U.S. patent application number 17/021242 was filed with the patent office on 2021-01-07 for genetic engineering of non-human animals for the production of chimeric antibodies.
The applicant listed for this patent is Ablexis, LLC. Invention is credited to Larry Green, Hiroaki Shizuya.
Application Number | 20210002385 17/021242 |
Document ID | / |
Family ID | |
Filed Date | 2021-01-07 |
![](/patent/app/20210002385/US20210002385A1-20210107-D00001.png)
![](/patent/app/20210002385/US20210002385A1-20210107-D00002.png)
![](/patent/app/20210002385/US20210002385A1-20210107-D00003.png)
![](/patent/app/20210002385/US20210002385A1-20210107-D00004.png)
![](/patent/app/20210002385/US20210002385A1-20210107-D00005.png)
United States Patent
Application |
20210002385 |
Kind Code |
A1 |
Green; Larry ; et
al. |
January 7, 2021 |
GENETIC ENGINEERING OF NON-HUMAN ANIMALS FOR THE PRODUCTION OF
CHIMERIC ANTIBODIES
Abstract
The invention provides non-human cells and mammals having a
genome encoding chimeric antibodies and methods of producing
transgenic cells and mammals. Certain aspects of the invention
include chimeric antibodies, humanized antibodies, pharmaceutical
compositions and kits. Certain aspects of the invention also relate
to diagnostic and treatment methods using the antibodies of the
invention.
Inventors: |
Green; Larry; (San Diego,
CA) ; Shizuya; Hiroaki; (South Pasadena, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ablexis, LLC |
San Diego |
CA |
US |
|
|
Appl. No.: |
17/021242 |
Filed: |
September 15, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15463806 |
Mar 20, 2017 |
|
|
|
17021242 |
|
|
|
|
15408114 |
Jan 17, 2017 |
10494445 |
|
|
15463806 |
|
|
|
|
13638522 |
Mar 5, 2013 |
9580491 |
|
|
PCT/US2011/030823 |
Mar 31, 2011 |
|
|
|
15408114 |
|
|
|
|
61361302 |
Jul 2, 2010 |
|
|
|
61319690 |
Mar 31, 2010 |
|
|
|
Current U.S.
Class: |
1/1 |
International
Class: |
C07K 16/46 20060101
C07K016/46; A01K 67/027 20060101 A01K067/027; C07K 16/00 20060101
C07K016/00; C07K 16/18 20060101 C07K016/18; C12N 15/85 20060101
C12N015/85 |
Claims
1. (canceled)
2. A mouse whose genome comprises a transgene encoding a
polypeptide comprising an immunoglobulin light chain variable
region, wherein the transgene comprises (1) a plurality of
immunoglobulin light chain variable (V) exons encoding feline
immunoglobulin light chain variable (V) polypeptides; (2)
non-coding sequences between the V exons; (3) a plurality of
immunoglobulin light chain joining (J) coding sequences encoding
feline immunoglobulin light chain joining (J) polypeptides; and (4)
non-coding sequences between the J coding sequences; wherein the
non-coding sequences between the V exons and the non-coding
sequences between the J coding sequences are derived from mouse
immunoglobulin light chain non-coding sequences and wherein the
transgene is capable of undergoing gene arrangement and thereby
upon expression to produce a polypeptide comprising the
immunoglobulin light chain variable region.
3. The mouse according to claim 2, wherein the non-coding sequences
between the V exons and the non-coding sequences between the J
coding sequences are selected from the group consisting of intronic
sequences and cis regulatory sequences.
4. The mouse according to claim 3, wherein the cis regulatory
sequences are selected from promoters, enhancers, recombination
signal sequences, splice acceptor sequences, and splice donor
sequences.
5. The mouse according to claim 2, wherein the V exons encode kappa
light chain V (V.kappa.) polypeptides or lambda light chain V
(V.lamda.) polypeptides.
6. The mouse according to claim 2, wherein the V exons encode kappa
light chain V (V.kappa.) polypeptides.
7. The mouse according to claim 2, wherein the V exons encode
lambda light chain V (V.lamda.) polypeptides
8. The mouse according to claim 2, wherein the transgene further
comprises a coding sequence encoding a feline or mouse
immunoglobulin light chain constant (C) polypeptide.
9. The mouse according to claim 8, wherein the coding sequence
encoding the immunoglobulin light chain C polypeptide encodes an
immunoglobulin light chain constant lambda (C.lamda.) polypeptide
or an immunoglobulin light chain constant kappa (C.kappa.)
polypeptide.
10. The mouse according to claim 2, wherein the transgene further
comprises a coding sequence encoding a mouse immunoglobulin light
chain constant (C) polypeptide.
11. The mouse according to claim 6, wherein the transgene further
comprises a coding sequence encoding a feline or mouse
immunoglobulin light chain constant kappa (C.kappa.)
polypeptide.
12. The mouse according to claim 7, wherein the transgene further
comprises a coding sequence encoding a feline or mouse
immunoglobulin light chain constant lambda (C) polypeptide.
13. The mouse according to claim 2, wherein the transgene further
comprises mouse non-coding sequences upstream of the V exons.
14. The mouse according to claim 13, wherein the non-coding
sequences upstream of the V exons are selected from promoters and
enhancers.
15. The mouse according to claim 2, wherein the transgene further
comprises mouse non-coding sequences downstream of the J coding
sequences.
16. The mouse according to claim 15, wherein the non-coding
sequences downstream of the J coding sequences are selected from
polyadenylation sites and 3' untranslated regions.
17. The mouse according to claim 12, wherein the transgene further
comprises a immunoglobulin light chain .lamda. 3' enhancer.
18. The mouse according to claim 12, wherein said transgene further
comprises an immunoglobulin light chain 3'LCR, or a functional
fragment thereof.
19. The mouse according to claim 18, wherein said immunoglobulin
light chain 3'LCR, or a functional fragment thereof, is from a
mammal selected from the group consisting of human, non-primate and
rat.
20. The mouse according to claim 2, wherein the genome further
comprises a second transgene encoding a feline immunoglobulin heavy
chain, or a portion thereof.
21. A non-human mammalian cell whose genome comprises a transgene
encoding a polypeptide comprising a feline immunoglobulin light
chain variable region, wherein the transgene comprises (1) a
plurality of immunoglobulin light chain variable (V) exons encoding
feline immunoglobulin light chain variable (V) polypeptides; (2)
non-coding sequences between the V exons; (3) a plurality of
immunoglobulin light chain joining (J) coding sequences encoding
feline immunoglobulin light chain joining (J) polypeptides; and (4)
non-coding sequences between the J coding sequences; wherein the
non-coding sequences between the V exons and the non-coding
sequences between the J coding sequences are derived from mouse
immunoglobulin light chain non-coding sequences and wherein the
transgene is capable of undergoing gene arrangement and thereby
upon expression to produce a polypeptide comprising the
immunoglobulin light chain variable region.
22. A method of producing an antibody, or antigen-binding fragment
thereof, the antibody or fragment comprising a feline
immunoglobulin light chain variable region polypeptide, comprising:
(a) immunizing the mouse according to claim 2; (b) recovering from
the mouse a genomic DNA or cDNA comprising a nucleotide sequence
encoding the feline immunoglobulin light chain variable region
polypeptide; and (c) recombinantly producing the feline
immunoglobulin light chain variable region polypeptide.
23. A chimeric antibody, or antigen binding fragment thereof,
comprising (i) a chimeric immunoglobulin light chain comprising a
feline immunoglobulin light chain variable region and a mouse
immunoglobulin light chain constant region; and (ii) an
immunoglobulin heavy chain.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit under 35 U.S.C. .sctn.
119(e) of U.S. Provisional Patent Application No. 61/319,690 filed
Mar. 31, 2010 and U.S. Provisional Patent Application No.
61/361,302 filed Jul. 2, 2010, where these two provisional
applications are incorporated herein by reference in their
entireties.
BACKGROUND
Technical Field
[0002] The present invention is directed generally to chimeric
immunoglobulin chains, antibodies and non-human animals and cells,
and the production thereof.
Description of the Related Art
[0003] Disease therapies utilizing monoclonal antibodies (mAbs)
have revolutionized medicine, and mAb-based drugs are now utilized
in the treatment of cancer, autoimmunity, inflammation, macular
degeneration, infections, etc. However, the available technologies
for generation and discovery of mAbs for use in the prevention and
treatment of diseases and disorders have significant drawbacks
including inefficiency, absence or loss of sufficient potency,
absence or loss of specificity and the induction of an immune
response against the therapeutic mAb. The first attempts to use
mAbs as therapeutics were hindered by the immunogenicity of the
mouse amino acid composition of the mAbs. When administered to
humans, the mouse amino acid sequence elicited a human anti-mouse
antibody (HAMA) response that dramatically reduced the potency and
pharmacokinetics of the drug as well as causing severe and
potentially fatal allergic reactions.
[0004] Additional methods to generate mAb therapeutics include
chimerized mAbs (cmAbs) created through recombinant DNA technology
combining a mouse-derived variable domain appended to a human
constant region. Other methods of generating antibodies involve
humanizing mAbs in vitro to further reduce the amount of mouse
amino acid sequence in a therapeutic mAb. Antibody-display
technologies developed to generate "fully-human" antibodies in
vitro have yet to adequately mimic the natural antibody maturation
process that occurs during an in vivo immune response (see pg.
1122-23, Lonberg, Nat. Biotech. (2005) 23:1117-1125.) mAbs
developed using these methods can elicit an immune response that
can reduce efficacy and/or be life-threatening, and these processes
are typically time-consuming and costly. Also, during the molecular
processes inherent in these methods, loss of affinity and epitope
shifting can occur, thereby reducing potency and introducing
undesirable changes in specificity.
[0005] Transgenic mice have been engineered to produce fully human
antibodies by introducing human antibody transgenes to functionally
replace inactivated mouse immunoglobulin (Ig) loci. However, many
of these transgenic mouse models lack important components in the
antibody development process, such as sufficient diversity in the
genes from which antibody variable regions are generated, the
ability to make IgD (Loset et al., J. Immunol., (2004)
172:2925-2934), important cis regulatory elements important for
class switch recombination (CSR), or a fully functional 3' locus
control region (LCR) (e.g., U.S. Pat. No. 7,049,426; and Pan et
al., Eur. J. Immunol. (2000) 30:1019-1029). Some transgenic mice
contain yeast artificial chromosomes or human miniloci as
integrated transgenes. Others carry transchromosomes that exhibit
various frequencies of mitotic and meiotic instability.
Furthermore, the fully human constant regions of these transgenic
mice function sub-optimally due to reduced activity in conjunction
with other endogenous and trans-acting components as compared to
wild-type mice, e.g., the BCR signal transduction apparatus,
(Ig.alpha. and Ig.beta.) and Fc receptors (FcR), respectively.
[0006] Knock-in mice have also been genetically engineered to
produce chimeric antibodies that are composed of human V domains
appended to mouse C domains that remain fully intact, with the
fully-intact portions comprising all genomic DNA downstream of the
J gene cluster (see U.S. Pat. Nos. 5,770,429 and 6,596,541 and U.S.
Patent Application Publication No. 2007/0061900). Human V regions
from these mice can be recovered and appended to human constant
region genes by molecular biological methods and expressed by
recombinant methods to produce fully-human antibodies. The
antibodies from these mice may exhibit reduction or loss of
activity, potency, solubility etc. when the human V region is
removed from the context of the mouse C domains with which it was
evolved and then appended to a human C region to make a fully human
antibody. Furthermore, because of the unique and differing
structures of the mouse immunoglobulin lambda locus versus that of
the human immunoglobulin lambda locus and because the endogenous 3'
enhancer of the mouse lambda locus may be defective, the described
knock-in approach would be expected to yield an inefficiently
functioning lambda locus.
[0007] Methods of transgene DNA construction for introduction into
eukaryotic, particularly metazoan, species have employed DNA
isolated from genomic libraries made from isolated natural DNA.
Engineering of the cloned natural DNA into the final desired design
for a transgene is typically achieved through processes of
recombination that are cumbersome, inefficient, slow and
error-prone and constrained by the availability of the DNAs present
in genomic libraries. In some instances, it is desirous to
construct a transgene from an organism, strain or specific
haplotype thereof for which a genomic library is not readily
available but for which either partial genomic sequence or
transcriptome sequence information is available. These hindrances
prevent the creation of transgenes comprising complexly
reconfigured sequences and/or transgenes designed to comprise
chimeric DNA sequence from different species or different strains
or different haplotypes of the same species. As a consequence, the
engineering of highly-tailored transgenes for eukaryotes,
particularly metazoans, is prevented.
[0008] Current methods of developing a therapeutic mAb can alter
functions of the antibody, such as solubility, potency and antigen
specificity, which were selected for during initial stages
development. In addition, mAbs generated by current methods have
the potential to elicit a dangerous immune response upon
administration. Current human and chimeric antibody producing mice
lack appropriate genetic content to function properly, e.g.,
genetic diversity, cis regulatory elements, trans acting regulatory
elements, signaling domains, genetic stability. It would be
beneficial to develop methods and compositions for the enhanced
generation and discovery of therapeutic antibodies and that retain
potency and specificity through the antibody generation, discovery,
development, and production process without eliciting an immune
response, as well as methods of producing such antibodies. Some of
the transgene compositions comprise DNA sequences so complexly
modified that construction of these improvements and derivation of
products therefrom have been prevented. While mice are preferred
because of their economy and established utility, a broad solution
across multiple species is desirable. The present invention
provides a solution for making and introducing such transgenes,
improving the genetic background into which these transgenes would
function if deployed in a mouse, and, in particular instances,
generating improved antibodies in transgenic animals.
BRIEF SUMMARY
[0009] The present invention relates to non-human animals and
cells, transgenes, antibodies, methods, compositions, including
pharmaceutical compositions, as well as kits of various embodiments
disclosed herein. More specifically, the present invention relates
to methods, compositions and kits relating to chimeric Ig chains
and antibodies produced by the non-human animals and cells and the
human antibodies and fragments thereof engineered from the variable
domains of said chimeric antibodies. In certain embodiments of the
invention, the non-human animals are mammals.
[0010] One embodiment of the invention relates to a method of
producing a cell comprising a genome that comprises a chimeric
immunoglobulin chain, wherein the immunoglobulin chain comprises a
non-endogenous variable domain and a chimeric constant region,
comprising the steps of (1) designing a DNA construct in silico,
wherein said construct comprises one or more non-endogenous V, (D)
and/or J gene segments and one or more non-endogenous constant
region gene segments; (2) producing said DNA construct; and (3)
introducing the construct into the genome of a cell. In certain
embodiments, the non-endogenous variable domain is human. In
another embodiment, the chimeric constant region comprises a mouse
constant domain gene segment. In one embodiment, the chimeric
constant region is encoded by a non-endogenous polynucleotide
sequence derived from two or more non-endogenous species, alleles
and/or haplotypes. In yet another embodiment, the non-endogenous
variable domain is encoded by a polynucleotide sequence derived
from two or more species, alleles and/or haplotypes. In certain
embodiments, the chimeric immunoglobulin chain is a light
chain.
[0011] In certain other embodiments, the chimeric immunoglobulin
chain is a heavy chain. In a related embodiment, the chimeric
constant region comprises a non-endogenous CH1 domain. In another
related embodiment, the method further comprises the steps of
designing a second DNA construct in silico, wherein said construct
comprises a non-endogenous immunoglobulin light chain; producing
said second DNA construct; and introducing the second construct
into the genome of a cell. In one embodiment, the non-endogenous
light chain comprises one or more human V.kappa. gene segments. In
another embodiment, the non-endogenous light chain further
comprises one or more human J.kappa. and C.kappa. gene segments. In
yet another embodiment, the non-endogenous light chain comprises 8
or more human V.lamda. gene segments. In a related embodiment, the
non-endogenous light chain further comprises 7 or more human
J.lamda.-C.lamda. gene segment pairs.
[0012] One embodiment relates to a non-human cell comprising a
genome that comprises a chimeric immunoglobulin chain, wherein the
immunoglobulin chain comprises a non-endogenous variable domain and
a chimeric constant region, wherein the cell is produced by a
method comprising the steps of (1) designing a DNA construct in
silico, wherein said construct comprises one or more non-endogenous
V, (D) and/or J gene segments and one or more non-endogenous
constant region gene segments; (2) producing said DNA construct;
and (3) introducing the construct into the genome of a cell.
Another embodiment encompasses a non-human animal generated from
the cell. Another embodiment provides a chimeric immunoglobulin
heavy chain produced by the non-human animal. Certain embodiments
provide a chimeric antibody produced by the non-human animal.
[0013] Another embodiment of the invention provides a chimeric
immunoglobulin heavy chain comprising a non-endogenous variable
domain and a chimeric constant region, wherein the non-endogenous
variable domain is derived from a non-human animal. In a related
embodiment, the chimeric constant region comprises a non-endogenous
CH1 domain. One embodiment provides a chimeric immunoglobulin heavy
chain comprising a non-endogenous variable domain and a chimeric
constant region, wherein the chimeric constant region is encoded by
a non-endogenous polynucleotide sequence derived from two or more
non-endogenous species, alleles and/or haplotypes. Another
embodiment provides a chimeric immunoglobulin heavy chain
comprising a non-endogenous variable domain and a chimeric constant
region, wherein said non-endogenous variable domain is encoded by a
polynucleotide sequence derived from two or more species, alleles
and/or haplotypes.
[0014] Yet another embodiment is directed to a polynucleotide
encoding the disclosed chimeric immunoglobulin heavy chain. In
particular embodiments, the polynucleotide comprises coding and
non-coding sequences. In certain embodiments, the polynucleotide is
synthetic. One embodiment relates to a construct comprising the
polynucleotide a polynucleotide encoding the disclosed chimeric
immunoglobulin heavy chain.
[0015] Another embodiment of the invention provides a chimeric
antibody, or an antigen-binding fragment thereof, comprising (1) a
chimeric immunoglobulin heavy chain, wherein the chimeric heavy
chain comprises a non-endogenous heavy chain variable domain and a
chimeric heavy chain constant region, and (2) a non-endogenous
immunoglobulin light chain, wherein the chimeric heavy chain
constant region is derived from two or more non-endogenous species,
alleles and/or haplotypes. Yet another embodiment provides a
chimeric antibody, or an antigen-binding fragment thereof,
comprising (1) a chimeric immunoglobulin heavy chain, wherein the
chimeric heavy chain comprises a non-endogenous heavy chain
variable domain and a chimeric heavy chain constant region, and (2)
a non-endogenous immunoglobulin light chain, and wherein said
non-endogenous heavy chain variable domain is derived from two or
more species, alleles and/or haplotypes. One embodiment relates to
a chimeric antibody, or an antigen-binding fragment thereof,
comprising a chimeric immunoglobulin heavy chain, wherein the
chimeric heavy chain comprises a non-endogenous variable domain and
a chimeric constant region, and wherein the variable domain is
derived from a non-human animal. In a related embodiment, the
disclosed chimeric antibody, or antigen-binding fragment thereof,
further comprises a non-endogenous light chain.
[0016] One embodiment of the invention provides a non-human cell
comprising a genome that comprises a chimeric immunoglobulin heavy
chain comprising a non-endogenous variable domain and a chimeric
constant region, wherein the non-endogenous variable domain is
derived from a non-human animal. In a related embodiment, the
genome of the cell further comprises a non-endogenous
immunoglobulin light chain. In particular embodiments, the genome
of the cell comprises a non-endogenous Ig.kappa. light chain and a
non-endogenous Ig, light chain. In certain embodiments, the cell
comprises an inactivated endogenous immunoglobulin locus. One
embodiment provides a chimeric antibody produced by the disclosed
cell.
[0017] Yet another embodiment provides a non-human cell comprising
a genome that comprises a chimeric immunoglobulin heavy chain
comprising a non-endogenous variable domain and a chimeric constant
region, wherein the constant region is derived from two or more
non-endogenous species, alleles and/or haplotypes. One embodiment
provides a non-human cell comprising a genome that comprises a
chimeric immunoglobulin heavy chain comprising a non-endogenous
variable domain and a chimeric constant region, wherein the
non-endogenous variable domain is derived from two or more species,
alleles and/or haplotypes. Another embodiment provides a non-human
cell comprising a genome that comprises a synthetic transgene
encoding a chimeric antibody, or an antigen-binding fragment
thereof, comprising (1) a chimeric immunoglobulin heavy chain,
wherein said chimeric heavy chain comprises a non-endogenous heavy
chain variable domain and a chimeric heavy chain constant region.
In certain embodiments, the genome of the disclosed cell further
comprises a non-endogenous immunoglobulin light chain. In one
embodiment, the genome of the cell comprises a non-endogenous
Ig.kappa. light chain and a non-endogenous Ig.lamda. light chain.
In particular embodiments, the cell comprises an inactivated
endogenous immunoglobulin locus. Another embodiment provides for a
chimeric antibody produced by the cell.
[0018] Another embodiment of the invention relates to a non-human
animal comprising a genome that comprises a chimeric immunoglobulin
heavy chain comprising a non-endogenous variable domain and a
chimeric constant region, wherein the non-endogenous variable
domain is derived from a non-human animal. In a related embodiment,
the genome of the animal further comprises a polynucleotide
sequence encoding a non-endogenous immunoglobulin light chain. In
certain embodiments, the genome of the animal comprises a
non-endogenous Ig.kappa. light chain and a non-endogenous Ig.lamda.
light chain. In another embodiment, the animal comprises an
inactivated endogenous immunoglobulin locus. In certain
embodiments, the animal is a mouse. Another embodiment provides a
chimeric antibody produced by the non-human animal.
[0019] Yet another embodiment of the invention provides a non-human
animal comprising a genome that comprises (1) a chimeric
immunoglobulin heavy chain, wherein the chimeric heavy chain
comprises a non-endogenous heavy chain variable domain and a
chimeric heavy chain constant region, and (2) a non-endogenous
immunoglobulin light chain, wherein the chimeric heavy chain
constant region is derived from two or more non-endogenous species,
alleles and/or haplotypes. Another embodiment provides a non-human
animal comprising a genome that comprises (1) a chimeric
immunoglobulin heavy chain, wherein the chimeric heavy chain
comprises a non-endogenous heavy chain variable domain and a
chimeric heavy chain constant region, and (2) a non-endogenous
immunoglobulin light chain, wherein the non-endogenous heavy chain
variable domain is derived from two or more species, alleles and/or
haplotypes. One embodiment provides a non-human animal comprising a
genome that comprises a synthetic transgene encoding a chimeric
antibody, or an antigen-binding fragment thereof, comprising (1) a
chimeric immunoglobulin heavy chain, wherein the chimeric heavy
chain comprises a non-endogenous heavy chain variable domain and a
chimeric heavy chain constant region. In particular embodiments,
the genome further comprises a non-endogenous immunoglobulin light
chain. In certain embodiments, the genome of the animal comprises a
non-endogenous Ig.kappa. light chain and a non-endogenous Ig.lamda.
light chain. In particular embodiments, the cell comprises an
inactivated endogenous immunoglobulin locus. Another embodiment
provides a chimeric antibody produced by the disclosed animal.
[0020] One embodiment of the invention provides a non-human animal
comprising an inactivated endogenous Ig locus, wherein the
endogenous Ig locus comprises a deletion that impairs formation of
a functional variable domain and formation of a constant region
capable of driving primary B cell development. In certain
embodiments, the endogenous immunoglobulin locus is a heavy chain
locus. In certain other embodiments, the endogenous immunoglobulin
locus is a light chain locus. Another embodiment provides a
non-human cell comprising an inactivated endogenous Ig locus,
wherein the endogenous Ig locus comprises a deletion that impairs
formation of a functional variable domain and formation of a
constant region capable of driving primary B cell development.
[0021] One embodiment provides a DNA construct comprising a first
flanking sequence, a transgene, and a second flanking sequence,
wherein the transgene comprises a polynucleotide sequence capable
of introducing a deletion in an endogenous Ig locus that impairs
formation of a functional variable domain and formation of a
constant region capable of supporting primary B cell development.
Another embodiment provides a kit comprising the DNA construct.
Another embodiment provides a method for inactivating an endogenous
immunoglobulin locus comprising impairing the formation of a
functional variable domain, and impairing the formation of a
constant region capable of driving primary B cell development.
[0022] Another embodiment of the invention provides a method of
producing an antibody display library comprising providing a
non-human animal having a genome that comprises a chimeric
immunoglobulin heavy chain, wherein the chimeric heavy chain
comprises a non-endogenous heavy chain variable domain and a
chimeric heavy chain constant region; recovering polynucleotide
sequences from the animal, wherein the polynucleotide sequences
encode immunoglobulin light chain variable regions and
non-endogenous immunoglobulin heavy chain variable regions; and
producing an antibody display library comprising the heavy chain
and light chain variable regions. One embodiment of the invention
provides an antibody display library comprising immunoglobulin
heavy chain variable regions generated by a non-human animal having
a genome that comprises a chimeric immunoglobulin heavy chain,
wherein the chimeric heavy chain comprises a non-endogenous heavy
chain variable domain and a chimeric heavy chain constant region,
wherein the variable regions are derived from chimeric
antibodies.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0023] FIG. 1 depicts homologous recombination of BAC C5 and BAC
P12 in E. coli.
[0024] FIG. 2 depicts the removal of the 70 kb repeat between the
two copies of the pBeloBAC vector using CRE-recombinase.
[0025] FIG. 3 depicts the insertion of Tpn-Zeo 15 kb from the
junction of the vector.
[0026] FIG. 4 depicts homologous recombination of BAC C5P12 and BAC
C20 in E. coli.
[0027] FIG. 5 depicts the removal of the 44 kb repeat between the
two copies of the pBeloBAC vector using CRE-recombinase.
DETAILED DESCRIPTION
Overview
[0028] The present invention includes chimeric antibodies,
non-human animals that produce chimeric or humanized antibodies,
methods of producing such non-human cells and animals, and
compositions and kits comprising the antibodies. In specific
embodiments of the invention, the non-human animals are
mammals.
[0029] Chimeric antibodies, and antigen-binding fragments thereof,
described herein comprise a non-endogenous variable domain and a
chimeric heavy chain constant region. In particular embodiments, an
IgH chain comprises one or more non-endogenous V, D and J gene
segments, a non-endogenous CH1 domain, and endogenous CH2 and CH3
domains. In certain embodiments, an antibody, or antigen-binding
fragment thereof, comprising the chimeric IgH chain described
herein further comprises an IgL chain having an amino acid sequence
encoded for by a non-endogenous nucleotide sequence. In other
embodiments, an antibody, or antigen-binding fragment thereof,
comprising the chimeric IgH chain described herein further
comprises an IgL chain having an amino acid sequence encoded for by
endogenous and non-endogenous nucleotide sequences.
[0030] Engineering the chimeric antibodies in this manner prevents
alteration in the V domain conformation resulting from the in vitro
switch from a first C region, particularly a CH1 domain and
optionally a portion of the hinge region from one species, e.g.,
mouse, with which it was evolved during the in vivo immune response
to a second C region, particularly a CH1 domain and optionally a
portion of the hinge region from another species, e.g., human. The
antibodies produced by the animals of the present invention do not
exhibit the reduction or loss of activity and potency seen in
antibodies from other chimeric antibody producing animals when, for
example, the human V region is appended to a human C region to make
a fully human antibody, which may be caused by altered conformation
of the VH domain resulting from the changing of the CH1 domain
and/or by differences in antigen binding because of changed length
or flexibility of the upper hinge regions (the peptide sequence
from the end of the CH1 to the first cysteine residue in the hinge
that forms an inter-heavy chain disulfide bond, and which are
variable in length and composition) when switching from mouse to
human constant region (Roux et al., J. Immunology (1997)
159:3372-3382 and references therein). The middle hinge region is
bounded by the cysteine residues that form inter-heavy chain
disulfide bonds.
Definitions
[0031] Before describing certain embodiments in detail, it is to be
understood that this invention is not limited to particular
compositions or biological systems, which can vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular illustrative embodiments only, and is not
intended to be limiting. The terms used in this specification
generally have their ordinary meaning in the art, within the
context of this invention and in the specific context where each
term is used. Certain terms are discussed below or elsewhere in the
specification, to provide additional guidance to the practitioner
in describing the compositions and methods of the invention and how
to make and use them. The scope and meaning of any use of a term
will be apparent from the specific context in which the term is
used. As such, the definitions set forth herein are intended to
provide illustrative guidance in ascertaining particular
embodiments of the invention, without limitation to particular
compositions or biological systems. As used in the present
disclosure and claims, the singular forms "a," "an," and "the"
include plural forms unless the context clearly dictates
otherwise.
[0032] As used herein, "antibody" and "immunoglobulin" (Ig) are
used interchangeably herein and refer to protein molecules produced
by B cells that recognize and bind specific antigens and that may
either be membrane bound or secreted. Antibodies may be monoclonal,
in that they are produced by a single clone of B cells and
therefore recognize the same epitope and have the same nucleic acid
and amino acid sequence, or polyclonal, in that they are produced
by multiple clones of B cells, recognize one or more epitopes of
the same antigen and typically have different nucleic acid and
amino acid sequences.
[0033] Antibody, or Ig, molecules are typically comprised of two
identical heavy chains and two identical light chains linked
together through disulfide bonds. There are two types of IgL,
Ig.kappa. and Ig.lamda.. Both heavy chains (IgH) and light chains
(IgL) contain a variable (V) region or domain and a constant (C)
region or domain. The portion of the IgH locus encoding the V
region comprises multiple copies of variable (V), diversity (D),
and joining (J) gene segments. The portion of the IgL loci,
Ig.kappa. and Ig.lamda., encoding the V region comprises multiple
copies of V and J gene segments. The V region encoding portion of
the IgH and IgL loci undergo gene rearrangement, e.g., different
combinations of gene segments arrange to form the IgH and IgL
variable regions, to develop diverse antigen specificity in
antibodies. The secreted form of the IgH C region is made up of
three C domains, CH1, CH2, CH3, optionally CH4 (C.mu.), and a hinge
region. The membrane-bound form of the IgH C region also has
membrane and intra-cellular domains. The IgH constant region
determines the isotype of the antibody, e.g. IgM, IgD, IgG1, IgG2,
IgG3, IgG4, IgA and IgE in humans. It will be appreciated that
non-human mammals encoding multiple Ig isotypes will be able to
undergo isotype class switching.
[0034] A "Fab" domain or fragment comprises the N-terminal portion
of the IgH, which includes the V region and the CH1 domain of the
IgH, and the entire IgL. A "F(ab').sub.2" domain comprises the Fab
domain and a portion of the hinge region, wherein the 2 IgH are
linked together via disulfide linkage in the middle hinge region.
Both the Fab and F(ab').sub.2 are "antigen-binding fragments." The
C-terminal portion of the IgH, comprising the CH2 and CH3 domains,
is the "Fc" domain. The Fc domain is the portion of the Ig
recognized by cell receptors, such as the FcR, and to which the
complement-activating protein, C1q, binds. The lower hinge region,
which is encoded in the 5' portion of the CH2 exon, provides
flexibility within the antibody for binding to FcR receptors.
[0035] As used herein "chimeric antibody" refers to an antibody
encoded by a polynucleotide sequence containing polynucleotide
sequences derived from two or more species.
[0036] A "humanized" antibody is a chimeric antibody that has been
engineered so as to comprise more human sequence than its parental
molecule. Humanized antibodies are less immunogenic after
administration to humans when compared to non-humanized antibodies
prepared from another species. For example, a humanized antibody
may comprise the variable region of a chimeric antibody appended to
a human constant region. Chimeric antibodies described herein can
be used to produce a fully human antibody.
[0037] As used herein "chimeric Ig chain" refers to an Ig heavy
chain or an Ig light chain encoded by a polynucleotide sequence
containing polynucleotide sequences derived from two or more
species. For example, a chimeric Ig heavy chain may comprise human
VH, DH, JH, and CH1 gene segments and mouse CH2 and CH3 gene
segments.
[0038] "Polypeptide," "peptide" or "protein" are used
interchangeably to describe a chain of amino acids that are linked
together by chemical bonds. A polypeptide or protein may be an IgH,
IgL, V domain, C domain, or an antibody.
[0039] "Polynucleotide" refers to a chain of nucleic acids that are
linked together by chemical bonds. Polynucleotides include, but are
not limited to, DNA, cDNA, RNA, mRNA, and gene sequences and
segments. Polynucleotides may be isolated from a living source such
as a eukaryotic cell, prokaryotic cell or virus, or may be derived
through in vitro manipulation by using standard techniques of
molecular biology, or by DNA synthesis, or by a combination of a
number of techniques.
[0040] "Locus" refers to a location on a chromosome that comprises
one or more genes or exons, such as an IgH or Ig.kappa. locus, the
cis regulatory elements, and the binding regions to which
trans-acting factors bind. As used herein, "gene" or "gene segment"
refers to the polynucleotide sequence encoding a specific
polypeptide or portion thereof, such as a VL domain, a CH1 domain,
an upper hinge region, or a portion thereof. As used herein, "gene
segment" and "exon" may be used interchangeably and refer to a
polynucleotide encoding a peptide, or a portion thereof. A gene, or
gene segment, may further comprise one or more introns,
transcriptional control elements, e.g., promoters, enhancers, or
other non-coding regions (e.g., cis regulatory elements, e.g., 5'
and/or 3' untranslated regions, poly-adenylation sites).
[0041] As used herein, the term "inactivated Ig locus" refers to an
Ig locus that does not encode a functional Ig chain. A "functional
variable region" produce from an Ig locus refers to a
polynucleotide sequence capable of undergoing V-(D)-J
recombination, being transcribed and said transcript being
translated into a variable region polypeptide that is capable of
being expressed on a cell surface. A "functional heavy chain
constant region" refers to a constant region capable of being
operationally joined to a variable region and driving primary B
cell development. Primary B cell development refers to the
development of B cells in the primary lymphoid organs, e.g., bone
marrow, and encompasses the transition from stem cell to immature B
cell, including the developmental stages of early pro-B cell (i.e.,
IgH D-J rearranging), late pro-B cell (i.e., IgH V-DJ rearranging),
large pre-B cell (i.e., expresses pre-B receptor), and small pre-B
cell (i.e., IgL V-J rearranging). By "driving" primary B cell
development, it is meant that the functional heavy chain constant
region is capable of, e.g., anchoring to the cell membrane, signal
transduction, and/or binding an Fc receptor. A "functional light
chain constant region" refers to a constant region capable of being
operationally joined to a variable region and binding to heavy
chain to advance B cell development beyond the small pre-B cell
stage.
[0042] "Impair" refers to the introduction of a deletion or
mutation that results in, e.g., a variable region that is no longer
functional or a constant region that is no longer function. For
example, homozygous deletion of C impairs an IgH from driving
primary B cell development in some mammals and strains thereof.
[0043] "Mutation" refers to a change in a naturally occurring
polynucleotide or polypeptide sequence. A mutation may result in a
functional change. Mutations include both the addition of
nucleotides and the deletion of nucleotides. "Deletion" refers to
the removal of one or more nucleotides from the naturally occurring
endogenous polynucleotide sequence. Deletions and additions may
introduce a frameshift mutation. Deletions may also remove entire
genes, gene segments or modules. In some instances, a deletion of
part of the naturally occurring endogenous sequence may coincide
with the addition of a non-endogenous sequence. For example, a
portion of the endogenous polynucleotide sequence may be removed,
i.e., deleted, upon homologous recombination with a polynucleotide
comprising a non-endogenous sequence, e.g., a selection marker. In
other aspects, a deletion of an endogenous polynucleotide sequence
may occur after the introduction of two non-endogenous recognition
sequence for a site-specific recombinase, e.g., a loxP site,
followed by exposure to the recombinase, e.g., CRE.
[0044] The term "endogenous" refers to a polynucleotide sequence
which occurs naturally within the cell or animal. "Orthologous"
refers to a polynucleotide sequence that encodes the corresponding
polypeptide in another species, e.g., a human CH1 domain and a
mouse CH1 domain. The term "syngeneic" refers to a polynucleotide
sequence that is found within the same species that may be
introduced into an animal of that same species, e.g., a mouse
V.kappa. gene segment introduced into a mouse. It should be noted
that the polynucleotide sequence from two individuals of the same
species but of different strains may have regions of significant
difference.
[0045] As used herein, the term "homologous" or "homologous
sequence" refers to a polynucleotide sequence that has a highly
similar sequence, or high percent identity (e.g. 30%, 40%, 50%,
60%, 70%, 80%, 90% or more), to another polynucleotide sequence or
segment thereof. For example, a DNA construct of the invention may
comprise a sequence that is homologous to a portion of an
endogenous DNA sequence to facilitate recombination at that
specific location. Homologous recombination may take place in
prokaryotic and eukaryotic cells.
[0046] As used herein, "flanking sequence" or "flanking DNA
sequence" refers to a DNA sequence adjacent to a non-endogenous DNA
sequence in a DNA construct that is homologous to an endogenous DNA
sequence or a previously recombined non-endogenous sequence, or a
portion thereof. DNA constructs of the invention may have one or
more flanking sequences, e.g., a flanking sequence on the 3' and 5'
end of the non-endogenous sequence or a flanking sequence on the 3'
or the 5' end of the non-endogenous sequence. The flanking sequence
may be homologous to an endogenous sequence within an endogenous
gene, or the flanking sequence may be homologous to an endogenous
sequence adjacent to (i.e., outside of) an endogenous gene.
[0047] The phrase "homologous recombination-competent cell" refers
to a cell that is capable of homologously recombining DNA fragments
that contain regions of overlapping homology. Examples of
homologous recombination-competent cells include, but are not
limited to, induced pluripotent stem cells, hematopoietic stem
cells, bacteria, yeast, various cell lines and embryonic stem (ES)
cells.
[0048] A "non-human animal" refers to any animal other than a human
such as, e.g., avians, reptiles and mammals. "Non-human mammal"
refers to an animal other than humans which belongs to the class
Mammalia. Examples of non-human mammals include, but are not
limited to, non-human primates, camelids, rodents, bovines, ovines,
equines, dogs, cats, goats, sheep, dolphins, bats, rabbits, and
marsupials. Preferred non-human mammals rely primarily on somatic
hypermutation and/or gene conversion to generate antibody
diversity, e.g., mouse, rabbit, pig, sheep, goat, camelids, rodents
and cow. Particularly preferred non-human mammals are mice.
[0049] The term "transgenic" refers to a cell or animal comprising
a non-endogenous polynucleotide sequence, e.g., a transgene derived
from another species, incorporated into its genome. For example, a
mouse which contains a human VH gene segment integrated into its
genome outside the endogenous mouse IgH locus is a transgenic
mouse; and a mouse which contains a human VH gene segment
integrated into its genome directly replacing an endogenous mouse
VH in the endogenous mouse IgH locus is a transgenic mouse,
sometimes also referred to as a "knock-in" mouse. In transgenic
cells and non-human mammals, the non-endogenous polynucleotide
sequence may either be expressed with the endogenous gene,
ectopically in the absence of the endogenous gene or in the absence
of the corresponding, or orthologous, endogenous sequence
originally found in the cell or non-human mammal.
[0050] As used herein, "replace" refers to both direct and
functional replacement. By "direct replacement" it is meant that an
endogenous DNA sequence is replaced with an engineered DNA sequence
that comprises a non-endogenous sequence at the location of the
endogenous sequence in the genome, such as by homologous
recombination. For example, the endogenous DNA sequence is removed
via homologous recombination, or the endogenous sequence remaining
between two incorporated non-endogenous sequences is deleted. By
"functional replacement" it is meant that the function (e.g., as
performed by the polypeptide produced from the engineered DNA
sequence) of an endogenous DNA sequence is carried out by a
non-endogenous DNA sequence. For example, an endogenous IgH locus
can be functionally replaced by a transgene that encodes a chimeric
IgH chain and that is inserted into the genome outside of the
endogenous IgH locus.
[0051] A "humanized" animal, as used herein refers to a non-human
animal, e.g., a mouse, that has a composite genetic structure that
retains gene sequences of the non-human animal, in addition to one
or more gene segments and or gene regulatory sequences of the
original genetic makeup having been replaced with analogous human
sequences.
[0052] As used herein, the term "vector" refers to a nucleic acid
molecule into which another nucleic acid fragment can be integrated
without loss of the vector's ability to replicate. Vectors may
originate from a virus, a plasmid or the cell of a higher organism.
Vectors are utilized to introduce foreign or recombinant DNA into a
host cell, wherein the vector is replicated.
[0053] A polynucleotide agent can be contained in a vector, which
can facilitate manipulation of the polynucleotide, including
introduction of the polynucleotide into a target cell. The vector
can be a cloning vector, which is useful for maintaining the
polynucleotide, or can be an expression vector, which contains, in
addition to the polynucleotide, regulatory elements useful for
expressing the polynucleotide and, where the polynucleotide encodes
an RNA, for expressing the encoded RNA in a particular cell, either
for subsequent translation of the RNA into a polypeptide or for
subsequent trans regulatory activity by the RNA in the cell. An
expression vector can contain the expression elements necessary to
achieve, for example, sustained transcription of the encoding
polynucleotide, or the regulatory elements can be operatively
linked to the polynucleotide prior to its being cloned into the
vector.
[0054] An expression vector (or the polynucleotide) generally
contains or encodes a promoter sequence, which can provide
constitutive or, if desired, inducible or tissue specific or
developmental stage specific expression of the encoding
polynucleotide, a poly-A recognition sequence, and a ribosome
recognition site or internal ribosome entry site, or other
regulatory elements such as an enhancer, which can be tissue
specific. The vector also can contain elements required for
replication in a prokaryotic or eukaryotic host system or both, as
desired. Such vectors, which include plasmid vectors and viral
vectors such as bacteriophage, baculovirus, retrovirus, lentivirus,
adenovirus, vaccinia virus, alpha virus and adeno-associated virus
vectors, are well known and can be purchased from a commercial
source (Promega, Madison Wis.; Stratagene, La Jolla Calif.;
GIBCO/BRL, Gaithersburg Md.) or can be constructed by one skilled
in the art (see, for example, Meth. Enzymol., Vol. 185, Goeddel,
ed. (Academic Press, Inc., 1990); Jolly, Canc. Gene Ther. 1:51-64,
1994; Flotte, J. Bioenerg. Biomemb 25:37-42, 1993; Kirshenbaum et
al., J. Clin. Invest 92:381-387, 1993; each of which is
incorporated herein by reference).
[0055] A DNA vector utilized in the methods of the invention can
contain positive and negative selection markers. Positive and
negative markers can be genes that when expressed confer drug
resistance to cells expressing these genes. Suitable selection
markers for E. coli can include, but are not limited to: Km
(Kanamycin resistant gene), tetA (tetracycline resistant gene) and
beta-lactamase (ampicillin resistant gene). Suitable selection
markers for mammalian cells in culture can include, but are not
limited to: hyg (hygromycin resistance gene), puro (puromycin
resistance gene) and G418 (neomycin resistance gene). The selection
markers also can be metabolic genes that can convert a substance
into a toxic substance. For example, the gene thymidine kinase when
expressed converts the drug gancyclovir into a toxic product. Thus,
treatment of cells with gancylcovir can negatively select for genes
that do not express thymidine kinase.
[0056] In a related aspect, the selection markers can be
"screenable markers," such as green fluorescent protein (GFP),
yellow fluorescent protein (YFP), red fluorescent protein (RFP),
GFP-like proteins, and luciferase.
[0057] Various types of vectors are available in the art and
include, but are not limited to, bacterial, viral, and yeast
vectors. A DNA vector can be any suitable DNA vector, including a
plasmid, cosmid, bacteriophage, p1-derived artificial chromosome
(PAC), bacterial artificial chromosome (BAC), yeast artificial
chromosome (YAC), or mammalian artificial chromosome (MAC). In
certain embodiments, the DNA vector is a BAC. The various DNA
vectors are selected as appropriate for the size of DNA inserted in
the construct. In one embodiment, the DNA constructs are bacterial
artificial chromosomes or fragments thereof.
[0058] The term "bacterial artificial chromosome" or "BAC" as used
herein refers to a bacterial DNA vector. BACs, such as those
derived from E. coli, may be utilized for introducing, deleting or
replacing DNA sequences of non-human mammalian cells or animals via
homologous recombination. E. coli can maintain complex genomic DNA
as large as 500 kb or greater in the form of BACs (see Shizuya and
Kouros-Mehr, Keio J Med. 2001, 50(1):26-30), with greater DNA
stability than cosmids or yeast artificial chromosomes. In
addition, BAC libraries of human DNA genomic DNA have more complete
and accurate representation of the human genome than libraries in
cosmids or yeast artificial chromosomes. BACs are described in
further detail in U.S. Application Nos. 10/659,034 and 61/012,701,
which are hereby incorporated by reference in their entireties.
[0059] DNA fragments comprising an Ig locus, or a portion thereof,
to be incorporated into the non-human mammal are isolated from the
same species of non-human mammal prior to humanization of the
locus. Multiple BACs containing overlapping fragments of an Ig
locus can be humanized and the overlapping fragments recombine to
generate a continuous IgH or IgL locus. The resulting chimeric Ig
locus comprises the human gene segments operably linked to the
non-human mammal Ig gene segments to produce a functional Ig locus,
wherein the locus is capable of undergoing gene rearrangement and
thereby producing a diversified repertoire of chimeric
antibodies.
[0060] These processes for recombining BACs and/or of engineering a
chimeric Ig locus or fragment thereof requires that a bacterial
cell, such as E. coli, be transformed with a BAC containing the
host Ig locus or a portion thereof. The BAC containing bacillus is
then transformed with a recombination vector comprising the desired
human Ig gene segment linked to flanking homology sequence shared
with the BAC containing the host Ig locus or portion thereof. The
shared sequence homology mediates homologous recombination and
cross-over between the human Ig gene segment on the recombination
vector and the non-human mammal Ig gene segment on the BAC.
Detection of homologously recombined BACs may utilize selectable
and/or screenable markers incorporated into the vector. Humanized
BACs can be readily isolated from the bacteria and used for
producing knock-in non-human cells. Methods of recombining BACs and
engineering insertions and deletions within DNA on BACs and methods
for producing genetically modified mice therefrom are documented.
See, e.g., U.S. Pat. No. 5,770,429; Fishwild, D. et al. (1996) Nat.
Biotechnol. 14:845-851; Valenzuela et al. Nature Biotech. (2003)
21:652-659; Testa et al. Nature Biotech. (2003) 21:443-447; and
Yang and Seed. Nature Biotech. (2003) 21:447-451.
[0061] The first recombination step may be carried out in a strain
of E. coli that is deficient for sbcB, sbcC, recB, recC or recD
activity and has a temperature sensitive mutation in recA. After
the recombination step, a recombined DNA construct is isolated, the
construct having the various sequences and orientations as
described.
[0062] The regions used for BAC recombineering should be a length
that allows for homologous recombination. For example, the flanking
regions may be from about 0.1 to 19 kb, and typically from about 1
kb to 15 kb, or about 2 kb to 10 kb.
[0063] The process for recombining BACs to make larger and/or
tailored BACs comprising portions of the Ig loci requires that a
bacterial cell, such as E. coli, be transformed with a BAC carrying
a first Ig locus, a portion thereof, or some other target sequence.
The BAC containing E. coli is then transformed with a recombination
vector (e.g., plasmid or BAC) comprising the desired Ig gene
segment to be introduced into the target DNA, e.g., one or more
human VH, DH and/or JH gene segments to be joined to a region from
the mouse IgH locus, both of which vectors have a region of
sequence identity. This shared region of identity in the presence
of functional recA in the E. coli mediates cross-over between the
Ig gene segment on the recombination vector and the non-human
mammal Ig gene segment on the BAC. Selection and resolution of
homologously recombined BACs may utilize selectable and/or
screenable markers incorporated into the vectors. Humanized and
chimeric BACs can be readily purified from the E. coli and used for
producing transgenic and knock-in non-human cells and animals by
introducing the DNA by various methods known in the art and
selecting and/or screening for either random or targeted
integration events.
[0064] Alternatively, the DNA fragments containing an Ig locus to
be incorporated into a non-human animal are derived from DNA
synthesized in vitro. The genomes of many organisms have been
completely sequenced (e.g., human, chimpanzee, rhesus monkey,
mouse, rat, dog, cat, chicken, guinea pig, rabbit, horse, cow,
alpaca) and are publicly available with annotation. For many other
organisms, there is publicly available information on the sequences
of the transcriptome. In particular but not limited to, the human
and mouse immunoglobulin loci have been studied and characterized
for the location and activity of coding gene segments and
non-coding regulatory elements.
[0065] The term "in silico," as used herein, refers to the use of a
computer or computer algorithm to model a naturally occurring or in
vitro process, and in particular, to assist in the design of a
nucleotide or polypeptide sequence and/or the synthetic production
of a nucleotide or polypeptide sequence using, all or in part, a
cell free system (e.g., using automated chemical synthesis). The
sequences of the Ig loci may be manipulated and recombined in
silico using commonly available software for nucleic acid sequence
analysis. In silico recombination may be within the same locus,
between two loci from the same species, or between loci from two or
more species. In silico recombination may be performed to design
either a functional sequence or a non-functional, inactivated
sequence. Precise nucleotide-by-nucleotide engineering allows for
precise manipulation of sequence composition that can be applied to
precisely engineer the function of the transgene and after
transcription and translation, result in precisely engineered
composition and function of the polypeptide product of the
locus.
[0066] Sequences of an Ig locus may also be recombined in silico
with those from a non-immunoglobulin locus, either from the same or
a different species. Such sequences include, but are not limited
to, genes for positive and negative drug selection markers such as
G418, hyg, puro and tk, site-specific recombinase recognition
sequences such lox P sites and its variants and frt sites, and
precisely demarcated sequences for driving homologous
recombination. After assembling the desired sequence in silico, it
may then be synthesized and assembled without errors (Kodumal et
al., Proc. Natl. Acad. Sci. (2004) 101:15573-15578). The synthesis,
assembly and sequencing of large DNAs are provided on a contractual
basis (e.g., DNA 2.0, Menlo Park, Calif.; Blue Heron Biotechnology,
Bothell, Wash.; and Eurogentec, San Diego, Calif.). Such synthetic
DNA sequences are carried in vectors such as plasmids and BACs and
can be transferred into other vectors such as YACs.
[0067] The term "construct" as used herein refers to a sequence of
DNA artificially constructed by genetic engineering, recombineering
or synthesis. Constructs include, for example, transgenes and
vectors (e.g., BACs, P1s, lambda bacteriophage, cosmids, plasmids,
YACs and MACs). In one embodiment, the DNA constructs are
linearized prior to introduction into a cell. In another
embodiment, the DNA constructs are not linearized prior to
introduction into a cell.
[0068] As used herein, "loxP" and "CRE" refer to a site-specific
recombination system derived from P1 bacteriophage. loxP sites are
34 nucleotides in length. When DNA is flanked on either side by a
loxP site and exposed to CRE mediated recombination, the
intervening DNA is deleted and the two loxP sites resolve to one.
The use of the CRE/lox system, including variant-sequence lox sites
and variants of CRE, for which genetic engineering in many species,
including mice, is well documented.
[0069] A similar system, employing frt sites and flp recombinase
from S. cerevisiae can be employed to similar effect. As used
herein, any implementation of CRE/loxP to mediate deletional events
in mammalian cells in culture can also be mediated by the flp/frt
system.
[0070] As used herein the terms "immunize," "immunization," and
"immunizing" refer to exposing the adaptive immune system of an
animal to an antigen. The antigen can be introduced using various
routes of administration, such as injection, inhalation, ingestion
or DNA immunization. Upon a second exposure to the same antigen,
the adaptive immune response, i.e. T cell and B cell responses, is
enhanced.
[0071] "Antigen" refers to a peptide, lipid, amino acid, nucleic
acid, saccharide, hapten or chemical entity that is recognized by
the adaptive immune system. Examples of antigens include, but are
not limited to, bacterial cell wall components, pollen, and rh
factor. "Target antigen" refers to an antigen, peptide, lipid,
saccharide, or amino acid, which is recognized by the adaptive
immune system that is chosen to produce an immune response against,
e.g., a specific infectious agent or endogenous or exogenous cell
or product thereof. Target antigens include, but are not limited
to, bacterial and viral components, tumor-specific antigens,
cytokines, cell surface molecules, any and all antigens against
which antibodies or other binding proteins have been made by in
vivo or in vitro methods, etc.
[0072] The term "pharmaceutical" or "pharmaceutical drug," as used
herein refers to any pharmacological, therapeutic or active
biological agent that may be administered to a subject or patient.
In certain embodiments the subject is an animal, and preferably a
mammal, most preferably a human.
[0073] The term "pharmaceutically acceptable carrier" refers
generally to any material that may accompany the pharmaceutical
drug and which does not cause an adverse reaction with the
subject's immune system.
[0074] The term "administering," as used herein, refers to any mode
of transferring, delivering, introducing, or transporting a
pharmaceutical drug or other agent, such as a target antigen, to a
subject. Such modes include oral administration, topical contact,
intravenous, intraperitoneal, intramuscular, intranasal, or
subcutaneous administration.
Non-Human Mammals and Cells Encoding Chimeric Ig Heavy Chains
[0075] Non-human animals and cells of the present invention
comprise one or more altered Ig loci (e.g., IgH, Ig.kappa., and/or
Ig.lamda.) comprising non-endogenous Ig gene segments that replace
the endogenous gene segments.
[0076] In certain embodiments, the altered loci directly replace
the endogenous gene segments. In other embodiments, the altered
loci functionally replace the endogenous gene segments.
[0077] The non-endogenous gene segments may be derived from any
species, and may include syngeneic gene segments. The
non-endogenous sequence may be derived from, for example, humans,
mice, non-human primates, camelids, rodents, bovines, ovines,
equines, dogs, cats, goats, sheep, dolphins, bats, rabbits, and
marsupials. As described above, the non-human cell or animal may be
any non-human animal. Accordingly, the transgenic cells and animals
described herein may comprise DNA sequences derived from any
combination of species, provided that the animal is a non-human
mammal. By way of example, chimeric mouse cells and mice comprising
human or camelid Ig polynucleotide sequences are envisioned. In
addition, the transgenic cell or animal may comprise non-endogenous
DNA from more than one species. For example, a transgenic mouse
genome can comprise both human and camelid DNA sequences.
[0078] The transgenic cells and animals described herein comprise
one or more non-endogenous V gene segments. In specific
embodiments, the preferred non-human animal is a mammal. In certain
embodiments, the cell or animal further comprises one or more
non-endogenous J gene segments. In another embodiment, a cell or
animal comprising a chimeric IgH chain optionally further comprises
one or more non-endogenous D gene segments.
[0079] In one embodiment, the cell or animal comprises a genome
encoding a chimeric IgH chain and a transgenic light chain. The
transgenic light chain may be an Ig.kappa. or an Ig.lamda. light
chain. In addition, the transgenic light chain may be chimeric, or
the transgenic light chain may comprise only non-endogenous amino
acid sequences. In particular embodiments, the cell or animal
comprises a genome encoding non-endogenous IgH, Ig.kappa. and
Ig.lamda. gene segments. The transgenic cells and mammals
comprising a chimeric IgH chain described herein comprise a
non-endogenous CH1 domain that replaces a CH1 domain in a specific
endogenous CH gene, e.g., C.mu., C.delta., or C.gamma.. In certain
embodiments, the non-endogenous CH1 domain is orthologous to the
endogenous CH region. In other embodiments, the non-endogenous
CH1-domain is not orthologous to the endogenous CH region. In
another embodiment, more than one endogenous CH1 domain is replaced
with a non-endogenous CH1 domain. In a related embodiment, all of
the endogenous CH1 domains are replaced with a non-endogenous CH1
domain. For example, an orthologous human CH1 may replace each of
the endogenous C.gamma. genes (e.g., human C.gamma.1 CH1 replaces
mouse C.gamma.1 CH1 and human C.gamma.2 CH1 replaces mouse
C.gamma.2 CH1 etc.). In another embodiment, the CH1 domain that
replaces the CH1 domain of each of the endogenous C.gamma. genes is
a single human IgG isotype more frequently used in therapeutic
mAbs, typically C.gamma.1, C.gamma.2 or C.gamma.4, so as to better
facilitate in vivo maturation of a human V domain in the context of
a more clinically relevant human CH1 domain.
[0080] Optionally, the upper hinge sequences of the endogenous C
genes may also be replaced with orthologous non-endogenous C hinge
sequences. Alternatively, the upper and middle hinge sequences of
the endogenous C genes may also be replaced with the orthologous
non-endogenous C hinge sequences, respectively. If human middle
hinge regions are used, the human C.gamma.4 middle hinge sequence
may be engineered to contain a proline at residue at position 229
rather than a serine in order to drive inter-heavy chain
dimerization via disulfide bonds. The lower hinge region, a part of
the CH2 domain, of the endogenous C.gamma. gene is not replaced in
order to facilitate optimal binding to an endogenous Fc.gamma.R.
These three optional engineering strategies provide a
non-endogenous heavy chain Fab domain, Fab domain plus upper hinge,
or F(ab').sub.2, respectively. If the upper are replaced with human
upper hinge regions, the variable region of the resulting antibody
is more likely to retain optimal characteristics upon conversion to
fully human IgG.
[0081] Another embodiment incorporates fully non-endogenous, e.g.,
human, Ig including the C regions comprising
CH1-hinge-CH2-CH3(-CH4) and the cognate syngeneic, e.g., mouse,
membrane and intracellular domains so as to provide native
intracellular signal transduction and to enable association of the
IgH in the B-cell receptor with Ig.alpha. and Ig.beta. and therein
allow endogenous-type signaling from the Ig.alpha., Ig.beta. and
IgG containing B-cell receptor. In yet another embodiment, the
membrane and intracellular domain of the heavy chain constant
region are from the same or non-cognate syngeneic heavy chain
isotypes. Such engineering of the constant region genes can be
readily accomplished using methods of the invention as detailed
below.
[0082] In yet another embodiment, the transgenic cells and animals
comprising a chimeric IgH chain described herein comprise constant
region encoded by a non-endogenous polynucleotide sequence derived
from two or more species. For example, a transgenic mouse having a
genome encoding a chimeric IgH chain constant region comprises a
human CH1 domain, human upper hinge regions, and rat CH2 and CH3
domains, is envisioned. In animals having a xenogeneic constant
region, it is preferred that the constant region is capable of
interacting with (e.g., binding) an endogenous FcR.
[0083] In yet another embodiment, the transgenic cells and animals
comprising a chimeric IgH chain described herein comprise constant
region encoded by a non-endogenous polynucleotide sequence and
endogenous polynucleotide sequence derived from two strains. For
example, a transgenic mouse having a genome encoding a chimeric IgH
chain constant region comprises a human CH1 domain, human upper
hinge regions, and Balb/c mouse CH2 and CH3 coding sequences
embedded into C57BL/6 ("B6") genomic DNA, comprising all B6 genetic
information except that Balb/c-sequence exons for CH2 and CH3
replace their B6 counterparts, is envisioned.
[0084] In one embodiment, the composite IgH sequence comprises at
least 3 kb upstream of the VH6 promoter through the D gene cluster
through 3' of JH6 and is all human and in germline configuration.
In another embodiment, the composite IgH sequence comprises at
least 3 kb upstream of the VH6 promoter through the D gene cluster
through 3' of JH6 and is all human and in germline configuration
except that the D gene cluster is replaced by all or part of that
of a xenogeneic species. In another aspect of the invention, there
are additional human VH genes upstream of human VH6. In yet another
aspect, the additional VH genes are in germline configuration. In
an alternative aspect, the additional VH genes are sizes less than
that in the human genome, unit sizes that comprise upstream
regulatory elements such as cis-regulatory elements and binding
sites for trans-acting factors, coding sequences, introns and 500
bp downstream of the last codon of each VH. In one aspect, the unit
size is 10 kb or less. In another aspect, the unit size is 5 kb or
less. In another aspect, the VH genes are selected from the subset
of commonly shared VH genes amongst human haplotypes. In another
aspect, VH genes, DH genes and JH genes are chosen to reflect a
specific allele such as the most prevalent allele in human
populations. In yet another aspect, the individual codons of the VH
gene are codon-optimized for efficient expression in a specific
non-human mammal. In another aspect the individual codons are
optimized to be a template for somatic hypermutation.
[0085] In another embodiment, the composite IgH sequence comprises
mouse DNA sequence starting at least 3 kb upstream of the promoter
for the functional VH gene nearest the D gene cluster, e.g., VH5-2,
through 3' of JH4 in germline configuration and into which the
coding sequences have been replaced, all or in part, by human
coding sequences, e.g., coding sequence for mouse VH5-2 is replaced
by coding sequence for human VH6-1, mouse DH coding sequences
replaced by human DH coding sequences and mouse JH coding sequences
replaced by human JH coding sequences. In instances in which the
number of human coding elements exceeds those in the mouse, e.g., 6
human JH coding sequences versus 4 mouse JH coding sequences, the
additional JH genes may be included by various means, e.g.,
inserting the additional human JH coding sequences with their cis
regulatory elements, such as recombination signal sequences
downstream of the JH4, or omitted altogether.
[0086] In other embodiments, the mouse VH coding sequences are
replaced, all or in part, by human VL coding sequences. In some
embodiments, the entire DH gene cluster is of mouse sequence. In
other embodiments, the entire DH gene cluster is of xenogeneic
species. In another aspect of the invention, there are additional
VH genes upstream of VH6 coding sequences, such that the all of the
sequence is mouse except that coding sequences of functional VH
genes are replaced with that of human VH genes.
[0087] In yet another aspect, the additional VH genes are in
germline configuration. In an alternative aspect, the additional VH
genes are sizes less than that in the mouse genome, unit sizes that
comprise upstream regulatory elements such as cis-regulatory
elements and binding sites for trans-acting factors, coding
sequences, introns and 500 bp downstream of the last codon of each
VH. In one aspect, the unit size is 10 kb or less. In another
aspect, the unit size is 5 kb or less.
[0088] In another aspect, the VH genes are selected from a subset
known to be functional, with the replacing human VH gene coding
sequence being from a known functional human VH gene and replacing
the mouse VH gene coding sequence of a known functional mouse VH
gene. In another aspect, the human VH coding sequences are selected
from the subset of commonly shared VH genes amongst human
haplotypes. In another aspect, the replacing VH coding sequences,
DH coding sequences and JH coding sequences are chosen to reflect a
specific allele such as the most prevalent allele in human
populations. In another aspect, some or all of the replacing VH
coding sequences, DH coding sequences and JH coding sequences are
from a xenogeneic species other than human. In yet another aspect,
the individual codons of the VH gene are codon-optimized for
efficient expression in a specific non-human mammal. In another
aspect the individual codons are optimized to be a template for
somatic hypermutation.
[0089] In another embodiment, the composite IgH sequence further
comprises 3' of the most 3' JH the mouse sequence immediately
downstream of mouse JH4 through E through C through C through
immediately 5' of the mouse C.gamma.3 promoter all in germline
configuration with the exception of the replacement of the CH1
domains of mouse C.mu. and C.delta. by their human counterparts. In
some instances, the mouse upper hinge regions are replaced by their
respective human upper hinge regions. In a further embodiment, the
mouse C.gamma. genes are configured in germline configuration with
the exception of the replacement of their CH1 domains by human CH1
domains.
[0090] In some instances, the mouse upper hinge regions are
replaced by human upper hinge regions. In some embodiments, the
mouse C.gamma.3 coding sequences are replaced by human CH1 and
mouse CH2, CH3, membrane and intracellular domains from C.gamma.1.
In another embodiment, the complete germline-configured mouse
C.gamma.3 sequence from the promoter upstream of the switch region
through the intracellular domains and 3' untranslated sequence and
poly(A) site are replaced by the complete corresponding sequences
from C.gamma.1 in germline configuration with human CH1 replacing
mouse CH1 from C.gamma.1 to effectively replace the complete
C.gamma.3 gene by chimeric C.gamma.1. In some embodiments, a mouse
constant coding sequence is replaced by human CH1 and mouse CH2,
CH3, membrane and intracellular domains from different mouse
constant region isotypes, e.g., CH2, CH3 and membrane domains from
mouse C.gamma.2a and intracellular domain from C.mu.. In still
other embodiments the sequence of the CH2 and CH3 domains are
furthered modified to modulate binding to Fc receptors, such as
diminished binding to the inhibitory receptor, Fc.gamma.R2b,
therein producing a stronger secondary immune response.
[0091] In another embodiment, the cell or non-human animal
comprises a locus encoding a human Ig light chain comprising a
human Ig.kappa. variable region. In a related embodiment, the Ig
light chain locus further comprises a human Ig.kappa. constant
region. In one embodiment, the composite Ig.kappa. sequence
comprises mouse DNA sequence from at least 3 kb upstream of the
promoter of the V.kappa. gene most proximal to mouse J.kappa.1
(V.kappa.3-1) through 3' of mouse J.kappa.5 and is in germline
configuration and into which the coding sequences have been
replaced, all or in part, by human coding sequences, e.g., coding
sequence for mouse V.kappa.3-1 is replaced by coding sequence for
human V.kappa.4-1 and mouse J.kappa. coding sequences replaced by
human J.kappa. coding sequences. In another embodiment the sequence
from J.kappa.5 through C.kappa. is mouse and in germline
configuration and into which the C.kappa. coding sequences have
been replaced, all or in part, by human coding sequences.
[0092] In another aspect there is a 3'LCR region and RS element
downstream of the C.kappa. gene. In one aspect, the 3' LCR and RS
elements are mouse and in germline configuration. In another aspect
of the invention, there are additional V.kappa. genes upstream of
the coding sequences for human V.kappa.4-1, such that all of the
sequence is mouse except that coding sequences of functional
V.kappa. genes are replaced with that of human V.kappa. genes.
[0093] In yet another aspect, the additional V.kappa. genes are in
germline configuration. In an alternative aspect, the additional
V.kappa. genes are sizes less than that in the mouse genome, unit
sizes that comprise upstream regulatory elements such as
cis-regulatory elements and binding sites for trans-acting factors,
coding sequences, introns and 500 bp downstream of the last codon
of each V.kappa.. In one aspect, the unit size is 10 kb or less. In
another aspect, the unit size is 5 kb or less. In another aspect,
the V.kappa. genes are selected from the subset of commonly shared
V.kappa. genes amongst human haplotypes. In another aspect,
V.kappa. genes and J.kappa. genes are chosen to reflect a specific
allele such as the most prevalent allele in human populations. In
yet another aspect, the individual codons of the V.kappa. gene are
codon-optimized for efficient expression in a specific non-human
mammal. In another aspect the individual codons are optimized to be
a template for somatic hypermutation.
[0094] In yet another embodiment, the human Ig light chain locus
comprises all or a portion of a human Ig.alpha., light chain locus
and an Ig.lamda. 3'LCR, or a functional fragment thereof. In one
embodiment, the human Ig.lamda. light chain locus comprises the
entire human Ig.lamda. locus. In another embodiment the human
Ig.lamda. light chain locus comprises human V.lamda. coding
sequences and 1 to 7 J.lamda.-C.lamda. coding sequence pairs,
wherein the human C.lamda. is replaced with syngeneic C.lamda.. In
yet another embodiment, the human Ig.lamda. light chain locus
comprises human V.lamda. coding sequences, 1 to 7 human J.lamda.
coding sequences, and a single human C.lamda. coding sequence,
wherein the human coding sequences resemble a human Ig.lamda. locus
configuration.
[0095] In particular embodiments, the Ig.lamda. 3' LCR, or a
functional fragment thereof, is from a mammal selected from the
group consisting of human, non-human primate, and rat. In one
embodiment the Ig.lamda. 3' LCR, or a functional fragment thereof,
is human. In particular embodiments, the Ig.lamda. 3' LCR, or a
functional fragment thereof, binds NF.kappa.b. In one embodiment,
the Ig.lamda. 3' LCR, or a functional fragment thereof, is from
mouse and has been mutagenized so as to restore binding of
NF.kappa.b. In other embodiments, the 3' LCR, or a functional
fragment thereof, in the human Ig.lamda. locus is an Ig.kappa. 3'
LCR, or functional fragment thereof.
[0096] In one embodiment, the composite Ig.lamda. sequence
comprises at least 3 kb upstream of the V.lamda. 3r promoter
through 3' of J.lamda.7-C.lamda.7 and is all human and in germline
configuration. In another aspect, the sequence from
J.lamda.7-C.lamda.7 through the 23' LCR is human and in germline
configuration. In another aspect of the invention, there are
additional human V.lamda. genes upstream of human V.lamda. 3r. In
yet another aspect, the additional V.lamda. genes are in germline
configuration. In an alternative aspect, the additional V.lamda.
genes are sizes less than that in the human genome, unit sizes that
comprise upstream regulatory elements such as cis-regulatory
elements and binding sites for trans-acting factors, coding
sequences, introns and 500 bp downstream of the last codon of each
V.lamda.. In one aspect, the unit size is 10 kb or less. In another
aspect, the unit size is 5 kb or less. In another aspect, the
V.lamda. genes are selected from the subset of commonly shared
V.lamda. genes amongst human haplotypes. In another aspect,
V.lamda. genes and J.lamda. genes are chosen to reflect a specific
allele such as the most prevalent allele in human populations. In
yet another aspect, the individual codons of the V.lamda. gene are
codon-optimized for efficient expression in a specific non-human
mammal. In another aspect the individual codons are optimized to be
a template for somatic hypermutation.
Production of Chimeric Cells and Animals
[0097] Specific embodiments of the invention provide methods of
producing the animals and cells. In antibody producing mammals, for
example, the endogenous Ig V, (D) and J genes are replaced by
non-endogenous (e.g., human) Ig gene segments. In certain
embodiments, the endogenous immunoglobulin (Ig) V, (D) and J genes
are directly replaced by non-endogenous orthologs. In other
embodiments, the endogenous genes are functionally replaced by
non-endogenous orthologs while the endogenous genes are inactivated
using various techniques as described herein and known in the
art.
[0098] For example, one or more constructs carrying large portions
of the non-endogenous V, D and J genes can replace all or a portion
of the endogenous V, D and J genes. In certain embodiments, this
can be done by homologously recombining the constructs into or
adjacent to each Ig locus. Accordingly, the constructs can replace
the endogenous sequences by sequential ("walking") replacement or
by introducing two constructs into or adjacent to the endogenous Ig
locus and subsequently removing intervening sequences.
[0099] An exemplary method of producing a cell having a genome that
comprises a chimeric immunoglobulin heavy chain, wherein the heavy
chain comprises a non-endogenous variable domain and a chimeric
constant region, comprises the steps of (1) producing a first DNA
construct, wherein the first construct comprises one or more
non-endogenous VH, DH and/or JH gene segments, a first and a second
flanking region, wherein the first flanking region is homologous to
a DNA sequence 5' of the endogenous immunoglobulin heavy chain
locus, and a first site specific recombination recognition sequence
near the 3' end of the first construct; (2) producing a second DNA
construct, wherein the second construct comprises one or more
non-endogenous constant region gene segments, a third and a fourth
flanking region, wherein the fourth flanking region is homologous
to a DNA sequence 3' of the endogenous immunoglobulin heavy chain
locus, and a second site specific recombination recognition
sequence near the 5' end of the second construct; (3) homologously
recombining the first and second constructs into the genome of a
cell; and (4) introducing a site-specific recombinase into the
cell, thereby removing an intervening sequence between the first
and second site-specific recombinase recognition sequences.
[0100] Alternatively, the constructs can be introduced into
non-human animal cells by transfection into cells in tissue culture
or by pro-nuclear microinjection into fertilized eggs, and the
non-endogenous sequences are randomly integrated into the genome. A
separate functional inactivation (i.e., "knock-out") of the
endogenous locus can be performed by gene targeting in mammalian
cells in culture using the methods known in the art or described
herein or by other methods such as the use of engineered
zinc-finger nucleases or meganucleases.
[0101] A construct carrying all or part of the IgH locus downstream
of JH can be engineered so that in each constant region gene, the
endogenous CH1 domain is replaced with a non-endogenous CH1 domain.
This can be accomplished by techniques known in the art, such as
recombination of BACs in E. coli or YACs in S. cerevisiae. Such
replacement can also be accomplished using sequential homologous
recombination driven knock-in replacement of the endogenous CH1
domain by the non-endogenous CH1 domain. Selectable markers used
for the selecting recombinants can be flanked by site-specific
recombinase recognition sequences, e.g. loxP sites and deleted via
subsequent exposure to the site-specific recombines, e.g. CRE.
Using different variant loxP sites to flank the selectable marker
at each step restricts the CRE-mediated deletion to only the
sequence between the specific loxP site and prevents longer-range
deletion to an already existing loxP site. Alternatively, a
construct carrying all or part of the IgH locus downstream of JH
can be engineered so that in each constant region gene, the
endogenous CH1 domain is replaced with a non-endogenous CH1 domain,
using the ability to precisely synthesize and assemble DNAs based
on published genome sequences of organisms such as humans and mice.
Such synthesis and assembly is known in the art and is practiced by
commercial entities (e.g., DNA2.0, Menlo Park, Calif.; Blue Heron
Biotechnology, Bothell, Wash.).
[0102] According to one method of producing a cell comprising a
chimeric heavy chain as described herein, a construct comprising
the endogenous IgH loci downstream of the J gene cluster, wherein
each retained C gene comprises a non-endogenous CH1-endogenous
CH2-CH3 (and CH4 for C.mu.), and membrane and intracellular domain
exons is generated and introduced into the genome of a non-human
cell. In certain embodiments, the construct is homologously
recombined into or adjacent to the endogenous IgH locus. In other
embodiments, the construct is randomly integrated into the genome
of the cell. The construct may further comprise one or more
non-endogenous V gene segments. In an alternative embodiment, the
construct comprising the constant region gene segments is
introduced into the genome of the cell either as a first
introduction step to be followed by replacement of the endogenous
V-D-J genes with non-endogenous V gene segments or in the opposite
order, i.e., introduction of non-endogenous V gene segments
followed by the introduction of the construct comprising the
constant region gene segments engineered as described herein.
[0103] When using more than one construct to introduce
non-endogenous Ig gene segments, the content of the Ig locus is not
restricted to only constant region gene segments on one construct
and variable region gene segments on the other. For example, a
construct comprising C gene segments may also comprise one or more
J gene segments, D gene segments and/or V gene segments. Similarly,
a construct comprising V gene segments may further comprise one or
more of D gene segments, J gene segments and/or C gene
segments.
[0104] Constructs carrying the constant region genes may be
engineered in vitro, in E. coli or S. cerevisiae or synthesized in
vitro prior to introduction into ES cells so as to delete any
unwanted or unneeded gene segments, such as the C.epsilon. and
C.alpha. genes. This would constrain the animals to making C.mu.
and C.delta. for primary immune responses and C.gamma. isotypes for
secondary, affinity-matured immune responses, from which
therapeutic antibody candidates would typically be recovered.
[0105] In addition, constructs include both coding and non-coding
polynucleotide sequences of which the non-coding polynucleotide
sequences may be either non-endogenous or syngeneic polynucleotide
sequences. For example, the endogenous (i.e., syngeneic) IgH 3'
locus control region (LCR), or a portion thereof, are included
downstream of the most 3' CH gene, E.mu., or a portion thereof is
included between the most 3' JH and C.mu., and all or a portion of
the S.mu. and S.gamma. regions, promoters upstream of gene segments
such as V gene segments and CH switch regions and recombination
signal sequences (RSS). In addition, it is advantageous to include
other intergenic regions that have been hypothesized to have gene
regulation function such as the intergenic region between the most
3' VH gene the start of the D cluster and the intergenic region
between C.delta. and the first Cg switch region. Corresponding
elements exist in the Ig light chain loci with documented function
and location, e.g., E.kappa., Ig.kappa. 3' LCR and Ed. Because the
endogenous mouse Ig light chain locus possesses defective 3' LCRs,
it is advantageous to use an orthologous functional Ig.kappa. 3'
LCR from another species, e.g., human, rodent other than mouse, or
to mutate the mouse 3' LCR to restore NF.kappa.b binding.
[0106] Similar strategies are employed for the endogenous Ig.kappa.
locus except that a complete non-endogenous C.kappa. gene can be
incorporated in the construct, thus producing fully non-endogenous
Ig.kappa. chains. A non-endogenous C locus could also be
incorporated in a similar manner. For example, a construct
comprising human V.kappa. and C.kappa. gene segments can be
generated that encodes a fully human Ig.kappa. chain. Similarly, a
construct comprising human V.lamda. and C gene segments can be
generated that encodes a fully human Ig.lamda. chain.
[0107] Yet another aspect of the invention comprises incorporating
fully human Ig loci, including human C regions, in place of the
complete endogenous Ig loci. In an additional embodiment, a cluster
of endogenous FcR genes is also replaced with an orthologous
cluster of human FcR genes using similar BAC-based genetic
engineering in homologous recombination competent cells, such as
mouse ES cells. The cluster of endogenous Fc.gamma.R genes can be
directly replaced in the same ES cell in which the human IgH locus
or portions thereof have replaced the endogenous locus or in a
separate ES cell. Alternatively, the cluster of endogenous
Fc.gamma.R genes can be functionally replaced in the same ES cell
in which the human IgH locus or portions thereof have replaced the
endogenous locus or in a separate ES cell. In the latter instance,
mice would be derived from said ES cells and bred with mice
carrying the engineered Ig locus (loci) so as to produce mice that
make human IgG antibodies that bind to human Fc.gamma.R in place of
mouse Fc.gamma.R genes. In either way fully human antibodies would
be produced and during an immune response would be able to engage
the human FcR receptors normally. Such transgenic animals would
also have the benefit of being useful for testing for the activity
and effector function of human therapeutic mAb candidates in models
of disease when bred onto the appropriate genetic background for
the model, i.e., SCID, nu/nu, nod, and lpr mice. Further, the human
target gene sequence can replace the endogenous gene using BAC
targeting technology in homologous recombination-competent cells,
providing models for target validation and functional testing of
the antibody. In this instance, the human CH genes may be
engineered to have cytoplasmic and/or membrane domain gene segments
from mouse or other orthologous species to facilitate native signal
transduction in the B cell. Alternatively to replacing the entire
endogenous Fc.gamma.R locus with the complete complement of
wild-type genes in the human Fc.gamma.R locus, certain Fc.gamma.R
could be mutated to have attenuated function or deleted entirely.
For example, mutation to render the inhibitory human Fc.gamma.R2b
inactive and having simultaneous inactivation of the mouse
orthologue would render the genetically engineered mouse carrying
both mutations more susceptible to developing autoreactive B cells,
with a consequent potential benefit of broadening the fully human
antibody response against antigens.
[0108] In addition, another aspect of the invention relates to the
design of the desired non-endogenous V region (e.g., human). In
particular, an entire V domain repertoire, or a portion thereof,
may be incorporated into the genome of the cell, or a tailored V
domain repertoire may be incorporated. For example, in certain
embodiments it is preferred to omit V domain gene segments that are
missing from some human haplotypes and instead tailoring the V
domain repertoire to be composed of only the functional V gene
segments common across all known human haplotypes. Doing so
provides antibody drug candidates with V domains that are better
immune tolerized across all potential patients, thereby preventing
the induction of a dangerous immune response upon administration of
the encoded antibody to a subject. One or more V domain gene
segments may be incorporated into the genome of a cell.
[0109] In certain embodiments of the invention, constructs
containing the desired Ig loci gene segments are used to
incorporate the genetic information into the target cell genome via
homologous recombination. In particular, the nature of BAC
engineering in E. coli provides additional opportunities to finely
tailor the immunoglobulin loci prior to introduction into competent
cells. BAC libraries and the complete sequence of the Ig loci are
available for many species. Synthetic constructs can also be finely
tailored as described herein.
[0110] The ability to finely tailor the constructs described herein
provides the ability to introduce specific non-endogenous and
syngeneic components. For example, the non-endogenous DH cluster
can be replaced or supplemented with D genes from other species,
such as from non-human primate, rabbit, rat, camelid, hamster etc.
D gene segments within the IgH loci can be defined from publicly
available sequence or genetic structure information, or by testing
using appropriate D specific probes or primers. The orthologous D
gene clusters or portions thereof can be homologously recombined
into the constructs or assembled in silico and then synthesized,
therein replacing or adding to the cluster of non-endogenous D gene
segments.
[0111] Because of the significant diversification that occurs in
making the complementarity determining region-3 (CDR3) and because
the structure of the V region is such that the CDR3 is relatively
solvent inaccessible, immunogenicity to the CDR3 sequence is of
less concern. Therefore, amino acids encoded by non-human D genes
incorporated into the CDR3 are less likely to be immunogenic upon
administration to a human. D genes derived from another species
could confer an advantage by producing novel CDR3 structures that
would expand the range of epitope specificities and affinities in a
panel of antigen-specific antibodies, therein broadening the
quality of activities mediated by a panel of mAbs.
[0112] Similarly, the JH gene cluster, i.e., one or more JH gene
segments, can be from a different non-endogenous species due to the
relative sequence conservation across mammals. The JH gene segment
may be derived from any animal, e.g., human, non-human primate,
rabbit, sheep, rat, hamster, camelid and mouse. In particular
embodiments, the JH gene segment is human.
[0113] In further aspects, after engineering the Ig loci
constructs, they are introduced into non-human mammalian cells and
are randomly integrated into the genome. Methods for introducing
one or more constructs comprising the altered Ig locus, or portion
thereof, include, for example, electroporation, lipofection,
calcium phosphate precipitation, E. coli spheroplast fusion, yeast
spheroplast fusion and microinjection, either into the pronucleus
of a fertilized egg to make transgenic animals directly or into
cells cultured in vitro. In certain embodiments, the construct is
engineered to carry a selectable marker gene, e.g., G418.sup.R,
hygromycin.sup.R, puromycin.sup.R, 5' of the most 5'V gene. A
selectable marker gene may also be 3' of the most 5'V gene. The
selectable marker gene may be flanked by site-specific recombinase
recognition sequences, which if brought into the presence of
recombinase, will recombine and delete the intervening selectable
marker. This is particularly important if a selectable marker
cassette is located 3' of the most 5' V gene and near an enhancer
sequence so as to not attenuate the function of the enhancer.
[0114] In certain embodiments, two or more constructs, such as
BACs, are introduced into the cell in a single step. If two
constructs are introduced into the cell simultaneously, they will
typically co-integrate. Some of the co-integrated constructs will
integrate in a functional head-to-tail fashion with, for example,
V, (D) and J segments operably oriented 5' of C region gene
segments. Co-introduced constructs can be any combination of BACs,
YACs, plasmids, bacteriophage, P1s etc. In some instance, there
will be a single-copy integration of the two constructs, creating a
single-copy transgene. In other instances, there will be a
multi-copy integration of the two constructs. Multi-copy
integration is not necessarily undesirable as it can yield
beneficial consequences, such as increased expression of the
transgene, resulting in more of the desired gene product. However,
if a single copy of the transgene is desired, there are in vitro
and in vivo processes for doing so.
[0115] For instance, if a site-specific recombinase sequence is
positioned at the 3' end of the 5' construct and if a site-specific
recombinase sequence is positioned at the 5' end of the 3'
construct, resulting co-integrants of the 5' construct and the 3'
construct both oriented in the 5' to 3' manner will have
site-specific recombinase sequences oriented so that any
intervening sequence between the terminal 5' construct and the
terminal 3' construct would be deleted upon exposure to the
site-specific recombinase, and thus the terminal 5' construct
becomes operably linked to the terminal 3' construct, resulting in
a single copy transgene. This process may be conducted either in
vitro in culture mammalian cells or in vivo in transgenic animals
expressing the recombinase (for example of resolving a multi-copy
single construct transgene into a single-copy transgene in vivo,
see Janssens et al. Proc. Natl. Acad. Sci. (2006)103: 15130-15135.)
Functional transgenes can also be made by pronuclear
co-microinjection of 3 or more constructs (see US Patent
Application Publication No. 2010/0077497.)
[0116] After introducing the Ig locus or loci described herein into
the genome of a cell to replace (e.g., functionally replace) an
endogenous Ig locus, or portion thereof, a non-human animal can be
produced. If the non-human mammalian cells are embryonic stem
cells, genetically engineered non-human mammals, such as mice and
rats, can be produced from the cells by methods such as blastocyst
microinjection followed by breeding of chimeric animals, morula
aggregation. If the cells are somatic cells, cloning methodologies,
such as somatic cell nuclear transfer, can be used to produce a
transgenic animal. Multi-stage breeding is used to produce animals
heterozygous or hemizygous for modified IgH and IgL loci (either
Ig.kappa. or Ig.lamda., or both Ig.kappa. and Ig.lamda.). Mice with
modified IgH and IgL loci can be further bred to produce mice
homozygous for IgH and lgL (either Ig.kappa. or Ig.lamda., or both
Ig.kappa. and Ig.lamda.).
[0117] The engineered Ig loci described herein will function in the
non-human animals. By using appropriate detection reagents, e.g.,
anti-human CH1 domain antibodies or anti-human CL antibodies, it is
possible to detect the antibodies produced by the engineered locus
even in the presence of antibodies expressed from an active
endogenous locus. Furthermore, in a mouse, for example, it is
possible to use allotypic sequences in the mouse portion of the
constant region of the transgene that are different from the
allotypic sequence of the constant region of the recipient mouse
strain, e.g., mouse IgH a allotypes (Balb/c) versus IgH b allotypes
(C57BL/6).
[0118] Inactivation of Endogenous Ig Loci
[0119] In certain embodiments, it may be desirable to functionally
inactivate one or more of the endogenous Ig loci in the recipient
non-human mammal. Various methods known in the art can be used to
inactivate the endogenous Ig loci. An animal comprising an
engineered Ig transgene is bred with an animal comprising one or
more inactivated endogenous loci to derive an animal capable of
expressing antibodies from the Ig transgene and without production
of the complete native immunoglobulin from the inactivated
endogenous loci with the Ig transgene therein functionally
replacing the inactivated endogenous locus.
[0120] The very fine tailoring of DNA sequences by combining in
silico recombination with in vitro DNA synthesis and assembly
technologies allows for the precise deletion and/or modification of
the homologous target sequences.
[0121] For instance, recombination signal sequences or splice donor
sequences for specific gene segments, e.g., J gene, may be altered
or deleted.
[0122] Components of an IgH locus that may be altered to down
modulate and/or abrogate locus function include the JH cluster
(complete deletion, removal of recombination signal sequences
(RSS), splice donor sequences or all or some of the above) (see,
for example, U.S. Pat. No. 5,939,598), E.mu., C.mu. and C.delta.,
the D gene cluster (complete deletion, removal of recombination
signal sequences (RSS), splice donor sequences or all or some of
the above), the VH genes (complete deletion, removal of
recombination signal sequences (RSS), splice donor sequences or all
or some of the above), and all of the constant region genes.
Placing a strong constitutive promoter such as PGK in the position
of critical enhancer elements such as E can have severe deleterious
consequences on locus function, effectively bringing about
inactivation.
[0123] Components of an Ig.kappa. locus that may be altered to down
modulate and/or abrogate locus function include the J cluster
(complete deletion, removal of recombination signal sequences
(RSS), splice donor sequences or all or some of the above),
E.kappa., C.kappa., and the V.kappa. genes (complete deletion,
removal of recombination signal sequences (RSS), splice donor
sequences or all or some of the above).
[0124] Components of the Ig.lamda. locus that may be altered to
down modulate and/or abrogate locus function include the J.lamda.
cluster (complete deletion, removal of recombination signal
sequences (RSS), splice donor sequences or all or some of the
above), E.lamda., C.lamda., and the V.lamda. genes (complete
deletion, removal of recombination signal sequences (RSS), splice
donor sequences or all or some of the above). Deletion of larger
sequence units such as the entire Ig.lamda. locus, the entire VH
gene repertoire of the IgH locus etc. may be effected by serial
insertion of site-specific recombination sequences (lox P or frt)
adjacent to the 5' and 3' ends of the sequence to be deleted
followed by transient expression of the relevant recombinase, e.g.,
CRE or FLP. Various methods known in the art can be used to
inactivate the endogenous Ig loci. See for example: Chen J., et al.
Int Immunol. 1993 June; 5(6):647-56; Jakobovits et al., Proc. Natl.
Acad. Sci. (1993) 90: 2551-2555; Nitschke et al. Proc. Natl. Acad.
Sci. (1993) 90: 1887-1891; U.S. Pat. No. 5,591,669; Afshar et al.,
J. Immunol. (2006) 176: 2439-2447; Perlot et al. Proc. Nat. Acad.
Sci. (2005) 97: 14362-14367; Roes and Rajewsky J. Exp Med. (1993)
177: 45-55; Lutz et al. Nature (1998) 393: 797-801; Ren et al.
Genomics (2004) 84: 686-695; Zou et al. EMBO J. (1993) 12: 811-820;
Takeda et al. EMBO J. (1993) 12: 2329-2336; Chen et al. EMBO J.
(1993) 12: 821-830; Zou et al., J. Immunol. (2003) 170: 1354-1361;
Zheng et al. Molec. Cell. Bio. (2000) 20: 648-655; Zhu et al. Proc.
Nat. Acad. Sci. (2000) 97: 1137-1142; Puech et al. Proc. Nat. Acad.
Sci. (2000) 97: 10090-10095; LePage et al. Proc. Natl. Acad. Sci.
(2000) 97: 10471-10476; Li et al. Proc. Nat. Acad. Sci. (1996) 93:
6158-6162.
[0125] In some embodiments, multiple deletions or multiple
mutations are introduced into an endogenous Ig locus to inactivate
the endogenous immunoglobulin locus, thereby solving the problem of
partially inactivating an endogenous Ig locus. In particular, the
two or more mutations independently impair both the formation of a
functional variable domain and the formation of a constant region
capable of driving primary B cell development. The mutations impair
primary B cell development because the resulting Ig sequence
prevent formation of an IgH capable of mediating signal
transduction (by itself or in association with Ig.alpha. and/or
Ig.beta.), e.g., gene rearrangement is blocked, transcription or
translation of a complete product fails, or the product cannot
signal.)
[0126] In one instance, the endogenous J genes, E.mu., C.mu. and
C.delta. are deleted. In another instance, the endogenous J genes,
E.mu., C.mu. and C.delta. are all replaced with a single
drug-resistance cassette that is transcriptionally active in ES
cells and B cells. An example of a drug-resistance cassette for use
in mice is the PGK-G418 neomycin resistance cassette comprising the
mouse pgk-1 promoter. Taken together, this deletion blocks V-D-J
recombination (J deletion and E replacement by an active expression
cassette) and primary B cell receptor signaling (deletion of C.mu.
and C.delta.) and anchoring in the B cell membrane. The
inactivation of multiple components produces multiple layers of
redundancy for inactivating the IgH locus at different
developmental stages, creating a failsafe against any one residual
activity rescuing B cell development.
[0127] Other combinations of deletions or mutations can also be
performed. For example, the entire C gene cluster may be deleted in
combination with a JH deletion. Deletion of the entire D gene
cluster in combination with C.mu. and C.delta. would also be
effective. Any combination of one or more mutations are
contemplated herein as long as the resulting mutations impair
formation of a functional variable region and formation of a
membrane-anchored heavy chain constant region capable of signal
transduction, either directly or in combination with the accessory
signal transducing proteins Ig.alpha. and/or Ig.beta..
[0128] Not all of each module needs to be deleted. For instance, a
portion of J, C.mu. or C.delta. genes may be left in the
immunoglobulin locus so long as one or more cis regulatory elements
such as recombination signal sequences (RSS), splice donor, and
splice acceptor sequences are deleted or mutated or the formation
of a functional open reading frame is obviated. Current
methodologies for mutating or synthesizing precise DNA sequences
enable the creation of very specific, even single nucleotide,
mutations to be introduced. This provides the benefit of allowing
for optimal positioning of the DNA arms driving homologous
recombination in ES cells while still inactivating the locus.
[0129] Deletion of portions of the IgH locus can be made in cells
using homologous recombination techniques that are now standard for
genetic engineering. Deletions may be made in one step or in
multiple steps, and they may be generated using one or more
constructs. The deletions could also be made using site-specific
recombinase systems such as Cre-lox or Flp-Frt. A combination of
homologous recombination and site-specific recombinase systems may
be used. Other systems such as engineered zinc-finger nucleases
injected into fertilized eggs may be used to engineer deletions
into the genes, in one or more steps to build up the number of
deleted or mutated modules of the IgH locus.
[0130] A similar strategy may be employed for inactivating the
endogenous immunoglobulin kappa light chain and/or lambda light
chain. The important modules for inactivation are conserved,
particularly the J, E (intronic enhancer) and C regions, between
all of the Ig loci. In embodiments regarding the inactivation of an
Ig light chain locus, inactivation of the constant region will
prevent the formation of a complete antibody molecule or a Fab
domain in that the light chain constant region is unable to form a
disulfide bond with the heavy chain.
[0131] For example, an endogenous Ig.kappa. locus can be
inactivated by replacing the J genes, E.kappa., and C.kappa. with a
single drug-resistance cassette that is transcriptionally active in
ES cells and B cells, such as the PGK-G418 neomycin resistance
cassette. Taken together, this deletion blocks V-J recombination (J
deletion and E.kappa. replacement by an active expression cassette)
and pairing of an Ig.kappa. light chain with a heavy chain in an
antibody (deletion of C.kappa.). Any combination of one or more
mutations are contemplated herein as long as the resulting
mutations impair formation of a functional variable region and
formation of a light chain constant region capable of forming a
disulfide bond with a heavy chain.
[0132] In another embodiment, all of the J-C pairs of the Ig, locus
3' of the V.lamda. gene segment can be deleted using a
site-specific recombinase system, such as Cre/loxP. The deletion of
all of the J segment genes prevents V-J rearrangement, and
therefore impairs the formation of a functional variable region.
The deletion of the C gene segments prevents the formation of a
functional constant region, thereby preventing the formation of a
constant region capable of forming a disulfide bond with any IgH
chain. In yet another embodiment, inactivation of the mouse
Ig.lamda. is achieved through two separate inactivations. The first
is inactivation is of V.lamda.1 and the second is inactivation is
of both V.lamda.2 and V.lamda.x. The second inactivation may be
done before the first inactivation. Inactivation may be achieved
through inactivation of RSS 5' or 3' of each of the V.lamda. genes,
inactivation of the promoters 5' of each gene, or inactivation of
the coding sequences. The inactivation may be through mutation,
either point, insertion or deletion, to render non-functional, or
complete deletion.
[0133] The endogenous Ig locus of a non-human cell may be
inactivated by homologous recombination with one or more constructs
designed to introduce the deletions or mutations capable of
impairing both the formation of a functional variable domain and
the formation of a constant region capable of driving primary B
cell development. Methods for effecting homologous recombination in
mouse and rat ES cells are known in the art. Upon homologous
recombination between the flanking regions located on the construct
and the corresponding homologous endogenous DNA sequences in the
cell, the desired deletions or mutations are incorporated into the
endogenous Ig locus.
[0134] Cells that have undergone a correct recombination event can
be screened for using positive and negative selection markers, such
as drug resistance. To further confirm homologous recombination,
genomic DNA is recovered from isolated clones and restriction
fragment length polymorphism (RFLP) analysis performed by a
technique such as Southern blotting with a DNA probe from the
endogenous loci, said probe mapping outside the replaced region.
RFLP analysis shows allelic differences between the two alleles,
the endogenous DNA and incoming DNA, when the homologous
recombination occurs via introduction of a novel restriction site
in the replacing DNA.
[0135] Various assays known in the art, including, but not limited
to, ELISA and fluorescence microscopy, may be used to confirm that
the mutations introduced into the endogenous Ig locus impair the
expression of a functional Ig heavy or light chain by the cell. An
absence of the endogenous Ig heavy or light chain indicates that
its expression is impaired. Other well known assays, such as
RT-PCR, can determine whether or not the modified locus is able to
be transcribed.
[0136] Cells having one or more inactivated Ig loci may be used to
produce transgenic non-human animals, e.g., mice, that have one or
more inactivated Ig loci. After engineering the mutated Ig locus
into non-human cells to delete or replace portions of the
endogenous Ig loci, genetically engineered non-human mammals, such
as mice, can be produced by now-standard methods such as blastocyst
microinjection followed by breeding of chimeric animals, morula
aggregation or cloning methodologies, such as somatic cell nuclear
transfer.
[0137] Breeding Strategies
[0138] Certain embodiments provide a method of producing a
non-human mammal having a genome encoding non-endogenous VH and CH1
gene segments and a non-endogenous Ig light chain locus comprising
the steps of breeding a non-human mammal comprising a chimeric Ig
heavy chain locus, wherein the Ig heavy chain locus comprises the
non-endogenous VH and CH1 gene segments, with a non-human mammal
comprising a non-endogenous Ig light chain locus; selecting
offspring having a genome comprising the chimeric Ig heavy chain
locus and the non-endogenous Ig light chain locus; further breeding
the offspring; and producing offspring having a genome homozygous
for the chimeric heavy and non-endogenous light chain loci. In
related embodiments, the genome of the mammal also encodes a
non-endogenous JH gene segment.
[0139] Further embodiments comprise selecting offspring having a
genome comprising the chimeric Ig heavy chain locus and the
non-endogenous Ig light chain locus; further breeding the offspring
with non-human mammals having functionally inactivated endogenous
Ig loci; and producing offspring and further breeding to produce
offspring having a genome homozygous for functionally inactivated
endogenous Ig loci, the chimeric heavy and the non-endogenous light
chain loci. In related embodiments, the genome of the mammal also
encodes a non-endogenous JH gene segment.
[0140] The genetic engineering strategies described herein can be
applied to engineering of mice and other animals so as to express
non-endogenous sequence V regions coupled with xenogeneic C
regions, or completely non-endogenous antibodies, or some
intermediate thereof. For animals for which there is a current lack
of ES cell technology for genetic engineering through blastocyst
microinjection or morula aggregation, the endogenous loci can be
modified in cells amenable to various cloning technologies or
developmental reprogramming (e.g., induced pluripotent stem cells,
IPS). The increased frequency of homologous recombination provided
by the BAC technology provides the ability to find doubly replaced
loci in the cells, and cloned animals derived therefrom would be
homozygous for the mutation, therein saving time and costs
especially when breeding large animals with long generation times.
Iterative replacements in the cultured cells could provide all the
requisite engineering at multiple loci and then direct production
of animals using cloning or IPS technology, without cross-breeding,
to produce the appropriate genotype. The ability to finely tailor
the introduced Ig genes and also finely specify the sites into
which they are introduced provides the ability to engineer
enhancements that provide better function. Engineered animals such
as goats, bovines, ovines, equines, rabbits, llamas, dogs etc. are
a source of fully human polyclonal antibodies.
[0141] Furthermore, if BACs are engineered in E. coli with DNA
components required for chromosome function, e.g., telomeres and a
centromere, preferably, but not required, of the recipient species
for optimal function, e.g., mouse telomeres and a mouse centromere,
they can be introduced into the recipient cell by electroporation,
microinjection etc. and function as artificial chromosomes. These
BAC-based artificial chromosomes also can be used as a foundation
for subsequent rounds of homologous recombination for building up
larger artificial chromosomes.
[0142] The engineered Ig locus or loci described herein provided on
vectors such as plasmids, BACs or YACs can also be used as standard
transgenes introduced via microinjection into the pronucleus of an
embryo such as mouse, rabbit, rat, or hamster. Several BACs, YACs,
plasmids or any combination thereof can be co-microinjected and
will co-integrate to make a functional locus. Various methods known
in the art and described herein can be used to inactivate the
endogenous Ig loci and the animals with an engineered Ig transgene
bred with those with one or more inactivated endogenous loci to
derive genotypes expressing antibodies from the transgene and
without production of the complete native immunoglobulin from the
inactivated endogenous loci.
Antibodies
[0143] A chimeric antibody, or antigen-binding fragment thereof, as
disclosed herein comprises a non-endogenous variable domain and a
chimeric heavy chain constant region. In particular, the chimeric
heavy chain constant region comprises a non-endogenous CH1 domain.
In certain embodiments, the chimeric antibody comprises a chimeric
heavy chain and a non-endogenous light chain. In other embodiments,
the chimeric antibody comprises a chimeric heavy chain and an
endogenous light chain. In one embodiment, the chimeric heavy chain
variable region is encoded by polynucleotide sequences derived from
two or more non-endogenous species.
[0144] In certain embodiments, the chimeric heavy chain comprises a
non-endogenous upper hinge region. In a related embodiment, the
chimeric heavy chain comprises non-endogenous upper and middle
hinge regions.
Eukaryotic Transgenes Comprising Sequences Designed in Silico and
Made Synthetically
[0145] The ability to obtain sequences, for genes, loci and full
genomes, and transcriptome sequences, either from public databases
with annotations, or derived using commercially available
sequencing technology, or derived through commercial operation
performing sequencing on a contractual basis, means that DNA
sequences can be readily manipulated in silico, e.g., taken apart
and reassembled, either within genes or loci, or between genes or
loci, across the same species, different strains of a species, or
across two or more different species. Heretofore eukaryotic
transgenes, particularly metazoans, have been constructed from DNAs
derived from a natural source. These natural source DNAs include
genomic DNA libraries cloned into various vectors such plasmid,
bacteriophage, P1s, cosmids, BACs, YACs and MACs and cDNA libraries
cloned into vectors, generally plasmids or bacteriophage.
Methodologies for recombining DNAs carried on these vectors and for
introducing small alterations such as site-directed mutations are
well known in the art and have been deployed to make transgenes
composed of sequence that overall conforms to the sequence of the
parental DNA in the library from which they were isolated. In some
instances, portions of DNA are missing from the library, or a
library from the desired animal, strain or haplotype thereof may be
unavailable and not able to be constructed using ordinary skill in
the art.
[0146] For example, there may be preferred allelic variants to be
included in a transgene and said allelic variant is not available
in any library and, furthermore, source nucleic acid such as RNA or
DNA may not be available. In complex loci with many genes or exons
and cis regulatory elements, it can be technically infeasible to
procure and recombine into one transgenes the DNAs encoding such if
they are from different species and strains or haplotypes. Thus,
the means by which to create DNA of complexly engineered,
particularly from a completely in silico design, is not possible
using standard methodologies.
[0147] Synthetic means of creating DNA sequences have been
described, are commercially available and can be used to make DNA
sequences based on a completely in silico design. However, whether
they can be introduced and, in particular, expressed in eukaryotes,
particularly metazoans, has heretofore been unknown.
[0148] Some of the Ig transgene constructs disclosed herein
comprise such complex sequence composition that they cannot be
engineered to the desired precision and accuracy by previously
described means. Further contemplated transgene structures include
a germline configured DNA in which are replaced only coding
sequences, all or a part thereof, by non-endogenous coding
sequences so that all of the cis regulatory sequences are
endogenous, retaining completely native gene regulation optimal
for, position-independent, copy-number dependent, tissue-specific,
developmental-specific gene regulation, e.g, an IgH sequence which
comprises completely mouse DNA except for sequences encoding human
VH, DH, JH, CH1 and upper hinge sequences replacing their mouse
orthologues; an Fc.gamma.R sequence which comprises completely
mouse DNA except for sequences encoding human Fc.gamma.R replacing
their mouse orthologues; an IgH sequence which comprises completely
mouse DNA except for sequences encoding camelid VH, DH, JH,
sequences replacing their mouse orthologues; an IgH sequence which
comprises completely mouse DNA except for sequences encoding human
VH, JH, CH1 and upper hinge sequences replacing their mouse
orthologues and non-human, non-mouse DH coding sequences, e.g.,
rabbit, replacing their mouse orthologues; an IgH sequence which
comprises completely mouse DNA except for sequences encoding VH,
DH, JH, from a species relevant to animal healthcare, e.g., canine,
feline, ovine, bovine, porcine, replacing their mouse orthologues;
an IgL sequence which comprises completely mouse DNA except for
sequences encoding human VL, JL, and optionally CL, replacing their
mouse orthologues; an IgL sequence which comprises completely mouse
DNA except for sequences encoding camelid VL, JL, and optionally
CL, replacing their mouse orthologues; an IgL sequence which
comprises completely mouse DNA except for sequences encoding VL,
JL, and optionally CL from a species relevant to animal healthcare,
e.g., canine, feline, ovine, bovine, porcine, replacing their mouse
orthologues. Other examples include deleting unneeded or
undesirable DNA sequences, e.g., V genes that are pseudogenes, V
genes that produce products that can misfold, V genes that are
absent from some human haplotypes, large tracts of non-regulatory
DNA, CH genes not therapeutically important. Other examples include
altering DNA sequences for optimizing transgene function or
producing a desired product therefrom, e.g., using the most
prevalent allele of a V gene, repairing the mouse Ig.lamda. 3'
enhancer to restore NF.kappa.b binding. Transgenes may comprise
parts that are synthetic and parts that are from natural sources.
DNAs for inactivating genes may also comprise synthetic DNA, all or
in part. Moreover, the method for transgene construction described
herein is not limited to immunoglobulin loci. Any transgene can be
constructed by the steps of first using in silico methods to
recombining and assemble the sequence from various DNA sequence and
second of employing available synthetic DNA methods to create the
physical DNA.
[0149] Methods of Producing Antibodies
[0150] An animal carrying the modified locus or loci can be
immunized with an antigen using various techniques available in the
art. Antigens may be selected for the treatment or prevention of a
particular disease or disorder, such as various types of cancer,
graft versus host disease, cardiovascular disease and associated
disorders, neurological diseases and disorders, autoimmune and
inflammatory disorders, and pathogenic infections. In other
embodiments, target antigens may be selected to develop an antibody
that would be useful as a diagnostic agent for the detection one of
the above diseases or disorders.
[0151] Antigen-specific repertoires can be recovered from immunized
animals by hybridoma technology, single-cell RT-PCR for selected B
cells, by antibody display technologies, and other methods known in
the art. For example, to recover human/mouse chimeric mAbs from
mouse-derived hybridomas, a human V-CH1-mouse hinge+CH2+CH3
antibody or a human V-CH1-upper/middle hinge-mouse lower
hinge+CH2+CH3 antibody (depending upon the IgH locus engineering)
is secreted into the culture supernatant and can be purified by
means known in the art such as column chromatography using protein
A, protein G, etc. The purified antibody can be used for further
testing and characterization of the antibody to determine potency
in vitro and in vivo, affinity etc.
[0152] In addition, since they can be detected with an antibody
specific for the endogenous constant region used as a secondary
agents, a human V-CH1 (upper/middle hinge)-non-human CH2-CH3 mAb
may be useful for immunochemistry assays of human tissues to assess
tissue distribution and expression of the target antigen. This
feature of the chimeric antibodies of the present invention allows
for specificity confirmation of the chimeric mAb over fully human
mAbs because of occasional challenges in using anti-human constant
region secondary detection agents against tissues that contain
normal human Ig and from the binding of human Fc regions to human
FcR expressed on cells in some tissues.
[0153] The non-endogenous variable regions of the mAbs can be
recovered and sequenced by standard methods. Either before or after
identifying lead candidate mAbs, the genes, either genomic DNA or
cDNAs, for the non-endogenous VH and VL domains can be recovered by
various molecular biology methods, such as RT-PCR, and then
appended to DNA encoding the remaining portion of the
non-endogenous constant region, thereby producing a fully
non-endogenous mAb. For example, a fully human mAb may be
generated. The DNAs encoding the now fully non-endogenous VH-CH and
non-endogenous VL-CL would be cloned into suitable expression
vectors known in the art or that can be custom-built and
transfected into mammalian cells, yeast cells such as Pichia, other
fungi etc. to secrete antibody into the culture supernatant. Other
methods of production such as ascites using hybridoma cells in
mice, transgenic animals that secrete the antibody into milk or
eggs, and transgenic plants that make antibody in the fruit, roots
or leaves can also be used for expression. The fully non-endogenous
recombinant antibody can be purified by various methods such as
column chromatography using protein A, protein G etc.
[0154] A purified antibody can be lyophilized for storage or
formulated into various solutions known in the art for solubility
and stability and consistent with safe administration into animals,
including humans. Purified recombinant antibody can be used for
further characterization using in vitro assays for efficacy,
affinity, specificity, etc., animal models for efficacy, toxicology
and pharmacokinetics etc. Further, purified antibody can be
administered to humans and non-human animals for clinical purposes
such as therapies and diagnostics for disease.
[0155] Various fragments of the non-endogenous V-CH1-(upper/middle
hinge)-endogenous CH2-CH3 mAbs can be isolated by methods including
enzymatic cleavage, recombinant technologies, etc. for various
purposes including reagents, diagnostics and therapeutics. The cDNA
for the repertoire of non-endogenous variable domains+CH1 or just
the non-endogenous variable domains can be isolated from the
engineered non-human mammals described above, specifically from RNA
from secondary lymphoid organs such as spleen and lymph nodes, and
the VH and VL cDNAs implemented into various antibody display
systems such as phage, ribosome, E. coli, yeast, mammalian etc. The
transgenic mammals may be immunologically naive or optimally may be
immunized against an antigen of choice. By using appropriate PCR
primers, such as 5' in the leader region or framework 1 of the
variable domain and 3' in the human CH1 of C.gamma. genes, the
somatically matured V regions can be recovered in order to display
solely the affinity-matured repertoire. The displayed antibodies
can be selected against the target antigen to efficiently recover
high-affinity antigen-specific Fv or Fabs, and are void of the
endogenous CH2-CH3 domains that would be present if mAbs were
recovered directly from the mammals. Moreover, it is not necessary
that the animals carrying the IgH and IgL transgene be functionally
inactivated for the endogenous Ig loci. Animals heterozygous for
IgH and IgL loci, or animals carrying the IgH and IgL transgenes
and heterozygous for inactivated endogenous IgH and IgL loci, which
produce the chimeric antibodies described herein as well as both
fully-endogenous antibodies (e.g., mouse antibodies) and mixed
endogenous and non-endogenous antibodies (e.g., human-mouse
antibodies), can also be used to generate antigen-specific
non-endogenous V-endogenous C mAbs (e.g., human V-mouse C mAbs).
Animals carrying just one Ig transgene, e.g., IgH, could be used as
a source of non-endogenous (e.g., human) VH domains (VH-CH1) and
other animals carrying just one different Ig transgene, e.g., IgL,
either Ig.kappa. or Ig.lamda., could be used as a source of
non-endogenous (e.g., human) VL domains (VL-CL) and then the VH-CH1
and VL-CL sequences combined into an antibody display library to
display fully human antibodies. In such animals, both, one or none
of the endogenous Ig loci may be activated. The animals may be
immunized so as to enable recovery of affinity-mature VH and VL.
For example, an antibody display library from two separate
mice--one with human VH-CH1-mouse CH2-CH3 and the other with human
V.kappa.-C.kappa.--could be used to recover fully human antibodies
using well-established techniques in molecular biology.
Methods of Use
[0156] Purified antibodies of the present invention may be
administered to a subject for the treatment or prevention of a
particular disease or disorder, such as various types of cancer,
graft versus host disease, cardiovascular disease and associated
disorders, neurological diseases and disorders, autoimmune and
inflammatory disorders, allergies, and pathogenic infections. In
preferred embodiments, the subject is human.
[0157] Antibody compositions are administered to subjects at
concentrations from about 0.1 to 100 mg/ml, preferably from about 1
to 10 mg/ml. An antibody composition may be administered topically,
intranasally, or via injection, e.g., intravenous, intraperitoneal,
intramuscular, intraocular, or subcutaneous. A preferred mode of
administration is injection. The administration may occur in a
single injection or an infusion over time, i.e., about 10 minutes
to 24 hours, preferably 30 minutes to about 6 hours. An effective
dosage may be administered one time or by a series of injections.
Repeat dosages may be administered twice a day, once a day, once a
week, bi-weekly, tri-weekly, once a month, or once every three
months, depending on the pharmacokinetics, pharmacodynamics and
clinical indications. Therapy may be continued for extended periods
of time, even in the absence of any symptoms.
[0158] A purified antibody composition may comprise polyclonal or
monoclonal antibodies. An antibody composition may contain
antibodies of multiple isotypes or antibodies of a single isotype.
An antibody composition may contain unmodified chimeric antibodies,
or the antibodies may have been modified in some way, e.g.,
chemically or enzymatically. An antibody composition may contain
unmodified human antibodies, or the human antibodies may have been
modified in some way, e.g., chemically or enzymatically. Thus an
antibody composition may contain intact Ig molecules or fragments
thereof, i.e., Fab, F(ab').sub.2, or Fc domains.
[0159] Administration of an antibody composition against an
infectious agent, alone or in combination with another therapeutic
agent, results in the elimination of the infectious agent from the
subject. The administration of an antibody composition reduces the
number of infectious organisms present in the subject 10 to 100
fold and preferably 1,000 fold, and more than 1,000 fold.
[0160] Similarly, administration of an antibody composition against
cancer cells, alone or in combination with another chemotherapeutic
agent, results in the elimination of cancer cells from the subject.
The administration of an antibody composition reduces the number of
cancer cells present in the subject 10 to 100 fold and preferably
1,000 fold, and more than 1,000 fold.
[0161] In certain aspects of the invention, an antibody may also be
utilized to bind and neutralize antigenic molecules, either soluble
or cell surface bound. Such neutralization may enhance clearance of
the antigenic molecule from circulation. Target antigenic molecules
for neutralization include, but are not limited to, toxins,
endocrine molecules, cytokines, chemokines, complement proteins,
bacteria, viruses, fungi, and parasites. Such an antibody may be
administered alone or in combination with other therapeutic agents
including other antibodies, other biological drugs, or chemical
agents.
[0162] It is also contemplated that an antibody of the present
invention may be used to enhance or inhibit cell surface receptor
signaling. An antibody specific for a cell surface receptor may be
utilized as a therapeutic agent or a research tool. Examples of
cell surface receptors include, but are not limited to, immune cell
receptors, adenosine receptors, adrenergic receptors, angiotensin
receptors, dopamine and serotonin receptors, chemokine receptors,
cytokine receptors, histamine receptors, etc. Such an antibody may
be administered alone or in combination with other therapeutic
agents including other antibodies, other biological drugs, or
chemical agents.
[0163] It is also contemplated that an antibody of the present
invention may be further modified to enhance therapeutic potential.
Modifications may include direct- and/or indirect-conjugation to
chemicals such as chemotherapeutic agents, radioisotopes, siRNAs,
double-stranded RNAs, etc. Other modifications may include Fc
regions engineered for either increased or decreased
antibody-dependent cellular cytotoxicity, either increased or
decreased complement-dependent cytotoxicity, or increased or
decreased circulating half-life.
[0164] In other embodiments, an antibody may be used as a
diagnostic agent for the detection one of the above diseases or
disorders. A chimeric antibody may be detected using a secondary
detection agent that recognizes a portion of the antibody, such as
an Fc or Fab domain. In the case of the constant region, the
portion recognized may be a CH1, CH2, or a CH3 domain. The C.kappa.
and C.lamda. domain may also be recognized for detection.
Immunohistochemical assays, such as evaluating tissue distribution
of the target antigen, may take advantage of the chimeric nature of
an antibody of the present invention. For example, when evaluating
a human tissue sample, the secondary detection agent reagent
recognizes the non-human portion of the Ig molecule, thereby
reducing background or non-specific binding to human Ig molecules
that may be present in the tissue sample.
Pharmaceutical Compositions and Kits
[0165] The present invention further relates to pharmaceutical
compositions and methods of use. The pharmaceutical compositions of
the present invention include an antibody, or an antigen-binding
fragment thereof, in a pharmaceutically acceptable carrier.
Pharmaceutical compositions may be administered in vivo for the
treatment or prevention of a disease or disorder. Furthermore,
pharmaceutical compositions comprising an antibody, or an
antigen-binding fragment thereof, of the present invention may
include one or more agents for use in combination, or may be
administered in conjunction with one or more agents.
[0166] The present invention also provides kits relating to any of
the antibodies, or antigen-binding fragments thereof, and/or
methods described herein. Kits of the present invention may be used
for diagnostic or treatment methods. A kit of the present invention
may further provide instructions for use of a composition or
antibody and packaging.
[0167] A kit of the present invention may include devices,
reagents, containers or other components. Furthermore, a kit of the
present invention may also require the use of an apparatus,
instrument or device, including a computer.
EXAMPLES
[0168] The following examples are provided as further illustrations
and not limitations of the present invention. The teachings of all
references, patents and published applications cited throughout
this application, as well as the Figures are hereby incorporated by
reference.
Example 1
Construction of BAC C5P12
[0169] A BAC vector is based on the F-factor found in E. coli. The
F-factor and the BAC vector derived from it are maintained as low
copy plasmids, generally found as one or two copies per cell
depending upon its life cycle. Both F-factor and BAC vector show
the fi+ phenotype, which excludes an additional copy of the plasmid
in the cell. By this mechanism, when E. coli already carries and
maintains one BAC, and then an additional BAC is introduced into
the E. coli, the cell maintains only one BAC, either the BAC
previously existing in the cell or the external BAC newly
introduced. This feature is extremely useful for selectively
isolating BACs homologously recombined as described below.
[0170] The homologous recombination in E. coli requires the
functional RecA gene product. In this example, the RecA gene had a
temperature-sensitive mutation (recA.sup.ts) so that the RecA
protein was only functional when the incubation temperature was
below 37.degree. C. When the incubation temperature was above
37.degree. C., the Rec A protein was non-functional or had greatly
reduced recombination activity. This temperature sensitive
recombination allowed manipulation of RecA function in E. coli so
as to activate conditional homologous recombination only when it
was desired. It is also possible to obtain, select or engineer
cold-sensitive mutations of Rec A protein such that the protein is
only functional above a certain temperature, e.g., 37.degree. C. In
that condition, the E. coli would be grown at a lower temperature,
albeit with a slower generation time, and recombination would be
triggered by incubating at above 37.degree. C. for a short period
of time to allow only a short interval of recombination.
[0171] Homologous recombination in E. coli was carried out by
providing overlapping DNA substrates that are found in two circular
BACs. BAC P12 (California Institute of Technology BAC library) was
182 kb in total size of which 172 kb was an insert of human genomic
DNA comprising human V.kappa. genes (from IGKV1-5 to IGKV1-12, FIG.
1). BAC P12 was carried by pBeloBAC2 vector that had a zeocin
resistant gene Zeo.sup.R. BAC C5 (California Institute of
Technology BAC library) carried a kanamycin resistance transposon
cassette (kan.sup.R) for selection in E. coli, KAN-2 (Epicentre
Biotechnologies). BAC C5 was 225 kb in total size of which 218 kb
was an insert of human genomic DNA comprising human V.kappa. genes
(from IGKV4-1 to IGK1-6), the J cluster, C.kappa. and 3' regulatory
elements. BACs C5 and P12 carried a 70 kb of homology in the insert
DNA. BAC C5 was carried in E. coli recAts.
[0172] Purified BAC P12 DNA was electroporated into E. coli
recA.sup.ts carrying BAC C5. The cells were incubated at 30.degree.
C., the permissive temperature for recA.sup.ts activity, for 30
minutes. E. coli carrying homologous recombinants of the two BACs
(kan.sup.Rzeo.sup.R) were selected by plating on plates of
selective low salt LB medium (Invitrogen) containing zeocin (50
ug/ml) and kanamycin (25 ug/ml) and incubated at 40.degree. C., a
non-permissive temperature for the recA.sup.ts activity. Homologous
recombinants in the 70 kb homology shared between C5 and P12
produced a single BAC of 407 kb in total size, of which 320 kb
represents the recombined inserts of C5 and P12 (FIG. 1). As
expected, the homologous recombination event created a duplication
of the 70 kb overlap, one copy of which was situated between the
repeated copies of the pBeloBAC vector sequences and the other copy
now joining the two fragments of human DNA from the Ig.kappa. locus
into one contiguous segment (FIG. 1). E. coli colonies that grew on
the double-selection plates exhibited kan.sup.Rzeo.sup.R, were
picked and BAC DNA isolated by miniprep. BAC DNA was digested with
Not and run on pulse-field gels. Clones exhibited the expected
pattern of bands (FIG. 2, left BAC map and left gel photo).
[0173] The 70 kb repeat between the two copies of the pBeloBAC
vector was excised by using CRE-recombinase acting on the two loxP
sites that exist on pBeloBAC (FIG. 2). Purified BAC (C5+P12) DNA
was treated with CRE recombinase (New England Biolabs) in vitro
according to the manufacturer's recommended conditions. The treated
DNA was introduced into a RecA deficient (recA-) strain of E. coli
via electroporation and the resulting bacteria plated on
chloramphenicol (Cm) containing plates and incubated at 37.degree.
C. All of the pBeloBAC vectors carry Cm.sup.R gene. The resolved
BAC had lost the duplication of the 70 kb overlap and the sequence
for pBeloBAC vector 2 (FIG. 2, right hand BAC map). The correctly
resolved BAC lost both markers of Zeo.sup.R and Km.sup.R.
[0174] E. coli colonies that grew on plates exhibited Cm.sup.R,
were picked and BAC DNA was isolated by miniprep. BAC DNA was
digested with NotI and run on pulse-field gels. Clones exhibited
the expected pattern of bands (FIG. 2, right BAC map and right gel
photo). The resolved BAC (C5P12) was 327 kb in total size of which
320 kb is human genomic DNA from, in 5' to 3' order, V.kappa.1-12
through the 3' cis regulatory regions, including 8 functional
V.kappa. genes, the entire J.kappa. cluster and C.kappa.. To make
the BAC (see, C5P12C20 in EXAMPLE 2), Tpn-Zeo was inserted at 15 kb
from the junction of the vector (FIG. 3). Tpn-Zeo was constructed
by inserting Zeo.sup.R gene into Transposon Construction Vector
(Epicentre Biotechnologies),
pMOD-3<R6K.gamma.ori/MCS>plasmid.
Example 2
Construction of a 489 Kb BAC Comprising the Majority of the Human
Ig.kappa. Locus
[0175] Homologous recombination in E. coli was carried out by
providing overlapping DNA substrates that are found in two circular
BACs. BAC C20 (California Institute of Technology BAC library) was
218 kb in total size of which 206 kb was an insert of human genomic
DNA comprising human V.kappa. genes. BAC C20 carried the KAN-2
kanamycin resistance transposon cassette (kan.sup.R) for selection
in E. coli. BAC C5P12 made in Example 1 carried a zeocin resistance
transposon cassette (zeo.sup.R) for selection in E. coli. C20 and
C5P12 carried a 44 kb of native homology in the insert DNA. BAC
C5P12 was carried in E. coli recAts.
[0176] Purified BAC C20 DNA was electroporated into E. coli
recA.sup.ts carrying BAC C5P12. The cells were incubated at
30.degree. C., the permissive temperature for recA.sup.ts activity,
for 30 minutes. The fi+ phenotype conferred by the pBeloBAC vector
prohibited the maintenance of more than one BAC in the cell,
resulting in a population of E. coli carrying only C20
(kan.sup.Rzeo.sup.S), only C5P12 (kan.sup.Szeo.sup.R), recombinants
between the two BACs (kan.sup.Rzeo.sup.R) or no BAC
(kan.sup.Szeo.sup.S). E. coli carrying homologous recombinants of
the two BACs were selected by plating on plates of selective low
salt LB medium (Invitrogen) containing zeocin (50 ug/ml) and
kanamycin (25 ug/ml) and incubated at 40.degree. C., a
non-permissive temperature for the recA.sup.ts activity.
[0177] Homologous recombinants in the 44 kb homology shared between
C20 and C5P12 produced a single BAC of 545 kb in total size ("C"),
of which 482 kb represents the recombined inserts of C20 and C5P12
(see FIG. 4). As expected, the homologous recombination event
created a duplication of the 44 kb overlap, one copy of which was
situated between the repeated copies of the pBeloBAC vector
sequences and the other copy now joining the two fragments of human
DNA from the Ig.kappa. locus into one contiguous segment (FIG.
4).
[0178] E. coli colonies that grew on the double-selection plates
exhibited kan.sup.Rzeo.sup.R, were picked and BAC DNA isolated by
miniprep. BAC DNA was digested with Not and run on pulse-field
gels. Clones exhibited the expected pattern of bands (FIG. 5, left
BAC map and left gel photo).
[0179] The 44 kb repeat between the two copies of the pBeloBAC
vector was excised by using CRE-recombinase acting on the two loxP
sites that exist on pBeloBAC (FIG. 5). Purified BAC C DNA was
treated with CRE recombinase (New England Biolabs) in vitro
according to the manufacturer's recommended conditions. The treated
DNA was introduced into a RecA deficient (recA-) strain of E. coli
via electroporation and the resulting bacteria plated on
zeocin/kanamycin double-selection plates as above and incubated at
37.degree. C.
[0180] The resolved BAC had lost the duplication of the 44 kb
overlap and the sequence for pBeloBAC vector 3 (FIG. 5, right hand
BAC map). E. coli colonies that grew on the double-selection plates
exhibited Km.sup.Rzeo.sup.R, were picked and BAC DNA isolated by
miniprep. BAC DNA was digested with NotI and run on pulse-field
gels. Clones exhibited the expected pattern of bands (FIG. 5, right
BAC map and right gel photo). The resolved BAC was 489 kb in total
size of which 467 kb is human genomic DNA from, in 5' to 3' order,
V.kappa.2-30 through the 3' cis regulatory regions, including 16
functional V.kappa. genes, the entire J.kappa. cluster and
C.kappa..
Example 3
In Silico Assembly of the Sequence of a Functional 194 Kb Synthetic
Human Ig Lambda Light Chain Transgene
[0181] The complete annotated sequence of the human immunoglobulin
lambda light chain locus (Ig;) is available. For example see
GenBank (http://www.ncbi.nlm.nih.gov/genbank/) Accession Number
NG_000002. Additional detailed information, including bibliographic
supporting scientific references is available at several public
domain websites including Vbase (http://vbase.mrc-cpe.cam.ac.uk/)
and IMGT (http://imgt.cines.fr/). This information includes data
for the genetic and phenotypic content of the human Ig locus, for
instance including, but not limited to, identification of expressed
gene sequences, pseudogenes, allelic variants, and which genes may
encode domains prone to misfolding.
[0182] Using such public information, it is possible to assemble
DNA sequences in silico using commonly available software for
manipulating DNA sequences (e.g., MacVector, DNASIS) that encode a
human Ig.lamda. light locus comprising only expressed human
V.lamda. genes in operational linkage with from one to all 7 human
J.lamda.-C.lamda. pairs and the complete functional human Ig.lamda.
3' enhancer (3' E). Cis regulatory elements controlling V.lamda.
gene expression may be captured on as little as 500 bp of DNA
immediately 5' to the start of the 5' untranslated region (UTR) and
500 bp or less DNA immediately 3' to the recombination signal
sequence (RSS) immediately 3' of the end of the coding sequence of
each V.lamda. gene. Preferably, a larger region of DNA 5' of the
start of the 5' UTR may be used to increase the distance between V
gene segments and to capture fully any and all cis regulatory
elements.
[0183] Furthermore, the region between the most 3' of the human
V.lamda. genes (V3-1) and J.lamda.1-C.lamda.1, the first
J.lamda.-C.lamda. pair, and through the 3' E is captured in
germline configuration. Alternatively, the distance of the sequence
between V.lamda.3-1 and J.lamda.1-C.lamda.1 and/or the distance
between J.lamda.7-C.lamda.7, the last J.lamda.-C.lamda. pair in the
human locus, and the 3' E may be truncated. The specific distances
are not so important as capturing of desired coding elements and
critical cis regulatory elements including splice acceptors and
splice donors, RSSs, intronic enhancer and 3' enhancer, preferably
all 3 DNAsel hypersensitive sites. Furthermore, the human Ig.alpha.
pseudogenes, J.lamda.4-C.lamda.4, J.lamda.5-C.lamda.5 and/or
J.lamda.6-C.lamda.6 may be excluded from the in silico assembled
sequence. There is a de minimus requirement for one functional
J.lamda.-C.lamda. pair, either J.lamda.1-C.lamda.1,
J.lamda.2-C.lamda.2, J.lamda.3-C.lamda.3 or
J.lamda.7-C.lamda.7.
[0184] Specific restriction enzyme sites may be introduced at the
end of the sequence. Specific restriction enzymes sites also may be
introduced or deleted internally through sequence insertion,
deletion or modification so long as they do not perturb gene
expression or coding. These enzymes sites may include sequences
useful for assembling the synthesized sequence in vitro, excising
the DNA from the vector or for various screening methodologies,
such as Southern blot of agarose gel using standard
electrophoresis, field inversion gel electrophoresis (FIGE), and
pulsed-field gel electrophoresis (PFGE).
[0185] Inserted sequences may also include primer binding sites to
facilitate PCR-based screening methods including qPCR, for the
desired and intact integration into the genome. Optionally, a
site-specific recombinase site(s) such as loxP or any of its
variants or frt are introduced to facilitate deletion of
intervening sequences to make a single-copy transgene, to
facilitate introduction of additional DNA via site specific
recombination, or to facilitate other genetic engineering designs
as known in the art. Also optionally included may be sequences for
drug-selection cassettes for mammalian cells such as a positive
selection marker for resistance to a drug such as hygromycin or a
negative selection cassette such as thymidine kinase.
[0186] Using the strategy outlined above, a core 191 kilobase
sequence ("Lambda Prime") was assembled in silico, comprising 29
functional human V) genes on approximately .about.5 kb units, all 7
human J.lamda.-C.lamda. pairs and the human 3' Ig.lamda. locus
enhancer, with the 57 kilobase sequence between V.lamda.3-1 and the
human 3' Enhancer in germline configuration. The 29 chosen human
V.lamda. genes were documented to be expressed in humans and
present in all known human haplotypes as determined by
investigation of the scientific literature. Sequences for the most
commonly used alleles that encode variable regions that fold
properly were chosen.
[0187] In instances in which two functional V.lamda. genes were
positioned in proximity of less than 5 kb distance in the human
germline configuration the entire sequence comprising the two
V.lamda. genes, from approximately 4 kb 5' of the 5' UTR of the
most 5' gene and approximately 500 bp 3' of the RSS of the most 3'
V.lamda. gene, was used. The coding and non-coding regions of the
sequence were sufficient to drive proper developmental regulation
and expression and to generate a diversity of human Ig.lamda. light
chains once introduced into the mouse genome.
[0188] A sequence for a hygromycin resistance expression cassette
was inserted 5' of the most 5' V.lamda. cassette. For ease of
excision from the BAC vector and for confirming intact integration,
rare cutting restriction enzymes were inserted into the sequence,
at the 5' end, recognition sequences for StuI/EcoRV/AsiSI/PvuI and
AgeI/PacI/AseI/BsaBI sites 5' and 3' of the hyg.sup.Rcassette,
respectively, and at the 3' end downstream of the 3' enhancer,
recognition sequences for AsiSI, AleI, EcoRI, Bsa BI were
inserted.
Example 4
In Silico Assembly of the Sequence of Two Synthetic Human Ig Lambda
Light Chain Transgenes Derived from the Sequence of Ig
Lambda-Prime
[0189] Using the Lambda-Prime sequence described in Example 3, two
additional transgenes were designed. A core 94 kilobase sequence
("Lambda 3") was assembled in silico, comprising 8 functional human
V.lamda. genes on approximately .about.5 kb units, all 7 human
J.lamda.-C.lamda. pairs and the human 3' Ig.lamda. locus enhancer,
with the 57 kilobase sequence between V.lamda.3-1 and the human 3'
Enhancer in germline configuration. The 8 chosen human V.lamda.
genes were documented to be expressed in humans. Sequences for the
most commonly used alleles that encode variable regions that fold
properly were chosen. The coding and non-coding regions of the
sequence were sufficient to drive proper developmental regulation
and expression and to generate a diversity of human Ig.lamda. light
chains once introduced into the mouse genome. An frt site was
inserted 5' of the most 5' V.lamda. gene cassette. For ease of
excision from the BAC vector and for confirming intact integration,
rare cutting restriction enzymes were inserted into the sequence,
at the 5' end, recognition sequences for ApaLI/AvrII/EcoRI sites
were inserted 5' of the frt site and a recognitions sequence for
FseI was inserted 3' of the frt site and, at the 3' end, the
recognition sequences for AsiSI, AleI, EcoRI, Bsa BI described in
Example 3 were retained.
[0190] Using the Lambda-Prime sequence described in Example 3, a
sequence ("Lambda 5") was assembled in silico comprising 21 human
V.lamda. genes with demonstrated expression and functionality, and
with no known non-functional alleles or haplotypic variation across
individual humans. The V.lamda. cassettes were generally
approximately 5 kb in size. The coding and non-coding regions of
the sequence were sufficient to drive proper development regulation
and expression and to generate a diversity of human Ig.lamda. light
chains once introduced into the mouse genome in operational linkage
with any DNA construct comprising at least one functional
J.lamda.-C.lamda. pair and preferably a functional 3' E. Sequences
for the most commonly used alleles that encode variable regions
that fold properly were chosen.
[0191] A sequence for a hygromycin resistance expression cassette
5' of the most 5' V.lamda. cassette as described in Example 3 was
retained. The sequence for an frt site was inserted 3' of the most
3' V.lamda. cassette. For ease of excision from the BAC vector and
for confirming intact integration, rare cutting restriction enzymes
were inserted into the sequence, StuI/EcoRV/AsiSI/PvuI sites 5' of
the hyg.sup.R cassette, AgeI/PacI/AseI/BsaBI sites 3' of the
hyg.sup.R cassette, as described in Example 3, and FseI/PvuI sites
5' of the frt site and KpnI/NheI sites 3' of the frt site.
Example 5
Synthesis and Assembly of DNAs Comprising the Lambda 3 and Lambda 5
Transgenes
[0192] DNAs of greater than approximately 30-40 kb in size are
carried on BACs. Example 2 documents the creation of a BAC 545 kb
in size. In addition, other cloning vectors capable of carrying
large pieces of DNA such as YACs, PACs, MACs, may be used. Genetic
engineering and physical recovery of large DNAs in all of these
vectors is well-documented in the literature.
[0193] Contract service providers synthesize and assemble very
large pieces of DNA. The DNA sequence of Lambda 3 was transmitted
to DNA2.0, Inc. (Menlo Park, Calif.). The sequence was synthesized
into physical DNA and assembled. The final fully assembled sequence
was carried in a BAC with pBeloBAC as the vector backbone. The full
BAC was sequenced by SeqWright, Inc. (Houston, Tex.) using 454
sequencing technology (454 Life Sciences, Roche). The sequence of
the synthetic Lambda 3 DNA was confirmed against the reference
sequence. Six sequence deviations from the in silico sequence were
likely 454 sequencing read errors due to long homopolymeric or
dipolymeric sequences. The deviations, even though very likely not
mutations in the actual physical synthetic DNA, mapped to
non-coding, non-regulatory regions.
[0194] The DNA sequence of Lambda 5 was transmitted to DNA2.0, Inc.
(Menlo Park, Calif.). The sequence was synthesized and assembled.
The final fully assembled sequence was carried in a BAC with
pBeloBAC as the vector backbone. The full BAC was sequenced by
SeqWright, Inc. (Houston, Tex.) using 454 sequencing technology
(454 Life Sciences, Roche). The sequence of the synthetic Lambda 5
DNA was confirmed against the reference sequence. Minimal
deviations from the in silico sequence were found. Any deviations
were likely 454 sequencing read errors due to long homopolymeric or
dipolymeric sequences. The deviations, even though very likely not
mutations in the actual physical synthetic DNA, map to non-coding,
non-regulatory regions.
Example 6
Generation of Transgenic Mice Carrying the Synthetic Lambda 3
Transgene
[0195] The Lambda 3 BAC was digested with FseI and AsiSI and the
synthetic human Lambda 3 insert purified from the vector sequence
by pulse-field gel electrophoresis. The 94 kb gel band containing
the Lambda 3 sequence was excised from the gel and purified from
the gel. The purified, concentrated DNA was microinjected into the
pronucleus of fertilized mouse eggs. Of 758 embryos transferred,
138 live mice were born. PCR assays to detect human Ig.lamda.
sequence comprising the 5' and 3' ends and in the middle of the
Lambda 3 transgene were used to screen DNAs isolated from tail
tissue from mouse pups to screen for the presence of DNA at the 5',
3' and the middle of the Lambda 3 transgene. Twenty-four mouse pups
were confirmed positive for all three PCR products. ELISA specific
for human Ig.lamda. was performed on serum samples from the founder
mice. Twenty independent founder mice were found to have
significant circulating levels of human Ig.lamda. in their serum,
confirming function of the Lambda 3 transgene. Founder mice were
bred to produce transgenic offspring.
Example 7
Expression of a Diversity of Human Ig Lambda Light Chains from the
Synthetic Human Lambda 3 Transgene in Mice
[0196] Samples of serum from the transgenic offspring of the
founder mice are confirmed to have the intact and expressed Lambda
3 transgene as described in Example 6.
[0197] Blood is drawn from Lambda 3 transgenic mice and collected
in heparinized tubes. Lymphocytes are separated and concentrated
via density gradient centrifugation over Lympholyte M. The
lymphocytes are treated with fluorochrome-conjugated antibodies
against a mouse B cell marker, e.g., B220 or CD19, and an antibody
specific for human Ig.lamda.. Mouse B cells expressing human
Ig.lamda. light chains on their surface are detected by FACs. The
percentage of human Ig.alpha. positive B cells ranges from 1 to 40%
or more. mRNA is isolated from lymphoid tissue, e.g., spleen, lymph
nodes, bone marrow, blood, of the Lambda 3 transgenic mice and
RT-PCR using primers specific for human V.lamda. and C.lamda. is
used to amplify the expressed repertoire of human variable regions
from the Lambda 3 transgene. The V.lamda. cDNAs are cloned into a
cloning vector such as TA (Invitrogen, Inc., Carlsbad, Calif.). The
human V.lamda. cDNAs are sequenced. All 8 V.lamda. genes and the
functional human J.lamda.-C.lamda. are shown to be represented in
the expressed repertoire. The sequence of the cDNAs have an
open-reading frame and encode fully human variable regions,
consistent with functional recombination of the V.lamda.-J.lamda.
and appropriate development regulation of the human Ig.lamda.
transgene.
[0198] Mice transgenic for Lambda 3 are immunized with antigen
using methods known in the art. mRNA is isolated from the secondary
lymphoid tissue, e.g., spleen, lymph nodes, of the Lambda 3
transgenic mice and RT-PCR using primers specific for human
V.lamda. and C.lamda. is used to amplify the expressed repertoire
of human variable regions from the Lambda 3 transgene. The V.lamda.
cDNAs are cloned into a cloning vector such as TA. The human
V.lamda. cDNAs are sequenced. The human V.lamda. regions are found
to be mutated as compared to the germline sequence, indicative of
somatic mutation events consistent with affinity maturation.
[0199] Taken together these data demonstrate that the Lambda 3
transgene is expressed in B cells, expresses a diversity of human
Ig.lamda. light chains, and is a template for somatic mutation
events indicative of it undergoing affinity maturation in the
secondary immune response.
Example 8
Generation of Transgenic Mice Carrying a 194 Kb Synthetic Human Ig
Lambda Transgene, Lambda-Prime, by Pronuclear Co-Microinjection
[0200] The synthetic, sequence confirmed Lambda 5 BAC is digested
with AsiSI and FseI, run on an agarose gel in PFGE and DNA
comprising the Lambda 5 sequence is isolated as in Example 6. This
DNA is co-microinjected with DNA comprising the Lambda 3 sequence,
isolated as in Example 6, into the pro-nucleus of fertilized mouse
eggs. The co-microinjected DNA co-integrates into the mouse genome,
with a significant proportion of the integration events comprising
Lambda 5 and Lambda 3 oriented in operable linkage, i.e., both are
oriented in the same 5' to 3' orientation respective to each other
and Lambda 5 is integrated 5' to Lambda 3, i.e., the 3' end of
Lambda 5 is juxtaposed to the 5' end of Lambda 3. Thus, the
contiguous human sequence of Lambda-Prime, 194 kb of synthetic DNA
in operable linkage is created, comprising 29 functional human
V.lamda. sequences, all human J.lamda.-C.lamda. and the human 3'
enhancer sequence. Intact integration in operably linkage is
confirmed by Southern blots of genomic DNAs cut with rare cutting
restriction enzymes, run on standard and PGFE gels and probed with
sequences specific to Lambda 5 and Lambda 3. Because the full
nucleotide sequence of an operably-linked co-integrated
Lambda-Prime sequence is fully known, in silico prediction of
restriction fragment patterns is readily accomplished to confirm
intact and operable linkage, as facilitated by the rare-cutting
restriction enzyme sites designed into the sequences as outlined in
Example 4. Transgene function is confirmed by ELISA for human
Ig.lamda. in the serum.
[0201] Founder mice are bred and transgenic offspring are produced.
Copy number is readily assessed by methods such as qPCR or
densitometric scanning of Southern blots of genomic DNA. If
desired, in lines in which multi-copy Lambda 5-3 transgenes are
integrated, transgenic mice are bred to transgenic mice expressing
FLP-recombinase. The frt sites present in the Lambda 5 and Lambda 3
transgenes recombine site-specifically, particular in the germline
cells. Gametes are produced that have a resolved single-copy
transgene of Lambda 5-Lambda 3 operably linked and these gametes
transmit the single-copy resolved Lambda-Prime sequence into the
next generation.
[0202] Transgenic mice, either multicopy or single copy, are tested
for Lambda Prime function as described in Example 7. The data
demonstrate that the Lambda-Prime transgene is expressed in B
cells, expresses a diversity of human Ig.lamda. light chains, and
is a template for somatic mutation events indicative of it
undergoing affinity maturation in the secondary immune
response.
Example 9
Generation of Transgenic Mice Carrying a 194 Kb Synthetic Human Ig
Lambda Transgene by Co-Transfection into ES Cells
[0203] DNAs comprising the Lambda 3 and the Lambda 5 sequences are
isolated as in Example 8. These DNAs are co-introduced into mouse
ES cells by a method such as lipofection or electroporation. The
presence of a positive-selectable maker cassette 5' of the most 5'
V.lamda. gene on Lambda 5, e.g., hygromycin, enables positive
selection for integration of Lambda 5. The co-introduced DNA
randomly co-integrates into the mouse genome, with a significant
proportion of the integration events comprising Lambda 5 and Lambda
3 oriented in operable linkage, i.e., both are oriented in the same
5' to 3' orientation respective to each other and Lambda 5 is
integrated 5' to Lambda 3', i.e., the 3' end of Lambda 5 is
juxtaposed to the 5' end of Lambda 3. Thus, the contiguous
Lambda-Prime sequence of 194 kb of synthetic DNA in operable
linkage is created.
[0204] Intact integration in operably linkage is confirmed by
Southern blots of genomic DNAs cut with rare cutting restriction
enzymes, run on standard and PGFE gels and probed with sequences
specific to Lambda 5 and Lambda 3. Because the full nucleotide
sequence of an operably-linked co-integrated Lambda-Prime sequence
is fully known, in silico prediction of restriction fragment
patterns is readily accomplished to confirm intact and operable
linkage, as facilitated by the rare-cutting restriction enzyme
sites designed into the sequences as outlined in Example 4. Copy
number may be readily assessed by methods such as qPCR or
densitometric scanning of Southern blots of genomic DNA. If
desired, in clones in which multi-copy Lambda 5-3 transgenes are
integrated, FLP-recombinase is transiently expressed in the clones.
The frt sites present in the Lambda 5 and Lambda 3 transgenes
recombine site-specifically. Clones are produced that have a
resolved single-copy transgene of Lambda 5-Lambda 3 operably
linked.
[0205] ES cells carrying the operably linked Lambda-Prime transgene
sequence are used to generate transgenic mice using
well-established methods. For examples, ES cells are microinjected
into mouse blastocysts, which are then implanted in pseudo-pregnant
foster females. Chimeric pups are born. Chimeric mice are bred and
the resulting offspring are screened for the presence of the
Lambda-Prime transgene.
[0206] Transgenic mice, either multicopy or single copy, are tested
for Lambda-Prime function as described in Example 7. The data
demonstrate that the Lambda-Prime transgene is expressed in B
cells, expresses a diversity of human Ig.alpha. light chains, and
is a template for somatic mutation events indicative of it
undergoing affinity maturation in the secondary immune
response.
Example 10
Synthesis and Assembly of a DNA Comprising the Lambda-Prime
Transgene and Generation of Transgenic Mice Therefrom
[0207] DNAs of greater than approximately 30-40 kb in size are
carried on BACs. Example 2 documents the creation of a BAC 545 kb
in size. In addition, other cloning vectors capable of carrying
large pieces of DNA such as YACs, PACs, MACs, may be used. Genetic
engineering and physical recovery of large DNAs in all of these
vectors is well-documented in the literature.
[0208] Contract service providers synthesize and assemble very
large pieces of DNA. The DNA sequence of Lambda-Prime is
transmitted to one search service provider, DNA2.0, Inc. (Menlo
Park, Calif.). The sequence is synthesized into physical DNA and
assembled. The final fully assembled sequence is carried in a BAC
with pBeloBAC as the vector backbone. The full BAC was sequenced by
sequencing service providers such as SeqWright, Inc. (Houston,
Tex.) using 454 sequencing technology (454 Life Sciences, Roche) or
standard shotgun sequencing. The sequence of the synthetic
Lambda-Prime DNA is confirmed against the reference sequence. Any
sequence deviations from the in silico sequence are likely
sequencing read errors due to long homopolymeric or dipolymeric
sequences. The deviations, even though very likely not mutations in
the actual physical synthetic DNA, map to non-coding,
non-regulatory regions.
[0209] Alternatively, Lambda 3 and Lambda 5 may be recombined in
vitro using techniques as described in Examples 1 and 2. They may
also be recombined using other methods of engineering BACs such as
recombineering, or standard restriction fragment ligation into
pBeloBAC following by transfection into E. coli.
[0210] Transgenic mice are generated as described in Examples 6 or
by introduction into ES cells such as by electroporation,
lipofection etc., as exemplified in Example 9. Transgenic mice,
either multicopy or single copy, are tested for Lambda-Prime
function as described in Example 8. The data demonstrate that the
Lambda-Prime transgene is expressed in B cells, expresses a
diversity of human Ig % light chains, and is a template for somatic
mutation events indicative of it undergoing affinity maturation in
the secondary immune response.
Example 11
Creation of a Human IgL Transgene Via Co-Introduction of Lambda 3
with Genomic DNA Comprising Additional Human V Lambda
Repertoire
[0211] Libraries of human genomic DNA are available commercially or
through licensing and are well-characterized. These include the
CalTech human genomic library carried on BACs and various human
genomic DNA libraries on YACs. The CalTech human library BAC clones
from libraries B, C and D may be ordered through Invitrogen
(Carlsbad, Calif.). Human genomic libraries are also available
carried on cosmids, phage, P1s, PACs etc. All of these vectors may
be modified prior to co-introduction using techniques readily
available in the art.
[0212] Because of the ready facility by which large fragments of
DNA may be sequenced, the genomic inserts on these BACs or YACs may
be sequenced confirmed using a contract service provider as
described above. The complete human DNA insert may be isolated.
Alternatively, a subfragment may be isolated using rare-cutting
restriction enzyme sites available in the genomic DNA. An example
of a suitable YAC is L1 (U.S. Pat. No. 7,435,871). Other YAC, BAC
and cosmid clones suitable for use are described in Kawasaki et
al., (Gen. Res. (1995) 5: 125-135) and Frippiat et al. (Hum. Mol.
Genet. (1995) 4: 983-991). One or more BACs or YACs comprising
additional human V.lamda. genes are co-introduced with the Lambda 3
construct. Optionally, the Lambda 3 DNA is co-introduced with two
or more other constructs with additional V.lamda. genes. The two or
more co-introduced constructs are confirmed to co-integrate in
operable linkage as outlined in Examples 8 and 9. Transgene
functionality is confirmed as in Example 7. Thus, a human Ig.lamda.
transgene may be partly synthetic and partly derived from a genomic
library, with the core J.lamda.-C.lamda. and 3' cis regulatory
sequences created by synthetic means and all or part of the
V.lamda. repertoire derived from a genomic library.
Example 12
Creation of a Human IgL Transgene Via Co-Introduction of Lambda 5
with a Genomic DNA Sequence Comprising at Least One Functional
Human JL-CL Pair
[0213] Libraries of human genomic DNA are available commercially or
through licensing and are well-characterized. These include the
CalTech human genomic library carried on BACs and various human
genomic DNA libraries on YACs. The CalTech human library BAC clones
from libraries B, C and D may be ordered through Invitrogen
(Carlsbad, Calif.). Human genomic libraries are also available
carried on cosmids, phage, P1s, PACs etc. All of these vectors may
be modified prior to co-introduction using techniques readily
available in the art.
[0214] Because of the ready facility by which large fragments of
DNA may be sequenced, the genomic inserts on these BACs or YACs may
be sequenced confirmed using a contract service provider as
described above. The complete human DNA insert may be isolated.
Alternatively, a subfragment may be isolated using rare-cutting
restriction enzyme sites available in the genomic DNA. An example
of a suitable YAC is L2 (U.S. Pat. No. 7,435,871), which contains
all 7 J.lamda.-C.lamda. pairs and the human 3' enhancer. Other YAC,
BAC and cosmid clones suitable for use are described in Kawasaki et
al., (Gen. Res. (1995) 5: 125-135) and Frippiat et al. (Hum. Mol.
Genet. (1995) 4: 983-991).
[0215] The core construct contains at least one functional human
J.lamda.-C.lamda. pair and preferably a functional 3' enhancer. The
Lambda 5 DNA is co-introduced with the isolated DNA of the core
construct and, optionally, one or more other constructs with
additional V.lamda. genes. The two or more co-introduced constructs
are confirmed to co-integrate in operable linkage as outlined in
Examples 8 and 8. Transgene functionality is confirmed as in
Example 7. Thus, a human Ig.lamda. transgene may be partly
synthetic and partly derived from a genomic library, with the core
J.lamda.-C.lamda. and 3' cis regulatory sequences derived from a
genomic library and all or part of the V.lamda. repertoire created
by synthetic means.
Example 13
Use of Cre-lox System to Recombine Transgenes
[0216] The sequence of the Lambda 3 transgene is designed in silico
as described in Example 4 with the alteration of an addition of a
loxP site or variant thereof replacing the sequence of the frt site
or being place adjacent to it, and a drug-resistance cassette
activity in mammalian cells such as puromycin-resistance is
inserted 5' to the loxP site, creating Lambda 3P. The Lambda 3P
sequence is synthesized and assembled into physical DNA as
described in Example 5. The Lambda 3P DNA is isolated from the
vector DNA, introduced into ES cells, puromycin-resistance colonies
selected for, picked and molecularly screened for intact
integration of Lambda 3P.
[0217] The sequence of the Lambda 5 transgene is designed in silico
as described in Example 4 with the alteration of an addition of a
loxP site or variant thereof replacing the sequence of the frt site
or being place adjacent to it, creating Lambda 3P. The Lambda 5P
sequence is synthesized and assembled into physical DNA as
described in Example 5 except that the BAC vector sequence, such as
pBeloBAC, has a deleted loxP site or carries a version incompatible
for recombination with that in the Lambda 5P sequence. The circular
Lambda 5P BAC DNA is isolated and co-transfected with CRE
recombinase into Lambda 3P ES clones. The CRE recombinase engenders
site-specific recombination between the loxP sites, resulting in
integration of the Lambda 5P DNA in operably linkage upstream of
the Lambda 3 DNA, therein reconstituting the Lambda Prime sequence.
Lambda 5P positive ES clones are selected for puromycin-resistance,
picked and molecularly screened for insertion of Lambda 5P into
Lambda 3P as described in Example 9. Transgenic mice are generated
from the ES cells and confirmed for Lambda Prime transgene function
as described in Example 9.
[0218] This process for insertion of additional V.lamda. repertoire
upstream of a functional core J.lamda.-C.lamda. sequence is
applicable for any vector existing as a circular DNA, e.g.,
plasmid, cosmid, BAC, or circularizable, such as a YAC, so long as
the loxP site is 3' of most 3' V.lamda. gene desired to be operably
linked to the J.lamda.-C.lamda. core sequence.
Example 15
Generation of Mice Transgenic Expressing Human Ig Lambda from a
Synthetic DNA Transgene Comprising a Highly Chimeric Human-Mouse
DNA Sequence
[0219] The annotated sequence of the mouse immunoglobulin lambda
light chain locus is available in the public domain, see Genbank
accession number NC_000082. Because of the unique structure of the
mouse Ig.lamda. locus, which is composed of two separates units
(see Selsing et al., Immunoglobulin Genes 1989 Acad. Press Ltd.,
pp. 111-122), the sequence of one of the mouse Ig; locus units is
selected. A 60,000 nucleotide (nt) sequence comprising 4 kb
upstream of the start codon of V.lamda.1 and 5 kb downstream of the
3' enhancer is isolated in silico. A sub-sequence of 4 kb upstream
of the start codon of mouse V.lamda.2 and 500 bp downstream of the
RSS is identified ("V.lamda. expression cassette"). FIG. 1 of
Ramsden and Wu (Proc. Nat. Acad. Sci. 199188: 10721-10725)
identifies the V.lamda.2 RSS and the RSS for J.lamda.3 and
J.lamda.1. The sequence of the 39 nucleotide RSS of V.lamda.1 of
mouse is replaced in the functional orientation with the functional
RSS from a human V.lamda., e.g., V.lamda.3-1. This approximately
5,000 nucleotide sequence comprising 4,000 nt upstream of the start
codon, human RSS and through 500 nt downstream of the RSS, is the
core V; expression construct.
[0220] The 28 nucleotide sequence of mouse J.lamda.3 and J.lamda.1
are replaced in the functional orientation with the functional RSS
from human J.lamda.3 and J.lamda.1. The coding sequences for mouse
J.lamda.3 and J.lamda.1 are replaced by the coding sequences for
human J.lamda.3 and J.lamda.1. The coding sequences for mouse
C.lamda.3 and C.lamda.1 are replaced by the coding sequences for
human C.lamda.3 and C.lamda.1. The coding sequence of mouse
V.lamda.1 is replaced with the coding sequence of a human V gene,
e.g., V.lamda.3-1. The sequence comprising the mouse 3' enhancer is
replaced with 7,562 nucleotide sequence comprising the 3 DNAsel
hypersensitive sites of the human Ig.lamda. 3' enhancer. This
sequence is the core chimeric Igl construct. Combriato and Klobeck
(J. Immunol. 2002 168:1259-1266) teach other sequence changes for
restoring optimal enhancer activity to the mouse 3' enhancer.
[0221] Additional V.lamda. repertoire is added in silico through
appending the core V.lamda. expression construct sequence 5' to the
core chimeric Ig.lamda. construct. In each appended V.lamda.
expression construct, the mouse V.lamda.1 coding sequence is
replaced with human V.lamda. coding sequence. The entire human
V.lamda. repertoire can be appended sequentially in silico yielding
a sequence of approximately 205,000 nt.
[0222] The sequence or two portions thereof is synthesized and
assembled into physical DNA is described in previous examples. The
DNA is used to construct transgenic mice as described in previous
examples. Transgenic mice are analyzed for transgene expression and
function as described in previous examples. The data demonstrate
that the transgene is expressed in B cells, expresses a diversity
of human Ig.lamda. light chains, and is a template for somatic
mutation events indicative of it undergoing affinity maturation in
the secondary immune response.
[0223] The preceding example illustrates the methodology by which
exquisitely precisely and complexly engineered sequences are
composed in silico and then a process for making transgenic animals
comprising that sequence. The methodology is not limited to the
described sequence.
Example 14
In Silico Assembly of the Sequence of a Functional Synthetic Human
Ig Kappa Light Chain Transgene
[0224] The methodologies described in the preceding examples are
broadly applicable for the in silico assembly and subsequent
synthesis of any sequence up to the cloning capacity of a BAC,
which as demonstrated in Example 2, is at least 500 kb. As
described in Example 3, a sequence encoding a human Ig.kappa.
transgene was assembled from publicly available information on the
sequence of the human and mouse Ig.kappa. loci. The annotated
sequence for the complete human Ig.kappa. locus was accessed from
Genbank, accession number NG_000834. The sequence comprises the
complete proximal V.kappa. cluster through the 3' regulatory
elements, 3' enhancer, Ed and RS. Additional detailed information,
including bibliographic supporting scientific references is
available at several public domain websites including Vbase
(http://vbase.mrc-cpe.cam.ac.uk/) and IMGT (http://imgt.cines.fr/).
This information includes data for the genetic and phenotypic
content of the human Ig.kappa. locus, for instance including, but
not limited to, identification of expressed gene sequences,
pseudogenes, allelic variants, and which genes may encode domains
prone to misfolding.
[0225] A 30,000 nt sequence comprising 4 kb upstream of human
V.kappa.4-1 through the complete human JR cluster through 1,000 nt
3' of human C.kappa. was in germline configuration. Appended in
silico 3' of human C.kappa. was a 25,600 nt germline configured
mouse DNA sequence comprising the Ig.kappa. 3' enhancer, Ed and RS.
This sequence served as the core Ig.kappa. expression cassette.
[0226] To expand the repertoire, sequence for additional V.kappa.
expression cassettes as units of .about.5,000 nt were added 5' of
V.kappa.4-1. In instances in which two functional V.kappa. genes
were positioned in proximity of less than 5 kb distance in the
human germline configuration the entire sequence comprising the two
V.kappa. genes, from approximately 4 kb 5' of the 5' UTR of the
most 5' gene and approximately 500 bp 3' of the RSS of the most 3'
V.kappa. gene, was used. As described in Example 3, recognition
sequences for specific restriction enzymes were introduced at the
ends of the sequence. Recognition sequences for specific
restriction enzymes were introduced and deleted internally through
sequence insertion, deletion or modification; these did not perturb
gene expression or coding.
Example 16
Generation of Mice Transgenic for a Locus Expressing Human
Ig.kappa., Said Locus Comprising Synthetic DNA
[0227] Using methodology described in any of the preceding Examples
1-2 and Examples 4-14 and the process for in silico assembly of
sequences described in Examples 3 and 15, physical DNA that encodes
human Ig.kappa. light chains is synthesized and used to create
transgenic mice. Transgenic mice are analyzed for transgene
expression and function as described in previous examples using
appropriate reagents for use in studying human Ig.kappa. expression
at the nucleic acid and protein levels. The data demonstrate that
the transgene is expressed in B cells, expresses a diversity of
human Ig.kappa. light chains, and is a template for somatic
mutation events indicative of it undergoing affinity maturation in
the secondary immune response.
Example 17
Generation of Mice Expressing Human Ig.kappa. from a Synthetic DNA
Transgene Comprising a Highly Chimeric Human-Mouse DNA Sequence
[0228] The annotated sequence of the human immunoglobulin kappa
light chain locus is publicly available, see Genbank accession
number NG_000834. The annotated sequence of the mouse
immunoglobulin kappa light chain locus is publicly available, see
Genbank accession number NC_005612. Other sources such as the IGMT
Repertoire website (http://imgt.cines.fr/) are used as a resource
on the map and functionality of individual components in the loci.
A mouse DNA sequence of approximately 50,000 bases, comprising a
V.kappa., preferably the most proximal mouse V.kappa., V.kappa.3-1,
the J.kappa. cluster, C.kappa. through 3' regulatory regions, is
isolated in silico. Though this sequence is preferably in germline
configuration, intergenic regions of DNA may be deleted to make a
smaller overall sequence so long as critical regulatory regions
such as the Ig.kappa. intronic enhancer, 3' enhancer, Ed and RS,
the sequence and location of which are all documented publicly, are
unpertubed.
[0229] The mouse exons for the V.kappa., J.kappa. and C.kappa. are
replaced with their human counterparts. The human V.kappa. exons
replacing the mouse V.kappa. exons may be V.kappa.4-1, which is
human V.kappa. most proximal to the human J.kappa. cluster but this
is not absolutely necessary. Any human V.kappa. exon sequences may
be used. It is noted that human V.kappa.4-1 and the next most
proximal V.kappa. gene, V.kappa.5-2, are inverted 3'-5' relative to
the human J.kappa. cluster in the germline configuration. The human
V.kappa. 4-1 exons would be oriented in the mouse V.kappa. context
in the 5'-3' orientation relative to the mouse J.kappa. locus in
the sequence constructed in silico. The mouse J.kappa. locus
comprises 5 J.kappa. sequences but J.kappa.3 may not be expressed
because of a non-canonical donor splice sequence. The human
J.kappa. locus comprises 5 J.kappa. sequences, all of which are
functional. Incorporation of the human J.kappa.3 exon would bring
with it the proper splice donor sequence, particular for splicing
to its counterpart splice acceptor sequence on human C.kappa..
[0230] Additional V.kappa. repertoire is added in silico through
identifying approximately 5 kb units comprising in proximal to
distal order the functional mouse V.kappa. genes. This number of 5
kb sequence units is equivalent to the number of human V.kappa.
genes to be represented in the transgenes. Mouse pseudogenes are
eliminated. In each appended V.kappa. expression construct, the
mouse V.kappa. coding sequence is replaced with human V.kappa.
coding sequence. The 5 kb unit may also be a repeated unit so that
identical non-coding sequences comprise each unit and the units are
only distinguished by the unique human V.kappa. exon sequence. Each
unit is appended onto the core sequence 5' to the preceding one,
sequentially building the sequence of the artificial locus,
proximally to distally.
[0231] The entire proximal human V.kappa. repertoire can be
appended sequentially in silico yielding a sequence of
approximately 140,000 bases. The inverted distal cluster of human
V.kappa. genes may also be included, though because they are
duplications of the genes in the proximal cluster, they contribute
to <10% of the expressed human Ig.kappa. repertoire, and because
they are missing in some human haplotypes, their inclusion is not
necessary and may be undesired for later antibody drug
development.
[0232] The sequence or two portions thereof is synthesized and
assembled into physical DNA is described in previous examples. The
DNA is used to construct transgenic mice as described in previous
examples. Transgenic mice are analyzed for transgene expression and
function as described in previous examples. The data demonstrate
that the transgene is expressed in B cells, expresses a diversity
of human Ig.kappa. light chains, and is a template for somatic
mutation events indicative of it undergoing affinity maturation in
the secondary immune response.
[0233] The preceding example illustrates the methodology by which
exquisitely precisely and complexly engineered sequences are
composed in silico and then a process for making transgenic animals
comprising that sequence. The methodology is not limited to the
described sequence.
[0234] The various embodiments described above can be combined to
provide further embodiments. All of the U.S. patents, U.S. patent
application publications, U.S. patent applications, foreign
patents, foreign patent applications and non-patent publications
referred to in this specification and/or listed in the Application
Data Sheet are incorporated herein by reference, in their entirety.
Aspects of the embodiments can be modified, if necessary to employ
concepts of the various patents, applications and publications to
provide yet further embodiments.
[0235] These and other changes can be made to the embodiments in
light of the above-detailed description. In general, in the
following claims, the terms used should not be construed to limit
the claims to the specific embodiments disclosed in the
specification and the claims, but should be construed to include
all possible embodiments along with the full scope of equivalents
to which such claims are entitled. Accordingly, the claims are not
limited by the disclosure.
* * * * *
References