U.S. patent application number 13/275017 was filed with the patent office on 2012-02-02 for monoclonal antibody production by ebv transformation of b cells.
This patent application is currently assigned to INSTITUTE FOR RESEARCH IN BIOMEDICINE. Invention is credited to Antonio Lanzavecchia.
Application Number | 20120027768 13/275017 |
Document ID | / |
Family ID | 9953683 |
Filed Date | 2012-02-02 |
United States Patent
Application |
20120027768 |
Kind Code |
A1 |
Lanzavecchia; Antonio |
February 2, 2012 |
MONOCLONAL ANTIBODY PRODUCTION BY EBV TRANSFORMATION OF B CELLS
Abstract
A method for producing a clone of an immortalised human B memory
lymphocyte, comprising the step of transforming human B memory
lymphocytes using Epstein Barr virus (EBV) in the presence of a
polyclonal B cell activator. The method is particularly useful in a
method for producing a clone of an immortalised human B memory
lymphocyte capable of producing a human monoclonal antibody with a
desired antigen specificity, comprising the steps of: (i) selecting
and isolating a human memory B lymphocyte subpopulation; (ii)
transforming the subpopulation with Epstein Ban virus (EBV) in the
presence of a polyclonal B cell activator; (iii) screening the
culture supernatant for antigen specificity; and (iv) isolating an
immortalised human B memory lymphocyte clone capable of producing a
human monoclonal antibody having the desired antigen
specificity.
Inventors: |
Lanzavecchia; Antonio;
(Bellinzona, CH) |
Assignee: |
INSTITUTE FOR RESEARCH IN
BIOMEDICINE
Bellinzona
CH
|
Family ID: |
9953683 |
Appl. No.: |
13/275017 |
Filed: |
October 17, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11719835 |
May 21, 2007 |
8071371 |
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PCT/IB04/01071 |
Feb 26, 2004 |
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13275017 |
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60516665 |
Oct 30, 2003 |
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Current U.S.
Class: |
424/147.1 ;
424/141.1; 530/388.1; 530/388.3 |
Current CPC
Class: |
A61K 2039/505 20130101;
C12P 21/00 20130101; C07K 16/20 20130101; C12P 21/005 20130101;
C07K 16/1027 20130101; Y02A 50/30 20180101; Y02A 50/58 20180101;
C07K 2317/14 20130101; C07K 2317/76 20130101; C12N 2510/02
20130101; C12N 5/0635 20130101; A61P 37/04 20180101; C07K 16/00
20130101; C12N 2510/04 20130101; C07K 2317/21 20130101; Y02A 50/466
20180101; C07K 16/1282 20130101; Y02A 50/412 20180101; A61P 31/14
20180101; C07K 16/10 20130101; A61P 37/02 20180101 |
Class at
Publication: |
424/147.1 ;
530/388.1; 530/388.3; 424/141.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 37/04 20060101 A61P037/04; A61P 31/14 20060101
A61P031/14; C07K 16/00 20060101 C07K016/00; C07K 16/10 20060101
C07K016/10 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 26, 2003 |
GB |
0304363.5 |
Aug 6, 2003 |
GB |
0318431.4 |
Oct 30, 2003 |
GB |
0325391.1 |
Claims
1. A monoclonal antibody, or an antigen binding fragment thereof,
produced by a method comprising: (i) contacting a memory B
lymphocyte with EBV and an agonist of a Pattern Recognition
Receptor that is expressed on memory B cells; (ii) culturing said
memory B lymphocyte; and (iii) obtaining, and optionally, isolating
said monoclonal antibody or fragment thereof, wherein said
monoclonal antibody or fragment thereof is directed against
respiratory syncytial virus (RSV).
2. A monoclonal antibody, or an antigen binding fragment thereof,
produced by a method comprising: (i) contacting memory B
lymphocytes with EBV and an agonist of a Pattern Recognition
Receptor that is expressed on memory B cells; (ii) culturing said
memory B lymphocytes; (iii) screening said memory B lymphocytes for
specificity against RSV; (iv) isolating an immortalized memory B
lymphocyte clone capable of producing a monoclonal antibody having
said specificity; (v) (a) (1) culturing said immortalized memory B
lymphocyte clone; and (2) obtaining, and optionally, isolating said
monoclonal antibody or fragment thereof, or (b) (1) obtaining
and/or sequencing nucleic acid that encodes an antibody, or
fragment thereof, from said immortalized memory B lymphocyte clone;
(2) inserting said nucleic acid into an expression host, or using
said nucleic acid sequence to prepare an expression host, in order
to permit expression of said antibody, or fragment thereof, in said
host; (3) expressing said antibody, or fragment thereof, in said
host; and (4) obtaining, and optionally, isolating said antibody or
fragment thereof, wherein said monoclonal antibody or fragment
thereof is directed against RSV.
3. A monoclonal antibody, or an antigen binding fragment thereof,
produced by a method comprising: (i) producing an immortalized
memory B lymphocyte comprising the steps of: (a) incubating or
pulsing a population of cells comprising a memory B lymphocyte with
EBV; (b) diluting said population of cells with culture medium; and
(c) contacting said diluted population of cells with a polyclonal B
cell activator, wherein said polyclonal B cell activator is an
agonist of a Pattern Recognition Receptor that is expressed on
memory B cells; (ii) (a) (1) culturing said immortalized memory B
lymphocyte; and (2) obtaining, and optionally, isolating said
monoclonal antibody or fragment thereof from said immortalized
memory B lymphocyte, or (b) (1) obtaining and/or sequencing nucleic
acid that encodes an antibody, or fragment thereof, from said
immortalized memory B lymphocyte; (2) inserting said nucleic acid
into an expression host, or using said nucleic acid sequence to
prepare an expression host, in order to permit expression of said
antibody, or fragment thereof, in said host; (3) expressing said
antibody, or fragment thereof, in said host; and (4) obtaining, and
optionally, isolating said antibody or fragment thereof, wherein
said monoclonal antibody or fragment thereof is directed against
RSV.
4. The monoclonal antibody, or an antigen binding fragment thereof,
of claim 3, wherein the method comprises the step of: increasing
the concentration of memory B lymphocytes in said population of
cells comprising memory B lymphocytes, or depleting undesired cells
from said population of cells comprising memory B lymphocytes prior
to the step of incubating or pulsing said population of cells with
EBV.
5. The monoclonal antibody, or an antigen binding fragment thereof,
of claim 2, wherein said nucleic acid or nucleic acid sequence is
manipulated before said inserting step to introduce restriction
sites, to change codon usage, or optimize transcription or
translation regulatory sequences.
6. The monoclonal antibody, or an antigen binding fragment thereof,
of claim 3, wherein said nucleic acid or nucleic acid sequence is
manipulated before said inserting step to introduce restriction
sites, to change codon usage, or optimize transcription or
translation regulatory sequences.
7. The monoclonal antibody, or an antigen binding fragment thereof,
of claim 1, wherein said Pattern Recognition Receptor is a Toll
Like Receptor ("TLR") that is expressed on memory B cells.
8. The monoclonal antibody, or an antigen binding fragment thereof,
of claim 2, wherein said Pattern Recognition Receptor is a Toll
Like Receptor ("TLR") that is expressed on memory B cells.
9. The monoclonal antibody, or an antigen binding fragment thereof,
of claim 3, wherein said Pattern Recognition Receptor is a Toll
Like Receptor ("TLR") that is expressed on memory B cells.
10. The monoclonal antibody, or an antigen binding fragment
thereof, of claim 4, wherein said Pattern Recognition Receptor is a
Toll Like Receptor ("TLR") that is expressed on memory B cells.
11. The monoclonal antibody, or an antigen binding fragment
thereof, of claim 1, wherein said Pattern Recognition Receptor is
Toll Like Receptor 7 (TLR7), Toll Like Receptor 9 (TLR9) and/or
Toll Like Receptor 10 (TLR10).
12. The monoclonal antibody, or an antigen binding fragment
thereof, of claim 2, wherein said Pattern Recognition Receptor is
Toll Like Receptor 7 (TLR7), Toll Like Receptor 9 (TLR9) and/or
Toll Like Receptor 10 (TLR10).
13. The monoclonal antibody, or an antigen binding fragment
thereof, of claim 3, wherein said Pattern Recognition Receptor is
Toll Like Receptor 7 (TLR7), Toll Like Receptor 9 (TLR9) and/or
Toll Like Receptor 10 (TLR10).
14. The monoclonal antibody, or an antigen binding fragment
thereof, of claim 4, wherein said Pattern Recognition Receptor is
Toll Like Receptor 7 (TLR7), Toll Like Receptor 9 (TLR9) and/or
Toll Like Receptor 10 (TLR10).
15. The monoclonal antibody, or an antigen binding fragment
thereof, of claim 1, wherein said agonist of said Pattern
Recognition Receptor is selected from the group consisting of: CpG
oligodeoxynucleotides that stimulate TLR9; imiquimod, R-848, and
other imidazoquinoline compounds that stimulate TLR7 and TLR8;
loxoribine, 7-thia-8-oxoguanosine, 7-deazaguanosine that stimulate
TLR7 and TLR8; monoclonal antibodies that mimic the effects of
these activators and other synthetic compounds that trigger TLR7,
TLR8 or TLR9.
16. The monoclonal antibody, or an antigen binding fragment
thereof, of claim 2, wherein said agonist of said Pattern
Recognition Receptor is selected from the group consisting of: CpG
oligodeoxynucleotides that stimulate TLR9; imiquimod, R-848, and
other imidazoquinoline compounds that stimulate TLR7 and TLR8;
loxoribine, 7-thia-8-oxoguanosine, 7-deazaguanosine that stimulate
TLR7 and TLR8; monoclonal antibodies that mimic the effects of
these activators and other synthetic compounds that trigger TLR7,
TLR8 or TLR9.
17. The monoclonal antibody, or an antigen binding fragment
thereof, of claim 3, wherein said agonist of said Pattern
Recognition Receptor is selected from the group consisting of: CpG
oligodeoxynucleotides that stimulate TLR9; imiquimod, R-848, and
other imidazoquinoline compounds that stimulate TLR7 and TLR8;
loxoribine, 7-thia-8-oxoguanosine, 7-deazaguanosine that stimulate
TLR7 and TLR8; monoclonal antibodies that mimic the effects of
these activators and other synthetic compounds that trigger TLR7,
TLR8 or TLR9.
18. The monoclonal antibody, or an antigen binding fragment
thereof, of claim 4, wherein said agonist of said Pattern
Recognition Receptor is selected from the group consisting of: CpG
oligodeoxynucleotides that stimulate TLR9; imiquimod, R-848, and
other imidazoquinoline compounds that stimulate TLR7 and TLR8;
loxoribine, 7-thia-8-oxoguanosine, 7-deazaguanosine that stimulate
TLR7 and TLR8; monoclonal antibodies that mimic the effects of
these activators and other synthetic compounds that trigger TLR7,
TLR8 or TLR9.
19. The monoclonal antibody, or an antigen binding fragment
thereof, of claim 1, wherein the agonist of said Pattern
Recognition Receptor is CpG 2006.
20. The monoclonal antibody, or an antigen binding fragment
thereof, of claim 2, wherein the agonist of said Pattern
Recognition Receptor is CpG 2006.
21. The monoclonal antibody, or an antigen binding fragment
thereof, of claim 3, wherein the agonist of said Pattern
Recognition Receptor is CpG 2006.
22. The monoclonal antibody, or an antigen binding fragment
thereof, of claim 4, wherein the agonist of said Pattern
Recognition Receptor is CpG 2006.
23. A pharmaceutical composition comprising the monoclonal
antibody, or an antigen binding fragment thereof, of claim 1, and a
pharmaceutically acceptable excipient.
24. A pharmaceutical composition comprising the monoclonal
antibody, or an antigen binding fragment thereof, of claim 2, and a
pharmaceutically acceptable excipient.
25. A pharmaceutical composition comprising the monoclonal
antibody, or an antigen binding fragment thereof, of claim 3, and a
pharmaceutically acceptable excipient.
26. A pharmaceutical composition comprising the monoclonal
antibody, or an antigen binding fragment thereof, of claim 4, and a
pharmaceutically acceptable excipient.
Description
[0001] All documents cited herein are incorporated by reference in
their entirety.
[0002] This application is a divisional of, and claims the benefit
of priority of U.S. application Ser. No. 11/719,835, filed Feb. 26,
2004, which is the National Phase application of International
Application No. PCT/IB2004/001071, filed Feb. 26, 2004, which
claims priority to GB Application Nos. 0304363.5, 0318431.4 and
0325391.1, filed Feb. 26, 2003, Aug. 6, 2003, and Oct. 30, 2003,
respectively, and to U.S. Application No. 60/516,665, filed Oct.
30, 2003, the disclosures of which are hereby incorporated by
reference, as if written herein, in their entirety.
TECHNICAL FIELD
[0003] This invention relates to monoclonal antibodies, to a method
for preparing immortalised memory B cells, to a method for
preparing immortalised memory B cells capable of producing a
monoclonal antibody with a desired antigen specificity and to the
use of antibodies produced by said immortalised memory B cells. The
invention is particularly useful for preparing human monoclonal
antibodies. In one embodiment, the invention relates to a method
for preparing immortalised human memory B cells capable of
producing antibodies specific for an infectious agent, more
particularly where the infectious agent is the severe acute
respiratory syndrome (SARS) virus. Further embodiments are
described below.
BACKGROUND ART
[0004] The success in generating murine monoclonal antibodies rests
on the efficient and selective fusion of antigen-stimulated B cell
blasts with a murine myeloma cell line followed by selection of
stable antibody producing hybrids (Kohler & Milstein 1975).
FR2817875 describes a modified version of this protocol where prior
to immortalisation, B lymphocytes are induced to differentiate by a
non-specific activating system and a cytokine. The B cell blasts
may be taken from the spleen or lymph nodes. However, the
difficulty in obtaining antigen-stimulated B cell blasts and the
lack of suitable fusion partners has hampered this approach in the
human system.
[0005] As an alternative approach to making human antibodies,
Epstein Barr Virus (EBV) has been used to immortalize human (and
primate) B cells producing specific antibodies. The EBV method has
been described in several publications since 1977 (Rosen et al.
1977; Steinitz et al. 1977; Steinitz et al. 1980; Kozbor &
Roder 1981; Lundgren et al. 1983; Rosen et al. 1983; Steinitz et
al. 1984; Lanzavecchia 1985). B cells are immortalized by infection
with EBV and growing clones secreting specific antibodies are
selected. The method does not require antigenic boost, since EBV
immortalizes also resting human B cells. However, the EBV-based
method has several limitations, namely the low efficiency of
immortalization, the low cloning efficiency of EBV-immortalized B
cells, the slow growth rate and, in some cases, low antibody
production. U.S. Pat. No. 4,997,764 describes a method of improving
the growth rate of the EBV immortalised cells comprising
transfecting EBV infected B cells with activated c-myc DNA. This
confers on the cells the ability to grow in semi-solid media and to
grow in hosts such as rats and mice. WO95/13089 describes the use
of GM-CSF and IL-3 to stimulate the release of antibody by B cells.
Bornkamm et al. (U.S. Pat. No. 5,798,230) have overcome the problem
of low production of antibodies by inactivating EBNA2. However,
this does not solve the problem of low efficiency of
immortalization. To circumvent these problems some authors carried
out an enrichment for antigen-specific B cells before EBV
immortalization using for instance biotinylated soluble antigens
(Casali et al. 1986). Others proposed the fusion of the
EBV-immortalised cells with mouse myelomas or human-mouse
heteromyelomas to exploit the higher growth rate and Ig secretion
of the hybrids (Kozbor et al. 1982; Bron et al. 1984; Thompson et
al. 1986). Claims that the cloning efficiency could be increased by
cell-derived growth factors such as thioredoxin have been made in a
publication (Ifversen et al. 1993), but these results have neither
been confirmed nor utilized, even by the same authors. In
conclusion, although the EBV method has in principle some
advantages, it has been abandoned because of the low efficiency of
immortalization and cloning.
[0006] Another reason why the EBV method has become obsolete is
that alternative approaches for making human or human-like
monoclonal antibodies became available through genetic engineering.
These include the humanization of murine antibodies, the isolation
of antibodies from libraries of different complexity and the
production of hybridomas using the classical method in mice
transgenic for human Ig loci (the "xeno-mouse"). The literature on
these alternative approaches is not reviewed here since is not
directly relevant to the present invention. However, it is worth
considering some limitations of these methods. Humanization of
murine monoclonal antibodies is a laborious and incomplete
procedure. Random antibody libraries represent an unbiased
repertoire and can therefore be used to select antibody
specificities against highly conserved antigens, but lead to
antibodies of low affinity. Libraries selected from antigen primed
B cells are enriched for a particular specificity, but do not
preserve the original VH-VL pairing and generally lead to
antibodies that have lower affinity for the antigen than those
present in the original antibody repertoire. The impact of this
technology has been limited. In contrast the xeno-mouse can be
efficiently immunized against an antigen of choice (especially if
this is a human antigen), but this system shares with the classical
murine hybridoma technology the limitation that the antibodies are
selected in a species other than human. Therefore these methods are
not suitable to produce antibodies with the characteristics of
those produced in the course of a physiological human immune
response. This applies to the antibody response to human pathogens
including HIV, the four Plasmodium species that cause malaria in
humans (P. falciparum, P. vivax, P. malariae and P. ovale), human
hepatitis B and C viruses, Measles virus, Ebola virus etc. (for an
exhaustive list see Fields et al. 1996). It also applies to
antibody responses to environmental allergens generated in allergic
patients, to tumour antigens generated in tumour bearing patients
and to self antigens in patients with autoimmune diseases.
[0007] There is therefore a need for an efficient method of
production of human monoclonal antibodies that have been selected
in the course of the natural immune response.
DISCLOSURE OF THE INVENTION
[0008] While the present invention is illustrated by embodiments
where human monoclonal antibodies are produced, the techniques
described herein are not so limited. The present invention can be
used for any species for which it is desired to produce monoclonal
antibodies efficiently.
[0009] The invention is based on the discovery that a polyclonal B
cell activator (such as CpG sequences) enhances the efficiency of
EBV immortalization and of cloning EBV-immortalized cells. This
increased efficiency represents a quantum leap that makes the EBV
technique suitable for the rapid isolation of large numbers of
human monoclonal antibodies from the memory repertoire with no need
for specific immunization or boosting. Antibodies are selected from
the physiological immunocompetent environment stimulated by natural
contact with a pathogen or antigen. The method is therefore
particularly useful to produce antibodies against antigenic
determinants that are specifically recognised by the human immune
system. These include neutralizing antibodies to human pathogens
and antibodies to allergens, tumour antigens, auto-antigens and
allo-antigens that are part of the memory repertoire of a given
individual. There is therefore no need for disease models to be
created or for immunization with purified antigens. The antibodies
of the current invention are also fully human (including native
post-translational modifications when expressed in B cells) and
exploit all the diversity generated in the course of a human immune
response (affinity maturation).
[0010] Thus, the invention provides inter alia a method for
producing immortalised human B memory lymphocytes, comprising the
step of transforming human B memory lymphocytes using Epstein Barr
virus (EBV) in the presence of a polyclonal B cell activator. The
method permits extremely high efficiency transformation for the
first time.
[0011] In a further aspect, the invention provides a method for
producing a clone of an immortalised human B memory lymphocyte
capable of producing a human monoclonal antibody with a desired
antigen specificity, comprising the steps of: [0012] (i)
transforming a population of cells comprising or consisting of
human memory B lymphocytes with Epstein Barr Virus (EBV) in the
presence of a polyclonal B cell activator; [0013] (ii) screening
the culture supernatant for antigen specificity; and [0014] (iii)
isolating an immortalised human B memory lymphocyte clone capable
of producing a human monoclonal antibody having the desired antigen
specificity.
[0015] The methods of the invention have already been used to clone
human memory B lymphocyte with up to 100% efficiency.
EBV-transformed B cell clones that produce neutralizing IgG
antibodies specific for measles virus, the Plasmodium species that
cause malaria in humans, tetanus toxoid, Toxoplasma gondii and
alloantigens have been produced using this procedure. Furthermore,
EBV-transformed B cell clones that produce neutralizing IgG
antibodies specific for the SARS coronavirus (CoV) have also been
produced. It will be appreciated that the method can be transferred
and used for the production of antibodies against any specificity
that is present in the human memory repertoire.
[0016] The sudden emergence of SARS in 2002 in China caused
hundreds of deaths in a number of countries. The causative agent
was unknown and as such there was no cure available. It has since
been discovered that the syndrome is caused by a new type of
coronavirus (Drosten et al. 2003; Ksiazek et al. 2003) and methods
to detect this virus and combat infections caused by it are
required.
[0017] In a preferred aspect, the invention provides a method for
producing a clone of immortalised human B memory lymphocytes
capable of producing a human monoclonal antibody specific for the
SARS virus, comprising the steps of: [0018] (i) transforming a
population of cells comprising or consisting of human memory B
lymphocytes with Epstein Barr Virus (EBV) in the presence of a
polyclonal B cell activator, [0019] (ii) screening the culture
supernatant for specificity for the SARS virus, and [0020] (iii)
isolating an immortalised human B memory lymphocyte clone capable
of producing a human monoclonal antibody having specificity for the
SARS virus.
[0021] In this specification, the term "antibody having specificity
for the SARS virus" means that an antibody molecule binds to the
coronavirus that is the causative agent of SARS with a greater
affinity compared to its binding affinity for other viruses.
[0022] Generally, the invention also provides a method for
producing immortalised human B memory lymphocytes, comprising a
step of transforming a population of human B memory lymphocytes,
wherein the method transforms .gtoreq.n % of the human B memory
lymphocytes in the population. The value of n is selected from 2,
3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100. After the
immortalised cells are produced then they can be screened to select
cells that produce antibodies with a desired specificity. Selected
cells can then be used for monoclonal antibody production. This
method preferably does not involve cellular fusion of the B memory
lymphocytes with other cells.
Polyclonal Activators
[0023] In this specification, the term "polyclonal activator" means
a molecule or compound or a combination thereof that activates B
lymphocytes irrespective of their antigenic specificity. A range of
different molecules may be used as the polyclonal activator.
[0024] Toll Like Receptors (TLRs) are pattern recognition receptors
of the innate immune system expressed on a variety of cells
including dendritic cells and B cells (Medzhitov & Janeway
2000, 2002). TLR agonists include microbial products and synthetic
compounds. Preferred polyclonal activators are agonists of the Toll
Like Receptors which are expressed on memory B cells, such as
TLR-7, TLR-9 and TLR-10 (Bernasconi et al. 2003). Such molecules
may be of microbial or cellular origin or synthetic.
[0025] Unmethylated DNA oligonucleotides (CpG) are TLR-9 agonists.
They stimulate dendritic cell maturation and activate B cell
proliferation and differentiation polyclonally, i.e. irrespective
of the antibody specificity (Krieg et al. 1995; Krieg 2002). The
biological effect of CpG is dependent on specific sequences and
chemical modifications (Krieg 2002). CpG oligonucleotides can be
used as polyclonal activators, and examples of suitable activators
are CpGs such as CpG 2006 (5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'; Hartmann
et al. 2000) and other oligonucleotide sequences that trigger
TLR-9. By "CpG" we mean sequences of unmethylated DNA
oligonucleotides. More particularly, the term "CpG" includes
single-stranded DNA molecules of between 5 and 100 nucleotides in
length (e.g. 10-80, 20-70, 30-60 nucleotides) that include one or
more instances (e.g. 2, 4, 6, 8, 10 or more) of the dinucleotide CG
sequence, with the C in the dinucleotide(s) being unmethylated.
[0026] Imidazoquinoline compounds, such as R-848 (resiquimod),
trigger TLR-7 and TLR-8 and stimulate dendritic cell maturation
(Hemmi et al. 2002). Such compounds can be used as polyclonal
activators with the invention e.g. R-848 (and its analogs) and
other synthetic compounds that trigger TLR-7 and TLR-8, including
but not limited to: imiquimod, loxoribine, and guanosine analogs
(e.g. 7-thia-8-oxoguanosine and 7-deazaguanosine).
[0027] Other polyclonal activators include other agonists of TLRs
and of other pattern recognition receptors (PRRs) that are
expressed on B memory cells, including monoclonal antibodies
specific for TLRs. Additional polyclonal activators include CD40L,
BAFF (B-cell activating factor, Schneider et al. 1999, also known
as tumor necrosis factor superfamilty member 13B, BLyS, or THANK),
antibodies specific for CD40 and other molecules expressed by
dendritic cells and activated T cells. In these cases the cells
themselves may be used as polyclonal activators.
[0028] Polyclonal activators may also include PAMPs
(pathogen-associated molecular patterns), such as
lipopolysaccharide (LPS), peptidoglycans, flagellins, zymosans and
other cell wall components found in pathogens. Other available
polyclonal activators include loxoribine, heat-killed Acholeplasma
ladilawii, heat-killed Listeria monocytogenes, lipoteichoic acids,
tripalmitoylated lipopeptides (e.g. Pam.sub.3CSK4), single-stranded
RNA (Diebold et al. 2004; Heil et al. 2004), double-stranded RNA,
poly(I:C), bacterial DNAs, etc. A detailed list of TLR agonists can
be found in Takeda et al. (2003). Some activators are not preferred
for use with human B cells e.g. LPS.
[0029] In a particularly preferred aspect, CpG 2006 is used as the
polyclonal activator.
[0030] Commercial suppliers of suitable polyclonal activators
include Invivogen.
[0031] Recently it has been shown that human memory B cells are
selectively stimulated by CpG (Bernasconi et al. 2002), and that
several TLRs are selectively expressed on human memory B cells but
not on naive B cells (Bernasconi et al. 2003, the entire contents
of which are herein incorporated by reference).
Transforming B Cells
[0032] In methods of the invention, cells can be transformed with
EBV in the presence of a polyclonal B cell activator.
Transformation with EBV is a standard technique and can easily be
adapted to include polyclonal B cell activators.
[0033] Additional stimulants of cellular growth and differentiation
may be added during the transformation step to further enhance the
efficiency. These stimulants may be cytokines such as IL-2 and
IL-15. In a particularly preferred aspect, IL-2 is added during the
immortalisation step to further improve the efficiency of
immortalisation, but its use is not essential.
[0034] The memory B cells to be transformed can come from various
sources (e.g. from whole blood, from peripheral blood mononuclear
cells (PBMCs), from blood culture, from bone marrow, from organs,
etc.), and suitable methods for obtaining human B cells are well
known in the art. Samples may include cells that are not memory B
cells e.g. other blood cells. A specific human memory B lymphocyte
subpopulation exhibiting a desired antigen specificity may be
selected before the transformation step by using methods known in
the art. In one embodiment, the human memory B lymphocyte
subpopulation has specificity for the SARS virus e.g. the B cells
are taken from a patient who is suffering or has recovered from
SARS. In another embodiment, B cells are taken from subjects with
Alzheimer's disease and include B cells with specificity for
.beta.-amyloid (e.g. Hock et al. (2002) Nature Medicine 8:1270-75;
Mattson & Chan (2003) Science 301:1847-9; etc.).
[0035] As an alternative to using EBV, other equivalent lymphocyte
transforming agents may be used, including other viruses that can
transform B cells. EBV is suitable for transforming the B cells of
most primates but, for other organisms, suitable viruses can be
selected.
Screening and Isolation of B Cells
[0036] Transformed B cells are screened for those having the
desired antigen specificity, and individual B cell clones can then
be produced from the positive cells.
[0037] The screening step may be carried out by ELISA, by staining
of tissues or cells (including transfected cells), a neutralisation
assay or one of a number of other methods known in the art for
identifying desired antigen specificity. The assay may select on
the basis of simple antigen recognition, or may select on the
additional basis of a desired function e.g. to select neutralising
antibodies rather than just antigen-binding antibodies, to select
antibodies that can change characteristics of targeted cells, such
as their signalling cascades, their shape, their growth rate, their
capability of influencing other cells, their response to the
influence by other cells or by other reagents or by a change in
conditions, their differentiation status, etc.
[0038] The cloning step for separating individual clones from the
mixture of positive cells may be carried out using limiting
dilution, micromanipulation, single cell deposition by cell sorting
or another method known in the art. Preferably the cloning is
carried out using limiting dilution.
[0039] The methods of the invention produce immortalised B cells
that produce antibodies having a desired antigen specificity. The
invention thus provides an immortalised B cell clone obtainable or
obtained by the methods of the invention. These B cells can be used
in various ways e.g. as a source of monoclonal antibodies, as a
source of nucleic acid (DNA or mRNA) encoding a monoclonal antibody
of interest, for delivery to patients for cellular therapy, for
research, etc.
[0040] The invention provides a composition comprising immortalised
B memory lymphocytes, wherein the lymphocytes produce antibodies,
and wherein the antibodies are produced at .gtoreq.10.sup.N ng per
clone per day. The invention also provides a composition comprising
clones of an immortalised B memory lymphocyte, wherein the clones
produce a monoclonal antibody of interest, and wherein the antibody
is produced at .gtoreq.10.sup.N ng per clone per day. The value of
N is selected from -3, -2, -1, 0, 1, 2, 3 or 4.
Antibodies of the Invention
[0041] The invention provides a monoclonal antibody obtainable or
obtained from B cell clones of the invention. The invention also
provides fragments of these monoclonal antibodies, particularly
fragments that retain the antigen-binding activity of the
antibodies.
[0042] In general, antibodies of the invention fall into two
categories: they either recognise self antigens or non-self
antigens. Antibodies that recognise self antigens can be used to
treat diseases caused by aberrant gene expression, including
cancers, and by aberrant protein processing, including Alzheimer's
disease. Antibodies that recognise non-self antigens can be used to
treat infectious diseases, including parasitic, viral and bacterial
infections. The invention can advantageously provide human
antibodies that recognise antigens of interest where it has not
previously been possible.
[0043] The methods of the invention produce antibodies with the
characteristics of those produced in the course of a physiological
human immune response i.e. antibody specificities that can only be
selected by the human immune system. This applies to the response
to human pathogens including HIV, the Plasmodium species that cause
human malaria, human hepatitis B and C viruses, Measles virus,
Ebola virus, the SARS virus, Pox virus, Bunyaviridae, Arenaviridae,
Bomaviridae, Reoviridae (including rotaviruses and orbiviruses),
Retroviridae (including HTLV-I, HTLV-II, HIV-1, HIV-2),
Polyomaviridae, Papillomaviridae, Adenoviridae, Parvoviridae,
Herpesviridae (including herpes simplex viruses 1 and 2,
cytomegaloviruses, varicella-zoster virus, herpesviruses 6A, 6B and
7), Poxyiridae, Hepadnaviridae, etc. (for an exhaustive list see
Fields et al. 1996). It also applies to antibody responses to
environmental allergens generated in allergic patients, to prion
proteins, to tumour antigens generated in tumour bearing patients
and to self-antigens in patients with autoimmune diseases, to
amyloid proteins, etc. These antibodies can be used as prophylactic
or therapeutic agents upon appropriate formulation or as a
diagnostic tool.
[0044] Particularly preferred monoclonal antibodies are those that
have specificity for the SARS virus. Thus the invention provides a
human monoclonal antibody that can neutralise the SARS
coronavirus.
[0045] In relation to any particular pathogen, a "neutralising
antibody" is one that can neutralise the ability of that pathogen
to initiate and/or perpetuate an infection in a host. The invention
provides a neutralising monoclonal human antibody, wherein the
antibody recognises an antigen from a pathogen selected from: human
immunodeficiency virus; hepatitis A virus; hepatitis B virus;
hepatitis C virus; herpes simplex virus type 1 or type 2; SARS
coronavirus; measles virus; mumps virus; rubella virus; rabies
virus; ebola virus; influenza virus; papillomavirus; vaccinia
virus; varicella-zoster virus; variola virus; polio virus; rhino
virus; respiratory syncytial virus; P. falciparum; P. vivax; P.
malariae; P. ovale; Corynebacterium diphtheriae; Clostridium
tetani; Clostridium botulinum; Bordetella pertussis; Haemophilus
influenzae; Neisseria meningitidis, serogroup A, B, C, W135 and/or
Y; Streptococcus pneumoniae; Streptococcus agalactiae;
Streptococcus pyogenes; Staphylococcus aureus; Bacillus anthracis;
Moraxella catarrhalis; Chlaymdia trachomatis; Chlamydia pneumoniae;
Yersinia pestis; Francisella tularensis; Salmonella species; Vibrio
cholerae; toxic E. coli; a human endogenous retrovirus; etc.
[0046] The invention also provides monoclonal human antibodies that
recognise proteins specifically expressed in tumours, in diseased
cardiovascular cells, during inflammatory responses, in
neurological disorders (including Alzheimer's disease e.g.
.beta.-amyloid proteins), in encephalopathies, etc. The invention
also provides monoclonal human antibodies that recognise narcotic
substances such as cocaine, heroin, benzoylecgonine, amphetamines,
etc.
[0047] Specific antigens that may be recognised by antibodies of
the invention include, but are not limited to: TNF-.alpha.,
.beta.-amyloid protein, SARS coronavirus spike protein, prion
protein PrP, complement C5, CBL, CD147, IL-8, HIV glycoprotein
gp120, VLA-4, CD11a, CD18, VEGF, CD40L, cellular adhesion molecules
such as ICAMs and VCAMs, CD80, integrins, TPL2, Her2, etc.
[0048] The invention also provides an antibody having two
polypeptide chains, wherein one or both of polypeptide chains
has/have a human VDJ sequence.
[0049] Monoclonal antibodies produced by the methods of the
invention may be further purified, if desired, using filtration,
centrifugation and various chromatographic methods such as HPLC or
affinity chromatography. Techniques for purification of monoclonal
antibodies, including techniques for producing pharmaceutical-grade
antibodies, are well known in the art.
[0050] Fragments of the monoclonal antibodies of the invention can
be obtained from the monoclonal antibodies so produced by methods
that include digestion with enzymes, such as pepsin or papain,
and/or by cleavage of disulfide bonds by chemical reduction.
Antibody "fragments" include Fab, F(ab').sub.2 and Fv fragments.
The invention also encompasses single-chain Fv fragments (scFv)
derived from the heavy and light chains of a monoclonal antibody of
the invention e.g. the invention includes a scFv comprising the
CDRs from an antibody of the invention.
[0051] The invention also provides a monoclonal human antibody with
neutralising activity, wherein the antibody can neutralise at a
concentration of 10.sup.-9M or lower (e.g. 10.sup.-10 M,
10.sup.-11M, 10.sup.-12M or lower).
[0052] Monoclonal antibodies are particularly useful in
identification and purification of the individual polypeptides or
other antigens against which they are directed. The monoclonal
antibodies of the invention have additional utility in that they
may be employed as reagents in immunoassays, radioimmunoassays
(RIA) or enzyme-linked immunosorbent assays (ELISA). In these
applications, the antibodies can be labelled with an
analytically-detectable reagent such as a radioisotope, a
fluorescent molecule or an enzyme. The monoclonal antibodies
produced by the above method may also be used for the molecular
identification and characterization (epitope mapping) of antigens
recognized by protected individuals in complex pathogens such as
plasmodia, the isolation of cross-reactive protective antibodies in
the case of highly variable pathogens such as those found in HIV
and for detecting pathogens and determining their variability.
[0053] Antibodies of the invention can be coupled to a drug for
delivery to a treatment site or coupled to a detectable label to
facilitate imaging of a site comprising cells of interest, such as
cancer cells. Methods for coupling antibodies to drugs and
detectable labels are well known in the art, as are methods for
imaging using detectable labels.
[0054] Antibodies of the invention may be attached to a solid
support.
[0055] Antibodies of the invention are preferably provided in
purified form. Typically, the antibody will be present in a
composition that is substantially free of other polypeptides e.g.
where less than 90% (by weight), usually less than 60% and more
usually less than 50% of the composition is made up of other
polypeptides.
[0056] Antibodies of the invention may be immunogenic in non-human
(or heterologous) hosts e.g. in mice. In particular, the antibodies
may have an idiotope that is immunogenic in non-human hosts, but
not in a human host. Antibodies of the invention for human use
include those that cannot be obtained from hosts such as mice,
goats, rabbits, rats, non-primate mammals, etc. and cannot be
obtained by humanisation or from xeno-mice.
[0057] Antibodies of the invention can be of any isotype (e.g. IgA,
IgG, IgM i.e. an .alpha., .gamma. or .mu. heavy chain), but will
generally be IgG. Within the IgG isotype, antibodies may be IgG1,
IgG2, IgG3 or IgG4 subclass. Antibodies of the invention may have a
.kappa. or a .lamda. light chain.
[0058] The invention also provides an immortalised B memory
lymphocyte cell (particularly a human cell), wherein the cell is
infected with EBV and encodes an antibody of the invention.
Pharmaceutical Compositions
[0059] The use of monoclonal antibodies as the active ingredient of
pharmaceuticals is now widespread, including the products
Herceptin.TM. (trastuzumab), Rituxan.TM., Campath.TM.,
Remicade.TM., ReoPro.TM. Mylotarg.TM., Zevalin.TM., Omalizumab,
Synagis.TM. (Palivizumab), Zenapax.TM. (daclizumab), etc. These
include antibodies that recognise human self-antigens (e.g.
Herceptin.TM. recognises the Her2 marker) and antibodies that
recognise pathogenic antigens (e.g. Synagis.TM. recognises an
antigen from respiratory syncytial virus).
[0060] The invention thus provides a pharmaceutical composition
containing the monoclonal antibodies of the invention and/or the
transformed B cells of the invention. A pharmaceutical composition
may also contain a pharmaceutically acceptable carrier for
administration of the antibody. The carrier should not itself
induce the production of antibodies harmful to the individual
receiving the composition and should not be toxic. Suitable
carriers may be large, slowly metabolised macromolecules such as
proteins, polypeptides, liposomes, polysaccharides, polylactic
acids, polyglycolic acids, polymeric amino acids, amino acid
copolymers and inactive virus particles.
[0061] Pharmaceutically acceptable salts can be used, for example
mineral acid salts, such as hydrochlorides, hydrobromides,
phosphates and sulphates, or salts of organic acids, such as
acetates, propionates, malonates and benzoates.
[0062] Pharmaceutically acceptable carriers in therapeutic
compositions may additionally contain liquids such as water,
saline, glycerol and ethanol. Additionally, auxiliary substances,
such as wetting or emulsifying agents or pH buffering substances,
may be present in such compositions. Such carriers enable the
pharmaceutical compositions to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries and suspensions,
for ingestion by the patient.
[0063] Preferred forms for administration include forms suitable
for parenteral administration, e.g. by injection or infusion, for
example by bolus injection or continuous infusion. Where the
product is for injection or infusion, it may take the form of a
suspension, solution or emulsion in an oily or aqueous vehicle and
it may contain formulatory agents, such as suspending,
preservative, stabilising and/or dispersing agents. Alternatively,
the antibody molecule may be in dry form, for reconstitution before
use with an appropriate sterile liquid.
[0064] Once formulated, the compositions of the invention can be
administered directly to the subject. It is preferred that the
compositions are adapted for administration to human subjects.
[0065] The pharmaceutical compositions of this invention may be
administered by any number of routes including, but not limited to,
oral, intravenous, intramuscular, intra-arterial, intramedullary,
intraperitoneal, intrathecal, intraventricular, transdermal,
transcutaneous (e.g. WO98/20734), topical, subcutaneous,
intranasal, enteral, sublingual, intravaginal or rectal routes.
Hyposprays may also be used to administer the pharmaceutical
compositions of the invention. Typically, the therapeutic
compositions may be prepared as injectables, either as liquid
solutions or suspensions. Solid forms suitable for solution in, or
suspension in, liquid vehicles prior to injection may also be
prepared.
[0066] Direct delivery of the compositions will generally be
accomplished by injection, subcutaneously, intraperitoneally,
intravenously or intramuscularly, or delivered to the interstitial
space of a tissue. The compositions can also be administered into a
lesion. Dosage treatment may be a single dose schedule or a
multiple dose schedule. Known antibody-based pharmaceuticals
provide guidance relating to frequency of administration e.g.
whether a pharmaceutical should be delivered daily, weekly,
monthly, etc. Frequency and dosage may also depend on the severity
of symptoms.
[0067] It will be appreciated that the active ingredient in the
composition will be an antibody molecule. As such, it will be
susceptible to degradation in the gastrointestinal tract. Thus, if
the composition is to be administered by a route using the
gastrointestinal tract, the composition will need to contain agents
which protect the antibody from degradation but which release the
antibody once it has been absorbed from the gastrointestinal
tract.
[0068] A thorough discussion of pharmaceutically acceptable
carriers is available in Remington's Pharmaceutical Sciences (Mack
Publishing Company, N.J. 1991) and in Gennaro (2000) Remington: The
Science and Practice of Pharmacy, 20th edition, ISBN:
0683306472.
[0069] Pharmaceutical compositions of the invention generally have
a pH between 5.5 and 8.5, preferably between 6 and 8, and more
preferably about 7. The pH may be maintained by the use of a
buffer. The composition may be sterile and/or pyrogen free. The
composition may be isotonic with respect to humans. Pharmaceutical
compositions of the invention are preferably supplied in
hermetically-sealed containers.
[0070] Pharmaceutical compositions will include an effective amount
of one or more antibodies of the invention and/or one or more
transformed B cells of the invention i.e. an amount that is
sufficient to treat, ameliorate, or prevent a desired disease or
condition, or to exhibit a detectable therapeutic effect.
Therapeutic effects also include reduction in physical symptoms.
The precise effective amount for any particular subject will depend
upon their size and health, the nature and extent of the condition,
and the therapeutics or combination of therapeutics selected for
administration. The effective amount for a given situation is
determined by routine experimentation and is within the judgment of
a clinician. For purposes of the present invention, an effective
dose will generally be from about 0.01 mg/kg to about 50 mg/kg, or
about 0.05 mg/kg to about 10 mg/kg of the compositions of the
present invention in the individual to which it is administered.
Known antibody-based pharmaceuticals provide guidance in this
respect e.g. Herceptin.TM. is administered by intravenous infusion
of a 21 mg/ml solution, with an initial loading dose of 4 mg/kg
body weight and a weekly maintenance dose of 2 mg/kg body weight;
Rituxan.TM. is administered weekly at 375 mg/m.sup.2; etc.
[0071] Compositions can include more than one (e.g. 2, 3, 4, 5,
etc.) antibody of the invention, particularly where such antibodies
bind to different antigens (or to different epitopes in the same
antigen) to provide a synergistic therapeutic effect.
[0072] Antibodies of the invention may be administered (either
combined or separately) with other therapeutics e.g. with
chemotherapeutic compounds, with radiotherapy, etc.
[0073] In compositions of the invention that include antibodies of
the invention, the antibodies preferably make up at least 50% by
weight (e.g. 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more) of the
total protein in the composition. The antibodies are thus in
purified form.
[0074] The invention provides a method of preparing a
pharmaceutical, comprising the steps of: (i) preparing a monoclonal
antibody of the invention; and (ii) admixing the purified antibody
with one or more pharmaceutically-acceptable carriers.
[0075] The invention also provides a method of preparing a
pharmaceutical, comprising the step of admixing a monoclonal
antibody with one or more pharmaceutically-acceptable carriers,
wherein the monoclonal antibody is a monoclonal antibody that was
obtained from a transformed B cell of the invention. Thus the
procedures for first obtaining the monoclonal antibody and then
preparing the pharmaceutical can be performed at very different
times by different people in different places (e.g. in different
countries).
[0076] As an alternative to delivering monoclonal antibodies or B
cells for therapeutic purposes, it is possible to deliver nucleic
acid (typically DNA) to a subject that encodes the monoclonal
antibody (or active fragment thereof) of interest, such that the
nucleic acid can be expressed in the subject in situ to provide a
desired therapeutic effect. Suitable gene therapy and nucleic acid
delivery vectors are known in the art.
Medical Treatments and Uses
[0077] The monoclonal antibodies of the invention or fragments
thereof may be used for the treatment of disease, for the
prevention of disease or for the diagnosis of disease. Preferably,
the monoclonal antibodies of the invention are used for the
prevention or treatment of SARS or for the diagnosis or SARS.
Methods of diagnosis may include contacting an antibody or an
antibody fragment with a sample. The methods of diagnosis may also
include the detection of an antigen/antibody complex.
[0078] The invention provides a composition of the invention for
use as a medicament. It also provides the use of an antibody of the
invention in the manufacture of a medicament for treatment of a
patient and/or diagnosis in a patient. It also provides a method
for treating a subject and/or of performing diagnosis on a subject,
comprising the step of administering to them a composition of the
invention. The subject is preferably a human. One way of checking
efficacy of therapeutic treatment involves monitoring disease
symptoms after administration of the composition of the invention.
Treatment can be a single dose schedule or a multiple dose
schedule. The invention is useful for treating infectious diseases,
cancers, inflammatory diseases, autoimmune diseases, etc.
[0079] Antibodies of the invention can be used in passive
immunisation.
[0080] Compositions of the invention will generally be administered
directly to a patient. Direct delivery may be accomplished by
parenteral injection (e.g. subcutaneously, intraperitoneally,
intravenously, intramuscularly, or to the interstitial space of a
tissue), or by rectal, oral (e.g. tablet, spray), vaginal, topical,
transdermal or transcutaneous, intranasal, ocular, aural, pulmonary
or other mucosal administration.
[0081] Compositions of the invention may be prepared in various
forms. For example, the compositions may be prepared as
injectables, either as liquid solutions or suspensions. Solid forms
suitable for solution in, or suspension in, liquid vehicles prior
to injection can also be prepared (e.g. a lyophilised composition,
like Synagis.TM. and Herceptin.TM., for reconsitution with sterile
water containing a preservative). The composition may be prepared
for topical administration e.g. as an ointment, cream or powder.
The composition may be prepared for oral administration e.g. as a
tablet or capsule, as a spray, or as a syrup (optionally
flavoured). The composition may be prepared for pulmonary
administration e.g. as an inhaler, using a fine powder or a spray.
The composition may be prepared as a suppository or pessary. The
composition may be prepared for nasal, aural or ocular
administration e.g. as drops. The composition may be in kit form,
designed such that a combined composition is reconstituted just
prior to administration to a patient. For example, a lyophilised
antibody can be provided in kit form with sterile water or a
sterile buffer.
[0082] Antibodies and fragments thereof as described in the present
invention may also be used in a kit for the diagnosis of tumour,
autoimmune or allergic disease.
Recombinant Expression
[0083] The immortalised memory B lymphocytes produced using the
method of the invention may also be used as a source of nucleic
acid for the cloning of antibody genes for subsequent recombinant
expression. Expression from recombinant sources is more common for
pharmaceutical purposes than expression from B cells or hybridomas
e.g. for reasons of stability, reproducibility, culture ease,
etc.
[0084] Thus the invention provides a method for preparing a
recombinant cell, comprising the steps of: (i) preparing an
immortalised B cell clone as described above; (ii) obtaining one or
more nucleic acids (e.g. heavy and/or light chain genes) from the B
cell clone that encodes the antibody of interest; and (iii)
inserting the nucleic acid into an expression host in order to
permit expression of the antibody of interest in that host.
[0085] Similarly, the invention provides a method for preparing a
recombinant cell, comprising the steps of: (i) preparing an
immortalised B cell clone as described above; (ii) sequencing
nucleic acid(s) from the B cell clone that encodes the antibody of
interest; and (iii) using the sequence information from step (ii)
to prepare nucleic acid(s) for inserting into an expression host in
order to permit expression of the antibody of interest in that
host.
[0086] The invention also provides a method of preparing a
recombinant cell, comprising the step of transforming a host cell
with one or more nucleic acids that encode a monoclonal antibody of
interest, wherein the nucleic acids are nucleic acids that were
derived from an immortalised B cell clone of the invention. Thus
the procedures for first preparing the nucleic acid(s) and then
using it to transform a host cell can be performed at different
times by different people in different places (e.g. in different
countries).
[0087] These recombinant cells of the invention can then be used
for expression and culture purposes. They are particularly useful
for expression of antibodies for large-scale pharmaceutical
production. They can also be used as the active ingredient of a
pharmaceutical composition. Any suitable culture techniques can be
used, including but not limited to static culture, roller bottle
culture, ascites fluid, hollow-fiber type bioreactor cartridge,
modular minifermenter, stirred tank, microcarrier culture, ceramic
core perfusion, etc.
[0088] Methods for obtaining and sequencing immunoglobulin genes
from B cells are well known in the art e.g. see chapter 4 of Kuby
Immunology (4th edition, 2000; ASIN: 0716733315).
[0089] The expression host is preferably a eukaryotic cell,
including yeast and animal cells, particularly mammalian cells
(e.g. CHO cells, human cells such as PER.C6 [Crucell; Jones et al.
Biotechnol Prog 2003, 19(1):163-8] or HKB-11 [Bayer; Cho et al.
Cytotechnology 2001, 37:23-30; Cho et al. Biotechnol Prog 2003,
19:229-32] cells, myeloma cells [U.S. Pat. Nos. 5,807,715 and
6,300,104], etc.), as well as plant cells. Preferred expression
hosts can glycosylate the antibody of the invention, particularly
with carbohydrate structures that are not themselves immunogenic in
humans. Expression hosts that can grow in serum-free media are
preferred. Expression hosts that can grow in culture without the
presence of animal-derived products are preferred.
[0090] The expression host may be cultured to give a cell line.
[0091] The invention provides a method for preparing one or more
nucleic acid molecules (e.g. heavy and light chain genes) that
encodes an antibody of interest, comprising the steps of: (i)
preparing an immortalised B cell clone according to the invention;
(ii) obtaining from the B cell clone nucleic acid that encodes the
antibody of interest. The invention also provides a method for
obtaining a nucleic acid sequence that encodes an antibody of
interest, comprising the steps of: (i) preparing an immortalised B
cell clone according to the invention; (ii) sequencing nucleic acid
from the B cell clone that encodes the antibody of interest.
[0092] The invention also provides a method of preparing nucleic
acid molecule(s) that encodes an antibody of interest, comprising
the step of obtaining the nucleic acid from a B cell clone that was
obtained from a transformed B cell of the invention. Thus the
procedures for first obtaining the B cell clone and then preparing
nucleic acid(s) from it can be performed at very different times by
different people in different places (e.g. in different
countries).
[0093] The invention provides a method for preparing an antibody
(e.g. for pharmaceutical use), comprising the steps of: (i)
transforming a population of human B memory lymphocytes and
selecting a transformed B cell that produces an antibody with a
desired specificity, as described above; (ii) obtaining and/or
sequencing one or more nucleic acids (e.g. heavy and light chain
genes) from the selected B cell the antibody of interest; (iii)
inserting the nucleic acid(s) into or using the nucleic acid(s) to
prepare an expression host that can express the antibody of
interest; (iv) culturing or sub-culturing the expression host under
conditions where the antibody of interest is expressed; and,
optionally, (v) purifying the antibody of the interest.
[0094] The invention also provides a method of preparing an
antibody comprising the steps of: culturing or sub-culturing an
expression host cell population under conditions where the antibody
of interest is expressed and, optionally, purifying the antibody of
the interest, wherein said expression host cell population has been
prepared by (i) providing nucleic acid(s) encoding a selected B
cell the antibody of interest that is produced by a population of B
memory lymphocytes prepared as described above, (ii) inserting the
nucleic acid(s) into an expression host that can express the
antibody of interest, and (iii) culturing or sub-culturing
expression hosts comprising said inserted nucleic acids to produce
said expression host cell population. Thus the procedures for first
preparing the recombinant expression host and then culturing it to
express antibody can be performed at very different times by
different people in different places (e.g. in different
countries).
[0095] The invention also provides a method of preparing a
pharmaceutical, comprising the step of admixing a monoclonal
antibody with one or more pharmaceutically-acceptable carriers,
wherein the monoclonal antibody is a monoclonal antibody that was
obtained from an expression host of the invention. Thus the
procedures for first obtaining the monoclonal antibody (e.g.
expressing it and/or purifying it) and then admixing it with the
pharmaceutical carrier(s) can be performed at very different times
by different people in different places (e.g. in different
countries).
[0096] Starting with a transformed B cell of the invention, various
steps of culturing, sub-culturing, cloning, sub-cloning,
sequencing, nucleic acid preparation etc. can be performed in order
to perpetuate the antibody expressed by the transformed B cell,
with optional optimisation at each step. In a preferred embodiment,
the above methods further comprise techniques of optimisation (e.g.
affinity maturation or optimisation) applied to the nucleic acids
encoding the antibody. The invention encompasses all cells, nucleic
acids, vectors, sequences, antibodies etc. used and prepared during
such steps.
[0097] In all these methods, the nucleic acid used in the
expression host may be manipulated between steps (ii) and (iii) to
insert, delete or amend certain nucleic acid sequences. Changes
from such manipulation include, but are not limited to, changes to
introduce restriction sites, to amend codon usage, to add or
optimise transcription and/or translation regulatory sequences,
etc. It is also possible to change the nucleic acid to alter the
encoded amino acids. For example, it may be useful to introduce one
or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid
substitutions, one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
etc.) amino acid deletions and/or one or more (e.g. 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, etc.) amino acid insertions into the antibody's
amino acid sequence. Such point mutations can modify effector
functions, antigen-binding affinity, post-translational
modifications, immunogenicity, etc., can introduce amino acids for
the attachment of covalent groups (e.g. labels) or can introduce
tags (e.g. for purification purposes). Mutations can be introduced
in specific sites or can be introduced at random, followed by
selection (e.g. molecular evolution).
SARS
[0098] Antibodies specific for the SARS virus may be particularly
useful for prophylaxis and may be administered to health care
workers or other people who may come into contact with SARS virus
infected patients. Such passive serotherapy may offer an immediate
cure of infected individuals as well as containment through
protection of contacts and medical personnel. Human sera containing
antibodies to the SARS virus are not available in sufficient
amounts, therefore the method of the invention provides an ideal
way of producing human neutralizing monoclonal antibodies. Such
antibodies may be used to develop a passive serotherapy against
this and other pathogens.
[0099] The invention therefore also provides a method of preventing
transmission of the SARS virus comprising administering an
effective amount of an antibody or antibody fragment specific for
the SARS virus. Stocks of antibody specific for the SARS virus
should therefore be maintained so that they are available for
immediate use in any further SARS outbreak.
Non-Human Species
[0100] The invention has been described above in relation to human
antibodies prepared from human B cells. It will be appreciated that
the invention is not technically restricted to use with human
cells, and can be used with any organism of interest e.g. to
provide antibodies for therapeutic or diagnostic veterinary use.
Organisms with B cells that can be transformed by the methods of
the invention include primates (monkeys, apes, gorillas, gibbons,
lemurs, chimpanzees, baboons, orang-utans, macaques, etc.) cows,
horses, goats, sheep, pigs, dogs, cats, camels, sharks, fish,
etc.
General
[0101] The term "comprising" encompasses "including" as well as
"consisting" e.g. a composition "comprising" X may consist
exclusively of X or may include something additional e.g. X+Y.
[0102] The word "substantially" does not exclude "completely" e.g.
a composition which is "substantially free" from Y may be
completely free from Y. Where necessary, the word "substantially"
may be omitted from the definition of the invention.
[0103] The term "about" in relation to a numerical value x means,
for example, x.+-.10%.
BRIEF DESCRIPTION OF DRAWINGS
[0104] FIG. 1 shows results of the ELISA using SARS virus-infected
Vero cells lysed in 3% SDS as antigen. Shown are the OD values of
serum ( 1/5000 dilution), supernatants of polyclonal cultures and
of independent B cell clones (1/2 dilution).
[0105] FIG. 2 shows the neutralizing titer of antibodies specific
for the SARS virus. From left to right: neutralization titer (Vero
cell assay) of convalescent serum; supernatants of polyclonal
cultures; positive clones isolated from the culture with highest
neutralizing titer.
[0106] FIG. 3 shows clonal analysis of the human antibody response
to the SARS virus spike protein. Culture supernatants were tested
for their capacity to stain BHK cells transfected with SARS virus
spike protein mRNA (Frankfurt isolate) and for their capacity to
neutralize the same strain of SARS virus. FIG. 3A shows the
correlation between neutralizing titer and staining of spike
protein by undiluted culture supernatant. FIG. 3B shows staining of
spike-transfected BHK cells by serial dilutions of supernatants
from eleven neutralizing cultures, with filled symbols showing the
maximum dilution where complete neutralization was observed.
[0107] FIG. 4 shows the characterization of the SARS neutralizing
antibody S3.1. Staining of spike-transfected BHK cells by purified
S3.1 (circles) and 6 months convalescent serum (squares). The
filled symbols indicate the maximum dilution where complete
neutralization was observed.
[0108] FIG. 5 shows immunoelectron microscopy of SARS coronavirus
in the presence of S3.1 antibody.
[0109] FIG. 6 is an overview of a method of the invention, from
human to monoclonal antibody.
MODES FOR CARRYING OUT THE INVENTION
[0110] The present invention permits the cloning of human memory B
lymphocytes with very high efficiency and achieves this by the
combination of two stimuli, namely: EBV, that immortalizes human B
cells with low efficiency and a polyclonal B cell activator that
enhances the efficiency of EBV-immortalization.
Example 1
Cloning of B Cells
[0111] Human memory B cells
(CD19.sup.+CD27.sup.+IgM.sup.-IgD.sup.-) were isolated from healthy
donors by cell sorting using methods well known in the art.
Different numbers of cells were seeded in replicate cultures in 96
well microplates in the presence of irradiated mononuclear cells
(5.times.10.sup.5/ml) and either EBV (supernatant of B95-8 cells)
alone or EBV in combination with CpG 2006 (2.5 .mu.g/ml) and
recombinant IL-2 (1000 U/ml). After 15 days the percentage of
cultures containing growing cells was scored. Frequencies were
determined by limiting dilution assays. Cultures were scored for
growing cells. Cloning efficiency using four different sources of B
cells were as follows:
TABLE-US-00001 Cloning efficiency B cell source EBV EBV + CpG +
IL-2 Exp 1 (CD19.sup.+ CD27.sup.+ IgM.sup.- IgD.sup.-) 1 in 200 1
in 1 Exp 2 (CD19.sup.+ CD27.sup.+ IgM.sup.- IgD.sup.-) 1 in 120 1
in 1.5 Exp 3 (CD19.sup.+ CD27.sup.+ IgM.sup.- IgD.sup.-) 1 in 60 1
in 1 Exp 4 (CD19.sup.+ CD27.sup.+) 1 in 90 1 in 1.6
[0112] No growth was observed in the absence of EBV.
[0113] Thus the methods of the invention allow virtually every
human memory B cell to be cloned (efficiency close to 100%). The
method is also suitable for subcloning.
Example 2
Production of Antibodies with a Desired Specificity
[0114] In a further experiment it was demonstrated that the
immortalisation can be used to exploit immunological memory to
produce human monoclonal antibodies of the desired specificity.
[0115] Mononuclear cells were isolated from 20 ml peripheral blood
obtained from a healthy blood donor. CD19.sup.+CD27.sup.+IgG.sup.+
human memory B cells were isolated by cell sorting and seeded at 10
cells/well in 96 well microplates in the presence of EBV, CpG 2006
(2.5 .mu.g/ml), recombinant IL-2 (1000 U/ml) and irradiated
mononuclear cells (5.times.10.sup.5/ml). Seeding only 10 cells per
well helps to increase cloning efficiency. After 15 days all
cultures contained growing cells. A sample of supernatant was
collected and tested in ELISA for total IgG antibodies and for IgG
specific for Toxoplasma gondii, tetanus toxoid and measles virus.
The supernatants were also tested in a measles virus neutralization
assay using Vero cells as targets.
[0116] Some of the positive cultures identified were subcloned by
limiting dilution to isolate specific clones producing the desired
monoclonal antibody. Cultures were subcloned at 0.5 cells/well in
the presence of CpG 2006 (2.5 .mu.g/ml), IL-2 (1000 U/ml) and
irradiated PBMC (5.times.10.sup.5/ml).
TABLE-US-00002 Antibody Positive Specific clones isolated/
(detection method) cultures.sup.# attempts made* IgG (ELISA)
180/180 Anti-Toxoplasma gondii (ELISA) 23/180 5/6 Anti-tetanus
toxoid (ELISA) 19/180 5/6 Anti-Measles virus (ELISA) 36/180 7/9
Anti-Measles virus (neutralization) 7/180 4/4 .sup.#For ELISA,
number of cultures with OD >0.8 in an assay with background
<0.2; for neutralisation assay, number with complete protection
from cytolitic effect of measles virus *Number of cases where at
least one antigen-specific clone could be isolated, relative to the
number of original cultures that were cloned. Cloning efficiency
varied from 50 to 100%.
[0117] EBV-transformed B cell clones that produce IgG antibodies to
measles virus, tetanus toxoid and Toxoplasma gondii could thus be
isolated, showing that human monoclonal antibodies with multiple
memory specificities can be prepared from a small sample of human
peripheral blood.
Example 3
Immortalised Memory B Cells that Express Antibodies Specific for
SARS Coronavirus
[0118] Blood samples were obtained from two patients with a
clinical record of SARS. Both patients had serum anti-SARS
antibodies as detected by two assays: (i) a neutralization assay
which detects neutralizing antibodies directed against surface
proteins of the SARS virus, likely the spike protein and (ii) an
ELISA assay, that detects antibodies binding to any denatured
protein of the SARS virus.
[0119] For the neutralization assay, serial dilutions of serum
obtained from the blood were added to microplate wells containing
Vero cells, followed by titrated amounts of SARS virus. After 2
days, the cytopathic effect was recorded by visual inspection. A
conventional ELISA was also developed using SARS virus infected
Vero cells lysed in 3% SDS as the antigen.
[0120] For the production of the clone of B cells producing
monoclonal antibodies specific for the SARS virus, blood from the
patient showing the higher titer of binding and neutralizing
antibodies was selected.
[0121] Memory B cells carrying surface IgG were isolated using
anti-human IgG microbeads, incubated with EBV (50% supernatant of
B95-8 cells) for 6 hours and were then plated at 10 cells/well in
96 well microplates in the presence of 2.5 .mu.g/ml CpG 2006 and
allogeneic irradiated PBMC (in these experiments IL-2 was omitted).
After two weeks the culture supernatants were screened for the
presence of specific antibodies.
[0122] Out of 1042 culture supernatants tested, 165 scored positive
in the ELISA assay (FIG. 1). 23 of these cultures were cloned as
above and specific clones were isolated from 16 of them. Some of
the human monoclonal antibodies isolated recognise the
nucleoprotein of the SARS virus in western blots, while some do
not, suggesting that they may recognise different viral proteins
(data not shown). None of these antibodies showed neutralizing
activity.
[0123] Out of 1042 culture supernatants tested in the
neutralization assay, seven showed low level neutralizing activity
while two (A11 and D8) showed high neutralizing titer ( 1/512 and
1/256 respectively). The All culture was cloned by limiting
dilution in the presence of CpG and irradiated PBMC and several B
cell clones with comparably high neutralizing activity were
isolated (FIG. 2). The All antibodies did not bind in the ELISA
assay, but stained surface spikes of the SARS virus as detected by
electron microscopy (data not shown).
[0124] Therefore, using this method it is possible to produce
neutralizing antibodies specific for an antigen using only a small
blood sample (around 10 ml) within a short timespan (30-40 days).
The method also allows selection of the best antibody from a large
pool, and is therefore a high throughput method.
[0125] The anti-SARS antibodies neutralise the SARS virus at
concentrations of .about.5 ng/ml. Neutralization of respiratory
syncytial virus (RSV, a common cause of respiratory tract
infections, especially in children) by commercially available
humanized antibodies produced by conventional techniques requires
around a 1000-fold higher antibody concentration (Johanson et al.
1997). Therefore the fully human antibodies produced by the method
of the invention appear to be around 1000-fold more effective. This
suggests that very small amounts of the antibody described here
should be sufficient to prevent or cure SARS infection.
Example 4
Screening SARS Virus Convalescent Patients for Antibodies
[0126] Peripheral blood was obtained from a patient at different
times after acute infection with SARS virus (2, 4 and 6 months
after infection). PBMC were isolated by gradient centrifugation.
IgG.sup.+ memory B cells were isolated by an improved method that
avoids triggering of the B cell receptor. Total B cells were
isolated from PBMC using CD22 microbeads (Miltenyi), which were
found to be even better than using CD19 microbeads. The cells were
stained with antibodies to human IgM, IgD and IgA and negative
cells carrying surface IgG were isolated by cell sorting. B cells
were pulsed with EBV (50% supernatant of B-95-8 cells) for 8 hours
and then plated at 10 cells/well in 96 well U-bottom microplates in
complete RPMI medium supplemented with 10% FCS, 2.5 .mu.g/ml CpG
2006 and irradiated PBMC (2.times.10.sup.5/ml). In this experiment
IL-2 was not used. After 2 weeks the culture supernatants were
screened for the presence of specific antibodies using the three
assays described below. Positive cultures were cloned by limiting
dilution in the presence of CpG 2006 and irradiated PBMC as above.
Positive clones were expanded and the antibody produced was
purified from culture supernatants by affinity chromatography on
protein A columns (Amersham).
[0127] The Frankfurt isolate of the SARS virus (Genbank accession
number AY310120) was used for three in vitro assays:
ELISA Vero cells were infected at a multiplicity of infection of
0.01 plaque forming units per cell. Cell culture supernatant
collected after 2 days was cleared by centrifugation at 3000 rpm, 5
min. The supernatant was subjected to centrifugation at 20,000 rpm
for 2 hours in a Beckman SW28 rotor through a 20% sucrose cushion.
The pellet was purified using a potassium tartrate/glycerol
gradient and resuspended in 500 .mu.l TNE buffer (10 mM Tris-HCl,
pH 7.4, 0.15 M NaCl, 2 mM EDTA) to a protein concentration of
approx 0.5 mg/ml. The antigen suspension used for the ELISA assay
was prepared by adding 1% SDS to the viral pellet followed by
boiling for 10 min. ELISA plates were coated with a 1:1000 dilution
of SARS virus antigen in 0.1 M phosphate buffer. Dilutions of sera
or culture supernatant were added and specific IgG1 antibodies were
detected using alkaline phosphatase goat anti-human IgG. Results
were expressed in arbitrary units (AU) relative to the 2 month
sample (=1000AU). Sera from 20 normal donors tested were negative
(<1 AU). Staining Antibodies specific for native spike protein
of SARS virus were detected by flow cytometry. Briefly, the SARS
virus spike gene was cloned in an appropriate vector and mRNA was
transcribed in vitro and used to transfect BHK cells by
electroporation. Transfectants were incubated with culture
supernatants or serum, washed and stained with APC-labelled goat
anti-human IgG antibody. This assay detects IgG1 antibodies
directed against native spike antigen, most of which have
neutralizing activity. Results were expressed in arbitrary units
(AU) relative to the 2 month sample (=1000AU). Sera from 20 normal
donors tested were negative (<1 AU). In vitro neutralization
Sera or culture supernatants were diluted in log 2 steps and mixed
with 75 TCID.sub.50 SARS virus in 25 .mu.l (virus titer was
determined according to the method of Karber). The mixture was
incubated for 45 min at room temperature. Subsequently, 50 .mu.l
trypsinized Vero cells (1.5.times.10.sup.5 per ml) were added and
incubated for 3 days at 37.degree. C. and finally, the
neutralization titer was determined by visual inspection to give
the serum dilution that neutralizes 75 TCID.sub.50 SARS virus. The
assays were performed in a biosafety level 4 laboratory.
[0128] Results were as follows:
TABLE-US-00003 Anti-SARS antibody detected by Months after
infection ELISA Spike staining Neutralization 2 1000 1000 1/128 4
650 700 1/128 6 300 400 1/128
[0129] While normal sera were negative, the patient's serum
collected at different time points after the onset of acute disease
scored positive in the three assays. Antibodies detected by ELISA
and those staining spike-transfected cells were highest 2 months
after infection and decreased to about one third by six months. In
contrast neutralizing antibodies remained constant with a titer of
1/128. The isotype of the antibodies detected in the Spike-binding
and ELISA assays was exclusively IgG1, no IgA or IgM antibodies
being detected (data not shown). Thus the post-infection serum of
this person had moderate titers of neutralizing antibodies to the
SARS virus and IgG antibodies that bound spike proteins and
detected denatured antigens in ELISA.
Example 5
Kinetics and Frequencies of Specific Memory B Cells
[0130] IgG.sup.+ memory B lymphocytes from the 2-month, 4-month and
6-month post-SARS sera were immortalized with EBV under conditions
where the number of B cells per culture was limiting, as described
above (10 B cells per well). This strategy allows analysis of the
product of only a few memory B cells per culture, thus ensuring
that the specific antibody detected in positive cultures is
monoclonal and, at the same time, increasing the probability of
isolating a clone producing the desired antibody by limiting
dilution. After two weeks of culture in the presence of EBV, CpG
2006 and irradiated feeder cells the culture supernatants were
screened for the presence of specific IgG antibodies using ELISA or
staining of spike transfectants. The frequency of cultures
screening positive in the SARS virus ELISA assay or staining SARS
virus spike transfectants were as follows:
TABLE-US-00004 Positive cultures/total cultures screened (%) Months
after infection ELISA Spike staining 2 275/480 (57.3%) Not
determined 4 123/480 (25.6%) 12/576 (2.1%) 6 44/480 (9/2%) 21/768
(2.7%)
[0131] The frequency of cultures producing antibodies detected by
the ELISA assay was very high 2 months after infection and
decreased by 4 and 6 months. The frequency of cultures producing
antibodies against native spike protein measured at 4 and 6 months
was lower. Importantly, the culture that scored positive for ELISA
antibodies were distinct from those containing antibodies to the
spike indicating that the two assays detect distinct
non-overlapping antibody specificities. Furthermore, a sizeable
proportion of IgG.sup.+ memory B cells are specific for the spike
protein.
[0132] Tests were then carried out to see whether there is a
correlation between spike binding and neutralizing activity. 56
culture supernatants which stained spike-transfected cells were
tested for their capacity to neutralize the same SARS virus isolate
from which the spike protein was cloned (FIG. 3A). Although the
antibodies with the highest staining showed high neutralizing
titers, there were some antibodies that neutralized efficiently in
spite of poor staining while others stained spike transfectants,
but failed to neutralize. Furthermore, when 11 supernatants with
high neutralizing titer were analyzed, no clear correlation between
staining and neutralization was evident (FIG. 3B). Taken together
these results indicate that at the clonal level the response to
spike is heterogeneous and that not all the anti-spike antibodies
produced in the course of the natural infection are capable of
neutralizing the virus.
Example 6
Isolation of Monoclonal Antibodies to SARS Virus
[0133] The results shown above prove that it is possible to
interrogate the human B cell memory repertoire with a variety of
assays to identify cultures producing an antibody of the desired
specificity. In these experiments, 29 of 38 attempts (76%) at
cloning positive cultures led to the isolation of one or more
clones producing antibodies of the selected specificity. The EBV
clones were stable and monoclonal antibodies were recovered in the
culture supernatant at concentrations of 10-20 .mu.g/ml. Of these
29, 21 were positive in the ELISA assay and 8 were both positive in
the spike staining assay and were able to neutralize SARS
virus.
[0134] Out of the 21 independent clones that scored positive in the
ELISA assay, 13 (62%) produced antibodies specific for the SARS
virus nucleoprotein (NP) as detected by Western blot, while 5 did
not recognize NP, but stained SARS virus infected cells, and the
remaining 3 reacted only in the ELISA assay. As expected, none of
these antibodies showed neutralizing activity.
[0135] Out of the 8 independent clones staining spike transfectants
and neutralizing SARS virus, one (S3.1, IgG1.kappa.) was selected
for in vivo neutralization assays. The monoclonal antibody from
this clone was purified from the culture supernatant and tested for
its capacity to stain spike-transfected cells and to neutralize
SARS virus (FIG. 4). S3.1 neutralized 75 TCID.sub.50 SARS virus at
concentrations of .about.300 ng/ml, and was up to 300 fold more
potent than convalescent serum. Furthermore S3.1 neutralized the
Frankfurt and Urbani isolates with the same efficiency (data not
shown), and decorated the spikes of SARS-CoV as detected by
immunoelectron microscopy (FIG. 5).
Example 7
S3.1 Neutralizes SARS Infection in an Animal Model
[0136] The in vivo neutralizing activity of the S3.1 monoclonal
antibody was tested in a mouse model of acute SARS infection.
Purified antibody was transferred to naive mice by intraperitoneal
injection to determine whether antibody alone could prevent
replication of SARS virus in the respiratory tract.
[0137] Drs. L. J. Anderson and T. G. Ksiazek from the Centers for
Disease Control and Prevention (CDC), Atlanta, Ga., provided SARS
virus (Urbani strain) for use in an in vivo neutralization assay.
The virus was isolated and passaged twice in Vero E6 cells at the
CDC and was passaged in Vero cells for two additional passages in
our laboratory to generate a virus stock with a titer of 10.sup.6.5
TCID.sub.50/ml. The Vero cells were maintained in OptiPro SFM
(Invitrogen). All work with infectious virus was performed inside a
biosafety cabinet, in a biosafety containment level 3 facility and
personnel wore powered air purifying respirators (3M HEPA AirMate,
Saint Paul, Minn.). The mouse studies were approved by the NIH
Animal Care and Use Committee and were carried out in an approved
animal biosafety level 3 facility. All personnel entering the
facility wore powered air purifying respirators.
[0138] Four-to-six week-old female BALB/c mice purchased from
Taconic (Germantown, N.Y.) were housed, 4 mice per cage. On day 0
mice, lightly anesthetized with isoflurane, were injected
intraperitoneally with 3 different doses (800, 200, 50 .mu.g) of
S3.1 antibody in 500 .mu.l or with the same volume of a polyclonal
human Ig that lacks neutralizing activity. 24 hours later mice were
intranasally challenged with 10.sup.4 TCID.sub.50 of SARS
coronavirus. After two additional days mice were sacrificed and
their lungs and nasal turbinates were removed and homogenized in a
5% w/v suspension in Leibovitz 15 medium (Invitrogen). Tissue
samples were assessed for infection, and virus titers were
determined as described above. Virus titers were expressed as
log.sub.10 TCID.sub.50 per gram of tissue:
TABLE-US-00005 Virus replication in challenged mice Lungs Nasal
turbinates Number Mean (.+-. SE) Number Mean (.+-. SE) Antibody
infected virus titer infected virus titer S3.1 800 .mu.g 0/4
.ltoreq.1.5 .+-. 0* 2/4 2.5 .+-. 0.47 S3.1 200 .mu.g 0/4
.ltoreq.1.5 .+-. 0* 4/4 3.4 .+-. 0.41 S3.1 50 .mu.g 2/4 3.2 .+-.
1.36 4/4 4.8 .+-. 0.75 Control 800 .mu.g 4/4 7.5 .+-. 0.1 4/4 6.4
.+-. 0.41 *The lower limit of detection of infectious virus in a
10% w/v suspension of lung homogenate was 1.5
log.sub.10TCID.sub.50/gm and in 5% w/v suspension of nasal
turbinates was 1.8 log.sub.10TCID.sub.50/gm. These values thus
indicate no detectable virus.
[0139] Mice that received S3.1 mAb were thus protected from
replication of challenge virus, particularly in the lower
respiratory tract. The differences in viral titers when compared to
the control were statistically significantly (p<0.05) in a
Student's t-test. Significant restriction of virus replication in
the upper respiratory tract was noted in the mice which received
the highest dose of S3.1 mAb.
Example 8
R-848
[0140] R-848 is an agonist of TLR7 and TLR8. This compound was
compared with CpG 2006 in terms of efficiency of EBV-induced
immortalization of human B cells. Memory B cells were isolated from
healthy donors using anti-CD19 or anti-CD22 magnetic microbeads
followed by negative depletion of cells carrying IgM, IgD and IgA
(or IgG). 48 replicate cultures were set up in 96 well U-bottomed
microplates by limiting dilution at 30, 10 and 3 B cells per well
in complete medium in the presence of irradiated mononuclear cells,
EBV (20% supernatant from B95-8 cells) and in the presence or
absence of 2.5 .mu.g/ml CpG 2006 or 2.5 .mu.g/ml R-848. The
frequency of cultures positive for cell growth and Ig production
was measured after 14 days and the efficiency of transformation was
calculated. Results were as follows:
TABLE-US-00006 B cell source EBV +CpG 2006 +R848 Donor AOS
CD19.sup.+ IgM.sup.- IgD.sup.- IgA.sup.- 1 in 150 1 in 3.5 1 in 2.5
Donor AOS CD22.sup.+ IgM.sup.- IgD.sup.- IgA.sup.- 1 in 300 1 in 2
1 in 1.5 Donor ASC CD19.sup.+ IgM.sup.- IgD.sup.- IgA.sup.- 1 in
320 1 in 2 1 in 2.4 Donor ASC CD19.sup.+ IgM.sup.- IgD.sup.-
IgG.sup.- 1 in 280 1 in 4 1 in 2.2 Donor ETR CD22.sup.+ IgM.sup.-
IgD.sup.- 1 in 230 1 in 1.9 1 in 2
[0141] R-848 and CpG 2006 are thus comparable in their capacity to
increase the efficiency of EBV-induced immortalization. In
addition, R-848 was comparable to CpG 2006 in the capacity to
increase the cloning efficiency of polyclonal EBV-immortalized B
cell lines. In the presence of R-848 the cloning efficiency of EBV
cell lines ranged from 25 to 100% in 10 independent
experiments.
Example 9
Isolation of High Affinity Antibodies Neutralizing SARS-CoV
[0142] A new series of monoclonal antibodies with SARS-CoV
neutralizing capacity was produced as described above from
immortalized memory B cells isolated from a convalescent patient
six months after infection. Serial dilutions of supernatants from
the B cell clones were tested for their antigen specificity (NP,
matrix (M) or spike proteins), and their capacity to neutralize the
cytopathic effect of SARS CoV (Frankfurt isolate) on Vero cells.
The concentration of monoclonal IgG was measured by ELISA in the
same culture supernatants. Neutralizing titers were expressed as
the final concentration of IgG (ng IgG per ml) in tissue culture
capable of completely neutralizing the virus (mean values of at
least three tests). Results were as follows:
TABLE-US-00007 B cell clone Isotype Specificity Neutralizing titer
S18.1 IgG, .kappa. NP -- S20.1 IgG, .lamda. NP -- S21.1 IgG,
.kappa. NP -- S23.4 IgG, .kappa. NP -- S24.1 IgG, .lamda. NP --
S13.1 IgG, .kappa. Not determined -- S5.1 IgG, .kappa. M -- S3.1
IgG, .kappa. Spike 300 S101.1 IgG, .kappa. Spike 40 S102.1 IgG,
.kappa. Spike 850 S103.3 IgG, .kappa. Spike 350 S104.1 IgG, .kappa.
Spike 150 S105.2 IgG, .kappa. Spike 150 S106.1 IgG, .kappa. Spike
45 S107.4 IgG, .kappa. Spike 75 S108.1 IgG, .kappa. Spike 40 S109.2
IgG, .kappa. Spike 80 S132.9 IgG, .kappa. Spike 200 S128.5 IgG,
.kappa. Spike 25 S127.6 IgG, .kappa. Spike 40 S124.4 IgG, .kappa.
Spike 40 S159.1 IgG, .lamda. Spike 25 S160.1 IgG, .kappa. Spike
15
[0143] Thus the invention is routinely able to provide antibodies
that can neutralize the virus at concentrations lower than
10.sup.-9 M and even down to 10.sup.-10 M (MW of human IgG is
.about.150 kDa, and so 150 ng/ml is .about.10.sup.-9M).
[0144] It will be understood that the invention has been described
by way of example only and modifications may be made whilst
remaining within the scope and spirit of the invention. In
particular, minor modifications that do not affect the
immunogenicity of the modified capsular saccharide of the present
invention are also encompassed.
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Sequence CWU 1
1
1124DNAArtificial SequenceSynthetic Construct 1tcgtcgtttt
gtcgttttgt cgtt 24
* * * * *