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Cancer Patent Abstract
The invention relates to the use of a binding member which binds
to Lewis.sup.y and Lewis.sup.b haptens in the treatment of tumours
and leukaemia. The binding member may be an antibody which binds
to Lewis.sup.y and Lewis.sup.b haptens and cancer cells and induces
cells death.
Cancer Patent Claims
The invention claimed is:
1. A method of treatment of a tumor in a patient, comprising administering
to the patient an effective amount of a naked antibody or fragment
thereof, which antibody or fragment thereof binds to both Lewis.sup.y
and Lewis.sup.b haptens and does not bind to H blood group antigen
and comprises a constant region of human antibody class IgM, IgA,
IgG.sub.2 or IgG.sub.3, and which antibody comprises the CDRs of
the antibody produced by the cell line deposited as ECACC Accession
No. 01050118.
2. The method of claim 1, wherein the antibody is produced by the
cell line deposited as ECACC Accession No. 01050118.
3. The method of claim 1, wherein the antibody or fragment thereof
binds to both Lewis.sup.y and Lewis.sup.b haptens in extended form,
wherein the extended forms are: ##STR00002##
4. A method of treating a patient comprising administering to the
patient an effective amount of a naked antibody or fragment thereof,
which antibody or fragment thereof binds to both Lewis.sup.y and
Lewis.sup.b haptens and does not bind to H blood group antigen and
comprises a constant region of human antibody class IgM, IgA, IgG.sub.2
or IgG.sub.3, and which antibody comprises CDRs of the antibody
produced by the cell line deposited as ECACC Accession No. 01050118
wherein the patient has leukemia.
5. The method of claim 1, wherein the cancer is one or more of
colorectal, breast, ovarian, gastric, lung, liver, skin, and myeloid
cancer.
6. The method of claim 1, wherein the patient is a mammal.
7. The method of claim 6, wherein the patient is a human.
Cancer Patent Description
This application is a 371 National Phase of International Application
Serial No. PCT/GB02/02182 filed May 10, 2002 which claims the benefit
of priority to United Kingdom Patent Application Serial No. 0111628,4
filed May 10, 2002 all of which are incorporated herein by reference
in their entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the use of binding members which
bind to both Lewis.sup.y and Lewis.sup.b haptens in the treatment
of tumours and leukaemia.
2. Description of the Related Art
The Lewis antigens, which include Lewis y, b, x and a antigens,
are blood group antigens. The Lewis.sup.y hapten is a difucosylated
tetrasaccharide (Fuc 1-2Gal.beta. 1-4(Fuc.alpha. 1-3)GIcNAc found
on type 2 blood group oligosaccharides. This antigen is a positional
isomer of the Lewis.sup.b hapten (Fuc1-2Gal.beta.1-3(Fuc.alpha.1-4)GINAc
and a fucosylated derivative of the Lewis.sup.x hapten. The Lewis.sup.y
hapten is a cell surface antigen epitope which is expressed by colorectal
tumours (Abe et al., Cancer Research, 46, 2639 (1986); Kim et al.,
Cancer Research, 46, 5985 (1986)).
The mouse monoclonal antibody C14 was raised to the C14gp200 antigen.
The mouse monoclonal antibody C14 recognises Lewis.sup.y hapten
(Brown et al, Biosci. Rep. 3, 163 (1983); Brown et al., Int. J.
Cancer, 33, 727) and binds to 78% of colorectal cancers (Durrant
et al., J. Natl. Cancer Inst., 81, 688 (1989)).
Other antibodies which bind to the Lewis.sup.y hapten are known.
For example, EP-B-0285059 discloses an antibody, BR-55, which reacts
with both Lewis.sup.y and B-7-2. B-7-2 has also been shown to be
associated with tumour cells (EP-B-0285059). EP-B-0285059 states
that the advantage of recognising two cancer-associated epitopes
is that it increases the chances of recognising more tumour cells
relative to normal cells. However, BR-55 relies on effector cells
in order to be able to kill cells.
In addition, US55869045 discloses an antibody, BR-96, which binds
to both Lewis.sup.y and Lewis.sup.x haptens. Although US5869045
teaches that antibodies which kill cells by themselves are rare,
BR-96, has been shown to have the ability to kill cancer cells in
unmodified form (US5869045). Since no other Lewis.sup.y antibody
has been reported to cause direct cytotoxicity, the activity of
BR-96 can be assumed to be related to its recognition of the Lewis.sup.x
hapten.
Antibodies which bind to both Lewis.sup.y and Lewis.sup.b antigens
are known. Studies have demonstrated that C14 monoclonal antibody
recognises and binds to both Lewis.sup.y and Lewis.sup.b (extended
and non-extended forms) antigens (Durrant et al., Hybridoma, 12,
647-660 (1993)). A C14 monoclonal antibody specific for both Lewis.sup.y
and Lewis.sup.b antigens was raised against primary colorectal tumour
cells using standard fusion protocols. The C14 antibody recognised
a range of solid tumours but as it was an IgM, it was not very useful
in reproducibly screening large numbers of serum samples. One of
the immunological characteristics of carbohydrate antigens is that
they usually elicit a T cell independent response, resulting in
the production of an IgM antibody.
Subsequently, an anti-idiotypic approach in mice was used to produce
an IgG variant of the C14 (IgM) monoclonal antibody. Rats were immunised
with C14 monoclonal antibody and rat anti-C14 monoclonal antibody
was purified. Immunisation of mice with the rat anti-C14 antiserum
and the C14gp200 antigen and subsequent fusion of the immune splenocytes
with a mouse myeloma produced five IgG (two IgG3s and three IgG
1s) monoclonal antibodies recognising the Lewis.sup.y and Lewis.sup.b
antigens (Durrant et al., Hybridoma, 12, 647-660 (1993)). Each of
the five IgGs (referred to as the "692" monoclonal antibodies)
demonstrated the same specificity as C14 (Durrant et al., Hybridoma,
12, 647-660 (1993)). These antibodies were shown by thin layer chromatography
and ELISA to bind to extended and non-extended Lewis.sup.y and Lewis.sup.b
haptens but not to Lewis.sup.x or H blood group hapten. The antibodies
bound to breast, lung, colorectal, gastric, and ovarian tumours
and myeloid leukaemia. Recognition of normal tissue was minimal
and restricted to weak staining of the upper gastrointestinal tract
basement membrane, mucin staining of stomach and fallopian tubes
and weak staining of liver capillaries.
BRIEF SUMMARY OF THE INVENTION
The present inventors have now, surprisingly, found that antibodies
which bind to both Lewis.sup.y and Lewis.sup.b haptens induce cell
death.
According to a first aspect, the present invention provides the
use of a naked binding member which binds to both Lewis.sup.y and
Lewis.sup.b haptens in the preparation of an agent for treating
cancer.
The present invention also provides a pharmaceutical composition
for the treatment of cancer, the composition comprising a naked
binding member that binds to both Lewis.sup.y and Lewis.sup.b haptens.
The present invention further provides a method of treatment of
a patient such as a mammal, such as a method of treatment of cancer
in a patient (preferably human) which comprises administering to
said patient an effective amount of a naked binding member which
binds to both Lewis.sup.y and Lewis.sup.b haptens.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 Thin layer chromatographs showing immunostaining of extended
Lewis.sup.b, extended Lewis.sup.y, H type 1 chain, H type 2 chain,
Lewis.sup.x, Lewis.sup.b, and Lewis.sup.y.
FIG. 2 ELISA analysis of SC101 binding to haptens.
FIG. 3 A graph demonstrating binding of monoclonal antibodies to
freshly disaggregated colorectal tumour cells.
FIG. 4 A graph demonstrating binding of monoclonal antibodies to
freshly disaggregated gastric tumour cells.
FIG. 5 A graph demonstrating binding of monoclonal antibodies to
freshly disaggregated ovarian tumour cells.
FIG. 6 A graph demonstrating binding of SC101/29 to a panel of
acute myeloid leukaemic cell lines.
FIG. 7 Scatter diagrams demonstrating size and granularity of colorectal
tumour cell line, C170, exposed to SC101/29 or control 791T/36 antibody.
FIG. 8 A histogram demonstrating the effect of SC101/29, or control
791T/36 antibody, on C170 tumour cells.
FIG. 9 Graphs demonstrating in vitro inhibition of cell growth.
FIG. 10 A graph demonstrating the effect of SC101/29 antibody on
cell growth.
FIG. 11 A graph demonstrating the effect of SC101/29 and cisplatin
on cells.
FIG. 12 A graph demonstrating the effect of cisplatin and SC101/29
on cell viability.
FIG. 13 A graph demonstrating the effect of 5 Fluorouracil and
SC101/29 antibody on cells.
FIG. 14 A graph demonstrating the effect of Tamoxifen and SC101/29
on C170 colorectal cells.
FIG. 15 A graph demonstrating the effects of 5-FU, Cisplatin, Doxorubicin,
Irinotecan, Mitomycin C, Oxaliplatin, Raltitrexed and Tamoxifen
on C170 cells either alone or in combination with SC101/29 antibody,
or with SC101/29 antibody alone.
FIG. 16 A graph demonstrating the effect of Tamoxifen and SC101/29
on MDA-MB-468 cells exposed to SC101/29 or control antibodies alone
or in combination with Tamoxifen.
FIG. 17 A graph demonstrating the effects of Tamoxifen and Doxorubicin
on MDA-MB-468 breast cells either alone or in combination with SC101/29
antibody or exposed to SC101/29 antibody alone.
FIG. 18 A graph demonstrating the effect of SC101, 5FU/leucovorin
and a combination of SC101 and 5FU/leucovorin on the growth of C170
xenografts growing in nude mice.
FIG. 19 A graph demonstrating the effect of SC101, 5FU/leucovorin
or the combination of SC101 and 5FU/leucovorin on the weights of
mice.
FIG. 20 A histogram demonstrating the effect of SC101, 5FU/leucovorin
or the combination of SC101 and 5FU on the final tumour weights
of C170 xenografts grown in nude mice.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, a "binding member" is a member of a pair
of molecules which have binding specificity for one another. The
binding member is, therefore, a specific binding member. The members
of a binding pair may be naturally derived or wholly or partially
synthetically produced. One member of the pair of molecules has
an area on its surface, which may be a protrusion or a cavity, which
specifically binds to and is therefore complementary to a particular
spatial and polar organisation of the other member of the pair of
molecules. Thus, the members of the pair have the property of binding
specifically to each other. Examples of types of binding pairs are
antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand,
enzyme-substrate. The present invention is concerned with antigen-antibody
type reactions, although a binding member of the invention may be
any moiety which can bind to both Lewis.sup.y and Lewis.sup.b haptens.
An "antibody" is an immunoglobulin, whether natural or
partly or wholly synthetically produced. The term also covers any
polypeptide, protein or peptide having a binding domain which is,
or is homologous to, an antibody binding domain. These can be derived
from natural sources, or they may be partly or wholly synthetically
produced. Examples of antibodies are the immunoglobulin isotypes
and their isotypic subclasses; fragments which comprise an antigen
binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies.
As used herein, "naked" means that the binding member
of the present invention is not bound to, or associated with, any
agent having anti-tumour properties.
The term "hapten" includes epitopes and antigens. Haptens
may be attached to a large carrier molecule such as a cell e.g a
tumour cell.
The binding member of the first aspect of the invention may be
an antibody such as a monoclonal or polyclonal antibody, or a fragment
thereof. The constant region of the antibody may be of any class
including, but not limited to, human classes IgG, IgA, IgM, IgD
and IgE. The antibody may belong to any sub class e.g. IgG1, IgG2,
IgG3 and IgG4. IgG1 is preferred. The antibody may be SC101 (corresponds
to, and used interchangeably with, "692" as described
in Durrant et al., Hybridoma, 12, 647-660 1993) for example, SC101/23,
SC101/29, SC101/33, SC101/42, SC101/43 or C14.
A cell line expressing an antibody which binds to both Lewis.sup.y
and Lewis.sup.b haptens, specifically the cell line SC101/29 has
been deposited with ECACC of CAMR, Salisbury Wilshire, SP4 0JG,
United Kingdom on May 1, 2001 and was assigned Accession no. 01050118.
These cell lines will be maintained at the ECACC during the pendency
of this application or any utility application filed there off of
and during the term of any patent issuing therefrom in accordance
with the rules of the United States Patent and Trademark Office
which allows access to the Patent Office during the pendency of
the application.
Early investigation by the inventors of the characteristics of
SC101 demonstrated that the antibody caused the death of tumour
cell-lines in suspension. The inventors have now found that the
antibody causes the specific onset of apoptosis or programmed cell-death
in colorectal tumour and leukaemia cell-lines and cells derived
from disaggregated tumour tissue. Several groups including Terada
and Nakanuma, Pathol. Int., 46, 764-770 (1996); Terada and Nakanuma,
American J. Pathol., 146, 67-74 (1995); Iwata et al., J. Pathol.,
179, 403-408 (1996); Yamada et al., Anticancer Research, 16, 735-740
(1996) have previously used anti-Lewis.sup.y antibodies to characterise
apoptotic cells; these results suggested that Lewis.sup.y was a
marker of apoptosis and predominantly over-expressed on dying cells.
These findings do not explain why the majority of viable tumour
cells also express this hapten or why a member (e.g an antibody)
which binds to Lewis.sup.y and Lewis.sup.b should induce apoptosis.
Recognition of normal tissue by the SC101 antibody is, surprisingly,
minimal compared to tumour cells thereby making the antibody an
effective anti-cancer agent. Since the Lewis.sup.y and Lewis.sup.b
antigens are expressed on tumour cells and also on normal cells,
this finding was contrary to expectation of the art. The minimal
binding of the SC101 antibody to normal tissues has the advantage
in that a higher dose of the antibody can be used in the treatment
of patients whilst avoiding any risk of toxicity to non-cancerous
cells.
As used herein, reference to "SC101" and "692"
includes sequences which show substantial homology with SC101 and/or
692. Preferably the degree of homology between SC101/692 complementary
determining regions (CDRs) and the CDRs of other antibodies will
be at least 60%, more preferably 70%, further preferably 80%, even
more preferably 90% or most preferably 95%.
The percent identity of two amino acid sequences or of two nucleic
acid sequences is determined by aligning the sequences for optimal
comparison purposes (e.g., gaps can be introduced in the first sequence
for best alignment with the sequence) and comparing the amino acid
residues or nucleotides at corresponding positions. The "best
alignments" is an alignment of two sequences which results
in the highest percent identity. The percent identity is determined
by the number of identical amino acid residues or nucleotides in
the sequences being compared (i.e., % identity=# of identical positions/total
# of positions.times.100).
The determination of percent identity between two sequences can
be accomplished using a mathematical algorithm known to those of
skill in the art. An example of a mathematical algorithm for comparing
two sequences is the algorithm of Karlin and Altschul (1990) Proc.
Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul
(1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST
programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410 have
incorporated such an algorithm. BLAST nucleotide searches can be
performed with the NBLAST program, score=100,wordlength=12 to obtain
nucleotide sequences homologous to nucleic acid molecules of the
invention. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences homologous
to protein molecules of the invention. To obtain gapped alignments
for comparison purposes, Gapped BLAST can be utilised as described
in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively,
PSI-Blast can be used to perform an iterated search which detects
distant relationships between molecules (Id.). When utilising BLAST,
Gapped BLAST, and PSI-Blast programs, the default parameters of
the respective programs (e.g., XBLAST and NBLAST) can be used. See
http://www.ncbi.nlm.nih.gov.
Another example of a mathematical algorithm utilised for the comparison
of sequences is the algorithm of Myers & Miller, CABIOS (1989).
The ALIGN program (version 2.0) which is part of the CGC sequence
alignment software package has incorporated such an algorithm. Other
algorithms for sequence analysis known in the art include ADVANCE
and ADAM as described in Torellis & Robotti (1994) Comput. Appl.
Biosci., 10:3-5; and FASTA described in Pearson & Lipman (1988)
Proc. Natl. Acad. Sci. 85:2444-8. Within FASTA, ktup is a control
option that sets the sensitivity and speed of the search.
Where high degrees of sequence identity are present there will
be relatively few differences in amino acid sequence. Thus for example
they may be less than 20, less than 10, or even less than 5 differences.
The present inventors have shown that SC101 and fragments and derivatives
thereof can be used as cancer therapeutics to inhibit the growth
or induce apoptosis of tumour cells as exemplified by the inhibition
of growth of tumour cell lines, apoptosis of tumour cell lines and
in vivo inhibition of tumour xenografts in nude mice (see the Examples).
Accordingly the invention further provides the use of naked "fragments"
or "derivatives" of SC101 or other polypeptides of the
"SC101" family which bind to both Lewis.sup.y and Lewis.sup.b
epitopes in the preparation of an agent for treating cancer. A preferred
group of fragments are those which include all or part of the CDR
regions of monoclonal antibody SC101.
The binding member may comprise one or more of the CDRs of the
antibody, or a fragment thereof, produced by the cell line deposited
as ECACC Accession No. 01050118.
The binding member may be the antibody produced by the cell line
deposited as ECACC Accession No. 01050118, or a fragment or derivative
thereof.
A fragment of SC101 or of a polypeptide of the SC101 family generally
means a stretch of amino acid residues of at least 5 to 7 contiguous
amino acids. Often at least about 7 to 9 contiguous amino acids,
typically at least about 9 to 13 contiguous amino acids, more preferably
at least about 20 to 30 or more contiguous amino acids and most
preferably at least about 30 to 40 or more consecutive amino acids.
A "derivative" of SC101 or of a polypeptide of the SC101
family, or of a fragment of SC101 family polypeptide, means a polypeptide
modified by varying the amino acid sequence of the protein, e.g.
by manipulation of the nucleic acid encoding the protein or by altering
the protein itself. Such derivatives of the natural amino acid sequence
may involve insertion, addition, deletion and/or substitution of
one or more amino acids, while providing a peptide capable of inducing
an anti-tumour T-cell response.
Preferably such derivatives involve the insertion, addition, deletion
and/or substitution of 25 or fewer amino acids, more preferably
of 15 or fewer, even more preferably of 10 or fewer, more preferably
still of 4 or fewer and most preferably of 1 or 2 amino acids only.
The present invention further provides products comprising a naked
binding member, which binds to both Lewis.sup.y and Lewis.sup.b
haptens, and an active agent as a combined preparation for simultaneous,
separate or sequential use in the treatment of cancer. Preferably,
the products contain a naked binding member, which binds to both
Lewis.sup.y and Lewis.sup.b haptens, and an active agent as a combined
preparation of simultaneous, separate or sequential use in the treatment
of cancer. Active agents may include chemotherapeutic agents including,
Doxorubicin, taxol, 5-Fluorouracil (5 FU), Leucovorin, Irinotecan,
Mitomycin C, Oxaliplatin, Raltitrexed, Tamoxifen and Cisplatin which
may operate synergistically with the binding member of the present
invention. Other active agents may include suitable doses of pain
relief drugs such as nonsteroidal anti-inflammatory drugs (e.g.
aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such as
morphine, or anti-emetics.
The ability of the binding member to synergise with an active agent
to enhance tumour killing may not be due to immune effector mechanisms
but rather may be a direct consequence of the binding member binding
to cell surface bound glycoproteins.
The binding member of the invention may carry a detectable label.
It is possible to take monoclonal and other antibodies and use
techniques of recombinant DNA technology to produce other antibodies
or chimeric molecules which retain the specificity of the original
antibody. Such techniques may involve introducing DNA encoding the
immunoglobulin variable region, or the complementary determining
regions (CDRs), of an antibody to the constant regions, or constant
regions plus framework regions, of a different immunoglobulin. See,
for instance, EP-A-184187, GB 2188638A or EP-A-239400. A hybridoma
or other cell producing an antibody may be subject to genetic mutation
or other changes, which may or may not alter the binding specificity
of antibodies produced.
As antibodies can be modified in a number of ways, the term "antibody"
should be construed as covering any binding member or substance
having a binding domain with the required specificity. Thus, this
term covers antibody fragments, derivatives, functional equivalents
and homologues of antibodies, including any polypeptide comprising
an immunoglobulin binding domain, whether natural or wholly or partially
synthetic. Chimeric molecules comprising an immunoglobulin binding
domain, or equivalent, fused to another polypeptide are therefore
included. Cloning and expression of chimeric antibodies are described
in EP-A-0120694 and EP-A-0125023.
A further aspect of the invention provides an antibody produced
by the cell line deposited as ECACC Accession No. 01050118.
It has been shown that fragments of a whole antibody can perform
the function of binding antigens. Examples of binding fragments
are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains;
(ii) the Fd fragment consisting of the VH and CH1 domains; (iii)
the Fv fragment consisting of the VL and VH domains of a single
antibody; (iv) the dAb fragment (Ward, E. S. et al., Nature 341:544-546
(1989)) which consists of a VH domain; (v) isolated CDR regions;
(vi) F(ab')2 fragments, a bivalent fragment comprising two linked
Fab fragments (vii) single chain Fv molecules (scFv), wherein a
VH domain and a VL domain are linked by a peptide linker which allows
the two domains to associate to form an antigen binding site (Bird
et al., Science 242:423-426 (1988); Huston et al., PNAS USA 85:5879-5883
(1988)); (viii) bispecific single chain Fv dimers (PCT/US92/09965)
and (ix) "diabodies", multivalent or multispecific fragments
constructed by gene fusion (WO94/13804; P. Hollinger et al., Proc.
Natl. Acad. Sci. USA 90:6444-6448 (1993)).
The term "antibody" includes antibodies which have been
"humanised". Methods for making humanised antibodies are
known in the art. Methods are described, for example, in Winter,
U.S. Pat. No. 5,225,539. A humanised antibody may be a modified
antibody having the hypervariable region of monoclonal antibody
SC101 and the constant region of a human antibody. Thus the binding
member may comprise a human constant region.
The variable region other than the hypervariable region may also
be derived from the variable region of a human antibody. The variable
region of the antibody outside of the hypervariable region may also
be derived from monoclonal antibody SC101. In such case, the entire
variable region is derived from murine monoclonal antibody SC101
and the antibody is said to be chimerised. Methods for making chimerised
antibodies are known in the art. Such methods include, for example,
those described in U.S. patents by Boss (Celltech) and by Cabilly
(Genentech). See U.S. Pat. Nos. 4,816,397 and 4,816,567, respectively.
The binding member of the first aspect of the invention binds to
Lewis.sup.y (Fuc 1-2Gal.beta. 1-4(Fuc.alpha.1-3)GIcNAc) and Lewis.sup.b
(Fuc1-2Gal.beta.1-3(Fuc.alpha.1-4)GlNAc) haptens which may be in
extended or non-extended form. The extended forms of Lewis.sup.y
and Lewis.sup.b are as follows:
##STR00001##
"Treatment" includes any regime that can benefit a human
or non-human animal. The treatment may be in respect of an existing
condition or may be prophylactic (preventative treatment).
"Treatment of cancer" includes treatment of conditions
caused by cancerous growth and includes the treatment of neoplastic
growths or tumours. Tumours may be benign or malignant. Tumours
may include colorectal, breast, ovarian, gastric, lung tumours,
liver, skin, myeloid (e.g. bone marrow) tumours. Treatment may also
be in respect of cancerous tissues or cell lines including, but
not limited to, leukaemic cells.
The binding member may, upon binding to Lewis.sup.y and Lewis.sup.b
haptens present on cancerous cells or tissues, including tumour
and non-tumour cells, induce apoptosis of cells and inhibit the
growth of cells.
Apoptosis is the process by which a cell actively commits suicide.
It is now well recognized that apoptosis is essential in many aspects
of normal development and is required for maintaining tissue homeostasis.
Cell death by suicide, sometimes referred to as programmed cell
death, is needed to destroy cells that represent a threat to the
integrity of the organism. There are two different mechanisms by
which a cell commits suicide by apoptosis. One is triggered by signals
arising from within the cell, the other by external signals (e.g
molecules) which bind to receptors at the cell surface.
Binding members of the present invention may be administered to
a patient in need of treatment via any suitable route, usually by
injection into the bloodstream. The precise dose will depend upon
a number of factors, including the precise nature of the member
(e.g. whole antibody, fragment or diabody), and the nature of the
detectable label attached to the member.
Binding members of the present invention will usually be administered
in the form of a pharmaceutical composition, which may comprise
at least one component in addition to the binding member.
Thus a further aspect provides pharmaceutical compositions according
to the present invention, and for use in accordance with the present
invention. Pharmaceutical compositions may comprise, in addition
to active ingredient, a pharmaceutically acceptable excipient, carrier,
buffer stabiliser or other materials well known to those skilled
in the art. Such materials should be non-toxic and should not interfere
with the efficacy of the active ingredient. The precise nature of
the carrier or other material will depend on the route of administration,
which may be oral, or by injection, e.g. intravenous.
The formulation may be a liquid, for example, a physiologic salt
solution containing non-phosphate buffer at pH 6.8-7.6, or a lyophilised
powder.
The compositions are preferably administered to an individual in
a "therapeutically effective amount", this being sufficient
to show benefit to the individual. The actual amount administered,
and rate and time-course of administration, will depend on the nature
and severity of what is being treated. Prescription of treatment,
e.g. decisions on dosage etc, is within the responsibility of general
practitioners and other medical doctors, and typically takes account
of the disorder to be treated, the condition of the individual patient,
the site of delivery, the method of administration and other factors
known to practitioners. The compositions of the invention are particularly
relevant to the treatment of existing cancer and in the prevention
of the recurrence of cancer after initial treatment or surgery.
Examples of the techniques and protocols mentioned above can be
found in Remington's Pharmaceutical Sciences, 16.sup.th edition,
Oslo, A. (ed), 1980.
The optimal dose can be determined by physicians based on a number
of parameters including, for example, age, sex, weight, severity
of the condition being treated, the active ingredient being administered
and the route of administration. In general, a serum concentration
of polypeptides and antibodies that permits saturation of receptors
is desirable. A concentration in excess of approximately 0.1 nM
is normally sufficient. For example, a dose of 100 mg/m.sup.2 of
antibody provides a serum concentration of approximately 20 nM for
approximately eight days.
As a rough guideline, doses of antibodies may be given weekly in
amounts of 10-300 mg/m.sup.2. Equivalent doses of antibody fragments
should be used at more frequent intervals in order to maintain a
serum level in excess of the concentration that permits saturation
of Lewis.sup.y/b haptens.
Some suitable routes of administration include intravenous, subcutaneous
and intramuscular administration. Intravenous administration is
preferred.
It is envisaged that injections (intravenous) will be the primary
route for therapeutic administration of the compositions although
delivery through a catheter or other surgical tubing is also used.
Liquid formulations may be utilised after reconstitution from powder
formulations.
For intravenous, injection, or injection at the site of affliction,
the active ingredient will be in the form of a parenterally acceptable
aqueous solution which is pyrogen-free and has suitable pH, isotonicity
and stability. Those of relevant skill in the art are well able
to prepare suitable solutions using, for example, isotonic vehicles
such as Sodium Chloride Injection, Ringer's Injection, Lactated
Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants
and/or other additives may be included, as required.
Pharmaceutical compositions for oral administration may be in tablet,
capsule, powder or liquid form. A tablet may comprise a solid carrier
such as gelatin or an adjuvant. Liquid pharmaceutical compositions
generally comprise a liquid carrier such as water, petroleum, animal
or vegetable oils, mineral oil or synthetic oil. Physiological saline
solution, dextrose or other saccharide solution or glycols such
as ethylene glycol, propylene glycol or polyethylene glycol may
be included.
The composition may also be administered via microspheres, liposomes,
other microparticulate delivery systems or sustained release formulations
placed in certain tissues including blood. Suitable examples of
sustained release carriers include semipermeable polymer matrices
in the form of shared articles, e.g. suppositories or microcapsules.
Implantable or microcapsular sustained release matrices include
polylactides (U.S. Pat. No. 3,773,919; EP-A-0058481) copolymers
of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, Biopolymers
22(1): 547-556, 1985), poly (2-hydroxyethyl-methacrylate) or ethylene
vinyl acetate (Langer et al, J. Biomed. Mater. Res. 15:167-277,
1981, and Langer, Chem. Tech. 12:98-105, 1982). Liposomes containing
the polypeptides are prepared by well-known methods: DE 3,218,121A;
Epstein et al, PNAS USA, 82: 3688-3692, 1985; Hwang et al, PNAS
USA, 77: 4030-4034, 1980; EP-A-0052522; E-A-0036676; EP-A-0088046;
EP-A-0143949;EP-A-0142541; JP-A-83-11808; U.S. Pat. Nos. 4,485,045
and 4,544,545. Ordinarily, the liposomes are of the small (about
200-800 Angstroms) unilamellar type in which the lipid content is
greater than about 30 mol. % cholesterol, the selected proportion
being adjusted for the optimal rate of the polypeptide leakage.
The composition may be administered in a localised manner to a
tumour site or other desired site or may be delivered in a manner
in which it targets tumour or other cells.
The dose of the composition will be dependent upon the properties
of the binding member, e.g. its binding activity and in vivo plasma
half-life, the concentration of the polypeptide in the formulation,
the administration route, the site and rate of dosage, the clinical
tolerance of the patient involved, the pathological condition afflicting
the patient and the like, as is well within the skill of the physician.
For example, doses of 300 .mu.g of antibody per patient per administration
are preferred, although dosages may range from about 10 .mu.g to
6 mg per dose. Different dosages are utilised during a series of
sequential inoculations; the practitioner may administer an initial
inoculation and then boost with relatively smaller doses of antibody.
This invention is also directed to optimise immunisation schedules
for enhancing a protective immune response against cancer.
The binding members of the present invention may be generated wholly
or partly by chemical synthesis. The binding members can be readily
prepared according to well-established, standard liquid or, preferably,
solid-phase peptide synthesis methods, general descriptions of which
are broadly available (see, for example, in J. M. Stewart and J.
D. Young, Solid Phase Peptide Synthesis, 2.sup.nd edition, Pierce
Chemical Company, Rockford, Ill. (1984), in M. Bodanzsky and A.
Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, N.Y.
(1984); and Applied Biosystems 430A Users Manual, ABI Inc., Foster
City, Calif.), or they may be prepared in solution, by the liquid
phase method or by any combination of solid-phase, liquid phase
and solution chemistry, e.g. by first completing the respective
peptide portion and then, if desired and appropriate, after removal
of any protecting groups being present, by introduction of the residue
X by reaction of the respective carbonic or sulfonic acid or a reactive
derivative thereof.
Another convenient way of producing a binding member according
to the present invention is to express nucleic acid encoding it,
by use of nucleic acid in an expression system.
Thus the present invention further provides the use of an isolated
nucleic acid encoding a naked binding member which binds to both
Lewis.sup.y and Lewis.sup.b haptens in the preparation of an agent
for treating cancer.
The present invention also provides a pharmaceutical composition
for the treatment of cancer, the composition comprising a naked
binding member which binds to both Lewis.sup.y and Lewis.sup.b haptens.
Nucleic acid includes DNA and RNA. In a preferred aspect, the present
invention provides a nucleic acid which codes for a binding member
of the invention as defined above. The skilled person will be able
to determine substitutions, deletions and/or additions to such nucleic
acids which will still provide a binding member of the present invention.
Nucleic acid sequences encoding a binding member in accordance
with the present invention can be readily prepared by the skilled
person using the information and references contained herein and
techniques known in the art (for example, see Sambrook, Fritsch
and Maniatis, "Molecular Cloning", A Laboratory Manual,
Cold Spring Harbor Laboratory Press, 1989, and Ausubel et al, Short
Protocols in Molecular Biology, John Wiley and Sons, 1992), given
the nucleic acid sequences and clones available. These techniques
include (i) the use of the polymerase chain reaction (PCR) to amplify
samples of such nucleic acid, e.g. from genomic sources, (ii) chemical
synthesis, or (iii) preparing cDNA sequences. DNA encoding antibody
fragments may be generated and used in any suitable way known to
those of skill in the art, including by taking encoding DNA, identifying
suitable restriction enzyme recognition sites either side of the
portion to be expressed, and cutting out said portion from the DNA.
The portion may then be operably linked to a suitable promoter in
a standard commercially available expression system. Another recombinant
approach is to amplify the relevant portion of the DNA with suitable
PCR primers. Modifications to the sequences can be made, e.g. using
site directed mutagenesis, to lead to the expression of modified
peptide or to take account of codon preferences in the host cells
used to express the nucleic acid.
The present invention also provides constructs in the form of plasmids,
vectors, transcription or expression cassettes which comprise at
least one nucleic acid as described above.
The present invention also provides a recombinant host cell which
comprises one or more constructs as above. Expression may conveniently
be achieved by culturing under appropriate conditions recombinant
host cells containing the nucleic acid. Following production by
expression a specific binding member may be isolated and/or purified
using any suitable technique, then used as appropriate.
Binding members encoding nucleic acid molecules and vectors according
to the present invention may be provided isolated and/or purified,
e.g. from their natural environment, in substantially pure or homogeneous
form, or, in the case of nucleic acid, free or substantially free
of nucleic acid or genes origin other than the sequence encoding
a polypeptide with the required function. Nucleic acid according
to the present invention may comprise DNA or RNA and may be wholly
or partially synthetic.
Systems for cloning and expression of a polypeptide in a variety
of different host cells are well known. Suitable host cells include
bacteria, mammalian cells, yeast and baculovirus systems. Mammalian
cell lines available in the art for expression of a heterologous
polypeptide include Chinese hamster ovary cells, HeLa cells, baby
hamster kidney cells, NSO mouse melanoma cells and many others.
A common, preferred bacterial host is E. coli.
The expression of antibodies and antibody fragments in prokaryotic
cells such as E. coli is well established in the art. For a review,
see for example Pluckthun, Bio/Technology 9:545-551 (1991). Expression
in eukaryotic cells in culture is also available to those skilled
in the art as an option for production of a binding member, see
for recent review, for example Reff, Curr. Opinion Biotech. 4:573-576
(1993); Trill et al., Curr. Opinion Biotech. 6:553-560 (1995).
Suitable vectors can be chosen or constructed, containing appropriate
regulatory sequences, including promoter sequences, terminator sequences,
polyadenylation sequences, enhancer sequences, marker genes and
other sequences as appropriate. Vectors may be plasmids, viral e.g.
phage, or phagemid, as appropriate. For further details see, for
example. Sambrook et al., Molecular Cloning: A Laboratory Manual:
2.sup.nd Edition, Cold Spring Harbor Laboratory Press (1989). Many
known techniques and protocols for manipulation of nucleic acid,
for example in preparation of nucleic acid constructs, mutagenesis,
sequencing, introduction of DNA into cells and gene expression,
and analysis of proteins, are described in detail in Ausubel et
al. eds., Short Protocols in Molecular Biology, 2.sup.nd Edition,
John Wiley & Sons (1992).
The nucleic acid may be introduced into a host cell by any suitable
means.
The introduction may employ any available technique. For eukaryotic
cells, suitable techniques may include calcium phosphate transfection,
DEAE-Dextran, electroporation, liposome-mediated transfection and
transduction using retrovirus or other virus, e.g. vaccinia or,
for insect cells, baculovirus. For bacterial cells, suitable techniques
may include calcium chloride transformation, electroporation and
transfection using bacteriophage.
Marker genes such as antibiotic resistance or sensitivity genes
may be used in identifying clones containing nucleic acid of interest,
as is well known in the art.
The introduction may be followed by causing or allowing expression
from the nucleic acid, e.g. by culturing host cells under conditions
for expression of the gene.
The nucleic acid of the invention may be integrated into the genome
(e.g. chromosome) of the host cell. Integration may be promoted
by inclusion of sequences which promote recombination with the genome
in accordance with standard techniques. The nucleic acid may be
on an extra-chromosomal vector within the cell, or otherwise identifiably
heterologous or foreign to the cell.
The present invention further provides a screening method comprising
the step of screening a library of candidate agents for the ability
to inhibit the binding of a naked binding member, as defined according
to the first aspect of the invention, to Lewis.sup.y and Lewis.sup.b
haptens.
The screening method may comprise any of the following steps: 1.
providing a naked binding member with the ability to bind to Lewis.sup.y
and Lewis.sup.b haptens; 2. providing candidate drugs; 3. screening
the candidate drugs by contacting the naked binding member with
one of the candidate drugs and determining the extent to which the
candidate drug inhibits binding of the naked binding member to Lewis.sup.y
and Lewis.sup.b haptens.
The screening method may additionally comprise the step of selecting
an agent which has the ability to inhibit the binding of the naked
binding member to Lewis.sup.y and Lewis.sup.b haptens, and optionally
modifying the agent to optimise the agent for administration as
a medicament.
The present invention further provides the use of an agent identified
by the screening method of the present invention in the manufacture
of a medicament for the treatment of cancer.
Preferred features of each aspect of the invention are as for each
of the other aspects mutatis mutandis.
The invention will now be described further in the following non-limiting
examples. Reference is made to the accompanying drawings in which:
FIG. 1 Thin layer chromatographs showing immunostaining of extended
Lewis.sup.b (lane 1), extended Lewis.sup.y (land 2), H type 1 chain
(lane 3), H type 2 chain (lane 4) Lewis.sup.x (lane 5), Lewis.sup.b
(lane 6) and Lewis.sup.y (lane 7). Plates are stained with either
Orcinol panel A or Mabs SC101/23 panel B, Mab SC101/29 panel C,
Mab SC101/33 panel D, Mab SC101/42 panel E and Mab C14 panel F.
FIG. 2 ELISA analysis of SCIOI binding to haptens. Binding of SC101/23
(O), SC101/29 (.circle-solid.), SC101/33 (.gradient.), SC101/42
(.quadrature.) and SC101/43 (.box-solid.) to a) Lewis.sup.y and
b) Lewis.sup.b haptens as determined by ELISA.
FIG. 3 A graph demonstrating binding of monoclonal antibodies to
freshly disaggregated colorectal tumour cells, as assayed by indirect
immunofluorescence and analysed by flow cytometry. Each point refers
to the mean fluorescence for an individual tumour. NCRC30, NCRC36
and SC104 (provided by Scancell Limited) are included as positive
controls to demonstate integrity of enzyme disaggregation.
FIG. 4 A graph demonstrating binding of monoclonal antibodies to
freshly disaggregated gastric tumour cells, as assayed by indirect
immunofluorescence and analysed by flow cytometry. Each point refers
to the mean fluorescence for an individual tumour. HLA/ABC, HLA-DR-AFB
and w6/32 (Serotech) are included as positive controls to demonstate
integrity of enzyme disaggregation.
FIG. 5 A graph demonstrating binding of monoclonal antibodies to
freshly disaggregated ovarian tumour cells, as assayed by indirect
immunofluorescence and analysed by flow cytometry. Each point refers
to the mean fluorescence for an individual tumour.
FIG. 6 A graph demonstrating binding of SC101/29 to a panel of
acute myeloid leukaemic (AML) cell lines (HL-60, KG1A, U937, TF1
(obtained from ECACC)). Cells were stained by indirect immunofluorescence
and analysed by flow cytometry. Results are expressed as a mean
linear fluorescence for each cell line.
FIG. 7 Scatter diagrams demonstrating size (forward scatter, FSCH)
and granularity (side scatter, SSCH) of colorectal tumour cell line,
C170, exposed to SC101/29 or control 791T/36 antibody. Cells were
analysed for size and granularity by flow cytometric analysis of
forward and side scatter. R1 gate defines viable healthy cells.
R2 gate defines dying cells with reduced size and granularity.
FIG. 8 A histogram demonstrating the effect of SC101/29, or control
791T/36 antibody, on C170 tumour cells. Cells were stained with
FITC labelled Annexin and propidium iodide and then analysed by
dual colour flow cytometry. A-C170 colorectal tumour cells, B-HT29
colorectal tumour cells C-HL60 myeloid leukaemic cells.
FIG. 9 Graphs demonstrating in vitro inhibition of cell growth.
Tumour cell lines a) C170 colorectal tumour cells b) HL-60 myeloid
leukaemic cells were exposed to SC101/29 or control 791T/36 monoclonal
antibody. The number of cells was determined by crystal violet staining
and optical density reading at 550 nm.
FIG. 10 A graph demonstrating the effect of SC101/29 antibody on
cell growth. HL-60 cells were incubated with SC101/29 antibody for
1 hr at room temperature and then the cells were stained with phycoerythrin
conjugated Apo2.7 mouse monoclonal antibody. Stained cells were
enumerated by flow cytometry. HL60 myeloid leukaemic cells undergo
apoptosis as measured by Apo2.7 staining when exposed to SC101/29
monodonal antibody.
FIG. 11 A graph demonstrating the effect of SC101/29 and cisplatin
on cells. Colorectal tumour cells were treated with various dilutions
of SC101/29 or control 791T/36 antibody. The cultures left overnight
in culture medium with or without cisplatin. The cultures were tested
for the presence of fragmented DNA by a CytoDeath ELISA (Roche).
The results were recorded by optical Density measurements at 405
nm.
FIG. 12 A graph demonstrating the effect of cisplatin and SC101/29
on cell viability. Colorectal tumour cells were exposed to cisplatin
and then SC101/29 or control 791T/36 antibody was added. The cells
were left for 4 days when the number of viable cells was determined
by crystal violet staining and optical density reading at 550 nm.
FIG. 13 A graph demonstrating the effect of 5 Fluorouracil and
SC101/29 antibody on cells. C170 cells were exposed to SC101/29
or control 791T/36 antibody and 5 FU. The number of cells was determined
by crystal violet staining and optical density reading at 550 nm.
FIG. 14 A graph demonstrating the effect of Tamoxifen and SC101/29
on C170 colorectal cells. C170 cells were exposed to SC101/29 or
control 791T/36 antibody or Tamoxifen or combinations thereof.
FIG. 15 A graph demonstrating the effects of 5-FU, Cisplatin,Doxorubicin,
Irinotecan, Mitomycin C, Oxaliplatin, Raltitrexed and Tamoxifen
on C170 cells either alone or in combination with SC101/29 antibody,
or with SC101/29 antibody alone.
FIG. 16 A graph demonstrating the effect of Tamoxifen and SC101/29
on MDA-MB-468cells exposed to SC101/29 or control antibodies alone
or in combination with Tamoxifen.
FIG. 17 A graph demonstrating the effects of Tamoxifen and Doxorubicin
on MDA-MB-468 breast cells either alone or in combination with SC101/29
antibody or exposed to SC101/29 antibody alone.
FIG. 18 A graph demonstrating the effect of SC101, 5 FU/leucovorin
and a combination of SC1010 and 5FU/leucovorin on the growth of
C170 xenografts growing in nude mice. Growth of C170 xenografts
was measured at days 12, 16, 19 and 23 by measurement of cross-sectional
area (mm.sup.2) when animals were treated with either SC101 ip(0.2
mg) (O), control antibody ip(0.2 mg) and 5FU/leucovorin (12.5 mg/Kgiv)
(.gradient.) or SC101 ip(0.2 mg) and 5FU/leucovorin (12.5 mg/Kg
iv, .gradient.) on days 1, 3, 5, 7, 21, 22.
FIG. 19 A graph demonstrating the effect of SC101, 5FU/leucovorin
or the combination of SC101 and 5FU/leucovorin on the weights of
mice. Animals were weighed on days 12, 16, 19 and 23 following treatment
with SC101 ip(0.2 mg) (O), control antibody ip(0.2 mg) and 5FU/leucovorin
(12.5 mg/Kgiv) (.gradient.) or SC101 ip(0.2 mg) and 5FU/leucovorin
(12.5 mg/Kg iv, .gradient.) on days 1, 3, 5, 7, 21, 22.
FIG. 20 A histogram demonstrating the effect of SC101, 5FU/leucovorin
or the combination of SC101 and 5FU on the final tumour weights
of C170 xenografts grown in nude mice. animals were treated with
either SC101 ip(0.2 mg) (O), control antibody ip(0.2 mg) and 5FU/leucovorin
(12.5 mg/Kgiv) (.gradient.) or SC101 ip(0.2 mg) and 5FU/leucovorin
(12.5 mg/Kg iv, .gradient.) on days 1, 3, 5, 7, 21, 22.
The invention is now described with reference to the following
non-limiting examples;
EXAMPLE 1
Binding Studies Using SC101 Monoclonal Antibody
Methods
Binding to Carbohydrates Separated by Thin Layer Chromatography
Purified glycolipid standards containing Lewis.sup.b, Lewis.sup.y,
trifucosyl Lewis.sup.b, trifucosyl Lewis.sup.y, H type 1 chain,
H type 2 chain, and Lewis.sup.x were spotted on the TLC plates were
run in a solvent system of Chloroform: methanol: 0.2% Calcium chloride:50:40:10.
The plates were then blocked with 5% BSA and then incubated with
either Orcinol, panel A or Mabs SC101/23 panel B, Mab SC101/29 panel
C, Mab SC101/33 panel D, Mab SC101/42 panel E and Mab C14 panel
F. After washing the plates, they were incubated with rabbit anti-mouse
IgG and IgM, followed by incubation with .sup.125I labelled protein
A. The bands were visualised by autoradiography.
Binding to Carbohydrates as Assayed by ELISA
Microtitre plates were coated with either purified Lewis.sup.b
or Lewis.sup.y glycolipids at (5 ug/ml). After blocking the plates
with 5% bovine serum albumin diluted in phosphate buffered saline,
purified monoclonal antibodies SC101/23, SC101/29, SC101/33, SC101/42
were added at different concentrations (0.15-20 .mu.g/ml) followed
by the additional of peroxidase conjugated goat anti-mouse IgG and
IgM. Bound enzyme was detected by optical density reading at 490
nm.
Binding to Freshly Disaggregated Tumour Cells
Colorectal tumours were collected and disaggregated with collagenase
(0.05%, Type IV) into highly viable single cell suspensions and
assayed for binding with SC101 by indirect immunofluorescence where
binding of SC101 was detected with a rabbit anti-mouse anti-serum
conjugated to FITC. Stained cells were analysed for fluorescence
by a FACS IV cell sorter. Fluroescein fluorescence was excited at
488 nm and collected via a 10 nm band pass filter centred at 515
nm and adjusted to standard conditions using fluorochrome labelled
latex beads. Fluorescence intensity is expressed as mean linear
fluorescence (MLF), calculated by multiplying the contents of each
channel by its channel number and dividing by the total number of
cells in the distribution. The FACS IV is set to selectively analyse
cells in the malignant cell size range. Each tumour was also stained
using normal mouse Ig and the MLF in this control was subtracted
from the values obtained with monoclonal antibody. However, the
mean binding of normal mouse Ig was 50.+-.25 and therefore tumours
were only described as positively staining if the MLF exceeds 50.+-.2s.d.i.e.
100. This was a conservative estimate as background of positively
stained cells was calculated as the number of cells with a fluorescence
that exceeded the value in which 95% of cells staining with normal
mouse immunoglobulin were observed.
Disaggregation of solid tumours yields a mixed population of cells
including red blood cells, lymphocytes, stromal cells, macrophages
and endothelial cells. The percentage of epithelial cells, as measured
by staining of cytokeratin with monoclonal antibody Cam 5.2, was
only 22.+-.13% (range 10-60). However, following forward angle light
scatter gating to selectively analyse cells in the malignant cell
size range 79.+-.4% (range 69-86) of the cells analysed were epithelial.
Furthermore the variation between tumours was considerably reduced.
The percentage of lymphocytes, as measured by staining with the
monoclonal antibody F10-89-4 (kindly provided by Peter Beverley,
Genera Institute), in the total nucleate population was 74.+-.16
(range 40-90). This was considerably reduced to 5.5.+-.5% (range
1-20) following FACS IV gating for malignant cell size. The percentage
of stromal cells in the population of cells analysed in the malignant
size range was 3.5.+-.3% (range 1-13).
Although the percentage of non-epithelial cells in the forward
light scatter gate was low and did not vary considerably between
tumours (21.+-.4%). This may have affected the mean linear fluorescence
of particular tumours or if they failed to stain contributed to
the heterogeneity of staining. Therefore only tumours in which >25%
(i.e. 21.+-.34% non epithelial cells) of the cells stained were
described as positive.
Binding to Leukaemic Cell Lines
5.times.10.sup.4 cells from well characterised myeloid leukaemic
cell-lines were incubated with various dilutions of SC101 antibody
and left for 1 hour on ice. Following extensive washing the cells
were mixed with a FITC labelled anti-mouse conjugate for a further
30 minutes on ice. After washing for a second time the cells were
fixed in proprietary cellfix and bound-guorescence measured by flow
cytometry.
Binding to Normal Tissues
Binding of SC101 antibody to normal tissues was determined by indirect
immunoperoxidase staining of post-mortem samples. Tissue sections
(5 .mu.m) of cryopreserved tumour and normal tissues were treated
with 0.3% H.sub.2O.sub.2 in 0.1% NaN.sub.3 for 15 min to inhibit
endogenous peroxidase. This was followed by incubation at room temperature
with 10% human serum and 1% BSA prepared in PBS, for 30 min, and
then the mouse SC101 monoclonal antibodies were added at saturating
levels which gave minimal non specific background staining for a
further 30 min. The bound antibody was detected with rabbit anti-nouse
Ig conjugated to peroxidase and following extensive washing the
slides were stained with 0.05% diaminobenzidine and 0.01% H.sub.2O.sub.2
in 0.05M Tris-HCl, pH7.6 and counter stained with haematoxylin.
Results
A monoclonal antibody C14 was raised in mice against primary colorectal
tumour cells. This antibody showed good tumour selectivity as it
binds to a cell surface antigen over-expressed by a range of tumours
and only present at low levels on normal cells. However C14 is an
IgM antibody and of limited value for tumour therapy. An antiserum
raised in rats to C14 was used to immunise mice and select 5 new
IgG monoclonal antibodies. These monoclonal antibodies are referred
to as SC101/23, SC101/29, SC101/33, SC101/42, SC101/43. These antibodies
were shown to recognise extended and non-extended Lewis.sup.y and
Lewis.sup.b antigens but not Lewis.sup.x or H blood group antigen
by thin layer chromatography (FIG. 1) and ELISA (FIG. 2).
FIG. 3 shows that the antigen recognised by SC101 shows a similar
distribution to antigens, CD55, CEA and cytokeratin, recognised
by antibodies NCRC30, NCRC36, SC104 and Cam 5.2. FIG. 4 shows that
the antigen recognised by SC101 shows a similar distribution to
antigens, CD55 and CEA, recognised by antibody HLA/DR, W6/32 and
cam 52. FIG. 5 shows that the antigen recognised by SC101 shows
a similar distribution to antigens CD55, CEA, MUC1 recognised by
HLA/ABC and cam52.
Epithelial cells are known to express cytokeratin. These results
show that as the majority of the tumours are cytokeratin positive,
SC101 recognises cells of epithelial tumour origin.
Staining disaggregated tumour cells with SC101 antibody demonstrates
that Lewis.sup.y and Lewis.sup.b antigens are over-expressed by
a wide variety of tumours including colorectal (FIG. 3), gastric
(FIG. 4), ovarian (FIG. 5), breast, lung and myeloid leukaemia (FIG.
6).
TABLE-US-00001 TABLE 1 Binding of SC101 monoclonal antibodies to
normal tissues TISSUES Binding of SC101 to tissues.sup.1 Rectum
No epithelial or stromal staining Descending colon No epithelial
or stromal staining Proximal Colon No epithelial or stromal staining
Ileium No epithelial or stromal staining Jejunum Basement membrane
of villous epithelium Duodenum Basement membrane staining of villi
in selected Areas. Mucin staining in acini Stomach Mucin staining
Liver Weak staining of capillaries No hepatic or reticulin staining
Abdominal wall, muscle and No staining connective tissue Skin (from
sebaceous cyst) Sebaceous gland with central staining Myometrium,
endometrium, No staining erosa and ovary Fallopian tubes Mucin staining
on surface epithelium
Recognition of normal tissue was minimal and restricted to weak
staining of the upper gastrointestinal tract basement membrane,
mucin staining of stomach and fallopian tubes and weak staining
of liver capillaries (Table 1).
A cell line expressing SC101/29 was deposited with ECACC under
Accession no. 01050118.
EXAMPLE 2
Exposure of Tumour Cells to SC101 Antibodies
Methods and Results
Experiment 1
5.times.10.sup.4 C170 (colorectal tumour) cells were incubated
with various dilutions of antibody and left for 1 hour on ice. Following
extensive washing the cells were mixed with a FITC labelled anti-mouse
conjugate for a further 30 minutes. After washing for a second time
the cells were fixed in proprietary cellfix and bound fluorescence
measured by flow cytometry. The profiles show the analyses of the
forward (FSCH) and side (SSH) scatter measurements made on the treated
cells revealing the alteration in cell size and granularity following
antibody treatment. During the routine characterisation of this
family of antibodies it was noted that disaggregated tumour cells
or cultured cell-lines exposed to SC101 rapidly shrank and increased
their granularity (see FIG. 7).
Experiment 2
1.times.10.sup.5 C170, HT29 or HL60 tumour cells in suspension
were incubated with various dilutions of SC101 antibody and appropriate
controls for 1 hour at room temperature. The cells were then washed,
resuspended in proprietary binding buffer and stained with FITC
labelled Annexin V and Propidium Iodide. The cells were then analysed
by dual colour flow cytometry to determine the number of cells staining
positive under the various conditions used. Cells staining with
Annexin alone are in the early stages of apoptosis whereas cells
staining with both Annexin and propidium iodide are in late stage
apoptosis or necrosis.
The results show that Annexin V binding is increased following
treatment of C170 cells with SC101 antibody but also reveals that
propidium Iodide staining is increased. This dual staining suggests
that the positive cells are entering late stage apoptosis. These
studies with Annexin-V showed that following a 1 h exposure of cells
to SC101, phosphatidylserine was exposed on the outer surface of
the cell membrane indicating the onset of apoptosis (see FIG. 8).
Experiment 3
3.times.10.sup.4 Colorectal C170 cells were aliquoted into individual
wells of a flat-bottomed 96-well plate and left to adhere overnight.
The following day the cells were treated with various dilutions
of SC101 antibody or appropriated controls and left for a further
5 days. The cultures were then washed and stained with crystal violet
to determine the number of viable cells left in each well. The results
were recorded by optical density at 490 nm and plotted in comparison
with suitable negative controls.
Significantly higher concentrations (compared to the concentrations
used in those experiments described above) of antibody were required
to inhibit the proliferation of adherent cell-lines growing as monolayer
cultures (see FIG. 9a). These results, suggest that deprivation
of matrix attachment and cell-cell signalling increases the sensitivity
of cells to treatment with SC101, enabling the antibody to induce
increased Apoptosis and eventual cell death. Numerous studies have
documented the importance of the cellular matrix in solid tumours
as it stimulates survival signals through adhesion molecules such
as the integrins. If this matrix support is removed these survival
signals are absent and the sensitivity of the cells to potentially
apoptotic stimuli is increased.
Experiment 4
In a further experiment, 3.times.10.sup.4 leukaemic HL-60 cells
were aliquoted into individual wells of a flat-bottomed 96-well
plate. The cells were then treated with various dilutions of SC101
antibody or appropriate controls and left for 5 days. The cultures
were then washed and stained with crystal violet to determine the
number of viable cells in each well. The results were recorded by
optical density at 490 nm and are plotted in comparison with suitable
negative controls. The results show that at higher concentrations
of SC101 HL-60 cell growth is significantly reduced compared with
control cultures. These studies showed that high doses of SC101
could also inhibit the proliferation of HL60 cells in culture (see
FIG. 9b).
Experiment 5
In a further experiment, 1.times.10.sup.5 HL-60 myeloid leukaemic
cells were incubated with SC101/29 monoclonal antibody appropriate
controls at various dilutions, washed and stained with PE conjugated
APO2.7 antibody. The cells were then analysed by flow cytometry
to determine the number of positively stained cells in each case.
The results shown in FIG. 10 show that SC101 antibody bound specific
myeloid leukaemic cell lines and under certain conditions could
induce apoptosis as measured by flow cytometry and Apo2.7, an antibody
known to recognise a mitochondrial antigen exposed at the onset
of Apoptosis.
EXAMPLE 3
Studies Using SC101/29 and Cisplatin
Methods and Results
Experiment 1
3.times.10.sup.4 Colorectal C170 cells were aliquoted into individual
wells of a flat-bottomed 96-well plate and left to adhere overnight
The following day the cells were treated with various dilutions
of SC101 antibody or appropriate controls for 1 hour at room temperature.
The cultures were then washed once and incubated in goat anti-mouse
antibody at 100 .mu.g/ml for 30 minutes at room temperature. The
cultures were then re-washed and left over night in culture medium
with or without cisplatin. The following day the cultures were washed,
lysed and tested for the presence of fragmented DNA by a Cyto Death
ELISA (Roche). The results were recorded by optical Density measurements
at 405 nm. FIG. 11 shows that cisplatin at 3 .mu.g/ml induces apoptosis
but that SC101 alone at concentrations up to 30 .mu.g/ml fails to
induce significant apoptosis. However when SC101 is added to cisplatin,
increased apoptosis is observed. These results demonstrate that
increased Apoptosis is observed when SC101 and Cisplatin are administered
together and that the increase in Apoptosis is greater than the
added effect of administering SC101 and Cisplatin separately.
The plots shown in FIG. 11 demonstrate the synergistic effect of
SC101 antibody on Cisplatin treated cells with significantly increased
levels of fragmented DNA indicative of increased apoptosis.
Experiment 2
Colorectal tumour cells were exposed to cisplatin (0-0.05 .mu.g/ml)
for 4 hrs at 37.degree. C. and then SC101/29 (0-50 .mu.g/ml) antibody
was added. The cells were left for 4 days when the number of viable
cells was determined by crystal violet staining and optical density
reading at 550 nm. FIG. 12 shows that cisplatin at 1 .mu.g/ml and
SC101/29 both induce 10% inhibition of C170 cell growth. The combination
of drug and antibody are synergistic, inducing a 50% reduction in
cell growth.
EXAMPLE 4
Studies Using SC101/29 and 5 Fluorouracil
Methods and Results
3.times.10.sup.3 C170 cells were plated into microtitre plates
and left overnight to adhere. 5FU was added to a final concentrations
of 0.25 and 0.5 .mu.g/ml .SC101/29 antibody was also added at concentration
of 1-50 .mu.g/ml. Cells were left for 5 days at 37.degree. C. prior
to staining with crystal violet and reading the optical density
reading at 550 nm to assess the number of cells. As shown in FIG.
13, a 10% in C170 cell growth is observed at a dose of 20 .mu.g/ml
of SC101/29. However, in combination with a non-toxic dose of 5
Fluorouracil (0.25 .mu.g/ml) there was a 70% reduction in cell growth.
These studies, in combination with the studies described in Example
3 demonstrate that SC101 increases the sensitivity of cultured C170
cells to the effects of Cisplatin and 5-Fluorouracil and that SC101,
Cisplatin and 5-Fluorouracil act synergistically in reducing cell
growth (inducing apoptosis).
EXAMPLE 5
Studies Using SC101/29 and Tamoxifen, Doxorubicin, Irinotecan,
Mitomycin C, Oxaliplatin and Raltitrexed.
Methods and Results
Experiment 1--C170 Colorectal Cells.
1.times.10.sup.3 Colorectal C170 cells were aliquoted into individual
wells of a flat bottomed 96-well plate and left to adhere overnight
at 37.degree. C. The following day the cells were treated with Tamoxifen
at final concentrations of 10, 3, 1, 0.3, 0.1 and 0 .mu.M. Against
each concentration of Tamoxifen the following concentrations of
SC101/29 were titrated: 10, 3, 1, 0.3 and 0 .mu.g/ml. As a negative
control 791T/36 at concentrations 100, 30, 10, 3 and 0 .mu.g/ml,
was titrated against each concentration of Tamoxifen used. Duplicate
wells were used. Cells were left for 5 days at 37.degree. C. prior
to the addition of MTS reagent to each well and optical density
reading at 490 nm. FIGS. 14a-e show the effect of SC101/29 alone
(a), 791T/36 alone (b), Tamoxifen alone (c), SC101/29 in combination
with Tamoxifen (d), and 791T/36 in combination with Tamoxifen (e)
on C170 growth at the concentrations stated. The minimum concentrations
of SC101/29 and Tamoxifen giving the maximum degree of synergy were
selected and plotted (FIG. 15). FIG. 15 also represents parallel
experiments measuring the synergistic/additive effect of SC101/29
in combination with 5-FU, Cisplatin, Doxorubicin, Irinotecan, Mitomycin
C, Oxaliplatin and Raltitrexed on C170 growth.
Experiment 2--MDA-MB468 Breast Cancer Cells.
Using the same method, the effect of SC101/29 alone and in combination
with Tamoxifen and Doxorubicin on the growth of the Breast carcinoma
cell line MDA-MB-468 was investigated. The effect of SC101/29 alone
and in combination with Tamoxifen is shown in FIGS. 16a-e Synergy
between SC101 and Tamoxifen or Doxorubicin is demonstrated by the
graph shown in FIG. 17. The concentrations of SC101/29 in combination
with Tamoxifen and Doxorubicin which yield the largest degree of
synergy is also shown by FIG. 17.
EXAMPLE 6
Xenograft Experiments
Method and Results
Mice were explanted with 3 mm 3 pieces of C170 xenografts. Groups
of mice were treated with 5FU/leucovorin (12.5 mg/Kg) by intravenous
infusion on days 1,3,5,7,21 and 22. On the same days mice were also
injected intra peritoneally with 0.2 mg of SC101/29 monoclonal antibody.
Control mice received either SC101/29 alone or control mouse IgG
antibody with 5FU/leucovorin. Tumour size was measured by callipers
and tumour cross sectional area calculated on days 12, 16, 19 and
23. At the termination of the experiment tumours were weighed to
assess anti-tumour efficacy. Animals were weighed to assess the
toxicity of treatment.
SC101 antibody significantly inhibited tumour growth at a dose
of 0.2 mg. 12.5 mg/Kg of 5FU/leucovorin also inhibited tumour growth
(FIG. 18 and FIG. 20). However, the combination was even more effective
(FIGS. 18 and 20). However, none of the treatments were toxic to
the animals as they showed normal weight gain (FIG. 19). These results
suggest that the anti-tumour efficacy of chemotherapy may be enhanced
by treatment with SC101 monoclonal antibodies.
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