Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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BRAIN SPECIFIC BINDING MENN~ERS
The present invention relates to specific binding members
directed to the brain, including specific binding members able
to pass through the blood brain barrier or which are directed
to areas of brain inflammation or blood brain barrier
breakdown. Panels and mixtures of antibody are provided, also
individual antibody molecules and VH and VL domains, along
with methods of obtaining specific binding members by
selection using libraries displayed on the surface of
filamentous bacteriophage.
Transport of many blood-borne molecules into the brain is
restricted when compared to other tissues in the body. This
has led to the concept of a blood brain barrier (BBB) which
isolates the brain from peripheral changes in physiology
(Bradbury MWB (1993) Experimental Physiology, 78: 453-472).
It has been shown that the endothelial cells which line the
capillaries of the brain are joined by extensive tight
junctions which restrict pericellular movement of
water-soluble molecules. These endothelial cells also express
specific transporter molecules on their surfaces which
selectively allow the passage of molecules which are essential
for brain function e.g. glucose, amino acids, transferrin.
The BBB presents a substantial hurdle for the delivery of many
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drugs, particularly proteins such as antibodies and cytokines
or growth factors into the central nervous system. With the
exception of lipid-soluble molecules, which have a molecule
weight from 400-600 Da, virtually all drugs that originate
from either biotechnology or classical small molecule
pharmacology undergo negligible transport through the intact
BBB. The identification of novel transporter molecules or
receptors associated with the BBB represents a potential
target for enhancing the uptake of drugs into the CNS, by for
example conjugation to the natural ligands of those receptors
or by selection of antibodies or drugs which bind directly to
the receptors.
Central Nervous System (CNS) diseases that may be treated in
this manner include sleep disorders (e. g. narcolepsy),
affective disorders, schizophrenia, regenerative disorders
such as Alzheimer's disease, Parkinson's disease, fronto-
temporal dementia, Huntington's chorea, demyelination diseases
(e. g. multiple sclerosis), spinal cord injury, brain tumours
(primary and secondary), viral encephalopathies (e. g. rabies
and cerebral HIV), motor neurone diseases, prion diseases,
hydrocephalus, stroke (primary haemorrhagic and ischaemic),
epilepsies, obsessive-compulsive disorders, anxieties and
phobias, drug abuse, alcohol and other substance abuses,
symptoms such as pain. In addition, consciousness may be
modulated, such as in general anaesthesia.
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The present invention relates to the selection and
characterisation of human antibodies from a phage display
antibody library which have been demonstrated to cross the BBB
or which localise to areas of inflammation in brain tissue.
Such antibodies have value and therapeutic potential as agents
to enhance drug delivery across the blood brain barrier, and
as tools to identify novel transporter or other blood brain
barrier or inflamed tissues marker molecules which may
themselves be targets for therapeutic intervention.
Some molecules on brain capillary endothelium are already
known to be specific receptors for circulating proteins or
plasma proteins. Human microvessels have been used to
characterise the human BBB receptors for insulin, insulin-like
growth factor I, insulin-like growth factor-II, transferrin
and leptin. (Reviewed by W Pardridge (1997) Journal of
Cerebral Blood Flow and Metabolism, 17: 713-731). The BBB
insulin, insulin-like growth factor, and transferrin receptor
systems have been shown to mediate the transcytosis of
peptides through the BBB in vivo (e. g. Duffy and Pardridge,
1987, Brain Res. 420: 32-38). The observation that peptide
receptors are present on brain capillaries endothelium, and
that some of these mediate peptide transcytosis through the
BBB led to the idea of covalently coupling nontransportable
drugs to a protein or peptide that normally undergoes
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receptor-mediated transcytosis through the BBB. Initial
experiments were carried out using cationized albumin which
undergoes absorptive-mediated transcytosis through the BBB
(Kumagai et al., 1987, J Biol. Chem 262: 15214-15219).
Subsequently murine mAbs to the rat transferrin receptor
(Friden et al., 1993, Science 259: 373-377) and the human
insulin receptor were developed (Pardrige et al., 1995, Pharm
Res 12: 807-816) which have BBB permeability.
The anti-human insulin receptor mAb 83-14 has been conjugated
to streptavidin and shown to retain its ability to be
transported (wu et al, 1997, J Clin Invest, 100: 1804-12)
allowing biotinylated compounds to be transported along with
it.
Little is known about the up or down-regulation of transport
systems at the BBB in disease states, but there are
indications that change at the level of expression occurs e.g.
the expression of ICAM-1 and ICAM-2 is upregulated in areas of
inflammation (Osborn, L. Cell 1990, 623-6). Following,
stimulation with lipopolysaccharide and interleukin -1 and -6
lymphocyte binding to the BBB was increased 3-fold and could
be blocked by mAbs specific for VLA4, CD18 and CDlla (de Vries
et al., J. Neuroimmunol 1994, 52; 1-8). Thus disease states
can yield important pointers towards achieving selectivity in
BBB transport by identifying up-regulated transport systems at
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the BBB. The picture that is emerging from in vitro and in
vivo studies in animal modes is that the cross-linking of
cell-surface adhesion molecules initiates an intracellular
signalling cascade that leads to changes in the cytoskeleton,
disassembly of the functional complex and, ultimately, an
increase in vessel permeability.
In one aspect, the present invention provides a mixture or
panel of at least 5, and preferably at least 10 different
specific binding members each comprising an antibody VH
variable domain and/or an antibody VL variable domain, wherein
an antibody VH variable domain has an amino acid sequence
selected from the group consisting of the G65 (SEQ ID NO. 2),
G67 (SEQ ID N0. 6), G73 (SEQ ID NO. 10), G76 (SEQ ID N0. 14),
G77 (SEQ ID N0. 18), G78 (SEQ ID N0. 22), G79 (SEQ ID NO. 24),
G81 (SEQ ID NO. 30), G83 (SEQ ID N0. 34), G85 (SEQ ID NO. 38),
G88 (SEQ ID N0. 42), G92 (SEQ ID N0. 46), G93 (SEQ ID N0. 50),
G95 (SEQ ID N0. 54), 6101 (SEQ ID N0. 58), 6102 (SEQ ID N0.
62), 6110 (SEQ ID N0. 66) and 6112 (SEQ ID NO. 70) VH domain
sequences disclosed herein, and/or an antibody VL variable
domain has an amino acid sequence selected from the group
consisting of the G65 (SEQ ID NO. 4), G67 (SEQ ID NO. 8), G73
(SEQ ID N0. 12), G76 (SEQ ID N0. 16), G77 (SEQ ID N0.20), G78
(SEQ ID N0. 24), G79 (SEQ ID N0. 28), G81 (SEQ ID N0. 32), G83
(SEQ ID NO. 36), G85 (SEQ ID N0. 40), G88 (SEQ ID NO. 44), G92
(SEQ ID N0. 48), G93 (SEQ ID N0. 52), G95 (SEQ ID NO. 56),
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6101 (SEQ ID N0. 60), 6102 (SEQ ID N0. 64), 6110 (SEQ ID N0.
68) and 6112 (SEQ ID N0. 72) VL domain sequences disclosed
herein, each specific binding member being able, when
displayed on the surface of filamentous bacteriophage
particles, to pass through a mammalian blood brain barrier,
preferably a human or rodent (e. g. rat) blood brain barrier.
The blood brain barrier may be an in vitro barrier such as
generated in accordance with Example 1 herein.
A mixture or panel of specific binding members may include at
least 11, 12, 13, 14, 15, 16, 17 or 18 of the VH or VL
domains. In such a mixture, preferably VH and VL domains are
paired according to corresponding nomenclature as used herein
(e.g. G65 VH (SEQ ID N0. 2) with G65 VL (SEQ ID NO. 4), G81 VH
(SEQ ID N0. 30) with G81 VL (SEQ ID N0. 32), and so on).
A panel or mixture of specific binding members provided by the
present invention is useful for selection of specific binding
members that cross the blood brain barrier. Additionally, a
panel or mixture may be employed together to deliver different
molecules or effector functions.
One or more specific binding members may be selected from a
panel or mixture e.g. by binding with antigen, which may be on
endothelial cells. Where the specific binding members are
displayed on filamentous bacteriophage, this allows nucleic
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acid encoding a selected specific binding member, or at least
a component of the member, to be readily isolated. Encoding
nucleic acid may then be manipulated and may be used to
produce more of the specific binding member or component
thereof by means of recombinant expression.
In a further, general aspect, the present invention provides a
method of obtaining one or more specific binding members, the
method including bringing into contact a library of specific
binding members displayed on the surface of filamentous
bacteriophage particles, and selecting one or more specific
binding members of the library able when displayed on
filamentous bacteriophage particles to pass through a
mammalian blood brain barrier. Selection on ability to pass
through a blood brain barrier may be followed by one or more
further rounds of selection for ability to bind a type of cell
of the brain or CNS, for instance endothelial cells, or cells
of the meninges, parenchyma, choroid plexus, cerebrum,
cerebellum, spinal cord or microglia.
A population or mixture of specific binding members which when
displayed on filamentous bacteriophage are able to pass
through a mammalian blood brain barrier is obtainable on
selection from a phage display library as disclosed. Such a
population or mixture may comprise at least 106 different
specific binding members. One or more further rounds of
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selection may be used to provide a sub-population of about
10,000 different specific binding members, 1,000, 100, a
mixture of 10-20 different specific binding members or fewer,
and individual specific binding members.
The invention further provides a plurality of different
antibody VH variable domains obtainable from a mixture, panel,
population or library as disclosed, and further provides a
plurality of different antibody VL variable domains obtainable
from such a mixture, panel, population or library.
Individual VH and VL domains of which sequences are shown
herein each represent individual aspects of the present
invention. A VH domain of which the sequence is disclosed
herein may be combined with a VL domain of which the sequence
is disclosed herein, or other VL domain, to provide a VH/VL
pairing representing an antigen-binding site of an antibody.
Similarly, a VL domain of which the sequence is disclosed
herein may be combined with a VH domain of which the sequence
is disclosed herein, or other VH domain. Pairings of VH and
VL domains represent further aspects of the present invention.
Preferred pairings are as identified by corresponding
nomenclature herein, e.g. G65 VH (SEQ ID NO. 2) with G65 VL
(SEQ ID NO. 4), G81 VH (SEQ ID N0. 30) with G81 VL (SEQ ID N0.
32), and so on.
Thus, individual aspects of the present invention provide a
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specific binding member comprising a VH/VL pairing selected
from the group consisting of G65, G67, G73, G76, G77, G78,
G79, G81, G83, G85, G88, G92, G93, G95, 6101, 6102, 6110 and
6112 (SEQ ID NO's are given above). Each VH domain and each
VL domain of each of these pairings represents a further
aspect of the present invention. Of the VH/VL pairings
disclosed herein, G67, G73, G76, G78, G79, G83, G85, G88, G92,
G93, G95 and 6110 represent preferred embodiments, being
cross-reactive with human tissue.
These as phage antibodies actively cross the blood brain
barrier and specifically recognise endothelial cells of the
brain, as shown by ELISA and immunocytochemistry. G93 (VH SEQ
ID N0. 50; VL SEQ ID N0. 52) and G73 (VH SEQ ID N0. 10; VL SEQ
ID N0. 12) are among particularly preferred embodiments of the
present invention; they show high levels of transport across
the blood brain barrier and cross react with antigens on human
endothelial cells.
As noted, further VH and VL domains, and pairings thereof, may
be obtained from a filamentous bacteriophage library,
employing selection for ability to pass through a blood brain
barrier.
Further aspects of the invention provide the D5 VH (SEQ ID N0.
74) and VL (SEQ ID NO. 76) domains of which the amino acid
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sequences are disclosed herein. D5 VH/VL is directed against
human serum amyloid protein (SAP) which passes through the
blood brain barrier. Experimental evidence included below
shows that D5 is actively transported across the blood brain
barrier.
One or more CDRs may be taken from any VH or VL domain
according to the present invention and incorporated into a
suitable framework. This is discussed further below.
Variants of the VH and VL domains and CDRs of which the
sequences are set out herein and which can be employed in
specific binding members for brain and/or endothelial cell
antigens can be obtained by means of methods of sequence
alteration or mutation and screening. Such methods are also
provided by the present invention.
In addition to antibody sequences, the specific binding member
may comprise other amino acids, e.g. forming a peptide or
polypeptide, such as a folded domain, or to impart to the
molecule another functional characteristic in addition to
ability to bind antigen. Specific binding members of the
invention may carry a detectable label, or may be conjugated
to a toxin or enzyme (e. g. via a peptidyl bond or linker).
Thus a VH domain or a VL domain according to the present
invention may be provided in a fusion with additional amino
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acids.
A further aspect of the present invention provides a method of
obtaining one or more specific binding members with a desired
property, the method including bringing into contact a library
or panel of specific binding members and selecting one or more
with the desired property. Such a method may employ phage
display technology, wherein the specific binding members in
the library or panel are displayed on the surface of
bacteriophage particles, each particle containing nucleic acid
encoding the specific binding member or a component thereof
(e.g. VH domain). Nucleic acid may be taken from a
bacteriophage particle containing nucleic acid encoding a
selected specific binding member or component thereof, and
nucleic acid with the sequence of the nucleic acid from the
particle can be used to provide (by means of recombinant
technology) the encoded product, or further nucleic acid with
the sequence, or a variant or derivative.
In further aspects, the invention provides an isolated nucleic
acid which comprises a sequence encoding a specific binding
member as defined above, and methods of preparing specific
binding members of the invention which comprise expressing
said nucleic acids under conditions to bring about expression
of said binding member, and recovering the binding member.
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Specific binding members according to the invention may be
used in a method of treatment or diagnosis of the human or
animal body, such as a method of treatment (which may include
prophylactic treatment) of a disease or disorder in a human
patient which comprises administering to said patient an
effective amount of a specific binding member of the
invention. The specific binding member may be unconjugated,
or may be conjugated to an active agent. Conditions treatable
in accordance with the present invention include neurological
diseases, including Alzheimer's disease, prion diseases, AIDS-
related dementia, and any disease involving inflammation
occurring within the brain or CNS.
These and other aspects of the invention are described in
further detail below.
TERMINOLOGY
Specific binding member
This describes a member of a pair of molecules which have
binding specificity for one another. The members of a
specific 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, or a cavity, which
specifically binds to and is therefore complementary to a
particular spatial and polar organisation of the other member
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of the pair of molecules. Thus the members of the pair have
the property of binding specifically to each other. Examples
of types of specific binding pairs are antigen-antibody,
biotin-avidin, hormone-hormone receptor, receptor-ligand,
enzyme-substrate. This application is concerned with
antigen-antibody type reactions.
Antibody
This describes an immunoglobulin whether natural or partly or
wholly synthetically produced. 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.
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 complementarity 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
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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 specific
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.
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 CHl 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
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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
(W094/13804; P. Holliger et al, Proc. Natl. Acad. Sci. USA 90
6444-6448, 1993). Fv, scFv or diabody molecules may be
stabilised by the incorporation of disulphide bridges linking
the VH and VL domains (Y. Reiter et al, Nature Biotech, 14,
1239-1245, 1996). Minibodies comprising a scFv joined to a
CH3 domain may also be made (S. Hu et al, Cancer Res., 56,
3055-3061, 1996).
Diabodies are multimers of polypeptides, each polypeptide
comprising a first domain comprising a binding region of an
immunoglobulin light chain and a second domain comprising a
binding region of an immunoglobulin heavy chain, the two
domains being linked (e.g. by a peptide linker) but unable to
associate with each other to form an antigen binding site:
antigen binding sites are formed by the association of the
first domain of one polypeptide within the multimer with the
second domain of another polypeptide within the multimer
(W094/13804).
Where bispecific antibodies are to be used, these may be
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conventional bispecific antibodies, which can be manufactured
in a variety of ways (Holliger, P. and Winter G. Current
Opinion Biotechnol. 4, 446-449 (1993)), e.g. prepared
chemically or from hybrid hybridomas, or may be any of the
bispecific antibody fragments mentioned above. It may be
preferable to use scFv dimers or diabodies rather than whole
antibodies. Diabodies and scFv can be constructed without an
Fc region, using only variable domains, potentially reducing
the effects of anti-idiotypic reaction.
Bispecific diabodies, as opposed to bispecific whole
antibodies, may also be particularly useful because they can
be readily constructed and expressed in E.coli. Diabodies
(and many other polypeptides such as antibody fragments) of
appropriate binding specificities can be readily selected
using phage display (W094/13804) from libraries. If one arm
of the diabody is to be kept constant, for instance, with a
specificity directed against antigen X, then a library can be
made where the other arm is varied and an antibody of
appropriate specificity selected. Bispecific whole antibodies
may be made by knobs-into-holes engineering (J. B. B. Ridgeway
et al, Protein Eng., 9, 616-621, 1996).
Antigen binding domain
This describes the part of an antibody which comprises the
area which specifically binds to and is complementary to part
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or all of an antigen. Where an antigen is large, an antibody
may only bind to a particular part of the antigen, which part
is termed an epitope. An antigen binding domain may be
provided by one or more antibody variable domains (e.g. a so-
called Fd antibody fragment consisting of a VH domain).
Preferably, an antigen binding domain comprises an antibody
light chain variable region (VL) and an antibody heavy chain
variable region (VH).
Speci fi c
This may be used to refer to the situation in which one member
of a specific binding pair will not show any significant
binding to molecules other than its specific binding
partner(s). The term is also applicable where e.g. an antigen
binding domain is specific for a particular epitope which is
carried by a number of antigens, in which case the specific
binding member carrying the antigen binding domain will be
able to bind to the various antigens carrying the epitope.
Comprise
This is generally used in the sense of include, that is to say
permitting the presence of one or more additional features or
components.
Isolated
This refers to the state in which specific binding members of
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the invention, or nucleic acid encoding such binding members,
will be in accordance with the present invention. Members and
nucleic acid will be free or substantially free of material
with which they are naturally associated such as other
polypeptides or nucleic acids with which they are found in
their natural environment, or the environment in which they
are prepared (e.g. cell culture) when such preparation is by
recombinant DNA technology practised in vitro or in vivo.
Members and nucleic acid may be formulated with diluents or
adjuvants and still for practical purposes be isolated - for
example the members will normally be mixed with gelatin or
other carriers if used to coat microtitre plates for use in
immunoassays, or will be mixed with pharmaceutically
acceptable carriers or diluents when used in diagnosis or
therapy. Specific binding members may be glycosylated, either
naturally or by systems of heterologous eukaryotic cells (e. g.
CHO or NSO (ECACC 85110503) cells, or they may be (for example
if produced by expression in a prokaryotic cell)
unglycosylated.
By "substantially as set out" it is meant that the relevant
CDR or VH or VL domain of the invention will be either
identical or highly similar to the specified regions of which
the sequence is set out herein. By "highly similar" it is
contemplated that from 1 to 5, preferably from 1 to 4 such as
1 to 3 or 1 or 2, or 3 or 4, substitutions may be made in the
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CDR and/or VH or~ VL domain.
The structure for carrying a CDR of the invention will
generally be of an antibody heavy or light chain sequence or
substantial portion thereof in which the CDR is located at a
location corresponding to the CDR of naturally occurring VH
and VL antibody variable domains encoded by rearranged
immunoglobulin genes. The structures and locations of
immunoglobulin variable domains may be determined by reference
to (Kabat, E.A. et al, Sequences of Proteins of Immunological
Interest. 4th Edition. US Department of Health and Human
Services. 1987, and updates thereof, now available on the
Internet (http://immuno.bme.nwu.edu)).
Preferably, a CDR amino acid sequence substantially as set out
herein is carried as a CDR in a human variable domain or a
substantial portion thereof. The VL CDR 3 and especially the
VH CDR3 sequences substantially as set out herein (e.g. see
Table 4) represent preferred embodiments of the present
invention and it is preferred that each of these is carried as
a respective VL CDR3 or VH CDR3 in a respective human light or
heavy chain variable domain or a substantial portion thereof.
Variable domains employed in the invention may be derived from
any germline or rearranged human variable domain, or may be a
synthetic variable domain based on consensus sequences of
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known human variable domains. A CDR-derived sequences of the
invention (e.g. CDR3) may be introduced into a repertoire of
variable domains lacking a CDR (e. g. CDR3), using recombinant
DNA technology.
For example, Marks et al (Bio/Technology, 1992, 10:779-783)
describe methods of producting repertoires of antibody
variable domains in which consensus primers directed at or
adjacent to the 5' end of the variable domain area are used in
conjunction with consensus primers to the third framework
region of human VH genes to provide a repertoire of VH
variable domains lacking a CDR3. Marks et al further describe
how this repertoire may be combined with a CDR3 of a
particular antibody. Using analogous techniques, the CDR3-
derived sequences of the present invention may be shuffled
with repertoires of VH or VL domains lacking a CDR3, and the
shuffled complete VH or VL domains combined with a cognate VL
or VH domain to provide specific binding members of the
invention. The repertoire may then be displayed in a suitable
host system such as the phage display system of W092/01047 so
that suitable specific binding members may be selected. A
repertoire may consist of from anything from 109 individual
members upwards, for example from 106 to 108 or 101° members.
Analogous shuffling or combinatorial techniques are also
disclosed by Stemmer (Nature, 1994, 370:389-391), who
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describes the technique in relation to a (3-lactamase gene but
observes that the approach may be used for the generation of
antibodies.
A further alternative is to generate novel VH or VL regions
carrying a CDR-derived sequences of the invention using random
mutagenesis of one or more selected VH and/or VL genes to
generate mutations within the entire variable domain. Such a
technique is described by Gram et a1 (1992, Proc. Natl. Acad.
Sci., USA, 89:3576-3580), who used error-prone PCR.
Another method which may be used is to direct mutagenesis to
CDR regions of VH or VL genes. Such techniques are disclosed
by Barbas et al, (1994, Proc. Natl. Acad. Sci., USA, 91:3809-
3813) and Schier et al (1996, J. Mol. Biol. 263:551-567).
All the above described techniques are known as such in the
art and in themselves do not form part of the present
invention. The skilled person will be able to use such
techniques to provide specific binding members of the
invention using routine methodology in the art.
A further aspect of the invention provides a method for
obtaining an antibody antigen binding domain with a desired
property, e.g. ability to cross the blood brain barrier and/or
specificity for a brain and/or endothelial cell antigen, the
method comprising providing by way of addition, deletion,
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substitution or insertion of one or more amino acids in the
amino acid sequence of a VH domain set out herein a VH domain
which is an amino acid sequence variant of the VH domain,
combining the VH domain thus provided with one or more VL
domains, and testing the VH/VL combination or combinations for
to identify an antibody antigen binding domain with the
desired property, e.g. ability to cross the blood brain
barrier and/or specificity for the antigen, and optionally
with one or more of preferred properties, e.g. ability to bind
areas of inflammation in the brain, breakdown of the blood
brain barrier. Said VL domain may have an amino acid sequence
which is substantially as set out herein.
An analogous method may be employed in which one or more
sequence variants of a VL domain disclosed herein are combined
with one or more VH domains.
A further aspect of the invention provides a method of
preparing a specific binding member with a desired property,
which method comprises:
(a) providing a starting repertoire of nucleic acids
encoding a VH domain which either include a CDR3 to be
replaced or lack a CDR3 encoding region;
(b) combining said repertoire with a donor nucleic acid
encoding an amino acid sequence substantially as set out
herein for a VH CDR3 such that said donor nucleic acid is
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23
inserted into the CDR3 region in the repertoire, so as to
provide a product repertoire of nucleic acids encoding a VH
domain;
(c) expressing the nucleic acids of said product
repertoire;
(d) selecting a specific binding member with the desired
property; and
(e) recovering said specific binding member or nucleic
acid encoding it.
Again, an analogous method may be employed in which a VL CDR3
of the invention is combined with a repertoire of nucleic
acids encoding a VL domain which either include a CDR3 to be
replaced or lack a CDR3 encoding region.
Similarly, one or more, or all three CDRs may be grafted into
a repertoire of VH or VL domains which are then screened for a
specific binding member or specific binding members with the
desired property.
As noted, the desired property may be any one or more of
ability to cross the blood brain barrier, bind an endothelial
cell or other brain cell antigen, bind areas of inlammation in
the brain or blood brain barrier breakdown, bind ICAM or bind
transferrin receptor.
A substantial portion of an immunoglobulin variable domain
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will comprise at least the three CDR regions, together with
their intervening framework regions. Preferably, the portion
will also include at least about 500 of either or both of the
first and fourth framework regions, the 50o being the C-
terminal 500 of the first framework region and the N-terminal
500 of the fourth framework region. Additional residues at
the N-terminal or C-terminal end of the substantial part of
the variable domain may be those not normally associated with
naturally occurring variable domain regions. For example,
construction of specific binding members of the present
invention made by recombinant DNA techniques may result in the
introduction of - or C-terminal residues encoded by linkers
introduced to facilitate cloning or other manipulation steps.
Other manipulation steps include the introduction of linkers
to join variable domains of the.invention to further protein
sequences including immunoglobulin heavy chains, other
variable domains (for example in the production of diabodies)
or protein labels as discussed in more details below.
Although in a preferred aspect of the invention specific
binding members comprising a pair of VH and VL domains are
preferred, single binding domains based on either VH or VL
domain sequences form further aspects of the invention. It is
known that single immunoglobulin domains, especially VH
domains, are capable of binding target antigens in a specific
manner.
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In the case of either of the single chain specific binding
domains, these domains may be used to screen for complementary
domains capable of forming a two-domain specific binding
member with the desired property.
This may be achieved by phage display screening methods using
the so-called hierarchical dual combinatorial approach as
disclosed in WO 92/01047 in which an individual colony
containing either an H or L chain clone is used to infect a
complete library of clones encoding the other chain (L or H)
and the resulting two-chain specific binding member is
selected in accordance with phage display techniques such as
those described in that reference. This technique is also
disclosed in Marks et al, ibid.
Specific binding members of the present invention may further
comprise antibody constant regions or parts thereof. For
example, a VL domain may be attached at its C-terminal end to
antibody light chain constant domains including human CK or C~,
chains, preferably C~, chains. Similarly, a specific binding
member based on a VH domain may be attached at its C-terminal
end to all or part of an immunoglobulin heavy chain derived
from any antibody isotype, e.g. IgG, IgA, IgE and IgM and any
of the isotype sub-classes, particularly IgGl and IgG4. IgG4
is preferred.
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Antibodies of the invention may be labelled with a detectable
or functional label. Detectable labels include radiolabels
such as 1311 or 99Tc, which may be attached to antibodies of the
invention using conventional chemistry known in the art of
antibody imaging. Labels also include enzyme labels such as
horseradish peroxidase. Labels further include chemical
moieties such as biotin which may be detected via binding to a
specific cognate detectable moiety, e.g. labelled avidin.
Antibodies of the present invention are designed to be used in
methods of diagnosis or treatment in human or animal subjects,
preferably human.
Accordingly, further aspects of the invention provide methods
of treatment comprising administration of a specific binding
member as provided, pharmaceutical compositions comprising
such a specific binding member, and use of such a specific
binding member in the manufacture of a medicament for
administration, for example in a method of making a medicament
or other composition comprising formulating the specific
binding member with at least one additional component, such as
a pharmaceutically acceptable excipient.
In accordance with the present invention, compositions
provided may be administered to individuals. Administration
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27
is preferably in a "therapeutically effective amount", this
being sufficient to show benefit to a patient. Such benefit
may be at least amelioration of at least one symptom. 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, eg decisions on
dosage etc, is within the responsibility of general
practioners and other medical doctors. Appropriate doses of
antibody are well known in the art; see Ledermann J.A. et al.
(1991) Int J. Cancer 47: 659-664; Bagshawe K.D. et al. (1991)
Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-
922.
A composition may be administered alone or in combination with
other treatments, either simultaneously or sequentially,
dependent upon the condition to be treated.
Antibodies of the present invention may be administered to a
patient in need of treatment via any suitable route, usually
by intravenous injection into the bloodstream. The precise
dose will depend upon a number of factors, including whether
the antibody is for diagnosis or for treatment, the size and
location of the area to be treated (e. g. wound), the precise
nature of the antibody (e.g. whole antibody, fragment or
diabody), and the nature of any detectable label or other
molecule attached to the antibody. A typical antibody dose
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will be in the range 0.5mg to 100g for systemic applications,
and 10~g to lmg for local applications. Typically, the
antibody will be a whole antibody, preferably the IgG4
subclass. This is a dose for a single treatment of an adult
patient, which may be proportionally adjusted for children and
infants, and also adjusted for other antibody formats in
proportion to molecular weight and pharmacokinetics.
Treatments may be by continuous infusion or may be repeated at
daily, twice-weekly, weekly or monthly intervals, as
appropriate. Treatment may be repeated within the same day,
e.g. for treatment of short-term problems such as epilepsy and
traumatic brain injury.
It is presently preferred that a whole antibody of the IgG4
subclass is used for systemic and local applications but for
local applications a scFv antibody may be particularly
valuable.
Specific 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
specific binding member.
Thus pharmaceutical compositions according to the present
invention, and for use in accordance with the present
invention, may comprise, in addition to active ingredient, a
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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.
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.
For 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 solution, Ringer's Injection solution, or Lactated
Ringer's Injection solution. Preservatives, stabilisers,
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buffers, antioxidants and/or other additives may be included,
as required.
A composition may be administered alone or in combination with
other treatments, either simultaneously or sequentially
dependent upon the condition to be treated. Other treatments
may include the administration of suitable doses of pain
relief drugs or anti-emetics.
The present invention provides a method comprising causing or
allowing binding of a-specific binding member as provided
herein to an antigen. As noted, such binding may take place
in vivo, e.g. following administration of a specific binding
member, or nucleic acid encoding a specific binding member, or
it may take place in vitro.
The amount of binding of specific binding member to an antigen
may be determined. Quantitation may be related to the amount
of the antigen in a test sample, which may be of diagnostic
interest.
The reactivities of antibodies on a sample may be determined
by any appropriate means. Radioimmunoassay (RIA) is one
possibility. Radioactive labelled antigen is mixed with
unlabelled antigen (the test sample) and allowed to bind to
the antibody. Bound antigen is physically separated from
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unbound antigen and the amount of radioactive antigen bound to
the antibody determined. The more antigen there is in the
test sample the less radioactive antigen will bind to the
antibody. A competitive binding assay may also be used with
non-radioactive antigen, using antigen or an analogue linked
to a reporter molecule. The reporter molecule may be a
fluorochrome, phosphor or laser dye with spectrally isolated
absorption or emission characteristics. Suitable
fluorochromes include fluorescein, rhodamine, phycoerythrin
and Texas Red. Suitable chromogenic dyes include
diaminobenzidine.
Other reporters include macromolecular colloidal particles or
particulate material such as latex beads that are coloured,
magnetic or paramagnetic, and biologically or chemically
active agents that can directly or indirectly cause detectable
signals to be visually observed, electronically detected or
otherwise recorded. These molecules may be enzymes which
catalyse reactions that develop or change colours or cause
changes in electrical properties, for example. They may be
molecularly excitable, such that electronic transitions
between energy states result in characteristic spectral
absorptions or emissions. They may include chemical entities
used in conjunction with biosensors. Biotin/avidin or
biotin/streptavidin and alkaline phosphatase detection systems
may be employed.
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The signals generated by individual antibody-reporter
conjugates may be used to derive quantifiable absolute or
relative data of the relevant antibody binding in samples
(normal and test).
The present invention also provides the use of a specific
binding member as above for measuring antigen levels in a
competition assay, that is to say a method of measuring the
level of antigen in a sample by employing a specific binding
member as provided by the present invention in a competition
assay. This may be where the physical separation of bound
from unbound antigen is not required. Linking a reporter
molecule to the specific binding member so that a physical or
optical change occurs on binding is one possibility. The
reporter molecule may directly or indirectly generate
detectable, and preferably measurable, signals. The linkage
of reporter molecules may be directly or indirectly,
covalently, e.g. via a peptide bond or non-covalently.
Linkage via a peptide bond may be as a result of recombinant
expression of a gene fusion encoding antibody and reporter
molecule.
The present invention also provides for measuring levels of
antigen directly, by employing a specific binding member
according to the invention for example in a biosensor system.
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The mode of determining binding is not a feature of the
present invention and those skilled in the art are able to
choose a suitable mode according to their preference and
general knowledge.
The present invention further extends to a specific binding
member which competes for binding to an antigen with any
specific binding member which both binds the antigen and
comprises a V domain including a CDR with amino acid
substantially as set out herein or a V domain with amino acid
sequence substantially as set out herein. Competition between
binding members may be assayed easily in vitro, for example by
tagging a specific reporter molecule to one binding member
which can be detected in the presence of other untagged
binding member(s), to enable identification of specific
binding members which bind the same epitope or an overlapping
epitope. Competition may be determined for example using the
ELISA, e.g. a whole cell ELISA using immobilised brain
endothelial cells, looking for inhibition of signal when cells
are pre-incubated with a potential competitor antibody.
In testing for competition a peptide fragment of the antigen
may be employed, especially a peptide including an epitope of
interest. A peptide may have the epitope sequence plus one or
more amino acids at either end, may be used. Such a peptide
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34
may be said to "consist essentially" of the specified
sequence. Specific binding members according to the present
invention may be such that their binding for antigen is
inhibited by a peptide with or including the sequence given.
In testing for this, a peptide with either sequence plus one
or more amino acids may be used.
Specific binding members which bind a specific peptide may be
isolated for example from a phage display library by panning
with the peptide(s).
The present invention further provides an isolated nucleic
acid encoding a specific binding member of the present
invention. Nucleic acid includes DNA and RNA. In a preferred
aspect, the present invention provides a nucleic acid which
codes for a CDR or VH or VL domain of the invention as defined
above.
The present invention also provides constructs in the form of
plasmids, vectors, transcription or expression cassettes which
comprise least one polynucleotide as above.
The present invention also provides a recombinant host cell
which comprises one or more constructs as above. A nucleic
acid encoding any CDR, VH or VL domain, or specific binding
member as provided itself forms an aspect of the present
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invention, as does a method of production of the encoded
product, which method comprises expression from encoding
nucleic acid therefor. Expression may conveniently be
achieved by culturing under appropriate conditions recombinant
host cells containing the nucleic acid. Following production
by expression a VH or VL domain, or specific binding member
may be isolated and/or purified using any suitable technique,
then used as appropriate.
Specific binding members, VH and/or VL domains, and 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.
Reference to a nucleotide sequence as set out herein
encompasses a DNA molecule with the specified sequence, and
encompasses a RNA molecule with the specified sequence in
which U is substituted for T, unless context requires
otherwise.
Systems for cloning and expression of a polypeptide in a
variety of different host cells are well known. Suitable host
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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. A suitable host where a coding
sequence includes a TAG suppressible stop codon, is a
suppressor strain such as the sup44 strain of TG1. In such a
strain, TAG codons are read as encoding glutamine.
Embodiments of the present invention where a suppressor strain
may be used in production of the encoded polypeptide by
expression from encoding nucleic acid comprising a TAG codon,
include where the coding sequence of any of SEQ ID NO's 37, 43
and 69 are used (respectively encoding G85 VH SEQ ID NO. 38,
G88 VL SEQ ID NO. 44, and 6112 VH SEQ ID N0. 70).
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 Pliickthun, A.
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 specific binding member, see
for recent reviews, for example Ref, M.E. (1993) Curr. Opinion
Biotech. 4: 573-576; Trill J.J. et al. (1995) Curr. Opinion
Biotech 6: 553-560.
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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, Molecular Cloning: a Laboratory Manual: 2nd edition,
Sambrook et al., 1989, Cold Spring Harbor Laboratory Press.
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 Short Protocols in Molecular Biology,
Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992.
The disclosures of Sambrook et al. and Ausubel et al. are
incorporated herein by reference.
Thus, a further aspect of the present invention provides a
host cell containing nucleic acid as disclosed herein. A
still further aspect provides a method comprising introducing
such nucleic acid into a host cell. 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
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bacterial cells, suitable techniques may include calcium
chloride transformation, electroporation and transfection
using bacteriophage.
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.
In one embodiment, the nucleic acid of the invention is
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 present invention also provides a method which comprises
using a construct as stated above in an expression system in
order to express a specific binding member or polypeptide as
above.
Aspects and embodiments of the present invention will now be
illustrated by way of example with reference to the following
experimentation.
ABBREVIATIONS
Immunocytochemistry (ICC)
Interleukin 1-(3 (IL-1(3)
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Enzyme linked immunosorbent assay (ELISA)
Serum amyloid protein (SAP)
Bovine serum albumin (BSA)
Blood brain barrier (BBB)
LIST OF EXAMPLES
EXAMPLE l: Generation of in vitro blood brain barrier cell
culture chambers.
EXAMPLE 2: Selection of antibody-expressing phage which cross
in vitro blood brain barriers.
EXAMPLE 3: Characterisation of selected antibodies by
endothelial cell ELISA and sequencing.
EXAMPLE 4: Demonstration of transport of clonal phage.
EXAMPLE 5: Rat brain immunocytochemistry of endothelial-cell
binding antibody clones.
EXAMPLE 6: Examination of ICC cross-reactivity of rat
endothelial cell-binding antibodies with a panel of human
tissues.
EXAMPLE 7: Selection of SAP-binding antibodies.
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EXAMPLE 8: Characterisation of SAP-binding antibodies on the
basis of sequence and ability to cross a blood brain barrier
in the presence or absence of SAP.
EXAMPLE 1 - GENERATION OF IN VITRO BLOOD BRAIN BARRIER CELL
CULTURE CHAMBERS
(a) Production of primary cell cultures to generate in vitro
blood brain barrier
(i) Preparation of cultures of astrocytes from rat cerebral
tissue
The protocol used for the preparation of separate cultures of
astrocytes was an adaptation of the method described by K.D.
McCarthy and J. De Vellis (J. Cell. Biol., 1980, vol. 85, 890-
902). Using similar methods, astroglial cultures obtained are
reported to appear to be >98% pure by immunocytochemistry
using an antibody to glial fibrillary acidic protein (GFAP).
A summary of this protocol for astrocyte cell culture has been
published (G.M.A. Wells., 1996, in Glia, vol. 18, p. 332 340).
The use of 12 to 24 hour old rat pups ensured the absence of
viable neurons in the cell suspension obtained from
dissociating the cerebral cortex and the ready dissociation of
cells by mechanical sieving techniques rather than using
trypsinisation. The length of the initial culture period was
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kept to between 7 and 9 days to ensure stratification of the
astrocytes, microglia and oligodendrocytes. The procedure for
the separation of individual glial cell cultures relies upon
the selective detachment of other cells from the layer of
firmly attached type 1 astrocytes when the primary cell
culture is exposed to sheer forces on shaking the cultures in
an orbital incubator overnight (approx. 16 hours) at 37°C.
The separated astroglial cell cultures can be maintained for
several weeks enabling investigation of this cell type.
Fifteen 12-24 hour old rat pups were placed in a chamber
containing halothane for approximately 5 minutes, or until
there was no movement from the animals. The neck of each
animal was broken by pinching firmly below the head. 3-4 of
the rat pups were decapitated and the heads were placed
upright on a pad of sterile filter paper so that blood from
the cut was absorbed. A head was dabbed on the filter paper to
remove excess blood, then turned upside down and the skin at
the base of the neck was grasped with fingers. Skin from the
back of the neck was cut to just above the eyes using.
dissection scissors, and the skin folded away from the skull.
Both sides of the skull were then cut from the back of the
neck to just above the eyes and peeled away with forceps. The
optic nerves were detached with a spatula, and the cerebral
hemispheres removed (without the cerebellum) from the cavity
with the same spatula and placed in a sterile 50 ml tube
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containing 20 ml DMEM (containing 4.5 g/1 glucose and 110 mg/1
sodium pyruvate - from Life Technologies) + L glutamine (Life
Technologies) + 20% foetal bovine serum (FBS) (Life
Technologies) + penicillin / streptomycin (Life Technologies).
The cerebral tissue in DMEM was poured from the 50 ml tube
into a sterile petri dish and each individual brain tissue was
removed with forceps onto a pad of sterile filter paper. The
cerebral hemispheres of each brain were parted and
individually rolled across the length of a fresh filter paper
to remove the meninges and blood vessels. These were then
placed in another petri dish containing 20 ml of fresh DMEM
containing 20o FBS. The brain tissue was chopped into very
small pieces using a single edged razor blade whilst immersed
in the DMEM in the petri dish. A 230,um re-usable metal sieve
in its cup shaped holder was placed on the top of a Sterilin
pot and 20 ml of fresh DMEM containing 20o FBS was pipetted
through it. The 20 ml of DMEM containing the chopped brain
tissue was then taken up from the petri dish in a 25 ml
pipette and pipetted onto the sieve. The chopped tissue was
forced through the sieve using the glass tissue. The filtered
cell suspension was divided equally between two 50 ml
centrifuge tubes and centrifuged for 10 minutes at 1000 rpm.
Supernatants were removed and the cell pellets resuspended by
gentle pipetting in a final volume of 20 ml DMEM containing
20o FBS.
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The cell suspension was then passed through a 100 ,um
disposable sieve placed on top of a 50 ml tube and washed
through with 10 ml DMEM containing 20o FBS. The filtered cell
suspension was then passed through a 70 ,um disposable sieve
placed on top of a fresh 50 ml tube and washed through with 10
ml DMEM containing 20% FBS. The resulting filtered cell
suspension was pipetted into a 75 cm tissue culture flask and
enough DMEM containing 20% FBS added so that there was a final
volume of 20 ml per brain used. The cell suspension was mixed
and divided into the relevant number of 75 cm flasks so that
each flask contained 20 ml of cell suspension. The top of
each flask was loosened and the flasks placed in a 37°C
incubator with a 5o CO atmosphere.
The flasks were left undisturbed for 4 days and then
inspected. The old medium and tissue debris was removed, the
cells fed with 20 ml of fresh DMEM containing 20% FBS and left
for a further 3 days in the incubator. On day 7 of the
primary culture flasks were checked for good coverage of total
cells (by this stage the astrocytes should be approaching
confluency). The medium was removed and replaced with 15 ml
of DMEM containing loo FBS. The flasks were returned to the
incubator with loosened tops and left for a further 24-48
hours. Astrocytes were passaged, with a 1:4 dilution of cells,
approximately every week (or upon reaching confluency) over a
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period of several weeks using DMEM containing loo FBS as the
standard growth medium.
(ii) Preparation of cultures of rat brain endothelial cells.
Attempts to establish highly enriched cultures of brain
endothelial cells (EC) from several species have resulted in a
variety of methods based on disruption of cerebral tissue
(usually by a combination of mechanical and enzymatic means),
followed by purification of microvessels by density
centrifugation, before plating out on tissue culture plastic
coated with extracellular matrix protein (for review see F.
Joo, 1992, in J. Neurochem, vol. 58, p. 1 17). The protocol
was adapted from the published methods of C. C. W. Hughes and
P. L. Lantos for the establishment of EC cultures from
microvessels derived from rat cerebral tissue. The use of
only dissected out cerebral grey matter reduces the amount of
myelin in the enzymatic digest of the tissue. Tissue
dissected from two Wistar rats results, digested and purified
using this protocol results in a primary cultures of
microvessels set up in two 6 well dishes from which individual
colonies of endothelial cells can be trypsinised out after 7
days in culture.
The cerebral tissue in DMEM was poured into a sterile petri
dish and each piece of tissue was removed onto a pad of
sterile filter paper. The cerebral hemispheres were parted and
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the meninges and large blood vessels were removed by peeling
off the outer layer of the tissue and rolling the tissue along
the length of the filter paper pad. The grey matter was
dissected away from the white matter, pooled together and
placed into a fresh petri dish containing sterile serum free
DMEM containing penicillin and streptomycin. This was then
chopped up into fine pieces using a single sided razor blade.
The chopped up tissue was then placed into a sterile 50 ml
centrifuge tube and pelleted at 200 g for 10 minutes at room
temperature. The supernatant was removed and the tissue was
resuspended in 10 ml of serum free DMEM containing penicillin
and streptomycin plus 1 mg/ml collagen (Boehringer Mannheim,
cat. no. 269 638) and 10 ,ug/ml DNase I (Sigma Type II, cat.
no. D4263). This was then placed on a shaking incubator at
37°C for 90 minutes. The tissue digest was then pelleted at
200 g for 10 minutes at room temperature and resuspended in 40
ml of DMEM containing 250 (w/v) bovine serum albumin (BSA,
Fraction V from Sigma, cat. no. A9647) with trituration until
it had a creamy texture. This was then spun at 1000 g for 20
minutes at room temperature to pellet the capillary fragments
(heavier) from myelin, astrocytes, neurons and other single
cell contaminants (lighter). The upper layer was carefully
removed making sure to remove all traces of the myelin etc.
The capillary pellet was resuspended in 10 ml serum free DMEM
and re-pelleted at 600 minutes at room temperature. The
resulting capillary pellet was then resuspended in 10 ml DMEM
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containing penicillin and streptomycin plus 1 mg/ml
collagenase/disease and incubated at 37°C for 3 hours with
shaking. The digest was then pelleted at 600 g for 5 minutes
at room temperature and resuspended with trituration in 10 ml
of endothelial cell growth medium (ECGM) which consists of
DMEM containing 4.5 g/1 glucose and 110 mg/ml sodium pyruvate
(Sigma) supplemented with 2 mM L-glutamine (Sigma), 100
units/ml penicillin plus 100 ,ug/ml streptomycin (Sigma), 200
plasma derived foetal calf serum (First Link (UK) Ltd., cat
no. 60 00 850) and 75 /.cg/ml endothelial cell growth supplement
(ECGS Sigma, cat. no. E 2759). The suspension was then passed
through successive 230, 100 and 40 ~cm sterile sieves with
gentle, washing of each sieve with 10 ml of fresh ECGM. The
resulting sieved suspension was plated out into 6 well plates
(3 4 ml per well) coated with type 1 collagen from rat tail
(Becton Dickinson, cat. no. 40236). The 6 well plates and all
flasks used below were coated with 100 ,ug/ml type 1 collagen
(diluted from stock concentration in sterile 0.02N acetic
acid) for 1 hour at room temperature with gentle rocking,
before removing the collagen and rinsing with PBS. Enough
diluted collagen was added to cover the entire surface area of
the tissue culture plastic (>5 ,ug/cmz ). This gave rise to the
primary enriched microvessel cultures from which endothelial
cells were further enriched from potential contaminants as
described below.
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The medium was replaced on the primary cultures with fresh
ECGM after 4 days. Large colonies of cells with endothelial
like morphology were observed after 7 days in culture having
grown out radially in spirals from pieces of microvessel
attached to the tissue culture plastic. Over a period of
several days, colonies of cells, which were thought to be
endothelial cells, were transferred aseptically into fresh 6
well plates (coated with collagen type 1) using small pieces
of filter paper soaked in trypsin/EDTA. After a brief period
in contact with the filter paper, the trypsinised EC stick to
the filter paper after detachment from the well and can be
transferred to another plate. Once these transferred cells
form colonies which had expanded to cover the entire surface
area of the 6 well dish over a period of 3-4 days (passage 1
cells), wells were trypsinised and placed into collagen coated
Tzs flasks (passage 2), cultured for a further 3-4 days and
then passaged into collagen coated T~5 flasks (passage 3).
Once the T75 flasks had reached confluency they were either
frozen down in liquid nitrogen or expanded with two 1_:5
passages to yield a large stock of flasks for preparation of
liquid nitrogen stocks.
(b)Preparation of in vitro blood brain model in tissue culture
inserts
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It has been found that brain ECs can be cultured successfully
on permeable filters that separate an upper from a lower
chamber in the commercially available tissue culture inserts.
As these are available in multi-well formats numerous
conditions can be tested simultaneously to determine the
integrity of the barrier or investigate specific transport
mechanisms. The electrical resistance measured across a
monolayer of cells that are connected to each other by tight
junctions is a sensitive indicator for the paracellular
permeability to ions. Thus the quality of the EC barriers can
be simply recorded to provide assurance for the model. It is
thought that type 1 astrocytes, which normally project to
brain capillary ECs, are able to provide some inductive
influences in making ECs less leaky by inducing the formation
of tight junctions of extremely high electrical resistance.
Various combinations of astrocytes and endothelial cells have
been tried in establishing in vitro models of the BBB. For
good reproducible results the culture of ECs in the upper
chamber on the permeable membranes and astrocytes in the lower
chamber on the base of the companion 24 well plate has been
demonstrated to yield barriers with a high transendothelial
cell resistance. This was the arrangement of cells used as
described in the protocol below.
Cultures of rat astrocytes and endothelial cells (EC) were set
up set up several days/weeks in advance from frozen stocks
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stored in liquid nitrogen. These were kept at as low passage
number as possible to preserve the normal characteristics of
the primary cells (astrocytes at less than passage 6, and
endothelial cells at less than passage 10). After thawing a
vial from liquid nitrogen storage, astrocytes were cultured in
astrocyte growth medium (AGM) consisting of DMEM containing
4.5 g/1 glucose and 110 mg/1 sodium pyruvate (Life
Technologies) supplemented with 2mM L-glutamine (Sigma), l00
foetal bovine serum (FBS)(Life Technologies), 100 units/ml
penicillin plus 100 ~g/ml streptomycin (Sigma). Likewise,
endothelial cells were cultured in endothelial cell growth
medium (ECGM) described in section B consisting of DMEM
containing 4.5 g/1 glucose and 110 mg/ml sodium pyruvate
(Sigma) supplemented with 2 mM L-glutamine (Sigma), 100
units/ml penicillin plus 100 ~g/ml streptomycin (Sigma), 200
plasma derived foetal calf serum (First Link (UK) Ltd., cat
no. 60-00-850) and 75 ~g/ml endothelial cell growth supplement
(ECGS-Sigma, cat. no. E 2759).
Initially in setting up the barriers in the tissue cultures,
astrocytes were placed in the lower chamber and allowed to
adhere before placing the tissue culture insert into the plate
and adding the endothelial cells to the upper chamber. A T1-,s
flask of astrocytes was trypsinised and the number of cells
counted in a haemocytometer. These were then diluted to 1.0 x
105 cells/ml in AGM and 1 ml of cell suspension was added to
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the wells of the 24 well lower plate. The astrocytes were
allowed to adhere to the surface of the lower chamber for at
least 2 hours in a COZ incubator. While the astrocytes were
settling down, the appropriate number of 8 ~.un inserts were
coated with 100 ~g/ml rat tail type 1 collagen before adding
the endothelial cells. The inserts were placed in a separate
lower plate and 1 ml of diluted collagen was added to the
lower chamber and 0.5 ml of collagen was added to the upper
chamber. The inserts were then left for at least 1 hour at
room temperature in a class II tissue culture cabinet. The
collagen was then removed and the inserts were rinsed in PBS.
Once the inserts had been coated with collagen and rinsed
with PBS, a T1,5 flask of EC was trypsinised and the number of
cells counted in a haemocytometer. These were then diluted to
2.0 x 105 cells/ml in AGM. The lower plate containing the
cultures of astrocytes was removed from the incubator and the
medium on the astrocytes was replaced with 1 ml of fresh AGM.
The coated inserts were then placed in the lower plate
containing the astrocyte cultures using forceps sterilised in
70o ethanol and 0.5m1 of the 2.0 x lOSEC/ml suspension was
added to the upper chamber. The plates were placed back in
the COZ incubator and not disturbed for at least 24 hours.
The next day the barrier cultures were inspected under the
microscope. The EC should have formed a near confluent
coating of the upper chamber, and, likewise, the astrocytes in
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the lower chamber should have spread out over most of the
surface area of the lower chamber. At this stage the
electrical resistance of EC barrier can be measured, however,
it takes several days before the resistance rises as the cells
need to reach confluency and then form tight junctions. The
medium in both the upper and lower chambers was replaced with
fresh AGM every 7 days and electrical resistance monitored
with time. High levels of electrical resistance are obtained
after at least 2 weeks in culture. These cultures have been
maintained for up to 8 weeks before any fall in electrical
resistance is observed.
(c) Measurement of electrical resistance of endothelial cell
barrier
Measurement of electrical resistance across the tissue culture
insert membrane can be performed under sterile conditions to
monitor the progress of the endothelial cell cultures in
forming a good model of the blood brain barrier in the
inserts. The use of the Endohm chamber electrodes for the 24
well inserts with its concentric electrodes situated above and
beneath the membrane reduces the background resistance to
between 20 and 30 SZ for an empty 8 ~m tissue culture insert.
This background figure was measured on each occasion and
subtracted from actual readings obtained with inserts
containing EC. Before measuring any resistances, the Endohm
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electrode was sterilised using 70o ethanol. The cup shaped
electrode was filled with 70o ethanol and the upper electrode
placed back in place. The ethanol was then tipped out and the
electrode drained. To remove any final traces of the 700
ethanol the electrode was then rinsed in sterile astrocyte
growth medium (AGM) a couple of times. 1 ml of fresh AGM was
added to the lower chamber of the electrode and the electrode
was ready for measuring the resistances of any inserts. The
resistance of an empty insert was measured at the same time as
the inserts containing EC and this was subtracted from all the
barrier insert resistances. To obtain standardised resistance
measurements, the resistance values recorded on the volt/ohm
meter were divided by the surface area of the membrane (for
the 24 well sized inserts used here this was 0.3cm) to give
values measured in ohms/cmz. Resistances were generally in the
500-600 ohm/cm2 range.
EXAMPLE 2 - SELECTION OF ANTIBODY-EXPRESSING PHAGE WHICH CROSS
IN VITRO BLOOD BRAIN BARRIERS
Antibody repertoire
A large single chain Fv library derived from lymphoid tissues
including tonsil, bone marrow and peripheral blood lymphocytes
was used.
Polyadenylated RNA was prepared from the B-cells of various
lymphoid tissues of 43 non-immunised donors using the
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"Quickprep mRNA Kit" (Pharmacia). First-strand cDNA was
synthesized from mRNA using a "First-strand cDNA synthesis"
kit (Pharmacia) using random hexamers to prime synthesis.
V-genes were amplified using family-specific primers for VH,
V~, and VK genes as previously described (Marks et al., (1991)
J. Mol. Biol. 222:581-597) and subsequently recombined
together with the (Gly9, Ser)3scFv linker by PCR assembly. The
VH-linker-VL antibody constructs were cloned into the Sfi I
and Not I sites of the phagemid vector, pCantab6. Ligation,
electroporation and plating out of the cells was as described
previously (Marks et al, supra). The library was made ca.
1000x larger than that described previously by bulking up the
amounts of vector and insert used and by performing multiple
electroporations. This generated a scFv repertoire that was
calculated to have ca. 1.3 x 101° individual recombinants which
by BstNI fingerprinting were shown to be extremely diverse.
Induction of phage antibody library
The phage antibody repertoire above was selected for
antibodies which cross the blood brain barrier.
The scFv repertoire was treated as follows in order to rescue
phagemid particles. 500 ml prewarmed (37 °-C)2YTAG (2YT media
supplemented with 100 ~g/ml ampicillin and 2o glucose) in a 21
conical flask was inoculated with approximately 3x101° cells
from a glycerol stock (-70°-C) culture of the library. The
culture was grown at 37°-C with good aeration until the OD6oo~m
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reached 0.7 (approximately 2 hours). M13K07 helper phage
(Stratagene) was added to the culture to a multiplicity of
infection (moi) of approximately 10 (assuming that an 600nm of 1
is equivalent to 5 x 108 cells per ml of culture). The
culture was incubated stationary at 37°-C for 15 minutes
followed by 45 minutes with light aeration (200 rpm) at the
same temperature. The culture was centrifuged and the
supernatant drained from the cell pellet. The cells were
resuspended in 500 ml 2YTAK (2YT media supplemented with 100
~g/ml ampicillin and 50 ~g/ml kanamycin), and the culture
incubated overnight at 30°-C with good aeration (300 rpm).
Phage particles were purified and concentrated by one
polyethylene glycol (PEG) precipitation (Sambrook, J.,
Fritsch, E.F., & Maniatis, T. (1990) Molecular Cloning - A
Laboratory Manual Cold Spring Harbor, New York) and
resuspended in 9m1 lOmM Tris containing 1 mM EDTA (TE). 4.0g
of CsCl was added to the phage stock and mixed gently to
dissolve. A 11.5m1 ultracentrifuge tube was filled with phage
at centrifuged at 40 000 rpm at 25gC for 24 hr. The
ultracentrifuge was stopped with the brake off and the clear
opalescent phage band collected using a pasteur pipette. Phage
were dialysed at 4°-C overnight against two changes of 11 of TE,
titred and stored at 4°-C.
Selection of phage from the Large phage library which cross in
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vitro blood brain barriers set up in chamber slides.
The in vitro BBB chambers provide a valuable system which can
be utilised as a means to functionally select for phage
antibodies which cross the in vitro barriers. The phage
antibody library can be placed in the top chamber of the in
vitro barrier and phage which are capable of passing through
the barrier and reaching the bottom reservoir can then be
collected, and the process repeated. This is the basis on
which the following selections were carried out.
(i) First round of selection
1 x 1012 library phage, or control phage which do not bind rat
endothelial cells, in 50 ,u1 TE were added to top reservoir of
endothelial culture chambers containing rat brain endothelial
cells growing as a monolayer in the presence of astrocytes as
described in Example 1. Chambers were incubated at either 37°-C
in a COZ incubator, or on ice on the bench. Media present in
the lower reservoir was collected and replaced with fresh
media at the appropriate temperature 30, 60 and 120 minutes
after addition of the library phage to the top chamber (wells
la, b, c and 7a, b, c). For other chambers transport was
allowed to continue for 30, 60 or 120 minutes and media.
collected only at the final time point. The number of phage
present in the media in the lower chamber (i.e. the number of
transported phage) was titred. Tu ring was achieved by adding
0.5 ml of the media recovered from the bottom chamber (out of
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a total of 1m1) to a 5m1 culture of exponentially growing E.
coli TG1 with light aeration in 2TY broth at 37°-C for 1 hour.
Infected TG1's were plated on 2TYAG medium in 243mm x 243mm
dishes. Dilutions of infected TGls were also plated out and
incubated at 30°-C overnight. Colony counts gave the phage
output titre.
Results of titration experiments from the first round of
selection are shown in Table 1.
Average initial resistance 606 S2cm2.
The phage population from well 7a was chosen as the input
population for a second round of selection.
(ii) Second round of selection
Colonies were scraped off the 7a 243mm x 243mm plate into 3 ml
of 2TY broth and 15% (v/v) glycerol added for storage at -70°-C.
Glycerol stock solutions from the first round of selection
were rescued using helper phage to derive phagemid particles
for the second round of selection. 250 ~1 of the 7a glycerol
stock was used to inoculate 50 ml 2YTAG broth, and incubated
in a 250 ml conical flask at 37°-C with good aeration until the
OD6oo nM reached 0.7 (approximately 2 hours). M13K07 helper
phage (moi=10) was added to the culture which was then
incubated stationary at 37QC for 15 minutes followed by 45
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minutes with light aeration (200rpm) at the same temperature.
The culture was centrifuged and the supernatant drained from
the cell pellet. The cells were resuspended in 50 ml
prewarmed 2YTAK, and the culture incubated overnight at 30°-C
with good aeration. Phage particles were purified and
concentrated by PEG precipitation (Sambrook et al., 1990) and
resuspended in TE to 10'3tu/ml, then caesium banded as
described in Example 2.
Library phage, pCantab6 phage (which is identical library
except it has only myc and his tags fused with the gene III
protein and no scFv) and the second round 7a phage were added
to top reservoirs of in vitro blood brain barrier culture
chambers using 1x1012 phage per chamber in 50 ~,l TE. Chambers
were incubated at 37°-C in a CO2 incubator. The lower reservoir
was removed at 30 min and 500 ~.1 of this used to replace the
top reservoir of a fresh barrier culture. After a further 30
min the lower reservoir of this barrier culture was removed
and 500 ~.1 of the media used to replace the top reservoir of a
fresh barrier culture. This was repeated one final time after
a further 30 min.
Colonies were plated out and titred as described above, and
the results of the titration experiments from the second round
of selection are shown in Table 2.
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Population 24 was chosen as the starting population for a
third round of selection.
(iii) Third round selection
Population 24 from the second round selection regime was
rescued and caesium banded as described above for the 7a
second round population. Library phage, pCANTAB6 phage,
population 7a and population 24 phage were added to the top
reservoir of precooled barrier cultures using 1 x lOlz phage
per chamber in 50 ~.1 TE. Chambers were incubated for 30 min on
ice and washed 3 times with 0.5 ml of ice cold media. Insert
and liquid within the upper chamber were then transferred to a
fresh chamber well containing 1 ml of media at 37°-C and the
chambers incubated at 37°-C in a COz incubator. The lower
reservoir was removed at 30 min and 500 ~.l of this used to
replace the top reservoir of a fresh barrier culture. After a
further 30 min the lower reservoir of this barrier culture was
removed and 500 ~1 of the media used to replace the top
reservoir of a fresh barrier culture. This was repeated one
final time after a further 30 min and the barrier culture
incubated for 30 min at 37qC.
Colonies were plated out and titred as described above (Table
3) .
Growth of single selected clones for immunoassay
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Individual colonies from the third round selection (chambers
25, 26 and 29) were used to inoculate 100 ~12YTAG into
individual wells of 96 well tissue culture plates (Corning).
Plates were incubated at 30°-C overnight with moderate shaking
(200 rpm). Glycerol to 15 o was added to each well and these
master plates stored at -70gC until ready for analysis.
EXAMPLE 3 - CHARACTERISATION OF SELECTED ANTIBODIES BY
ENDOTHELIAL CELL ELISA AND SEQUENCING
Endothelial cell phage ELISA
Selected phage were analysed by phage ELISA for their ability
to bind to culture rat endothelial cells. Phage ELISAs were
carried out as follows: individual clones were picked into a
96 well tissue culture plate containing 100 ~1 2YTAG. Plates
were incubated at 37°-C for 6 hours. M13K07 helper phage was
added to each well to an moi of 10 and incubated with gentle
shaking for 45 min at 37°-C. The plates were centrifuged at 2000
rpm for 10 min and the supernatant removed. Cell pellets were
resuspended in 100 ~1 2TYA with kanamycin (50 ~g/ml) and
incubated at 30°-C overnight. Each plate was centrifuged at 2000
rpm and the 100 ~,1 phage-containing supernatant from each well
recovered and blocked in 20 ~1 6x PBS containing 18o marvel
stationary at room temperature for 1 hour. Meanwhile, 96 well
tissue culture plates, containing either 1 x 105 endothelial
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cells per well or control uncoated plates, were blocked for 2
h stationary at room temperature in PBS containing 3o Marvel
(3MPBS). These plates were then washed three times with PBS
and 50 ~1 preblocked phage added to each well. The plates
were incubated stationary at room temperature for 1 h after
which the phage solutions were poured off. The plates were
washed by incubating for 2 min in three changes of PBS at room
temperature.
To the ELISA plate well, 100 ~l of a 1 in 5000 dilution of the
anti-gene8-HRP conjugate (Pharmacia) in 3MPBS was added and
the plates incubated at room temperature stationary for 1 h.
Each plate was washed as described. 100 ~1 of TMB substrate
was then added to each well, and incubated at room temperature
for approximately 30 minutes, after which the colour reaction
was stopped by the addition of 50 ~1 of 1M H2S09. The
absorbance signal generated by each clone was assessed by
measuring the optical density at 405 nm using a microtitre
plate reader. Clones were chosen for further analysis if the
ELISA signal generated on the cell-coated plate was at least
double that of the uncoated plate. Of 96 clones screened from
the third round of selection 20 were positive by endothelial
cell ELISA.
Sequencing of anti-endothelial cell ScFv Antibodies
The nucleotide sequences of the rat endothelial cell binding
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antibodies were determined by first using vector-specific
primers to amplify the inserted DNA from each clone. Cells
from an individual colony on a 2YTAG agar plate were used as
the template for a polymerase chain reaction (PCR)
amplification of the inserted DNA using the primers
pUCl9reverse and fdtetseq. Amplification conditions consisted
of 30 cycles of 94°-C for 1 min, 55°-C for 1 min and 72°-
C for
2min, followed by 10 min at 72°-C. The PCR products were
purified using a PCR Clean-up Kit (Promega) in to a final
volume of 50 ~lHzO. Between 2 and 5 ~1 of each insert
preparation was used as the template for sequencing using the
Taq Dye-terminator cycle sequencing system (Applied
Biosystems). The primers pUCl9reverse and fdtseq were used to
sequence the heavy and light chain of each clone respectively.
The diversity of the CDR3s and the germline families is shown
in Table 4. Detailed sequence data is included below.
EXAMPLE 4 - DEMONSTRATION OF TRANSPORT OF CLONAL PHAGE.
Four phage antibody clones, that gave positive endothelial
cell ELISA results and gave endothelial specific staining by
ICC on rat brain, were tested for their ability to cross the
in vitro blood brain barrier as clonal preparations. Phage
were prepared by rescue and caesium banding as described
Example 2. 1x108 phage in 50 ~1 TE were added to the upper
reservoir of in vitro blood brain barrier chamber cultures
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which had been pre-chilled on ice. The phage were allowed to
bind to the endothelial cell layer for 30 min on ice and the
cell layer then washed 3 x with 1m1 ice cold media. The
chamber inserts were transferred to media prewarmed to 37-°C and
incubated at 37°-C in a COZ incubator to allow transport to
proceed. Phage crossing the barrier were titred, as described
above, following the 30 min on ice then following 30 at 37°-C
(Table 5).
In the cases of the four test clones (G65, G73, G77 and G93)
the number of phage actively crossing the barrier after 30
minutes at 37-°C compared with the number of phage crossing
non-specifically at 4°-C was greatly increased. G65 showed a
12-fold increase over background transport (No. phage crossing
at 37°-C / No. of phage crossing at 4°-C), G73 a 2.6-fold
increase, G77 a 12.8 fold increase and G93 a 4.3-fold
increase. The control phage pCantab6 which expresses no
surface scFv showed no increase in transport at 37°-C over
transport at 4-°C .
EXAMPLE 5 - RAT BRAIN IMMUNOCYTOCHEMISTRY OF ENDOTHELIAL-CELL
BINDING ANTIBODY CLONES
The twenty clones identified by cell ELISA as binding to
activated rat endothelial cells were also tested by
immunocytochemistry (ICC) for their ability to bind to
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inflamed rat brain sections.
Preparation of rat brain sections.
3 week-old Lewis rats were anaesthetised with avertin (1m1 /
100g) and placed in a sterotaxic frame. Injections of 1~1 of
interleukin-1(3 (10 units) were placed into,the striatum. An
incision was made in the scalp to expose bone and a 2-mm
diameter burr hole was drilled through the skull to allow the
tip of a finely drawn calibrated glass capillary tube to be
inserted. Rats were deeply anaesthetised with sodium
pentobarbitone and transcardialy perfused with 100m1 of saline
followed by 200m1 of Karnovsky's fixative (1.250
gluteraldehyde and 1.250 paraformaldehyde in phosphate
buffer). The animals were recovered from the anaesthetic
before being killed. The brain was removed and cryoprotected
in 30o sucrose overnight at 4°-C before being embedded in
Tissue-Tek (Miles Inc, Elkhart, USA) and quickly frozen in
liquid nitrogen. 5 micron cryosections were taken of treated
brains.
Preparation of phage for ICC
Phage clones were inoculated into lml 2TYGA in a deep well
microtitre plate and grown at 37°-C with aeration for 5 hours.
M13K07 helper phage was added to each well at an moi of 10 and
incubated with gentle shaking for 45 min at 37sC. The plates
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were centrifuged at 2000 rpm for 10 min and the supernatant
removed. Cell pellets were resuspended in lml 2TYKA and
incubated at 25gC overnight. Plates were centrifuged at 2000
rpm for 10 min and the phage supernatant collected from each
well ready for use in ICC.
ICC protocol
Rat brain sections were fixed by immersion in acetone at
ambient temperature for 15 min, and washed twice for 3 min in
PBST. Sections were blocked in 5 ~g/ml streptavidin in PBST
for 15 min, washed 3 times 3 min in PBST and incubated in 10
~g/ml biotin in PBST for 15 min. Sections were washed 5 times
3 min in PBST, then twice for 10 min in wash containing to
BSA. to BSA was added to the phage supernatants and phage
incubated on the sections for 2 hr at ambient temperature.
Slides were washed 5 times for 3 min in PBST and incubated
with an anti-M13-HRP conjugate (Pharmacia) diluted 1/500 in
PBST containing BSA. Sections were washed 5 times 3 min in
PBST and a biotin tyramine amplification step then carried
out. Biotin tyramine amplification consisted of incubation of
the section with biotin tyramine diluted 1/600 in 50mM
Tris-HC1, pH 7.4 containing 0.030 hydrogen peroxide for 10 min
at room temperature, after which the slides was washed in
twice for 3 min in PBST. Sections were then incubated for 30
min in SABC-HRP complex (DAKO K0377) diluted in PBST, and then
washed 5 times for 3 min in PBST. Sections were stained by
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incubation with 3-amino-9-ethyl-carbazole (AEC, Sigma) at a
concentration of 2.4 mg/ml in dimethylformamide. ACE
incubation was for 3 min followed by washing in 0.1% tween,
and this was repeated twice. Slides were further washed in
0.1% tween twice for 5 min and the slides then counterstaining
with haemotoxylin (DAKO) for 10 sec. Seven changes of 3 min
washing in water were then carried out and the sections coated
in aqueous mount.
Summary of results.
18 of the 20 ELISA positive clones gave staining of vessel
walls in rat brain sections. Profiles varied in intensity of
staining. No staining was observed in tissues other than
vessels. Control sections in which phage antibody was omitted
gave no specific staining of the rat brains.
EXAMPLE 6 - EXAMINATION OF ICC CROSS-REACTIVITY OF RAT
ENDOTHELIAL-BINDING PHAGE ANTIBODY CLONES ON A PANEL OF HUMAN
TISSUES
The rat endothelial cell clones positive by ICC on rat brain
were tested on a range of normal and diseased human tissues
for cross-reactivity.
Preparation of phage and ICC.
Phage were prepared for ICC as described in Example 5. The
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panel of human tissues prepared for ICC consisted of:
Normal tissues Diseased tissues
Tonsil Adenocarcinoma of breast
Kidney Cirrhosis of liver
Cerebellum Lung carcinoma
Spinal cord Crohn's disease
Peripheral nerve Ulcerative colitis
Cerebrum
Striated muscle
Testis
Skin
Spleen
Lung
Breast
Liver
Heart
Myometrium
ICC results
ICC was carried out exactly as described in Example 5. Six
(G65, G77, 6102, G81, 6101, 6112) out of the 18 clones tested
gave no specific staining on the panel of human tissues.
clones gave staining in the cerebellum: G73 was cerebellum
specific; 6110 stained adenocarcinoma and striated muscle in
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addition to cerebellum; G95 stained smooth muscle in addition
to cerebellum; G93 stained spinal cord in addition to
cerebellum; G92 stained spinal cord and cerebrum in addition
to cerebellum.
G76 stained peripheral nerve and cell bodies in spinal cord.
Of the remaining clones a number of other staining patterns
were observed. G88 gave specific staining of lung carcinoma.
G83 stained kidney glomeruli and leydig cells of the testis.
G92 gave staining of the leydig cells of the testis, plus
other punctate staining of the testis, and some staining of
striated muscle.
ICC staining patterns are summarised in Table 6.
EXAMPLE 7 - SELECTION OF SAP-BINDING ANTIBODIES
Serum Amyloid Protein (SAP) is specifically transported across
the blood brain barrier. Phage antibodies binding to. human
SAP (hSAP) were isolated, as outlined below, and a phage
antibody clone (D5) which gave a strong signal by ELISA on
hSAP was examined for its ability to be transported across an
in vitro BBB.
Isolation of Phage binding to hSAP.
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hSAP was coated onto the surface of a Maxisorp tube using 1 ml
of 10~g/ml in PBS overnight at 4°-C. After coating the Maxisorp
tube was rinsed three times with PBS and filled to the brim
with 30 (w/v) skimmed milk powder in PBS (3MPBS) and blocked
for 1h at 37qC. The library phage were blocked at room
temperature for 1h with 3MPBS. The coated Maxisorp tube was
rinsed three times with PBS, and 1 ml of pre-blocked phage was
added to the Maxisorp tube. Phage was incubate for 1 h at 37-°C,
and the tube then rinsed twenty times with PBST, then twenty
times with PBS. The bound phage were eluted by the addition
of 1 ml of freshly made 100 mM triethylamine to the Maxisorp
tube. The tube was incubated (stationary) at room temperature
for 10 min and eluted phage were transferred to a 1.5 ml
microcentrifuge tube. The phage were neutralised by the
addition of 500.1 1 M Tris buffer pH 7.4. A single colony of
E. coli. TG1, taken from a minimal agar plate, was used to
inoculate 50 ml of 2TY broth and incubated with shaking at 37°-C
until an A.6oo of 1.0 had been achieved. 5 ml of the
exponentially growing TG1 were placed in a 50 ml Falcon tube,
7501 (half) of eluted phage added and infection carried out
by incubation at 37°-C for 30 min stationary and for 30 min
shaking (<200 r.p.m.). The remainder of the eluted phage was
stored at 4gC as a back-up. The infected cells were centrifuged
3500 r.p.m. for 10 min and the cell pellet resuspended in 0.6
ml 2TY broth and spread on one 243 x 243 mm 2TYAG agar plate.
Colonies were grown at 30°-C overnight.
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The colonies were scraped into 10 ml of 2TY broth in a 50 ml
Falcon tube, 5 ml of sterile 50o v/v glycerol was added and
mixed by placing tube on an end-over-end rotator for 10 min at
room temperature. 25 ml of 2TYG + 100~.g/ml ampicillin was
inoculated with around 501 of the above plate scrape and
grown at 37°-C to an A6oo of 0.5 to 1Ø M13K07 helper phage was
added to a final concentration of 5 x 10g pfu/ml and the cells
infected at 37°-C for 30 min stationary and for 30 min shaking
(<200 r.p.m.). The cells were transferred to a 50 ml Falcon
tube and centrifuged at 3500 r.p.m. for 10 min then the
bacterial pellet resuspended in 25 ml prewarmed 2TY with
kanamycin (50~g/ml) and ampicillin (100~.g/ml). This was
transferred to a fresh 250 ml flask and grown overnight with
rapid shaking (300 r.p.m.) at 30°-C to produce phage particles.
Approximately 1 ml of culture was transferred to a 1.5 ml
microcentrifuge tube and centrifuged at 13000 r.p.m. in a
microfuge. The phage-containing supernatant was transferred to
a fresh tube and used as the input population for a second
round of selection which was performed exactly as described
above.
Screening of selected clones by phage ELISA on hSAP.
Individual colonies were picked into 100.1 of 2TYAG in a 96
well microtitre plate and grown at 30°-C shaking at 100 rpm
overnight. The cultures in this plate were used to inoculate a
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fresh plate and 501 of 50% v/v glycerol was added to the
original plate and it was stored frozen at -70~C. The fresh
plate was grown at 37gC for 5-6 hrs until the cultures were
turbid. To each well of the replica plate 101 of M13K07 in
2TYG + 100ug/ml ampicillin (at 5 x 101° pfu/ml, an m.o.i. of
10) was added. The plate was incubated for 30 min without
shaking at 37°-C and then for 30 min with shaking (100 r.p.m.)
at 37°-C to allow superinfection of the helper phage. The
cultures were pelleted at 2,000 rpm for 10 min and the
supernatant discarded. The bacterial pellets were resuspended
in 1001 of 2TYAK and grown overnight at 30°-C shaking at 100
rpm.
The cultures were centrifuged at 2,000 rpm for 10 min and the
supernatant transferred to a fresh plate. The phage was
blocked by the addition of 201 of 6 x PBS/18% Marvel, mixed
by pipetting and incubated for 1 h at room temp.
Antigen plates were prepared by adding 1001 of 10~g/ml hSAP
in PBS to microtitre plate wells and incubating overnight at
4°-C. Plates were washed 3 x in PBS and blocked for 1 h in 30
MPBS at 37°-C. Phage ELISAs were performed as described in
Example 3.
Approximately 30% of the phage antibody clones screened by
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71
ELISA were found to be positive in terms of binding to hSAP
compared to BSA or an uncoated ELISA plate. One clone, D5,
which gave the highest ELISA signal was chosen for further
transport studies. Clone D5 was sequenced (as described in
Example 3) and the relevant sequences are included below (SEQ
ID N0. 73 encoding VH domain SEQ ID NO. 74; SEQ ID N0. 75
encoding VL domain SEQ ID N0. 76).
EXAMPLE 8: CHARACTERISATION OF THE SAP-BINDING PHAGE ANTIBODY
DS ON THE BASIS OF ITS ABILITY TO CROSS AN IN VITRO BBB IN THE
PRESENCE OR ABSENCE OF SAP
Experiments were carried out in serum free media to avoid
competition between any endogenous SAP and the added hSAP.
Phage, either D5 or pCANTAB6, were added to the upper
reservoir of ice cold BBB cultures in the presence or absence
of 10~g/ml hSAP at 108, 106, or 10' phage per chamber. The
cultures were incubated on ice for 30 min and the liquid in
the lower reservoir then removed for titring. The upper
chamber was washed 3 x with 0.5 ml of ice-cold media.then
transferred to a fresh well and incubated at 37°-C in a COZ
incubator for 30 min. Liquid from the lower reservoir was
again removed and the phage titre present determined by
titration (Table 7).
Transport of the D5 phage is increased approximately two fold
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in the presence of hSAP whereas the transport of pCANTAB6 is
reduced slightly in the presence of hSAP. Overall the D5 phage
were transported 10-fold more efficiently than the pCantba6
phage in the absence of SAP, and 100-fold more efficiently in
the presence of SAP. This provides indication that the D5
phage are being actively transported, and the higher level of
D5 transport in the absence of SAP may be a result of
endogenous SAP which is pre-bound to the rat endothelial
cells.
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Table 1.
37C
Timewell phage titre Resistance
(min) S2/cmZ
30 l a library 2.5x 10"
60 1b library 2.5x10"
120 lc library 2.5x10 310
60 2 library 2.5x10"
120 3 library 2.5x10
30 4 Fat67 1.5x10
60 5 Fat67 2.5x10"
120 6 Fat67 2.5x 10 453
4C
Timewell phage titre Resistance
(min) S2/cmz
30 7a library 4x10"
60 7b library 4x10
120 7c library 3x10 500
60 8 library 4x10
120 9 library 4x 10
30 10 Fat67 1x10
60 I1 Fat67 1x10"
120 12 Fat67 2x 10 500
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74
Table 2.
WellInput phage Output titreInitial resistance
~?Jcm2
16 Library 3.8x10' 750
17 1/2 of 16 20 816.7
output
18 1/2 of 17 0 670
output
19 1/2 of 18 0 573.3
output
20 pCANTAB6 3x10 750
21 1/2 of 20 0 790
output
22 1/2 of 21 0 716.7
output
23 1/2 of 22 0 716.7
output
24 Population 6x10 690
7a
25 1/2 of 24 34 790
output
26 1/2 of 25 0 530
output
27 1/2 of 26 0 N.D.
output
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Table 3.
WellInput phage Time Output titreInitial Final resistance
(min) resistancef?J cmZ
d2/cm2
16 Library 30 1.2x10' 603 583
17 1/2 of 16 output30 300 647 496
18 1/2 of 17 output30 5 647 750
19 1/2 of 18 output30 0 613 690
20 pCANTAB6 30 1.5x10' 660 570
21 1/2 of 20 output30 0 647 633
22 I/2 of 21 output30 0 593 706
23 1/2 of 22 output30 0 587 720
24 Population 7a 30 2.6x10' 637 643
25 1/2 of 24 output30 2.4x10' 620 660
26 I/2 of 25 output30 8 647 753
27 1/2 of 26 output30 0 640 750
28 Population 24 30 5.2x10a 670 663
29 1/2 of 28 output30 1x10" 620 650
30 I/2 of 29 output30 0 627 720
31 1/2 of 30 output30 0 680 676
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76
Table 4.
CloneVL VL CDR3 SEQ VH VH CDR3 SEQ
ID ID
NO. NO.
G65 DPL16 NSRDSSGNHVV 77 DP-49 TGEYSGYDTSGVEL 78
G67 DPL16 HSRDSSGNHVL 79 DP-35 DFVATMVRGAPTRVLRS 80
G73 DPL16 NSRDSSGNHVV 81 V3-30 TGEYSGYDTSGVEL 82
G76 DPL16 NSRDSSGNHVV 83 DP71 GPLRLRAFDL 84
G77 DPL8 QSYDSSLSNMI 85 DP-5 ASETRVMVDDVFNV 86
G78 DPL16 NSRDSSGNHVV 87 DP71 GPLRLRAFD 88
G79 DPL16 HSRDSSGNHVL 89 DP-7 RSGALGGLIPLNYFDY 90
G81 DPL11 NSRDSSGNHVV 91 VII-5+ VTNGHWYYFDY 92
G83 Ll2a+ QQSYSTPWT 93 DP-71 AASLSCTGSSCRYNYFDP 94
G85 DPK4 ENYNSVPLS 95 DP-72 GDGSDYYAMDY 96
G88 Ll2a+ QQYSNYPLT 97 2M27 EGTAIHRAFDI 98
G92 DPL2 ASWDDSLNGRV 99 DP-71 VGVVVTGRGAFDI 100
G93 DPL11 SSYTTRGTRV 101 DP7 GRGARDDGFDV 102
G95 Ll2a+ QQYSNYPLT 103 DP-75 GEIFDY 104
6101DPL16 HSRDSSGNHVL 105 V2-1 GTSFSSSWYSNYFFYYYIDL106
6102DPL11 SSYTTRSTRV 107 DP-63 VGQYNYLHAFYLEY 108
6110Ll2a+ QPYSNDALT 109 DP-75 GRSLTIG 110
6112Ll2a+ QQSYSTPWT 111 DP-73 RWKGHFDY 112
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77
Table 5.
Phage Time Output Resistance
titre S2/cm
BeforeAfter
Clone 65 30 min @ 4C 1 x 10' 613.3
30 min @ 37C 1.2x103 493.3
Clone 73 30 min @ 4C 3.6x10" 640.0
30 min @ 37C 9.2x10 486.7
Clone 77 30 min @ 4C 4.7x10' 593.3
30 min @ 37C 6.0x103 506.7
Clone 93 30 min @ 4C 8.4x10' 586.7
30 min @ 37C 3.6x103 470.0
pCANTAB6 30 min @ 4C 1.5x10" 626.7
30 min @ 37C 8.5x103 526.7
CA 02393292 2002-05-31
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78
a~
U II
>~ ~ ai
rr~ . . , , , . . . . + . , + , + .
, H
ria , , , . , . , . , ,
, , , , , ,
0
U
II
, , , , , , , , , , , , , , , , II
, U~
on
~o ~ . , , , , , , , , . . . + + , +
,
a~ .fl
o .~
U
~ .'~
v
, , , , , , , , , , , , , , , , ,,.,
+ ~ U
O
U ~ ~
II
~ a
, , , . . , . . . . , , . + , e2 II
~U~
o
'b ~
~, a
. , , , . + + , . , , . o
, , , , , ...
o .~ ~
II
H , , , . . . . . . . + , . . . .
.
U
O
~ b4
, , , , , , , + , , , + + , , ,
~
II
c~ U
U , . , , , . . + , + + . , + + + 11
.
~ o
d ~
a
.~ a
~ .x
~
" , . " , . . " , . . " . .
+ + + + + + + + + + + + + + + + ~ o
+
.-r N o II II
O V7 l~ O ~ O .-~ t!7 M 01 ~ t~ M ~
N ~!1 M ~~ 00
'O (w .~ 00 .-..m.., 00 O~ (~ I~ N
U ~D 00 01 01 I~ .~ 00 ~ H
C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 ~
C7 C7 C7 C7 C7 C7
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79
Table 7.
Phage Titre added Output after
30 min at 37
C
DS 1x10 6.2x10"
1x10 5.6x10'
1x10" 45
DS + SAP 1x10 1.1x10'
1x10 1.5x10'
1x10" 50
pCANTAB6 1 x 10 2.6x 10'
1x10 20
1x10" 0
pCANTAB6 1 x 10 2.0x 10'
+ SAP 1x10 15
1x10" 0
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SEQUENCE LISTING
G65 VH
SEQ ID N0. 1 encoding SEQ ID N0. 2
CAGCTGGTGCAGTGTGGGGAGTCGTGGTACAGCCTGGGGGGGTCCCTGAGACTCTACTGT
Q L V Q C G E S W Y S L G G S L R L Y C
GCATGCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGC
A C S G F T F S S Y G M H W V R Q A P G
AAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTATTAAATACTATGCAGAC
K G L E W V A V I S Y D G S I K Y Y A D
TCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAA
S V K G R F T I S R D N S K N T L Y L Q
ATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGCGAACTGGTGAATAT
M N S L R A E D T A V Y Y C A R T G E Y
AGTGGCTACGATACGAGTGGTGTGGAGCTCTGGGGCCAGGGAACCCTGGTCACCGTCTCC
S G Y D T S G V E L W G Q G T L V T V S
TCA
S
G65 VL
SEQ ID NO. 3 encoding SEQ ID N0. 4
TCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACA
S E L T Q D P A V S V A L G Q T V R I T
TGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAG
C Q G D S L R S Y Y A S W Y Q Q K P G Q
GCCCCTGTACTTGTCATCTGTGGTAATAACACCCGGCCCTCAGGGATCCCAGACCGATTC
A P V L V I C G N N T R P S G I P D R F
TCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCCTCACTGGGGCTCAGGCGGAAGAT
S G S S S G N T A S L T L T G A Q A E D
GAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACCATGTGGTATTCGGCGGA
E A D Y Y C N S R D S S G N H V V F G G
GGGACCAAGCTGACCGTCCTAGGT
G T K L T V L G
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2
G67 VH
SEQ ID N0. 5 encoding SEQ ID N0. 6
GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTC
E V Q L V Q S G G G L V K P G G S L R L
TCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGCGTTGGGTCCGCCAGGCG
S C A A S G F T F S D Y Y M R W V R Q A
CCAGGGAAGGGGCTGGAGTGGCTTTCATACATTAGTCCTGATTCTAGTATCACAAAATAC
P G K G L E W L S Y I S P D S S I T K Y
GCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACACGCTGTAT
A D S V K G R F T I S R D N A K N T L Y
CTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGATTTC
L Q M N S L R A E D T A V Y Y C A K D F
GTAGCTACTATGGTTCGGGGAGCCCCCACTAGGTACTTCGATCTCTGGGGCAGAGGGACA
V A T M V R G A P T R Y F D L W G R G T
ATGGTCACCGTCTCGAGT
M V T V S S
G67 VL
SEQ ID NO. 7 encoding SEQ ID N0. 8
TCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACT
S E L T Q D P A V S V A L G Q T V R I T
TGCCAAGGAGACAGTCTCAGAAGCTATTACACAAACTGGTTCCAGCAGAAGCCAGGACAG
C Q G D S L R S Y Y T N W F Q Q K P G Q
GCCCCTCTACTTGTCGTCTATGCTAAAAATAAGCGGCCCTCAGGGATCCCAGACCGATTC
A P L L V V Y A K N K R P S G I P D R F
TCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGAT
S G S S S G N T A S L T I T G A Q A E D
GAGGCTGACTATTACTGTCATTCCCGGGACAGCAGTGGTAACCATGTGCTTTTCGGCGGA
E A D Y Y C H S R D S S G N H V L F G G
GGGACCAAGCTGACCGTCCTAGGT
G T K L T V L G
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3
G73 VH
SEQ ID N0. 9 encoding SEQ ID NO. 10
CAGGTGCAGCTGGTGCAATCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTC
Q V Q L V Q S G G G V V Q P G R S L R L
TTCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCT
F C A A S G F T F S S Y G M H W V R Q A
CCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTATTAAATACTAT
P G K G L E W V A V I S Y D G S I K Y Y
GCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTAT
A D S V K G R F T I S R D N S K N T L Y
CTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCGGTGTATTACTGTGCGCGAACTGGT
L Q M N S L R A E D T A V Y Y C A R T G
GAATATAGTGGCTACGATACGAGTGGTGTGGAGCTCTGGGGCAGGAGGACAATGGTCACC
E Y S G Y D T S G V E L W G R R T M V T
GTCTCTTCA
V S S
G73 VL
SEQ ID N0. 11 encoding SEQ ID N0. 12
TCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACA
S E L T Q D P A V S V A L G Q T V R I T
TGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAG
C Q G D S L R S Y Y A S W Y Q Q K P G Q
GCCCCTGTACTTGTCATCTATGGTAATAACAACCGGCCCTCAGGGATCCCAGACCGATTC
A P V L V I Y G N N N R P S G I P D R F
TCTGGCTCCAGCTCAGGATACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGAT
S G S S S G Y T A S L T I T G A Q A E D
GAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACCATGTGGTATTCGGCGGA
E A D Y Y C N S R D S S G N H V V F G G
GGGACCAAGCTGACCGTCCTAGGT
G T K L T V L G
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4
G7 6 VH
SEQ ID N0. 13 encoding SEQ ID N0. 19
CAGGTGCAGCTACAGCAGTGGGGGCCAGGACAGGTGAAGCCTTGGGAGACCCTGTCACTC
Q V Q L Q Q W G P G Q V K P W E T L S L
ACCTGCAGTATCTCTGGTGGCTACATCAATTCGCACTACTGGAATTGGATCAGACAGCCC
T C S I S G G Y I N S H Y W N W I R Q P
CCAGGCAAGGGACTCGAGTGGATTGGATATATCTATTACAGTGGGACCACCAACTACAAC
P G K G L E W I G Y I Y Y S G T T N Y N
CCCTCCCTCAAGAGTCGAGTCTCTATATCAATAGACACGTCCAAGAACCAGGTCTCCCTA
P S L K S R V S I S I D T S K N Q V S L
AAGTTGAGTTCTGTGATCGCCGCAGACACGGCCGTCTATTTTTGTGCGAGAGGTCCATTA
K L S S V I A A D T A V Y F C A R G P L
CGATTACGCGCTTTTGATCTGTGGGGCCAAGGCACCCTGGTCACCGTCTCGAGT
R L R A F D L W G Q G T L V T V S S
G7 6 VL
SEQ ID N0. 15 encoding SEQ ID N0. 16
TCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACA
S E L T Q D P A V S V A L G Q T V R I T
TGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAG
C Q G D S L R S Y Y A S W Y Q Q K P G Q
GCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTC
A P V L V I Y G K N N R P S G I P D R F
TCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGAT
S G S S S G N T A S L T I T G A Q A E D
GAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACCATGTGGTATTCGGCGGA
E A D Y Y C N S R D S S G N H V V F G G
GGGACCAAGCTGACCGTCCTAGGT
G T K L T V L G
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G77 VH
SEQ ID N0. 17 encoding SEQ ID N0. 18
GAGGTCCAGCTGGGACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTC
E V Q L G Q S G A E V K K P G A S V K V
TCCTGTAAGGTTTCCGGAGACACCCTCAGTGAATTCTCCATACACTGGGTGCGACAGGCT
S C K V S G D T L S E F S I H W V R Q A
CCTGGAAAAGGCCTTGAGTGGATGGGAGGTTTCGATCCTGAAGATGTTGAGATTACCTAC
P G K G L E W M G G F D P E D V E I T Y
GCACAGAAGTTTCAGGGCAGAGTCACCATGACCGAGGACACATCTACAGAGACAGCCTAC
A Q K F Q G R V T M T E D T S T E T A Y
ATGGAGCTGCGCAGCCTGAGACCTGAGGACACGGCCGTGTATTATTGTGCAACTGCCTCT
M E L R S L R P E D T A V Y Y C A T A S
GAGACCCGGGTCATGGTTGATGATGTTTTTAATGTCTGGGGCCGAGGGACAATGGTCACC
E T R V M V D D V F N V W G R G T M V T
GTCTCGAGT
V S S
G77 VL
SEQ ID NO. 19 encoding SEQ ID NO. 20
CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGGGACCCCCGGGCAGAGAGTCACCATC
Q S V L T Q P P S V S G T P G Q R V T I
TCTTGTTCTGGAGGCAGATCCAACATCGGCAGTAATACTGTAAAGTGGTATCAGCAGCTC
S C S G G R S N I G S N T V K W Y Q Q L
CCAGGAACAGCCCCCAAACTCCTCATCTATGGTAACAGCAATCGGCCCTCAGGGGTCCCT
P G T A P K L L I Y G N S N R P S G V P
GACCGCTTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAG
D R F S G S K S G T S A S L A I S G L Q,
GCTGAGGATGAGGCTCATTATTACTGCCAGTCCTATGACAGCAGCCTGAGTAATATGATA
A E D E A H Y Y C Q S Y D S S L S N M I
TTCGGCGAAGGGACCAAGCTGACCGTCCTAGGT
F G E G T K L T V L G
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6
G7 8 VH
SEQ ID N0. 21 encoding SEQ ID N0. 22
CAGGGGCAGCTACAGGAGTGGGGGCCAAGACAGGTGAAGCCTTGGGAGACCCTGGCACTC
Q G Q L Q E W G P R Q V K P W E T L A L
ACCTGCAGTATCTTTGGTGGCTACATCAATTCGTACTACTGGAATTGGATCACACAGCCC
T C S I F G G Y I N S Y Y W N W I T Q P
TCAGGCAAGGGACTCGAGTGGATTGGATATATCTATTACAGTGGGACCACCAACTACAAC
S G K G L E W I G Y I Y Y S G T T N Y N i
CCCTCCCTCAAGAGTCGAGTCTCTATATCAATAGACACGTACAAGAACCAGGTCTCCCTA
P S L K S R V S I S I D T Y K N Q V S L
AAGTTGAGTTCTGAGATCTGCCGCAGTACACGTGCCGTCTATTTTTGTGCGAGAGGTCCA
K L S S E I C R S T R A V Y F C A R G P
TTACGATTACGCGCTTTTGATCTGTGGGGCCAAGGCACCCTGGTCTCCGTCTCGAGT
L R L R A F D L W G Q G T L V S V S S
G78 VL
c
SEQ ID N0. 23 encoding SEQ ID N0. 24
GAGCTGACTCAGGACCTTGCTGTGTCTGTGGCCTTGGCACTGACAGTCAGGATCACATGC
E L T Q D L A V S V A L A L T V R I T C
CAAGTAGACAGCCTCAATAGCTATTATGCCAGCTGGTGCCAGCAGAAGCCAGCACAGGCC
Q V D S L N S Y Y A S W C Q Q K P A Q A
CCTGCACTTGTCATCTATGGTAAAAACTCCCGGCCCTCAGGGATCCCAGCCCGATTCTCT
P A L V I Y G K N S R P S G I P A R F S
GGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTAGGGCTCAGGCGGAAGATGAG
G S S S G N T A S L T I T R A Q A E D E
GCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACCATGTGGTATTCGGCGGAGGG
A D Y Y C N S R D S S G N H V V F G G G
ACCAAGCTGACCGTCCTAGGT
T K L T V L G
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7
G7 9 VH
SEQ ID N0. 25 encoding SEQ ID N0. 26
CAGGTGCAGCTGGTGCAGTCTGCGGGTGACCTGCAGAAGCCTGGGGCCTCAGTGAAGGTT
Q V Q L V Q S A G D L Q K P G A S V K V
TCCTGCAAGACATTTGGATACAGCTTCAGCAGTTACCATATACACTGGGTGAGACAGGCC
S C K T F G Y S F S S Y H I H W V R Q A
CCTGGACAAGGGCTTGAGTGGATGGGGATAATCGACCCTCGTGGTGGCAGTACAAGTTAC
P G Q G L E W M G I I D P R G G S T S Y
GCACAGAAGTTCCAGGGCAGAGTCACCATGACCGCTGACACGTCCACAGGCACAGTGTAT
A Q K F Q G R V T M T A D T S T G T V Y
ATGGAACTGAGCAGCCTGAAATCTGACGACACGTCCATATATTACTGTGCAAGGAGGTCC
M E L S S L K S D D T S I Y Y C A R R S
GGGGCTCTTGGGGGACTAATTCCCCTTAACTACTTTGACTACTGGGGCCGGGGGACAATG
G A L G G L I P L N Y F D Y W G R G T M
GTCACCGTCTCGAGT
V T V S S
G7 9 VL
SEQ ID N0. 27 encoding SEQ ID NO. 28
TCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACT
S E L T Q D P A V S V A L G Q T V R I T
TGCCAAGGAGACAGTCTCAGAAGCTATTACACAAACTGGTTCCAGCAGAAGCCAGGACAG
C Q G D S L R S Y Y T N W F Q Q K P G Q
GCCCCTCTACTTGTCGTCTATGCTAAAAATAAGCGGCCCTCAGGGATCCCAGACCGATTC
A P L L V V Y A K N K R P S G I P D R F
TCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGAT
S G S S S G N T A S L T I T G A Q A E D
GAGGCTGACTATTACTGTCATTCCCGGGACAGCAGTGGTAACCATGTGCTTTTCGGCGGA
E A D Y Y C H S R D S S G N H V L F G G
GGGACCAAGCTGACCGTCCTAGGT
G T K L T V L G
CA 02393292 2002-05-31
WO 01/44300 PCT/GB00/04501
8
G81 VH
SEQ ID N0. 29 encoding SEQ ID N0. 30
CAGGTCACCTTGAAGGAGTCTGGGCCTACGCTGGTGAAACCCACACAGACCCTTACGCTG
Q V T L K E S G P T L V K P T Q T L T L
ACCTGCACCTTCTCTGGGTTCTCACTCAGGACTACTGGAGTGGGCGTGGGCTGGGTCCGT
T C T F S G F S L R T T G V G V G W V R
CAGCCCCCAGGAGAGGCCCTGGAGTCGCTTGCGATCATTTATTGGAATGATGATAAGCGC
Q P P G E A L E S ~ A I I Y W N D D K R
AACAGCCCATCTCTGAAGAGAAGGCTCACCATCACCAAGGACACCTCCAGAAACCAGGTG
N S P S L K R R L T I T K D T S R N Q V
GTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCCCGCGTG
V L T M T N M D P V D T A T Y Y C A R V
ACCAATGGTCACTGGTACTACTTTGACTACTGGGGCAGAGGCACCCTGGTCACCGTCTCG
T N G H W Y Y F D Y W G R G T L V T V S
AGT
S
G81 VL
SEQ ID N0. 31 encoding SEQ ID N0. 32
CAGTCTGTGCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATC
Q S V L T Q P A S V S G S P G Q S I T I
TCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAA
S C T G T S S D V G G Y N Y V S W Y Q Q
CACCCAGGCAAAGCCCCCAAACTCATGATTTATGAGGGCAGTAAGCGGCCCTCAGGGGTT
H P G K A P K L M I Y E G S K R P S G V
TCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCTTGACCATCACTGGGGCT
S N R F S G S K S G N T A S L T I T G A
CAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACCATGTG
Q A E D E A D Y Y C N S R D S S G N H V
GTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT
V F G G G T K L T V L G
CA 02393292 2002-05-31
WO 01/44300 PCT/GB00/04501
9
G83 VH
SEQ ID N0. 33 encoding SEQ ID N0. 34
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTC
Q V Q L Q E S G P G L V K P S E T L S L
ACCTGCACTGTCTCTGGTGGTTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCC
T C T V S G G S I S S Y Y W S W I R Q P
CCAGGGAAGGGACTGGAGTGGATTGGGCATATCTATTACAGTGGGAACACCAACTACAAC
P G K G L E W I G H I Y Y S G N T N Y N
CCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCGAAGAACCAGTTCTCCCTG
P S L K S R V T I S V D T S K N Q F S L
AAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGGGCGGCGAGT
K L S S V T A A D T A V Y Y C A R A A S
CTATCTTGTACTGGTAGCAGCTGCAGGTACAACTACTTCGACCCCTGGGGCCGAGGAACC
L S C T G S S C R Y N Y F D P W G R G T
CTGGTCACCGTCTCGAGT
L V T V S S
G83 VL
SEQ ID N0. 35 encoding SEQ ID N0. 36
GACATCGTGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACC
D I V M T Q S P S T L S A S V G D R V T
ATCACTTGCCGGGCCAGTCAGGGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCA
I T C R A S Q G I S S W L A W Y Q Q K P
GGGAGAGCCCTTAAGGTCTTGATCTATAAGGCATGGCCTTTAGAAAGTGGGGTCCCATCA
G R A L K V L I Y K A W P L E S G V P S
AGGTTCACCGACAGTGGATCTGGGGCAGATTTCACTCTCACCATCAGCAGTTTGCAACCT
R F T D S G S G A D F T L T I S S L Q P
GAAGATTTTGCAACTTATTACTGTCAGCAGAGTTACAGTACTCCGTGGACGTTCGGCCAA
E D F A T Y Y C Q Q S Y S T P W T F G Q
GGGACCAAGCTGAAGATCAAACGCCGG
G T K L K I K R R
CA 02393292 2002-05-31
WO 01/44300 PCT/GB00/04501
G85 V~,
SEQ ID N0. 37 encoding SEQ ID N0. 38
CAGCTGCAGCTGCAGGAGTCCGGGGCTGAGCTTGTGAAGCCTGGGGCTTCAGTGAAAATG
Q L Q L Q E S G A E L V K P G A S V K M
TCCTGCAAGGCTTCTGGCTATACCTTCGCCAGCTACTGGATAAACTGGATGTAGCAGAGG
S C K A S G Y T F A S Y W I N W M Q Q R
CCTGGACAAGGCCTTGAGTGGATTGGACATATTTATCCTGTTAGAAGTATTACTAAGTAC
P G Q G L E W I G H I Y P V R S I T K Y
AATGAGAAGTTCAAGAGTAAGGCCACACTGTCTCTAGACACATCCTCCAGCACAGCCTAC
N E K F K S K A T L S L D T S S S T A Y
ATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTATTGTTCAAGAGGGGAC
M Q L S S L T S E D S A V Y Y C S R G D
GGCAGTGATTATTATGCTATGGACTACTGGGGCCAGGGGACAATGGTCACCGTCTCTTCA
G S D Y Y A M D Y W G Q G T M V T V S S
G85 VL
SEQ ID N0. 39 encoding SEQ ID N0. 40
GATGTTGTGATGACTCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACC
D V V M T Q S P S S V S A S V G D R V T
ATCACTTGTCGGGCGAGTCAGGGTATTAGCAGTTATTTAGCCTGGTATCAGCAAAAACCA
I T C R A S Q G I S S Y L A W Y Q Q K P
GGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAATCAGGGGTCCCATCT
G K A P K L L I Y A A S T L Q S G V P S
CGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCACCCTGCAGCCT
R F S G S G S G T D F T L T I S T L Q P
GAAGATGTTGCAACTTATTACTGTGAAAACTATAACAGTGTCCCGCTCAGTTTCGGCGGA
E D V A T Y Y C E N Y N S V P L S F G G
GGGACCAAGCTGGAGATCAGACGT
G T K L E I R R
CA 02393292 2002-05-31
WO 01/44300 PCT/GB00/04501
11
G88 VH
SEQ ID NO. 41 encoding SEQ ID N0. 42
GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTC
E V Q L V Q S G A E V K K P G S S V K V
TCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCC
S C K A S G G T F S S Y A I S W V R Q A
CCTGGACAAGGGCTTGAGTGGATGGGAAGGGATCATCCCTATCTTTGGACAGCAAACTAC
P G Q G L E W M G R D H P Y L W T A N Y
GCACAGAAGTTCCAGGCAGAGTCACGATTACCGCGGGACGAATCCACGAGCACAGCCTAC
A Q K F Q A E S R L P R D E S T S T A Y
ATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGAAGGG
M E L S S L R S E D T A V Y Y C A R E G
ACTGCTATCCACCGCGCTTTTGATATCTGGGGGCAGGGGACCACGGTCACCGTCTCGAGC
T A I H R A F D I W G Q G T T V T V S S
G88 VL
SEQ ID NO. 43 encoding SEQ ID NO. 94
GACATCTAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTATTGGAGACAGAGTCACC
D I Q M T Q S P S T L S A S I G D R V T
ATCACCTGCCGGGCCAGTGAGGGTATTTATCACTGGTTGGCCTGGTATCAGCAGAAGCCA
I T C R A S E G I Y H W L A W Y Q Q K P
GGGAAAGCCCCTAAACTCCTGATCTATAAGGCCTCTAGTTTAGCCAGTGGGGCCCCATCA
G K A P K L L I Y K A S S L A S G A P S
AGGTTCAGCGGCAGTGGATTTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCT
R F S G S G F G T D F T L T I S S L Q P
GATGATTTTGCAACTTATTACTGCCAACAATATAGTAATTATCCGCTCACTTTCGGCGGA
D D F A T Y Y C Q Q Y S N Y P L T F G G
GGGACCAAGCTGGAGATCAAACGT
G T K L E I K R
CA 02393292 2002-05-31
WO 01/44300 PCT/GB00/04501
12
G92 VH
SEQ ID NO. 45 encoding SEQ ID N0. 46
GAGGTGCAGCTGGTGGAGTCTGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTC
E V Q L V E S G P G L V K P S E T L S L
ACCTGCACTGTCTCTGGTGGTTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCC
T C T V S G G S I S S Y Y W S W I R Q P
CCAGGGAAGGGACTGGAGTGGATTGGCTATATCTATTACAGTGGGAGCACCAACTACAAC
P G K G L E W I G Y I Y Y S G S T N Y N
CCCCCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCATTTCTCCCTG
P P L K S R V T I S V D T S K N H F S L
AAGCTGAACTCTGTGACCGCCGCAGACACGGCCGTGTATTACTGTGCGAGAGTTGGGGTG
K L N S V T A A D T A V Y Y C A R V G V
GTGGTGACTGGGAGAGGTGCTTTTGATATCTGGGGGCCAAGGACAATGGTCACCGTCTCG
V V T G R G A F D I W G P R T M V T V S
AGT
S
G92 VL
SEQ ID N0. 47 encoding SEQ ID NO. 48
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATC
Q S V L T Q P P S A S G T P G Q R V T I
TCTTGTTCTGGAAGCAGCTCCAACATCGGGAGTAACACTGTAAACTGGTACCAGCGACTC
S C S G S S S N I G S N T V N W Y Q R L
CCAGGAGCGGCCCCCCAACTCCTCATCTACAATAATGACCAGCGGCCCTCAGGGATCCCT
P G A A P Q L L I Y N N D Q R P S G I P
GACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGGCTCCCTGGTCATCAGTGGGCTCCAG
D R F S G S K S G T S G S L V I S G L Q
TCTGAAGATGAGGCTGATTACTACTGTGCGTCATGGGATGACAGTCTGAATGGCCGGGTG
S E D E A D Y Y C A S W D D S L N G R V
TTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT
F G G G T K L T V L G
CA 02393292 2002-05-31
WO 01/44300 PCT/GB00/04501
13
G93 VH
SEQ ID N0. 49 encoding SEQ ID N0. 50
CAGGTGCAGTTACAGCAGTCGGGGGCTGCGGTGAAGACGCCTGGGGCCTCAGTCAAAGTT
Q V Q L Q Q S G A A V K T P G A S V K V
TCCTGTAAGGCATCTGGATATCCCTTCATCACCTACAACATGCACTGGGTGCGGCAGGCC
S C K A S G Y P F I T Y N M H W V R Q A
CCTGGACAGGGCTTTGAGTGGATGGGAATAATCGACCCAAGTGGTGGTCGCACAACGTAC
P G Q G F E W M G I I D P S G G R T T Y
TCAAAGAACTTCCAGGGCAGACTCACCGTGACCAGGGAGACGTCAACGACCACGGTCACC
S K N F Q G R L T V T R E T S T T T V T
ATGGAGCTGAGTGGCCTGAGATCTGAGGACACGGCCCTGTATTTCTGTGCGAGAGGGCGC
M E L S G L R S E D T A L Y F C A R G R
GGTGCGAGGGATGATGGTTTTGATGTCTGGGGCCAGGGGACAATGGTCACCGTCTCTTCA
G A R D D G F D V W G Q G T M V T V S S
G93 VL
SEQ ID N0. 51 encoding SEQ ID N0. 52
CAGTCTGTGCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATC
Q S V L T Q P A S V S G S P G Q S I T I
TCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAA
S C T G T S S D V G G Y N Y V S W Y Q Q
CACCCAGGCAAAGCCCCCAAACTCATGATTTATGAGGGCAGTAAGCGGCCCTCAGGGGTT
H P G K A P K L M I Y E G S K R P S G V
TCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACAATCTCTGGGCTC
S N R F S G S K S G N T A S L T I S G L
CAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATACAACCAGGGGCACTCGAGTT
Q A E D E A D Y Y C S S Y T T R G T R V
TTCGGCGGAGGGACCAAGCTGACCGTCCTAGGG
F G G G T K L T V L G
CA 02393292 2002-05-31
WO 01/44300 PCT/GB00/04501
14
G95 VH
SEQ ID N0. 53 encoding SEQ ID N0. 59
CAGGTACAGCTGCAGCAGTCAGGACCTGAGCTGGCGAGTCCTGGGGCATCAGTGACACTG
Q V Q L Q Q S G P E L A S P G A S V T L
TCCTGCAAGGCTTCTGGCTACACATTTACTGACCATATTATGAATTGGGTAAAAAAGAGG
S C K A S G Y T F T D H I M N W V K K R
CCTGGACAGGGCCTTGAGTGGATTGGAAGGATTTATCCAGTAAGTGGTGAAAGTAACTAC
P G Q G L E W I G R I Y P V S G E S N Y
AATCAAAAGTTCATGGGCAAGGCCACATTCTCTGTAGACCGGTCCTCCAGCACGGTGTAT
N Q K F M G K A T F S V D R S S S T V Y
ATGGTGTTGAACAGCCTGACATCTGAAGACCCTGCTGTCTATTACTGTGCAAGGGGGGAG
M V L N S L T S E D P A V Y Y C A R G E
ATCTTTGACTATTGGGCCGGGGGACCACGGTCACCGTCTCCTTCA
I F D Y W A G G P R S P S P S
G95 VL
SEQ ID N0. 55 encoding SEQ ID NO. 56
GACATCCAGATGACCCAGTCTCCTTCCACCCCGTCTGCATCTATTGGAGACAGAGTCACC
D I Q M T Q S P S T P S A S I G D R V T
ATCACCTGCCGGGCCAGTGAGGGTATTTATCACTGGTTGGCCTGGTATCAGCAGAAGCCA
I T C R A S E G I Y H W L A W Y Q Q K P
GGGAAAGCCCCTAAACTCCTGATCTATAAGGCCTCTAGTTTAGCCAGTGGGGCCCCATCA
G K A P K L L I Y K A S S L A S G A P S
AGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCT
R F S G S G S G T D F T L T I S S L Q P
GATGATTTTGCAACTTATTACTGCCAACAATATAGTAATTATCCGCTCACTTTCGGCGGA
D D F A T Y Y C Q Q Y S N Y P L T F G G
GGGACACGACTGGAGATTAAACGT
G T R L E I K R
CA 02393292 2002-05-31
WO 01/44300 PCT/GB00/04501
IS
6101 VH
SEQ ID N0. 57 encoding SEQ ID NO. 58
CAGGTGCAGCTGCAGGAGTCGGGCCCGGGACTGCTGAAGGCTTCGGAGACCCTGTCCCTC
Q V Q L Q E S G P G L L K A S E T L S L
GTTTGCAGTGTCTCTGGCGCCTCCGTCGACAGTGACCAGTTCTACTGGGTCTGGATCCGT
V C S V S G A S V D S D Q F Y W V W I R
CAGCCCCCAGGGAAAGGACTAGAGTGGATTGGGACTGCCTATTATAGTGGGAGCAGCCAC
Q P P G K G L E W I G T A Y Y S G S S H
TACAACCCGTCCCTCAACAATCGGGTCACCATATCTGTAGACACGTCCAAAGACCTCTTC
Y N P S L N N R V T I S V D T S K D L F
TCCCTGAGTCTGAACTCTGTGACCGTCGCAGATACGGCTGTGTATTACTGTGCGAGGGGG
S L S L N S V T V A D T A V Y Y C A R G
ACTTCATTTAGCAGCAGTTGGTACAGTAATTACTTCTTCTATTATTACATTGACCTCTGG
T S F S S S W Y S N Y F F Y Y Y I D L W
GGCAAGGGAACCCTGGTCACCGTCTCGAGT
G K G T L V T V S S
6101 VL
SEQ ID N0. 59 encoding SEQ ID N0. 60
TCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACT
S E L T Q D P A V S V A L G Q T V R I T
TGCCAAGGAGACAGTCTCAGAAGCTATTACACAAACTGGTTCCAGCAGAAGCCAGGACAG
C Q G D S L R S Y Y T N W F Q Q K P G Q
GCCCCTCTACTTGTCGTCTATGCTAAAAATAAGCGGCCCTCAGGGATCCCAGACCGATTC
A P L L V V Y A K N K R P S G I P D R F
TCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGAT
S G S S S G N T A S L T I T G A Q A E D
GAGGCTGACTATTACTGTCATTCCCGGGACAGCAGTGGTAACCATGTGCTTTTCGGCGGA
E A D Y Y C H S R D S S G N H V L F G G
GGGACCAAGCTGACCGTCCTAGGA
G T K L T V L G
CA 02393292 2002-05-31
WO 01/44300 PCT/GB00/04501
16
6102 VH
SEQ ID NO. 61 encoding SEQ ID N0. 62
CAGGTGCAGCTACAGCAGTGGGGCGCGGGAGTACTGGAGCCTTCGGAGACCCTGTCCCTC
Q V Q L Q Q W G A G V L E P S E T L S L
ACCTGCGGTGTTTCTGGTGCGTCTTTGGATGGTCACTACTGGGCCTGGATCCGCCAGTCC
T C G V S G A S L D G H Y W A W I R Q S
CCAGGGACGGGGCTGGAATGGATTGCTGAAATCAATTCAAGTGGCAGCACCAACTACAAC
P G T G L E W I A E I N S S G S T N Y N
CCGTCCCTCAAGAGCCGGGTCGCCATATCAATGGACACGTCTAAGAACTCCTTGTCCCTG
P S L K S R V A I S M D T S K N S L S L
AACTTGAAGTCTGTGACCGCCGCGGACACGGCTGTATATTACTGTGCGAGAGTCGGGCAA
N L K S V T A A D T A V Y Y C A R V G Q
TACAATTATCTCCACGCGTTCTACTTAGAATACTGGGGCCGAAGGACAATGGTCACCGTC
Y N Y L H A F Y L E Y W G R R T M V T V
TCTTCA
S S
6102 VL
SEQ ID NO. 63 encoding SEQ ID NO. 64
CAGTCTGTGCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGGCAGTCGATCACCATC
Q S V L T Q P A S V S G S P G Q S I T I
TCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAA
S C T G T S S D V G G Y N Y V S W Y Q Q
CACCCAGGCAAAGCCCCCAAACTCATGATTTATGAGGGCAGTAAGCGGCCCTCAGGGGTT
H P G K A P K L M I Y E G S K R P S G V
TCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACAATCTCTGGGCTC
S N R F S G S K S G N T A' S L T I S G L
CAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATACAACCAGGAGCACTCGAGTT
Q A E D E A D Y Y C S S Y T T R S T R V
TTCGGCGGAGGGACCAAGCTGACCGTCCTAGGA
F G G G T K L T V L G
CA 02393292 2002-05-31
WO 01/44300 PCT/GB00/04501
17
G110VH
SEQ ID N0. 65 encoding SEQ ID NO. 66
CAGGTACAGCTGCAGCAGTCAGGACCTGAGCTGGCGAGTCCCGGGGCATCAGTGACACTG
Q V Q L Q Q S G P E L A S P G A S V T L
TCCTGCAAGGCTTCTGGCGACACACTTACTGACCATATTATGAATTGGGTAAAACAGAGG
S C K A S G D T L T D H I M N W V K Q R
CCTGGACAGGGCCTTGAGTGGATTGGAAGGATTTATCCAGTAAGTGGTGAAAGTAACTAC
P G Q G L E W I G R I Y P V S G E S N Y
AATCAAAAGTTCATGGGCAAGGCCACATTCTCTGTAGACCGGTCCTCCAGCACGGTGTAT
N Q K F M G K A T F S V D R S S S T V Y
ACGGTGTTGAACAGCCTGACATCTGAAGACCCTGCTGTCTATTACTGTGGAAGGGGGGAG
T V L N S L T S E D P A V Y Y C G R G E
ATCTTTGACTATTGGGGGCAAGGGACCACGGTCACCGTYTCGAGT
I F D Y W G Q G T T V T V S S
6110 VL
SEQ ID N0. 67 encoding SEQ ID NO. 68
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTATTGGAGACAGAGTCACC
D I Q M T Q S P S T L S A S I G D R V T
ATCACCTGCCGGGCCAGTGAGGGTATTTATCACTGGTTGGCCTGGTATCAGCAGAAGCCA
I T C R A S E G I Y H W L A W Y Q Q K P
GGGAAAGCCCCTAAACTCCTGATCTATAAGGCCTCTAGTTTAGCCAGTGGGGCCCCATCA
G K A P K L L I Y K A S S L A S G A P S
AGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCT
R F S G S G S G T D F T L T I S S L Q P
GATGATTTTGCAACTTATTACTGCCAACCATATAGTAATGATGCGCTCACTTTCGGCGGA
D D F A T Y Y C Q P Y S N D A L T F G G
GGGACACGACTGGAGATTAAACGT
G T R L E I K R
CA 02393292 2002-05-31
WO 01/44300 PCT/GB00/04501
18
6112 VH
SEQ ID N0. 69 encoding SEQ ID NO. 70
CAGGTCAACTTAAGGGAGTCTGGGGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATC
Q V N L R E S G A E V K K P G E S L K I
TCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGGCTGGGTGCGCCAGATG
S C K G S G Y S F T S Y W I G W V R Q M
CCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATAC
P G K G L E W M G I I Y P G D S D T R Y
AGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTAC
S P S F Q G Q V T I S A D K S I S T A Y
CTGTAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACGGTGG
L Q W S S L K A S D T A M Y Y C A R R W
AAAGGGCACTTTGACTACTGGGGCCAAGGCACCTTGGTCACCGTCTCGAGT
K G H F D Y W G Q G T L V T V S S
6112 VL
SEQ ID NO. 71 encoding SEQ ID N0. 72
GACATCGTGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACC
D I V M T Q S P S T L S A S V G D R V T
ATCACTTGCCGGGCCAGTCAGGGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCA
I T C R A S Q G I S S W L A W Y Q Q K P
GGGAGAGCCCCTAAGGTCTTGATCTATAAGGCATCTACTTTAGAAAGTGGGGTCCCATCA
G R A P K V L I Y K A S T L E S G V P S
AGGTTCAGCGGCAGTGGATCTGGGACAGATTTCGCTCTYACCATCAGCAGTCTGCAACCT
R F S G S G S G T D F A L T I S S L Q P
GAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCGTGGACGTTCGGCCAA
E D F A T Y Y C Q Q S Y S T P W T F G Q
GGGACCAAGCTGGAGGTCAAACGT
G T K L E V K R
CA 02393292 2002-05-31
WO 01/44300 PCT/GB00/04501
19
D5 VH
SEQ ID N0. 73 encoding SEQ ID N0. 74
CAGGTGCAGCTGGTGGAGGTCGGAGGCTTGGTCAAGCCTGGAGGGTCCTTAAGAAGTTCT
Q V Q L V E V G G L V K P G G S L R S S
TCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATTAGTTGGATCCGTCAGGCT
S C A A S G F T F S D Y Y I S W I R Q A
CCAGGGAAGGGCTTGAAGTGGGTTGCAAACATTAGTAACAGTGGCAATACCATACACTAC
P G K G L K W V A N I S N S G N T I H Y
GCAGACTCTGTCAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAGGTCAATGTAT
A D S V K G R F T I S R D N A K R S M Y
CTGCAAATGAACAGCCTGAGAGCCGACGACACGGCCGTCTATTACTGTGCGAGGGTGCGA
L Q M N S L R A D D T A V Y Y C A R V R
TCAATTCGGAATAGTCGCTGGTTCGACCCCTGGGGCCAAGGAACCCTGGTCACCGTCTCC
S I R N S R W F D P W G Q G T L V T V S
TCG
S
D5 VL
SEQ ID N0. 75 encoding SEQ ID N0. 76
GAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGC
E L T Q D P A V S V A L G Q T V R I T C
CAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACGGGCC
Q G D S L R S Y Y A S W Y Q Q K P G R A
CCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCT
P V L V I Y G K N N R P S G I P D R F S
GGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAG
G S S S G N T A S L T I T G A Q A E D E
GCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACCATGTGGTATTCGGCGGAGGG
A D Y Y C N S R D S S G N H V V F G G G
ACCAAGCTGACCGTCCTAGG
T K L T V L G
