisera composition” as used
herein includes affinity purified polyclonal antibody preparations.
A variety of antigens can be used to immunize a transgenic animal. Such antigens
include but are not limited to, microorganisms, e.g. viruses and unicellular organisms (such as
bacteria and fungi), alive, attenuated or dead, fragments of the microorganisms, or antigenic
molecules isolated from the microorganisms.
Preferred bacterial antigens for use in immunizing an animal include purified antigens
from Staphylococcus aureus such as capsular polysaccharides type 5 and 8, recombinant versions
of virulence factors such as alpha-toxin, adhesin binding proteins, collagen binding proteins, and
fibronectin binding proteins. Preferred bacterial antigens also include an attenuated version of S.
aureus, Pseudomonas aeruginosa, enterococcus, enterobacter, and Klebsiella pneumoniae, or
culture supernatant from these bacteria cells. Other bacterial antigens which can be used in
immunization include purified lipopolysaccharide (LPS), capsular antigens, capsular
polysaccharides and/or recombinant versions of the outer membrane proteins, fibronectin
binding proteins, endotoxin, and exotoxin from Pseudomonas aeruginosa, enterococcus,
enterobacter, and Klebsiella pneumoniae.
Preferred antigens for the generation of antibodies against fungi include attenuated
version of fungi or outer membrane proteins thereof, which fungi include, but are not limited to,
Candida albicans, Candida parapsilosis, Candida tropicalis, and Cryptococcus neoformans.
Preferred antigens for use in immunization in order to generate antibodies against
viruses include the envelop proteins and attenuated versions of viruses which include, but are not
limited to respiratory synctial virus (RSV) (particularly the F-Protein), Hepatitis C virus (HCV),
Hepatits B virus (HBV), cytomegalovirus (CMV), EBV, and HSV.
Antibodies specific for cancer can be generated by immunizing transgenic animals
with isolated tumor cells or tumor cell lines as well as tumor-associated antigens which include,
but are not limited to, Herneu antigen (antibodies against which are useful for the treatment of
breast cancer); CD20, CD22 and CD53 antigens (antibodies against which are useful for the
treatment of B cell lymphomas), prostate specific membrane antigen (PMSA) (antibodies against
which are useful for the treatment of prostate cancer), and 17-1A molecule (antibodies against
which are useful for the treatment of colon cancer).
The antigens can be administered to a transgenic animal in any convenient manner,
with or without an adjuvant, and can be administered in accordance with a predetermined
schedule.
For making a monoclonal antibody, spleen cells are isolated from the immunized
transgenic animal and used either in cell fusion with transformed cell lines for the production of
hybridomas, or cDNAs encoding antibodies are cloned by standard molecular biology techniques
and expressed in transfected cells. The procedures for making monoclonal antibodies are well
established in the art. See, e.g., European Patent Application 0 583 980 A1 (“Method For
Generating Monoclonal Antibodies From Rabbits”), U.S. Patent No. 4,977,081 (“Stable Rabbit-
Mouse Hybridomas And Secretion Products Thereof”), WO 97/16537 (“Stable Chicken B-cell
Line And Method of Use Thereof”), and EP 0 491 057 B1 (“Hybridoma Which Produces Avian
Specific Immunoglobulin G”), the disclosures of which are incorporated herein by reference. In
vitro production of monoclonal antibodies from cloned cDNA molecules has been described by
Andris-Widhopf et al., “Methods for the generation of chicken monoclonal antibody fragments
by phage display”, J Immunol Methods 242:159 (2000), and by Burton, D. R., “Phage display”,
Immunotechnology 1:87 (1995).
Once chimeric monoclonal antibodies with human idiotypes have been generated,
such chimeric antibodies can be easily converted into fully human antibodies using standard
molecular biology techniques. Fully human monoclonal antibodies are not immunogenic in
humans and are appropriate for use in the therapeutic treatment of human subjects.
Antibodies of the description include heavy chain-only antibodies
In one embodiment, transgenic animals which lack a functional Ig light chain locus,
and comprising an artificial heavy chain locus, are immunized with antigen to produce heavy
chain-only antibodies that specifically bind to antigen.
In one embodiment, the description includes monoclonal antibody producing cells
derived from such animals, as well as nucleic acids derived therefrom. Also described are
hybridomas derived therefrom. Also described are fully human heavy chain-only antibodies, as
well as encoding nucleic acids, derived therefrom.
Teachings on heavy chain-only antibodies are found in the art. For example, see PCT
publications WO02085944, WO02085945, WO2006008548, and WO2007096779. See also US
,840,526; US 5,874,541; US 6,005,079; US 6,765,087; US 5,800,988; EP 1589107; WO
9734103; and US 6,015,695.
Pharmaceutical Compositions
In a further embodiment of the present description, purified monoclonal or polyclonal
antibodies are admixed with an appropriate pharmaceutical carrier suitable for administration to
patients, to provide pharmaceutical compositions.
Patients treated with the pharmaceutical compositions of the description are
preferably mammals, more preferably humans, though veterinary uses are also contemplated.
Pharmaceutically acceptable carriers which can be employed in the present
pharmaceutical compositions can be any and all solvents, dispersion media, isotonic agents and
the like. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the
recipient or to the therapeutic effectiveness of the antibodies contained therein, its use in the
pharmaceutical compositions of the present description is appropriate.
The carrier can be liquid, semi-solid, e.g. pastes, or solid carriers. Examples of
carriers include oils, water, saline solutions, alcohol, sugar, gel, lipids, liposomes, resins, porous
matrices, binders, fillers, coatings, preservatives and the like, or combinations thereof.
Methods of Treatment
In a further embodiment of the present description, methods are included for treating
a disease in a vertebrate, preferably a mammal, preferably a primate, with human subjects being
an especially preferred embodiment, by administering a purified antibody composition of the
description desirable for treating such disease.
The antibody compositions can be used to bind and neutralize or modulate an
antigenic entity in human body tissues that causes or contributes to disease or that elicits
undesired or abnormal immune responses. An "antigenic entity" is herein defined to encompass
any soluble or cell surface bound molecules including proteins, as well as cells or infectious
disease-causing organisms or agents that are at least capable of binding to an antibody and
preferably are also capable of stimulating an immune response.
Administration of an antibody composition against an infectious agent as a
monotherapy or in combination with chemotherapy results in elimination of infectious particles.
A single administration of antibodies decreases the number of infectious particles generally 10 to
100 fold, more commonly more than 1000-fold. Similarly, antibody therapy in patients with a
malignant disease employed as a monotherapy or in combination with chemotherapy reduces the
number of malignant cells generally 10 to 100 fold, or more than 1000-fold. Therapy may be
repeated over an extended amount of time to assure the complete elimination of infectious
particles, malignant cells, etc. In some instances, therapy with antibody preparations will be
continued for extended periods of time in the absence of detectable amounts of infectious
particles or undesirable cells.
Similarly, the use of antibody therapy for the modulation of immune responses may
consist of single or multiple administrations of therapeutic antibodies. Therapy may be
continued for extended periods of time in the absence of any disease symptoms.
The subject treatment may be employed in conjunction with chemotherapy at dosages
sufficient to inhibit infectious disease or malignancies. In autoimmune disease patients or
transplant recipients, antibody therapy may be employed in conjunction with immunosuppressive
therapy at dosages sufficient to inhibit immune reactions.
EXAMPLES
In mice transgenic for human immunoglobulin (Ig) loci, suboptimal efficacy in
delivery of fully human antibodies has been attributed to imperfect interaction between the
constant regions of human membrane IgH chains and the mouse cellular signaling machinery. To
) carrying a chimeric
obviate this problem, we here describe a humanized rat strain (OmniRat
human/rat IgH locus [comprising 22 human V s, all human D and J segments in natural
configuration but linked to the rat C locus] together with fully human light-chain loci [12 V κs
linked to J κ-C κ and 16 V λs linked to J λ-C λ]. The endogenous rat Ig loci were silenced by
designer zinc finger nucleases. Following immunization, OmniRats perform as efficiently as
normal rats in yielding high affinity serum IgG. Monoclonal antibodies, comprising fully human
variable regions with sub-nanomolar antigen affinity and carrying extensive somatic mutations,
are readily obtainable – similarly to the yield of conventional antibodies from normal rats.
MATERIALS AND METHODS
Construction of modified human Ig loci on YACs and BACs
a) IgH loci
The human IgH V genes were covered by 2 BACs: BAC6-VH3-11 containing the
authentic region spanning from VH4-39 to VH3-23 followed by VH3-11 (modified from a
commercially available BAC clone 3054M17 CITB) and BAC3 containing the authentic region
spanning from VH3-11 to VH6-1 (811L16 RPCI-11). A BAC termed Annabel was constructed
by joining rat CH region genes immediately downstream of the human VH6Ds-JHs region
(Figure 1). All BAC clones containing part of the human or rat IgH locus were purchased from
Invitrogen.
Both BAC6-VH3-11 and Annabel were initially constructed in S. cerevisiae as
circular YACs (cYACs) and further checked and maintained in E. coli as BACs. Construction
details can be found at www.ratltd.net.
Unlike YACs, BAC plasmid preps yield large quantities of the desired DNA. To
convert a linear YAC into a cYAC or to assemble DNA fragments with overlapping ends into a
single cYAC in S. cerevisiae, which can also be maintained as a BAC in E. coli, two self-
replicating S. cerevisiae/E. coli shuttle vectors, pBelo-CEN-URA, and pBelo-CEN-HYG were
constructed. Briefly, S. cerevisiae CEN4 was cut out as an AvrII fragment from pYAC-RC
ligated to SpeI – linearised pAP599 . The resulting plasmid contains CEN4 cloned in between S.
cerevisiae URA3 and a hygromycin-resistance expression cassette (HygR). From this plasmid,
an ApaLI–BamHI fragment containing URA3 followed by CEN4 or a PmlI–SphI fragment
containing CEN4 followed by HygR was cut out, and ligated to ApaLI and BamHI or HpaI and
SphI doubly digested pBACBelo11 (New England Biolabs) to yield pBelo-CEN-URA and
pBelo-CEN-HYG.
To construct BAC6-VH3-11, initially two fragments, a 115 kb NotI-PmeI and a 110
kb RsrII-SgrAI, were cut out from the BAC clone 3054M17 CITB. The 3’ end of the former
fragment overlaps 22 kb with the 5’ end of the latter. The NotI-PmeI fragment was ligated to a
NotI-BamHI YAC arm containing S. cerevisiae CEN4 as well as TRP1/ARS1 from pYAC-RC,
and the RsrII-SgrAI fragment was ligated to a SgrAI-BamHI YAC arm containing S. cerevisiae
URA3, also from pYAC-RC. Subsequently, the ligation mixture was transformed into S.
cerevisiae AB1380 cells via spheroplast transformation , and URA+TRP+ yeast clones were
selected. Clones, termed YAC6, containing the linear region from human VH4-39 to VH3-23
were confirmed by Southern blot analysis. YAC6 was further extended by addition of a 10.6 kb
fragment 3’ of VH3-23, and conversion to a cYAC. The 10.6 kb extension contains the human
VH3-11 and also occurs at the 5’ end of BAC3. We constructed pBeloHYG-YAC6+BAC3(5’)
for the modification of YAC6. Briefly, 3 fragments with overlapping ends were prepared by
PCR: 1) a ‘stuff’ fragment containing S. cerevisiae TRP1-ARS1 flanked by HpaI sites with 5’
tail matching the sequence upstream of VH4-39 and 3’ tail matching downstream of VH3-23 in
YAC6 (using long oligoes 561 and 562, and pYAC-RC as template), 2) the 10.6 kb extension
fragment with a 5’ tail matching the sequence downstream of VH3-23 as described above and a
unique AscI site at its 3’ end (using long oligoes 570 and 412, and human genomic DNA as
template), and 3) pBelo-CEN-HYG vector with the CEN4 joined downstream with a homology
tail matching the 3’ end of the 10.6 extension fragment and the HygR joined upstream with a tail
matching the sequence upstream of VH4-39 as described above (using long oligoes 414 and 566,
and pBelo-CEN-HYG as template). Subsequently, the 3 PCR fragments were assembled into a
small cYAC conferring HYGR and TRP+ in S. cerevisiae via homologous recombination
associated with spheroplast transformation, and this cYAC was further converted into the BAC
pBeloHYG-YAC6+BAC3(5’). Finally, the HpaI-digested pBeloHYG-YAC6+BAC3(5’) was
used to transform yeast cells carrying YAC6, and through homologous recombination cYAC
BAC6-VH3-11 conferring only HYGR was generated. Via transformation, see below, this cYAC
was introduced as a BAC in E. coli. The human VH genes in BAC6-VH3-11 were cut out as a ~
182 kb AsiSI (occurring naturally in the HygR) – AscI fragment, and the VH genes in BAC3
were cut out as a ~173 kb NotI- fragment (Figure 1 top).
For the assembly of the C region with the VH overlap, the human VH6Ds-JHs
region had to be joined with the rat genomic sequence immediately downstream of the last JH
followed by rat Cs to yield a cYAC/BAC. To achieve this, 5 overlapping restriction as well as
PCR fragments were prepared; a 6.1 kb fragment 5’ of human VH6-1 (using oligoes 383 and
384, and human genomic DNA as template), an ~78 kb PvuI-PacI fragment containing the
human VH6Ds-JHs region cut out from BAC1 (RP11645E6), a 8.7 kb fragment joining the
human JH6 with the rat genomic sequence immediately downstream of the last JH and
containing part of rat µ coding sequence (using oligos 488 and 346, and rat genomic DNA as
template), an ~ 52 kb NotI-PmeI fragment containing the authentic rat µ, δ and γ2c region cut out
from BAC M5 (CH230-408M5) and the pBelo-CEN-URA vector with the URA3 joined
downstream with a homology tail matching the 3’ end of the rat γ2c region and the CEN4 joined
upstream with a tail matching the 5’ region of human VH6-1 as described (using long oligoes
385 and 550, and pBelo-CEN-URA as template). Correct assembly via homologous
recombination in S. cerevisiae was analysed by PCR and purified cYAC from the correct clones
was converted into a BAC in E. coli.
For the assembly of Annabel parts of the above cYAC/BAC containing humanVH6-
1-Ds-JHs followed by the authentic rat µ, δ and γ2c region, as well as PCR fragments were used.
Five overlapping fragments contained the 6.1 kb fragment at the 5’ end of human VH6-1 as
described above, an ~83 kb SpeI fragment comprising human VH6Ds-JHs immediately
followed by the rat genomic sequence downstream of the last JH and containing part of rat Cµ, a
.2 kb fragment joining the 3’ end of rat µ with the 5’ end of rat γ1 (using oligos 490 and 534,
and rat genomic DNA as template), an ~118 kb NotI-SgrAI fragment containing the authentic rat
γ1, γ2b, ε, α and 3’E IgH enhancer region cut out from BAC I8 (CH230-162I08), and the pBelo-
CEN-URA vector with the URA3 joined downstream with a homology tail matching the 3’ end
of rat 3’E and the CEN4 joined upstream with a tail matching the 5’ end of human VH6-1 as
described above. There is a 10.3 kb overlap between the human VH6-1 regions in both the BAC3
and Annabel. The human VH6-1 -Ds - JHs followed by the rat CH region together with the S.
cerevisiae URA3 in Annabel can be cut out as a single ~183 kb NotI-fragment (see Figure 1 top).
BAC6-VH3-11, BAC3 and Annabel were checked extensively by restriction analysis
and partial sequencing for their authenticity.
b) IgL loci
The human Ig λ locus on a ~410 kb YAC was obtained by recombination assembly of
a V λ YAC with 3 C λ containing cosmids . Rearrangement and expression was verified in
transgenic mice derived from ES cells containing one copy of a complete human Ig λ YAC .
This Ig λ YAC was shortened by the generation of a circular YAC removing ~100kb of the
region 5’ of V λ3-27. The vector pYAC-RC was digested with ClaI and BspEI to remove URA3
and ligated with a ClaI/NgoMIV fragment from pAP 599 containing HYG. PCR of the region
containing the yeast centromere and hygromycin marker gene from the new vector (pYAC-RC-
HYG) was carried out with primers with 5’ ends homologous to a region 5’ of V λ3-27 (primer
276) and within the ADE2 marker gene in the YAC arm (primer 275). The PCR fragment (3.8
kb) was integrated into the Ig λ YAC using a high efficiency lithium acetate transformation
method and selection on hygromycin containing YPD plates. DNA was prepared from the
clones (Epicentre MasterPure Yeast DNA purification kit) and analysed for the correct junctions
by PCR using the following oligos: 243 + 278 and Hyg end R + 238. Plugs were made and
yeast chromosomes removed by PFGE (0.8% agarose (PFC) (Biorad) gel [6V/cm, pulse times of
60s for 10hr and 10s for 10hr, 8C) leaving the circular yeast artificial chromosome caught in the
agarose block . The blocks were removed and digested with NruI. Briefly, blocks were
preincubated with restriction enzyme buffer in excess at a 1X final concentration for 1 hr on ice.
Excess buffer was removed leaving just enough to cover the plugs, restriction enzyme was added
to a final concentration of 100U/ml and the tube incubated at 37C for 4-5hrs. The linearized
YAC was ran out of the blocks by PFGE, cut out from the gel as a strip and purified as described
below.
For the human Ig κ locus 3 BACs were chosen (RP11-344F17, RP11-1134E24 and
RP11-156D9, Invitrogen), which covered a region over 300 kb from 5’ V κ1-17 to 3’ KDE . In
digests and sequence analyses three overlapping fragments were identified: from V κ1-17 to V κ3-
7 (150 kb NotI with ~14 kb overlap), from V κ3-7 to 3’ of C κ (158 kb NotI with ~40 kb overlap)
and from C κ to 3’ of the KDE (55 kb PacI with 40 kb overlap). Overlapping regions may
generally favour joint integration when co-injected into oocytes .
Gel analyses and DNA purification
Purified YAC and BAC DNA was analysed by restriction digest and separation on
conventional 0.7% agarose gels . Larger fragments, 50-200 kb, were separated by PFGE
(Biorad Chef MapperTM) at 80C, using 0.8% PFC Agaraose in 0.5% TBE, at 2-20 sec switch
time for 16 h, 6V/cm, 10mA. Purification allowed a direct comparison of the resulting fragments
with the predicted size obtained from the sequence analysis. Alterations were analysed by PCR
and sequencing.
Linear YACs, circular YACs and BAC fragments after digests, were purified by
electro-elution using ElutrapTM (Schleicher and Schuell) from strips cut from 0.8% agarose
gels run conventionally or from pulsed-field-gel electrophoresis (PFGE). The DNA
concentration was usually several ng/µl in a volume of ~100µl. For fragments up to ~200 kb the
DNA was precipitated and re-dissolved in micro-injection buffer (10 mM Tris-HCl pH 7.5, 100
mM EDTA pH 8 and 100 mM NaCl but without Spermine/Spermidine) to the desired
concentration.
The purification of circular YACs from yeast was carried out using Nucleobond AX
silica-based anion-exchange resin (Macherey-Nagel, Germany). Briefly, spheroplasts were made
. The cells then underwent alkaline lysis, binding to
using zymolyase or lyticase and pelleted
AX100 column and elution as described in the Nucleobond method for a low-copy plasmid.
Contaminating yeast chromosomal DNA was hydolyzed using Plamid –Safe™ ATP-Dependent
DNase (Epicentre Biotechnologies) followed by a final cleanup step using SureClean (Bioline).
An aliquot of DH10 electrocompetent cells (Invitrogen) was then transformed with the circular
YAC to obtain BAC colonies. For microinjection, the insert DNA (150-200 kb), was separated
from BAC vector DNA(~10 kb) using a filtration step with sepharose 4B-CL .
Derivation of rats and breeding
Purified DNA encoding recombinant immunoglobulin loci was resuspended in
microinjection buffer with 10 mM Spermine and 10 mM Spemidine. The DNA was injected into
fertilized oocytes at various concentrations from 0.5 to 3 ng/µl.
Plasmid DNA or mRNA encoding ZFNs specific for rat immunoglobulin genes were
injected into fertilized oocytes at various concentrations from 0.5 to 10 ng/ul.
Microinjections were performed at Caliper Life Sciences facility. Outbred SD/Hsd
(WT) strain animals were housed in standard microisolator cages under approved animal care
protocols in animal facility that is accredited by the Association for the Assessment and
Accreditation for Laboratory Animal Care (AAALAC). The rats were maintained on a 14-10 h
light/dark cycle with ad libitum access to food and water. Four to five week old SD/Hsd female
rats were injected with 20-25 IU PMSG (Sigma-Aldrich) followed 48 hours later with 20-25 IU
hCG (Sigma-Aldrich) before breeding to outbred SD/Hsd males. Fertilized 1-cell stage embryos
were collected for subsequent microinjection. Manipulated embryos were transferred to
pseudopregnant SD/Hsd female rats to be carried to parturition.
Multi-feature human Ig rats (human IgH, Ig κ and Ig λ in combination with rat J KO, κ
KO and λ KO) and WT, as control, were analyzed at 10–18 weeks of age. The animals were bred
at Charles River under specific pathogen-free conditions.
PCR and RT-PCR
Transgenic rats were identified by PCR from tail or ear clip DNA using a Genomic
DNA Mini Kid (Bioline). For IgH PCRs < 1kb GoTaq Green Master mix was used (Promega)
under the following conditions: 94°C 2 mins, 32 x (94°C 30 secs, 54-670C (see supplemental
Table 1 for primers and specific annealing temperatures) 30 secs, 72°C 1 min), 72°C 2 mins. For
IgH PCRs >1kb KOD polymerase (Novagen) was used under the following conditions: 95°C 2
mins, 32 x (95°C 20 secs, 56-620C (supplementary Table 1) 20 secs, 70°C 90 secs), 70°C 2
mins. For Ig κ and Ig λ PCR, all <1kb, the above condition were used except extension at 72oC
for 50 secs.
RNA was extracted from Blood using the RiboPure Blood Kit (Ambion) and RNA
extraction from spleen, bone marrow or lymph nodes used RNASpin mini kit. (GE Healthcare).
cDNA was made using Oligo dT and Promega Reverse Transcriptase at 42°C for 1 hour.
GAPDH PCR reactions (oligos 429-430) determined the concentration.
RT-PCRs were set up using VH leader primers with rat µCH2 or rat γCH2 primers
(supplementary Table 1). Amplification with GoTaq Green Master mix were 94°C 2 mins, 34 x
(94°C 30 secs, 55-65oC 30 secs, 72°C 50-60 secs), 72°C 2 mins. PCR products of the expected
size were either purified by gel or QuickClean (Bioline) and sequenced directly or cloned into
pGemT (Promega).
Protein purification
IgM was purified on anti-IgM affinity matrix (BAC B.V., Netherlands, CaptureSelect
#2890.05) as described in the protocol. Similarly, human Ig κ and Ig λ was purified on anti-L
chain affinity matrix (CaptureSelect anti-Ig κ #0833 and anti-Ig λ # 0849) according to the
protocol.
For rat IgG purification protein A and protein G agarose was used (Innova,
Cambridge, UK, #851-0024 and #895-0024). Serum was incubated with the resin and binding
facilitated at 0.1 M sodium phosphate pH 7 for protein G and pH 8 for protein A under gentle
mixing. Poly-prep columns (Bio-Rad) were packed with the mixture and washed extensively
with PBS pH7.4. Elution buffer was 0.1 M Sodium Citrate pH 2.5 and neutralization buffer was
1 M Tris-HCl pH 9
Electrophoresis was performed on 4-15% SDS-PAGE and Coomassie brilliant blue
was used for staining. MW standards were HyperPage Prestained Protein Marker (#BIO-33066,
Bioline).
Flow cytometry analysis and FISH
Cell suspensions were washed and adjusted to 5x105 cells/100 µl in PBS-1% BSA-
0.1% Azide. Different B-cell subsets were identified using mouse anti-rat IgM FITC-labelled
mAb (MARM 4, Jackson Immunoresearch Laboratories) in combination with anti-B cell CD45R
(rat B220)-PE-conjugated mAb (His 24, BD biosciences) or anti-IgD-PE-conjugated mAb
(MARD-3, Abd Serotec). A FACS CantoII flow cytometer and FlowJo software (Becton
Dickinson, Pont de Claix, France) was used for the analysis.
Fluorescence in situ hybridisation was carried out on fixed blood lymphocytes using
purified IgH and IgL C-region BACs as described.
Immunization, cell fusion and affinity measurement
Immunizations were performed with 125 µg PG in CFA, 150 µg hGHR in CFA, 200
µg Tau/KLH in CFA, 150 µg HEL in CFA, 150 µg OVA in CFA at the base of the tail and
medial iliac lymph node cells were fused with mouse P3X63Ag8.653 myeloma cells 22 days
later as described . For multiple immunizations protein, 125 µg PG or HEL, or 100 µg hGHR or
CD14 in GERBU adjuvant (www.Gerbu.com), was administered intraperitoneally as follows:
day 0, day 14, day 28 and day 41 without adjuvant, followed by spleen cell fusion with
P3x63Ag8.653 cells 4 days later.
Binding kinetics were analyzed by surface Plasmon resonance using a Biacore 2000
with the antigens directly immobilized as described .
Supplementary Table 1
* **
PCR and RT-PCR conditions to detect human IgH and IgL integration and expression
IgH Primers Annealing Temp (Tm-5) Fragment size
Hyg (5’ BAC6) Hyg 3’ F - 459 54°C ~400bp
V4-34 (BAC6) 205-206 65°C ~1kb
V4-28 (BAC6) 203-204 65°C ~1kb
V3-11 (overlap BAC6461 60°C ~500bp
BAC3)
V1-8 (BAC3) 371-372 60°C ~300bp
V4-4 (BAC3) 393-396 60°C ~750bp
V6-1 (BAC3- 359-360 65°C ~350bp
Annabel)
JH (Annabel) 368-369 62°C ~250bp
583-535 62°C ~3kb
µ-γ1 (Annabel)
Ura (3’ Annabel) 241-253 56°C ~3kb
Primers Annealing Temp (Tm-5) Fragment size
KDE 313-314 66°C ~600bp
cKappa 307-308 64°C ~600bp
V4-1 333-334 60°C ~300bp
V1-5 329-330 64°C ~400bp
V1-6 331-332 60°C ~300bp
V3-7 309-310 66°C ~700bp
V3-15 311-312 66°C ~500bp
Igλ Primers Annealing Temp (Tm-5) Fragment size
V3-27 215-216 67°C ~400bp
V3-19 213-214 67°C ~700bp
V2-14 211-212 67°C ~400bp
V middle 168-169 65°C ~500bp
JLambda 162-163 67°C ~800bp
cLambda 170-171 67°C ~500bp
Enhancer 172-173 67°C ~400bp
For DNA extraction from ear and tail clips the Genomic DNA Mini Kit (Bioline) was used.
For PCRs 1kb or less in size GoTaq Green Master mix (Promega) was used under the following
conditions: 94°C 2 mins, 32 x (94°C 30 secs, Tm-5 (below) 30 secs, 72°C 1 min [50 sec for
Igκ/λ]), 72°C 2 mins. Annealing temperatures were set at the lowest primer Tm– 5 C
(www.sigmagenosys.com/calc/DNACalc.asp). For PCRs >1kb KOD polymerase (Novagen)
was used under the following conditions: 95°C 2 mins, 32 x (95°C 20 secs, Tm-5 20 secs, 70°C
90 secs), 70°C 2mins.
IgH Primer Annealing Temp (Tm-5) Fragment size
VH1 Leader 390 65°C
VH2 Leader 391 65°C
VH3 Leader 392 65°C
VH4 Leader 393 60°C
VH6 Leader 394 65°C
VH4-39 Leader 761 55°C
Rat µCH2 345 ~1kb
Rat CH2 682 ~800bp
Ig Primer Annealing Temp (Tm-5) Fragment size
HuVK1 Leader 400/474 63°C
HuVK3 Leader 401/475 63°C
HuVK4 Leader 476 63°C
HuVK5 Leader 477 63°C
Hu C region 402 ~600bp
Ig Primer Annealing Temp (Tm-5) Fragment size
HuVL2 Leader 388/478 58°C
HuVL3 Leader 398/479/480/482/483/481/484 58°C
HuVL4 Leader 485 58°C
Hu C region 387 ~600bp
RNA was extracted from Blood using the RiboPure Blood Kit (Ambion). RNA extracted from
spleen, bone marrow or lymph nodes used the RNASpin mini kit (GE Healthcare). cDNA was
made using Oligo dT and Promega Reverse Transcriptase at 42°C 1 hour. PCRs using the GoTaq
Green Master mix were set up as follows: 94°C 2 mins, 34 x (94°C 30 secs, Tm-5 30 secs, 72°C
1 min [50 sec for Igκ/λ]), 72°C 2 mins.
Primers
Number Oligonucleotide sequence 5'-3'
162 GGGGCCAAGGCCCCGAGAGATCTCAGG
163 CACTGGGTTCAGGGTTCTTTCCACC
168 GTGGTACAGAAGTTAGAGGGGATGTTGTTCC
169 TCTTCTACAAGCCCTTCTAAGAACACCTGG
170 AGCACAATGCTGAGGATGTTGCTCC
171 ACTGACCCTGATCCTGACCCTACTGC
172 AAACACCCCTCTTCTCCCACCAGC
173 CGCTCATGGTGAACCAGTGCTCTG
203 GCTATTTAAGACCCACTCCCTGGCA
204 AAAACCTGCAGCAAGGATGTGAGG
205 GCTCCTTCAGCACATTTCCTACCTGGA
206 CCATATATGGCAAAATGAGTCATGCAGG
211 CTCTGCTGCTCCTCACCCTCCTCACTCAGG
212 GAGAGTGCTGCTGCTTGTATATGAGCTGCA
213 TGGCTCACTCTCCTCACTCTTTGCATAGGTT
214 GATGGTTACCACTGCTGTCCCGGGAGTTAC
215 ATCCCTCTCCTGCTCCCCCTCCTCATTCTCTG
216 TGATGGTCAAGGTGACTGTGGTCCCTGAGCTG
238 AACAAGTGCGTGGAGCAG
241 GTACTGTTGACATTGCGAAGAGC
243 TGGTTGACATGCTGGCTAGTC
253 TGTCTGGCTGGAATACACTC
275 AAATGAGCTTCAAATTGAGAAGTGACGCAAGCATCAATGGTATAATGTCC
Number Oligonucleotide sequence 5'-3'
AGAGTTGTGAGGCCTTGGGGACTGTGTGCCGAACATGCTC
276 CCAGCACTGTTCAATCACAGTATGATGAGCCTAATGGGAATCCCACTAGG
CTAGTCTAGTCACCACATTAAAGCACGTGGCCTCTTATCG
278 TGACCATTGCTTCCAAGTCC
307 GAGGAAAGAGAGAAACCACAGGTGC
308 CACCCAAGGGCAGAACTTTGTTACT
309 TGTCCAGGTATGTTGAAGAATGTCCTCC
310 TGGACTCTGTTCAACTGAGGCACCAG
311 GGCCTTCATGCTGTGTGCAGACTA
312 CAGGTCGCACTGATTCAAGAAGTGAGT
313 TTCAGGCAGGCTCTTACCAGGACTCA
314 TGCTCTGACCTCTGAGGACCTGTCTGTA
329 TCACGTGACTGTGATCCCTAGAA
330 CACTGTTATGCCAACTGAACAGC
331 CGTAGCAGTCCCCATCTGTAATC
332 ATGTCAGAGGAGCAGGAGAGAGA
333 CACGCCTCACATCCAATATGTTA
334 ATACCCTCCTGACATCTGGTGAA
345 GCTTTCAGTGATGGTCAGTGTGCTTATGAC
346 TGGAAGACCAGGAGATATTCAGGGTGTC
359 TTGCTTAACTCCACACCTGCTCCTG
360 TGCTTGGAACTGGATCAGGCAGTC
368 CACCCTGGTCACCGTCTCC
369 AGACAGTGACCAGGGTGCCAC
371 TGAGGAACGGATCCTGGTTCAGTC
372 ATCTCCTCAGCCCAGCACAGC
383 CCTCCCATGATTCCAACACTG
384 CTCACCGTCCACCACTGCTG
385 CTGTGCCACAAACATGCAAAGATAAGTTCCATGTGACAAGTCTGAACTCA
GTGTTGGAATCATGGGAGGCGGCCGCGTTATCTATGCTGTCTCACCATAG
387 TGCTCAGGCGTCAGGCTCAG
388 TGCTCAGGCGTCAGGCTCAG
390 ATGGACTGGACCTGGAGGATCC
391 TCCACGCTCCTGCTGCTGAC
392 ATGGAGTTTGGGCTGAGCTGG
393 TGAAACACCTGTGGTTCTTCC
394 TCATCTTCCTGCCCGTGCTGG
396 GACTCGACTCTTGAGGGACG
398 ATGTGGCCACAGGCTAGCTC
400 ATGAGGGTCCCCGCTCAG
401 ATGGAAGCCCCAGCTCAGC
402 CCTGGGAGTTACCCGATTGG
412 GGCGCGCCAAGCATCATGTCCTACCTGGCTG
414 CAAAGTACGTGGCACCTCCCTCGTCTTTCTTCCTCCTGCTCCAGCCAGGTA
GGACATGATGCTTGGCGCGCCGTTATCTATGCTGTCTCACCATAG
Number Oligonucleotide sequence 5'-3'
429 CAGTGCCAGCCTCGTCTCAT
430 AGGGGCCATCCACAGTCTTC
448 CTTCACTGTGTGTTCTTGGGATAC
459 GTGTAATGCTTTGGACGGTGTGTTAGTCTC
461 GCATAGCGGCGCGCCAAGCATCATGTCCTACCTGGCTG
474 GACATGAGAGTCCTCGCTCAGC
475 AAGCCCCAGCGCAGCTTC
476 ATGGTGTTGCAGACCCAGGTC
477 GTCCCAGGTTCACCTCCTCAG
478 TCCTCASYCTCCTCACTCAGG
479 CGTCCTTGCTTACTGCACAG
480 AGCCTCCTTGCTCACTTTACAG
481 CCTCCTCAYTYTCTGCACAG
482 GCTCACTCTCCTCACTCTTTGC
483 CCTCCTCTCTCACTGCACAG
484 GCCACACTCCTGCTCCCACT
485 ATGGCCTGGGTCTCCTTCTAC
488 ATTACTACTACTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTC
ACCGTCTCCTCAGGAAGAATGGCCTCTCCAGGTC
490 CTGTCGTTGAGATGAACCCCAATGTGAG
534 GGAACTGATGTGATCTCAGTCACACAGCTAATGCAAAGGTCAGCAGGCT
GTTTACTGCCTGGAGGTTCATCGCCCAATTCCAAAGTCAC
535 CTAGTCTGCATGGGTCTCCGCAAAC
550 CTGGTATAATCATAAGTCTCCACTTAATAGTTCTGTAGACAGAATCTTCAT
TTAGACTTACAGACCGCGGCCGCACCGCAGGGTAATAACTG
561 GCAACCCTTCTTGCCACTCATGTCCCAGCTCTCACCATGTGACATAGCCTG
TTAACAATTCGGTCGAAAAAAGAAAAGGAGAG
562 AATGTTCTTAGTATATATAAACAAGCTACTCCCAATTCATAGTCAACTAA
GTTAACATTCCACATGTTAAAATAGTGAAGGAG
566 TTAACAGGCTATGTCACATGGTGAGAGCTGGGACATGAGTGGCAAGAAG
GGTTGCCAGACTCCCCCTTTACCTCTATATCGTGTTC
570 CTTAGTTGACTATGAATTGGGAGTAGCTTGTTTATATATACTAAGAACATT
TGTCAGAAGCTCTTTCTTGTTTATTCCCAGTTTGC
583 CATGTCCGTATGTTGCATCTGC
682 GGGAAGATGAAGACAGATG
761 TGGAGTGGATTGGGAGT
RESULTS
The human IgH and IgL loci
Construction of the human Ig loci employed established technologies to assemble
16, 19, 24-26
large DNA segments using YACs and BACs . As multiple BAC modifications in E. coli
frequently deleted repetitive regions such as switch sequences and enhancers, a method was
developed to assemble sequences with overlapping ends in S. cerevisiae as circular YAC
(cYAC) and, subsequently, converting such a cYAC into a BAC. Advantages of YACs include
their large size, the ease of homologous alterations in the yeast host and the sequence stability,
while BACs propagated in E. coli offer the advantages of easy preparation and large yield.
Additionally, detailed restriction mapping and sequencing analysis can be better achieved in
BACs than in YACs.
Sequence analysis and digests identified gene clusters of interest and ensured locus
integrity and functionality to secure DNA rearrangement and switching over a wide region. The
layout of the human IgH (human VH, D and JH segments followed by rat C genes), Ig κ and Ig λ
loci are depicted in Fig. 1a-c. As shown previously, overlapping regions may generally favor
joint integration when co-injected into oocytes . Thereby, insertion of BAC6-VH3-11, a 182 kb
AsiSI-AscI fragment, with BAC3, a 173 kb NotI fragment, and BAC3-1N12M5I8 (Hu-Rat
Annabel), a 193 kb NotI fragment, led to the reconstitution of a fully functional transgenic IgH
loci in the rat genome. Similarly, the human Ig κ locus was integrated by homologous overlaps.
The human Ig λ locus was isolated intact as a ~300 kb YAC and also fully inserted into a rat
chromosome. The integration success was identified by transcript analysis which showed
V(D)J-C recombinations from the most 5’ to the most 3’ end of the locus injected. Multiple
copies were identified by qPCR (not shown) and it is likely that head to tail integrations
occurred. In all cases, transgenic animals with single-site integrations were generated by
breeding.
Breeding to homozygosity
The derivation of transgenic rats by DNA microinjection into oocytes, their breeding
and immunization is comparable to the mouse. However, ZFN technology to obtain gene knock-
11, 13
outs has only been reported recently . Silencing of the rat IgH locus by J deletion using ZFN
KO technology has been described and a manuscript describing silencing of the rat IgL loci,
targeting of C κ and deletion of J-C λ genes, is in preparation. We derived multiple founders with
integrated human Ig loci and silenced endogenous Ig production; all analyzed by PCR and FISH
with complete trans-locus integration selected and interbred (Table 2). Several founder rats
carried low translocus copy numbers; with the rat C-gene BAC in OmniRat likely to be fully
integrated in 5 copies as determined by qPCR of Cµ and C α products (not shown). Identification
by FISH of single position insertion in many lines confirmed that spreading or multiple
integration of BAC mixtures were rare; an advantage for breeding to homozygosity, which was
achieved.
Table 2: Generated rat lines: transgenic integration, knock-out and gene usage
human human ZFN
human V rat C FISH
Igk Igl KO
BAC6-
BAC3 (Annabel) BACs Igl YAC rat
rat line VH3-11 J KO Ig κ KO Ig γ KO
173 kb 193 kb 300 kb 300 kb chromosome
182 kb
HC14 √ √ √ 5q22
homozygous
OmniRat √ √ √ √ √ √ √ √
LC#79 √ 17
LC#6.2 √ 6q23
#117 √ 6q32
#23 √ 4
#35 √ 11
Rats carrying the individual human transloci - IgH, Ig κ and Ig λ - were crossbred
successfully to homozygosity with Ig locus KO rats. This produced a highly efficient new multi-
feature line (OmniRats™) with human V -D-J regions of over 400 kb containing 22 functional
V s and a rat C region of ~116 kb. DNA rearrangement, expression levels, class-switching and
hypermutation was very similar between the different founders and comparable to wt rats. This is
probably the result of the associated rat constant region accommodating several Cs and with the
3’E (enhancer control) region in authentic configuration.
B-cell development in the knock-out background
To assess whether the introduced human Ig loci were capable of reconstituting normal
B-cell development flow cytometric analyses were performed. Particular differentiation stages
were analyzed in spleen and bone marrow lymphocytes (Fig. 2), which previously showed a lack
of B-cell development in JKO/JKO rats , and no corresponding IgL expression in κKO/ κKO as
well as in λKO/ λKO animals (data not shown). Most striking was the complete recovery of B-
cell development in OmniRats compared to wt animals, with similar numbers of B220(CD45R)
lymphocytes in bone marrow and spleen. IgM expression in a large proportion of CD45R B-
cells marked a fully reconstituted immune system. Size and shape separation of spleen cells was
indistinguishable between OmniRats and wt animals and thus successfully restored in the
transgenic rats expressing human idiotypes with rat C region. Moreover, the small sIgG
lymphocyte population was present in OmniRats (Fig. 2 right).
The analysis of other OmniRat lymphocyte tissues showed that they were
indistinguishable from wt controls and, for example, T-cell subsets were fully retained (data not
shown), which further supports the notion that optimal immune function has been completely
restored.
Diverse human H- and L-chain transcripts
Extensive transcriptional analysis was carried out using blood lymphocytes or spleen
cells from transgenic rats with functional endogenous Ig loci. RT-PCR from specific human V
group forward to Cµ or C γ reverse primers, showed human V DJ usage. For L-chain analysis
group specific human V κ or V λ forward primers were used with C κ or C λ reverse primers. The
results (Table 3) showed the use of all integrated human V genes regarded as functional in
combination with diverse use of D segments and all J segments.
The analysis of class-switch and hypermutation (Fig. 3) in the JKO/JKO background
showed that these essential and highly desirable mechanisms are fully operative in OmniRats.
Amplification of IgG switch products from PBLs revealed an extensive rate of mutation (>2 aa
changes) in the majority of cells, ~80%, and in near equal numbers of γ1 and γ2b H-chains. A
small percentage of trans-switch sequences, γ2a and 2c, were also identified (Fig. 3), which
supports the observation that the translocus is similarly active, but providing human (V -D-J )s,
as the endogenous IgH locus . The number of mutated human Ig γ and Ig κ L-chain sequences is
~30% and thus considerably lower than IgG H-chains. The reason is the general amplification of
L-chain from all producing cells rather than from IgG or differentiated plasma cells.
Ig levels in serum
To gain unambiguous information about antibody production we compared quality
and quantity of serum Ig from OmniRats and normal wt animals. Purification of IgM and IgG
separated on SDS-PAGE under reducing conditions (Fig. 4) showed the expected size - ~75 kDa
for µ, ~55 kDa for γ H-chains, and ~25 kDa for L-chains – which appeared indistinguishable
between OmniRats and wt animals. The Ig yield from serum was determined to be between 100-
300 µg/ml for IgM and 1-3 mg/ml for IgG for both, several OmniRats and wt animals. However,
as rat IgG purification on protein A or G is seen as suboptimal , rat Ig levels may be under
represented. Taken into consideration that these young (~3 months old) rats were housed in
pathogen-free facilities and had not been immunized, this compares well with the IgM levels of
, 31
0.5-1 mg/ml and IgG levels of several mgs/ml reported for rats kept in open facilities .
Interestingly, we were able to visualize class-specific mobility of rat IgG isotypes on SDS-PAGE
as demonstrated for monoclonals . In IgG separations (Fig. 4b) a distinct lower γH-chain band
is visible in wt but not OmniRat Ig. This band has been attributed to γ2a H-chains, which are not
present in the OmniRat (HC14) translocus. As the IgG levels are similar between OmniRats and
wt animals we assume class-switching is similarly efficient. The reason that the lack of C γ2a in
OmniRats is not limiting may be that several copies of the transgenic locus favorably increase
the level of switch products. Purification of human Ig κ and Ig λ by capturing with anti-L-chain
was also successful (Fig. 4c and d) and predicted H- and L-chain bands were of the expected
size. Confirmation of the IgM/G titers was also obtained by ELISA, which determined wt and
OmniRat isotype distribution and identified comparable amounts of IgG1 and IgG2b (not
shown).
A direct comparison of human Ig L-chain titers in solid phase titrations (Fig. 4e
and f) revealed 5-10 fold lower levels in OmniRats than in human serum. However, this was
expected as human control serum from mature adults can sometimes contain over 10-times
higher Ig levels than in children up to their teens , which would be similar to the human Ig κ and
Ig λ titers in young rats. Interestingly, wt rats produce very little endogenous Ig λ while transgenic
rats can efficiently express both types of human L-chain, Ig κ and Ig λ.
Fully human antigen-specific IgG
Several cell fusions were carried out, using either a rapid one-immunization scheme
and harvesting lymph nodes or, alternatively, using booster immunizations and spleen cells
(Table 2). For example, a considerable number of stable hybridomas were obtained after one
immunization with human progranulin (PG) and myeloma fusion 22 days later. Here cell growth
was observed in ~3,520 and ~1,600 wells in SD control and OmniRat hybridoma clones,
respectively. Anti-progranulin specific IgG, characterized by biosensor measurements, was
produced by 148 OmniRat clones. Limiting dilution, to exclude mixed wells, and repeat affinity
measurements revealed that OmniRat clones retain their antigen specificity. A comparison of
association and dissociation rates of antibodies from SD and OmniRat clones showed similar
affinities between 0.3 and 74 nM (Table 4 and data not shown). Single immunizations with
human growth hormone receptor (hGHR), TAU receptor coupled to keyhole limpet hemocyanin
(TAU/KLH), hen egg lysozyme (HEL) or ovalbumin (OVA), followed by lymph node fusions
also produced many high affinity human antibodies often at similar numbers compared to wt.
Furthermore, conventional booster immunizations with human PG, hGHR, human
CD14 and HEL resulted in high affinities (pM range) of IgG with human idiotypes. OmniRats
always showed the expected 4- to 5-log titer increase of antigen-specific serum IgG, similar to
and as pronounced as wt rats (Table 4a). Although the results could vary from animal to animal,
comparable numbers of hybridomas producing antigen-specific antibodies with similarly high
affinities were obtained from wt animals (SD and other strains) and OmniRats. A summary of
individual IgG producing lymph node and spleen cell fusion clones, showing their diverse human
V -D-J , human V κ-J κ or V λ-J � characteristics and affinities are presented in Table 4b. The
immunization and fusion results showed that affinities well below 1 nM (determined by
biosensor analysis) were frequently obtained from OmniRats immunized with PG, CD14, Tau,
HEL and OVA antigens. In summary, antigen-specific hybridomas from OmniRats could be as
easily generated as from wt animals yielding numerous mAbs with sub-nanomolar affinity even
after a single immunization.
Table 4a Diverse antigen-specific rat IgG hybridomas with fully human idiotypes
Animal Antigen Cells* fusions titer hybrids IgGs** Kd***
SD PG LN 1 38400 3520 38 0.3-1.0 nM
OmniRat PG LN 1 12800 1600 148 0.7-2.4 nM
SD PG SP 1 51200 8000 29 ND
OmniRat PG SP 1 51200 36000 24 ND
OmniRat hGHR LN 3 4800 704-1024 18, 3, 2 ND
SD hGHR SP 1 204800 53760 230 <0.07-0.4 nM
OmniRat hGHR SP 1 76800 53760 7 0.16-2.4 nM
OmniRat CD14 SP 2 102400 2800-3500 54, 14 <0.1-0.2 nM
TAU/K
SD LN 1 20000 1728 99# 0.6-2.4 nM
TAU/K
OmniRat LN 1 4800 1880 118# 0.5-3.2 nM
SD HEL LN 1 12800 1564 26 0.02-0.1 nM
OmniRat HEL LN 3 25600 288-640 0, 2, 7 0.6-1.5 nM
SD HEL SP 1 6400 30720 0 ND
SD OVA LN 1 9600 1488 10 1.1-4.8 nM
0, 30,
OmniRat OVA LN 4 8000 512-2240 0.7-1.5 nM
0, 1
*cell numbers were 3-9 x 10 per fusion
** antigen specificity confirmed by biosensor
analysis
*** range of 5 highest affinities
# 8 mAbs were specific for Tau-peptide
Table 4b
OmniRats (HC14/Huκ and/or Huλ/JKOJKO/KKOKKO) and control SD rats were immunized with human
progranulin (PG), human growth hormone receptor (hGHR), human CD14, Tau-peptide (TAU-KLH), hen egg
lysozyme (HEL), ovalbumin (OVA) or β-galactosidase (β-gal). Lymph nodes (LN) or spleen cells (SP) were fused
after single or multiple administration of antigen, respectively.
DISCUSSION
A combination of human and rat genes to assemble a novel IgH locus has resulted in
highly efficient near normal expression of antibodies with human idiotypes. Moreover,
integration of the human Ig κ and Ig � loci revealed that chimeric Ig with fully human specificity
is readily produced and that association of rat C-regions with human L-chains is not detrimental.
Advantages of using part of the rat IgH locus are that species-specific C regions and enhancer
control elements are kept in their natural configuration, with essentially only the diverse human
V D J region being transplanted. Furthermore, expression of antibodies with rat Fc-regions
allow normal B-cell receptor assembly and optimal activation of the downstream signaling
pathway essential for the initiation of highly efficient immune responses. In particular, the
quality of an immune response to antigen challenge relies on combined actions of many receptor
associated signaling and modifier components (see:
www.biocarta.com/pathfiles/h_bcrpathway.asp).
The approach of using YACs and BACs, and interchanging between the two, has
the advantage of both, speed and the ability to check integrity when making constructs of large
regions by overlapping homology. Several founder rats carried low translocus copy numbers;
with the rat C-gene BAC in OmniRat likely to be fully integrated in 5 copies as determined by
qPCR of Cµ and Cα products (not shown). Identification by FISH of single position insertion in
many lines (see Table 1d) confirmed that spreading or multiple integration of BAC mixtures
were rare; an advantage for breeding to homozygosity, which was achieved. Little was known
whether extensive overlapping regions would integrate, such as to maintain the full functionality,
essential for DNA rearrangement. Previously, overlapping integration has been reported but for
24, 33
much smaller regions (<100 kb) and our results suggest that desired integration by homology
or in tandem is a frequent event. This eases the transgenic technology substantially as no
laborious integration of large YACs into stem cells and subsequent animal derivation therefrom
18, 19 11, 12
has to be performed . In addition, ZFN technology, also performed via DNA injection ,
produced Ig KO strains easily and may well be the future technology of choice for gene
disruptions and replacement. Silenced endogenous Ig gene expression in OmniRats, containing
human-rat IgH and human IgL loci, has the advantage that no interfering or undesired rat Ig
could give rise to mixed products. Interestingly, immunization and hybridoma generation in
OmniRats still producing wt Ig revealed that many products were fully human, human-rat IgH
and human IgL, despite incomplete Ig KOs. Here, despite the extensive number of wt V genes, it
was remarkable that the introduced human genes amplified readily and thus showed to be
efficient expression competitors. This is in line with the observation of generally good
expression levels of all our integrated transgenes, which favorably compete with the endogenous
loci. Previously in mice expressing a human antibody repertoire, Ig KOs were essential as little
8, 18
expression of human products was found when wt Ig is released .
It is possible that the production of fully human Ig loci even in Ig KO mice is
suboptimal as strain specific cis-acting sequences are required for high-level expression. In the
mouse an enhancer region downstream of Cα plays a vital role in class-switch recombination
and it is likely that elements in that region may facilitate hypermutation . This may be the
reason why immune responses and generation of diverse hybridomas at high frequency may be
, 36
difficult in mice carrying even a large fully human locus . As the chimeric human-rat IgH
locus facilitates near wt differentiation and expression levels in OmniRats, it can be concluded
that the endogenous rat C region and indeed the ~30 kb enhancer sequence 3’ of Cα are
providing optimal locus control to express and mature human V genes. Another region, Cδ with
its 3’ control motif cluster , has been removed from the chimeric C-region BAC since silencing
37 and refs therein
or a lack of IgD did not appear to reduce immune function . Normally, mature
IgM IgD B-cells down-regulate IgD upon antigen contact, which initiates class-switch
recombination . Thus, switching may be increased without IgD control, which is supported by
our finding that IgG transcripts and serum levels are significantly lower when the Cδ region is
retained in transgenic constructs (data not shown).
The production of specific IgG in OmniRats is particularly encouraging as we found
that in various immunizations mAbs with diversity in sequence and epitope, comparable to what
was produced in wt controls, could be isolated via spleen and lymph node fusion. V-gene, D and
J diversity was as expected and nearly all segments were found to be used productively as
predicted . This was in stark contrast to mice carrying fully human transloci where clonal
expansion from a few precursor B-cells produced little diversity . Since the number of
transplanted V-genes is only about half of what is used in humans we anticipated to find
restricted immune responses and limited diversity when comparing OmniRats with wt animals.
However, this was not the case and a comparison of CDR3 diversity in over 1000 clones
(sequences can be provided) revealed the same extensive junctional differences in OmniRats as
in wt animals. The few identical gene-segment combinations were further diversified by N-
sequence additions or deletion at the V to D and/or D to J junctions and also by
hypermutation. Thus, it is clear that the rat C region sequence is highly efficient in controlling
DNA rearrangement and expression of human V DJ . Extensive diversity was also seen for the
22, 23, 38
introduced human Ig κ and Ig λ loci, similar to what has previously been shown in mice .
Hence, substantially reduced efficiency in the production of human antibodies from mice has
been overcome in OmniRats, which diversify rearranged H-chains reliably and extensively by
class-switch and hypermutation to yield high affinity antibodies in bulk rather than occasionally.
The yield of transgenic IgG and the level of hypermutation, impressively utilized in antigen-
specific mAbs, showed that clonal diversification and production level are similar between
OmniRats and wt animals. Routine generation of high affinity specificities in the subnanomolar
range was even accomplished by different single immunizations and again compares favorably
with wt animals; results that have not been shown in transgenic mice producing human antibody
repertoires from entirely human loci .
In summary, to maximize human antibody production an IgH locus that uses
human genes for antibody specificity but rodent genes for control of differentiation and high
expression should be regarded essential. L-chain flexibility is a bonus as it permits highly
efficient human IgH/IgL assembly even when wt Ig is present. For therapeutic applications
chimeric H-chains can be easily converted into fully human Abs by C-gene replacement without
compromising the specificity.
All patents and patent publications referred to herein are hereby incorporated by
reference.
Certain modifications and improvements will occur to those skilled in the art upon a
reading of the foregoing description. It should be understood that all such modifications and
improvements have been deleted herein for the sake of conciseness and readability but are
properly within the scope of the following claims.
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50. Kishiro, Y., Kagawa, M., Naito, I. & Sado, Y. A novel method of preparing rat-
monoclonal antibody-producing hybridomas by using rat medial iliac lymph node cells. Cell
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Claims (16)
1. A chimeric polynucleotide comprising, in 5’ to 3’ order, a human immunoglobulin (Ig) variable (V) region gene, a human Ig diversity (D) region gene, at least one human immunoglobulin (Ig) joining (J) region gene, an Ig constant region gene, and a rat 3' enhancer comprising the sequence set forth as SEQ ID NO:1.
2. The chimeric polynucleotide as in claim 1, wherein the constant region gene is selected from the group consisting of a human constant region and a rat constant region gene.
3. The chimeric polynucleotide according to claim 1 or claim 2, wherein the Ig constant region (C) gene comprises a rat constant (C) region gene.
4. The chimeric polynucleotide of any one of the preceding claims, wherein the Ig constant region gene comprises an Ig constant (C) region gene selected from the group consisting of C μ and Cy.
5. The chimeric polynucleotide according to any one of the preceding claims, comprising a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO:10 or a portion thereof.
6. The chimeric polynucleotide according to any one of claims 1 to 5 wherein said human Ig V region comprises at least one human V region gene isolatable from BAC6-V 3-11 (BAC6 (SEQ ID NO:8) extended by VH3-11 to provide a 10.6kb overlap with BAC3 (SEQ ID NO:9)) and/or SEQ ID NO:9.
7. The chimeric polynucleotide as in any one of the preceding claims, comprising nucleic acid sequences (a) and (b) in 5’ to 3’ order: a) a human Ig V region comprising human Ig V region genes in natural configuration isolatable from BAC6-VH3-11 (BAC6 (SEQ ID NO:8) extended by VH3-11 to provide a 10.6kb overlap with BAC3 (SEQ ID NO:9)) and/or SEQ ID NO:9; and b) a human Ig J region comprising human Ig J region genes in natural configuration isolatable from bacterial artificial chromosome (BAC) having the sequence set out in SEQ ID NO:10.
8. The chimeric polynucleotide as in any one of claims 1-7 wherein each of the human immunoglobulin variable region, the human immunoglobulin diversity region, the human immunoglobulin joining region, the immunoglobulin constant region, and the rat 3’ enhancer are in the relative positions shown in .
9. The chimeric polynucleotide according to claim 8, comprising a nucleic acid sequence having at least 85% sequence identity to the nucleic acid set forth as SEQ ID NO:2.
10. The chimeric polynucleotide according to claim 8, comprising a nucleic acid sequence having at least 85% sequence identity to the nucleic acid set forth as SEQ ID NO:11.
11. The chimeric polynucleotide according to any one of claims 1 to 10, wherein said Ig V- D-J regions are functional and capable of gene rearrangement.
12. The chimeric polynucleotide according to any one of claims 1 to 10, wherein said V-D-J regions are rearranged and form a complete exon encoding a heavy chain variable domain.
13. An isolated rodent cell comprising a) the chimeric polynucleotide according to any one of claims 1-11, or b) the chimeric polynucleotide according to claim 12.
14. The isolated rodent cell according to claim 13(a), comprising a polynucleotide encoding a functional immunoglobulin comprising Ig V-, J- and (C) region genes and a nucleic acid sequence having at least 85% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:7.
15. The chimeric polynucleotide according to any one of claims 1 to 12, substantially as herein described with reference to any example thereof and with reference to the accompanying figures.
16. The isolated rodent cell according to claim 13 or 14, substantially as herein described with reference to any example thereof and with reference to the accompanying figures. HC14 fi1es_ST25
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NZ749259A NZ749259B2 (en) | 2012-12-14 | 2013-12-13 | Polynucleotides encoding rodent antibodies with human idiotypes and animals comprising same |
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Application Number | Priority Date | Filing Date | Title |
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US201261737371P | 2012-12-14 | 2012-12-14 | |
US61/737,371 | 2012-12-14 | ||
PCT/US2013/075157 WO2014093908A2 (en) | 2012-12-14 | 2013-12-13 | Polynucleotides encoding rodent antibodies with human idiotypes and animals comprising same |
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NZ709608B2 true NZ709608B2 (en) | 2021-01-06 |
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