U.S. patent application number 09/736792 was filed with the patent office on 2001-08-30 for tumour necrosis factor binding ligands.
Invention is credited to Aston, Roger, Rathjen, Deborah Ann.
Application Number | 20010018507 09/736792 |
Document ID | / |
Family ID | 27424270 |
Filed Date | 2001-08-30 |
United States Patent
Application |
20010018507 |
Kind Code |
A1 |
Rathjen, Deborah Ann ; et
al. |
August 30, 2001 |
Tumour necrosis factor binding ligands
Abstract
The present invention relates to ligands which bind to human
tumor necrosis factor alpha (TNF) in a manner such that upon
binding of these ligands to TNF the biological activity of TNF is
modified. In preferred forms the ligand binds to TNF in a manner
such that the induction of endothelial procoagulant activity of the
TNF is inhibited; the binding of TNF to receptors on endothelial
cells is inhibited; the induction of fibrin deposition in the tumor
and tumor regression activities of the TNF are enhanced; and the
cytotoxicity and receptor binding activities of the TNF are
unaffected or enhanced on tumor cells. The ligand is preferably an
antibody, F(ab) fragment, single domain antibody (dABs) single
chain antibody or a serum binding protein. It is preferred,
however, that the ligand is a monoclonal antibody or F(ab) fragment
thereof.
Inventors: |
Rathjen, Deborah Ann; (New
South Wales, AU) ; Aston, Roger; (Gloucester,
GB) |
Correspondence
Address: |
Rebecca Shortle
Morrison & Foerster LLP
755 Page Mill Road
Palo Alto
CA
94304-1018
US
|
Family ID: |
27424270 |
Appl. No.: |
09/736792 |
Filed: |
December 13, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09736792 |
Dec 13, 2000 |
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09364039 |
Jul 30, 1999 |
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09364039 |
Jul 30, 1999 |
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08823893 |
Mar 17, 1997 |
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5959087 |
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08823893 |
Mar 17, 1997 |
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08344133 |
Nov 23, 1994 |
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5644034 |
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08344133 |
Nov 23, 1994 |
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07828956 |
Feb 18, 1992 |
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07828956 |
Feb 18, 1992 |
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PCT/AU90/00337 |
Aug 7, 1990 |
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Current U.S.
Class: |
530/387.1 |
Current CPC
Class: |
C07K 2317/24 20130101;
A61K 38/00 20130101; C07K 2317/21 20130101; C07K 2319/00 20130101;
C07K 16/241 20130101; A61K 47/68 20170801; A61K 47/6813 20170801;
C07K 2317/622 20130101 |
Class at
Publication: |
530/387.1 |
International
Class: |
C07K 016/24 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 7, 1989 |
AU |
PJ5662 |
Nov 24, 1989 |
AU |
PJ7576 |
Aug 7, 1990 |
US |
PCT/AU90/00337 |
Claims
We claim:
1. A ligand capable of binding to human TNF, the ligand being
characterised in that when it binds to TNF the in vivo tumour
regression activity of the TNF is enhanced.
2. A ligand capable of binding to human TNF, the ligand being
characterised in that when it binds to TNF the in vivo tumour
regression activity of the TNF is enhanced; the ligand binding to
the TNF such that the epitope of the TNF defined by the topographic
region of residues 1-30, 117-128 and 141-153 is substantially
prevented from binding to naturally occurring biologically active
ligands.
3. A ligand which binds to human TNF in the topographic regions of
residues 1-30, 117-128 and 141-153.
4. A ligand as claimed in claim 3 in which the ligand binds to
human TNF in the topographic regions of residues 1-26, 117-128 and
141-153.
5. A ligand as claimed in claim 1 in which the ligand is selected
from the group consisting of antibodies, F(ab) fragments, single
domain antibodies (dABs) restructured antibodies, single chain
antibodies and serum binding proteins.
6. A ligand as claimed in claim 5 in which the ligand is a
monoclonal antibody or F(ab) fragment thereof.
7. A ligand as claimed in claim 1 in which the ligand is MAb 32
(ECACC 89080302).
8. A composition comprising TNF in combination with a ligand as
claimed in claim 1 in which the ligand is bound to the TNF.
Description
[0001] This is a continuation-in-part of application Ser. No
07/828,956 filed Aug. 7. 1990, the disclosure of which is
incorporated herein by cross-reference.
FIELD OF THE INVENTION
[0002] The present invention relates to ligands which bind to human
tumour necrosis factor alpha (TNF) in a manner such that upon
binding the biological activity of TNF is modified. The type of
modification shown here is distinct from previous descriptions of
antibodies which bind to TNF alpha and inhibit all TNF alpha
activity. The new discovery shows how the different activities of
TNF alpha can be selectively inhibited or enhanced. In addition,
the present invention-relates to a composition comprising a
molecule bound to TNF and to methods of therapy utilising TNF and
molecules active against TNF.
BACKGROUND OF THE INVENTION
[0003] Tumor necrosis factor alpha (TNF) is a product of activated
macrophages first observed in the serum of experimental animals
presensitized with Bacillus Calmette-Guerin or Corynebacterium
parvum and challenged with endotoxin (LPS). Following the
systematic administration of TNF haemorrhagic necrosis was observed
in some transplantable tumours of mice while in vitro TNF caused
cytolytic or cytostatic effects on tumour cell lines.
[0004] In addition to its host-protective effect, TNF has been
implicated as the causative agent of pathological changes in
septicemia, cachexia and cerebral malaria. Passive immunization of
mice with a polyclonal rabbit serum against TNF has been shown to
protect mice against the lethal effects of LPS endotoxin, the
initiating agent of toxic shock, when administered prior to
infection.
[0005] The gene encoding TNF has been cloned allowing the
usefulness of this monokine as a potential cancer therapy agent to
be assessed. While TNF infusion into cancer patients in stage 1
clinical trials has resulted in tumour regression, side-effects
such as thrombocytopaenia, lymphocytopaenia, hepatotoxicity, renal
impairment and hypertension have also been reported. These quite
significant side-effects associated with the clinical use of TNF
are predictable in view of the many known effects of TNF, some of
which are listed in Table 1.
TABLE 1
Biological Activities of TNF
[0006] ANTI-TUMOUR
[0007] ANTI-VIRAL
[0008] ANTI-PARASITE
[0009] Function
[0010] cytotoxic action on tumour cells
[0011] pyrogenic activity
[0012] angiogenic activity
[0013] inhibition of lipoprotein lipase
[0014] activation of neutrophils
[0015] osteoclast activation
[0016] induction of endothelial, monocyte and tumour cell
[0017] procoagulant activity
[0018] induction of surface antigens on endothelial cells
[0019] induction of IL-6
[0020] induction of c-myc and c-fos
[0021] induction of EGF receptor
[0022] induction of IL-1
[0023] induction of TNF synthesis
[0024] induction of GM-CSF synthesis
[0025] increased prostaglandin and collagenase synthesis
[0026] induction of acute phase protein C3
[0027] Of particular importance is the activation of coagulation
which occurs as a consequence of TNF activation of endothelium and
also peripheral blood monocytes. Disseminated intravascular
coagulation is associated with toxic shock and many cancers
including gastrointestinal cancer, cancer of the pancreas,
prostate, lung, breast and ovary, melanoma, acute leukaemia,
myeloma, myeloproliferative syndrome and myeloblastic leukaemia.
Clearly modifications of TNF activity such that tumour regression
activity remains intact but other undesirable effects such as
activation of coagulation are removed or masked would lead to a
more advantageous cancer therapy, while complete abrogation of TNF
activity is sought for successful treatment of toxic shock.
[0028] Segregation of hormonal activity through the use-of
site-specific antibodies (both polyclonal and monoclonal) can
result in enhanced hormonal activity (Aston et al, 1989, Mol.
Immunol. 2&, 435). To date few attempts have been made to
assign antigenicity or function to particular regions of the TNF
molecule for which the three-dimensional structure is now known.
Assignment of function to such regions would permit the development
of MAbs and other ligands of therapeutic use. Polyclonal antibodies
to amino acids 1 to 15 have been reported to block Hela R19 cell
receptor binding by TNF (Socher et al, 1987, PNAS 84, 8829) whilst
monoclonal antibodies recognising undefined conformational epitopes
on TNF have been shown to inhibit TNF cytotoxicity in vitro
(Bringman and Aggarwal, 1987, Hybridoma 6, 489). However, the
effects of these antibodies on other TNF activities is unknown.
DESCRIPTION OF THE PRESENT INVENTION
[0029] The present inventors have produced panels of monoclonal
antibodies active against human TNF and have characterised them
with respect to their effects on the anti-tumour effect of TNF
(both in vitro and in viva), TNF receptor binding, activation of
coagulation (both in vitro and in viva) and defined their
topographic specificities. This approach has led the inventors to
show that different topographic regions of TNF alpha are associated
with different activities. Therefore the inventors enable the
identification of antibodies or ligands which selectively enhance
or inhibit TNF alpha activity, thereby providing for improved
therapeutic agents and regimes including TNF alpha.
[0030] In a first aspect the present invention consists in a ligand
capable of binding to human TNF, the ligand being characterised in
that when it binds to TNF the following biological activities of
the TNF are inhibited:--
[0031] 1. Tumour regression;
[0032] 2. Induction of endothelial procoagulant;
[0033] 3. Induction of tumour fibrin deposition;
[0034] 4. Cytotoxicity; and
[0035] 5. Receptor binding.
[0036] In a preferred embodiment of all aspects the present
invention the ligand is selected from the group consisting of
antibodies, F(ab) fragments, restructured antibodies (CDR grafted
humanised antibodies) single domain antibodies (dAbs), single chain
antibodies, serum binding proteins, receptors and natural
inhibitors. The ligand may also be a protein or peptide which has
been synthesised and which is analogous to one of the foregoing
fragments. However, it is presently preferred that the ligand is a
monoclonal antibody or F(ab) fragment thereof.
[0037] In a second aspect the present invention consists in a
ligand capable of binding to human TNF, the ligand being
characterized in that when it binds to TNF the induction of
endothelial procoagulant, tumour regression, induction of tumour
fibrin deposition, cytotoxicity and receptor binding activities of
the TNF are inhibited, the ligand binding to the TNF such that the
epitope of the TNF defined by the topographic regions of residues
1-18, 58-65, 115-125 and 138-149, or the topographic region of
residues 1-18, 108-128, or the topographic region of residues
56-79, 110-127 and 135-155 is substantially prevented from binding
to naturally occurring biologically active ligands.
[0038] In a third aspect the present invention consists in a ligand
which binds to human TNF in at least two regions selected from the
group consisting predominantly of the topographic region of
residues 1-20, the topographic region of residues 56-77, the
topographic region of residues 108-127 and the topographic region
of residues 138-149.
[0039] In a preferred embodiment of the third aspect of the present
invention the ligand binds to human TNF in the topographic regions
of residues 1-18, 58-65, 115-125 and 138-149. Such sequence regions
are topographically represented in FIG. 23.
[0040] In a further preferred embodiment of the third aspect of the
present invention the ligand binds to human TNF in the topographic
regions of residues 1-18 and 108-128. Such sequence regions are
topographically represented in FIG. 24.
[0041] In a further preferred embodiment of the second aspect of
the present invention the ligand binds to human TNF in the
topographic regions of residues 56-79, 110-127 and 136-155. Such
sequence regions are topographically represented in FIG. 25.
[0042] In a particularly preferred embodiment of the first, second
and third aspects of the present invention the ligand is a
monoclonal antibody selected from the group consisting of the
monoclonal antibodies designated MAb 1, MAb 47 and MAb 54. Samples
of the hybridoma cell lines which produce MAb 11 MAb 54 and MAb 47
have been deposited with the European Collection of Animal Cell
Cultures (ECACC), Vaccine Research and Production Laboratory,
Public Health Laboratory Service, Centre for Applied Microbiology
and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, United
Kingdom. MAb 1 was deposited-on Aug. 13, 989 and accorded accession
No. 89080301; MAb 54 was deposited on Aug. 31, 1989 and accorded
accession No. 89083103; MAb 47 was deposited on Dec. 14, 1989 and
accorded accession No. 89121402.
[0043] In a fourth aspect the present invention consists in a
composition comprising TNF in combination with the ligand of the
first, second or third aspect of the present invention,
characterised in that the ligand is bound to the TNF.
[0044] In a fifth aspect the present invention consists in a method
of treating toxic shock comprising administering either the ligand
of the first, second or third aspect of the present invention or
the composition of the fourth aspect of the present invention.
[0045] In a sixth aspect the present invention consists in a ligand
capable of binding to human TNF, the ligand being characterised in
that when it binds to TNF the induction of endothelial procoagulant
activity of the TNF is inhibited; binding of TNF to receptors on
endothelial cells is inhibited; the induction of tumour fibrin
deposition and tumour regression activities of the TNF are
enhanced; the cytotoxicity is unaffected and tumour receptor
binding activities of the TNF are unaffected or enhanced.
[0046] In a seventh aspect the present invention consists in a
ligand capable of binding to human TNF, the ligand being
characterized in that-when it binds to TNF the induction of
endothelial procoagulant activity of the TNF is inhibited; the
binding of the TNF to receptors on endothelial cells is inhibited,
the induction of tumour fibrin deposition and tumour regression
activities of the TNF are enhanced; and the cytotoxicity and
receptor binding activities of the TNF are unaffected; the ligand
binding to the TNF such that the epitope of the TNF defined by the
topographic regions of residues 1-30, 117-128 and 141-153 is
substantially prevented from binding to naturally occurring
biologically active ligands.
[0047] In an eighth aspect the present invention consists of a
ligand which binds to human TNF in the topographic regions of
residues 1-30, 117-128 and 141-153.
[0048] In a preferred embodiment of the eighth aspect of the
present invention the ligand binds to human TNF in the topographic
regions of residues 1-26, 117-128 and 141-153. Such sequence
regions are topographically represented in FIG. 26.
[0049] In a preferred embodiment of the sixth, seventh and eighth
aspects of the present invention the ligand is the monoclonal
antibody designated MAb 32. A sample of the hybridoma producing MAb
32 was deposited with The European Collection of Animal Cell
Cultures (ECACC), Vaccine Research and Production Laboratory,
Public Health Laboratory Service, Centre for Applied Microbiology
and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, United
Kingdom on Aug. 3, 1989 and was accorded accession No.
89080302.
[0050] In a ninth aspect the present invention consists in a
composition comprising TNF in combination with a ligand of the
sixth, seventh or eighth aspects of the present invention
characterised in that the ligand is bound to TNF. No previous
documentation of administering MAbs with TNF in order to modify
activity of the administered cytokine exists.
[0051] In a tenth aspect the present invention consists in a method
of treating tumours the growth of which is inhibited by TNF,
comprising administering either the ligand of the sixth, seventh or
eighth aspects of the present invention or the composition of the
ninth aspect of the present invention.
[0052] In an eleventh aspect the present invention consists in a
ligand which binds to residues 1-18 of human TNF (peptide 301).
[0053] In a twelfth aspect the present invention consists in a
ligand capable of binding to human TNF, the ligand being
characterized in that when it binds to TNF the induction of
endothelial procoagulant activity of the TNF is inhibited; the
binding of TNF to receptors on endothelial cells is inhibited; the
induction of tumour fibrin deposition and tumour regression
activities of the TNF are enhanced; the cytotoxicity of the TNF are
unaffected and tumour receptor binding activities of the TNF are
unaffected or enhanced, the ligand binding to TNF such that the
epitope of the TNF defined by the topographic region of residues
1-18 is substantially prevented from binding to naturally occurring
biologically active ligands.
[0054] In a thirteenth aspect the present invention consists in a
composition comprising TNF in combination with a ligand of the
eleventh or twelfth aspects of the present invention characterized
in that the ligand is bound to the TNF.
[0055] In a fourteenth aspect the present invention consists in a
method of treating tumours the growth of which is inhibited by TNF,
comprising administering either the ligand of the eleventh or
twelfth aspect of the present invention or the composition of the
thirteenth aspect of the present invention.
[0056] In a fifteenth aspect the present invention consists in a
ligand capable of binding to human TNF, the ligand being
characterised in that when it binds to TNF the cytotoxicity and
tumour regression activities of the TNF are unaffected; the
induction of endothelial procoagulant and induction of tumour
fibrin deposition activities of the TNF are inhibited and receptor
binding activities of the TNF are unaffected.
[0057] In a sixteenth aspect the present invention consists in a
ligand capable of binding to human TNF, the ligand being
characterized in that when it binds to TNF the cytotoxicity and
tumour regression activies of the TNF are unaffected; the induction
of endothelial procoagulant and induction of tumour fibrin
deposition activities of the TNF are inhibited and the tumour
receptor binding activities of the TNF are unaffected, the ligand
binding to TNF such that the epitope of the TNF defined by the
topographic regions of residues 22-40, 49-97, 110-127 and 136-153
is substantially prevented from binding to naturally occurring
biologically active ligands.
[0058] In a seventeenth aspect the present invention consists in a
ligand which binds to human TNF in the topographic regions of
residues 22-40, 49-97, 110-127 and 136-153. Such sequence regions
are topographically represented in FIG. 27.
[0059] In a preferred embodiment of the seventeenth aspect of the
present invention the ligand binds to human TNF in the topographic
regions of residues 22-40, 49-96, 110-127 and 136-153. These
regions being proximate in the 3D structure of TNF alpha.
[0060] In a preferred embodiment of the fifteenth, sixteenth and
seventeenth aspects of the present invention the ligand is the
monoclonal antibody designated MAb 42. A sample of the hybridoma
cell line producing MAb 42 was deposited with The European
Collection of Animal Cell Cultures (ECACC), Vaccine Research and
Production Laboratory, Public Health Laboratory Service, Centre for
Applied Microbiology and Research, Porton Down, Salisbury,
Wiltshire SP4 OJG, United Kingdom on Aug. 3, 1989 and was accorded
accession No. 89080304.
[0061] In all eighteenth aspect the present invention consists in a
composition comprising TNF in combination with the ligand of the
fifteenth, sixteenth or seventeenth aspects of the present
invention, characterised in that the ligand is bound to the
TNF.
[0062] In a nineteenth aspect the present invention consists in a
method of treating tumours inhibited by the action of TNF
comprising administering the ligand of the fifteenth, sixteenth or
seventeenth aspects of the present invention or the composition of
the eighteenth aspect of the present invention.
[0063] In a twentieth aspect the present invention consists in a
ligand capable of binding to human TNF, the ligand being
characterised in that when it binds to TNF the tumour fibrin
deposition activity of the TNF is enhanced; the induction of
endothelial procoagulant activity of the TNF is unaffected and the
cytotoxicity, tumour regression and receptor binding activities of
the TNP are inhibited.
[0064] In a twenty-first aspect the present invention consists in a
ligand capable of binding to human TNF, the ligand being
characterized in that when it binds to TNF the tumour fibrin
deposition activity of the TNF is enhanced; the induction of
endothelial procoagulant activity of the TNF is unaffected and the
cytotoxicity, tumour regression and tumour receptor binding
activities of the TNF are inhibited, the ligand binding to TNF such
that the epitope of the TNF defined by the topographic regions of
residues 12-22, 36-45, 96-105 and 132-157 is substantially
prevented from binding to naturally occurring biologically active
ligands.
[0065] In a twenty-second aspect the present invention consists in
a ligand which binds to human TNF in the topographic regions of
residues 12-22, 36-45, 96-105 and 132-157. These regions are
proximate in the 3D structure of TNF and are topographically
represented in FIG. 28.
[0066] In a preferred embodiment of the twentieth, twenty-first and
twenty-second aspects of the present invention the ligand is the
monoclonal antibody designated MAb 25. A sample of the hybridoma
cell line producing MAb 25 was deposited with the European
Collection of Animal Cell Cultures (ECACC), Vaccine Research and
Production Laboratory, Public Health Laboratory Service, Centre for
Applied Microbiology and Research, Porton Down, Salisbury,
Wiltshire SP4 OJG, United Kingdom on Dec. 14, 1989 and was accorded
accession No. 89121401.
[0067] In a twenty-third aspect the present invention consists in a
ligand capable of binding to human TNF, the ligand being
characterised in that when it binds to TNF the tumour fibrin
deposition activity of the TNF is enhanced and the cytotoxicity,
tumour regression, induction of endothelial procoagulant and
receptor binding activities of the TNF are inhibited.
[0068] In a twenty-fourth aspect the present invention consists in
a ligand capable of binding to human TNF, the ligand being
characterized in that when it binds to TNF the tumour fibrin
deposition activity of the TNF is enhanced and the cytotoxicity,
tumour regression, induction of endothelial procoagulant and tumour
receptor binding activities of the TNF are inhibited, the ligand
binding to the TNF such that the epitope of the TNF defined by the
topographic regions of residues 1-20 and 76-90 is substantially
prevented from binding to naturally occurring biologically active
ligands.
[0069] In a twenty-fifth aspect the present invention consists in a
ligand which binds to human TNF in the topographic regions of
residues 1-20 and 76-90. These regions are proximate in the 3D
structure of TNF and are topographically represented in FIG.
29.
[0070] In a preferred embodiment of the twenty-fifth aspect of the
present invention the ligand binds to TNF in the topographic
regions of residues 1-18 and 76-90.
[0071] In a preferred embodiment of the twenty-third, twenty-fourth
and twenty-fifth aspects of the present invention the ligand is the
monoclonal antibody designated MAb 21. A sample of the hybridoma
cell line producing MAb 21 was deposited with the European
Collection of Animal Cell Cultures (ECACC), Vaccine Research and
Production Laboratory, Public Health Laboratory Service, Centre for
Applied Microbiology and Research, Porton Down, Salisbury,
Wiltshire SP4 OJG, United Kingdom on Jan. 25, 1990 and was accorded
accession No. 90012432.
[0072] In a twenty-sixth aspect the present invention consists in a
ligand capable of binding to human TNF, the ligand being
characterised in that when it binds to TNF the fibrin deposition
activity of the TNF is unaffected and the cytotoxicity, tumour
regression, induction of endothelial procoagulant and tumour
receptor binding activities of the TNF are inhibited.
[0073] In a twenty-seventh aspect the present invention consists in
a ligand capable of binding to human TNF, the ligand being
characterized in that when it binds to TNF the tumour fibrin
deposition activity of the TNF is unaffected and the cytotoxicity,
tumour regression, induction of endothelial procoagulant and
receptor binding activities of the TNF are inhibited, the ligand
binding to the TNF such that the epitope of the TNF defined by the
topographic regions of residues 22-40, 69-97, 105-128 and 135-155
is substantially prevented from binding to naturally occurring
biologically active ligands.
[0074] In a twenty-eighth aspect the present invention consists in
a ligand which binds to human TNF in the topographic regions of
residues 22-40, 69-97, 105-128 and 135-155. These regions are
proximate in the 3D structure of TNF and are topographically
represented in FIG. 30.
[0075] In a preferred embodiment of the twenty-sixth,
twenty-seventh and twenty-eighth aspects of the present invention
the ligand is the monoclonal antibody designated MAb 53. A sample
of the hybridoma cell line producing MAb 53 was deposited with the
European Collection of Animal Cell Cultures (ECACC), Vaccine
Research and Production Laboratory, Public Health Laboratory
Service, Centre for Applied Microbiology and Research, Porton Down,
Salisbury, Wiltshire SP4 OJG, United Kingdom on Jan. 25, 1990 and
was accorded accession No. 90012433.
[0076] In a twenty-ninth aspect the present invention consists in a
ligand capable of binding to human TNF, the ligand being
characterised in that when it binds to the TNF tumour fibrin
deposition, induction of endothelial procoagulant, cytotoxicity,
tumour regression and receptor binding activities of the TNF are
unaffected.
[0077] In a thirtieth aspect the present invention consists in a
ligand capable of binding to human TNF, the ligand being
characterised in that when it binds to TNF the tumour fibrin
deposition, induction of endothelial procoagulant, cytotoxicity,
tumour regression and receptor binding activities of the TNF are
unaffected, the ligand binding to TNF such that the epitope of the
TNF defined by the topographic regions of residues 22-31 and
146-157 is substantially prevented from binding to naturally
occurring biologically active ligands.
[0078] In a thirty-first aspect the present invention consists in a
ligand which binds to human TNF in the topographic regions of
residues 22-31 and 146-157. These regions are proximate in the 3D
structure of TNF and are typographically represented in FIG.
31.
[0079] In a preferred embodiment of the twenty-ninth, thirtieth and
thirty-first aspects of the present invention the ligand is the
monoclonal antibody designated MAb 37. A sample of the hybridoma
cell line producing MAb 37 was deposited with the European
Collection of Animal Cell Cultures (ECACC), Vaccine Research and
Production Laboratory, Public Health Laboratory Service, Centre for
Applied Microbiology and Research, Porton Down, Salisbury,
Wiltshire SP4 OJG, United Kingdom on Aug. 3, 1989 and was accorded
accession No. 89080303.
[0080] In a thirty-second aspect the present invention consists in
a ligand capable of binding to human TNF, the ligand being
characterised in that when it binds to TNF the induction of
endothelial procoagulant activity of the TNF is unaffected and the
cytotoxicity, tumour regression, tumour fibrin deposition, and
receptor binding activities of the TNF are inhibited.
[0081] In a thirty-third aspect the present invention consists in a
ligand capable of binding to human TNF, the ligand being
characterised in that when it binds to TNF the induction of
endothelial procoagulant activity of the TNF is unaffected and the
cytotoxicity, tumour regression, tumour fibrin deposition and
receptor binding activities of the TNF are inhibited, the ligand
binding to the TNF such that the epitope of the TNF defined by the
topographic regions of residues 22-40 and 49-98 is substantially
prevented from binding to naturally occurring biologically active
ligands.
[0082] In a thirty-fourth aspect the present invention consists in
a ligand which binds to human TNF in at least one of the regions
selected from the group consisting of the topographic region of
residues 22-40, the topographic region of residues 49-98 and the
topographic region of residues 69-97.
[0083] In a preferred embodiment of the thirty-fourth aspect of the
present invention the ligand binds to human TNF in the
topographical region of residues 49-98. This region is
topographically represented in FIG. 32.
[0084] In a further preferred embodiment of the thirty-fourth
aspect of the present invention-the ligand binds to human TNF in
the topographic regions of residues 22-40 and 70-87. These regions
are proximate in the 3D structure of TNF and are topographically
represented in FIG. 33.
[0085] In a preferred embodiment of the thirty-second, thirty-third
and thirty-fourth aspects of the present invention the ligand is
monoclonal antibody MAb 11 or MAb 12.
[0086] In a thirty-fifth aspect the present invention consists in a
ligand capable of binding to human TNF, the ligand being
characterised in that when it binds to TNF the induction of
endothelial procoagulant activity of the TNF is inhibited.
[0087] In a thirty-sixth aspect the present invention consists in a
ligand capable of binding to human TNF, the ligand being
characterised in that when it binds to TNF the induction of
endothelial procoagulant activity of the TNF is inhibited, the
ligand binding to TNF such that the epitope of the TNF defined by
the topographical region of residues 108-128 is prevented from
binding to naturally occurring biologically active ligands.
[0088] In a thirty-seventh aspect the present invention consists in
a ligand which binds to human TNF in the topographical region of
residues 108-128.
[0089] In a preferred embodiment of the thirty-fifth, thirty-sixth
and thirty-seventh aspects of the present invention the ligand is
selected from the group consisting of monoclonal antibodies
designated MAb 1, MAb 32, MAb 42, MAb 47, MAb 53 and MAb 54.
[0090] The biological activities of TNF referred to herein by the
terms "Tumour Regression", "Induction of Endothelial Procoagulant",
"Induction of Tumour Fibrin Deposition", "Cytotoxicity" and
"Receptor Binding" are to be determined by the methods described
below.
[0091] The term "single domain antibodies" as used herein is used
to denote those antibody fragments such as described in Ward et al
(Nature, Vol. 341, 1989, 544-546) as suggested by these
authors.
[0092] In order that the nature of the present invention may be
more clearly understood, preferred forms thereof will now be
described with reference to the following example and accompanying
figures in which:--
[0093] FIG. 1 shows the results of a titration; assay with MAb 1
against TNF;
[0094] FIG. 2 shows TNF MAb 1 scatchard plot and affinity
determination;
[0095] FIG. 3 shows the effect of anti-TNF monoclonal antibodies 1
and 32 on TNF cytotoxicity in WEHI-164 cells;
[0096] FIG. 4 shows the effect of MAb 1 on TNF-induced regression
of a Meth A solid tumour;
[0097] FIG. 5 shows the effect of MAbs 1 and 25 on TNF-induced Meth
A Ascites tumour regression;
[0098] FIG. 6 shows the effect of anti-TNF MAbs on induction of
endothelial cell procoagulant activity by TNF;
[0099] FIG. 7 shows incorporation of labelled fibrinogen into
tumours of tumour-bearing mice and the effect of anti-TNF MAbs;
[0100] FIG. 8 is a schematic representation of epitopes on TNF;
[0101] FIG. 9 shows the effect of anti-TNF MAbs on TNF-induced
regression of WEHI-164 tumours;
[0102] FIG. 10 shows the enhancement of TNF regression activity by
MAb 32 in two experiments;
[0103] FIG. 11 shows the enhancement of TNF-induced tumour
regression by MAb 32--dose response at day 1 and day 2;
[0104] FIG. 12 shows binding of radio labelled TNF to receptors on
bovine aortic endothelial cells;
[0105] FIG. 13 shows receptor binding studies of TNF completed with
MAb 32 (), control antibody and MAb 47 () on melanoma cell line
M4-18E;
[0106] FIG. 14 shows receptor binding studies of TNF completed with
MAb 32 (), control antibody and XAb 47 () on melanoma cell line
IGR3;
[0107] FIG. 15 shows receptor binding studies of TNF completed with
MAb 32 (), control antibody and MAb 47 () on bladder carcinoma cell
line 5637;
[0108] FIG. 16 shows receptor binding studies of TNF completed with
MAb 32 (), control antibody and MAb 47 () on breast carcinoma cell
line MCF7;
[0109] FIG. 17 shows receptor binding studies of TNF completed with
MAb 32 (), control antibody and MAb 47 () on colon carcinoma cell
line B10;
[0110] FIG. 18 shows the effect on TNF-mediated tumour regression
in vivo by MAb 32 (.box-solid.) control MAb and MAb 47 (*);
[0111] FIG. 19 shows the effect on TNF-mediated tumour regression
in vivo by control MAb, MAb 32 and univalent Fab' fragments of MAb
32;
[0112] FIG. 20 shows the effect on TNF induced tumour regression by
control MAb (.box-solid.), MAb 32 and peptide 301 antiserum
[0113] FIG. 21 shows MAb 32 reactivity with overlapping peptides of
10 AA length; and
[0114] FIG. 22 shows a schematic three dimensional representation
of the TNF molecule.
[0115] FIG. 23 shows topographically the region of residues 1-20,
56-77, 108-127 and 138-149;
[0116] FIG. 24 shows topographically the region of residues 1-18
and 108-128;
[0117] FIG. 25 shows topographically the region of residues 56-79,
110-127 and 136-155;
[0118] FIG. 26 shows topographically the region of residues 1-26,
117-128 and 141-153;
[0119] FIG. 27 shows topographically the region of residues 22-40,
49-97, 110-127 and 136-153;
[0120] FIG. 28 shows topographically the region of residues 12-22,
36-45, 96-105 and 132-157;
[0121] FIG. 29 shows topographically the region of residues 1-20
and 76-90;
[0122] FIG. 30 shows topographically the region of residues 22-40,
69-97, 105-128 and 135-155;
[0123] FIG. 31 shows topographically the region of residues 22-31
and 146-157;
[0124] FIG. 32 shows topographically the region of residues
49-98;
[0125] FIG. 33 shows topographically the region of residues 22-40
and 70-87;
[0126] FIG. 34 shows results of an ELISA using samples containing
varying levels of TNF; and
[0127] FIG. 35 shows the effect of VHP3-V.lambda.A2 on anti-tumour
activity of TNF.
[0128] Animals and Tumour Cell Lines
[0129] In all experiments BALB/C female mice aged 10-12 weeks
obtained from the CSIRO animal facility were used. Meth A solid
tumour and Meth A ascites tumour cell lines were obtained from the
laboratory of Dr. Lloyd J. Old (Sloan Kettering Cancer Centre) and
the WEHI-164 fibrosarcoma line was obtained from Dr. Geeta Chauhdri
(John Curtin School of Medical Research, Australian National
University).
[0130] Fusions and Production of Hybridomas
[0131] Mice were immunised with 10 ug human recombinant TNF
intra-peritoneally in Freund's complete adjuvant. One month later
10 ug TNF in Freund's incomplete adjuvant was administered. Six
weeks later and four days prior to fusion selected mice were
boosted with 10 ug TNF in PBS. Spleen cells from immune mice were
fused with the myeloma Sp2/0 according to the procedure of Rathjen
and Underwood (1986, Mol. Immunol. 21, 441). Cell lines found to
secrete anti-TNF antibodies by radioimmunoassay were subcloned by
limiting dilution on a feeder layer of mouse peritoneal
macrophages. Antibody subclasses were determined by ELISA
(Misotest, Commonwealth Serum Laboratories).
[0132] Radioimmunoassay
[0133] TNF was iodinated using lactoperoxidase-according to
standard procedures. Culture supernatants from hybridomas (50 ul)
were incubated with 125I TNF (20,000 cpm in 50 ul) overnight at
4.degree. C. before the addition of 100 ul Sac-Cel (donkey
anti-mouse/rat immunoglobulins coated cellulose, Wellcome
Diagnostics) and incubated for a further 20 minutes at room
temperature (20.degree. C.). Following this incubation 1 ml of PBS
was added and the tubes centrifuged at 2,500 rpm for 5 minutes. The
supernatant was decanted and the pellet counted for bound
radioactivity.
[0134] Antibody-Antibody Competition Assays
[0135] The comparative specificites of the monoclonal antibodies
were determined in competition assays using either immobilized
antigen (LACT) or antibody (PACT) (Aston and Ivanyi, 1985, Pharmac.
Therapeut. 27, 403).
[0136] PACT
[0137] Flexible microtitre trays were coated with monoclonal
antibody (sodium sulphate precipitated globulins from mouse ascites
fluid, 100 micrograms perrml in sodium bicarbonate buffer, 0.05M,
pH 9.6) overnight at 4.degree. C. prior to blocking non-specific
binding sites with 1% bovine serum albumin in PBS (BSA/PBS). The
binding of 125I TNF to immobilised antibody was determined in the
presence of varying concentrations of a second anti-TNF monoclonal
antibody. Antibody and TNF were added simultaneously and incubated
for 24 hours prior to washing with PBS (4 times) and counting wells
for bound radioactivity. 100% binding was determined in the absence
of heterologous monoclonal antibody while 100% competition was
determined in the presence of excess homologous monoclonal
antibody. All dilutions were prepared in BSA/PBS.
[0138] LACT
[0139] The binding of protein A purified, radiolabelled monoclonal
antibodies to TNF coated microtitre wells was determined in the
presence of varying concentrations of a second monoclonal antibody.
Microtitre plates were coated with TNF (50 micrograms per ml) as
described above. Quantities of competing antibodies (50
microlitres) were pre-incubated on plates for 4 hour at 4.degree.
C. prior to addition of 125I monoclonal antibody (30,000 cpm) for a
further 24 hours. Binding of counts to wells was determined after
four washes with PBS. 100% binding was determined in the absence of
competing antibody while 100% competition was determined in the
presence of excess unlabelled monoclonal antibody.
[0140] WEHI-164 Cytotoxicity Assay
[0141] Bioassay of recombinant TNF activity was performed according
to Espevik and Nissen-Meyer (1986, J. Immunol. Methods 95, 99). The
effect of the monoclonal antibody on TNF activity was determined by
the addition of the monoclonal antibody to cell cultures at
ABT90.
[0142] Tumour Regression Experiments
[0143] Modulation of TNF-induced tumour regression activity by
monoclonal antibodies was assessed in three tumour models: the
subcutaneous tumours WEHI-164 and Meth A sarcoma and the ascitic
Meth A tumour. Subcutaneous tumours were induced by the injection
of approximately 5.times.10.sup.5 cells. This produced tumours of
between 10-15 mm approximately 14 days later. Mice were injected
intra-peritoneally with human recombinant TNF (10 micrograms) plus
monoclonal antibody (200 microlitres ascites globulin) for four
consecutive days. Control groups received injections of PBS alone
or TNF plus monoclonal antibody against bovine growth hormone. At
the commencement of each experiment tumour size was measured with
calipers in the case of solid tumours or tumour-bearing animals
weighed in the case of ascites mice. These measurements were taken
daily throughout the course of the experiment.
[0144] Radio-Receptor Assays
[0145] WEHI-164 cells grown to confluency were scrape harvested and
washed once with 1% BSA in Hank's balanced salt solution (HBSS,
Gibco). 100 ul of unlabelled TNF (1-10,000 ng/tube) or monoclonal
antibody (10 fold dilutions commencing 1 in 10 to 1 in 100,000 of
ascitic globulin) was added to 50 ul 125I TNF (50,000 cpm). WEHI
cells were then added (200 microlitres containing 2.times.10.sup.6
cells). This mixture was incubated in a shaking water bath at
37.degree. C. for 3 hours. At the completion of this incubation 1
ml of HBSS was added and the cells spun at 16,000 rpm for 30
seconds. The supernatant was discarded and bound 125I TNF in the
cell pellet counted. All dilutions were prepared in HBSS containing
1% BSA.
[0146] Procoagulant Induction by TNF on Endothelial Cells
[0147] Bovine aortic endothelial cells (passage 10) were grown in
RPMI-1640 containing 10% foetal calf serum (FCS), penicillin,
streptomycin, and 2-mercaptoethanol at 37.degree. C. in 5%
CO.sub.2. For induction of procoagulant activity by TNF the cells
were trypsinised and plated into 24-well Costar trays according to
the protocol of Bevilacqua et al., 1986 (PNAS 83, 4533). TNF (0-500
units/culture) and monoclonal antibody (1 in 250 dilution of
ascitic globulin) was added after washing of the confluent cell
monolayer with HBSS. After 4 hours the cells were scrape harvested,
frozen and sonicated. Total cellular procoagulant activity was
determined by the recalcification time of normal donor
platelet-poor plasma performed at 37.degree. C., 100 microlitres of
citrated platelet-poor plasma was added to 100 ul of cell lysate
and 100 ul of calcium chloride (30 mM) and the time taken for clot
formation recorded. In some experiments tumour cell culture
supernatant was added to endothelial cells treated with TNF and/or
monoclonal antibody (final concentration of 1 in 2).
[0148] Incorporation of 1251 Fibrinogen into Tumours of Mice
Treated with TNF and Monoclonal Antibody
[0149] In order to examine the effect of TNF and monoclonal
antibodies on fibrin formation in vivo, BALB/c mice were injected
subcutaneously with WEHI-164 cells (105 cells/animal). After 7-14
days, when tumours reached a size of approximately 1 cm in
diameter, animals were injected intra-peritoneally with TNF (10
ug/animal) and 125I human fibrinogen (7.5 ug/animal, 122 uCi/mg
Amersham) either alone or in the presence of monoclonal antibody to
human TNF (200 ul/animal ascitic globulin). Monoclonal antibody
against bovine growth hormone was used as control monoclonal
antibody. Two hours after TNF infusion incorporation of 125I
fibrinogen into mouse tissue was determined by removing a piece of
tissue, weighing it and counting the sample in a gamma counter.
[0150] In all 13 monoclonal antibodies reacting with human TNF were
isolated. These monoclonal antibodies were designated MAb 1, MAb
11, MAb 12, MAb 20, MAb 21, MAb 25, MAb 31, MAb 32, MAb 37, MAb 42,
MAb 47, MAb 53 and MAb 54. The effect of these monoclonal
antibodies on the bioactivity of human TNF is set out in Table
2.
[0151] As can be seen from Table 2, whilst some monoclonal
antibodies inhibit both anti-tumour activity and activation of
coagulation by human TNF (MAb 1, 47 and 54) not all antibodies
which inhibit the anti-tumour activity inhibit activation of
coagulation either in vitro or in vivo (MAb 11, 12, 25 and 53).
Indeed MAb 21 which inhibited tumour regression enhanced the
activation of coagulation in vivo.
1TABLE 2 EFFECT OF MONOCLONAL ANTIBODIES ON TNF BIOACTIVITY
MONOCLONAL ANTIBODY TNF BIOACTIVITY 1 11 12 20 21 25 31 32 37 42 47
53 54 Cytotoxicity - - - 0 - - 0 0 0 0 - - - Tumour Regression - -
- 0 - - 0 + 0 0 - - - Induction of - 0 0 - - 0 0 - 0 - - - -
Procoagulant (Endothelial Fibrin Deposition - - - + + + + + 0 - - 0
- (tumour) Receptor Binding - - - 0 - - 0 +/ 0 0 - - - (WEHI - 164)
0* + Enhancement 0 No effect - Inhibition *Depending on MAb
concentration in the case of WEHI-164 tumour cells and tumour type
(see FIGS. 3, 13-17).
[0152] MAbs 1, 47 and 54, which have been shown in competition
binding studies to share an epitope on TNF, can be seen to have
highly desirable characteristics in treatment of toxic shock and
other conditions of bacterial, viral and parasitic infection where
TNF levels are high requiring complete neutralisation of TNF. Other
monoclonal antibodies such as MAb 32 are more appropriate as agents
for coadministration with TNF during cancer therapy since they do
not inhibit tumour regression but do inhibit activation of
coagulation. This form of therapy is particularly indicated in
conjunction with cytotoxic drugs used in cancer therapy which may
potentiate activation of coagulation by TNF (e.g. vinblastin,
acyclovir, IFN alpha, IL-2, actinomycin D, AZT, radiotherapy,
adriamycin, mytomycin C, cytosine arabinoside, dounorubicin,
cis-platin, vincristine, 5-flurouracil, bleomycin, (Watanabe N et
al 1988 Immunopharmacol. Immunotoxicol. 10 117-127) or in diseases
where at certain stages TNF levels are low (e.g. AIDS) and where
individuals may have AIDS associated cancer e.g. Kaposi sarcoma,
non-Hodgkins lymphoma and squamous cell carcinoma.
[0153] Monoclonal antibody MAb 1 has been found to have the
following characteristics:--
[0154] 1. Binds human recombinant TNF alpha, but not human
lymphotoxin (TNF beta) or human interferon. Similarly MAb 1 does
not cross-react with recombinant murine TNF (FIG. 1).
[0155] 2. MAb 1 is of the immunoglobulin type IgG1, K with an
apparent affinity of 4.4.times.10.sup.-9 moles/litre (FIG. 2).
[0156] 3. MAb neutralises the cytotoxic effect of recombinant human
TNF on WEHI-164 mouse fibrosarcoma cells in culture. One microgram
of MAb 1 neutralizes approximately 156.25 units of TNF In vitro
(FIG. 3).
[0157] 4. MAb 1 neutralises the tumour regression activity of TNF
in the following mouse tumour models in vivo; WEHI-164 subcutaneous
solid tumour, the Meth A subcutaneous solid tumour and the Meth A
ascites tumour (FIGS. 4, 5 and 9).
[0158] 5. Mab1 prevents cerebral damage caused by human TNF in mice
infected with malarial parasites.
[0159] 6. In radioreceptor assays MAb 1 prevents binding of TNF to
receptors on WEHI-164 cells (Table 3).
[0160] 7. MAb 1 inhibits the induction of procoagulant activity
(tissue factor) on cultured bovine aortic endothelial cells (FIG.
6).
[0161] 8. MAb 1 reduces the uptake of 125I fibrinogen into tumours
of mice treated with TNF (FIG. 7).
[0162] 9. MAb 1 competes for binding of 125I TNF and thus shares an
overlapping epitope with the following monoclonal antibodies: 21,
25, 32, 47, 54 and 37.
[0163] 10. MAb 1 does not compete for binding of 125I TNF with the
following monoclonal antibodies: 11, 12, 42, 53, 31 and 20 (FIG.
8).
2TABLE 3 RADIORECEPTOR ASSAY: INHIBITION OF TNF BINDING TO WEHI-164
CELLS BY MAb 1 TREATMENT % SPECIFIC BINDING MAb 1 1/10 0 1/100 21
1/1,000 49 1/10,000 73 1/100,000 105 cold TNF (ng/tube) 10,000 0
5,000 0 1,000 0 500 10 100 11 10 64 1 108 0 100
[0164] MAb 32 is an IgG2b,K antibody with an affinity for human TNF
alpha of 8.77.times.10.sup.-9 moles/litre as determined by
Scatchard analysis. This monoclonal antibody does not react with
either human TNF beta (lymphotoxin) or mouse TNF alpha.
[0165] As shown in FIG. 3 MAb 32 does not inhibit TNF cytotoxicity
in vitro as determined in the WEHI-164 assay.
[0166] Monoclonal antibody 32 variably enhances TNF-induced tumour
regression activity against WEHI-164 fibrosarcoma tumours implanted
subcutaneously into BALB/c mice at a TNF dose of 10 ug/day (see
FIGS. 10 and 11). This feature is not common to all monoclonal
antibodies directed against TNF (FIG. 9) but resides within the
binding site specificity of MAb 32 (FIG. 8) which may allow greater
receptor mediated uptake of TNF into tumour cells (see Table
4).
3TABLE 4 BINDING OF TNF TO RECEPTORS ON WEHI-164 CELLS IN THE
PRESENCE OF MAb 32 % BINDING.sup.125 I-TNF MAB DILUTION CONTROL MAB
MAB 32 1/10 36 141 1/100 74 88 1/1000 101 83 1/10,000 92 82
1/100,000 97 93
[0167] Enhancement of TNF activity by MAb 32 at lower doses of TNF
is such that at least tenfold less TNF is required to achieve the
same degree of tumour regression (see FIGS. 11 and 18). The results
for day 1, 2.5 ug and 1 ug TNF and day 2, 5 ug, 2.5 ug and lug are
statistically significant in a t-test at p<0.01 level. This
level of enhancement also increases the survival rate of recipients
since the lower dose of TNF used is not toxic. FIG. 19 shows that
univalent Fab fragments of MAb 32 also cause enhancement of
TNF-induced tumour regression in the same manner as whole MAb 32
(see below).
[0168] MAb 32 inhibits the expression of clotting factors on
endothelial cells normally induced by incubation of the cultured
cells with TNF (see FIG. 6). This response may be mediated by a
previously unidentified TNF receptor which is distinct to the
receptor found on other cells.
[0169] Conversely, MAb 32 enhances the in vivo activation of
coagulation within the tumour bed as shown by the incorporation of
radiolabelled fibrinogen (FIG. 7). This may be due to activation of
monocytes/macrophage procoagulant and may provide further insight
into the mechanism of TNF-induced tumour regression.
[0170] The results obtained with MAb 32 are shown in comparison to
other anti-TNF MAbs in Table 2.
[0171] The ability of MAb 32 and MAb 47 to inhibit the binding of
TNF to endothelial cells was also assessed. Bovine aortic
endothelial (BAE) cells (passage 11) were plated in 24-well culture
dishes (Corning) which had been pre-coated with gelatin (0.2%) and
grown to confluence-in McCoys 5A (modified) medium supplemented
with 20% foetal calf serum. For the radio-receptor assay all
dilutions (of cold TNF and MAbs) were made in this medium. The BAE
cells were incubated for one hour in the presence of either cold
TNF (0 to 100 ng) or MAb (ascites globulins diluted 1/100 to
1/100,000) and iodinated TNF (50,000 cpm). At the end of this time
the medium was withdrawn and the cells washed before being lysed
with 1M sodium hydroxide. The cell lysate was then counted for
bound radioactive TNF. Specific binding of labelled TNF to the
cells was then determined.
[0172] The results obtained in this assay with-MAb 32, MAb 47 and a
control MAb are set out in FIG. 12.
[0173] The results obtained in the clotting assay using BAE cells
cultured in the presence of TNF and anti-TNF MAb correlate with the
results obtained in the BAE radioreceptor assay i.e. MAbs which
inhibit the induction of clotting factors on the surface of
endothelial cells (as shown by the increase in clotting time
compared to TNF alone) also inhibit the binding of TNF to its
receptor. This is exemplified by MAbs 32 and 47.
[0174] MAb 32, which does not inhibit TNF binding to WEHI-164
cells, does inhibit binding of TNF to endothelial cells. This
result provides support for the hypothesis that distinct functional
sites exist on the TNF molecule and that these sites interact with
distinct receptor subpopulations on different cell types. Thus
ligands which bind to defined regions of TNF are able to modify the
biological effects of TNF by limiting its binding to particular
receptor subtypes.
[0175] As shown in FIG. 12 MAb 47 is a particularly potent
inhibitor of TNF interaction with endothelial cells, the percentage
specific binding at a dilution of 1/100 to 1/10,000 being
effectively zero.
[0176] Receptor Binding Studies of Human TNF Complexed With MAB 32
on Human Carcinoma Cell Lines In Vitro
[0177] MAb 32 has been shown to enhance the anti-tumour activity of
human TNF. The mechanisms behind the enhancement may include
restriction of TNF binding to particular (tumour) receptor subtypes
but not others (endothelial) with subsequent decrease in TNF
toxicity to non-tumour cells. This mechanism does not require
enhanced uptake of TNF by tumour cells in in vitro assays. In
addition, MAb 32 also potentiates the binding of human TNF directly
to TNF receptors on certain human carcinoma cell lines.
[0178] Materials and Methods
[0179] The following human carcinoma cell lines have been assayed
for enhanced receptor-mediated uptake of TNF in the presence of MAb
32: B10, CaCo, HT 29, SKC01 (all colon carcinomas), 5637 (Bladder
carcinoma), MM418E (melanoma), IGR3 (melanoma), MCP 7 (breast
carcinoma). The cells were propogated in either RPMI-1640 (MM418E)
DMEM (CaCo and IGR 3) or Iscoves modified DMEM (B10, HT 29, SK01,
S637, MCF 7) supplemented with 10% foetal calf serum,
penecillin/streptomycin and L-glutamine. Receptor assays were
performed as previously described for endothelial cells except that
the incubation time with iodinated TNF was extended to 3 hours for
all but the B10 cells for which the radiolabel was incubated for 1
hour.
[0180] Results
[0181] Enhanced TNF uptake was observed in the presence of MAb32 by
the melanoma cell lines tested MM418E and IGR 3 (FIGS. 13 and 14),
the bladder carcinoma 5637 (FIG. 15), and the breast carcinoma MCF
7 (FIG. 16). MAb 32 did not affect TNF-receptor interaction in any
of the other cell lines as shown by B 10 (FIG. 17) MAb 47, which
has been shown to inhibit TNF binding to WEHI-164 cells and
endothelial cells, and which also inhibits TNF-mediated tumour
regression was found to markedly inhibit TNF binding to all the
cell lines tested (FIGS. 13-17).
[0182] Conclusions
[0183] Receptor binding analyses have indicated a second mechanism
whereby MAb 32 may potentiate the anti-tumour activity of TNF. This
second pathway for enhancement of TNF results from increased uptake
of TNF by tumour all receptors in the presence of MAb 32.
[0184] Enhancement of TNF-Mediated Tumour Regression in Vivo By MAB
32 or Univalent FAB" Fragments of MAB 32
[0185] Tumour regression studies were carried out as described
above in mice carrying WEHI-164 subcutaneous tumours (N=5
animals/group). Tumour size was determined daily during the course
of the experiment. The results obtained using MAb 32 are set out in
FIG. 22 and show the mean +/- SD% change in tumour area at the
completion of treatment (day 2) (.box-solid. MAb 32: control MAbs
*MAb 47). Differences observed between control MAb-TNF and MAb
32-TNF treated groups are statistically significant in a T-test at
the p-<0.01 level.
[0186] The results using the univalent Fab' fragments of MAb 32 are
shown in FIG. 19. Tumour size was determined daily during the
course of the experiment. The results show the mean SD% change in
tumour area at the completion of treatment (day 2). Differences
between the control MAb-10 F and MAb 32-TNF treated groups are
statistically significant in a T-test at the P-<0.01 level.
[0187] TNF Induced Tumour Regression: Effect of Anti-Peptide 301
Sera
[0188] FIG. 20 shows the percent change in tumour area in
tumour-bearing mice treated for three days with TNF plus control
MAb (antibody against bovine growth hormone), TNF plus MAb 32 or
TNF plus antiserum (globulin fraction) against peptide 301. In an
unpaired T-test the control group is significantly different from
both of the test groups (MAb 32, antiserum 301) while the MAb 32
and peptide antiserum 301 groups are not significantly different
from each other. (control vs MAb 32, p<0.002; control vs
antipeptide 301, p<0.025). Thus antisera raised using a peptide
which comprises part of the MAb 32 specificity, also causes TNF
enhancement of tumour regression.
[0189] As shown in FIG. 9 competition binding studies has shown
that the thirteen monoclonal antibodies can be sub-divided into two
main groups, namely MAbs 1, 21, 47, 54, 37, 32 and 25 and MAbs 11,
12, 53 and 42. Experiments were then conducted to identify-the
regions on human TNF recognised by these monoclonal antibodies.
[0190] Identification of Regions on Human TNF Recognised by
Monoclonal Antiodies
[0191] Methods
[0192] 1. Overlapping peptides of 7 and 10 amino acid residues long
were synthesized on polypropylene pins according to the method of
Geysen et al., 1984, PNAS 81, 3998-4002. The overlap was of 6 and 9
residues respectively and collectively the peptides covered the
entire TNF amino acid sequence. The peptides were tested for
reactivity with the MAbs by ELISA. MAbs which had TNF reactivity
absorbed from them by prior incubation with whole TNF were also
tested for reactivity with the peptides and acted as a negative
control.
[0193] 2. Longer peptides of TNF were synthesized as described
below. These peptides were used to raise antisera in sheep using
the following protocol. Merino sheep were primed with TNF peptide
conjugated to ovalbumin and emulsified in Freunds Complete adjuvant
and boosted at 4 weekly intervals with peptide-ovalbumin and sera
assayed for the presence of anti-TNF antibody by radioimmunoassay.
Of the peptides shown only peptides 275, 301, 305, 306 and 307
elicited sera reacting with whole TNF. The positive sera were then
used in competitive binding assays (PACT assays) with the MAbs.
[0194] The following peptides were synthesised and are described
using the conventional three letter code for each amino acid with
the TNF sequence region indicated in brackets.
[0195] Peptide 275
[0196] H-Ala-Lys-Pro-Trp-Tyr-Glu-Pro-Ile-Tyr-Leu-OH (111-120)
[0197] Peptide 301
[0198]
H-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp-Lys-Pro-Val-Ala-His-Val-V-
al-Ala-OH (1-18)
[0199] Peptide302
[0200]
H-Leu-Arg-Asp-Asn-Gln-Leu-Val-Val-Pro-Ser-Glu-Gly-Leu-Tyr-Leu-Ile-O-
H (43-58)
[0201] Peptide 304
[0202] H-Leu-Phe-Lys-Gly-Gln-Gly-Cys-Pro-Ser-Thr-H
is--Val-Leu-Leu-Thr-His- -Thr-Ile-Ser-Arg-Ile-OH (63-83)
[0203] Peotide 305
[0204]
H-Leu-Ser-Ala-Glu-Ile-Asn-Arg-Pro-Asp-Tyr-Leu-Asp-Phe-Ala-Glu-Ser-G-
ly-Gln-Val-OH (132-150)
[0205] Peotide 306
[0206] H-Val-Ala-His-Val-Val-Ala-Asn-Pro-Gln-Ala-Glu-Gly-Gln-Leu-OH
(13-26)
[0207] Peotide 307
[0208]
H-Ala-Glu-Gly-Gln-Leu-Gln-Trp-Leu-Asn-Arg-Arg-Ala-Asn-Ala-Leu-Leu-A-
la-Asn-Gly-OH (22-40)
[0209] Peptide 308
[0210]
H-Gly-Leu-Tyr-Leu-Ile-Tyr-Ser-Gln-Val-Leu-Phe-Lys-Gly-Gln-Gly-OH
(54-68)
[0211] Peptide 309
[0212]
H-His-Val-Leu-Leu-Thr-His-Thr-Ile-Ser-Arg-Ile-Ala-Val-Ser-Thr-Gln-T-
hr-Lys-Val-Asn-Leu-Leu-COOH (73-94)
[0213] Peptide 323
[0214] H-Thr-Ile-Ser-Arg-Ile-Ala-Val-Ser-Thr-Gln-Thr-OH (79-89)
[0215] These peptides were synthesised using the following general
protocol.
[0216] All peptide were synthesised using the Fmoc-polyamide method
of solid phase peptide synthesis (Atherton et al, 1978,
J.Chem.Soc.Chem.Commun., 13, 537-539). The solid resin used was
PepSyn KA which is a polydimethylacrylamide gel on Kieselguhr
support with 4-hydroxymethylphenoxy- acetic acid as the
functionalised linker (Atherton et al., 1975, J.Am.Chem. Soc. 27,
6584-6585).
[0217] The carboxy terminal amino acid was attached to the solid
support by a DCC/DMAP-mediated symmetrical-anhydride
esterification.
[0218] All Fmoc-groups were removed by piperidine/DMF wash and
peptide bonds were formed either via pentafluorophenyl active
esters or directly by BOP/NMM/HOBt (Castro's reagent) (Fournier et
al, 1989, Int.J.Peptide Protein Res., 33, 133-139) except for
certain amino acids as specified in Table 5.
[0219] Side chain protection chosen for the amino acids was removed
concomittantly during cleavage with the exception of Acm on
cysteine which was left on after synthesis.
4 TABLE 5 Amino Acid Protecting Group Coupling Method Arg Mtr or
Pmc Either Asp OBut Either Cys Acm (permanent) Either Glu OBut
Either His Boc OPfp only Lys Boc Either Ser But BOP only Thr But
BOP only Tyr But Either Trp none Either Asn none OPfp only Gln none
OPfp only
[0220] Cleavage and Purification
[0221] Peptide 301, 302, 305 are cleaved form the resin with 95%
TFA and 5% thioanisole (1.5 h) and purified on reverse phase C4
column, (Buffer A --0.1% aqueous TFA, Buffer B--80% ACN 20% A).
[0222] Peptide 303, 304 are cleaved from the resin with 95% TFA and
5% phenol (5-6 h) and purified on reverse phase C4 column. (Buffers
as above).
[0223] Peptide 306, 308 are cleaved from the resin with 95% TFA and
5% water (1.5 h) and purified on reverse phase C4 column. (Buffers
as above).
[0224] Peptide 309 Peptide was cleaved from the resin with 95% TFA
and 5% thioanisole and purified on reverse phase C4 column.
(Buffers as above).
[0225] Peptide 307 Peptide was cleaved from the resin with a
mixture of 93% TFA, 3.1% Anisole, 2.97% Ethylmethylsulfide and
0.95% Ethanedithiol (3 h) and purified on reverse phase C4 column.
(Buffers as above).
[0226] Results
[0227] Typical results of MAb ELISA using the 7 and 10 mers are
shown in FIG. 21. Together with the results of PACT assays using
the sheep anti-peptide sera (shown in Table 6) the following
regions of TNF contain the binding sites of the anti-TNF MAbs.
[0228] MAb 1 : residues 1-18, 58-65, 115-125, 138-149
[0229] MAb 11: residues 49-98
[0230] MAb 12: residues 22-40, 70-87
[0231] MAb 21: residues 1-18, 76-90
[0232] MAb 25: residues 12-22, 36-45, 96-105, 132-157
[0233] MAb 32: residues 1-26, 117-128, 141-153
[0234] MAb 37: residues 22-31, 146-157
[0235] MAb 42: residues 22-40, 49-96, 110-127, 136-153
[0236] MAb 47: residues 1-18, 108-128
[0237] MAb 53: residues 22-40, 69-97, 105-128, 135-15-5
[0238] MAb 54: residues 56-79, 110-127, 136-155
5TABLE 6 COMPETITIVE BINDING OF TNF BY ANTI-TNF MONOCLONES IN THE
PRESENCE OF ANTI PEPTIDE SERA MAB/PEPTIDE SERA 275 301 305 306 307
1 - + - - - 11 - +/- - - - 12 - + - - ++ 21 - ++ - - - 25 - + - - -
32 - ++++ + + - 37 - + +/- - + 47 - + - - - 53 - + - - + 54 - + - -
- 42 - + + - + Note 1: - indicates no competition, + indicates
slight competition at high concentration of anti-peptide antisera
(1/50), ++++ indicates trong competition by anti-peptide sera equal
to that of the homolgous MAb. Note 2: Only peptide which elicited
sera recongising whole TNF were used in this assay.
[0239] As will be understood by persons skilled in this field the
ligands of the present invention can be used in assays of
biological fluids for detecting the presence of and quantifying the
concentration of TNF in a sample. One means by which this may be
achieved is by using the ligands of the present invention in
conventional ELISAs. Set out below is an example of such an
assay.
6 TNF ELISA REAGENTS CARBONATE COATING BUFFER, pH 9.6
Na.sub.2CO.sub.3 1.6 g NaHCO.sub.3 2.9 g Add 800 mL dH.sub.2O, pH
to 9.6 then make to 1 L with dH.sub.2O BLOCKING BUFFER BSA 1 g PBS
100 mL Add BSA to PBS and allow to dissolve fully before using.
Store at 4.degree. C. WASH BUFFER (0.05% Tween/PBS) Tween 20 0.5 g
PBS 1 L Add Tween to PBS and mix thoroughly before use CITRATE
BUFFER Citric Acid. 2.1 g in 50 mL Add solutions together 1H.sub.2O
dH.sub.2O and adjust TriSodium 1.47 g in 50 mL pH to 4.0-4.2
Citrate 2H.sub.2O dH.sub.2O NB: All incubations can be carried out
at 4.degree. C. overnight OR at room temperature for 2 hrs OR at
37.degree. C. for 1 hr.
[0240] Method
[0241] Coat ELISA plates with equal proportions of Mab1, MAb32 and
MAb54 to human TNF in carbonate coating buffer. The total
immunoglobulin concentration should be 20 .mu.g/mL and 100 .mu.L is
added to each well. Cover plates and incubate.
[0242] Wash plates 3.times. with PBS/Tween.
[0243] Incubate plates with 250 .mu.L/well blocking buffer
[0244] Wash plates 3.times. with PBS/Tween.
[0245] Add 100 .mu.L sample or TNF standards, diluted in blocking
buffer where required, to plates, then cover and incubate.
[0246] Wash plates 3.times. with PBS/Tween.
[0247] add 100 .mu.L biotinylated antibody mix (equal proportions
of biotinylated monoclonal antibodies 11 & 42 to human TNF) at
a final concentration of 10 .mu.g/mL in blocking buffer to each
well, cover and incubate.
[0248] Wash plates 3.times. with PES/Tween.
[0249] Add 100 .mu.L/well streptavidin-peroxidase (Amersham product
no. RPN 1231) at 1/2,000 in blocking buffer, then cover and
incubate.
[0250] Wash plates 3.times. with PBS/Tween.
[0251] Add 100 .mu.L/well biotinylated anti-stretpavidin monoclonal
antibody (Jackson Immunoresearch) at 1/40,000 in blocking buffer,
cover and incubate.
[0252] Wash plates 3.times. with PBS/Tween.
[0253] Add 100 .mu.L/well streptavidin-peroxidase at 1/2,000 in
blocking buffer, cover and incubate.
[0254] Wash plates 3.times. with PBS/Tween.
[0255] Add 100 .mu.L/well peroxidase substrate (ABTS) at 1 mg/mL in
citrate buffer containing 0.3 .mu.L/ml H.sub.2O.sub.2 and leave to
incubate at room temperature for up to 1 hour.
[0256] NB: Substrate solution should be prepared immediately prior
to use.
[0257] Read absorbance at 405 nm, and compare sample readings with
TNF standard curve to determine TNF levels.
7 BIOTINYLATION OF IgG 50 mM BIOCARBONATE BUFFER, pH 8.5
Na.sub.2CO.sub.3 1.6 g NaHCO.sub.3 2.9 g In 1 L dH2O, adjust pH
with HCl 0.1 PHOSPHATE BUFFER, pH 7.0
[0258] Method
[0259] Prepare immunoglobulins by purifying on a protein A column,
then freeze-drying.
[0260] Reconstitute the immunoglobulins with 50 mM bicarbonate
buffer to a concentration of 20 mg/mL in a clean glass test
tube.
[0261] Add 0.4 mg biotin per 20 mg Ig directly to the tube.
[0262] Place the test tube on ice and incubate for 2 hours.
[0263] Remove the unreacted biotin by centrifuging at 1000 g for
15-30 minutes in a Centricon-30 microconcentrator. Dilute the
sample in 0.1M phosphate buffer and repeat the centrifugration
twice.
[0264] Make the sample up to the original volume with phosphate
buffer, add 0.1% NaN.sub.3 and store at 4.degree. C. until
used.
[0265] The results obtained in such an assay using samples
containing known amounts of TNF is shown in FIG. 34.
[0266] As mentioned above the specific mouse monoclonal antibodies
disclosed in this application can be humanised if required. A
number of methods of obtaining humanised antibodies are set out in
PCT/GB92/01755 (WO93/06213). A humanised version of MAb32
designated VHP3-V.lambda.A2 was produced by the method disclosed in
PCT/GB92/01755. Briefly, this antibody was produced as follows:
[0267] 1 Cloning and Display of the V Genes of MAb 32 Phage
[0268] Cloning of the V-Genes of MAb32:
[0269] The genes of the mouse MAb32 antibody (IgG2b, Kappa) were
rescued by PCR essentially as described (Clackson et al., 1991,
supra, Clackson et al in "PCR: a practical approach, eds Mr Phenox
et al, IRL Press, Oxford pp 187-214) using the primers VH1BACk and
VH1FOR2 for the VH gene and Vk2BACK and VK4FOR for the VL gene and
the polymerase chain reaction (PCR, R. K. Saiki et al., 1985,
Science 230, p1350). The mouse VH and Vk genes were assembled for
expression as scFv fragments by PCR assembly (Clarckson et al.,
supra) amplified with VH1BACKSfi and VFFOR4NOT and ligated into
phagemid pHEN1 (H. R. Hoogenboom et al., 1991 Nucl. Acids. Res. 19,
pp4133-4137) as a SfiI-NotI cut restriction fragment, and
electroporated into E.coli HB2151 cells. Of 96 clones analysed by
ELISA (see below), 9 secreted TNF-binding soluble scFv fragments.
Sequencing revealed in all clones a mouse VH of family IIB and a
mouse Vk of family VI (E. A. Kabat et al., 1991 Sequences of
Proteins of Immunological Interest, US Public Health Services).
Nucleotide mutations which were probably introduced by the PCR were
detected by comparing the 9 sequences, and a clone with consensus
sequence and binding activity (scFv-MAb32) chosen for further
clroning experiments.
[0270] Recloning of the MAb32 V-genes for soluble expression:
[0271] The murine V-genes were recloned for soluble expression of
heavy (Fd, VHCH1) or light chain, by linking the mouse V-genes to
the human CH1 (of the mu-isotype) or human Ck gene respectively by
splice overlap extension. The mouse Vk gene was amplified from
scFv-MAb32 DNA with oligonucleotides MOJK1FORNX (binds in joining
region of V-gene and MVKBASFI (binds in 5' region and adds Sfil
restriction site); the human Ck was obtained by PCR from a
mouse-human chimaeric light chain gene (of NQ10.12.5, described in
Hoogenboom et al., 1991 supra), with oligonucleotides
MOVK-HUCK-BACK (binds in 5' of human Ck and is partially
complementary with mouse Jk 1 region) and HUCKNOT16NOMYC (sits in
3'0 end of human Ck, retains the terminal cysteine, and tags on a
NotI restriction site) as in Clarkson et al, 1991 using a two
fragment assembly. For linkage of the DNA fragments, the two PCR
fragments were mixed and amplified with MVKBASFI and
HUCKNOT16NOMYC. The chimaeric VkCk gene was subsequently cloned as
a SfiI-NotI fragment in pUCl9 derivative containing the pelB signal
peptide sequence and appropriate cloning sites for soluble
expression of the light chain (pUC19-pelB-myc). Similarly, the
mouse VH gene (amplified from scFv-MAb32 with LMB3 and VH1FOR-2)
was combined by splicing by overlap extension PCR with the human
u-CH1 domain (amplified from human IgM-derived cDNA (Marks et al.,
1991, supra WO 92/01047) with Mo-VH-Ku-CH1 and HCM1FONO, and cloned
as SfiI-NotI fragment into a pUC19-pelB-myc for soluble expression
of a tagged chain.
[0272] Display of the MAb32 Antibody on Phage:
[0273] The chimaeric light chain was displayed on phage fd by
reamplification of the mouse/human chimaeric chain with HUCKCYSNOT
and MVKBAAPA and cloning into fd-tet-DOG1 as an ApaLI-NotI
fragment. Cells harbouring a plasmid with the heavy Fd chain gene
were grown in 2xTY containing AMP-GLU (1%) to logarithmic phase
(OD600 of 0.5) and infected with a 20-fold excess of light-chain
displaying phage. After 45 min at 37.degree. C. without shaking and
45 min at 37.degree. C. with shaking in the 2xTY, ampicillin (100
.mu.g/ml). Glucose 1% medium, a sample was diluted into 50-fold
volume of prewarmed (37.degree. C.) 2.times.TY, ampicillin (100
.mu.g/ml) and tetracyclin (15 .mu.g/ml), grown for 1 hr at
37.degree. C. and then overnight at 30.degree. C. (shaking). Phage
particles collected from the supernatant of such culture displayed
TNF-binding Fab fragments anchored through the light chain on their
surface.
[0274] Similarly, the reversed configuration was made. The heavy
chain VHCH1 fragment was cloned into fd-tet-DOG1 (after
amplification of the Fd chain gene from the mouse/human chimeric
construct with VH1BACKAPA and HCM1FONO), and phage used to infect
cells capable of producing soluble light chain. Phage particles
collected from the supernatant of such culture displayed
TNF-binding Fab fragments anchored through the heavy chain VHCH1
fragment on their surface.
[0275] Properties of MAb 32 Fragments Displayed on Phage:
[0276] The V-genes of the murine antibody MAb32 were cloned by
amplifying the hybridoma V-genes, cloning the VH and Vk genes as
scFv fragments in phagemid pHEN1 as above. Antibody scFv fragments
which bind to TNF were identified by ELISA. The mouse VH gene was
recloned in pUC19-pelB-myc for soluble expression as a mouse VH
linked to human mu-CH1, while the light chain was recloned with the
human Ck domain in vector fd-tet-DOG1 as a fusion with g3p. When
cells harbouring the heavy chain construct were infected with the
fd-phage carrying the light chain, phage particles emerged which
carried light chain-g3p associated with the fd heavy chain. Indeed,
binding to TNF and the 301 peptide was retained, as judged by ELISA
with phage displaying the mouse-human chimaeric Fab fragment. In
the phage ELISA, the background signal of phage carrying the light
chain only was a lightly higher than wild-type fd-tet-DOG1 phage,
but always lower than the signal obtained with Fab-displaying
phage. Similarly, TNF binding phage was made with the heavy chain
VHCH1 fragment anchored on phage, and the light chain provided as a
soluble fragment. Hence, MAb32 is functional in the dual
combinatorial format in both display orientations.
[0277] 2 Chain shuffling by Epitope Imprinted Selection (EIS)
Construction of One Chain-Libraries:
[0278] Kappa, lambda light chain and Mu-specific CDNA was made from
the mRNA prepared from the peripheral blood lymphocytes from two
healthy donors essentially as in Marks et al., 1991, supra. The
first-strand cDNA synthesis was performed with oligonucleotides
HCM1FO, HUCLCYS and HUCKCYS for Mu-specific, lambda and kappa
libraries respectively. The VH-CH 1 repertoire was amplified from
this cDNA with oligonucleotides HCM1FO and six family specific
VHBACK primers (as in Marks et al., 1991, supra), reamplified with
a NotI-tagged forward primer (HCMLFONO) and ApaLI tagged VHBACK
primers (6 primers HUVH1BAAPA to HuVH6BAAPA). Similarly, the light
chain repertoires were amplified with HUCLCYS or HUCKCYS forward
primers and HUV.lambda.LBACK to HuV.lambda.6BACK or HuVk1BACK to
HuVk6BACK back primers described in Marks et al., 1991, supra and
PCT/GB91/01134 (WO 92/01047). In each case described in this
section the lambda and kappa chain variable repertoires were
amplified separately. The amplified repertoires were reamplified
with ApaLI and NotI tagged versions of these oligonucleotides (13
back primers HuV.lambda.1BAAPA to Hu.lambda.6BAAPA or HuVk1BAAPA to
HuVkBAAPA and two forward primers HuCLCYSNOT and HuCKCYSNOT,
respectively). All three repertoires were cloned into vector
fd-tet-DOG1 as ApaLI-NotI fragments, and electroporated into E.coli
MC1061 cells, to obtain libraries of 1.0.times.10.sup.7 clones for
V.lambda.CA, 1.4.times.10.sup.6 clones for VkCk, and
5.times.10.sup.6 clones for IgM-derived VHCH1. The presence of
insert was checked and the frequency of inserts in the library
found to be higher than 95% in all three cases.
[0279] Selecting a Human VL Using the Mouse VH Domain as Docking
Chain:
[0280] In a first chain shuffling experiment, the mouse VH (linked
to the human CH1 domain), expressed from pUC19-pelB-myc, was paired
as Fab fragment with a library of 10.sup.7 different human
V.lambda.C.lambda. domains. Phage displaying the antibody fragments
were subjected to rounds of panning on TNF-coated tubes. By
following the titre of the eluted phage, the extent of selection
was monitored. After 4 rounds (with a 100-fold increase in the
titre of eluted phage), 24 out of 28 individual clones were found
to be binding to TNF in an ELISA with phage expressing Fab
fragments (all with the mouse VH-human CH1). Phage only displaying
the selected human V.lambda.C.lambda. domains gave a background
similar to phage displaying only the chimaeric mouse Vk-human Ck.
Sixteen clones taken after the first round of selection were found
to be negative.
[0281] Only three different BstN1 fingerprints were found amongst
the 24 binders, with one pattern dominating (21/24). Light chains
V.lambda.A2, V.lambda.C4 and V.lambda.D1 were found with
frequencies of 21/24, 2/24 and 1/24 respectively. Sequencing
revealed that all three light chains are derived from the same
germline gene, a human V.lambda.1-1-1. Clone V.lambda.C4 has 1,
clone V.lambda.D1 has 2 and clone V.lambda.A2 7 amino-acid residue
differences from the germline. However, clone V.lambda.A2 uses a
framework-l region which more closely resembled the germline
sequence of a related V.lambda.1, humv1117, and therefore may be
the result of a cross-over. The germline character of the clones
was also noted in the CDR3 sequence, with minimal variation in
sequence and no length variation between the three clones.
Apparently, only a very limited number of genes with very similar
sequences fix the stringent requirements (being compatible with the
mouse VH and forming an antigen-binding pair).
[0282] Selecting a Human VH Using the Selected Human VL Domains as
Docking Chains:
[0283] Three selected V.lambda. genes were recloned in
pUC19-pelB-myc for soluble expression as V.lambda.C.lambda. chains.
E.coli cells harbouring the three light chain plasmids were mixed,
infected with a phage library of human VHCH1 genes, expressed from
the fd-tet-DOC1 library described earlier and the library subjected
to rounds of panning on TNF-coated Immuno tubes. Clones were picked
after 5 rounds, when the titre of eluted phage increased 100-fold.
Fifteen out of 20 clones analysed by BstNI fingerprint of the DNA
insert used one of two pattens (with approximately the same
frequency). The 15 clones when combining their heavy chain VHCH1
fragments with the V.lambda.A2 light chain gave stronger phage
ELISA signals than when combined with the V.lambda.C4 or
V.lambda.D1 light chain. Background signals obtained with phage
displaying the heavy chain VHCH1 fragment only were similar to the
signal of the murine VH-human CH1.
[0284] Sequencing revealed that the two patterns could be assigned
to three unique human VH sequences (clones VHP1/2/3, with clone
VHP1 having a BstNI fingerprint which is nearly identical to that
of clone VHP2). Like the selected light chain genes, the selected
heavy chain genes are derived from the same germline VH gene
(germline DP-51 from the VH3 family, Tomlinson et al., J. Mol.
Biol. 227, pp776-798 1992), with minimal residue differences. The
selected human V-genes were aligned to their closest germline
homologue; identical residues in the selected genes are represented
by hyphens. Framework 4 of the V.sub.H genes was truncated at 4th
residue. Clone VHP1 was most likely a cross-over between DP-51 and
a related germline, DP-47. All three selected VH-genes had
relatively short CDR3 loops (8, 9 and 10 residues), but shared
little homology in this sequence.
[0285] Specificity of Binding of the Selected V-Gene Pairs:
[0286] A specificity ELISA with MAb32 and soluble ScFv fragments on
a number of antigens showed that MAb32, its ScFv-derivative and
three of the humanised TNF-binders (as ScFv-fragments) bind
specifically to TNF. No significant binding was obtained to ELISA
plates coated with keyhole limpet haemocyanin, ovalbumin,
cytochrome c. bovine serum albumin, human thyroglobulin, or
2-phenyloxazol-5-one-BSA or to plastic only. Fully humanised clones
were obtained which bound to both peptide 301 and TNF.
[0287] In addition, to show that the human scFv fragments compete
with the original antibody for binding to TNF, the binding of the
scFv constructs in a competition ELISA with the Fab fragment
derived by proteolytic cleavage of MAb32 was analysed. Single chain
Fv fragments were incubated on a TNF-coated surface with increasing
amounts of the Fab fragment and the amount of bound scFv detected
in ELISA. Each of the scFv fragments competed with the FabMAb32 for
binding to TNF, including both the original scFv-MAb32 and the
humanised scFv fragments.
[0288] Thus the fine specificity of MAb32 for peptide 301 of TNF
was retained through the humanisation process.
[0289] Affinity of Binding of the Selected V Gene Pairs:
[0290] MAb32 and purified, monomeric forms of the recombinant mouse
scFv-MAb32 and the human scFv antibodies VHP1-V.lambda.A2.
VHP2-V.lambda.A2 and VHP3-V.lambda.A2, were subjected to
competition ELISA for the determination of the relative affinity
for TNF. Antibodies were incubated on a TNF-coated surface in the
presence of increasing amounts of soluble TNF. All the clones
showed a roughly similar decrease in the ELISA signal over the same
range of increasing TNF concentrations (with an IC50 in the 10 nN
to 100 nM range).
[0291] MAb32 and VHP3V.lambda.A2 fragments were also analysed for
binding properties using the Pharmacia BIAcore. TNF was indirectly
immobilised on the surface, and the binding of antibody monitored.
On the TNF surface, the Fab fragment from MAb32 by proteolytic
cleavage and the scFv Mhb32 showed very similar fast off rates
(approximately 10.sup.-2s.sup.-1). The human VHP3-V.lambda.A2
antibody has an off rate in the same range as the original
scFv-MAb32. On rates for antibody protein interactions were in the
range seen for the interaction between other proteins and their
receptors, and cover a 100 fold range between 10.sup.4 and 10.sup.6
M.sup.-1S.sup.-1 (Mason D. W. and Williams, A. F., 1986, Kinetics
of Antibody Reactions and the Analysis of Cell Surface Antigens,
Blackwell, oxford; Pecht, I., 1992 in Sela, M. (ed), Dynamic
Aspects of Antibody Function, Academic Press Inc., New York, Vol. 6
pp 1-68). Assuming the on rates of the antibody TNF interactions
are typical of antibody protein interactions, the off rate derived
by the BIACore analysis is consistent with the affinity indicated
by the competition ELISA (Kd.congruent.10.sup.-7to 10.sup.-8M).
[0292] Thus, these determinations are consistent with scFvMAb32 and
the humanised scFv clone VHP3-V.lambda.A2 having a similar affinity
and thus with the retention of affinity, as well as specificity,
through epitope imprinted selection.
[0293] Conclusion
[0294] We have shown that a mouse antibody can be rebuilt into a
human antibody with the same specificity by the process of epitope
imprinted selection (EIS).
[0295] A library of human light chains were shuffled with a mouse
VH domain, binding combinations selected and then used in a second
shuffle as "docking domains" for a library of human VH genes.
Completely human antibodies were isolated from such "genuine" human
library. The antibodies were shown to bind retain binding
specificity. Alternatively, the mouse VL was used as docking chain
for selecting human VH partners. Such VH domains can be used to
find human VL genes, or alternatively, can be combined with human
VL domains selected with the mouse VH domain. Indeed, binding
activity was obtained by combining two independently selected
V-genes, pointing towards potential additivity of the EIS
procedure.
[0296] The EIS approach may serve to humanise antibodies more
rapidly than by CDR-grafting (Riechmann et al., 1988, supra), as
this method requires very often a detailed knowledge of the 3-D
structure of the antibody. However, the EIS method can be extended
to for example antibody repertoires obtained by phage selection
from immunised rodents. Following immunisation with antigen, a
repertoire of V-genes with high affinity and specificity may be
selected and then used in an epitope imprinted selection (see
example 4) to generate a range of human antibodies of high affinity
and enriched for the desired specificity.
Enhancement of TNF-Induced Tumour Regression by Antibody
VHP3-V.lambda.A2, the Human Equivalent of MAB 32
[0297] BALB/c mice were inoculated with WEHI-164 tumour cells as
described above. After development of subcutaneous tumours the mice
were treated daily with TNF (1 or 10 .mu.g) alone or in combination
with purified P3A2 (50.mu.) by intraperitoneal injection. Tumour
size was measured throughout the course of the treatment
period.
[0298] Results are shown in FIG. 35.
[0299] VHP3-V.mu.A2 enhanced the anti-tumour activity of TNF at
both the 1 and 10 .mu.g levels.
[0300] Conclusions
[0301] Mapping of the regions recognised by each of the MAbs has
indicated that MAbs in group I (MAbs 1, 21, 47, 54, 37, 32 and 25)
as shown on the schematic diagram bind TNF in the region of
residues 1-18 with the exception of MAbs 37 and 54, while MAbs in
group II of the schematic diagram (MAbs 11, 12, 53 and 42) bind TNF
in the region of residues 70-96 which encompasses a so-called
pallendromic loop on the TNF 3-D structure. MAbs which inhibit the
induction of endothelial cell procoagulant activity (MAbs 1, 32,
42, 47, 54 and 53) all bind in the region of residues 108-128 which
again contains a loop structure in the 3-D model and may indicate
that this region interacts with TNF receptors which are found on
endothelial cells but not tumour cells. MAb 32 which potentiates
the in vivo tumour regression and anti-viral activity of TNF is the
only antibody which binds all the loop regions associated with
residues 1-26, 117-128, and 141-153 and hence binding of these
regions is crucial for enhanced TNF bioactivity with concommittant
reduction of toxicity for normal cells.
[0302] As is apparent from Table 2 MAb 1, 47 and 54 have the same
effect on the bioactivity of TNF. From the results presented above
it is noted that these three monoclonals bind to similar regions of
the TNF molecule. Accordingly, it is believed that a ligand which
binds to TNF in at least two regions selected from the group
consisting predominately of the region of residues 1-20, the region
of residues 56-77, the region of residues 108-128 and the region of
residues 138-149 will effect the bioactivity of TNF in a manner
similar to that of MAbs 1, 47 and 54. Similarly, it is believed
that a ligand which binds to TNF predominately in the regions of
residues 1-20 and 76-90 will have the same effect on the
bioactivity of TNF as MAb 21. A ligand which binds to TNF
predominately in the regions of residues 22-40 and 69-97 will have
the same effect on bioactivity of TNF as MAb 12. A ligand which
binds to TNF predominately in the regions of residues 1-30,
117-128, and 141-153 would be expected to have the same effect on
the bioactivity of TNF as MAb 32 and a ligand which binds to TNF
predominately in the regions of residues 22-40, 49-97, 110-127 and
136-153 would be expected to have the same effect on the
bioactivity of TNF as MAb 42. A ligand which binds to TNF
predominately in the regions of residues 22-31 and 146-157 would be
expected to have the same effect on the bioactivity of TNF as MAb
37 and a ligand which binds to TNF predominately in the regions of
residues 22-40, 69-97, 105-128 and 135-155 would be expected to
have the same effect on the bioactivity of TNF as MAb 53.
[0303] The present inventors have quite clearly shown that the
bioactivity of TNF can be altered by the binding of a ligand to the
TNF, and that the effect on the bioactivity is a function of the
specificity of the ligand. For example, the binding of MAb 32 to
TNF in the regions of residues 1-26, 117-128 and 141-153 results in
the induction of endothelial procoagulant activity of the TNF and
binding of TNF to receptors on endothelial cells being inhibited;
the induction of tumour fibrin deposition and tumour regression
activities of the TNF being enhanced; the cytotoxicity being
unaffected and the tumour receptor binding activities of the TNF
being unaffected or enhanced. It is believed that this effect on
the bioactivity of the TNF may be due to the prevention of the
binding of the epitope of the TNF recognised by MAb 32 to naturally
occurring biologically active ligands. Accordingly, it is believed
that a similar effect to that produced by MAb 32 could also be
produced by a ligand which binds to a region of TNF in a manner
such that the epitope recognised by MAb 32 is prevented from
binding to naturally occurring biologically active ligands. This
prevention of binding may be due to steric hindrance or other
mechanisms.
[0304] Accordingly, it is intended that the prevention of the
binding of epitopes recognised by the various monoclonal antibodies
described herein to naturally occurring biologically active ligands
is within the scope of the present invention.
* * * * *