U.S. patent application number 12/657255 was filed with the patent office on 2010-11-11 for carrier molecules.
This patent application is currently assigned to Agen Biomedical Limited. Invention is credited to Francis J. Carr, Anita A. Hamilton.
Application Number | 20100286375 12/657255 |
Document ID | / |
Family ID | 26972062 |
Filed Date | 2010-11-11 |
United States Patent
Application |
20100286375 |
Kind Code |
A1 |
Carr; Francis J. ; et
al. |
November 11, 2010 |
Carrier molecules
Abstract
The present invention relates generally to carrier molecules
derived from one animal or avian species or strains and which are
substantially non-immunogenic when exposed to an immune system from
a species or strain of another animal or avian creature. More
particularly, the present invention provides deimmunized
immunointeractive molecules and even more particular deimmunized
antibodies for use in diagnostic and therapeutic applications.
Inventors: |
Carr; Francis J.; (Aberdeen,
GB) ; Hamilton; Anita A.; (Aberdeen, GB) |
Correspondence
Address: |
ROBINS & PASTERNAK
1731 EMBARCADERO ROAD, SUITE 230
PALO ALTO
CA
94303
US
|
Assignee: |
Agen Biomedical Limited
|
Family ID: |
26972062 |
Appl. No.: |
12/657255 |
Filed: |
January 15, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12231381 |
Sep 2, 2008 |
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12657255 |
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11432223 |
May 10, 2006 |
7459143 |
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12231381 |
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10184300 |
Jun 26, 2002 |
7087724 |
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11432223 |
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60301154 |
Jun 26, 2001 |
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60300947 |
Jun 27, 2001 |
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Current U.S.
Class: |
530/389.3 |
Current CPC
Class: |
C07K 2317/56 20130101;
A61P 9/10 20180101; C07K 2317/24 20130101; A61P 7/00 20180101; A61P
7/02 20180101; A61P 35/00 20180101; C07K 16/18 20130101; A61K
2039/505 20130101 |
Class at
Publication: |
530/389.3 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 26, 2002 |
AU |
PCT/AU02/00827 |
Claims
1. An isolated antibody specific for an epitope on human D-dimer
which recognizes cross-linked fibrin but not fibrinogen, wherein
said antibody is selected from the group consisting of: (i) an
antibody that comprises a binding site for said D-dimer epitope,
wherein one or more amino acid residues in a T-cell epitope present
in the variable (v)-domain of said antibody are mutated to
eliminate or reduce the ability of peptide fragments of said
v-domain to associate with MHC class II molecules; and (ii) an
antibody that comprises complementary determining regions (CDRs)
that define a binding site for said D-dimer epitope, interspersed
between framework regions that comprise a human antibody light and
heavy chain variable region.
2. The isolated antibody of claim 1 wherein said antibody is
specific for an epitope recognized by anti-fibrin murine monoclonal
antibody 3B6.
3. The isolated antibody of claim 1, wherein the antibody comprises
an H-chain selected from the group consisting of Hv5 (3B6DIVHv5;
SEQ ID NO:1), Hv6 (3B6DIVHv6; SEQ ID NO:2) and Hv7 (3B6DIVHv7; SEQ
ID NO:3).
4. The isolated antibody of claim 1, wherein the antibody comprises
an L-chain selected from the group consisting of Kv1 (3B6DIVKv1;
SEQ ID NO:4), Kv4 (3B6DIVKv4; SEQ ID NO:5) and Kv7 (3B6DIVKv7; SEQ
ID NO:6).
5. The isolated antibody of claim 1, wherein said antibody
comprises a combination of an H-chain and an L-chain selected from
the group consisting of VHv5NKv1, VHv6NKv1, VHv7NKv1, VHv5/VKv7,
VHv6NKv7, VHv6/VKv4, VHv7NKv4, VHv7NKv7 and VHv5NKv4.
6. The isolated antibody of claim 1, wherein said antibody
comprises a combination of an H and an L chain v-domain comprising
a combination of amino acid sequences selected from the group
consisting of SEQ ID NO:1/SEQ ID NO:4, SEQ ID NO:2/SEQ ID NO:4, SEQ
ID NO:3/SEQ ID NO:4, SEQ ID NO:1/SEQ ID NO:6, SEQ ID NO:2/SEQ ID
No:6, SEQ ID NO:2/SEQ ID NO:5, SEQ ID NO:3/SEQ ID NO:5, SEQ ID
NO:3/SEQ ID NO:6, and SEQ ID NO:1/SEQ ID NO:5 or combinations of
amino acid sequences having at least 70% sequence identity to the
contiguous sequence of one or both amino acid sequences in each of
the above listed pairs.
7. The antibody of claim 6, wherein said antibody is an Fab'
fragment.
8. The antibody of claim 1, wherein said antibody is selected by
binding to D-dimer that is not passively bound to a substrate.
9. The antibody of claim 1, wherein said antibody is selected by
binding to D-dimer in solution.
10. A deimmunized antibody specific for an epitope on human D-dimer
which recognizes cross-linked fibrin but not fibrinogen, wherein
said antibody comprises (a) an H-chain selected from the group
consisting of Hv5 (3B6DIVHv5; SEQ ID NO:1), Hv6 (3B6DIVHv6; SEQ ID
NO:2) and Hv7 (3B6DIVHv7; SEQ ID NO:3), and (b) an L-chain selected
from the group consisting of Kv1 (3B6DIVKv1; SEQ ID NO:4), Kv4
(3B6DIVKv4; SEQ ID NO:5) and Kv7 (3B6DIVKv7; SEQ ID NO:6), and
further wherein said antibody is selected by binding to D-dimer in
solution.
11. The deimmunized antibody of claim 10, wherein said antibody
comprises a combination of an H and an L chain v-domain comprising
a combination of amino acid sequences encoded by a combination of
nucleotide sequences selected from the group consisting of SEQ ID
NO:7/SEQ ID NO:10, SEQ ID NO:8/SEQ ID NO:10, SEQ ID NO:9/SEQ ID
NO:10, SEQ ID NO:7/SEQ ID NO:12, SEQ ID NO:8/SEQ ID NO:12, SEQ ID
NO:8/SEQ ID NO:11, SEQ ID NO:9/SEQ ID NO:11, SEQ ID NO:9/SEQ ID
NO:12, and SEQ ID NO:7/SEQ ID NO:11.
12. The deimmunized antibody of claim 10, wherein said antibody
comprises a combination of an H and an L chain v-domain comprising
a combination of amino acid sequences selected from the group
consisting of SEQ ID NO:1/SEQ ID NO:4, SEQ ID NO:2/SEQ ID NO:4, SEQ
ID NO:3/SEQ ID NO:4, SEQ ID NO:1/SEQ ID NO:6, SEQ ID NO:2/SEQ ID
No:6, SEQ ID NO:2/SEQ ID NO:5, SEQ ID NO:3/SEQ ID NO:5, SEQ ID
NO:3/SEQ ID NO:6, and SEQ ID NO:1/SEQ ID NO:5.
13. The deimmunized antibody of claim 10, wherein said antibody is
an Fab' fragment.
14. An isolated antibody wherein said antibody comprises human
constant regions, and variable regions specific for an epitope
recognized by anti-fibrin murine monoclonal antibody 3B6.
15. The antibody of claim 14, wherein said antibody comprises a
combination of heavy and light chain v-regions from VHv5 and
VKv1.
16. The antibody of claim 15, wherein said antibody comprises a
heavy chain v-region comprising amino acid residues 26-36, 51-71
and 99-107 of SEQ ID NO:1.
17. The antibody of claim 15, wherein said antibody comprises a
light chain v-region comprising amino acid residues 24-34, 50-56
and 89-97 of SEQ ID NO:4.
18. The antibody of claim 15, wherein said antibody comprises a
heavy chain v-region comprising amino acid residues 26-36, 51-71
and 99-107 of SEQ ID NO:1 and a light chain v-region comprising
amino acid residues 24-34, 50-56 and 89-97 of SEQ ID NO:4.
19. An isolated antibody that comprises human constant regions
specific for an epitope recognized by anti-fibrin murine monoclonal
antibody 3B6, and variable regions specific for an epitope
recognized by anti-fibrin murine monoclonal antibody 3B6.
20. A deimmunized antibody specific for an epitope on human D-dimer
which recognizes cross-linked fibrin but not fibrinogen, wherein
said antibody comprises a combination of an H and an L chain
v-domain comprising a combination of amino acid sequences selected
from the group consisting of SEQ ID NO:1/SEQ ID NO:4, SEQ ID
NO:2/SEQ ID NO:4, SEQ ID NO:3/SEQ ID NO:4, SEQ ID NO: 1/SEQ ID
NO:6, SEQ ID NO:2/SEQ ID No:6, SEQ ID NO:2/SEQ ID NO:5, SEQ ID
NO:3/SEQ ID NO:5, SEQ ID NO:3/SEQ ID NO:6, and SEQ ID NO: 1/SEQ ID
NO:5, or combinations of amino acid sequences having a conservative
amino acid substitution in one or both amino acid sequences in each
of the above listed pairs.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
12/231,381, filed Sep. 2, 2008, which is a continuation of U.S.
Ser. No. 11/432,223, filed May 10, 2006, now U.S. Pat. No.
7,459,143, which is a continuation of U.S. Ser. No. 10/184,300,
filed Jun. 26, 2002, now U.S. Pat. No. 7,087,724, from which
applications priority is claimed under 35 U.S.C. .sctn.120. U.S.
Ser. No. 10/184,300 claims the benefit under 35 U.S.C.
.sctn.119(e)(1) of U.S. Provisional No. 60/301,154, filed Jun. 26,
2001 and U.S. Provisional No. 60/300,947, filed Jun. 27, 2001. The
foregoing applications are incorporated herein by reference in
their entireties.
[0002] The present invention relates generally to carrier molecules
derived from one animal or avian species or strains and which are
substantially non-immunogenic when exposed to an immune system from
a species or strain of another animal or avian creature. More
particularly, the present invention provides deimmunized
immunointeractive molecules and even more particular deimmunized
antibodies for use in diagnostic and therapeutic applications.
BACKGROUND OF THE INVENTION
[0003] Bibliographic details of the publications referred to in
this specification are collected at the end of the description.
[0004] Reference to any prior art in this specification is not, and
should not be taken as, an acknowledgment or any form of suggestion
that this prior art forms part of the common general knowledge in
any country.
[0005] Fibrinogen is a large protein molecule which normally
circulates in blood plasma in a dissolved state. In the presence of
thrombin, fibrinogen molecules form long thread-like polymers or
networks called fibrin which is the primary ingredient of blood
clots.
[0006] Upon digestion with plasmin, fibrinogen forms fragments
designated A-E. Fragments D and E are the predominant fragments and
there is about twice as much D as there is of B. Fibrinogen has a
trinodular shape where fragment E is a central component and
fragment D is a terminal component.
[0007] Plasmin digests of fibrin and fibrinogen can be
differentiated from each other using polyacrylamide gel
electrophoresis (PAGE). Cross-linking of fibrin with Factor XIIIa
forms dimers of fragment D called D-dimer. Factor XIIIa is an
enzyme which introduces covalent bonds between adjacent monomers in
fibrin (Budzynski et al., Blood 54(4): 794-804, 1979). Factor XIIIa
is activated by the thrombin-catalyzed removal of a peptide from a
precursor in plasma and in blood platelets. D-dimer is a molecule
of about 189,000 daltons which consists essentially of two fragment
D moieties derived from different fibrin molecules covalently bound
by cross-link bonds between the .gamma. chain remnants of
fibrinogen. Fibrinogen itself comprises six chains with two copies
of an .alpha., .beta. and .gamma. chain.
[0008] Another complex (DD)E is formed by plasmin degradation of
cross-linked human fibrin and comprises a combination of two D
fragments and fragment E.
[0009] Other cross-linked derivatives are described by Graeff and
Halfer (Graeff and Halfer, "Detection and Relevance of Cross-linked
Fibrin Derivatives in Blood", Seminars in Thrombosis and Hemostatis
8(1), 1982) and include high molecular weight cross-linked
derivatives such as DY, YY, XD, XY, DXD and YXD.
[0010] Normal haemostasis or coagulation of blood involves
maintaining intravascular constituents in a liquid phase or
suspension while concomitantly permitting local deposition of solid
phase blood components in areas of vessel damage. In health, it has
been assumed, but never experimentally demonstrated, that a balance
exists between a low-grade intravascular deposition of fibrin and
its removal by fibrinolysis or cellular phagocytosis.
[0011] Early clinical observations revealed that some severely ill
patients developed signs of haemorrhage and massive bruising and
had prolonged clotting times and thrombocytopenia. At postmortem,
in some cases, fibrin thrombi were demonstrated in the
microvasculature. The diffuse nature of these thrombi gave rise to
disseminated intravascular coagulation (DIC) also known as
consumptive coagulopathy. Subsequently, the thrombin were
associated with conditions such as deep vein thrombosis (DVT) and
pulmonary embolism (PE).
[0012] Conditions such as DIC, DVT and PE involve activation of the
coagulation system resulting in platelet consumption, thrombin
generation, fibrin deposition and secondary fibrinolysis. The net
biologic effect of this process reflects a balance between fibrin
deposition and fibrin clearance. The resulting clinical
manifestations may be haemorrhage, when depletion of coagulation
factors predominates, or ischemic tissue damage, due to the effects
of vascular occlusion amongst other conditions.
[0013] DIC, DVT and PE have been reported as a secondary phenomenon
in a wide variety of disorders, particularly those accompanied by a
combination of shock, acidosis and hypoxemia. The well-recognized
clinical associations are sepsis, major trauma, malignancy and
obstetric disorders. Recently, DVT has been recognized as a
particular problem during prolonged air travel or other prolonged
immobility. In any event, activation of the coagulation sequence
results in consumption of coagulation protein and platelets,
leading to fibrin deposition in the micro-circulation.
[0014] Ideally, a definitive diagnosis of conditions such as DIC,
DVT and PE requires the direct demonstration of diffuse fibrin
deposition. The practical difficulty of obtaining multiple direct
biopsy evidence to differentiate between localized and generalized
fibrin formation has led to the development of indirect tests that
are substituted as diagnostic end points. However, these tests are
not specific for the syndrome of intravascular fibrin deposition.
Their specificity is further reduced by the action of other enzymes
that although not able to convert fibrinogen to fibrin can cause
similar alterations to thrombin on the other coagulation factors
involved in thrombosis. All of the indirect tests are based on the
principle that thrombin is the only enzyme (snake venoms excluded)
capable of converting fibrinogen to fibrin in mammals.
[0015] Also, apart from the paracoagulation tests that detect the
presence of circulating soluble fibrin monomer complexes, none of
the more specific thrombin specific tests is readily available or
useful for immediate clinical application in the diagnosis of these
fibrin-associated conditions. These tests include the FPA
(fibrinopeptide A) test where FPA is measured by a specific RIA
procedure, fibrin monomer assays, fibrinogen gel exclusion
chromatography and tests for FPB (fibrinopeptide B) or thrombin
increasable FPB.
[0016] Tests with biochemical non-specificity for thrombin action
include the prothrombin time (PT), thromboplastin time (A PTT) and
thrombin clotting time (TCT) tests. Although frequently useful in
practice, it must be recognized that information obtained from
these tests is non-specific in nature, acting as a measure of
clotting factor depletion regardless of etiology.
[0017] Coagulation factor assays have also been found to be
relatively non-specific and these include assays for cofactors V
and VIII as well as tests for fibrinogen levels.
[0018] Tests for fibrin-fibrinogen degradation products so far have
not proved to be specific for the action of plasmin on fibrin and
may yield positive results where there has been fibrinogenolysis
without prior thrombin action on the fibrinogen molecule. These
tests include tests for fragments D and E.
[0019] Tests for thrombin-mediated platelet interaction or release
have been found to be non-specific in nature. These include
platelet count, platelet survival and tests of platelet
release.
[0020] The use of radio labeled fibrinogen in relation to
identifying clotting factors have also been attempted but found to
be time consuming and difficult to perform.
[0021] Thus, the efficacy of a diagnostic test lies in its ability
to indicate the presence or absence of disease. There are well
recognized essential design principles for studies determining the
efficacy of a diagnostic test which enables the four indices of
sensitivity, specificity, positive predictive value and negative
predictive value to be determined. The first requirement is the
adoption of a suitable standard for diagnosis. Ideally, this
standard should be slightly more than a clinical definition and
should be as specific as possible for the disease entity. An
inherent difficulty in relation to DVT and PE in particular is that
many of the routinely available laboratory tests also lack
diagnostic specificity. A low platelet count supports the
likelihood of these conditions but may occur as an isolated finding
secondary to infection. Similar limitations apply to many of the
coagulation assays. Hypofibrinogenemia does not distinguish between
primary fibrinolysis, due either to the action of plasmin or
elastases and secondary fibrinolysis following the
thrombin-mediated conversion of fibrinogen to fibrin.
Alternatively, sensitive tests of thrombin action are available but
there are obvious drawbacks with their clinical use. An example is
the FPA assay which, although specific for thrombin action, is
exquisitely sensitive and may detect localized intravascular
coagulation yielding a positive result in uncomplicated venous
thrombosis. The clinical significance of an elevated FPA level,
even with a positive paracoagulation test, is then at issue,
particularly if the platelet count, global clotting tests and
fibrinogen level are normal.
[0022] For these reasons, sensitivity, specificity and predictive
values cannot be determined in a standard fashion. The clinical
presentation of these disorders is complex and unpredictable. The
application of the available tests for diagnosis are, therefore,
best considered in relation to the different clinical syndromes of
intravascular coagulation.
[0023] Murine monoclonal antibody 3B6 was disclosed (U.S. Pat. No.
4,758,524). This antibody is specific for D-dimer and represents
the first clot-specific antibody. The ability to use this antibody,
however, in humans as a systemic diagnostic agent is limited due to
the immunogenicity of the molecule. There is a need, therefore, to
modify the 3B6 antibody to reduce its immunogenicity in non-murine
animals and humans.
SUMMARY OF THE INVENTION
[0024] Throughout this specification, unless the context requires
otherwise, the word "comprise", or variations such as "comprises"
or "comprising", will be understood to imply the inclusion of a
stated element or integer or group of elements or integers but not
the exclusion of any other element or integer or group of elements
or integers.
[0025] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e. to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0026] In work leading up to the present invention, deimmunization
technology was used to reduce the immunogenicity of the 3B6
antibody. This has enabled the development of a thrombosis imaging
diagnostic procedure for use in humans. Furthermore, the
deimmunized form of the 3B6 antibody permits its use as a clot
targeting agent to deliver clot dissolution or clot growth
prevention agents such as anti-coagulants to the site of a clot.
The deimmunized molecules of the present invention act, therefore,
as carriers of diagnostic and therapeutic agents to a target site
such as a clot. The molecules may also have their own diagnostic or
therapeutic properties. The development of a deimmunized form of
the 3B6 antibody has application for a range of conditions such as
DVT and PE. Furthermore, the deimmunized 3B6 antibodies can be used
in combination with computer assisted tomographic nuclear medicine
or planar imaging techniques such as CT, MRI or ultrasound.
[0027] The present invention provides, therefore, a carrier
molecule generally in the form of an immunointeractive molecule and
in particular a monoclonal antibody rendered chimeric and/or
mutated relative to a parent molecule such that it exhibits reduced
capacity for immunogenicity in a target host, such as a human. The
process of chimerism or mutation is referred to herein as
deimmunization. In a particularly preferred embodiment, the
immunointeractive molecule such as the monoclonal antibody is
humanized to reduce its immunogenicity in humans. Deimmunization
may be conducted in different ways but in a preferred embodiment,
one or more amino acids in the variable (v) region of a monoclonal
antibody are mutated (e.g. substituted) to reduce MHC II
recognition of peptides derived from this region. In other words,
the deimmunization process is aimed at reducing a T cell
epitope-mediated immune response to the antibody. The most
preferred antibody of the present invention is a deimmunized form
of murine monoclonal antibody 3B6 which exhibits specificity for
D-dimer. The generation of a deimmunized form of 3B6 permits
development inter alia of a systemic clot targeting agent for blood
clots in humans. This permits its use as an imaging agent and as a
vehicle to deliver clot dissolution or clot growth prevention
agents such as to the site of a clot.
[0028] The deimmunized antibody acts, therefore, alone or as a
carrier for a range of diagnostic and/or therapeutic agents.
[0029] Accordingly, one aspect of the present invention provides a
variant of an immunointeractive molecule comprising a portion
having specificity for cross-linked fibrin derivatives and which
portion is derived from an immunointeractive molecule obtainable
from one animal or avian creature wherein the variant exhibits
reduced immunogenicity in another animal or avian creature from the
same or different species.
[0030] Preferably, the immunointeractive molecule is a variant
monoclonal antibody comprising a portion having specificity for
cross-linked fibrin derivatives.
[0031] More preferably, the monoclonal antibody is a variant of a
murine-derived monoclonal antibody having specificity for
human-derived D-dimer and other cross-linked fibrin derivatives and
non-reactivity with fibrinogen or fibrinogen degradation products
inclusive of fragments D and E wherein the variant murine-derived
monoclonal antibody is substantially non-immunogenic in a
human.
[0032] Preferably, the antibody is a deimmunized antibody molecule
having specificity for an epitope recognized by monoclonal antibody
3B6 and comprises at least one of the complementary determining
regions (CDRs) of the variable domain derived from the 3B6
monoclonal antibody and the remaining immunoglobulin-derived parts
of the deimmunized antibody molecule are derived from an
immunoglobulin or an analogue thereof from the host for which the
antibody is to be deimmunized.
[0033] The present invention provides, therefore, a deimmunized
antibody molecule having specificity for an epitope recognized by
monoclonal antibody 3B6 wherein at least one of the complementary
determining regions (CDRs) of the variable domain of said
deimmunized antibody is derived from the 3B6 monoclonal antibody
wherein one or more amino acids in a variable region of said 3B6
antibody is mutated to reduce MHC class II recognition of peptides
derived from this region.
[0034] The present invention further provides a variant of murine
monoclonal antibody 3B6 deimmunized for use in humans comprising
one or more amino acid mutations in the v-region of the 3B6
antibody designed to eliminate or reduce peptide fragments of the
v-region associating with MHC class II molecule.
[0035] The deimmunized immunointeractive molecules are useful alone
or as carriers of diagnostic and/or therapeutic agents to a target
site such as a blood clot.
[0036] Accordingly, the present invention contemplates a method for
detecting a blood clot in a human patient by introducing into the
patient a deimmunized form of murine monoclonal antibody 3B6 or an
antigen-binding fragment thereof labeled with a reporter molecule
allowing dissemination of the labeled antibody throughout the
circulatory system and then subjecting the patient to reporter
molecule-detection means to identify the location of the antibody
in a clot.
[0037] In an alternative embodiment, the deimmunized form of 3B6 is
not labeled but a second antibody having anti-immunoglobulin
specificity is labeled. This antibody forms a labeled complex with
the first mentioned antibody.
[0038] As a carrier, the deimmunized carrier may deliver any clot
binding molecule to the site of a clot as well as other diagnostic
or therapeutic agents.
[0039] The present invention contemplates, therefore, the use of a
deimmunized murine monoclonal antibody specific for D-dimer or
other cross-linked fibrin derivatives in the manufacture of clot
imaging agent.
[0040] Yet another aspect of the present invention contemplates a
method for facilitating the dissolution or removal of a blood clot
in a human, said method comprising administering to said human a
clot dissolution or clot growth prevention-effective amount of a
variant-murine-derived monoclonal antibody having specificity for
human-derived D-dimer and other cross-linked fibrin derivatives and
non-reactivity with fibrinogen or fibrinogen degradation products
inclusive of fragments D and E wherein said variant murine-derived
monoclonal antibody is substantially non-immunogenic in a human
wherein said monoclonal antibody further comprises a clot
dissolution or clot growth prevention agent fused, bound or
otherwise associated thereto.
[0041] Still another aspect of the present invention is directed to
the use of a variant murine-derived monoclonal antibody having
specificity for human-derived D-dimer and other cross-linked fibrin
derivatives and non-reactivity with fibrinogen or fibrinogen
degradation products inclusive of fragments D and E wherein said
variant murine-derived monoclonal antibody is substantially
non-immunogenic in a human and said antibody further comprising a
clot dissolution or clot growth prevention agent fused, bound or
otherwise attached thereto in the manufacture of a medicament for
the dissolution of a blot clot in a human.
[0042] A preferred molecule is a variant murine monoclonal antibody
3B6 deimmunized for use in humans and comprising a combination of
heavy and light chain v-regions comprising the amino acid sequences
encoded by nucleotide sequences selected from SEQ ID NO:7/SEQ ID
NO:10, SEQ ID NO:8/SEQ ID NO:10, SEQ ID NO:9/SEQ ID NO:10, SEQ ID
NO:7/SEQ ID NO:12, SEQ ID NO:8/SEQ ID NO:12, SEQ ID NO:8/SEQ ID
NO:11, SEQ ID NO:9/SEQ ID NO:11, SEQ ID NO:9/SEQ ID NO:12 and SEQ
ID NO:7/SEQ ID NO:11 or combinations of amino acid sequences
encoded by nucleotide sequences having at least 70% similarity to
one or both amino acid sequences in each of the above listed pairs
or nucleotide sequences capable of hybridizing to low stringency
conditions to one or both nucleotide sequences or their
complementary forms in each of the above listed pairs.
[0043] The variant 3B6 antibody deimmunized for use preferably
comprises a combination of heavy and light chain v-regions
comprising the amino acid sequences selected from SEQ ID NO:1/SEQ
ID SEQ ID NO:2/SEQ ID NO:4, SEQ ID NO:3/SEQ ID NO:4, SEQ ID
NO:1/SEQ ID NO:6, SEQ ID NO:2/SEQ ID NO:6, SEQ ID NO:2/SEQ ID NO:5,
SEQ ID NO:3/SEQ ID NO:5, SEQ ID NO:3/SEQ ID NO:6 and SEQ ID
NO:1/SEQ ID NO:5 or combinations of amino acid sequences having at
least 70% similarity to one or both amino acid sequences in each of
the above listed pairs.
[0044] Another preferred variant of 3B6 comprises a combination of
heavy and light chain v-regions selected from VHv5/VKv1, VHv6/VKv1,
VHv7/VKv1, VHv5/VKv7, VHv6/VKv7, VHv6/VKv4, VHv7/VKv4, VHv7/VKv7
and VHv5/VKv4.
[0045] Another aspect of the present invention contemplates a
method for detecting a blood clot in a human patient by introducing
into the patient a deimmunized form of murine monoclonal antibody
3B6 or an antigen-binding fragment thereof labeled with a reporter
molecule allowing dissemination of the labeled antibody throughout
the circulatory system and then subjecting the patient to a
computer assisted tomographic nuclear medicine scan to visualize
the clot.
[0046] Yet another aspect of the present invention contemplates a
method for detecting a blood clot in a human patient by introducing
into the patient a deimmunized form of murine monoclonal antibody
3B6 or an antigen-binding fragment thereof labeled with a reporter
molecule allowing dissemination of the labeled antibody throughout
the circulatory system and then subjecting said patient to planar
clot imaging to visualize the clot.
[0047] The deimmunized immunointeractive molecule of the present
invention is also useful to anchor an anti-coagulant at a
particular site. This aspect provides, therefore, tissue-specific
anchoring of a diagnostic or therapeutic agent to, for example, a
clot. Furthermore, the immunointeractive molecule may be engineered
to have multiple specificities. For example, a bi-specific
deimmunized antibody is contemplated which comprises one
specificity for a clot and another specificity to a site of a clot
(e.g. to a cell receptor). This enables the antibody to remain at
the site of the clot.
[0048] In an alternative, a multi-step treatment is contemplated
where, for example, the deimmunized 3B6 or other interactive
molecule conjugated to an anti-coagulant is administered to target
the clot and forms a complex and then a second antibody directed to
the first antibody and/or the anti-coagulant is administered to
enhance or monitor the first complex.
[0049] The deimmunized immunointeracative molecule may also be used
to determine the kinetics of clot dissipation or clot
disappearance. This is useful to predict even earlier the
appearance or disappearance of clots and, hence, aids in
determining the kinetics of when to initiate second treatments such
as anti-coagulant treatments.
[0050] The present invention further provides conjugates comprising
the deimmunized immunointeractive molecules and imaging and/or
therapeutic tags. Examples of imaging tags include MRI, ultrasound
and CT tags. Examples of therapeutic tags include radioactive
isotopes; anti-clotting agents and cytokines.
[0051] A summary of sequence identifiers used throughout the
subject specification is provided in Table 1.
TABLE-US-00001 TABLE 1 Summary of Sequence Identifiers SEQUENCE ID
NO: DESCRIPTION 1 Amino acid of 3B6DIVHv5 2 Amino acid of 3B6DIVHv6
3 Amino acid of 3B6DIVHv7 4 Amino acid of 3B6DIVKv1 5 Amino acid of
3B6DIVKv4 6 Amino acid 3B6DIVKv7 7 Nucleotide sequence encoding
3B6DIVHv5 8 Nucleotide sequence encoding 3B6DIVHv6 9 Nucleotide
sequence encoding 3B6DIVHv7 10 Nucleotide sequence encoding
3B6DIVKv1 11 Nucleotide sequence encoding 3B6DIVKv4 12 Nucleotide
sequence encoding 3B6DIVKv7
BRIEF DESCRIPTION OF THE FIGURES
[0052] FIG. 1 is a representation showing (A) a schematic of the
3B6 monoclonal antibody; (B) a photograph of 3B6 binding to blood
clots (.times.4,2000 magnification).
[0053] FIG. 2 is a schematic representation of a 3B6 antibody
labeled with a nuclear tag (.sup.99mTc).
[0054] FIG. 3A is a diagrammatic representation showing
administration of 3B6 .sup.99mTc to the circulatory system of a
human.
[0055] FIG. 3B is a photographic representation showing
visualization of blood clots in the anterior thighs as radiation
from .sup.99mTc concentrates at the clot site.
[0056] FIGS. 4A, 4B and 4C are graphical representations showing
D-dimer capture assay using deimmunized 3B6 monoclonal
antibodies.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0057] The present invention is predicated in part on the
application of biochemical techniques to render an
immunointeractive molecule derived from one animal or avian
creature substantially non-immunogenic in another animal or avian
creature of the same or different species. The biochemical process
is referred to herein as "deimmunization". Reference herein to
"deimmunization" includes processes such as complementary
determinant region (CDR) grafting, "reshaping" with respect to a
framework region of an immunointeractive molecule and variable (v)
region mutation, all aimed at reducing the immunogenicity of an
immunointeractive molecule in a particular host. In the present
case, the preferred immunointeractive molecule is an antibody such
as a polyclonal or monoclonal antibody. In a most preferred
embodiment, the immunointeractive molecule is a monoclonal
antibody, derived from one animal or avian creature and which
exhibits reduced immunogenicity in another animal or avian creature
from the same or different species.
[0058] The present invention relates generally to carrier molecules
derived from one animal or avian species or strains and which are
substantially non-immunogenic when exposed to an immune system from
a species or strain of another animal or avian creature. The
carrier molecules may exhibit useful diagnostic or therapeutic
properties per se or may be used to deliver active compounds (e.g.
anti-clotting agents, radioactive isotopes) to a target site.
Generally, the carrier molecules are immunointeractive molecules.
More particularly, the present invention is directed to a
deimmunized including a non-murine mammalianized form of a
murine-derived monoclonal antibody substantially incapable of
inducing an immune response against itself in a non-murine animal
and in particular a human. Even more particularly, the present
invention provides a deimmunized form of murine monoclonal antibody
3B6 such that it is incapable of, or exhibits reduced capacity for,
inducing a substantial immune response against itself or its
derivatives when administered to a human. The deimmunized
immunointeractive molecules and in particular antibodies of the
present invention have a range of useful diagnostic and therapeutic
applications such as in the detection of blood clots in the
circulatory system of a human and as a clot targeting agent to
deliver a clot dissolution or clot growth prevention agent such as
an anticoagulant. The deimmunized including humanized forms of the
subject monoclonal antibody are particularly useful in the
diagnosis and treatment of conditions such as deep vein thrombosis
and pulmonary embolism. The molecules of the present invention are
particularly useful as agents for delivering active compounds to a
target site. Such active compounds include clot binding
molecules.
[0059] Accordingly, one aspect of the present invention provides a
variant of an immunointeractive molecule, said variant comprising a
portion having specificity for cross-linked fibrin derivatives and
which portion is derived from an immunointeractive molecule
obtainable from one animal or avian creature wherein said variant
exhibits reduced immunogenicity in another animal or avian creature
from the same or different species.
[0060] As stated above, the preferred form of immunointeractive
molecule is an antibody and in particular a monoclonal
antibody.
[0061] Accordingly, another aspect of the present invention
provides a variant monoclonal antibody comprising a portion having
specificity for cross-linked fibrin derivatives and which portion
is derived from a monoclonal antibody obtainable from a first
animal or avian creature wherein said variant exhibits reduced
immunogenicity in a second animal or avian creature from the same
or different species.
[0062] In a particularly preferred embodiment, a monoclonal
antibody is obtained from a murine animal and is deimmunized with
respect to another murine animal or a different species of animal
such as a human. The murine monoclonal antibody is raised in the
murine animal to non-denatured D-dimer which is derived from
fibrinogen. The latter molecule is digested by plasmin and
generates a range of fragments designated fragments A to E.
Cross-linking of fibrin with Factor XIIIa forms dimers of fragment
D referred to as "D-dimer". The D-dimer is a molecule of about
189,000 daltons and comprises two fragment D moieties bound by
cross-linked bonds between the .gamma. chain remnants of
fibrinogen.
[0063] Accordingly, another aspect of the present invention is
directed to a variant murine-derived monoclonal antibody having
specificity for human-derived D-dimer and other cross-linked fibrin
derivatives and non-reactivity with fibrinogen or fibrinogen
degradation products inclusive of fragments D and E wherein said
variant murine-derived monoclonal antibody is substantially
non-immunogenic in a human.
[0064] Reference to "substantially non-immunogenic" includes
reduced immunogenicity compared to a parent antibody, i.e. an
antibody before exposure to deimmunization processes. The term
"immunogenicity" includes an ability to provoke, induce or
otherwise facilitate a humoral and/or T-cell mediated response in a
host animal. Particularly convenient immunogenic criteria include
the ability for amino acid sequences derived from a variable (v)
region of an antibody to interact with MHC class II molecules
thereby stimulating or facilitating a T-cell mediating response
including a T-cell-assisted humoral response. The immunointeractive
molecule and in particular a monoclonal antibody contemplated by
the present invention includes reference to a clot targeting
agent.
[0065] The preferred murine-derived monoclonal antibody is referred
to herein as monoclonal antibody 3B6 which is described in U.S.
Pat. No. 4,758,524.
[0066] Accordingly, in a particularly preferred embodiment, the
present invention provides a deimmunized form of monoclonal
antibody 3B6 wherein said deimmunized 3B6 is substantially
non-immunogenic in humans.
[0067] Again, "substantially non-immunogenic" in this context means
a reduced capacity of the deimmunized 3B6 monoclonal antibody to
induce or facilitate an immune response against itself (following
initial or subsequent administration) in a human compared to murine
monoclonal antibody 3B6, prior to deimmunization.
[0068] Although the preferred invention is particularly directed to
a deimmunized form of 3B6 with respect to humans, the present
invention extends to this antibody or another antibody with a
similar specificity for D-dimer and/or other cross-linked fibrin
derivatives deimmunized for any other animal or avian species.
[0069] Reference herein to other cross-linked fibrin derivatives
includes, for example, in addition to D-dimer, derivatives of
D-dimer and a complex comprising D and E fragments. The latter
includes (DD)E and is formed by plasmin degradation of cross-linked
human fibrin and comprises a combination of two D fragments and
fragment E. Other cross-linked derivatives include DY, YY, XD, XY,
DXD and YXD where the letters represent fragments of fibrinogen
formed following degradation by plasmin wherein X and Y are
different and are selected from fragments A to C and E. Preferably,
the deimmunized antibody of the subject invention is derived from
an antibody specific for D-dimer and other cross-linked fibrin
derivatives but which does not cross-react with fibrinogen,
fibrinogen degradation products inclusive of fragment D and
fragment E. Preferably, the antibody-producing clones are selected
using solution phase D-dimer molecules rather than immobilized
D-dimer although clones selected by either form of D-dimer are
contemplated by the present invention.
[0070] Preferably, the deimmunized antibody exhibits an affinity
for its target antigen which is similar to the affinity exhibited
by murine monoclonal antibody 3B6.
[0071] By "affinity" in relation to the interaction between an
individual antigen binding site on an antigen-binding molecule and
its corresponding site on the antigen includes the strength of this
interaction.
[0072] By "antibody" is meant a protein of the immunoglobulin
family that is capable of combining, interacting or otherwise
associating with an antigen. An antibody is, therefore, an
antigen-binding molecule. An "antibody" is an example of an
immunointeractive molecule and includes a polyclonal or monoclonal
antibody. The preferred immunointeractive molecules of the present
invention are monoclonal antibodies. An antibody includes parts
thereof including Fab portions and antigen-binding
determinants.
[0073] The term "antigen" is used herein in its broadest sense to
refer to a substance that is capable of reacting in and/or inducing
an immune response. Reference to an "antigen" includes an antigenic
determinant or epitope. The antigen in the present context is
regarded as the immunointeractive molecule and, more particularly,
a monoclonal antibody.
[0074] Any molecule that has binding affinity for a target antigen
is referred to as an "antigen-binding molecule". It will be
understood that this term extends to immunoglobulins (e.g.
polyclonal or monoclonal antibodies), immunoglobulin fragments and
non-immunoglobulin derived protein frameworks that exhibit
antigen-binding activity. The terms "antibody" and "antigen-binding
molecules" include deimmunized forms of these molecules.
[0075] That part of an antigenic molecule against which a
particular immune response is directed is referred to as an
"antigenic determinant" or "epitope" and includes a hapten.
Typically, in an animal, antigens present several or even many
antigenic determinants simultaneously. A "hapten" is a substance
that can combine specificity with an antibody but cannot or only
poorly induces an immune response unless bound to a carrier. A
hapten typically comprises a single antigenic determinant or
epitope.
[0076] As stated above, although the preferred antibodies of the
present invention are deimmunized forms of murine monoclonal
antibodies for use in humans, the subject invention extends to
antibodies from any source and deimmunized for use in any host.
Examples of animal and avian sources and hosts include humans,
primates, livestock animals (e.g. sheep, cows, horses, pigs,
donkeys), laboratory test animals (e.g. mice, rabbits, guinea pigs,
hamsters), companion animals (e.g. dogs, cats), poultry bird (e.g.
chickens, ducks, geese, turkeys) and game birds (e.g. pheasants).
The deimmunized antibodies or part thereof may also be generated in
on-animal tissues such as plants. Plants are particularly useful as
a source of single chain antibodies.
[0077] Another aspect of the present invention contemplates a
method for generating a deimmunized monoclonal antibody having
specificity for antigenic determinants on human D-dimer or other
cross-linked fibrin derivatives, said method comprising:
[0078] (i) obtaining a cross-linked fibrin derivative or extract
containing same from a human;
[0079] (ii) generating an antibody in a non-human animal specific
to said cross-linked fibrin derivative but which does not
cross-react with fragment D; and
[0080] (iii) subjecting said non-human derived antibody to
deimmunization means.
[0081] The cross-linked fibrin derivative may be derived from any
suitable antigenic extract including plasmin-mediated degradation
of fibrin clots or by simultaneous action of thrombin, Factor Ma
and plasmin on fibrinogen with transient clot formation and
subsequent clot lysis. In the latter method, the fibrinogen is
converted to fibrin by the action of thrombin and Factor Ma and
subsequently digested with plasmin. It will, of course, be
appreciated that the fibrin derivative or extract containing same
may be obtained from an animal source other than human. The
antigenic source is conveniently from a biological sample.
[0082] A sample that may be extracted, untreated, treated, diluted
or concentrated from an animal is included in the term "biological
sample".
[0083] The above method of obtaining the crude antigenic fraction
represents an in vitro method. A suitable in vivo method includes
obtaining sera or other body fluid containing the cross-linked
fibrin derivative from an animal including human and subjecting the
body fluid to a PAGE process wherein substantially pure
cross-linked fibrin derivative is isolated.
[0084] Alternatively, cross-linked fibrin derivatives may be
purified from serum obtained from patients suffering severe
thrombotic disorders based on a technique using gel filtration in
combination with ion exchange chromatography as described by
Willner et al., Biochemistry 21: 2687-2692, 1982.
[0085] The antigen (i.e. D-dimer or other cross-linked fibrin
derivative) can be separated from the biological sample by any
suitable means. For example, the separation may take advantage of
any one or more of the antigen's surface charge properties, size,
density, biological activity and its affinity for another entity
(e.g. another protein or chemical compound to which it binds or
otherwise associates). Thus, for example, separation of the antigen
from the biological fluid may be achieved by any one or more of
ultra-centrifugation, ion-exchange chromatography (e.g. anion
exchange chromatography, cation exchange chromatography),
electrophoresis (e.g. polyacrylamide gel electrophoresis,
isoelectric focussing), size separation (e.g., gel filtration,
ultra-filtration) and affinity-mediated separation (e.g.
immunoaffinity separation including, but not limited to, magnetic
bead separation such as Dynabead.TM. separation,
immunochromatography, immuno-precipitation). Choice of the
separation technique(s) employed may depend on the biological
activity or physical properties of the particular antigen.
[0086] Preferably, the separation of the antigen from the
biological fluid preserves conformational epitopes present on the
antigen surface and, thus, suitably avoids techniques that cause
denaturation of the antigen. Persons of skill in the art will
recognize the importance of maintaining or mimicking as close as
possible physiological conditions peculiar to the antigen (e.g. the
biological fluid from which they are obtained) to ensure that the
antigenic determinants or active site/s on the antigen, which are
exposed to the animal, are structurally identical to that of the
native antigen. This ensures the raising of appropriate antibodies
in the immunised animal that would recognize the native antigen. In
a preferred embodiment of this type, the antigen is separated from
the biological fluid using any one or more of affinity separation;
gel filtration and ultra-filtration.
[0087] Immunization and subsequent production of monoclonal
antibodies can be carried out using standard protocols as for
example described by Kohler and Milstein (Nature 256: 495-499,
1975; Kohler and Milstein, Eur. J. Immunol. 6(7): 511-519, 1976),
Coligan et al. (Current Protocols in Immunology, John Wiley &
Sons, Inc., 1991-1997) or Toyama et al. ("Monoclonal Antibody,
Experiment Manual", published by Kodansha Scientific, 1987).
Essentially, an animal is immunized with an antigen-containing
biological fluid or fraction thereof by standard methods to produce
antibody-producing cells, particularly antibody-producing somatic
cells (e.g. B lymphocytes). These cells can then be removed from
the immunized animal for immortalization. The antigen may need to
first be associated with a larger molecule. The latter is any
substance of typically high molecular weight to which a non- or
poorly immunogenic substance (e.g. a hapten) is naturally or
artificially linked to enhance its immunogenicity.
[0088] Immortalization of antibody-producing cells may be carried
out using methods, which are well-known in the art. For example,
the immortalization may be achieved by the transformation method
using Epstein-Barr virus (EBV) (Kozbor et al., Methods in
Enzymology 121: 140, 1986): In a preferred embodiment,
antibody-producing cells are immortalized using the cell fusion
method (described in Coligan et al., 1991-1997, supra), which is
widely employed for the production of monoclonal antibodies. In
this method, somatic antibody-producing cells with the potential to
produce antibodies, particularly B cells, are fused with a myeloma
cell line. These somatic cells may be derived from the lymph nodes,
spleens and peripheral blood of primed animals, preferably rodent
animals such as mice and rats. In the exemplary embodiment of this
invention mice, spleen cells are used. It would be possible,
however, to use rat, rabbit, sheep or goat cells, or cells from
other animal species instead.
[0089] Specialized myeloma cell lines have been developed from
lymphocytic tumours for use in hybridoma-producing fusion
procedures (Kohler and Milstein, 1976, supra; Shulman et al.,
Nature 276: 269-270, 1978; Volk et al., J. Virol. 42(1): 220-227,
1982). These cell lines have been developed for at least three
reasons. The first is to facilitate the selection of fused
hybridomas from infused and similarly indefinitely self-propagating
myeloma cells. Usually, this is accomplished by using myelomas with
enzyme deficiencies that render them incapable of growing in
certain selective media that support the growth of hybridomas. The
second reason arises from the inherent ability of lymphocytic
tumour cells to produce their own antibodies. To eliminate the
production of tumour cell antibodies by the hybridomas, myeloma
cell lines incapable of producing endogenous light or heavy
immunoglobulin chains are used. A third reason for selection of
these cell lines is for their suitability and efficiency for
fusion.
[0090] Many myeloma cell lines may be used for the production of
fused cell hybrids, including, e.g. P3X63-Ag8, P3X63-AG8.653,
P3/NS1-Ag4-1 (NS-1), Sp2/0-Ag14 and S194/5.XXO.Bu.1. The P3X63-Ag8
and NS-1 cell lines have been described by Kohler and Milstein
(1976, supra). Shulman et al. (1978, supra) developed the
Sp2/0-Ag14 myeloma line. The S194/5.XXO.Bu.1 line was reported by
Trowbridge (J. Exp. Med. 148(1): 313-323, 1978).
[0091] Methods for generating hybrids of antibody-producing spleen
or lymph node cells and myeloma cells usually involve mixing
somatic cells with myeloma cells in a 10:1 proportion (although the
proportion may vary from about 20:1 to about 1:1), respectively, in
the presence of an agent or agents (chemical, viral or electrical)
that promotes the fusion of cell membranes. Fusion methods have
been described (Kohler and Milstein, 1975, supra; Kohler and
Milstein, 1976, supra; Getter et al., Somatic Cell Genet. 3:
231-236, 1977; Volk et al., 1982, supra). The fusion-promoting
agents used by those investigators were Sendai virus and
polyethylene glycol (PEG).
[0092] Because fusion procedures produce viable hybrids at very low
frequency (e.g. when spleens are used as a source of somatic cells,
only one hybrid is obtained for roughly every 1.times.10.sup.5
spleen cells), it is preferable to have a means of selecting the
fused cell hybrids from the remaining unfused cells, particularly
the unfused myeloma cells. A means of detecting the desired
antibody-producing hybridomas among other resulting fused cell
hybrids is also necessary. Generally, the selection of fused cell
hybrids is accomplished by culturing the cells in media that
support the growth of hybridomas but prevent the growth of the
unfused myeloma cells, which normally would go on dividing
indefinitely. The-somatic cells used in the fusion do not maintain
long-term viability in in vitro culture and hence do not pose a
problem. In the example of the present invention, myeloma cells
lacking hypoxanthine phosphoribosyl transferase (HPRT-negative)
were used. Selection against these cells is made in
hypoxanthindaminopterin/thymidine (HAT) medium, a medium in which
the fused cell hybrids survive due to the HPRT-positive genotype of
the spleen cells. The use of myeloma cells with different genetic
deficiencies (drug sensitivities, etc.) that can be selected
against in media supporting the growth of genotypically competent
hybrids is also possible.
[0093] Several weeks are required to selectively culture the fused
cell hybrids. Early in this time period, it is necessary to
identify those hybrids which produce the desired antibody, so that
they may subsequently be cloned and propagated. Generally, around
10% of the hybrids obtained produce the desired antibody, although
a range of from about 1 to about 30% is not uncommon. The detection
of antibody-producing hybrids can be achieved by any one of several
standard assay methods, including enzyme-linked immunoassay and
radioimmunoassay techniques as, for example, described in Kennet et
al. ((eds) Monoclonal Antibodies and Hybridomas: A New Dimension in
Biological Analyses, pp. 376-384, Plenum Press, New York, 1980). In
a particularly preferred embodiment, an enzyme linked immunosorbent
assay (ELISA) is performed to selected antibody producing clones
using solution phase D-dimer.
[0094] Once the desired fused cell hybrids have been selected and
cloned into individual antibody-producing cell lines, each cell
line may be propagated in either of two standard ways. A suspension
of the hybridoma cells can be injected into a histocompatible
animal. The injected animal will then develop tumours that secrete
the specific monoclonal antibody produced by the fused cell hybrid.
The body fluids of the animal, such as serum or ascites fluid, can
be tapped to provide monoclonal antibodies in high concentration.
Alternatively, the individual cell lines may be propagated in vitro
in laboratory culture vessels. The culture medium containing high
concentrations of a single specific monoclonal antibody can be
harvested by decantation, filtration or centrifugation, and
subsequently purified.
[0095] The cell lines are tested for their specificity to detect
the antigen of interest by any suitable immunodetection means. For
example, cell lines can be aliquoted into a number of wells and
incubated and the supernatant from each well is analyzed by
enzyme-linked immunosorbent assay (ELISA), indirect fluorescent
antibody technique, or the like. The cell line(s) producing a
monoclonal antibody capable of recognizing the target antigen but
which does not recognize non-target epitopes are identified and
then directly cultured in vitro or injected into a histocompatible
animal to form tumours and to produce, collect and purify the
required antibodies.
[0096] Thus, the present invention provides in a first step
monoclonal antibodies which specifically interact with D-dimer or
other cross-linked fibrin derivative.
[0097] As indicated above, non-animal cells such as a plant, yeast
and/or microbial cells may be used to generate typically
single-chain antibodies. In this embodiment, such cells are
engineered to express nucleic acid molecules which encode a chain
of an antibody.
[0098] The monoclonal antibody is then subjected to deimmunization
means. Such a process may take any of a number of forms including
the preparation of chimeric antibodies which have the same or
similar specificity as the monoclonal antibodies prepared according
to the present invention. Chimeric antibodies are antibodies whose
light and heavy chain genes have been constructed, typically by
genetic engineering, from immunoglobulin variable and constant
region genes belonging to different species. Thus, in accordance
with the present invention, once a hybridoma producing the desired
monoclonal antibody is obtained, techniques are used to produce
interspecific monoclonal antibodies wherein the binding region of
one species is combined with a non-binding region of the antibody
of another species (Liu et al., Proc. Natl. Acad. Sci. USA 84:
3439-3443, 1987). For example, the CDRs from a non-human (e.g.
murine) monoclonal antibody can be grafted onto a human antibody,
thereby "humanizing" the murine antibody (European Patent
Publication No. 0 239 400; Jones et al., Nature 321: 522-525, 1986;
Verhoeyen et al., Science 239: 1534-1536, 1988; Riechmann et al.,
Nature 332: 323-327, 1988). In this case, the deimmunizing process
is specific for humans. More particularly, the CDRs can be grafted
onto a human antibody variable region with or without human
constant regions. The non-human antibody providing the CDRs is
typically referred to as the "donor" and the human antibody
providing the framework is typically referred to as the "acceptor".
Constant regions need not be present, but if they are, they must be
substantially identical to human immunoglobulin constant regions,
i.e. at least about 85-90%, preferably about 95% or more identical.
Hence, all parts of a humanized antibody, except possibly the CDRs,
are substantially identical to corresponding parts of natural human
immunoglobulin sequences. Thus, a "humanized antibody" is an
antibody comprising a humanized light chain and a humanized heavy
chain immunoglobulin. A donor antibody is said to be "humanized",
by the process of "humanization", because the resultant humanized
antibody is expected to bind to the same antigen as the donor
antibody that provides the CDRs. Reference herein to "humanized"
includes reference to an antibody deimmunized to a particular host,
in this case, a human host.
[0099] It will be understood that the deimmunized antibodies may
have additional conservative amino acid substitutions which have
substantially no effect on antigen binding or other immunoglobulin:
functions. Exemplary conservative substitutions may be made
according to Table 2.
TABLE-US-00002 TABLE 2 ORIGINAL EXEMPLARY RESIDUE SUBSTITUTIONS Ala
Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro
His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu,
Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile,
Leu
[0100] Exemplary methods which may be employed to produce
deimmunized antibodies according to the present invention are
described, for example, in Richmann et al., 1988, supra; U.S. Pat.
Nos. 6,056,957, 6,180,370 and 6,180,377 and Chothia et al., J. Mol.
Biol. 196: 901, 1987.
[0101] Thus, in one embodiment, the present invention contemplates
a deimmunized antibody molecule having specificity for an epitope
recognized by monoclonal antibody 3B6 wherein at least one or at
least two or at least three or at least four or at least five of
the complementary determining regions (CDRs) of the variable domain
of said deimmunized antibody is derived from said 3B6 monoclonal
antibody and the remaining immunoglobulin-derived parts of the
deimmunized antibody molecule are derived from an immunoglobulin or
an analogue thereof from the host for which the antibody is to be
deimmunized.
[0102] This aspect of the present invention involves manipulation
of the framework region of a non-human antibody.
[0103] Preferably, the deimmunized antibody is a humanized form of
murine 3B6.
[0104] One preferred deimmunization process is referred to herein a
variable(v)-region grafting and results in a chimeric antibody. The
resulting antibody comprises one or more amino acid substitutions
within the v-region when compared to the present (e.g. murine)
antibody. The rationale for making v-region changes is to further
the potential for an induced immune response in the intended host
(e.g. a human). The basis of deimmunization is predicated in part
on the assumption that a substantive immune response to an
introduced antibody requires a T-cell mediated response. The
trigger for the T-cell response is the presentation of processed
peptides emanating from the introduced antibody on the surface of
antigen presenting cells (APCs). The APCs present such peptides in
association with surface MHC class II molecules. The deimmunized
approach is, therefore, based on:
[0105] (i) predicting peptide sequences capable of associating with
MHC class II molecules; and
[0106] (ii) changing strategic residues to eliminate the ability of
the peptide to associate with the MHC class II molecule.
[0107] Accordingly, another aspect of the present invention
provides a variant of murine monoclonal antibody 3B6 deimmunized
for use in humans, said variant comprising one or more amino acid
mutations in the v-region of said 3B6 antibody to eliminate or
reduce peptide fragments of said v-region associating with MHC
class II molecules.
[0108] One or more amino acid substitutions, additions and/or
deletions or one or more nucleotide substitutions, additions and/or
deletions is encompassed by the term "mutation" or "mutations".
[0109] In a particularly preferred embodiment, the deimmunized
antibodies are generated by co-transfection of different
combinations of three deimmunized H-chain genes and three
deimmunized L-chain genes. The resulting variants are derived from
different combinations encoded by H-chain and L-chain genes.
Preferred H-chains are Hv5, Hv6 and Hv7. These are referred to
herein as 3B6DIVHv5 (SEQ ID NO:1), 3B6DIVHv6 (SEQ ID NO:2) and
3B6DIVHv7 (SEQ ID NO:3). Preferred L-chains are Kv1, Kv4 and Kv7.
These are referred to herein as 3B6DIVKv1 (SEQ ID NO:4), 3B6DIVKv4
(SEQ ID NO:5) and 3B6DIVKv7 (SEQ ID NO:6). Particularly useful
combinations include VHv5/VKv1, VHv6/VKv1, VHv7/VKv1, VHv5/VKv7,
VHv6/VKv7, VHv6/VKv4, VHv7/VKv4, VHv7/VKv7 and VHv5/VKv4. The
sequence identifier numbers (SEQ ID NOS:) in parentheses represent
the amino acid sequences of the particular chains. Corresponding
nucleotide sequences encoding each of SEQ ID NOS:1-6 are
represented by SEQ ID NOS:7-12.
[0110] All such combinations of H and L chains are also encompassed
by the present invention.
[0111] Accordingly, the present invention provides a variant murine
monoclonal antibody 3B6 deimmunized for use in humans, said variant
comprising a combination of heavy and light chain v-regions
comprising the amino acid sequences encoded by nucleotide sequences
selected from SEQ ID NO:7/SEQ ID NO:10, SEQ ID NO:8/SEQ ID NO:10,
SEQ ID NO:9/SEQ ID NO:10, SEQ ID NO:7/SEQ ID NO:12, SEQ ID NO:8/SEQ
ID NO:12, SEQ ID NO:8/SEQ ID NO:11, SEQ ID NO:9/SEQ ID NO:11, SEQ
ED NO:9/SEQ ID NO:12 and SEQ ID NO:7/SEQ ID NO:11 or combinations
of amino acid sequences encoded by nucleotide sequences having at
least 70% similarity to one or both amino acid sequences in each of
the above listed pairs or nucleotide sequences capable of
hybridizing to low stringency conditions to one or both nucleotide
sequences or their complementary forms in each of the above listed
pairs.
[0112] All such combinations of H and L chains are also encompassed
by the present invention.
[0113] Accordingly, the present invention provides a variant murine
monoclonal antibody 3B6 deimmunized for use in humans, said variant
comprising a combination of heavy and light chain v-regions
comprising the amino acid sequences selected from SEQ ID NO:1/SEQ
ID NO:4, SEQ ID NO:2/SEQ ID NO:4, SEQ ID NO:3/SEQ ID NO:4, SEQ ID
NO:1/SEQ ID NO:6, SEQ ID NO:2/SEQ ID NO:6, SEQ ID NO:2/SEQ ID NO:5,
SEQ NO:3/SEQ ID NO:5, SEQ ID NO:3/SEQ ID NO:6 and SEQ ID NO:1/SEQ
ID NO:5 or combinations of amino acid sequences encoded by
nucleotide sequences having at least 70% similarity to one or both
amino acid sequences in each of the above listed pairs.
[0114] More particularly, the present invention provides a variant
of a murine monoclonal antibody 3B6 deimmunized for use in humans,
said variant comprising a combination of heavy and light chain
v-regions selected from VHv5NKv1, VHv6/VKv1, VHv7NKv1, VHv5/VKv7,
VHv6/VKv7, VHv6/VKv4, VHv7/VKv4, VHv7NKv7 and VHv5/VKv4.
[0115] The term "similarity" as used herein includes exact identity
between compared sequences at the nucleotide or amino acid level.
Where there is non-identity at the nucleotide level, "similarity"
includes differences between sequences which result in different
amino acids that are nevertheless related to each other at the
structural, functional, biochemical and/or conformational levels.
Where there is non-identity at the amino acid level, "similarity"
includes amino acids that are nevertheless related to each other at
the structural, functional, biochemical and/or conformational
levels. In a particularly preferred embodiment, nucleotide and
sequence comparisons are made at the level of identity rather than
similarity.
[0116] Terms used to describe sequence relationships between two or
more polynucleotides or polypeptides include "reference sequence",
"comparison window", "sequence similarity", "sequence identity",
"percentage of sequence similarity", "percentage of sequence
identity", "substantially similar" and "substantial identity". A
"reference sequence" is at least 12 but frequently 15 to 18 and
often at least 25 or above, such as 30 monomer units, inclusive of
nucleotides and amino acid residues, in length. Because two
polynucleotides may each comprise (1) a sequence (i.e. only a
portion of the complete polynucleotide sequence) that is similar
between the two polynucleotides, and (2) a sequence that is
divergent between the two polynucleotides, sequence comparisons
between two (or more) polynucleotides are typically performed by
comparing sequences of the two polynucleotides over a "comparison
window" to identify and compare local regions of sequence
similarity. A "comparison window" refers to a conceptual segment of
typically 12 contiguous residues that is compared to a reference
sequence. The comparison window may comprise additions or deletions
(i.e. gaps) of about 20% or less as compared to the reference
sequence (which does not comprise additions or deletions) for
optimal alignment of the two sequences. Optimal alignment of
sequences for aligning a comparison window may be conducted by
computerised implementations of algorithms (GAP, BESTFIT, FASTA,
and TFASTA in the Wisconsin Genetics Software Package Release 7.0,
Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or
by inspection and the best alignment (i.e. resulting in the highest
percentage homology over the comparison window) generated by any of
the various methods selected. Reference also may be made to the
BLAST family of programs as, for example, disclosed by Altschul et
al. (Nucl. Acids Res. 25: 3389-3402. 1997). A detailed discussion
of sequence analysis can be found in Unit 19.3 of Ausubel et al.
("Current Protocols in Molecular Biology" John Wiley & Sons
Inc, 1994-1998, Chapter 15).
[0117] The terms "sequence similarity" and "sequence identity" as
used herein refers to the extent that sequences are identical or
functionally or structurally similar on a nucleotide-by-nucleotide
basis or an amino acid-by-amino acid basis over a window of
comparison. Thus, a "percentage of sequence identity", for example,
is calculated by comparing two optimally aligned sequences over the
window of comparison, determining the number of positions at which
the identical nucleic acid base (e.g. A, T, C, G, I) or the
identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val,
Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and
Met) occurs in both sequences to yield the number of matched
positions, dividing the number of matched positions by the total
number of positions in the window of comparison (i.e., the window
size), and multiplying the result by 100 to yield the percentage of
sequence identity. For the purposes of the present invention,
"sequence identity" will be understood to mean the "match
percentage" calculated by the DNASIS computer program (Version 2.5
for windows; available from Hitachi Software engineering Co., Ltd.,
South San Francisco, Calif., USA) using standard defaults as used
in the reference manual accompanying the software. Similar comments
apply in relation to sequence similarity.
[0118] Mutations and derivatives contemplated by the present
invention include redundant mutations in nucleotide sequences which
do not result in a change in amino acid sequence.
[0119] Reference herein to a low stringency includes and
encompasses from at least about 0 to at least about 15% v/v
formamide and from at least about 1 M to at least about 2 M salt
for hybridization, and at least about 1 M to at least about 2 M
salt for washing conditions. Generally; low stringency is at from
about 25-30.degree. C. to about 42.degree. C. The temperature may
be altered and higher temperatures used to replace formamide and/or
to give alternative stringency conditions. Alternative stringency
conditions may be applied where necessary, such as medium
stringency, which includes and encompasses from at least about 16%
v/v to at least about 30% v/v formamide and from at least about 0.5
M to at least about 0.9 M salt for hybridization, and at least
about 0.5 M to at least about 0.9 M salt for washing conditions, or
high stringency, which includes and encompasses from at least about
31% v/v to at least about 50% v/v formamide and from at least about
0.01 M to at least about 0.15 M salt for hybridization, and at
least about 0.01 M to at least about 0.15 M salt for washing
conditions. In general, washing is carried out T.sub.m=69.3+0.41
(G+C) % (Marmur and Doty, J. Mol. Biol. 5: 109, 1962). However, the
T.sub.m of a duplex DNA decreases by 1.degree. C. with every
increase of 1% in the number of mismatch base pairs (Bonner and
Laskey, J. Mol. Biol. 5: 109, 1962). Formamide is optional in these
hybridization conditions. Accordingly, particularly preferred
levels of stringency are defined as follows: low stringency is
6.times.SSC buffer, 0.1% w/v SDS at 25-42.degree. C.; a moderate
stringency is 2.times.SSC buffer, 0.1% w/v SDS at a temperature in
the range 20.degree. C. to 65.degree. C.; high stringency is
0.1.times.SSC buffer, 0.1% w/v SDS at a temperature of at least
65.degree. C.
[0120] As used herein, the term "CDR" includes CDR structural loops
which covers to the three light chain and the three heavy chain
regions in the variable portion of an antibody framework region
which bridge .beta. strands on the binding portion of the molecule.
These loops have characteristic canonical structures (Chothia et
al., J. Mol. Biol. 227: 799, 1992; Kabat et al., "Sequences of
Proteins of Immunological Interest", U.S. Department of Health and
Human Services, 1983).
[0121] An immunoglobulin light or heavy chain variable region,
which is interrupted by three hypervariable regions, also called
CDRs, is referred to herein as a "framework region". The extent of
the framework region and CDRs have been precisely defined (see, for
example, Krebber et al., J. Immunol. Methods 201(1): 35-55, 19).
The sequences of the framework regions of different light or heavy
chains are relatively conserved within a species. As used herein, a
"human framework region" is a framework region that is
substantially identical (about 85% or more, usually 90-95% or more)
to the framework region of a naturally occurring human
immunoglobulin. The framework region of an antibody, that is the
combined framework regions of the constituent light and heavy
chains, serves to position and align the CDRs. The CDRs are
primarily responsible for binding to an epitope of an antigen.
[0122] As used herein, the term "heavy chain variable region" means
a polypeptide which is from about 110 to 125 amino acid residues in
length, the amino acid sequence of which corresponds to that of a
heavy chain of a monoclonal antibody of the invention, starting
from the amino-terminal (N-terminal) amino acid residue of the
heavy chain. Likewise, the term "light chain variable region" means
a polypeptide which is from about 95 to 130 amino acid residues in
length, the amino acid sequence of which corresponds to that of a
light chain of a monoclonal antibody of the invention, starting
from the N-terminal amino acid residue of the light chain.
Full-length immunoglobulin "light chains" (about 25 Kd or 214 amino
acids) are encoded by a variable region gene at the
NH.sub.2-terminus (about 110 amino acids) and a K or X constant
region gene at the COOH-terminus. Full-length immunoglobulin "heavy
chains" (about 50 Kd or 446 amino acids), are similarly encoded by
a variable region gene (about 116 amino acids) and one of the other
aforementioned constant region genes, e.g. .gamma. (encoding about
330 amino acids).
[0123] The term "immunogenicity" is used herein in its broadest
sense to include the property of evoking an immune response within
an organism. Immunogenicity typically depends partly upon the size
of the substance in question, and partly upon how unlike host
molecules it is. It is generally considered that highly conserved
proteins tend to have rather low immunogenicity.
[0124] The term "immunoglobulin" is used herein to refer to a
protein consisting of one or more polypeptides substantially
encoded by immunoglobulin genes. The recognized immunoglobulin
genes include the .kappa., .lamda., .alpha., .gamma.(IgG.sub.1,
IgG.sub.2, IgG.sub.3, IgG.sub.4), .delta., .epsilon. and .mu.
constant region genes, as well as the myriad immunoglobulin
variable region genes. One form of immunoglobulin constitutes the
basic structural unit of an antibody. This form is a tetramer and
consists of two identical pairs of immunoglobulin chains, each pair
having one light and one heavy chain. In each pair, the light and
heavy chain variable regions are together responsible for binding
to an antigen, and the constant regions are responsible for the
antibody effector functions. In addition to antibodies,
immunoglobulins may exist in a variety of other forms including,
for example, Fv, Fab, Fab' and (Fab').sub.2.
[0125] Reference herein to "immuno-interactive" includes reference
to any interaction, reaction, or other form of association between
molecules and in particular where one of the molecules is, or
mimics, a component of the immune system. An "immunointeractive
molecule" includes an antibody, antibody fragment, synthetic
antibody or a T-cell associated binding molecule (TABM).
[0126] By "isolated" is meant material that is substantially or
essentially free from components that normally accompany it in its
native state.
[0127] A sample of biological fluid that is isolated from, or
derived from, a particular source of the host is described as being
"obtained from".
[0128] The invention also contemplates the use and generation of
fragments of monoclonal antibodies produced by the method of the
present invention including, for example, Fv, Fab, Fab' and
F(ab).sub.2 fragments. Such fragments may be prepared by standard
methods as for example described by Coligan et al. (1991-1997,
supra).
[0129] The present invention also contemplates synthetic or
recombinant antigen-binding molecules with the same or similar
specificity as the monoclonal antibodies of the invention. Antigen
binding molecules of this type may comprise a synthetic stabilized
Fv fragment. Exemplary fragments of this type include single chain
Fv fragments (sFv, frequently termed scFv) in which a peptide
linker is used to bridge the N terminus or C terminus of a V.sub.H
domain with the C terminus or N-terminus, respectively, of a
V.sub.L domain. ScFv lack all constant parts of whole antibodies
and are not able to activate complement. Suitable peptide linkers
for joining the V.sub.H and V.sub.L domains are those which allow
the V.sub.H and V.sub.L domains to fold into a single polypeptide
chain having an antigen binding site with a three dimensional
structure similar to that of the antigen binding site of a whole
antibody from which the Fv fragment is derived. Linkers having the
desired properties may be obtained by the method disclosed in U.S.
Pat. No. 4,946,778. However, in some cases a linker is absent.
ScFvs may be prepared, for example, in accordance with methods
outlined in Krebber et al. (1997, supra). Alternatively, they may
be prepared by methods described in U.S. Pat. No. 5,091,513,
European Patent No 239,400 or the articles by Winter and Milstein
(Nature 349: 293, 1991) and Pluckthun et al. (In Antibody
engineering: A practical approach 203-252, 1996).
[0130] Alternatively, the synthetic stabilised Fv fragment
comprises a disulphide stabilized Fv (dsFv) in which cysteine
residues are introduced into the V.sub.H and V.sub.L domains such
that in the fully folded Fv molecule the two residues will form a
disulphide bond therebetween. Suitable methods of producing dsFv
are described, for example, in (Glockshuber et al., Biochem. 29:
1363-1367, 1990; Reiter et al., J. Biol. Chem. 269: 18327-18331,
1994; Reiter et al., Biochem. 33: 5451-5459, 1994; Reiter et al.,
Cancer Res. 54: 2714-2718, 1994; Webber et al., Mol. Immunol. 32:
249-258, 1995).
[0131] Also contemplated as synthetic or recombinant
antigen-binding molecules are single variable region domains
(termed dAbs) as, for example, disclosed in (Ward et al., Nature
341: 544-546, 1989; Hamers-Casterman et al., Nature 363: 446-448,
1993; Davies & Riechmann, FEBS Lett. 339: 285-290, 1994).
[0132] Alternatively, the synthetic or recombinant antigen-binding
molecule may comprise a "minibody". In this regard, minibodies are
small versions of whole antibodies, which encode in a single chain
the essential elements of a whole antibody. Suitably, the minibody
is comprised of the V.sub.H and V.sub.L domains of a native
antibody fused to the hinge region and CH3 domain of the
immunoglobulin molecule as, for example, disclosed in U.S. Pat. No.
5,837,821.
[0133] In an alternate embodiment, the synthetic or recombinant
antigen binding molecule may comprise non-immunoglobulin derived,
protein frameworks. For example, reference may be made to Ku &
Schutz (Proc. Natl. Acad. Sci. USA 92: 6552-6556, 1995) which
discloses a four-helix bundle protein cytochrome b562 having two
loops randomized to create complementarity determining regions
(CDRs), which have been selected for antigen binding.
[0134] The synthetic or recombinant antigen-binding molecule may be
multivalent (i.e. having more than one antigen binding site). Such
multivalent molecules may be specific for one or more antigens.
Multivalent molecules of this type may be prepared by dimerization
of two antibody fragments through a cysteinyl-containing peptide
as, for example disclosed by (Adams et al., Cancer Res. 53:
4026-4034, 1993; Cumber et al., J. Immunol. 149: 120-126, 1992).
Alternatively, dimerization may be facilitated by fusion of the
antibody fragments to amphiphilic helices that naturally dimerize
(Plunckthun, Biochem. 31: 1579-1584, 1992) or by use of domains
(such as leucine zippers jun and fos) that preferentially
heterodimerize (Kostelny et al., J. Immunol. 148: 1547-1553, 1992).
In further embodiment, a multi-step process is employed such as
first administering a deimmunized antibody and then an
anti-antibody with, for example, a reporter molecule.
[0135] The present invention further encompasses chemical analogues
of amino acids in the variant antibodies. The use of chemical
analogues of amino acids is useful inter alia to stabilize the
molecules when administered to a subject. The analogues of the
amino acids contemplated herein include, but are not limited to,
modifications of side chains, incorporation of unnatural amino
acids and/or their derivatives during peptide, polypeptide or
protein synthesis and the use of crosslinkers and other methods
which impose conformational constraints on the proteinaceous
molecule or their analogues.
[0136] Examples of side chain modifications contemplated by the
present invention include modifications of amino groups such as by
reductive alkylation by reaction with an aldehyde followed by
reduction with NaBH.sub.4; amidination with methylacetimidate;
acylation with acetic anhydride; carbamoylation of amino groups
with cyanate; trinitrobenzylation of amino groups with
2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino
groups with succinic anhydride and tetrahydrophthalic anhydride;
and pyridoxylation of lysine with pyridoxal-5-phosphate followed by
reduction with NaBH.sub.4.
[0137] The guanidine group of arginine residues may be modified by
the formation of heterocyclic condensation products with reagents
such as 2,3-butanedione, phenylglyoxal and glyoxal.
[0138] The carboxyl group may be modified by carbodiimide
activation via O-acylisourea formation followed by subsequent
derivitisation, for example, to a corresponding amide.
[0139] Sulphydryl groups may be modified by methods such as
carboxymethylation with iodoacetic acid or iodoacetamide; performic
acid oxidation to cysteic acid; formation of a mixed disulphides
with other thiol compounds; reaction with maleimide, maleic
anhydride or other substituted maleimide; formation of mercurial
derivatives using 4-chloromercuribenzoate,
4-chloromercuriphenylsulphonic acid, phenylmercury chloride,
2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation
with cyanate at alkaline pH.
[0140] Tryptophan residues may be modified by, for example,
oxidation with N-bromosuccinimide or alkylation of the indole ring
with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine
residues on the other hand, may be altered by nitration with
tetranitromethane to form a 3-nitrotyrosine derivative.
[0141] Modification of the imidazole ring of a histidine residue
may be accomplished by alkylation with iodoacetic acid derivatives
or N-carbethoxylation with diethylpyrocarbonate.
[0142] Examples of incorporating unnatural amino acids and
derivatives during peptide synthesis include, but are not limited
to, use of norleucine, 4-amino butyric acid,
4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid,
t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine,
4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or
D-isomers of amino acids. A list of unnatural amino acid,
contemplated herein is shown in Table 3.
TABLE-US-00003 TABLE 3 Non-conventional Non-conventional amino acid
Code amino acid Code .alpha.-aminobutyric acid Abu
L-N-methylalanine Nmala .alpha.-amino-.alpha.-methylbutyrate Mgabu
L-N-methylarginine Nmarg aminocyclopropane- Cpro
L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid
Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys
aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate
L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa
L-Nmethylhistidine Nmhis cyclopentylalanine Cpen
L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine Nmleu
D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp
L-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine
Nmnle D-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid
Dglu L-N-methylornithine Nmorn D-histidine Dhis
L-N-methylphenylalanine Nmphe D-isoleucine Dile L-N-methylproline
Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys
L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophan
Nmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine
Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine
Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine
Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine
Dtyr .alpha.-methyl-aminoisobutyrate Maib D-valine Dval
.alpha.-methyl-.gamma.-aminobutyrate Mgabu D-.alpha.-methylalanine
Dmala .alpha.-methylcyclohexylalanine Mchexa
D-.alpha.-methylarginine Dmarg .alpha.-methylcylcopentylalanine
Mcpen D-.alpha.-methylasparagine Dmasn
.alpha.-methyl-.alpha.-napthylalanine Manap
D-.alpha.-methylaspartate Dmasp .alpha.-methylpenicillamine Mpen
D-.alpha.-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu
D-.alpha.-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg
D-.alpha.-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn
D-.alpha.-methylisoleucine Dmile N-amino-.alpha.-methylbutyrate
Nmaabu D-.alpha.-methylleucine Dmleu .alpha.-napthylalanine Anap
D-.alpha.-methyllysine Dmlys N-benzylglycine Nphe
D-.alpha.-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln
D-.alpha.-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn
D-.alpha.-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu
D-.alpha.-methylproline Dmpro N-(carboxymethyl)glycine Nasp
D-.alpha.-methylserine Dmser N-cyclobutylglycine Ncbut
D-.alpha.-methylthreonine Dmthr N-cycloheptylglycine Nchep
D-.alpha.-methyltryptophan Dmtrp N-cyclohexylglycine Nchex
D-.alpha.-methyltyrosine Dmty N-cyclodecylglycine Ncdec
D-.alpha.-methylvaline Dmval N-cylcododecylglycine Ncdod
D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct
D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro
D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund
D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm
D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe
D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg
D-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine Nthr
D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser
D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis
D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp
D-N-methyllysine Dnmlys N-methyl-.gamma.-aminobutyrate Nmgabu
N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet
D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen
N-methylglycine Nala D-N-methylphenylalanine Dnmphe
N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro
N-(1-methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr
D-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nval
D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
D-N-methylvaline Dnmval N-methylpenicillamine Nmpen
.gamma.-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr
L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg
penicillamine Pen L-homophenylalanine Hphe L-.alpha.-methylalanine
Mala L-.alpha.-methylarginine Marg L-.alpha.-methylasparagine Masn
L-.alpha.-methylaspartate Masp L-.alpha.-methyl-t-butylglycine
Mtbug L-.alpha.-methylcysteine Mcys L-methylethylglycine Metg
L-.alpha.-methylglutamine Mgln L-.alpha.-methylglutamate Mglu
L-.alpha.-methylhistidine Mhis L-.alpha.-methylhomophenylalanine
Mhphe L-.alpha.-methylisoleucine Mile N-(2-methylthioethyl)glycine
Nmet L-.alpha.-methylleucine Mleu L-.alpha.-methyllysine Mlys
L-.alpha.-methylmethionine Mmet L-.alpha.-methylnorleucine Mnle
L-.alpha.-methylnorvaline Mnva L-.alpha.-methylornithine Morn
L-.alpha.-methylphenylalanine Mphe L-.alpha.-methylproline Mpro
L-.alpha.-methylserine Mser L-.alpha.-methylthreonine Mthr
L-.alpha.-methyltryptophan Mtrp L-.alpha.-methyltyrosine Mtyr
L-.alpha.-methylvaline Mval L-N-methylhomophenylalanine Nmhphe
N-(N-(2,2-diphenylethyl)carbamylmethyl)glycine Nnbhm
N-(N-(3,3-diphenylpropyl)carbamylmethyl)glycine Nnbhe
1-carboxy-1-(2,2-diphenyl- Nmbc ethylamino)cyclopropane
[0143] Crosslinkers can be used, for example, to stabilize 3D
conformations, using homo-bifunctional crosslinkers such as the
bifunctional imido esters having (CH.sub.2).sub.n spacer groups
with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and
hetero-bifunctional reagents which usually contain an
amino-reactive moiety such as N-hydroxysuccinimide and another
group specific-reactive moiety such as maleimido or dithio moiety
(SH) or carbodiimide (COOH). In addition, peptides can be
conformationally constrained by, for example, incorporation of
C.sub..alpha. and N.sub..alpha.-methylamino acids, introduction of
double bonds between C.sub..alpha. and C.sub..beta. atoms of amino
acids and the formation of cyclic peptides or analogues by
introducing covalent bonds such as forming an amide bond between
the N and C termini, between two side chains or between a side
chain and the N or C terminus.
[0144] A monoclonal antibody obtained before deimmunization may be
identified by any number of means including the steps of
[0145] (a) coating a surface with antigen selected from
cross-linked fibrin derivative or extract containing same or
fibrinogen degradation product;
[0146] (b) contacting the antigen in step (a) with monoclonal
antibody derived from fibrin cross-linked derivative prepared as
described above; and
[0147] (c) subjecting the complex formed in step (b) to a signal
amplification step.
[0148] Suitably, in step (a), a well plate may be utilized in which
cross-linked fibrin derivatives such as D-dimer and/or fibrinogen
degradation product (preferably obtained from a procedure wherein
fibrinogen was suitably digested with thrombin to obtain fragment
D, fragment E and optionally fragments X and Y) was applied to the
individual wells.
[0149] Subsequently, monoclonal antibody derived from a
cross-linked fibrin derivative was then added to each well. An
appropriate signal amplification step which may be applied is an
EIA step wherein an appropriate enzyme conjugate may be coupled to
the complex and substrate subsequently added. Alternatively, RIA,
FIA, agglutination, adherence or chemiluminescence may be used as
appropriate signal amplification steps.
[0150] The purpose of the screening assay procedure referred to
above is to ensure that the cells being tested are producing
antibody specific to the relevant cross-linked fibrin derivative,
but not to fragment D.
[0151] There should be minimal reaction with fibrinogen or
fibrinogen degradation products and a positive reaction with the
derivative. The term "minimal" includes no reactivity but extends
to basal levels such as compared to an antibody-directed to
fibrinogen per se. Consequently, a minimal reaction includes
sub-optimal reactivity compared to a fibrinogen-specific
antibody.
[0152] The present invention also includes within its scope an
assay to detect linked fibrin derivatives including the steps
of:
[0153] (1) contacting a monoclonal antibody specific to
cross-linked fibrin derivatives but not fragment D with a
biological sample suspected of containing an antigen derived from a
cross-linked fibrin derivative or comprising a cross-linked fibrin
derivative per se; and
[0154] (2) subjecting the complex formed in step (1) to a signal
amplification step.
[0155] In the above-mentioned assay, the cross-linked fibrin
derivative is suitably D-dimer, D.sub.2E or any other derivative of
a high molecular weight nature as described above. The monoclonal
antibody is prepared as described previously which is relevant to
the particular cross-linked fibrin derivative being assayed.
[0156] The presence of the cross-linked fibrin derivative may be
used as a suitable diagnostic aid for prethrombotic, thrombotic or
other conditions that involve the formation and lysis of
fibrin.
[0157] The deimmunized monoclonal antibody of the present invention
is particularly useful for blood clot imaging as well as for
targeting blood clots in order to bring the clot into contact with
enzymes or other chemical agents capable of dissolving, wholly or
partially, the clot.
[0158] With respect to clot imaging, a reporter molecule is
attached to the deimmunized monoclonal antibody or to an antibody
having specificity for the deimmunized antibody or a portion or
conjugate thereon and this is then introduced to a host, such as a
human. By detecting the reporter molecule, blood clots can be
visualized. One particularly useful form of reporter molecule is a
nuclear tag. Nuclear tags contemplated for use in the present
invention include but not limited to a bifunctional metal ion
chelate. The chelate may be attached to the antibody itself or
multiple chelates may be attached to the protein via dendrimers.
Particularly preferred nuclear tags are .sup.99mTc, .sup.18F,
.sup.64Cu, .sup.67Ga, .sup.68Ga, .sup.77Br, .sup.97Ru, .sup.111In,
.sup.123I, .sup.124I, .sup.131I and .sup.188Re. The most preferred
nuclear tag is .sup.99mTc. Preferably, the host is a human and,
hence, it is necessary for the 3B6 murine monoclonal antibody to be
deimmunized.
[0159] Alternative forms of immunoscintigraphy may be obtained
using isotopes such as a .sup.68Ga or .sup.124I or other PET
isotopes. Such technology may be described as "immuno-PET". The
technology has advantages over .gamma. camera scintigraphy and may
provide high resolution images of blood clots especially in areas
of the body less amenable to conventional diagnostic means such as
lungs or small clots in the calf or pelvis.
[0160] Accordingly, the present invention provides a conjugate
molecule comprising a deimmunized immunointeractive molecule such
as a deimmunized antibody and one or both of an imaging tag or a
therapeutic agent.
[0161] Preferred imaging tags are MRI-, ultrasound- and/or CT-type
tags such as but not limited to .sup.99mTc, .sup.18F, .sup.64Cu,
.sup.67Ga, .sup.68Ga, .sup.77Br, .sup.97Ru, .sup.111In, .sup.123I,
.sup.124I, .sup.131I and .sup.188Re.
[0162] Preferred therapeutic tags include cytokines, anti-clotting
agents, wound-repairing agents and anti-infection agents.
[0163] Another aspect of the present invention contemplates a
method for detecting a blood clot in a human patient, said method
comprising introducing into said patient a deimmunized form of
murine monoclonal antibody 3B6 or an antigen-binding fragment
thereof labeled with a reporter molecule allowing dissemination of
the labeled antibody throughout the circulatory system and then
subjecting said patient to reporter molecule-detection means to
identify the location of the antibody in a clot.
[0164] Preferably, the reporter molecule is a nuclear tag.
[0165] Preferably, the nuclear tag is .sup.99mTc, .sup.18F,
.sup.64Cu, .sup.67Ga, .sup.68Ga, .sup.77Br, .sup.97Ru, .sup.111In,
.sup.123I, .sup.124I, .sup.131I and .sup.188Re.
[0166] Preferably, the nuclear tag is .sup.99mTc.
[0167] The present invention further contemplates the use of a
deimmunized murine monoclonal antibody specific for D-dimer or
other cross-linked fibrin derivatives in the manufacture of clot
imaging agent.
[0168] Preferably, the murine monoclonal antibody is 3B6 or a
homologue thereof.
[0169] Preferably, the clot imaging tag is for use in humans.
[0170] The same antibody may also carry multiple tages such as
multiple anti-coagulant agents and/or reporter molecules.
Alternatively, or in addition, multiple anti-antibodies may be
administered each carrying a different tag.
[0171] The present clot targeting antibody may be used alone or in
combination with other imaging protocols. One such protocol is
planar imaging such as but not limited to CT, MRI or
ultrasound.
[0172] Accordingly, another aspect of the present invention
contemplates a method for detecting a blood clot in a human
patient, said method comprising introducing into said patient a
deimmunized form of murine monoclonal antibody 3B6 or an
antigen-binding fragment thereof labeled with a reporter molecule
allowing dissemination of the labeled antibody throughout the
circulatory system and then subjecting said patient to planar clot
imaging.
[0173] Preferably, the planar imaging is MRI or CT scanning.
Ultrasound may also be used in the imaging process.
[0174] Accordingly, another aspect of the present invention
contemplates a method for detecting a blood clot in a human
patient, said method comprising introducing into said patient a
deimmunized form of murine monoclonal antibody 3B6 or an
antigen-binding fragment thereof labeled with a reporter molecule
allowing dissemination of the labeled antibody throughout the
circulatory system and then subjecting said patient to a computer
assisted tomographic nuclear medicine scan to visualize the
clot.
[0175] Preferably, the reporter molecule is a nuclear tag.
[0176] Preferably, the nuclear tag is .sup.99mTc, .sup.18F,
.sup.64Cu, .sup.67Ga, .sup.68Ga, .sup.77Br, .sup.97Ru, .sup.111In,
.sup.123I, .sup.124I, .sup.131I and .sup.188Re.
[0177] Preferably, the nuclear tag is .sup.99mTc.
[0178] The clot imaging agents of the present invention are also
useful as therapeutic agents. In particular, the clot targeting
agents are fused, bound or otherwise associated with a clot
dissolution or clot growth prevention agent such as an
anticoagulant molecule.
[0179] Accordingly, another aspect of the present invention
contemplates a method for facilitating the dissolution or removal
of a blood clot in a human, said method comprising administering to
said human a clot dissolution or clot growth prevention-effective
amount of a variant murine-derived monoclonal antibody having
specificity for human-derived D-dimer and other cross-linked fibrin
derivatives and non-reactivity with fibrinogen or fibrinogen
degradation products inclusive of fragments D and E wherein said
variant murine-derived monoclonal antibody is substantially
non-immunogenic in a human wherein said monoclonal antibody further
comprises a clot dissolution or clot growth prevention agent fused,
bound or otherwise associated thereto.
[0180] Yet another aspect of the present invention is directed to
the use of a variant murine-derived monoclonal antibody having
specificity for human-derived D-dimer and other cross-linked fibrin
derivatives and non-reactivity with fibrinogen or fibrinogen
degradation products inclusive of fragments D and E wherein said
variant murine-derived monoclonal antibody is substantially
non-immunogenic in a human and said antibody further comprising a
clot dissolution or clot growth prevention agent fused, bound or
otherwise attached thereto in the manufacture of a medicament for
the dissolution of a blot clot in a human.
[0181] In an alternative embodiment, multiple deimmunized
antibodies may be used. In one example, a deimmunized 3B6 antibody
is administered alone and then deimmunized anti-immunoglobulin
antibodies each carrying an agent such as a diagnostic or
therapeutic agent which will target a clot-3B6 complex. Yet another
alternative is to engineer antibodies with multiple (e.g. bi-)
specificities. In this case, one specificity may be to the clot and
another to the site of the clot (e.g. to a cell receptor). This may
also be accomplished using multiple antibodies.
[0182] The present invention further contemplates compositions
comprising the clot targeting agents of the present invention and
one or more pharmaceutically acceptable carriers and/or
diluents.
[0183] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions as well as lyophilized forms of antibody
preparations together with stabilizing agents such as sugar,
proteins or other compounds or molecules which facilitate the
radiolabeling process. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dilution medium comprising, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol and liquid polyethylene glycol, and the like), suitable
mixtures thereof and vegetable oils. The proper fluidity can be
maintained, for example, by the use of superfactants. The
prevention of the action of microorganisms can be brought about by
various anti-bacterial and anti-fungal agents, for example,
parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the
like. In many cases, it will be preferable to include isotonic
agents, for example, sugars or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by
the use in the compositions of agents delaying absorption, for
example, aluminium monostearate and gelatin.
[0184] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with the active ingredient and optionally other active
ingredients as required, followed by filtered sterilization or
other appropriate means of sterilization.
[0185] Pharmaceutically acceptable carriers and/or diluents include
any and all solvents, dispersion media, coatings, anti-bacterial
and anti-fungal agents, isotonic and absorption delaying agents and
the like. The use of such media and agents for pharmaceutical
active substances is well known in the art and except insofar as
any conventional media or agent is incompatible with the active
ingredient, their use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0186] The clot targeting agents of the present invention are
useful for the diagnosis and/or treatment of thrombin-associated
conditions such as DVT, PE and DIC.
[0187] Yet another aspect of the present invention contemplates a
method for treating a subject with cancer associated with fibrin.
In this embodiment, antibodies to the D-dimer epitope may be used
to deliver cytotoxic agents such as an isotope that emits .beta. or
.gamma. emission or combinations thereof. Such isotopes, include
but are not limited to .sup.131I, yttrium-90, rhenium-186,
rhenium-188, lutetium-117 and copper-67. Fibrin associated with a
cancer includes a fibrin encapsulated tumor.
[0188] The deimmunized immunointeractive molecules of the present
invention are, therefore, carriers for any clot binding agents or
clot dissolving agents or for any agents which have useful
diagnostic or therapeutic properties. The deimmunized
immunointeractive molecules of the present invention are also
useful for determining the kinetics of clot dissolution,
dissipation and/or disappearance. One this information is
available, clot dissolving or imaging agents can very quickly be
administered.
[0189] The present invention is further described by the following
non-limiting Examples.
Example 1
Cell Fusion and Selection of Hybrids
[0190] Spleens were removed aseptically from 2 immunized mice
killed by cervical dislocation three days after an injection of
D-dimer. Previously, the mice had been immunized with three
injections of fibrin lysate digested with proteolytic enzymes
thrombin and plasmin as reported in the aforementioned Graeff and
Halter reference. Two spleens were placed in a 60 mm Petri dish
(Falcon, 3001, Oxnard, Calif.) containing 5 ml complete medium (85%
RPMI 1640, 15% w/v fetal calf serum, 100 I.U./ml penicillin, 100
.mu.g/ml streptomycin and 2.times.10.sup.-3 M glutamine; Gibco,
Grand Island, N.Y.). A cell suspension was prepared by
decapsulating the spleen with 2.times.18 gauge needles attached to
3 ml disposable syringes with the last cm of the tip bent through
an angle of 60.degree.. The cell suspension was then aspirated into
a 10 ml syringe fitted with a 22 gauge needle and ejected with
moderate pressure. This operation was performed twice before
filtering the cells into a Falcon 2001 tube through a fine mesh
stainless steel screen to remove larger cell clumps and debris.
[0191] The cell suspension was allowed to stand for 5 minutes at
room temperature to allow smaller clumps and membrane fragments to
settle before transferring the cell suspension to a fresh Falcon
2001 tube. The cells were centrifuged at 350G for 5 minutes at room
temperature and the supernatant was decanted from the first cell
pellet to a fresh tube and spun at 700G for five minutes to give a
second cell pellet and the two pellets were pooled and resuspended
in 5 ml complete medium. The spleen white blood cells (SWBC) were
then counted and their viability estimated by Turks and Trypan blue
stains, respectively, and 100.times.10.sup.6 viable SWBC were
placed in separate Falcon 2001 tubes in a total volume of 5 ml
complete medium. The NS-1 myeloma cells to be used for fusion, were
washed once by centrifugation at 380G for 15 minutes at room
temperature and adjusted to 5.times.10.sup.6 viable cells/ml in
complete medium.
[0192] Twenty-five.times.10.sup.6 NS-1 and 100.times.10.sup.5
immune SWBC were mixed and spun at 350G for 5 minutes at room
temperature. The supernatant was decanted, the remaining medium was
carefully removed with a Pasteur pipette and 2 ml of a 42% w/v
solution of polyethylene glycol (PEG, MW1540) (Baker Chemical Co.,
New Jersey). In RPMI 1640 containing 15% v/v dimethyl sulfoxide
(DMSO) at 37.degree. C. was added with a 5 ml glass disposable
pipette (Corning Glass, Corning, N.Y.) and the cells were
resuspended with the same 5 ml pipette for 30 seconds with the aid
of an electric pipetter (Pipet-aid Drummond Scientific Co.,
Broomall, Pa.). The PEG-cell suspension was allowed to stand for a
further 30 seconds at room temperature before adding 5 ml complete
medium, dropwise, with a Pasteur pipette, over a period of 90
seconds with constant flicking of the tube, sufficient to ensure
complete mixing with the viscous PEG solution. A further 5 ml
complete medium was immediately added and mixed by inversion and
the cell suspension was allowed to stand for a further 150 seconds
at room temperature before centrifugation at 350G for 5.minutes at
room temperature. The supernatant was decanted and the cell pellet
was gently resuspended in 5 ml complete medium using a 5 ml pipette
with the electric pipetter, extreme care was taken not to break up
all cell clumps. Using a Tridak stepper (Bellco Glass Inc.,
Vineland, N.J.), 0.05 ml of the cell suspension was added to each
well of 4 Costar 24 well plates (Costar 3524, Cambridge, Mass.)
containing 1.times.10.sup.6 normal BALE/c mouse SWBC as feeder
cells in 1 ml complete medium containing 10.sup.-4 M Hypoxanthine
(Sigma), 4.times.10.sup.-7 M Aminopterin (Sigma),
1.6.times.10.sup.-5 M Thymidine (Sigma) and 4.times.10.sup.-5 M
2-Mercaptoethanol (HAT medium), hereinafter referred to as
1.degree. fusion plates.
[0193] The 1.degree. fusion plates were then placed in a humidified
5% CO.sub.2 95% air atmosphere at 37.degree. C. The cells were
first fed either on days 5 or 7 and thereafter when necessary, with
0.5 ml fresh HAT medium. Generally, on day 10, 0.5 ml of the medium
was removed for the screening assay from each well showing
hybridoma growth and 0.5 ml fresh HAT medium was replaced. A number
of the strongest growth wells were chosen for maintenance on the
basis of the screening assay. The chosen wells were allowed to grow
to confluency in the original original well (1.degree. well), then
each was split in half and transferred to a fresh well (2.degree.
well) of a 24 well Costar plate (2.degree. plate). The wells were
checked daily and expanded to a second, third or fourth well of the
2.degree. Costar plate when necessary. From days 14-28, cells were
fed with HT medium. When there was strong growth in at least two
wells of the 2.degree. plate, supernatant from one well of each
clonotype was chosen for rescreening and a number of specific
antibody producing clonotypes were chosen from the results of the
second screening assay to produce monoclonal antibody secreting
cell lines by limiting dilution.
Example 2
Cloning of Hybridomas
[0194] One 2.degree. well of each chosen clonotype was resuspended
and the number of viable cells per well was estimated by Trypan
blue exclusion. Immediately before plating each clonotype, the
relevant series of dilutions were made in HT medium or complete
medium (if the cells were older than 28 days post fusion) to give a
frequency of 0.5 cells/0.05 ml. This volume was then added with a
Tridak stepper to each well of a 96 well flat bottomed tissue
culture plate (Flow Laboratories, Mississauga, Ontario, Canada) (LD
plate) containing 1.times.10.sup.5 normal mouse spleen feeder cells
in 0.1 ml HT or complete medium the LD plates were then placed in a
37.degree. C. humidified 5% CO.sup.2, 95% air atmosphere and
screened for clonal growth 7-10 days later. From each positive
growth well, 0.1 ml supernatant was removed for screening and these
wells were fed for the first time with 0.1-0.15 ml HT or complete
medium. On the basis of the LD screening assay, a minimum of two of
the "better" specific antibody-producing clones were finally
selected for expansion to mass culture.
[0195] Alternatively, if it was desired to obtain a large amount of
Mab, female BALB/c mice were given an intraperitoneal injection of
0, 5 ml 2, 5, 10, 14, tetramethylpentadecane (Pristane, Aldrich
Chemical Corp., Milwaukee, Wisonsin) 14 days prior to the injection
of 2.times.10.sup.6 viable hybridoma cells and ascites fluids were
collected from the mice 12-14 days after injection of the cells.
The ascitic fluid was clarified by centrifugation and MAb recovered
by precipitation with 45% ammonium sulphate and stored at either
4.degree. C. or -70.degree. C. in phosphate buffered saline (PBS)
containing 0.01% sodium azide.
Example 3
Monoclonal Antibody Screening Assay
[0196] The wells of a 96 well U bottomed microtest plate
(Disposable Products Pty. Ltd., Adelaide, South Australia) were
coated by adding 50 .mu.l of either D-dimer (5 .mu.g/ml) or
Fibrinogen degradation products (5 .mu.g/ml in PBS for one hour at
room temperature (25.degree. C.). Excess antigen was removed by
inverting and tapping the plate and the plate was then washed three
times with PBS containing 0.05% w/v Tween 20 (Sigma Chemical Corp.,
St Louis, Mo.). Clones secreting MAb to D-dimer or Fibrinogen
degradation products were then detected by adding 50 .mu.l of
tissue culture supernatant to each well and incubating for one hour
at room temperature. Unbound MAb was removed by inversion and
tapping and the plate was washed three times with PBS/Tween. One
hundred .mu.l of a 1/1000 dilution of peroxidase conjugated rabbit
anti-mouse immunoglobulin (Dakopatts, Copenhagen, Denmark) in
PBS/Tween was added and allowed to incubate a further one hour at
room temperature. The plate was again inverted and washed three
times with PBS/Tween and 100 .mu.l of activated substrate
(immediately before use, 10 .mu.l of 3% solution of hydrogen
peroxide was added to 10 ml of a substrate solution containing 50
mM citrate, 2.5 mM of O-tolidine dihydrochloride (O-tolidine, Sigma
Chemical Co., recrystallized from dilute HCl) 0.025 mM EDTA pH 4.5)
was added to each well. The colour reaction was stopped after 10
minutes by the addition of 50 .mu.l of 3M HCl which caused a colour
change from blue to yellow and the absorbance was recorded at 450
nm on a Titertek multiskan.
Example 4
Identification of 3B6 Variable Region Sequences
[0197] The murine hybridoma 3B6 was propagated in RPMI 1640 medium
supplemented with 15% w/v fetal calf serum. Total RNA was prepared
from 10.sup.7 hybridoma cells. V.sub.H and V.sup.K cDNA was
prepared using reverse transcriptase and mouse .kappa. constant
region and mouse IgG constant region primers. The first strand
cDNAs were amplified by PCR using a variety of mouse signal
sequence primers (6 sets for V.sub.H and 7 sets for V.sub.K). The
amplified DNAs were gel-purified and cloned into the vector
pGem.RTM. T Easy (Promega). The V.sub.H and V.sub.K clones obtained
were screened for inserts of the expected size by PCR and the DNA
sequence of selected clones determined by the dideoxy chain
termination method.
[0198] Productive V.sub.H and V.sub.K genes were identified by
sequence analysis. The location of the complementarity determining
regions (CDRs) was determined with reference to other antibody
sequences (43). The 3B6 V.sub.H can be assigned to mouse heavy
chains sub-group IA. The 3B6 V.sub.K can be assigned to mouse K
chains sub-group I.
Example 5
Analysis of 3B6 Variable (v) Region Sequences with Potential T Cell
Epitopes
[0199] 3B6 V.sub.H and V.sub.K sequences were analyzed for the
presence of potential T cell epitopes using procedures described
previously (Carr et al., International Patent Publication No. WO
98/52976). The peptides identified as potential T cell epitopes
(MHC class II binding peptides) were modified in silico and the
modified sequence re-analyzed to ensure loss of potential MHC class
II binding and verify that further MHC class II binding motifs had
not been generated in the surrounding sequence. Alternatively, the
sequence was modified to convert the MHC class II binding motif to
one found in the human germ line. Single, generally conservative,
amino acid substitutions were tested and substitutions made with
due regard to overall antibody structure. A number of variant
sequences are compiled for the V.sub.H and V.sub.K, each containing
different numbers of substitutions.
Example 6
Designer Variant 3B6 Variable (v) Regions Sequences with Reduced
Numbers of Potential T Cell Epitopes
[0200] The heavy and light (v) regions designed according the
scheme of Example 5 were constructed in vitro by the method of
overlapping PCR recombination described (Daugherty et al., Nucleic
Acids Research 19: 2471-2476, 1991). The cloned murine V.sub.H and
V.sub.K genes were used as templates for mutagenesis of the
framework regions to the required humanized sequences. Sets of
mutagenic primer pairs were synthesized encompassing the regions to
be altered. Adjacent primers included 15 by of homologous sequence.
A first round of PCR using these primers produced 5 to 8
overlapping DNA fragments encompassing the designed (v) region
gene. The vectors V.sub.H-PCR1 and V.sub.K-PCR1 (Orlandi et al.,
Proc. Natl. Acad. Sci. USA 86: 3833-3837, 1989) were used as
templates to introduce 5' flanking sequence including the leader
signal peptide, leader intron and the murine immunoglobulin
promoter, and 3' flanking sequence including the splice site and
intron sequences, in an additional two overlapping fragments. The
DNA fragments produced were combined in a second round of PCR using
outer flanking primers to obtain PCR products of the required full
length. These PCR products were cloned into the vector pUC19 for
DNA sequence determination. Clones were selected that contained the
expected sequence alterations and the entire DNA sequence was
confirmed to be correct for each desired V.sub.H and V.sub.K. The
heavy and light chain genes were transferred to the expression
vectors pSV gpt and pSVhyg with human IgG1 or .kappa. constant
regions as described (Tempest et al., Biotechnology 9: 266-271,
1991). The vectors V.sub.H-PCR1 and V.sub.K-PCR1 (Orlandi et al.,
1989, supra) were used as templates to introduce 5' flanking
sequence including the leader signal peptide, leader intron and the
murine immunoglobulin promoter, and 3' flanking sequence including
the splice site and intron sequences.
Example 7
Expression and Purification of Variant 3B6 Antibodies
[0201] The variant 3B6 heavy and light chain expression vectors
were co-transfected in different combinations by electroporation
into NS0, a non-immunoglobulin producing mouse myeloma, obtained
from the European Collection of Animal Cell Cultures, Porton, U.K.
(ECACC No 85110505). Colonies expressing the gpt gene were selected
in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 0.8
.mu.g/ml mycophenolic acid and 250 .mu.g/ml xanthine. Production of
human antibody by transfected cell clones was measured by ELISA for
human IgG (48). Cell lines secreting antibody were selected and
expanded. Variant 3B6 antibodies were purified using Prosep.RTM.-A
(Bioprocessing Ltd, Conset, U.K.).
Example 8
Functional Testing of Variant 3B6 Antibodies
[0202] Variant antibodies were tested for D-dimer binding using
ELISA based assays broadly as described in Example 3. Binding
specificity was confirmed using the human fibrinogen binding assay.
In a preferred embodiment, however, the D-dimer was used in
solution phase. In this assay, the 3B6 antibodies were coated on
the ELISA plate at 0.5 .mu.g/well, to capture D-dimer in solution.
D-dimer was applied at 10 .mu.g/ml (500 ng/well) and doubling
dilutions. The revealing antibody was HRPO conjugated mouse
monoclonal anti-D (Dimertest EIA Tag; Agen) and the results were
developed by OPD substrate and read a 492 nm. The deimmunized 3B6
antibodies are compared to the murine and chimeric 3B6 antibodies
and the previous lead deimmunized antibody 3B6 DIVH1/DIVK1. The
results are shown in FIGS. 4A, 4B and 4C. The use of solution phase
D-dimer proved better than solid phase D-dimer in the selection of
clones and is a preferred aspect of the present invention.
Example 9
Thromboviewing Using 3B6-99 mTc
[0203] The 3B6 monoclonal antibody from mice and deimmunized form
for humans is represented in FIG. 1A and exhibits specificity for
fibrin which is a major part of blood clots (FIG. 1B). A clot
imaging concept is developed by labelling the 3B6 monoclonal
antibody with a nuclear tag, in this case, .sup.99mTc (FIG. 2).
Administration of the labeled 3B6 deimmunized monoclonal antibody
in humans (FIG. 3A). Visualization of clots in the circulatory
system such as blood clots in the anterior thighs (FIG. 3B) occurs
by binding of the monoclonal antibody to fibrin resulting in
concentration of radiation at the clot site.
Example 10
Thromboviewing Using 3B6-.sup.99mTc
[0204] Pulmonary emboli (0.1-0.5 g) were created in anesthetized
dogs by embolization of pre-formed thrombi made by infusion of
thrombin and human fibrinogen through balloon catheters placed in
the femoral veins. Purified Fab' fragments (0.35 mg) of a chimeric
(human/murine) derivative of a fibrin-specific monoclonal antibody
were labeled with a 15 mCi of .sup.99mTc. One hour after
embolization, the radiolabeled antibody preparation was injected
through a peripheral intravenous catheter. Eight hours after
antibody injection, imaging scans were performed to visualize the
emboli.
[0205] .sup.99mTc labeled antibody fragments cleared from the
circulation with a t.sub.1/3 of one hour for both subjects. In
subject 1, two small emboli in the right lower lobe (combined mass,
0.187 g) were visible. The clot/blood radioactivity ratio was 38:1.
In subject 2, one embolus in the right lower lobe (mass, 0.449 g)
was visible. Clot/blood radioactivity ratio was 27:2. A small
embolus (0.091 g) was discovered in the right ventricle of subject
1. The clot/blood radioactivity ratio was 45:1. No adverse effects
were noted from either antibody administration or scanning
methodology.
[0206] Infusion of radiolabeled anti-fibrin antibody fragments
followed by imaging produces images of emboli, even relatively
small emboli in the periphery of the lung. The images are reliable
and require minimal training to interpret. The technique can be
used to image deep vein thrombi in the same setting. This agent is
well tolerated by the subjects. There is no need for breath-holding
or cardiac gating. It uses no nephrotoxic intravenous contrast dye.
The radiation dose is similar to the dose used for
ventilation/perfusion scans. This: technology may simplify and
clarify the diagnosis of PE and DVT, using technology available in
most medical centres.
[0207] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations of any two or more of said steps or features.
[0208] All publications, patents, patent applications and
provisional applications referred to herein are incorporated herein
by reference in their entirety.
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Sequence CWU 1
1
121118PRTArtificial sequencemisc_feature()..()Synthetic construct
of 3B6DIVHv5 1Asp Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Lys
Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr Val Thr Gly Tyr Ser
Ile Thr Ser Asp 20 25 30Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly
Lys Gly Leu Glu Trp 35 40 45Met Gly Tyr Ile Thr Tyr Ser Gly Thr Thr
Ser Tyr Asn Pro Ser Leu 50 55 60Thr Ser Arg Ile Ser Ile Ser Arg Asp
Thr Ser Lys Asn Gln Phe Phe65 70 75 80Leu Gln Leu Asn Ser Leu Thr
Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95Ala Arg Glu Trp Phe Pro
Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Leu Thr Val
Ser Ser 1152118PRTArtificial sequencemisc_feature()..()Synthetic
construct of 3B6DIVHv6 2Asp Val Gln Leu Lys Glu Ser Gly Pro Gly Leu
Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr Val Thr Gly
Tyr Ser Ile Thr Ser Asp 20 25 30Tyr Ala Trp Asn Trp Ile Arg Gln Pro
Pro Gly Lys Gly Leu Glu Trp 35 40 45Met Gly Tyr Ile Thr Tyr Ser Gly
Thr Thr Ser Tyr Asn Pro Ser Leu 50 55 60Thr Ser Arg Ile Ser Ile Ser
Arg Asp Thr Ser Lys Asn Gln Phe Phe65 70 75 80Leu Gln Leu Asn Ser
Val Thr Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95Ala Arg Glu Trp
Phe Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Leu
Thr Val Ser Ser 1153118PRTArtificial
sequencemisc_feature()..()Synthetic construct of 3B6DIVHv7 3Asp Val
Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Lys Pro Thr Gln1 5 10 15Thr
Leu Thr Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20 25
30Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp
35 40 45Met Gly Tyr Ile Thr Tyr Ser Gly Thr Thr Ser Tyr Asn Pro Ser
Leu 50 55 60Thr Ser Arg Ile Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln
Phe Phe65 70 75 80Leu Gln Leu Asn Ser Val Thr Ser Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95Ala Arg Glu Trp Phe Pro Tyr Tyr Phe Asp Tyr
Trp Gly Gln Gly Thr 100 105 110Thr Leu Thr Val Ser Ser
1154107PRTArtificial sequencemisc_feature()..()Synthetic construct
of 3B6DIVKv1 4Asp Ile Val Met Thr Gln Ser Gln Lys Ser Met Ser Thr
Ser Val Gly1 5 10 15Asp Arg Val Ser Ile Ser Cys Lys Ala Ser Gln Asn
Val Gly Thr Pro 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Glu Gln Ser
Pro Lys Leu Leu Ile 35 40 45Tyr Ser Thr Ser Thr Arg Tyr Pro Gly Val
Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Asn Leu Gln Ala65 70 75 80Glu Asp Val Ala Asp Tyr Phe
Cys Gln Gln Tyr Ser Leu Tyr Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr
Lys Val Glu Leu Lys 100 1055107PRTArtificial
sequencemisc_feature()..()Synthetic construct of 3B6DIVKv4 5Asp Ile
Val Met Thr Gln Ser Gln Lys Ser Met Ser Thr Ser Val Gly1 5 10 15Asp
Arg Val Ser Ile Ser Cys Lys Ala Ser Gln Asn Val Gly Thr Pro 20 25
30Val Ala Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile
35 40 45Tyr Ser Thr Ser Thr Arg Tyr Pro Gly Val Pro Asp Arg Phe Thr
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Leu
Gln Ser65 70 75 80Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser
Leu Tyr Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr Lys Val Glu Leu Lys
100 1056107PRTArtificial sequencemisc_feature()..()Synthetic
construct of 3B6DIVKv7 6Asp Ile Val Met Thr Gln Ser Gln Lys Ser Met
Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Ser Ile Ser Cys Lys Ala Ser
Gln Asn Val Gly Thr Pro 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Glu
Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Ser Thr Ser Thr Arg Tyr Pro
Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Asn Leu Gln Ala65 70 75 80Glu Asp Val Ala Asp
Tyr Phe Cys Gln Gln Tyr Ser Leu Tyr Pro Leu 85 90 95Thr Phe Gly Ala
Gly Thr Lys Leu Glu Leu Lys 100 1057354DNAArtificial
sequencemisc_feature()..()Synthetic construct of 3B6DIVHv5
7gatgtgcagc ttaaggagtc gggacctggc ctggttaaac ctactcagac tctgaccctc
60acctgcactg tcactggcta ctcaatcacc agtgattatg cctggaactg gatacggcag
120ccaccaggaa agggactgga gtggatgggc tacataacct acagtggtac
cactagctac 180aacccatctc tcacaagtcg aatctctatc tctcgcgaca
catccaagaa ccagttcttc 240ctgcagttga attctctgac ttctgaggac
acagccacat attactgtgc aagagagtgg 300tttccttact actttgacta
ctggggccaa ggcaccactc tcacagtctc ttca 3548354DNAArtificial
sequencemisc_feature()..()Synthetic construct of 3B6DIVHv6
8gatgtgcagc ttaaggagtc gggacctggc ctggttaaac ctactcagac tctgaccctc
60acctgcactg tcactggcta ctcaatcacc agtgattatg cctggaactg gatacggcag
120ccaccaggaa agggactgga gtggatgggc tacataacct acagtggtac
cactagctac 180aacccatctc tcacaagtcg aatctctatc tctcgcgaca
catccaagaa ccagttcttc 240ctgcagttga attctgtgac ttctgaggac
acagccacat attactgtgc aagagagtgg 300tttccttact actttgacta
ctggggccaa ggcaccactc tcacagtctc ttca 3549354DNAArtificial
sequencemisc_feature()..()Synthetic construct of 3B6DIVHv7
9gatgtgcagc ttaaggagtc gggacctggc ctggttaaac ctactcagac tctgaccctc
60acctgcactg tcactggcta ctcaatcacc agtgattatg cctggaactg gatacggcag
120tttccaggaa acaaactgga gtggatgggc tacataacct acagtggtac
cactagctac 180aacccatctc tcacaagtcg aatctctatc tctcgcgaca
catccaagaa ccagttcttc 240ctgcagttga attctgtgac ttctgaggac
acagccacat attactgtgc aagagagtgg 300tttccttact actttgacta
ctggggccaa ggcaccactc tcacagtctc ttca 35410321DNAArtificial
sequencemisc_feature()..()Synthetic construct of 3B6DIVKv1
10gacattgtga tgacccagtc tcaaaaatcc atgtccacat cagtaggaga cagggtcagc
60atctcctgca aggccagtca gaatgtgggt actcctgtag cctggtatca gcagaaacca
120gaacaatctc ctaaacttct gatttactcg acatccactc ggtaccctgg
agtccctgat 180cgcttcactg gcagtggatc tgggacagat ttcactctca
ccatcagcaa tctgcaggct 240gaagacgtgg cagattattt ctgccagcaa
tatagcctct atcctctcac gttcggtgct 300gggaccaagg tggagctgaa a
32111321DNAArtificial sequencemisc_feature()..()Synthetic construct
of 3B6DIVKv4 11gacattgtga tgacccagtc tcaaaaatcc atgtccacat
cagtaggaga cagggtcagc 60atctcctgca aggccagtca gaatgtgggt actcctgtag
cctggtatca gcagaaacca 120gaacaatctc ctaaacttct gatttactcg
acatccactc ggtaccctgg agtccctgat 180cgcttcactg gcagtggatc
tgggacagat ttcactctca ccatcagcaa tctgcagtct 240gaagacctgg
cagattattt ctgccagcaa tatagcctct atcctctcac gttcggtgct
300gggaccaagg tggagctgaa a 32112321DNAArtificial
sequencemisc_feature()..()Synthetic construct of 3B6DIVKv7
12gacattgtga tgacccagtc tcaaaaatcc atgtccacat cagtaggaga cagggtcagc
60atctcctgca aggccagtca gaatgtgggt actcctgtag cctggtatca gcagaaacca
120gaacaatctc ctaaacttct gatttactcg acatccactc ggtaccctgg
agtccctgat 180cgcttcactg gcagtggatc tgggacagat ttcactctca
ccatcagcaa tctgcaggct 240gaagacgtgg cagattattt ctgccagcaa
tatagcctct atcctctcac gttcggtgct 300gggaccaagc tggagctgaa a 321
* * * * *