U.S. patent application number 14/418089 was filed with the patent office on 2015-07-16 for complex-specific antibodies and antibody fragments and its use.
The applicant listed for this patent is MorphoSys AG. Invention is credited to Christian Frisch, Achim Knappik, Haertle Stefan.
Application Number | 20150197579 14/418089 |
Document ID | / |
Family ID | 50101254 |
Filed Date | 2015-07-16 |
United States Patent
Application |
20150197579 |
Kind Code |
A1 |
Stefan; Haertle ; et
al. |
July 16, 2015 |
Complex-specific antibodies and antibody fragments and its use
Abstract
The present invention provides antibodies and fragments thereof
that specifically detect the complex of a specific cognate
antigen-binding moiety, in particular antibodies, and its antigen.
The antibodies of the present disclosure do not bind either said
cognate antigen binding moiety or said antigen alone and thus can
be used e.g. to directly detect antigen-bound antigen-binding
moieties. Further disclosed are methods for use of said antibodies
and antibody fragments.
Inventors: |
Stefan; Haertle;
(Mammendorf, DE) ; Frisch; Christian; (Munich,
DE) ; Knappik; Achim; (Moorenweis, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MorphoSys AG |
Martinsried/Planegg |
|
DE |
|
|
Family ID: |
50101254 |
Appl. No.: |
14/418089 |
Filed: |
August 8, 2013 |
PCT Filed: |
August 8, 2013 |
PCT NO: |
PCT/EP2013/066625 |
371 Date: |
January 29, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61684836 |
Aug 20, 2012 |
|
|
|
Current U.S.
Class: |
506/9 ; 435/7.92;
436/501; 530/387.2 |
Current CPC
Class: |
C07K 16/4241 20130101;
C07K 2317/32 20130101; C07K 16/42 20130101; C07K 2317/30 20130101;
C07K 2317/55 20130101; C07K 16/241 20130101; G01N 33/686 20130101;
C07K 16/243 20130101 |
International
Class: |
C07K 16/42 20060101
C07K016/42; G01N 33/68 20060101 G01N033/68; C07K 16/24 20060101
C07K016/24 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 17, 2012 |
DE |
12180835.6 |
Claims
1. An isolated monoclonal antibody or fragment thereof, which
specifically binds to the complex of a specific cognate antibody
and its antigen.
2. The isolated monoclonal antibody or fragment thereof of claim 1,
which specifically binds to the complex of a specific cognate
antibody and its antigen and does not bind either said specific
cognate antibody or said antigen alone.
3. The isolated monoclonal antibody or fragment thereof of claim 1,
wherein the epitope of said isolated monoclonal antibody or
fragment thereof includes one or more amino acids of a variable
region of the specific cognate antibody.
4. The isolated monoclonal antibody or fragment thereof of claim 1,
wherein said isolated monoclonal antibody or fragment thereof is a
humanized or human monoclonal antibody or fragment.
5. The isolated monoclonal antibody or fragment thereof of claim 1,
wherein said specific cognate antibody is a murine, chimeric,
humanized of human monoclonal antibody or fragment thereof.
6. The isolated monoclonal antibody or fragment thereof of claim 5,
wherein the specific cognate antibody or fragment thereof is
selected from a list that consists but is not limited to
Adalimumab, MOR103, Rituximab, Trastuzumab, Alemtuzumab,
Bevacizumab, Cetuximab, Gemtuzumab, Infliximab, Ranibizumab,
Ustekinumab, Golimumab, Natalizumab, Ofatumumab, Omalizumab,
Panitumumab.
7. The isolated monoclonal antibody or fragment thereof of claim 1,
wherein the antigen is a cytokine or a receptor.
8. Use of an isolated antibody or fragment thereof of claim 1 for
the detection of a complex of a specific cognate antibody and its
antigen in a sample.
9. A method of detecting the complex of a specific cognate antibody
and its antigen in a sample, the method comprising the steps of a)
contacting said sample with an isolated antibody or fragment of
claim 1 and b) detecting said isolated antibody or fragment bound
to said complex.
10. The method of claim 9, wherein said complex is detected using
EIA, ELISA, RIA, indirect competitive immunoassay, direct
competitive immunoassay, noncompetitive immunoassay, sandwich
immunoassay, agglutination assay or MSD.
11. The method of claim 9, wherein said complex of a specific
cognate antibody and its antigen is selected from a group that
consists but is not limited to Adalimumab/TNF-.alpha.,
MOR103/GM-CSF, Trastuzumab/Her2/c-neu, Alemtuzumab/CD52,
Bevacizumab/VEGF-A, Cetuximab/EGF-R, Gemtuzumab/CD33,
Infliximab/TNF-a, Ranibizumab/VEGF-A, Ustekinumab/IL-12,
Ustekinumab/IL-23, Golimumab/TNF-.alpha., Natalizumab/a4-integrin,
Ofatumumab/CD20, Rituximab/CD20, Omalizumab/lgE (Fc region),
Panitumumab/EGFR.
12. A method to identify an isolated monoclonal antibody, or
fragment thereof, of claim 1, said method comprising (a) screening
a library of antibodies or antibody fragments against a complex of
a specific cognate antibody and its antigen in the presence of the
unbound antigen and an antibody that has the same isotype as the
specific cognate antibody, (b) isolating said complex of a specific
cognate antibody and its antigen and the bound antibodies or
antibody fragments, and (c) identifying and isolating said
antibodies or antibody fragments.
13. An isolated monoclonal antibody of fragment thereof obtainable
by the method according to claim 12.
14. A kit comprising one or more antibodies, or fragments thereof,
according to claim 1 and at least one reagent or device necessary
for the detection of a complex of a specific cognate antibody and
its antigen.
15. The kit of claim 14, wherein said one or more antibodies are
selected from the group of antibodies or fragments thereof which
bind to one of the complexes selected from a group that consists
but is not limited to Adalimumab/TNF-a, MOR103/GM-CSF,
Trastuzumab/Her2/c-neu, Alemtuzumab/CD52, BevacizumabA EGF-A,
Cetuximab/EGF-R, Gemtuzumab/CD33, Infliximab TNF-a,
RanibizumabA/EGF-A, Ustekinumab/IL-12, Ustekinumab/IL-23,
Golimumab/TNF-a, Natalizumab/a4-integrin, Ofatumumab/CD20,
Rituximab/CD20, Omalizumab/lgE (Fc region), Panitumumab/EGFR.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. provisional
application Ser. No. 61/684,836 filed Aug. 20, 2012, which is
incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Immunoglobulins, such as antibodies, are of continued and
increasing interest for the pharmaceutical industry. Since 2000,
the therapeutic market for monoclonal antibodies has grown
exponentially and in 2007, eight of the 20 best-selling
biotechnology drugs in the U.S. were therapeutic monoclonal
antibodies each having world wide annual sales of more than 5
billion USD.
[0003] Currently, a significant number of antibodies and also
derivatives and fragments of immunoglobulins are in pre-clinical
and clinical development. Before entry into humans, the drug under
investigation has to be analysed and characterized in extensive
discovery and pre-clinical testing. Important criteria like
toxicological, pharmacokinetic and pharmacodynamic characteristics
need to be explored for the establishment of a safe and potent drug
profile. In order to quantify and monitor the therapeutic antibody
levels, many of these studies require the use of drug-specific
agents for the specific detection of the therapeutic antibody in a
sample matrix like e.g. the sera or any body liquid from a patient
or an experimental animal.
[0004] Drug-specific agents include e.g. antibodies that only
detect human or humanized immunoglobulin and can therefore be used
to quantify a human or humanized therapeutic antibody in a sample
derived from a non-human experimental host (see e.g. WO2006066912;
U.S. Ser. No. 11/792,910 which is incorporated by reference in its
entirety). One step further is the use of anti-idiotypic antibodies
or antibody fragments, which are specific for a unique structure
within the therapeutic antibody. Therefore, anti-idiotypic
antibodies can be used to detect a specific therapeutic antibody or
antibody fragment in a sample matrix irrespective of the host the
sample is isolated from (see e.g. WO2009032128). However, because
the vast majority of anti-idiotypic antibodies bind to one or more
of the unique CDRs of the therapeutic antibody and the CDRs define
the paratope that specifically interacts with the antigen of the
therapeutic antibody, only the detection and monitoring of free,
non antigen-bound therapeutic antibodies are possible.
[0005] US 2012/0157663 describes so-called "domino antibodies"
which have the capacity to bind to an antibody only if the antibody
is bound to the respective antigen. The antibodies of US
2012/0157663 are generated via a specific hybridoma-based screening
technology. Common to all domino antibodies is that the epitope of
the domino antibody on the target antibody is formed through a
conformational change upon binding of the target antibody to its
respective antigen. The epitope is located in the constant region
of the target antibody (e.g. the constant region of the light
chain) and does not include any parts of the antigen nor the CDR
region of the target antibody. In contrast, the complex-specific
antibodies and antibody fragments of the present invention bind to
at least certain parts of the CDR regions of the target antibody.
Therefore, although domino antibodies only recognize target
antibodies when the target antibodies are bound to their respective
antigen, domino antibodies also bind to other target antibodies
with the same antigen specificity, i.e. they are pan-specific in
this respect. In contrast, the antibodies and antibody fragments of
the present invention are specific for one single target antibody,
and they only bind to this target antibody when the target antibody
has bound to its antigen.
[0006] Since a therapeutic antibody that was applied to a patient
is always balanced between different states within the periphery of
the host's body, the monitoring and proportion of these different
states provides mandatory information for the safety of the
therapeutic antibody. These different states are balanced according
to the law of mass action and comprise total antibody, unbound
antibody and bound antibody and said balance is dependent e.g. on
the affinity of the therapeutic antibody and also the concentration
of the antigen in the body. Furthermore, due to the relatively slow
clearance of therapeutic antibodies from the body, the therapeutic
antibody bound to its antigen often leads to an increase of antigen
levels upon its administration for a longer term (Charles P. (1999)
Journal of Immunology 163; 1521-1528). In the presence of the
therapeutic antibody the bound antigen is neutralized and
predominantly is not bioactive. However, this phenomenon must be
monitored and is important e.g. to assess the risk of an abrupt
withdrawal of the drug.
[0007] Taken together the specific detection of total antibody,
unbound antibody and bound antibody is of particular interest and
importance for the profiling and later approval of a therapeutic
antibody (Kuang B. (2010) Bioanalysis, 2(6):1125-40). Only a few
anti-idiotypic antibodies are exemplified which are able to bind to
the unbound therapeutic antibody and are also able to bind to the
complex (therapeutic antibody bound to its antigen) and therefore
are useful to detect the total antibody load. Such a non-paratopic
anti-idiotypic antibody is disclosed in WO2009032128.
[0008] In contrast, almost all anti-idiotypic antibodies are
directed to the CDRs of the target antibody and therefore only
detect unbound antibody (see e.g. Tornetta M. (2007) Journal of
Immunological Methods 328, 34-44).
[0009] However, neither the use of the CDR-specific anti-idiotypic
antibody nor the use of the non-paratopic anti-idiotypic antibody
enables the direct detection and quantification of the drug-antigen
complex only. In order to quantify the bound antibody, various
ELISA-based assays are established but always require the use of
secondary, e.g. anti-human Fc, antibodies for indirect detection.
The use of Fc-specific detection antibodies requires an extra step
and extensive washing to capture and isolate the complex from the
sera immunoglobulin and therefore these assays are susceptible for
background noise and signal variations.
[0010] Accordingly alternative more sensitive and robust approaches
are needed to detect and quantify antibody-antigen complexes.
SUMMARY OF THE INVENTION
[0011] The present invention discloses antibodies and antibody
fragments that specifically detect and bind to the complex of a
cognate antibody and its antigen. The antibodies of the present
disclosure do not bind either said cognate antigen binding moiety
or said antigen alone and thus can be used to directly detect bound
therapeutic antibodies without using secondary Fc-specific
antibodies.
[0012] The present invention also discloses antibodies and antibody
fragments that specifically detect and bind to the complex of a
specific cognate antibody and its antigen. In particular, the
antibodies and antibody fragments of the present invention do not
bind to the complex of other cognate antibodies with the same
antigen specificity.
[0013] These complex-specific antibodies enable superior methods to
quantify antibody-antigen complexes but also free or unbound drug
in samples isolated from human patients or experimental animals.
More sensitive and more robust assays, like e.g. ELISA set-ups, are
disclosed herein and provide alternative and improved assays for
pharmacokinetic studies. Furthermore, the quantification assays
disclosed herein can be used to develop point of care tests using,
for instance, lateral flow techniques to monitor drug levels.
[0014] The present disclosure further discloses the use of said
antibodies in assays for the detection of said complexes.
Furthermore, the present disclosure discloses methods to identify
antibodies that specifically detect the complex of a specific
cognate antibody and its antigen.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 depicts the results of 7 antibodies tested for
specific binding in an ELISA against a series of unrelated and
related antigens and the Adalimumab/TNF-.alpha. complex. 5 .mu.g/mL
of each of the antigens were coated on a microtiter plate over
night. After washing and blocking with 5% BSA,
anti-Adalimumab/TNF-.alpha. antibodies in Fab-FH format (20 .mu.L
from a 2 .mu.g/mL solution) were added. Detection was performed
using an HRP-labeled anti-His antibody and QuantaBlu fluorogenic
peroxidase substrate.
[0016] FIG. 2 depicts the results of a titration of AdaTNF#5 on
different immobilized antigens in ELISA. Over the concentration
range tested (0.03 to 2000 ng/mL), AdaTNb/F#5 bound only to the
Adalimumab/TNF-.alpha. complex but not to its single components and
other antigens.
[0017] FIG. 3 depicts results of purified AdaTNF#5 converted into a
full length human IgG1 tested on various antigens in ELISA.
Purified AdaTNF#5-hIgG1 conjugated to HRP specifically binds
complex of Adalimumab and TNF-.alpha..
[0018] FIG. 4 depicts results of a pharmacokinetic ELISA assay.
Human TNF-.alpha. was coated on a microtiter plate and increasing
concentrations of Adalimumab were spiked into 10% human serum and
were applied to the pre-coated plate. After washing, the
anti-Adalimumab/TNF-.alpha. hIgG1 antibody AdaTNF#5 (conjugated to
HRP) was added at 2 .mu.g/ml. Detection was performed by adding
QuantaBlu.RTM. fluorogenic peroxidase substrate. AdaTNF#5 bound to
Adalimumab/TNF-.alpha. complex in a dose-dependent fashion in the
presence of human serum.
[0019] FIG. 5 depicts results of IFX-TNF#1, IFX-TNF#2 and IFX-TNF#3
tested on various antigens in ELISA. Therefore 5 .mu.g/mL of each
of the antigens were coated on a microtiter plate and
anti-Infliximab/TNF-.alpha. antibodies in Fab-FH format (20 .mu.L
from a 2 .mu.g/mL solution) were added. IFX-TNF#1 proofed to
specifically detect the Infliximab/TNF-.alpha. complex (FIG.
5).
[0020] FIG. 6 depicts results of a screening ELISA to detect
anti-MOR103/GM-CSF complex antibodies. 5 .mu.g/mL of each of the
antigens (BSA, GST, MOR03207 and MOR103) were coated on a
microtiter plate. Furthermore MOR103/GM-CSF complex was immobilized
on the plate as well. M103GmCSF#1, M103GmCSF#2 and M103GmCSF#3
specifically detect the MOR103/GM-CSF complex but not GM-CSF or
MOR103 alone.
[0021] FIG. 7 depicts results of an ELISA testing M103GmCSF#1 for
target selectivity. Either MOR103 alone, the biotinylated GM-CSF or
the biotinylated GM-CSF bound to MOR103 were coated on an
Avidin-coated plate. His-tagged M103GmCSF#1 Fab was added in
increasing concentrations and was detected. M103GmCSF#1 showed high
selectivity for binding to the drug-target complex and not to the
individual proteins (drug and target).
[0022] FIG. 8 depicts results of a MSD.RTM. (Meso Scale Discovery)
based ligand binding assay to quantify MOR103/GM-CSF complexes in
human serum. MOR103/GM-CSF complexes were supplemented with human
serum and titrated on a Multi-array.RTM. 96-well plate Standard
plate. ECL-labeled M103GmCSF#1 IgG was used to detect the
MOR103/GM-CSF complexes. Throughout the titration curve the
MOR103/GM-CSF complexes were specifically detected in the presence
of 50% human sera in a dose-dependent manner.
DETAILED DESCRIPTION OF THE INVENTION
[0023] Accordingly, in one aspect, the disclosure pertains to an
isolated monoclonal antibody or fragment thereof, which
specifically binds to the complex of a specific cognate antigen
binding moiety and its antigen. In one embodiment the isolated
monoclonal antibody or fragment thereof, specifically binds to the
complex of a specific cognate antigen binding moiety and its
antigen and does not bind either said cognate antigen-binding
moiety or said antigen alone.
[0024] Isolated Monoclonal Antibody or Fragment Thereof
[0025] In another aspect, the disclosure pertains to an isolated
monoclonal antibody or fragment thereof wherein the isolated
monoclonal antibody or fragment thereof specifically binds to the
complex of a specific cognate antigen binding moiety and its
antigen with an EC.sub.50 concentration of less than 100 nM, 90 nM,
80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM,
7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM or 1 nM.
[0026] In another aspect, the disclosure pertains to an isolated
monoclonal antibody or fragment thereof wherein the isolated
monoclonal antibody or fragment thereof specifically binds to the
complex of a specific cognate antigen binding moiety and its
antigen with a dissociation constant (K.sub.D) of less than
1.times.10.sup.7 M.sup.-1, 10.sup.8 M.sup.-1, 10.sup.9 M.sup.-1,
10.sup.10 M.sup.-1, 10.sup.11 M.sup.-1, 10.sup.12 M.sup.-1 or
10.sup.13 M.sup.-1.
[0027] In one aspect, the disclosure pertains to an isolated
monoclonal antibody or fragment thereof wherein the isolated
monoclonal antibody or fragment thereof is a monoclonal antibody or
a polyclonal antibody. In one embodiment said isolated antibody or
fragment thereof is a human or humanized antibody. In one
embodiment said isolated antibody or fragment thereof is a chimeric
antibody. In one embodiment said isolated antibody or fragment
thereof comprises a human heavy chain constant region and a human
light chain constant region. In one embodiment said isolated
antibody is an IgG isotype. In another embodiment the antibodies
can be of any isotype (e.g., IgG, IgE, IgM, IgD, and IgA), class
(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or derivative thereof
(e.g. IgG1f LALA). In one embodiment the antibodies are of IgG1f
LALA isotype. In one embodiment said isolated antibody or fragment
thereof is selected from the group consisting of a Fab, F(ab2)',
F(ab)2' and scFV. In one embodiment the isolated antibody is
selected from the group consisting of a monoclonal antibody, a
polyclonal antibody, a chimeric antibody, a humanized antibody, and
a synthetic antibody. In one embodiment, the antibody or fragment
thereof is a human or humanized antibody.
[0028] The Cognate Antigen Binding Moiety
[0029] In one aspect, the disclosure pertains to an isolated
monoclonal antibody or fragment thereof, which specifically binds
to the complex of a cognate antigen binding moiety and its antigen.
In another aspect, the disclosure pertains to an isolated
monoclonal antibody or fragment thereof, which specifically binds
to the complex of a specific cognate antigen binding moiety and its
antigen. In one embodiment said cognate antigen binding moiety, or
said specific cognate antigen binding moiety, is a cognate antibody
or a fragment thereof. In one embodiment said cognate antibody or
fragment thereof, or said specific cognate antibody or fragment
thereof, is a therapeutic antibody or a therapeutic antibody
fragment. In another embodiment said cognate antibody or fragment
thereof, or said specific cognate antibody or fragment thereof, is
a diagnostic antibody or a diagnostic antibody fragment.
[0030] In a preferred embodiment the cognate antibody or fragment
thereof is a specific cognate monoclonal antibody or fragment
thereof. A "specific cognate monoclonal antibody" refers to one,
and only one, monoclonal antibody that specifically binds to its
antigen. The target antibodies of so-called "domino antibodies"
(see US 2012/0157663) are not specific cognate antibodies under
this definition, since domino antibodies bind to the constant
region of their target antibodies and therefore are not specific
for one single antibody, but for all, or at least numerous,
antibodies with a certain target specificity.
[0031] In a preferred embodiment the cognate antibody or fragment
thereof is a cognate monoclonal antibody or fragment thereof.
[0032] In one embodiment said cognate monoclonal antibody or
fragment thereof is a human or humanized antibody. In one
embodiment said cognate monoclonal antibody or fragment thereof is
a chimeric antibody. In one embodiment said cognate monoclonal
antibody or fragment thereof comprises a human heavy chain constant
region and a human light chain constant region. In one embodiment
said cognate monoclonal antibody is an IgG isotype. In another
embodiment the cognate antibodies can be of any isotype (e.g., IgG,
IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1
and IgA2) or derivative thereof (e.g. IgG1f LALA). In one
embodiment the cognate antibodies are of IgG1f LALA isotype.
[0033] In one embodiment said cognate monoclonal antibody or
fragment thereof is selected from the group consisting of a Fab,
F(ab2)', F(ab)2' and scFV. In one embodiment said cognate
monoclonal antibody or fragment thereof is selected from the group
consisting of a monoclonal antibody, a polyclonal antibody, a
chimeric antibody, a humanized antibody, and a synthetic antibody.
In one embodiment, the cognate antibody or fragment thereof is a
human or humanized antibody.
[0034] In one aspect, the disclosure pertains to an isolated
monoclonal antibody or fragment thereof, which specifically binds
to the complex of a cognate antigen binding moiety and its antigen,
or a specific cognate antigen binding moiety and its antigen,
wherein the cognate antigen binding moiety is an antibody-derived
scaffold. In one embodiment the antibody-derived scaffold is
selected from the group consisting of a scFv, a tetravalent
antibody, a cross-linked Fab or a IgG. In one embodiment, the
cognate antibody or fragment thereof is a single chain
antibody.
[0035] In one aspect, the disclosure pertains to an isolated
monoclonal antibody or fragment thereof, which specifically binds
to the complex of a cognate antigen binding moiety and its antigen,
or the complex of a specific cognate antigen binding moiety and its
antigen, wherein the cognate antigen binding moiety is selected
from the group consisting of single domain antibodies, maxibodies,
minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR,
camelid antibodies, ankyrins, domain antibodies, lipocalins, small
modular immuno-pharmaceuticals, maxybodies, Protein A and
affilins.
[0036] In one aspect, the disclosure pertains to an isolated
monoclonal antibody or fragment thereof, which specifically binds
to the complex of a cognate antigen binding moiety and its antigen,
or the complex of a cognate antigen binding moiety and its antigen,
wherein the cognate antigen binding moiety is selected from a list
that consists but is not limited to Adalimumab, MOR103, Rituximab,
Trastuzumab, Alemtuzumab, Bevacizumab, Cetuximab, Gemtuzumab,
Infliximab, Ranibizumab, Ustekinumab, Golimumab, Natalizumab,
Ofatumumab, Omalizumab, Panitumumab.
[0037] The Antigen
[0038] In one aspect, the disclosure pertains to an isolated
monoclonal antibody or fragment thereof, which specifically binds
to the complex of a specific cognate antigen binding moiety and its
antigen wherein the antigen is a protein. In a preferred embodiment
the protein is a human protein. The epitope of the isolated
monoclonal antibody or fragment of the present invention includes
one or more amino acids of a variable region of the specific
cognate antibody. Therefore, in certain aspects the present
disclosure pertains to an isolated monoclonal antibody or fragment
thereof of, wherein the epitope of said isolated monoclonal
antibody or fragment thereof includes one or more amino acids of a
variable region of the specific cognate antibody. In other aspects,
the present disclosure pertains to an isolated monoclonal antibody
or fragment thereof, wherein the epitope of said isolated
monoclonal antibody or fragment thereof includes one or more amino
acids of a variable region of a specific cognate antibody and one
or more amino acids of the antigen of said specific cognate
antibody.
[0039] In other aspects, the present disclosure pertains to an
isolated monoclonal antibody or fragment thereof, wherein the
epitope of said isolated monoclonal antibody or fragment thereof
includes stretches from both, a specific cognate antibody and the
antigen of said specific cognate antibody.
[0040] In one embodiment the protein is associated with a specific
disorder. In one embodiment the protein is a useful target for a
specific biological therapy in a specific disorder. In one
embodiment the protein is a useful target for a specific drug. In
one embodiment the protein is a useful target for a therapeutic
antibody or fragment thereof. In one embodiment the protein is a
useful target for a diagnostic antibody or fragment thereof. In one
embodiment the protein is a cytokine. In one embodiment the protein
is a receptor.
[0041] In one embodiment the protein is associated with an
inflammatory disease, autoimmune disease, viral, bacterial and
parasitic infection, malignancy, neurodegenerative disease or any
tumour-associated disease. In one embodiment the protein is
associated with cancer.
[0042] In one embodiment In one embodiment the protein is selected
from a list that consists but is not limited to TNF-.alpha.,
TNF-.beta., VEGF-A, .alpha.4-integrin, CD20, IgE (Fc region), EGFR,
GM-CSF, CD19, M-CSF, CD38, MIF, DDT, IL-17A, IL-17C, IL-1.alpha.,
IL1-.beta., IL-6, IL-12, IL-23, Her2/c-neu, CD52, CD33.
[0043] In one aspect, the disclosure pertains to an isolated
monoclonal antibody or fragment thereof, which specifically binds
to the complex of a cognate antigen binding moiety and its antigen
wherein the complex is selected from a group that consists but is
not limited to Adalimumab/TNF-.alpha., MOR103/GM-CSF,
Trastuzumab/Her2/c-neu, Alemtuzumab/CD52, BevacizumabNEGF-A,
Cetuximab/EGF-R, Gemtuzumab/CD33, RanibizumabNEGF-A,
Ustekinumab/IL-12, Ustekinumab/IL-23, Golimumab/TNF-.alpha.,
Natalizumab/.alpha.4-integrin, Ofatumumab/CD20, Rituximab/CD20,
Omalizumab/IgE (Fc region), Panitumumab/EGFR.
[0044] Use of the Isolated Monoclonal Antibody or Fragment
Thereof
[0045] In one aspect, the disclosure pertains to the use of an
isolated antibody or fragment thereof for the detection of a
complex of a cognate antigen binding moiety and its antigen, or a
complex of a specific cognate antigen binding moiety and its
antigen, in a sample, wherein said isolated antibody or fragment
thereof specifically binds to the complex of a cognate antigen
binding moiety and its antigen, or the complex of a specific
cognate antigen binding moiety and its antigen, and does not bind
either said cognate antigen binding moiety or said antigen
alone.
[0046] In another aspect, the disclosure pertains to a method of
detecting the complex of a cognate antigen-binding moiety and its
antigen, or the complex of a specific cognate antigen-binding
moiety and its antigen, in a sample using an isolated antibody or
fragment thereof which specifically binds to the complex of a
cognate antigen binding moiety and its antigen, or to the complex
of a cognate antigen binding moiety and its antigen, and does not
bind either said cognate antigen binding moiety or said antigen
alone.
[0047] In one aspect, the disclosure pertains to a method of
detecting the complex of a cognate antigen-binding moiety and its
antigen, or the complex of a specific cognate antigen-binding
moiety and its antigen, in a sample, the method comprising the
steps of [0048] a) contacting said sample with an isolated antibody
or fragment thereof wherein said isolated antibody or fragment
thereof specifically binds said complex and does not bind either
said cognate antigen binding moiety or said antigen alone [0049] b)
detecting said isolated antibody or fragment bound to said
complex.
[0050] In another aspect, the disclosure pertains to a method of
detecting the complex of a cognate antigen-binding moiety and its
antigen, or a specific cognate antigen-binding moiety and its
antigen, in a sample, the method comprising the steps of [0051] a)
contacting said sample with an isolated antibody or fragment
thereof wherein said an isolated antibody or fragment thereof
specifically binds said complex and does not bind either said
cognate antigen binding moiety or said antigen alone [0052] b)
detecting said isolated antibody or fragment bound to said complex,
and [0053] c) correlating said isolated antibody or fragment bound
to said complex with the concentration of the antigen-bound cognate
antigen-binding moiety.
[0054] In another aspect, the disclosure pertains to a method of
detecting the complex of a cognate antibody and its antigen, or the
complex of a specific cognate antibody and its antigen, in a
sample, the method comprising the steps of [0055] a) providing the
sample to be analysed, [0056] b) contacting said sample with an
isolated antibody or fragment thereof wherein said an isolated
antibody or fragment thereof specifically binds said complex and
does not bind either said cognate antibody or said antigen alone,
[0057] c) detecting said isolated antibody or fragment bound to
said complex, and [0058] d) correlating said isolated antibody or
fragment bound to said complex with the concentration of the
antigen-bound cognate antibody.
[0059] In one embodiment said cognate antigen binding moiety is a
cognate antibody or fragment thereof. In a preferred embodiment the
cognate antibody or fragment thereof is a cognate monoclonal
antibody or fragment thereof. In one embodiment said cognate
monoclonal antibody fragment thereof is a human or humanized
antibody. In one embodiment said cognate monoclonal antibody
fragment thereof is a chimeric antibody. In one embodiment said
cognate monoclonal antibody fragment thereof comprises a human
heavy chain constant region and a human light chain constant
region. In one embodiment said cognate monoclonal antibody is an
IgG isotype. In another embodiment the cognate antibodies can be of
any isotype (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1,
IgG2, IgG3, IgG4, IgA1 and IgA2) or derivative thereof (e.g. IgG1f
LALA). In one embodiment the cognate antibodies are of IgG1f LALA
isotype.
[0060] In one embodiment said cognate monoclonal antibody fragment
thereof is selected from the group consisting of a Fab, F(ab2)',
F(ab)2' and scFV. In one embodiment said cognate monoclonal
antibody fragment thereof is selected from the group consisting of
a monoclonal antibody, a polyclonal antibody, a chimeric antibody,
a humanized antibody, and a synthetic antibody. In one embodiment,
the cognate antibody fragment thereof is a human or humanized
antibody.
[0061] In one embodiment said complex of a specific cognate
antibody and its antigen is selected from a group that consists but
is not limited to Adalimumab/TNF-.alpha., MOR103/GM-CSF,
Trastuzumab/Her2/c-neu, Alemtuzumab/CD52, Bevacizumab/VEGF-A,
Cetuximab/EGF-R, Gemtuzumab/CD33, Infliximab/TNF-.alpha.,
Ranibizumab/VEGF-A, Ustekinumab/IL-12, Ustekinumab/IL-23,
Golimumab/TNF-.alpha., Natalizumab/.alpha.4-integrin,
Ofatumumab/CD20, Rituximab/CD20, Omalizumab/IgE (Fc region),
Panitumumab/EGFR.
[0062] In one aspect, the disclosure pertains to a method of
detecting an antigen-binding moiety in a sample, the method
comprising the steps of [0063] a) immobilizing the antigen of the
cognate antigen-binding moiety [0064] b) bringing said immobilized
antigen in contact with said sample [0065] c) detecting the complex
formed between said cognate antigen-binding moiety and its antigen
with an isolated antibody or fragment thereof which specifically
binds to said complex and does not bind either said cognate antigen
binding moiety or said antigen alone.
[0066] In another aspect, the disclosure pertains to a method of
detecting an unbound cognate antigen-binding moiety in a sample,
the method comprising the steps of [0067] a) immobilizing the
antigen of the cognate antigen-binding moiety [0068] b) bringing
said immobilized antigen in contact with said sample [0069] c)
detecting the complex formed between said cognate antigen-binding
moiety and its antigen with an isolated antibody or fragment
thereof which specifically binds to said complex and does not bind
either said cognate antigen binding moiety or said antigen
alone.
[0070] In another aspect, the disclosure pertains to a method of
detecting an unbound cognate antigen-binding moiety in a sample,
the method comprising the steps of [0071] a) immobilizing the
antigen of the cognate antigen-binding moiety [0072] b) bringing
said immobilized antigen in contact with said sample [0073] c)
detecting the complex formed between said cognate antigen-binding
moiety and its antigen with an isolated antibody or fragment
thereof which specifically binds to said complex and does not bind
either said cognate antigen binding moiety or said antigen alone,
and [0074] d) correlating the complex formed in b) with the
concentration of the unbound cognate antigen-binding moiety in the
sample.
[0075] In one embodiment the method, wherein said detection is
accomplished by a means selected from the group consisting of EIA,
ELISA, RIA, indirect competitive immunoassay, direct competitive
immunoassay, non-competitive immunoassay, sandwich immunoassay,
agglutination assay and MSD (Meso Scale Discovery). In a preferred
embodiment said detection is accomplished by a sandwich ELISA. In
another preferred embodiment said detection is accomplished by a
MSD (Meso Scale Discovery) assay.
[0076] In one embodiment the sample is a tissue or a liquid sample.
In a further embodiment the liquid sample is Saliva, urine, whole
blood, plasma or serum. In a preferred embodiment the sample is
obtained from an experimental animal or a human. In a more
preferred embodiment the sample is whole blood, plasma or serum
obtained from a human.
[0077] In one aspect, the disclosure pertains to a method to
identify an isolated monoclonal antibody, or fragment thereof which
specifically binds to the complex of a cognate antigen binding
moiety and its antigen and does not bind either said cognate
antigen binding moiety or said antigen alone, said method
comprising [0078] (a) screening a library of antibodies or antibody
fragments against a complex of a cognate antibody and its antigen,
or a complex of a specific cognate antibody and its antigen, in the
presence of the unbound antigen and an antibody that has the same
isotype as the cognate antibody, [0079] (b) isolating said complex
of a cognate antibody and its antigen, or said complex of a
specific cognate antibody and its antigen, and the bound
antigen-binding moiety, and [0080] (c) identifying and isolating
said antigen-binding moiety.
[0081] In another aspect, the disclosure pertains to a method to
identify an isolated monoclonal antibody, or fragment thereof which
specifically binds to the complex of a cognate antigen binding
moiety and its antigen and does not bind either said cognate
antigen binding moiety or said antigen alone, said method
comprising [0082] (a) screening a library of antibodies or antibody
fragments against a complex of a cognate antibody and its antigen,
or said complex of a specific cognate antibody and its antigen, in
the presence of the unbound antigen and an antibody that has the
same isotype and the same framework as the cognate antibody, [0083]
(b) isolating said complex of a cognate antibody and its antigen,
or said complex of a specific cognate antibody and its antigen, and
the bound antigen-binding moiety, and [0084] (c) identifying and
isolating said antigen-binding moiety.
[0085] In one aspect, the disclosure pertains to a kit comprising
one or more antibodies, or fragments thereof, which specifically
bind to the complex of a cognate antigen binding moiety and its
antigen, or the complex of a specific cognate antigen binding
moiety and its antigen, and do not bind either said cognate antigen
binding moiety or said antigen alone and at least one reagent or
device necessary for the detection of such a complex.
[0086] In another aspect, the disclosure pertains to a kit
comprising one or more antibodies, or fragments thereof, which
specifically bind to the complex of a cognate antigen binding
moiety and its antigen, or the complex of a specific cognate
antigen binding moiety and its antigen, and at least one reagent or
device necessary for the detection of one or more different
complexes of a cognate antigen binding moiety and its antigen of a
specific cognate antigen binding moiety and its antigen. In another
aspect, the disclosure pertains to a kit comprising an antibody or
fragment thereof, which specifically binds to the complex of a
cognate antigen binding moiety and its antigen, or the complex of a
specific cognate antigen binding moiety and its antigen, and at
least one reagent or device necessary for the detection of said
complex. In one embodiment said device is a lateral flow
device.
[0087] In another aspect, the disclosure pertains to a lateral flow
device, comprising one or more antibodies, or fragments thereof,
which specifically bind to the complex of a cognate antigen binding
moiety and its antigen or to the complex of a specific cognate
antigen binding moiety and its antigen. In one embodiment said one
or more antibodies or fragments thereof, specifically bind to the
complex of a cognate antigen binding moiety and its antigen, or to
the complex of a specific cognate antigen binding moiety and its
antigen, and do not bind either said cognate antigen binding moiety
or said antigen alone. In a further embodiment said one or more
antibodies are selected from the group of antibodies or fragments
thereof which bind to one of the complexes selected from a group
that consists but is not limited to Adalimumab/TNF-.alpha.,
MOR103/GM-CSF, Trastuzumab/Her2/c-neu, Alemtuzumab/CD52,
Bevacizumab/VEGF-A, Cetuximab/EGF-R, Gemtuzumab/CD33,
Infliximab/TNF-.alpha., Ranibizumab/VEGF-A, Ustekinumab/IL-12,
Ustekinumab/IL-23, Golimumab/TNF-.alpha.,
Natalizumab/.alpha.4-integrin, Ofatumumab/CD20, Rituximab/CD20,
Omalizumab/IgE (Fc region), Panitumumab/EGFR.
[0088] In another aspect, the disclosure pertains to an isolated
nucleic acid encoding a monoclonal antibody or fragment thereof,
which specifically binds to the complex of a cognate antigen
binding moiety and its antigen.
[0089] In another aspect, the disclosure pertains to a vector
comprising an isolated nucleic acid encoding a monoclonal antibody
or fragment thereof, which specifically binds to the complex of a
cognate antigen binding moiety and its antigen.
[0090] In another aspect, the disclosure pertains to a host cell
comprising a vector comprising an isolated nucleic acid encoding a
monoclonal antibody or fragment thereof, which specifically binds
to the complex of a cognate antigen binding moiety and its antigen.
In one embodiment the host cell is a prokaryotic or eukaryotic host
cell. In a preferred embodiment the host cell is a mammalian host
cell.
[0091] In another aspect, the disclosure pertains to an isolated
monoclonal antibody or fragment thereof that cross-competes with an
antibody described in Table 1. In a certain embodiment, isolated
monoclonal antibody or fragment thereof that cross-competes with an
antibody described in Table 1 and reduces the specific binding of
one of the antibodies described in Table 1 by at least 20%, 30%,
40%, 50%, 60%, 70%, 80% or 90% in an ELISA-based
cross-competition.
[0092] In another aspect, the disclosure pertains to an isolated
monoclonal antibody or fragment thereof that interacts with (e.g.,
by binding, stabilizing, spatial distribution) the same epitope as
an antibody described in Table 1.
[0093] In one aspect, the disclosure pertains to an isolated
monoclonal antibody or fragment thereof comprising 6 CDRs defined
by Kabat of any of the antibodies in Table 1. In another aspect,
the disclosure pertains to an isolated monoclonal antibody or
fragment thereof comprising 6 CDRs defined by Kabat of each of the
antibodies in Table 1.
[0094] In one aspect, the disclosure pertains to an isolated
monoclonal antibody or fragment thereof comprising a VH and a VL of
any of the antibodies in Table 1.
[0095] In another aspect, the disclosure pertains to a nucleic acid
encoding an isolated monoclonal antibody or fragment thereof
wherein the nucleic acid comprises a VH and a VL of any of the
antibodies in Table 1.
[0096] In another aspect, the disclosure pertains to a nucleic acid
encoding an isolated monoclonal antibody or fragment thereof having
at least 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity to
nucleic acids described in Table 1.
DEFINITIONS
[0097] The term "antigen binding moiety", as used herein, refers to
a moiety which comprises a polypeptide that confers the ability to
specifically bind to a given antigen. For example, antibodies,
antibody fragment, antibody derivatives, antibody-like scaffolds
and alternative scaffolds comprise at least one antigen binding
moiety. Antigen binding moieties can also be incorporated into
single domain antibodies, maxibodies, minibodies, intrabodies,
diabodies, triabodies, tetrabodies, v-NAR and scFv (see, e.g.,
Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9,
1126-1136). Further examples of molecules comprising antigen
binding moieties are given herein below and include fibronectin
(Adnexus, fully owned by Bristol-Myers Squibb, Waltham, Mass.),
camelid antibodies, ankyrins (Molecular Partners AG, Zurich,
Switzerland), domain antibodies (Domantis, Ltd., Cambridge, Mass.,
and Ablynx nv, Zwijnaarde, Belgium), lipocalins (Pieris Proteolab
AG, Freising, Germany), small modular immuno-pharmaceuticals
(Trubion Pharmaceuticals Inc., Seattle, Wash.), maxybodies (Avidia,
Inc., Mountain View, Calif.), Protein A (Affibody AG, Sweden), and
affilins (gamma-crystallin or ubiquitin) (Scil Proteins GmbH,
Halle, Germany).
[0098] The term "antigen-binding region" as used herein refers to a
domain of an antigen binding moiety that is responsible for the
specific binding between an antigen binding moiety and an antigen.
For example, the antigen-binding region of an antibody or a
fragment thereof is formed by amino acid residues of the N-terminal
variable regions of the heavy chain (abbreviated herein as VH) and
the light chain (abbreviated herein as VL). The variable regions of
the VH and the VL each comprise three hypervariable regions, termed
complementary determining regions (CDR). The 3 CDRs of the VH and
the 3 CDRs of the VL are three-dimensionally disposed relative to
each other to form an antigen binding surface.
[0099] The term "antibody" as used herein includes whole antibodies
and any fragment or single chains thereof. A naturally occurring
"antibody" is a protein comprising at least two heavy (H) chains
and two light (L) chains inter-connected by disulfide bonds. Each
heavy chain is comprised of a heavy chain variable region
(abbreviated herein as VH) and a heavy chain constant region. The
heavy chain constant region is comprised of three domains, CH1, CH2
and CH3. Each light chain is comprised of a light chain variable
region (abbreviated herein as VL) and a light chain constant
region. The light chain constant region is comprised of one domain,
CL. The VH and VL regions can be further subdivided into regions of
hypervariability, termed complementary determining regions (CDR),
interspersed with regions that are more conserved, termed framework
regions (FR). Each VH and VL is composed of three CDRs and four FRs
arranged from amino-terminus to carboxy-terminus in the following
order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions
of the antibodies may mediate the binding of the immunoglobulin to
host tissues or factors, including various cells of the immune
system (e.g., effector cells) and the first component (C1q) of the
classical complement system. The antibodies can be of any isotype
(e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2,
IgG3, IgG4, IgA1 and IgA2), subclass or modified version thereof
(e.g. IgG1f LALA).
[0100] The term "fragment" of an antibody refers to one or more
fragments of an antibody that retain the ability to specifically
bind to an antigen. Examples of binding fragments encompassed
within the term "fragment" include a Fab fragment, a monovalent
fragment consisting of the VL, VH, CL and CH1 domains; a
F(ab).sub.2 fragment, a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region; an Fd
fragment consisting of the VH and CH1 domains; an Fv fragment
consisting of the VL and VH domains of a single arm of an antibody;
a single domain antibody (dAb) fragment (Ward et al., (1989) Nature
341:544-546), which consists of a VH domain; and an isolated
complementary determining region (CDR) and a single chain Fragment
(scFv) in which the VL and VH regions pair to form monovalent
molecules (known as single chain Fv (scFv); see, e.g., Bird et al.,
(1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl.
Acad. Sci. 85:5879-5883). Although the two domains VL and VH are
coded for by separate genes, they can be joined, using recombinant
methods, by an artificial peptide linker that enables them to be
made as a single protein chain. Such single chain antibodies
include one or more antigen binding moieties. These antibody
fragments are obtained using conventional techniques known to those
of skill in the art, and the fragments are screened for utility in
the same manner as are intact antibodies.
[0101] An "antigen" is defined as any molecule or complex of any
molecules that is bound specifically by an antigen-binding
moiety.
[0102] The term "complex" refers to an association between at least
two moieties (e.g. chemical or biochemical) that have an affinity
for one another. "Protein complex" or "polypeptide complex" refers
to a complex comprising at least one or more polypeptides. As used
herein, a complex comprises of a cognate antigen-binding moiety and
its antigen. In one embodiment the complex is an antibody-antigen
complex. In a preferred embodiment the complex is an
antibody-antigen complex, comprising of a therapeutic antibody and
its antigen
[0103] The term "cognate" refers to components that function
together, or have some aspect of specificity for each other, e.g.,
an orthogonal tRNA and an orthogonal aminoacyl-tRNA synthetase or
an antibody and an antigen. The components can also be referred to
as being complementary. As used herein, a cognate antigen-binding
moiety is an antigen-binding moiety that specifically binds to its
antigen.
[0104] The terms "heavy chain variable region CDR1" and "H-CDR1"
are used interchangeably, as are the terms "heavy chain variable
region CDR2" and "H-CDR2", the terms "heavy chain variable region
CDR3" and "H-CDR3", the terms "light chain variable region CDR1"
and "L-CDR1"; the terms "light chain variable region CDR2" and
"L-CDR2" and the terms "light chain variable region CDR3" and
"L-CDR3"
[0105] The term "human antibody", as used herein, is intended to
include antibodies having variable regions in which both the
framework and CDR regions are derived from sequences of human
origin. As used herein, a human antibody comprises heavy or light
chain variable regions or full length heavy or light chains. In
certain cases, a human antibody may be at least 60%, 70%, 80%, 90%,
or at least 95%, or even at least 96%, 97%, 98%, or 99% identical
in amino acid sequence to the amino acid sequence encoded by the
germline immunoglobulin gene. Thereby said human antibody can be
obtained from technology platforms which comprise antibodies
derived from human germline genes either generated by
PCR-amplification of VH/VL repertoire isolated from B-cells or are
generated synthetically. Technology platforms include library based
approaches comprising human immunoglobulin genes displayed on
phage, ribosome or yeast. Respective display technologies are
standard in the scientific community. Furthermore immunization of a
transgenic mouse carrying human immunoglobulin repertoire is
another approach to generate human antibodies against an antigen of
interest. Antibodies or fragments thereof selected from an antibody
library based on the MorphoSys HuCAL.RTM. concept (Knappik et al.,
(2000) J Mol Biol 296:57-86) are considered as fully human.
[0106] The terms "monoclonal antibody" as used herein refer to a
preparation of antibody molecules of single molecular composition.
A monoclonal antibody composition displays a unique binding site
having a unique binding specificity and affinity for particular
epitopes.
[0107] A "humanized" antibody is an antibody that retains the
reactivity of a non-human antibody while being less immunogenic in
humans. This can be achieved, for instance, by retaining the
non-human CDR regions and replacing the remaining parts of the
antibody with their human counterparts (i.e., the constant region
as well as the framework portions of the variable region). See,
e.g., Morrison et al (1994) Proc. Natl. Acad. Sci. USA,
81:6851-6855; Morrison and Oi (1988) Adv. Immunol., 44:65-92;
Verhoeyen et al. (1988) Science, 239:1534-1536; Padlan, Molec
(1991) Immun., 28:489-498; and Padlan, Molec (1994) Immun.,
31:169-217. Other examples of human engineering technology include,
but are not limited to Xoma technology disclosed in U.S. Pat. No.
5,766,886.
[0108] The term "chimeric antibody" is an antibody molecule in
which (a) the constant region, or a portion thereof, is altered,
replaced or exchanged so that the antigen binding site (variable
region) is linked to a constant region of a different or altered
class, effector function and/or species, or an entirely different
molecule which confers new properties to the chimeric antibody,
e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b)
the variable region, or a portion thereof, is altered, replaced or
exchanged with a variable region having a different or altered
antigen specificity. For example, a mouse antibody can be modified
by replacing its constant region with the constant region from a
human immunoglobulin. Due to the replacement with a human constant
region, the chimeric antibody can retain its specificity in
recognizing the antigen while having reduced antigenicity in human
as compared to the original mouse antibody.
[0109] The term "isolated" refers to a compound which can be e.g.
an antibody or an antigen binding moiety that is substantially free
of other antibodies or antigen binding moieties having different
antigenic specificities. Moreover, an isolated antibody antigen
binding moiety may be substantially free of other cellular material
and/or chemicals.
[0110] The terms "polypeptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymers. Unless otherwise indicated, a particular
polypeptide sequence also implicitly encompasses conservatively
modified variants thereof.
[0111] The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); hepatic
growth factor; fibroblast growth factor; prolactin; placental
lactogen; tumor necrosis factor-.alpha. and -.beta.;
mullerian-inhibiting substance; mouse gonadotropin-associated
peptide; inhibin; activin; vascular endothelial growth factor A-F
(e.g. VEGF-A); integrin (e.g. .alpha.4-integrin; thrombopoietin
(TPO); nerve growth factors such as NGF-.beta.; platelet-growth
factor; transforming growth factors (TGFs) such as TGF-.alpha. and
TGF-.beta.; insulin-like growth factor-I and -II; erythropoietin
(EPO); osteoinductive factors; interferons such as
interferon-.alpha., -.beta., and -.gamma.; colony stimulating
factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-I, IL-I.alpha., IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL17-family members
(e.g. IL-17C); a tumor necrosis factor such as TNF-.alpha. or
TNF-.beta.; and other polypeptide factors including LIF, kit ligand
(KL), MIF, D-DT. As used herein, the term cytokine includes
proteins from natural sources or from recombinant cell culture and
biologically active equivalents of the native sequence
cytokines.
[0112] The term "receptor" is a generic term for proteins having
the ability to affect biological activity, in e.g., a cell, as a
result of interaction with a specific ligand or binding partner.
Cell membrane bound receptors are characterized by an extracellular
ligand-binding domain, one or more membrane spanning or
transmembrane domains, and an intracellular effector domain that is
typically involved in signal transduction. Ligand binding to cell
membrane receptors causes changes in the extracellular domain that
are communicated across the cell membrane, direct or indirect
interaction with one or more intracellular proteins, and alters
cellular properties, such as enzyme activity, cell shape, or gene
expression profile. Receptors may also be untethered to the cell
surface and may be cytosolic, nuclear, or released from the cell
altogether. In general, receptors can be membrane bound, cytosolic
or nuclear; monomeric (e.g., thyroid stimulating hormone receptor,
beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,
growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF I
receptor, erythropoietin receptor and IL-6 receptor). More
particular examples of receptors include, but are not limited to
further clusters of differentiation (e.g. CD20, CD19, CD38, CD52,
CD33), immunoglobulin (e.g. IgE (Fc region)), epidermal growth
factor receptors (e.g. EGFR) or receptor tyrosine kinases (RTK)s
(e.g. Her2/c-neu, Her3, Her4).
[0113] The term "isotype" refers to the antibody class (e.g., IgM,
IgE, IgG such as IgG1 or IgG4) that is provided by the heavy chain
constant region genes. Isotype also includes modified versions of
one of these classes, where modifications have been made to alter
the Fc function, for example, to enhance or reduce effector
functions or binding to Fc receptors. For example IgG1f LALA is a
modified version of the IgG isotype having significantly reduced
effector functions. Specific substitutions of amino acids reduced
the binding affinity for Fc gamma RI receptor as compared with
unmodified antibody. IgG1f LALA is described in U.S. Ser. No.
08/479,752 (SCOTGEN BIOPHARMACEUTICALS INC.) which is incorporated
by reference in its entirety. In certain embodiments of the present
disclosure the antigen-binding moieties of are antibodies and are
of the type IgG, IgM, IgA, IGE or IgD. In specific embodiments the
antibodies are of the type IgG. In certain embodiments of the
present disclosure the antibodies are of the subtype IgG1, IgG2,
IgG3 or IgG4. In specific embodiments the antibodies are of the
subtype IgG1 or IgG4. In other specific embodiments the antibodies
are of the subtype IgG1 or IgG1f LALA.
[0114] The phrase "specifically binds" to an antigen refers to a
binding reaction that is determinable in the presence of an antigen
in a heterogeneous population of proteins and other biologics.
Thereby the phrases "recognizing an antigen" and "specific for an
antigen" are used interchangeably herein with the term "binds
specifically to an antigen". Specific binding of an antigen binding
moiety, like e.g. a monoclonal antibody, to an antigen can be
determined by various established methods known in the art and
include ELISA, FACS, Western Blot, Immuno Blot, MSD, BIAcore and
SET. In the present disclosure an antigen binding moiety is deemed
to be specific for an antigen if the antigen binding moiety is
demonstrated to be able to bind to a specific antigen at least
2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least
6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least
10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at
least 500-fold, at least 1000-fold over background. Thereby the
background is determined by an antigen binding moiety which is
known to be unspecific for the selected antigens or by comparison
to binding to an unrelated antigen.
[0115] "Cross competes" means the ability of an antibody or other
antigen-binding moieties to interfere with the binding of other
antibodies or antigen-binding moieties to a specific antigen in a
standard competitive binding assay. The ability or extent to which
an antibody or other antigen-binding moieties is able to interfere
with the binding of another antibody or antigen-binding moieties to
a specific antigen, and, therefore whether it can be said to
cross-compete according to the invention, can be determined using
standard competition binding assays. One suitable assay involves
the use of the Biacore technology (e.g. by using the BIAcore 3000
instrument (Biacore, Uppsala, Sweden)), which can measure the
extent of interactions using surface plasmon resonance technology.
Another assay for measuring cross-competing uses an ELISA-based
approach. A high throughput process for "epitope binning"
antibodies based upon their cross-competition is described in
International Patent Application No. WO 2003/48731
[0116] The term "epitope" includes any protein determinant capable
of specific binding to an antibody or otherwise interacting with a
molecule. Epitopic determinants generally consist of chemically
active surface groupings of molecules such as amino acids or
carbohydrate or sugar side chains and can have specific
three-dimensional structural characteristics, as well as specific
charge characteristics. An epitope may be "linear" or
"conformational." The term "linear epitope" refers to an epitope
with all of the points of interaction between the protein and the
interacting molecule (such as an antibody) occur linearally along
the primary amino acid sequence of the protein (continuous). The
term "conformational epitope" refers to an epitope in which
discontinuous amino acids that come together in three dimensional
conformation. In a conformational epitope, the points of
interaction occur across amino acid residues on the protein that
are separated from one another.
[0117] "Binds the same epitope as" means the ability of an antibody
or other antigen-binding moiety to bind to a specific antigen and
having the same epitope as the exemplified antibody. The epitopes
of the exemplified antibody and other antibodies can be determined
using epitope mapping techniques. Epitope mapping techniques are
well known in the art. For example, conformational epitopes are
readily identified by determining spatial conformation of amino
acids such as by, e.g., hydrogen/deuterium exchange, x-ray
crystallography and two-dimensional nuclear magnetic resonance.
[0118] The term "affinity" as used herein refers to the strength of
interaction between an antigen binding moiety, like e.g. a
monoclonal antibody and an antigen at single antigenic sites.
Within each antigenic site, the variable region of the antibody
"arm" interacts through weak non-covalent forces with antigen at
numerous sites; the more interactions, the stronger the
affinity.
[0119] The term "KD", as used herein, refers to the dissociation
constant, which is obtained from the ratio of Kd to Ka (i.e. Kd/Ka)
and is expressed as a molar concentration (M). KD values for
antigen binding moieties like e.g. monoclonal antibodies can be
determined using methods well established in the art. Methods for
determining the KD of an antigen binding moiety like e.g. a
monoclonal antibody are SET (soluble equilibrium titration) or
surface plasmon resonance using a biosensor system such as a
Biacore.RTM. system. In the present disclosure an antigen-binding
moiety typically has a dissociation rate constant (KD) (koff/kon)
of less than 5.times.10.sup.-2M, less than 10.sup.-2M, less than
5.times.10.sup.-3M, less than 10.sup.-3M, less than
5.times.10.sup.-4M, less than 10.sup.-4M, less than
5.times.10.sup.-5M, less than 10.sup.-5M, less than
5.times.10.sup.-6M, less than 10.sup.-6M, less than
5.times.10.sup.-7M, less than 10.sup.-7M, less than
5.times.10.sup.-8M, less than 10.sup.-5M, less than
5.times.10.sup.-9M, less than 10.sup.-9M, less than
5.times.10.sup.-10M, less than 10.sup.-10M, less than
5.times.10.sup.-11M, less than 10.sup.11M, less than
5.times.10.sup.-12M, less than 10.sup.-12M, less than
5.times.10.sup.-13M, less than 10.sup.-13M, less than
5.times.10.sup.-14M, less than 10.sup.-14M, less than
5.times.10.sup.-15M, or less than 10.sup.-15M or lower.
[0120] A "disorder" is any condition that would benefit from
medical treatment by using e.g. a therapeutic antibody or other
antigen-binding moieties. Non-limiting examples of disorders
include autoimmune disease, inflammation, cell proliferative
disorders; B cell lymphomas, non-leukemias and lymphoid
malignancies; neuronal, glial, astrocytal, hypothalamic and other
glandular, macrophagal, epithelial, stromal and blastocoelic
disorders; and inflammatory, immunologic, or infectious diseases.
The terms "cell proliferative disorder" and "proliferative
disorder" refer to disorders that are associated with some degree
of abnormal cell proliferation. In one embodiment, the disorder is
cancer.
[0121] As used herein, the term "autoimmune disease" refers
generally to diseases which are characterized as having a component
of self-recognition. Examples of autoimmune diseases include, but
are not limited to, Autoimmune hepatitis, Multiple Sclerosis,
Systemic Lupus Erythematosus, Idiopathic Thrombocytopenic Purpura,
Myasthenia Gravis, Type I diabetes, Rheumatoid Arthritis,
Psoriasis, Hashimoto's Thyroiditis, Grave's disease, Ankylosing
Spondylitis Sjogrens Disease, CREST syndrome, Scleroderma, IgA
Neprhopathy, Bullous Pemphigoid, Pemphigous Vulgaris,
ANCA-Associated Vasculitis, Antiphospholipid Syndrome and many
more. Most autoimmune diseases are also chronic inflammatory
diseases. This is defined as a disease process associated with
long-term (>6 months) activation of inflammatory cells
(leukocytes). The chronic inflammation leads to damage of patient
organs or tissues. Many diseases are chronic inflammatory
disorders, but are not known to have an autoimmune basis. For
example, Atherosclerosis, Congestive Heart Failure, Crohn's
disease, Ulcerative Colitis, Polyarteritis nodosa, Whipple's
Disease, Primary Sclerosing Cholangitis and many more.
[0122] The term "cancer" refers to the physiological condition in
mammals that is typically characterized by unregulated cell
growth/proliferation. Examples of cancer include, but are not
limited to: carcinoma, lymphoma, blastoma, and leukemia. More
particular examples of cancers include, but are not limited to:
colorectal cancer, chronic lymphocytic leukemia (CLL), lung,
including non small cell (NSCLC), breast, ovarian, cervical,
endometrial, prostate, colorectal, intestinal carcinoid, bladder,
gastric, pancreatic, hepatic (hepatocellular), hepatoblastoma,
esophageal, pulmonary adenocarcinoma, mesothelioma, synovial
sarcoma, osteosarcoma, head and neck squamous cell carcinoma,
juvenile nasopharyngeal angiofibromas, liposarcoma, thyroid,
melanoma, basal cell carcinoma (BCC), medulloblastoma and desmoid.
Cancers of particular interest for treatment by the subject methods
include gliomas, medulloblastomas, colon cancer, colorectal cancer,
melanoma, breast cancer, lung cancer, liver cancer, and gastric
cancer.
[0123] The term "therapeutic antibody" relates to any antibody
preparation which is intended for use in a human being. Preferably
such therapeutic antibody will be a monoclonal antibody. Further
preferred such monoclonal antibody will be obtained from a great
ape or be a human monoclonal antibody. Preferably, it will be a
human monoclonal antibody. Also preferred such therapeutic
monoclonal antibody will be a humanized monoclonal antibody.
Therapeutic antibodies are being used widely for the treatment of
various disorders such as oncological diseases (e.g. hematological
and solid malignancies including non-Hodgkin's lymphoma, breast
cancer, and colorectal cancer), immunological diseases, central
nervous diseases, vascular diseases, or infectious diseases. Such
antibodies are, in one embodiment antibodies against TNF-.alpha.,
TNF-.beta., VEGF-A, .alpha.4-integrin, CD20, IgE (Fc region), EGFR,
GM-CSF, CD19, M CSF, CD38, MIF, DDT, IL-17C, IL-12, Her2/c-neu,
CD52, CD33. Such antibodies are e.g. Adalimumab, MOR103, Rituximab,
Trastuzumab, Alemtuzumab, Bevacizumab, Cetuximab, Gemtuzumab,
Infliximab, Ranibizumab, Ustekinumab, Golimumab, Natalizumab,
Ofatumumab, Omalizumab, and Panitumumab.
[0124] The term "sample" as used within this application denotes,
but is not limited to, any quantity of a substance from a living
thing or formerly living thing. Such living things include, but are
not limited to, humans, mice, monkeys, rats, rabbits, and other
animals. Such substances include, but are not limited to, Saliva,
urine, whole blood, serum or plasma from an individual. The most
widely used sources of sample in clinical routine are whole blood,
plasma or serum. In a preferred embodiment the sample was isolated
from a patient. In more preferred embodiment the sample was
isolated from a human.
[0125] The term "patient" as used herein denotes a mammal.
Preferably, a patient according to the invention is a human.
[0126] As used herein, the term "bound" refers to binding or
attachment that may be covalent, e.g., by chemically coupling, or
non-covalent, e.g., ionic interactions, hydrophobic interactions,
hydrogen bonds, etc. In a preferred embodiment the binding or
attachment is a non-covalent interaction. In one example the term
bound refers to the attachment of a cognate antigen binding moiety
to its antigen
[0127] The term "antigen-bound" antigen-binding moiety is used to
indicate the antigen-binding moiety as present in the circulation
of an experimental animal or a patient that is bound to its
antigen. In a further embodiment the antigen-bound antigen-binding
moiety is an antibody. In a further embodiment the antigen-bound
antigen-binding moiety is a therapeutic antibody.
[0128] The term "unbound" antigen-binding moiety is used to
indicate the antigen-binding moiety as present in the circulation
of an experimental animal or a patient that is not bound to its
antigen. In a further embodiment the antigen-binding moiety is an
antibody or fragment thereof. In a further embodiment the
antigen-binding moiety is a therapeutic antibody.
[0129] The term "collection" or "library" means at least two
members. The term "member" includes, but is not limited to nucleic
acids encoding antibodies or fragments thereof or the antibodies or
fragments thereof themselves.
[0130] The term "library" refers to a set of entities comprising
two or more entities having diversity as described herein. For
example, a "library of antibodies or antibody fragments" refers to
a set of polynucleotides comprising two or more polynucleotides
encoding antibodies or antibody fragments and having diversity as
described herein. For example the commercially available phage
display libraries, like e.g. the MorphoSys HuCAL PLATINUM.RTM.
library can be used.
[0131] As used herein, the term "diversity" refers to a variety or
a noticeable heterogeneity.
[0132] The term "framework" means an antibody variable domain as
defined by Kabat et al. (1991) as the part of the variable domain
which serves as a scaffold for the antigen binding loops of this
variable domain. Examples of the framework regions include FR1,
FR2, FR3, and FR4 of either the variable heavy or variable light
chains
[0133] The term "isotype" refers to the antibody class (e.g., IgM,
IgE, IgG such as IgG1 or IgG4) that is provided by the heavy chain
constant region genes. Isotype also includes modified versions of
one of these classes, where modifications have been made to alter
the Fc function, for example, to enhance or reduce effector
functions or binding to Fc receptors.
[0134] By an "anti-idiotypic antibody" is meant an antibody that
specifically binds to the antigen-binding region of another
antibody and, therefore, is specifically bound by the other
antibody. The anti-idiotype antibody can mimic the epitope normally
recognized by another antibody. An idiotype is the genetically
determined variation of structures in the variable regions of
immunoglobulins. The precise genetic basis of idiotype variability
has only been partially explained. However, idiotype variation
involves the amino acid sequence and protein structure (so-called
determinants) especially in the area of the antigen-binding region,
also referred to as the idiotope. The term "idiotype" designates
the complete set of determinants of a variable region of an
antibody molecule.
[0135] The term "amino acid" refers to naturally occurring and also
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refer to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an alpha carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0136] The term "nucleic acid" is used herein interchangeably with
the term "polynucleotide" and refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form. The term encompasses nucleic acids containing
known nucleotide analogs or modified backbone residues or linkages,
which are synthetic, naturally occurring, and non-naturally
occurring, which have similar binding properties as the reference
nucleic acid, and which are metabolized in a manner similar to the
reference nucleotides. Examples of such analogs include, without
limitation, phosphorothioates, phosphoramidates, methyl
phosphonates, chiral-methyl phosphonates, 2-O-methyl
ribonucleotides, peptide-nucleic acids (PNAs). Unless otherwise
indicated, a particular nucleic acid sequence also implicitly
encompasses conservatively modified variants thereof (e.g.,
degenerate codon substitutions) and complementary sequences, as
well as the sequence explicitly indicated. Specifically, as
detailed below, degenerate codon substitutions may be achieved by
generating sequences in which the third position of one or more
selected (or all) codons is substituted with mixed-base and/or
deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res.
19:5081; Ohtsuka et al. (1985) J. Biol. Chem. 260:2605-2608; and
Rossolini et al. (1994) Mol. Cell. Probes 8:91-98).
[0137] The term "recombinant host cell" (or simply "host cell")
refers to a cell into which a recombinant expression vector has
been introduced. It should be understood that such terms are
intended to refer not only to the particular subject cell but to
the progeny of such a cell. Because certain modifications may occur
in succeeding generations due to either mutation or environmental
influences, such progeny may not, in fact, be identical to the
parent cell, but are still included within the scope of the term
"host cell" as used herein.
[0138] The term "vector", as used herein, refers to a molecular
vehicle used to transfer foreign genetic material into another
cell. The vector itself is generally a DNA sequence that consists
of an insert (sequence of interest) and a larger sequence that
serves as the "backbone" of the vector. The purpose of a vector to
transfer genetic information to another cell is typically to
isolate, multiply, or express the insert in the target cell.
[0139] As used herein the term "lateral flow" refers to liquid flow
along the plane of a substrate or carrier, e.g., a lateral flow
membrane. In general, lateral flow devices comprise a strip (or a
plurality of strips in fluid communication) of material capable of
transporting a solution by capillary action, i.e., a wicking or
chromatographic action, wherein different areas or zones in the
strip(s) contain assay reagents, which are either diffusively or
non-diffusively bound to the substrate, that produce a detectable
signal as the solution is transported to or migrates through such
zones. Typically, such assays comprise an application zone adapted
to receive a liquid sample, a reagent zone spaced laterally from
and in fluid communication with the application zone, and a
detection zone spaced laterally from and in fluid communication
with the reagent zone. The reagent zone can comprise a compound
(e.g. an antibody) that is mobile in the liquid and capable of
interacting with an analyte in the sample, e.g., to form an
analyte-reagent complex, and/or with a molecule bound in the
detection zone. The detection zone may comprise a binding molecule
(e.g. an antibody) that is immobilized on the strip and is capable
of interacting with the analyte and/or the reagent and/or an
analyte-reagent complex to produce a detectable signal. Such assays
can be used to detect an analyte in a sample through direct
(sandwich assay) or competitive binding. Examples of lateral flow
devices are provided in U.S. Pat. No. 6,194,220, U.S. Pat. No.
5,998,221 and U.S. Pat. No. 5,798,273.
TABLE-US-00001 TABLE 1 Sequences ANTIBODY-ID/ SEQ-ID NUMBER REGION
SEQUENCE AdaTNF#1 VH1A VL.kappa.3 SEQ ID NO: 1 HCDR1 GGTFSTYAIS
(Kabat) SEQ ID NO: 2 HCDR2 WMGGIIPIFGTANYAQKFQG (Kabat) SEQ ID NO:
3 HCDR3 DYFSSIGWVVYYGPMDY (Kabat) SEQ ID NO: 4 LCDR1 RASQSVSSPYLA
(Kabat) SEQ ID NO: 5 LCDR2 LLIYDVSSRAT (Kabat) SEQ ID NO: 6 LCDR3
QQYTSTPP (Kabat) SEQ ID NO: 7 VL
DIVLTQSPATLSLSPGERATLSCRASQSVSSPYLAWYQQ
KPGQAPRLLIYDVSSRATGIPARFSGSGSGTDFTLTISSL
EPEDFAVYYCQQYTSTPPTFGQGTKVEIKRT SEQ ID NO: 8 VH
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYAISWVR
QAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTS
TAYMELSSLRSEDTAVYYCARDYFSSIGWVVYYGPMDY WGQGTLVTVSS SEQ ID NO: 9 DNA
VL gatatcgtgctgacccagagcccggcgaccctgagcctgag
cccgggtgaacgtgccaccctgagctgcagagcgagccagt
ctgtttcttctccgtacctggcttggtaccagcagaaaccg
ggccaggccccgcgtctattaatctacgacgtttcttctcg
tgcgaccggcattccggcgcgttttagcggcagcggatccg
gcaccgatttcaccctgaccattagcagcctggaaccggaa
gactttgcggtgtattattgccagcagtacacttctactcc
gccgacctttggccagggcacgaaagttgaaattaaacgta cg SEQ ID NO: 10 DNA VH
caggtgcaattggtgcagagcggtgccgaagtgaaaaaacc
gggcagcagcgtgaaagttagctgcaaagcatccggaggga
cgttttctacttacgctatctcttgggtgcgccaggccccg
ggccagggcctcgagtggatgggcggtatcatcccgatctt
cggcactgcgaactacgcccagaaatttcagggccgggtga
ccattaccgccgatgaaagcaccagcaccgcctatatggaa
ctgagcagcctgcgcagcgaagatacggccgtgtattattg
cgcgcgtgactacttctcttctatcggttgggttgtttact
acggtccgatggattactggggccaaggcaccctggtgact gttagctca AdaTNF#5 VH1A
VL.kappa.1 SEQ ID NO: 11 HCDR1 GGTFSTNAIS (Kabat) SEQ ID NO: 12
HCDR2 WMGGINPHLGHADYAQKFQG (Kabat) SEQ ID NO: 13 HCDR3
GWYYIGSNPSMYPNYFDP (Kabat) SEQ ID NO: 14 LCDR1 RASQTISSYLN (Kabat)
SEQ ID NO: 15 LCDR2 LLIYTASNLQS (Kabat) SEQ ID NO: 16 LCDR3
QQVLHLPH (Kabat) SEQ ID NO: 17 VL
DIQMTQSPSSLSASVGDRVTITCRASQTISSYLNWYQQK
PGKAPKLLIYTASNLQSGVPSRFSGSGSGTDFTLTISSLQ
PEDFATYYCQQVLHLPHTFGQGTKVEIKRT SEQ ID NO: 18 VH
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSTNAISWVR
QAPGQGLEWMGGINPHLGHADYAQKFQGRVTITADEST
STAYMELSSLRSEDTAVYYCARGWYYIGSNPSMYPNYF DPWGQGTLVTVSS SEQ ID NO: 19
DNA VL gatatccagatgacccagagcccgagcagcctgagcgccag
cgtgggcgatcgcgtgaccattacctgcagagccagccaga
ctatttcttcttacctgaactggtaccagcagaaaccgggc
aaagcgccgaaactattaatctacactgcttctaacctgca
aagcggcgtgccgagccgctttagcggcagcggatccggca
ccgatttcaccctgaccattagctctctgcaaccggaagac
tttgcgacctattattgccagcaggttctgcatctgccgca
tacctttggccagggcacgaaagttgaaattaaacgtacg SEQ ID NO: 20 DNA VH
Caggtgcaattggtgcagagcggtgccgaagtgaaaaaacc
gggcagcagcgtgaaagttagctgcaaagcatccggaggga
cgttttctactaacgctatctcttgggtgcgccaggccccg
ggccagggcctcgagtggatgggcggtatcaacccgcatct
gggccatgcggactacgcccagaaatttcagggccgggtga
ccattaccgccgatgaaagcaccagcaccgcctatatggaa
ctgagcagcctgcgcagcgaagatacggccgtgtattattg
cgcgcgtggttggtactacatcggttctaacccgtctatgt
acccgaactacttcgatccgtggggccaaggcaccctggtg actgttagctca IFX-TNF#1
VH3-23 VL.kappa.3 SEQ ID NO: 21 HCDR1 GFTFSSYGMH (Kabat) SEQ ID NO:
22 HCDR2 WVSYIYYGGSDTYYADSVKG (Kabat) SEQ ID NO: 23 HCDR3
GMYYLYDQPAFDY (Kabat) SEQ ID NO: 24 LCDR1 SGDNIRSDYVH (Kabat) SEQ
ID NO: 25 LCDR2 LVIYDKSERPS (Ka bat) SEQ ID NO: 26 LCDR3 QAADTWSTIV
(Kabat) SEQ ID NO: 27 VL DIELTQPPSVSVAPGQTARISCSGDSLGDYYVHWYQQK
PGQAPVLVIYADNNRPSGIPERFSGSNSGNTATLTISGT
QAEDEADYYCQTYDDRSSPVFGGGTKLTVLGQ SEQ ID NO: 28 VH
QVQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMSWV
RQAPGKGLEWVSGIGSYTYYADSVKGRFTISRDNSKNT
LYLQMNSLRAEDTAVYYCARLSQTGVMDYWGQGTLVT VSS SEQ ID NO: 29 DNA VL
gatatcgaactgacccagccgccttcagtgagcgttgcacc
aggtcagaccgcgcgtatctcgtgtagcggcgattctcttg
gtgattattatgttcattggtaccagcagaaacccgggcag
gcgccagttcttgtgatttatgctgataataatcgtccctc
aggcatcccggaacgctttagcggatccaacagcggcaaca
ccgcgaccctgaccattagcggcactcaggcggaagacgaa
gcggattattattgccagacttatgatgatcgttcttctcc
tgtgtttggcggcggcacgaagttaaccgtcctaggtcag SEQ ID NO: 30 DNA VH
caggtgcaattggtggaaagcggcggcggcctggtgcaacc
gggcggcagcctgcgtctgagctgcgcggcctccggattta
cctttaattcttatgctatgtcttgggtgcgccaagcccct
gggaagggtctcgagtgggtgagcggtatcggtagctatac
ctattatgcggatagcgtgaaaggccgttttaccatttcac
gtgataattcgaaaaacaccctgtatctgcaaatgaacagc
ctgcgtgcggaagatacggccgtgtattattgcgcgcgtct
ttctcagactggtgttatggattattggggccaaggcaccc tggtgacggttagctca
EXAMPLES
Generation of Fab Fragments and Antibodies that are Specific for an
Antibody/Antigen Complex
[0140] For the selection of antibodies specifically recognizing the
antibody/antigen complex a commercially available phage display
library, the MorphoSys HuCAL PLATINUM.RTM. library was used. Said
antibody library is based on the HuCAL.RTM. concept (Knappik et
al., (2000) J Mol Biol 296:57-86) and employs the CysDisplay.RTM.
technology for displaying the Fab on the phage surface
(WO2001/05950 to Lohning). However, any other available antibody
library would be suitable to identify complex-specific
antibodies.
[0141] To identify antibody/antigen complex antibodies specific
panning strategies had been developed to target antibody/antigen
complexes. Thereby recombinant, purified antigen and its respective
recombinant therapeutic antibody were used for the panning.
According to the described examples below, for 3 different
antibody/antigen complexes, antibodies that only bind to that
specific complex were identified. All described panning strategies
and antigens were used for the antibody selection process. Each
panning strategy comprised of at least 3 individual rounds of
panning and contained unique antigens, antigen concentrations and
washing stringency.
Example 1
Generation and Characterization of Fab Fragments and Antibodies
that are Specific for the Adalimumab/TNF-.alpha. Complex
[0142] To identify antibodies which specifically bind to the
Adalimumab/TNF-.alpha. complex recombinant TNF-.alpha. (BioLegend
570108, Lot B137143) coupled to magnetic beads and Adalimumab were
used as antigens for a solution panning approach.
[0143] a) Panning
[0144] For the solution panning TNF-.alpha. was coupled covalently
to the Epoxy M-450 magnetic beads (Dynabeads M-450, Dynal) and the
phage preparation of a phage display antibody library are washed
and blocked with Chemiblocker (Chemicon).
[0145] To provide Adalimumab/TNF-.alpha. complexes the recombinant
TNF-.alpha. which was coupled to magnetic beads was pre-incubated
with Adalimumab. For the first panning round, in the presence of
Chemiblocker (Chemicon) and Tween/PBS, phage-antibodies were added
to the Adalimumab/TNF-.alpha. complexes in the presence of purified
human IgG1kappa (50 .mu.g/mL), 10% human serum, 50 .mu.g/ml
Rituximab (IgG1kappa) and 1 .mu.g/ml TNF-.alpha. to adsorb all
phage-antibodies which are specific to IgG1kappa and free
TNF-.alpha. or that cross-react with any components of human serum.
After incubation on a rotator over night at 2 to 8.degree. C. the
phage-antigen mixture was transferred to tubes and the magnetic
beads were captured using a magnetic separator. The supernatant was
carefully removed from the beads and remaining beads were washed
PBST.
[0146] Subsequent panning round 2 and 3 were performed in a similar
fashion with increasing concentrations of free TNF-.alpha. (5
.mu.g/ml in 2nd panning round; 25 .mu.g/ml in 3rd panning round) to
increase stringency and discard antibodies which cross-react to
free TNF-.alpha..
[0147] Upon each round of panning the remaining phages were eluted,
and eluted phages were used immediately for infection of E. coli
TG1 bacteria. After rescue of the phages by using helper phage the
polyclonal amplified phage output was titered again and used in
consecutive selection steps. After the 3rd round of panning the DNA
of the eluted antigen-specific phages was isolated from the
infected bacteria and the Fab-encoding DNA was subcloned via PCR
into specific Fab expression vectors. After transformation of TG1-F
bacteria, using the Fab-encoding vectors, single clone expression
and preparation of periplasmic extracts containing HuCAL-Fab
fragments were performed. Fab-containing periplasmic extracts were
used for the initial screening and characterization.
[0148] For further characterization purified Fabs had been used.
Expression of Fab fragments in TG-1 cells was carried out in shaker
flask cultures using 500 ml of 2.times. YT medium supplemented with
1 mM chloramphenicol and 0.1% glucose. Expression was induced by
addition of 0.75 mM IPTG for 20 h at 30.degree. C. Cells were
disrupted using a lysis buffer containing lysozyme, Bugbuster and
Benzonase and Fab fragments isolated by Ni-NTA chromatography
(Bio-Rad, Germany). Protein concentrations were determined by
UV-spectrophotometry. Purity of Fab fragments was analyzed in
denatured, reduced state using SDS-PAGE and in native state by
HP-SEC.
[0149] In order to express full length IgGs, variable domain
fragments of heavy (VH) and light chains (VL) were subcloned from
Fab expression vectors into appropriate pMORPH.RTM._hIg vectors for
human IgG2, human IgG4, human IgG4_Pro, and human IgG1f LALA.
[0150] b) Screening
[0151] Primary screening was done by ELISA. 368 clones were
randomly picked from the output of the above described panning
procedure and grown in the 384 well ELISA plates. After induction
of antibody expression (0.75 mM IPTG for 20 h at 30.degree. C.) and
lysis of the cells by using lysozyme, the cell lysates containing
the antibodies were tested in ELISA.
[0152] Therefore the following antigens, Adalimumab/TNF-.alpha.
complex, purified human IgG1/kappa from myeloma cell line;
Rituximab and free recombinant TNF-.alpha. were coated on an ELISA
plate.
[0153] Altogether 12 clones that were positive (signal at least
5-fold over background) on the complex but did not bind to the
other antigens were identified. Thereupon the 12 clones were
sequenced to identify unique antibodies. Seven unique sequences
could be identified. These 7 clones were expressed and purified and
were then characterized.
[0154] c) Characterization
[0155] First the 7 antibodies were tested for specific binding in
an ELISA against a series of unrelated and related antigens and the
Adalimumab/TNF-.alpha. complex, wherein either Adalimumab was
coated on the plate and TNF-.alpha. was subjected for the formation
of the complex or the other way around. Therefore 5 .mu.g/mL of
each of the antigens were coated on a microtiter plate over night.
After washing and blocking with 5% BSA, anti-Adalimumab/TNF-.alpha.
antibodies in Fab-FH format (20 .mu.L from a 2 .mu.g/mL solution)
were added. Detection was performed using an HRP-labeled anti-His
antibody and QuantaBlu fluorogenic peroxidase substrate. AdaTNF#1
and AdaTNF#5 proofed to be highly specific for the complex (FIG.
1).
[0156] In a next step, AdaTNF#5 was tested in more detail by
titrating the antibody on different immobilized antigens. Over the
concentration range tested (0.03 to 2000 ng/mL), the antibody bound
only to the Adalimumab/TNF-.alpha. complex but not to an
infliximab/TNF-.alpha. complex, not to unbound Adalimumab or free
TNF-.alpha., and not to other antigens (FIG. 2)
[0157] AdaTNF#5 was converted into a full length human IgG1 format,
expressed in a human cell line and purified via protein A
chromatography for further analysis. First it was tested whether
the antibody in (bivalent) IgG1 format still shows the same
specificity. Antigens were coated at 5 .mu.g/mL on a microtiter
plate over night. After washing and blocking with 5% BSA,
HRP-conjugated AdaTNF#5-hIgG1 (20 .mu.L from a 2 .mu.g/mL solution
in HiSpec buffer) was added. Detection was performed using the
QuantaBlu.RTM. fluorogenic peroxidase substrate. Purified
AdaTNF#5-hIgG1 conjugated to HRP specifically binds complex of
Adalimumab and TNF-.alpha.. (FIG. 3)
[0158] For further characterization, the monovalent intrinsic
affinity of AdaTNF#5 was measured as kD=67 nM by real time, label
free molecular interaction analysis using an Attana A200 instrument
on an immobilized Adalimumab-TNF-.alpha. complex. It was then
tested whether the complex specific antibody AdaTNF#5 can be used
to determine Adalimumab spiked in human serum. Human TNF-.alpha.
was coated at 5 .mu.g/mL on a microtiter plate and incubated over
night. After washing and blocking with 5% BSA in PBST, increasing
concentrations of Adalimumab were spiked into 10% human serum and
were applied to the pre-coated plate. After washing, the
anti-Adalimumab/TNF-.alpha. hIgG1 antibody AdaTNF#5 (conjugated to
HRP) was added at 2 .mu.g/mL. Detection was performed by adding
QuantaBlu.RTM. fluorogenic peroxidase substrate. AdaTNF#5 bound to
Adalimumab/TNF-.alpha. complex in a dose-dependent fashion (FIG.
4). Therefore, this novel specificity can be used to develop highly
sensitive quantification assays that are not dependent on the most
often used bridging format.
Example 2
Generation and Characterization of Fab Fragments and Antibodies
that are Specific for the Infliximab/INF-.alpha. Complex
[0159] To identify antibodies which specifically bind to the
Infliximab/TNF-.alpha. complex recombinant TNF-.alpha. (BioLegend
570108, Lot B137143) coupled to magnetic beads and Infliximab were
used as antigens for a solution panning approach.
[0160] Panning and screening was performed as described in Example
1. Upon primary screening 3 unique antibodies were identified to
bind to the Infliximab/INF-.alpha. complex and were expressed,
purified and subjected to further characterization studies.
[0161] IFX-TNF#1, IFX-TNF#2 and IFX-TNF#3 were tested for specific
binding in an ELISA against a series of unrelated and related
antigens and the Infliximab/TNF-.alpha. complex. Therefore 5
.mu.g/mL of each of the antigens were coated on a microtiter plate
over night. After washing and blocking with 5% BSA,
anti-Infliximab/TNF-.alpha. antibodies in Fab-FH format (20 .mu.L
from a 2 .mu.g/mL solution) were added. Detection was performed
using an HRP-labeled anti-His antibody and QuantaBlu fluorogenic
peroxidase substrate. IFX-TNF#1 proofed to be highly specific for
the complex (FIG. 5).
Example 3
Generation and Characterization of Fab Fragments and Antibodies
that are Specific for the MOR103/GM-CSF Complex
[0162] To identify antibodies which specifically bind to the
MOR103/GM-CSF complex recombinant biotinylated human GM-CSF and
MOR103 were used as antigens for a solid phase panning
approach.
[0163] a) Panning
[0164] According to the panning procedures as described in Example
1 the phage preparations were prepared.
[0165] To provide MOR103/GM-CSF complexes the recombinant GM-CSF
was immobilized on a streptavidin coated plate and MOR103 was added
for the formation of complexes.
[0166] All 3 rounds of panning were performed in the presence of
100 .mu.g/ml of MOR3207 (lysozyme specific human IgG1) and 5
.mu.g/ml of free GM-CSF. As described in Example 1 the DNA of the
isolated phages were subcloned into Fab-expression vectors and
respective clones were subjected to primary screening.
[0167] b) Screening
[0168] Primary screening was done by ELISA. 368 clones were
randomly picked from the output of the above described panning
procedure and grown in the 284 well ELISA plates. After induction
of antibody expression (0.75 mM IPTG for 20 h at 30.degree. C.) and
lysis of the cells by using lysozyme, the cell lysates containing
the antibodies were tested in ELISA.
[0169] Therefore the following antigens, MOR103/GM-CSF complex,
purified human MOR3207 (IgG1/kappa); MOR103 and free recombinant
human GM-CSF were coated on an ELISA plate.
[0170] Altogether 3 unique clones that were positive (signal at
least 5-fold over background) on the complex but did not bind to
the other antigens were identified. Thereupon selected clones were
expressed and purified and were tested for specific binding in an
ELISA against a series of unrelated and related antigens and the
MOR103/GM-CSF complex.
[0171] 5 .mu.g/mL of each of the antigens (BSA, GST, MOR03207 and
MOR103) were coated on a microtiter plate over night. For the
immobilisation of biotinylated GM-CSF (GM-CSF-bio), Neutravidin was
coated at 5 .mu.g/mL over night and after blocking with 5% BSA
GM-CSF-bio was added. The respective MOR103/GM-CSF-bio complex was
formed by adding MOR103 at 5 .mu.g/ml to the immobilised
GM-CSF-bio. After washing and blocking with 5% BSA, anti
MOR103/GM-CSF antibodies in Fab format containing a C-terminal
6-His tag (20 .mu.L from a 2 .mu.g/mL solution) were added,
detected thereafter using an anti-His detection antibody and
quantified after washing using QuantaBlu fluorogenic peroxidase
substrate.
[0172] All 3 antibodies (M103GmCSF#1, M103GmCSF#2, and M103GmCSF#3)
turned out to specifically detect the MOR103/GM-CSF complex but not
GM-CSF or MOR103 alone (FIG. 6). Some binding of M103GmCSF#1 to
MOR103 was observed but could not be confirmed in a subsequent
titration ELISA.
[0173] c) Characterization
[0174] M103GmCSF#1 was further characterized. Either MOR103 alone,
the biotinylated GM-CSF or the biotinylated GM-CSF bound to MOR103
were coated on an Avidin-coated plate. His-tagged M103GmCSF#1 Fab
was added in increasing concentrations and was detected using a
His-specific POD conjugated secondary antibody and quantified using
QuantaBlu fluorogenic peroxidase substrate. M103GmCSF#1 showed high
selectivity for binding to the drug-target complex and not to the
individual proteins (drug and target) (FIG. 7). Monovalent affinity
of M103GmCSF#1 Fab to drug-target complex: kD=4.9 nM.
Example 4
Assay to Detect Antibody/Antigen Complex in Human Sera Using an
Antibody Specific for the Complex of a Cognate Antibody and its
Antigen
[0175] To monitor and quantify specific antibody/antigen complexes
from human sera or plasma a robust pharmacokinetic detection assay
was established based on MSD.RTM. (Meso Scale Discovery)
technology.
[0176] In brief, 2 .mu.g/ml of rat anti-human GM-CSF solved in PBS
was coated to the respective wells of a Multi-array.RTM. 96-well
plate Standard plate (Meso Scale Discovery; Cat: L11XA-3).
[0177] The next day MOR103 (hIgG1.lamda.; DSM: P19292; CMC2; conc.:
2.0 mg/mL) and GM-CSF (Bayer; NDC50419-002-33 Lot: B16891; conc.:
0.25 mg/mL) were diluted in LCB buffer (LowCross-Buffer, Candor
Bioscience GmbH Cat#100500, Lot#100C434c). To form MOR103/GM-CSF
complexes MOR103 and GM-CSF were mixed in a 5:1 ratio and
supplemented with 100% Human serum (pooled, male; Sigma, Cat:
H4522; Lot: 11M0605). After 1 h incubation the MOR103 and GM-CSF
mix was diluted 2-fold with LCB buffer leading to a final serum
concentration of 50% and transferred from the pre-incubation plate
to the pre-coated Multi-array.RTM. 96-well plate and further
incubated for 1 h at room temperature. The assay plate was washed
using PBST and 400 ng/ml of ECL-labelled M103GmCSF#1 IgG (Lot:
110801.sub.--11 STE11*1; ECL-labelling: Lot: 110831.sub.--5 AUN51;
conc.:1.7 mg/mL in PBS) was added for 1 h to detect MOR103/GM-CSF
complexes and was subsequently quantified using MSD read buffer T
(Cat: R92TC-1) and measured in a MSD Sector Imager 6000 (Meso Scale
Discovery, Gaithersburg, Md., USA).
[0178] Throughout the titration curve the MOR103/GM-CSF complexes
were specifically detected in the presence of human sera in
dose-dependent manner and with a back accuracy to a fitted curve of
at least 96-104% for each of the concentrations (see below, FIG.
8)
TABLE-US-00002 Precission Nominal Duplikate Calibration Conc.
Analysis Accuracy Sample [pM] [%] [%] St01 500 2.3 100.3 St02 250
0.5 99.5 St03 125 0.4 99.5 St04 62.5 3.1 100.2 St05 31.25 3.3 100.4
St06 15.63 2.2 99.7 St07 7.81 2.8 103.7 St08 3.91 2.6 96.5 St09
1.95 4.1 97.3 St10 0.98 0 104.8 St11 0.49 20 99.8
[0179] The disclosure having been fully described, it is further
illustrated by the following examples and claims, which are
illustrative and are not meant to be further limiting.
[0180] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
disclosure. It will be appreciated, however, that no matter how
detailed the foregoing may appear in text, the disclosure may be
practiced in many ways and the disclosure should be construed in
accordance with the appended claims and any equivalents thereof.
Sequence CWU 1
1
30110PRTArtificial SequenceAdaTNF#1 HCDR1 1Gly Gly Thr Phe Ser Thr
Tyr Ala Ile Ser 1 5 10 220PRTArtificial SequenceAdaTNF#1 HCDR2 2Trp
Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln 1 5 10
15 Lys Phe Gln Gly 20 317PRTArtificial SequenceAdaTNF#1 HCDR3 3Asp
Tyr Phe Ser Ser Ile Gly Trp Val Val Tyr Tyr Gly Pro Met Asp 1 5 10
15 Tyr 412PRTArtificial SequenceAdaTNF#1 LCDR1 4Arg Ala Ser Gln Ser
Val Ser Ser Pro Tyr Leu Ala 1 5 10 511PRTArtificial
SequenceAdaTNF#1 LCDR2 5Leu Leu Ile Tyr Asp Val Ser Ser Arg Ala Thr
1 5 10 68PRTArtificial SequenceAdaTNF#1 LCDR3 6Gln Gln Tyr Thr Ser
Thr Pro Pro 1 5 7110PRTArtificial SequenceAdaTNF#1 VL 7Asp Ile Val
Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Pro 20 25
30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45 Ile Tyr Asp Val Ser Ser Arg Ala Thr Gly Ile Pro Ala Arg
Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Tyr Thr Ser Thr Pro 85 90 95 Pro Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg Thr 100 105 110 8126PRTArtificial
SequenceAdaTNF#1 VH 8Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Ser Thr Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro
Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asp Tyr Phe Ser Ser Ile Gly Trp Val Val Tyr Tyr Gly Pro
100 105 110 Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125 9330DNAArtificial SequenceAdaTNF#1 VL 9gatatcgtgc
tgacccagag cccggcgacc ctgagcctga gcccgggtga acgtgccacc 60ctgagctgca
gagcgagcca gtctgtttct tctccgtacc tggcttggta ccagcagaaa
120ccgggccagg ccccgcgtct attaatctac gacgtttctt ctcgtgcgac
cggcattccg 180gcgcgtttta gcggcagcgg atccggcacc gatttcaccc
tgaccattag cagcctggaa 240ccggaagact ttgcggtgta ttattgccag
cagtacactt ctactccgcc gacctttggc 300cagggcacga aagttgaaat
taaacgtacg 33010378DNAArtificial SequenceAdaTNF#1 VH 10caggtgcaat
tggtgcagag cggtgccgaa gtgaaaaaac cgggcagcag cgtgaaagtt 60agctgcaaag
catccggagg gacgttttct acttacgcta tctcttgggt gcgccaggcc
120ccgggccagg gcctcgagtg gatgggcggt atcatcccga tcttcggcac
tgcgaactac 180gcccagaaat ttcagggccg ggtgaccatt accgccgatg
aaagcaccag caccgcctat 240atggaactga gcagcctgcg cagcgaagat
acggccgtgt attattgcgc gcgtgactac 300ttctcttcta tcggttgggt
tgtttactac ggtccgatgg attactgggg ccaaggcacc 360ctggtgactg ttagctca
3781110PRTArtificial SequenceAdaTNF#5 HCDR1 11Gly Gly Thr Phe Ser
Thr Asn Ala Ile Ser 1 5 10 1220PRTArtificial SequenceAdaTNF#5 HCDR2
12Trp Met Gly Gly Ile Asn Pro His Leu Gly His Ala Asp Tyr Ala Gln 1
5 10 15 Lys Phe Gln Gly 20 1318PRTArtificial SequenceAdaTNF#5 HCDR3
13Gly Trp Tyr Tyr Ile Gly Ser Asn Pro Ser Met Tyr Pro Asn Tyr Phe 1
5 10 15 Asp Pro 1411PRTArtificial SequenceAdaTNF#5 LCDR1 14Arg Ala
Ser Gln Thr Ile Ser Ser Tyr Leu Asn 1 5 10 1511PRTArtificial
SequenceAdaTNF#5 LCDR2 15Leu Leu Ile Tyr Thr Ala Ser Asn Leu Gln
Ser 1 5 10 168PRTArtificial SequenceAdaTNF#5 LCDr3 16Gln Gln Val
Leu His Leu Pro His 1 5 17109PRTArtificial SequenceAdaTNF#5 VL
17Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser Ser
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Thr Ala Ser Asn Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Val Leu His Leu Pro His 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg Thr 100 105 18127PRTArtificial
SequenceAdaTNF#5 VH 18Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Ser Thr Asn 20 25 30 Ala Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro
His Leu Gly His Ala Asp Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Trp Tyr Tyr Ile Gly Ser Asn Pro Ser Met Tyr Pro Asn
100 105 110 Tyr Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 125 19327DNAArtificial SequenceAdaTNF#5 VL 19gatatccaga
tgacccagag cccgagcagc ctgagcgcca gcgtgggcga tcgcgtgacc 60attacctgca
gagccagcca gactatttct tcttacctga actggtacca gcagaaaccg
120ggcaaagcgc cgaaactatt aatctacact gcttctaacc tgcaaagcgg
cgtgccgagc 180cgctttagcg gcagcggatc cggcaccgat ttcaccctga
ccattagctc tctgcaaccg 240gaagactttg cgacctatta ttgccagcag
gttctgcatc tgccgcatac ctttggccag 300ggcacgaaag ttgaaattaa acgtacg
32720381DNAArtificial SequenceAdaTNF#5 VH 20caggtgcaat tggtgcagag
cggtgccgaa gtgaaaaaac cgggcagcag cgtgaaagtt 60agctgcaaag catccggagg
gacgttttct actaacgcta tctcttgggt gcgccaggcc 120ccgggccagg
gcctcgagtg gatgggcggt atcaacccgc atctgggcca tgcggactac
180gcccagaaat ttcagggccg ggtgaccatt accgccgatg aaagcaccag
caccgcctat 240atggaactga gcagcctgcg cagcgaagat acggccgtgt
attattgcgc gcgtggttgg 300tactacatcg gttctaaccc gtctatgtac
ccgaactact tcgatccgtg gggccaaggc 360accctggtga ctgttagctc a
3812110PRTArtificial SequenceIFX-TNF#1 HCDR1 21Gly Phe Thr Phe Ser
Ser Tyr Gly Met His 1 5 10 2220PRTArtificial SequenceIFX-TNF#1
HCDR2 22Trp Val Ser Tyr Ile Tyr Tyr Gly Gly Ser Asp Thr Tyr Tyr Ala
Asp 1 5 10 15 Ser Val Lys Gly 20 2313PRTArtificial
SequenceIFX-TNF#1 HCDR3 23Gly Met Tyr Tyr Leu Tyr Asp Gln Pro Ala
Phe Asp Tyr 1 5 10 2411PRTArtificial SequenceIFX-TNF#1 LCDR1 24Ser
Gly Asp Asn Ile Arg Ser Asp Tyr Val His 1 5 10 2511PRTArtificial
SequenceIFX-TNF#1 LCDR2 25Leu Val Ile Tyr Asp Lys Ser Glu Arg Pro
Ser 1 5 10 2610PRTArtificial SequenceIFX-TNF#1 LCDR3 26Gln Ala Ala
Asp Thr Trp Ser Thr Ile Val 1 5 10 27109PRTArtificial
SequenceIFX-TNF#1 VL 27Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser
Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Ser Cys Ser Gly Asp
Ser Leu Gly Asp Tyr Tyr Val 20 25 30 His Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asp Asn Asn Arg
Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly
Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu 65 70 75 80 Asp
Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Asp Asp Arg Ser Ser Pro 85 90
95 Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105
28115PRTArtificial SequenceIFX-TNF#1 VH 28Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ser Tyr 20 25 30 Ala Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Gly Ile Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg 50
55 60 Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln
Met 65 70 75 80 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
Ala Arg Leu 85 90 95 Ser Gln Thr Gly Val Met Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 29327DNAArtificial
SequenceIFX-TNF#1 VL 29gatatcgaac tgacccagcc gccttcagtg agcgttgcac
caggtcagac cgcgcgtatc 60tcgtgtagcg gcgattctct tggtgattat tatgttcatt
ggtaccagca gaaacccggg 120caggcgccag ttcttgtgat ttatgctgat
aataatcgtc cctcaggcat cccggaacgc 180tttagcggat ccaacagcgg
caacaccgcg accctgacca ttagcggcac tcaggcggaa 240gacgaagcgg
attattattg ccagacttat gatgatcgtt cttctcctgt gtttggcggc
300ggcacgaagt taaccgtcct aggtcag 32730345DNAArtificial
SequenceIFX-TNF#1 VH 30caggtgcaat tggtggaaag cggcggcggc ctggtgcaac
cgggcggcag cctgcgtctg 60agctgcgcgg cctccggatt tacctttaat tcttatgcta
tgtcttgggt gcgccaagcc 120cctgggaagg gtctcgagtg ggtgagcggt
atcggtagct atacctatta tgcggatagc 180gtgaaaggcc gttttaccat
ttcacgtgat aattcgaaaa acaccctgta tctgcaaatg 240aacagcctgc
gtgcggaaga tacggccgtg tattattgcg cgcgtctttc tcagactggt
300gttatggatt attggggcca aggcaccctg gtgacggtta gctca 345
* * * * *