U.S. patent application number 17/368266 was filed with the patent office on 2022-01-06 for manipulation of immunoglobulin gene diversity and multi-antibody therapeutics.
The applicant listed for this patent is Kymab Limited. Invention is credited to Jasper Clube, Nicholas England, Glenn Friedrich, E-Chiang Lee.
Application Number | 20220000085 17/368266 |
Document ID | / |
Family ID | 1000005843892 |
Filed Date | 2022-01-06 |
United States Patent
Application |
20220000085 |
Kind Code |
A1 |
Friedrich; Glenn ; et
al. |
January 6, 2022 |
MANIPULATION OF IMMUNOGLOBULIN GENE DIVERSITY AND MULTI-ANTIBODY
THERAPEUTICS
Abstract
The invention provides improved non-human vertebrates and
non-vertebrate cells capable of expressing antibodies comprising
human variable region sequences. The present invention is directed
to the provision of long HCDR3s from non-human vertebrates and
cells. The present invention is also directed to the provision of
novel V, D and J pairings in immunoglobulin heavy and light chain
loci. Novel, biased antibody diversities and potentially expanded
diversities are provided. The invention also provides for novel and
potentially expanded diversity or diversity that is biased towards
variable gene usage common to antibodies useful for treating and/or
preventing certain diseases or conditions, such as infectious
diseases. The invention also provides methods of generating
antibodies using such vertebrates, as well as the antibodies per
se, therapeutic compositions thereof and uses.
Inventors: |
Friedrich; Glenn;
(Cambridge, GB) ; Lee; E-Chiang; (Cambridge,
GB) ; Clube; Jasper; (Cambridge, GB) ;
England; Nicholas; (Cambridge, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kymab Limited |
Cambridge |
|
GB |
|
|
Family ID: |
1000005843892 |
Appl. No.: |
17/368266 |
Filed: |
July 6, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14220074 |
Mar 19, 2014 |
11051497 |
|
|
17368266 |
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PCT/GB2012/052298 |
Sep 18, 2012 |
|
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14220074 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/18 20130101;
C07K 16/08 20130101; A01K 2217/072 20130101; C07K 16/1045 20130101;
C12N 2800/204 20130101; C07K 16/1242 20130101; A01K 2217/15
20130101; A01K 67/0278 20130101; C07K 16/12 20130101; A01K 2227/105
20130101; C07K 16/1018 20130101; C07K 16/1217 20130101; C07K
2317/24 20130101; C07K 16/462 20130101; C12N 15/8509 20130101; C07K
16/46 20130101; C07K 2317/56 20130101; C07K 16/088 20130101; C07K
16/085 20130101; A01K 2267/01 20130101; C07K 16/1232 20130101 |
International
Class: |
A01K 67/027 20060101
A01K067/027; C07K 16/18 20060101 C07K016/18; C07K 16/46 20060101
C07K016/46; C12N 15/85 20060101 C12N015/85; C07K 16/08 20060101
C07K016/08; C07K 16/12 20060101 C07K016/12 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 19, 2011 |
GB |
1116120.5 |
Sep 19, 2011 |
GB |
1116122.1 |
Feb 24, 2012 |
GB |
1203257.9 |
Mar 15, 2012 |
GB |
1204592.8 |
Mar 29, 2012 |
GB |
1205702.2 |
May 18, 2012 |
GB |
1208749.0 |
Jul 2, 2012 |
GB |
1211692.7 |
Claims
1. A method of producing an antigen specific antibody or antigen
binding fragment thereof, said antibody comprising a human
immunoglobulin heavy (IgH) chain, wherein said human IgH chain
comprises a human IgH chain variable region and a human IgH chain
constant region, and said fragment comprising a human IgH chain
variable region, the method comprising: expressing the antibody or
antigen binding fragment thereof from a host cell comprising
nucleic acid encoding said human IgH chain variable region and said
human IgH chain constant region of said antibody, or comprising
nucleic acid encoding said human IgH chain variable region of said
fragment, wherein said human IgH chain variable region of said
antigen-specific antibody or antigen binding fragment is of a B
cell, or a hybridoma thereof, of a transgenic mouse contacted with
said antigen; wherein said transgenic mouse comprises a germline
comprising: (i) an immunoglobulin heavy (IgH) chain locus
comprising a plurality of human VH, one or more human D and one or
more human JH gene segments at an endogenous locus upstream of and
operably linked to a constant region; wherein said plurality of
human VH gene segments is selected from at least two of the group
consisting of: IGHV3-7*01, IGHV3-9*01, IGHV7-4-1*01, IGHV1-3*01,
IGHV4-4*02, IGHV3-13*01, IGHV3-23*04 and VH3-20*d01, and where one
or more human D gene segments is selected from the group consisting
of: IGHD2-2*01, IGHD3-9*01, IGHD3-10*01, IGHD6-13*01, IGHD4-17*01,
IGHD6-19*01, IGHD3-22*01 and IGHD1-26*01, wherein each of said
selected human VH gene segments of said plurality of human VH gene
segments is capable of joining with a human D gene segment and a
human JH gene segment to encode a variable region, wherein each of
said selected human VH gene segments of said plurality of human VH
gene segments is capable of joining with a human D gene segment and
a human JH segment to encode a variable region comprising a HCDR3
of 20 or more amino acids in length in said mouse; (ii) an
immunoglobulin kappa light (Ig.kappa.) chain locus comprising one
or more human Ig.kappa. variable (V.kappa.) gene segments and one
or more human Ig.kappa. joining (J.kappa.) gene segments upstream
of and operatively linked to a constant region; wherein said one or
more V.kappa. gene segments is selected from the group consisting
of a V.kappa.I gene family member, .kappa.1-15A (KL012), a
V.kappa.II family member, a V.kappa.III family member, V.kappa.II
A2, V.kappa.II A2a and V.kappa. A27 (Humkv325), such that said
transgenic mouse is capable, upon contact with an antigen, of
producing a plurality of antibodies specific for said antigen
comprising an immunoglobulin heavy chain comprising a human VH
region and a C region; wherein said plurality of antibodies
specific for said antigen comprises a human VH region having an
HCDR-3 length of at least 20 amino acids.
2. The method of claim 1, wherein said one or more human D gene
segments comprises IGHD2-2*02, IGHD3-9*01, IGHD3-10*01,
IGHD6-13*01, IGHD4-17*01, IGHD6-19*01, IGHD3-22*01 and
IGHD1-26*01.
3. The method of claim 1, wherein the C region of the IgH locus is
a mouse C region.
4. The method of claim 1, wherein said B cells of said transgenic
mouse contacted with antigen are isolated.
5. The method of claim 4, comprising the step of isolating
antigen-specific antibody from said isolated B cells.
6. The method of claim 4, further comprising the step of isolating
from said B cells nucleic acid encoding said human VH region and
said mouse C region of said chimeric immunoglobulin heavy
chain.
7. The method of claim 6, further comprising the step of replacing
in said nucleic acid encoding said human VH region and said mouse C
region of said chimeric immunoglobulin heavy chain polypeptide, the
portion encoding said mouse C region with nucleic acid encoding a
human C region, thereby forming a nucleic acid encoding said human
VH region and said human C region.
8. The method of claim 4, further comprising isolating an antibody
that binds said antigen, comprising (i) immortalizing said isolated
B cells expressing said antigen-specific antibody or progeny
thereof, optionally producing hybridomas therefrom; and (ii)
isolating an antibody expressed by the immortalized B cells of
(i).
9. The method of claim 1, wherein before said contact with antigen,
said mouse is naive.
10. The method of claim 1, wherein said antibody is an IgG-type
antibody.
11. The method of claim 1, wherein said antigen is an antigen of an
infectious disease pathogen.
12. The method of claim 1, wherein the antigen is of a viral
pathogen.
13. The method of claim 1, wherein expression of mouse Ig heavy
chain comprising a mouse IgH variable region and a mouse IgH
constant region is inactive in said mouse.
14. A method of producing an antigen specific antibody or antigen
binding fragment thereof, said antibody comprising a human
immunoglobulin heavy (IgH) chain, wherein said human IgH chain
comprises a human IgH chain variable region and a human IgH chain
constant region, and said fragment comprising a human IgH chain
variable region, the method comprising expressing the antibody or
antigen binding fragment thereof from a host cell comprising
nucleic acid encoding said human IgH chain variable region and said
human IgH chain constant region of said antibody, or comprising
nucleic acid encoding said human IgH chain variable region of said
fragment, wherein said human IgH chain variable region of said
antigen-specific antibody or antigen binding fragment is of a B
cell, or a hybridoma thereof, of a transgenic mouse contacted with
said antigen; wherein said transgenic mouse comprises a germline
comprising: (i) wherein said immunoglobulin heavy (IgH) chain locus
comprising a plurality of human VH, one or more human D and one or
more human JH gene segments at an endogenous locus upstream of and
operably linked to a constant region; wherein IGHV3-7*01,
IGHV3-9*01, IGHV7-4-1*01, IGHV1-3*01, IGHV4-4*02, IGHV3-13*01,
IGHV3-23*04, and VH3-20*d01, and where one or more human D gene
segments is selected from the group consisting of: IGHD2-2*01,
IGHD3-9*01, IGHD3-10*01, IGHD6-13*01, IGHD4-17*01, IGHD6-19*01,
IGHD3-22*01 and IGHD1-26*01, wherein said plurality of human VH
gene segments comprise human VH gene segments capable of joining
with a human D gene segment and a human JH gene segment to encode a
variable region, wherein said plurality of human VH gene segments
comprise human VH gene segments capable of joining with a human D
gene segment and a human JH segment to encode a variable region
comprising a HCDR3 of 20 or more amino acids in length in said
mouse; (ii) an immunoglobulin kappa light (Ig.kappa.) chain locus
comprising one or more human Ig.kappa. variable (V.kappa.) gene
segments and one or more human Ig.kappa. joining (J.kappa.) gene
segments upstream of and operatively linked to a constant region;
wherein said one or more V.kappa. gene segments is selected from
the group consisting of a V.kappa.I gene family member,
.kappa.1-15A (KL012), a V.kappa.II family member, a V.kappa.III
family member, V.kappa.II A2, V.kappa.II A2a and V.kappa. A27
(Humkv325), such that said transgenic mouse is capable, upon
stimulation with an antigen, of producing a plurality of antibodies
specific for said antigen comprising an immunoglobulin heavy chain
comprising a human VH region and a C region; wherein said plurality
of antibodies specific for said antigen comprises a human VH region
having an HCDR-3 length of at least 20 amino acids.
15. The method of claim 14, wherein said one or more human D gene
segments comprises IGHD2-2*02, IGHD3-9*01, IGHD3-10*01,
IGHD6-13*01, IGHD4-17*01, IGHD6-19*01, IGHD3-22*01 and
IGHD1-26*01.
16. The method of claim 14, wherein the C region of the IgH locus
is a mouse C region.
17. The method of claim 14, wherein said B cells of said transgenic
mouse contacted with antigen are isolated.
18. The method of claim 17, further comprising the step of
isolating antigen-specific antibody from said isolated B cells.
19. The method of claim 17, further comprising the step of
isolating from said B cells nucleic acid encoding said human VH
region and said mouse C region of said chimeric immunoglobulin
heavy chain polypeptide.
20. The method of claim 19, further comprising the step of
replacing in said nucleic acid encoding said human VH region and
said mouse C region of said chimeric immunoglobulin heavy chain
polypeptide, the portion encoding said mouse C region with nucleic
acid encoding a human C region, thereby forming a nucleic acid
encoding said human VH region and said human C region.
21. The method of claim 17, further comprising isolating an
antibody that binds said antigen, comprising (i) immortalizing said
isolated B cells expressing said antigen-specific antibody or
progeny thereof, optionally producing hybridomas therefrom; and
(ii) isolating an antibody expressed by the immortalized B cells of
(i).
22. The method of claim 14, wherein before said contact with
antigen, said mouse is naive.
23. The method of claim 14, wherein said antibody is an IgG-type
antibody.
24. The method of claim 14, wherein said antigen is an antigen of
an infectious disease pathogen.
25. The method of claim 14, wherein the antigen is of a viral
pathogen.
26. The method of claim 14, wherein expression of mouse Ig heavy
chain comprising a mouse IgH variable region and a mouse IgH
constant region is inactive in said mouse.
Description
CROSS REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 14/220,074 filed Mar. 19, 2014, which is a continuation of
international application PCT/GB2012/052298 filed Sep. 18, 2012,
which claims priority to GB1116122.1 filed Sep. 19, 2011,
GB1116120.5 filed Sep. 19, 2011, GB1203257.9 filed Feb. 24, 2012,
GBBab filed Mar. 15, 2012, GB1205702.2 filed Mar. 29, 2012,
GB1208749.0 filed May 18, 2012 and GB1211692.7 filed Jul. 2, 2012,
all of which are herein incorporated by reference.
[0002] The attached sequence listing titled
39080-18401_seq_listing.txt (size 2 kb and created on Jul. 6,
2021), is hereby incorporated by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to the provision of antibodies
with long HCDR3 lengths. The present invention is also directed to
the provision of novel V, D and J pairings in immunoglobulin heavy
and light chain loci. Novel, biased antibody diversities and
potentially expanded diversities are provided. The invention also
provides for novel and potentially expanded diversity or diversity
that is biased towards variable gene usage common to antibodies
useful for treating and/or preventing certain diseases or
conditions, such as infectious diseases. This ability to bias the
antibody repertoire also provides methods of simplifying the
production of antibody mixtures, such as polyclonal antibody
therapeutics useful for the treatment and/or prevention of
infectious diseases where a polyclonal approach to target multiple
pathogen antigens is desirable. To this end, the present invention
also provides bispecific antibodies that are capable of binding to
more than one antigen (eg, multiple infectious antigens expressed
by the same pathogen), thus providing advantages (such as
manufacturing, dosing and administration advantages) not possible
with polyclonal antibody mixtures.
[0004] The present invention provides vertebrates and cells, such
as transgenic mice or rats or transgenic mouse or rat cells.
Furthermore, the invention relates to methods of using the
vertebrates to isolate antibodies or nucleotide sequences encoding
antibodies. Antibodies, nucleotide sequences, pharmaceutical
compositions and uses are also provided by the invention.
BACKGROUND
[0005] The state of the art provides non-human vertebrates (eg,
mice and rats) and cells comprising transgenic immunoglobulin loci,
such loci comprising human variable (V), diversity (D) and/or
joining (J) segments, and optionally human constant regions.
Alternatively, endogenous constant regions of the host vertebrate
(eg, mouse or rat constant regions) are provided in the transgenic
loci. Methods of constructing such transgenic vertebrates and use
of these to generate antibodies and nucleic acids thereof following
antigen immunisation are known in the art, eg, see U.S. Pat. No.
7,501,552 (Medarex), U.S. Pat. No. 5,939,598 (Abgenix), U.S. Pat.
No. 6,130,364 (Abgenix), WO02/066630 (Regeneron), WO2011004192
(Genome Research Limited), WO2009076464, WO2009143472 and
WO2010039900 (Ablexis), the disclosures of which are explicitly
incorporated herein. Such transgenic loci in the art include
varying amounts of the human V(D) J repertoire.
[0006] Existing transgenic immunoglobulin loci tend to be based on
a single human DNA source. The potential diversity of human
antibody variable regions in non-human vertebrates bearing such
transgenic loci is thus confined by the repertoire used.
[0007] It would be desirable to provide for novel and potentially
expanded repertoire and diversity of human variable regions in
transgenic immunoglobulin loci and non-human vertebrates harbouring
these, as well as in antibodies produced following immunisation of
such animals.
SUMMARY OF THE INVENTION
[0008] The present inventors have discovered, by way of
construction of transgenic non-human vertebrates, immunisation,
antibody heavy chain collection and deep bioinformatics analysis,
how to rationally design for VH domains, heavy chains and
antibodies having long HCDR3s. These are useful for addressing
antigens (such as infectious disease pathogen antigens, receptors
and enzyme clefts) where a longer CDR better addresses the
target.
[0009] The present inventors also realised the possibility of
providing combinations of V, D and J gene segments in new ways to
provide synthetic gene segment combinations in immunoglobulin loci
that are not found in nature or in state-of-the-art loci. The
inventors realised the importance of this to provide for novel and
potentially expanded repertoire and diversity of human variable
regions in transgenic immunoglobulin loci and non-human vertebrates
harbouring these, as well as in antibodies produced following
immunisation of such animals. In one aspect, the inventors realised
that it would be desirable to bias the novel repertoire for the
production of antibodies having improved affinity and/or
biophysical characteristics, and/or wherein the range of epitope
specificities produced by means of such repertoire is novel,
provides for antibodies to epitopes that have hitherto been
intractable by prior transgenic immunoglobulin loci or difficult to
address. For example, the inventors envisaged a specific
application to bias the novel repertoire for the production of
antibodies useful in the therapy and/or prevention of infectious
disease.
[0010] To this end, in a first configuration of the invention,
there is provided A non-human vertebrate (optionally a mouse or a
rat) or vertebrate cell whose genome comprises:
(a) An immunoglobulin heavy chain locus comprising one or more
human V gene segments, one or more human D gene segments and one or
more human J gene segments upstream of a constant region;
optionally wherein the heavy chain locus is according to any
configuration of the invention described below; and (b) An
immunoglobulin light chain locus comprising either (i) one or more
human VH gene segments and one or more human J gene segments
upstream of a constant region (optionally a rearranged VHJLCL or
VHJ.lamda.CL, wherein the CL is C.lamda. or C.kappa.); or (ii) one
or more human VL gene segments, one or more human D gene segments
and one or more human JH gene segments upstream of a constant
region (optionally a rearranged VLDJHCL or V.lamda.DJHCL, wherein
the CL is C.lamda. or C.kappa.); or (iii) one or more human VL gene
segments selected from the group consisting of: a V.lamda.II gene
family member, V.lamda.VII 4A, V.lamda.II 2.1, V.lamda.VII 4A, a
V.lamda.1 gene family member, a VX3gene family member, IGLV1S2,
V.lamda.3-cML70, Ialh2, Ialvl, Ia3h3, Kv325, a V.kappa.I gene
family member, .kappa.I-15A (KL012), V.kappa.II family member, a
V.kappa.III family member, a V.kappa.I gene family member,
.kappa.I-15A (KL012), V.kappa.II A2 (optionally the A2a allele),
V.kappa. A27 (Humkv325) and a gene segment at least 80% identical
thereto, and one or more human JL gene segments upstream of a
constant region;
[0011] Wherein the gene segments in the heavy chain locus are
operably linked to the constant region thereof, and the gene
segments in the light chain locus are operably linked to the
constant region thereof, so that upon immunisation the mouse is
capable of producing an antibody comprising heavy chains produced
by recombination of the heavy chain locus and light chains produced
by recombination of the light chain locus.
[0012] In one aspect,
in (b)(i) the V gene segment repertoire of the light chain locus
comprises or consists of one or more VH gene segments selected from
the group consisting of: a VHIII gene family member (optionally, a
VHIIIa or VHIIIb family member), a VHIV gene family member, VHIII
9.1 (VH3-15), VHIII VH26 (VH3-23), VH3-21, LSG6.1, LSG12.1, DP77
(V3-21), VH H11, VH1GRR, ha3h2, VHI-ha1c1, VHIII-VH2-1, VH4.18,
ha4h3, Hv1051, 71-2, Hv1110, VH4.11, 71-4, VH251, VH1-69 and a gene
segment at least 80% identical thereto; or in (b)(iii) the light
chain locus V gene segment repertoire consists of one VL gene
segment type (optionally and one or mutants thereof), wherein the
VL gene segment is selected from said group of VL gene
segments.
[0013] In a second configuration of the present invention, there is
provided
[0014] A non-human vertebrate (optionally a mouse or a rat) or
vertebrate cell whose genome comprises:
(a) An immunoglobulin heavy chain locus comprising one or more
human V gene segments, one or more human D gene segments and one or
more human J gene segments upstream of a constant region; and (b)
(i) An unrearranged immunoglobulin light chain locus comprising one
or more human VH gene segments and one or more human J gene
segments upstream of a constant region, wherein each human VH gene
segment is a human gene segment identical to (or mutant of) a human
VH gene segment used to produce a rearranged VDJ encoding a heavy
chain variable region of a human antibody from an
antibody-expressing cell wherein said antibody binds to an antigen
of an infectious disease pathogen (optionally the variable regions
of said antibody being identical to an antibody from a human
individual suffering, susceptible to, or recovered from, a disease
or condition caused or mediated by an organism harbouring or
secreting said antigen; or from a human individual harbouring said
organism); or (ii) An immunoglobulin light chain locus comprising a
rearranged VJ region or VDJ region upstream of a constant region,
wherein the nucleotide sequence of the recombined region is
identical to a nucleotide sequence produced by the recombination of
a human J gene segment and optionally a human D gene segment with a
human VH gene segment that is identical to (or mutant of) the human
VH gene segment used to produce a rearranged VDJ encoding a heavy
chain variable region of a human antibody from an
antibody-expressing cell wherein said antibody binds to an antigen
of an infectious disease pathogen (optionally the variable regions
of said antibody being identical to an antibody from a human
individual suffering, susceptible to, or recovered from, a disease
or condition caused or mediated by an organism harbouring or
secreting said antigen; or from a human individual harbouring said
organism); (c) Wherein the gene segments in the heavy chain locus
are operably linked to the constant region thereof, and the gene
segments or VJ or VDJ in the light chain locus are operably linked
to the constant region thereof, so that upon immunisation the mouse
is capable of producing an antibody comprising heavy chains
produced by recombination of the heavy chain locus and light chains
derived from the light chain locus; (d) Optionally when (b)(i)
applies, each said VH gene segment in the light chain locus is
selected from the group consisting of: a VHIII gene family member
(optionally, a VHIIIa or VHIIIb family member), a VHIV gene family
member, VHIII 9.1 (VH3-15), VHIII VH26 (VH3-23), VH3-21, LSG6.1,
LSG12.1, DP77 (V3-21), VH H11, VH1GRR, ha3h2, VHI-ha1c1,
VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1110, VH4.11, 71-4,
VH251, VH1-69 and a gene segment at least 80% identical thereto;
(e) Optionally when (b)(ii) applies, the nucleotide sequence of the
recombined region is identical to a nucleotide sequence produced by
the recombination of a human J gene segment and optionally a human
D gene segment with a human VH gene segment selected from the group
consisting of: a VHIII gene family member (optionally, a VHIIIa or
VHIIIb family member), a VHIV gene family member, VHIII 9.1
(VH3-15), VHIII VH26 (VH3-23), VH3-21, LSG6.1, LSG12.1, DP77
(V3-21), VH H11, VH1GRR, ha3h2, VHI-ha1c1, VHIII-VH2-1, VH4.18,
ha4h3, Hv1051, 71-2, Hv1110, VH4.11, 71-4, VH251, VH1-69 and a gene
segment at least 80% identical thereto.
[0015] In one aspect, the V gene segment repertoire of the light
chain locus comprises or consists of one human VH gene segment;
optionally germline VH and one or more polymorphic variants
thereof, eg, where each polymorphic variant differs from the
germline VH nucleotide sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
positions. In one aspect, the V gene segment repertoire of the
light chain locus comprises or consists of human VH1-69 gene
segment; optionally germline VH1-69 and one or more polymorphic
variants thereof, eg, where each polymorphic variant differs from
the germline VH1-69 nucleotide sequence by 1, 2, 3, 4, 5, 6, 7, 8,
9 or 10 positions. An example of constructing an immunoglobulin
locus comprising VH1-69 and polymorphic variants is given below. By
using a particular gene segment (eg, one commonly found in human
antibodies raised in humans against an infection or other
condition) and one or more polymorphic variants thereof, it is
possible to provide a repertoire of genes and yet still bias the
antibody gene repertoire to a gene segment that is relevant to the
disease (eg, an infectious disease, such as a bacterial or viral
disease, eg, influenza). This provides a useful pool of genes from
which to ultimately generate and isolate a lead antibody for a
therapeutic/prophylactic against the disease in question. In an
example, the polymorphic variants are natural variants seen in
human beings or human populations. The skilled person will know of
sources of human antibody gene sequences, such as IMGT
(www.imgt.org), GenBank (www.ncbi.nlm.nih.gov/genbank) and the 1000
Genomes databases (www.1000genomes.org). Bioinformatics tools for
database manipulation are also readily available and known to the
skilled person, eg, as publicly available from the 1000 Genomes
Project/EBI (www.1000genomes.org)
[0016] In another aspect, the genome of said vertebrate or cell is
homozygous for light chain locus (b)(i) or (ii); optionally
wherein:
[0017] the V gene segment repertoire of the light chain loci
consists of one or more human VH gene segments selected from the
group consisting of: a VHIII gene family member (optionally, a
VHIIIa or VHIIIb family member), a VHIV gene family member, VHIII
9.1 (VH3-15), VHIII VH26 (VH3-23), VH3-21, LSG6.1, LSG12.1, DP77
(V3-21), VH H11, VH1GRR, ha3h2, VHI-ha1c1, VHIII-VH2-1, VH4.18,
ha4h3, Hv1051, 71-2, Hv1f10, VH4.11, 71-4, VH251, VH1-69 and a gene
segment at least 80% identical thereto; or [0018] the recombined VJ
or VDJ repertoire of the light chain loci consists of sequences
identical to one or more nucleotide sequences produced by the
recombination of a human VH gene segment selected from the group
consisting of: a VHIII gene family member (optionally, a VHIIIa or
VHIIIb family member), a VHIV gene family member, VHIII 9.1
(VH3-15), VHIII VH26 (VH3-23), VH3-21, LSG6.1, LSG12.1, DP77
(V3-21), VH H11, VH1GRR, ha3h2, VHI-ha1c1, VHIII-VH2-1, VH4.18,
ha4h3, Hv1051, 71-2, Hv1f10, VH4.11, 71-4, VH251, VH1-69 and a gene
segment at least 80% identical thereto, with a human J gene segment
and optionally a human D gene segment.
[0019] In another aspect, each immunoglobulin light chain locus of
said vertebrate or cell is according to (b)(i) and comprises only a
single human VH gene segment selected from the group consisting of:
a VHIII gene family member (optionally, a VHIIIa or VHIIIb family
member), a VHIV gene family member, VHIII 9.1 (VH3-15), VHIII VH26
(VH3-23), VH3-21, LSG6.1, LSG12.1, DP77 (V3-21), VH H11, VH1GRR,
ha3h2, VHI-ha1c1, VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1f10,
VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80%
identical thereto, optionally wherein the genome of the vertebrate
or cell is homozygous for said light chain so that all light chain
loci comprise the same, single human VH gene segment.
[0020] The invention provides a first method of isolating an
antibody that binds a predetermined antigen, the method
comprising
(a) providing a vertebrate (optionally a mouse or rat) according to
any preceding configuration or aspect; (b) immunising said
vertebrate with said antigen (optionally wherein the antigen is an
antigen of an infectious disease pathogen); (c) removing B
lymphocytes from the vertebrate and selecting one or more B
lymphocytes expressing antibodies that bind to the antigen; (d)
optionally immortalising said selected B lymphocytes or progeny
thereof, optionally by producing hybridomas therefrom; and (e)
isolating an antibody (eg, and IgG-type antibody) expressed by the
B lymphocytes.
[0021] A second method is provided comprising carrying out the
first method and the step of isolating from said B lymphocytes
nucleic acid encoding said antibody that binds said antigen;
optionally exchanging the heavy chain constant region nucleotide
sequence of the antibody with a nucleotide sequence encoding a
human or humanised heavy chain constant region and optionally
affinity maturing the variable region of said antibody; and
optionally inserting said nucleic acid into an expression vector
and optionally a host.
[0022] An aspect provides method of producing a polyclonal antibody
mixture, the method comprising carrying out the first method by
separately immunising first and second vertebrates (optionally
first and second mice or first and second rats) with antigen and
combining the anti-antigen antibodies isolated from each vertebrate
(or mutants or derivatives of said antibodies) to produce a
polyclonal antibody mixture; optionally wherein the following apply
separately or in combination ((i) and (ii); or (i) and (iii)):
(i) the vertebrates are immunised with the same antigen or
different antigens (optionally wherein the different antigens are
expressed by the same pathogenic organism (or a family member
thereof)); (ii) prior to immunisation the light chain loci of the
vertebrates contain the identical VH gene repertoire (optionally a
single VH gene) and optionally the identical J repertoire;
optionally the light chain loci of the mammals are identical prior
to immunisation; (iii) prior to immunisation the light chain loci
of the vertebrates contain the identical rearranged VJ or VDJ
repertoire (optionally a single VJ or VDJ); optionally the light
chain loci of the vertebrates are identical prior to
immunisation.
[0023] An aspect provides method of producing a polyclonal antibody
mixture, the method comprising carrying out the first method by
immunising one or a plurality of vertebrates (optionally mice or
rats) with first and second antigens and combining the anti-antigen
antibodies isolated from each vertebrate (or mutants or derivatives
of said antibodies) to produce a polyclonal antibody mixture;
optionally wherein the following apply separately or in combination
((i) and (ii); or (i) and (iii)):
(i) the antigens are expressed by the same pathogenic organism (or
a family member thereof)); (ii) prior to immunisation the light
chain loci of the vertebrates contain the identical VH gene
repertoire (optionally a single VH gene) and optionally the
identical J repertoire; optionally the light chain loci of the
mammals are identical prior to immunisation; (iii) prior to
immunisation the light chain loci of the vertebrates contain the
identical rearranged VJ or VDJ repertoire (optionally a single VJ
or VDJ); optionally the light chain loci of the vertebrates are
identical prior to immunisation.
[0024] An aspect provides method of producing host cells capable of
expressing a polyclonal antibody mixture, the method comprising, in
the second method:--
(a) immunising one or a plurality of vertebrates (optionally mice
or rats) with first and second antigens (optionally wherein the
different antigens are expressed by the same pathogenic organism
(or a family member thereof)); (b) isolating nucleic acid encoding
first and second anti-antigen antibodies from B lymphocytes from
said vertebrates; (c) determining the nucleotide sequences of the
heavy and light chain variable regions of the first antibody; (d)
determining the nucleotide sequence of the heavy variable region
and optionally the light chain variable region of the second
antibody; (e) inserting the heavy chain variable region coding
sequence of each antibody into a heavy chain expression vector;
optionally wherein the constant region coding sequence of each
heavy chain is exchanged for a nucleotide sequence that encodes a
human or humanised constant region; (f) inserting the light chain
variable region coding sequence of the first antibody into a light
chain expression vector; optionally wherein the constant region
coding sequence of the light chain of the first antibody is
exchanged for a nucleotide sequence that encodes a human or
humanised constant region; (g) optionally inserting the light chain
variable region coding sequence of the second antibody into a light
chain expression vector; optionally wherein the constant region
coding sequence of the light chain of the second antibody is
exchanged for a nucleotide sequence that encodes a human or
humanised constant region; and (h) introducing each expression
vector into a host cell and co-expressing antibody chains in a
mixture of said host cells to produce antibodies, each antibody
comprising one or both of said heavy chain variable regions and a
light chain; optionally wherein the expression vectors are
introduced together into the same host cell (eg, a CHO or HEK293
cell) so that the cell is capable of expressing antibody light
chains and heavy chains, such that the cell or a plurality of the
host cells express antibodies, each comprising one or both of said
heavy chain variable regions and a light chain; (i) optionally:
prior to immunisation the light chain loci of the vertebrates
contain the identical VH gene repertoire (optionally a single VH
gene segment) and optionally the identical J repertoire (optionally
a single J gene segment); optionally the light chain loci of the
vertebrates are identical prior to immunisation; or prior to
immunisation the light chain loci of the vertebrates contain the
identical rearranged VJ or VDJ repertoire (optionally a single VJ
or VDJ); optionally the light chain loci of the vertebrates are
identical prior to immunisation.
[0025] The invention also provides a method of producing a
monoclonal or polyclonal antibody mixture, optionally for use in
medicine, optionally for the treatment and/or prevention of an
infectious disease, wherein optionally wherein each antibody binds
an antigen of an infectious disease pathogen, preferably the same
antigen. The invention also provides the use of an isolated,
monoclonal or polyclonal antibody, or a mutant or derivative
antibody thereof in the manufacture of a medicament for the
treatment and/or prevention of an infectious disease, optionally
wherein the infectious disease is a disease caused by a bacterial
or viral pathogen.
[0026] The invention further provides an isolated antibody (eg,
IgG-type antibody) obtainable or obtained by a method of the
invention, or a mutant or derivative antibody thereof wherein (i)
the isolated antibody comprises two copies of the heavy chain
variable region of said first antibody paired with two copies of
the light chain variable region of said first antibody; or (ii) the
isolated antibody comprises two copies of the heavy chain variable
region of said second antibody paired with two copies of the light
chain variable region of said first antibody; or (iii) the isolated
antibody is a bispecific antibody comprising one copy of the heavy
chain variable region of said first antibody paired with a copy of
the light chain variable region of the first antibody, and one copy
of the heavy chain variable region of said the antibody paired with
a copy of the light chain variable region of the first antibody,
optionally wherein the bispecific antibody binds to said first and
second antigens are expressed by the same pathogenic organism (or a
family member thereof; optionally for use in medicine, optionally
for the treatment and/or prevention of an infectious disease.
[0027] In an aspect, there is provided a nucleotide sequence
encoding an antibody of the invention, optionally wherein the
nucleotide sequence is part of a vector.
[0028] In an aspect, there is provided a pharmaceutical composition
comprising the antibody or antibodies of the invention and a
diluent, excipient or carrier.
[0029] In a third configuration of the invention, there is provided
A non-human vertebrate (optionally a mouse or a rat) or vertebrate
cell whose genome comprises:
(a) An immunoglobulin heavy chain locus comprising either:-- (i)
one or more human VL gene segments, one or more human D gene
segments and one or more human J gene segments upstream of a
constant region (optionally a rearranged VLDJHCH or V.lamda.DJHCH);
or (ii) one or more human VH gene segments selected from the group
consisting of: a VHIII gene family member (optionally, a VHIIIa or
VHIIIb family member), a VHIV gene family member, VHIII 9.1
(VH3-15), VHIII VH26 (VH3-23), VH3-21, LSG6.1, LSG12.1, DP77
(V3-21), VH H11, VH1GRR, ha3h2, VHI-ha1c1, VHIII-VH2-1, VH4.18,
ha4h3, Hv1051, 71-2, Hv1110, VH4.11, 71-4, VH251, VH1-69 and a gene
segment at least 80% identical; one or more human D gene segments
and one or more human JH gene segments upstream of a constant
region; and (b) An immunoglobulin light chain locus comprising one
or more human V gene segments and one or more human J gene segments
upstream of a constant region, optionally wherein the light chain
locus is according to (b)(i) or (b)(ii) of the first configuration
of the invention;
[0030] Wherein the gene segments in the heavy chain locus are
operably linked to the constant region thereof, and the gene
segments in the light chain locus are operably linked to the
constant region thereof, so that upon immunisation the mouse is
capable of producing an antibody comprising heavy chains produced
by recombination of the heavy chain locus and light chains produced
by recombination of the light chain locus.
[0031] In a fourth configuration of the invention, there is
provided
[0032] A non-human vertebrate (optionally a mouse or a rat) or
vertebrate cell whose genome comprises:
(a) (i) An unrearranged immunoglobulin heavy chain locus comprising
one or more human VL gene segments, one or more human D gene
segments and one or more JH gene segments upstream of a constant
region, wherein each human VL gene segment is a human gene segment
identical to (or mutant of) a human VL gene segment used to produce
a rearranged VJ encoding a light chain variable region of a human
antibody from an antibody-expressing cell wherein said antibody
binds to an antigen of an infectious disease pathogen (optionally
the variable regions of said antibody being identical to an
antibody from a human individual suffering, susceptible to, or
recovered from, a disease or condition caused or mediated by an
organism harbouring or secreting said antigen; or from a human
individual harbouring said organism); or (ii) An immunoglobulin
heavy chain locus comprising a rearranged VJ region or VDJ region
upstream of a constant region, wherein the nucleotide sequence of
the recombined region is identical to a nucleotide sequence
produced by the recombination of a human J gene segment and
optionally a human D gene segment with a human VL gene segment that
is identical to (or mutant of) the human VL gene segment used to
produce a rearranged VJ encoding a light chain variable region of a
human antibody from an antibody-expressing cell wherein said
antibody binds to an antigen of an infectious disease pathogen
(optionally the variable regions of said antibody being identical
to an antibody from a human individual suffering, susceptible to,
or recovered from, a disease or condition caused or mediated by an
organism harbouring or secreting said antigen; or from a human
individual harbouring said organism); (b) An immunoglobulin light
chain locus comprising one or more human V gene segments and one or
more human J gene segments upstream of a constant region; and (c)
Wherein the gene segments in the light chain locus are operably
linked to the constant region thereof, and the gene segments or VJ
or VDJ in the heavy chain locus are operably linked to the constant
region thereof, so that upon immunisation the mouse is capable of
producing an antibody comprising light chains produced by
recombination of the light chain locus and heavy chains derived
from the heavy chain locus; (d) Optionally when (a)(i) applies,
each said VL gene segment in the heavy chain locus is selected from
the group consisting of a VL gene segment selected from the group
consisting of a V.lamda.II gene family member, V.lamda.VII 4A,
V.lamda.II 2.1, V.lamda.VII 4A, a V.lamda.1 gene family member, a
V.lamda.3gene family member, IGLV1S2, V.lamda.3-cML70, Ialh2,
Ialvl, Ia3h3, Kv325, a V.kappa.I gene family member, .kappa.I-15A
(KL012), V.kappa.II family member, a V.kappa.III family member, a
V.kappa.I gene family member, .kappa.I-15A (KL012), V.kappa.II A2
(optionally the A2a allele), V.kappa. A27 (Humkv325) and a gene
segment at least 80% identical thereto; (e) Optionally when (a)(ii)
applies, the nucleotide sequence of the recombined region is
identical to a nucleotide sequence produced by the recombination of
a human J gene segment and optionally a human D gene segment with a
human VL gene segment selected from the group consisting of a VL
gene segment selected from the group consisting of a V.lamda.II
gene family member, V.lamda.VII 4A, V.lamda.II 2.1, V.lamda.VII 4A,
a V.lamda.1 gene family member, a V.lamda.3gene family member,
IGLV1S2, VX3-cML70, Ialh2, Ialvl, Ia3h3, Kv325, a V.kappa.I gene
family member, .kappa.I-15A (KL012), V.kappa.II family member, a
V.kappa.III family member, a V.kappa.I gene family member,
.kappa.I-15A (KL012), V.kappa.II A2 (optionally the A2a allele), VK
A27 (Humkv325) and a gene segment at least 80% identical
thereto.
[0033] In one aspect of the fourth configuration of the invention,
the genome of said vertebrate or cell is homozygous for heavy chain
locus (a)(i) or (ii); optionally wherein:
[0034] the V gene segment repertoire of the heavy chain loci
consists of one or more human VL gene segments selected from the
group consisting of a VL gene segment selected from the group
consisting of a V.lamda.II gene family member, V.lamda.VII 4A,
V.lamda.II 2.1, V.lamda.VII 4A, a V.lamda.1 gene family member, a
VX3gene family member, IGLV1S2, VX3-cML70, Ialh2, Ialvl, Ia3h3,
Kv325, a V.kappa.I gene family member, .kappa.I-15A (KL012),
V.kappa.II family member, a V.kappa.III family member, a V.kappa.I
gene family member, .kappa.I-15A (KL012), V.kappa.II A2 (optionally
the A2a allele), VK A27 (Humkv325) and a gene segment at least 80%
identical thereto; or
[0035] the recombined VJ or VDJ repertoire of the heavy chain loci
consists of sequences identical to one or more nucleotide sequences
produced by the recombination of a human VL gene segment selected
from the group consisting of a VL gene segment selected from the
group consisting of a V.lamda.II gene family member, V.lamda.VII
4A, V.lamda.II 2.1, V.lamda.VII 4A, a V.lamda.1 gene family member,
a VX3gene family member, IGLV1S2, VX3-cML70, Ialh2, Ialvl, Ia3h3,
Kv325, a V.kappa.I gene family member, .kappa.I-15A (KL012),
V.kappa.II family member, a V.kappa.III family member, a V.kappa.I
gene family member, .kappa.I-15A (KL012), V.kappa.II A2 (optionally
the A2a allele), V.kappa. A27 (Humkv325) and a gene segment at
least 80% identical thereto with a human J gene segment and
optionally a human D gene segment.
[0036] The invention provides a monoclonal or polyclonal antibody
composition prepared by immunisation of at least one vertebrate
(eg, mouse or rat) according to any preceding configuration or
aspect with an antigen, optionally wherein the antigen is an
antigen of an infectious disease pathogen, optionally wherein the
same antigen is used to immunise all the vertebrates; optionally
wherein the antibody or antibodies are IgG-type.
[0037] The invention also provides an isolated chimaeric antibody
for treating and/or preventing an infectious disease or condition,
the antibody comprising a non-human vertebrate (optionally a mouse
or rat) heavy chain constant regions and human variable regions
that bind an antigen of an infectious disease pathogen, wherein the
antibody is obtainable or obtained in a method comprising
immunisation of a non-human vertebrate of the invention with said
antigen.
[0038] The invention also provides an isolated human antibody for
treating and/or preventing an infectious disease or condition, the
antibody comprising human heavy chain constant regions and human
variable regions that bind an antigen of an infectious disease
pathogen, wherein the antibody is obtainable or obtained in a
method comprising affinity maturation of antibody variable regions
in vivo in a transgenic non-human vertebrate (eg, mouse or rat)
when said variable regions are operably linked to heavy chain
constant regions of said vertebrate (eg, mouse or rat heavy chain
constant regions) by (a) immunisation of a vertebrate of the
invention with said antigen, (b) isolation of nucleic acid encoding
a chimaeric antibody according to the invention, (c) replacing the
nucleotide sequences of the nucleic acid that encode the non-human
vertebrate heavy chain constant regions with nucleotide sequence
encoding human heavy chain constant regions to produce nucleic acid
encoding a human antibody; (d) expressing the human antibody in
vitro (optionally from CHO or HEK293 cells harbouring the human
nucleic acid) and (e) isolating the human antibody (optionally with
further affinity maturation of the antibody and/or producing a
derivative thereof).
[0039] An aspect provides a mixture of first and second human
antibodies, each antibody being capable of binding to an antigen of
an infectious disease pathogen (optionally wherein the first
antibody binds a first antigen and the second antibody binds a
second antigen, said antigens being from the same pathogen; or
wherein the antigens are the same). In an embodiment, a common
light chain is used which enables simplified manufacture of the
antibody mixture. Thus, there is provided in the mixture, the light
chain amino acid sequence of the first antibody that is identical
to the light chain amino acid sequence of the second antibody, or
has up to 15 amino acid changes therefrom.
[0040] The invention further provides a host cell comprising one or
more expression vectors encoding 3 or more first and second
antibody heavy and light chains.
[0041] In a fifth configuration of the invention, there is provided
A synthetic immunoglobulin locus comprising one or more variable
and J gene segments (and optionally one or more D gene segments)
operably linked 5' of a constant region, wherein the locus
comprises a 5' to 3' V(D)J arrangement selected from the group
consisting of immunoglobulin locus can be constructed with one or
more of the following arrangements (5' to 3'):-- [0042] (a) [V
(heavy, lambda or kappa)]--[two-turn RSS]--[one-turn
RSS]--[D]--[JH], wherein said RSSs are in an opposite orientation;
[0043] (b) [VH]--[D]--[two-turn RSS]--[one-turn RSS]--[J lambda],
wherein said RSSs are in an opposite orientation; [0044] (c)
[VH]--[D]--[one-turn RSS]--[two-turn RSS]--[J kappa], wherein said
RSSs are in an opposite orientation; [0045] (d) [VH or V
kappa]-[two-turn RSS]--[one-turn RSS]--[J lambda], wherein said
RSSs are in an opposite orientation; [0046] (e) [V kappa]-[one-turn
RSS]--[two-turn RSS]--[JH or J lambda], wherein said RSSs are in an
opposite orientation; [0047] (f) [V (heavy, lambda or
kappa)]--[one-turn RSS]--[two-turn RSS]--[D]--[JH], wherein said
RSSs are in an opposite orientation; [0048] (g)
[VH]--[D]--[one-turn RSS]--[two-turn RSS]--[J lambda], wherein said
RSSs are in an opposite orientation; [0049] (h)
[VH]--[D]--[two-turn RSS]--[one-turn RSS]--[J kappa], wherein said
RSSs are in an opposite orientation; [0050] (i) [VH or V
kappa]-[one-turn RSS]--[two-turn RSS]--[J lambda], wherein said
RSSs are in an opposite orientation; [0051] (j) [V kappa]-[two-turn
RSS]--[one-turn RSS]--[JH or J lambda], wherein said RSSs are in an
opposite orientation.
[0052] In a sixth configuration, the invention also provides means
for generating VH domains, heavy chains and antibodies having a
long HCDR3 length. In this context, the invention provides:--
[0053] A non-human vertebrate (eg, a mouse or a rat) or a non-human
vertebrate cell (eg, a mouse cell or a rat cell) whose genome
comprises a human immunoglobulin D gene segment repertoire that is
biased to the human D2 and/or D3 family or biased to one, more or
all human D gene segments selected from the group D1-26, D2-2,
D3-9, D3-10, D3-22, D4-17, D6-13 and D6-19.
[0054] A non-human vertebrate (eg, a mouse or a rat) or a non-human
vertebrate cell (eg, a mouse cell or a rat cell), optionally
according to any preceding claim, whose genome comprises a human
immunoglobulin VH gene segment repertoire that is biased to one,
more or all of gene segments selected from the group VH1-2, VH1-3,
VH1-8, VH1-18, VH5-51, VH1-69, VH2-5, VH3-7, VH3-9, VH3-11, VH3-13,
VH3-20, VH3-21, VH3-23, VH4-4, VH6-1 and VH7-4-1.
[0055] A non-human vertebrate (eg, a mouse or a rat) or a non-human
vertebrate cell (eg, a mouse cell or a rat cell) whose genome
comprises a human immunoglobulin JH gene segment repertoire that is
biased to human JH6.
[0056] A monoclonal or polyclonal antibody composition or a
population of antibody-producing cells for producing such
composition, wherein the composition or population is prepared by
immunising at least one vertebrate according to any preceding claim
with an antigen, wherein the antibody or antibodies have human
heavy chain variable regions comprising non-human vertebrate
AID-pattern somatic hypermutations, (eg, mouse or rat AID-pattern
mutations) when compared to corresponding human germline V, D and J
sequences and/or non-human (eg, mouse or rat) terminal
deoxynucleotidyl transferase (TdT)-pattern junctional mutations
when compared to corresponding human germline V, D and J sequences;
wherein the composition comprises at least one antigen-specific
antibody having a HCDR3 length of at least 20 amino acids
(according to IMGT).
[0057] A repertoire of antibody heavy chains (eg, provided by
antibodies) comprising one or more heavy chains whose variable
domain HCDR3 has a length of at least 20 amino acids (according to
IMGT) and derived from the recombination of a human VH, D and JH,
wherein the VH is selected from the group
VH1-2*02, VH1-3*01, VH1-8*01, VH1-18*01, VH2-5*10, VH3-7*01,
VH3-9*01, VH3-11*01, VH3-13*01, VH3-21*03, VH3-23*04, VH4-4*02,
VH6-1*01 and VH7-4-1*01 and
[0058] the D is selected from the group
D2-2*02, D3-9*01, D3-10*01 and D3-22*01, or
D2-2*02, D3-9*01 and D3-10*01, or
D3-9*01 and D3-10*01, or
D1-26, D2-2, D3-9, D3-10, D3-22, D4-17, D6-13 and D6-19, or
D1-26*01, D2-2*02, D3-9*01, D3-10*01, D3-22*01, D4-17*01, D6-13*01
and D6-19*01, or
D2-2, D3-9, D3-10, D3-22, D4-17, D6-13 and D6-19, or D2-2*02,
D3-9*01, D3-10*01, D3-22*01, D4-17*01, D6-13*01 and D6-19*01,
or
D1-26, D2-2, D3-10 and D6-19, or
D2-2, D3-9 and D3-10;
[0059] and optionally the JH is JH6 (eg, JH6*02);
Wherein
[0060] (a) the heavy chain variable domain has been produced in
vivo in a non-human vertebrate (eg, a mouse or a rat); and/or (b)
the heavy chain variable domain comprises non-human vertebrate
AID-pattern somatic hypermutations, (eg, mouse or rat AID-pattern
mutations) when compared to corresponding human germline V, D and J
sequences and/or non-human (eg, mouse or rat) terminal
deoxynucleotidyl transferase (TdT)-pattern junctional mutations
when compared to corresponding human germline V, D and J
sequences.
BRIEF DESCRIPTION OF THE FIGURES
[0061] FIGS. 1 to 3: Schematic illustrating a protocol for
producing recombineered BAC vectors to add V gene segments into a
mouse genome;
[0062] FIG. 4: Schematic illustrating a protocol for adding V gene
segments to a mouse genome using sequential recombinase mediated
cassette exchange (sRMCE);
[0063] FIG. 5 (in 4 parts): Alignment of 13 IGHV1-69 alleles
showing the variable (V) coding region only. Nucleotides that
differ from VH1-69 allele *01 are indicated at the appropriate
position whereas identical nucleotides are marked with a dash.
Where nucleotide changes result in amino acid differences, the
encoded amino acid is shown above the corresponding triplet. Boxed
regions correspond to CDR1, CDR2 and CDR3 as indicated; and
[0064] FIG. 6: RSS structure and recombination schematic.
DETAILED DESCRIPTION OF THE INVENTION
[0065] A source for human V, D and J gene segments is Bacterial
Artificial Chromosomes (RPCI-11 BACs) obtained from Roswell Park
Cancer Institute (RPCI)/Invitrogen. See
http://bacpac.chori.org/hmale11.htm, which describes the BACs as
follows:--
"RPCI--11 Human Male BAC Library
[0066] The RPCI-11 Human Male BAC Library (Osoegawa et al., 2001)
was constructed using improved cloning techniques (Osoegawa et al.,
1998) developed by Kazutoyo Osoegawa. The library was generated by
Kazutoyo Osoegawa. Construction was funded by a grant from the
National Human Genome Research Institute (NHGRI, NIH)
(#1R01RG01165-03). This library was generated according to the new
NHGRI/DOE "Guidance on Human Subjects in Large-Scale DNA Sequencing
. . . .
[0067] "Male blood was obtained via a double-blind selection
protocol. Male blood DNA was isolated from one randomly chosen
donor (out of 10 male donors)". [0068] Osoegawa K, Mammoser A G, Wu
C, Frengen E, Zeng C, Catanese J J, de Jong P J; Genome Res. 2001
March; 11(3):483-96; "A bacterial artificial chromosome library for
sequencing the complete human genome"; [0069] Osoegawa, K., Woon,
P. Y., Zhao, B., Frengen, E., Tateno, M., Catanese, J. J, and de
Jong, P. J. (1998); "An Improved Approach for Construction of
Bacterial Artificial Chromosome Libraries"; Genomics 52, 1-8.
[0070] As a source of antibody gene segment sequences, the skilled
person will also be aware of the following available databases and
resources (including updates thereof):--
1.1. The Kabat Database (G. Johnson and T. T. Wu, 2002;
http://www.kabatdatabase.com). Created by E. A. Kabat and T. T. Wu
in 1966, the Kabat database publishes aligned sequences of
antibodies, T-cell receptors, major histocompatibility complex
(MHC) class I and II molecules, and other proteins of immunological
interest. A searchable interface is provided by the SeqhuntII tool,
and a range of utilities is available for sequence alignment,
sequence subgroup classification, and the generation of variability
plots. See also Kabat, E. A., Wu, T. T., Perry, H., Gottesman, K.,
and Foeller, C. (1991) Sequences of Proteins of Immunological
Interest, 5th ed., NIH Publication No. 91-3242, Bethesda, Md.,
which is incorporated herein by reference, in particular with
reference to human gene segments for use in the present invention.
1.2. KabatMan (A. C. R. Martin, 2002;
http://www.bioinf.org.uk/abs/simkab.html). This is a web interface
to make simple queries to the Kabat sequence database. 1.3. IMGT,
the International ImMunoGeneTics Information System.RTM.; M. -P.
Lefranc, 2002; http://imqt.cines.fr). IMGT is an integrated
information system that specializes in antibodies, T cell
receptors, and MHC molecules of all vertebrate species. It provides
a common portal to standardized data that include nucleotide and
protein sequences, oligonucleotide primers, gene maps, genetic
polymorphisms, specificities, and two-dimensional (2D) and
three-dimensional (3D) structures. IMGT includes three sequence
databases (IMGT/LIGM-DB, IMGT/MHC-DB, IMGT/PRIMERDB), one genome
database (IMGT/GENE-DB), one 3D structure database
(IMGT/3Dstructure-DB), and a range of web resources ("IMGT
Marie-Paule page") and interactive tools. 1.4. V-BASE (I. M.
Tomlinson, 2002; http://www.mrc-cpe.cam.ac.uk/vbase). V-BASE is a
comprehensive directory of all human antibody germline variable
region sequences compiled from more than one thousand published
sequences. It includes a version of the alignment software DNAPLOT
(developed by Hans-Helmar Althaus and Werner Muller) that allows
the assignment of rearranged antibody V genes to their closest
germline gene segments. 1.5. Antibodies--Structure and Sequence (A.
C. R. Martin, 2002; http://www.bioinf.org.uk/abs). This page
summarizes useful information on antibody structure and sequence.
It provides a query interface to the Kabat antibody sequence data,
general information on antibodies, crystal structures, and links to
other antibody-related information. It also distributes an
automated summary of all antibody structures deposited in the
Protein Databank (PDB). Of particular interest is a thorough
description and comparison of the various numbering schemes for
antibody variable regions. 1.6. AAAAA--AHo's Amazing Atlas of
Antibody Anatomy (A. Honegger, 2001;
http://www.unizh.ch/.about.antibody). This resource includes tools
for structural analysis, modeling, and engineering. It adopts a
unifying scheme for comprehensive structural alignment of antibody
and T-cell-receptor sequences, and includes Excel macros for
antibody analysis and graphical representation. 1.7. WAM--Web
Antibody Modeling (N. Whitelegg and A. R. Rees, 2001;
http://antibody.bath.ac.uk). Hosted by the Centre for Protein
Analysis and Design at the University of Bath, United Kingdom.
Based on the AbM package (formerly marketed by Oxford Molecular) to
construct 3D models of antibody Fv sequences using a combination of
established theoretical methods, this site also includes the latest
antibody structural information. 1.8. Mike's Immunoglobulin
Structure/Function Page (M. R. Clark, 2001;
http://www.path.cam.ac.uk/.about.mrc7/mikeimages.html) These pages
provide educational materials on immunoglobulin structure and
function, and are illustrated by many colour images, models, and
animations. Additional information is available on antibody
humanization and Mike Clark's Therapeutic Antibody Human Homology
Project, which aims to correlate clinical efficacy and
anti-immunoglobulin responses with variable region sequences of
therapeutic antibodies. 1.9. The Antibody Resource Page (The
Antibody Resource Page, 2000; http://www.antibodyresource.com).
This site describes itself as the "complete guide to antibody
research and suppliers." Links to amino acid sequencing tools,
nucleotide antibody sequencing tools, and hybridoma/cell-culture
databases are provided. 1.9. Humanization bY Design (J. Saldanha,
2000; http://people.cryst.bbk.ac.uk/.about.ubcq07s). This resource
provides an overview on antibody humanization technology. The most
useful feature is a searchable database (by sequence and text) of
more than 40 published humanized antibodies including information
on design issues, framework choice, framework back-mutations, and
binding affinity of the humanized constructs.
[0071] See also Antibody Engineering Methods and Protocols, Ed.
Benny K C Lo, Methods in Molecular Biology.TM., Human Press. Also
at
http://www.blogsua.com/pdf/antibody-engineering-methods-and-protocolsanti-
body-engineering-methods-and-protocols.pdf
[0072] In one embodiment throughout the present text, "germline"
refers to the canonical germline gene segment sequence.
[0073] The present invention is directed to the provision of novel
V, D and J pairings in immunoglobulin heavy and light chain loci.
Novel, biased antibody diversities and potentially expanded
diversities are provided. One aspect of the invention exploits the
natural pairing of compatible recombination signal sequences (RSSs)
during antibody V(D)J recombination in vivo, and this aspect of the
invention provides new, synthetic combinations of V, D and J gene
segments using the observation of RSS compatibility.
[0074] Another aspect of the invention is based on the observation
of V, D and J usage bias in naturally-occurring human antibodies
raised against infectious disease pathogens. The invention is
useful for manipulating the antibody gene diversity in transgenic
non-human animals, thus providing for novel and potentially
expanded diversity or diversity that is biased towards variable
gene usage common to antibodies useful for treating and/or
preventing certain diseases or conditions, such as infectious
diseases. This ability to bias the antibody repertoire also
provides methods of simplifying the production of antibody
mixtures, such as polyclonal antibody therapeutics useful for the
treatment and/or prevention of infectious diseases where a
polyclonal approach to target multiple pathogen antigens is
desirable. To this end, the present invention also provides
bispecific antibodies that are capable of binding to more than one
antigen (eg, multiple infectious antigens expressed by the same
pathogen), thus providing advantages (such as manufacturing, dosing
and administration advantages) not possible with polyclonal
antibody mixtures.
[0075] The present invention provides vertebrates and cells, such
as transgenic mice or rats or transgenic mouse or rat cells.
Furthermore, the invention relates to methods of using the
vertebrates to isolate antibodies or nucleotide sequences encoding
antibodies. Antibodies, nucleotide sequences, pharmaceutical
compositions and uses are also provided by the invention.
To this End, the Present Invention Provides, in a First
Configuration
[0076] A non-human vertebrate (optionally a mouse or a rat) or
vertebrate cell whose genome comprises: [0077] (a) An
immunoglobulin heavy chain locus comprising one or more human V
gene segments (optionally a plurality of VH), one or more human D
gene segments and one or more human J gene segments upstream of a
constant region; optionally wherein the heavy chain locus is
according to (a) of the second configuration described below; and
[0078] (b) An immunoglobulin light chain locus comprising either
[0079] (i) one or more human VH gene segments and one or more human
J gene segments upstream of a constant region (optionally a
rearranged V.sub.HJ.sub.LC.sub.L or V.sub.HJ.sub.AC.sub.L, wherein
the C.sub.L is C.sub..lamda. or C.sub.K); or [0080] (ii) one or
more human VL gene segments, one or more human D gene segments and
one or more human J.sub.H gene segments upstream of a constant
region (optionally a rearranged V.sub.LDJ.sub.HC.sub.L or
V.sub..lamda.DJ.sub.HC.sub.L, wherein the C.sub.L is C.sub..lamda.
or C.sub.K); or [0081] (iii) one or more human VL gene segments
selected from the group consisting of: a V.sub..lamda.II gene
family member, V.sub..lamda.VII 4A, V.sub..lamda.II 2.1,
V.sub..lamda.VII 4A, a V.sub..lamda.1 gene family member, a
V.sub..lamda.3 gene family member, IGLV1S2, V.sub..lamda.3-cML70,
Ialh2, Ialvl, Ia3h3, Kv325, a V.kappa.I gene family member,
.kappa.I-15A (KL012), V.sub..kappa.II family member, a
V.sub..kappa.III family member, a V.kappa.I gene family member,
.kappa.I-15A (KL012), V.sub..kappa.II A2 (optionally the Ata
allele), V.sub..kappa. A27 (Humkv325) and a gene segment at least
80% identical thereto, and one or more human J.sub.L gene segments
upstream of a constant region; optionally the one or more VL gene
segments are selected from List A1, A2, A1.1, A1.2, A2.1, A2.2,
A2.3 or A2.4 below. [0082] Wherein the gene segments in the heavy
chain locus are operably linked to the constant region thereof, and
the gene segments in the light chain locus are operably linked to
the constant region thereof, so that upon immunisation the mouse is
capable of producing an antibody comprising heavy chains produced
by recombination of the heavy chain locus and light chains produced
by recombination of the light chain locus.
[0083] This configuration of the invention, thus, provides for the
possibility of novel, synthetic antibody and gene repertoires in a
transgenic non-human vertebrate, such as a mouse or rat. Such new
repertoires are desirable, since they provide for the possibility
of a novel pool of antibodies from which lead antibodies can be
selected following immunisation of the vertebrate with a
predetermined antigen. This, therefore, provides for a pool from
which antibodies with desirable characteristics can be isolated,
for example, antibodies with relatively high affinity for specific
target antigen binding. It is desirable to isolate high affinity
antibodies directly from the immunised vertebrate, since this can
provide for an antibody lead that is potentially useful as a
therapeutic and/or prophylactic medicament without the need for
further extensive affinity maturation (eg, by in vitro antibody
display such as ribosome display or phage display). Modification of
the effector portions of the antibody can be made as desired (eg,
humanisation of the constant region), without the need to
manipulate the sequences of the variable regions. Alternatively, or
additionally, the pool of antibodies may allow for selection of a
lead antibody with desirable biophysical characteristics and/or
epitope specificity. The latter may be important for finding lead
antibodies against epitopes that have not previously raised
therapeutic and/or prophylactic antibodies or epitopes that are
difficult to reach by antibodies generated by antibody gene
diversities generated by prior non-human vertebrates bearing
transgenic immunoglobulin loci, eg, those based on the single human
genome represented by the RPCI-11 BACs.
[0084] The cells of the invention (according to any aspect or
configuration) is, for example, a B-cell, hybridoma or a stem cell,
optionally an embryonic stem cell or haematopoietic stem cell. In
one aspect the ES cell is derived from the mouse C57BL/6N,
C57BL/6J, 129S5 or 129Sv strain. In one aspect the non-human
vertebrate is a rodent, suitably a mouse, and cells of the
invention, are rodent cells or ES cells, suitably mouse ES cells.
The ES cells of the present invention can be used to generate
animals using techniques well known in the art, which comprise
injection of the ES cell into a blastocyst followed by implantation
of chimaeric blastocystys into females to produce offspring which
can be bred and selected for homozygous recombinants having the
required insertion. In one aspect the invention relates to a
transgenic animal comprised of ES cell-derived tissue and host
embryo derived tissue. In one aspect the invention relates to
genetically-altered subsequent generation animals, which include
animals having a homozygous recombinants for the VDJ and/or VJ
regions.
[0085] Vertebrates bearing one or more light chain loci according
to (b)(i) and (ii) provide for novel and potentially expanded
antibody and gene repertoires by exploiting synthetic,
non-naturally-occurring, combinations of immunoglobulin gene
segments (V, D, J, C). In this respect, the present inventors have
realised the desirability and possibility of providing for antibody
and gene repertoires that mix heavy chain gene segments with those
of light chain loci. This is based on observations of the
inventors: Firstly, nature suggests the possibility of functional
antibodies having VH-VH or VL-VL pairings (as opposed to more
classical VH-VL pairings). For example, reference is made to heavy
chain antibodies of Camelidae which produce antibodies with paired
VH domains and is devoid of light chain VL domains (eg, see Nature.
1993 Jun. 3; 363(6428):446-8; Naturally occurring antibodies devoid
of light chains; Hamers-Casterman C, Atarhouch T, Muyldermans S,
Robinson G, Hamers C, Songa E B, Bendahman N, Hamers R). These
antibodies function to specifically bind antigen, such antibodies
being found in the blood of such Camelidae (eg, llamas, camels,
alpacas). Such antibodies with VH pairs can also be synthetically
produced to provide therapeutic and prophylactic medicaments (eg,
see WO1994004678, WO2004041862, WO2004041863). Transgenic mice also
can produce such heavy chain antibodies and the in vivo production
of the antibodies allows the mouse's immune system to select for
VH-VH pairings, sometimes selecting for such pairings in which
mutations have been introduced in vivo by the mouse to accommodate
the pairing (WO2010109165A2). Thus, the inventors realised that the
adoption of an in vivo antibody production system (rather than an
in vitro system such as phage or ribosome display of antibodies) is
desirable to accommodate the synthetic immunoglobulin gene segment
combinations that are now contemplated by the present
invention.
[0086] A second observation of the present inventors lies in the
architecture of naturally-occurring immunoglobulin loci, and in
particular the arrangement of recombination signal sequences (RSSs)
that mediate V(D)J recombination in vivo (see, eg, Cell. 2002
April; 109 Suppl:S45-55. The mechanism and regulation of
chromosomal V(D)J recombination; Bassing C H, Swat W, Alt F W, the
disclosure of which is incorporated herein by reference). As
illustrated in FIG. 6, two types of RSS element have been
identified: a one-turn RSS (12-RSS) and a two-turn RSS (23-RSS). In
natural VJ recombination in the lambda light chain locus,
recombination if effected between a two-turn RSS that lies 3' of a
V lambda and a one-turn RSS that lies 5' of a J lambda, the RSSs
being in opposite orientation. In natural VJ recombination in the
kappa light chain locus, recombination if effected between a
one-turn RSS that lies 3' of a V kappa and a two-turn RSS that lies
5' of a J kappa, the RSSs being in opposite orientation. In natural
VD recombination in the heavy chain locus, recombination if
effected between a two-turn RSS that lies 3' of a VH and a one-turn
RSS that lies 5' of a D, the RSSs being in opposite orientation. In
natural DJ recombination in the heavy chain locus, recombination if
effected between a one-turn RSS that lies 3' of a D and a two-turn
RSS that lies 5' of a JH, the RSSs being in opposite orientation.
Thus, generally a two-turn RSS is compatible with a one-turn RSS in
the opposite orientation. The inventors realised that they could
use this observation in constructing transgenic immunoglobulin loci
such that a 5' gene segment can recombine with a 3' gene segment
(eg, a V with a J; or a V with a D) when there is provided a
two-turn RSS and a one-turn RSS in the opposite orientation, with
each RSS adjacent a respective one of the gene segments. Thus, the
inventors realised in one embodiment that an immunoglobulin locus
can be constructed with one or more of the following arrangements
(5' to 3'):-- [0087] (k) [V (heavy, lambda or kappa)]--[two-turn
RSS]--[one-turn RSS]--[D]--[JH], wherein said RSSs are in an
opposite orientation; [0088] (l) [VH]--[D]--[two-turn
RSS]--[one-turn RSS]--[J lambda], wherein said RSSs are in an
opposite orientation; [0089] (m) [VH]--[D]--[one-turn
RSS]--[two-turn RSS]--[J kappa], wherein said RSSs are in an
opposite orientation; [0090] (n) [VH or V kappa]-[two-turn
RSS]--[one-turn RSS]--[J lambda], wherein said RSSs are in an
opposite orientation; [0091] (o) [V kappa]-[one-turn
RSS]--[two-turn RSS]--[JH or J lambda], wherein said RSSs are in an
opposite orientation; [0092] (p) [V (heavy, lambda or
kappa)]--[one-turn RSS]--[two-turn RSS]--[D]--[JH], wherein said
RSSs are in an opposite orientation; [0093] (q)
[VH]--[D]--[one-turn RSS]--[two-turn RSS]--[J lambda], wherein said
RSSs are in an opposite orientation; [0094] (r)
[VH]--[D]--[two-turn RSS]--[one-turn RSS]--[J kappa], wherein said
RSSs are in an opposite orientation; [0095] (s) [VH or V
kappa]-[one-turn RSS]--[two-turn RSS]--[J lambda], wherein said
RSSs are in an opposite orientation; [0096] (t) [V kappa]-[two-turn
RSS]--[one-turn RSS]--[JH or J lambda], wherein said RSSs are in an
opposite orientation.
[0097] The skilled person will realise that standard molecular
biology techniques can be used to provide vectors comprising
synthetic combinations of RSS with V, D or J for use in this aspect
of the invention, such that the vectors can be used to build a
transgenic immunoglobulin locus (eg, using homologous recombination
and/or recombinase mediated cassette exchange as known in the art,
eg, see U.S. Pat. No. 7,501,552 (Medarex), U.S. Pat. No. 5,939,598
(Abgenix), U.S. Pat. No. 6,130,364 (Abgenix), WO02/066630
(Regeneron), WO2011004192 (Genome Research Limited), WO2009076464,
WO2009143472 and WO2010039900 (Ablexis), the disclosures of which
are explicitly incorporated herein. For example, such synthetic
combinations with RSS and gene segments can be made using standard
recombineering techniques in E. coli to construct BAC vectors
harbouring the synthetic combination prior to insertion in
embryonic stem cells using homologous recombination or RMCE (eg,
using cre/lox site-specific recombination). Details of
recombineering can be found at www.genebridges.com and in EP1034260
and EP1204740 the disclosures of which are explicitly incorporated
herein.
[0098] In one embodiment of (b)(i), all of the light chain locus V
gene segments are human VH gene segments (optionally with one or
more human V lambda gene segments).
[0099] In one embodiment of (b)(i), the constant region is a mouse,
rat or human CL, eg, C.lamda.. In one embodiment, the J and
constant regions are provided by one or more human
J.lamda.C.lamda..
[0100] Although having utility generally to any antigen and disease
setting, vertebrates bearing one or more light chain loci according
to (b)(iii) are useful, in particular, for generating antibody
leads against infectious disease pathogens. In this respect, the
present inventors have realised the desirability and possibility of
providing for antibody and gene repertoires that are biased to
immunoglobulin gene segments commonly found in natural antibody
reactions of humans to infectious disease pathogens. The inventors
realised that it would be desirable to provide for vertebrates,
cells, methods etc for the production of therapeutic and/or
prophylactic antibodies based on natural human immune responses to
antigens, such as antigens of infectious disease pathogens. In this
respect, the literature observes frequently used immunoglobulin
gene segments to raise anti-infective responses in humans (Table
1).
TABLE-US-00001 TABLE 1 Immunoglobulin Gene Usage in Human Antibody
Responses to Infectious Disease Pathogens REFERENCES V GENE ANTIGEN
ORGANISM [Human Ab Source] BACTERIAL PATHOGENS KAPPA V GENES
Haemophilus influenzae type b Haemophilus influenzae 1. Lonberg,
Nat Biotech 2005; Vk II germline gene A2 + JK3 polysaccharide (Hib
PS) [human PBMCs] Vk II family gene + JK4 2. Adderson et al, J Clin
Invest 1992; 94% identical to the A27 [Human PBLs] (Humkv325) germ
line gene 3. Chung et al, J Immunol 1993 a V.kappa.I gene family
member; .kappa.I- 4. Nadel et al, J Immunol 1998 15A (KL012) 5.
Feeney et al, J Clin Invest 1996 LAMBDA V GENES 6. Lucas et al,
Infect Immun 1994; Four V.lamda. VII family members that [Human
PBLs] are 96-98% identical to 7. Adderson et al, J Clin Invest
1993; each other [Human PBLs] V.lamda. II family members (82, 89
and 8. Granoff et al, J Clin Invest 1993; 91% homologous to
V.lamda.2.1 gene) [Human PBLs] +VHIII segments closely 9. Azmi et
al, Infect Immun 1994; homologous to germline tonsil cells gene 9.1
V.sub..lamda.VII 4A All with J.lamda. homologous to germline
J.lamda.2 and J.lamda.3 VH GENES VH 96% identical to the VH germ
line gene segment DP77 (V3-21) LSG6.1, LSG12.1, V.sub.HIII VH26,
V.sub.HIII 9.1 VH and VL COMBINATIONS V.sub.HIII 9.1 +
V.sub..lamda.VII 4A V.sub.HIII 9.1 + V.sub..lamda.II 2.1 V.sub.HIII
9.1 + V.sub..kappa.II A2 V.sub.HIII VH26 + V.sub..lamda.II 2.1
V.sub.HIII 9.1; V.sub.HIII H11; V.sub.HIII VH26 Polysaccharide
capsule E coli K1 9. Azmi et al, Infect Immun 1994 of E coli
.kappa.I 15A K1 V.lamda.2.1 Meningococcal B polysaccharide;
Neisseria meningitidis Group B
Poly[.alpha.(2.fwdarw.8)-N-acetylneuramic acid VIRAL PATHOGENS
VHIII or VHIV family member Herpes family virus 10. Huang et al, J
Clin Invest 1992; V.lamda.I or V.lamda.3 member HSV 120-kD
glycoprotein Herpes simplex virus (HSV); [human tonsils] HSV-1;
HSV-2 VH26 + Dk1 + JH6 with IGLV1S2 + 116-, 105-, 64-kD
glycoproteins Varicella zoster virus (VZV) J.lamda.2 of VZV VH4.18
VH2-1 (VH3) + D region Dxp'1 + JH5 with V.lamda.3 cML70 + J.lamda.3
VH1GRR + JH3 + Dn4r or D2r with IGLV1S2 + J.lamda.2 For VZV Abs:
ha3h2 (VH3) with IaIh2 (V.lamda.); or ha1c1 (VH1) with IaIvI
(V.lamda.1) For VZV Abs: ha4h3 (VH4) with Ia3h3 (V.lamda.3) Hv1051
(VH) Cytomegalovirus (CMV) 10. Huang et al, J Clin Invest 1992;
Kv325 (Vk) 71-2 (VH) HIV 10. Huang et al, J Clin Invest 1992;
Hv1f10 (VH) 11. Wang & Palese, Science 2011 VH4.11 71-4 (VH)
VH251 VH1-69 VH1-69 Haemagglutinin (HA) Influenza virus, eg, Group
1 12. Ekiert et al, Science 2009 and/or Group 2 Infulenza A 13.
Throsby et al, PLoS One 2008 virus; eg, H1N1, H2N2, or 14. Sui et
al, Nat Struct Mol Biol 2009 H3N2 or H7N2 or H7N7 15. Ekiert et al,
Science 2011 influenza virus
REFERENCES
[0101] 1. Nat Biotechnol. 2005 September; 23(9):1117-25; Human
antibodies from transgenic animals; Lonberg N. [0102] 2. J Clin
Invest. 1992 March; 89(3):729-38; Immunoglobulin light chain
variable region gene sequences for human antibodies to Haemophilus
influenzae type b capsular polysaccharide are dominated by a
limited number of V kappa and V lambda segments and VJ
combinations; Adderson E E, Shackelford P G, Insel R A, Quinn A,
Wilson P M, Carroll W L. [0103] 3. J Immunol. 1993 Oct. 15;
151(8):4352-61; Clonal characterization of the human IgG antibody
repertoire to Haemophilus influenzae type b polysaccharide. V. In
vivo expression of individual antibody clones is dependent on Ig CH
haplotypes and the categories of antigen; Chung G H, Scott M G, Kim
K H, Kearney J, Siber G R, Ambrosino D M, Nahm M H. [0104] 4. J
Immunol. 1998 Dec. 1; 161(11):6068-73; Decreased frequency of
rearrangement due to the synergistic effect of nucleotide changes
in the heptamer and nonamer of the recombination signal sequence of
the V kappa gene Alb, which is associated with increased
susceptibility of Navajos to Haemophilus influenzae type b disease;
Nadel B, Tang A, Lugo G, Love V, Escuro G, Feeney A J. [0105] 5. J
Clin Invest. 1996 May 15; 97(10):2277-82; A defective Vkappa A2
allele in Navajos which may play a role in increased susceptibility
to Haemophilus influenzae type b disease; Feeney A J, Atkinson M J,
Cowan M J, Escuro G, Lugo G. [0106] 6. Infect Immun. 1994
September; 62(9):3873-80; Variable region sequences of a protective
human monoclonal antibody specific for the Haemophilus influenzae
type b capsular polysaccharide; Lucas A H, Larrick J W, Reason D C.
[0107] 7. J Clin Invest. 1993 June; 91(6):2734-43; Restricted
immunoglobulin VH usage and VDJ combinations in the human response
to Haemophilus influenzae type b capsular polysaccharide.
Nucleotide sequences of monospecific anti-Haemophilus antibodies
and polyspecific antibodies cross-reacting with self antigens;
Adderson E E, Shackelford P G, Quinn A, Wilson P M, Cunningham M W,
Insel R A, Carroll W L. [0108] 8. J Clin Invest. 1993 March;
91(3):788-96; Variable region expression in the antibody responses
of infants vaccinated with Haemophilus influenzae type b
polysaccharide-protein conjugates. Description of a new lambda
light chain-associated idiotype and the relation between idiotype
expression, avidity, and vaccine formulation. The Collaborative
Vaccine Study Group; Granoff D M, Shackelford P G, Holmes S J,
Lucas A H. [0109] 9. Infect Immun. 1994 May; 62(5):1776-86;
Variable region sequences and idiotypic expression of a protective
human immunoglobulin M antibody to capsular polysaccharides of
Neisseria meningitidis group B and Escherichia coli K1; Azmi F H,
Lucas A H, Raff H V, Granoff D M. [0110] 10. J Clin Invest. 1992
December; 90(6):2197-208; Sequence analyses of three immunoglobulin
G anti-virus antibodies reveal their utilization of
autoantibody-related immunoglobulin Vh genes, but not V lambda
genes; Huang D F, Olee T, Masuho Y, Matsumoto Y, Carson D A, Chen P
P. [0111] 11. Science. 2011 Aug. 12; 333(6044):834-5, Biochemistry.
Catching a moving target, Wang T T, Palese P [0112] 12. Science.
2009 Apr. 10; 324(5924):246-51. Epub 2009 Feb. 26; Antibody
recognition of a highly conserved influenza virus epitope; Ekiert D
C, Bhabha G, Elsliger M A, Friesen R H, Jongeneelen M, Throsby M,
Goudsmit J, Wilson I A. [0113] 13. PLoS One. 2008; 3(12):e3942.
Epub 2008 Dec. 16; Heterosubtypic neutralizing monoclonal
antibodies cross-protective against H5N1 and H1N1 recovered from
human IgM+ memory B cells; Throsby M, van den Brink E, Jongeneelen
M, Poon L L, Alard P, Cornelissen L, Bakker A, Cox F, van Deventer
E, Guan Y, Cinatl J, ter Meulen J, Lasters I, Carsetti R, Peiris M,
de Kruif J, Goudsmit J. [0114] 14. Nat Struct Mol Biol. 2009 March;
16(3):265-73. Epub 2009 Feb. 22, Structural and functional bases
for broad-spectrum neutralization of avian and human influenza A
viruses, Sui J, Hwang W C, Perez S, Wei G, Aird D, Chen L M,
Santelli E, Stec B, Cadwell G, Ali M, Wan H, Murakami A, Yammanuru
A, Han T, Cox N J, Bankston L A, Donis R O, Liddington R C, Marasco
W A. [0115] 15. Science. 2011 Aug. 12; 333(6044):843-50. Epub 2011
Jul. 7, A highly conserved neutralizing epitope on group 2
influenza A viruses, Ekiert D C, Friesen R H, Bhabha G, Kwaks T,
Jongeneelen M, Yu W, Ophorst C, Cox F, Korse H J, Brandenburg B,
Vogels R, Brakenhoff J P, Kompier R, Koldijk M H, Cornelissen L A,
Poon L L, Peiris M, Koudstaal W, Wilson I A, Goudsmit J.
[0116] In one embodiment, in (b)(i) the J gene segments of the
light chain locus are J.sub..lamda. gene segments and optionally
the constant region of the light chain locus is a lambda constant
region; or in (b)(ii) the VL is a V.sub..lamda. hand optionally the
constant region of the light chain locus is a lambda constant
region. Alternatively, the constant region is C kappa.
[0117] In one embodiment, in (b)(i) the V gene segment repertoire
of the light chain locus comprises or consists of one or more VH
gene segments selected from the group consisting of: a V.sub.HIII
gene family member (optionally, a VHIIIa or VHIIIb family member),
a V.sub.HIV gene family member, V.sub.HIII 9.1 (VH3-15), V.sub.HIII
VH26 (VH3-23), V.sub.H3-21, LSG6.1, LSG12.1, DP77 (V3-21), V.sub.H
H11, VH1GRR, ha3h2, V.sub.HI-ha1c1, V.sub.HIII-VH2-1, VH4.18,
ha4h3, Hv1051, 71-2, Hv1110, VH4.11, 71-4, VH251, VH1-69 and a gene
segment at least 80% identical thereto. These gene segments are
useful because they expand the repertoire in vivo to VH gene
segments that are found in natural human immune responses to
antigens, such as antigens of infectious disease pathogens. This is
useful, for example, when the vertebrate is immunised with an
antigen of an infectious disease pathogen, for generation and
isolation of an antibody for treating and/or preventing a disease
or condition mediated by said pathogen. In one example, in (b)(i)
the V gene segment repertoire of the light chain locus comprises or
consists of only VH gene segment selected from the group consisting
of: a V.sub.HIII gene family member (optionally, a VHIIIa or VHIIIb
family member), a V.sub.HIV gene family member, V.sub.HIII 9.1
(VH3-15), V.sub.HIII VH26 (VH3-23), V.sub.H3-21, LSG6.1, LSG12.1,
DP77 (V3-21), V.sub.H H11, VH1GRR, ha3h2, V.sub.HI-ha1c1,
V.sub.HIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1110, VH4.11,
71-4, VH251, VH1-69 and a gene segment at least 80% identical
thereto. This is useful to bias the immune response of the
vertebrate (and thus resultant lead antibodies) to a predetermined
gene segment, eg, one known to be commonly used in natural human
immune responses to antigens, such as antigens of infectious
disease pathogens. For example, VH1-69 is commonly used to produce
antibodies in humans against Influenza virus (see Table 1); it is
possible, therefore, to confine the single VH segment to VH1-69 in
embodiment (b)(i) of the invention.
[0118] In one embodiment, in (b)(iii) the light chain locus V gene
segment repertoire consists of only one (optionally only two, three
or four) VL gene segment type (optionally and one or mutants
thereof), wherein the VL gene segment is selected from said group
of VL gene segments. This is useful to bias the immune response of
the vertebrate (and thus resultant lead antibodies) to a
predetermined gene segment, eg, one known to be commonly used in
natural human immune responses to antigens, such as antigens of
infectious disease pathogens.
[0119] In one embodiment, in (a) said constant region is a heavy
chain endogenous non-human vertebrate (optionally host mouse or
rat) constant region and/or in (b) said constant region is a light
chain endogenous non-human vertebrate (optionally host mouse or
rat) constant region.
[0120] In one embodiment in any configuration of the invention, the
genome has been modified to prevent or reduce the expression of
fully-endogenous antibody. Examples of suitable techniques for
doing this can be found in PCT/GB2010/051122, U.S. Pat. Nos.
7,501,552, 6,673,986, 6,130,364, WO2009/076464, EP1399559 and U.S.
Pat. No. 6,586,251, the disclosures of which are incorporated
herein by reference. In one embodiment, the non-human vertebrate
VDJ region of the endogenous heavy chain immunoglobulin locus, and
optionally VJ region of the endogenous light chain immunoglobulin
loci (lambda and/or kappa loci), have been inactivated. For
example, all or part of the non-human vertebrate VDJ region is
inactivated by inversion in the endogenous heavy chain
immunoglobulin locus of the mammal, optionally with the inverted
region being moved upstream or downstream of the endogenous Ig
locus (see, eg, WO2011004192, the disclosure of which is
incorporated herein by reference). For example, all or part of the
non-human vertebrate VJ region is inactivated by inversion in the
endogenous kappa chain immunoglobulin locus of the mammal,
optionally with the inverted region being moved upstream or
downstream of the endogenous Ig locus. For example, all or part of
the non-human vertebrate VJ region is inactivated by inversion in
the endogenous lambda chain immunoglobulin locus of the mammal,
optionally with the inverted region being moved upstream or
downstream of the endogenous Ig locus. In one embodiment the
endogenous heavy chain locus is inactivated in this way as is one
or both of the endogenous kappa and lambda loci.
[0121] Additionally or alternatively, the vertebrate has been
generated in a genetic background which prevents the production of
mature host B and T lymphocytes, optionally a RAG-1-deficient
and/or RAG-2 deficient background. See U.S. Pat. No. 5,859,301 for
techniques of generating RAG-1 deficient animals.
[0122] Thus, in one embodiment of any configuration or aspect of
the invention herein, endogenous heavy and light chain expression
has been inactivated.
In a Second Configuration of the Invention, there is Provided
[0123] A non-human vertebrate (optionally a mouse or a rat) or
vertebrate cell whose genome comprises: [0124] (a) An
immunoglobulin heavy chain locus comprising one or more human V
gene segments (eg, a plurality of VH), one or more human D gene
segments and one or more human J gene segments upstream of a
constant region; and [0125] (b) (i) An unrearranged immunoglobulin
light chain locus comprising one or more human VH gene segments and
one or more human J gene segments upstream of a constant region,
wherein each human VH gene segment is a human gene segment
identical to (or mutant of, eg, having up to 15 or 10 nucleotide
changes from the human gene segment) a human VH gene segment (eg, a
germline VH gene segment; eg, a gene segment selected from List A1,
A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4 below) used to produce a
rearranged VDJ encoding a heavy chain variable region of a human
antibody from an antibody-expressing cell wherein said antibody
binds to an antigen of an infectious disease pathogen (optionally
the variable regions of said antibody being identical to an
antibody from a human individual suffering, susceptible to, or
recovered from, a disease or condition caused or mediated by an
organism harbouring or secreting said antigen; or from a human
individual harbouring said organism); or [0126] (ii) An
immunoglobulin light chain locus comprising a rearranged VJ region
or VDJ region upstream of a constant region, wherein the nucleotide
sequence of the recombined region is identical to a nucleotide
sequence produced by the recombination of a human J gene segment
and optionally a human D gene segment with a human VH gene segment
that is identical to (or mutant of; eg, having up to 15 or 10
nucleotide changes from the human gene segment)) the human VH gene
segment (eg, germline VH gene segment; eg, a gene segment selected
from List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4 below) used
to produce a rearranged VDJ encoding a heavy chain variable region
of a human antibody from an antibody-expressing cell wherein said
antibody binds to an antigen of an infectious disease pathogen
(optionally the variable regions of said antibody being identical
to an antibody from a human individual suffering, susceptible to,
or recovered from, a disease or condition caused or mediated by an
organism harbouring or secreting said antigen; or from a human
individual harbouring said organism); [0127] (c) Wherein the gene
segments in the heavy chain locus are operably linked to the
constant region thereof, and the gene segments or VJ or VDJ in the
light chain locus are operably linked to the constant region
thereof, so that upon immunisation the mouse is capable of
producing an antibody comprising heavy chains produced by
recombination of the heavy chain locus and light chains derived
from the light chain locus; [0128] (d) Optionally when (b)(i)
applies, each said VH gene segment in the light chain locus is
selected from the group consisting of: a VHIII gene family member
(optionally, a VHIIIa or VHIIIb family member), a VHIV gene family
member, VHIII 9.1 (VH3-15), VHIII VH26 (VH3-23), VH3-21, LSG6.1,
LSG12.1, DP77 (V3-21), VH H11, VH1GRR, ha3h2, VHI-ha1c1,
VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1110, VH4.11, 71-4,
VH251, VH1-69 and a gene segment at least 80% identical thereto;
optionally each VH gene segment is selected from List A1, A2, A1.1,
A1.2, A2.1, A2.2, A2.3 or A2.4 below. [0129] (e) Optionally when
(b)(ii) applies, the nucleotide sequence of the recombined region
is identical to a nucleotide sequence produced by the recombination
of a human J gene segment and optionally a human D gene segment
with a human VH gene segment selected from the group consisting of:
a VHIII gene family member (optionally, a VHIIIa or VHIIIb family
member), a VHIV gene family member, VHIII 9.1 (VH3-15), VHIII VH26
(VH3-23), VH3-21, LSG6.1, LSG12.1, DP77 (V3-21), VH H11, VH1GRR,
ha3h2, VHI-ha1c1, VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1110,
VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80%
identical thereto; optionally each VH gene segment is selected from
List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4 below.
[0130] In one embodiment, the antigen is an antigen expressed by a
bacterial or viral infectious disease pathogen, eg, any of the
pathogens listed in Table 1. For example, the antigen is an antigen
selected from the antigens listed in Table 1.
[0131] In one embodiment of any aspect, configuration or embodiment
of the invention herein, the "human individual harbouring said
organism" is a patient that has natural resistance to the pathogen
and produces antibodies that bind to the pathogen or an antigen
expressed thereby.
[0132] In one embodiment of the second configuration, the i gene
segments of the light chain locus are J.sub..lamda. gene segments
and optionally the constant region of the light chain locus is a
lambda constant region. Alternatively, the constant region is C
kappa.
[0133] In one embodiment of the second configuration, the V gene
segment repertoire of the light chain locus comprises or consists
of one or more VH gene segments selected from the group consisting
of: a V.sub.HIII gene family member (optionally, a VHIIIa or VHIIIb
family member), a V.sub.HIV gene family member, V.sub.HIII 9.1
(VH3-15), V.sub.HIII VH26 (VH3-23), V.sub.H3-21, LSG6.1, LSG12.1,
DP77 (V3-21), V.sub.H H11, VH1GRR, ha3h2, V.sub.HI-ha1c1,
V.sub.HIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1110, VH4.11,
71-4, VH251, VH1-69 and a gene segment at least 80% identical
thereto. These gene segments are useful because they expand the
repertoire in vivo to VH gene segments that are found in natural
human immune responses to antigens, such as antigens of infectious
disease pathogens. This is useful, for example, when the vertebrate
is immunised with an antigen of an infectious disease pathogen, for
generation and isolation of an antibody for treating and/or
preventing a disease or condition mediated by said pathogen. In one
example, in (b)(i) the V gene segment repertoire of the light chain
locus comprises or consists of only VH gene segment selected from
the group consisting of: a V.sub.HIII gene family member
(optionally, a VHIIIa or VHIIIb family member), a V.sub.HIV gene
family member, V.sub.HIII 9.1 (VH3-15), V.sub.HIII VH26 (VH3-23),
V.sub.H3-21, LSG6.1, LSG12.1, DP77 (V3-21), V.sub.H H11, VH1GRR,
ha3h2, V.sub.HI-ha1c1, V.sub.HIII-VH2-1, VH4.18, ha4h3, Hv1051,
71-2, Hv1110, VH4.11, 71-4, VH251, VH1-69 and a gene segment at
least 80% identical thereto. This is useful to bias the immune
response of the vertebrate (and thus resultant lead antibodies) to
a predetermined gene segment, eg, one known to be commonly used in
natural human immune responses to antigens, such as antigens of
infectious disease pathogens. For example, VH1-69 is commonly used
to produce antibodies in humans against Influenza virus (see Table
1); it is possible, therefore, to confine the single VH segment to
VH1-69 in embodiment (b)(i) of the invention.
[0134] In one embodiment of the second configuration, in (a) said
constant region is a heavy chain endogenous non-human vertebrate
(optionally host mouse or rat) constant region.
[0135] In one embodiment of the second configuration, in (b) said
constant region is a light chain endogenous non-human vertebrate
(optionally host mouse or rat) constant region.
[0136] In one embodiment of the second configuration, the genome of
said vertebrate or cell is homozygous for light chain locus (b)(i)
or (ii); optionally wherein:
[0137] the V gene segment repertoire of the light chain loci
consists of one or more human VH gene segments selected from the
group consisting of: a VHIII gene family member (optionally, a
VHIIIa or VHIIIb family member), a VHIV gene family member, VHIII
9.1 (VH3-15), VHIII VH26 (VH3-23), VH3-21, LSG6.1, LSG12.1, DP77
(V3-21), VH H11, VH1GRR, ha3h2, VHI-ha1c1, VHIII-VH2-1, VH4.18,
ha4h3, Hv1051, 71-2, Hv1f10, VH4.11, 71-4, VH251, VH1-69 and a gene
segment at least 80% identical thereto; or
[0138] the recombined VJ or VDJ repertoire of the light chain loci
consists of sequences identical to one or more nucleotide sequences
produced by the recombination of a human VH gene segment selected
from the group consisting of: a VHIII gene family member
(optionally, a VHIIIa or VHIIIb family member), a VHIV gene family
member, VHIII 9.1 (VH3-15), VHIII VH26 (VH3-23), VH3-21, LSG6.1,
LSG12.1, DP77 (V3-21), VH H11, VH1GRR, ha3h2, VHI-ha1c1,
VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1f10, VH4.11, 71-4,
VH251, VH1-69 and a gene segment at least 80% identical thereto,
with a human J gene segment and optionally a human D gene segment.
In one embodiment, all of the light chain locus V gene segments are
from this group.
[0139] In one embodiment of the second configuration, endogenous
heavy and light chain expression has been inactivated, and wherein
light chain loci according to the second configuration are the only
functional light chain loci in the genome of the vertebrate or
cell.
[0140] In one embodiment of the second configuration, each
immunoglobulin light chain locus of said vertebrate or cell is
according to (b)(i) and comprises only a single human VH gene
segment selected from the group consisting of: a VHIII gene family
member (optionally, a VHIIIa or VHIIIb family member), a VHIV gene
family member, VHIII 9.1 (VH3-15), VHIII VH26 (VH3-23), VH3-21,
LSG6.1, LSG12.1, DP77 (V3-21), VH H11, VH1GRR, ha3h2, VHI-ha1c1,
VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1f10, VH4.11, 71-4,
VH251, VH1-69 and a gene segment at least 80% identical thereto,
optionally wherein the genome of the vertebrate or cell is
homozygous for said light chain so that all light chain loci
comprise the same, single human VH gene segment. In this embodiment
(and generally in other embodiments, configurations and aspects of
the invention), confinement of heavy and/or light chain locus
architecture is useful for biasing or controlling the antibody and
gene repertoire, eg, to mirror human immune responses as mentioned
above. Provision of a single light or heavy chain variable (and
optionally D and/or J) gene segment (or only this with closely
related mutants thereof)--or confinement in embodiments below to a
single rearranged V(D)J region or single heavy or light chain--is
advantageous for simplifying the expression and production of
therapeutic/prophylactic antibodies since this restricts the number
of antibody species produced during downstream manufacture. A
common heavy or light chain is advantageous to enable co-expression
of a plurality (eg, two, three or more) different antibodies in the
same expression medium, for example from the same host cell. See,
eg, EP1523496 (Merus B V) and WO2011097603 (Regeneron
Pharmaceuticals, Inc).
[0141] In one embodiment of the second configuration, each
immunoglobulin light chain locus of said vertebrate or cell is
according to (b)(ii) and comprises only a single rearranged VJ or
VDJ region, optionally wherein the genome of the vertebrate or cell
is homozygous for said light chain so that all light chain loci
comprise the same, single rearranged VJ or VDJ region.
[0142] In one embodiment of the second configuration, each
immunoglobulin light chain locus further comprises a VH gene
segment or rearranged region that is a mutant (eg, having up to 15
or 10 nucleotide changes from the VH gene segment) respectively of
said selected human VH gene segment or rearranged region,
optionally wherein the genome of the vertebrate or cell is
homozygous for said light chain mutant VH gene segment or
rearranged region.
[0143] In one embodiment of the second configuration, each
immunoglobulin light chain locus comprises only two or three human
VH gene segments selected from said group, optionally wherein the
genome of the vertebrate or cell is homozygous for said two or
three light chain human VH gene segments.
[0144] In one embodiment of the second configuration, each
immunoglobulin light chain locus comprises only two or three of
said rearranged VJ or VDJ regions, optionally wherein the genome of
the vertebrate or cell is homozygous for said two or three light
chain rearranged VJ or VDJ regions.
[0145] The invention provides a monoclonal or polyclonal antibody
composition prepared by immunisation of at least one vertebrate
(eg, mouse or rat) according to any configuration, aspect or
embodiment of the invention, optionally wherein the antigen is an
antigen of an infectious disease pathogen (eg, a bacterial or viral
pathogen antigen or an antigen listed in Table 1), optionally
wherein the same antigen is used to immunise all the vertebrates;
optionally wherein the antibody or antibodies are IgG-type (eg,
IgG1).
[0146] The invention provides a first method of isolating an
antibody that binds a predetermined antigen (eg, a bacterial or
viral pathogen antigen or an antigen listed in Table 1), the method
comprising [0147] (a) providing a vertebrate (optionally a mouse or
rat) according to according to any configuration, aspect or
embodiment of the invention; [0148] (b) immunising (eg, using a
standard prime-boost method) said vertebrate with said antigen
(optionally wherein the antigen is an antigen of an infectious
disease pathogen); [0149] (c) removing B lymphocytes from the
vertebrate and selecting one or more B lymphocytes expressing
antibodies that bind to the antigen; [0150] (d) optionally
immortalising said selected B lymphocytes or progeny thereof,
optionally by producing hybridomas therefrom; and [0151] (e)
isolating an antibody (eg, and IgG-type antibody) expressed by the
B lymphocytes.
[0152] In a first embodiment of the first method of the invention,
the method comprises the step of isolating from said B lymphocytes
nucleic acid encoding said antibody that binds said antigen;
optionally exchanging the heavy chain constant region nucleotide
sequence of the antibody with a nucleotide sequence encoding a
human or humanised heavy chain constant region and optionally
affinity maturing the variable region of said antibody; and
optionally inserting said nucleic acid into an expression vector
and optionally a host. The skilled person will be aware of standard
molecular biology techniques to do this. For example, see Harlow,
E. & Lane, D. 1998, 5.sup.th edition, Antibodies: A Laboratory
Manual, Cold Spring Harbor Lab. Press, Plainview, N.Y.; and
Pasqualini and Arap, Proceedings of the National Academy of
Sciences (2004) 101:257-259 for standard immunisation. Joining of
the variable regions of an antibody to a human constant region can
be effected by techniques readily available in the art, such as
using conventional recombinant DNA and RNA technology as will be
apparent to the skilled person. See e.g. Sambrook, J and Russell,
D. (2001, 3'd edition) Molecular Cloning: A Laboratory Manual (Cold
Spring Harbor Lab. Press, Plainview, N.Y.).
[0153] In one embodiment of the first method of the invention, the
method comprises further making a mutant or derivative of the
antibody.
[0154] A method of producing a polyclonal antibody mixture is
provided, the method comprising carrying out the first method of
the invention by separately immunising first and second vertebrates
(optionally first and second mice or first and second rats) with
antigen and combining the anti-antigen antibodies isolated from
each vertebrate (or mutants or derivatives of said antibodies) to
produce a polyclonal antibody mixture; optionally wherein the
following apply separately or in combination ((i) and (ii); or (i)
and (iii)):
(i) the vertebrates are immunised with the same antigen or
different antigens (optionally wherein the different antigens are
expressed by the same pathogenic organism (or by family members or
different strains of the organism)); (ii) prior to immunisation the
light chain loci of the vertebrates contain the identical VH gene
repertoire (optionally a single VH gene) and optionally the
identical J repertoire; optionally the light chain loci of the
mammals are identical prior to immunisation; (iii) prior to
immunisation the light chain loci of the vertebrates contain the
identical rearranged VJ or VDJ repertoire (optionally a single VJ
or VDJ); optionally the light chain loci of the vertebrates are
identical prior to immunisation.
[0155] A method of producing a polyclonal antibody mixture is
provided, the method comprising carrying out the first method of
the invention by immunising one or a plurality of vertebrates
(optionally mice or rats) with first and second antigens and
combining the anti-antigen antibodies isolated from each vertebrate
(or mutants or derivatives of said antibodies) to produce a
polyclonal antibody mixture; optionally wherein the following apply
separately or in combination ((i) and (ii); or (i) and (iii)):
(i) the antigens are expressed by the same pathogenic organism (or
by family members or different strains of the organism)); (ii)
prior to immunisation the light chain loci of the vertebrates
contain the identical VH gene repertoire (optionally a single VH
gene) and optionally the identical J repertoire; optionally the
light chain loci of the mammals are identical prior to
immunisation; (iii) prior to immunisation the light chain loci of
the vertebrates contain the identical rearranged VJ or VDJ
repertoire (optionally a single VJ or VDJ); optionally the light
chain loci of the vertebrates are identical prior to
immunisation.
The Invention Provides a Second Method:
[0156] A method of producing host cells (eg, Chinese Hamster Ovary
(CHO) or HEK293 cells) capable of expressing a polyclonal antibody
mixture is provided, the method comprising, in a method according
to said first embodiment of the first method of the invention:--
[0157] (a) immunising one or a plurality of vertebrates (optionally
mice or rats) with first and second antigens (optionally wherein
the different antigens are expressed by the same pathogenic
organism (or a family member thereof)); [0158] (b) isolating
nucleic acid encoding first and second anti-antigen antibodies from
B lymphocytes from said vertebrates; [0159] (c) determining the
nucleotide sequences of the heavy and light chain variable regions
(optionally the entire heavy and/or light chain sequences) of the
first antibody; [0160] (d) determining the nucleotide sequence of
the heavy variable region and optionally the light chain variable
region of the second antibody; [0161] (e) inserting the heavy chain
variable region coding sequence of each antibody into a heavy chain
expression vector; optionally wherein the constant region coding
sequence of each heavy chain is exchanged for a nucleotide sequence
that encodes a human or humanised constant region; [0162] (f)
inserting the light chain variable region coding sequence of the
first antibody into a light chain expression vector; optionally
wherein the constant region coding sequence of the light chain of
the first antibody is exchanged for a nucleotide sequence that
encodes a human or humanised constant region; [0163] (g) optionally
inserting the light chain variable region coding sequence of the
second antibody into a light chain expression vector; optionally
wherein the constant region coding sequence of the light chain of
the second antibody is exchanged for a nucleotide sequence that
encodes a human or humanised constant region; and [0164] (h)
introducing each expression vector into a host cell and
co-expressing antibody chains in a mixture of said host cells to
produce antibodies, each antibody comprising one or both of said
heavy chain variable regions and a light chain; optionally wherein
the expression vectors are introduced together into the same host
cell (eg, a CHO or HEK293 cell) so that the cell is capable of
expressing antibody light chains and heavy chains, such that the
cell or a plurality of the host cells express antibodies (eg, two,
three, four or more different antibodies), each comprising one or
both of said heavy chain variable regions and a light chain; [0165]
(i) optionally: [0166] prior to immunisation the light chain loci
of the vertebrates contain the identical VH gene repertoire
(optionally a single VH gene segment) and optionally the identical
J repertoire (optionally a single J gene segment); optionally the
light chain loci of the vertebrates are identical prior to
immunisation; or prior to immunisation the light chain loci of the
vertebrates contain the identical rearranged VJ or VDJ repertoire
(optionally a single VJ or VDJ); optionally the light chain loci of
the vertebrates are identical prior to immunisation. [0167] (j)
optionally: [0168] producing a monoclonal or polyclonal antibody
mixture, by expressing a monoclonal antibody or polyclonal mixture
of said antibodies; optionally followed by isolating an antibody
comprising the heavy chain variable region of the first and/or
second antibodies.
[0169] The invention also provides a monoclonal or polyclonal
antibody mixture so produced or a derivative antibody or mixture
thereof, eg, where one or more constant region has been changed
(eg, replaced with a different constant region such as a human
constant region; or mutated to enhance or ablate Fc effector
function). (optionally the entire heavy and/or light chain
sequences)
[0170] In any of the methods of the invention, optionally each
vertebrate used for immunisation is provided by
(a) isolating from a human blood or tissue (eg, B lymphocytes
(PBLs), peripheral blood mononuclear cells (PBMCs), bone marrow,
spleen, tonsil or lymph node) sample a B lymphocyte that expresses
an antibody that binds a predetermined antigen (eg, an antigen
expressed by an infectious disease pathogen; optionally wherein
said serum or tissue was from a human individual suffering,
susceptible to, or recovered from, a disease or condition caused or
mediated by an organism harbouring or secreting said antigen; or
from a human individual harbouring said organism); (b) determining
which human germline VH gene segment was recombined in the human to
produce the nucleotide sequence of said B lymphocyte that encodes
the heavy chain variable region of the antibody; (c) constructing a
transgenic vertebrate wherein said human germline VH gene segment
is provided in a light chain locus thereof according the first or
second configuration of the invention; and (d) providing said
transgenic vertebrate for immunisation in the first method of the
invention.
[0171] The term "Human blood" herein includes a human blood product
minus one or more non-B lymphocyte cellular populations, provided
that the product retains antibody-producing cells, eg, PBLs.
[0172] In an embodiment of the first method of the invention, each
vertebrate used for immunisation is provided by
(a) isolating from a human blood or tissue (eg, B lymphocytes,
PBMCs, bone marrow, spleen, tonsil or lymph node) sample a B
lymphocyte that expresses an antibody that binds a predetermined
antigen (eg, an antigen expressed by an infectious disease
pathogen; optionally wherein said serum or tissue was from a human
individual suffering, susceptible to, or recovered from, a disease
or condition caused or mediated by an organism harbouring or
secreting said antigen; or from a human individual harbouring said
organism); (b) determining a nucleotide sequence of said B
lymphocyte that encodes a rearranged VDJ or VJ region of the
antibody; (c) constructing a transgenic vertebrate wherein said
rearranged VDJ or VJ region is provided in a light chain locus
thereof according to the first or second configuration of the
invention; and (d) providing said transgenic vertebrate for
immunisation in the first method of the invention.
Common Light Chain Antibodies & Bispecifics (eg, to Two
Pathogen Antigens for Infectious Diseases)
[0173] The invention provides an isolated antibody (eg, IgG-type,
such as IgG1-type, antibody) obtainable or obtained by the second
method of the invention (including step (j), or a mutant or
derivative antibody thereof wherein (i) the isolated antibody
comprises two copies of the heavy chain variable region of said
first antibody paired with two copies of the light chain variable
region of said first antibody; or (ii) the isolated antibody
comprises two copies of the heavy chain variable region of said
second antibody paired with two copies of the light chain variable
region of said first antibody; or (iii) the isolated antibody is a
bispecific antibody comprising one copy of the heavy chain variable
region of said first antibody paired with a copy of the light chain
variable region of the first antibody, and one copy of the heavy
chain variable region of said the antibody paired with a copy of
the light chain variable region of the first antibody, optionally
wherein the bispecific antibody binds to said first and second
antigens recited in claim 24; optionally for use in medicine,
optionally for the treatment and/or prevention of an infectious
disease.
[0174] In an aspect of the invention, there is provided a
monoclonal or polyclonal antibody mixture (eg, IgG-type antibody or
antibodies), wherein the monoclonal antibody or mixture is
according to any configuration, aspect, embodiment or example of
the invention, or a mutant or derivative antibody thereof
optionally for use in medicine, optionally for the treatment and/or
prevention of an infectious disease, wherein optionally wherein
each antibody binds an antigen of an infectious disease pathogen,
preferably the same antigen.
[0175] In an aspect of the invention, there is provided the use of
an isolated, monoclonal or polyclonal antibody according to any
configuration, aspect, embodiment or example of the invention, or a
mutant or derivative antibody thereof in the manufacture of a
medicament for the treatment and/or prevention of an infectious
disease, optionally wherein the infectious disease is a disease
caused by a bacterial or viral pathogen.
[0176] An example of a mutant antibody is one that bears up to 15
or 10 amino acid mutations in its variable regions relative to an
isolated antibody (eg, IgG-type, such as IgG1-type, antibody)
obtainable or obtained by the second method of the invention
(including step (j). An example of a derivative is one that has
been modified to replace a constant region with a different
constant region such as a human constant region; or mutated to
enhance or ablate Fc effector function.
[0177] Examples of infectious diseases are diseases caused or
mediated by a bacterial or viral pathogen, eg, a pathogen listed in
Table 1. Examples of antigens are those listed in Table 1.
[0178] For example, the infectious disease is selected from the
group consisting of a disease caused by a pathogen selected from
the group consisting of Haemophilus influenza, E. coli, Neisseria
meningitidis, a herpes family virus, cytomegalovirus (CMV), HIV and
influenza virus.
[0179] The invention further provides a nucleotide sequence
encoding an antibody according to any configuration, aspect,
embodiment or example of the invention, optionally wherein the
nucleotide sequence is part of a vector.
[0180] The invention further provides a pharmaceutical composition
comprising the antibody or antibodies of any configuration, aspect,
embodiment or example of the invention and a diluent, excipient or
carrier.
In a Third Configuration of the Invention, there is Provided
[0181] A non-human vertebrate (optionally a mouse or a rat) or
vertebrate cell whose genome comprises: [0182] (a) An
immunoglobulin heavy chain locus comprising either:-- [0183] (i)
one or more human VL gene segments, one or more human D gene
segments and one or more human J gene segments upstream of a
constant region (optionally a rearranged V.sub.LDJ.sub.HC.sub.H or
V.sub..lamda.DJ.sub.HC.sub.H); or [0184] (ii) one or more human VH
gene segments selected from the group consisting of: a VHIII gene
family member (optionally, a VHIIIa or VHIIIb family member), a
VHIV gene family member, VHIII 9.1 (VH3-15), VHIII VH26 (VH3-23),
VH3-21, LSG6.1, LSG12.1, DP77 (V3-21), VH H11, VH1GRR, ha3h2,
VHI-ha1c1, VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1110,
VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80%
identical; one or more human D gene segments and one or more human
J.sub.H gene segments upstream of a constant region; optionally
each VH gene segment (and optionally each D) is selected from List
A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4 below; and [0185] (b)
An immunoglobulin light chain locus comprising one or more human V
gene segments (eg, a plurality of VL) and one or more human J gene
segments upstream of a constant region, optionally wherein the
light chain locus is according to (b)(i) or (b)(ii) of the first
configuration of the invention;
[0186] Wherein the gene segments in the heavy chain locus are
operably linked to the constant region thereof, and the gene
segments in the light chain locus are operably linked to the
constant region thereof, so that upon immunisation the mouse is
capable of producing an antibody comprising heavy chains produced
by recombination of the heavy chain locus and light chains produced
by recombination of the light chain locus.
[0187] In one example, in (a)(i) all of the heavy chain locus V
gene segments are human VL gene segments.
[0188] In one embodiment of the third configuration, the V gene
segment repertoire of the light chain locus comprises or consists
of one or more VL gene segments selected from the group consisting
of a V.sub..lamda.II gene family member, V.sub..lamda.VII 4A, WI
2.1, V.sub..lamda.VII 4A, a V.sub..lamda.1 gene family member, a
V.sub..lamda.3 gene family member, IGLV1S2, V.sub..lamda.3-cML70,
Ialh2, Ialvl, Ia3h3, Kv325, a V.kappa.I gene family member,
.kappa.I-15A (KL012), V.sub..kappa.II family member, a
V.sub..kappa.III family member, a V.kappa.I gene family member,
.kappa.I-15A (KL012), V.sub..kappa.II A2 (optionally the Ata
allele), V.sub..kappa. A27 (Humkv325) and a gene segment at least
80% identical thereto; optionally each VL gene segment is selected
from List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4 below;
and/or wherein or in (a)(ii) the heavy chain locus V gene segment
repertoire consists of only one (or two, three or four) VH gene
segment type (optionally and one or mutants thereof), wherein the
VH gene segment is selected from said group of VH gene segments.
This is useful to bias the immune response of the vertebrate (and
thus resultant lead antibodies) to a predetermined gene segment,
eg, one known to be commonly used in natural human immune responses
to antigens, such as antigens of infectious disease pathogens.
[0189] In one embodiment of the third configuration, in (a) said
constant region is a heavy chain endogenous non-human vertebrate
(optionally host mouse or rat) constant region and/or in (b) said
constant region is a light chain endogenous non-human vertebrate
(optionally host mouse or rat) constant region.
[0190] In one embodiment of the third configuration, endogenous
heavy and light chain expression has been inactivated.
A Fourth Configuration of the Present Invention Provides
[0191] A non-human vertebrate (optionally a mouse or a rat) or
vertebrate cell whose genome comprises: [0192] (a) (i) An
unrearranged immunoglobulin heavy chain locus comprising one or
more human VL gene segments, one or more human D gene segments and
one or more J.sub.H gene segments upstream of a constant region,
wherein each human VL gene segment is a human gene segment
identical to (or mutant of, eg, having up to 15 or 10 nucleotide
changes from the human gene segment) a human VL gene segment (eg, a
germline VL gene segment; eg, a VL gene segment selected from List
A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4 below) used to produce
a rearranged VJ encoding a light chain variable region of a human
antibody from an antibody-expressing cell wherein said antibody
binds to an antigen of an infectious disease pathogen (optionally
the variable regions of said antibody being identical to an
antibody from a human individual suffering, susceptible to, or
recovered from, a disease or condition caused or mediated by an
organism harbouring or secreting said antigen; or from a human
individual harbouring said organism); or
[0193] (ii) An immunoglobulin heavy chain locus comprising a
rearranged VJ region or VDJ region upstream of a constant region,
wherein the nucleotide sequence of the recombined region is
identical to a nucleotide sequence produced by the recombination of
a human J gene segment and optionally a human D gene segment with a
human gene segment identical to (or mutant of, eg, having up to 15
or 10 nucleotide changes from the human gene segment) a human VL
gene segment (eg, a germline VL gene segment; eg, a VL gene segment
selected from List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4
below) used to produce a rearranged VJ encoding a light chain
variable region of a human antibody from an antibody-expressing
cell wherein said antibody binds to an antigen of an infectious
disease pathogen (optionally the variable regions of said antibody
being identical to an antibody from a human individual suffering,
susceptible to, or recovered from, a disease or condition caused or
mediated by an organism harbouring or secreting said antigen; or
from a human individual harbouring said organism); [0194] (b) An
immunoglobulin light chain locus comprising one or more human V
gene segments (eg, a plurality of VL) and one or more human J gene
segments upstream of a constant region; and [0195] (c) Wherein the
gene segments in the light chain locus are operably linked to the
constant region thereof, and the gene segments or VJ or VDJ in the
heavy chain locus are operably linked to the constant region
thereof, so that upon immunisation the mouse is capable of
producing an antibody comprising light chains produced by
recombination of the light chain locus and heavy chains derived
from the heavy chain locus; [0196] (d) Optionally when (a)(i)
applies, each said VL gene segment in the heavy chain locus is
selected from the group consisting of a VL gene segment selected
from the group consisting of a V.lamda.II gene family member,
V.lamda.VII 4A, V.lamda.II 2.1, V.lamda.VII 4A, a V.lamda.I gene
family member, a V.lamda.3gene family member, IGLV1S2,
V.lamda.3-cML70, Ialh2, Ialvl, Ia3h3, Kv325, a V.kappa.I gene
family member, .kappa.I-15A (KL012), V.kappa.II family member, a
V.kappa.III family member, a V.kappa.I gene family member,
.kappa.I-15A (KL012), V.kappa.II A2 (optionally the A2a allele),
V.kappa.A27 (Humkv325) and a gene segment at least 80% identical
thereto; optionally each VL gene segment is selected from List A1,
A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4 below; [0197] (e)
Optionally when (a)(ii) applies, the nucleotide sequence of the
recombined region is identical to a nucleotide sequence produced by
the recombination of a human J gene segment and optionally a human
D gene segment with a human VL gene segment selected from the group
consisting of a V.lamda.II gene family member, V.lamda.VII 4A,
V.lamda.II 2.1, V.lamda.VII 4A, a V.lamda.1 gene family member, a
VX3gene family member, IGLV1S2, VX3-cML70, Ialh2, Ialvl, Ia3h3,
Kv325, a V.kappa.I gene family member, .kappa.I-15A (KL012),
V.kappa.II family member, a V.kappa.III family member, a V.kappa.I
gene family member, .kappa.I-15A (KL012), V.kappa.II A2 (optionally
the A2a allele), V.kappa. A27 (Humkv325) and a gene segment at
least 80% identical thereto.
[0198] The group of VL gene segments is useful to bias the immune
response of the vertebrate (and thus resultant lead antibodies) to
a predetermined gene segment, eg, one known to be commonly used in
natural human immune responses to antigens, such as antigens of
infectious disease pathogens.
[0199] In an embodiment of the fourth configuration, the VL gene
segments of the heavy chain locus are V.sub..lamda. gene
segments.
[0200] In an embodiment of the fourth configuration, in (a) said
constant region is a heavy chain endogenous non-human vertebrate
(optionally host mouse or rat) constant region.
[0201] In an embodiment of the fourth configuration, in (b) said
constant region is a light chain endogenous non-human vertebrate
(optionally host mouse or rat) constant region.
[0202] In an embodiment of the fourth configuration, the genome of
said vertebrate or cell is homozygous for heavy chain locus (a)(i)
or (ii); optionally wherein:
[0203] the V gene segment repertoire of the heavy chain loci
consists of one or more (or consists only of) human VL gene
segments selected from the group consisting of a VL gene segment
selected from the group consisting of a WI gene family member,
V.sub..lamda.VII 4A, V.sub..lamda.II 2.1, V.sub..lamda.VII 4A, a
V.sub..lamda.1 gene family member, a V.sub..lamda.3 gene family
member, IGLV1S2, V.sub..lamda.3-cML70, Ialh2, Ialvl, Ia3h3, Kv325,
a V.kappa.I gene family member, .kappa.I-15A (KL012),
V.sub..kappa.II family member, a V.sub..kappa.III family member, a
V.kappa.I gene family member, .kappa.I-15A (KL012), V.sub..kappa.II
A2 (optionally the A2a allele), V.sub..kappa. A27 (Humkv325) and a
gene segment at least 80% identical thereto; or
[0204] the recombined VJ or VDJ repertoire of the heavy chain loci
consists of sequences identical to one or more nucleotide sequences
produced by the recombination of a human VL gene segment selected
from the group consisting of a VL gene segment selected from the
group consisting of a V.lamda.II gene family member, V.lamda.VII
4A, V.lamda.II 2.1, V.lamda.VII 4A, a V.lamda.1 gene family member,
a V.lamda.3gene family member, IGLV1S2, V.lamda.3-cML70, Ialh2,
Ialvl, Ia3h3, Kv325, a V.kappa.I gene family member, .kappa.I-15A
(KL012), V.kappa.II family member, a V.kappa.III family member, a
V.kappa.I gene family member, .kappa.I-15A (KL012), V.kappa.II A2
(optionally the A2a allele), V.kappa. A27 (Humkv325) and a gene
segment at least 80% identical thereto with a human J gene segment
and optionally a human D gene segment.
[0205] In an embodiment of the fourth configuration, endogenous
heavy and light chain expression has been inactivated, and wherein
heavy chain loci according to the fourth configuration are the only
functional heavy chain loci in the genome of the vertebrate or
cell.
[0206] In an embodiment of the fourth configuration, each
immunoglobulin heavy chain locus of said vertebrate or cell is
according to (a)(i) and comprises only a single human VL gene
segment selected from the group consisting of a VL gene segment
selected from the group consisting of a V.lamda.II gene family
member, V.lamda.VII 4A, V.lamda.II 2.1, V.lamda.VII 4A, a V.lamda.1
gene family member, a V.lamda.3gene family member, IGLV1S2,
V.lamda.3-cML70, Ialh2, Ialvl, Ia3h3, Kv325, a V.kappa.I gene
family member, .kappa.I-15A (KL012), V.kappa.II family member, a
V.kappa.III family member, a V.kappa.I gene family member,
.kappa.I-15A (KL012), V.kappa.II A2 (optionally the Ata allele),
V.kappa. A27 (Humkv325) and a gene segment at least 80% identical
thereto, optionally wherein the genome of the vertebrate or cell is
homozygous for said heavy chain so that all heavy chain loci
comprise the same, single human VL gene segment.
[0207] In an embodiment of the fourth configuration, each
immunoglobulin heavy chain locus of said vertebrate or cell is
according to (a)(ii) and comprises only a single rearranged VJ or
VDJ region, optionally wherein the genome of the vertebrate or cell
is homozygous for said heavy chain so that all heavy chain loci
comprise the same, single rearranged VJ or VDJ region.
[0208] In an embodiment of the fourth configuration, each
immunoglobulin heavy chain locus further comprises a VL gene
segment or rearranged region that is a mutant respectively of said
selected human VL gene segment or rearranged region, optionally
wherein the genome of the vertebrate or cell is homozygous for said
light chain mutant VL gene segment or rearranged region.
[0209] In all configurations, aspects, examples and embodiments of
the invention, where a "mutant" is mentioned, this includes a
mutant sequence that is identical to a reference sequence (eg,
reference VH, VL, VJ or VDJ) but with 1, 2, 3, 4, 5, 6, 7, 8, 9 or
10 nucleotide or amino acid changes therefrom.
[0210] In an embodiment of the fourth configuration, each
immunoglobulin heavy chain locus comprises only two or three human
VL gene segments selected from said group, optionally wherein the
genome of the vertebrate or cell is homozygous for said two or
three heavy chain human VL gene segments.
[0211] In an embodiment of the fourth configuration, each
immunoglobulin heavy chain locus comprises only two or three of
said rearranged VJ or VDJ regions, optionally wherein the genome of
the vertebrate or cell is homozygous for said two or three heavy
chain rearranged VJ or VDJ regions.
[0212] The invention provides a monoclonal or polyclonal antibody
composition prepared by immunisation of at least one vertebrate
(eg, mouse or rat) according to the third or fourth embodiment of
the invention with an antigen, optionally wherein the antigen is an
antigen of an infectious disease pathogen, optionally wherein the
same antigen is used to immunise all the vertebrates; optionally
wherein the antibody or antibodies are IgG-type.
[0213] The invention provides a third method: A method of isolating
an antibody (eg, IgG-type, such as IgG1) that binds a predetermined
antigen, the method comprising
(a) providing a vertebrate (optionally a mouse or rat) according to
the third or fourth embodiment of the invention; (b) immunising
(eg, using standard prime-boost) said vertebrate with said antigen
(optionally wherein the antigen is an antigen of an infectious
disease pathogen); (c) removing B lymphocytes from the vertebrate
and selecting one or more B lymphocytes expressing antibodies that
bind to the antigen; (d) optionally immortalising said selected B
lymphocytes or progeny thereof, optionally by producing hybridomas
therefrom; and (e) isolating an antibody (eg, and IgG-type
antibody) expressed by the B lymphocytes; (f) Optionally, the third
method comprises the step of isolating from said B lymphocytes
nucleic acid encoding said antibody that binds said antigen;
optionally exchanging the heavy chain constant region nucleotide
sequence of the antibody with a nucleotide sequence encoding a
human or humanised heavy chain constant region and optionally
affinity maturing the variable region of said antibody; and
optionally inserting said nucleic acid into an expression vector
and optionally a host.
[0214] Optionally, the third method further comprises making a
mutant or derivative of the antibody.
[0215] The invention provides a fourth method: A method of
producing a polyclonal antibody mixture, the method comprising
carrying out the third method by separately immunising first and
second vertebrates (optionally first and second mice or first and
second rats) with antigen (eg, any antigen disclosed herein) and
combining the anti-antigen antibodies isolated from each vertebrate
(or mutants or derivatives of said antibodies) to produce a
polyclonal antibody mixture; optionally wherein the following apply
separately or in combination ((i) and (ii); or (i) and (iii)):
(i) the vertebrates are immunised with the same antigen or
different antigens (optionally wherein the different antigens are
expressed by the same pathogenic organism (or different family
members thereof or different strains of the organism)); (ii) prior
to immunisation the heavy chain loci of the vertebrates contain the
identical VL gene repertoire (optionally a single VL gene) and
optionally the identical D and/or J repertoire; optionally the
heavy chain loci of the mammals are identical prior to
immunisation; (iii) prior to immunisation the heavy chain loci of
the vertebrates contain the identical rearranged VJ or VDJ
repertoire (optionally a single VJ or VDJ); optionally the heavy
chain loci of the vertebrates are identical prior to
immunisation.
[0216] The invention provides a fifth method: A method of producing
a polyclonal antibody mixture, the method comprising carrying out
the third method by immunising one or a plurality of vertebrates
(optionally mice or rats) with first and second antigens and
combining the anti-antigen antibodies isolated from each vertebrate
(or mutants or derivatives of said antibodies) to produce a
polyclonal antibody mixture; optionally wherein the following apply
separately or in combination ((i) and (ii); or (i) and (iii)):
(i) the antigens are expressed by the same pathogenic organism (or
different family members thereof or different strains of the
organism); (ii) prior to immunisation the heavy chain loci of the
vertebrates contain the identical VL gene repertoire (optionally a
single VL gene) and optionally the identical D and/or J repertoire;
optionally the heavy chain loci of the mammals are identical prior
to immunisation; (iii) prior to immunisation the heavy chain loci
of the vertebrates contain the identical rearranged VJ or VDJ
repertoire (optionally a single VJ or VDJ); optionally the heavy
chain loci of the vertebrates are identical prior to
immunisation.
[0217] The invention provides a sixth method: A method of producing
host cells capable of expressing a polyclonal antibody mixture, the
method comprising, in the third method wherein step (f) is carried
out:-- [0218] (a) immunising one or a plurality of vertebrates
(optionally mice or rats) with first and second antigens
(optionally wherein the different antigens are expressed by the
same pathogenic organism (or a family member thereof)); [0219] (b)
isolating nucleic acid encoding first and second anti-antigen
antibodies from B lymphocytes from said vertebrates; [0220] (c)
determining the nucleotide sequences of the heavy and light chain
variable regions (optionally the entire heavy and/or light chain
sequences) of the first antibody; [0221] (d) determining the
nucleotide sequence of the light variable region and optionally the
heavy chain variable region of the second antibody; [0222] (e)
inserting the light chain variable region coding sequence of each
antibody into a light chain expression vector; optionally wherein
the constant region coding sequence of each light chain is
exchanged for a nucleotide sequence that encodes a human or
humanised constant region; [0223] (f) inserting the heavy chain
variable region coding sequence of the first antibody into a heavy
chain expression vector; optionally wherein the constant region
coding sequence of the heavy chain of the first antibody is
exchanged for a nucleotide sequence that encodes a human or
humanised constant region; [0224] (g) optionally inserting the
heavy chain variable region coding sequence of the second antibody
into a heavy chain expression vector; optionally wherein the
constant region coding sequence of the heavy chain of the second
antibody is exchanged for a nucleotide sequence that encodes a
human or humanised constant region; and [0225] (h) introducing each
expression vector into a host cell and co-expressing antibody
chains in a mixture of said host cells to produce antibodies, each
antibody comprising one or both of said light chain variable
regions and a heavy chain; optionally wherein the expression
vectors are introduced together into the same host cell (eg, a CHO
or HEK293 cell) so that the cell is capable of expressing antibody
light chains and heavy chains, such that the cell or a plurality of
the host cells express antibodies (eg, two, three or four different
antibodies), each comprising one or both of said light chain
variable regions and a heavy chain; [0226] (i) optionally: [0227]
prior to immunisation the heavy chain loci of the vertebrates
contain the identical VL gene repertoire (optionally a single VL
gene segment) and optionally the identical D and/or J repertoire
(optionally a single D and J gene segment); optionally the heavy
chain loci of the vertebrates are identical prior to immunisation;
or [0228] prior to immunisation the heavy chain loci of the
vertebrates contain the identical rearranged VJ or VDJ repertoire
(optionally a single VJ or VDJ); optionally the heavy chain loci of
the vertebrates are identical prior to immunisation.
[0229] The invention also provides a monoclonal or polyclonal
antibody mixture so produced or a derivative antibody or mixture
thereof, eg, where one or more constant region has been changed
(eg, replaced with a different constant region such as a human
constant region; or mutated to enhance or ablate Fc effector
function).
[0230] The invention provides a seventh method: A method of
producing a monoclonal antibody or polyclonal antibody mixture, the
method comprising carrying out the sixth method and expressing a
monoclonal antibody or polyclonal mixture of said antibodies;
optionally followed by isolating an antibody comprising the light
chain variable region of the first and/or second antibodies.
[0231] Optionally, each vertebrate used for immunisation is
provided by
(a) isolating from a human blood or tissue (eg, B lymphocytes,
PBMCs, bone marrow, spleen, tonsil or lymph node) sample a B
lymphocyte that expresses an antibody that binds a predetermined
antigen (eg, an antigen expressed by an infectious disease
pathogen; optionally wherein said serum or tissue was from a human
individual suffering, susceptible to, or recovered from, a disease
or condition caused or mediated by an organism harbouring or
secreting said antigen; or from a human individual harbouring said
organism); (b) determining which human germline VL gene segment was
recombined in the human to produce the nucleotide sequence of said
B lymphocyte that encodes the light chain variable region of the
antibody; (c) constructing a transgenic vertebrate wherein said
human germline VL gene segment is provided in a heavy chain locus
thereof according to the third or fourth configuration of the
invention; and (d) providing said transgenic vertebrate for
immunisation in the fourth, fifth or sixth method of the
invention.
[0232] In another embodiment, each vertebrate used for immunisation
is provided by
(a) isolating from a human blood or tissue (eg, B lymphocytes,
PBMCs, bone marrow, spleen, tonsil or lymph node) sample a B
lymphocyte that expresses an antibody that binds a predetermined
antigen (eg, an antigen expressed by an infectious disease
pathogen; optionally wherein said serum or tissue was from a human
individual suffering, susceptible to, or recovered from, a disease
or condition caused or mediated by an organism harbouring or
secreting said antigen; or from a human individual harbouring said
organism); (b) determining a nucleotide sequence of said B
lymphocyte that encodes a rearranged VDJ or VJ region of the
antibody; (c) constructing a transgenic vertebrate wherein said
rearranged VDJ or VJ region is provided in a heavy chain locus
thereof according the third or fourth configuration of the
invention; and (d) providing said transgenic vertebrate for
immunisation in the method of the fourth, fifth or sixth method of
the invention.
Common Heavy Chain Antibodies & Bispecifics (eg, to Two
Pathogen Antigens for Infectious Diseases)
[0233] The invention provides an isolated antibody (eg, IgG-type
antibody) obtainable or obtained by the seventh method, or a mutant
or derivative antibody thereof wherein (i) the isolated antibody
comprises two copies of the heavy chain variable region of said
first antibody paired with two copies of the light chain variable
region of said first antibody; or (ii) the isolated antibody
comprises two copies of the heavy chain variable region of said
second antibody paired with two copies of the light chain variable
region of said first antibody; or (iii) the isolated antibody is a
bispecific antibody comprising one copy of the heavy chain variable
region of said first antibody paired with a copy of the light chain
variable region of the first antibody, and one copy of the heavy
chain variable region of said the antibody paired with a copy of
the light chain variable region of the first antibody, optionally
wherein the bispecific antibody binds to said first and second
antigens described above; optionally for use in medicine,
optionally for the treatment and/or prevention of an infectious
disease.
[0234] The invention provides a monoclonal or polyclonal antibody
mixture (eg, IgG-type antibody or antibodies), wherein the
monoclonal antibody or mixture comprises or consists of antibodies
produced by the fourth, fifth, sixth or seventh method, or a mutant
or derivative antibody thereof optionally for use in medicine,
optionally for the treatment and/or prevention of an infectious
disease, wherein optionally wherein each antibody binds an antigen
of an infectious disease pathogen, preferably the same antigen.
[0235] The following embodiments relate to antibodies, host cells,
nucleic acids and compositions and apply to such elements obtained
or obtainable by any previous configuration or method of the
invention:--
[0236] The invention provides an isolated chimaeric antibody for
treating and/or preventing an infectious disease or condition, the
antibody comprising a non-human vertebrate (optionally a mouse or
rat) heavy chain constant regions and human variable regions that
bind an antigen of an infectious disease pathogen, wherein the
antibody is obtainable or obtained in a method comprising
immunisation of a vertebrate according to of any one of the first
to seventh methods of the invention with said antigen. The antigen
is, for example, any antigen mentioned above. The disease or
condition is, for example, any disease or condition mentioned
above.
[0237] The invention provides an isolated human antibody for
treating and/or preventing an infectious disease or condition, the
antibody comprising human heavy chain constant regions and human
variable regions that bind an antigen of an infectious disease
pathogen, wherein the antibody is obtainable or obtained in a
method comprising affinity maturation of antibody variable regions
in vivo in a transgenic non-human vertebrate (eg, mouse or rat)
when said variable regions are operably linked to heavy chain
constant regions of said vertebrate (eg, mouse or rat heavy chain
constant regions) by (a) immunisation of a vertebrate of any
configuration of the invention with said antigen, (b) isolation of
nucleic acid encoding a chimaeric antibody as described above, (c)
replacing the nucleotide sequences of the nucleic acid that encode
the non-human vertebrate heavy chain constant regions with
nucleotide sequence encoding human heavy chain constant regions to
produce nucleic acid encoding a human antibody; (d) expressing the
human antibody in vitro (optionally from CHO or HEK293 cells
harbouring the human nucleic acid) and (e) isolating the human
antibody (optionally with further affinity maturation of the
antibody and/or producing a derivative thereof). The invention
provides a mixture of first and second such human antibodies (an
optionally also third and optionally fourth antibodies), each
antibody being capable of binding to an antigen of an infectious
disease pathogen (optionally wherein the first antibody binds a
first antigen and the second antibody binds a second antigen, said
antigens being from the same pathogen; or wherein the antigens are
the same). Optionally, the light chain amino acid sequence of the
first antibody is identical to the light chain amino acid sequence
of the second antibody, or has up to 15 amino acid changes
therefrom. The advantages of such a common (or closely-related)
chain are explained above, and include relative ease of
manufacture.
[0238] The antigen is, for example, any antigen mentioned above.
The disease or condition is, for example, any disease or condition
mentioned above. The pathogen is, for example, any pathogen
mentioned above.
[0239] The invention provides an antibody comprising human variable
domains that bind a predetermined antigen (eg, an antigen expressed
by a bacterial or viral pathogen), wherein the variable domain
sequences are encoded by rearranged VDJ and VJ regions, each of the
VDJ and/or VJ being a hybrid region produced by the in vivo
rearrangement of human heavy and light chain variable region gene
segments (V and J and optionally D segments); optionally wherein
the antibody comprises human constant regions.
[0240] The invention provides a method of producing an isolated
human antibody for treating and/or preventing an infectious disease
or condition, the antibody comprising human heavy chain constant
regions and human variable regions that bind an antigen of an
infectious disease pathogen, wherein the method comprises affinity
maturing antibody variable regions in vivo in a transgenic
non-human vertebrate (eg, mouse or rat) when said variable regions
are operably linked to heavy chain constant regions of said
vertebrate (eg, mouse or rat heavy chain constant regions) by (a)
immunisation of a vertebrate of any configuration of the invention
with said antigen, (b) isolation of nucleic acid encoding a
chimaeric antibody as described above, (c) replacing the nucleotide
sequences of the nucleic acid that encode the non-human vertebrate
heavy chain constant regions with nucleotide sequence encoding
human heavy chain constant regions to produce nucleic acid encoding
a human antibody; (d) expressing the human antibody in vitro
(optionally from CHO or HEK293 cells harbouring the human nucleic
acid) and (e) isolating the human antibody (optionally with further
affinity maturation of the antibody and/or producing a derivative
thereof). The antigen is, for example, any antigen mentioned above.
The disease or condition is, for example, any disease or condition
mentioned above. The pathogen is, for example, any pathogen
mentioned above.
[0241] The invention provides the use of any isolated, monoclonal
or polyclonal antibody or mixture of the invention as described
above, in the manufacture of a medicament for the treatment and/or
prevention of an infectious disease, optionally wherein the
infectious disease is a disease caused by a bacterial or viral
pathogen. The disease or condition is, for example, any disease or
condition mentioned above. The pathogen is, for example, any
pathogen mentioned above. For example, the infectious disease is
selected from the group consisting of a disease caused by a
pathogen selected from the group consisting of Haemophilus
influenza, E. coli, Neisseria meningitidis, a herpes family virus,
cytomegalovirus (CMV), HIV and influenza virus.
[0242] The invention provides first and second nucleotide sequences
(eg, DNA, RNA, mRNA, cDNA) encoding the heavy and light chains of
an antibody according to any configuration, aspect, example or
embodiment of the invention or at least the variable regions
thereof, optionally wherein each nucleotide sequence is part of a
vector.
[0243] The invention provides a host cell comprising one or more
expression vectors encoding the heavy chains of the first and
second antibodies mentioned above, and the light chain of the first
antibody mentioned above (and optionally also the light chain of
the second antibody). Again, reference is made to the discussion
above about the advantages of having a common antibody chain for
the production of antibody mixtures.
[0244] The invention provides a pharmaceutical composition
comprising the antibody or antibodies of any configuration, aspect,
example or embodiment of the invention and a diluent, excipient or
carrier; optionally wherein the composition is provided in a
container connected to an IV needle or syringe or in an IV bag. The
skilled person will know standard diluents, excipients and carriers
suitable for pharmaceutical application.
[0245] Throughout this description, where it is mentioned "at least
80% identical", there is contemplated in the alternative one of the
following identities: at least 85%, 90, 95, 96, 97, 98 or 99
identical and the disclosure herein contemplates that one or more
of these identities may be recited in a claim herein in place of
"at least 80% identical".
Tailoring V(D)J Incorporation into Immunoglobin Loci for the
Generation of Antibodies Against Infectious Disease
[0246] In the various configurations, aspects, embodiments and
examples above, the invention provides the skilled addressee with
the possibility of choosing immunoglobulin gene segments in a way
that tailors or biases the repertoire for application to generating
antibodies to treat and/or prevent infectious diseases. The
inventors have categorised the following groups of gene segments
for use in the invention according to the desired application of
resultant antibodies.
List A:
[0247] Immunoglobulin Gene Segments for Antibodies that Bind an
Antigen Expressed by a Pathogen [0248] (a) a VL gene segment
selected from the group consisting of a WI gene family member,
V.sub..lamda.VII 4A, V.sub..lamda.II 2.1, V.sub..lamda.VII 4A, a
V.sub..lamda.1 gene family member, a V.sub..lamda.3 gene family
member, IGLV1S2, V.sub..lamda.3-cML70, Ialh2, Ialvl, Ia3h3, Kv325,
a V.kappa.I gene family member, .kappa.I-15A (KL012),
V.sub..kappa.II family member, a V.sub..kappa.III family member, a
V.kappa.I gene family member, .kappa.I-15A (KL012), V.sub..kappa.II
A2 (optionally the A2a allele), V.sub..kappa. A27 (Humkv325) and a
gene segment at least 80% identical thereto. [0249] (b) a
V.sub..lamda. gene segment selected from a WI gene family member,
V.sub..lamda.VII 4A, WI 2.1, V.sub..lamda.VII 4A, a V.sub..lamda.1
gene family member, a V.sub..lamda.3 gene family member, IGLV1S2,
V.sub..lamda.3-cML70, Ialh2, Ialvl, Ia3h3 and a gene segment at
least 80% identical thereto. [0250] (c) a V.sub..kappa. gene
segment selected from Kv325, a V.kappa.I gene family member,
.kappa.I-15A (KL012), V.sub..kappa.II family member, a
V.sub..kappa.III family member, a V.kappa.I gene family member,
.kappa.I-15A (KL012), V.sub..kappa.II A2 (optionally the A2a
allele), V.sub..kappa. A27 (Humkv325) and a gene segment at least
80% identical thereto. [0251] (d) a V.sub.H gene segment a
V.sub.HIII gene family member (optionally, a VHIIIa or VHIIIb
family member), a V.sub.HIV gene family member, V.sub.HIII 9.1
(VH3-15), V.sub.HIII VH26 (VH3-23), V.sub.H3-21, LSG6.1, LSG12.1,
DP77 (V3-21), V.sub.H H11, VH1GRR, ha3h2, V.sub.HI-ha1c1,
V.sub.HIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1f10, VH4.11,
71-4, VH251, VH1-69 and a gene segment at least 80% identical
thereto. [0252] (e) a J.sub..lamda. gene segment selected from
J.sub..lamda.2, J.sub..lamda.3 and a gene segment at least 80%
identical thereto. [0253] (f) a D gene segment selected from Dk1,
Dxp'1, Dn4r, D2r and a gene segment at least 80% identical
thereto.
List A1:
[0254] Immunoglobulin Gene Segments for Antibodies that Bind an
Antigen Expressed by a Bacterial Pathogen [0255] (a) a
V.sub..lamda. gene segment selected from a WI gene family member,
V.sub..lamda.VII 4A, V.sub..lamda.II 2.1, V.sub..lamda.VII 4A and a
gene segment at least 80% identical thereto. [0256] (b) a
V.sub..kappa. gene segment selected from a V.kappa.I gene family
member, .kappa.I-15A (KL012), V.sub..kappa.II family member, a
V.sub..kappa.III family member, a V.kappa.I gene family member,
.kappa.I-15A (KL012), V.sub..kappa.II A2 (optionally the Ata
allele), V.sub..kappa. A27 (Humkv325) and a gene segment at least
80% identical thereto. [0257] (c) a V.sub.H gene segment a VH3 gene
family member (optionally, a VHIIIa or VHIIIb family member),
V.sub.HIII 9.1 (VH3-15), V.sub.HIII VH26 (VH3-23), V.sub.H3-21,
LSG6.1, LSG12.1, DP77 (V3-21), V.sub.H H11 and a gene segment at
least 80% identical thereto. [0258] (d) a J.sub..lamda. gene
segment selected from J.sub..lamda.2, J.sub..lamda.3 and a gene
segment at least 80% identical thereto. [0259] (e) a J.sub.H gene
segment selected from J.sub.H2, J.sub.H3, J.sub.H4 and a gene
segment at least 80% identical thereto.
List A1.1:
[0260] Immunoglobulin Gene Segments for Antibodies that Bind an
Antigen Expressed by H Influenza [0261] (a) a V.sub..lamda. gene
segment selected from a WI gene family member, V.sub..lamda.VII 4A,
V.sub..lamda.II 2.1, V.sub..lamda.VII 4A and a gene segment at
least 80% identical thereto. [0262] (b) a V.sub..kappa. gene
segment selected from a V.sub..kappa.II family member, a
V.sub..kappa.III family member, a V.kappa.I gene family member,
.kappa.I-15A (KL012), V.sub..kappa.II A2 (optionally the Ata
allele), V.sub..kappa. A27 (Humkv325) and a gene segment at least
80% identical thereto. [0263] (c) a V.sub.H gene segment a VH3 gene
family member (optionally, a VHIIIb family member), V.sub.HIII 9.1
(VH3-15), V.sub.HIII VH26 (VH3-23), V.sub.H3-21, LSG6.1, LSG12.1,
DP77 (V3-21) and a gene segment at least 80% identical thereto.
[0264] (d) a J.sub..lamda. gene segment selected from
J.sub..lamda.2, J.sub..lamda.3 and a gene segment at least 80%
identical thereto.
List A1.2:
[0265] Immunoglobulin Gene Segments for Antibodies that Bind an
Antigen Expressed by E Coli or Neisseria Meningitidis [0266] (a) a
V.sub.H gene segment a VH3 gene family member (optionally a VHIIIa
or VHIIIb member), V.sub.HIII 9.1 (VH3-15), V.sub.H H11, V.sub.HIII
VH26 (VH3-23) a gene segment at least 80% identical thereto, eg,
V.sub.HIII 9.1+J.sub.H3; or V.sub.H H11+J.sub.H4; or V.sub.HIII
VH26+J.sub.H2. [0267] (b) a V.sub..kappa. gene segment selected
from a V.kappa.I gene family member, .kappa.I-15A (KL012) and a
gene segment at least 80% identical thereto. [0268] (c) a
V.sub..lamda. gene segment selected from a WI gene family member,
V.sub..lamda.II 2.1 and a gene segment at least 80% identical
thereto. [0269] (d) a J.sub.H gene segment selected from J.sub.H2,
J.sub.H3, J.sub.H4 and a gene segment at least 80% identical
thereto.
A2:
[0270] Immunoglobulin Gene Segments for Antibodies that Bind an
Antigen Expressed by a Viral Pathogen [0271] (a) a V.sub.H gene
segment selected from a V.sub.HIII gene family member, a V.sub.HIV
gene family member, V.sub.HIII-VH26 (VH3-23), VH1GRR, ha3h2,
V.sub.HI-ha1c1, V.sub.HIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2,
Hv1f10, VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80%
identical thereto. [0272] (b) a V.sub..lamda. gene segment selected
from a V.sub..lamda.1 gene family member, a V.sub..lamda.3 gene
family member, IGLV1S2, V.sub..lamda.3-cML70, Ialh2, Ialvl, Ia3h3
and a gene segment at least 80% identical thereto. [0273] (c) a Vk
gene segment selected from Kv325 and a gene segment at least 80%
identical thereto. [0274] (d) a J.sub.H gene segment selected from
J.sub.H3, J.sub.HS, J.sub.H6 and a gene segment at least 80%
identical thereto. [0275] (e) a D gene segment selected from Dk1,
Dxp'1, Dn4r, D2r and a gene segment at least 80% identical thereto.
[0276] (f) a J.sub..lamda. gene segment selected from
J.sub..lamda.2, J.sub..lamda.3 and a gene segment at least 80%
identical thereto.
A2.1:
[0277] Immunoglobulin Gene Segments for Antibodies that Bind an
Antigen Expressed by Herpes Virus Family (eg, VZV or HSV) [0278]
(a) a V.sub.H gene segment selected from a V.sub.HIII gene family
member, a V.sub.HIV gene family member, V.sub.HIII-VH26 (VH3-23),
VH1GRR, ha3h2, V.sub.HI-ha1c1, V.sub.HIII-VH2-1, VH4.18, ha4h3, and
a gene segment at least 80% identical thereto. [0279] (b) a
V.sub..lamda. gene segment selected from a V.sub..lamda.1 gene
family member, a V.sub..lamda.3 gene family member, IGLV1S2,
V.sub..lamda.3-cML70, Ialh2, Ialvl, Ia3h3 and a gene segment at
least 80% identical thereto. [0280] (c) a J.sub.H gene segment
selected from J.sub.H3, J.sub.HS, J.sub.H6 and a gene segment at
least 80% identical thereto. [0281] (d) a D gene segment selected
from Dk1, Dxp'1, Dn4r, D2r and a gene segment at least 80%
identical thereto. [0282] (e) a J.sub..lamda. gene segment selected
from J.sub..lamda.2, J.sub..lamda.3 and a gene segment at least 80%
identical thereto. A2.2: Immunoglobulin Gene Segments for
Antibodies that Bind an Antigen Expressed by CMV [0283] (a) a
V.sub.H gene segment selected from Hv1051 and a gene segment at
least 80% identical thereto. [0284] (b) a Vk gene segment selected
from Kv325 and a gene segment at least 80% identical thereto.
A2.3:
[0285] Immunoglobulin Gene Segments for Antibodies that Bind an
Antigen Expressed by HIV [0286] (a) a V.sub.H gene segment selected
from 71-2, Hv1110, VH4.11, 71-4, VH251, VH1-69 and a gene segment
at least 80% identical thereto.
A2.4:
[0287] Immunoglobulin Gene Segments for Antibodies that Bind an
Antigen Expressed by Influenza Virus [0288] (a) a V.sub.H gene
segment selected from VH1-69 and a gene segment at least 80%
identical thereto.
[0289] Thus,
[0290] Where one wishes to generate an antibody or antibody mixture
to treat and/or prevent an infectious disease, one or more V, D
and/or or all J gene segments used in any configuration, aspect,
method, example or embodiment of the invention can be selected from
List A1. Thus, for example in (a) of the first configuration of the
invention, the recited heavy chain V gene segment is selected from
the VH gene segments in List A, optionally with a D in that
list.
[0291] Where one wishes to generate an antibody or antibody mixture
to treat and/or prevent an infectious disease caused or mediated by
a bacterial pathogen, one or more or all V, D and/or J gene
segments used in any configuration, aspect, method, example or
embodiment of the invention can be selected from List A1.
[0292] Where one wishes to generate an antibody or antibody mixture
to treat and/or prevent an infectious disease caused or mediated by
a viral pathogen, one or more or all V, D and/or J gene segments
used in any configuration, aspect, method, example or embodiment of
the invention can be selected from List A2.
[0293] Where one wishes to generate an antibody or antibody mixture
to treat and/or prevent an infectious disease caused or mediated by
H. influenza, one or more or all V, D and/or J gene segments used
in any configuration, aspect, method, example or embodiment of the
invention can be selected from List A1.1.
[0294] Where one wishes to generate an antibody or antibody mixture
to treat and/or prevent an infectious disease caused or mediated by
E Coli or Neisseria meningitidis, one or more or all V, D and/or J
gene segments used in any configuration, aspect, method, example or
embodiment of the invention can be selected from List A1.2.
[0295] Where one wishes to generate an antibody or antibody mixture
to treat and/or prevent an infectious disease caused or mediated by
Herpes Virus Family (eg, VZV or HSV), one or more or all V, D
and/or J gene segments used in any configuration, aspect, method,
example or embodiment of the invention can be selected from List
A2.1.
[0296] Where one wishes to generate an antibody or antibody mixture
to treat and/or prevent an infectious disease caused or mediated by
CMV, one or more or all V, D and/or J gene segments used in any
configuration, aspect, method, example or embodiment of the
invention can be selected from List A2.2.
[0297] Where one wishes to generate an antibody or antibody mixture
to treat and/or prevent an infectious disease caused or mediated by
HIV, one or more or all V, D and/or J gene segments used in any
configuration, aspect, method, example or embodiment of the
invention can be selected from List A2.3.
[0298] Where one wishes to generate an antibody or antibody mixture
to treat and/or prevent an infectious disease caused or mediated by
Influenza Virus, one or more or all V, D and/or J gene segments
used in any configuration, aspect, method, example or embodiment of
the invention can be selected from List A2.4.
[0299] Optionally each VH segment in the locus of the invention is
selected from List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or
A2.4.
[0300] Optionally each VL segment in the locus of the invention is
selected from List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4
[0301] Optionally each D segment in the locus of the invention is
selected from List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or
A2.4.
[0302] Optionally each J.sub.L segment in the locus of the
invention is selected from List A1, A2, A1.1, A1.2, A2.1, A2.2,
A2.3 or A2.4.
Long HCDR3 Binding Sites & Tailoring Gene Segments to Pathogens
& Other Antigens
[0303] This aspect of the invention relates to the development of
vertebrates, cells, methods and antibodies with relatively long
HCDR3 binding sites. There is also provided embodiments in which
genomes and antibodies are tailored in terms of their gene segments
usage to address infectious disease pathogen antigens or other
antigens which are advantageously addressed with a longer HCDR3
length for binding or neutralisation. Antibodies may be raised in
the vertebrates by immunisation with a non-pathogen target antigen,
eg, an antigen bearing an epitope in a cleft requiring a long CDR
for contact, or an antigen from a pathogen that causes or is
implicated in harmful human disease or conditions. Examples are
bacterial or viral pathogens and the target antigen may be a
bacterial cell-surface antigen or a viral surface-exposed antigen
(eg, coat protein). Additionally or alternatively, the antigen may
be an antigen that is released (eg, secreted) from a pathogenic
bacterium or virus. The invention is not limited to addressing
pathogen antigens, but is also useful for addressing other antigens
where a long CDR3 would be useful for binding (eg, an enzyme active
site or receptor cleft).
[0304] Antibodies with long HCDR3 (at least 20 amino acids
according to IMGT nomenclature) have been shown to effectively
neutralise a variety of pathogens including HIV, Influenza virus,
malaria and Africa trypanosomes. Reference is also made to
naturally-occurring Camelid (eg, llama or camel) heavy chain-only
antibodies which bear long HCDR3s for reaching relatively
inaccessible epitopes (see, eg, EP0937140). Long HCDR3s can form
unique stable subdomains with extended loop structure that towers
above the antibody surface to confer fine specificity. In some
cases, the long HCDR3 itself is sufficient for epitope binding and
neutralization (Liu, L et al; Journal of Virology. 2011. 85:
8467-8476, incorporated herein by reference). The unique structure
of the long HCDR3 allows it to bind to cognate epitopes within
inaccessible structure or extensive glycosylation on a pathogen
surface. In human peripheral blood, there is around 3.5% of naive B
antibodies or 1.9% of memory B IgG antibodies containing the HCDR3s
with lengths of more than 24 amino acids (Briney, B S et al,
referenced given below) (FIG. 1 of Briney, B S et al). The usage
analysis indicates that these antibodies have the preference to use
human VH1-69, D2-2, D3-3, D2-15 and JH6 segments (FIGS. 2-5 of
Briney, B S et al). There are around 20% of all HCDR3 length
antibodies using JH6. However, in those antibodies with more than
24 amino acids of HCDR3, there are 70% using JH6 (FIG. 2 of Briney,
B S et al). Human VH5-51 is also commonly used for anti-HIV
antibodies (see Gorny et al, PLoS One. 2011; 6(12):e27780. Epub
2011 Dec. 2.
[0305] Human anti-V3 HIV-1 monoclonal antibodies encoded by the
VH5-51/VL lambda genes define a conserved antigenic structure,
incorporated herein by reference).
[0306] Supplementing these observations, the inventors have found
(see examples) that other selected human heavy chain variable
region gene segments (V, D, J) recombine in transgenic non-human
vertebrates to produce long HCDR3 (at least 20 amino acids).
[0307] Thus, as explained further in the examples, the inventors
constructed transgenic IgH loci in ES cells, wherein the loci
purposely included selected human heavy chain variable region gene
segments (V, D, J) that recombine to produce long HCDR3 (at least
20 amino acids). From the ES cells, the inventors generated
transgenic non-human vertebrates (both naive and immunised with a
range of different target antigen types--disease pathogen and human
antigenic species), isolated antibodies and heavy chain sequences
based on the selected gene segments as well as B-cells expressing
these and made hybridomas expressing antigen-specific antibodies
that are based on the selected gene segments.
[0308] There is a need in the art for genetically modified
non-human animals that prefer to make human antibodies that have
long HCDR3s, as well as antibodies that can be selected from such
animals wherein the antibodies can address target epitopes more
easily accessed by long HCDR3s. Long CDRH3 is also useful for
penetrating highly glycan-covered epitope sites (eg, virus epitopes
or any glycoprotein targets, eg, see Nature. 2011 Dec. 14;
480(7377):324-5. doi: 10.1038/480324a; Vaccinology: "A sweet cleft
in HIV's armour", Sattentau Q J, incorporated herein by reference),
and the target antigen can comprise such a target epitope.
[0309] The present invention provides vertebrates that can
artificially simulate those naturally-occurring human long HCDR3
antibodies, and can provide antibody, heavy chain and variable
domain repertoires from which can be selected an antibody, heavy
chain or variable domain having a long HCDR3 (eg, having a HCDR3
length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino
acids (according to IMGT). The invention provides for the
combination of human VH, D and J gene repertoires upstream of
non-human vertebrate (eg, mouse or rat, eg, endogenous mouse or
rat) constant region in heavy chain loci comprised by the
vertebrate genomes. This enables the recombination, maturation and
selection of the human gene segments in the context of endogenous
or other non-vertebrate constant regions which enhances the
development of good sized antibody, heavy chain and variable domain
repertoires from which to select long HCDR3-type binding sites.
Thus, in an example of any configuration of the invention, the
human gene segments are provided in a heavy chain locus upstream of
a non-human vertebrate (eg, endogenous) constant region. Similarly
any antibody of the invention comprises human variable domains and
non-human vertebrate (eg, endogenous) domains. The latter can be
replaced by human constant domains after selection and
isolation.
[0310] For example, the following antibodies of the invention are
contemplated (eg, produced in a vertebrate of this aspect of the
invention by a method disclosed herein) or a copy or derivative of
an antibody so produced:--
[0311] An isolated, synthetic or recombinant antibody comprising
human heavy chain variable domains having a HCDR3 length of 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids (according
to IMGT), the heavy chain variable domains being derived from the
recombination of a human VH gene segment selected from a VH group
disclosed herein with a human D gene segment and a human JH gene
segment (optionally a JH6), wherein the antibody binds a target
antigen; wherein the heavy chain variable domains have non-human
vertebrate AID-pattern somatic hypermutations, (eg, mouse or rat
AID-pattern mutations) when compared to corresponding human
germline V, D and J sequences and/or non-human (eg, mouse or rat)
terminal deoxynucleotidyl transferase (TdT)-pattern junctional
mutations when compared to corresponding human germline V, D and J
sequences. In an example, the antibody of the invention has a HCDR3
length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids
(according to IMGT). In an example, the antigen is an antigen of a
pathogen that causes or is implicated in a human infectious disease
or condition, eg, a pathogen listed in Table 1. In an example, the
antibody specifically binds an active site or cleft of an antigen
(eg, an enzyme active site or receptor cleft). This can be
determined, eg, using standard X-ray crystallography of a complex
of the antibody (or heavy chain or VH domain) with the cognate
antigen, as is known to the skilled person.
[0312] Mouse AID-pattern somatic hypermutations and/or mouse
dTd-pattern mutations can be provided, for example, wherein VH
domain is produced in a mouse comprising mouse AID and/or mouse TdT
(eg, endogenous AID or TdT). See also Annu. Rev. Biochem. 2007.
76:1-22; Javier M. Di Noia and Michael S. Neuberger, "Molecular
Mechanisms of Antibody Somatic Hypermutation" (in particular FIG. 1
and associated discussion on AID hotspots in mouse); and Curr Opin
Immunol. 1995 Apr.; 7(2):248-54, "Somatic hypermutation", Neuberger
M S and Milstein C (in particular, discussion on hotspots in
mouse), the disclosures of which are incorporated herein by
reference. Such mice can be made using corresponding mouse ES cell
technology.
[0313] In an example, the antibody specifically binds to a HIV
antigen. Several naturally-occurring human antibodies are known to
be neutralising of HIV and have rather long HCDR3 lengths (20 amino
acids or more according to IMGT; see Breden et al, PLoS One. 2011
Mar. 30; 6(3):e16857; "Comparison of antibody repertoires produced
by HIV-1 infection, other chronic and acute infections, and
systemic autoimmune disease" (incorporated herein by
reference)--VH1-69 preferred for long HCDR3). See also PLoS One.
2012; 7(5):e36750. Epub 2012 May 9; "Human peripheral blood
antibodies with long HCDR3s are established primarily at original
recombination using a limited subset of germline genes"; Briney B S
e al (incorporated herein by reference). Thus, it is desirable to
provide antibodies of the invention that have similarly long HCDR3
lengths. The antibody of the invention is, in one example, provided
for treating and/or preventing HIV infection, eg, chronic HIV
infection, in a human. The invention also provides a method of
treating and/or preventing HIV infection, eg, chronic HIV
infection, in a human, the method comprising administering a
pharmaceutically acceptable dose of the antibody of the invention.
The dose can be split into one or more administration aliquots, eg,
administered over a time course according to a medically-determined
regimen, as the skilled person will be able to determine.
[0314] In an example, the antibody specifically binds to Hemophilus
influenza type b polysaccharide. The antibody of the invention is,
in one example, provided for treating and/or preventing Hemophilus
influenza infection, eg, chronic Hemophilus influenza infection, in
a human. The invention also provides a method of treating and/or
preventing Hemophilus influenza infection, eg, chronic Hemophilus
influenza infection, in a human, the method comprising
administering a pharmaceutically acceptable dose of the antibody of
the invention. The dose can be split into one or more
administration aliquots, eg, administered over a time course
according to a medically-determined regimen, as the skilled person
will be able to determine.
[0315] In an example, the antibody specifically binds to a
rotavirus antigen (eg, protein 6 or 7). The antibody of the
invention is, in one example, provided for treating and/or
preventing rotavirus infection, eg, chronic rotavirus infection, in
a human. The invention also provides a method of treating and/or
preventing rotavirus infection, eg, chronic rotavirus infection, in
a human, the method comprising administering a pharmaceutically
acceptable dose of the antibody of the invention. The dose can be
split into one or more administration aliquots, eg, administered
over a time course according to a medically-determined regimen, as
the skilled person will be able to determine.
[0316] In an example, the antibody specifically binds to a
cytomegalovirus antigen (eg, cytomegalovirus gB antigen). The
antibody of the invention is, in one example, provided for treating
and/or preventing cytomegalovirus infection, eg, chronic
cytomegalovirus infection, in a human. The invention also provides
a method of treating and/or preventing cytomegalovirus infection,
eg, chronic cytomegalovirus infection, in a human, the method
comprising administering a pharmaceutically acceptable dose of the
antibody of the invention. The dose can be split into one or more
administration aliquots, eg, administered over a time course
according to a medically-determined regimen, as the skilled person
will be able to determine.
[0317] The invention also provides a vertebrate or cell for
expressing such an antibody; thus the invention provides a
non-human vertebrate (eg, a mouse or a rat) or a non-human
vertebrate cell (eg, a mouse cell or a rat cell) whose genome
comprises a human immunoglobulin VH gene segment repertoire that is
biased to one, more or all human VH gene segments selected from a
VH group disclosed herein.
[0318] The invention also provides a method of isolating an
antibody that binds a HIV antigen, Hemophilus influenza type b
polysaccharide, cytomegalovirus antigen or rotavirus antigen, the
method comprising
(a) providing the human VH biased vertebrate of the invention; (b)
immunising said vertebrate with said HIV antigen, Hemophilus
influenza type b polysaccharide, cytomegalovirus antigen or
rotavirus antigen; (c) removing B lymphocytes from the vertebrate
and selecting one or more B lymphocytes expressing antibodies that
bind to the antigen; (d) optionally immortalising said selected B
lymphocytes or progeny thereof, optionally by producing hybridomas
therefrom; and (e) isolating an antibody (eg, and IgG-type
antibody) expressed by the B lymphocytes, wherein the antibody has
a HCDR3 length of 20 amino acids or more.
[0319] Optionally, the method further comprises the step of
isolating from said B lymphocytes nucleic acid encoding said
antibody that binds said antigen; optionally exchanging the heavy
chain constant region nucleotide sequence of the antibody with a
nucleotide sequence encoding a human or humanised heavy chain
constant region and optionally affinity maturing the variable
region of said antibody; and optionally inserting said nucleic acid
into an expression vector and optionally a host.
[0320] Optionally, the method further comprises making a copy,
mutant or derivative (eg, humanised version) of the antibody
produced by the method.
[0321] This aspect of the invention also provides
[0322] A pharmaceutical composition comprising the anti-HIV
antibody, for treating and/or preventing HIV in a human (eg, an
infant human).
[0323] A pharmaceutical composition comprising the anti-Hemophilus
influenza type b polysaccharide antibody, for treating and/or
preventing Haemophilus influenza in a human (eg, an infant
human).
[0324] A pharmaceutical composition comprising the anti-rotavirus
antibody, for treating and/or preventing rotavirus in a human (eg,
an infant human).
[0325] A pharmaceutical composition comprising the
anti-cytomegalovirus antibody, for treating and/or preventing
cytomegalovirus in a human (eg, an infant human).
[0326] The invention also provides a method of generating such an
antibody (eg, any one of embodiments (i) et seq above) by
immunising a vertebrate of the invention with the target antigen
and isolating the antibody from the vertebrate, optionally also
making a copy or derivative of the antibody. In a further step, a
B-cell capable of expressing the antibody is isolated from the
vertebrate. In a further step, a nucleic acid encoding the antibody
(or a VH domain thereof) is isolated from the vertebrate (eg, a
nucleic acid PCR cloned from a B-cell isolated from the
vertebrate).
[0327] In an example, the antibody of the invention is a
neutralising antibody. In an example, the antibody of the invention
has a HCDR3 length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or
more amino acids (according to IMGT). In an example, the antibody
of the invention has a HCDR3 length of 20, 21, 22, 23, 24, 25, 26,
27, 28, 29 or 30 amino acids (according to IMGT). In an example,
the antibody of the invention is isolated from a non-human
vertebrate (eg, a mouse or a rat), for example a vertebrate of the
invention; or the antibody is a copy or derivative (eg, humanised
version) thereof. In an example, the antibody of the invention has
non-human vertebrate constant regions (eg, mouse or rat constant
regions); these may be replaced using standard recombinant DNA
technology with human constant regions, so the invention also
provides for human versions of the antibodies recited above,
wherein the human antibody comprises human variable and constant
regions, wherein the variable regions bind the antigen. In an
example, the antibody of the has lambda-type human light chain
variable domains. In another example, the antibody of the invention
has kappa-type human light chain variable domains.
[0328] Antibody competition can be determined, for example, by
ELISA or surface plasmon resonance (SPR; eg, by competition
Biacore.TM. or Proteon) as is standard.
[0329] The invention also provides the following embodiments
(recited below as numbered clauses):--
D Bias
[0330] 1. A non-human vertebrate (eg, a mouse or a rat) or a
non-human vertebrate cell (eg, a mouse cell or a rat cell) whose
genome comprises a human immunoglobulin D gene segment repertoire
that is biased to the human D2 and/or D3 family or biased to one,
more or all human D gene segments selected from the group D1-26,
D2-2, D3-9, D3-10, D3-22, D4-17, D6-13 and D6-19.
[0331] For example, the repertoire consists of only human D gene
segments from the D2 and/or D3 family.
[0332] Optionally the repertoire is biased to one or more of human
D2-2, D2-15, D3-3, D3-9, D3-10 and D3-22, or the repertoire
consists of one, more or all of these D gene segments. These
produce long HCDR3 lengths (eg, see Table 2 and references cited
herein).
[0333] For example, the repertoire is biased to one or more of
human of D2-2*02, D3-9*01, D3-10*01 and D3-22*01, or the repertoire
consists of one, more or all of these D gene segments.
[0334] For example, the repertoire is biased to one or more of
human D2-2*02, D3-9*01 and D3-10*01, or the repertoire consists of
one, more or all of these D gene segments.
[0335] For example, the repertoire is biased to D3-9*01 and
D3-10*01, or consists of one, more or all of these D gene
segments.
[0336] Optionally the repertoire consists of one, more or all of
human D1-26, D2-2, D3-9, D3-10, D3-22, D4-17, D6-13 and D6-19.
These produce long HCDR3 lengths (eg, see Table 2).
[0337] Optionally the repertoire is biased to one or more of human
D1-26*01, D2-2*02, D3-9*01, D3-10*01, D3-22*01, D4-17*01, D6-13*01
and D6-19*01, or the repertoire consists of one, more or all of
these D gene segments.
[0338] Optionally the repertoire is biased to one or more of human
D2-2, D3-9, D3-10, D3-22, D4-17, D6-13 and D6-19, or the repertoire
consists of one, more or all of these D gene segments. Optionally
the repertoire is biased to one or more of human D2-2*02, D3-9*01,
D3-10*01, D3-22*01, D4-17*01, D6-13*01 and D6-19*01, or the
repertoire consists of one, more or all of these D gene segments.
These produce long HCDR3 lengths in naive repertoires (eg, see
Table 2).
[0339] Optionally the repertoire is biased to one or more of human
D1-26, D2-2, D3-10 and D6-19, or the repertoire consists of one,
more or all of these D gene segments. Optionally the repertoire is
biased to one or more of human D1-26*01, D2-2*02, D3-10*01 and
D6-19*01, or the repertoire consists of one, more or all of these D
gene segments. These produce long HCDR3 lengths in immunised
repertoires (eg, see Table 2).
[0340] Optionally the repertoire is biased to one or more of human
D2-2, D3-9 and D3-10, or the repertoire consists of one, more or
all of these D gene segments. Optionally the repertoire is biased
to one or more of human D2-2*02, D3-9*01 and D3-10*01, or the
repertoire consists of one, more or all of these D gene segments.
These produce long HCDR3 lengths in antigen-specific repertoires
(eg, see Table 2).
[0341] IMGT nomenclature is used for all gene segments.
[0342] Throughout this text, Genbank is a reference to Genbank
release number 185.0 or 191.0; the 1000 Genomes database is Phase
1, release v3, 16 Mar. 2012; the Ensembl database is assembly
GRCh37.p8 (10/04/2012); the IMGT database is available at
www.imgt.org. The sequences of all VH gene segments explicitly
mentioned herein are disclosed herein in their entirety (for
possible inclusion in clauses in conjunction with any aspect of the
invention as clauseed), such sequences being those in the IMGT and
1000 Genomes databases.
[0343] In one embodiment, the genome comprises an IgH locus
comprising a targeted insertion of said human D gene segments. In
an example, the IgH locus comprises (in 5' to 3' order) one or more
human VH gene segments, said D gene segment repertoire, one or more
human JH gene segments and a constant region (eg, wherein the
constant region is a human constant region or a non-human (eg,
endogenous, eg, mouse or rat) constant region).
[0344] In another embodiment, the genome comprises said human D
gene segments randomly inserted therein. This can be effected, eg,
by incorporating human DNA borne by YACS into the genome of ES
cells (followed optionally by generation of a non-human vertebrate
therefrom, as is standard).
[0345] Optionally, the human D gene segment repertoire further
comprises no more than 5 additional human D gene segments, for
example, the repertoire includes 1, 2, 3, 4 or 5 additional human D
gene segments. [0346] 2. The vertebrate or cell of clause 1,
wherein the D gene segment repertoire consists of or substantially
consists of one, two or three human gene segments selected from the
group D1-26, D2-2, D3-9, D3-10, D3-22, D4-17, D6-13 and D6-19.
[0347] 3. The vertebrate or cell of clause 1 or 2, wherein the
genome comprises an unrearranged immunoglobulin heavy chain locus
comprising (in 5' to 3' order) human VH, D and JH gene segments and
said human D gene segments recited in clause 1 are spaced from the
VH gene segment(s) by no more than four other D gene segments (eg,
by no D gene segments). [0348] This provides for bias wherein
proximal D gene segments (those more 3', ie, closer to the constant
region) are likely to be more frequently used than those segments
from distal (ie, 5' or further away from the constant region).
[0349] 4. The vertebrate or cell of any preceding clause, wherein
the genome comprises an unrearranged immunoglobulin heavy chain
locus comprising said human D gene segments and there are no other
D gene segments in the locus between said human D gene segments.
[0350] This is another way of biasing the repertoire of D gene
segments. Thus, the desired Ds are provided in tandem, aimed to
promote use in recombination. [0351] 5. The vertebrate or cell of
any preceding clause, wherein the genome comprises three or more
copies of a human D gene segment selected from D1-26, D2-2, D2-15,
D3-3, D3-9, D3-10, D3-22, D4-17, D6-13 and D6-19. [0352] For
example, the genome comprises three or more copies of a human D
gene segment selected from D1-26, D2-2, D3-9, D3-10, D3-22, D4-17,
D6-13 and D6-19. [0353] This is another way of biasing the
repertoire of D gene segments. [0354] 6. The vertebrate or cell of
clause 5, wherein the genome comprises first and second human D
gene segments selected from D1-26, D2-2, D2-15, D3-3, D3-9, D3-10,
D3-22, D4-17, D6-13 and D6-19 when the first D gene segment is
present as three or more copies and wherein the second D gene
segment is present as three or more copies. [0355] For example, the
first and second gene segments are selected from D1-26, D2-2, D3-9,
D3-10, D3-22, D4-17, D6-13 and D6-19. [0356] The various gene
segment biasing techniques described herein can be performed using
conventional DNA manipulation in the construction of transgenic
vertebrates or cells of the invention, which techniques (eg,
recombineering and recombinant DNA technology) will be known to the
skilled person. For example, BACs can be constructed using these
techniques in which the desired combination of human gene segments
is provided, and these BACs can be introduced into ES cells for
incorporation of the human gene segments into the genomes thereof
(eg, by targeted insertion into Ig loci). The ES cells can be used
to generate transgenic vertebrates as is standard and cells (eg,
B-cells) can be isolated from these wherein the genome is as per
the invention.
[0357] In one embodiment, the biased D gene segment(s) are selected
from the IMGT database of variants or the 1000 Genomes database.
[0358] 7. The vertebrate or cell of any preceding clause, wherein
the D gene segments are selected from D2-2*02, D3-9*01, D3-10*01
and D3-22*01, or selected from D1-26*01, D2-2*02, D3-9*01,
D3-10*01, D3-22*01, D4-17*01, D6-13*01 and D6-19*01.
VH Bias
[0358] [0359] 8. A non-human vertebrate (eg, a mouse or a rat) or a
non-human vertebrate cell (eg, a mouse cell or a rat cell),
optionally according to any preceding clause, whose genome
comprises a human immunoglobulin VH gene segment repertoire that is
biased to one, more or all of gene segments selected from the group
VH1-2, VH1-3, VH1-8, VH1-18, VH5-51, VH1-69, VH2-5, VH3-7, VH3-9,
VH3-11, VH3-13, VH3-20, VH3-21, VH3-23, VH4-4, VH6-1 and VH7-4-1.
[0360] These produce long HCDR3 lengths (see Table 2 and references
cited herein). [0361] For example, the VH repertoire is biased to
one, more or all of VH1-2, VH1-3, VH1-8, VH1-18, VH2-5, VH3-7,
VH3-9, VH3-11, VH3-13, VH3-20, VH3-21, VH3-23, VH4-4, VH6-1 and
VH7-4-1. These produce long HCDR3 lengths (see Table 2), or the
repertoire consists of one, more or all of these VH gene segments.
For example, the VH repertoire is biased to one, more or all of
VH1-2*02, VH1-3*01, VH1-8*01, VH1-18*01, VH2-5*10, VH3-7*01,
VH3-9*01, VH3-11*01, VH3-13*01, VH3-21*03, VH3-23*04, VH4-4*02,
VH6-1*01 and VH7-4-1*01, or the repertoire consists of one, more or
all of these VH gene segments. [0362] For example, the VH
repertoire is biased to one, more or all of VH1-2*02, VH1-8*01,
VH1-18*01, VH1-3*01, VH2-5*10, VH3-7*01, VH3-9*01, VH3-13*01,
VH3-21*03, VH3-23*04, VH4-4*02, VH6-1*01 and VH7-4-1*01, or the
repertoire consists of one, more or all of these VH gene segments.
These produce long HCDR3 lengths in naive repertoires (see Table
2). [0363] For example, the VH repertoire is biased to one, more or
all of VH4-4*02, VH3-11*01 and VH3-7*01, or the repertoire consists
of one, more or all of these VH gene segments. These produce long
HCDR3 lengths in immunised repertoires (see Table 2). [0364] For
example, the VH repertoire is biased to one, more or all of
VH1-3*01, VH1-8*01, VH3-7*01, VH3-9*01, VH3-11*01 and VH4-4*02, or
the repertoire consists of one, more or all of these VH gene
segments. These produce long HCDR3 lengths in antigen-specific
repertoires (see Table 2).
[0365] Optionally, the human VH gene segment repertoire further
comprises no more than 5 additional human VH gene segments, for
example, the repertoire includes 1, 2, 3, 4 or 5 additional human
VH gene segments.
[0366] In one embodiment, the genome comprises an IgH locus
comprising a targeted insertion of said human VH gene segments. In
an example, the IgH locus comprises (in 5' to 3' order) said VH
gene segment repertoire, one or more human D gene segments, one or
more human JH gene segments and a constant region (eg, wherein the
constant region is a human constant region or a non-human (eg,
endogenous, eg, mouse or rat) constant region).
[0367] In another embodiment, the genome comprises said human VH
gene segments randomly inserted therein. This can be effected, eg,
by incorporating human DNA borne by YACS into the genome of ES
cells (followed optionally by generation of a non-human vertebrate
therefrom, as is standard). [0368] 9. The vertebrate or cell of
clause 8, wherein the VH gene segment repertoire substantially
consists of or substantially consists of one, two or three human
gene segments selected from VH1-2, VH1-3, VH1-8, VH1-18, VH5-51,
VH1-69, VH2-5, VH3-7, VH3-9, VH3-11, VH3-13, VH3-20, VH3-21,
VH3-23, VH4-4, VH6-1 and VH7-4-1. [0369] For example, the VH gene
segment repertoire substantially consists of or substantially
consists of one, two or three human gene segments selected from the
group consisting of VH1-2*02, VH1-3*01, VH1-8*01, VH1-18*01,
VH2-5*10, VH3-7*01, VH3-9*01, VH3-11*01, VH3-13*01, VH3-21*03,
VH3-23*04, VH4-4*02, VH6-1*01 and VH7-4-1*01. [0370] 10. The
vertebrate or cell of clause 8 or 9, wherein the genome comprises
an unrearranged immunoglobulin heavy chain locus comprising (in 5'
to 3' order) human VH, D and JH gene segments and said human VH
gene segments are spaced from the D gene segment(s) by no more than
four other VH gene segments (eg, by no VH gene segments). [0371]
This provides for bias wherein proximal VH gene segments (those
more 3', ie, closer to the constant region) are likely to be more
frequently used than those segments from distal (ie, 5' or further
away from the constant region). [0372] 11. The vertebrate or cell
of any one of clauses 8 to 10, wherein the genome comprises an
unrearranged immunoglobulin heavy chain locus comprising said human
VH gene segments and there are no other VH gene segments in the
locus between said human VH gene segments. [0373] This is another
way of biasing the repertoire of VH gene segments. [0374] 12. The
vertebrate or cell of any one of clauses 8 to 11, wherein the
genome comprises three or more copies of a human VH gene segment
selected from the group consisting of VH1-2, VH1-3, VH1-8, VH1-18,
VH5-51, VH1-69, VH2-5, VH3-7, VH3-9, VH3-11, VH3-13, VH3-20,
VH3-21, VH3-23, VH4-4, VH6-1 and VH7-4-1. [0375] For example, the
genome comprises three or more copies of a human VH gene segment
selected from the group consisting of VH1-2*02, VH1-3*01, VH1-8*01,
VH1-18*01, VH2-5*10, VH3-7*01, VH3-9*01, VH3-11*01, VH3-13*01,
VH3-21*03, VH3-23*04, VH4-4*02, VH6-1*01 and VH7-4-1*01. [0376]
This is another way of biasing the repertoire of VH gene segments.
[0377] 13. The vertebrate or cell of clause 12, wherein the genome
comprises first and second human VH gene segments selected from the
group consisting of VH1-2, VH1-3, VH1-8, VH1-18, VH5-51, VH1-69,
VH2-5, VH3-7, VH3-9, VH3-11, VH3-13, VH3-20, VH3-21, VH3-23, VH4-4,
VH6-1 and VH7-4-1 when the first VH gene segment is present as
three or more copies and wherein the second VH gene segment is
present as three or more copies. [0378] For example, the genome
comprises first and second human VH gene segments selected from the
group consisting of VH1-2*02, VH1-3*01, VH1-8*01, VH1-18*01,
VH2-5*10, VH3-7*01, VH3-9*01, VH3-11*01, VH3-13*01, VH3-21*03,
VH3-23*04, VH4-4*02, VH6-1*01 and VH7-4-1*01 when the first VH gene
segment is present as three or more copies and wherein the second
VH gene segment is present as three or more copies. [0379] In an
embodiment, all or substantially all of VH gene segments are
present as three or more copies each. [0380] The various gene
segment biasing techniques described herein can be performed using
conventional DNA manipulation in the construction of transgenic
vertebrates or cells of the invention, which techniques (eg,
recombineering and recombinant DNA technology) will be known to the
skilled person. For example, BACs can be constructed using these
techniques in which the desired combination of human gene segments
is provided, and these BACs can be introduced into ES cells for
incorporation of the human gene segments into the genomes thereof
(eg, by targeted insertion into Ig loci). The ES cells can be used
to generate transgenic vertebrates as is standard and cells (eg,
B-cells) can be isolated from these wherein the genome is as per
the invention.
[0381] In one embodiment, the biased D gene segment(s) are selected
from the IMGT database of variants or the 1000 Genomes database.
[0382] 14. The vertebrate or cell of any one of clauses 8 to 13,
wherein the VH gene segments are selected from the group consisting
of VH1-2*02, VH1-3*01, VH1-8*01, VH1-18*01, VH2-5*10, VH3-7*01,
VH3-9*01, VH3-11*01, VH3-13*01, VH3-21*03, VH3-23*04, VH4-4*02,
VH6-1*01 and VH7-4-1*01. [0383] In an embodiment, the genome
comprises a human immunoglobulin VH gene segment repertoire that is
biased to VH1-69. [0384] In an embodiment, the human immunoglobulin
VH gene segment repertoire substantially consists of one or more
human VH1-69 gene segments. [0385] The gene segments are provided
in one or more immunoglobulin loci. For example, the gene segment
repertoire (D and/or VH) is provided in both IgH loci (ie, in a
homozygous state). [0386] 15. The vertebrate or cell of any one of
clauses 8 to 16, comprising an immunoglobulin heavy chain locus
comprising two or more copies of a VH gene segment selected from
said group. [0387] Thus, at least one of said copies is closer to
the constant region of the locus than the germline distance in a
human from a human constant region. The aim is to provide by bias
by providing more than one copy on the same locus. Also as at least
one of the copies is closer (more proximal to) the constant region
and J-C intron (which includes regulatory elements such as the Emu
enhancer region), this may favour use of the gene segment, thus
contributing to the desired bias. [0388] Optionally, the genome is
homozygous for the heavy chain locus. [0389] Optionally the two or
more copies of gene segments are identical (eg, all VH1-69*01,
using IMGT nomenclature). In another example, copies are variants
of each other, eg, naturally-occurring human variants.
Alternatively, synthetic variants may be used with or without a
naturally-occurring variant. [0390] In any embodiment of the
invention, the vertebrate is naive or immunised with a target
antigen. [0391] 16. The vertebrate or cell of any clause, wherein
the genome comprises a human JH gene segment repertoire consisting
of one or more human JH6 gene segments. [0392] This biases the JH
repertoire for the production of long HCDR3, since this is the
longest naturally-occurring human JH gene segment type and is
commonly found in naturally-occurring human antibodies having long
HCDR3. [0393] For example, the repertoire comprises two or more
different JH6 variants. In an example, the repertoire comprises two
or more JH6*02 variants (IMGT nomenclature). [0394] 17. The
vertebrate or cell of any preceding clause, wherein the genome
comprises a human immunoglobulin JH gene segment repertoire that is
biased to JH6, optionally JH6*02. [0395] 18. The vertebrate or cell
of clause 17, wherein the JH gene segment repertoire consists or
substantially consists of three or more human JH6 gene segments.
[0396] 19. The vertebrate or cell of any preceding clause, wherein
the sequence of each of said human gene segments is a human
germline gene segment sequence. [0397] 20. The vertebrate or cell
of any preceding clause, wherein one, more or all of the selected
gene segments are present in the genome as two or more copies, the
copies being variants of each other. [0398] Thus, one, more or all
of the human V, D and JH gene segments of said genome is present in
two or more variant versions, such as naturally-occurring human
variants, eg, variants found in the 1000 Genomes database and/or
IMGT database. In another example, one or more of the variants may
be a synthetic variant. [0399] 21. The vertebrate or cell of any
preceding clause, wherein said human gene segments are provided by
homozygous immunoglobulin heavy chain loci. [0400] In an example,
no other (non-human) active heavy chain VH, D or JH gene segments
are present in heavy chain loci of the genome. Additionally, in an
example no active non-human light chain VL or JL gene segments are
present in the genome. [0401] This is useful for ensuring that
endogenous (non-human) variable region expression is inactivated.
Thus, all heavy chains produced by the vertebrate or cell will have
human variable regions, which is useful for producing drugs for
administration to humans. [0402] 22. A non-human vertebrate (eg, a
mouse or a rat) or a non-human vertebrate cell (eg, a mouse cell or
a rat cell) whose genome comprises a human immunoglobulin VH gene
segment repertoire, one or more human D gene segments and one or
more human JH gene segments, wherein the VH repertoire does not
comprise one, more or all VH gene segments selected from the group
VH1-2, VH1-3, VH1-8, VH1-18, VH5-51, VH1-69, VH2-5, VH3-7, VH3-9,
VH3-11, VH3-13, VH3-20, VH3-21, VH3-23, VH4-4, VH6-1 and VH7-4-1.
[0403] 23. A non-human vertebrate (eg, a mouse or a rat) or a
non-human vertebrate cell (eg, a mouse cell or a rat cell) whose
genome comprises a human immunoglobulin D gene segment repertoire,
one or more human VH gene segments and one or more human JH gene
segments, wherein the D repertoire does not comprise one, more or
all D gene segments selected from the group D1-26, D2-2, D3-9,
D3-10, D3-22, D4-17, D6-13 and D6-19. [0404] In instances it has
been observed in that the art that certain human gene usage may
dominate the immune response to infectious disease pathogen
antigens or other antigens. While this may yield many specific
antibodies, typically these may not be neutralising and thus the
immune response is relatively ineffective. This may happen, for
example, where the antigen is a decoy antigen expressed by the
pathogen. The present embodiments of the invention where specific
gene segments are omitted are useful for avoiding dominance of
certain human gene segments, such as those omitted from the genome.
In this way, the genome human gene segment repertoire is biased
away from the dominance and this enables better use and sampling of
the remaining human gene segment sequence space, thereby providing
the chance of producing antibodies that may not be normally raised
in a natural setting. Antigen specific antibodies can be selected
from vertebrates and cells with such genomes. In some examples,
this may yield neutralising antibodies. [0405] It is advantageous
to include a plurality of different human VH gene segments, making
up the human VH gene segment repertoire. This provides for good
diversities of rearranged human variable regions from which to
select leads. It is possible, for example, to include an otherwise
complete, functional repertoire of human VH gene segments. To this
end, the human VH gene segment repertoire comprises, in one
example, a plurality of human VH gene segments, eg, at least 7, 10,
15, 20, 15, 30, 35, 40 or 45 different human VH gene segments. This
can be achieved, for example, using BACs harbouring stretches of
unrearranged human variable region DNA comprising VH gene
segments--homologous recombination and/or sRMCE being used to
insert several stretches of such DNA from serial BACs into an
endogenous heavy chain locus upstream of the constant region
thereof in the genome of a non-human vertebrate ES cell (eg, mouse
or rat ES cell), followed by development of one or more progeny
vertebrates from such cells (and optional breeding to homozygosity
of the heavy chain locus). In one embodiment, human DNA is inserted
that includes a first human VH (eg, VH1-69 and/or VH1-2) and
flanking VH gene segments upstream and downstream of these. In a
second ES cell genomic manipulation, the first VH is deleted from
the genome, eg, using standard homolgous recombination techniques
as is known in the art. In this way, one or more VH gene segments
usually upstream and/or downstream of the deleted gene segment(s)
in a wild-type human germline genome are retained so that they can
be available to contribute to the subsequent rearranged human V
region repertoire that is used for selection of leads. In another
example, the initial insertion of human DNA is made using stretches
of DNA that already omit the first VH (eg, by deleting such
stretches using recombineering of BACs in E. coli, as is known in
the art). Similar techniques can be used (with appropriate BACs)
for the omission of human D and/or J gene segments.
[0406] Thus, in an embodiment, VH gene segments that normally are
upstream and/or downstream of the omitted human VH gene segments
(or omitted D or J as per other embodiments) in a wild-type
germline human genome are included in the vertebrate or cell of the
invention. For example, the human VH gene segment repertoire of the
genome in the invention does not comprise VH1-69, but does comprise
one, two, three or four human VH gene segments selected from
VH2-10, VH3-72, VH3-73 and VH3-74. These are gene segments that are
immediately upstream of VH1-69 in a wild-type human germline heavy
chain locus (see IMGT). For example, additionally or alternatively
the human VH gene segment repertoire of the genome in the invention
does not comprise VH1-69, but does comprise one, two, three, four
or more (or all of) human VH gene segments selected from VH3-66,
VH3-64, VH4-61, VH4-59, VH1-58, VH3-53, VH3-49, VH3-48, VH1-46 and
VH1-45. These are gene segments that are immediately downstream of
VH1-69 in a wild-type human germline heavy chain locus (see IMGT).
Additionally or alternatively, the human VH gene segment repertoire
of the genome in the invention does not comprise VH1-69, but does
comprise one, two, three, four or more (or all of) human VH gene
segments selected from VH2-5, 7-41, 4-4, 1-3, 1-2 and 6-1.
Additionally or alternatively, the human VH gene segment repertoire
of the genome in the invention does not comprise VH1-69, but does
comprise one, two, three, four, 5, 6, 7, 8, 9, 10 or more (or all
of) human VH gene segments selected from VH2-5, 7-41, 4-4, 1-3,
1-2, 6-1, 3-7, 1-8, 3-9, 3-11 and 3-13. Additionally or
alternatively, the human VH gene segment repertoire of the genome
in the invention does not comprise VH1-69, but does comprise one,
two, three, four, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or
more (or all of) human VH gene segments selected from VH2-5, 7-41,
4-4, 1-3, 1-2, 6-1, 3-7, 1-8, 3-9, 3-11, 3-13, 3-15, 1-18, 3-20,
3-21, 3-23, 1-24 and 2-26. Additionally or alternatively, the human
VH gene segment repertoire of the genome in the invention does not
comprise VH1-69, but does comprise VH6-1 (which is commonly used in
human immune responses, VH6-1 being the most proximal to the
constant region in a wild-type human germline heavy chain locus)
and/or VH3-23 (which is commonly used in human immune responses).
In embodiment (eg, for generating VH, heavy chains or antibodies
for treating and/or preventing an infectious disease, eg, HIV
infection, in a human), VH1-2 is omitted in the genome or locus. In
this case one, two, three or all human VH gene segments immediately
5' and 3' of VH1-2 in a wild-type germline human IgH locus (eg, see
IMGT) are included in the genome, such as comprised by the same IgH
locus upstream of human D and JH gene segments and a constant
region. [0407] 24. The vertebrate or cell of clause 22 or 23,
wherein the genome comprises a human JH gene segment repertoire
that does not comprise JH6.
JH Bias
[0407] [0408] 25. A non-human vertebrate (eg, a mouse or a rat) or
a non-human vertebrate cell (eg, a mouse cell or a rat cell) whose
genome comprises a human immunoglobulin JH gene segment repertoire
that is biased to human JH6. [0409] In an example, the repertoire
is biased to human JH6*02 (IMGT nomenclature). [0410] So, the
inventors made a choice of human JH6*02 on the basis of [0411] (i)
Containing YYG and YYGXDX motifs that is conserved across several
vertebrate species; [0412] (ii) Provision of one less TAC codon
than other human JH6 variant (an AID hotspot that risks stop
codons) and a choice instead of a codon that preserves the YYG and
YYGXDX motifs; [0413] (iii) Avoidance of a GGCA AID hotspot in the
region of the HCDR3/FW4 junction; and [0414] (iv) Common occurrence
(and thus conservation and acceptability) in humans of the JH6*02
variant. [0415] 26. The vertebrate or cell of clause 25, wherein
the genome comprises an unrearranged immunoglobulin heavy chain
locus comprising a plurality of human JH6 gene segments; optionally
wherein the genome is homozygous for said locus. [0416] In an
example, the plurality comprises or consists of a plurality of
JH6*02 gene segments. [0417] 27. The vertebrate or cell of clause
26, wherein the heavy chain locus comprises (in 5' to 3' order)
human VH, D and JH gene segments and said JH6 gene segments are
spaced from the D gene segment(s) by no more than two other JH gene
segments. [0418] 28. The vertebrate or cell of clause 25, 26 or 27,
wherein are no other JH gene segments in the locus between said
human JH6 gene segments. [0419] 29. A non-human vertebrate (eg, a
mouse or a rat) or a non-human vertebrate cell (eg, a mouse cell or
a rat cell) whose genome comprises a human immunoglobulin JH gene
segment repertoire that consists of one or more human JH6 gene
segments. [0420] In an example, all of the gene segments are JH6*02
gene segments. [0421] 30. The vertebrate or cell of any one of
clauses 25 to 29, wherein all of said gene segments are human
germline gene segments. [0422] 31. The vertebrate or cell of any
one of clauses 25 to 30, comprising different variant JH6 gene
segments. [0423] In an example, the variants are all
naturally-occurring (eg, appearing in the IMGT or 1000 Genome
databases). In an other example, one or more variant is synthetic.
[0424] 32. The vertebrate or cell of any one of clauses 25 to 31,
wherein said gene segments are provided by homozygous
immunoglobulin heavy chain loci.
[0425] In one embodiment, the biased JH gene segment(s) are
selected from the IMGT database of variants or the 1000 Genomes
database. [0426] 33. A monoclonal or polyclonal antibody
composition or a population of antibody-producing cells for
producing such composition, wherein the composition or population
is prepared by immunising at least one vertebrate according to any
preceding clause with an antigen, wherein the antibody or
antibodies have human heavy chain variable regions comprising
non-human vertebrate AID-pattern somatic hypermutations, (eg, mouse
or rat AID-pattern mutations) when compared to corresponding human
germline V, D and J sequences and/or non-human (eg, mouse or rat)
terminal deoxynucleotidyl transferase (TdT)-pattern junctional
mutations when compared to corresponding human germline V, D and J
sequences; wherein the composition comprises at least one
antigen-specific antibody having a HCDR3 length of at least 20
amino acids (according to IMGT). [0427] As will be readily apparent
to the skilled person, AID and TdT mutations can be determined
using bioinformatics analysis to find the closest matching human
germline gene segment(s) that correspond to a given variable domain
sequence, aligning the sequences and determining the differences.
AID has known hotspots for mutation (eg, see Annu. Rev. Biochem.
2007. 76:1-22; Javier M. Di Noia and Michael S. Neuberger,
"Molecular Mechanisms of Antibody Somatic Hypermutation" (in
particular FIG. 1 and associated discussion on AID hotspots in
mouse); and Curr Opin Immunol. 1995 April; 7(2):248-54, "Somatic
hypermutation", Neuberger M S and Milstein C (in particular,
discussion on hotspots in mouse), the disclosures of which are
incorporated herein by reference). By carrying out the standard
bioinformatics analysis, TdT mutations (eg, to provide junctional
mutations and diversity) can be determined, as will be familiar to
the skilled person. [0428] Corresponding human germline V, D and J
sequences can be according to the IMGT database or 1000 Genomes
database, for example. [0429] For example, the HCDR3 length is at
least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids.
[0430] For example, the HCDR3 length is from 20 to 23 or 24 to 30,
eg, from 28 to 30 amino acids. [0431] For example, the cells are B
cells (eg, immortalised B cells) or hybridomas. [0432] Optionally
the antibodies of any aspect of the invention comprise human light
chain variable regions. For example, the human light chain variable
regions have non-human vertebrate AID-pattern somatic
hypermutations, (eg, mouse or rat AID-pattern mutations) when
compared to corresponding human germline V, D and J sequences
and/or non-human (eg, mouse or rat) terminal deoxynucleotidyl
transferase (TdT)-pattern junctional mutations when compared to
corresponding human germline V, D and J sequences. [0433] 34. An
isolated antibody that specifically binds an antigen, the antibody
comprising human heavy chain variable regions and non-human
constant regions, wherein the variable regions are derived from the
recombination in a non-human vertebrate of (i) a human VH gene
segment selected from the group recited in clause 8 with (ii) a
human D gene segment selected from the group recited in clause 1
and with a human JH gene segment (optionally JH6); wherein the
antibody has a HCDR3 length of at least 20 amino acids (according
to IMGT); and non-human vertebrate AID-pattern somatic
hypermutations, (eg, mouse or rat AID-pattern mutations) when
compared to corresponding human germline V, D and J sequences
and/or non-human (eg, mouse or rat) terminal deoxynucleotidyl
transferase (TdT)-pattern junctional mutations when compared to
corresponding human germline V, D and J sequences. [0434] In
examples, the VH is selected from the group VH1-2*02, VH1-3*01,
VH1-8*01, VH1-18*01, VH2-5*10, VH3-7*01, VH3-9*01, VH3-11*01,
VH3-13*01, VH3-21*03, VH3-23*04, VH4-4*02, VH6-1*01 and VH7-4-1*01
and/or [0435] the D is selected from the group [0436] D2-2*02,
D3-9*01, D3-10*01 and D3-22*01, or [0437] D2-2*02, D3-9*01 and
D3-10*01, or [0438] D3-9*01 and D3-10*01, or [0439] D1-26, D2-2,
D3-9, D3-10, D3-22, D4-17, D6-13 and D6-19, or [0440] D1-26*01,
D2-2*02, D3-9*01, D3-10*01, D3-22*01, D4-17*01, D6-13*01 and
D6-19*01, or [0441] D2-2, D3-9, D3-10, D3-22, D4-17, D6-13 and
D6-19, or D2-2*02, D3-9*01, D3-10*01, D3-22*01, [0442] D4-17*01,
D6-13*01 and D6-19*01, or [0443] D1-26, D2-2, D3-10 and D6-19, or
[0444] D2-2, D3-9 and D3-10. [0445] 35. The antibody of clause 34,
wherein the antibody is obtained or obtainable from a vertebrate
according to any one of clauses 1 to 32. [0446] In an embodiment,
the antibody is obtained from said vertebrate, or is a copy of such
an antibody. [0447] 36. A method of isolating an antibody that
binds a predetermined antigen, the method comprising [0448] (a)
providing a vertebrate (optionally a mouse or rat) according to any
one of clauses 1 to 32; [0449] (b) immunising said vertebrate with
said antigen; [0450] (c) removing B lymphocytes from the vertebrate
and selecting one or more B lymphocytes expressing antibodies that
bind to the antigen; [0451] (d) optionally immortalising said
selected B lymphocytes or progeny thereof, optionally by producing
hybridomas therefrom; and [0452] (e) isolating an antibody (eg, and
IgG-type antibody) expressed by the B lymphocytes. [0453] 37. The
method of clause 36, wherein in step (e) wherein the antibody has a
HCDR3 length of at least 20 amino acids (according to IMGT). [0454]
The length can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30
amino acids (according to IMGT), eg, from 20 to 23 amino acids (a
produced in the examples). [0455] 38. The method of clause 36 or
37, comprising the step of isolating from said B lymphocytes
nucleic acid encoding said antibody that binds said antigen;
optionally exchanging the heavy chain constant region nucleotide
sequence of the antibody with a nucleotide sequence encoding a
human or humanised heavy chain constant region and optionally
affinity maturing the variable region of said antibody; and
optionally inserting said nucleic acid into an expression vector
and optionally a host. [0456] 39. The method of clause 36, 37 or
38, further comprising making a copy, mutant or derivative (eg,
humanised version) of the antibody produced by the method. [0457]
Humanisation can entail making the constant regions human. [0458]
40. The antibody composition, cell population, antibody or method
of any one of clauses 33 to 39, wherein the antigen is an antigen
of an infectious disease pathogen; optionally wherein the pathogen
is a virus or bacterium. [0459] 41. The antibody composition, cell
population, antibody or method of clause 40, wherein pathogen is
selected from the group consisting of Haemophilus influenza, E.
coli, Neisseria meningitidis, a herpes family virus,
cytomegalovirus (CMV), HIV and influenza virus. [0460] 42. The
antibody composition, cell population, antibody or method of any
one of clauses 33 to 41, wherein the antigen is a HIV gp120 antigen
or a HIV gp41 antigen. [0461] 43. The antibody composition, cell
population, antibody or method of any one of clauses 33 to 40,
wherein the antigen comprises an active site or cleft, wherein the
antibody having a HCDR3 length of at least 20 amino acids
specifically binds to the active site or cleft of the antigen.
[0462] 44. A pharmaceutical composition comprising an antibody or
antibody composition according to any one of clauses 33 to 35 and
40 to 43, or an antibody produced by the method of any one of
clauses 36 to 38, for treating and/or preventing an infectious
disease in a human (eg, wherein the infectious disease is caused by
a pathogen selected from the group consisting of Haemophilus
influenza, E. coli, Neisseria meningitidis, a herpes family virus,
cytomegalovirus (CMV), HIV and influenza virus). [0463] 45. A
repertoire of antibody heavy chains (eg, provided by antibodies)
comprising one or more heavy chains whose variable domain HCDR3 has
a length of at least 20 amino acids (according to IMGT) and derived
from the recombination of a human VH, D and JH, wherein [0464] the
VH is selected from the group [0465] VH1-2*02, VH1-3*01, VH1-8*01,
VH1-18*01, VH2-5*10, VH3-7*01, VH3-9*01, VH3-11*01, VH3-13*01,
VH3-21*03, VH3-23*04, VH4-4*02, VH6-1*01 and VH7-4-1*01 and [0466]
the D is selected from the group [0467] D2-2*02, D3-9*01, D3-10*01
and D3-22*01, or [0468] D2-2*02, D3-9*01 and D3-10*01, or [0469]
D3-9*01 and D3-10*01, or [0470] D1-26, D2-2, D3-9, D3-10, D3-22,
D4-17, D6-13 and D6-19, or [0471] D1-26*01, D2-2*02, D3-9*01,
D3-10*01, D3-22*01, D4-17*01, D6-13*01 and D6-19*01, or [0472]
D2-2, D3-9, D3-10, D3-22, D4-17, D6-13 and D6-19, or D2-2*02,
D3-9*01, D3-10*01, D3-22*01, [0473] D4-17*01, D6-13*01 and
D6-19*01, or [0474] D1-26, D2-2, D3-10 and D6-19, or [0475] D2-2,
D3-9 and D3-10; [0476] and optionally the JH is JH6 (eg, JH6*02);
[0477] Wherein [0478] (a) the heavy chain variable domain has been
produced in vivo in a non-human vertebrate (eg, a mouse or a rat);
and/or [0479] (b) the heavy chain variable domain comprises
non-human vertebrate AID-pattern somatic hypermutations, (eg, mouse
or rat AID-pattern mutations) when compared to corresponding human
germline V, D and J sequences and/or non-human (eg, mouse or rat)
terminal deoxynucleotidyl transferase (TdT)-pattern junctional
mutations when compared to corresponding human germline V, D and J
sequences. [0480] In an example, the heavy chain (or all heavy
chains in the repertoire) comprise non-human vertebrate constant
regions (eg, mouse or rat constant regions). For example, the
constant regions are gamma-type constant regions (eg, gamma-1,
gamma-2 or gamma-4 type). [0481] In an example, the repertoire is a
naive repertoire. This is shown in the examples section herein.
[0482] In an example, the repertoire is an immunised repertoire.
This is shown in the examples section herein. [0483] In an example,
the repertoire is an antigen-specific repertoire (eg, provided by a
plurality of hybridomas). This is shown in the examples section
herein. [0484] The repertoire can be provided by B cells (eg,
immortalised B cells). [0485] The repertoire can be provided by
hybridomas. [0486] In an example, the vectors are harboured by host
cells (eg, CHO or HEK293 cells or yeast cells). [0487] The HCDR3
length can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino
acids (according to IMGT), eg, from 20 to 23 amino acids (a
produced in the examples). [0488] In an example, in (a) the
vertebrate is a vertebrate according to the invention. [0489] 46. A
nucleic acid collection encoding the heavy chain repertoire of
clause 45. [0490] In an example, the nucleic acids are provided in
respective vectors (eg, expression vectors, eg, E coli or CHO or
HEK293 vectors). [0491] 47. A method of obtaining an
antigen-specific heavy chain (eg, provided by an antibody), the
method comprising exposing the repertoire of clause 45 to a
predetermined antigen and selecting one or more heavy chains that
specifically bind to the antigen, wherein one or more heavy chains
is isolated that has a HCDR3 length of at least 20 amino acids.
[0492] Optionally, when the heavy chain has a non-human constant
region, this is swapped for a human constant region, as is
conventional in the art. Thus, the invention provides a human
antibody heavy chain so produced (eg, provided in combination with
a human light chain to produce a human antibody which is useful for
human therapeutic and/or prophylactic use, eg, to treat and/or
prevent an infectious disease in a human patient).
[0493] In an example of the vertebrate or cell of any aspect of the
invention, the genome comprises an immunoglobulin light chain locus
comprising one or more human V gene segments and one or more human
J gene segments upstream of a constant region (eg, a human or a
mouse lambda or kappa constant region).
[0494] For rearrangement and expression of heavy chains, the locus
comprises control elements, such as an E.mu. and S.mu. between the
J gene segment(s) and the constant region as is known by the
skilled person. In one example, a mouse E.mu. and S.mu. is included
in the heavy chain locus between the JH repertoire and the constant
region (ie, in 5' to 3' order the locus comprises the JH gene
segment(s), E.mu. and S.mu. and constant region). In an example,
the E.mu. and S.mu. are E.mu. and S.mu. of a mouse 129-derived
genome (eg, a 129Sv-derived genome, eg, 129Sv/EV (such as
129S7Sv/Ev (such as from AB2.1 or AB2.2 cells obtainable from
Baylor College of Medicine, Texas, USA) or 129S6Sv/Ev))); in
another example, the E.mu. and S.mu. are E.mu. and S.mu. of a mouse
C57BL/6-derived genome. In this respect, the locus can be
constructed in the IgH locus of the genome of a cell selected from
AB2.1, AB2.2, VGF1, CJ7 and FH14. VGF1 cells were established and
described in Auerbach W, Dunmore J H, Fairchild-Huntress V, et al;
Establishment and chimera analysis of 129/SvEv- and C57BL/6-derived
mouse embryonic stem cell lines. Biotechniques 2000; 29:1024-8, 30,
32, incorporated herein by reference.
[0495] Additionally or alternatively, the constant region (or at
least a C.mu.; or C.mu. and gamma constant regions thereof) is a
constant region (or C.mu.; or C.mu. and gamma constant regions
thereof) is of a genome described in the paragraph immediately
above.
[0496] A suitable source of human DNA sequences or gene segments
will be readily apparent to the skilled person. For example, it is
possible to collect a DNA sample from a consenting human donor (eg,
a cheek swab sample as per the Example herein) from which can be
obtained suitable DNA sequences for use in constructing a locus of
the invention. Other sources of human DNA are commercially
available, as will be known to the skilled person. Alternatively,
the skilled person is able to construct gene segment sequence by
referring to one or more databases of human Ig gene segment
sequences disclosed herein.
[0497] In an example, the genome comprises all or some of the
following human VH gene segments
TABLE-US-00002 IGHV6-1 IGHV3-7 IGHV1-8 IGHV3-9 IGHV3-11 IGHV3-13
IGHV1-18 IGHV3-30 IGHV4-31 IGHV4-39 IGHV4-59
[0498] Optionally also (i) and/or (ii)
TABLE-US-00003 (i) IGHV1-2 IGHV2-5 and IGHV3-21 (ii) IGHV1-2
IGHV2-5 IGHV3-21 IGHV1-24 (ii) IGHV1-2*04 IGHV2-5*10 and
IGHV3-21*03 (iv) IGHV1-2*02 IGHV2-5*01 IGHV3-21*01 and
IGHV1-24*01
[0499] For example, the genome comprises all or some of the
following human VH gene segment variants
TABLE-US-00004 IGHV6-1*01 IGHV3-7*01 IGHV1-8*01 IGHV3-9*01
IGHV3-11*01 IGHV3-13*01 IGHV1-18*01 IGHV3-30*18 IGHV4-31*03
IGHV4-39*01 and IGHV4-59*01;
[0500] Optionally also (iii) or (iv)
[0501] For example, the genome comprises all or some of the
following human JH gene segment variants
TABLE-US-00005 IGHJ2*01 IGHJ3*02 IGHJ4*02 IGHJ5*02 and IGHJ6*02
[0502] For example, the genome comprises all or some of the
following human D gene segments
TABLE-US-00006 IGHD1-1 IGHD2-2 IGHD3-9 IGHD3-10 IGHD5-12 IGHD6-13
IGHD1-14 IGHD2-15 IGHD3-16 IGHD4-17 IGHD6-19 IGHD2-21 IGHD5-24
IGHD1-26 and IGHD7-27
and optionally also (v) or (vi)
TABLE-US-00007 (v) IGHD3-3 (vi) IGHD3-3 IGHD4-4 IGHD5-5 IGHD6-6
IGHD1-7 IGHD2-8 and IGHD2-8
[0503] It will be understood that particular embodiments described
herein are shown by way of illustration and not as limitations of
the invention. The principal features of this invention can be
employed in various embodiments without departing from the scope of
the invention. Those skilled in the art will recognize, or be able
to ascertain using no more than routine study, numerous equivalents
to the specific procedures described herein. Such equivalents are
considered to be within the scope of this invention and are covered
by the claims. All publications and patent applications mentioned
in the specification are indicative of the level of skill of those
skilled in the art to which this invention pertains. All
publications and patent applications are herein incorporated by
reference to the same extent as if each individual publication or
patent application was specifically and individually indicated to
be incorporated by reference. The use of the word "a" or "an" when
used in conjunction with the term "comprising" in the claims and/or
the specification may mean "one," but it is also consistent with
the meaning of "one or more," "at least one," and "one or more than
one." The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for
the device, the method being employed to determine the value, or
the variation that exists among the study subjects.
[0504] As used in this specification and claim(s), the words
"comprising" (and any form of comprising, such as "comprise" and
"comprises"), "having" (and any form of having, such as "have" and
"has"), "including" (and any form of including, such as "includes"
and "include") or "containing" (and any form of containing, such as
"contains" and "contain") are inclusive or open-ended and do not
exclude additional, unrecited elements or method steps
[0505] The term "or combinations thereof" as used herein refers to
all permutations and combinations of the listed items preceding the
term. For example, "A, B, C, or combinations thereof is intended to
include at least one of: A, B, C, AB, AC, BC, or ABC, and if order
is important in a particular context, also BA, CA, CB, CBA, BCA,
ACB, BAC, or CAB. Continuing with this example, expressly included
are combinations that contain repeats of one or more item or term,
such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth.
The skilled artisan will understand that typically there is no
limit on the number of items or terms in any combination, unless
otherwise apparent from the context.
[0506] Any part of this disclosure may be read in combination with
any other part of the disclosure, unless otherwise apparent from
the context.
[0507] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. All such similar substitutes and modifications apparent
to those skilled in the art are deemed to be within the spirit,
scope and concept of the invention as defined by the appended
claims.
[0508] The present invention is described in more detail in the
following non limiting Examples (Examples 1-3 being prophetic).
Example 4 is a worked example.
EXAMPLES
Example 1
Recombineered BAC Vectors to add Polymorphic V-regions to the Mouse
Genome
[0509] FIG. 1 through 3 depict recombineering methods (see
references above) that can be used to introduce polymorphic V-gene
regions into genomic DNA. In one embodiment, a genomic fragment
from the human heavy chain region is inserted into a bacterial
artificial chromosome (BAC) vector by standard techniques.
Preferably, such a BAC, which can range in size from 20-kb to
200-kb or more, can be isolated from libraries of BACs by standard
techniques including sequence searches of commercially available
libraries or by hybridization to bacterial colonies containing BACs
to identify those with a BAC of interest.
[0510] A BAC is chosen that has several VH gene segments; in FIG.
1, these are generically identified as VH[a] through VH[z] for
example. One skilled in the art will readily identify appropriate
genomic fragments, for example, an approximately 120-kb fragment
from human VH5-78 through VH1-68 which includes 5 endogenous active
VH gene segments and 7 VH psuedogenes. Using recombineering
techniques, the endogenous VH gene segments can be replaced by
polymorphic VH or VL gene segments. In this example, two steps are
required. The first step replaces the V-region coding exon of an
endogenous VH gene segment with a positive-negative selection
operon, in this example, an operon encoding an ampicillin
resistance gene (Amp) and a streptomycin-sensitizing ribosomal
protein (rpsL). Certain strains of bacteria can be selected for the
absence of the rpsL gene by resistance to streptomycin. Short
stretches of DNA homologous to sequences flanking the endogenous VH
gene exon are placed 5' and 3' of the rpsL-Amp operon. In the
presence of appropriate recombination factors per standard
recombineering techniques (see references above) recombination
between the operon fragment and the BAC will result in replacement
of the endogenous VH gene exon with the operon (FIG. 1a) which are
selected by resistance to ampicillin. The second step uses the same
homologous sequences in order to replace the inserted operon with a
desired polymorphic VH gene segment. In this example, a human
VH1-69 gene is inserted (FIGS. 1b and 1c). In particular the *02
allele of VH1-69 is used [ref IMGT and FIG. 5]. Successful
integrations of the polymorphic VH gene segment are selected in
bacteria that become resistant to streptomycin due to the loss of
the operon, specifically the rpsL portion.
[0511] In this example, the two step process as described can be
repeated for each of the endogenous VH gene segments or for as many
endogenous gene segments that one wishes to replace with
polymorphic V gene segments (FIG. 1d).
[0512] As is apparent, any polymorphic V gene segment can be
inserted in this manner and any endogenous V gene segment can act
as a target, including pseudogenes. V gene segments in each of the
heavy chain and two light chain loci can be replaced using this
technique with appropriate genomic fragments available as BAC
inserts.
[0513] FIG. 2 depicts another method for creating a genomic
fragment encoding polymorphic V gene segments. In this example,
polymorphic V gene segments are inserted into a region of genomic
DNA devoid of other genes, control elements or other functions.
Such `desert` regions can be selected based on sequence analysis
and corresponding DNA fragments cloned into BACs or identified in
existing BAC libraries. Starting with such a genomic fragment,
recombineering techniques can be used to insert polymorphic V gene
segments at intervals of, for example, 10-kb. In this example, a
150-kb genomic fragment might accommodate insertion of up to 15
polymorphic V gene segments. Insertion of the segments is a
two-step process. The first recombineering step inserts the
rpsL-Amp operon at a specific site. Sequences homologous to a
specific site are used to flank the operon. These are used by the
recombineering system to insert the element specifically into the
BAC genomic fragment and positive events are selected by resistance
to ampicillin (FIG. 2a). The second step replaces the operon in the
genomic fragment with a polymorphic V gene segment by a similar
recombineering step using the same sequence homology (FIG. 2b). In
this example, both exons and promoter element of a polymorphic VH
gene segment are inserted, resulting in replacement of the rpsL-Amp
operon and therefore resistance to streptomycin (FIG. 2c).
[0514] The two step technique for inserting polymorphic V gene
segments into a specific site on the genomic fragment can be
repeated multiple times resulting in a BAC genomic fragment with
several polymorphic gene segments, including their promoter
elements. It is apparent that the examples shown in FIGS. 1 and 2
can be combined wherein the technique for insertion can be used to
add extra polymorphic V gene segments to a BAC genomic fragment as
depicted in FIG. 1. One might choose to add these extra segments to
an IG genomic fragment since such a fragment would be more amenable
to proper IG gene expression once inserted into a non-human
mammal's genome. It is known that a genomic fragment can have
elements such as enhancers or elements that contribute to certain
chromatin conformations, both important in wild-type gene
expression.
[0515] FIG. 3 depicts an additional method to create genomic
fragments with polymorphic V gene segments. This method depends
upon the efficiency with which short (around 50 to 150 bases,
preferably 100 bases) single stranded DNA fragments recombine with
a homologous sequence using recombineering (Nat Rev Genet. 2001
October; 2(10):769-79; Recombineering: a powerful new tool for
mouse functional genomics; Copeland N G, Jenkins N A, Court D L).
The recombinases used in recombineering preferentially bind and use
such short single-stranded fragments of DNA as a substrate for
initiating homologous recombination. The efficiency can be as high
as 10-2, that is, a positive event can be found in approximately
100 randomly picked (not selected) clones resulting from
recombineering. A positive event in this example occurring when one
or more single nucleotide changes introduced into the
single-stranded fragment get transferred to the BAC insert
containing V gene segments and surrounding genomic DNA, said
nucleotide change or changes occurring at a homologous sequence on
the BAC.
[0516] Polymorphic V gene segments can differ from endogenous V
gene segments by only 1 or 2, or up to 10 or 15 nucleotide changes,
for example. An example of such nucleotide polymorphisms are
depicted in FIG. 5. Short single stranded regions that encompass
the polymorphic nucleotide changes can be chemically synthesized
using standard techniques. The resulting single stranded DNA
fragments are introduced into bacteria and via recombineering
techniques approximately 1 in 100 BAC fragments will have
incorporated the polymorphic nucleotides via homologous
incorporation of the single stranded fragment (FIG. 3a). BACs with
the desired nucleotide change can be identified by screening for
example several hundred individual clones by polymerase chain
reaction (PCR) amplification and sequencing, both by standard
techniques. In the example, two nucleotide changes will convert a
VH1-69*01 gene segment into a VH1-69*02 gene segment (FIG. 3b).
[0517] It is clear that this process can be repeated for multiple
endogenous V gene segments contained on a single BAC genomic
fragment. In addition, the techniques depicted in FIG. 2 can be
used to add additional polymorphic V gene segments by insertion
into regions between existing V gene segments. As would be evident
to one skilled in the art, a combination of these techniques can be
used to create numerous variations of both polymorphic and
endogenous human V gene segments. And it would be evident that
several different genomic fragments with engineered polymorphic V
gene segments and endogenous human V gene segments can be combined
to create even more variations.
Example 2
Adding Polymorphic V-regions to the Genome using SRMCE of Modified
BACs
[0518] Modified BACs with polymorphic V gene segments created using
the methods described in Example 1 can be used to alter the genome
of non-human mammals. These alterations can result in an intact IG
locus in which normal immunoglobin region recombination results in
VDJ or VJ combinations which includes the human V gene segments. An
example of how such an animal can be created is by altering the
genome of, for example, mouse embryonic stem (ES) cells using the
strategy outlined in FIG. 4.
[0519] One technique to integrate modified BACs with polymorphic V
gene segments into a genome is sequential recombinase mediated
cassette exchange (SRMCE). The technique is described in
WO2011004192 (Genome Research Limited), which is incorporated here
in its entirety by reference.
[0520] SRMCE provides for a locus modified with a `landing pad`
inserted at a specific location. This insertion can either be de
novo via homologous recombination or as a consequence of a previous
BAC insertion. In this example, the landing pad is inserted in the
mouse IGH locus between the most 3' J gene segment and the Cu gene
segment and a previous BAC insertion via SRMCE techniques have
resulted in the addition of 5 human V gene segments and 2 V region
pseudogenes. The landing pad has elements as shown in FIG. 4 that
will allow the selection of correct insertion of a second targeting
BAC fragment. The specificity of this insertion is provided by cre
recombinase-mediated exchange between permissive lox sites. A lox
site is permissive for recombination only with a compatible lox
site. In this example, the loxP site will only recombine with loxP
and lox2272 will only recombine with lox2272. This provides
directionality to the insertion of the BAC fragment as depicted in
FIGS. 4b and 4c.
[0521] ES cell clones with correct insertions are selected from a
pool of clones without insertions or with non-productive insertions
by resistance to puromycin. Resistance to puromycin results from
the juxtaposition of an active promoter element, PGK, with the
puroTK coding region. Correct insertions are verified by standard
techniques including PCR of junctions, PCR of internal elements,
Southern blotting, comparative genomic hybridization (CGH),
sequencing and etc. In the example, correct lox2272-lox2272 and
loxP-loxP recombination also results in two intact sets of piggyBac
elements that did not exist prior to insertion. An intact piggyBac
element is comprised of a set of inverted repeats which are
depicted in the figure by "PB5'" and "PB3'". An appropriated
oriented set of piggyBac elements are the substrate of piggyBac
transposase which can catalyse recombination between the elements,
resulting in deletion of intervening sequences as well as both
elements. The DNA remaining after a piggyBac transposition is left
intact and is lacking any remnant of the piggyBac element. In the
example, ES cell clones with successful piggyBac transposition are
selected by loss of the active puroTK element which renders the
cells resistant to the drug FIAU (FIGS. 4c and 4d).
[0522] The final product of the SRMCE method in this example is a
IGH locus with several polymorphic V gene segments inserted along
with a set of endogenous unmodified VH gene segments between
sequences of the mouse genome on the 5' side and the mouse IGH
constant region gene segments on the 3' side. The polymorphic V
gene segments are positioned such that they can participate in the
recombination events associated with B cell maturation yielding VDJ
gene segments. These gene segments can then be transcribed and
spliced to the mouse constant region. Translation of these
transcripts will result in the production of an antibody heavy
chain encoded by the polymorphic V gene segment, a human DH gene
segment, a human JH gene segment and a mouse constant heavy chain
gene segment.
[0523] As is well known to those skilled in the art, an ES cell
clone can be used to create a line of genetically modified mice via
injection of said cells into a mouse blastocyst embryo,
transferring the injected embryo to a suitable recipient and
breeding the chimeric offspring that result. The modified gene
locus can be propagated through breeding and made either
heterozygous or homozygous depending on the genetic cross.
[0524] It is evident from the structure of the IGH locus provided
in this example and by knowledge of the mechanisms involved in B
cell receptor (BCR) and antibody gene rearrangements that a large
set of different combinations of polymorphic V gene segments with
various DH and JH gene segments will result and these can
contribute to a large repertoire of functional antibody genes in a
population of B cells in genetically modified animals. In this
example, several different human VH1-69 polymorphs are incorporated
to provide superhuman VH diversity. This particular VH gene segment
is known to be prevalent in antibodies that bind infectious disease
pathogens (such as influenza virus) and therefore the antibody
repertoire of a mouse with the genetic modification of this example
would be expected to produce antibodies with a bias in favour of
those that bind infectious disease pathogens. The repertoire, in
other words, would have a larger subset of antibodies with superior
affinities for pathogen antigens. Examples of such pathogens
include influenza virus, hepatitis C virus (HCV) and human
immunodeficiency virus-1 (HIV-1) (see also table above).
Example 3
[0525] "Alignment of 13 VH1-69 Alleles"
[0526] Building a more diverse antibody repertoire by incorporating
additional V gene segment polymorphs requires availability of
polymorphic alleles of V gene segments. One source of such alleles
include sequence databases. In this example, 13 distinct alleles of
the VH1-69 gene segment are provided. These allele sequences and
comparisons are drawn from the "IMmunoGeneTics" IMGT Information
System (www.imgt.com) database. FIG. 5 is a diagram of the
alignment of alleles *02 through *13 with the *01 allele. The
VH1-69*01 nucleotide and amino acid sequence is provided at the top
of the figure. Where the remaining alleles are identical to the *01
allele sequence a dash is inserted below the sequence. Nucleotide
differences are noted alongside the appropriate allele and if the
sequence change results in a protein coding change, the amino acid
change is indicated above the triplet.
[0527] FIG. 5 depicts between 1 and 4 amino acid changes for each
allele in comparison to the *01 allele. All of the amino acid
changes occur in the part of the heavy chain protein encoding the
complementarity determining regions (CDRs). These regions are
responsible for antigen specificity and the affinity of the
antibody for the antigen. It is evident that providing additional
polymorphic CDRs in a repertoire of antibodies will increase the
likelihood of there being an antibody with superior binding
characteristics for various antigens. In several reports, it has
been observed that the VH1-69-encoded variable region of the heavy
chain is often found in antibodies that bind influenza virus, HCV
and HIV-1 antigens (see table above). Therefore incorporating the
polymorphic V gene segments of this example into a transgenic
animal model using the methods of Examples 1 and 2 would likely
result in an antibody repertoire in said transgenic animal with
more antibodies that bind to antigens associated with these and
other pathogens. And as is known in the art, a larger repertoire
increases the probability of finding monoclonal antibodies using,
for example, hybridoma technology, that bind with high affinity and
specificity to a desired antigen.
[0528] This disclosure therefore describes in these examples a
transgenic mouse model which can be immunized with pathogen or
other antigens. Plasma B cells from such an immunized mouse can be
used to make a hybridoma library that can be screened for
production of antibodies that bind the pathogen antigens. This
library will be superior to libraries from traditional transgenic
mice for finding such antibodies given the addition of polymorphic
VH1-69 gene segments to the IGH locus in said transgenic mouse.
[0529] These examples are not limiting to the human polymorphic V
gene segments that can be chosen or to the methods used to
introduce them into an animal model. The method can be used to
construct a transgenic locus with immunoglobulin D and/or J
segments. The V, D, J segments can be from a plurality of human
sources (optionally more than one human ethnic population).
Example 4
Transgenic Mice, B-Cells, Hybridomas, Antibodies & Heavy Chains
Based on Human JH6*02
[0530] A functional human gene segment repertoire (from V.sub.H2-26
to J.sub.H6, see the IMGT database for the structure of the human
IgH locus;
http://www.imgt.org/IMGTrepertoire/index.php?section=LocusGenes&repertoir-
e=locus&species=human&group=IGK) was sectored by the
inventors to produce two different transgenic heavy chain alleles
(denoted S2F and S3F) and corresponding mice. The transgenic
alleles were expressed in the mice and the heavy chain repertoires
were assessed at the RNA transcript level. Deep sequence analysis
was carried out using Bioinformatics methods to assess V, D and JH
gene usage, including in variable domain sequences having a HCDR3
length of at least 20 amino acids. Endogenous, mouse variable
region gene segments were inactivated by inversion (as per the
method described in WO2011004192, this disclosure being
incorporated herein by reference).
Sequencing of Human Donor DNA Samples: Identification of Conserved
JH6*02 Variant
[0531] DNA samples from 9 anonymised consenting human donors were
obtained by taking cheek swabs.
[0532] The samples were processed and the DNA Samples were
extracted follow the protocol of QIAamp DNA Mini Kit (Cat. No.
51304, Qiagen).
[0533] PCR reactions were set up to amplify the JH6 region and PCR
products were sequenced (PCR Oligos sequence: Fwd.
5'-AGGCCAGCAGAGGGTTCCATG-3' (SEQ ID NO: 444), Rev.
5'-GGCTCCCAGATCCTCAAGGCAC-3' (SEQ ID NO: 445)).
[0534] Sequence analysis was carried out by comparing to the JH6
reference sequence from IMGT annotated database
(http://www.imgt.org/), and this identified that all 9 donor
genomes contained the human JH6*02 variant, with this variant being
in the homozygous state in 7 out of the 9 donors. The inventors
also consulted the genomic sequences publicly available for Jim
Watson and Craig Venter at Ensembl human genome database
[http://www.ensembl.org/]. These too contained the human JH6*02
variant. This confirmed to the inventors that human JH6*02 is a
common, conserved variant in humans, and thus a good candidate for
construction of a transgenic IgH locus as per the invention
Identification of Suitable Human DNA Sequence BACs
[0535] A series of human bacterial artificial chromosome (BAC)
clones were identified from Ensemble
(http://www.ensembl.org/index.html) or UCSC
(http://genome.ucsc.edu/) human database searches based on gene
name (IGH) or location (chromosome 14: 106026574-107346185). Seven
human RP11 BAC clones were selected, RP11-1065N8 BAC carrying human
JH6*02. In total, the following BACs were identified as sources of
human IgH locus DNA: RP11-1065N8, RP11-659B19, RP11-14117,
RP-112H5, RP11-101G24, RP11-12F16 and RP11-47P23.
[0536] With a similar approach, different BAC clones (eg, different
RP11 clone IDs or different sources from RP11) or genetically
engineered BACs can be selected for insertion into the mouse IGH
locus to provide different sets of human repertoires in the
transgenic mouse.
Construction of Transgenic IgH Loci
[0537] Insertion of human heavy gene segments from a 1st IGH BAC
(RP11-1065N8) into the IGH locus of mouse AB2.1 ES cells (Baylor
College of Medicine) was performed to create a heavy chain allele
denoted the S1 allele. The inserted human sequence corresponds to
the sequence of human chromosome 14 from position 106494908 to
position 106328951 and comprises functional heavy gene segments
V.sub.H2-5, V.sub.H7-4-1, V.sub.H4-4, V.sub.H1-3, V.sub.H1-2,
V.sub.H6-1, D1-1, D2-2, D3-9, D3-10, D4-11, D5-12, D6-13, D1-14,
D2-15, D3-16, D4-17, D5-18, D6-19, D1-20, D2-21, D3-22, D4-23,
D5-24, D6-25, D1-26, D7-27, J.sub.H1, J.sub.H2, J.sub.H3, J.sub.H4,
J.sub.H5 and J.sub.H6 (in 5' to 3' order), wherein the JH6 was
chosen to be the human JH6*02 variant. The insertion was made
between positions 114666435 and 114666436 on mouse chromosome 12,
which is upstream of the mouse C.mu. region. The mouse V.sub.H, D
and J.sub.H gene segments were retained in the locus, immediately
upstream of (5' of) the inserted human heavy chain DNA.
[0538] A second allele, S2 was constructed in which more human
functional V.sub.H gene segments were inserted upstream (5') of the
5'-most V.sub.H inserted in the S1 allele by the sequential
insertion of human DNA from a second BAC (BAC2). The inserted human
sequence from BAC2 corresponds to the sequence of human chromosome
14 from position 106601551 to position 106494909 and comprises
functional heavy chain gene segments V.sub.H3-13, V.sub.H3-11,
V.sub.H3-9, V.sub.H1-8, V.sub.H3-7. The mouse V.sub.H, D and
J.sub.H gene segments were retained in the locus, immediately
upstream of (5' of) the inserted human heavy chain DNA. In a
subsequent step, these were inverted to inactivate them, thereby
producing S2F mice in which only the human heavy chain variable
region gene segments are active.
[0539] A third allele, S3 was constructed in which more human
functional V.sub.H gene segments were inserted upstream (5') of the
5'-most V.sub.H inserted in the S2 allele by the sequential
insertion of human DNA from a third BAC (BAC3). The inserted
sequence corresponds to the sequence of human chromosome 14 from
position 106759988 to position 106609301, and comprises functional
heavy chain gene segments, V.sub.H2-26, V.sub.H1-24, V.sub.H3-23,
V.sub.H3-21, V.sub.H3-20, V.sub.H1-18, and V.sub.H3-15. The mouse
V.sub.H, D and J.sub.H gene segments were retained in the locus,
immediately upstream of (5' of) the inserted human heavy chain DNA.
In a subsequent step, these were inverted to inactivate them,
thereby producing S3F mice in which only the human heavy chain
variable region gene segments are active.
[0540] Mice bearing either the S2F or S3F insertion into an
endogenous heavy chain locus were generated from the ES cells using
standard procedures. The other endogenous heavy chain locus was
inactivated in the mice by insertion of an inactivating sequence
comprising neo.sup.R into the mouse J.sub.H-C.mu. intron (to
produce the "HA" allele).
[0541] Specifically, the following alleles were included:--
VH1-2*02, VH1-3*01, VH1-8*01, VH1-18*01, VH2-5*10, VH3-7*01,
VH3-9*01, VH3-11*01, VH3-13*01, VH3-21*03, VH3-23*04, VH4-4*02,
VH6-1*01 and VH7-4-1*01
D1-26*01, D2-2*02, D3-9*01, D3-10*01, D3-22*01, D4-17*01, D6-13*01
and D6-19*01
Immunisation Procedure
[0542] Transgenic mice of the S2F or S3F genotype were primed with
20-40 ug recombinant proteins obtained commercially or produced in
house with Antigen 1 (OVA (Sigma A7641); Antigen 2 (a human
infectious disease pathogen antigen) and Antigen 3 (a human
antigen) via the ip route in complete Freunds adjuvant (Sigma F
5881) and 10 ug/animal CpG (CpG oligo; Invivogen, San Diego,
Calif., USA) and then boosted twice in about two weekly intervals
with about half the amount of antigen in incomplete Freunds
adjuvant (Sigma F 5506) and 10 ug/animal CpG. Final boosts were
administered two weeks later iv without any adjuvant and contained
5-10 ug protein in PBS.
Hybridoma Fusion Procedure
[0543] Spleens were taken 3 days after the final boost and
spleenocytes were treated with CpG (25 .mu.m final concentration)
for and left until the following day. Cells were then fused with
SP0/2 Ag14 myeloma cells (HPA Cultures Cat No 85072401) using a BTX
ECM2001 electrofusion instrument. Fused cells were left to recover
for 20 minutes then seeded in a T75 flask until next morning. Then
the cells were spun down and plated out by dilution series on
96-well culture plates and left for about 10 days before screening.
Media was changed 1-3 times during this period.
Screening
[0544] Culture supernatants of the hybridoma wells above were
screened using homogenious time resolved fluorescence assay (htrf)
using Europium cryptate labelled anti-mouse IgG (Cisbio anti-mouse
Ig Europium Cryptate) and a biotin tagged target antigen with a
commercially available streptavidin conjucated donor (Cisbio;
streptaviding conjugated D2) or by IgG-specific 384 well ELISA.
Positive wells identified by htrf were scaled to 24-well plates or
immediately counterscreened using an IgG-specific detection ELISA
method. Positives identified by primary ELISA screen were
immediately expanded to 24-well plates. Once cultures were expanded
to 24-well stage and reached confluency, supernatants were
re-tested using htrf or IgG-specific ELISA to confirm binding to
target antigen. Supernatant of such confirmed cultures were then
also analysed by surface plasmon resonance using a BioRad ProteOn
XPR36 instrument. For this, antibody expressed in the hybridoma
cultures was captured on a biosensor GLM chip (BioRad 176-512)
which had an anti-mouse IgG (GE Healthcare BR-1008-38)) covalently
coupled the biosensor chip surface. The antigen was then used as
the analyte and passed over the captured hybridoma antibody
surface. For Antigen 2 and Antigen 3, concentrations of 256 nM, 64
nM, 16 nM, 4 nM and 1 nM were typically used, for Antigen 1,
concentrations of 1028 nM, 256 nM, 64 nM, 16 nM and 4 nM were
typically used, binding curves were double referenced using a 0 nM
injection (i.e. buffer alone). Kinetics and overall affinities were
determined using the 1:1 model inherent to the BioRad ProteOn XPR36
analysis software.
[0545] Any clones with confirmed binding activity were used for
preparing total RNA and followed by PCR to recover the heavy chain
variable region sequences. Standard 5'-RACE was carried out to
analyse RNA transcripts from the transgenic heavy chain loci in the
S2F and S3F mice. Additionally, deep sequence analysis of almost
2000 sequences produced by the mice was carried out.
Bionformatics Analysis
[0546] Sequences for analysis were obtained from two different
methods: [0547] The first is from RNA extracted from the spleen:
first cDNA strand was synthesized using an oligo based on the Cmu
region of the mouse IGH locus as a PCR template. PCR was performed
using this oligo with an oligo dT-anchor primer. Then PCR product
was cloned into pDrive vector (Qiagen) and then sequenced. [0548]
The second is from hybridomas generated through electro-fusion:
total RNA was extracted from hybridoma lines of interest using
standard Trizol methods and frozen at -80.degree. C. for long term
storage. cDNA was generated from 100 ng total RNA using standard
Superscript III reverse transcriptase and a gene-specific reverse
primer binding to all mouse IgG isotypes for heavy chain and a
mouse kappa constant region primer for the light chain
amplification. 2-3 ul of cDNA were then used as template in a PCR
reaction using Pfu DNA polymerase and a panel of degenerate forward
primers annealing to the leader sequence of the human
immunoglobulin variable domain as well as one mouse pan-IgG reverse
primer. PCR products were run out of a 1% agarose gel and bands of
approximately 350-450 basepairs extracted and purified. DNA was
then sequenced.
[0549] The sequences from the first method can either be from IgM
from Naive mice or IgG from immunised mice. The samples from the
second method are all from IgG from immunised mice, and specific to
the immunising antigen. Almost 2000 sequences were analysed.
[0550] The sequences were obtained as a pair of forward and reverse
reads. These were first trimmed to remove low-quality base calls
from the ends of the reads (trimmed from both ends until a 19
nucleotide window had an average quality score of 25 or more). The
reads were combined together by taking the reverse complement of
the reverse read, and aligning it against the forward read. The
alignment scoring was 5 for a match, -4 for a mismatch, a gap open
penalty of 10 and a gap extension penalty of 1. A consensus
sequence was then produced by stepping through the alignment and
comparing bases. When there was a disagreement the base with the
highest quality value from sequencing was used.
[0551] The BLAST+ (Basic Local Alignment Search Tool) (Camacho C.,
Coulouris G., Avagyan V., Ma N., Papadopoulos J., Bealer K., &
Madden T. L. (2008) "BLAST+: architecture and applications." BMC
Bioinformatics 10:421 http://www.ncbi.nlm.nih.gov/pubmed/20003500)
program `blastn` was then used to find the germline J and V
segments used in each sequence. A wordsize of 30 was used for V
matching, and 15 for J matching. The database searched against was
constructed from the NGS sequencing of the BACs which were used to
generate the Kymouse.
[0552] If a sequence matched both a V and a J segment, the sequence
between the two was then compared to a database of germline D
segments in the mouse using `blastn` with a wordsize of 4 and the
options `blastn-short` and `ungapped`. This was used to assign a D
segment, if possible. The CDR3 was identified by searching for the
conserved "TATTACTGT" sequence in the V segment, and the "CTGGGG"
in the J segment. If these motifs were not found, then up to 4
mismatches were allowed. The IMGT definition of CDR3 was used, so
the CDR3 length is calculated from after the "TGT" in the V to
before the "TGG" in the J. Sequences with an out of frame junction
(those which do not have a CDR3 nucleotide length divisible by 3)
or which contained a stop codon ("TAA", "TAG" or "TGA") were
excluded.
[0553] The identity of the matching V, J and D segments as well as
the CDR3 length from this assignment were then saved as a table for
downstream analysis. The ratio of IGHJ6*02 used increased from the
naive to immunised mice, as well as being enriched in the
sub-population of sequences with a long HCDR3 (defined as
consisting of 20 or more amino acids):
TABLE-US-00008 All HCDR3 > 20 Total Total JH6*02% Count JH6*02%
Count % HCDR3 > 20 Naive 22.31% 1340 91.11% 45 3.36% Immunised
37.50% 256 66.67% 9 3.52% Hybridoma 36.13% 119 63.64% 11 9.24%
[0554] This shows that the JH6*02 gene segment is selected for by
immunisation, as the proportion of JH6*02 usage increases after
immunisation. JH6*02 is also used in the majority of antibodies
with a long HCDR3 length, which is desirable for targets which are
specifically bound by long HCDR3 length antibodies.
[0555] Additionally, the analysis revealed that certain VH and D
gene segments frequently yielded HCDR3s of long length (in all of
naive, immunised and antigen-specific repertoires of heavy chains).
See Table 2.
TABLE-US-00009 TABLE 2 Long HCDR3s from Naive Repertoires Average
CDR3 V Length Count IGHV1-2*02 21 3 IGHV1-18*01 21 5 IGHV3-7*01 22
3 IGHV6-1*01 21 5 IGHV3-9*01 20 2 IGHV2-5*10 20 1 IGHV7-4-1*01 21 3
IGHV1-3*01 21 5 IGHV4-4*02 20 3 IGHV3-13*01 22 1 IGHV3-23*04 20 1
IGHV1-8*01 21 10 IGHV3-21*03 23 3 Average CDR3 D Length Count
IGHD2-2*02 20 1 IGHD3-9*01 21 13 IGHD3-10*01 21 26 IGHD6-13*01 20 1
IGHD4-17*01 22 2 IGHD6-19*01 23 1 IGHD3-22*01 20 1 CDR3 Length (All
Naive) Count 20 23 21 10 22 7 23 3 24 1 26 1 Long HCDR3s from
Immunised Repertoires Average CDR3 V Length Count IGHV4-4*02 20 1
IGHV3-11*01 23 2 IGHV3-7*01 21 6 Average D CDR3Length Count
IGHD2-2*02 22 2 IGHD3-10*01 22 5 IGHD6-19*01 20 1 IGHD1-26*01 20 1
CDR3 Length (All Immunised) Count 20 4 21 1 22 2 24 1 25 1 Long
HCDR3s from Antigen-Specific Repertoires Average CDR3 V Length
Count IGHV4-4*02 20 2 IGHV1-3*01 21 3 IGHV3-11*01 21 1 IGHV3-7*01
22 1 IGHV1-8*01 22 2 IGHV3-20*d01 22 1 IGHV3-9*01 20 1 Average CDR3
D Length Count IGHD2-2*02 22 1 IGHD3-9*01 21 1 IGHD3-10*01 21 9
CDR3 Length (All Antigen- Specific) Count 20 4 22 2 21 4 24 1
Sequence CWU 1
1
21296DNAHomo sapiens 1caggtgcagc tggtgcagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60tcctgcaagg cttctggagg caccttcagc agctatgcta
tcagctgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggaggg
atcatcccta tctttggtac agcaaactac 180gcacagaagt tccagggcag
agtcacgatt accgcggacg aatccacgag cacagcctac 240atggagctga
gcagcctgag atctgaggac acggccgtgt attactgtgc gagaga 296298PRTHomo
sapiens 2Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe
Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn
Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
* * * * *
References