U.S. patent application number 10/568246 was filed with the patent office on 2006-11-23 for methods for obtaining antibodies.
Invention is credited to Meryn Ruth Griffiths, Alastair David Griffiths Lawson.
Application Number | 20060263353 10/568246 |
Document ID | / |
Family ID | 34219619 |
Filed Date | 2006-11-23 |
United States Patent
Application |
20060263353 |
Kind Code |
A1 |
Lawson; Alastair David Griffiths ;
et al. |
November 23, 2006 |
Methods for obtaining antibodies
Abstract
The invention is directed towards a method of enriching a
population of cells in those cells that produce an antibody which
recognises an antigen of interest. In particular, an untagged
antigen is used in conjunction with a polyclonal antibody to
isolate cells recognizing said antigen.
Inventors: |
Lawson; Alastair David
Griffiths; (Slough Berkshire, GB) ; Griffiths; Meryn
Ruth; (Slough Berkshire, GB) |
Correspondence
Address: |
KLAUBER & JACKSON
411 HACKENSACK AVENUE
HACKENSACK
NJ
07601
US
|
Family ID: |
34219619 |
Appl. No.: |
10/568246 |
Filed: |
August 12, 2004 |
PCT Filed: |
August 12, 2004 |
PCT NO: |
PCT/GB04/03523 |
371 Date: |
July 28, 2006 |
Current U.S.
Class: |
424/133.1 ;
435/320.1; 435/326; 435/69.1; 435/7.2; 530/387.1; 536/23.53 |
Current CPC
Class: |
G01N 33/582 20130101;
G01N 33/56972 20130101 |
Class at
Publication: |
424/133.1 ;
435/007.2; 435/069.1; 435/320.1; 435/326; 530/387.1;
536/023.53 |
International
Class: |
A61K 39/395 20060101
A61K039/395; G01N 33/567 20060101 G01N033/567; C07H 21/04 20060101
C07H021/04; C12P 21/06 20060101 C12P021/06; C07K 16/18 20060101
C07K016/18 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 20, 2003 |
GB |
0319587.2 |
Feb 6, 2004 |
GB |
0402641.5 |
Claims
1. A method of enriching a population of cells in those cells which
produce an antibody that recognises an antigen of interest,
comprising: a) bringing said population into contact with an
antibody that recognises a marker which is essentially unique to
those cells present in the population which are capable of
producing an antibody, said antibody being attached to a first
fluorescent label; b) bringing said population into contact with
the antigen of interest; c) bringing said population into contact
with a sample comprising an antibody that recognises said antigen,
said antibody being attached to a second fluorescent label; and d)
separating from the population those cells which are detectable by
virtue of being associated with the first and second fluorescent
labels.
2. The method of claim 1, wherein parts a), b), and c) are
performed simultaneously and the performance optionally comprises
at least one wash step.
3. The method of claim 1, wherein parts a) and b), or a) and c), or
b) and c), are performed simultaneously and optionally comprise at
least one wash step.
4. The method of claim 1, wherein parts a), b) and c) are performed
consecutively in any order, and wherein each performance optionally
comprises at least one wash step.
5. The method of claim 1, wherein part a) additionally comprises
bringing said population into contact with an antibody that
recognises a second marker essentially unique to those cells
present in the population which are capable of producing an
antibody, said antibody being labelled with a third label.
6. The method of claim 1, wherein the separation of the cells
producing an antibody that recognises the antigen of interest is
performed using fluorescence activated cell sorting.
7. A method according to claim 1 additionally comprising: a)
culturing a plurality of those cells associated with the
antigen-antibody-particle complex; b) screening the cultured cells
to identify those cells capable of producing an antibody which
recognises an antigen of interest; and c) isolating said antibody
directly or indirectly from the cells.
8. An antibody prepared directly or indirectly according to the
method of claim 1.
9. A pharmaceutical composition comprising an antibody according to
claim 8 and a pharmaceutically acceptable carrier.
10. (canceled)
11. (canceled)
12. The method of claim 2, wherein the separation of the cells
producing an antibody that recognises the antigen of interest is
performed using fluorescence activated cell sorting.
13. The method of claim 4, wherein the separation of the cells
producing an antibody that recognises the antigen of interest is
performed using fluorescence activated cell sorting.
14. The method of claim 5, wherein the separation of the cells
producing an antibody that recognises the antigen of interest is
performed using fluorescence activated cell sorting.
15. A method according to claim 2 additionally comprising: a)
culturing a plurality of those cells associated with the
antigen-antibody-particle complex; b) screening the cultured cells
to identify those cells capable of producing an antibody which
recognises an antigen of interest; and c) isolating said antibody
directly or indirectly from the cells.
16. A method according to claim 4 additionally comprising: a)
culturing a plurality of those cells associated with the
antigen-antibody-particle complex; b) screening the cultured cells
to identify those cells capable of producing an antibody which
recognises an antigen of interest; and c) isolating said antibody
directly or indirectly from the cells.
17. A method according to claim 5 additionally comprising: a)
culturing a plurality of those cells associated with the
antigen-antibody-particle complex; b) screening the cultured cells
to identify those cells capable of producing an antibody which
recognises an antigen of interest; and c) isolating said antibody
directly or indirectly from the cells.
18. A method according to claim 6 additionally comprising: a)
culturing a plurality of those cells associated with the
antigen-antibody-particle complex; b) screening the cultured cells
to identify those cells capable of producing an antibody which
recognises an antigen of interest; and c) isolating said antibody
directly or indirectly from the cells.
19. An antibody prepared directly or indirectly according to the
method of claim 5.
20. An antibody prepared directly or indirectly according to the
method of claim 7.
Description
[0001] The present invention relates to improved methods for the
selection of cells producing antibodies specific for an antigen of
interest.
[0002] Antibodies are a particular class of proteins which have
been developed for therapeutic and diagnostic purposes. The
isolation of cells producing antibody specific for an antigen of
interest is historically performed using hybridoma technology.
Other methods include isolating antibodies from bacterially
expressed libraries which are limited by: (i) restrictions to the
practical limits of the size of libraries; and (ii) the requirement
for the antibody to be expressed and properly folded in bacteria. A
number of alternative methods have been designed to enable high
affinity antibodies generated during in vivo immune responses to be
isolated from any species (Babcook et al., 1996, Proc. Natl. Acad.
Sci, 93, 7843-7848; WO 92/02551; de Wildt et al., 1997, J. Immunol.
Methods, 207:61-67 and in Lagerkvist, et al., 1995, BioTechniques
18(5):862-869).
[0003] Methods for the detection and isolation of cells producing
antibody specific for an antigen of interest are well-known in the
art and include isolation of antibody producing cells by binding to
biotinylated antigen and capture on streptavidin-beads, panning
against antigen-coated plastic surfaces, resetting with
antigen-coated red blood cells, flow cytometric analysis and single
cell sorting where the antigen is fluorescently labelled. The major
drawback of these methods is that the presentation of antigen is
generally random such that masking of the antigenic epitope, which
is specifically recognised by the antibody producing, cell can
occur. In particular, where the antigen of interest is a protein,
the labelling of the antigen for example with a fluorescent label,
is a chemical modification of the surface of the antigen that can
decrease the affinity of an antibody-antigen interaction or prevent
interaction. Where the antigens of interest are small proteins or
short peptides the introduction of a chemical modification in the
form of a label, such as a fluorescent label, may interfere such
that little or no binding of antigen to specific antibody
occurs.
[0004] Similar problems exist where the antigen of interest is
provided tagged as a fusion protein. The incorporation of an
additional sequence on the C-terminus or N-terminus of a protein
can result in aberrant folding such that the antigen is not folded
into a native conformation. As such, antigenic epitopes exposed
within, for example, a host immunised with native antigen may not
be accessible on a fusion protein of the same antigen. Thus, at the
step of enrichment of cells producing antibody specific for the
native antigen, those cells producing antibody to epitopes which
are masked or altered in the fusion protein will not be detected
and isolated.
[0005] Using unlabelled or untagged antigen is therefore
particularly advantageous in that this avoids any modification of
the antigen which can modify or mask interaction sites and which,
in turn, can result in absence of detection of an antibody specific
for the antigen of interest. Accordingly, provided is a method of
enriching a population of cells in those cells which produce an
antibody that recognises an antigen of interest, comprising: [0006]
a) bringing said population into contact with an antibody that
recognises a marker which is essentially unique to those cells
present in the population which are capable of producing an
antibody, said antibody being attached to a first fluorescent
label; [0007] b) bringing said population into contact with the
antigen of interest; [0008] c) bringing said population into
contact with a sample comprising an antibody that recognises said
antigen, said antibody being attached to a second fluorescent
label; and [0009] d) separating from the population those cells
which are detectable by virtue of being associated with the first
and second fluorescent labels.
[0010] The terms `cells which are capable of producing an antibody`
or `antibody producing cell` include any cell secreting an
antibody, such as a B-lymphocyte, a plasma cell, a plasmablast, an
activated B cell or a memory B cell. Such cells may produce
antibody of any affinity, for example high affinity antibody or
lower affinity antibody. The methods of the invention are not
dependent on the affinity of the antibody produced by such cells. A
population comprising antibody producing cells for use in the
invention may be obtained from an animal which has either been
immunized with an antigen of interest, or which has developed an
immune response to an antigen as a result of disease. For example
but without limitation, the population can comprise a peripheral
blood cell sample, spleen cells or cells derived from a lymph node.
Other populations comprising antibody producing cells for use in
the present invention may include a population of hybridoma cells,
a population comprising any transformed cell, and in particular, a
population comprising any mammalian cells which express
immunoglobulin genes or parts thereof. Examples of such mammalian
cells include but are not limited to NS0, CHO, COS and 293 cells.
In a preferred embodiment, the populations of antibody producing
cells for use in the present invention produce a range of
antibodies with different binding specificities. In another
embodiment, the population of cells comprising at least one cell
producing an antibody that recognises an antigen of interest is
derived from several sources, for example but without limitation
from several lymph nodes which may be from one or more animals. It
will also be apparent that samples or populations of cells derived
from two or more animals can be pooled for use in the methods of
the invention. In a particular embodiment, the population of cells
is derived from a human who has been exposed to an antigen of
interest or who has developed an immune response to an antigen as a
result of a disease or condition. In such a case, the sample
comprising the population of cells for enrichment is preferably a
peripheral blood sample or one or more lymph nodes.
[0011] Preferably, the population is suspended in an appropriate
medium for use in the methods of the invention. An appropriate
medium for the assay will be one that provides at least the minimum
requirements for short-term maintenance of cellular integrity and
cellular structures, such as an isotonic buffer. One example, but
without limitation, is immune cell medium comprising Roswell Park
Memorial Institute medium (RPMI)+10% foetal bovine serum; 50 .mu.M
2-.beta.-mercaptoethanol; 2 mM glutamine; 20 mM Hepes; and 1.times.
Penicillin and Streptomycin. Under such conditions the antibody
producing cells produce and secrete antibodies.
[0012] A population comprising antibody producing cells may be
depleted of unwanted cells, such as for example but without
limitation, red blood cells, T cells, macrophages or other cells if
so desired. In the methods of the invention the population of cells
provided, at least one of which is capable of producing an
antibody, is preferably depleted of any red blood cells for example
using centrifugation or red cell lysis as known in the art.
[0013] The term `antibody` includes any recombinant or naturally
occurring immunoglobulin molecule such as a member of the IgG class
e.g. IgG1, including a monoclonal or polyclonal antibody, any
antigen binding immunoglobulin fragment, such as Fv, Fab' and
F(ab').sub.2 fragments, and any derivatives thereof, such as single
chain Fv fragments. The term antibody that `recognises an antigen`
includes an antibody that binds to, or is specific for, an antigen
of interest. Most preferably, an antibody binds to the antigen of
interest and does not bind to or recognise other unrelated
antigens.
[0014] In the methods of the invention, the antibody of part a)
above recognises a marker which is essentially unique to those
cells capable of producing an antibody, for example a marker
essentially unique to B cells. `Essentially unique` includes a
marker that is predominantly present on those cells capable of
producing antibody compared to other cells types, but not
necessarily to the exclusion of all other cell types. Hence, such a
marker may also be present on one or two or even three or more
other cell types. Examples of preferred markers include without
limitation, CD5, CD9, CD10, CD19, CD20, CD21, CD22, CD45, CD45 RC,
although one skilled in the art will recognise that many of the
`CD` markers present on B cells can be used. In one example, the
antibody of part a) above is a monoclonal antibody. Cells
expressing a marker recognised by an antibody of part a) above will
be distinguished by virtue of having the first label attached.
Those cells that are not capable of producing antibody will be
distinguished by being unlabelled or negative. In a further
embodiment, those cells, within a population, which arc capable of
producing an antibody may be attached to two labels. In such a
case, an antibody that recognises a second, different marker
essentially unique to said cells is labelled with a third label,
i.e. a label that is different from those which are attached to the
antibodies of parts a) and b), above. Those antibody producing
cells within the population provided will thus be distinguished by
having two labels attached. Thus, the selection of those cells
which carry the chosen markers is made possible by distinguishing
from those which carry only one marker, or no marker in which case
said cells will be unlabelled or negative.
[0015] In the methods of the invention, the antibody of part b)
above is most preferably a polyclonal antibody that is from the
same source as that of the population of cells for enrichment. In a
preferred example, the source of the population of cells is an
animal immunised with an untagged antigen of interest and the
polyclonal antibody is prepared from a sample of blood from said
immunised animal. In another embodiment, the antibody of part b)
above is present in a pool of polyclonal sera; for example it is
present in a pool of sera from at least two animals which have been
immunised with the antigen of interest. It will be apparent to one
skilled in the art that the polyclonal antibody may be present as a
serum sample, but is more preferably prepared as an IgG fraction.
Methods for producing an IgG fraction are well known in the art and
include affinity chromatography such as Protein A or Protein G
affinity chromatography, and ammonium sulphate or caprylic acid
precipitation. The polyclonal antibody may be a whole IgG or a
fragment thereof such as a Fab', F(ab').sub.2 or Fab fragment.
Fragments may be produced using any method known in the art, for
example using papain or pepsin digestion. In an alternative
embodiment, the antibody of part b) above is a monoclonal
antibody.
[0016] Labels of use in the methods of the invention are
fluorescent labels. Appropriate fluorescent labels are well known
in the art, as are methods for performing the labelling of
antibodies with such labels. Such labels can include, but are not
limited to, Alexa Fluor 488, R-phycoerythrin, Aqua, Texas-Red,
FITC, rhodamine, a rhodamine derivative, fluorescein, a fluorescein
derivative, cascade blue, Cy5 or Cy3. Preferably, the first or
second fluorescent label is Alexa Fluor 488 or R-phycoerythrin. It
is understood that the first and second labels, and any subsequent
labels, if used, are different labels. In a preferred embodiment,
the methods of the invention utilise two labels, i.e. a first and
second fluorescent label. Labelled antibodies are preferably used
at, for example but without limitation, 1-5 .mu.g/ml and incubation
of such antibodies with a cell sample is preferably performed in
the cold for an appropriate length of time, for example 60 mins. It
will be apparent to one skilled in the art that the concentration
of antibody may be less or more than the stated values, above, and
hence may range from, for example 0.1 .mu.g/ml or less to 10
.mu.g/ml or more.
[0017] In the methods of the invention the antigen is provided in
an untagged form. The term `antigen` includes any substance that
can be recognised by an antibody, including proteins, glycoproteins
and carbohydrates. Preferably these antigens include biologically
active proteins, such as hormones, cytokines, and their cell
surface receptors, bacterial or parasitic cell membranes or
purified components thereof, and viral antigens. In a particular
embodiment, the antigen is presented on the surface of a cell. Such
an antigen may be endogenous or recombinant. Most preferably, the
antigen is available in a pure form obtained either by direct
purification from the native source or by recombinant expression
and purification of said antigen. A pure form includes antigens
that are at least 75%, 80%, 85% or 90% free from contaminants, and
preferably at least 95% or 99% free from contaminants. Most
preferably, antigens are 100% pure or have no detectable
contaminants. In one embodiment, the antigen is expressed
recombinantly as a fusion protein and the fusion tag has been
removed prior to use of the antigen. Removal of such tags is well
known in the art and such removal may leave a small number of
residual amino acid residues which are not normally present at the
N-terminus or C-terminus of the antigen in its native state, e.g.
residues from a linker region and/or the region of cleavage of the
tag. Thus, the term `untagged` antigen includes antigens which have
had a tag removed regardless of whether or not additional amino
acid residues remain attached to the antigen as a result of the
cleavage. Examples of such tags are known in the art and expression
vectors incorporating nucleic acid code for such tags are
commercially available for example, but not limited to, myc, FLAG
or His tags.
[0018] Preferably, incubation of the antigen at a concentration of
approximately 1 .mu.M to 1 pM in the presence of a population of,
for example but without limitation, approximately 10.sup.7 or
10.sup.8 white blood cells some of which are capable of producing
antibody is performed on ice for approximately 60 min. It is
understood that the number of cells may be less or more than
10.sup.7, and may be 10.sup.6, 10.sup.5, 10.sup.4 or less, or
10.sup.9 or more as desired. The concentration of antigen may also
be varied and may be chosen as appropriate and understood by one
skilled in the art. In one embodiment, low concentrations of
antigen can be used to bias towards selecting B cells that produce
high affinity antibody.
[0019] In a preferred embodiment, parts a), b), and c) are
performed simultaneously, the first and second labels are
fluorescent labels and the performance is optionally followed by at
least one wash step. In another embodiment parts a) and b), or a)
and c), or b) and c), are performed simultaneously and optionally
comprise at least one wash step. Wash steps may be performed by any
means known in the art, for example using phosphate buffered saline
(PBS) or other appropriate media. The population of cells may be
separated from any incubation or wash media using, for example but
without limitation, centrifugation. Alternatively, cells may be
left to settle under gravity followed by removal of the washing
buffer or other media.
[0020] In another embodiment, parts a), b) and c) are performed
consecutively, in any order, such that the performance of part a)
may be followed by the performance of part b), and then part c). It
will be apparent to a person skilled in the art that any
permutation is possible, e.g. part a), then part c) followed by
part b), or part b) then part a) followed by part c), etc. It will
also be apparent that one or more washing steps as described above
may be carried out after the performance of any of parts a) to d)
or combination thereof.
[0021] In the methods of the invention, separation of those cells
that are attached to both a first and a second label from a
population of cells is most preferably performed using fluorescence
activated cell sorting (FACS). Alternatively, said cells are
identified using a fluorescence microscope. Cells identified using
the latter method may be isolated by micromanipulation. Thus, an
enriched population of cells producing an antibody that recognises
an antigen of interest can be prepared using the methods of the
invention. In one preferred embodiment, the enriched population of
cells is subject to at least one washing step. Alternatively, the
enriched population of cells is not washed.
[0022] An enriched population of cells obtained using any of the
methods of the invention may be further separated, if desired, to
obtain and clone one or more single antibody producing cells using
any method known in the art. Methods to obtain and clone single
antibody producing cells include methods such as, but not limited
to, the selected lymphocyte antibody method (SLAM) described in WO
92/02551 and Babcook et al., 1996, Proc. Natl. Acad. Sci USA
93:7843-7848. Other techniques include those described by de Wildt
et al., 1997, J. Immunol. Methods, 207:61-67 and Lagerkvist et al.,
1995, BioTechniques 18(5):862-869. The above methods rely on the
isolation of individual antibody producing cells which are then
clonally expanded, followed by screening for those clones which are
producing an antibody that recognises an antigen of interest, and,
if desired, the subsequent identification of the sequence of their
variable heavy (V.sub.H)and light (V.sub.L) chain genes.
[0023] Alternatively, the enriched population of cells producing
antibody that recognises an antigen of interest may be plated into
one or more wells and screened for antibody production followed
sequence identification, if desired, as above. In such a case, the
enriched population of cells is divided such that a plurality of
cells is placed in the well for culture. It is preferred that the
wells are seeded with between 2 and 100 B cells; more preferably
with between 2 and 75 B cells; more preferably between 5 and 50 B
cells; more preferably between 5 and 25 B cells; more preferably
between 5 and 15 B cells; more preferably between 8 and 12 B cells;
yet more preferably about 10 B cells/well.
[0024] In one embodiment, the B cells are cultured for about, or at
least, 4, 5, 6, 7, 8, 9 or 10 days or up to one month. Preferably,
the B cells are cultured for about 5 to 10 days, more preferably
for about 6 to 9, or 6 to 8 days.
[0025] Most preferably, the cells are cultured under conditions
suitable for the clonal expansion of the B cells. Clonal expansion
results in a greater quantity of antibody being produced and higher
levels of mRNA expression. Clonal expansion is preferably performed
in the presence of an antigen to which the antibody with the
desired function binds which may assist in the isolation of higher
affinity antibodies via in vitro affinity maturation.
[0026] Conditions suitable for the clonal expansion of B cells are
well known in the art. Important conditions include the culture
medium, the time for which the cells are cultured, temperature and
atmospheric CO.sub.2.
[0027] Preferably, the B cells are cultured with irradiated EL-4
cells in T cell conditioned media. More preferably, the B cells are
cultured with irradiated mutant murine EL-4 thymoma cells, EL-4/B5,
in conjunction with human T-cell/macrophage supernatant as a source
of proliferation and differentiation factors. The EL-4/B5 cells
activate the B-cells via a MHC-nonrestricted direct cell-cell
interaction. The activation signal itself is not mitogenic but
sensitizes the B cells to respond to one (IL-2) or several
cytokines present in human T-cell supernatant.
[0028] Once the cells have been cultured, a plurality of the
cultured cells may be screened to ascertain the presence of cells
capable of producing an antibody having the desired function.
Preferably, this involves screening the culture supernatant of said
plurality of cultured cells. Where cells have been cultured in a
series of wells, the wells can be individually assayed (e.g. by
taking culture supernatant from the wells) for the presence of
cells capable of producing an antibody recognising the antigen of
interest to thereby identify one or more wells which are positive
for the presence of cells capable of producing an antibody having
the desired function. Said antibodies can then be obtained from a
positive well. The antibodies can be synthesized directly or
indirectly from the cells present in the well.
[0029] Accordingly, provided is a method of enriching a population
of cells in those cells which produce an antibody that recognises
an antigen of interest, comprising: [0030] a) bringing said
population into contact with an antibody that recognises a marker
which is essentially unique to those cells present in the
population which are capable of producing an antibody, said
antibody being attached to a first fluorescent label; [0031] b)
bringing said population into contact with the antigen of interest;
[0032] c) bringing said population into contact with a sample
comprising an antibody that recognises said antigen, said antibody
being attached to a second fluorescent label; [0033] d) separating
from the population those cells which are detectable by virtue of
being associated with the first and second fluorescent labels;
[0034] e) culturing a plurality of those cells associated with the
antigen-antibody-particle complex; [0035] f) screening the cultured
cells to identify those cells capable of producing an antibody
which recognises an antigen of interest; and [0036] g) isolating
said antibody directly or indirectly from the cells.
[0037] Screening for cells producing antibodies recognising the
antigen of interest may be performed by any means known in the art,
such as by enzyme-linked immunosorbent assay (ELISA) or by
screening for a functional activity such as neutralisation of
antigen activity, or antagonistic or agonistic activities. Such
assays are known in the art, for example, functional screening of
receptor/ligand binding. Antibodies may be selected based on
binding affinities such as, for example, may be determined using a
BIAcore machine, or using a competitive radioimmunoassay.
[0038] The desired antibody, i.e. the antibody recognising the
antigen of interest, may be isolated directly or indirectly from
the cultured cells or from descendants thereof. Direct isolation
can be achieved by purification of secreted antibody from the
clonal culture supernatant using standard, well known techniques.
Alternatively, indirect isolation is performed. Such antibodies are
synthesised by isolating the V.sub.L and V.sub.H chain gene regions
or the entire genes may be cloned and used to produce recombinant
antibodies that recognise the antigen of interest.
[0039] Such antibodies can include functionally active fragments,
derivatives or analogues and may be, but are not limited to, bi-,
tri- or tetra-valent antibodies, humanized or chimeric antibodies,
single chain antibodies, Fab fragments, Fab' and F(ab').sub.2
fragments, fragments produced by a Fab expression library,
anti-idiotypic (anti-Id) antibodies, epitope-binding fragments and
derivatives of any of the above, e.g. single chain FV fragments.
Humanized antibodies are antibody molecules from non-human species
having one or more complementarity determining regions (CDRs) from
the non-human species and a framework region from a human
immunoglobulin molecule (see, e.g. U.S. Pat. No. 5,585,089). The
methods for creating these antibody molecules are well known in the
art (see for example, Boss et al., U.S. Pat. No. 4,816,397; Cabilly
et al., U.S. Pat. No. 6,331,415; Shrader et al., WO 92/02551; Ward
et al., 1989, Nature, 341, 544; Orlandi et al., 1989,
Proc.Natl.Acad.Sci. USA, 86, 3833; Riechmann et al., 1988, Nature,
322, 323; Bird et al, 1988, Science, 242, 423; Queen et al., U.S.
Pat. No. 5,585,089; Adair, WO91/09967; Mountain and Adair, 1992,
Biotechnol. Genet. Eng. Rev, 10, 1-142).
[0040] Chimeric antibodies are those antibodies encoded by
immunoglobulin genes that have been genetically engineered so that
the light and heavy chain genes are composed of immunoglobulin gene
segments belonging to different species. These chimeric antibodies
are likely to be less antigenic. Bivalent antibodies may be made by
methods known in the art (Milstein et al., 1983, Nature
305:537-539; WO 93/08829, Traunecker et al., 1991, EMBO J.
10:3655-3659). Bi-, tri- and tetra-valent antibodies may comprise
multiple specificities or may be monospecific (see for example WO
92/22853).
[0041] The types of expression systems available to produce these
antibody molecules include bacterial, yeast, insect and mammalian
expression systems, the methods for which are well known in the art
(Verma et al., 1998, Journal of Immunological Methods, 216,
165-181). Techniques for the production of single chain antibodies,
such as those described in U.S. Pat. No. 4,946,778 can also be
adapted to produce single chain antibodies to the antigen of
interest. Also, transgenic mice or other organisms, including other
mammals, may be used to express humanized antibodies.
[0042] Antibodies obtained using the above methods may be used
without further modification, or if desired following modification
including conjugation to one or more reporter or effector
molecules, for any suitable diagnostic or therapeutic purpose. An
antibody, optionally conjugated to a therapeutic moiety, can be
used therapeutically alone or in combination with a cytotoxic
factor(s) and/or cytokine(s). In particular, antibodies can be
conjugated to a therapeutic agent, such as a cytotoxic agent, a
radionuclide or drug moiety to modify a given biological response.
The therapeutic agent is not to be construed as limited to
classical chemical therapeutic agents. For example, the therapeutic
agent may be a drug moiety which may be a protein or polypeptide
possessing a desired biological activity. Such moieties may
include, for example and without limitation, a toxin such as abrin,
ricin A, pseudomonas exotoxin, or diphtheria toxin, a protein such
as tumour necrosis factor, .alpha.-interferon, .beta.-interferon,
nerve growth factor, platelet derived growth factor or tissue
plasminogen activator, a thrombotic agent or an anti-angiogenic
agent, e.g. angiostatin or endostatin, or, a biological response
modifier such as a lymphokine, interleukin-1 (IL-1), interleukin-2
(IL-2), interleukin-6 (IL-6), granulocyte macrophage colony
stimulating factor (GM-CSF), granulocyte colony stimulating factor
(G-CSF), nerve growth factor (NGF) or other growth factor.
[0043] Therapeutic agents also include cytotoxins or cytotoxic
agents including any agent that is detrimental to (e.g. kills)
cells. Examples include taxol, cytochalasin B, gramicidin D,
ethidium bromide, emetine, mitomycin, etoposide, tenoposide,
vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin
D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Therapeutic agents also include, but are not limited to,
antimetabolites (e.g. methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g. mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g. daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g. dactinomycin (formerly actinomycin), bleomycin,
mithramycin, anthramycin (AMC), calicheamicins or duocarmycins),
and anti-mitotic agents (e.g. vincristine and vinblastine).
[0044] Other therapeutic moieties may include radionuclides such as
.sup.111In and .sup.90Y, Lu.sup.177, Bismuth.sup.213,
Califorium.sup.252, Iridium.sup.192 and
Tunsten.sup.188/Rhenium.sup.188; or drugs such as but not limited
to, alkylphosphocholines, topoisomerase I inhibitors, taxoids and
suramin.
[0045] Techniques for conjugating such therapeutic agents to
antibodies are well known in the art (see, e.g. Arnon et al.,
"Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer
Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et
al., eds., 1985 pp. 243-56, ed. Alan R. Liss, Inc; Hellstrom et
al., "Antibodies For Drug Delivery", in Controlled Drug Delivery,
2nd Ed., Robinson et al., eds., 1987, pp. 623-53, Marcel Dekker,
Inc.; Thorpe, "Antibody Carriers Of Cytotoxic Agents in Cancer
Therapy: A Review", in Monoclonal Antibodies '84: Biological And
Clinical Applications; Pinchera et al., 1985, eds., pp. 475-506;
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabelled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
1985, pp. 303-16, Academic Press; Thorpe et al., 1982 "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 and Dubowchik et al., 1999, Pharmacology
and Therapeutics, 83, 67-123).
[0046] The antibodies for use in the invention include analogues
and derivatives that are modified, for example but without
limitation, by the covalent attachment of any type of molecule.
Preferably, said attachment does not impair immunospecific binding.
In one aspect, an antibody can be conjugated to a second antibody
to form an antibody heteroconjugate (see U.S. Pat. No.
4,676,980).
[0047] In other embodiments, the invention provides the therapeutic
use of fusion proteins of the antibodies (or functionally active
fragments thereof), for example but without limitation, where the
antibody or fragment thereof is fused via a covalent bond (e.g. a
peptide bond), at optionally the N-terminus or the C-terminus, to
an amino acid sequence of another protein (or portion thereof;
preferably at least a 10, 20 or 50 amino acid portion of the
protein). Preferably the antibody, or fragment thereof, is linked
to the other protein at the N-terminus of the constant domain of
the antibody. In another aspect, an antibody fusion protein may
facilitate depletion or purification of a polypeptide as described
herein, increase half-life in vivo, and enhance the delivery of an
antigen across an epithelial barrier to the immune system.
[0048] Where the fusion protein is an antibody fragment linked to
an effector or reporter molecule, this may be prepared by standard
chemical or recombinant DNA procedures. For instance, it may have a
macrocycle for chelating a heavy metal atom, or a toxin, such as
ricin, attached to it by a covalent bridging structure. A preferred
effector group is a polymer molecule, which may be attached to the
modified Fab fragment to increase its half-life in vivo.
[0049] The polymer molecule may, in general, be a synthetic or a
naturally occurring polymer, for example an optionally substituted
straight or branched chain polyalkylene, polyalkenylene or
polyoxyalkylene polymer or a branched or unbranched polysaccharide,
e.g. a homo- or hetero-polysaccharide.
[0050] Particular optional substituents which may be present on the
above-mentioned synthetic polymers include one or more hydroxy,
methyl or methoxy groups. Particular examples of synthetic polymers
include optionally substituted straight or branched chain
poly(ethyleneglycol), poly(propyleneglycol) poly(vinylalcohol) or
derivatives thereof, especially optionally substituted
poly(ethyleneglycol) such as methoxypoly(ethyleneglycol) or
derivatives thereof.
[0051] Particular naturally occurring polymers include lactose,
amylose, dextran, glycogen or derivatives thereof.
[0052] "Derivatives" as used herein is intended to include reactive
derivatives, for example thiol-selective reactive groups such as
maleimides and the like. The reactive group may be linked directly
or through a linker segment to the polymer. It will be appreciated
that the residue of such a group will in some instances form part
of the product as the linking group between the antibody fragment
and the polymer.
[0053] The size of the polymer may be varied as desired, but will
generally be in an average molecular weight range from 500 Da to
50000 Da, preferably from 5000 to 40000 Da and more preferably from
25000 to 40000 Da. The polymer size may in particular be selected
on the basis of the intended use of the product. Thus, for example,
where the product is intended to leave the circulation and
penetrate tissue, for example for use in the treatment of a tumour,
it may be advantageous to use a small molecular weight polymer, for
example with a molecular weight of around 5000 Da. For applications
where the product remains in the circulation, it may be
advantageous to use a higher molecular weight polymer, for example
having a molecular weight in the range from 25000 Da to 40000
Da.
[0054] Particularly preferred polymers include a polyalkylene
polymer, such as a poly(ethyleneglycol) or, especially, a
methoxypoly(ethyleneglycol) or a derivative thereof, and especially
with a molecular weight in the range from about 25000 Da to about
40000 Da.
[0055] Each polymer molecule attached to the modified antibody
fragment may be covalently linked to the sulphur atom of a cysteine
residue located in the fragment. The covalent linkage will
generally be a disulphide bond or, in particular, a sulphur-carbon
bond.
[0056] Where desired, the antibody fragment may have one or more
effector or reporter molecules attached to it. The effector or
reporter molecules may be attached to the antibody fragment through
any available amino acid side-chain or terminal amino acid
functional group located in the fragment, for example any free
amino, imino, hydroxyl or carboxyl group.
[0057] An activated polymer may be used as the starting material in
the preparation of polymer-modified antibody fragments as described
above. The activated polymer may be any polymer containing a thiol
reactive group such as an .alpha.-halocarboxylic acid or ester,
e.g. iodoacetamide, an imide, e.g. maleimide, a vinyl sulphone or a
disulphide. Such starting materials may be obtained commercially
(for example from Nektar, formerly Shearwater Polymers Inc.,
Huntsville, Ala., USA) or may be prepared from commercially
available starting materials using conventional chemical
procedures. Particular PEG molecules include 20K methoxy-PEG-amine
(obtainable from Nektar, formerly Shearwater; Rapp Polymere; and
SunBio) and M-PEG-SPA (obtainable from Nektar, formerly
Shearwater).
[0058] Standard chemical or recombinant DNA procedures in which the
antibody fragment is linked either directly or via a coupling agent
to the effector or reporter molecule either before or after
reaction with the activated polymer as appropriate may be used.
Particular chemical procedures include, for example, those
described in WO 93/06231, WO 92/22583, WO 90/09195, WO 89/01476, WO
99/15549 and WO 03/031581. Alternatively, where the effector or
reporter molecule is a protein or polypeptide the linkage may be
achieved using recombinant DNA procedures, for example as described
in WO 86/01533 and EP 0392745.
[0059] Most preferably antibodies are attached to
poly(ethyleneglycol) (PEG) moieties. Preferably, a modified Fab
fragment is PEGylated, i. e. has PEG (poly(ethyleneglycol))
covalently attached thereto, e.g. according to the method disclosed
in EP 0948544 [see also "Poly(ethyleneglycol) Chemistry,
Biotechnical and Biomedical Applications", 1992, J. Milton Harris
(ed), Plenum Press, New York, "Poly(ethyleneglycol) Chemistry and
Biological Applications", 1997, J. Milton Harris and S. Zalipsky
(eds), American Chemical Society, Washington D.C. and
"Bioconjugation Protein Coupling Techniques for the Biomedical
Sciences", 1998, M. Aslam and A. Dent, Grove Publishers, New York;
Chapman, A. 2002, Advanced Drug Delivery Reviews 2002, 54:531-545].
In one embodiment, a PEG modified Fab fragment has a maleimide
group covalently linked to a single thiol group in a modified hinge
region. A lysine residue may be covalently linked to the maleimide
group. To each of the amine groups on the lysine residue may be
attached a methoxypoly(ethyleneglycol) polymer having a molecular
weight of approximately 20,000 Da. The total molecular weight of
the entire effector molecule may therefore be approximately 40,000
Da.
[0060] Antibodies isolated directly or indirectly according to the
methods of the invention are useful in diagnosis. Thus, provided is
a method of screening for and/or diagnosis or prognosis of a
disease in a subject, and/or monitoring the effectiveness of
therapy for said disease, which comprises the step of detecting
and/or quantifying in a biological sample obtained from said
subject, the expression of an antigen recognised by an antibody
isolated according to the methods of the invention. In particular,
the step of detecting comprises contacting the sample with the
antibody and detecting whether binding has occurred between the
antibody and the antigen in the sample.
[0061] Repertoires of such antibodies, or fragments thereof, are
also useful in bacteriophage libraries.
[0062] Antibodies prepared directly or indirectly as a result of
using the methods of the invention also find use in the treatment
and/or prophylaxis of a disease or condition depending on the
antigen of interest selected. For example but without limitation,
an antigen restricted to expression on the surface of tumour cells
may be selected for immunisation of one or more animals.
Accordingly, in a further aspect, the invention includes an
antibody or fragment thereof isolated according to any one of the
methods described, above. In one embodiment, such antibodies are
humanised (see, for example, Adair et al., 1992, Immunol Rev.
130:5-40 and WO 91/09967). Therefore, according to the invention
provided is the use of an antibody prepared using the methods of
the invention for the manufacture of a medicament for the treatment
and/or prophylaxis of a condition or disease associated with
aberrant expression or aberrant activity of the antigen of
interest, i.e. recognised by an antibody identified using the
methods of the invention. Also provided is a method for the
treatment and/or prophylaxis of a disease associated with aberrant
expression or aberrant activity of an antigen recognised by an
antibody isolated according to the methods of the invention
comprising administering a therapeutically effective amount of a
composition comprising said antibody. Such a disease or condition
includes cancers, autoimmune disorders or inflammatory disorders.
For such use the antibodies will generally be administered in the
form of a pharmaceutical composition.
[0063] Thus, according to the invention there is provided a
pharmaceutical composition comprising an antibody that recognises
an antigen of interest and a pharmaceutically acceptable diluent,
excipient and/or carrier.
[0064] When a reference is made herein to a method of treating or
preventing a disease or condition using a particular antibody or
combination of antibodies, it is to be understood that such a
reference is intended to include the use of that antibody or
combination of antibodies in the preparation of a medicament for
the treatment and/or prophylaxis of the disease or condition.
[0065] The composition will usually be supplied as part of a
sterile, pharmaceutical composition that will normally include a
pharmaceutically acceptable carrier. This composition may be in any
suitable form (depending upon the desired method of administering
it to a patient).
[0066] Antibodies of the invention may be administered to a subject
by any of the routes conventionally used for drug administration,
for example they may be administered parenterally, orally or by
inhalation. The most suitable route for administration in any given
case will depend on the particular antibody, the disease or
condition involved, the subject, and the nature and severity of the
disease or condition and the physical condition of the subject.
[0067] The antibodies may be administered in combination, e.g.
simultaneously, sequentially or separately, with one or more other
therapeutically active compounds, e.g. anti-tumour or
anti-inflammatory compounds.
[0068] Pharmaceutical compositions may be conveniently presented in
unit dose forms containing a predetermined amount of an antibody of
the invention per dose. Such a unit may contain for example but
without limitation, 750 mg/kg to 0.1 mg/kg depending on the
condition being treated, the route of administration and the age,
weight and condition of the subject.
[0069] Pharmaceutically acceptable carriers for use in the
invention may take a wide variety of forms depending, e.g. on the
route of administration.
[0070] Compositions for oral administration may be liquid or solid.
Oral liquid preparations may be in the form of, for example,
aqueous or oily suspensions, solutions, emulsions, syrups or
elixirs, or may be presented as a dry product for reconstitution
with water or other suitable vehicle before use. Oral liquid
preparations may contain suspending agents as known in the art.
[0071] In the case of oral solid preparations such as powders,
capsules and tablets, carriers such as starches, sugars,
microcrystalline cellulose, diluents, granulating agents,
lubricants, binders, disintegrating agents, and the like may be
included. Because of their ease of administration, tablets and
capsules represent the most advantageous oral dosage unit form in
which case solid pharmaceutical carriers are generally employed. In
addition to the common dosage forms set out above, antibodies of
the invention may also be administered by controlled release means
and/or delivery devices. Tablets and capsules may comprise
conventional carriers or excipients such as binding agents for
example, syrup, acacia, gelatin, sorbitol, tragacanth, or
polyvinylpyrrolidone; fillers, for example lactose, sugar,
maize-starch, calcium phosphate, sorbitol or glycine; tableting
lubricants, for example magnesium stearate, talc, polyethylene
glycol or silica; disintegrants, for example potato starch; or
acceptable wetting agents such as sodium lauryl sulphate. The
tablets may be coated by standard aqueous or non-aqueous techniques
according to methods well known in normal pharmaceutical
practice.
[0072] Pharmaceutical compositions of the present invention
suitable for oral administration may be presented as discrete units
such as capsules, cachets or tablets, each containing a
predetermined amount of the antibody, as a powder or granules, or
as a solution or a suspension in an aqueous liquid, a non-aqueous
liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion.
Such compositions may be prepared by any of the methods of pharmacy
but all methods include the step of bringing into association the
antibody with the carrier, which constitutes one or more necessary
ingredients. In general, the compositions are prepared by uniformly
and intimately admixing the antibody with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product into the desired presentation. For example, a tablet may be
prepared by compression or moulding, optionally with one or more
accessory ingredients.
[0073] Pharmaceutical compositions suitable for parenteral
administration may be prepared as solutions or suspensions of the
antibodies of the invention in water suitably mixed with a
surfactant such as hydroxypropylcellulose. Dispersions can also be
prepared in glycerol, liquid polyethylene glycols, and mixtures
thereof in oils. Under ordinary conditions of storage and use,
these preparations contain a preservative to prevent the growth of
microorganisms.
[0074] The pharmaceutical forms suitable for injectable use include
aqueous or non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the composition isotonic with the blood of the intended
recipient, and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents. Extemporaneous
injection solutions, dispersions and suspensions may be prepared
from sterile powders, granules and tablets.
[0075] Pharmaceutical compositions can be administered with medical
devices known in the art. For example, in a preferred embodiment, a
pharmaceutical composition of the invention can be administered
with a needleless hypodermic injection device, such as the devices
disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335;
5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of
well-known implants and modules useful in the present invention
include: U.S. Pat. No. 4,487,603, which discloses an implantable
micro-infusion pump for dispensing medication at a controlled rate;
U.S. Pat. No. 4,486,194, which discloses a therapeutic device for
administering medicaments through the skin; U.S. Pat. No.
4,447,233, which discloses a medication infusion pump for
delivering medication at a precise infusion rate; U.S. Pat. No.
4,447,224, which discloses a variable flow implantable infusion
apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196,
which discloses an osmotic drug delivery system having
multi-chamber compartments; and U.S. Pat. No. 4,475,196, which
discloses an osmotic drug delivery system. Many other such
implants, delivery systems, and modules are known to those skilled
in the art.
[0076] In certain embodiments, the pharmaceutical compositions of
the invention can be formulated to ensure proper distribution in
vivo. For example, the blood-brain barrier excludes many highly
hydrophilic compounds and it may be preferable to deliver
pharmaceutical compositions in liposomes. Thus, in one embodiment
of the invention, the antibodies of the invention are formulated in
liposomes; in a more preferred embodiment, the liposomes include a
targeting moiety. In a most preferred embodiment, the therapeutic
compounds in the liposomes are delivered by bolus injection to a
site proximal to the problem area, e.g. proximal to a tumour. For
methods of manufacturing liposomes, see, e.g. U.S. Pat. Nos.
4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one
or more moieties which are selectively transported into specific
cells or organs, thus enhancing targeted drug delivery (see, e.g.
Ranade, V V. 1989, J. Clin. Pharmacol. 29:685). Exemplary targeting
moieties include folate or biotin (see, e.g. U.S. Pat. No.
5,416,016.); mannosides (Umezawa et al., 1988, Biochem. Biophys.
Res. Commun. 153:1038); antibodies (Bloeman, P G. et al., 1995,
FEBS Lett. 357:140; M. Owais et al., 1995, Antimicrob. Agents
Chemother. 39:180); surfactant protein A receptor (Briscoe et al.,
1995, Am. J. Physiol. 1233:134), different species of which may
comprise the formulations of the inventions, as well as components
of the invented molecules; p 120 (Schreier et al., 1994, J. Biol.
Chem. 269:9090); see also Keinanen, K. & Laukkanen, M L. 1994,
FEBS Lett. 346:123; Killion, J J. & Fidler, I J. 1994,
Immunomethods 4:273. The compositions maybe presented in unit-dose
or multi-dose containers, for example in sealed ampoules and vials
and to enhance stability, may be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid carrier, for example water for injections, immediately prior
to use. The sterile liquid carrier may be supplied in a separate
vial or ampoule and can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (e.g. glycerol,
propylene glycol and liquid polyethylene glycol), suitable mixtures
thereof, and vegetable oils. Advantageously, agents such as a local
anaesthetic, preservative and buffering agents can be included in
the sterile liquid carrier.
[0077] The dosage to be administered of an antibody will vary
according to the particular antibody, the disease or condition
involved, the subject, and the nature and severity of the disease
and the physical condition of the subject, and the selected route
of administration; the appropriate dosage can be readily determined
by a person skilled in the art. For the treatment and/or
prophylaxis of a disease or condition in humans and animals
pharmaceutical compositions comprising antibodies can be
administered to patients (e.g., human subjects) at therapeutically
or prophylactically effective dosages (e.g. dosages which result in
tumour growth inhibition and/or tumour cell migration inhibition)
using any suitable route of administration, such as injection and
other routes of administration known in the art for antibody-based
clinical products.
[0078] The compositions may contain from 0.1% by weight, preferably
from 10-60%, or more, by weight, of the antibody of the invention,
depending on the method of administration.
[0079] It will be recognized by one of skill in the art that the
optimal quantity and spacing of individual dosages of an antibody
of the invention will be determined by the nature and extent of the
disease or condition being treated, the form, route and site of
administration, and the age and condition of the particular subject
being treated, and that a physician will ultimately determine
appropriate dosages to be used. This dosage may be repeated as
often as appropriate. If side effects develop the amount and/or
frequency of the dosage can be altered or reduced, in accordance
with normal clinical practice.
[0080] FIG. 1 shows neutralisation of human IL-1.beta. signalling
with recombinant antibodies 97 and 100. Neutralization (reduction
in luminescence) of IL-1.beta. by increasing concentrations of
antibody 97 is shown by the closed diamonds, by increasing
concentrations of antibody 100 by closed squares. Controls were
included: the open circle shows background luminescence, the open
triangle shows luciferase production in the presence of 3 pg/ml
IL-1.beta. alone, the open square shows luciferase production in
the presence of 15 pg/ml IL-1.beta. alone, and the open diamond
shows luciferase production in the presence of 30 pg/ml IL-1.beta.
alone.
[0081] Preferred features of each embodiment of the invention are
as for each of the other embodiments mutatis mutandis. All
publications, including but not limited to patents and patent
applications cited in this specification are herein incorporated by
reference as if each individual publication were specifically and
individually indicated to be incorporated by reference herein as
though fully set forth.
[0082] The invention will now be described with reference to the
following experimental section, which is merely illustrative and
should not in any way be construed as limiting the scope of the
present invention.
EXPERIMENTAL
Positive Selection of Antigen-Specific B Cells by Flow
Cytometry
Immunisation
[0083] Five Sprague Dawley Rats were immunised with 10 .mu.g
recombinant human IL-1.beta. (Peprotech #200-01B) in 200 .mu.l,
50:50 complete Freund's adjuvant and Sterile PBS. The animals were
then boosted at week 4 and week 7 with 15 .mu.g recombinant human
IL- 1.beta. in 200 .mu.l, 50:50 incomplete Freund's adjuvant and
Sterile PBS. A test bleed was taken for each of the animals and the
sera was assayed for human IL-1.beta. neutralising activity (see
below). The animals were then boosted a further three times at
approximately 4 weekly intervals with 15 .mu.g recombinant human
IL-1.beta. in 200 .beta.l, 50:50 incomplete Freund's Adjuvant and
Sterile PBS. Two weeks after the last boost, blood and spleens were
harvested.
Preparation of Rat Lymphocytes and Splenocytes (the Populations for
Enrichment)
[0084] Lymphocytes were separated from whole blood by firstly
diluting the samples at approximately 1:10 in sterile PBS. Then 8
ml of diluted blood was layered onto 5 ml of Mammalian
Lympholyte.RTM. (Cedarlane Laboratories Ltd. #CL5120) in 15 ml
Falcon tubes. The samples were then centrifuge at 800 g for 20 min
giving a well-defined, white lymphocyte layer at the interface
between the liquids. This lymphocyte containing band and the upper
`blood plasma` containing layer were transferred into a new 15 ml
falcon tube and centrifuged at 800 g for 10 min to pellet the
lymphocytes. The supernatant was collected and stored at 4.degree.
C. for future use, whilst the cells were washed 3 times in immune
medium (RPMI, 10% FCS, 2% HEPES, 1% Glutamine, and 1.times.
Pen/Strep). Following the final wash the cells were frozen in 90%
FCS and 10%DMSO and stored in liquid nitrogen until use.
[0085] Individual rat spleens were transferred into warmed immune
media and cut into small pieces. The pieces were pushed through a
mesh strainer to disperse the splenocytes. These were pelleted by
centrifugation, the supernatant was removed and the cells washed
thoroughly in immune media. The cell pellets were frozen in 90% FCS
and 10% DMSO aliquots and stored in liquid nitrogen until use.
Protein G Purification of Anti-Human IL-1.beta. IgG from Rat Serum
and Plasma
[0086] A pool of a total of 44 ml of plasma and serum from three of
the immunised rats was used to prepare an IgG fraction for
labelling with Alexa Fluor 488 dye. The serum/plasma pool volume
was made up to 100 ml with PBS and passed through a protein G
column (18 ml) with a minimum contact time with the Protein G beads
of 20 min/ml of the pool. Following loading the column was washed
with 5-10 column volumes of PBS. IgG was eluted in 0.1M
glycine-HCl, pH 2.7 and neutralised using 2M Tris-HCl, pH 8.5
immediately. The IgG pool was concentrated and diafiltered into PBS
using an Amicon stirred cell (Millipore, Cat. No. YM1013632).
Purification and Labelling of Polyclonal Rat Anti-Human
IL-1.beta.
[0087] A total of 30 .mu.l of 10 mM Alexa Fluor 488 O-succinimide
ester (Molecular Probes Inc. Product No. 20000) in dry DMSO (Perbio
Science UK Ltd) was added dropwise approximately 1 ml of IgG (4.5
mg total) with vortexing. The reaction was allowed to proceed in
the dark at 37.degree. C. for 3 hr. The reaction mixture was
separated by application to a PD10 column (Amersham Biosciences) in
PBS (the PD10 column was prepared by blocking with 20% PEG, 20000
MW followed by equilibration in PBS).
Cell Labelling
[0088] An aliquot of splenocytes from the human IL-1.beta.
immunised rats was recovered from liquid nitrogen storage,
transferred into 10 ml of warmed immune media, centrifuged and
washed in immune media before being transferred into a T175 tissue
culture flask in 60 ml immune media. The splenocytes were cultured
overnight (16 hr) at 37.degree. C. to deplete all adherent cell
types from the cell mix. Cells that remained in suspension were
counted and washed thoroughly in cold PBS before use.
[0089] Cell labelling was carried out in a 15 ml Falcon-tube that
had been blocked with blocking buffer (5% FCS, 0.1 mM EDTA in PBS)
on ice at 4.degree. C. for 30 min. A total of approximately
3.times.10.sup.7 cells was incubated with 60 .mu.g/ml ChromPure.TM.
rat IgG from normal rat sera (Jackson InmunoResearch #012-000-003)
and 17 ng/ml human IL-1.beta. (Peprotech #200-01B) in blocking
buffer for 1 hr on ice. Following this incubation the cells were
washed 3 times with 15 ml ice-cold blocking buffer. After the final
wash cells were resuspended in 500 .mu.l blocking buffer containing
2 .mu.g/ml purified polyclonal rat anti human IL-1.mu. conjugated
with Alexa Fluor 488 and 100 .mu.l PE-conjugated mouse anti-rat
CD45RA (Serotech #MCA340PE), and incubated in the dark at 4.degree.
C. for 1 hr. The cells were then washed 3 times with 15 ml ice-cold
blocking buffer. After the final wash the cells were resuspended to
give a final concentration of 1.times.10.sup.7 cells per ml in PBS
containing 0.1 mM EDTA. The sample was passed through a cell filter
(50 .mu.m Syringe Filcons BD Bioscience #340603) to remove any
debris or cell aggregates that may cause an obstruction during the
flow cytometry.
FACS Analysis
[0090] FACS analysis and sorting was performed using a MoFlo.RTM.
Cytomation and the Cytomation Summit software. A specific
population of live cells stained with PE conjugated to the anti-rat
CD45RA detected in the FL2 channel, which was also positively
stained with FITC labelled anti-human IL-1.beta. in the FL1 channel
was identified. This double-labelled population represented a
population enriched in B cells displaying surface IgG specific for
human IL-1.beta..
Plating and Culture
[0091] The enriched population of cells was seeded at 5 cells per
well across 12.times.96-well tissue culture treated plates already
containing a B cell stimulating culture mix. This B cell
stimulating culture mix consisted of 50,000 irradiated ELA.B5 cells
and 4% rabbit TSN (rabbit T cells stimulated culture supernatant)
in a total volume of 200 .mu.l per well. The cells were then
cultured for 7 days before the culture supernatant was assayed for
human IL-1.beta. specific antibody secretion.
Screening--Primary ELISA Screen
[0092] An ELISA screen of the culture supernatant was used to
identify the wells that contained B cells secreting anti-human
IL-1.beta. specific antibodies. The assays were carried out in
12.times.96-well Nunc Maxisorp plates (Fisher #DIS-971-010P) coated
overnight at 4.degree. C. with 50 .mu.l/well of 1 .beta.g/ml
polyclonal goat anti-human IL-1.beta.in coating buffer (50 mM
Sodium Bicarbonate, pH 9.6). The coating buffer was removed and 50
.mu.l of 500 ng/ml hIL-1.beta. made up in PEG blocking buffer (1%
PEG 20000 MW, 0.1% Tween in PBS) was added to each of the wells and
the plates were incubated for 1 hr at room temperature. Following
this incubation the wells were washed 3 times with wash buffer (PBS
containing 0.1% Tween 20). The plates were then blocked with 100
.mu.l PEG blocking buffer for 1 hr at room temperature before being
washed 3 more times in wash buffer. The 40 .mu.l of PEG blocking
buffer was added to each of the wells before adding 10 .mu.l of
culture supernatant from the B cell culture plates. The assay was
then allowed to incubate for 1 hr at room temperature. The wells
were then washed 3 times with wash buffer before adding 50 .mu.l
peroxidase conjugated goat F(ab').sub.2 anti-rat IgG Fc.gamma.
specific antibody (Jackson ImmunoResearch #112-036-071) diluted
1:3000 in PEG blocking buffer. The plates were once again incubated
for 1 hr at room temperature before being washed 3 times with wash
buffer. Finally, 100 .mu.l TMB (Roche #784974) substrate was added
to each of the wells and the colourless to blue colour change
revealed positive wells. The plates were then read on the Multiskan
(Labsystems) plate reader, using the Ascent software at 630/490 nm,
for data analysis. In particular, two positive antibodies were
selected for neutralising ability (see below).
Neutralisation Assay--Biacore Analysis of Antibody Blocking of
IL-1.beta./Receptor Docking
[0093] BIA (Biomolecular Interaction Analysis) was performed using
a BIAcore 2000 (BIAcore AB). Immobilised anti-rat IgG, Fc fragment
specific (Jackson ImmunoResearch) captured IgG from a 40 .mu.l
injection of culture supernatant at a flow rate of 10 .mu.l/min in
HBS-EP buffer. For the kinetic analysis human IL1.beta. was
titrated over the captured antibody at various concentrations.
Results are shown in Table 1. The sensorgrams for IL-1.beta.
binding were double referenced by using a blocked flow cell and a
buffer blank. Kinetic parameters were calculated using
BIAevaluation 2.1, 3.0 or 3.1 software. TABLE-US-00001 TABLE 1
Sample Ka e.sup.5 Kd e.sup.-5 KD pM 97 3.9 3.27 83.8 100 3.61 3.05
84.5
Specific Antibody Gene Cloning
[0094] B cells from positive wells identified following the primary
ELISA screen which were specific for IL-1.beta. that also showed
potential neutralising activity on the Biacore screen were selected
for cloning. For this step the entire contents of the well was
harvested and washed in PBS before being pelleted and resuspended
into 15 .mu.l of PBS. The cell sample was then split in 3 .mu.l
aliquots and transferred to 0.5 ml PCR tubes. cDNA was prepared by
reverse transcription using a Superscript.TM. III Reverse
Transcriptase kit (Invitrogen cat. #18080-044) in a 20 .mu.l total
volume. Primary PCR fragments were prepared by adding 2 .mu.l of
the reverse transcription reaction and the appropriate primary
primers in conjunction with a TaqPlus Precision PCR system
(Stratagene cat. #600211) in a total volume of 50 .mu.l as
described below. A secondary PCR reaction was performed with 2
.mu.l of the primary PCR product, the appropriate secondary primers
in conjunction with Precision PCR buffer, dNTPs and TaqPlus
Precision in a total volume of 50 .mu.l as described below.
[0095] Thermal Cycler Programs: TABLE-US-00002 Reverse
Transcription 1. 50.degree. C. 60 minutes 2. 70.degree. C. 15
minutes Primary PCR 1. 94.degree. C. 3 minutes 2. 94.degree. C. 30
seconds 3. 50.degree. C. 30 seconds 4. 72.degree. C. 1 minute 5. go
to step 2 40 cycles total 6. 72.degree. C. 5 minutes 7. 4.degree.
C. hold Secondary Nested PCR 1. 94.degree. C. 3 minutes 2.
94.degree. C. 30 seconds 3. 55.degree. C. 30 seconds 4. 72.degree.
C. 1 minute 5. go to step 2 40 cycles total 6. 72.degree. C. 5
minutes 7. 4.degree. C. hold
[0096] The PCR fragments were purified using Qiagen's Qiaquick 8
PCR purification kit (catalogue No. 28144) and eluted in 80 .mu.l
elution buffer. The heavy chain variable fragments were digested
with XhoI and HindIII and ligated into the corresponding sites of
the expression vector phFabHC (mammalian expression construct
containing human .gamma.1 CH1). The kappa light chain fragments
were digested with BsiWI and HindIII and ligated into the
corresponding sites of expression vector pMR10-HS (mammalian
expression construct containing human C.kappa.). This results in
the formation of heavy and light chain rat-human chimeric antibody
genes. 4.times.VH and 4.times.VL clones (plasmid DNA from
individual transformed colonies) from each cell pellet aliquot were
sequenced. Consensus sequences were identified and pairs of
relevant clones were used for transient expression in CHO
cells.
Expression in Mammalian Cells
[0097] Monolayers of CHO cells in 6-well plates (1.2.times.10.sup.6
cells/well) were transfected with 2 .mu.g each of heavy and light
chain plasmid DNA using Invitrogen's Lipofectamine 2000 (catalogue
No. 11668-019) transfection reagent according to the manufacturer's
instructions. The cells were incubated for 5 days at 37.degree. C.,
then the culture supernatants were harvested and assayed for the
presence of Fab, their ability to neutralize human IL-1 and their
binding affinity for human IL-1.beta. (Biacore analysis, as
described above).
Anti-IL-1.beta. Reporter Gene Bioassay
[0098] A549 cells which had been stably transfected with the
E-selectin promoter linked to the luciferase gene (hereinafter
referred to as A549-ES-Luc cells) were grown in RPMI 1640
(phenol-free) containing 10% FCS, 2 mM Glutamine and 1 mg/ml G418.
These A549-ES-Luc cells express IL-1RI receptors. Cells were plated
out into white opaque 96-well plates (Packard) at 15,000 cells/well
and allowed to adhere overnight at 37.degree. C./5% CO2. Samples
for assay were set up in individual assay tubes, each tube
containing culture supernatant (from transfected CHO cells, above)
or control culture supernatant (untransfected CHO cell growth
medium). Each tube additionally contained recombinant human
IL-1.beta. (Preprotech) at a final concentration of 30 pg/ml. Tubes
were incubated for 30 minutes at room temperature. A549-ES-Luc
growth media was removed and replaced with 100 .mu.l of the room
temperature incubate. Cells were incubated for 4 hours at
37.degree. C. in 5% CO.sub.2. Control wells that did not contain
IL-1.beta. were included to allow the correction for basal
luciferase activity in these cells. Luciferase expression was then
assayed using a luciferase reporter gene assay kit (LucLite from
Packard). FIG. 1 shows the results of the neutralisation of IL-1RI
signalling by the transiently expressed antibodies, numbers 97 and
100. Both antibodies neutralised IL-1RI signalling as shown by the
loss of luciferase production with increasing antibody
concentration.
* * * * *