U.S. patent application number 15/128604 was filed with the patent office on 2017-07-20 for monoclonal antibodies to growth and differentiation factor 15 (gdf-15), and uses thereof for treating cancer cachexia and cancer.
The applicant listed for this patent is JULIUS-MAXIMILIANS-UNIVERSITAT WURZBURG. Invention is credited to Markus JUNKER, Dirk PUHRINGER, Tina SCHAFER, Jorg WISCHHUSEN.
Application Number | 20170204174 15/128604 |
Document ID | / |
Family ID | 52745885 |
Filed Date | 2017-07-20 |
United States Patent
Application |
20170204174 |
Kind Code |
A1 |
WISCHHUSEN; Jorg ; et
al. |
July 20, 2017 |
MONOCLONAL ANTIBODIES TO GROWTH AND DIFFERENTIATION FACTOR 15
(GDF-15), AND USES THEREOF FOR TREATING CANCER CACHEXIA AND
CANCER
Abstract
The present invention relates to monoclonal anti-human-GDF-15
antibodies. The antibodies include chimeric antibodies and
humanized antibodies. The invention also relates to monoclonal
anti-human-GDF-15 antibodies including murine antibodies, chimeric
antibodies and humanized antibodies for use in methods for the
treatment of cancer cachexia and also for the treatment of cancer.
The invention also provides pharmaceutical compositions, kits,
methods and uses and cell lines capable of producing the monoclonal
antibodies of the invention.
Inventors: |
WISCHHUSEN; Jorg; (Wurzburg,
DE) ; JUNKER; Markus; (Wurzburg, DE) ;
SCHAFER; Tina; (Wurzburg, DE) ; PUHRINGER; Dirk;
(Rimpar, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
JULIUS-MAXIMILIANS-UNIVERSITAT WURZBURG |
Wurzburg |
|
DE |
|
|
Family ID: |
52745885 |
Appl. No.: |
15/128604 |
Filed: |
March 26, 2015 |
PCT Filed: |
March 26, 2015 |
PCT NO: |
PCT/EP2015/056654 |
371 Date: |
September 23, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/73 20130101;
A61K 2039/505 20130101; C07K 2317/24 20130101; C07K 2317/92
20130101; C07K 16/22 20130101; C07K 2317/94 20130101; C07K 2317/76
20130101; C07K 2317/565 20130101; C07K 2317/34 20130101; A61P 35/00
20180101 |
International
Class: |
C07K 16/22 20060101
C07K016/22 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 26, 2014 |
GB |
1405475.3 |
Mar 26, 2014 |
GB |
1405477.9 |
Claims
1.-60. (canceled)
61. A monoclonal antibody capable of binding to human GDF-15, or an
antigen-binding portion thereof, wherein the heavy chain variable
domain comprises a CDR3 region comprising the amino acid sequence
of SEQ ID NO: 5 or an amino acid sequence at least 90% identical
thereto, and wherein the light chain variable domain comprises a
CDR3 region comprising the amino acid sequence of SEQ ID NO: 7 or
an amino acid sequence at least 85% identical thereto, wherein the
constant domain of the heavy chain comprises the amino acid
sequence of SEQ ID No: 29, or an amino acid sequence at least 85%
identical thereto, and wherein the constant domain of the light
chain comprises the amino acid sequence of SEQ ID No: 32, or an
amino acid sequence at least 85% identical thereto.
62. A monoclonal antibody capable of binding to human GDF-15, or an
antigen-binding portion thereof, wherein the binding is binding to
a conformational or discontinuous epitope on human GDF-15 comprised
by the amino acid sequences of SEQ ID No: 25 and SEQ ID No: 26,
wherein the constant domain of the heavy chain comprises the amino
acid sequence of SEQ ID No: 29, or an amino acid sequence at least
85% identical thereto, and wherein the constant domain of the light
chain comprises the amino acid sequence of SEQ ID No: 32, or an
amino acid sequence at least 85% identical thereto.
63. The antibody or antigen-binding portion thereof of any one of
claims 61-62, wherein the antibody is a humanized antibody.
64. The antibody or antigen-binding portion thereof according to
any one of claims 61-63 for use in medicine.
65. An antibody or antigen-binding portion thereof according to any
one of claims 61-63, for use in a method for treating cancer
cachexia in a mammal.
66. An antibody or antigen-binding portion thereof according to any
one of claims 61-63, for use in a method for treating cancer in a
mammal.
67. A kit comprising the antibody or antigen-binding portion
thereof of any of claims 61-63.
68. An expression vector comprising a nucleotide sequence encoding
the antibody or antigen-binding portion thereof according to any of
claims 61-63.
69. A cell line capable of producing an antibody or antigen-binding
portion thereof according to any one of claims 61-63.
70. A monoclonal antibody capable of binding to human GDF-15, or an
antigen-binding portion thereof, wherein the heavy chain variable
domain comprises a CDR3 region comprising the amino acid sequence
of SEQ ID NO: 5 or an amino acid sequence at least 90% identical
thereto, and wherein the light chain variable domain comprises a
CDR3 region comprising the amino acid sequence of SEQ ID NO: 7 or
an amino acid sequence at least 85% identical thereto, for use in a
method for treating cancer cachexia in a mammal.
71. A monoclonal antibody capable of binding to human GDF-15, or an
antigen-binding portion thereof, wherein the binding is binding to
a conformational or discontinuous epitope on human GDF-15 comprised
by the amino acid sequences of SEQ ID No: 25 and SEQ ID No: 26, for
use in a method for treating cancer cachexia in a mammal.
72. The antibody or antigen-binding portion thereof of any one of
claim 65-66 or 70-71 for the use according to any one of claim
65-66 or 70-71, wherein the method is a method for both treating
cancer and treating cancer cachexia in the same mammal.
73. The antibody or antigen-binding portion thereof of any one of
claim 65-66 or 70-72 for the use according to any one of claim
65-66 or 70-72, wherein the cancer cells of the mammal endogenously
express GDF-15 and/or the cancer cells of the mammal stimulate
endogenous expression of GDF-15 in non-cancerous cells of the
mammal.
74. The antibody or antigen-binding portion thereof of any one of
claim 65-66 or 70-73 for the use according to any one of claim
65-66 or 70-73, wherein the cancer cells of the mammal endogenously
express GDF-15.
75. The antibody or antigen-binding portion thereof of any one of
claim 65-66 or 70-74 for the use according to any one of claim
65-66 or 70-74, wherein the mammal is a human patient.
76. The antibody or antigen-binding portion thereof of any one of
claims 71-75 for the use according to any one of claims 71-75, or
the antibody or antigen-binding portion thereof of any one of
claims 62-63, wherein the heavy chain variable domain comprises a
CDR3 region comprising the amino acid sequence of SEQ ID NO: 5 or
an amino acid sequence at least 90% identical thereto, and wherein
the light chain variable domain comprises a CDR3 region comprising
the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence
at least 85% identical thereto.
77. The antibody or antigen-binding portion thereof of any one of
claims 70 and 72-75 for the use according to any one of claims 70
and 72-75, or the antibody or antigen-binding portion thereof of
any one of claims 61 and 63, wherein the binding is binding to a
conformational or discontinuous epitope on human GDF-15 that is
comprised by the amino acid sequences of SEQ ID No: 25 and SEQ ID
No: 26.
78. The antibody or antigen-binding portion thereof of any one of
claims 70-77 for the use according to any one of claims 70-77, or
the antibody or antigen-binding portion thereof of any one of
claims 61-63, wherein the heavy chain variable domain comprises a
CDR1 region comprising the amino acid sequence of SEQ ID NO: 3 and
a CDR2 region comprising the amino acid sequence of SEQ ID NO: 4,
and wherein the light chain variable domain comprises a CDR1 region
comprising the amino acid sequence of SEQ ID NO: 6 and a CDR2
region comprising the amino acid sequence ser-ala-ser.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to monoclonal
anti-human-GDF-15 antibodies, pharmaceutical compositions, kits,
methods and uses and the cell lines capable of producing the
monoclonal antibodies described herein. The present invention
further relates to antibodies to human GDF-15 capable of inhibiting
cancer growth, treating cancer-induced weight loss and cancer
cachexia.
BACKGROUND
[0002] To date, many cancers are still areas of unmet medical
needs, and accordingly, means to more effectively treat cancer, and
to treat cancer in a broader range of cancers are needed.
[0003] In addition to the suffering caused by the cancer itself,
many patients suffer from cancer cachexia, a medical condition
triggered by the cancer that typically involves weight loss and
loss of skeletal muscle mass. Cancer cachexia accounts for more
than 20 percent of all cancer-related deaths (Murphy K T and Lynch
G S: Update on emerging drugs for cancer cachexia. Expert Opin
Emerg Drugs. 2009 December; 14(4):619-32.).
[0004] Thus, in order to improve the treatment and prognosis of
cancers which lead to cancer cachexia, treatment regimens that
target both of these medical conditions are needed. To date, most
of the emerging drugs for treatments of cancer cachexia are drugs
that target cachexia but not the cancer itself (see Murphy K T and
Lynch G S: Update on emerging drugs for cancer cachexia. Expert
Opin Emerg Drugs. 2009 December; 14(4):619-32.). Only very few
drugs are effective against both the cancer and cancer cachexia,
and therefore, complex treatment regimens that combine anti-cancer
drugs and anti-cancer cachexia drugs are oftentimes needed.
Accordingly, there is still an unmet medical need for drugs that
can be used to effectively treat both cancer and cancer cachexia in
a broad range of cancers.
[0005] Many types of cancer are known to express growth factors,
including factors such as VEGF, PDGF, TGF-.beta. and GDF-15.
[0006] GDF-15, growth and differentiation factor-15, is a divergent
member of the TGF-.beta. superfamily. It is a protein which is
intracellularly expressed as a precursor, subsequently processed
and eventually becomes secreted from the cell into the environment.
Both the active, fully processed (mature) form and the precursor of
GDF-15 can be found outside cells. The precursor covalently binds
via its COOH-terminal amino acid sequence to the extracellular
matrix (Bauskin A R et al., Cancer Research 2005) and thus resides
on the exterior of a cell. The active, fully processed (mature)
form of GDF-15 is soluble and is found in blood sera. Thus, the
processed form of GDF-15 may potentially act on any target cell
within the body that is connected to the blood circulation,
provided that the potential target cell expresses a receptor for
the soluble GDF-15 ligand.
[0007] During pregnancy, GDF-15 is found under physiological
conditions in the placenta. However, many malignant cancers
(especially aggressive brain cancers, melanoma, lung cancer,
gastrointestinal tumors, colon cancer, pancreatic cancer, prostate
cancer and breast cancer (Mimeault M and Batra S K, J. Cell Physiol
2010)) exhibit increased GDF-15 levels in the tumor as well as in
blood serum. Likewise, correlations have been described between
high GDF-15 expression and chemoresistance (Huang C Y et al., Clin.
Cancer Res. 2009) and between high GDF-15 expression and poor
prognosis, respectively (Brown D A et al., Clin. Cancer Res.
2009).
[0008] GDF-15 is expressed in gliomas of different WHO grades as
assessed by immunohistochemistry (Roth et al., Clin. Cancer Res.
2010). Further, Roth et al. stably expressed short hairpin.
RNA-expressing DNA constructs targeting endogenous GDF-15 or
control constructs in SMA560 glioma cells. When using these
pre-established stable cell lines, they observed that tumor
formation in mice bearing GDF-15 knockdown SMA560 cells was delayed
compared to mice bearing control constructs.
[0009] Patent application PCT/EP2013/070127 relates to monoclonal
anti-GDF-15 antibodies, in particular to an antibody produced by
the hybridoma cell line B1-23 deposited with the Deutsche Sammlung
fur Mikroorganismen and Zellkulturen GmbH (DSMZ) under the
accession No. DSM ACC3142 under the Budapest treaty.
PCT/EP2013/070127 also relates to uses of the anti-GDF-15
antibodies.
[0010] Patent applications WO 2005/099746 and WO 2009/021293 relate
to an anti-human-GDF-15 antibody (Mab26) capable of antagonizing
effects of human GDF-15 on tumor-induced weight loss in vivo in
mice: In these documents, immunologically compromised mice were
administered with human tumor cells (prostate carcinoma cells
DU145) transfected with plasmids overexpressing human GDF-15. Tumor
cells carrying plasmids lacking a GDF-15 sequence served as a
negative control. Those mice expressing xenograft GDF-15 exhibited
a tumor-induced weight loss (clinical term: cachexia) and anorexia.
A single intraperitoneal administration of 1 mg of Mab26 from WO
2005/099746 resulted in a complete reversal of tumor-induced weight
loss. WO 2005/099746 and WO 2009/021293 do not disclose effects of
an anti-human-GDF-15 antibody on tumor growth. Moreover, these
documents are silent as to whether anti-human-GDF-15 antibodies
could lead to an increase in body weight of the treated mice
compared to their body weight before the onset of cachexia.
[0011] Similarly, Johnen H et al. (Nature Medicine, 2007) reported
effects of an anti-human-GDF-15 monoclonal antibody on
cancer-induced anorexia and weight loss but did not observe any
effects of the anti-human-GDF-15 antibody on the size of the tumor
formed by the cancer, even when the antibody was administered at a
high dosage of 1 mg, and thus the antibody did not inhibit growth
of the cancer.
[0012] Accordingly, to date, there was still a need in the art for
means to effectively treat cancer and cancer cachexia, and for
means to treat cancer and cancer cachexia in a broader range of
cancers.
[0013] It is therefore an object of the invention to obtain means
that can be used to effectively treat cancer cachexia, and to also
effectively treat cancer, and means that can be used to treat
cancer cachexia, and to also effectively treat cancer in a broader
range of cancers.
[0014] In an effort to find means to achieve these objects, the
present inventors have surprisingly found that a monoclonal
antibody to human GDF-15 can be used to treat cancer cachexia and
to also treat cancer of human xenograft tumors in mice.
[0015] Additionally, an antibody to human GDF-15 in accordance with
the present invention has an equilibrium dissociation constant of
about 790 pM for recombinant GDF-15 even without additional
affinity maturation, which is a higher affinity compared to most
known therapeutic antibodies.
[0016] Thus, the antibody to human GDF-15 according to the present
invention has superior properties compared to antibodies known from
the art, and is particularly useful for inhibiting cancer growth
and cancer cachexia. The antibody of the present invention is
therefore useful for treating cancer and for treating cancer
cachexia. Accordingly, the present invention was completed.
BRIEF DESCRIPTION OF THE INVENTION
[0017] The present invention solves the above-mentioned objects by
providing the monoclonal antibodies, pharmaceutical compositions,
kits, uses and the cell lines capable of producing the monoclonal
antibodies described herein.
[0018] In particular, the present inventors surprisingly show that
monoclonal antibodies to human GDF-15 and antigen binding portions
thereof according to the invention are capable of inhibiting cancer
cachexia and/or cancer growth. This was unexpected because those
monoclonal antibodies to GDF-15 that were previously known from the
art (WO 2005/099746, WO 2009/021293 and Johnen H et al., Nature
Medicine, 2007) were only known to cause a reversal of
cancer-induced weight loss (i.e. a reversal of a secondary symptom
induced by the GDF-15 expressed by the cancer), but were shown to
fail at inhibiting growth of the cancer.
[0019] By showing that the monoclonal antibodies to human GDF-15
according to the invention can be used to treat cancer-induced
weight loss and/or cancer cachexia and treat cancer, the present
inventors also surprisingly show that human GDF-15 protein can be
targeted by the antibodies of the invention in a way that both
cancer growth is inhibited and cancer-induced weight loss and
cancer cachexia is treated. It is expected that the same mechanisms
of cancer growth inhibition and treatment of cancer-induced weight
loss and cancer cachexia are applicable to a large number of
cancers that overexpress human GDF-15 including the cancers listed
below.
[0020] The monoclonal antibodies and antigen-binding portions
thereof according to the invention are derived from a murine
anti-GDF-15 antibody, mAb-B1-23, which was described in
PCT/EP2013/070127 and deposited with the Deutsche Sammlung fur
Mikroorganismen and Zellkulturen GmbH (DSMZ) under the accession
No. DSM ACC3142 under the Budapest treaty. The anti-human GDF-15
mAb-B1-23 antibodies according to the invention can be generated by
replacing constant domains of the murine antibody mAb-B1-23 with
the constant domains of a human IgG1 antibody.
[0021] Surprisingly, it was observed that a chimeric and a
humanized B1-23 antibody according to the invention showed no
tendency to aggregate. These antibody properties according to the
invention are expected to increase the bioavailability of these
antibodies and to be advantageous for clinical formulation of these
antibodies.
[0022] Thus, the present invention relates to a monoclonal antibody
capable of binding to human GDF-15, or an antigen-binding portion
thereof, wherein the heavy chain variable domain comprises a CDR3
region comprising the amino acid sequence of SEQ ID NO: 5 or an
amino acid sequence at least 90% identical thereto, and wherein the
light chain variable domain comprises a CDR3 region comprising the
amino acid sequence of SEQ ID NO: 7 or an amino acid sequence at
least 85% identical thereto wherein the constant domain of the
heavy chain comprises the amino acid sequence of SEQ ID No: 29, or
an amino acid sequence at least 85%, preferably at least 90%, more
preferably at least 95% identical thereto, and wherein the constant
domain of the light chain comprises the amino acid sequence of SEQ
ID No: 32, or an amino acid sequence at least 85%, preferably at
least 90%, more preferably at least 95% identical thereto.
[0023] The present invention also relates to a monoclonal antibody
capable of binding to human GDF-15, or an antigen-binding portion
thereof, wherein the heavy chain variable domain comprises a CDR3
region comprising the amino acid sequence of SEQ ID NO: 5 or an
amino acid sequence at least 90% identical thereto, and wherein the
light chain variable domain comprises a CDR3 region comprising the
amino acid sequence of SEQ ID NO: 7 or an amino acid sequence at
least 85% identical thereto, for use in a method for treating
cancer cachexia in a mammal. The method comprises administering the
antibody or antigen-binding portion thereof to said mammal.
Additionally, the present invention relates to a corresponding
method for treatment.
[0024] Further, the invention also relates to a monoclonal antibody
capable of binding to human GDF-15, or an antigen-binding portion
thereof, wherein the binding is binding to a conformational or
discontinuous epitope on human GDF-15 comprised by the amino acid
sequences of SEQ ID No: 25 and SEQ ID No: 26, wherein the constant
domain of the heavy chain comprises the amino acid sequence of SEQ
ID No: 29, or an amino acid sequence at least 85%, preferably at
least 90%, more preferably at least 95% identical thereto, and
wherein the constant domain of the light chain comprises the amino
acid sequence of SEQ ID No: 32, or an amino acid sequence at least
85%, preferably at least 90%, more preferably at least 95%
identical thereto.
[0025] Further, the invention also relates to a monoclonal antibody
capable of binding to human GDF-15, or an antigen-binding portion
thereof, wherein the binding is binding to a conformational or
discontinuous epitope on human GDF-15 comprised by the amino acid
sequences of SEQ ID No: 25 and SEQ ID No: 26, for use in a method
for treating cancer cachexia in a mammal. The method comprises
administering the antibody or antigen-binding portion thereof to
said mammal. Additionally, the present invention relates to a
corresponding method for treatment.
[0026] The invention also relates to a pharmaceutical composition
comprising the antibody or antigen-binding portion thereof
according to the invention.
[0027] The invention also relates to an antibody or antigen-binding
portion thereof according to the invention for use in medicine.
[0028] Further, the invention relates to an antibody or
antigen-binding portion thereof or a pharmaceutical composition
according to the invention for use in a method for treating cancer
in a mammal. The method comprises administering the antibody or
antigen-binding portion thereof or the pharmaceutical composition
to said mammal.
[0029] Further, the invention relates to an antibody or
antigen-binding portion thereof or a pharmaceutical composition
according to the invention for use in a method for treating cancer
cachexia in a mammal. The method comprises administering the
antibody or antigen-binding portion thereof or the pharmaceutical
composition to said mammal.
[0030] Additionally, the invention relates to a kit comprising the
pharmaceutical composition according to the invention.
[0031] The invention also relates to an expression vector
comprising a nucleotide sequence encoding the antibody or
antigen-binding portion thereof according to the invention.
[0032] Further, the invention relates to a cell line capable of
producing an antibody or antigen-binding portion thereof according
to the invention.
[0033] Thus, by providing monoclonal antibodies to human GDF-15,
the present invention provides means for the treatment of cancer
cachexia and a cancer growth inhibitor that meets the above-defined
needs in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1: NKG2D Expression on NK Cells after Treatment with or
without GDF-15. The cell surface expression of NKG2D was determined
on NK cells after treatment with the indicated cytokines in the
presence or absence of the anti-GDF-15 antibody mAb B1-23. The
figure displays specific fluorescence intensities determined by
flow cytometry, quantified relative to an unspecific control
antibody.
[0035] FIG. 2: Akt Phosphorylation in the Ovarian Carcinoma Cell
Line SK-OV-3. In order to quantify the Western Blot for the ovarian
carcinoma cell line SK-OV-3, the ratio of phosphorylated Akt to the
total amount of Akt was calculated and normalized to the untreated
control.
[0036] FIG. 3: JNK1/2 Phosphorylation in Immune Cells. In order to
quantify the Western Blot, the ratio of phosphorylated JNK1/2 to
the total amount of JNK was calculated and normalized to the
untreated control.
[0037] FIG. 4: An anti-tumor effect of murine B1-23 in vivo.
Balb/c.sup.nu/nu nude mice were used in a xenograft setting with
the melanoma cell line UACC-257. The tumor size of the animal
cohort treated with B1-23 (open squares) was significantly
decreased, compared to the PBS control group (filled solid
circles). Significance was defined as p<0.05 as assessed by
Wilcoxon's log-rank test.
[0038] FIG. 5: Treatment of cancer cachexia with anti-GDF-15
antibodies. The figure shows a comparison of the mean body weight
of all treated Balb/c.sup.nu/nu nude mice, which were inoculated
with UACC-257 cells. The changes of the body weight are depicted in
percent as compared to the starting body weight on day 0, for a
period of 38 days.
[0039] FIG. 6: Coomassie stain of antibodies used in the study No.
140123.
[0040] FIG. 7: Improved solubility of the chimeric and the
humanized antibody at physiological pH.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0041] Unless otherwise defined below, the terms used in the
present invention shall be understood in accordance with their
common meaning known to the person skilled in the art.
[0042] The term "antibody" as used herein refers to any functional
antibody that is capable of specific binding to the antigen of
interest, as generally outlined in chapter 7 of Paul, W. E. (Ed.).:
Fundamental Immunology 2nd Ed. Raven Press, Ltd., New York 1989,
which is incorporated herein by reference. Without particular
limitation, the term "antibody" encompasses antibodies from any
appropriate source species, including chicken and mammalian such as
mouse, goat, non-human primate and human. Preferably, the antibody
is a humanized antibody. The antibody is preferably a monoclonal
antibody which can be prepared by methods well-known in the art.
The term "antibody" encompasses an IgG-1, -2, -3, or -4, IgE, IgA,
IgM, or IgD isotype antibody. The term "antibody" encompasses
monomeric antibodies (such as IgD, IgE, IgG) or oligomeric
antibodies (such as IgA or IgM). The term "antibody" also
encompasses--without particular limitations--isolated antibodies
and modified antibodies such as genetically engineered antibodies,
e.g. chimeric antibodies.
[0043] The nomenclature of the domains of antibodies follows the
terms as known in the art. Each monomer of an antibody comprises
two heavy chains and two light chains, as generally known in the
art. Of these, each heavy and light chain comprises a variable
domain (termed V.sub.H for the heavy chain and V.sub.L for the
light chain) which is important for antigen binding. These heavy
and light chain variable domains comprise (in an N-terminal to
C-terminal order) the regions FR1, CDR1, FR2, CDR2, FR3, CDR3, and
FR4 (FR, framework region; CDR, complementarity determining region
which is also known as hypervariable region). The identification
and assignment of the above-mentioned antibody regions within the
antibody sequence is generally in accordance with Kabat et al.
(Sequences of proteins of immunological interest, U.S. Dept. of
Health and Human Services, Public Health Service, National
Institutes of Health, Bethesda, Md. 1983), or Chothia et al.
(Conformations of immunoglobulin hypervariable regions. Nature.
1989 Dec. 21-28; 342(6252):877-83.), or may be performed by using
the IMGT/V-QUEST software described in Giudicelli et al.
(IMGT/V-QUEST, an integrated software program for immunoglobulin
and T cell receptor V-J and V-D-J rearrangement analysis. Nucleic
Acids Res. 2004 Jul. 1; 32 (Web Server issue):W435-40.), which is
incorporated herein by reference. Preferably, the antibody regions
indicated above are identified and assigned by using the
IMGT/V-QUEST software.
[0044] A "monoclonal antibody" is an antibody from an essentially
homogenous population of antibodies, wherein the antibodies are
substantially identical in sequence (i.e. identical except for
minor fraction of antibodies containing naturally occurring
sequence modifications such as amino acid modifications at their N-
and C-termini). Unlike polyclonal antibodies which contain a
mixture of different antibodies directed to numerous epitopes,
monoclonal antibodies are directed to the same epitope and are
therefore highly specific. The term "monoclonal antibody" includes
(but is not limited to) antibodies which are obtained from a
monoclonal cell population derived from a single cell clone, as for
instance the antibodies generated by the hybridoma method described
in Kohler and Milstein (Nature, 1975 Aug. 7; 256(5517):495-7) or
Harlow and Lane ("Antibodies: A Laboratory Manual" Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1988). A
monoclonal antibody may also be obtained from other suitable
methods, including phage display techniques such as those described
in Clackson et al. (Nature. 1991 Aug. 15; 352(6336):624-8) or Marks
et al. (J Mol Biol. 1991 Dec. 5; 222(3):581-97). A monoclonal
antibody may be an antibody that has been optimized for
antigen-binding properties such as decreased Kd values, optimized
association and dissociation kinetics by methods known in the art.
For instance, Kd values may be optimized by display methods
including phage display, resulting in affinity-matured monoclonal
antibodies. The term "monoclonal antibody" is not limited to
antibody sequences from particular species of origin or from one
single species of origin. Thus, the meaning of the term "monoclonal
antibody" encompasses chimeric monoclonal antibodies such as
humanized monoclonal antibodies.
[0045] "Humanized antibodies" are antibodies which contain human
sequences and a minor portion of non-human sequences which confer
binding specificity to an antigen of interest (e.g. human GDF-15).
Typically, humanized antibodies are generated by replacing
hypervariable region sequences from a human acceptor antibody by
hypervariable region sequences from a non-human donor antibody
(e.g. a mouse, rabbit, rat donor antibody) that binds to an antigen
of interest (e.g. human GDF-15). In some cases, framework region
sequences of the acceptor antibody may also be replaced by the
corresponding sequences of the donor antibody. In addition to the
sequences derived from the donor and acceptor antibodies, a
"humanized antibody" may either contain other (additional or
substitute) residues or sequences or not. Such other residues or
sequences may serve to further improve antibody properties such as
binding properties (e.g. to decrease Kd values) and/or immunogenic
properties (e.g. to decrease antigenicity in humans). Non-limiting
examples for methods to generate humanized antibodies are known in
the art, e.g. from Riechmann et al. (Nature. 1988 Mar. 24;
332(6162):323-7) or Jones et al. (Nature. 1986 May 29-Jun. 4;
321(6069):522-5).
[0046] The term "human antibody" relates to an antibody containing
human variable and constant domain sequences. This definition
encompasses antibodies having human sequences bearing single amino
acid substitutions or modifications which may serve to further
improve antibody properties such as binding properties (e.g. to
decrease Kd values) and/or immunogenic properties (e.g. to decrease
antigenicity in humans). The term "human antibody" excludes
humanized antibodies where a portion of non-human sequences confers
binding specificity to an antigen of interest.
[0047] An "antigen-binding portion" of an antibody as used herein
refers to a portion of an antibody that retains the capability of
the antibody to specifically bind to the antigen (e.g. GDF-15),
i.e. the "antigen-binding portion" is capable of competing with the
antibody for specific binding to the antigen. The "antigen-binding
portion" may contain one or more fragments of the antibody. Without
particular limitation, it can be produced by any suitable method
known in the art, including recombinant DNA methods and preparation
by chemical or enzymatic fragmentation of antibodies.
Antigen-binding portions may be Fab fragments, F(ab') fragments,
F(ab').sub.2 fragments, single chain antibodies (scFv),
single-domain antibodies, diabodies or any other portion(s) of the
antibody that allow(s) to retain binding to the antigen.
[0048] An "antibody" (e.g. a monoclonal antibody) or an
"antigen-binding portion" may have been derivatized or be linked to
a different molecule. For example, molecules that may be linked to
the antibody are other proteins (e.g. other antibodies), a
molecular label (e.g. a fluorescent, luminescent, colored or
radioactive molecule), a pharmaceutical and/or a toxic agent. The
antibody or antigen-binding portion may be linked directly (e.g. in
form of a fusion between two proteins), or via a linker molecule
(e.g. any suitable type of chemical linker known in the art).
[0049] As used herein, the terms "binding" or "bind" refer to
specific binding to the antigen of interest (e.g. human GDF-15).
Preferably, the Kd value is less than 100 nM, more preferably less
than 50 nM, still more preferably less than nM, still more
preferably less than 5 nM and most preferably less than 2 nM.
[0050] The term "epitope" as used herein refers to a small portion
of an antigen that forms the binding site for an antibody.
[0051] In the context of the present invention, binding or
competitive binding of antibodies or their antigen-binding portions
to the antigen of interest (e.g. human GDF-15) is measured by using
surface plasmon resonance measurements as a reference standard
assay, as described below.
[0052] The terms "K.sub.D" or "K.sub.D value" relate to the
equilibrium dissociation constant as known in the art. In the
context of the present invention, these terms relate to the
equilibrium dissociation constant of an antibody with respect to a
particular antigen of interest (e.g. human GDF-15). The equilibrium
dissociation constant is a measure of the propensity of a complex
(e.g. an antigen-antibody complex) to reversibly dissociate into
its components (e.g. the antigen and the antibody). For the
antibodies according to the invention, K.sub.D values (such as
those for the antigen human GDF-15) are generally determined by
using surface plasmon resonance measurements as described
below.
[0053] The term "cancer growth" as used herein relates to any
measureable growth of the cancer. For cancers forming solid tumors,
"cancer growth" relates to a measurable increase in tumor volume
over time. If the cancer has formed only a single tumor, "cancer
growth" relates only to the increase in volume of the single tumor.
If the cancer has formed multiple tumors such as metastases,
"cancer growth" relates to the increase in volume of all measurable
tumors. For solid tumors, the tumor volume can be measured by any
method known in the art, including magnetic resonance imaging and
computed tomography (CT scan).
[0054] For leukemias which are characterized by the presence of
cancerous cells of the blood system in blood, "cancer growth"
relates to a measurable increase in the number of cancer cells per
blood volume. In order to carry out such measurements, cancer cells
can be identified from blood samples by using any method known in
the art, including cell morphology measurements, or staining of
tumor cell marker proteins such as tumor marker cell surface
proteins, e.g. by staining with specific antibodies, and the cancer
cells can be counted.
[0055] Terms such as "inhibiting cancer growth" as used herein
refer to a measurable inhibition of cancer growth in patient
treated with the antibody. Preferably, the inhibition is
statistically significant. Inhibition of cancer growth may be
assessed by comparing cancer growth in a group of patients treated
in accordance with the present invention to a control group of
untreated patients, or by comparing a group of patients that
receive a standard cancer treatment of the art plus a treatment
according to the invention with a control group of patients that
only receive a standard cancer treatment of the art. Such studies
for assessing the inhibition of cancer growth are designed in
accordance with accepted standards for clinical studies, e.g.
double-blinded, randomized studies with sufficient statistical
power. The term "inhibiting cancer growth" includes an inhibition
of cancer growth where the cancer growth is inhibited partially
(i.e. where the cancer growth in the patient is delayed compared to
the control group of patients), an inhibition where the cancer
growth is inhibited completely (i.e. where the cancer growth in the
patient is stopped), and an inhibition where cancer growth is
reversed (i.e. the cancer shrinks).
[0056] An "isolated antibody" as used herein is an antibody that
has been identified and separated from the majority of components
(by weight) of its source environment, e.g. from the components of
a hybridoma cell culture or a different cell culture that was used
for its production (e.g. producer cells such as CHO cells that
recombinantly express the antibody). The separation is performed
such that it sufficiently removes components that may otherwise
interfere with the suitability of the antibody for the desired
applications (e.g. with a therapeutic use of the anti-human GDF-15
antibody according to the invention). Methods for preparing
isolated antibodies are known in the art and include Protein A
chromatography, anion exchange chromatography, cation exchange
chromatography, virus retentive filtration and ultrafiltration.
Preferably, the isolated antibody preparation is at least 70% pure
(w/w), more preferably at least 80% pure (w/w), still more
preferably at least 90% pure (w/w), still more preferably at least
95% pure (w/w), and most preferably at least 99% pure (w/w), as
measured by using the Lowry protein assay.
[0057] A "diabody" as used herein is a small bivalent
antigen-binding antibody portion which comprises a heavy chain
variable domain linked to a light chain variable domain on the same
polypeptide chain linked by a peptide linker that is too short to
allow pairing between the two domains on the same chain. This
results in pairing with the complementary domains of another chain
and in the assembly of a dimeric molecule with two antigen binding
sites. Diabodies may be bivalent and monospecific (such as
diabodies with two antigen binding sites for human GDF-15), or may
be bivalent and bispecific (e.g. diabodies with two antigen binding
sites, one being a binding site for human GDF-15, and the other one
being a binding site for a different antigen). A detailed
description of diabodies can be found in Holliger P et al.
("Diabodies": small bivalent and bispecific antibody fragments."
Proc Natl Acad Sci USA. 1993 Jul. 15; 90(14):6444-8.).
[0058] A "single-domain antibody" (which is also referred to as
"Nanobody.TM.") as used herein is an antibody fragment consisting
of a single monomeric variable antibody domain. Structures of and
methods for producing single-domain antibodies are known from the
art, e.g. from Holt L J et al. ("Domain antibodies: proteins for
therapy." Trends Biotechnol. 2003 November; 21(11):484-90.),
Saerens D et al. ("Single-domain antibodies as building blocks for
novel therapeutics." Curr Opin Pharmacol. 2008 October; 8(5):600-8.
Epub 2008 Aug. 22.), and Arbabi Ghahroudi M et al. ("Selection and
identification of single domain antibody fragments from camel
heavy-chain antibodies." FEBS Lett. 1997 Sep. 15;
414(3):521-6.).
[0059] The term "higher" as used herein means that a value (e.g. a
GDF-15 level) in a patient sample is higher than a value in a
corresponding control sample or group of control samples.
Preferably, the difference is statistically significant.
[0060] The term "elevated GDF-15 levels" as used herein means that
the human patient has higher GDF-15 levels in blood serum before
administration of the antibody or antigen-binding portion thereof
or the pharmaceutical composition according to the invention, when
compared to median GDF-15 levels in blood sera of healthy human
control individuals as a reference.
[0061] A preferred median reference for GDF-15 level in blood sera
of healthy human control individuals is <0.8 ng/ml. The expected
range is between 0.2 ng/ml and 1.2 ng/ml in healthy human controls
(Reference: Tanno T et al.: "Growth differentiation factor 15 in
erythroid health and disease." Curr Opin Hematol. 2010 May; 17(3):
184-190.). Preferably, the levels are 1.2-fold higher, more
preferably 1.5-fold higher, still more preferably 2-fold higher and
most preferably 5-fold higher.
[0062] The term "prior to administration" as used herein means the
period of time immediately before administration of the antibody,
fragment thereof or the pharmaceutical composition according to the
invention. Preferably, the term "prior to administration" means a
period of 30 days immediately before administration; most
preferably a period of one week immediately before
administration.
[0063] The terms "significant", "significantly", etc. as used
herein refer to a statistically significant difference between
values.
[0064] The terms "cancer" and "cancer cell" is used herein in
accordance with their common meaning in the art (see for instance
Weinberg R. et al.: The Biology of Cancer. Garland Science: New
York 2006. 850p.).
[0065] The term "cancer-induced weight loss" is used herein in
accordance with its common meaning in the art. Cancer-induced
weight loss is frequently seen as an adverse effect in individuals
having cancer (see, for instance Fearon K. et al.: Definition and
classification of cancer cachexia: an international consensus.
Lancet Oncol. 2011 May; 12(5):489-95.; Tisdale M J.: Mechanisms of
cancer cachexia. Physiol Rev. 2009 April; 89(2):381-410.). The term
"cancer-induced weight loss" relates to the body weight loss
induced by the cancer. Additional body weight loss in addition to
the cancer-induced weight loss--e.g. body weight loss induced by
cancer treatments such as surgery, chemotherapy and
radiotherapy--can also occur in individuals having cancer. It is
understood that the meaning of the term "cancer-induced weight
loss" does not include this additional body weight loss. However,
this does not exclude the possibility that the antibodies of the
present invention--in addition to their effects on cancer-induced
weight loss and on cancer growth--may have beneficial effects
against such additional body weight loss, e.g. by reverting or
partly reverting such additional weight loss, or by preventing or
partly preventing such additional body weight loss.
[0066] Body weight can easily be measured by weighing, and body
weight is typically expressed in units of mass such as kg.
[0067] The term "cancer cachexia" is used herein in accordance with
its common meaning in the art (see, for instance Fearon K. et al.:
Definition and classification of cancer cachexia: an international
consensus. Lancet Oncol. 2011 May; 12(5):489-95.; Tisdale M J.:
Mechanisms of cancer cachexia. Physiol Rev. 2009 April;
89(2):381-410.). The most common symptom of cancer cachexia is
cancer-induced weight loss. Thus, according to one definition,
cancer cachexia is characterized by an ongoing loss of skeletal
muscle mass (with or without loss of fat mass) that cannot be fully
reversed by conventional nutrition. In human patients, cancer
cachexia can be defined by a weight loss of more than 5% during the
past 6 months, or by a body mass index of less than 20 g/m.sup.2
and any degree of ongoing weight loss that is higher than 2%, or by
sarcopenia (i.e. degenerative loss of muscle mass) and an ongoing
weight loss that is higher than 2% (see, Fearon K. et al.:
Definition and classification of cancer cachexia: an international
consensus. Lancet Oncol. 2011 May; 12(5):489-95.). A further
symptom of cancer cachexia can be a depletion of adipose
tissue.
[0068] With respect to cancer cachexia, a "treatment" according to
the present invention may be a treatment for preventing and/or a
treatment for inhibiting or reverting cancer cachexia. Typically, a
treatment for preventing cancer cachexia is a treatment that is
given prophylactically at a stage of the cancer disease where no
cancer cachexia has yet occurred. A treatment for inhibiting cancer
cachexia is typically a treatment that is given at a stage of the
cancer disease where some cancer cachexia has occurred, in order to
inhibit a further progression of the cancer cachexia. A treatment
for reverting cancer cachexia is typically a treatment that is
started at a stage of the cancer where some cancer cachexia has
occurred, and which reverts the cancer cachexia. The effect of the
treatment can be a partial effect, i.e. a partial prevention, a
partial inhibition or a partial reversion of cancer cachexia, or a
complete effect, i.e. a complete prevention, a complete inhibition
or a complete reversion of cancer cachexia. Preferably, according
to the present invention, the effect of the treatment is a complete
prevention, a complete inhibition or a complete reversion of cancer
cachexia. More preferably, the effect of the treatment according to
the present invention is a complete prevention or a complete
reversion of cancer cachexia.
[0069] As used herein, the term "complete(ly)" in connection with a
treatment of cancer cachexia according to the invention means that
in case of a treatment for preventing, no cancer cachexia occurs in
the treated individual during and/or following the treatment. In
case of a treatment for inhibiting cancer cachexia, the term
"complete(ly)" means that no further progression of the cancer
cachexia occurs in the treated individual during and/or following
the treatment. In case of a treatment for reverting cancer
cachexia, the term "complete(ly)" means that during or following
the treatment, the cancer cachexia is completely reverted such that
no cancer cachexia is present in the treated individual.
[0070] With respect to these effects of the treatment according to
the invention, the term "no cancer cachexia" means that by using
standard methods for measurements and for diagnosis known in the
art, no cancer cachexia is detectable. Likewise, the term "no
further progression of the cancer cachexia" means that by using
standard methods for measurements and for diagnosis known in the
art, no further progression of cancer cachexia is detectable. The
methods known in the art and referred to herein are for instance
described in Fearon K C.: Cancer cachexia: developing multimodal
therapy for a multidimensional problem. Eur J Cancer. 2008 May;
44(8):1124-32; Fearon K. et al.: Definition and classification of
cancer cachexia: an international consensus. Lancet Oncol. 2011
May; 12(5):489-95.; or Tisdale M J.: Mechanisms of cancer cachexia.
Physiol Rev. 2009 April; 89(2):381-410.
[0071] In addition to completely preventing or completely reverting
cancer cachexia, the treatment methods and products for use in
these methods according to the invention may increase the body
weight of the treated mammal compared to its body weight before the
onset of cancer cachexia. As used herein, the term "before the
onset of cancer cachexia" means a point in time during the course
of the cancer disease, after which cancer cachexia becomes
measurable by the methods known in the art such as the methods
referred to above.
[0072] Preferably, the above-defined effects of the cancer cachexia
treatment according to the invention are statistically significant
when assessed against a suitable control group whereas individual
patients who are treated would not show significant cachexia.
[0073] In accordance with the present invention, each occurrence of
the term "comprising" may optionally be substituted with the term
"consisting of".
[0074] Methods and Techniques
[0075] Generally, unless otherwise defined herein, the methods used
in the present invention (e.g. cloning methods or methods relating
to antibodies) are performed in accordance with procedures known in
the art, e.g. the procedures described in Sambrook et al.
("Molecular Cloning: A Laboratory Manual.", 2.sup.nd Ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989),
Ausubel et al. ("Current Protocols in Molecular Biology." Greene
Publishing Associates and Wiley Interscience; New York 1992), and
Harlow and Lane ("Antibodies: A Laboratory Manual" Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1988), all of
which are incorporated herein by reference.
[0076] Molecular weight is measured by methods known in the art
such as mass spectrometry. It is expressed in Dalton (Da) or
Kilodalton (kDa).
[0077] Binding of monoclonal anti-human-GDF-15 antibodies according
to the invention is generally assessed by employing surface plasmon
resonance measurements using a Bio-Rad.RTM. ProteOn.TM. XPR36
system and Bio-Rad.RTM. GLC sensor chips as described for murine
anti-human GDF-15 mAb-B1-23 in Example 1.
[0078] Sequence alignments of sequences according to the invention
are performed by using the BLAST algorithm (see Altschul et al.
(1990) "Basic local alignment search tool." Journal of Molecular
Biology 215. p. 403-410.; Altschul et al.: (1997) Gapped BLAST and
PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res. 25:3389-3402.). Preferably, the following
parameters are used: Max target sequences 10; Word size 3; BLOSUM
62 matrix; gap costs: existence 11, extension 1; conditional
compositional score matrix adjustment. Thus, when used in
connection with sequences, terms such as "identity" or "identical"
refer to the identity value obtained by using the BLAST
algorithm.
[0079] Monoclonal antibodies according to the invention can be
produced by any method known in the art, including but not limited
to the methods referred to in Siegel D L ("Recombinant monoclonal
antibody technology." Transfus Clin Biol. 2002 January;
9(1):15-22.). In a preferred embodiment, an antibody according to
the invention is produced by the hybridoma cell line 81-23
deposited with the Deutsche Sammlung far Mikroorganismen and
Zellkulturen GmbH (DSMZ) under the accession No. DSM ACC3142 under
the Budapest treaty. The deposit was filed on Sep. 29, 2011.
[0080] Cell proliferation can be measured by suitable methods known
in the art, including (but not limited to) visual microscopy,
metabolic assays such as those which measure mitochondrial redox
potential (e.g. MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
assay; Resazurin staining which is also known as Alamar Blue.RTM.
assay), staining of known endogenous proliferation biomarkers (e.g.
Ki-67), and methods measuring cellular DNA synthesis (e.g. BrdU and
[.sup.3H]-Thymidine incorporation assays).
[0081] Immunosuppression can be measured by suitable methods known
in the art, including (but not limited to) immune cell
proliferation, cytokine secretion, intracellular cytokine staining
by flow cytometry, cytokine measurement by qRT-PCR, redirected
target cell lysis, further cytotoxicity or degranulation assays,
downregulation of activating immune cell receptors (like NKG2D),
upregulation of inhibitory immune cell receptors, immunological
synapse formation, immune cell infiltration. For the term
immunosuppression to apply, an effect shall be measurable in at
least one of these or in any other suitable assay. The lack of
effect in a specific test does not imply a general absence of
immunosuppression.
[0082] Human GDF-15 levels can be measured by any method known in
the art, including measurements of GDF-15 mRNA levels by methods
including (but not limited to) quantitative real-time PCR (qRT-PCR)
for human GDF-15 mRNA using primers specific to human GDF-15, mRNA
in situ hybridization with probes specific to human GDF-15, mRNA
deep sequencing methods; and including measurements of GDF-15
protein levels by methods including (but not limited to) mass
spectrometry for proteins or peptides derived from human GDF-15,
Western Blotting using antibodies specific to human GDF-15, flow
cytometry using antibodies specific to human GDF-15, strip tests
using antibodies specific to human GDF-15, or immunocytochemistry
using antibodies specific to human GDF-15. For such methods using
antibodies specific to human GDF-15, the anti-human GDF-15
antibodies of the present invention are preferred, and the antibody
of the invention produced by the hybridoma cell line B1-23
deposited with the Deutsche Sammlung far Mikroorganismen and
Zellkulturen GmbH (DSMZ) under the accession No. DSM ACC3142 is
most preferred.
EMBODIMENTS OF THE INVENTION
[0083] As described above, the inventors show that human GDF-15
protein can be targeted by an antibody in accordance with the
invention in a way that cancer cachexia and cancer-induced weight
loss can be treated and that also cancer growth is inhibited.
[0084] When taking into account the present invention, it becomes
clear that the anti-GDF-15 antibodies known from WO 2005/099746, WO
2009/021293 and Johnen H et al., Nature Medicine, 2007 only inhibit
one of the effects of human GDF-15 (i.e. cancer-induced weight
loss), but fail to inhibit other effects of human GDF-15 such as
those related to cancer growth. In view of the present invention,
one possible explanation for this failure is that the antibodies
known from the above documents may only interfere with transport of
human GDF-15 across the blood-brain barrier (by forming a large
complex that cannot be transported across the blood-brain barrier)
but are incapable of binding human GDF-15 in a way that renders it
generally unable to interact with its receptor (e.g. a receptor
residing on cells outside the brain). Furthermore, and different
from the antibodies of the present invention, the anti-GDF-15
antibodies known from WO 2005/099746, WO 2009/021293 and Johnen H
et al., Nature Medicine, 2007 did not lead to a detectable increase
in the body weight of the mammals compared to its body weight
before the onset of cancer cachexia.
[0085] Accordingly, the effects of the antibodies for use according
to the invention are unexpected in view of the art.
[0086] The following properties of the antibodies of the present
invention are expected to contribute to their capability of
inhibiting the effects of human GDF-15 more completely, including
the treatment of cachexia and the inhibition of cancer growth:
[0087] Broad Binding Specificity to Forms of Human GDF-15
[0088] The antibodies of the present invention are capable of
binding to mature recombinant human GDF-15 (represented by SEQ ID
No: 8) and are therefore capable of binding to active, fully
processed (mature) human GDF-15.
[0089] Additionally, by performing staining experiments with the
murine mAb-B1-23 antibody according to the invention on human
cells, the inventors show that the mAb-B1-23 antibody according to
the invention is capable of binding to the human GDF-15 precursor
on human cells.
[0090] Thus, it is expected that binding and effects of the
antibodies according to the present invention, in particular the
inhibition of cancer growth, are not generally limited to effects
on a particular form of human GDF-15.
[0091] As to the effects of human GDF-15 on cancer cachexia, these
effects may be caused a subset of forms human GDF-15, for instance
to soluble forms human GDF-15, which are capable of passing the
blood-brain barrier. As exemplified in the Examples of the present
invention, all of the tested anti-GDF-15 antibodies according to
the invention can be used to treat cancer-induced cachexia. Thus,
the antibodies according to the present invention can interfere
with the forms of human GDF-15 which are responsible for cancer
cachexia.
[0092] High Binding Affinity
[0093] The antibodies and antigen binding portions thereof
according to the invention have high binding affinity, as
demonstrated by the mAb-B1-23 antibody according to the invention
which has an equilibrium dissociation constant of about 790 pM for
recombinant human GDF-15. Notably, such affinity values are
superior to most of the existing therapeutic antibodies, e.g. to
the therapeutic antibody Rituximab which has an equilibrium
dissociation constant of about 8 nM.
[0094] High binding affinity will ensure that the antibody to human
GDF-15 according to the invention stably binds to human GDF-15,
such that effects of human GDF-15 including effects on cancer
growth are effectively inhibited. Likewise, stable binding of the
antibodies according to the invention is expected to ensure that
forms of human GDF-15 which cause cancer cachexia cannot carry out
their pathological function. This may for instance be due to an
antibody-dependent sequestration of these forms of human GDF-15
from their possible site of action in the brain. Such binding and
sequestration may for instance take place at the site of the
cancer, or the antibodies according to the invention may interfere
with the transport of human-GDF-15 across the blood
brain-barrier.
[0095] Binding to a Discontinuous or Conformational Epitope
[0096] The antibodies and antigen binding portions thereof
according to the invention bind to a discontinuous or
conformational epitope, as demonstrated below for a murine
mAb-B1-23 antibody according to the invention.
[0097] Binding of antibodies and antigen binding portions thereof
according to the invention to a discontinuous or conformational
GDF-15 epitope may help to keep human GDF-15 in a specific
conformation. This conformation-specificity may be advantageous to
keep GDF-15 in a form that cannot be released from the tumor, or
that cannot cross the blood brain-barrier and cause cancer cachexia
at a possible site of action in the brain. Additionally, such
binding to a discontinuous or conformational GDF-15 epitope may
contribute to the effective inhibition of effects of human GDF-15
including effects on cancer growth, e.g. by keeping GDF-15 in a
conformation that cannot functionally interact with its
receptor.
[0098] Thus, the invention relates to the following
embodiments:
A) ANTIBODIES, VECTORS AND CELL LINES
[0099] Concretely, the invention relates to a monoclonal antibody
capable of binding to human GDF-15, or an antigen-binding portion
thereof, wherein the heavy chain variable domain comprises a CDR3
region comprising the amino acid sequence of SEQ ID NO: 5 or an
amino acid sequence at least 90% identical thereto, and wherein the
light chain variable domain comprises a CDR3 region comprising the
amino acid sequence of SEQ ID NO: 7 or an amino acid sequence at
least 85% identical thereto, wherein the constant domain of the
heavy chain comprises the amino acid sequence of SEQ ID No: 29, or
an amino acid sequence at least 85%, preferably at least 90%, more
preferably at least 95% identical thereto, and wherein the constant
domain of the light chain comprises the amino acid sequence of SEQ
ID No: 32, or an amino acid sequence at least 85%, preferably at
least 90%, more preferably at least 95% identical thereto.
[0100] In a preferred aspect of this embodiment, the constant
domain of the heavy chain comprises the amino acid sequence of SEQ
ID No: 29, or an amino acid sequence at least 98%, preferably at
least 99% identical thereto, and the constant domain of the light
chain comprises the amino acid sequence of SEQ ID No: 32, or an
amino acid sequence at least 98%, preferably at least 99% identical
thereto.
[0101] In another preferred aspect of this embodiment, the constant
domain of the heavy chain comprises an amino acid sequence at least
85% identical to the amino acid sequence of SEQ ID No: 29, and the
constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 85%,
preferably at least 90%, more preferably at least 95%, still more
preferably at least 98%, and most preferably at least 99% identical
thereto.
[0102] In another preferred aspect of this embodiment, the constant
domain of the heavy chain comprises an amino acid sequence at least
90% identical to the amino acid sequence of SEQ ID No: 29, and the
constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 85%,
preferably at least 90%, more preferably at least 95%, still more
preferably at least 98%, and most preferably at least 99% identical
thereto.
[0103] In another preferred aspect of this embodiment, the constant
domain of the heavy chain comprises an amino acid sequence at least
95% identical to the amino acid sequence of SEQ ID No: 29, and the
constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 85%,
preferably at least 90%, more preferably at least 95%, still more
preferably at least 98%, and most preferably at least 99% identical
thereto.
[0104] In another preferred aspect of this embodiment, the constant
domain of the heavy chain comprises an amino acid sequence at least
98% identical to the amino acid sequence of SEQ ID No: 29, and the
constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 85%,
preferably at least 90%, more preferably at least 95%, still more
preferably at least 98%, and most preferably at least 99% identical
thereto.
[0105] In another preferred aspect of this embodiment, the constant
domain of the heavy chain comprises an amino acid sequence at least
99% identical to the amino acid sequence of SEQ ID No: 29, and the
constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 85%,
preferably at least 90%, more preferably at least 95%, still more
preferably at least 98%, and most preferably at least 99% identical
thereto.
[0106] In another preferred aspect of this embodiment, the constant
domain of the heavy chain comprises the amino acid sequence of SEQ
ID No: 29, or an amino acid sequence at least 85%, preferably at
least 90%, more preferably at least 95%, still more preferably at
least 98%, and most preferably at least 99% identical thereto, and
the constant domain of the light chain comprises an amino acid
sequence at least 85% identical to the amino acid sequence of SEQ
ID No: 32.
[0107] In another preferred aspect of this embodiment, the constant
domain of the heavy chain comprises the amino acid sequence of SEQ
ID No: 29, or an amino acid sequence at least 85%, preferably at
least 90%, more preferably at least 95%, still more preferably at
least 98%, and most preferably at least 99% identical thereto, and
the constant domain of the light chain comprises an amino acid
sequence at least 90% identical to the amino acid sequence of SEQ
ID No: 32.
[0108] In another preferred aspect of this embodiment, the constant
domain of the heavy chain comprises the amino acid sequence of SEQ
ID No: 29, or an amino acid sequence at least 85%, preferably at
least 90%, more preferably at least 95%, still more preferably at
least 98%, and most preferably at least 99% identical thereto, and
the constant domain of the light chain comprises an amino acid
sequence at least 95% identical to the amino acid sequence of SEQ
ID No: 32.
[0109] In another preferred aspect of this embodiment, the constant
domain of the heavy chain comprises the amino acid sequence of SEQ
ID No: 29, or an amino acid sequence at least 85%, preferably at
least 90%, more preferably at least 95%, still more preferably at
least 98%, and most preferably at least 99% identical thereto, and
the constant domain of the light chain comprises an amino acid
sequence at least 98% identical to the amino acid sequence of SEQ
ID No: 32.
[0110] In another preferred aspect of this embodiment, the constant
domain of the heavy chain comprises the amino acid sequence of SEQ
ID No: 29, or an amino acid sequence at least 85%, preferably at
least 90%, more preferably at least 95%, still more preferably at
least 98%, and most preferably at least 99% identical thereto, and
the constant domain of the light chain comprises an amino acid
sequence at least 99% identical to the amino acid sequence of SEQ
ID No: 32.
[0111] In this embodiment, most preferably, the constant domain of
the heavy chain comprises the amino acid sequence of SEQ ID No: 29,
and the constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32.
[0112] In an alternative embodiment, the invention relates to a
monoclonal antibody capable of binding to human GDF-15, or an
antigen-binding portion thereof, wherein the heavy chain variable
domain comprises a CDR3 region comprising the amino acid sequence
of SEQ ID NO: 5 or an amino acid sequence that differs by not more
than one amino acid from the amino acid sequence of SEQ ID NO: 5,
and wherein the light chain variable domain comprises a CDR3 region
comprising the amino acid sequence of SEQ ID NO: 7 or an amino acid
sequence or an amino acid sequence that differs by not more than
one amino acid from the amino acid sequence of SEQ ID NO: 7,
wherein the constant domain of the heavy chain comprises the amino
acid sequence of SEQ ID No: 29, or an amino acid sequence at least
85%, preferably at least 90%, more preferably at least 95%
identical thereto, and wherein the constant domain of the light
chain comprises the amino acid sequence of SEQ ID No: 32, or an
amino acid sequence at least 85%, preferably at least 90%, more
preferably at least 95% identical thereto.
[0113] In a preferred aspect of this embodiment, the constant
domain of the heavy chain comprises the amino acid sequence of SEQ
ID No: 29, or an amino acid sequence at least 98%, preferably at
least 99% identical thereto, and the constant domain of the light
chain comprises the amino acid sequence of SEQ ID No: 32, or an
amino acid sequence at least 98%, preferably at least 99% identical
thereto.
[0114] In another preferred aspect of this embodiment, the constant
domain of the heavy chain comprises an amino acid sequence at least
85% identical to the amino acid sequence of SEQ ID No: 29, and the
constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 85%,
preferably at least 90%, more preferably at least 95%, still more
preferably at least 98%, and most preferably at least 99% identical
thereto.
[0115] In another preferred aspect of this embodiment, the constant
domain of the heavy chain comprises an amino acid sequence at least
90% identical to the amino acid sequence of SEQ ID No: 29, and the
constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 85%,
preferably at least 90%, more preferably at least 95%, still more
preferably at least 98%, and most preferably at least 99% identical
thereto.
[0116] In another preferred aspect of this embodiment, the constant
domain of the heavy chain comprises an amino acid sequence at least
95% identical to the amino acid sequence of SEQ ID No: 29, and the
constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 85%,
preferably at least 90%, more preferably at least 95%, still more
preferably at least 98%, and most preferably at least 99% identical
thereto.
[0117] In another preferred aspect of this embodiment, the constant
domain of the heavy chain comprises an amino acid sequence at least
98% identical to the amino acid sequence of SEQ ID No: 29, and the
constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 85%,
preferably at least 90%, more preferably at least 95%, still more
preferably at least 98%, and most preferably at least 99% identical
thereto.
[0118] In another preferred aspect of this embodiment, the constant
domain of the heavy chain comprises an amino acid sequence at least
99% identical to the amino acid sequence of SEQ ID No: 29, and the
constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 85%,
preferably at least 90%, more preferably at least 95%, still more
preferably at least 98%, and most preferably at least 99% identical
thereto.
[0119] In another preferred aspect of this embodiment, the constant
domain of the heavy chain comprises the amino acid sequence of SEQ
ID No: 29, or an amino acid sequence at least 85%, preferably at
least 90%, more preferably at least 95%, still more preferably at
least 98%, and most preferably at least 99% identical thereto, and
the constant domain of the light chain comprises an amino acid
sequence at least 85% identical to the amino acid sequence of SEQ
ID No: 32.
[0120] In another preferred aspect of this embodiment, the constant
domain of the heavy chain comprises the amino acid sequence of SEQ
ID No: 29, or an amino acid sequence at least 85%, preferably at
least 90%, more preferably at least 95%, still more preferably at
least 98%, and most preferably at least 99% identical thereto, and
the constant domain of the light chain comprises an amino acid
sequence at least 90% identical to the amino acid sequence of SEQ
ID No: 32.
[0121] In another preferred aspect of this embodiment, the constant
domain of the heavy chain comprises the amino acid sequence of SEQ
ID No: 29, or an amino acid sequence at least 85%, preferably at
least 90%, more preferably at least 95%, still more preferably at
least 98%, and most preferably at least 99% identical thereto, and
the constant domain of the light chain comprises an amino acid
sequence at least 95% identical to the amino acid sequence of SEQ
ID No: 32.
[0122] In another preferred aspect of this embodiment, the constant
domain of the heavy chain comprises the amino acid sequence of SEQ
ID No: 29, or an amino acid sequence at least 85%, preferably at
least 90%, more preferably at least 95%, still more preferably at
least 98%, and most preferably at least 99% identical thereto, and
the constant domain of the light chain comprises an amino acid
sequence at least 98% identical to the amino acid sequence of SEQ
ID No: 32.
[0123] In another preferred aspect of this embodiment, the constant
domain of the heavy chain comprises the amino acid sequence of SEQ
ID No: 29, or an amino acid sequence at least 85%, preferably at
least 90%, more preferably at least 95%, still more preferably at
least 98%, and most preferably at least 99% identical thereto, and
the constant domain of the light chain comprises an amino acid
sequence at least 99% identical to the amino acid sequence of SEQ
ID No: 32.
[0124] In this embodiment, most preferably, the constant domain of
the heavy chain comprises the amino acid sequence of SEQ ID No: 29,
and the constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32.
[0125] Further, a monoclonal antibody capable of binding to human
GDF-15, or an antigen-binding portion thereof is provided, wherein
the heavy chain variable domain comprises a CDR3 region comprising
the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence
at least 90% identical thereto, and wherein the light chain
variable domain comprises a CDR3 region comprising the amino acid
sequence of SEQ ID NO: 7 or an amino acid sequence at least 85%
identical thereto. Preferably, the constant domain of the heavy
chain of this monoclonal antibody or antigen-binding portion
thereof comprises the amino acid sequence of SEQ ID No: 29, or an
amino acid sequence at least 85%, preferably at least 90%, more
preferably at least 95% identical thereto, and the constant domain
of the light chain of this monoclonal antibody or antigen-binding
portion thereof comprises the amino acid sequence of SEQ ID No: 32,
or an amino acid sequence at least 85%, preferably at least 90%,
more preferably at least 95% identical thereto. More preferably,
the constant domain of the heavy chain comprises the amino acid
sequence of SEQ ID No: 29, or an amino acid sequence at least 98%,
preferably at least 99% identical thereto, and the constant domain
of the light chain comprises the amino acid sequence of SEQ ID No:
32, or an amino acid sequence at least 98%, preferably at least 99%
identical thereto. Still more preferably, the constant domain of
the heavy chain comprises the amino acid sequence of SEQ ID No: 29,
and the constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32.
[0126] Further, a monoclonal antibody capable of binding to human
GDF-15, or an antigen-binding portion thereof is provided, wherein
the heavy chain variable domain comprises a CDR3 region comprising
the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence
that differs by not more than one amino acid from the amino acid
sequence of SEQ ID NO: 5, and wherein the light chain variable
domain comprises a CDR3 region comprising the amino acid sequence
of SEQ ID NO: 7 or an amino acid sequence or an amino acid sequence
that differs by not more than one amino acid from the amino acid
sequence of SEQ ID NO: 7. Preferably, the constant domain of the
heavy chain of this monoclonal antibody or antigen-binding portion
thereof comprises the amino acid sequence of SEQ ID No: 29, or an
amino acid sequence at least 85%, preferably at least 90%, more
preferably at least 95% identical thereto, and the constant domain
of the light chain of this monoclonal antibody or antigen-binding
portion thereof comprises the amino acid sequence of SEQ ID No: 32,
or an amino acid sequence at least 85%, preferably at least 90%,
more preferably at least 95% identical thereto. More preferably,
the constant domain of the heavy chain comprises the amino acid
sequence of SEQ ID No: 29, or an amino acid sequence at least 98%,
preferably at least 99% identical thereto, and the constant domain
of the light chain comprises the amino acid sequence of SEQ ID No:
32, or an amino acid sequence at least 98%, preferably at least 99%
identical thereto. Still more preferably, the constant domain of
the heavy chain comprises the amino acid sequence of SEQ ID No: 29,
and the constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32.
[0127] In a second embodiment in accordance with the above
embodiments, the heavy chain variable domain of the monoclonal
antibody or antigen-binding portion thereof comprises a CDR3 region
comprising the amino acid sequence of SEQ ID NO: 5, or the light
chain variable domain comprises a CDR3 region comprising the amino
acid sequence of SEQ ID NO: 7.
[0128] In a third embodiment in accordance with the above
embodiments, the heavy chain variable domain of the monoclonal
antibody or antigen-binding portion thereof comprises a CDR3 region
comprising the amino acid sequence of SEQ ID NO: 5, and the light
chain variable domain comprises a CDR3 region comprising the amino
acid sequence of SEQ ID NO: 7.
[0129] In a fourth embodiment in accordance with the above
embodiments, the heavy chain variable domain of the monoclonal
antibody or antigen-binding portion thereof comprises a CDR1 region
comprising the amino acid sequence of SEQ ID NO: 3 and a CDR2
region comprising the amino acid sequence of SEQ ID NO: 4, and the
light chain variable domain of the monoclonal antibody or
antigen-binding portion thereof comprises a CDR1 region comprising
the amino acid sequence of SEQ ID NO: 6 and a CDR2 region
comprising the amino acid sequence ser-ala-ser.
[0130] In a fifth embodiment in accordance with the above
embodiments, the antibody is a humanized antibody. Preferably, all
of the variable domains of the humanized antibody are humanized
variable domains.
[0131] In a further embodiment in accordance with the above
embodiments, the heavy chain variable domain of the monoclonal
antibody or antigen-binding portion thereof comprises the amino
acid sequence of SEQ ID No: 28, or an amino acid sequence at least
90%, preferably at least 95%, more preferably at least 98%, still
more preferably at least 99% identical thereto, and the light chain
variable domain of the monoclonal antibody or antigen-binding
portion thereof comprises the amino acid sequence of SEQ ID No: 31,
or an amino acid sequence at least 90%, preferably at least 95%,
more preferably at least 98%, still more preferably at least 99%
identical thereto. In the most preferred aspect of this embodiment,
the heavy chain variable domain comprises the amino acid sequence
of SEQ ID No: 28, and the light chain variable domain comprises the
amino acid sequence of SEQ ID No: 31.
[0132] In a further preferred embodiment in accordance with the
above embodiments, the heavy chain of the monoclonal antibody or
antigen-binding portion thereof comprises the amino acid sequence
of SEQ ID No: 27, and the light chain of the monoclonal antibody or
antigen-binding portion thereof comprises the amino acid sequence
of SEQ ID No: 30.
[0133] In a another preferred embodiment in accordance with the
above embodiments, the heavy chain variable domain of the
monoclonal antibody or antigen-binding portion thereof comprises
the amino acid sequence of SEQ ID No: 34, or an amino acid sequence
at least 75%, more preferably at least 80%, more preferably at
least 85%, more preferably at least 90%, more preferably at least
95%, more preferably at least 98%, still more preferably at least
99% identical thereto, and the light chain variable domain of the
monoclonal antibody or antigen-binding portion thereof comprises
the amino acid sequence of SEQ ID No: 37, or an amino acid sequence
at least 80%, more preferably at least 85%, more preferably at
least 90%, more preferably at least 95%, more preferably at least
98%, still more preferably at least 99% identical thereto. In the
most preferred aspect of this embodiment in accordance with the
above embodiments, the heavy chain variable domain comprises the
amino acid sequence of SEQ ID No: 34, and the light chain variable
domain comprises the amino acid sequence of SEQ ID No: 37.
[0134] In still another embodiment in accordance with the above
first to third embodiment, the heavy chain variable domain
comprises a region comprising an FR1, a CDR1, an FR2, a CDR2 and an
FR3 region and comprising the amino acid sequence of SEQ ID NO: 1
or a sequence 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identical thereto, and the light chain variable domain
comprises a region comprising an FR1, a CDR1, an FR2, a CDR2 and an
FR3 region and comprising the amino acid sequence of SEQ ID NO: 2
or a sequence 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99.COPYRGT.s identical thereto.
[0135] In a preferred embodiment in accordance with the above first
to third embodiment, the heavy chain variable domain comprises a
region comprising an FR1, a CDR1, an FR2, a CDR2 and an FR3 region
and comprising the amino acid sequence of SEQ ID NO: 1 or a
sequence 95% identical thereto, and the light chain variable domain
comprises a region comprising an FR1, a CDR1, an FR2, a CDR2 and an
FR3 region and comprising the amino acid sequence of SEQ ID NO: 2
or a sequence 95% identical thereto.
[0136] In a more preferred embodiment in accordance with the above
first to third embodiment, the heavy chain variable domain
comprises a region comprising an FR1, a CDR1, an FR2, a CDR2 and an
FR3 region and comprising the amino acid sequence of SEQ ID NO: 1
or a sequence 98% identical thereto, and the light chain variable
domain comprises a region comprising an FR1, a CDR1, an FR2, a CDR2
and an FR3 region and comprising the amino acid sequence of SEQ ID
NO: 2 or a sequence 98% identical thereto.
[0137] In a still more preferred embodiment in accordance with the
above first to third embodiment, the heavy chain variable domain
comprises a region comprising an FR1, a CDR1, an FR2, a CDR2 and an
FR3 region and comprising the amino acid sequence of SEQ ID NO: 1,
and the light chain variable domain comprises a region comprising
an FR1, a CDR1, an FR2, a CDR2 and an FR3 region and comprising the
amino acid sequence of SEQ ID NO: 2.
[0138] Further, a monoclonal antibody capable of binding to human
GDF-15, or an antigen-binding portion thereof is provided, wherein
the heavy chain variable domain comprises a CDR1 region comprising
the amino acid sequence of SEQ ID NO: 3 and a CDR2 region
comprising the amino acid sequence of SEQ ID NO: 4, and wherein the
light chain variable domain comprises a CDR1 region comprising the
amino acid sequence of SEQ ID NO: 6 and a CDR2 region comprising
the amino acid sequence of SEQ ID NO: 7. In a preferred aspect of
this embodiment, the antibody may have CDR3 sequences as defined in
any of the embodiments of the invention described above.
[0139] In another embodiment, the a monoclonal antibody capable of
binding to human GDF-15, or an antigen-binding portion thereof is
provided, wherein the antibody or antigen-binding portion thereof
is capable of inhibiting cancer growth in a mammal, preferably a
human patient.
[0140] In another embodiment in accordance with the above
embodiments, the invention relates to an antigen-binding portion
capable of binding to human GDF-15, wherein the antigen-binding
portion is a single-domain antibody (also referred to as
"Nanobody.TM."). In one aspect of this embodiment, the
single-domain antibody comprises the CDR1, CDR2, and CDR3 amino
acid sequences of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5,
respectively. In another aspect of this embodiment, the
single-domain antibody comprises the CDR1, CDR2, and CDR3 amino
acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 7,
respectively. In a preferred aspect of this embodiment, the
single-domain antibody is a humanized antibody.
[0141] Preferably, the antibodies of the invention capable of
binding to human GDF-15 or the antigen-binding portions thereof
have an equilibrium dissociation constant for human GDF-15 that is
equal to or less than 100 nM, less than 20 nM, preferably less than
10 nM, more preferably less than 5 nM and most preferably between
0.1 nM and 2 nM.
[0142] In another embodiment of the invention, the antibody capable
of binding to human GDF-15 or the antigen-binding portion thereof
binds to the same human GDF-15 epitope as the antibody to human
GDF-15 obtainable from the cell line B1-23 deposited with the
Deutsche Sammlung fur Mikroorganismen und Zellkulturen GmbH (DMSZ)
under the accession No. DSM ACC3142. As described herein, antibody
binding to human GDF-15 in accordance with the present invention is
assessed by surface plasmon resonance measurements as a reference
standard method, in accordance with the procedures described in
Example 1. Binding to the same epitope on human GDF-15 can be
assessed similarly by surface plasmon resonance competitive binding
experiments of the antibody to human GDF-15 obtainable from the
cell line B1-23 and the antibody that is expected to bind to the
same human GDF-15 epitope as the antibody to human GDF-15
obtainable from the cell line B1-23.
[0143] In a very preferred embodiment, the antibody is the
monoclonal antibody capable of binding to human GDF-15 obtainable
from the cell line B1-23 deposited with the Deutsche Sammlung fur
Mikroorganismen und Zellkulturen GmbH (DMSZ) under the accession
No. DSM ACC3142 or an antigen-binding portion thereof.
[0144] In a preferred embodiment, the antibody capable of binding
to human GDF-15 or the antigen-binding portion thereof according to
the invention is a humanized monoclonal antibody or an
antigen-binding portion thereof. For any given non-human antibody
sequence in accordance with the invention (i.e. a donor antibody
sequence), humanized monoclonal anti-human-GDF-15 antibodies of the
invention or antigen-binding portions thereof can be generated in
accordance with techniques known in the art, as described
above.
[0145] In a very preferred embodiment, the monoclonal antibody
capable of binding to human GDF-15 or antigen-binding portion
thereof is a humanized antibody derived from the monoclonal
antibody capable of binding to human GDF-15 obtainable from the
cell line B1-23 deposited with the Deutsche Sammlung fur
Mikroorganismen and Zeilkulturen GmbH (DMSZ) under the accession
No. DSM ACC3142, or an antigen-binding portion thereof. In a
non-limiting aspect of this embodiment, the heavy chain variable
domain of the humanized antibody or antigen-binding portion thereof
comprises a CDR3 region comprising the amino acid sequence of SEQ
ID NO: 5, and the light chain variable domain of the humanized
antibody or antigen-binding portion thereof comprises a CDR3 region
comprising the amino acid sequence of SEQ ID NO: 7. In a further
non-limiting aspect of this embodiment, the heavy chain variable
domain of the humanized antibody or antigen-binding portion thereof
comprises or further comprises a CDR1 region comprising the amino
acid sequence of SEQ ID NO: 3 and a CDR2 region comprising the
amino acid sequence of SEQ ID NO: 4, and the light chain variable
domain of the humanized antibody or antigen-binding portion thereof
comprises or further comprises a CDR1 region comprising the amino
acid sequence of SEQ ID NO: 6 and a CDR2 region comprising the
amino acid sequence of SEQ ID NO: 7.
[0146] Further, a monoclonal antibody capable of binding to human
GDF-15, or an antigen-binding portion thereof is provided, wherein
the binding is binding to a conformational or discontinuous epitope
on human GDF-15 comprised by the amino acid sequences of SEQ ID No:
25 and SEQ ID No: 26. In a preferred aspect of this embodiment, the
antibody or antigen-binding portion thereof is an antibody or
antigen-binding portion thereof as defined in any one of the above
embodiments.
[0147] In another embodiment of the invention in accordance with
the above embodiments, the antibody capable of binding to human
GDF-15 or the antigen-binding portion thereof is a diabody. In one
aspect of this embodiment, the diabody is bivalent and
monospecific, with two identical antigen binding sites for human
GDF-15. In a second, alternative aspect of this embodiment, the
diabody is bivalent and bispecific, with one antigen binding site
being a binding site for human GDF-15, and the other antigen
binding site being a binding site for a different antigen.
Non-limiting examples for the different antigen according to this
second aspect of this embodiment are i) cell surface antigens that
are co-expressed with GDF-15 at high levels on the same cancer
(e.g. at higher levels compared to a control sample of the same
patient obtained from a non-cancerous part of the tissue which is
the tissue of origin of the cancer), and ii) cell surface antigens
on cells of the immune system which are known as useful antigens
for the recruitment of cells of the immune system to the tumor.
[0148] In still another embodiment of the invention in accordance
with the above embodiments, the antibody capable of binding to
human GDF-15 or the antigen-binding portion thereof is linked to a
drug. In non-limiting aspects of this embodiment, the drug can be a
known anticancer agent and/or an immune-stimulatory molecule. Known
anticancer agents include alkylating agents such as cisplatin,
carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide,
chlorambucil, and ifosfamide; anti-metabolites such as azathioprine
and mercaptopurine; alkaloids such as vinca alkaloids (e.g.
vincristine, vinblastine, vinorelbine, and vindesine), taxanes
(e.g. paclitaxel, docetaxel) etoposide and teniposide;
topoisomerase inhibitors such as camptothecins (e.g. irinotecan and
topotecan); cytotoxic antibiotics such as actinomycin,
anthracyclines, doxorubicin, daunorubicin, valrubicin, idarubicin,
epirubicin, bleomycin, plicamycin and mitomycin; and radioisotopes.
Linking of the antibodies or the antigen-binding portions thereof
of the invention to anticancer agents is expected to result in
stronger cancer tumor growth inhibition compared to the antibody
without the anticancer agent, because the resulting conjugate will
accumulate at the site of the tumor due to the presence of GDF-15
in the tumor, leading to the accumulation of the anticancer agent
at the site of the tumor and to enhanced effects of the anticancer
agent on the tumor.
[0149] In a further embodiment in accordance with the above
embodiments, the antibody capable of binding to human GDF-15 or the
antigen-binding portion thereof is modified by an amino acid tag.
Non-limiting examples of such tags include Polyhistidin (His-)
tags, FLAG-tag, Hemagglutinin (HA) tag, glycoprotein D (gD) tag,
and c-myc tag. Tags may be used for various purposes. For instance,
they may be used to assist purification of the antibody capable of
binding to human GDF- or the antigen-binding portion thereof, or
they may be used for detection of the antibody or the
antigen-binding portion thereof (e.g. when used in diagnostic
assays). Preferably, such tags are present at the C-terminus or
N-terminus of the antibody capable of binding to human GDF-15 or
the antigen-binding portion thereof.
[0150] In a preferred embodiment of the present invention in
accordance with the above embodiments, the antibody capable of
binding to human GDF-15 or the antigen-binding portion thereof is
capable of inhibiting cancer growth in a mammal, preferably a human
patient.
[0151] In another preferred embodiment of the present invention in
accordance with the above embodiments, the human GDF-15 is
recombinant human. GDF-15 having the amino acid sequence
represented by SEQ ID No: 8.
[0152] In still another preferred embodiment of the present
invention in accordance with the above embodiments, the binding of
the antibody capable of binding to human GDF-15 or the
antigen-binding portion thereof is a binding to a conformational or
discontinuous epitope on human GDF-15.
[0153] Preferably, the monoclonal antibodies of the present
invention capable of binding to human GDF-15 or the antigen-binding
portions thereof are isolated antibodies.
[0154] In a preferred embodiment of the above antibodies or
antigen-binding portions thereof according to the invention, the
antibody has a size of more than 100 kDa, preferably more than 110
kDa, more preferably more than 120 kDa, still more preferably more
than 130 kDa, and most preferably more than 140 kDa. Preferably,
the antibody is a full-length antibody, more preferably a
full-length IgG antibody.
[0155] The invention also relates to an expression vector
comprising a nucleotide sequence encoding the antibody or
antigen-binding portion thereof as defined above.
[0156] Further, the present invention also provides a cell line
capable of producing an antibody or antigen-binding portion thereof
according to the present invention.
[0157] In one embodiment, the cell line can be derived from any
cell line that is known in that art and suitable for the production
of antibodies or antigen-binding portions thereof.
[0158] In a preferred embodiment, the cell line is the cell line
B1-23 deposited with the Deutsche Sammlung fur Mikroorganismen and
Zellkulturen GmbH (DMSZ) under the accession No. DSM ACC3142.
[0159] In another preferred embodiment, the cell line contains an
expression vector according to the invention as defined above.
B) PHARMACEUTICAL COMPOSITIONS
[0160] In a further embodiment, the present invention relates to a
pharmaceutical composition comprising any of the antibodies or
antigen-binding portions thereof as defined above.
[0161] Pharmaceutical compositions in accordance with the present
invention are prepared in accordance with known standards for the
preparation of pharmaceutical compositions containing antibodies
and portions thereof.
[0162] For instance, the compositions are prepared in a way that
they can be stored and administered appropriately, e.g. by using
pharmaceutically acceptable components such as carriers, excipients
or stabilizers.
[0163] Such pharmaceutically acceptable components are not toxic in
the amounts used when administering the pharmaceutical composition
to a patient. The pharmaceutical acceptable components added to the
pharmaceutical compositions may depend on the particular intended
use of the pharmaceutical compositions and the route of
administration.
[0164] In general, the pharmaceutically acceptable components used
in connection with the present invention are used in accordance
with knowledge available in the art, e.g. from Remington's
Pharmaceutical Sciences, Ed. A R Gennaro, 20th edition, 2000,
Williams & Wilkins, PA, USA.
C) THERAPEUTIC METHODS AND PRODUCTS FOR USE IN THESE METHODS
[0165] The present invention further relates to a method for
treating cancer cachexia in a mammal. The method comprises
administering an antibody or antigen-binding portion thereof as
defined above, or a pharmaceutical composition as defined above to
said mammal. Alternatively, the present invention relates to an
antibody or antigen-binding portion thereof as defined above, or a
pharmaceutical composition as defined above for use in these
methods. In a very preferred aspect of these embodiments, the
mammal is a human patient.
[0166] The present invention further relates to a method for
treating cancer in a mammal. The method comprises administering an
antibody or antigen-binding portion thereof as defined above, or a
pharmaceutical composition as defined above to said mammal.
Alternatively, the present invention relates to an antibody or
antigen-binding portion thereof as defined above, or a
pharmaceutical composition as defined above for use in these
methods. In a very preferred aspect of these embodiments, the
mammal is a human patient.
[0167] When taking into account the present invention, it becomes
clear that the anti-GDF-15 antibodies known from WO 2005/099746, WO
2009/021293 and Johnen H et al., Nature Medicine, 2007 only inhibit
cancer-induced weight loss, but fail to inhibit other effects of
human GDF-15 such as those related to cancer growth.
[0168] The present invention relates to several surprising
advantages compared to the effects observed in the art.
[0169] In particular, one main benefit of the invention lies in
that the anti-GDF-15 antibodies disclosed herein can be used to
more effectively treat cancer-induced weight loss and/or cancer
cachexia.
[0170] For instance, the treatment with the antibodies according to
the invention can completely prevent cancer cachexia (when given
prophylactically) or completely reverse cancer cachexia (when given
after the onset of cancer cachexia).
[0171] Moreover, the antibodies according to the invention can even
increase the body weight of the treated mammal during a
prophylactic treatment for the prevention of cachexia. Likewise, it
is expected that in the course of a therapeutic treatment started
after the onset of cancer cachexia, the antibodies according to the
invention can not only reverse the loss in body weight, but also
increase the body weight of the treated mammal compared to its body
weight before the onset of cancer cachexia.
[0172] This unexpected effect of the antibodies according to the
invention may be beneficial in various clinical situations. For
instance, administration of many ingredients that are
pharmaceutically active against cancer (e.g. various
chemotherapeutic drugs) can lead to a loss of body weight of
mammals including human patients. Such an additional loss in body
weight could be counteracted by the increase in body weight due to
the administration of the antibodies according to the invention.
Therefore, the uses of the antibodies according to the invention
may be particularly advantageous and safe for combination regimens
with additional chemotherapeutic drugs. Similarly, the uses of the
antibodies according to the invention may be particularly
advantageous for mammals such as human patients that already had a
low body weight prior to the onset of cancer and/or prior to the
onset of cancer cachexia. Patients with a low body weight may for
instance be cachectic patients, e.g. patients with a
body-mass-index of less than 18 kg/m.sup.2.
[0173] Moreover, unexpectedly, according to the invention, the
antibodies are not only effective for the treatment of cancer
cachexia, but also effective for the treatment of cancer.
[0174] Thus, the treatment methods and products for use of the
antibodies according to the invention are expected to be
particularly beneficial for the treatment of cancer patient
sub-groups which suffer from cancer-induced weight loss and/or
cancer cachexia, respectively.
[0175] However, the effects according to the invention are also
expected to be advantageous for the treatment of a complete patient
group of a cancer referred to herein: By using the antibodies
according to the invention that are effective both against the
cancer itself and against cancer-induced weight loss and/or cancer
cachexia, cancer and cancer cachexia treatments may be simplified
by using the same treatment for all cancer patients, irrespective
of whether or not they suffer from cancer-induced weight loss
and/or cancer cachexia. This is because due to the dual effects of
the antibodies against cancer and cancer cachexia, it is expected
that these antibodies will obviate the need for additional drugs
for the treatment of cancer cachexia.
[0176] Likewise, due to the dual effects of the antibodies in
accordance with the invention, it may also become unnecessary to
diagnose cancer-induced weight loss and/or cancer cachexia. Hence
it is expected that the overall costs of therapy and diagnosis will
be reduced.
[0177] Therefore, in a preferred embodiment of the above methods,
or antibodies, antigen-binding portions thereof or pharmaceutical
compositions for use in these methods according to the invention,
the method for treating cancer cachexia is a method for completely
preventing or completely reverting cancer cachexia. In a more
preferred embodiment of this method, or the antibodies,
antigen-binding portions thereof or pharmaceutical compositions for
use in this method, the method for treating cancer cachexia is a
method for completely preventing cancer cachexia. In an alternative
more preferred embodiment of this method, or the antibodies,
antigen-binding portions thereof or pharmaceutical compositions for
use in this method, the method for treating cancer cachexia is a
method for completely reverting cancer cachexia.
[0178] In a preferred embodiment of the above methods, or
antibodies, antigen-binding portions thereof or pharmaceutical
compositions for use in these methods according to the invention,
only mammals suffering from both
[0179] i) the cancer, and
[0180] ii) cancer cachexia [0181] are treated in the method.
[0182] In a preferred embodiment of the above methods, or
antibodies, antigen-binding portions thereof or pharmaceutical
compositions for use in these methods according to the invention,
the method increases body weight of the mammal compared to its body
weight before the onset of cancer cachexia. Preferably, the
increase in body weight of the mammal is at least 1.5%, preferably
at least 2.5%, more preferably at least 5% compared to its body
weight before the onset of cancer cachexia.
[0183] In a preferred embodiment of the above methods, or
antibodies, antigen-binding portions thereof or pharmaceutical
compositions for use in these methods according to the invention,
the method is a method for both treating cancer and treating cancer
cachexia in the same mammal.
[0184] In a preferred embodiment of the above methods, or
antibodies, antigen-binding portions thereof or pharmaceutical
compositions for use in these methods according to the invention,
the antibody has a size of more than 100 kDa, preferably more than
110 kDa, more preferably more than 120 kDa, still more preferably
more than 130 kDa, and most preferably more than 140 kDa.
Preferably, the antibody is a full-length antibody, more preferably
a full-length IgG antibody.
[0185] In a further preferred embodiment of the above methods, or
antibodies, antigen-binding portions thereof or pharmaceutical
compositions for use in these methods according to the invention,
the antibody has an Fc portion which is capable of binding to the
Fc receptor.
[0186] In a preferred embodiment of the above methods, or
antibodies, antigen-binding portions thereof or pharmaceutical
compositions for use in these methods according to the invention,
the cancer cells of the mammal endogenously express GDF-15 and/or
the cancer cells of the mammal stimulate endogenous expression of
GDF-15 in non-cancerous cells of the mammal.
[0187] In a preferred embodiment of the above methods, or
antibodies, antigen-binding portions thereof or pharmaceutical
compositions for use in these methods according to the invention,
the cancer cells of the mammal are characterized in that they
endogenously express GDF-15.
[0188] In a preferred embodiment of the above methods, or
antibodies, antigen-binding portions thereof or pharmaceutical
compositions for use in these methods according to the invention,
the mammal is human patient.
[0189] In a preferred embodiment of the above methods, or
antibodies, antigen-binding portions thereof or pharmaceutical
compositions for use in these methods according to the invention,
the human GDF-15 is recombinant human GDF-15 having the amino acid
sequence represented by SEQ ID No: 8.
[0190] In a preferred embodiment of the above methods, or
antibodies, antigen-binding portions thereof or pharmaceutical
compositions for use in these methods, the human patient has
elevated GDF-15 levels in blood serum before administration. In a
patient sub-group having elevated GDF-15 levels in blood serum, the
treatment methods according to the invention are expected to be
particularly effective at inhibiting cancer growth. In the most
preferred aspect of this embodiment, GDF-15 levels are GDF-15
protein levels measured using the antibody according to the
invention obtainable from the hybridoma cell line B1-23 deposited
with the Deutsche Sammlung fur Mikroorganismen and Zellkulturen
GmbH (DSMZ) under the accession No. DSM ACC3142, preferably
measured by immunochemistry.
[0191] In another embodiment of the above methods, or antibodies,
antigen-binding portions thereof or pharmaceutical compositions for
use in these methods, the antibody or antigen-binding portion
thereof is the sole ingredient pharmaceutically active against
cancer used in the method.
[0192] In an alternative embodiment of the above methods, or
antibodies, antigen-binding portions thereof or pharmaceutical
compositions for use in these methods, the antibody or
antigen-binding portion thereof is used in combination with one or
more further ingredients pharmaceutically active against cancer. In
one aspect of this embodiment, the one or more further ingredients
pharmaceutically active against cancer is a known anticancer agent
and/or an immune-stimulatory molecule as defined above. Thus, the
anticancer agent can for instance be selected from alkylating
agents such as cisplatin, carboplatin, oxaliplatin,
mechlorethamine, cyclophosphamide, chlorambucil, and Ifosfamide;
anti-metabolites such as azathioprine and mercaptopurine; alkaloids
such as vinca alkaloids (e.g. vincristine, vinblastine,
vinorelbine, and vindesine), taxanes (e.g. paclitaxel, docetaxel)
etoposide and teniposide; topoisomerase inhibitors such as
camptothecins (e.g. irinotecan and topotecan); cytotoxic
antibiotics such as actinomycin, anthracyclines, doxorubicin,
daunorubicin, valrubicin, idarubicin, epirubicin, bleomycin,
plicamycin and mitomycin; and radioisotopes. Due to the increasing
effect of the antibodies according to the invention on body weight
of the mammals including human patients, these combined uses of the
antibodies or antigen-binding portions thereof and the ingredients
pharmaceutically active against cancer are expected to be
particularly safe, because they may compensate a possible
additional weight loss resulting from the administration of the
ingredients pharmaceutically active against cancer.
[0193] In a preferred embodiment of the above methods, or
antibodies, antigen-binding portions thereof or pharmaceutical
compositions for use in these methods, the cancer is selected from
the group consisting of brain cancers including glioma, cancers of
the nervous system, melanoma, lung cancer, lip and oral cavity
cancer, hepatic carcinoma, leukemia, Hodgkin lymphoma, Non-Hodgkin
lymphoma, bladder cancer, cervix uteri cancer, corpus uteri cancer,
testis cancer, thyroid cancer, kidney cancer, gallbladder cancer,
multiple myeloma, nasopharynx cancer, larynx cancer, pharynx
cancer, oesophagus cancer, gastrointestinal tumors including
stomach and colorectal cancer, pancreatic cancer, prostate cancer,
ovarian cancer and breast cancer, preferably from the group
consisting of melanoma, prostate cancer, breast cancer, brain
cancers including glioma, colorectal cancer, stomach cancer,
oesophagus cancer and ovarian cancer, and most preferably is
melanoma. In one embodiment the cancer is selected from the above
group, which further comprises endometrial cancer, such as
endometrial carcinoma, breast cancer including subtypes of breast
cancer, in particular triple-negative breast cancer and bladder
cancer such as urothelial cell carcinoma.
[0194] In another preferred embodiment of the above methods, or
antibodies, antigen-binding portions thereof or pharmaceutical
compositions for use in these methods, the tumor or tumors formed
by the cancer have higher human GDF-15 levels prior to
administration compared to a control sample of the same patient
obtained from a non-cancerous part of the tissue which is the
tissue of origin of the cancer, preferably 1.2-fold higher levels,
more preferably 1.5-fold higher levels, still more preferably
2-fold higher levels and most preferably 5-fold higher levels. In a
patient sub-group having higher GDF-15 levels in the tumor or
tumors formed by the cancer compared to the above control sample,
the treatment methods according to the invention are expected to be
particularly effective at inhibiting cancer growth.
[0195] In a very preferred embodiment of the above methods, or
antibodies, antigen-binding portions thereof or pharmaceutical
compositions for use in these methods, the method for treating
cancer comprises inhibiting cancer growth. In a preferred aspect of
this embodiment, cancer growth is stopped. In a more preferred
aspect, the cancer shrinks.
[0196] In a preferred embodiment of the above methods, or
antibodies, antigen-binding portions thereof or pharmaceutical
compositions for use in these methods, the method for treating
cancer comprises the induction of killing of cancer cells by NK
cells and CD8+ T cells in the human patient. Due to their
capability of preventing GDF-15 mediated down-regulation of the
known immune surveillance regulator NKG2D, the antibodies or
antigen-binding portions thereof according to the invention are
expected to restore immune surveillance and induce the killing of
cancer cells by NK cells and CD8+ T cells, in addition to effects
of the antibodies or antigen-binding portions thereof that are
independent of the immune system.
D) KITS
[0197] The present invention also provides kits comprising the
pharmaceutical compositions as defined above.
[0198] In one embodiment, the kits are kits for use in the methods
according to the invention as defined above.
[0199] In further embodiments, the present invention also provides
a diagnostic kit comprising any of the antibodies or
antigen-binding portions thereof according to the invention.
[0200] In one embodiment, the diagnostic kit may be used to detect
whether the tumor or tumors of a cancer patient formed by the
cancer have higher human GDF-15 levels compared to a control sample
of the same patient obtained from a non-cancerous part of the
tissue which is the tissue of origin of the cancer.
[0201] In another embodiment, the diagnostic kit may be used to
detect whether a human cancer patient has elevated GDF-15 levels in
blood serum.
E) SEQUENCES
[0202] The amino acid sequences referred to in the present
application are as follows (in an N-terminal to C-terminal order;
represented in the one-letter amino acid code):
[0203] SEQ ID No: 1 (Region of the Heavy Chain Variable Domain
comprising an FR1, a CDR1, an FR2, a CDR2 and an FR3 region from
the Polypeptide Sequence of monoclonal anti-human GDF-15
mAb-B1-23):
TABLE-US-00001 QVKLQQSGPGILQSSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWL
AHIYWDDDKRYNPTLKSRLTISKDPSRNQVFLKITSVDTADTATYYC
[0204] SEQ ID No: 2 (Region of the Light Chain Variable Domain
comprising an FR1, a CDR1, an FR2, a CDR2 and an FR3 region from
the Polypeptide Sequence of monoclonal anti-human GDF-15
mAb-B1-23):
TABLE-US-00002 DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWFLQKPGQSPKALIYS
ASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC
[0205] SEQ ID No: 3 (Heavy Chain CDR1 Region Peptide Sequence of
monoclonal anti-human GDF-15 mAb-B1-23):
TABLE-US-00003 GFSLSTSGMG
[0206] SEQ ID No: 4 (Heavy Chain CDR2 Region Peptide Sequence of
monoclonal anti-human GDF-15 mAb-B1-23):
TABLE-US-00004 IYWDDDK
[0207] SEQ ID No: 5 (Heavy Chain CDR3 Region Peptide Sequence of
monoclonal anti-human GDF-15 mAb-B1-23):
TABLE-US-00005 ARSSYGAMDY
[0208] SEQ ID No; 6 (Light Chain CDR1 Region Peptide Sequence of
monoclonal anti-human GDF-15 mAb-B1-23):
TABLE-US-00006 QNVGTN
[0209] Light Chain CDR2 Region Peptide Sequence of monoclonal
anti-human GDF-15 mAb-B1-23:
TABLE-US-00007 SAS
[0210] SEQ ID No: 7 (Light Chain CDR3 Region Peptide Sequence of
monoclonal anti-human GDF-15 mAb-B1-23):
TABLE-US-00008 QQYNNFPYT
[0211] SEQ ID No: 8 (recombinant mature human GDF-15 protein):
TABLE-US-00009 GSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGAC
PSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSL
QTYDDLLAKDCHCI
[0212] SEQ ID No: 9 (human GDF-15 precursor protein):
TABLE-US-00010 MPGQELRTVNGSQMLLVLLVLSWLPHGGALSLAEASRASFPGPSELHSED
SRFRELRKRYEDLLTRLRANQSWEDSNTDLVPAPAVRILTPEVRLGSGGH
LHLRISRAALPEGLPEASRLHRALFRLSPTASRSWDVTRPLRRQLSLARP
QAPALHLRLSPPPSQSDQLLAESSSARPQLELHLRPQAARGRRRARARNG
DHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRA
ANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDL LAKDCHCI
[0213] SEQ ID No: 10 (human GDF-15 precursor protein N-terminal and
C-terminal GSGS linker):
TABLE-US-00011 GSGSGSGMPGQELRTVNGSQMLLVLLVLSWLPHGGALSLAEASRASFPGP
SELHSEDSRFRELRKRYEDLLTRLRANQSWEDSNTDLVPAPAVRILTPEV
RLGSGGHLHLRISRAALPEGLPEASRLHRALFRLSPTASRSWDVTRPLRR
QLSLARPQAPALHLRLSPPPSQSDQLLAESSSARPQLELHLRPQAARGRR
RARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGA
CPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVS
LQTYDDLLAKDCHCIGSGSGSG
TABLE-US-00012 DYKDDDDKGG
[0214] SEQ ID No: 11 (Flag peptide):
TABLE-US-00013 YPYDVPDYAG
[0215] SEQ ID No: 12 (HA peptide):
[0216] SEQ ID No: 13 (peptide derived from human GDF-15):
TABLE-US-00014 ELHLRPQAARGRR
[0217] SEQ ID No; 14 (peptide derived from human GDF-15):
TABLE-US-00015 LHLRPQAARGRRR
[0218] SEQ ID No: 15 (peptide derived from human GDF-15):
TABLE-US-00016 HLRPQAARGRRRA
[0219] SEQ ID No: 16 (peptide derived from human GDF-15):
TABLE-US-00017 LRPQAARGRRRAR
[0220] SEQ ID No: 17 (peptide derived from human GDF-5):
TABLE-US-00018 RPQAARGRRRARA
[0221] SEQ ID No: 18 (peptide derived from human GDF-15):
TABLE-US-00019 PQAARGRRRARAR
[0222] SEQ ID No: 19 (peptide derived from human GDF-15):
TABLE-US-00020 QAARGRRRARARN
[0223] SEQ ID No: 20 (peptide derived from human GDF-15):
TABLE-US-00021 MHAQIKTSLHRLK
[0224] SEQ ID No: 25 (GDF-15 peptide comprising part of the GDF-15
Epitope that binds to B1-23):
TABLE-US-00022 EVQVTMCIGACPSQFR
[0225] SEQ ID No: 26 (GDF-15 peptide comprising part of the GDF-15
Epitope that binds to B1-23):
TABLE-US-00023 TDTGVSLQTYDDLLAKDCHCI
[0226] The nucleic acid sequences referred to in the present
application are as follows (in a 5' to 3' order; represented in
accordance with the standard nucleic acid code):
[0227] SEQ ID No: 21 (DNA nucleotide sequence encoding the amino
acid sequence defined in SEQ ID No: 1):
TABLE-US-00024 CAAGTGAAGCTGCAGCAGTCAGGCCCTGGGATATTGCAGTCCTCCCAGAC
CCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGTACTTCTGGTA
TGGGTGTGAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGGAGTGGCTG
GCACACATTTACTGGGATGATGACAAGCGCTATAACCCAACCCTGAAGAG
CCGGCTCACAATCTCCAAGGATCCCTCCAGAAACCAGGTATTCCTCAAGA
TCACCAGTGTGGACACTGCAGATACTGCCACATACTACTGT
[0228] SEQ ID No: 22 (DNA nucleotide sequence encoding the amino
acid sequence defined in SEQ ID No: 2):
TABLE-US-00025 GACATTGTGCTCACCCAGTCTCCAAAATTCATGTCCACATCAGTAGGAGA
CAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTGG
CCTGGTTTCTACAGAAACCAGGGCAATCTCCTAAAGCACTTATTTACTCG
GCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAACGTGCAGTCTGAAGACTTGG
CAGAGTATTTCTGT
[0229] SEQ ID No: 23 (DNA nucleotide sequence encoding the amino
acid sequence defined in SEQ ID No: 5):
TABLE-US-00026 GCTCGAAGTTCCTACGGGGCAATGGACTAC
[0230] SEQ ID No: 24 (DNA nucleotide sequence encoding the amino
acid sequence defined in SEQ ID No: 7):
TABLE-US-00027 CAGCAATATAACAACTTTCCGTACACG
[0231] SEQ ID No: 27 (amino acid sequence of the heavy chain of the
H1L5 humanized B1-23 anti-GDF-15 antibody):
TABLE-US-00028 QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWL
AHIYWDDDKRYNPTLKSRLTITKDPSKNQVVLTMTNMDPVDTATYYCARS
SYGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0232] SEQ ID No: 28 (amino acid sequence of the heavy chain
variable domain of the H1L5 humanized B1-23 anti-GDF-15
antibody):
TABLE-US-00029 QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWL
AHIYWDDDKRYNPTLKSRLTITKDPSKNQVVLTMTNMDPVDTATYYCARS
SYGAMDYWGQGTLVTVSS
[0233] SEQ ID No: 29 (amino acid sequence of the heavy chain
constant domain of the H1L5 humanized B1-23 anti-GDF-15
antibody):
TABLE-US-00030 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0234] SEQ ID No: 30 (amino acid sequence of the light chain of the
H1L5 humanized B1-23 anti-GDF-15 antibody):
TABLE-US-00031 DIVLTQSPSFLSASVGDRVTITCKASQNVGTNVAWFQQKPGKSPKALIYS
ASYRYSGVPDRFTGSGSGTEFTLTISSLQPEDFAAYFCQQYNNFPYTFGG
GTKLEIKRAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC
[0235] SEQ ID No: 31 (amino acid sequence of the light chain
variable domain of the H1L5 humanized B1-23 anti-GDF-15
antibody):
TABLE-US-00032 DIVLTQSPSFLSASVGDRVTITCKASQNVGTNVAWFQQKPGKSPKALIYS
ASYRYSGVPDRFTGSGSGTEFTLTISSLQPEDFAAYFCQQYNNFPYTFGG GTKLEIKR
[0236] SEQ ID No: 32 (amino acid sequence of the light chain
constant domain of the H1L5 humanized B1-23 anti-GDF-15
antibody):
TABLE-US-00033 APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC
[0237] SEQ ID No: 33 (amino acid sequence of the heavy chain of the
chimeric B1-23 anti-GDF-15 antibody):
TABLE-US-00034 QVKLQQSGPGILQSSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWL
AHIYWDDDKRYNPTLKSRLTISKDPSRNQVFLKITSVDTADTATYYCARS
SYGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0238] SEQ ID No: 34 (amino acid sequence of the heavy chain
variable domain of the chimeric B1-23 anti-GDF-15 antibody):
TABLE-US-00035 QVKLQQSGPGILQSSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWL
AHIYWDDDKRYNPTLKSRLTISKDPSRNQVFLKITSVDTADTATYYCARS
SYGAMDYWGQGTSVTVSS
[0239] SEQ ID No: 35 (amino acid sequence of the heavy chain
constant domain of the chimeric B1-23 anti-GDF-15 antibody):
TABLE-US-00036 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0240] SEQ ID No: 36 (amino acid sequence of the light chain of the
chimeric B1-23 anti-GDF-15 antibody);
TABLE-US-00037 DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWFLQKPGQSPKALIYS
ASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNNFPYTFGG
GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
[0241] SEQ ID No: 37 (amino acid sequence of the light chain
variable domain of the chimeric B1-23 anti-GDF-15 antibody):
TABLE-US-00038 DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWFLQKPGQSPKALIYS
ASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNNFPYTFGG GTKLEIKRTVA
[0242] SEQ ID No: 38 (amino acid sequence of the light chain
constant domain of the chimeric B1-23 anti-GDF-15 antibody):
TABLE-US-00039 APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC
F) EXAMPLES
[0243] The present invention is illustrated by the following
non-limiting Examples:
Example 1: Generation and Characterization of the Murine GDF-15
Antibody B1-23, and Generation of Chimeric and Humanized
Antibodies
[0244] The antibody B1-23 was generated in a GDF-15 knock out
mouse. Recombinant human GDF-15 (SEQ ID No: 8) was used as the
immunogen.
[0245] The hybridoma cell line B1-23 producing mAb-B1-23 was
deposited with the Deutsche Sammlung fur Mikroorganismen and
Zellkulturen GmbH (DMSZ) under the accession No. DSM ACC3142, in
accordance with the Budapest Treaty.
[0246] By means of a commercially available test strip system,
B1-23 was identified as an IgG2a (kappa chain) isotype. Using
surface plasmon resonance measurements, the dissociation constant
(Kd) was determined as follows:
[0247] Binding of the monoclonal anti-human-GDF-15 antibody
anti-human GDF-15 mAb-B1-23 was measured by employing surface
plasmon resonance measurements using a Bio-Rad.RTM. ProteOn.TM.
XPR36 system and Bio-Rad.RTM. GLC sensor chips:
[0248] For preparing the biosensors recombinant mature human GDF-15
protein was immobilized on flow cells 1 and 2. On one flow cell
recombinant GDF-15 derived from Baculvirus-transfected insect cells
(HighFive insect cells) and on the other recombinant protein
derived from expression in E. coli was used. The GLC sensor chip
was activated using Sulfo-NHS (N-Hydroxysulfosuccinimide) and EDC
(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride)
(Bio-Rae ProteOn.TM. Amine Coupling Kit) according to the
manufacturer's recommendation, the sensor surface was subsequently
loaded with the proteins up to a density of about 600 RU (1 Ru=1 pg
mm.sup.-2). The non-reacted coupling groups were then quenched by
perfusion with 1M ethanolamine pH 8.5 and the biosensor was
equilibrated by perfusing the chip with running buffer (10M HEPES,
150 mM NaCl, 3.4 mM EDTA, 0.005% Tween.RTM.20, pH 7.4, referred to
as HBS150). As controls two flow cells were used, one empty with no
protein coupled and one coupled with an non-physiological protein
partner (human Interleukin-5), which was immobilized using the same
coupling chemistry and the same coupling density. For interaction
measurements anti-human GDF-15 mAb-B1-23 was dissolved in HBS150
and used in six different concentrations as analyte (concentration:
0.4, 0.8, 3, 12, 49 and 98 nM). The analyte was perfused over the
biosensor using the one-shot kinetics setup to avoid intermittent
regeneration, all measurements were performed at 25.degree. C. and
using a flow rate of 1041 min.sup.-1. For processing the bulk face
effect and unspecific binding to the sensor matrix was removed by
subtracting the SPR data of the empty flow cell (flow cell 3) from
all other SPR data. The resulting sensogram was analyzed using the
software ProteOn Manager version 3.0. For analysis of the binding
kinetics a 1:1 Langmuir-type interaction was assumed. For the
association rate constant a value of 5.4.+-.0.06.times.10.sup.5
M.sup.-1 s.sup.-1 (k.sub.on) and for the dissociation rate constant
a value of 4.3.+-.0.03.times.10.sup.-4 s.sup.-1 (k.sub.off) could
be determined (values are for the interaction of anti-human GDF-15
mAb-B1-23 with GDF-15 derived from insect cell expression). The
equilibrium dissociation constant was calculated using the equation
K.sub.D=k.sub.off/k.sub.on to yield a value of about 790 pM.
Affinity values for the interaction of GDF-15 derived from E. coli
expression and the anti-human GDF-15 mAb-B1-23 differ by less than
a factor of 2, rate constants for GDF-15 derived from insect cells
and E. coli deviate by about 45% and are thus within the accuracy
of SPR measurements and likely do not reflect a real difference in
affinity. Under the conditions used the anti-human GDF-15 mAb-B1-23
shows no binding to human interleukin-5 and thus confirms the
specificity of the interaction data and the anti-human GDF-15
mAb-B1-23.
[0249] The amino acid sequence of recombinant human GDF-15 (as
expressed in Baculovirus-transfected insect cells) is:
TABLE-US-00040 (SEQ ID No: 8) GSARNGDHCP LGPGRCCRLH TVRASLEDLG
WADWVLSPRE VQVTMCIGAC PSQFRAANMH AQIKTSLHRL KPDTVPAPCC VPASYNPMVL
IQKTDTGVSL QTYDDLLAKD CHCI
[0250] Thus, using surface plasmon resonance measurements, the
dissociation constant (Kd) of 790 pM was determined. As a
comparison: the therapeutically used antibody Rituximab has a
significantly lower affinity (Kd=8 nM).
[0251] From the murine anti-human GDF-15 mAb-B1-23, a chimeric
anti-human GDF-15 mAb-B1-23 antibody according to the invention was
generated by replacing constant domains of the murine antibody with
the constant domains of a human IgG1 antibody (trastuzumab
backbone). The amino acid sequence of the heavy chain of this
chimeric antibody is shown in SEQ ID No: 33, and the amino acid
sequence of the light chain of this chimeric antibody is shown in
SEQ ID No: 36.
[0252] From the chimeric anti-human GDF-15 mAb-B1-23, a humanized
anti-human GDF-15 mAb-B1-23 antibody according to the invention was
developed by humanizing the variable domains of the chimeric
antibody, i.e. by replacing the framework regions of the chimeric
antibody with human sequences. The amino acid sequence of the heavy
chain of this humanized antibody is shown in SEQ ID No: 27, and the
amino acid sequence of the light chain of this humanized antibody
is shown in SEQ ID No: 30. This antibody is referred to as H1L5
anti-GDF-15 antibody or humanized B1-23-H1L5 antibody or H1L5
antibody.
[0253] In order to generate the above-mentioned chimeric
anti-human. GDF-15 mAb-B1-23 antibody and the humanized B1-23-H1L5
antibody as indicated above, the cDNAs encoding the antibody
sequences were optimized, and the genes were synthesized. The gene
sequences were then cloned into a cloning/expression vector system.
From these vectors, plasmid DNA with low endotoxin levels was
synthesized.
[0254] The plasmid DNA was then transiently transfected into CHO
cells, followed by an analysis and quantification of antibody
expression using a protein A biosensor. The cDNA of candidate
cultures for antibody expression was sequenced. The obtained
monoclonal antibodies were analyzed (see Examples 7 to 9).
Example 2: Antagonization of GDF-15 Mediated Effects with mAB
B1-23
[0255] a) The NKG2D (Natural Killer Group 2D) receptor, which is
expressed on NK cells and CD8+ T cells, is known to play an
important role in the immune surveillance against tumors.
Transformed as well as viral infected cells express ligands, which
bind to the NKG2D receptor, thereby activating the cytotoxic
effector functions of the described immune cells. In that way
transformed cells can be detected and eliminated by the immune
system. After treatment of immune cells with either recombinant
human GDF-15 or tumor cell secreted GDF-15 in vitro for 72 hours,
the expression level of NKG2D on the cell surface of lymphocytes
was downregulated (FIG. 1).
[0256] After 72 hours incubation the immune cells were stained with
the following FACS-antibodies: anti CD3, anti CD56, anti-NKG2D.
Using this antibody combination, the experiment focused on NK cells
and their NKG2D surface expression. The low NKG2D level on immune
cells led to an impaired tumor/target cell lysis. The GDF-15
mediated downregulation of NKG2D was prevented by mAb B1-23.
[0257] It is therefore concluded that human GDF-15 downregulates
expression of NKG2D on the cell surface of lymphocytes and thereby
downregulates immune surveillance against tumors. By binding to
human GDF-15, the antibodies of the present invention are capable
of preventing GDF-15 mediated downregulation of NKG2D and should be
capable of restoring immune surveillance and inducing the killing
of cancer cells by NK cells and CD8+ T cells. Given that the CDR
regions of the mAb B1-23 antibody correspond to CDR regions of the
chimeric and humanized antibodies, it is expected that the
functional properties including the binding properties of these
antibodies are similar.
[0258] b) The treatment of the ovarian cancer cell line SK-OV-3
with recombinant GDF-15 led to the phosphorylation of AKT. AKT is a
molecule, which is part of the PI3K-pathway and contributes to the
activation and proliferation of cells. In this experiment SK-OV-3
cells were treated with 10 ng/ml recombinant GDF-15 for 10 min at
37.degree. C., 5% CO2. 5 minutes preincubation of 2 .mu.g mAb-B1-23
with 10 ng/ml GDF-15 at 37.degree. C. blocked the GDF-15 mediated
AKT-phosphorylation (FIG. 2). This showed the neutralizing effect
of mAb-B1-23.
[0259] c) Treatment of immune cells with recombinant. GDF-15 led to
the phosphorylation of JNK, a kinase, which is activated either by
cytokines or by stress. Antagonization of 10 ng/ml GDF-15 with 2
.mu.g mAb-B1-23 (5 minute preincubation at 37.degree.) blocked the
GDF-15 mediated JNK1/2-phosphorylation (FIG. 3).
Example 3: Inhibition of Cancer Cell Proliferation Using mAb
B1-23
[0260] Data generated with B1-23 showed an antiproliferative effect
of the antibody on cancer cells in vitro. The strongest
antiproliferative effect was observed using the prostate cancer
cell line LnCap, which produces lots of GDF-15. A metabolic assay
(Alamar Blue.RTM. assay) showed a decrease of proliferation of 30%
after 72 hrs when mAb-B1-23 was present, compared with the control
group, where the antibody was not applied. Since cytotoxic effects
of the antibody have been excluded in different assays, this effect
proves a significantly decreased cell division rate after blockade
of GDF-15.
Example 4: B1-23 Inhibits Growth of Tumors In Vivo
[0261] The following in vivo study was carried out:
[0262] To assess an anti-tumor effect of B1-23 in vivo,
Balb/c.sup.nu/nu nude mice were used in a xenograft setting with
the melanoma cell line UACC-257. The mice were treated either with
the antibody B1-23 or with PBS. Each treatment cohort contained 10
Balb/c.sup.nu/nu nude mice.
[0263] Prior to injection, the UACC-257 melanoma cells were grown
in complete medium, excluding any contamination. The cells were
harvested when 70-80% confluence was reached in the cell culture
flask. Cells were then washed with PBS and counted.
1.times.10.sup.7 viable cells were suspended in PBS.
[0264] The first injection/treatment was administered in 6 week old
Balb/c.sup.nu/nu nude mice. The inoculation area of the mice was
cleaned with ethanol. The UACC 257 cells were mixed and drawn into
a syringe without a needle, in order to avoid negative pressure on
the tumor cells. The cell suspension containing 1.times.10.sup.7
cells in PBS was injected subcutaneously (s.c.) into the lower
flank of the mice.
[0265] The intraperitoneal (i.p.) injection of either B1-23 (25
mg/kg body weight) or the same volume of PBS started immediately
after the tumor cell inoculation (defined as day 1) and was
administered twice a week. The tumors were grown for 48 days. The
tumor diameters were measured with a caliper and the tumor volume
in mm.sup.3 was calculated by the formula:
Volume=(width).sup.2.times.length/2
[0266] The results which were obtained from the study are shown in
FIG. 4.
[0267] As demonstrated in the Figure, the tumor size of the animal
cohort treated with B1-23 was significantly decreased, compared to
the PBS control group.
[0268] Given that the CDR regions of the mAb B1-23 antibody
correspond to CDR regions of the chimeric and humanized antibodies,
it is expected that the functional properties including anti-cancer
effects of these antibodies are similar.
Example 5: mAb B1-23 Recognizes a Conformational or a Discontinuous
Epitope of Human GDF-15
[0269] Epitope Mapping: Monoclonal mouse antibody GDF-15 against
13mer linear peptides derived from GDF-15
[0270] Antigen: GDF-15:
[0271]
GSGSGSGMPGQELRTVNGSQMLLVLLVLSWLPHGGALSLAEASRASFPGPSELHSEDSRFR
ELRKRYEDLLTRLRANQSWEDSNTDLVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLP
EASRLHRALFRLSPTASRSWDVTRPLRRQLSLARPQAPALHLRLSPPPSQSDQLLAESSSA
RPQLELHLRPQAARGRRRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQV
TMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYD
DLLAKDCHCIGSGSGSG (322 amino acids with linker)(SEQ ID No: 10)
[0272] The protein sequence was translated into 13mer peptides with
a shift of one amino acid. The C- and N-termini were elongated by a
neutral GSGS linker to avoid truncated peptides (bold letters).
[0273] Control Peptides:
[0274] Flag: DYKDDDDKGG (SEQ ID No: 3), 78 spots; HA: YPYDVPDYAG
(SEQ ID No:14), 78 spots (each array copy)
[0275] Peptide Chip Identifier:
[0276] 000264_01 (10/90, Ala2Asp linker)
[0277] Staining Conditions:
[0278] Standard buffer: PBS, pH 7.4+0.05% Tween.RTM.20
[0279] Blocking buffer: Rockland blocking buffer MB-070
[0280] Incubation buffer: Standard buffer with 10% Rockland
blocking buffer MB-070
[0281] Primary sample: Monoclonal mouse antibody GDF-15 (1
.mu.g/.mu.l):
[0282] Staining in incubation buffer for 16 h at 4.degree. C. at a
dilution of 1:100 and slight shaking at 500 rpm
[0283] Secondary antibody: Goat anti-mouse IgG (H+L) IRDye680,
staining in incubation buffer with a dilution of 1:5000 for 30 min
at room temperature (RT)
[0284] Control antibodies: Monoclonal anti-HA (12CA5)-LL-Atto 680
(1:1000), monoclonal anti-FLAG(M2)-FluoProbes752 (1:1000); staining
in incubation buffer for 1 h at RT
[0285] Scanner:
[0286] Odyssey.RTM. Imaging System, LI-COR Biosciences
[0287] Settings: offset: 1 mm; resolution: 21 .mu.m; intensity
green/red: 7/7
[0288] Results:
[0289] After 30 min pre-swelling in standard buffer and 30 min in
blocking buffer, the peptide array with 10, 12 and 15mer
B7H3-derived linear peptides was incubated with secondary goat
anti-mouse IgG (H+L) IRDye680 antibody only at a dilution of 1:5000
for 1 h at room temperature to analyze background interactions of
the secondary antibody. The PEPperCHIP.RTM. was washed 2.times.1
min with standard buffer, rinsed with dist. water and dried in a
stream of air. Read-out was done with Odyssey.RTM. Imaging System
at a resolution of 21 .mu.m and green/red intensities of 7/7; We
observed a weak interaction of arginine-rich peptides
(ELHLRPQAARGRR (SEQ ID No:15), LHLRPQAARGRRR (SEQ ID No:16),
HLRPQAARGRRRA (SEQ ID No:17), LRPQAARGRRRAR (SEQ ID No:18),
RPQAARGRRRARA (SEQ ID No:19), PQAARGRR (SEQ ID No:20) and
QAARGRRRARARN (SEQ ID No:21)) that are known as frequent binders,
and with the basic peptide MHAQIKTSLHRLK (SEQ ID No:22) due to
ionic interactions with the charged antibody dye.
[0290] After pre-swelling for 10 min in standard buffer, the
peptide microarray was incubated overnight at 4.degree. C. with
monoclonal mouse antibody GDF-15 at a dilution of 1:100. Repeated
washing in standard buffer (2.times.1 min) was followed by
incubation for 30 min with the secondary antibody at a dilution of
1:5000 at room temperature. After 2.times.10 sec. washing in
standard buffer and short rinsing with dist. water, the
PEPperCHIP.RTM. was dried in a stream of air. Read-out was done
with Odyssey.RTM. Imaging System at a resolution of 21 .mu.m and
green/red intensities of 7/7 before and after staining of control
peptides by anti-HA and anti-FLAG(M2) antibodies.
[0291] It was shown that none of the linear 13mer peptides derived
from GDF-15 interacted with monoclonal mouse antibody GDF-15 even
at overregulated intensities. Staining of Flag and HA control
peptides that frame the array, however, gave rise to good and
homogeneous spot intensities.
SUMMARY
[0292] The Epitope Mapping of monoclonal mouse GDF-15 antibody
against GDF-15 did not reveal any linear epitope with the 13mer
peptides derived from the antigen. According to this finding it is
very likely that monoclonal mouse antibody GDF-recognizes a
conformational or a discontinuous epitope with low affinity of
partial epitopes. Due to the obvious absence of any GDF-15 signal
above the background staining of the secondary antibody only,
quantification of spot intensities with PepSlide.RTM. Analyzer and
subsequent peptide annotation were omitted.
Example 6: Structural Identification of Peptide Ligand Epitopes by
Mass Spectrometric Epitope Excision and Epitope Extraction
[0293] The epitope of recombinant human GDF-15 which binds to the
antibody B1-23 was identified by means of the epitope excision
method and epitope extraction method (Suckau et al. Proc Nati Acad
Sci USA. 1990 December; 87(24): 9848-9852.; R. Stefanescu et al.,
Eur. J. Mass Spectrom. 13, 69-75 (2007)).
[0294] For preparation of the antibody column, the antibody B1-23
was added to NHS-activated 6-aminohexanoic acid coupled sepharose.
The sepharose-coupled antibody B1-23 was then loaded into a 0.8 ml
microcolumn and washed with blocking and washing buffers.
[0295] Epitope Extraction Experiment:
[0296] Recombinant human GDF-15 was digested with trypsin for 2 h
at 37.degree. C. (in solution), resulting in different peptides,
according to the trypsin cleavage sites in the protein. After
complete digestion, the peptides were loaded on the affinity column
containing the immobilized antibody B1-23. Unbound as well as
potentially bound peptides of GDF-15 were used for mass
spectrometry analysis. An identification of peptides by means of
mass spectrometry was not possible. This was a further indicator
that the binding region of GDF-15 in the immune complex B1-23
comprises a discontinuous or conformational epitope. In case of a
continuous linear epitope, the digested peptides should bind its
interaction partner, unless there was a trypsin cleavage site in
the epitope peptide. A discontinuous or conformational epitope
could be confirmed by the epitope excision method described in the
following part.
[0297] Epitope Excision Experiment:
[0298] The immobilized antibody B1-23 on the affinity column was
then incubated with recombinant GDF-15 for 2 h. The formed immune
complex on the affinity column was then incubated with trypsin for
2 h at 37.degree. C. The cleavage resulted in different peptides
derived from the recombinant GDF-15. The immobilized antibody
itself is proteolytically stable. The resulting peptides of the
digested GDF-15 protein, which were shielded by the antibody and
thus protected from proteolytic cleavage, were eluted under acidic
conditions (TFA, pH2), collected and identified by mass
spectrometry.
[0299] The epitope excision method using MS/MS identification
resulted in the following peptides:
TABLE-US-00041 Position in Peptide sequence Mass Ion/Charge
EVQVTMCIGACPSQFR 40-55 1769.91 590.50 (3+) (SEQ ID No: 25)
TDTGVSLQTYDDLLAKDCHCI 94-114 2310.96 771:33 (3+) (SEQ ID No:
26)
[0300] The part of human GDF-15, which binds the antibody B1-23,
comprises a discontinuous or conformational epitope. Mass
spectrometry identified 2 peptides in the GDF-15 protein, which are
responsible for the formation of the immune complex. These peptides
are restricted to the positions 40-(EVQVTMCIGACPSQFR) and 94-114
(TDTGVSLQTYDDLLAKDCHCI) in the GDF-15 amino acid sequence. Thus,
these two peptides comprise an epitope of the GDF-15 protein that
binds to the antibody B1-23.
[0301] Again, since the CDR regions of the mAb B1-23 antibody
correspond to CDR regions of the chimeric and humanized antibodies,
it is expected that the binding properties of these antibodies are
similar.
Example 7: Treatment of Cancer-Induced Weight Loss with Anti-GDF-15
Antibodies
[0302] In the underlying animal study No. 140123, 10
Balb/c.sup.nu/nu mice per treatment group were subcutaneously
inoculated with 10.times.10.sup.6 UACC-257 cells per animal in a
1:1 volume ratio with matrigel (100 .mu.l cells+100 .mu.l
matrigel). The animals were treated on the same day with the
respective antibodies, as indicated below:
TABLE-US-00042 Study groups 1-6 Amounts of substances (10 animals
per group) (for 45 days) 1. Dacarbazine* 80 mg (reference, Lot.
No.: C120522C) 2. PBS (SIGMA, Lot. No.: RNBD0341) 30 ml 3. B1-23
anti-GDF-15 antibody 75 mg (murine, Lot. No.: 515980) 4. Chimeric
B1-23 anti-GDF-15 antibody 75 mg (chimeric; Lot.: PR0057) 5. H1L5
anti-GDF-15 antibody 75 mg (humanized B1-23, Lot.: PR3176) 6. B12
Isotype control antibody 75 mg (Isotype antibody, Lot. No.: ID3195)
*Detidemac 500 mg (exp.: March 2015)
[0303] The dacarbazine group (group 1) served as a reference
group/positive control for tumor growth arrest (cytostatic drug for
the treatment of malignant melanoma in humans).
[0304] The PBS group (group 2) served as a growth control/vehicle
control group, because all used substances of the other groups were
administered in PBS.
[0305] The group of the murine B1-23 lead candidate antibody (group
3) served as reference group for a comparison with the chimeric
B1-23 antibody and with the humanized B1-23 H1L5 (groups 4 and
5).
[0306] Group 4 is the group of the chimerized B1-23 lead candidate
antibody, which contains murine variable domains and constant
domains of a human IgG1 antibody (trastuzumab backbone).
[0307] Group 5 is the group of the H1L5 humanized B1-23 lead
candidate antibody, which contains humanized frameworks within the
murine variable regions and constant domains of a human IgG1
antibody (trastuzumab backbone).
[0308] Group 6 is the group of the B12 isotype antibody. For this
isotype control group, the antibody B12 (Lot. No.: ID3195) was
produced by the company Evitria AG. B12 binds to an HIV antigen and
should therefore neither bind to antigens in nude mice nor to
antigens of the human tumor. 312 was selected as a highly suitable
isotype control, because the immunoglobulin backbone of B12 also
consists of the human IgG1 antibody trastuzumab and is therefore
almost identical to the chimeric B1-23 and the H1L5 humanized 31-23
antibodies, except for their variable regions.
[0309] The study was carried out in a double-blinded manner for the
treatment with the antibodies and for the treatment with PBS.
[0310] In groups 1, 2 and 6 which did not receive anti-GDF-15
antibodies, more than 10% body weight loss was observed (i.e.
weight loss to a relative body weight of less than 90% compared to
day 0). In contrast, in the groups which had received treatment
with the anti-GDF-15 antibodies B1-23, chimeric B1-23 and humanized
B1-23-H1L5, respectively, an increase in body weight was observed
(FIG. 5).
[0311] Thus, surprisingly, treatment with all of the tested
anti-GDF-15 antibodies completely prevents cancer-induced weight
loss in mice. This effect was significant for all of the groups
treated with anti-GDF-15 antibodies (two-way ANOVA; p<0.05).
[0312] It is also noteworthy that the mice of the groups that did
not receive treatment with anti-GDF-15 antibodies exhibited a
weight loss of more than 10%. In humans, a weight loss of as little
as 5% over a period of 6 months is considered as being indicative
of cancer cachexia (Fearon K. et al.: Definition and classification
of cancer cachexia: an international consensus. Lancet Oncol. 2011
May; 12(5):489-95.). Given the larger weight loss of the mice
observed in the present study which even exceeded 10%, it is
expected that the mice in the study, which did not receive
treatment with anti-GDF-15 antibodies, not only exhibited weight
loss but also exhibited cancer cachexia. This effect is completely
prevented by the anti-GDF-15 antibodies tested. It is therefore
expected that the anti-GDF-15 antibodies in accordance with the
invention are capable of both treating cancer-induced weight loss
and treating cancer cachexia.
[0313] Notably, the extent of weight loss did not correlate with
the respective tumor size (r.sup.2=10.sup.-6). If the prevention of
weight loss were only a secondary effect resulting from the
inhibition of cancer growth and the smaller tumor sizes, a
correlation between tumor size and weight loss would be expected.
Thus, the lack of such correlation shows that uses of the
anti-GDF-15 antibodies according to the invention result in two
independent treatment effects: [0314] an inhibition of cancer
growth, and [0315] a prevention of weight loss as an additional
effect, which is independent from the inhibition of cancer growth,
and which is expected to reflect a prevention of cancer
cachexia.
[0316] Despite their mechanistic independence, it was observed that
these effects can occur simultaneously in the same animals.
[0317] In addition to evaluating the mean body weight of the mice,
the feed consumption of the mice was evaluated by pairwise
comparisons of the study groups (Table 1). Notably, the feed
consumption of the mice in the anti-GDF-15 antibody groups (B1-23,
chimeric B1-23 and humanized B1-23-H1L5) was significantly higher
than the feed consumption of the mice in the groups which did not
receive the anti-GDF-15 antibodies.
TABLE-US-00043 TABLE 1 Table 1: Comparative evaluation of the feed
consumption between the different treatment groups. For the
measured time intervals (day 17-20, day 20-24, day 24-27, day
27-31, day 31-34), the average feed consumption per mouse and day
was calculated for each respective group. The values are indicated
together with their standard deviation. Feed consumption vs. vs.
per mouse chimeric humanized. vs. and day B1-23 B1-23 B1-23
dacarbazine 2.8 .+-. 0.2 g ** ** ** PBS 2.6 .+-. 0.4 g ** ** **
Chimeric 3.5 .+-. 0.2 g -- n.s. n.s. B1-23 B12 2.7 .+-. 0.2 g ***
*** *** humanized 3.4 .+-. 0.2 g n.s. -- n.s. B1-23 B1-23 3.6 .+-.
0.2 g n.s. n.s. -- (*p < 0.05; ** p < 0.01; *** p < 0.001
as assessed by unpaired two-sided Student's t-test)
[0318] The quality of the humanized anti-GDF-15 antibody B1-23-H1L5
used in the study was tested by using gel electrophoresis and
coomassie staining of the antibodies (see FIG. 6). Notably, the
band of the humanized anti-GDF-15 antibody B1-23-H1L5 was sharp and
clear, whereas the bands of the murine B1-23 anti-GDF-15 antibody
and the B12 control antibody appeared less sharp and at a higher
molecular weight. This suggests that the humanized anti-GDF-15
antibody B1-23-H1L5 is not prone to aggregation, and that some
aggregation may have shifted the molecular weight of the other
antibodies to higher values.
[0319] Additionally, by using a colorimetric assay, it was
confirmed that all anti-GDF-15 antibodies used in the study bound
to GDF-15 in a concentration-dependent manner. To determine the
binding of B1-23 antibody variants to GDF-15, a colorimetric ELISA
experiment was performed. The B12 antibody served as an isotype
antibody, which does not bind to human GDF-15. Therefore, Maxisorp
96 well plates (Nunc) were coated with hrGDF-15 (25 ng protein per
well, 50 .mu.l volume) over night at 4.degree. C. The following
day, plates were washed to remove unbound protein (3 times with 150
.mu.l of PBS 0.05%) Tween.RTM. and non-specific binding sites were
blocked with 150 .mu.l of PBS 1% BSA for 2 hours at room
temperature. Again, plates were washed and different variants of
B1-23 test antibodies were applied (50 .mu.l volume). To inquire
specificity of the antibody binding, endpoint dilution was
performed starting from 333 ng/ml and 1:3 serial dilution. As
background control, PBS 1% BSA was applied. Following binding for 1
hour at room temperature, wells were washed as described above. As
secondary antibody HRP conjugated Anti-human IgG (Life
technologies, 1:5000) was applied for 1 hour at room temperature.
Wells were washed as described above to remove unbound secondary
antibody. For detection, 50 .mu.l of peroxide substrate (TMB 1:100
in 0.1 M sodium acetate pH6) were added and following 10 minutes of
incubation, 50 .mu.l of stop solution (2N H.sub.2SO.sub.4) were
added. As negative controls, wells without GDF-15 coating and wells
without secondary antibody were included. For analysis, optical
densitiy at 450 nm was quantified using the ELISA reader (Tecan
Sunrise) and the corresponding Magellan software. It was observed
that in comparison to the B12 antibody, the humanized H1L5
antibody, the chimeric B1-23 antibody and the murine B1-23 antibody
exhibited a clearly concentration-dependent binding to GDF-15.
Example 8: Determination of Kd Values of Anti-GDF-15 Antibodies
[0320] The Kd values of different anti-GDF-15 antibodies were
compared using Surface Acoustic Wave (SAW) gold chip biosensors
technology (SAW Instruments GmbH, Schwertberger Str. 16, D-53177
Bonn, Germany):
TABLE-US-00044 Antibody: Kd value (nM) B1-23 anti-GDF-15 antibody
28.8 nM (murine, IgG2a) Chimeric B1-23 anti-GDF-15 antibody 14 nM
(chimerized, human IgG1) H1L5 humanized B1-23 5.62 nM (humanized,
human IgG1) Rituxumab 1116 nM (control antibody) Herceptin No
binding (control antibody)
[0321] The murine antibody (B1-23) as well as the chimeric B1-23
antibody were present in purified form. The H1L5 humanized B1-23
antibody was a serum-free CHO cell culture supernatant. The Kd
value of the murine B1-23 deviates from the Kd values determined by
Biacore.RTM. analyses (surface plasmon resonance) by a factor of
35.
[0322] This deviation may--apart from the differences in the
measurement methods--be explained by a reduced availability of free
murine B1-23 antibody, since it was found that this antibody can
form aggregates in its form as mouse antibody. The solution of the
murine B1-23 antibody was therefore stabilized by addition of 0.2%
BSA. Therefore, binding of the antibody to albumin may have reduced
the availability of the murine B1-23 antibody and could explain the
differences in the affinity values obtained by the different
measurement methods. Compared to the murine 81-23 antibody, the
H1L5 humanized B1-23 antibody surprisingly showed no tendency to
aggregate (see also Example 9 below).
[0323] In the present assay, the chimeric 81-23 anti-GDF-15
antibody and the H1L5 humanized B1-23 antibody exhibited affinities
to human GDF-15 which were about 2-fold and 5-fold higher,
respectively, than the affinity of the murine B1-23
anti-GDF-antibody. Thus, the chimeric B1-23 anti-GDF-15 antibody
and the H1L5 humanized B1-23 antibody are high affinity
antibodies.
Example 9: Aggregation Studies of the Anti-GDF-15 Antibodies
[0324] In order to test the aggregation properties of anti-GDF-15
antibodies, antibody samples were shaken for 48 hours at room
temperature in microcentrifuge tubes, and subsequently, the tubes
were visually analyzed for aggregated antibody precipitates.
[0325] It was observed that compared to the murine B1-23 antibody,
the H1L5 humanized B1-23 antibody surprisingly showed no tendency
to aggregate, even when the antibody was only present in
phosphate-buffered saline (PBS), and when no stabilizing proteins
such as BSA were present.
[0326] Moreover, in freeze/thaw and dilution experiments, it was
observed that the H1L5 humanized B1-23 antibody did not aggregate
during any of the dilution steps or freeze/thaw cycles.
[0327] Additionally, the following experiment was carried out (see
FIG. 7):
[0328] 5 mg of antibody (B1-23, chimeric B1-23="ChimB1-23", H1L5)
were loaded on Proteus.TM. protein A columns, eluted and collected
in a TRIS Buffer at physiological pH. After elution, the quality of
the purified antibodies was assessed by Coomassie Brilliant Blue
gel analysis. The concentration of the eluted antibodies was
measured photometrically as well as in a Bradford assay
(Roti-Quant, Carl Roth, Karlsruhe, Germany). All 3 antibody
solutions (B1-23, ChimB1-23, H1L5) showed similar concentrations
and were adjusted to 0.5 mg/ml. The antibodies were then 10 fold
concentrated via spin columns (Centricon, MWCO 30). After this
step, turbidity indicated the presence of precipitates in the
sample containing B1-23, whereas ChimB1-23 and H1L5 showed no signs
of aggregation. All concentrated eluates were then centrifuged for
5 min at 13000 rpm in order to precipitate antibody aggregates. The
remaining amount of soluble antibodies was finally determined via
Bradford assay from the supernatant.
[0329] These properties of the antibodies are expected to be
advantageous for clinical formulation of the antibodies.
G) INDUSTRIAL APPLICABILITY
[0330] The antibodies, antigen-binding portions thereof,
pharmaceutical compositions and kits according to the present
invention may be industrially manufactured and sold as products for
the claimed methods and uses (e.g. for treating cancer cachexia and
cancer), in accordance with known standards for the manufacture of
pharmaceutical products. Accordingly, the present invention is
industrially applicable.
Preferred Embodiments
[0331] 1. A monoclonal antibody capable of binding to human GDF-15,
or an antigen-binding portion thereof, wherein the heavy chain
variable domain comprises a CDR3 region comprising the amino acid
sequence of SEQ ID NO: 5 or an amino acid sequence at least 90%
identical thereto, and wherein the light chain variable domain
comprises a CDR3 region comprising the amino acid sequence of SEQ
ID NO: 7 or an amino acid sequence at least 85% identical thereto,
wherein the constant domain of the heavy chain comprises the amino
acid sequence of SEQ ID No: 29, or an amino acid sequence at least
85%, preferably at least 90%, more preferably at least 95%
identical thereto, and wherein the constant domain of the light
chain comprises the amino acid sequence of SEQ ID No: 32, or an
amino acid sequence at least 85%, preferably at least 90%, more
preferably at least 95% identical thereto. [0332] 2. A monoclonal
antibody capable of binding to human GDF-15, or an antigen-binding
portion thereof, wherein the binding is binding to a conformational
or discontinuous epitope on human GDF-15 comprised by the amino
acid sequences of SEQ ID No: 25 and SEQ ID No: 26, wherein the
constant domain of the heavy chain comprises the amino acid
sequence of SEQ ID No: 29, or an amino acid sequence at least 85%,
preferably at least 90%, more preferably at least 95% identical
thereto, and wherein the constant domain of the light chain
comprises the amino acid sequence of SEQ ID No: 32, or an amino
acid sequence at least 85%, preferably at least 90%, more
preferably at least 95% identical thereto. [0333] 3. The antibody
or an antigen-binding portion thereof of item 1 or 2, wherein the
constant domain of the heavy chain comprises the amino acid
sequence of SEQ ID No: 29, or an amino acid sequence at least 98%,
preferably at least 99% identical thereto, and wherein the constant
domain of the light chain comprises the amino acid sequence of SEQ
ID No: 32, or an amino acid sequence at least 98%, preferably at
least 99% identical thereto. [0334] 4. The antibody or an
antigen-binding portion thereof of any of items 1 to 3, wherein the
constant domain of the heavy chain comprises the amino acid
sequence of SEQ ID No: 29, and wherein the constant domain of the
light chain comprises the amino acid sequence of SEQ ID No: 32.
[0335] 5. The antibody or antigen-binding portion thereof of any
one of items 1-4, wherein the antibody is a humanized antibody.
[0336] 6. The antibody or antigen-binding portion thereof of item
5, wherein all of the variable domains of the antibody are
humanized variable domains. [0337] 7. The antibody or
antigen-binding portion thereof of any one of items 1-6, wherein
the heavy chain variable domain comprises the amino acid sequence
of SEQ ID No: 28, or an amino acid sequence at least 90%,
preferably at least 95%, more preferably at least 98%, still more
preferably at least 99% identical thereto, and wherein the light
chain variable domain comprises the amino acid sequence of SEQ ID
No: 31, or an amino acid sequence at least 90%, preferably at least
95%, more preferably at least 98%, still more preferably at least
99% identical thereto. [0338] 8. The antibody or antigen-binding
portion thereof of any one of items 1-7, wherein the heavy chain
variable domain comprises the amino acid sequence of SEQ ID No: 28,
and wherein the light chain variable domain comprises the amino
acid sequence of SEQ ID No: 31. [0339] 9. The antibody or
antigen-binding portion thereof of any one of items 1-8, wherein
the heavy chain comprises the amino acid sequence of SEQ ID No: 27,
and wherein the light chain comprises the amino acid sequence of
SEQ ID No: 30. [0340] 10. The antibody or antigen-binding portion
thereof of any one of items 1-4, wherein the heavy chain variable
domain comprises the amino acid sequence of SEQ ID No: 34, or an
amino acid sequence at least 75%, more preferably at least 90%,
more preferably at least 95%, more preferably at least 98%, still
more preferably at least 99% identical thereto, and wherein the
light chain variable domain comprises the amino acid sequence of
SEQ ID No: 37, or an amino acid sequence at least 80%, more
preferably at least 90%, more preferably at least 95%, more
preferably at least 98%, still more preferably at least 99%
identical thereto. [0341] 11. The antibody or antigen-binding
portion thereof of item 10, wherein the heavy chain variable domain
comprises the amino acid sequence of SEQ ID No: 34, and wherein the
light chain variable domain comprises the amino acid sequence of
SEQ ID No: 37. [0342] 12. The antibody or antigen-binding portion
thereof of any one of items 1-11, wherein the heavy chain variable
domain comprises a CDR1 region comprising the amino acid sequence
of SEQ ID NO: 3 and a CDR2 region comprising the amino acid
sequence of SEQ ID NO: 4, and wherein the light chain variable
domain comprises a CDR1 region comprising the amino acid sequence
of SEQ ID NO: 6 and a CDR2 region comprising the amino acid
sequence ser-ala-ser. [0343] 13. The antibody or antigen-binding
portion thereof of any one of items 2-12, wherein the heavy chain
variable domain comprises a CDR3 region comprising the amino acid
sequence of SEQ ID NO: 5 or an amino acid sequence at least 90%
identical thereto, and wherein the light chain variable domain
comprises a CDR3 region comprising the amino acid sequence of SEQ
ID NO: 7 or an amino acid sequence at least 85% identical thereto.
[0344] 14. The antibody or antigen-binding portion thereof of any
one of items 1 and 3-12, wherein the binding is binding to a
conformational or discontinuous epitope on human GDF-15 that is
comprised by the amino acid sequences of SEQ ID No: 25 and SEQ ID
No: 26. [0345] 15. The antibody or antigen-binding portion thereof
of any one of items 1-14, wherein the antibody has a size of more
than 100 kDa, preferably more than 110 kDa, more preferably more
than 120 kDa, still more preferably more than 130 kDa, and most
preferably more than 140 kDa. [0346] 16. The antibody or
antigen-binding portion thereof of item 15, wherein the antibody is
a full-length antibody. [0347] 17. The antibody or antigen-binding
portion thereof of item 16, wherein the antibody is a full-length
IgG antibody, preferably a full-length IgG1 antibody. [0348] 18.
The antibody or antigen-binding portion thereof of any one of items
1 to 17, wherein the antibody has an Fc portion which is capable of
binding to the Fc receptor. [0349] 19. The antibody or
antigen-binding portion thereof of any one of items 1 to 18,
wherein the human GDF-15 is recombinant human GDF-15 having the
amino acid sequence represented by SEQ ID No: 8. [0350] 20. An
antibody or antigen-binding portion thereof according to any one of
items 1 to 19 for use in medicine. [0351] 21. An antibody or
antigen-binding portion thereof according to any one of items 1 to
19, for use in a method for treating cancer cachexia in a mammal.
[0352] 22. An antibody or antigen-binding portion thereof according
to any one of items 1 to 19, for use in a method for treating
cancer in a mammal. [0353] 23. An antibody or antigen-binding
portion thereof according to any one of items 21 to 22 for use
according to any one of items 21 to 22, wherein the method is a
method for both treating cancer and treating cancer cachexia in the
same mammal. [0354] 24. The antibody or antigen-binding portion
thereof of any one of items 21 to 23 for the use according to any
one of items 21 to 23, wherein the mammal is a human patient.
[0355] 25. The antibody or antigen-binding portion thereof of item
21 or 23-24 for the use according to item 21 or 23-24, wherein the
method for treating cancer cachexia is a method for completely
preventing or completely reverting cancer cachexia. [0356] 26. The
antibody or antigen-binding portion thereof of item 25 for the use
according to item 25, wherein the method for treating cancer
cachexia is a method for completely preventing cancer cachexia.
[0357] 27. The antibody or antigen-binding portion thereof of item
25 for the use according to item 25, wherein the method for
treating cancer cachexia is a method for completely reverting
cancer cachexia. [0358] 28. The antibody or antigen-binding portion
thereof of any one of items 21-27 for the use according to any one
of items 21-27, wherein in the method, only mammals suffering from
both [0359] i) the cancer, and [0360] ii) cancer cachexia [0361]
are treated. [0362] 29. The antibody or antigen-binding portion
thereof of any one of items 21 or 23-28 for the use according to
any one of items 21 or 23-28, wherein the method increases body
weight of the mammal compared to its body weight before the onset
of cancer cachexia. [0363] 30. The antibody or antigen-binding
portion thereof of item 29 for the use according to item 29,
wherein the increase in body weight of the mammal is at least 1.5%,
preferably at least 2.5%, more preferably at least 5% compared to
its body weight before the onset of cancer cachexia. [0364] 31. The
antibody or antigen-binding portion thereof of any one of items 21
to 30 for the use according to any one of items 21 to 30, wherein
the cancer cells of the mammal endogenously express GDF-15 and/or
the cancer cells of the mammal stimulate endogenous expression of
GDF-15 in non-cancerous cells of the mammal. [0365] 32. The
antibody or antigen-binding portion thereof of any one of items 21
to 31 for the use according to any one of items 21 to 31, wherein
the cancer cells of the mammal endogenously express GDF-15. [0366]
33. The antibody or antigen-binding portion thereof of any one of
items 22-32 for the use according to any one of items 22-32,
wherein the method for treating cancer is a method comprising
inhibition of cancer growth. [0367] 34. The antibody or
antigen-binding portion thereof of any one of items 22-33 for the
use according to any one of items 22-33, wherein the method for
treating cancer comprises the induction of killing of cancer cells
by NK cells and CD8+ T cells in the human patient. [0368] 35. The
antibody or antigen-binding portion thereof of any one of items
21-34 for the use according to any one of items 21-34, wherein the
human patient has elevated GDF-15 levels in blood serum before
administration. [0369] 36. The antibody or antigen-binding portion
thereof of any one of items 21-35 for the use according to any one
of items 21-35, wherein the antibody or antigen-binding portion
thereof is [0370] A) the sole ingredient pharmaceutically active
against cancer used in the method, or [0371] B) used in combination
with one or more further ingredients pharmaceutically active
against cancer. [0372] 37. The antibody or antigen-binding portion
thereof of any one of items 21-36 for the use according to any one
of items 21-36, wherein the cancer is selected from the group
consisting of brain cancers including glioma, cancers of the
nervous system, melanoma, lung cancer, lip and oral cavity cancer,
hepatic carcinoma, leukemia, Hodgkin lymphoma, Non-Hodgkin
lymphoma, bladder cancer, cervix uteri cancer, corpus uteri cancer,
testis cancer, thyroid cancer, kidney cancer, gallbladder cancer,
multiple myeloma, nasopharynx cancer, larynx cancer, pharynx
cancer, oesophagus cancer, gastrointestinal tumors including
stomach and colorectal cancer, pancreatic cancer, prostate cancer,
ovarian cancer and breast cancer, preferably from the group
consisting of melanoma, prostate cancer, breast cancer, brain
cancers including glioma, colorectal cancer, stomach cancer,
oesophagus cancer and ovarian cancer, and most preferably is
melanoma. [0373] 38. The antibody or antigen-binding portion
thereof of any one of items 21-37 for the use according to any one
of items 21-37, wherein prior to administration, the tumor or
tumors formed by the cancer have higher human GDF-15 levels
compared to a control sample of the same patient obtained from a
non-cancerous part of the tissue which is the tissue of origin of
the cancer, preferably 1.2-fold higher levels, more preferably
1.5-fold higher levels, still more preferably 2-fold higher levels
and most preferably 5-fold higher levels. [0374] 39. The antibody
or antigen-binding portion thereof of item 38 for the use according
to item 38, wherein the antibody or antigen-binding portion thereof
is used in combination with one or more further ingredients
pharmaceutically active against cancer, and wherein the one or more
further ingredients pharmaceutically active against cancer are
selected from the group consisting of: alkylating agents;
anti-metabolites; alkaloids, taxanes; topoisomerase inhibitors;
cytotoxic antibiotics; and radioisotopes. [0375] 40. The antibody
or antigen-binding portion thereof of item 39 for the use according
to item 39, wherein the one or more further ingredients
pharmaceutically active against cancer are selected from the group
consisting of: cisplatin, carboplatin, oxaliplatin,
mechlorethamine, cyclophosphamide, chlorambucil, and ifosfamide;
azathioprine and mercaptopurine; vincristine, vinblastine,
vinorelbine, and vindesine, paclitaxel, docetaxel, etoposide and
teniposide; irinotecan and topotecan; actinomycin, anthracyclines,
doxorubicin, daunorubicin, valrubicin, idarubicin, epirubicin,
bleomycin, plicamycin and mitomycin. [0376] 41. A kit comprising
the antibody or antigen-binding portion thereof of any one of items
1-19. [0377] 42. The kit of item 41 for a use according to any one
of items 21 to 40. [0378] 43. An expression vector comprising a
nucleotide sequence encoding the antibody or antigen-binding
portion thereof according to any of items 1-19. [0379] 44. A cell
line capable of producing an antibody or antigen-binding portion
thereof according to any one of items 1 to 19. [0380] 45. A
monoclonal antibody capable of binding to human GDF-15, or an
antigen-binding portion thereof, wherein the heavy chain variable
domain comprises a CDR3 region comprising the amino acid sequence
of SEQ ID NO: 5 or an amino acid sequence at least 90% identical
thereto, and wherein the light chain variable domain comprises a
CDR3 region comprising the amino acid sequence of SEQ ID NO: 7 or
an amino acid sequence at least 85% identical thereto, for use in a
method for treating cancer cachexia in a mammal. [0381] 46. A
monoclonal antibody capable of binding to human GDF-15, or an
antigen-binding portion thereof, wherein the binding is binding to
a conformational or discontinuous epitope on human GDF-15 comprised
by the amino acid sequences of SEQ ID No: 25 and SEQ ID No: 26, for
use in a method for treating cancer cachexia in a mammal. [0382]
47. The antibody or antigen-binding portion thereof of item 45 or
46 for the use according to item 45 or 46, wherein the method for
treating cancer cachexia is a method for completely preventing or
completely reverting cancer cachexia. [0383] 48. The antibody or
antigen-binding portion thereof of item 47 for the use according to
item 47, wherein the method for treating cancer cachexia is a
method for completely preventing cancer cachexia.
[0384] 49. The antibody or antigen-binding portion thereof of item
47 for the use according to item 47, wherein the method for
treating cancer cachexia is a method for completely reverting
cancer cachexia. [0385] 50. The antibody or antigen-binding portion
thereof of any one of items 45-49 for the use according to any one
of items 45-49, wherein in the method, only mammals suffering from
both [0386] iii) the cancer, and [0387] iv) cancer cachexia [0388]
are treated. [0389] 51. The antibody or antigen-binding portion
thereof of any one of items 49-50 for the use according to any one
of items 49-50, wherein the method increases body weight of the
mammal compared to its body weight before the onset of cancer
cachexia. [0390] 52. The antibody or antigen-binding portion
thereof of item 51 for the use according to item 51, wherein the
increase in body weight of the mammal is at least 1.50, preferably
at least 2.5%, more preferably at least 5% compared to its body
weight before the onset of cancer cachexia. [0391] 53. The antibody
or antigen-binding portion thereof of any one of items 45-52 for
the use according to any one of items 45-52, wherein the method is
a method for both treating cancer and treating cancer cachexia in
the same mammal. [0392] 54. The antibody or antigen-binding portion
thereof of any one of items 45-53 for the use according to any one
of items 45-53, wherein the antibody has a size of more than 100
kDa, preferably more than 110 kDa, more preferably more than 120
kDa, still more preferably more than 130 kDa, and most preferably
more than 140 kDa. [0393] 55. The antibody or antigen-binding
portion thereof of item 54 for the use according to item 54,
wherein the antibody is a full-length antibody. [0394] 56. The
antibody or antigen-binding portion thereof of item 55 for the use
according to item 55, wherein the antibody is a full-length IgG
antibody. [0395] 57. The antibody or antigen-binding portion
thereof of any one of items 45 to 56 for the use according to any
one of items 45 to 56, wherein the antibody has an Fc portion which
is capable of binding to the Fc receptor. [0396] 58. The antibody
or antigen-binding portion thereof of any one of items 45 to 57 for
the use according to any one of items 45 to 57, wherein the cancer
cells of the mammal endogenously express GDF-15 and/or the cancer
cells of the mammal stimulate endogenous expression of GDF-15 in
non-cancerous cells of the mammal. [0397] 59. The antibody or
antigen-binding portion thereof of any one of items 45 to 58 for
the use according to any one of items 45 to 58, wherein the cancer
cells of the mammal endogenously express GDF-15. [0398] 60. The
antibody or antigen-binding portion thereof of any one of items 45
to 59 for the use according to any one of items 45 to 59, wherein
the mammal is a human patient. [0399] 61. The antibody or
antigen-binding portion thereof of any one of items 45 to 60 for
the use according to any one of items 45 to 60, wherein the human
GDF-15 is recombinant human GDF-15 having the amino acid sequence
represented by SEQ ID No: 8. [0400] 62. The antibody or
antigen-binding portion thereof of any one of items 46-61 for the
use according to any one of items 46-61, wherein the heavy chain
variable domain comprises a CDR3 region comprising the amino acid
sequence of SEQ ID NO: 5 or an amino acid sequence at least 90%
identical thereto, and wherein the light chain variable domain
comprises a CDR3 region comprising the amino acid sequence of SEQ
ID NO: 7 or an amino acid sequence at least 85% identical thereto.
[0401] 63. The antibody or antigen-binding portion thereof of any
one of items 45 and 47-61 for the use according to any one of items
45 and 47-61, wherein the binding is binding to a conformational or
discontinuous epitope on human GDF-15 that is comprised by the
amino acid sequences of SEQ ID No: 25 and SEQ ID No: 26. [0402] 64.
The antibody or antigen-binding portion thereof of any one of items
53-63 for the use according to any one of items 53-63, wherein the
method for treating cancer is a method comprising inhibition of
cancer growth. [0403] 65. The antibody or antigen-binding portion
thereof of any one of items 53-64 for the use according to any one
of items 53-64, wherein the method for treating cancer comprises
the induction of killing of cancer cells by NK cells and CD8+ T
cells in the human patient. [0404] 66. The antibody or
antigen-binding portion thereof of any one of items 45-65 for the
use according to any one of items 45-65, wherein the heavy chain
variable domain comprises a CDR1 region comprising the amino acid
sequence of SEQ ID NO: 3 and a CDR2 region comprising the amino
acid sequence of SEQ ID NO: 4, and wherein the light chain variable
domain comprises a CDR1 region comprising the amino acid sequence
of SEQ ID NO: 6 and a CDR2 region comprising the amino acid
sequence ser-ala-ser. [0405] 67. The antibody or antigen-binding
portion thereof of any one of items 45-66 for the use according to
any one of items 45-66, wherein the constant domain of the heavy
chain comprises the amino acid sequence of SEQ ID No: 29, or an
amino acid sequence at least 85%, preferably at least 90%, more
preferably at least 95% identical thereto, and wherein the constant
domain of the light chain comprises the amino acid sequence of SEQ
ID No: 32, or an amino acid sequence at least 85%, preferably at
least 90%, more preferably at least 95% identical thereto. [0406]
68. The antibody or antigen-binding portion thereof of any one of
items 45-67 for the use according to any one of items 45-67,
wherein the constant domain of the heavy chain comprises the amino
acid sequence of SEQ ID No: 29, or an amino acid sequence at least
98%, preferably at least 99% identical thereto, and wherein the
constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 98%,
preferably at least 99% identical thereto. [0407] 69. The antibody
or antigen-binding portion thereof of any one of items 45-68 for
the use according to any one of items 45-68, wherein the constant
domain of the heavy chain comprises the amino acid sequence of SEQ
ID No: 29, and wherein the constant domain of the light chain
comprises the amino acid sequence of SEQ ID No: 32. [0408] 70. The
antibody or antigen-binding portion thereof of any one of items
45-69 for the use according to any one of items 45-69, wherein the
antibody is a humanized antibody, and wherein all of the variable
domains of the antibody are humanized variable domains. [0409] 71.
The antibody or antigen-binding portion thereof of any one of items
45-70 for the use according to any one of items 45-70, wherein the
heavy chain variable domain comprises the amino acid sequence of
SEQ ID No: 28, or an amino acid sequence at least 90%, preferably
at least 95%, more preferably at least 98%, still more preferably
at least 99% identical thereto, and wherein the light chain
variable domain comprises the amino acid sequence of SEQ ID No: 31,
or an amino acid sequence at least 90%, preferably at least 95%,
more preferably at least 98%, still more preferably at least 99%
identical thereto. [0410] 72. The antibody or antigen-binding
portion thereof of any one of items 45-71 for the use according to
any one of items 45-71, wherein the heavy chain variable domain
comprises the amino acid sequence of SEQ ID No: 28, and wherein the
light chain variable domain comprises the amino acid sequence of
SEQ ID No: 31. [0411] 73. The antibody or antigen-binding portion
thereof of any one of items 45-72 for the use according to any one
of items 45-72, wherein the heavy chain comprises the amino acid
sequence of SEQ ID No: 27, and wherein the light chain comprises
the amino acid sequence of SEQ ID No: 30. [0412] 74. The antibody
or antigen-binding portion thereof of any one of items 45-69 for
the use according to any one of items 45-69, wherein the heavy
chain variable domain comprises the amino acid sequence of SEQ ID
No: 34, or an amino acid sequence at least 75%, more preferably at
least 90%, more preferably at least 95%, more preferably at least
98%, still more preferably at least 99% identical thereto, and
wherein the light chain variable domain comprises the amino acid
sequence of SEQ ID No: 37, or an amino acid sequence at least 80%,
more preferably at least 90%, more preferably at least 95%, more
preferably at least 98%, still more preferably at least 99%
identical thereto. [0413] 75. The antibody or antigen-binding
portion thereof of item 74 for the use according to item 74,
wherein the heavy chain variable domain comprises the amino acid
sequence of SEQ ID No: 34, and wherein the light chain variable
domain comprises the amino acid sequence of SEQ ID No: 37. [0414]
76. The antibody or antigen-binding portion thereof of any one of
items 45-66 for the use according to any one of items 45-66,
wherein the antibody is the antibody to human GDF-15 obtainable
from the cell line B1-23 deposited with the Deutsche Sammlung fur
Mikroorganismen and Zellkulturen GmbH (DMSZ) under the accession
No. DSM ACC3142 or an antigen-binding portion thereof. [0415] 77.
The antibody or antigen-binding portion thereof of any one of items
45-75 for the use according to any one of items 45-75, wherein the
heavy chain variable domain comprises a CDR3 region comprising the
amino acid sequence of SEQ ID NO: 5, or wherein the light chain
variable domain comprises a CDR3 region comprising the amino acid
sequence of SEQ ID NO: 7. [0416] 78. The antibody or
antigen-binding portion thereof of any one of items 45-75 and 77
for the use according to any one of items 45-75 and 77, wherein the
heavy chain variable domain comprises a CDR3 region comprising the
amino acid sequence of SEQ ID NO: 5, and wherein the light chain
variable domain comprises a CDR3 region comprising the amino acid
sequence of SEQ ID NO: 7. [0417] 79. The antibody or
antigen-binding portion thereof of any one of items 45-69 and 77-78
for the use according to any one of items 45-69 and 77-78, wherein
the heavy chain variable domain comprises a region comprising an
FR1, a CDR1, an FR2, a CDR2 and an FR3 region and comprising the
amino acid sequence of SEQ ID NO: 1 or a sequence 95% identical
thereto, and wherein the light chain variable domain comprises a
region comprising an FR1, a CDR1, an FR2, a CDR2 and an FR3 region
and comprising the amino acid sequence of SEQ ID NO: 2 or a
sequence 95% identical thereto. [0418] 80. The antibody or
antigen-binding portion thereof of any one of items 45-69 and 77-79
for the use according to any one of items 45-69 and 77-79, wherein
the heavy chain variable domain comprises a region comprising an
FR1, a CDR1, an FR2, a CDR2 and an FR3 region and comprising the
amino acid sequence of SEQ ID NO: 1 or a sequence 98% identical
thereto, and wherein the light chain variable domain comprises a
region comprising an FR1, a CDR1, an FR2, a CDR2 and an FR3 region
and comprising the amino acid sequence of SEQ ID NO: 2 or a
sequence 98% identical thereto. [0419] 81. The antibody or
antigen-binding portion thereof of any one of items 45-80 for the
use according to any one of items 45-80, wherein the antibody or
antigen-binding portion thereof has an equilibrium dissociation
constant for human GDF-15 that is equal to or less than 20 nM,
preferably less than 10 nM, more preferably less than 5 nM and most
preferably between 0.1 nM and 2 nM. [0420] 82. The antibody or
antigen-binding portion thereof of any one of items 45-75 and 77-81
for the use according to any one of items 45-75 and 77-81, wherein
the antibody or antigen-binding portion thereof binds to the same
human GDF-15 epitope as the antibody to human GDF-15 obtainable
from the cell line B1-23 deposited with the Deutsche Sammlung fur
Mikroorganismen and Zellkulturen GmbH (DMSZ) under the accession
No. DSM ACC3142. [0421] 83. The antibody or antigen-binding portion
thereof of any one of items 45-82 for the use according to any one
of items 45-82, wherein the human patient has elevated GDF-15
levels in blood serum before administration. [0422] 84. The
antibody or antigen-binding portion thereof of any one of items
45-83 for the use according to any one of items 45-83, wherein the
antibody or antigen-binding portion thereof is [0423] A) the sole
ingredient pharmaceutically active against cancer used in the
method, or [0424] B) used in combination with one or more further
ingredients pharmaceutically active against cancer. [0425] 85. The
antibody or antigen-binding portion thereof of any one of items
45-84 for the use according to any one of items 45-84, wherein the
cancer is selected from the group consisting of brain cancers
including glioma, cancers of the nervous system, melanoma, lung
cancer, lip and oral cavity cancer, hepatic carcinoma, leukemia,
Hodgkin lymphoma, Non-Hodgkin lymphoma, bladder cancer, cervix
uteri cancer, corpus uteri cancer, testis cancer, thyroid cancer,
kidney cancer, gallbladder cancer, multiple myeloma, nasopharynx
cancer, larynx cancer, pharynx cancer, oesophagus cancer,
gastrointestinal tumors including stomach and colorectal cancer,
pancreatic cancer, prostate cancer, ovarian cancer and breast
cancer, preferably from the group consisting of melanoma, prostate
cancer, breast cancer, brain cancers including glioma, colorectal
cancer, stomach cancer, oesophagus cancer and ovarian cancer, and
most preferably is melanoma. [0426] 86. The antibody or
antigen-binding portion thereof of any one of items 45-85 for the
use according to any one of items 45-85, wherein prior to
administration, the tumor or tumors formed by the cancer have
higher human GDF-15 levels compared to a control sample of the same
patient obtained from a non-cancerous part of the tissue which is
the tissue of origin of the cancer, preferably 1.2-fold higher
levels, more preferably 1.5-fold higher levels, still more
preferably 2-fold higher levels and most preferably 5-fold higher
levels. [0427] 87. The antibody or antigen-binding portion thereof
of item 84 for the use according to item 84, wherein the antibody
or antigen-binding portion thereof is used in combination with one
or more further ingredients pharmaceutically active against cancer,
and wherein the one or more further ingredients pharmaceutically
active against cancer are selected from the group consisting of:
alkylating agents; anti-metabolites; alkaloids, taxanes;
topoisomerase inhibitors; cytotoxic antibiotics; and radioisotopes.
[0428] 88. The antibody or antigen-binding portion thereof of item
87 for the use according to item 87, wherein the one or more
further ingredients pharmaceutically active against cancer are
selected from the group consisting of: cisplatin, carboplatin,
oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, and
ifosfamide; azathioprine and mercaptopurine; vincristine,
vinblastine, vinorelbine, and vindesine, paclitaxel, docetaxel,
etoposide and teniposide; irinotecan and topotecan; actinomycin,
anthracyclines, doxorubicin, daunorubicin, valrubicin, idarubicin,
epirubicin, bleomycin, plicamycin and mitomycin.
REFERENCES
[0428] [0429] Arbabi Ghahroudi M et al.: "Selection and
identification of single domain antibody fragments from camel
heavy-chain antibodies." FEES Lett. 1997 Sep. 15; 414(3):521-6.
[0430] Ausubel et al.: "Current Protocols in Molecular Biology."
Greene Publishing Associates and Wiley Interscience; New York 1992.
[0431] Bauskin A R et al.: "The propeptide mediates formation of
stromal stores of PROMIC-1: role in determining prostate cancer
outcome." Cancer Res. 2005 Mar. 15; 65(6):2330-6. [0432] Brown D A
et al.: "Macrophage inhibitory cytokine 1: a new prognostic marker
in prostate cancer." Clin Cancer Res. 2009 Nov. 1; 15(21):6658-64.
[0433] Chothia C et al.: Conformations of immunoglobulin
hypervariable regions. Nature. 1989 Dec. 21-28; 342(6252):877-83.
[0434] Clackson T et al.: "Making antibody fragments using phage
display libraries." Nature. 1991 Aug. 15; 352(6336):624-8. [0435]
Fearon K C.: Cancer cachexia: developing multimodal therapy for a
multidimensional problem. Eur J Cancer. 2008 May; 44(8):1124-32
[0436] Fearon K. et al.: Definition and classification of cancer
cachexia: an international consensus. Lancet Oncol. 2011 May;
12(5):489-95. [0437] Giudicelli V et al.: IMGT/V-QUEST, an
integrated software program for immunoglobulin and T cell receptor
V-J and V-D-J rearrangement analysis. Nucleic Acids Res. 2004 Jul.
1; 32 (Web Server issue):W435-40. [0438] Harlow and Lane:
"Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. 1988. [0439] Holliger P et al.:
"Diabodies": small bivalent and bispecific antibody fragments."
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J et al.: "Domain antibodies: proteins for therapy." Trends
Biotechnol. 2003 November; 21(11):484-90. [0441] Huang C Y et al.:
"Molecular alterations in prostate carcinomas that associate with
in vivo exposure to chemotherapy: identification of a
cytoprotective mechanism involving growth differentiation factor
15." Clin Cancer Res. 2007 Oct. 1; 13(19):5825-33. [0442] Johnen H
et al.: "Tumor-induced anorexia and weight loss are mediated by the
TGF-beta superfamily cytokine MIC-1." Nat Med. 2007 November;
13(11):1333-40. [0443] Jones P T et al.: "Replacing the
complementarity-determining regions in a human antibody with those
from a mouse." Nature. 1986 May 29-Jun. 4; 321(6069):522-5. [0444]
Kabat et al.: Sequences of proteins of immunological interest, U.S.
Dept. of Health and Human Services, Public Health Service, National
Institutes of Health, Bethesda, Md. 1983. [0445] Kohler G and
Milstein C: "Continuous cultures of fused cells secreting antibody
of predefined specificity." Nature. 1975 Aug. 7; 256(5517):495-7.
[0446] Marks J D et al.: "By-passing immunization. Human antibodies
from V-gene libraries displayed on phage." J Mol Biol. 1991 Dec. 5;
222(3):581-97. [0447] Mimeault M and Batra S K: "Divergent
molecular mechanisms underlying the pleiotropic functions of
macrophage inhibitory cytokine-1 in cancer." J Cell Physiol. 2010
September; 224(3):626-35. [0448] Murphy K T and. Lynch G S: Update
on emerging drugs for cancer cachexia. Expert Opin Emerg Drugs.
2009. December; 14(4):619-32. [0449] Paul, W. E. (Ed.).:
"Fundamental Immunology" 2nd Ed. Raven Press, Ltd., New York 1989.
[0450] PCT/EP2013/070127 [0451] Remington's Pharmaceutical
Sciences, Ed. A R Gennaro, 20th edition, 2000, Williams &
Wilkins, P A, USA. [0452] Riechmann L et al.: "Reshaping human
antibodies for therapy." Nature. 1988 Mar. 24; 332(6162):323-7.
[0453] Roth P et al.: "GDF-15 contributes to proliferation and
immune escape of malignant gliomas." Clin Cancer Res. 2010 Aug. 1;
16(15):3851-9. [0454] Saerens D et al.: "Single-domain antibodies
as building blocks for novel therapeutics." Curr Opin Pharmacol.
2008 October; 8(5):600-8. Epub 2008 Aug. 22. [0455] Sambrook et
al.: "Molecular Cloning: A Laboratory Manual.", 2nd Ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989.
[0456] Siegel D L: "Recombinant monoclonal antibody technology."
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al. Proc Nati Acad Sci USA. 1990 December; 87(24): 9848-9852.
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2005/099746 [0462] WO 2009/021293
Sequence CWU 1
1
38197PRTMus musculus 1Gln Val Lys Leu Gln Gln Ser Gly Pro Gly Ile
Leu Gln Ser Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser
Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Ser Trp Ile
Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile
Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Thr 50 55 60 Leu Lys Ser
Arg Leu Thr Ile Ser Lys Asp Pro Ser Arg Asn Gln Val 65 70 75 80 Phe
Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90
95 Cys 288PRTMus musculus 2Asp Ile Val Leu Thr Gln Ser Pro Lys Phe
Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Val Thr Cys Lys
Ala Ser Gln Asn Val Gly Thr Asn 20 25 30 Val Ala Trp Phe Leu Gln
Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile 35 40 45 Tyr Ser Ala Ser
Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80
Glu Asp Leu Ala Glu Tyr Phe Cys 85 310PRTMus musculus 3Gly Phe Ser
Leu Ser Thr Ser Gly Met Gly 1 5 10 47PRTMus musculus 4Ile Tyr Trp
Asp Asp Asp Lys 1 5 510PRTMus musculus 5Ala Arg Ser Ser Tyr Gly Ala
Met Asp Tyr 1 5 10 66PRTMus musculus 6Gln Asn Val Gly Thr Asn 1 5
79PRTMus musculus 7Gln Gln Tyr Asn Asn Phe Pro Tyr Thr 1 5
8114PRTArtificial Sequencerecombinant mature human GDF-15 protein
8Gly Ser Ala Arg Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys 1
5 10 15 Cys Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp
Ala 20 25 30 Asp Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met
Cys Ile Gly 35 40 45 Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn Met
His Ala Gln Ile Lys 50 55 60 Thr Ser Leu His Arg Leu Lys Pro Asp
Thr Val Pro Ala Pro Cys Cys 65 70 75 80 Val Pro Ala Ser Tyr Asn Pro
Met Val Leu Ile Gln Lys Thr Asp Thr 85 90 95 Gly Val Ser Leu Gln
Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His 100 105 110 Cys Ile
9308PRTHomo sapiens 9Met Pro Gly Gln Glu Leu Arg Thr Val Asn Gly
Ser Gln Met Leu Leu 1 5 10 15 Val Leu Leu Val Leu Ser Trp Leu Pro
His Gly Gly Ala Leu Ser Leu 20 25 30 Ala Glu Ala Ser Arg Ala Ser
Phe Pro Gly Pro Ser Glu Leu His Ser 35 40 45 Glu Asp Ser Arg Phe
Arg Glu Leu Arg Lys Arg Tyr Glu Asp Leu Leu 50 55 60 Thr Arg Leu
Arg Ala Asn Gln Ser Trp Glu Asp Ser Asn Thr Asp Leu 65 70 75 80 Val
Pro Ala Pro Ala Val Arg Ile Leu Thr Pro Glu Val Arg Leu Gly 85 90
95 Ser Gly Gly His Leu His Leu Arg Ile Ser Arg Ala Ala Leu Pro Glu
100 105 110 Gly Leu Pro Glu Ala Ser Arg Leu His Arg Ala Leu Phe Arg
Leu Ser 115 120 125 Pro Thr Ala Ser Arg Ser Trp Asp Val Thr Arg Pro
Leu Arg Arg Gln 130 135 140 Leu Ser Leu Ala Arg Pro Gln Ala Pro Ala
Leu His Leu Arg Leu Ser 145 150 155 160 Pro Pro Pro Ser Gln Ser Asp
Gln Leu Leu Ala Glu Ser Ser Ser Ala 165 170 175 Arg Pro Gln Leu Glu
Leu His Leu Arg Pro Gln Ala Ala Arg Gly Arg 180 185 190 Arg Arg Ala
Arg Ala Arg Asn Gly Asp His Cys Pro Leu Gly Pro Gly 195 200 205 Arg
Cys Cys Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly 210 215
220 Trp Ala Asp Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys
225 230 235 240 Ile Gly Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn Met
His Ala Gln 245 250 255 Ile Lys Thr Ser Leu His Arg Leu Lys Pro Asp
Thr Val Pro Ala Pro 260 265 270 Cys Cys Val Pro Ala Ser Tyr Asn Pro
Met Val Leu Ile Gln Lys Thr 275 280 285 Asp Thr Gly Val Ser Leu Gln
Thr Tyr Asp Asp Leu Leu Ala Lys Asp 290 295 300 Cys His Cys Ile 305
10322PRTArtificial Sequencehuman GDF-15 precursor protein +
N-terminal and C-terminal GSGS linker 10Gly Ser Gly Ser Gly Ser Gly
Met Pro Gly Gln Glu Leu Arg Thr Val 1 5 10 15 Asn Gly Ser Gln Met
Leu Leu Val Leu Leu Val Leu Ser Trp Leu Pro 20 25 30 His Gly Gly
Ala Leu Ser Leu Ala Glu Ala Ser Arg Ala Ser Phe Pro 35 40 45 Gly
Pro Ser Glu Leu His Ser Glu Asp Ser Arg Phe Arg Glu Leu Arg 50 55
60 Lys Arg Tyr Glu Asp Leu Leu Thr Arg Leu Arg Ala Asn Gln Ser Trp
65 70 75 80 Glu Asp Ser Asn Thr Asp Leu Val Pro Ala Pro Ala Val Arg
Ile Leu 85 90 95 Thr Pro Glu Val Arg Leu Gly Ser Gly Gly His Leu
His Leu Arg Ile 100 105 110 Ser Arg Ala Ala Leu Pro Glu Gly Leu Pro
Glu Ala Ser Arg Leu His 115 120 125 Arg Ala Leu Phe Arg Leu Ser Pro
Thr Ala Ser Arg Ser Trp Asp Val 130 135 140 Thr Arg Pro Leu Arg Arg
Gln Leu Ser Leu Ala Arg Pro Gln Ala Pro 145 150 155 160 Ala Leu His
Leu Arg Leu Ser Pro Pro Pro Ser Gln Ser Asp Gln Leu 165 170 175 Leu
Ala Glu Ser Ser Ser Ala Arg Pro Gln Leu Glu Leu His Leu Arg 180 185
190 Pro Gln Ala Ala Arg Gly Arg Arg Arg Ala Arg Ala Arg Asn Gly Asp
195 200 205 His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His Thr
Val Arg 210 215 220 Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val
Leu Ser Pro Arg 225 230 235 240 Glu Val Gln Val Thr Met Cys Ile Gly
Ala Cys Pro Ser Gln Phe Arg 245 250 255 Ala Ala Asn Met His Ala Gln
Ile Lys Thr Ser Leu His Arg Leu Lys 260 265 270 Pro Asp Thr Val Pro
Ala Pro Cys Cys Val Pro Ala Ser Tyr Asn Pro 275 280 285 Met Val Leu
Ile Gln Lys Thr Asp Thr Gly Val Ser Leu Gln Thr Tyr 290 295 300 Asp
Asp Leu Leu Ala Lys Asp Cys His Cys Ile Gly Ser Gly Ser Gly 305 310
315 320 Ser Gly 1110PRTArtificial SequenceFlag peptide 11Asp Tyr
Lys Asp Asp Asp Asp Lys Gly Gly 1 5 10 1210PRTArtificial SequenceHA
peptide 12Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Gly 1 5 10
1313PRTArtificial Sequencepeptide derived from human GDF-15 13Glu
Leu His Leu Arg Pro Gln Ala Ala Arg Gly Arg Arg 1 5 10
1413PRTArtificial Sequencepeptide derived from human GDF-15 14Leu
His Leu Arg Pro Gln Ala Ala Arg Gly Arg Arg Arg 1 5 10
1513PRTArtificial Sequencepeptide derived from human GDF-15 15His
Leu Arg Pro Gln Ala Ala Arg Gly Arg Arg Arg Ala 1 5 10
1613PRTArtificial Sequencepeptide derived from human GDF-15 16Leu
Arg Pro Gln Ala Ala Arg Gly Arg Arg Arg Ala Arg 1 5 10
1713PRTArtificial Sequencepeptide derived from human GDF-15 17Arg
Pro Gln Ala Ala Arg Gly Arg Arg Arg Ala Arg Ala 1 5 10
1813PRTArtificial Sequencepeptide derived from human GDF-15 18Pro
Gln Ala Ala Arg Gly Arg Arg Arg Ala Arg Ala Arg 1 5 10
1913PRTArtificial Sequencepeptide derived from human GDF-15 19Gln
Ala Ala Arg Gly Arg Arg Arg Ala Arg Ala Arg Asn 1 5 10
2013PRTArtificial Sequencepeptide derived from human GDF-15 20Met
His Ala Gln Ile Lys Thr Ser Leu His Arg Leu Lys 1 5 10 21291DNAMus
musculus 21caagtgaagc tgcagcagtc aggccctggg atattgcagt cctcccagac
cctcagtctg 60acttgttctt tctctgggtt ttcactgagt acttctggta tgggtgtgag
ctggattcgt 120cagccttcag gaaagggtct ggagtggctg gcacacattt
actgggatga tgacaagcgc 180tataacccaa ccctgaagag ccggctcaca
atctccaagg atccctccag aaaccaggta 240ttcctcaaga tcaccagtgt
ggacactgca gatactgcca catactactg t 29122264DNAMus musculus
22gacattgtgc tcacccagtc tccaaaattc atgtccacat cagtaggaga cagggtcagc
60gtcacctgca aggccagtca gaatgtgggt actaatgtgg cctggtttct acagaaacca
120gggcaatctc ctaaagcact tatttactcg gcatcctacc ggtacagtgg
agtccctgat 180cgcttcacag gcagtggatc tgggacagat ttcactctca
ccatcagcaa cgtgcagtct 240gaagacttgg cagagtattt ctgt 2642330DNAMus
musculus 23gctcgaagtt cctacggggc aatggactac 302427DNAMus musculus
24cagcaatata acaactttcc gtacacg 272516PRTHomo sapiens 25Glu Val Gln
Val Thr Met Cys Ile Gly Ala Cys Pro Ser Gln Phe Arg 1 5 10 15
2621PRTHomo sapiens 26Thr Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp
Asp Leu Leu Ala Lys 1 5 10 15 Asp Cys His Cys Ile 20
27448PRTArtificial Sequenceamino acid sequence of the heavy chain
of the H1L5 humanized B1-23 anti-GDF-15 antibody 27Gln Ile Thr Leu
Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu
Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30
Gly Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35
40 45 Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro
Thr 50 55 60 Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Pro Ser Lys
Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp
Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Ser Tyr Gly Ala Met
Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165
170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410
415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 435 440 445 28118PRTArtificial Sequenceamino acid sequence
of the heavy chain variable domain of the H1L5 humanized B1-23
anti-GDF-15 antibody 28Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu
Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser
Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Ser Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile
Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Thr 50 55 60 Leu Lys Ser
Arg Leu Thr Ile Thr Lys Asp Pro Ser Lys Asn Gln Val 65 70 75 80 Val
Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90
95 Cys Ala Arg Ser Ser Tyr Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110 Leu Val Thr Val Ser Ser 115 29330PRTArtificial
Sequenceamino acid sequence of the heavy chain constant domain of
the H1L5 humanized B1-23 anti-GDF-15 antibody 29Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305
310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
30211PRTArtificial Sequenceamino acid sequence of the light chain
of the H1L5 humanized B1-23 anti-GDF-15 antibody 30Asp Ile Val Leu
Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn 20 25 30
Val Ala Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Ala Leu Ile 35
40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr
Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Ala Tyr Phe Cys Gln Gln Tyr
Asn Asn Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys Arg Ala Pro Ser Val 100 105 110 Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly Thr Ala Ser 115 120 125 Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln 130 135 140 Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val 145 150 155 160
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu 165
170 175 Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys
Glu 180 185 190 Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
Phe Asn Arg 195 200 205 Gly Glu Cys 210 31108PRTArtificial
Sequenceamino acid sequence of the light chain variable domain of
the H1L5 humanized B1-23 anti-GDF-15 antibody 31 Asp Ile Val Leu
Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn 20 25 30
Val Ala Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Ala Leu Ile 35
40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr
Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Ala Tyr Phe Cys Gln Gln Tyr
Asn Asn Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys Arg 100 105 32103PRTArtificial Sequenceamino acid sequence
of the light chain constant domain of the H1L5 humanized B1-23
anti-GDF-15 antibody 32Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser 1 5 10 15 Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu 20 25 30 Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser 35 40 45 Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 50 55 60 Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 65 70 75 80 Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 85 90
95 Ser Phe Asn Arg Gly Glu Cys 100 33448PRTArtificial Sequenceamino
acid sequence of the heavy chain of the chimeric B1-23 anti-GDF-15
antibody 33Gln Val Lys Leu Gln Gln Ser Gly Pro Gly Ile Leu Gln Ser
Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser
Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Ser Trp Ile Arg Gln Pro
Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Tyr Trp Asp
Asp Asp Lys Arg Tyr Asn Pro Thr 50 55 60 Leu Lys Ser Arg Leu Thr
Ile Ser Lys Asp Pro Ser Arg Asn Gln Val 65 70 75 80 Phe Leu Lys Ile
Thr Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala
Arg Ser Ser Tyr Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110
Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115
120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235
240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360
365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 34118PRTArtificial
Sequenceamino acid sequence of the heavy chain variable domain of
the chimeric B1-23 anti-GDF-15 antibody 34Gln Val Lys Leu Gln Gln
Ser Gly Pro Gly Ile Leu Gln Ser Ser Gln 1 5 10 15 Thr Leu Ser Leu
Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met
Gly Val Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45
Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Thr 50
55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Pro Ser Arg Asn Gln
Val 65 70 75 80 Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Thr Ala
Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Ser Tyr Gly Ala Met Asp Tyr
Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115
35330PRTArtificial Sequenceamino acid sequence of the heavy chain
constant domain of the chimeric B1-23 anti-GDF-15 antibody 35Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10
15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145
150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 325 330 36214PRTArtificial Sequenceamino acid sequence of the
light chain of the chimeric B1-23 anti-GDF-15 antibody 36Asp Ile
Val Leu Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15
Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn 20
25 30 Val Ala Trp Phe Leu Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu
Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg
Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Glu Tyr Phe Cys Gln
Gln Tyr Asn Asn Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150
155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
37111PRTArtificial Sequenceamino acid sequence of the light chain
variable domain of the chimeric B1-23 anti-GDF-15 antibody 37 Asp
Ile Val Leu Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly 1 5 10
15 Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn
20 25 30 Val Ala Trp Phe Leu Gln Lys Pro Gly Gln Ser Pro Lys Ala
Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp
Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Glu Tyr Phe Cys
Gln Gln Tyr Asn Asn Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 38103PRTArtificial
Sequenceamino acid sequence of the light chain constant domain of
the chimeric B1-23 anti-GDF-15 antibody 38Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 1 5 10 15 Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 20 25 30 Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 35 40 45
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 50
55 60 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val 65 70 75 80 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys 85 90 95 Ser Phe Asn Arg Gly Glu Cys 100
* * * * *