U.S. patent application number 14/485140 was filed with the patent office on 2015-03-12 for combination therapy of antibodies against human csf-1r and antibodies agains human pd-l1.
This patent application is currently assigned to HOFFMANN-LA ROCHE INC.. The applicant listed for this patent is Hoffmann-La Roche Inc.. Invention is credited to Frank Herting, Sabine Hoves, Carola Ries, Katharina Wartha.
Application Number | 20150073129 14/485140 |
Document ID | / |
Family ID | 49123797 |
Filed Date | 2015-03-12 |
United States Patent
Application |
20150073129 |
Kind Code |
A1 |
Herting; Frank ; et
al. |
March 12, 2015 |
Combination therapy of antibodies against human CSF-1R and
antibodies agains human PD-L1
Abstract
The present invention relates to the combination therapy of
specific antibodies which bind human CSF-1R with specific
antibodies which bind human PD-L1.
Inventors: |
Herting; Frank; (Penzberg,
DE) ; Hoves; Sabine; (Habach, DE) ; Ries;
Carola; (Penzberg, DE) ; Wartha; Katharina;
(Germering, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc. |
Little Falls |
NJ |
US |
|
|
Assignee: |
HOFFMANN-LA ROCHE INC.
Little Falls
NJ
|
Family ID: |
49123797 |
Appl. No.: |
14/485140 |
Filed: |
September 12, 2014 |
Current U.S.
Class: |
530/387.3 ;
530/387.7 |
Current CPC
Class: |
C07K 2317/76 20130101;
C07K 16/2866 20130101; C07K 16/30 20130101; A61K 2039/507 20130101;
C07K 2317/52 20130101; C07K 16/2827 20130101; A61P 35/04 20180101;
A61K 2039/505 20130101; A61P 35/00 20180101; C07K 2317/565
20130101; C07K 16/2809 20130101 |
Class at
Publication: |
530/387.3 ;
530/387.7 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 12, 2013 |
EP |
13184120.7 |
Claims
1. An antibody which binds to human CSF-1R wherein the antibody is
administered in combination with an antibody which binds to human
PD-L1 for use in the treatment of cancer, for use in the prevention
or treatment of metastasis, for use in the treatment inflammatory
diseases, for use in the treatment of bone loss, for use in
treating or delaying progression of an immune related disease such
as tumor immunity, or for use in stimulating an immune response or
function, such as T cell activity, wherein the antibody which binds
to human CSF-1R used in the combination therapy is characterized in
comprising a) a heavy chain variable domain VH of SEQ ID NO:23 and
a light chain variable domain VL of SEQ ID NO:24, or b) a heavy
chain variable domain VH of SEQ ID NO:31 and a light chain variable
domain VL of SEQ ID NO:32, or c) a heavy chain variable domain VH
of SEQ ID NO:39 and a light chain variable domain VL of SEQ ID
NO:40, or d) a heavy chain variable domain VH of SEQ ID NO:47 and a
light chain variable domain VL of SEQ ID NO:48, or e) a heavy chain
variable domain VH of SEQ ID NO:55 and a light chain variable
domain VL of SEQ ID NO:56; and the antibody which binds to human
PD-L1 used in the combination therapy is characterized in
comprising a) a heavy chain variable domain VH of SEQ ID NO:89 and
a light chain variable domain VL of SEQ ID NO:92, or b) a heavy
chain variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:93, or c) a heavy chain variable domain VH
of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID
NO:94, or d) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:95, or e) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:96, or f) a heavy chain variable domain VH
of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID
NO:97, or g) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:98, or h) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:99, or i) a heavy chain variable domain VH
of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID
NO:100, or j) a heavy chain variable domain VH of SEQ ID NO:90 and
a light chain variable domain VL of SEQ ID NO:101, or k) a heavy
chain variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:102, or l) a heavy chain variable domain VH
of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID
NO:103, or m) a heavy chain variable domain VH of SEQ ID NO:90 and
a light chain variable domain VL of SEQ ID NO:104, or n) a heavy
chain variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:105, or o) a heavy chain variable domain VH
of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID
NO:106, or p) a heavy chain variable domain VH of SEQ ID NO:91 and
a light chain variable domain VL of SEQ ID NO:107.
2. The antibody according to claim 1, for use in the treatment of
cancer.
3. The antibody according to claim 2, for use in the treatment of
breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma
cancer, bladder cancer, renal cancer, kidney cancer, liver cancer,
head and neck cancer, colorectal cancer, pancreatic cancer, gastric
carcinoma cancer, esophageal cancer, mesotheliioma, prostate
cancer, leukemia, lymphomas, myelomas.
4. The antibody according to claim 1, for use in the prevention or
treatment of metastasis.
5. The antibody according to claim 1, for use in the treatment of
bone loss.
6. The antibody according to claim 1, for use in the treatment of
inflammatory diseases.
7. The antibody according to claim 1, for use in treating or
delaying progression of an immune related disease such as tumor
immunity.
8. The antibody according to claim 1, for use in stimulating an
immune response or function, such as T cell activity.
9. An antibody which binds to human CSF-1R wherein the antibody is
administered in combination with an antibody which binds to human
PD-L1, for use in i) the inhibition of cell proliferation in CSF-1R
ligand-dependent and/or CSF-1 ligand-independent CSF-1R expressing
tumor cells; ii) the inhibition of cell proliferation of tumors
with CSF-1R ligand-dependent and/or CSF-1R ligand-independent
CSF-1R expressing macrophage infiltrate; iii) the inhibition of
cell survival (in CSF-1R ligand-dependent and/or CSF-1R
ligand-independent) CSF-1R expressing monocytes and macrophages;
and/or iv) the inhibition of cell differentiation (in CSF-1R
ligand-dependent and/or CSF-1R ligand-independent) CSF-1R
expressing monocytes into macrophages, wherein the antibody which
binds to human CSF-1R used in the combination therapy is
characterized in comprising a) a heavy chain variable domain VH of
SEQ ID NO:23 and a light chain variable domain VL of SEQ ID NO:24,
or b) a heavy chain variable domain VH of SEQ ID NO:31 and a light
chain variable domain VL of SEQ ID NO:32, or c) a heavy chain
variable domain VH of SEQ ID NO:39 and a light chain variable
domain VL of SEQ ID NO:40, or d) a heavy chain variable domain VH
of SEQ ID NO:47 and a light chain variable domain VL of SEQ ID
NO:48, or e) a heavy chain variable domain VH of SEQ ID NO:55 and a
light chain variable domain VL of SEQ ID NO:56; and the antibody
which binds to human PD-L1 used in the combination therapy is
characterized in comprising a) a heavy chain variable domain VH of
SEQ ID NO:89 and a light chain variable domain VL of SEQ ID NO:92,
or b) a heavy chain variable domain VH of SEQ ID NO:90 and a light
chain variable domain VL of SEQ ID NO:93, or c) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:94, or d) a heavy chain variable domain VH
of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID
NO:95, or e) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:96, or f) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:97, or g) a heavy chain variable domain VH
of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID
NO:98, or h) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:99, or i) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:100, or j) a heavy chain variable domain VH
of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID
NO:101, or k) a heavy chain variable domain VH of SEQ ID NO:90 and
a light chain variable domain VL of SEQ ID NO:102, or l) a heavy
chain variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:103, or m) a heavy chain variable domain VH
of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID
NO:104, or n) a heavy chain variable domain VH of SEQ ID NO:90 and
a light chain variable domain VL of SEQ ID NO:105, or o) a heavy
chain variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:106, or p) a heavy chain variable domain VH
of SEQ ID NO:91 and a light chain variable domain VL of SEQ ID
NO:107.
10. An antibody which binds to human CSF-1R, for use in the
treatment of a patient having a CSF-1R expressing tumor or having a
tumor with CSF-1R expressing macrophage infiltrate, wherein the
tumor is characterized by an increase of CSF-1R ligand and wherein
the anti-CSF-1R antibody is administered in combination with an
antibody which binds to human PD-L1, wherein the antibody which
binds to human CSF-1R used in the combination therapy is
characterized in comprising a) a heavy chain variable domain VH of
SEQ ID NO:23 and a light chain variable domain VL of SEQ ID NO:24,
or b) a heavy chain variable domain VH of SEQ ID NO:31 and a light
chain variable domain VL of SEQ ID NO:32, or c) a heavy chain
variable domain VH of SEQ ID NO:39 and a light chain variable
domain VL of SEQ ID NO:40, or d) a heavy chain variable domain VH
of SEQ ID NO:47 and a light chain variable domain VL of SEQ ID
NO:48, or e) a heavy chain variable domain VH of SEQ ID NO:55 and a
light chain variable domain VL of SEQ ID NO:56; and the antibody
which binds to human PD-L1 used in the combination therapy is
characterized in comprising a) a heavy chain variable domain VH of
SEQ ID NO:89 and a light chain variable domain VL of SEQ ID NO:92,
or b) a heavy chain variable domain VH of SEQ ID NO:90 and a light
chain variable domain VL of SEQ ID NO:93, or c) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:94, or d) a heavy chain variable domain VH
of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID
NO:95, or e) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:96, or f) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:97, or g) a heavy chain variable domain VH
of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID
NO:98, or h) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:99, or i) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:100, or j) a heavy chain variable domain VH
of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID
NO:101, or k) a heavy chain variable domain VH of SEQ ID NO:90 and
a light chain variable domain VL of SEQ ID NO:102, or l) a heavy
chain variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:103, or m) a heavy chain variable domain VH
of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID
NO:104, or n) a heavy chain variable domain VH of SEQ ID NO:90 and
a light chain variable domain VL of SEQ ID NO:105, or o) a heavy
chain variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:106, or p) a heavy chain variable domain VH
of SEQ ID NO:91 and a light chain variable domain VL of SEQ ID
NO:107.
11. The antibody according to any one of the preceding claims,
characterized in that said antibodies are of human IgG1 subclass or
human IgG4 subclass.
12. The antibody according to any one of the preceding claims,
characterized in that said antibodies have reduced or minimal
effector function.
13. The antibody according to any one of the preceding claims,
characterized in that the minimal effector function results from an
effectorless Fc mutation.
14. The antibody according to any one of the preceding claims,
characterized in that the effectorless Fc mutation is L234A/L235A
or L234A/L235A/P329G or N297A or D265A/N297A.
Description
[0001] The present invention relates to the combination therapy of
specific antibodies which bind human CSF-1R with specific
antibodies which bind human PD-L1.
BACKGROUND OF THE INVENTION
CSF-1R and CSF-1R Antibodies
[0002] The human CSF-1 receptor (CSF-1R; colony stimulating factor
1 receptor; synonyms: M-CSF receptor; Macrophage colony-stimulating
factor 1 receptor, Fms proto-oncogene, c-fms, SEQ ID NO: 62) is
known since 1986 (Coussens, L., et al., Nature 320 (1986) 277-280).
CSF-1R is a growth factor and encoded by the c-fms proto-oncogene
(reviewed e.g. in Roth, P., and Stanley, E. R., Curr. Top.
Microbiol. Immunol. 181 (1992) 141-167).
[0003] CSF-1R is the receptor for CSF-1 (colony stimulating factor
1, also called M-CSF, macrophage colony-stimulating factor) and
mediates the biological effects of this cytokine (Sherr, C. J., et
al., Cell 41 (1985) 665-676). The cloning of the colony stimulating
factor-1 receptor (CSF-1R) (also called c-fms) was described for
the first time in Roussel, M. F., et al., Nature 325 (1987)
549-552. In that publication, it was shown that CSF-1R had
transforming potential dependent on changes in the C-terminal tail
of the protein including the loss of the inhibitory tyrosine 969
phosphorylation which binds Cbl and thereby regulates receptor down
regulation (Lee, P. S., et al., Embo J. 18 (1999) 3616-3628).
Recently a second ligand for CSF-1R termed interleukin-34 (IL-34)
was identified (Lin, H., et al, Science 320 (2008) 807-811).
[0004] Currently two CSF-1R ligands that bind to the extracellular
domain of CSF-1R are known. The first one is CSF-1 (colony
stimulating factor 1, also called M-CSF, macrophage; SEQ ID NO: 86)
and is found extracellularly as a disulfide-linked homodimer
(Stanley, E. R. et al., Journal of Cellular Biochemistry 21 (1983)
151-159; Stanley, E. R. et al., Stem Cells 12 Suppl. 1 (1995)
15-24). The second one is IL-34 (Human IL-34; SEQ ID NO: 87) (Hume,
D. A., et al, Blood 119 (2012) 1810-1820). The main biological
effects of CSF-1R signaling are the differentiation, proliferation,
migration, and survival of hematopoietic precursor cells to the
macrophage lineage (including osteoclast). Activation of CSF-1R is
mediated by its CSF-1R ligands, CSF-1 (M-CSF) and IL-34. Binding of
CSF-1 (M-CSF) to CSF-1R induces the formation of homodimers and
activation of the kinase by tyrosine phosphorylation (Li, W. et al,
EMBO Journal. 10 (1991) 277-288; Stanley, E. R., et al., Mol.
Reprod. Dev. 46 (1997) 4-10).
[0005] The biologically active homodimer CSF-1 binds to the CSF-1R
within the subdomains D1 to D3 of the extracellular domain of the
CSF-1 receptor (CSF-1R-ECD). The CSF-1R-ECD comprises five
immunoglobulin-like subdomains (designated D1 to D5). The
subdomains D4 to D5 of the extracellular domain (CSF-1R-ECD) are
not involved in the CSF-1 binding (Wang, Z., et al Molecular and
Cellular Biology 13 (1993) 5348-5359). The subdomain D4 is involved
in dimerization (Yeung, Y-G., et al Molecular & Cellular
Proteomics 2 (2003) 1143-1155; Pixley, F. J., et al., Trends Cell
Biol. 14 (2004) 628-638).
[0006] Further signaling is mediated by the p85 subunit of PI3K and
Grb2 connecting to the PI3K/AKT and Ras/MAPK pathways,
respectively. These two important signaling pathways can regulate
proliferation, survival and apoptosis. Other signaling molecules
that bind the phosphorylated intracellular domain of CSF-1R include
STAT1, STAT3, PLCy, and Cbl (Bourette, R. P. and Rohrschneider, L.
R., Growth Factors 17 (2000) 155-166).
[0007] CSF-1R signaling has a physiological role in immune
responses, in bone remodeling and in the reproductive system. The
knockout animals for either CSF-1 (Pollard, J. W., Mol. Reprod.
Dev. 46 (1997) 54-61) or CSF-1R (Dai, X. M., et al., Blood 99
(2002) 111-120) have been shown to have osteopetrotic,
hematopoietic, tissue macrophage, and reproductive phenotypes
consistent with a role for CSF-1R in the respective cell types.
[0008] Sherr, C. J., et al., Blood 73 (1989) 1786-1793 relates to
some antibodies against CSF-1R that inhibit the CSF-1 activity.
Ashmun, R. A., et al., Blood 73 (1989) 827-837 relates to CSF-1R
antibodies. Lenda, D., et al., Journal of Immunology 170 (2003)
3254-3262 relates to reduced macrophage recruitment, proliferation,
and activation in CSF-1-deficient mice results in decreased tubular
apoptosis during renal inflammation. Kitaura, H., et al., Journal
of Dental Research 87 (2008) 396-400 refers to an anti-CSF-1
antibody which inhibits orthodontic tooth movement. WO 2001/030381
mentions CSF-1 activity inhibitors including antisense nucleotides
and antibodies while disclosing only CSF-1 antisense nucleotides.
WO 2004/045532 relates to metastases and bone loss prevention and
treatment of metastatic cancer by a CSF-1 antagonist disclosing as
antagonist anti-CSF-1-antibodies only. WO 2005/046657 relates to
the treatment of inflammatory bowel disease by
anti-CSF-1-antibodies. US 2002/0141994 relates to inhibitors of
colony stimulating factors. WO 2006/096489 relates to the treatment
of rheumatoid arthritis by anti-CSF-1-antibodies. WO 2009/026303
and WO 2009/112245 relate to certain anti-CSF-1R antibodies binding
to CSF-1R within the first three subdomains (D1 to D3) of the
Extracellular Domain (CSF-1R-ECD). WO2011/123381(A1) relates to
antibodies against CSF-1R. WO2011/070024 relate to certain
anti-CSF-1R antibodies binding to CSF-1R within the dimerization
domain (D4 to D5).
PD-L1 and PD-L1 antibodies
[0009] Co-stimulation or the provision of two distinct signals to
T-cells is a widely accepted model of lymphocyte activation of
resting T lymphocytes by antigen-presenting cells (APCs). Lafferty
et al., Aust. J. Exp. Biol. Med. Sci. 53: 27-42 (1975).
[0010] This model further provides for the discrimination of self
from non-self and immune tolerance. Bretscher et al., Science 169:
1042-1049 (1970); Bretscher, P. A., P.N.A.S. USA 96: 185-190
(1999); Jenkins et al., J. Exp. Med. 165: 302-319 (1987). The
primary signal, or antigen specific signal, is transduced through
the T-cell receptor (TCR) following recognition of foreign antigen
peptide presented in the context of the major
histocompatibility-complex (MHC). The second or co-stimulatory
signal is delivered to T-cells by co-stimulatory molecules
expressed on antigen-presenting cells (APCs), and induce T-cells to
promote clonal expansion, cytokine secretion and effector function.
Lenschow et al., Ann. Rev. Immunol. 14:233 (1996). In the absence
of co-stimulation, T-cells can become refractory to antigen
stimulation, do not mount an effective immune response, and further
may result in exhaustion or tolerance to foreign antigens.
[0011] The simple two-signal model can be an oversimplification
because the strength of the TCR signal actually has a quantitative
influence on T-cell activation and differentiation. Viola et al.,
Science 273: 104-106 (1996); Sloan-Lancaster, Nature 363: 156-159
(1993). Moreover, T-cell activation can occur even in the absence
of co-stimulatory signal if the TCR signal strength is high. More
importantly, T-cells receive both positive and negative secondary
co-stimulatory signals. The regulation of such positive and
negative signals is critical to maximize the host's protective
immune responses, while maintaining immune tolerance and preventing
autoimmunity.
[0012] Negative secondary signals seem necessary for induction of
T-cell tolerance, while positive signals promote T-cell activation.
While the simple two-signal model still provides a valid
explanation for naive lymphocytes, a host's immune response is a
dynamic process, and co-stimulatory signals can also be provided to
antigen-exposed T-cells.
[0013] The mechanism of co-stimulation is of therapeutic interest
because the manipulation of co-stimulatory signals has shown to
provide a means to either enhance or terminate cell-based immune
response. Recently, it has been discovered that T cell dysfunction
or anergy occurs concurrently with an induced and sustained
expression of the inhibitory receptor, programmed death 1
polypeptide (PD-1). As a result, therapeutic targeting PD-1 and
other molecules which signal through interactions with PD-1, such
as programmed death ligand 1 (PD-L1) and programmed death ligand 2
(PD-L2) are an area of intense interest. The inhibition of PD-L1
signaling has been proposed as a means to enhance T cell immunity
for the treatment of cancer (e.g., tumor immunity) and infection,
including both acute and chronic (e.g., persistent) infection.
However, as an optimal therapeutic directed to a target in this
pathway has yet to be commercialized, a significant unmet medical
need exists. Antibodies against PD-L1 are described e.g. in WO
2010/077634.
SUMMARY OF THE INVENTION
[0014] The invention comprises the combination therapy of an
antibody which binds to human CSF-1R with an antibody which binds
to human PD-L1 for use in the treatment of cancer, for use in the
prevention or treatment of metastasis, for use in the treatment
inflammatory diseases, for use in the treatment of bone loss, for
use in treating or delaying progression of an immune related
disease such as tumor immunity, or for use in stimulating an immune
response or function, such as T cell activity.
[0015] The invention further comprises the use of antibody which
binds to human CSF-1R for the manufacture of a medicament for use
in the treatment of cancer, for use in the treatment inflammatory
diseases, for use in the treatment of bone loss, for use in
treating or delaying progression of an immune related disease such
as tumor immunity, or for use in stimulating an immune response or
function, such as T cell activity, wherein the antibody is
administered in combination with an antibody which binds to human
PD-L1.
[0016] The antibody which binds to human CSF-1R used in the
combination therapy is characterized in comprising [0017] a) a
heavy chain variable domain VH of SEQ ID NO:23 and a light chain
variable domain VL of SEQ ID NO:24, or [0018] b) a heavy chain
variable domain VH of SEQ ID NO:31 and a light chain variable
domain VL of SEQ ID NO:32, or [0019] c) a heavy chain variable
domain VH of SEQ ID NO:39 and a light chain variable domain VL of
SEQ ID NO:40, or [0020] d) a heavy chain variable domain VH of SEQ
ID NO:47 and a light chain variable domain VL of SEQ ID NO:48, or
[0021] e) a heavy chain variable domain VH of SEQ ID NO:55 and a
light chain variable domain VL of SEQ ID NO:56; and the antibody
which binds to human PD-L1 used in the combination therapy is
characterized in comprising [0022] a) a heavy chain variable domain
VH of SEQ ID NO:89 and a light chain variable domain VL of SEQ ID
NO:92, or [0023] b) a heavy chain variable domain VH of SEQ ID
NO:90 and a light chain variable domain VL of SEQ ID NO:93, or
[0024] c) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:94, or [0025] d) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:95, or [0026] e) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:96, or [0027] f) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:97, or [0028] g) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:98, or
[0029] h) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:99, or [0030] i) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:100, or [0031] j) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:101, or [0032] k) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:102, or [0033] l) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:103, or
[0034] m) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:104, or [0035] n) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:105, or [0036] o) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:106, or [0037] p) a heavy chain variable
domain VH of SEQ ID NO:91 and a light chain variable domain VL of
SEQ ID NO:107. In one embodiment the antibody is for use in the
treatment of cancer. [0038] In one embodiment the antibody is for
use in the prevention or treatment of metastasis. [0039] In one
embodiment the antibody is, for use in the treatment of bone loss.
[0040] In one embodiment the antibody is for use in the treatment
of inflammatory diseases. In one embodiment the antibody is for use
in treating or delaying progression of an immune related disease
such as tumor immunity. In one embodiment the antibody is for use
in stimulating an immune response or function, such as T cell
activity. [0041] The invention further comprises antibody which
binds to human CSF-1R wherein the antibody is administered in
combination with an antibody which binds to human PD-L1 for use in
[0042] i) the inhibition of cell proliferation in CSF-1R
ligand-dependent and/or CSF-1 ligand-independent CSF-1R expressing
tumor cells; [0043] ii) the inhibition of cell proliferation of
tumors with CSF-1R ligand-dependent and/or CSF-1R
ligand-independent CSF-1R expressing macrophage infiltrate; [0044]
iii) the inhibition of cell survival (in CSF-1R ligand-dependent
and/or CSF-1R ligand-independent) CSF-1R expressing monocytes and
macrophages; and/or [0045] iv) the inhibition of cell
differentiation (in CSF-1R ligand-dependent and/or CSF-1R
ligand-independent) CSF-1R expressing monocytes into macrophages;
[0046] wherein the antibody is administered in combination with an
antibody which binds to human PD-L1; [0047] wherein the antibody
which binds to human CSF-1R used in the combination therapy is
characterized in comprising [0048] a) a heavy chain variable domain
VH of SEQ ID NO:23 and a light chain variable domain VL of SEQ ID
NO:24, or [0049] b) a heavy chain variable domain VH of SEQ ID
NO:31 and a light chain variable domain VL of SEQ ID NO:32, or
[0050] c) a heavy chain variable domain VH of SEQ ID NO:39 and a
light chain variable domain VL of SEQ ID NO:40, or [0051] d) a
heavy chain variable domain VH of SEQ ID NO:47 and a light chain
variable domain VL of SEQ ID NO:48, or [0052] e) a heavy chain
variable domain VH of SEQ ID NO:55 and a light chain variable
domain VL of SEQ ID NO:56; [0053] and the antibody which binds to
human PD-L1 used in the combination therapy is characterized in
comprising [0054] a) a heavy chain variable domain VH of SEQ ID
NO:89 and a light chain variable domain VL of SEQ ID NO:92, or
[0055] b) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:93, or [0056] c) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:94, or [0057] d) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:95, or [0058] e) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:96, or [0059] f) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:97, or
[0060] g) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:98, or [0061] h) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:99, or [0062] i) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:100, or [0063] j) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:101, or [0064] k) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:102, or
[0065] l) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:103, or [0066] m) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:104, or [0067] n) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:105, or [0068] o) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:106, or [0069] p) a heavy chain variable domain VH of SEQ
ID NO:91 and a light chain variable domain VL of SEQ ID NO:107.
[0070] The invention further comprised an antibody which binds to
human CSF-1R, for use in the treatment of a patient having a CSF-1R
expressing tumor or having a tumor with CSF-1R expressing
macrophage infiltrate, wherein the tumor is characterized by an
increase of CSF-1R ligand and wherein the anti-CSF-1R antibody is
administered in combination with an antibody which binds to human
PD-L1, [0071] wherein the antibody which binds to human CSF-1R used
in the combination therapy is characterized in comprising [0072] a)
a heavy chain variable domain VH of SEQ ID NO:23 and a light chain
variable domain VL of SEQ ID NO:24, or [0073] b) a heavy chain
variable domain VH of SEQ ID NO:31 and a light chain variable
domain VL of SEQ ID NO:32, or [0074] c) a heavy chain variable
domain VH of SEQ ID NO:39 and a light chain variable domain VL of
SEQ ID NO:40, or [0075] d) a heavy chain variable domain VH of SEQ
ID NO:47 and a light chain variable domain VL of SEQ ID NO:48, or
[0076] e) a heavy chain variable domain VH of SEQ ID NO:55 and a
light chain variable domain VL of SEQ ID NO:56; [0077] and the
antibody which binds to human PD-L1 used in the combination therapy
is characterized in comprising [0078] a) a heavy chain variable
domain VH of SEQ ID NO:89 and a light chain variable domain VL of
SEQ ID NO:92, or [0079] b) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:93, or
[0080] c) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:94, or [0081] d) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:95, or [0082] e) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:96, or [0083] f) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:97, or [0084] g) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:98, or
[0085] h) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:99, or [0086] i) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:100, or [0087] j) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:101, or [0088] k) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:102, or [0089] l) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:103, or
[0090] m) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:104, or [0091] n) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:105, or [0092] o) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:106, or [0093] p) a heavy chain variable
domain VH of SEQ ID NO:91 and a light chain variable domain VL of
SEQ ID NO:107. [0094] In one embodiment the antibodies are of human
IgG1 subclass or human IgG4 subclass. [0095] The invention further
comprises: [0096] A) a method for [0097] i) the inhibition of cell
proliferation in CSF-1R ligand-dependent and/or CSF-1R
ligand-independent CSF-1R expressing tumor cells; [0098] ii) the
inhibition of cell proliferation of tumors with CSF-1R
ligand-dependent and/or CSF-1R ligand-independent CSF-1R expressing
macrophage infiltrate; [0099] iii) the inhibition of cell survival
(in CSF-1R ligand-dependent and/or CSF-1R ligand-independent)
CSF-1R expressing monocytes and macrophages; and/or [0100] iv) the
inhibition of cell differentiation (in CSF-1R ligand-dependent
and/or CSF-1R ligand-independent) CSF-1R expressing monocytes into
macrophages; [0101] wherein an antibody which binds to human
CSF-1R, is administered in combination with an antibody which binds
to human PD-L1, [0102] or [0103] B) a method of treatment of a
patient having a CSF-1R expressing tumor or having a tumor with
CSF-1R expressing macrophage infiltrate, wherein the tumor is
characterized by an increase of CSF-1R ligand and wherein an
antibody which binds to human CSF-1R is administered in combination
with an antibody which binds to human PD-L1, [0104] wherein the
antibody which binds to human CSF-1R used in the combination
therapy is characterized in comprising [0105] a) a heavy chain
variable domain VH of SEQ ID NO:23 and a light chain variable
domain VL of SEQ ID NO:24, or [0106] b) a heavy chain variable
domain VH of SEQ ID NO:31 and a light chain variable domain VL of
SEQ ID NO:32, or [0107] c) a heavy chain variable domain VH of SEQ
ID NO:39 and a light chain variable domain VL of SEQ ID NO:40, or
[0108] d) a heavy chain variable domain VH of SEQ ID NO:47 and a
light chain variable domain VL of SEQ ID NO:48, or [0109] e) a
heavy chain variable domain VH of SEQ ID NO:55 and a light chain
variable domain VL of SEQ ID NO:56; [0110] and the antibody which
binds to human PD-L1 used in the combination therapy is
characterized in comprising [0111] a) a heavy chain variable domain
VH of SEQ ID NO:89 and a light chain variable domain VL of SEQ ID
NO:92, or [0112] b) a heavy chain variable domain VH of SEQ ID
NO:90 and a light chain variable domain VL of SEQ ID NO:93, or
[0113] c) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:94, or [0114] d) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:95, or [0115] e) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:96, or [0116] f) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:97, or [0117] g) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:98, or
[0118] h) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:99, or [0119] i) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:100, or [0120] j) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:101, or [0121] k) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:102, or [0122] l) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:103, or
[0123] m) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:104, or [0124] n) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:105, or [0125] o) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:106, or [0126] p) a heavy chain variable
domain VH of SEQ ID NO:91 and a light chain variable domain VL of
SEQ ID NO:107.
[0127] The term "ligand independent" as used herein refers to a
ligand-independent signaling through the extracellular ECD (and
does not include the ligand independent signaling mediated by
activating point mutations in the intracellular kinase domain). In
one embodiment CSF-1R ligand in this context refers a CSF-1R ligand
selected from human CSF-1 (SEQ ID No: 86) and human IL-34 (SEQ ID
No: 87); in one embodiment the CSF-1R ligand is human CSF-1 (SEQ ID
No: 86); in one embodiment the CSF-1R ligand is human IL-34 (SEQ ID
No: 87)).
[0128] The invention comprises the combination treatment of a
patient having a CSF-1R expressing tumor or having a tumor with
CSF-1R expressing macrophage infiltrate, wherein the tumor is
characterized by an increase of CSF-1R ligand (in one embodiment
the CSF-1R ligand is selected from human CSF-1 (SEQ ID No: 86) and
human IL-34 (SEQ ID No: 87); in one embodiment the CSF-1R ligand is
human CSF-1 (SEQ ID No: 86); in one embodiment the CSF-1R ligand is
human IL-34 (SEQ ID No: 87)) (detectable in serum, urine or tumor
biopsies), wherein an antibody which binds to human CSF-1R as
described herein is administered in combination with an anti-PD-L1
antibody as described herein. The term "increase of CSF-1R ligand"
refers to the overexpression of human CSF-1R ligand (in one
embodiment the CSF-1R ligand is selected from human CSF-1 (SEQ ID
No: 86) and human IL-34 (SEQ ID No: 87); in one embodiment the
CSF-1R ligand is human CSF-1 (SEQ ID No: 86); in one embodiment the
CSF-1R ligand is human IL-34 (SEQ ID No: 87)) (compared to normal
tissue) before treatment or overexpression of human CSF-1R ligand
induced by treatment with anti-CSF-1R antibody (and compared to the
expression levels before treatment). In certain embodiments, the
term "increase" or "above" refers to a level above the reference
level or to an overall increase of 5%, 10%, 20%, 25%, 30%, 40%,
50%, 60%, 70%, 80%, 85%, 90%, 95%, 100% or greater, in CSF-1R
ligand level detected by the methods described herein, as compared
to the CSF-1R ligand level from a reference sample. In certain
embodiments, the term increase refers to the increase in CSF-1R
ligand level wherein, the increase is at least about 1.5-, 1.75-,
2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 40-, 50-,
60-, 70-, 75-, 80-, 90-, or 100-fold higher as compared to the
CSF-1R ligand level e.g. predetermined from a reference sample. In
one preferred embodiment the term increased level relates to a
value at or above a reference level.
[0129] The combination therapies of the antibodies described herein
show benefits for patients in need of a CSF-1R targeting therapy.
The specific anti-CSF-1R antibodies according to the invention show
efficient antiproliferative activity against ligand-independent and
ligand-dependent proliferation and are especially useful inter alia
in the treatment of cancer and metastasis in combination with the
specific anti-PD-L1 antibodies described herein.
DESCRIPTION OF THE FIGURES
[0130] FIG. 1a-b 1a: Human Monocytes differentiated into
macrophages with coculture of GM-CSF or CSF-1 (100 ng/ml ligand).
After 6 days differentiation addition of hMab 2F11-e7. Cell
viability was measured at day 7 of antibody treatment in a CTG
Viability Assay (CellTiterGlo.RTM. Promega). Calculation of % cell
viability: RLU signals from treated cells divided by RLU signal
from untreated control without antibody, (n=4). [0131] 1b: Human
Monocytes differentiated into macrophages with GM-CSF (M1) or M-CSF
(M2) for 7 days. Phenotype analyzed by indirect fluorescence
analysis-staining with anti CD163-PE, anti CD80-PE or anti
HLA-DR/DQ/DP-Zenon-Alexa647 labeled. The number in each histogram
corresponds to mean ratio fluorescence intensity (MRFI); calculated
ratio between mean fluorescence intensity (MFI) of cells stained
with the selected antibody (empty histogram) and of corresponding
isotype control (negative control; gray filled histogram)
(mean.+-.SD; n.gtoreq.5).
[0132] FIG. 2a-d CSF-1 levels in Cynomolgus monkey after
application of different dosages of anti-CSF-1R antibody hMab
2F11-e7.
[0133] FIG. 3 In the presence of TAMs, T cell expansion induced by
activation of CD3 and CD28 was suppressed: TAM were isolated from
MC38 tumors and co-cultured at the ratios indicated with
CFSE-labeled CD8+ T cells in the presence of CD3/CD28 stimulation.
T cell proliferation was analyzed after 3 days using bead
quantification of CFSElow dividing cells. One representative
experiment out of two is depicted as means+SEM of triplicate
wells.
[0134] FIG. 4 Anti tumor Efficacy of <mouse CSF1R>
antibody/<PD-L1> antibody combination in the MC38 mouse CRC
in vivo model (Kaplan-Meier Plot for Progression of tumor
volume>700 mm3).
[0135] FIG. 5 Anti tumor Efficacy of <mouse CSF1R>
antibody/<PD-L1> antibody combination in the subcutaneous
syngeneic CT26.WT colon carcinoma in vivo model (Kaplan-Meier Plot
for Progression of tumor volume>700 mm3).
DETAILED DESCRIPTION OF THE INVENTION
[0136] Many tumors are characterized by a prominent immune cell
infiltrate, including macrophages. Initially, the immune cells were
thought to be part of a defense mechanism against the tumor, but
recent data support the notion that several immune cell populations
including macrophages may, in fact, promote tumor progression.
Macrophages are characterized by their plasticity. Depending on the
cytokine microenvironment, macrophages can exhibit so-called M1 or
M2-subtypes. M2 macrophages are engaged in the suppression of tumor
immunity. They also play an important role in tissue repair
functions such as angiogenesis and tissue remodeling which are
coopted by the tumor to support growth. In contrast to tumor
promoting M2 macrophages, M1 macrophages exhibit antitumor activity
via the secretion of inflammatory cytokines and their engagement in
antigen presentation and phagocytosis (Mantovani, A. et al., Curr.
Opin. Immunol. 2 (2010) 231-237).
[0137] By secreting various cytokines such as colony stimulating
factor 1 (CSF-1) and IL-10, tumor cells are able to recruit and
shape macrophages into the M2-subtype, whereas cytokines such as
granulocyte macrophage colony stimulating factor (GM-CSF),
IFN-gamma program macrophages towards the M1 subtype. Using
immunohistochemistry, it is possible to distinguish between a
macrophage subpopulation co-expressing CD68 and CD163, which is
likely to be enriched for M2 Macrophages, and a subset showing the
CD68+/MHC II+, or CD68+/CD80+ immunophenotype, likely to include M1
macrophages. Cell shape, size, and spatial distribution of CD68 and
CD163 positive macrophages is consistent with published hypotheses
on a tumor-promoting role of M2 macrophages, for example by their
preferential location in tumor intersecting stroma, and vital tumor
areas. In contrast, CD68+/MHC class II+ macrophages are
ubiquitously found. Their hypothetical role in phagocytosis is
reflected by clusters of the CD68+/MHC class II+, but
CD163-immunophenotype near apoptotic cells and necrotic tumor
areas.
[0138] The subtype and marker expression of different macrophage
subpopulations is linked with their functional state. M2
macrophages can support tumorigenesis by: [0139] a) enhancing
angiogenesis via the secretion of angiogenic factors such as VEGF
or bFGF, [0140] b) supporting metastasis formation via secretion of
matrix metalloproteinases(MMPs), growth factors and migratory
factors guiding the tumor cells to the blood stream and setting up
the metastatic niche (Wyckoff, J. et al., Cancer Res. 67 (2007)
2649-2656), [0141] c) playing a role in building an
immunosuppressive milieu by secreting immunosuppressive cytokines
such as IL-4, 11-13, IL-1ra and IL-10, which in turn regulate T
regulatory cell function. Conversely CD4 positive T cells have been
shown to enhance the activity of tumor promoting macrophages in
preclinical models (Mantovani, A. et al., Eur. J. Cancer 40 (2004)
1660-1667; DeNardo, D. et al., Cancer Cell 16 (2009) 91-102).
[0142] Accordingly, in several types of cancer (e.g. breast,
ovarian, Hodgkin's lymphoma) the prevalence of M2 subtype tumor
associated macrophages (TAMS) has been associated with poor
prognosis (Bingle, L. et al., J. Pathol. 3 (2002) 254-265; Orre,
M., and Rogers, P. A., Gynecol. Oncol. 1 (1999) 47-50; Steidl, C.
et al., N. Engl. J. Med. 10 (2010) 875-885). Recent data show a
correlation of CD163 positive macrophage infiltrate in tumors and
tumor grade (Kawamura, K. et al., Pathol. Int. 59 (2009) 300-305).
TAMs isolated from patient tumors had a tolerant phenotype and were
not cytotoxic to tumor cells (Mantovani, A. et al., Eur. J. Cancer
40 (2004) 1660-1667). However, infiltration of TAMs in the presence
of cytotoxic T cells correlates with improved survival in non small
cell lung cancer and hence reflects a more prominent M1 macrophage
infiltrate in this tumor type (Kawai, O. et al., Cancer 6 (2008)
1387-1395).
[0143] Recently, a so-called immune signature comprising high
numbers of macrophages and CD4 positive T cells, but low numbers of
cytotoxic CD8 positive T cells was shown to correlate with reduced
overall survival (OS) in breast cancer patients and to represent an
independent prognostic factor (DeNardo, D. et al., Cancer Discovery
1 (2011) 54-67).
[0144] Consistent with a role for CSF-1 in driving the
pro-tumorigenic function of M2 macrophages, high CSF-1 expression
in rare sarcomas or locally aggressive connective tissue tumors,
such as pigmented villonodular synovitis (PVNS) and tenosynovial
giant cell tumor (TGCT) due in part to a translocation of the CSF-1
gene, leads to the accumulation of monocytes and macrophages
expressing the receptor for CSF-1, the colony-stimulating factor 1
receptor (CSF-1R) forming the majority of the tumor mass (West, R.
B. et al., Proc. Natl. Acad. Sci. USA 3 (2006) 690-695). These
tumors were subsequently used to define a CSF-1 dependent
macrophage signature by gene expression profiling. In breast cancer
and leiomyosarcoma patient tumors this CSF-1 response gene
signature predicts poor prognosis (Espinosa, I. et al., Am. J.
Pathol. 6 (2009) 2347-2356; Beck, A. et al., Clin. Cancer Res. 3
(2009) 778-787).
[0145] CSF-1R belongs to the class III subfamily of receptor
tyrosine kinases and is encoded by the c-fms proto-oncogene.
Binding of CSF-1 or IL-34 induces receptor dimerization, followed
by autophosphorylation and activation of downstream signaling
cascades. Activation of CSF-1R regulates the survival,
proliferation and differentiation of monocytes and macrophages
(Xiong, Y. et al., J. Biol. Chem. 286 (2011) 952-960).
[0146] In addition to cells of the monocytic lineage and
osteoclasts, which derive from the same hematopoietic precursor as
the macrophage, CSF-1R/c-fms has also been found to be expressed by
several human epithelial cancers such as ovarian and breast cancer
and in leiomyosarcoma and TGCT/PVNS, albeit at lower expression
levels compared to macrophages. As with TGCT/PVNS, elevated levels
of CSF-1, the ligand for CSF-1R, in serum as well as ascites of
ovarian cancer patients have been correlated with poor prognosis
(Scholl, S. et al., Br. J. Cancer 62 (1994) 342-346; Price, F. et
al., Am. J. Obstet. Gynecol. 168 (1993) 520-527). Furthermore, a
constitutively active mutant form of CSF 1R is able to transform
NIH3T3 cells, one of the properties of an oncogene (Chambers, S.,
Future Oncol 5 (2009) 1429-1440).
[0147] Preclinical models provide validation of CSF-1R as an
oncology target. Blockade of CSF-1 as well as CSF-1R activity
results in reduced recruitment of TAMs. Chemotherapy resulted in
elevated CSF-1 expression in tumor cells leading to enhanced TAM
recruitment. Blockade of CSF-1R in combination with paclitaxel
resulted in activation of CD8 positive cytotoxic T cells leading to
reduced tumor growth and metastatic burden in a spontaneous
transgenic breast cancer model (DeNardo, D. et al., Cancer
Discovery 1 (2011) 54-67).
[0148] The human CSF-1R (CSF-1 receptor; synonyms: M-CSF receptor;
Macrophage colony-stimulating factor 1 receptor, Fms
proto-oncogene, c-fms, SEQ ID NO: 22)) is known since 1986
(Coussens, L., et al., Nature 320 (1986) 277-280). CSF-1R is a
growth factor and encoded by the c-fms proto-oncogene (reviewed
e.g. in Roth, P. and Stanley, E. R., Curr. Top. Microbiol. Immunol.
181 (1992) 141-167).
[0149] CSF-1R is the receptor for the CSF-1R ligands CSF-1
(macrophage colony stimulating factor, also called M-CSF) (SEQ ID
No.: 86) and IL-34 (SEQ ID No.: 87) and mediates the biological
effects of these cytokines (Sherr, C. J., et al., Cell 41 (1985)
665-676; Lin, H., et al., Science 320 (2008) 807-811). The cloning
of the colony stimulating factor-1 receptor (also called c-fms) was
described for the first time in Roussel, M. F., et al., Nature 325
(1987) 549-552. In that publication, it was shown that CSF-1R had
transforming potential dependent on changes in the C-terminal tail
of the protein including the loss of the inhibitory tyrosine 969
phosphorylation which binds Cbl and thereby regulates receptor down
regulation (Lee, P. S., et al., Embo J. 18 (1999) 3616-3628).
[0150] CSF-1R is a single chain, transmembrane receptor tyrosine
kinase (RTK) and a member of the family of immunoglobulin (Ig)
motif containing RTKs characterized by 5 repeated Ig-like
subdomains D1-D5 in the extracellular domain (ECD) of the receptor
(Wang, Z., et al Molecular and Cellular Biology 13 (1993)
5348-5359). The human CSF-1R Extracellular Domain (CSF-1R-ECD) (SEQ
ID NO: 64) comprises all five extracellular Ig-like subdomains
D1-D5. The human CSF-1R fragment delD4 (SEQ ID NO: 65) comprises
the extracellular Ig-like subdomains D1-D3 and D5, but is missing
the D4 subdomain. The human CSF-1R fragment D1-D3 (SEQ ID NO: 66)
comprises the respective subdomains D1-D3. The sequences are listed
without the signal peptide MGSGPGVLLL LLVATAWHGQ G (SEQ ID NO: 67).
The human CSF-1R fragment D4-D3 (SEQ ID NO: 85) comprises the
respective subdomains D4-D3.
[0151] Currently two CSF-1R ligands that bind to the extracellular
domain of CSF-1R are known. The first one is CSF-1 (colony
stimulating factor 1, also called M-CSF, macrophage; human CSF-1,
SEQ ID NO: 86) and is found extracellularly as a disulfide-linked
homodimer (Stanley, E. R. et al., Journal of Cellular Biochemistry
21 (1983) 151-159; Stanley, E. R. et al., Stem Cells 12 Suppl. 1
(1995) 15-24). The second one is IL-34 (human IL-34; SEQ ID NO: 87)
(Hume, D. A., et al, Blood 119 (2012) 1810-1820). Thus in one
embodiment the term "CSF-1R ligand" refers to human CSF-1 (SEQ ID
NO: 86) and/or human IL-34 (SEQ ID NO: 87).
[0152] For experiments often the active 149 amino acid (aa)
fragment of human CSF-1 (aa 33-181 of SEQ ID NO: 86) is used. This
active 149 aa fragment of human CSF-1 (aa 33-181 of SEQ ID NO: 86)
is contained in all 3 major forms of CSF-1 and is sufficient to
mediate binding to CSF-1R (Hume, D. A., et al, Blood 119 (2012)
1810-1820).
[0153] The main biological effects of CSF-1R signaling are the
differentiation, proliferation, migration, and survival of
hematopoietic precursor cells to the macrophage lineage (including
osteoclast). Activation of CSF-1R is mediated by its CSF-1R
ligands, CSF-1 (M-CSF) and IL-34. Binding of CSF-1 (M-CSF) to
CSF-1R induces the formation of homodimers and activation of the
kinase by tyrosine phosphorylation (Li, W. et al, EMBO Journal. 10
(1991) 277-288; Stanley, E. R., et al., Mol. Reprod. Dev. 46 (1997)
4-10).
[0154] The intracellular protein tyrosine kinase domain is
interrupted by a unique insert domain that is also present in the
other related RTK class III family members that include the
platelet derived growth factor receptors (PDGFR), stem cell growth
factor receptor (c-Kit) and fins-like cytokine receptor (FLT3). In
spite of the structural homology among this family of growth factor
receptors, they have distinct tissue-specific functions.
[0155] CSF-1R is mainly expressed on cells of the monocytic lineage
and in the female reproductive tract and placenta. In addition
expression of CSF-1R has been reported in Langerhans cells in skin,
a subset of smooth muscle cells (Inaba, T., et al., J. Biol. Chem.
267 (1992) 5693-5699), B cells (Baker, A. H., et al., Oncogene 8
(1993) 371-378) and microglia (Sawada, M., et al., Brain Res. 509
(1990) 119-124). Cells with mutant human CSF-1R ((SEQ ID NO: 23)
are known to proliferate independently of ligand stimulation.
[0156] As used herein, "binding to human CSF-1R" or "specifically
binding to human CSF-1R" or "which binds to human CSF-1R" or
"anti-CSF-1R antibody" refers to an antibody specifically binding
to the human CSF-1R antigen with a binding affinity of KD-value of
1.0.times.10.sup.-8 mol/l or lower, in one embodiment of a KD-value
of 1.0.times.10.sup.-9 mol/l or lower. The binding affinity is
determined with a standard binding assay, such as surface plasmon
resonance technique (BIAcore.RTM., GE-Healthcare Uppsala, Sweden).
Thus an "antibody binding to human CSF-1R" as used herein refers to
an antibody specifically binding to the human CSF-1R antigen with a
binding affinity of KD 1.0.times.10.sup.-8 mol/l or lower (in one
embodiment 1.0.times.10.sup.-8 mol/l-1.0.times.10.sup.-13 mol/1),
in on embodiment of a KD 1.0.times.10.sup.-9 mol/l or lower (in one
embodiment 1.0.times.10.sup.-9 mol/1-1.0.times.10.sup.-13
mol/1).
PD-1/PD-L1/PD-L2 Pathway:
[0157] An important negative co-stimulatory signal regulating T
cell activation is provided by programmed death--1 receptor
(PD-1)(CD279), and its ligand binding partners PD-L1 (B7-H1, CD274;
SEQ ID NO: 88) and PD-L2 (B7-DC, CD273). The negative regulatory
role of PD-1 was revealed by PD-1 knock outs (Pdcd1-/-), which are
prone to autoimmunity. Nishimura et al., Immunity 11: 141-51
(1999); Nishimura et al., Science 291: 319-22 (2001). PD-1 is
related to CD28 and CTLA-4, but lacks the membrane proximal
cysteine that allows homodimerization. The cytoplasmic domain of
PD-1 contains an immunoreceptor tyrosine-based inhibition motif
(ITIM, V/IxYxxL/V). PD-1 only binds to PD-L1 and PD-L2. Freeman et
al., J. Exp. Med. 192: 1-9 (2000); Dong et al., Nature Med. 5:
1365-1369 (1999); Latchman et al., Nature Immunol. 2: 261-268
(2001); Tseng et al., J. Exp. Med. 193: 839-846 (2001).
[0158] PD-1 can be expressed on T cells, B cells, natural killer T
cells, activated monocytes and dendritic cells (DCs). PD-1 is
expressed by activated, but not by unstimulated human CD4+ and CD8+
T cells, B cells and myeloid cells. This stands in contrast to the
more restricted expression of CD28 and CTLA-4. Nishimura et al.,
Int. Immunol. 8: 773-80 (1996); Boettler et al., J. Virol. 80:
3532-40 (2006). There are at least 4 variants of PD-1 that have
been cloned from activated human T cells, including transcripts
lacking (i) exon 2, (ii) exon 3, (iii) exons 2 and 3 or (iv) exons
2 through 4. Nielsen et al., Cell. Immunol. 235: 109-16 (2005).
With the exception of PD-1 .DELTA.ex3, all variants are expressed
at similar levels as full length PD-1 in resting peripheral blood
mononuclear cells (PBMCs). Expression of all variants is
significantly induced upon activation of human T cells with
anti-CD3 and anti-CD28. The PD-1 .DELTA.ex3 variants lacks a
transmembrane domain, and resembles soluble CTLA-4, which plays an
important role in autoimmunity. Ueda et al., Nature 423: 506-11
(2003). This variant is enriched in the synovial fluid and sera of
patients with rheumatoid arthritis. Wan et al., J. Immunol. 177:
8844-50 (2006).
[0159] The two PD-1 ligands differ in their expression patterns.
PD-L1 is constitutively expressed on mouse T and B cells, CDs,
macrophages, mesenchymal stem cells and bone marrow-derived mast
cells. Yamazaki et al., J. Immunol. 169: 5538-45 (2002). PD-L1 is
expressed on a wide range of nonhematopoietic cells (e.g., cornea,
lung, vascular epithelium, liver nonparenchymal cells, mesenchymal
stem cells, pancreatic islets, placental synctiotrophoblasts,
keratinocytes, etc.) [Keir et al., Annu. Rev. Immunol. 26: 677-704
(2008)], and is upregulated on a number of cell types after
activation. Both type I and type II interferons IFN's) upregulate
PD-L1. Eppihimer et al., Microcirculation 9: 133-45 (2002);
Schreiner et al., J. Neuroimmunol. 155: 172-82 (2004). PD-L1
expression in cell lines is decreased when MyD88, TRAF6 and MEK are
inhibited. Liu et al., Blood 110: 296-304 (2007). JAK2 has also
been implicated in PD-L1 induction. Lee et al., FEBS Lett. 580:
755-62 (2006); Liu et al., Blood 110: 296-304 (2007). Loss or
inhibition of phosphatase and tensin homolog (PTEN), a cellular
phosphatase that modified phosphatidylinositol 3-kinase (PI3K) and
Akt signaling, increased post-transcriptional PD-L1 expression in
cancers. Parsa et al., Nat. Med. 13: 84-88 (2007).
[0160] PD-L2 expression is more restricted than PD-L1. PD-L2 is
inducibly expressed on DCs, macrophages, and bone marrow-derived
mast cells. PD-L2 is also expressed on about half to two-thirds of
resting peritoneal B1 cells, but not on conventional B2 B cells.
Zhong et al., Eur. J. Immunol. 37: 2405-10 (2007). PD-L2+B1 cells
bind phosphatidylcholine and may be important for innate immune
responses against bacterial antigens. Induction of PD-L2 by
IFN-gamma is partially dependent upon NF-.kappa.B. Liang et al.,
Eur. J. Immunol. 33: 2706-16 (2003). PD-L2 can also be induced on
monocytes and macrophages by GM-CF, IL-4 and IFN-gamma. Yamazaki et
al., J. Immunol. 169: 5538-45 (2002); Loke et al., PNAS 100:5336-41
(2003).
[0161] PD-1 signaling typically has a greater effect on cytokine
production than on cellular proliferation, with significant effects
on IFN-gamma, TNF-alpha and IL-2 production. PD-1 mediated
inhibitory signaling also depends on the strength of the TCR
signaling, with greater inhibition delivered at low levels of TCR
stimulation. This reduction can be overcome by costimulation
through CD28 [Freeman et al., J. Exp. Med. 192: 1027-34 (2000)] or
the presence of IL-2 [Carter et al., Eur. J. Immunol. 32: 634-43
(2002)].
[0162] Evidence is mounting that signaling through PD-L1 and PD-L2
may be bidirectional. That is, in addition to modifying TCR or BCR
signaling, signaling may also be delivered back to the cells
expressing PD-L1 and PD-L2. While treatment of dendritic cells with
a naturally human anti-PD-L2 antibody isolated from a patient with
Waldenstrom's macroglobulinemia was not found to upregulate MHC II
or B7 costimulatory molecules, such cells did produce greater
amount of proinflammatory cytokines, particularly TNF-alpha and
IL-6, and stimulated T cell proliferation. Nguyen et al., J. Exp.
Med. 196: 1393-98 (2002). Treatment of mice with this antibody also
(1) enhanced resistance to transplanted b16 melanoma and rapidly
induced tumor-specific CTL. Radhakrishnan et al., J. Immunol. 170:
1830-38 (2003); Radhakrishnan et al., Cancer Res. 64: 4965-72
(2004); Heckman et al., Eur. J. Immunol. 37: 1827-35 (2007); (2)
blocked development of airway inflammatory disease in a mouse model
of allergic asthma. Radhakrishnan et al., J. Immunol. 173: 1360-65
(2004); Radhakrishnan et al., J. Allergy Clin. Immunol. 116: 668-74
(2005).
[0163] Further evidence of reverse signaling into dendritic cells
("DC's") results from studies of bone marrow derived DC's cultured
with soluble PD-1 (PD-1 EC domain fused to Ig constant
region--"s-PD-1"). Kuipers et al., Eur. J. Immunol. 36: 2472-82
(2006). This sPD-1 inhibited DC activation and increased IL-10
production, in a manner reversible through administration of
anti-PD-1.
[0164] Additionally, several studies show a receptor for PD-L1 or
PD-L2 that is independent of PD-1. B7.1 has already been identified
as a binding partner for PD-L1. Butte et al., Immunity 27: 111-22
(2007). Chemical crosslinking studies suggest that PD-L1 and B7.1
can interact through their IgV-like domains. B7.1:PD-L1
interactions can induce an inhibitory signal into T cells. Ligation
of PD-L1 on CD4+ T cells by B7.1 or ligation of B7.1 on CD4+ T
cells by PD-L1 delivers an inhibitory signal. T cells lacking CD28
and CTLA-4 show decreased proliferation and cytokine production
when stimulated by anti-CD3 plus B7.1 coated beads. In T cells
lacking all the receptors for B7.1 (i.e., CD28, CTLA-4 and PD-L1),
T cell proliferation and cytokine production were no longer
inhibited by anti-CD3 plus B7.1 coated beads. This indicates that
B7.1 acts specifically through PD-L1 on the T-cell in the absence
of CD28 and CTLA-4. Similarly, T cells lacking PD-1 showed
decreased proliferation and cytokine production when stimulated in
the presence of anti-CD3 plus PD-L1 coated beads, demonstrating the
inhibitory effect of PD-L1 ligation on B7.1 on T cells. When T
cells lacking all known receptors for PD-L1 (i.e., no PD-1 and
B7.1), T cell proliferation was no longer impaired by anti-CD3 plus
PD-L1 coated beads. Thus, PD-L1 can exert an inhibitory effect on T
cells either through B7.1 or PD-1.
[0165] The direct interaction between B7.1 and PD-L1 suggests that
the current understanding of costimulation is incomplete, and
underscores the significance to the expression of these molecules
on T cells. Studies of PD-L1-/- T cells indicate that PD-L1 on T
cells can downregulate T cell cytokine production. Latchman et al.,
Proc. Natl. Acad. Sci. USA 101: 10691-96 (2004). Because both PD-L1
and B7.1 are expressed on T cells, B cells, DCs and macrophages,
there is the potential for directional interactions between B7.1
and PD-L1 on these cells types. Additionally, PD-L1 on
non-hematopoietic cells may interact with B7.1 as well as PD-1 on T
cells, raising the question of whether PD-L1 is involved in their
regulation. One possible explanation for the inhibitory effect of
B7.1:PD-L1 interaction is that T cell PD-L1 may trap or segregate
away APC B7.1 from interaction with CD28.
[0166] As a result, the antagonism of signaling through PD-L1,
including blocking PD-L1 from interacting with either PD-1, B7.1 or
both, thereby preventing PD-L1 from sending a negative
co-stimulatory signal to T-cells and other antigen presenting cells
is likely to enhance immunity in response to infection (e.g., acute
and chronic) and tumor immunity. In addition, the anti-PD-L1
antibodies of the present invention, may be combined with
antagonists of other components of PD-1:PD-L1 signaling, for
example, antagonist anti-PD-1 and anti-PD-L2 antibodies.
[0167] The term "human PD-L1" refers to the human protein PD-L1
(SEQ ID NO: 88, PD-1 signaling typically). As used herein, "binding
to human PD-L1" or "specifically binding to human PD-L1" or "which
binds to human PD-L1" or "anti-PD-L1 antibody" refers to an
antibody specifically binding to the human PD-L1 antigen with a
binding affinity of KD-value of 1.0.times.10.sup.-8 mol/l or lower,
in one embodiment of a KD-value of 1.0.times.10.sup.-9 mol/l or
lower. The binding affinity is determined with a standard binding
assay, such as surface plasmon resonance technique (BIAcore.RTM.,
GE-Healthcare Uppsala, Sweden). Thus an "antibody binding to human
PD-L1" as used herein refers to an antibody specifically binding to
the human PD-L1 antigen with a binding affinity of KD
1.0.times.10.sup.-8 mol/l or lower (in one embodiment
1.0.times.10.sup.-8 mol/1-1.0.times.10.sup.-13 mol/1), in on
embodiment of a KD 1.0.times.10.sup.-9 mol/l or lower (in one
embodiment 1.0.times.10.sup.-9 mol/1-1.0.times.10.sup.-13
mol/1).
[0168] In one embodiment the antibody which binds to human CSF-1R
used in the combination therapy described herein is selected from
the group consisting of
hMab 2F11-c11, hMab 2F11-d8, hMab 2F11-e7, hMab 2F11-f12, and hMab
2F11-g1.
[0169] These antibodies are described in WO2011/070024 and are
characterized in comprising the following VH and VL sequences as
described herein:
TABLE-US-00001 TABLE 1 amino acid sequence of amino acid sequence
of anti-CSF-1R the heavy chain variable the light chain variable
antibody domain VH, SEQ ID NO: domain VL, SEQ ID NO: hMab 2F11-c11
23 24 hMab 2F11-d8 31 32 hMab 2F11-e7 39 40 hMab 2F11-f12 47 48
hMab 2F11-g1 55 56
[0170] In one embodiment the antibody which binds to human PD-L1
used in the combination therapy described herein is selected from
the group consisting of:
243.55.S70, 243.55.H1, 243.55.H12, 243.55.H37, 243.55.H70,
243.55.H89, 243.55.S1, 243.55.5, 243.55.8, 243.55.30, 243.55.34,
243.55.S37, 243.55.49, 243.55.51, 243.55.62, and 243.55.84.
[0171] These antibodies are described in WO 2010/77634 (sequences
are shown in FIG. 11 of WO 2010/77634) and are characterized in
comprising the following VH and VL sequences as described
herein:
TABLE-US-00002 TABLE 2 amino acid sequence of amino acid sequence
of anti-PD-L1 the heavy chain variable the light chain variable
antibody domain VH, SEQ ID NO: domain VL, SEQ ID NO: 243.55.S70 89
92 243.55.H1 90 93 243.55.H12 90 94 243.55.H37 90 95 243.55.H70 90
96 243.55.H89 90 97 243.55.S1 90 98 243.55.5 90 99 243.55.8 90 100
243.55.30 90 101 243.55.34 90 102 243.55.S37 90 103 243.55.49 90
104 243.55.51 90 105 243.55.62 90 106 243.55.84 91 107
[0172] In one embodiment of the invention the antibody which binds
to human CSF-1R used in the combination therapy described herein is
characterized in comprising [0173] a) a heavy chain variable domain
VH of SEQ ID NO:23 and a light chain variable domain VL of SEQ ID
NO:24, or [0174] b) a heavy chain variable domain VH of SEQ ID
NO:31 and a light chain variable domain VL of SEQ ID NO:32, or
[0175] c) a heavy chain variable domain VH of SEQ ID NO:39 and a
light chain variable domain VL of SEQ ID NO:40, or [0176] d) a
heavy chain variable domain VH of SEQ ID NO:47 and a light chain
variable domain VL of SEQ ID NO:48, or [0177] e) a heavy chain
variable domain VH of SEQ ID NO:55 and a light chain variable
domain VL of SEQ ID NO:56; and the antibody which binds to human
PD-L1 used in the combination therapy is characterized in
comprising [0178] a) a heavy chain variable domain VH of SEQ ID
NO:89 and a light chain variable domain VL of SEQ ID NO:92, or
[0179] b) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:93, or [0180] c) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:94, or [0181] d) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:95, or [0182] e) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:96, or [0183] f) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:97, or
[0184] g) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:98, or [0185] h) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:99, or [0186] i) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:100, or [0187] j) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:101, or [0188] k) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:102, or
[0189] l) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:103, or [0190] m) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:104, or [0191] n) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:105, or [0192] o) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:106, or [0193] p) a heavy chain variable domain VH of SEQ
ID NO:91 and a light chain variable domain VL of SEQ ID NO:107.
[0194] In one embodiment the antibody which binds to human CSF-1R
used in the combination therapy is characterized in comprising
[0195] a heavy chain variable domain VH of SEQ ID NO:23 and a light
chain variable domain VL of SEQ ID NO:24.
[0196] In one embodiment the antibody which binds to human CSF-1R
used in the combination therapy is characterized in comprising
[0197] a heavy chain variable domain VH of SEQ ID NO:31 and a light
chain variable domain VL of SEQ ID NO:32.
[0198] In one embodiment the antibody which binds to human CSF-1R
used in the combination therapy is characterized in comprising
[0199] a heavy chain variable domain VH of SEQ ID NO:39 and a light
chain variable domain VL of SEQ ID NO:40.
[0200] In one embodiment the antibody which binds to human CSF-1R
used in the combination therapy is characterized in comprising
[0201] a heavy chain variable domain VH of SEQ ID NO:47 and a light
chain variable domain VL of SEQ ID NO:48.
[0202] In one embodiment the antibody which binds to human PD-L1
used in the combination therapy is characterized in comprising
a heavy chain variable domain VH of SEQ ID NO:89 and a light chain
variable domain VL of SEQ ID NO:92.
[0203] In one embodiment the antibody which binds to human PD-L1
used in the combination therapy is characterized in comprising
a heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:93.
[0204] In one embodiment the antibody which binds to human PD-L1
used in the combination therapy is characterized in comprising
a heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:94.
[0205] In one embodiment the antibody which binds to human PD-L1
used in the combination therapy is characterized in comprising
a heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:95.
[0206] In one embodiment the antibody which binds to human PD-L1
used in the combination therapy is characterized in comprising
a heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:96.
[0207] In one embodiment the antibody which binds to human PD-L1
used in the combination therapy is characterized in comprising
a heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:97.
[0208] In one embodiment the antibody which binds to human PD-L1
used in the combination therapy is characterized in comprising
a heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:98.
[0209] In one embodiment the antibody which binds to human PD-L1
used in the combination therapy is characterized in comprising
a heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:99.
[0210] In one embodiment the antibody which binds to human PD-L1
used in the combination therapy is characterized in comprising
a heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:100.
[0211] In one embodiment the antibody which binds to human PD-L1
used in the combination therapy is characterized in comprising
a heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:101.
[0212] In one embodiment the antibody which binds to human PD-L1
used in the combination therapy is characterized in comprising
a heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:102.
[0213] In one embodiment the antibody which binds to human PD-L1
used in the combination therapy is characterized in comprising
a heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:103.
[0214] In one embodiment the antibody which binds to human PD-L1
used in the combination therapy is characterized in comprising
a heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:104.
[0215] In one embodiment the antibody which binds to human PD-L1
used in the combination therapy is characterized in comprising
a heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:105.
[0216] In one embodiment the antibody which binds to human PD-L1
used in the combination therapy is characterized in comprising
a heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:106.
[0217] In one embodiment the antibody which binds to human PD-L1
used in the combination therapy is characterized in comprising
a heavy chain variable domain VH of SEQ ID NO:91 and a light chain
variable domain VL of SEQ ID NO:107.
[0218] In one preferred embodiment of the invention the antibody
which binds to human CSF-1R used in the combination therapy
described herein is characterized in comprising
a heavy chain variable domain VH of SEQ ID NO:39 and a light chain
variable domain VL of SEQ ID NO:40, and the antibody which binds to
human PD-L1 used in the combination therapy is characterized in
comprising a heavy chain variable domain VH of SEQ ID NO:89 and a
light chain variable domain VL of SEQ ID NO:92.
[0219] The term "epitope" denotes a protein determinant of human
CSF-1R or PD-L1 capable of specifically binding to an antibody.
Epitopes usually consist of chemically active surface groupings of
molecules such as amino acids or sugar side chains and usually
epitopes have specific three dimensional structural
characteristics, as well as specific charge characteristics.
Conformational and nonconformational epitopes are distinguished in
that the binding to the former but not the latter is lost in the
presence of denaturing solvents.
[0220] The "variable domain" (light chain variable domain VL, heavy
chain variable domain VH) as used herein denotes each of the pair
of light and heavy chain domains which are involved directly in
binding the antibody to the antigen. The variable light and heavy
chain domains have the same general structure and each domain
comprises four framework (FR) regions whose sequences are widely
conserved, connected by three "hypervariable regions" (or
complementary determining regions, CDRs). The framework regions
adopt a beta-sheet conformation and the CDRs may form loops
connecting the beta-sheet structure. The CDRs in each chain are
held in their three-dimensional structure by the framework regions
and form together with the CDRs from the other chain the antigen
binding site. The antibody's heavy and light chain CDR3 regions
play a particularly important role in the binding
specificity/affinity of the antibodies according to the invention
and therefore provide a further object of the invention.
[0221] The term "antigen-binding portion of an antibody" when used
herein refer to the amino acid residues of an antibody which are
responsible for antigen-binding. The antigen-binding portion of an
antibody comprises amino acid residues from the "complementary
determining regions" or "CDRs". "Framework" or "FR" regions are
those variable domain regions other than the hypervariable region
residues as herein defined. Therefore, the light and heavy chain
variable domains of an antibody comprise from N- to C-terminus the
domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Especially, CDR3
of the heavy chain is the region which contributes most to antigen
binding and defines the antibody's properties. CDR and FR regions
are determined according to the standard definition of Kabat et
al., Sequences of Proteins of Immunological Interest, 5th ed.,
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991) and/or those residues from a "hypervariable loop".
[0222] The terms "nucleic acid" or "nucleic acid molecule", as used
herein, are intended to include DNA molecules and RNA molecules. A
nucleic acid molecule may be single-stranded or double-stranded,
but preferably is double-stranded DNA.
[0223] The term "amino acid" as used within this application
denotes the group of naturally occurring carboxy alpha-amino acids
comprising alanine (three letter code: ala, one letter code: A),
arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D),
cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E),
glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine
(leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe,
F), proline (pro, P), serine (ser, S), threonine (thr, T),
tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).
[0224] The "Fc part" of an antibody is not involved directly in
binding of an antibody to an antigen, but exhibit various effector
functions. A "Fc part of an antibody" is a term well known to the
skilled artisan and defined on the basis of papain cleavage of
antibodies. Depending on the amino acid sequence of the constant
region of their heavy chains, antibodies or immunoglobulins are
divided in the classes: IgA, IgD,
[0225] IgE, IgG and IgM, and several of these may be further
divided into subclasses (isotypes), e.g. IgG1, IgG2, IgG3, and
IgG4, IgA1, and IgA2. According to the heavy chain constant regions
the different classes of immunoglobulins are called a, .delta.,
.epsilon., .gamma., and .mu., respectively. The Fc part of an
antibody is directly involved in ADCC (antibody-dependent
cell-mediated cytotoxicity) and CDC (complement-dependent
cytotoxicity) based on complement activation, C1 q binding and Fc
receptor binding. Complement activation (CDC) is initiated by
binding of complement factor C1q to the Fc part of most IgG
antibody subclasses. While the influence of an antibody on the
complement system is dependent on certain conditions, binding to
C1q is caused by defined binding sites in the Fc part. Such binding
sites are known in the state of the art and described e.g. by
Boackle, R. J., et al., Nature 282 (1979) 742-743; Lukas, T. J., et
al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R., and Cebra, J.
J., Mol. Immunol. 16 (1979) 907-917; Burton, D. R., et al., Nature
288 (1980) 338-344; Thommesen, J. E., et al., Mol. Immunol. 37
(2000) 995-1004; Idusogie, E. E., et al., J. Immunol. 164 (2000)
4178-4184; Hezareh, M., et al., J. Virology 75 (2001) 12161-12168;
Morgan, A., et al., Immunology 86 (1995) 319-324; EP 0 307 434.
Such binding sites are e.g. L234, L235, D270, N297, E318, K320,
K322, P331 and P329 (numbering according to EU index of Kabat, E.
A., see below). Antibodies of subclass IgG1, IgG2 and IgG3 usually
show complement activation and C1q and C3 binding, whereas IgG4 do
not activate the complement system and do not bind C1q and C3.
[0226] In one embodiment the antibody according to the invention
comprises an Fc part derived from human origin and preferably all
other parts of the human constant regions. As used herein the term
"Fc part derived from human origin" denotes a Fc part which is
either a Fc part of a human antibody of the subclass IgG1, IgG2,
IgG3 or IgG4, preferably a Fc part from human IgG1 subclass, a
mutated Fc part from human IgG1 subclass (in one embodiment with a
mutation on L234A+L235A), a Fc part from human IgG4 subclass or a
mutated Fc part from human IgG4 subclass (in one embodiment with a
mutation on S228P). In one preferred embodiment the human heavy
chain constant region is SEQ ID NO: 58 (human IgG1 subclass), in
another preferred embodiment the human heavy chain constant region
is SEQ ID NO: 59 (human IgG1 subclass with mutations L234A and
L235A), in another preferred embodiment the human heavy chain
constant region is SEQ ID NO: 60 (human IgG4 subclass), and in
another preferred embodiment the human heavy chain constant region
is SEQ ID NO: 61 (human IgG4 subclass with mutation S228P). In one
embodiment said antibodies have reduced or minimal effector
function. In one embodiment the minimal effector function results
from an effectorless Fc mutation. In one embodiment the
effectorless Fc mutation is L234A/L235A or L234A/L235A/P329G or
N297A or D265A/N297A. In one embodiment the effectorless Fc
mutation is selected for each of the antibodies independently of
each other from the group comprising (consisting of) L234A/L235A,
L234A/L235A/P329G, N297A and D265A/N297A.
[0227] In one embodiment the antibodies described herein are of
human IgG class (i.e. of IgG1, IgG2, IgG3 or IgG4 subclass).
[0228] In a preferred embodiment the antibodies described herein
are of human IgG1 subclass or of human IgG4 subclass. In one
embodiment the described herein are of human IgG1 subclass. In one
embodiment the antibodies described herein are of human IgG4
subclass.
[0229] In one embodiment the antibody described herein is
characterized in that the constant chains are of human origin. Such
constant chains are well known in the state of the art and e.g.
described by Kabat, E. A., (see e.g. Johnson, G. and Wu, T. T.,
Nucleic Acids Res. 28 (2000) 214-218). For example, a useful human
heavy chain constant region comprises an amino acid sequence of SEQ
ID NO: 58. For example, a useful human light chain constant region
comprises an amino acid sequence of a kappa-light chain constant
region of SEQ ID NO: 57.
[0230] The invention comprises a method for the treatment of a
patient in need of therapy, characterized by administering to the
patient a therapeutically effective amount of an antibody according
to the invention.
[0231] The invention comprises the use of an antibody according to
the invention for the described therapy.
[0232] One preferred embodiment of the invention are the CSF-1R
antibodies of the present invention for use in the treatment of
"CSF-1R mediated diseases" or the CSF-1R antibodies of the present
invention for use for the manufacture of a medicament in the
treatment of "CSF-1R mediated diseases", which can be described as
follows:
[0233] There are 3 distinct mechanisms by which CSF-1R signaling is
likely involved in tumor growth and metastasis. The first is that
expression of CSF-ligand and receptor has been found in tumor cells
originating in the female reproductive system (breast, ovarian,
endometrium, cervical) (Scholl, S. M., et al., J. Natl. Cancer
Inst. 86 (1994) 120-126; Kacinski, B. M., Mol. Reprod. Dev. 46
(1997) 71-74; Ngan, H. Y., et al., Eur. J. Cancer 35 (1999)
1546-1550; Kirma, N., et al., Cancer Res 67 (2007) 1918-1926) and
the expression has been associated with breast cancer xenograft
growth as well as poor prognosis in breast cancer patients. Two
point mutations were seen in CSF-1R in about 10-20% of acute
myelocytic leukemia, chronic myelocytic leukemia and myelodysplasia
patients tested in one study, and one of the mutations was found to
disrupt receptor turnover (Ridge, S. A., et al., Proc. Natl. Acad.
Sci USA 87 (1990) 1377-1380). However the incidence of the
mutations could not be confirmed in later studies (Abu-Duhier, F.
M., et al., Br. J. Haematol. 120 (2003) 464-470). Mutations were
also found in some cases of hepatocellular cancer (Yang, D. H., et
al., Hepatobiliary Pancreat. Dis. Int. 3 (2004) 86-89) and
idiopathic myelofibrosis (Abu-Duhier, F. M., et al., Br. J.
Haematol. 120 (2003) 464-470). Recently, in the GDM-1 cell line
derived from a patient with myelomonoblastic leukemia the Y571D
mutation in CSF-1R was identified (Chase, A., et al., Leukemia 23
(2009) 358-364).
[0234] Pigmented villonodular synovitis (PVNS) and Tenosynovial
Giant cell tumors (TGCT) can occur as a result of a translocation
that fuses the M-CSF gene to a collagen gene COL6A3 and results in
overexpression of M-CSF (West, R. B., et al., Proc. Natl. Acad.
Sci. USA 103 (2006) 690-695). A landscape effect is proposed to be
responsible for the resulting tumor mass that consists of monocytic
cells attracted by cells that express M-CSF. TGCTs are smaller
tumors that can be relatively easily removed from fingers where
they mostly occur. PVNS is more aggressive as it can recur in large
joints and is not as easily controlled surgically.
[0235] The second mechanism is based on blocking signaling through
M-CSF/CSF-1R at metastatic sites in bone which induces
osteoclastogenesis, bone resorption and osteolytic bone lesions.
Breast, multiple myeloma and lung cancers are examples of cancers
that have been found to metastasize to the bone and cause
osteolytic bone disease resulting in skeletal complications. M-CSF
released by tumor cells and stroma induces the differentiation of
hematopoietic myeloid monocyte progenitors to mature osteoclasts in
collaboration with the receptor activator of nuclear factor kappa-B
ligand-RANKL. During this process, M-CSF acts as a permissive
factor by giving the survival signal to osteoclasts (Tanaka, S., et
al., J. Clin. Invest. 91 (1993) 257-263) Inhibition of CSF-1R
activity during osteoclast differentiation and maturation with an
anti-CSF-1R antibody is likely to prevent unbalanced activity of
osteoclasts that cause osteolytic disease and the associated
skeletal related events in metastatic disease. Whereas breast, lung
cancer and multiple myeloma typically result in osteolytic lesions,
metastasis to the bone in prostate cancer initially has an
osteoblastic appearance in which increased bone forming activity
results in `woven bone` which is different from typical lamellar
structure of normal bone. During disease progression bone lesions
display a significant osteolytic component as well as high serum
levels of bone resorption and suggests that anti-resorptive therapy
may be useful. Bisphosphonates have been shown to inhibit the
formation of osteolytic lesions and reduced the number of
skeletal-related events only in men with hormone-refractory
metastatic prostate cancer but at this point their effect on
osteoblastic lesions is controversial and bisphosphonates have not
been beneficial in preventing bone metastasis or hormone responsive
prostate cancer to date. The effect of anti-resorptive agents in
mixed osteolytic/osteoblastic prostate cancer is still being
studied in the clinic (Choueiri, M. B., et al., Cancer Metastasis
Rev. 25 (2006) 601-609; Vessella, R. L. and Corey, E., Clin. Cancer
Res. 12 (20 Pt 2) (2006) 6285s-6290s).
[0236] The third mechanism is based on the recent observation that
tumor associated macrophages (TAM) found in solid tumors of the
breast, prostate, ovarian and cervical cancers correlated with poor
prognosis (Bingle, L., et al., J. Pathol. 196 (2002) 254-265;
Pollard, J. W., Nat. Rev. Cancer 4 (2004) 71-78). Macrophages are
recruited to the tumor by M-CSF and other chemokines. The
macrophages can then contribute to tumor progression through the
secretion of angiogenic factors, proteases and other growth factors
and cytokines and may be blocked by inhibition of CSF-1R signaling.
Recently it was shown by Zins et al (Zins, K., et al., Cancer Res.
67 (2007) 1038-1045) that expression of siRNA of Tumor necrosis
factor alpha (TNF alpha), M-CSF or the combination of both would
reduce tumor growth in a mouse xenograft model between 34% and 50%
after intratumoral injection of the respective siRNA. SiRNA
targeting the TNF alpha secreted by the human SW620 cells reduced
mouse M-CSF levels and led to reduction of macrophages in the
tumor. In addition treatment of MCF7 tumor xenografts with an
antigen binding fragment directed against M-CSF did result in 40%
tumor growth inhibition, reversed the resistance to
chemotherapeutics and improved survival of the mice when given in
combination with chemotherapeutics (Paulus, P., et al., Cancer Res.
66 (2006) 4349-4356).
[0237] TAMs are only one example of an emerging link between
chronic inflammation and cancer. There is additional evidence for a
link between inflammation and cancer as many chronic diseases are
associated with an increased risk of cancer, cancers arise at sites
of chronic inflammation, chemical mediators of inflammation are
found in many cancers; deletion of the cellular or chemical
mediators of inflammation inhibits development of experimental
cancers and long-term use of anti-inflammatory agents reduce the
risk of some cancers. A link to cancer exists for a number of
inflammatory conditions among-those H. pylori induced gastritis for
gastric cancer, Schistosomiasis for bladder cancer, HHVX for
Kaposi's sarcoma, endometriosis for ovarian cancer and prostatitis
for prostate cancer (Balkwill, F., et al., Cancer Cell 7 (2005)
211-217). Macrophages are key cells in chronic inflammation and
respond differentially to their microenvironment. There are two
types of macrophages that are considered extremes in a continuum of
functional states: M1 macrophages are involved in Type 1 reactions.
These reactions involve the activation by microbial products and
consequent killing of pathogenic microorganisms that result in
reactive oxygen intermediates. On the other end of the extreme are
M2 macrophages involved in Type 2 reactions that promote cell
proliferation, tune inflammation and adaptive immunity and promote
tissue remodeling, angiogenesis and repair (Mantovani, A., et al.,
Trends Immunol. 25 (2004) 677-686). Chronic inflammation resulting
in established neoplasia is usually associated with M2 macrophages.
A pivotal cytokine that mediates inflammatory reactions is TNF
alpha that true to its name can stimulate anti-tumor immunity and
hemorrhagic necrosis at high doses but has also recently been found
to be expressed by tumor cells and acting as a tumor promoter
(Zins, K., et al., Cancer Res. 67 (2007) 1038-1045; Balkwill, F.,
Cancer Metastasis Rev. 25 (2006) 409-416). The specific role of
macrophages with respect to the tumor still needs to be better
understood including the potential spatial and temporal dependence
on their function and the relevance to specific tumor types.
[0238] Thus one embodiment of the invention are the CSF-1R
antibodies described herein in for use in the treatment of cancer
in combination with an anti-PD-L1 antibody as described herein. The
term "cancer" as used herein may be, for example, lung cancer, non
small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer,
bone cancer, pancreatic cancer, skin cancer, cancer of the head or
neck, cutaneous or intraocular melanoma, uterine cancer, ovarian
cancer, rectal cancer, cancer of the anal region, stomach cancer,
gastric cancer, colon cancer, breast cancer, uterine cancer,
carcinoma of the fallopian tubes, carcinoma of the endometrium,
carcinoma of the cervix, carcinoma of the vagina, carcinoma of the
vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the
small intestine, cancer of the endocrine system, cancer of the
thyroid gland, cancer of the parathyroid gland, cancer of the
adrenal gland, sarcoma of soft tissue, cancer of the urethra,
cancer of the penis, prostate cancer, cancer of the bladder, cancer
of the kidney or ureter, renal cell carcinoma, carcinoma of the
renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer,
neoplasms of the central nervous system (CNS), spinal axis tumors,
brain stem glioma, glioblastoma multiforme, astrocytomas,
schwanomas, ependymonas, medulloblastomas, meningiomas, squamous
cell carcinomas, pituitary adenoma, lymphoma, lymphocytic leukemia,
including refractory versions of any of the above cancers, or a
combination of one or more of the above cancers. In one preferred
embodiment such cancer is a breast cancer, colorectal cancer,
melanoma, head and neck cancer, lung cancer or prostate cancer. In
one preferred embodiment such cancer is a breast cancer, ovarian
cancer, cervical cancer, lung cancer or prostate cancer. In another
preferred embodiment such cancer is breast cancer, lung cancer,
colon cancer, ovarian cancer, melanoma cancer, bladder cancer,
renal cancer, kidney cancer, liver cancer, head and neck cancer,
colorectal cancer, pancreatic cancer, gastric carcinoma cancer,
esophageal cancer, mesothelioma, prostate cancer, leukemia,
lymphoma, myelomas. In one preferred embodiment such cancers are
further characterized by CSF-1 or CSF-1R expression or
overexpression. One further embodiment the invention are the CSF-1R
antibodies of the present invention for use in the simultaneous
treatment of primary tumors and new metastases. Thus another
embodiment of the invention are the CSF-1R antibodies of the
present invention for use in the treatment of periodontitis,
histiocytosis X, osteoporosis, Paget's disease of bone (PDB), bone
loss due to cancer therapy, periprosthetic osteolysis,
glucocorticoid-induced osteoporosis, rheumatoid arthritis, psiratic
arthritis, osteoarthritis, inflammatory arthridities, and
inflammation.
[0239] Rabello, D., et al., Biochem. Biophys. Res. Commun. 347
(2006) 791-796 has demonstrated that SNPs in the CSF1 gene
exhibited a positive association with aggressive periodontitis: an
inflammatory disease of the periodontal tissues that causes tooth
loss due to resorption of the alveolar bone.
[0240] Histiocytosis X (also called Langerhans cell histiocytosis,
LCH) is a proliferative disease of Langerhans dendritic cells that
appear to differentiate into osteoclasts in bone and extra osseous
LCH lesions. Langerhans cells are derived from circulating
monocytes. Increased levels of M-CSF that have been measured in
sera and lesions where found to correlate with disease severity (da
Costa, C. E., et al., J. Exp. Med. 201 (2005) 687-693). The disease
occurs primarily in a pediatric patient population and has to be
treated with chemotherapy when the disease becomes systemic or is
recurrent.
[0241] The pathophysiology of osteoporosis is mediated by loss of
bone forming osteoblasts and increased osteoclast dependent bone
resorption. Supporting data has been described by Cenci et al
showing that an anti-M-CSF antibody injection preserves bone
density and inhibits bone resorption in ovariectomized mice (Cenci,
S., et al., J. Clin. Invest. 105 (2000) 1279-1287). Recently a
potential link between postmenopausal bone loss due to estrogen
deficiency was identified and found that the presence of TNF alpha
producing T-cell affected bone metabolism (Roggia, C., et al.,
Minerva Med. 95 (2004) 125-132). A possible mechanism could be the
induction of M-CSF by TNF alpha in vivo. An important role for
M-CSF in TNF-alpha-induced osteoclastogenesis was confirmed by the
effect of an antibody directed against M-CSF that blocked the TNF
alpha induced osteolysis in mice and thereby making inhibitors of
CSF-1R signaling potential targets for inflammatory arthritis
(Kitaura, H., et al., J. Clin. Invest. 115 (2005) 3418-3427).
[0242] Paget's disease of bone (PDB) is the second most common bone
metabolism disorder after osteoporosis in which focal abnormalities
of increased bone turnover lead to complications such as bone pain,
deformity, pathological fractures and deafness. Mutations in four
genes have been identified that regulate normal osteoclast function
and predispose individuals to PDB and related disorders: insertion
mutations in TNFRSF11A, which encodes receptor activator of nuclear
factor (NF) kappaB (RANK)-a critical regulator of osteoclast
function, inactivating mutations of TNFRSF11B which encodes
osteoprotegerin (a decoy receptor for RANK ligand), mutations of
the sequestosome 1 gene (SQSTM1), which encodes an important
scaffold protein in the NFkappaB pathway and mutations in the
valosin-containing protein (VCP) gene. This gene encodes VCP, which
has a role in targeting the inhibitor of NFkappaB for degradation
by the proteasome (Daroszewska, A. and Ralston, S. H., Nat. Clin.
Pract. Rheumatol. 2 (2006) 270-277). Targeted CSF-1R inhibitors
provide an opportunity to block the deregulation of the RANKL
signaling indirectly and add an additional treatment option to the
currently used bisphosphonates.
[0243] Cancer therapy induced bone loss especially in breast and
prostate cancer patients is an additional indication where a
targeted CSF-1R inhibitor could prevent bone loss (Lester, J. E.,
et al., Br. J. Cancer 94 (2006) 30-35). With the improved prognosis
for early breast cancer the long-term consequences of the adjuvant
therapies become more important as some of the therapies including
chemotherapy, irradiation, aromatase inhibitors and ovary ablation
affect bone metabolism by decreasing the bone mineral density,
resulting in increased risk for osteoporosis and associated
fractures (Lester, J. E., et al., Br. J. Cancer 94 (2006) 30-35).
The equivalent to adjuvant aromatase inhibitor therapy in breast
cancer is androgen ablation therapy in prostate cancer which leads
to loss of bone mineral density and significantly increases the
risk of osteoporosis-related fractures (Stoch, S. A., et al., J.
Clin. Endocrinol. Metab. 86 (2001) 2787-2791).
[0244] Targeted inhibition of CSF-1R signaling is likely to be
beneficial in other indications as well when targeted cell types
include osteoclasts and macrophages e.g. treatment of specific
complications in response to joint replacement as a consequence of
rheumatoid arthritis. Implant failure due to periprosthetic bone
loss and consequent loosing of prostheses is a major complication
of joint replacement and requires repeated surgery with high
socioeconomic burdens for the individual patient and the
health-care system. To date, there is no approved drug therapy to
prevent or inhibit periprosthetic osteolysis (Drees, P., et al.,
Nat. Clin. Pract. Rheumatol. 3 (2007) 165-171).
[0245] Glucocorticoid-induced osteoporosis (GIOP) is another
indication in which a CSF-1R inhibitor could prevent bone loss
after longterm glucocorticosteroid use that is given as a result of
various conditions among those chronic obstructive pulmonary
disease, asthma and rheumatoid arthritis (Guzman-Clark, J. R., et
al., Arthritis Rheum. 57 (2007) 140-146; Feldstein, A. C., et al.,
Osteoporos. Int. 16 (2005) 2168-2174).
[0246] Rheumatoid arthritis, psioratic arthritis and inflammatory
arthridities are in itself potential indications for CSF-1R
signaling inhibitors in that they consist of a macrophage component
and to a varying degree bone destruction (Ritchlin, C. T., et al.,
J. Clin. Invest. 111 (2003) 821-831). Osteoarthritis and rheumatoid
arthritis are inflammatory autoimmune disease caused by the
accumulation of macrophages in the connective tissue and
infiltration of macrophages into the synovial fluid, which is at
least partially mediated by M-CSF. Campbell, I., K., et al., J.
Leukoc. Biol. 68 (2000) 144-150, demonstrated that M-CSF is
produced by human joint tissue cells (chondrocytes, synovial
fibroblasts) in vitro and is found in synovial fluid of patients
with rheumatoid arthritis, suggesting that it contributes to the
synovial tissue proliferation and macrophage infiltration which is
associated with the pathogenesis of the disease Inhibition of
CSF-1R signaling is likely to control the number of macrophages in
the joint and alleviate the pain from the associated bone
destruction. In order to minimize adverse effects and to further
understand the impact of the CSF-1R signaling in these indications,
one method is to specifically inhibit CSF-1R without targeting a
myriad other kinases, such as Raf kinase.
[0247] Recent literature reports correlate increased circulating
M-CSF with poor prognosis and atherosclerotic progression in
chronic coronary artery disease (Saitoh, T., et al., J. Am. Coll.
Cardiol. 35 (2000) 655-665; Ikonomidis, I., et al., Eur. Heart. J.
26 (2005) p. 1618-1624); M-CSF influences the atherosclerotic
process by aiding the formation of foam cells (macrophages with
ingested oxidized LDL) that express CSF-1R and represent the
initial plaque (Murayama, T., et al., Circulation 99 (1999)
1740-1746).
[0248] Expression and signaling of M-CSF and CSF-1R is found in
activated microglia. Microglia, which are resident macrophages of
the central nervous system, can be activated by various insults,
including infection and traumatic injury. M-CSF is considered a key
regulator of inflammatory responses in the brain and M-CSF levels
increase in HIV-1, encephalitis, Alzheimer's disease (AD) and brain
tumors. Microgliosis as a consequence of autocrine signaling by
M-CSF/CSF-1R results in induction of inflammatory cytokines and
nitric oxides being released as demonstrated by e.g. using an
experimental neuronal damage model (Hao, A. J., et al.,
Neuroscience 112 (2002) 889-900; Murphy, G. M., Jr., et al., J.
Biol. Chem. 273 (1998) 20967-20971). Microglia that have increased
expression of CSF-1R are found to surround plaques in AD and in the
amyloid precursor protein V717F transgenic mouse model of AD
(Murphy, G. M., Jr., et al., Am. J. Pathol. 157 (2000) 895-904). On
the other hand op/op mice with fewer microglia in the brain
resulted in fibrillar deposition of A-beta and neuronal loss
compared to normal control suggesting that microglia do have a
neuroprotective function in the development of AD lacking in the
op/op mice (Kaku, M., et al., Brain Res. Brain Res. Protoc. 12
(2003) 104-108).
[0249] Expression and signaling of M-CSF and CSF-1R is associated
with inflammatory bowel disease (IBD) (WO 2005/046657). The term
"inflammatory bowel disease" refers to serious, chronic disorders
of the intestinal tract characterized by chronic inflammation at
various sites in the gastrointestinal tract, and specifically
includes ulcerative colitis (UC) and Crohn's disease.
[0250] Thus another embodiment of the invention are the CSF-1R
antibodies being characterized by the above mentioned amino acid
sequences and amino acid sequence in combination with an anti-PD-L1
antibody being characterized by the above mentioned amino acid
sequences and amino acid sequence for use in the treatment of
periodontitis, histiocytosis X, osteoporosis, Paget's disease of
bone (PDB), bone loss due to cancer therapy, periprosthetic
osteolysis, glucocorticoid-induced osteoporosis, rheumatoid
arthritis, psiratic arthritis, osteoarthritis, inflammatory
arthridities, and inflammation. [0251] The invention comprises the
combination therapy with an antibody binding to human CSF-1R being
characterized by the above mentioned amino acid sequences and amino
acid sequence fragments with an anti-PD-L1 antibody being
characterized by the above mentioned amino acid sequences and amino
acid sequence fragments for the treatment of cancer. [0252] The
invention comprises the combination therapy with an antibody
binding to human CSF-1R being characterized by the above mentioned
amino acid sequences and amino acid sequence fragments with an
anti-PD-L1 antibody being characterized by the above mentioned
amino acid sequences and amino acid sequence fragments for the
treatment of bone loss. [0253] The invention comprises the
combination therapy with an antibody binding to human CSF-1R being
characterized by the above mentioned amino acid sequences and amino
acid sequence fragments with an anti-PD-L1 antibody being
characterized by the above mentioned amino acid sequences and amino
acid sequence fragments for the prevention or treatment of
metastasis. [0254] The invention comprises the combination therapy
of antibody binding to human CSF-1R being characterized by the
above mentioned amino acid sequences and amino acid sequence
fragments with an anti-PD-L1 antibody being characterized by the
above mentioned amino acid sequences and amino acid sequence
fragments for treatment of inflammatory diseases. [0255] The
invention comprises the combination therapy of antibody binding to
human CSF-1R being characterized by the above mentioned amino acid
sequences and amino acid sequence fragments with an anti-PD-L1
antibody being characterized by the above mentioned amino acid
sequences and amino acid sequence fragments for use in treating or
delaying progression of an immune related disease such as tumor
immunity. [0256] The invention comprises the combination therapy of
antibody binding to human CSF-1R being characterized by the above
mentioned amino acid sequences and amino acid sequence fragments
with an anti-PD-L1 antibody being characterized by the above
mentioned amino acid sequences and amino acid sequence fragments
for use in stimulating an immune response or function, such as T
cell activity. [0257] The invention comprises the use of an
antibody characterized in comprising the antibody binding to human
CSF-1R being characterized by the above mentioned amino acid
sequences and amino acid sequence fragments for the combination
treatment of cancer with an anti-PD-L1 antibody or alternatively
for the manufacture of a medicament for the combination treatment
of cancer with an anti-PD-L1 antibody as described herein. [0258]
The invention comprises the use of an antibody characterized in
comprising the antibody binding to human CSF-1R being characterized
by the above mentioned amino acid sequences and amino acid sequence
fragments for the combination treatment of bone loss with an
anti-PD-L1 antibody as described herein or alternatively for the
manufacture of a medicament for the combination treatment of bone
loss with an anti-PD-L1 antibody as described herein. [0259] The
invention comprises the use of an antibody characterized in
comprising the antibody binding to human CSF-1R being characterized
by the above mentioned amino acid sequences and amino acid sequence
fragments for the prevention or treatment of metastasis in the
combination with an anti-PD-L1 antibody as described herein or
alternatively for the manufacture of a medicament for the
prevention or treatment of metastasis in the combination with an
anti-PD-L1 antibody as described herein. [0260] The invention
comprises the use of an antibody characterized in comprising the
antibody binding to human CSF-1R being characterized by the above
mentioned amino acid sequences and amino acid sequence fragments
for combination treatment of inflammatory diseases with an
anti-PD-L1 antibody as described herein or alternatively for the
manufacture of a medicament for the combination treatment of
inflammatory diseases with an anti-PD-L1 antibody as described
herein. [0261] The invention comprises the use of an antibody
characterized in comprising the antibody binding to human CSF-1R
being characterized by the above mentioned amino acid sequences and
amino acid sequence fragments for use in treating or delaying
progression of an immune related disease such as tumor immunity in
combination with an anti-PD-L1 antibody as described herein or
alternatively for the manufacture of a medicament for use in
treating or delaying progression of an immune related disease such
as tumor immunity in combination with an anti-PD-L1 antibody as
described herein. [0262] The invention comprises the use of an
antibody characterized in comprising the antibody binding to human
CSF-1R being characterized by the above mentioned amino acid
sequences and amino acid sequence fragments for use in stimulating
an immune response or function, such as T cell activity in
combination with an anti-PD-L1 antibody as described herein or
alternatively for the manufacture of a medicament for use in
stimulating an immune response or function, such as T cell activity
in combination with an anti-PD-L1 antibody as described herein.
[0263] In one preferred embodiment of the invention the antibody
which binds to human CSF-1R used in the above described combination
treatments and medical uses of different diseases is characterized
in comprising
[0264] a heavy chain variable domain VH of SEQ ID NO:39 and a light
chain variable domain VL of SEQ ID NO:40, and
the antibody which binds to human PD-L1 used in such combination
treatments is characterized in comprising a heavy chain variable
domain VH of SEQ ID NO:89 and a light chain variable domain VL of
SEQ ID NO:92.
[0265] The antibodies described herein are preferably produced by
recombinant means. Such methods are widely known in the state of
the art and comprise protein expression in prokaryotic and
eukaryotic cells with subsequent isolation of the antibody
polypeptide and usually purification to a pharmaceutically
acceptable purity. For the protein expression nucleic acids
encoding light and heavy chains or fragments thereof are inserted
into expression vectors by standard methods. Expression is
performed in appropriate prokaryotic or eukaryotic host cells, such
as CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells,
yeast, or E. coli cells, and the antibody is recovered from the
cells (from the supernatant or after cells lysis).
[0266] Recombinant production of antibodies is well-known in the
state of the art and described, for example, in the review articles
of Makrides, S. C., Protein Expr. Purif. 17 (1999) 183-202; Geisse,
S., et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R. J.,
Mol. Biotechnol. 16 (2000) 151-161; Werner, R. G., Drug Res. 48
(1998) 870-880.
[0267] The antibodies may be present in whole cells, in a cell
lysate, or in a partially purified, or substantially pure form.
Purification is performed in order to eliminate other cellular
components or other contaminants, e.g. other cellular nucleic acids
or proteins, by standard techniques, including alkaline/SDS
treatment, CsCl banding, column chromatography, agarose gel
electrophoresis, and others well known in the art. See Ausubel, F.,
et al., ed. Current Protocols in Molecular Biology, Greene
Publishing and Wiley Interscience, New York (1987).
[0268] Expression in NS0 cells is described by, e.g., Barnes, L.
M., et al., Cytotechnology 32 (2000) 109-123; Barnes, L. M., et
al., Biotech. Bioeng. 73 (2001) 261-270. Transient expression is
described by, e.g., Durocher, Y., et al., Nucl. Acids. Res. 30
(2002) E9. Cloning of variable domains is described by Orlandi, R.,
et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P.,
et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; Norderhaug,
L., et al., J. Immunol. Methods 204 (1997) 77-87. A preferred
transient expression system (HEK 293) is described by Schlaeger,
E.-J. and Christensen, K., in Cytotechnology 30 (1999) 71-83, and
by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996) 191-199.
[0269] The heavy and light chain variable domains according to the
invention are combined with sequences of promoter, translation
initiation, constant region, 3' untranslated region,
polyadenylation, and transcription termination to form expression
vector constructs. The heavy and light chain expression constructs
can be combined into a single vector, co-transfected, serially
transfected, or separately transfected into host cells which are
then fused to form a single host cell expressing both chains.
[0270] The control sequences that are suitable for prokaryotes, for
example, include a promoter, optionally an operator sequence, and a
ribosome binding site. Eukaryotic cells are known to utilize
promoters, enhancers and polyadenylation signals.
[0271] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading frame. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0272] The monoclonal antibodies are suitably separated from the
culture medium by conventional immunoglobulin purification
procedures such as, for example, protein A-Sepharose,
hydroxylapatite chromatography, gel electrophoresis, dialysis, or
affinity chromatography. DNA and RNA encoding the monoclonal
antibodies are readily isolated and sequenced using conventional
procedures. The hybridoma cells can serve as a source of such DNA
and RNA. Once isolated, the DNA may be inserted into expression
vectors, which are then transfected into host cells such as HEK 293
cells, CHO cells, or myeloma cells that do not otherwise produce
immunoglobulin protein, to obtain the synthesis of recombinant
monoclonal antibodies in the host cells.
[0273] As used herein, the expressions "cell", "cell line", and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived
therefrom without regard for the number of transfers. It is also
understood that all progeny may not be precisely identical in DNA
content, due to deliberate or inadvertent mutations. Variant
progeny that have the same function or biological activity as
screened for in the originally transformed cell are included.
[0274] In another aspect, the present invention provides a
composition, e.g. a pharmaceutical composition, containing one or a
combination of monoclonal antibodies, or the antigen-binding
portion thereof, of the present invention, formulated together with
a pharmaceutically acceptable carrier.
[0275] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and
absorption/resorption delaying agents, and the like that are
physiologically compatible. Preferably, the carrier is suitable for
injection or infusion.
[0276] A composition of the present invention can be administered
by a variety of methods known in the art. As will be appreciated by
the skilled artisan, the route and/or mode of administration will
vary depending upon the desired results.
[0277] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the preparation of
sterile injectable solutions or dispersion. The use of such media
and agents for pharmaceutically active substances is known in the
art. In addition to water, the carrier can be, for example, an
isotonic buffered saline solution.
[0278] Regardless of the route of administration selected, the
compounds of the present invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, are formulated into pharmaceutically acceptable
dosage forms by conventional methods known to those of skill in the
art.
[0279] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient (effective amount). The selected
dosage level will depend upon a variety of pharmacokinetic factors
including the activity of the particular compositions of the
present invention employed, or the ester, salt or amide thereof,
the route of administration, the time of administration, the rate
of excretion of the particular compound being employed, other
drugs, compounds and/or materials used in combination with the
particular compositions employed, the age, sex, weight, condition,
general health and prior medical history of the patient being
treated, and like factors well known in the medical arts.
[0280] The term "a method of treating" or its equivalent, when
applied to, for example, cancer refers to a procedure or course of
action that is designed to reduce or eliminate the number of cancer
cells in a patient, or to alleviate the symptoms of a cancer. "A
method of treating" cancer or another proliferative disorder does
not necessarily mean that the cancer cells or other disorder will,
in fact, be eliminated, that the number of cells or disorder will,
in fact, be reduced, or that the symptoms of a cancer or other
disorder will, in fact, be alleviated. Often, a method of treating
cancer will be performed even with a low likelihood of success, but
which, given the medical history and estimated survival expectancy
of a patient, is nevertheless deemed to induce an overall
beneficial course of action.
[0281] The terms "administered in combination with" or
"co-administration", "co-administering", "combination therapy" or
"combination treatment" refer to the administration of the
anti-CSF-1R as described herein, and the anti-PD-L1 antibody as
described herein e.g. as separate formulations/applications (or as
one single formulation/application). The co-administration can be
simultaneous or sequential in either order, wherein preferably
there is a time period while both (or all) active agents
simultaneously exert their biological activities. Said antibody and
said further agent are co-administered either simultaneously or
sequentially (e.g. intravenous (i.v.) through a continuous
infusion. When both therapeutic agents are co-administered
sequentially the dose is administered either on the same day in two
separate administrations, or one of the agents is administered on
day 1 and the second is co-administered on day 2 to day 7,
preferably on day 2 to 4. Thus in one embodiment the term
"sequentially" means within 7 days after the dose of the first
component, preferably within 4 days after the dose of the first
component; and the term "simultaneously" means at the same time.
The terms "co-administration" with respect to the maintenance doses
of anti-CSF-1R antibody and/or anti-PD-L1 antibody mean that the
maintenance doses can be either co-administered simultaneously, if
the treatment cycle is appropriate for both drugs, e.g. every week.
Or the further agent is e.g. administered e.g. every first to third
day and said antibody is administered every week. Or the
maintenance doses are co-administered sequentially, either within
one or within several days.
[0282] It is self-evident that the antibodies are administered to
the patient in a "therapeutically effective amount" (or simply
"effective amount") which is the amount of the respective compound
or combination that will elicit the biological or medical response
of a tissue, system, animal or human that is being sought by the
researcher, veterinarian, medical doctor or other clinician.
[0283] The amount of co-administration and the timing of
co-administration will depend on the type (species, gender, age,
weight, etc.) and condition of the patient being treated and the
severity of the disease or condition being treated. Said
anti-CSF-1R antibody and further agent are suitably co-administered
to the patient at one time or over a series of treatments e.g. on
the same day or on the day after.
[0284] Depending on the type and severity of the disease, about 0.1
mg/kg to 50 mg/kg (e.g. 0.1-20 mg/kg) of said anti-CSF-1R antibody
and/or anti-PD-L1 antibody; is an initial candidate dosage for
co-administration of both drugs to the patient The invention
comprises the use of the antibodies according to the invention for
the treatment of a patient suffering from cancer, especially from
colon, lung or pancreas cancer.
[0285] Depending on the type and severity of the disease, about 0.1
mg/kg to 50 mg/kg (e.g. 0.1-20 mg/kg) of said anti-CSF-1R antibody
and/or anti-PD-L1 antibody; is an initial candidate dosage for
co-administration of both drugs to the patient The invention
comprises the use of the antibodies according to the invention for
the treatment of a patient suffering from cancer, especially from
colon, lung or pancreas cancer.
[0286] In addition to the anti-CSF-1R antibody in combination with
the anti-PD-L1 antibody also a chemotherapeutic agent can be
administered.
[0287] In one embodiment such additional chemotherapeutic agents,
which may be administered with anti-CSF-1R antibody as described
herein and the anti-PD-L1 antibody as described herein, include,
but are not limited to, anti-neoplastic agents including alkylating
agents including: nitrogen mustards, such as mechlorethamine,
cyclophosphamide, ifosfamide, melphalan and chlorambucil;
nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), and
semustine (methyl-CCNU); Temodal.TM. (temozolamide),
ethylenimines/methylmelamine such as thriethylenemelamine (TEM),
triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM,
altretamine); alkyl sulfonates such as busulfan; triazines such as
dacarbazine (DTIC); antimetabolites including folic acid analogs
such as methotrexate and trimetrexate, pyrimidine analogs such as
5-fluorouracil (5FU), fluorodeoxyuridine, gemcitabine, cytosine
arabinoside (AraC, cytarabine), 5-azacytidine,
2,2'-difluorodeoxycytidine, purine analogs such as
6-merca.rho.topurine, 6-thioguamne, azathioprine, T-deoxycoformycin
(pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine
phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA); natural
products including antimitotic drugs such as paclitaxel, vinca
alkaloids including vinblastine (VLB), vincristine, and
vinorelbine, taxotere, estramustine, and estramustine phosphate;
pipodophylotoxins such as etoposide and teniposide; antibiotics
such as actinomycin D, daunomycin (rubidomycin), doxorubicin,
mitoxantrone, idarubicin, bleomycins, plicamycin (mithramycin),
mitomycin C, and actinomycin; enzymes such as L-asparaginase;
biological response modifiers such as interferon-alpha, IL-2, G-CSF
and GM-CSF; miscellaneous agents including platinum coordination
complexes such as oxaliplatin, cisplatin and carboplatin,
anthracenediones such as mitoxantrone, substituted urea such as
hydroxyurea, methylhydrazine derivatives including
N-methylhydrazine (MIH) and procarbazine, adrenocortical
suppressants such as mitotane (o, p-DDD) and aminoglutethimide;
hormones and antagonists including adrenocorticosteroid antagonists
such as prednisone and equivalents, dexamethasone and
aminoglutethimide; Gemzar.TM. (gemcitabine), progestin such as
hydroxyprogesterone caproate, medroxyprogesterone acetate and
megestrol acetate; estrogen such as diethylstilbestrol and ethinyl
estradiol equivalents; antiestrogen such as tamoxifen; androgens
including testosterone propionate and fluoxymesterone/equivalents;
antiandrogens such as flutamide, gonadotropin-releasing hormone
analogs and leuprolide; and non-steroidal antiandrogens such as
flutamide. Therapies targeting epigenetic mechanism including, but
not limited to, histone deacetylase inhibitors, demethylating
agents (e.g., Vidaza) and release of transcriptional repression
(ATRA) therapies can also be combined with the antigen binding
proteins. In one embodiment the chemotherapeutic agent is selected
from the group consisting of taxanes (like e.g. paclitaxel (Taxol),
docetaxel (Taxotere), modified paclitaxel (e.g., Abraxane and
Opaxio), doxorubicin, sunitinib (Sutent), sorafenib (Nexavar), and
other multikinase inhibitors, oxaliplatin, cisplatin and
carboplatin, etoposide, gemcitabine, and vinblastine. In one
embodiment the chemotherapeutic agent is selected from the group
consisting of taxanes (like e.g. taxol (paclitaxel), docetaxel
(Taxotere), modified paclitaxel (e.g. Abraxane and Opaxio). In one
embodiment, the additional chemotherapeutic agent is selected from
5-fluorouracil (5-FU), leucovorin, irinotecan, or oxaliplatin. In
one embodiment the chemotherapeutic agent is 5-fluorouracil,
leucovorin and irinotecan (FOLFIRI). In one embodiment the
chemotherapeutic agent is 5-fluorouracil, and oxaliplatin
(FOLFOX).
[0288] Specific examples of combination therapies with additional
chemotherapeutic agents include, for instance, therapies taxanes
(e.g., docetaxel or paclitaxel) or a modified paclitaxel (e.g.,
Abraxane or Opaxio), doxorubicin), capecitabine and/or bevacizumab
(Avastin) for the treatment of breast cancer; therapies with
carboplatin, oxaliplatin, cisplatin, paclitaxel, doxorubicin (or
modified doxorubicin (Caelyx or Doxil)), or topotecan (Hycamtin)
for ovarian cancer, the therapies with a multi-kinase inhibitor,
MKI, (Sutent, Nexavar, or 706) and/or doxorubicin for treatment of
kidney cancer; therapies with oxaliplatin, cisplatin and/or
radiation for the treatment of squamous cell carcinoma; therapies
with taxol and/or carboplatin for the treatment of lung cancer.
[0289] Therefore, in one embodiment the additional chemotherapeutic
agent is selected from the group of taxanes (docetaxel or
paclitaxel or a modified paclitaxel (Abraxane or Opaxio),
doxorubicin, capecitabine and/or bevacizumab for the treatment of
breast cancer.
[0290] In one embodiment the CSF-1R antibody/PD-L1 antibody
combination therapy is no chemotherapeutic agents are
administered.
[0291] The invention comprises also a method for the treatment of a
patient suffering from such disease.
[0292] The invention further provides a method for the manufacture
of a pharmaceutical composition comprising an effective amount of
an antibody according to the invention together with a
pharmaceutically acceptable carrier and the use of the antibody
according to the invention for such a method.
[0293] The invention further provides the use of an antibody
according to the invention in an effective amount for the
manufacture of a pharmaceutical agent, preferably together with a
pharmaceutically acceptable carrier, for the treatment of a patient
suffering from cancer.
[0294] The invention also provides the use of an antibody according
to the invention in an effective amount for the manufacture of a
pharmaceutical agent, preferably together with a pharmaceutically
acceptable carrier, for the treatment of a patient suffering from
cancer.
[0295] The following examples, sequence listing and figures are
provided to aid the understanding of the present invention, the
true scope of which is set forth in the appended claims. It is
understood that modifications can be made in the procedures set
forth without departing from the spirit of the invention.
TABLE-US-00003 Description of the Sequences SEQ ID NO: 1 heavy
chain CDR3, Mab 2F11 SEQ ID NO: 2 heavy chain CDR2, Mab 2F11 SEQ ID
NO: 3 heavy chain CDR1, Mab 2F11 SEQ ID NO: 4 light chain CDR3, Mab
2F11 SEQ ID NO: 5 light chain CDR2, Mab 2F11 SEQ ID NO: 6 light
chain CDR1, Mab 2F11 SEQ ID NO: 7 heavy chain variable domain, Mab
2F11 SEQ ID NO: 8 light chain variable domain, Mab 2F11 SEQ ID NO:
9 heavy chain CDR3, Mab 2E10 SEQ ID NO: 10 heavy chain CDR2, Mab
2E10 SEQ ID NO: 11 heavy chain CDR1, Mab 2E10 SEQ ID NO: 12 light
chain CDR3, Mab 2E10 SEQ ID NO: 13 light chain CDR2, Mab 2E10 SEQ
ID NO: 14 light chain CDR1, Mab 2E10 SEQ ID NO: 15 heavy chain
variable domain, Mab 2E10 SEQ ID NO: 16 light chain variable
domain, Mab 2E10 SEQ ID NO: 17 heavy chain CDR3, hMab 2F11-c11 SEQ
ID NO: 18 heavy chain CDR2, hMab 2F11-c11 SEQ ID NO: 19 heavy chain
CDR1, hMab 2F11-c11 SEQ ID NO: 20 light chain CDR3, hMab 2F11-c11
SEQ ID NO: 21 light chain CDR2, hMab 2F11-c11 SEQ ID NO: 22 light
chain CDR1, hMab 2F11-c11 SEQ ID NO: 23 heavy chain variable
domain, hMab 2F11-c11 SEQ ID NO: 24 light chain variable domain,
hMab 2F11-c11 SEQ ID NO: 25 heavy chain CDR3, hMab 2F11-d8 SEQ ID
NO: 26 heavy chain CDR2, hMab 2F11-d8 SEQ ID NO: 27 heavy chain
CDR1, hMab 2F11-d8 SEQ ID NO: 28 light chain CDR3, hMab 2F11-d8 SEQ
ID NO: 29 light chain CDR2, hMab 2F11-d8 SEQ ID NO: 30 light chain
CDR1, hMab 2F11-d8 SEQ ID NO: 31 heavy chain variable domain, hMab
2F11-d8 SEQ ID NO: 32 light chain variable domain, hMab 2F11-d8 SEQ
ID NO: 33 heavy chain CDR3, hMab 2F11-e7 SEQ ID NO: 34 heavy chain
CDR2, hMab 2F11-e7 SEQ ID NO: 35 heavy chain CDR1, hMab 2F11-e7 SEQ
ID NO: 36 light chain CDR3, hMab 2F11-e7 SEQ ID NO: 37 light chain
CDR2, hMab 2F11-e7 SEQ ID NO: 38 light chain CDR1, hMab 2F11-e7 SEQ
ID NO: 39 heavy chain variable domain, hMab 2F11-e7 SEQ ID NO: 40
light chain variable domain, hMab 2F11-e7 SEQ ID NO: 41 heavy chain
CDR3, hMab 2F11-f12 SEQ ID NO: 42 heavy chain CDR2, hMab 2F11-f12
SEQ ID NO: 43 heavy chain CDR1, hMab 2F11-f12 SEQ ID NO: 44 light
chain CDR3, hMab 2F11-f12 SEQ ID NO: 45 light chain CDR2, hMab
2F11-f12 SEQ ID NO: 46 light chain CDR1, hMab 2F11-f12 SEQ ID NO:
47 heavy chain variable domain, hMab 2F11-f12 SEQ ID NO: 48 light
chain variable domain, hMab 2F11-f12 SEQ ID NO: 49 heavy chain
CDR3, hMab 2F11-g1 SEQ ID NO: 50 heavy chain CDR2, hMab 2F11-g1 SEQ
ID NO: 51 heavy chain CDR1, hMab 2F11-g1 SEQ ID NO: 52 light chain
CDR3, hMab 2F11-g1 SEQ ID NO: 53 light chain CDR2, hMab 2F11-g1 SEQ
ID NO: 54 light chain CDR1, hMab 2F11-g1 SEQ ID NO: 55 heavy chain
variable domain, hMab 2F11-g1 SEQ ID NO: 56 light chain variable
domain, hMab 2F11-g1 SEQ ID NO: 57 human kappa light chain constant
region SEQ ID NO: 58 human heavy chain constant region derived from
IgG1 SEQ ID NO: 59 human heavy chain constant region derived from
IgG1 mutated on L234A and L235A SEQ ID NO: 60 human heavy chain
constant region derived from IgG4 SEQ ID NO: 61 human heavy chain
constant region derived from IgG4 mutated on S228P SEQ ID NO: 62
human wildtype CSF-1R (wt CSF-1R) (including signal sequence) SEQ
ID NO: 63 human mutant CSF-1R L301S Y969F (including signal
sequence) SEQ ID NO: 64 human CSF-1R Extracellular Domain (domains
D1-D5) SEQ ID NO: 65 human CSF-1R fragment delD4 SEQ ID NO: 66
human CSF-1R fragment domains D1-D3 SEQ ID NO: 67 signal peptide
SEQ ID NO: 68 Primer SEQ ID NO: 69 heavy chain CDR3, Mab 1G10 SEQ
ID NO: 70 heavy chain CDR2, Mab 1G10 SEQ ID NO: 71 heavy chain
CDR1, Mab 1G10 SEQ ID NO: 72 light chain CDR3, Mab 1G10 SEQ ID NO:
73 light chain CDR2, Mab 1G10 SEQ ID NO: 74 light chain CDR1, Mab
1G10 SEQ ID NO: 75 heavy chain variable domain, Mab 1G10 SEQ ID NO:
76 light chain variable domain, Mab 1G10 SEQ ID NO: 77 heavy chain
CDR3, Mab 2H7 SEQ ID NO: 78 heavy chain CDR2, Mab 2H7 SEQ ID NO: 79
heavy chain CDR1, Mab 2H7 SEQ ID NO: 80 light chain CDR3, Mab 2H7
SEQ ID NO: 81 light chain CDR2, Mab 2H7 SEQ ID NO: 82 light chain
CDR1, Mab 2H7 SEQ ID NO: 83 heavy chain variable domain, Mab 2H7
SEQ ID NO: 84 light chain variable domain, Mab 2H7 SEQ ID NO: 85
human CSF-1R fragment domains D4-D5 SEQ ID NO: 86 human CSF-1
(including signal sequence) SEQ ID NO: 87 human IL-34 (including
signal sequence) SEQ ID NO: 88 human PD-L1 (including signal
sequence) SEQ ID NO: 89 heavy chain variable domain VH variant 1,
anti-PD-L1 243.55 SEQ ID NO: 90 heavy chain variable domain VH
variant 2, anti-PD-L1 243.55 SEQ ID NO: 91 heavy chain variable
domain VH variant 3, anti-PD-L1 243.55 SEQ ID NO: 92 light chain
variable domain VL variant 1, anti-PD-L1 243.55 SEQ ID NO: 93 light
chain variable domain VL variant 2, anti-PD-L1 243.55 SEQ ID NO: 94
light chain variable domain VL variant 3, anti-PD-L1 243.55 SEQ ID
NO: 95 light chain variable domain VL variant 4, anti-PD-L1 243.55
SEQ ID NO: 96 light chain variable domain VL variant 5, anti-PD-L1
243.55 SEQ ID NO: 97 light chain variable domain VL variant 6,
anti-PD-L1 243.55 SEQ ID NO: 98 light chain variable domain VL
variant 7, anti-PD-L1 243.55 SEQ ID NO: 99 light chain variable
domain VL variant 8, anti-PD-L1 243.55 SEQ ID NO: 100 light chain
variable domain VL variant 9, anti-PD-L1 243.55 SEQ ID NO: 101
light chain variable domain VL variant 10, anti-PD-L1 243.55 SEQ ID
NO: 102 light chain variable domain VL variant 11, anti-PD-L1
243.55 SEQ ID NO: 103 light chain variable domain VL variant 12,
anti-PD-L1 243.55 SEQ ID NO: 104 light chain variable domain VL
variant 13, anti-PD-L1 243.55 SEQ ID NO: 105 light chain variable
domain VL variant 14, anti-PD-L1 243.55 SEQ ID NO: 106 light chain
variable domain VL variant 15, anti-PD-L1 243.55 SEQ ID NO: 107
light chain variable domain VL variant 16, anti-PD-L1 243.55
In the following embodiment of the invention are described: [0296]
1. A) An antibody which binds to human CSF-1R wherein the antibody
is administered in combination with an antibody which binds to
human PD-L1 for use in the treatment of cancer, for use in the
prevention or treatment of metastasis, for use in the treatment
inflammatory diseases, for use in the treatment of bone loss, for
use in treating or delaying progression of an immune related
disease such as tumor immunity, or for use in stimulating an immune
response or function, such as T cell activity; or [0297] B) the use
of an antibody which binds to human CSF-1R for the manufacture of a
medicament for use in the treatment of cancer, for use in the
prevention or treatment of metastasis, for use in the treatment
inflammatory diseases, for use in the treatment of bone loss, for
use in treating or delaying progression of an immune related
disease such as tumor immunity, or for use in stimulating an immune
response or function, such as T cell activity, wherein the antibody
is administered in combination with an antibody which binds to
human PD-L1; [0298] wherein the antibody which binds to human
CSF-1R used in the combination therapy is characterized in
comprising [0299] a) a heavy chain variable domain VH of SEQ ID
NO:23 and a light chain variable domain VL of SEQ ID NO:24, or
[0300] b) a heavy chain variable domain VH of SEQ ID NO:31 and a
light chain variable domain VL of SEQ ID NO:32, or [0301] c) a
heavy chain variable domain VH of SEQ ID NO:39 and a light chain
variable domain VL of SEQ ID NO:40, or [0302] d) a heavy chain
variable domain VH of SEQ ID NO:47 and a light chain variable
domain VL of SEQ ID NO:48, or [0303] e) a heavy chain variable
domain VH of SEQ ID NO:55 and a light chain variable domain VL of
SEQ ID NO:56; [0304] and the antibody which binds to human PD-L1
used in the combination therapy is characterized in comprising
[0305] a) a heavy chain variable domain VH of SEQ ID NO:89 and a
light chain variable domain VL of SEQ ID NO:92, or [0306] b) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:93, or [0307] c) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:94, or [0308] d) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:95, or [0309] e) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:96, or
[0310] f) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:97, or [0311] g) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:98, or [0312] h) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:99, or [0313] i) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:100, or [0314] j) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:101, or
[0315] k) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:102, or [0316] l) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:103, or [0317] m) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:104, or [0318] n) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:105, or [0319] o) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:106, or
[0320] p) a heavy chain variable domain VH of SEQ ID NO:91 and a
light chain variable domain VL of SEQ ID NO:107. [0321] 2. Use of a
combination of [0322] A) an antibody which binds to human CSF-1R,
comprising [0323] a) a heavy chain variable domain VH of SEQ ID
NO:23 and a light chain variable domain VL of SEQ ID NO:24, or
[0324] b) a heavy chain variable domain VH of SEQ ID NO:31 and a
light chain variable domain VL of SEQ ID NO:32, or [0325] c) a
heavy chain variable domain VH of SEQ ID NO:39 and a light chain
variable domain VL of SEQ ID NO:40, or [0326] d) a heavy chain
variable domain VH of SEQ ID NO:47 and a light chain variable
domain VL of SEQ ID NO:48, or [0327] e) a heavy chain variable
domain VH of SEQ ID NO:55 and a light chain variable domain VL of
SEQ ID NO:56; [0328] and [0329] B) an antibody which binds to human
PD-L1 comprising [0330] a) a heavy chain variable domain VH of SEQ
ID NO:89 and a light chain variable domain VL of SEQ ID NO:92, or
[0331] b) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:93, or [0332] c) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:94, or [0333] d) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:95, or [0334] e) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:96, or [0335] f) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:97, or
[0336] g) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:98, or [0337] h) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:99, or [0338] i) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:100, or [0339] j) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:101, or [0340] k) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:102, or
[0341] l) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:103, or [0342] m) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:104, or [0343] n) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:105, or [0344] o) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:106, or [0345] p) a heavy chain variable domain VH of SEQ
ID NO:91 and a light chain variable domain VL of SEQ ID NO:107.
[0346] for the manufacture of a medicament for use in the treatment
of cancer, for use in the prevention or treatment of metastasis,
for use in the treatment inflammatory diseases, for use in the
treatment of bone loss, for use in treating or delaying progression
of an immune related disease such as tumor immunity, or for use in
stimulating an immune response or function, such as T cell
activity, wherein the antibody is administered in combination with
an antibody which binds to human PD-L1 [0347] 3. The antibody or
use according to any one of embodiments 1 or 2, for use in the
treatment of cancer. [0348] 4. The antibody or use according to
embodiment 3, for use in the treatment of breast cancer, lung
cancer, colon cancer, ovarian cancer, melanoma cancer, bladder
cancer, renal cancer, kidney cancer, liver cancer, head and neck
cancer, colorectal cancer, pancreatic cancer, gastric carcinoma
cancer, esophageal cancer, mesotheliioma, prostate cancer,
leukemia, lymphomas, myelomas. [0349] 5. The antibody or use
according to any one of embodiments 1 or 2, for use in the
prevention or treatment of metastasis. [0350] 6. The antibody or
use according to any one of embodiments 1 or 2, for use in the
treatment of bone loss. [0351] 7. The antibody or use according to
any one of embodiments 1 or 2, for use in the treatment of
inflammatory diseases. [0352] 8. The antibody or use according to
any one of embodiments 1 or 2, for use in treating or delaying
progression of an immune related disease such as tumor immunity.
[0353] 9. The antibody or use according to any one of embodiments 1
or 2 for use in stimulating an immune response or function, such as
T cell activity. [0354] 10. A) An antibody which binds to human
CSF-1R wherein the antibody is administered in combination with an
antibody which binds to human PD-L1 for use in [0355] i) the
inhibition of cell proliferation in CSF-1R ligand-dependent and/or
CSF-1 ligand-independent CSF-1R expressing tumor cells; [0356] ii)
the inhibition of cell proliferation of tumors with CSF-1 R
ligand-dependent and/or CSF-1R ligand-independent CSF-1R expressing
macrophage infiltrate; [0357] iii) the inhibition of cell survival
(in CSF-1R ligand-dependent and/or CSF-1R ligand-independent)
CSF-1R expressing monocytes and macrophages; and/or [0358] iv) the
inhibition of cell differentiation (in CSF-1R ligand-dependent
and/or CSF-1R ligand-independent) CSF-1R expressing monocytes into
macrophages; [0359] or [0360] B) use of an antibody which binds to
human CSF-1R for the manufacture of a medicament for use in [0361]
i) the inhibition of cell proliferation in CSF-1R ligand-dependent
and/or CSF-1 ligand-independent CSF-1R expressing tumor cells;
[0362] ii) the inhibition of cell proliferation of tumors with
CSF-1R ligand-dependent and/or CSF-1R ligand-independent CSF-1R
expressing macrophage infiltrate; [0363] iii) the inhibition of
cell survival (in CSF-1R ligand-dependent and/or CSF-1R
ligand-independent) CSF-1R expressing monocytes and macrophages;
and/or [0364] iv) the inhibition of cell differentiation (in CSF-1R
ligand-dependent and/or CSF-1R ligand-independent) CSF-1R
expressing monocytes into macrophages, [0365] wherein the antibody
is administered in combination with an antibody which binds to
human PD-L1; [0366] wherein the antibody which binds to human
CSF-1R used in the combination therapy is characterized in
comprising [0367] a) a heavy chain variable domain VH of SEQ ID
NO:23 and a light chain variable domain VL of SEQ ID NO:24, or
[0368] b) a heavy chain variable domain VH of SEQ ID NO:31 and a
light chain variable domain VL of SEQ ID NO:32, or [0369] c) a
heavy chain variable domain VH of SEQ ID NO:39 and a light chain
variable domain VL of SEQ ID NO:40, or [0370] d) a heavy chain
variable domain VH of SEQ ID NO:47 and a light chain variable
domain VL of SEQ ID NO:48, or [0371] e) a heavy chain variable
domain VH of SEQ ID NO:55 and a light chain variable domain VL of
SEQ ID NO:56; [0372] and the antibody which binds to human PD-L1
used in the combination therapy is characterized in comprising
[0373] a) a heavy chain variable domain VH of SEQ ID NO:89 and a
light chain variable domain VL of SEQ ID NO:92, or [0374] b) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:93, or [0375] c) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:94, or [0376] d) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:95, or [0377] e) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:96, or
[0378] f) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:97, or [0379] g) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:98, or [0380] h) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:99, or [0381] i) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:100, or [0382] j) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:101, or
[0383] k) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:102, or [0384] l) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:103, or [0385] m) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:104, or [0386] n) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:105, or [0387] o) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:106, or
[0388] p) a heavy chain variable domain VH of SEQ ID NO:91 and a
light chain variable domain VL of SEQ ID NO:107. [0389] 11. A) An
antibody which binds to human CSF-1R, for use in the treatment of a
patient having a CSF-1R expressing tumor or having a tumor with
CSF-1R expressing macrophage infiltrate, wherein the tumor is
characterized by an increase of CSF-1R ligand and wherein the
anti-CSF-1R antibody is administered in combination with an
antibody which binds to human PD-L1, or [0390] B) use of an
antibody which binds to human CSF-1R, for the manufacture of a
medicament for use in the treatment of a patient having a CSF-1R
expressing tumor or having a tumor with CSF-1R expressing
macrophage infiltrate, wherein the tumor is characterized by an
increase of CSF-1R ligand and wherein the anti-CSF-1R antibody is
administered in combination with an antibody which binds to human
PD-L1, [0391] wherein the antibody which binds to human CSF-1R used
in the combination therapy is characterized in comprising [0392] a)
a heavy chain variable domain VH of SEQ ID NO:23 and a light chain
variable domain VL of SEQ ID NO:24, or [0393] b) a heavy chain
variable domain VH of SEQ ID NO:31 and a light chain variable
domain VL of SEQ ID NO:32, or [0394] c) a heavy chain variable
domain VH of SEQ ID NO:39 and a light chain variable domain VL of
SEQ ID NO:40, or [0395] d) a heavy chain variable domain VH of SEQ
ID NO:47 and a light chain variable domain VL of SEQ ID NO:48, or
[0396] e) a heavy chain variable domain VH of SEQ ID NO:55 and a
light chain variable domain VL of SEQ ID NO:56; [0397] and the
antibody which binds to human PD-L1 used in the combination therapy
is characterized in comprising [0398] a) a heavy chain variable
domain VH of SEQ ID NO:89 and a light chain variable domain VL of
SEQ ID NO:92, or [0399] b) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:93, or
[0400] c) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:94, or [0401] d) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:95, or [0402] e) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:96, or [0403] f) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:97, or [0404] g) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:98, or
[0405] h) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:99, or [0406] i) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:100, or
[0407] j) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:101, or [0408] k) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:102, or [0409] l) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:103, or [0410] m) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:104, or [0411] n) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:105, or
[0412] o) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:106, or [0413] p) a
heavy chain variable domain VH of SEQ ID NO:91 and a light chain
variable domain VL of SEQ ID NO:107. [0414] 12. The antibody or use
according any one of the preceding embodiments, [0415] wherein the
antibody which binds to human CSF-1R used in the combination
therapy is characterized in comprising [0416] c) a heavy chain
variable domain VH of SEQ ID NO:39 and a light chain variable
domain VL of SEQ ID NO:40, or [0417] and wherein the antibody which
binds to human PD-L1 used in the combination therapy is
characterized in comprising a) a heavy chain variable domain VH of
SEQ ID NO:89 and a light chain variable domain VL of SEQ ID NO:92.
[0418] 13. The antibody or use according any one of the preceding
embodiments, characterized in that said antibodies are of human
IgG1 subclass or human IgG4 subclass. [0419] 14. The antibody or
use according to any one of the preceding embodiments,
characterized in that said antibodies have reduced or minimal
effector function. [0420] 15. The antibody or use according to any
one of the preceding embodiments, wherein the minimal effector
function results from an effectorless Fc mutation. [0421] 16. The
antibody or use according to any one of the preceding embodiments,
wherein the effectorless Fc mutation is L234A/L235A or
L234A/L235A/P329G or N297A or D265A/N297A. [0422] 17. A) A method
for [0423] i) the inhibition of cell proliferation in CSF-1R
ligand-dependent and/or CSF-1R ligand-independent CSF-1R expressing
tumor cells; [0424] ii) the inhibition of cell proliferation of
tumors with CSF-1R ligand-dependent and/or CSF-1R
ligand-independent CSF-1R expressing macrophage infiltrate; [0425]
iii) the inhibition of cell survival (in CSF-1R ligand-dependent
and/or CSF-1R ligand-independent) CSF-1R expressing monocytes and
macrophages; and/or [0426] iv) the inhibition of cell
differentiation (in CSF-1R ligand-dependent and/or CSF-1R
ligand-independent) CSF-1R expressing monocytes into macrophages;
[0427] wherein an antibody which binds to human CSF-1R, is
administered in combination with an antibody which binds to human
PD-L1, [0428] or [0429] B) a method of treatment of a patient
having a CSF-1R expressing tumor or having a tumor with CSF-1R
expressing macrophage infiltrate, wherein the tumor is
characterized by an increase of CSF-1R ligand and wherein an
antibody which binds to human CSF-1R is administered in combination
with an antibody which binds to human PD-L1, [0430] wherein the
antibody which binds to human CSF-1R used in the combination
therapy is characterized in comprising [0431] a) a heavy chain
variable domain VH of SEQ ID NO:23 and a light chain variable
domain VL of SEQ ID NO:24, or [0432] b) a heavy chain variable
domain VH of SEQ ID NO:31 and a light chain variable domain VL of
SEQ ID NO:32, or [0433] c) a heavy chain variable domain VH of SEQ
ID NO:39 and a light chain variable domain VL of SEQ ID NO:40, or
[0434] d) a heavy chain variable domain VH of SEQ ID NO:47 and a
light chain variable domain VL of SEQ ID NO:48, or [0435] e) a
heavy chain variable domain VH of SEQ ID NO:55 and a light chain
variable domain VL of SEQ ID NO:56; [0436] and the antibody which
binds to human PD-L1 used in the combination therapy is
characterized in comprising [0437] a) a heavy chain variable domain
VH of SEQ ID NO:89 and a light chain variable domain VL of SEQ ID
NO:92, or [0438] b) a heavy chain variable domain VH of SEQ ID
NO:90 and a light chain variable domain VL of SEQ ID NO:93, or
[0439] c) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:94, or [0440] d) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:95, or [0441] e) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:96, or [0442] f) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:97, or [0443] g) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:98, or
[0444] h) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:99, or [0445] i) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:100, or [0446] j) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:101, or [0447] k) a heavy chain variable
domain VH of SEQ ID NO:90 and a light chain variable domain VL of
SEQ ID NO:102, or [0448] l) a heavy chain variable domain VH of SEQ
ID NO:90 and a light chain variable domain VL of SEQ ID NO:103, or
[0449] m) a heavy chain variable domain VH of SEQ ID NO:90 and a
light chain variable domain VL of SEQ ID NO:104, or [0450] n) a
heavy chain variable domain VH of SEQ ID NO:90 and a light chain
variable domain VL of SEQ ID NO:105, or [0451] o) a heavy chain
variable domain VH of SEQ ID NO:90 and a light chain variable
domain VL of SEQ ID NO:106, or [0452] p) a heavy chain variable
domain VH of SEQ ID NO:91 and a light chain variable domain VL of
SEQ ID NO:107.
EXAMPLES
Example 1
Inhibition of CSF-1-Induced CSF-1R Phosphorylation in NIH3T3-CSF-1R
Recombinant Cells
[0453] 4.5.times.10.sup.3 NIH 3T3 cells, retrovirally infected with
an expression vector for full-length CSF-1R, were cultured in DMEM
(PAA Cat. No.E15-011), 2 mM L-glutamine (Sigma, Cat. No. G7513, 2
mM Sodium pyruvate, 1.times. nonessential amino acids, 10% FKS
(PAA, Cat. No. A15-649) and 100 .mu.g/ml PenStrep (Sigma, Cat. No.
P4333 [10 mg/ml]) until they reached confluency. Thereafter cells
were washed with serum-free DMEM media (PAA Cat. No. E15-011)
supplemented with sodium selenite [5 ng/ml] (Sigma, Cat. No.
S9133), transferrin [10 .mu.g/ml] (Sigma, Cat. No. T8158), BSA [400
.mu.g/ml] (Roche Diagnostics GmbH, Cat. No. 10735078), 4 mM
L-glutamine (Sigma, Cat. No. G7513), 2 mM sodium pyruvate (Gibco,
Cat. No. 11360), 1.times. nonessential amino acids (Gibco, Cat:
11140-035), 2-mercaptoethanol [0.05 mM] (Merck, Cat. No. M7522),
100 .mu.g/ml and PenStrep (Sigma, Cat. No. P4333) and incubated in
30 .mu.l of the same medium for 16 hours to allow for receptor
up-regulation. 10 .mu.l of diluted anti-CSR-1R antibodies were
added to the cells for 1.5 h. Then cells were stimulated with 10
.mu.l of 100 ng/ml hu CSF-1 (active 149 aa fragment of human CSF-1
(aa 33-181 of SEQ ID NO: 86); Biomol, DE, Cat. No. 60530) for 5
min. After the incubation, supernatant was removed, cells were
washed twice with 80 .mu.l of ice-cold PBS and 50 .mu.l of freshly
prepared ice-cold lysis buffer (150 mM NaCl/20 mM Tris pH 7.5/1 mM
EDTA/1 mM EGTA/1% Triton X-100/1 protease inhibitor tablet (Roche
Diagnostics GmbH Cat. No. 1 836 170) per 10 ml buffer/10 .mu.l/ml
phosphatase inhibitor cocktail 1 (Sigma Cat. No. P-2850, 100.times.
Stock), 10 .mu.l/ml protease inhibitor 1 (Sigma Cat. No. P-5726,
100.times. Stock)/10 .mu.l/ml 1 M NaF) was added. After 30 minutes
on ice the plates were shaken vigorously on a plateshaker for 3
minutes and then centrifuged 10 minutes at 2200 rpm (Heraeus
Megafuge 10).
[0454] The presence of phosphorylated and total CSF-1 receptor in
the cell lysate was analyzed with Elisa. For detection of the
phosphorylated receptor the kit from R&D Systems (Cat. No.
DYC3268-2) was used according to the instructions of the supplier.
For detection of total CSF-1R 10 .mu.l of the lysate was
immobilized on plate by use of the capture antibody contained in
the kit. Thereafter 1:750 diluted biotinylated anti CSF-1R antibody
BAF329 (R&D Systems) and 1:1000 diluted streptavidin-HRP
conjugate was added. After 60 minutes plates were developed with
freshly prepared ABTS.RTM. solution and the absorbance was
detected. Data were calculated as % of positive control without
antibody and the ratio value phospho/total receptor expressed. The
negative control was defined without addition of M-CSF-1. Anti
CSF-1R SC 2-4A5 (Santa Cruz Biotechnology, US, see also Sherr, C.
J. et al., Blood 73 (1989) 1786-1793), which inhibits the
ligand-receptor interaction, was used as reference control.
TABLE-US-00004 TABLE 3 Calculated IC50 values for the inhibition of
CSF-1 receptor phosphorylation. IC50 CSF-1R Phosphorylation CSF-1R
Mab [ng/ml] Mab 2F11 219.4 Mab 2E10 752.0 Mab 2H7 703.4 Mab 1G10
56.6 SC-2-4A5 1006.6
Example 2
Growth Inhibition of NIH3T3-CSF-1R Recombinant Cells in 3D Culture
Under Treatment with Anti-CSF-1R Monoclonal Antibodies
(CellTiterGlo-Assay)
[0455] NIH 3T3 cells, retrovirally infected with either an
expression vector for full-length wildtype CSF-1R (SEQ ID NO: 62)
or mutant CSF-1R L301S Y969F (SEQ ID NO: 63), were cultured in DMEM
high glucose media (PAA, Pasching, Austria) supplemented with 2 mM
L-glutamine, 2 mM sodium pyruvate and non-essential amino acids and
10% fetal bovine serum (Sigma, Taufkirchen, Germany) on poly-HEMA
(poly(2-hydroxyethylmethacrylate)) (Polysciences, Warrington, Pa.,
USA)) coated dishes to prevent adherence to the plastic surface.
Cells are seeded in medium replacing serum with 5 ng/ml sodium
selenite, 10 mg/ml transferrin, 400 .mu.g/ml BSA and 0.05 mM
2-mercaptoethanol. When treated with 100 ng/ml hu CSF-1 (active 149
aa fragment of human CSF-1 (aa 33-181 of SEQ ID NO: 86); Biomol,
DE, Cat. No. 60530) wtCSF-1R (expressing cells form dense spheroids
that grow three dimensionally, a property that is called anchorage
independence. These spheroids resemble closely the three
dimensional architecture and organization of solid tumors in situ.
Mutant CSF-1R recombinant cells are able to form spheroids
independent of the CSF-1 ligand. The anti-CSF-1R antibody according
to the invention hMab 2F11-e7 and the anti-CSF-1R antibodies 1.2.SM
(ligand displacing CSF-1R antibody described in WO 2009/026303),
CXIIG6 (ligand displacing CSF-1R antibody described in WO
2009/112245), the goat polyclonal anti-CSF-1R antibody ab10676
(abcam), and SC 2-4A5 (Santa Cruz Biotechnology, US-- see also
Sherr, C. J. et al., Blood 73 (1989) 1786-1793) and Mab
R&D-Systems 3291 were investigated. Reference control Mab
R&D-Systems 3291 did not show inhibition of mutant CSF-1R
recombinant cell proliferation.
[0456] Spheroid cultures were incubated for 3 days in the presence
of different concentrations of antibody in order to determine an
IC30 (concentration with 30 percent inhibition of cell viability).
Maximum concentration was 20 .mu.g/ml The CellTiterGlo assay was
used to detect cell viability by measuring the ATP-content of the
cells.
TABLE-US-00005 TABLE 4 wtCSF-1R Mutant CSF-1R CSF-1R Mab IC.sub.30
[.mu.g/ml] IC.sub.30 [.mu.g/ml] hMab 2F11-e7 4.91 0.54 1.2.SM 1.19
>20 .mu.g/ml (-19% inhibition at 20 .mu.g/ml = 19% stimulation)
CXIIG6 >20 .mu.g/ml (21% >20 .mu.g/ml (-36% inhibition at 20
inhibition at 20 .mu.g/ml = .mu.g/ml) 36% stimulation) ab10676
14.15 >20 .mu.g/ml (0% inhibition at 20 .mu.g/ml) SC 2-4A5 16.62
2.56
Example 3
Inhibition of Human Macrophage Differentiation Under Treatment with
Anti-CSF-1R Monoclonal Antibodies (CellTiterGlo-Assay)
[0457] Human monocytes were isolated from peripheral blood using
the RosetteSep.TM. Human Monocyte Enrichment Cocktail (StemCell
Tech.--Cat. No. 15028). Enriched monocyte populations were seeded
into 96 well microtiterplates (2.5.times.10.sup.4 cells/well) in
100 .mu.l RPMI 1640 (Gibco--Cat. No. 31870) supplemented with 10%
FCS (GIBCO--Cat. No. 011-090014M), 4 mM L-glutamine (GIBCO--Cat.
No. 25030) and 1.times. PenStrep (Roche Cat. No. 1 074 440) at
37.degree. C. and 5% CO.sub.2 in a humidified atmosphere. When 150
ng/ml huCSF-1 was added to the medium, a clear differentiation into
adherent macrophages could be observed. This differentiation could
be inhibited by addition of anti-CSF-1R antibodies. Furthermore,
the monocyte survival is affected and could be analyzed by
CellTiterGlo (CTG) analysis. From the concentration dependent
inhibition of the survival of monocytes by antibody treatment, an
IC.sub.50 was calculated (see Table below).
TABLE-US-00006 TABLE 5 CSF-1R Mab IC.sub.50 [.mu.g/ml] Mab 2F11
0.08 Mab 2E10 0.06 Mab 2H7 0.03 Mab 1G10 0.06 SC 2-4A5 0.36
[0458] In a separate test series humanized versions of Mab 2 F11,
e.g. hMab 2F11-c11, hMab 2F11-d8, hMab 2F11-e7, hMab 2F11412,
showed IC50 values of 0.07 .mu.g/ml (hMab 2F11-c11), 0.07 .mu.g/ml
(hMab 2F11-d8), 0.04 .mu.g/ml (hMab 2F11-e7) and 0.09 .mu.g/ml
(hMab 2F11-f12).
Example 4
Inhibition of Human Macrophage Differentiation Under Treatment with
Anti-CSF-1R Monoclonal Antibodies (CellTiterGlo-Assay)
[0459] Human monocytes were isolated from peripheral blood using
the RosetteSep.TM. Human Monocyte Enrichment Cocktail (StemCell
Tech.--Cat. No. 15028). Enriched monocyte populations were seeded
into 96 well microtiterplates (2.5.times.10.sup.4 cells/well) in
100 .mu.l RPMI 1640 (Gibco--Cat. No. 31870) supplemented with 10%
FCS (GIBCO--Cat. No. 011-090014M), 4 mM L-glutamine (GIBCO--Cat.
No. 25030) and 1.times. PenStrep (Roche Cat. No. 1 074 440) at
37.degree. C. and 5% CO.sub.2 in a humidified atmosphere. When 150
ng/ml huCSF-1 was added to the medium, a clear differentiation into
adherent macrophages could be observed. This differentiation could
be inhibited by addition of anti-CSF-1R antibodies. Furthermore,
the monocyte survival is affected and could be analyzed by
CellTiterGlo (CTG) analysis. From the concentration dependent
inhibition of the survival of monocytes by antibody treatment, an
IC.sub.50 was calculated. Humanized versions of Mab 2 F11, e.g.
hMab 2F11-c11, hMab 2F11-d8, hMab 2F11-e7, hMab 2F11412, showed
IC50 values of 0.07 .mu.g/ml (hMab 2F11-c11), 0.07 .mu.g/ml (hMab
2F11-d8), 0.04 .mu.g/ml (hMab 2F11-e7) and 0.09 .mu.g/ml (hMab
2F11-f12).
Example 5
Inhibition of Human M1 and M2 Macrophage Differentiation Under
Treatment with Anti-CSF-1R Monoclonal Antibodies
(CellTiterGlo-Assay)
[0460] Human monocytes were isolated from peripheral blood using
the RosetteSep.TM. Human Monocyte Enrichment Cocktail (StemCell
Tech.--Cat. No. 15028). Enriched monocyte populations were seeded
into 96 well microtiterplates (2.5.times.10.sup.4 cells/well) in
100 .mu.l RPMI 1640 (Gibco--Cat. No. 31870) supplemented with 10%
FCS (GIBCO--Cat. No. 011-090014M), 4 mM L-glutamine (GIBCO--Cat.
No. 25030) and 1.times. PenStrep (Roche Cat. No. 1 074 440) at
37.degree. C. and 5% CO.sub.2 in a humidified atmosphere. When 100
ng/ml huCSF-1 was added for 6 days to the medium, a clear
differentiation into adherent, M2 macrophages with elongated
morphology could be observed. When 100 ng/ml huGM-CSF was added to
the medium for 6 days, a clear differentiation into adherent, M1
macrophages with round morphology could be observed. This
differentiation was associated with the expression of certain
markers such as CD163 for M2 macrophages and CD80 or high MHC class
II for M1 macrophages as assessed by flow cytometry. Cells were
washed with PBS and, if adherent, detached using a 5 mM EDTA
solution in PBS (20 min at 37.degree. C.). Cells were then well
resuspended, washed with staining buffer (5% FCS in PBS) and
centrifuged at 300.times.g for 5 min. Pellets were resuspended in 1
ml staining buffer and cells counted in a Neubauer chamber.
Approximately 1.times.10e5 cells were transferred in each FACS
tube, centrifuged at 300.times.g for 5 min and resuspended in
staining buffer. Fc.gamma. receptors were blocked by incubation
with 1 .mu.g human IgG/2.5.times.10e4 cells (JIR Cat. No.
009-000-003) in staining buffer for 20 min on ice. Cells were then
mixed with 1.5 .mu.l antibody/2.5.times.10e4 cells for CD80 and
CD163 detection whereas 5 .mu.l antibody/2.5.times.10e4 cells for
MHC class II detection was used: PE labeled mouse anti human CD163
(BD Bioscience Cat. No. 556018), PE labeled mouse anti human CD80
(BD Bioscience Cat. No. 557227) and Alexa 647 labeled mouse anti
human MHC class II (Dako-Cat. No. M0775). The Alexa 647 label was
conjugated to the antibody by using the Zenon Alexa 647 mouse IgG
labeling kit (Invitrogen Cat. No. Z25008) After a 1-hour incubation
on ice cells were washed twice with staining buffer, resuspended
and measured at a FACS Canto II.
[0461] Exclusively M2 macrophage differentiation which is
characterized by the expression of CD163, absence of CD80 and low
MHC class II expression could be inhibited by addition of humanized
anti-CSF-1R antibody hMab 2F11-e7. Furthermore, the M2 but not M1
macrophage survival is affected and could be analyzed by
CellTiterGlo (CTG) analysis. Concentration dependent inhibition of
the survival of macrophages by antibody treatment for 7 days is
depicted in FIG. 1a. Expression of M1 and M2 macrophage markers
assessed by flow cytometry is shown in FIG. 1b.
Example 6
CSF-1 Level Increase During CSF-1R Inhibition in Cynomolgus
Monkey
[0462] Serum CSF-1 levels provide a pharmacodynamic marker of
CSF-1R neutralizing activity of anti-human CSF-1R dimerization
inhibitor hMab 2F11-e7. One male and one female cynomolgus monkey
per dosage group (1 and 10 mg/kg) were intravenously administered
anti-CSF1R antibody hMab 2F11-e7. Blood samples for analysis of
CSF-1 levels were collected 1 week before treatment (pre-dose), 2,
24, 48, 72, 96, 168 hours post-dose and weekly for two additional
weeks. CSF-1 levels were determined using a commercially available
ELISA kit (Quantikine.RTM. human M-CSF) according to the
manufacturer's instructions (R&D Systems, UK). Monkey CSF-1
level were determined by comparison with CSF-1 standard curve
samples provided in the kit.
[0463] Administration of hMab 2F11-e7 induced a dramatic increase
in CSF-1 by.about.1000-fold, which depending on the dose
administered lasted for 48 hr (1 mg/kg) or 15 days (10 mg/kg).
Hence, a dimerization inhibitor for CSF-1R offers the advantage to
not directly compete with the dramatically upregulated ligand for
binding to the receptor in contrast to a ligand displacing
antibody. (Results are shown in FIG. 2)
Example 7
Relationship Between M2 Subtype Tumor Associated Macrophages (TAMs)
and T Cells--Rationale for Combining Anti-CSF-R1 Antibody and a T
Cell Engaging Agents
[0464] To investigate the functional relationship between TAMs and
T cells we isolated TAMs from the MC38 tumor and cocultured them
with CD8+ T cells.
TAM Suppression Assay
[0465] TAMs were enriched from single cell suspensions of MC38
tumors after enzymatic digest using a two-step protocol: Single
cells were stained with CD11b-FITC (clone M1/70) and positively
enriched over MACS columns by anti-FITC beads (Miltenyi). Upon
removal from the column, anti-FITC beads were detached using
release buffer protocol as provided the manufacturer. Finally, TAM
were isolated by adding anti-Ly6G and anti-Ly6C positive selection
beads in order to remove granulocytic and monocytic cells from TAM
preparations. Final cell purity was analyzed and was usually
>90%. Subsequently, TAM were titrated in the indicated ratios to
total CD3+ T cells labeled with CFSE in U-bottom plates coated with
anti-CD3 and soluble anti-CD28 was added. Cell proliferation was
determined from CFSElow cells using blank Sphero beads as
previously described after 3 days of incubation (Hoves, S. et al.
Monocyte-derived human macrophages mediate anergy in allogeneic T
cells and induce regulatory T cells. J. Immunol. 177, 2691-2698
(2006)). In the presence of TAMs, T cell expansion induced by
activation of CD3 and CD28 was suppressed. (see FIG. 3).
Example 8
Inhibition of Tumor Growth Under Treatment with Anti-CSF-1R
Monoclonal Antibody in Combination with PD-L1 Antibody in
Subcutaneous Syngeneic MC38 Colon Carcinoma Model
[0466] Cells of the murine colorectal adenocarcinoma cell line MC38
(obtained from Beckman Research Institute of the City of Hope,
Calif., USA) were cultured in Dulbecco's Modified Eagle Medium
(DMEM, PAN Biotech) supplemented with 10% FCS and 2 mM L-glutamine
at 37.degree. C. in a water saturated atmosphere at 5% CO2. At the
day of inoculation, MC38 tumor cells were harvested with PBS from
culture flasks and transferred into culture medium, centrifuged,
washed once and re-suspended in PBS. For injection of cells, the
final titer was adjusted to l.times.10.sup.7 cells/ml. Subsequently
100 .mu.l of this suspension (1.times.10.sup.6 cells) were
inoculated subcutaneously into 7-9 weeks old female C57BL/6N mice
(obtained from Charles River, Sulzfeld, Germany). Treatment with
control antibody (MOPC-21; Bio X Cell, West Lebanon), anti-murine
CSF-1R mAb<mouse CSF1R> antibody at a weekly dose of 30 mg/kg
i.p. alone or in combination with a mouse crossreactive anti PD-L1
antibody (10 mg/kg i.p., 6.times. q3d) started after tumors were
established and had reached an average size of 100 mm.sup.3. Tumor
volume was measured twice a week and animal weights were monitored
in parallel.
[0467] In first experiment monotherapy with <mouse CSF1R>
antibody did not inhibit primary tumor growth when compared to
control antibody treatment (TGI: 0%, TCR: 1.07 CI: 0.80-1.43,
median time to progression>700 mm.sup.3: 21 days). Anti-PD-L1
monotherapy had an effect on MC38 primary tumor growth (TGI: 83%,
TCR: 0.27 CI: 0.09-0.49, median time to progression>700
mm.sup.3: 32 days). Addition of <mouse CSF1R> antibody to
anti-PD-L1 therapy led to a slightly improved anti-tumor efficacy
compared to anti-PD-L1 treatment alone (TGI: 83%, TCR: 0.28 CI:
0.09-0.51 median time to progression>700 mm.sup.3: 37 days) (see
table below).
TABLE-US-00007 TABLE 6 Anti tumor Efficacy of <mouse
anti-CSF1R> antibody/<anti-PD-L1> antibody combination in
the MC38 mouse CRC in vivo model 95% CI Median time TGI TCR vs.
group to progression Group (day 21) (day 12) 1 TV > 700 mm.sup.3
Control (Mouse IgG1) -- -- -- 21 <mouse CSF1R> 0% 1.07 (1.43-
21 0.80) <anti-PD-L1> 83% 0.27 (0.49- 32 0.09) <mouse
CSF1R>/<anti- 83% 0.28 (0.51- 37 PD-L1> 0.09)
[0468] Median time of progression>700 mm.sup.3 was 21 days for
control (mouse IgG1) treated animals. Monotherapy with <mouse
CSF1R> antibody did not inhibit primary tumor growth when
compared to control antibody treatment (median time to
progression>700 mm.sup.3: 21 days). Anti-PD-L1 monotherapy had
an effect on MC38 primary tumor growth (median time to
progression>700 mm.sup.3: 32 days). Addition of <mouse
CSF1R> antibody to anti-PD-L1 therapy led to a slightly improved
anti-tumor efficacy compared to PD-L1 treatment alone (median time
to progression>700 mm.sup.3: 37 days) (see table below and FIG.
4).
TABLE-US-00008 TABLE 7 Anti tumor Efficacy of <mouse
anti-CSF1R> antibody/<anti- PD-L1> antibody combination in
the MC38 mouse CRC in vivo model (Median time to progression >
700 mm.sup.3) Median time to progression Group TV > 700 mm.sup.3
Control (Mouse IgG1) 21 <mouse CSF1R> 21 <anti-PD-L1>
32 <mouse CSF1R>/<anti- 37 PD-L1>
[0469] In analogous experiments, but starting treatment at
different tumor sizes (e.g. starting treatment when the tumor has
reached a volume above and below 100 mm.sup.3 (different groups are
evaluated) and in a further experiment also using different anti
PD-L1 antibodies described in table 2, the inhibition of tumor
growth under treatment with anti-CSF-1R monoclonal antibody in
combination with anti-PD-L1 antibody in subcutaneous syngeneic MC38
colon carcinoma model is evaluated.
Example 9
Inhibition of Tumor Growth Under Treatment with Anti-CSF-1R
Monoclonal Antibody in Combination with PD-L1 Antibody in
Subcutaneous Syngeneic CT26.WT Colon Carcinoma Model
[0470] Cells of the murine colorectal adenocarcinoma cell line
CT26.WT tumor cells (obtained from ATCC) were cultured in
Dulbecco's Modified Eagle Medium (DMEM, PAN Biotech) supplemented
with 10% FCS and 2 mM L-glutamine at 37.degree. C. in a water
saturated atmosphere at 5% CO2. At the day of inoculation, CT26.WT
tumor cells were harvested with PBS from culture flasks and
transferred into culture medium, centrifuged, washed once and
re-suspended in PBS. For injection of cells, the final titer was
adjusted to 1.times.10.sup.7 cells/ml. Subsequently 100 IA of this
suspension (1.times.10.sup.6 cells) were inoculated subcutaneously
into 11-13 weeks old female Balb/c mice (obtained from Charles
River, Sulzfeld, Germany). Treatment with control antibody
(MOPC-21; Bio X Cell, West Lebanon), anti-murine CSF-1R mAb
<mouse CSF1R> antibody at a weekly dose of 30 mg/kg i.p.
alone or in combination with a mouse crossreactive anti PD-L1
antibody (10 mg/kg i.p., 6.times. q3d) started after tumors were
established and had reached an average size of 150 mm.sup.3. While
treatment in monotherapy groups started on day 9 after tumor cell
inoculation, treatment in combination group was sequential (day 9:
start of treatment with anti-murine CSF-1R mAb; day 11: start of
treatment with anti PD-L1 antibody). Tumor volume was measured
twice a week and animal weights were monitored in parallel. Results
are shown in Figure
[0471] Median time to progression.gtoreq.700 mm3 was 17 days for
IgG control treatment group, 16 days for <mouse anti-CSF1R>
antibody monotherapy group, 18 days for <anti-PD-L1> antibody
monotherapy group and 18 days for <mouse
anti-CSF1R>/<anti-PD-L1> antibody combination group.
[0472] While all animals in control or monotherapy groups needed to
be terminated due to progressive tumor burden one animal of the
<mouse anti-CSF1R>/<anti-PD-L1> antibody combination
group experienced tumor shrinkage and remained tumor-free until
study termination on day 79 after tumor inoculation.
Sequence CWU 1
1
10718PRTMus musculus 1Asp Gln Arg Leu Tyr Phe Asp Val 1 5 216PRTMus
musculus 2Val Ile Trp Thr Asp Gly Gly Thr Asn Tyr Asn Ser Pro Phe
Met Ser 1 5 10 15 35PRTMus musculus 3Thr Tyr Asp Ile Ser 1 5
48PRTMus musculus 4Gly Gln Ser Phe Ser Tyr Pro Thr 1 5 57PRTMus
musculus 5Gly Ala Ser Asn Arg Tyr Thr 1 5 611PRTMus musculus 6Lys
Ala Ser Glu Asp Val Asn Thr Tyr Val Ser 1 5 10 7116PRTMus musculus
7Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1
5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Thr
Tyr 20 25 30 Asp Ile Ser Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu
Glu Trp Leu 35 40 45 Gly Val Ile Trp Thr Asp Gly Gly Thr Asn Tyr
Asn Ser Pro Phe Met 50 55 60 Ser Arg Leu Ser Ile Arg Lys Asp Asn
Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Arg Leu Gln Thr
Asp Asp Thr Ala Ile Tyr Tyr Cys Val 85 90 95 Arg Asp Gln Arg Leu
Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val 100 105 110 Thr Val Ser
Ser 115 8106PRTMus musculus 8Asn Ile Val Met Thr Gln Ser Pro Lys
Ser Met Ser Met Ser Val Gly 1 5 10 15 Glu Arg Val Thr Leu Asn Cys
Lys Ala Ser Glu Asp Val Asn Thr Tyr 20 25 30 Val Ser Trp Tyr Gln
Gln Gln Pro Glu Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala
Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Gly
Gly Ser Thr Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70
75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Gly Gln Ser Phe Ser Tyr Pro
Thr 85 90 95 Phe Gly Thr Gly Thr Lys Leu Glu Ile Lys 100 105
97PRTMus musculus 9Asp Pro Arg Leu Tyr Phe Asp 1 5 1016PRTMus
musculus 10Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Gly Phe
Met Ser 1 5 10 15 115PRTMus musculus 11Ser Phe Asp Ile Ser 1 5
128PRTMus musculus 12Gly Gln Thr Phe Ser Tyr Pro Thr 1 5 137PRTMus
musculus 13Gly Ala Ser Asn Arg Tyr Thr 1 5 1411PRTMus musculus
14Lys Ala Ser Glu Asp Val Val Thr Tyr Val Ser 1 5 10 15116PRTMus
musculus 15Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro
Ser Lys 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Ser Ser
Leu Asp Ser Phe 20 25 30 Asp Ile Ser Trp Ile Arg Gln Ser Pro Gly
Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Thr Gly Gly Gly
Thr Asn Tyr Asn Ser Gly Phe Met 50 55 60 Ser Arg Leu Arg Ile Thr
Lys Asp Asn Ser Lys Ser Gln Val Leu Leu 65 70 75 80 Lys Met Asn Ser
Leu Gln Ser Asp Asp Thr Ala Ile Tyr Tyr Cys Val 85 90 95 Arg Asp
Pro Arg Leu Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val 100 105 110
Thr Val Ser Ser 115 16106PRTMus musculus 16Asn Ile Val Met Thr Gln
Ser Pro Lys Ser Met Ser Met Ser Val Gly 1 5 10 15 Glu Arg Val Thr
Leu Ser Cys Lys Ala Ser Glu Asp Val Val Thr Tyr 20 25 30 Val Ser
Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45
Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50
55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln
Ala 65 70 75 80 Glu Asp Leu Ala Asp Tyr Tyr Cys Gly Gln Thr Phe Ser
Tyr Pro Thr 85 90 95 Phe Gly Thr Gly Thr Lys Leu Glu Ile Lys 100
105 178PRTArtificialheavy chain CDR3, hMab 2F11-c11 17Asp Gln Arg
Leu Tyr Phe Asp Val 1 5 1816PRTArtificialheavy chain CDR2, hMab
2F11-c11 18Val Ile Trp Thr Asp Gly Gly Thr Asn Tyr Asn Ser Pro Phe
Met Ser 1 5 10 15 195PRTArtificialheavy chain CDR1, hMab 2F11-c11
19Thr Tyr Asp Ile Ser 1 5 208PRTArtificiallight chain CDR3, hMab
2F11-c11 20Gly Gln Ser Phe Ser Tyr Pro Thr 1 5
217PRTArtificiallight chain CDR2, hMab 2F11-c11 21Gly Ala Ser Asn
Arg Tyr Thr 1 5 2211PRTArtificiallight chain CDR1, hMab 2F11-c11
22Arg Ala Ser Glu Asp Val Asn Thr Tyr Val Ser 1 5 10
23116PRTArtificialheavy chain variable domain, hMab 2F11-c11 23Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Leu Thr Thr Tyr
20 25 30 Asp Ile Ser Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45 Gly Val Ile Trp Thr Asp Gly Gly Thr Asn Tyr Asn
Ser Pro Phe Met 50 55 60 Ser Arg Val Thr Ile Thr Lys Asp Glu Ser
Thr Ser Thr Ala Tyr Met 65 70 75 80 Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Arg Asp Gln Arg Leu Tyr
Phe Asp Val Trp Gly Gln Gly Thr Thr Val 100 105 110 Thr Val Ser Ser
115 24106PRTArtificiallight chain variable domain, hMab 2F11-c11
24Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asp Val Asn Thr
Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gly Gln Ser Phe Ser Tyr Pro Thr 85 90 95 Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys 100 105 258PRTArtificialheavy chain CDR3, hMab
2F11-d8 25Asp Gln Arg Leu Tyr Phe Asp Val 1 5
2616PRTArtificialheavy chain CDR2, hMab 2F11-d8 26Val Ile Trp Thr
Asp Gly Gly Ala Asn Tyr Ala Gln Lys Phe Gln Gly 1 5 10 15
275PRTArtificialheavy chain CDR1, hMab 2F11-d8 27Thr Tyr Asp Ile
Ser 1 5 288PRTArtificiallight chain CDR3, hMab 2F11-d8 28Gly Gln
Ser Phe Ser Tyr Pro Thr 1 5 297PRTArtificiallight chain CDR2, hMab
2F11-d8 29Gly Ala Ser Asn Arg Tyr Thr 1 5 3011PRTArtificiallight
chain CDR1, hMab 2F11-d8 30Lys Ala Ser Glu Asp Val Asn Thr Tyr Val
Ser 1 5 10 31116PRTArtificialheavy chain variable domain, hMab
2F11-d8 31Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser
Leu Thr Thr Tyr 20 25 30 Asp Ile Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Val Ile Trp Thr Asp Gly Gly
Ala Asn Tyr Ala Gln Lys Phe Gln 50 55 60 Gly Arg Val Thr Ile Thr
Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met 65 70 75 80 Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp
Gln Arg Leu Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val 100 105 110
Thr Val Ser Ser 115 32106PRTArtificiallight chain variable domain,
hMab 2F11-d8 32Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu
Asp Val Asn Thr Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr
Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gly Gln Ser Phe Ser Tyr Pro Thr 85 90 95 Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 338PRTArtificialheavy
chain CDR3, hMab 2F11-e7 33Asp Gln Arg Leu Tyr Phe Asp Val 1 5
3416PRTArtificialheavy chain CDR2, hMab 2F11-e7 34Val Ile Trp Thr
Asp Gly Gly Thr Asn Tyr Ala Gln Lys Leu Gln Gly 1 5 10 15
355PRTArtificialheavy chain CDR1, hMab 2F11-e7 35Ser Tyr Asp Ile
Ser 1 5 368PRTArtificiallight chain CDR3, hMab 2F11-e7 36Gln Gln
Ser Phe Ser Tyr Pro Thr 1 5 377PRTArtificiallight chain CDR2, hMab
2F11-e7 37Ala Ala Ser Asn Arg Tyr Thr 1 5 3811PRTArtificiallight
chain CDR1, hMab 2F11-e7 38Arg Ala Ser Glu Asp Val Asn Thr Tyr Val
Ser 1 5 10 39116PRTArtificialheavy chain variable domain, hMab
2F11-e7 39Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Ser Tyr 20 25 30 Asp Ile Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Val Ile Trp Thr Asp Gly Gly
Thr Asn Tyr Ala Gln Lys Leu Gln 50 55 60 Gly Arg Val Thr Met Thr
Thr Asp Thr Ser Thr Ser Thr Ala Tyr Met 65 70 75 80 Glu Leu Arg Ser
Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp
Gln Arg Leu Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val 100 105 110
Thr Val Ser Ser 115 40106PRTArtificiallight chain variable domain,
hMab 2F11-e7 40Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu
Asp Val Asn Thr Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Asn Arg Tyr
Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Ser Phe Ser Tyr Pro Thr 85 90 95 Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 418PRTArtificialheavy
chain CDR3, hMab 2F11-f12 41Asp Gln Arg Leu Tyr Phe Asp Val 1 5
4216PRTArtificialheavy chain CDR2, hMab 2F11-f12 42Val Ile Trp Thr
Asp Gly Gly Thr Asn Tyr Asn Ser Pro Phe Met Ser 1 5 10 15
435PRTArtificialheavy chain CDR1, hMab 2F11-f12 43Thr Tyr Asp Ile
Ser 1 5 448PRTArtificiallight chain CDR3, hMab 2F11-f12 44Gly Gln
Ser Phe Ser Tyr Pro Thr 1 5 457PRTArtificiallight chain CDR2, hMab
2F11-f12 45Gly Ala Ser Ser Leu Gln Ser 1 5 4611PRTArtificiallight
chain CDR1, hMab 2F11-f12 46Arg Ala Ser Glu Asp Val Asn Thr Tyr Val
Ser 1 5 10 47116PRTArtificialheavy chain variable domain, hMab
2F11-f12 47Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser
Leu Thr Thr Tyr 20 25 30 Asp Ile Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Val Ile Trp Thr Asp Gly Gly
Thr Asn Tyr Asn Ser Pro Phe Met 50 55 60 Ser Arg Val Thr Ile Thr
Lys Asp Glu Ser Thr Ser Thr Ala Tyr Met 65 70 75 80 Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Arg Asp
Gln Arg Leu Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val 100 105 110
Thr Val Ser Ser 115 48106PRTArtificiallight chain variable domain,
hMab 2F11-f12 48Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu
Asp Val Asn Thr Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gly Gln Ser Phe Ser Tyr Pro Thr 85 90 95 Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 498PRTArtificialheavy
chain CDR3, hMab 2F11-g1 49Asp Gln Arg Leu Tyr Phe Asp Val 1 5
5016PRTArtificialheavy chain CDR2, hMab 2F11-g1 50Val Ile Trp Thr
Asp Gly Gly Thr Asn Tyr Asn Ser Pro Leu Lys Ser 1 5 10 15
515PRTArtificialheavy chain CDR1, hMab 2F11-g1 51Thr Tyr Asp Ile
Ser 1 5 528PRTArtificiallight chain CDR3, hMab 2F11-g1 52Gly Gln
Ser Phe Ser Tyr Pro Thr 1 5 537PRTArtificiallight chain CDR2, hMab
2F11-g1 53Gly Ala Ser Ser Arg Ala Thr 1 5 5411PRTArtificiallight
chain CDR1, hMab 2F11-g1 54Arg Ala Ser Glu Asp Val Asn Thr Tyr Leu
Ala 1 5 10 55116PRTArtificialheavy chain variable domain, hMab
2F11-g1 55Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser
Leu Thr Thr Tyr 20 25 30 Asp Ile Ser Trp Ile Arg Gln Pro Pro Gly
Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Trp Thr Asp Gly Gly
Thr Asn Tyr Asn Ser Pro Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp
Gln Arg Leu Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val 100 105 110
Thr Val Ser Ser 115 56106PRTArtificiallight chain variable domain,
hMab 2F11-g1 56Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu
Asp Val Asn Thr Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ser Arg Ala
Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe
Ala Val Tyr Tyr Cys Gly Gln Ser Phe Ser Tyr Pro Thr 85 90 95 Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 57107PRTHomo sapiens
57Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1
5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu
Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys 100 105 58330PRTHomo sapiens 58Ala 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 Asp Glu 225 230 235 240 Leu 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
59330PRTArtificialhuman heavy chain constant region derived from
IgG1 mutated on L234A and L235A 59Ala 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 Ala Ala 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 Asp Glu 225 230 235 240 Leu 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
60327PRTHomo sapiens 60 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser 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 Lys Thr 65 70 75 80 Tyr
Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro
100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215
220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser
Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser
Leu Ser Leu Gly Lys 325 61327PRTArtificialhuman heavy chain
constant region derived from IgG4 mutated onS228P 61Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr
Ser Glu Ser 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 Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro
Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165
170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290
295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 62972PRTHomo
sapiens 62Met Gly Pro Gly Val Leu Leu Leu Leu Leu Val Ala Thr Ala
Trp His 1 5 10 15 Gly Gln Gly Ile Pro Val Ile Glu Pro Ser Val Pro
Glu Leu Val Val 20 25 30 Lys Pro Gly Ala Thr Val Thr Leu Arg Cys
Val Gly Asn Gly Ser Val 35 40 45 Glu Trp Asp Gly Pro Pro Ser Pro
His Trp Thr Leu Tyr Ser Asp Gly 50 55 60 Ser Ser Ser Ile Leu Ser
Thr Asn Asn Ala Thr Phe Gln Asn Thr Gly 65 70 75 80 Thr Tyr Arg Cys
Thr Glu Pro Gly Asp Pro Leu Gly Gly Ser Ala Ala 85 90 95 Ile His
Leu Tyr Val Lys Asp Pro Ala Arg Pro Trp Asn Val Leu Ala 100 105 110
Gln Glu Val Val Val Phe Glu Asp Gln Asp Ala Leu Leu Pro Cys Leu 115
120 125 Leu Thr Asp Pro Val Leu Glu Ala Gly Val Ser Leu Val Arg Val
Arg 130 135 140 Gly Arg Pro Leu Met Arg His Thr Asn Tyr Ser Phe Ser
Pro Trp His 145 150 155 160 Gly Phe Thr Ile His Arg Ala Lys Phe Ile
Gln Ser Gln Asp Tyr Gln 165 170 175 Cys Ser Ala Leu Met Gly Gly Arg
Lys Val Met Ser Ile Ser Ile Arg 180 185 190 Leu Lys Val Gln Lys Val
Ile Pro Gly Pro Pro Ala Leu Thr Leu Val 195 200 205 Pro Ala Glu Leu
Val Arg Ile Arg Gly Glu Ala Ala Gln Ile Val Cys 210 215 220 Ser Ala
Ser Ser Val Asp Val Asn Phe Asp Val Phe Leu Gln His Asn 225 230 235
240 Asn Thr Lys Leu Ala Ile Pro Gln Gln Ser Asp Phe His Asn Asn Arg
245 250 255 Tyr Gln Lys Val Leu Thr Leu Asn Leu Asp Gln Val Asp Phe
Gln His 260 265 270 Ala Gly Asn Tyr Ser Cys Val Ala Ser Asn Val Gln
Gly Lys His Ser 275 280 285 Thr Ser Met Phe Phe Arg Val Val Glu Ser
Ala Tyr Leu Asn Leu Ser 290 295 300 Ser Glu Gln Asn Leu Ile Gln Glu
Val Thr Val Gly Glu Gly Leu Asn 305 310 315 320 Leu Lys Val Met Val
Glu Ala Tyr Pro Gly Leu Gln Gly Phe Asn Trp 325 330 335 Thr Tyr Leu
Gly Pro Phe Ser Asp His Gln Pro Glu Pro Lys Leu Ala 340 345 350 Asn
Ala Thr Thr Lys Asp Thr Tyr Arg His Thr Phe Thr Leu Ser Leu 355 360
365 Pro Arg Leu Lys Pro Ser Glu Ala Gly Arg Tyr Ser Phe Leu Ala Arg
370 375 380 Asn Pro Gly Gly Trp Arg Ala Leu Thr Phe Glu Leu Thr Leu
Arg Tyr 385 390 395 400 Pro Pro Glu Val Ser Val Ile Trp Thr Phe Ile
Asn Gly Ser Gly Thr 405 410 415 Leu Leu Cys Ala Ala Ser Gly Tyr Pro
Gln Pro Asn Val Thr Trp Leu 420 425 430 Gln Cys Ser Gly His Thr Asp
Arg Cys Asp Glu Ala Gln Val Leu Gln 435 440 445 Val Trp Asp Asp Pro
Tyr Pro Glu Val Leu Ser Gln Glu Pro Phe His 450 455 460 Lys Val Thr
Val Gln Ser Leu Leu Thr Val Glu Thr Leu Glu His Asn 465 470 475 480
Gln Thr Tyr Glu Cys Arg Ala His Asn Ser Val Gly Ser Gly Ser Trp 485
490 495 Ala Phe Ile Pro Ile Ser Ala Gly Ala His Thr His Pro Pro Asp
Glu 500 505 510 Phe Leu Phe Thr Pro Val Val Val Ala Cys Met Ser Ile
Met Ala Leu 515 520 525 Leu Leu Leu Leu Leu Leu Leu Leu Leu Tyr Lys
Tyr Lys Gln Lys Pro 530 535 540 Lys Tyr Gln Val Arg Trp Lys Ile Ile
Glu Ser Tyr Glu Gly Asn Ser 545 550 555 560 Tyr Thr Phe Ile Asp Pro
Thr Gln Leu Pro Tyr Asn Glu Lys Trp Glu 565 570 575 Phe Pro Arg Asn
Asn Leu Gln Phe Gly Lys Thr Leu Gly Ala Gly Ala 580 585 590 Phe Gly
Lys Val Val Glu Ala Thr Ala Phe Gly Leu Gly Lys Glu Asp 595 600 605
Ala Val Leu Lys Val Ala Val Lys Met Leu Lys Ser Thr Ala His Ala 610
615 620 Asp Glu Lys Glu Ala Leu Met Ser Glu Leu Lys Ile Met Ser His
Leu 625 630 635 640 Gly Gln His Glu Asn Ile Val Asn Leu Leu Gly Ala
Cys Thr His Gly 645 650 655 Gly Pro Val Leu Val Ile Thr Glu Tyr Cys
Cys Tyr Gly Asp Leu Leu 660 665 670 Asn Phe Leu Arg Arg Lys Ala Glu
Ala Met Leu Gly Pro Ser Leu Ser 675 680 685 Pro Gly Gln Asp Pro Glu
Gly Gly Val Asp Tyr Lys Asn Ile His Leu 690 695 700 Glu Lys Lys Tyr
Val Arg Arg Asp Ser Gly Phe Ser Ser Gln Gly Val 705 710 715 720 Asp
Thr Tyr Val Glu Met Arg Pro Val Ser Thr Ser Ser Asn Asp Ser 725 730
735 Phe Ser Glu Gln Asp Leu Asp Lys Glu Asp Gly Arg Pro Leu Glu Leu
740 745 750 Arg Asp Leu Leu His Phe Ser Ser Gln Val Ala Gln Gly Met
Ala Phe 755 760 765 Leu Ala Ser Lys Asn Cys Ile His Arg Asp Val Ala
Ala Arg Asn Val 770 775 780 Leu Leu Thr Asn Gly His Val Ala Lys Ile
Gly Asp Phe Gly Leu Ala 785 790 795 800 Arg Asp Ile Met Asn Asp Ser
Asn Tyr Ile Val Lys Gly Asn Ala Arg 805 810 815 Leu Pro Val Lys Trp
Met Ala Pro Glu Ser Ile Phe Asp Cys Val Tyr 820 825 830 Thr Val Gln
Ser Asp Val Trp Ser Tyr Gly Ile Leu Leu Trp Glu Ile 835 840 845 Phe
Ser Leu Gly Leu Asn Pro Tyr Pro Gly Ile Leu Val Asn Ser Lys 850 855
860 Phe Tyr Lys Leu Val Lys Asp Gly Tyr Gln Met Ala Gln Pro Ala Phe
865 870 875 880 Ala Pro Lys Asn Ile Tyr Ser Ile Met Gln Ala Cys Trp
Ala Leu Glu
885 890 895 Pro Thr His Arg Pro Thr Phe Gln Gln Ile Cys Ser Phe Leu
Gln Glu 900 905 910 Gln Ala Gln Glu Asp Arg Arg Glu Arg Asp Tyr Thr
Asn Leu Pro Ser 915 920 925 Ser Ser Arg Ser Gly Gly Ser Gly Ser Ser
Ser Ser Glu Leu Glu Glu 930 935 940 Glu Ser Ser Ser Glu His Leu Thr
Cys Cys Glu Gln Gly Asp Ile Ala 945 950 955 960 Gln Pro Leu Leu Gln
Pro Asn Asn Tyr Gln Phe Cys 965 970 63972PRTArtificialmutant CSF-1R
L301S Y969F 63Met Gly Pro Gly Val Leu Leu Leu Leu Leu Val Ala Thr
Ala Trp His 1 5 10 15 Gly Gln Gly Ile Pro Val Ile Glu Pro Ser Val
Pro Glu Leu Val Val 20 25 30 Lys Pro Gly Ala Thr Val Thr Leu Arg
Cys Val Gly Asn Gly Ser Val 35 40 45 Glu Trp Asp Gly Pro Pro Ser
Pro His Trp Thr Leu Tyr Ser Asp Gly 50 55 60 Ser Ser Ser Ile Leu
Ser Thr Asn Asn Ala Thr Phe Gln Asn Thr Gly 65 70 75 80 Thr Tyr Arg
Cys Thr Glu Pro Gly Asp Pro Leu Gly Gly Ser Ala Ala 85 90 95 Ile
His Leu Tyr Val Lys Asp Pro Ala Arg Pro Trp Asn Val Leu Ala 100 105
110 Gln Glu Val Val Val Phe Glu Asp Gln Asp Ala Leu Leu Pro Cys Leu
115 120 125 Leu Thr Asp Pro Val Leu Glu Ala Gly Val Ser Leu Val Arg
Val Arg 130 135 140 Gly Arg Pro Leu Met Arg His Thr Asn Tyr Ser Phe
Ser Pro Trp His 145 150 155 160 Gly Phe Thr Ile His Arg Ala Lys Phe
Ile Gln Ser Gln Asp Tyr Gln 165 170 175 Cys Ser Ala Leu Met Gly Gly
Arg Lys Val Met Ser Ile Ser Ile Arg 180 185 190 Leu Lys Val Gln Lys
Val Ile Pro Gly Pro Pro Ala Leu Thr Leu Val 195 200 205 Pro Ala Glu
Leu Val Arg Ile Arg Gly Glu Ala Ala Gln Ile Val Cys 210 215 220 Ser
Ala Ser Ser Val Asp Val Asn Phe Asp Val Phe Leu Gln His Asn 225 230
235 240 Asn Thr Lys Leu Ala Ile Pro Gln Gln Ser Asp Phe His Asn Asn
Arg 245 250 255 Tyr Gln Lys Val Leu Thr Leu Asn Leu Asp Gln Val Asp
Phe Gln His 260 265 270 Ala Gly Asn Tyr Ser Cys Val Ala Ser Asn Val
Gln Gly Lys His Ser 275 280 285 Thr Ser Met Phe Phe Arg Val Val Glu
Ser Ala Tyr Ser Asn Leu Ser 290 295 300 Ser Glu Gln Asn Leu Ile Gln
Glu Val Thr Val Gly Glu Gly Leu Asn 305 310 315 320 Leu Lys Val Met
Val Glu Ala Tyr Pro Gly Leu Gln Gly Phe Asn Trp 325 330 335 Thr Tyr
Leu Gly Pro Phe Ser Asp His Gln Pro Glu Pro Lys Leu Ala 340 345 350
Asn Ala Thr Thr Lys Asp Thr Tyr Arg His Thr Phe Thr Leu Ser Leu 355
360 365 Pro Arg Leu Lys Pro Ser Glu Ala Gly Arg Tyr Ser Phe Leu Ala
Arg 370 375 380 Asn Pro Gly Gly Trp Arg Ala Leu Thr Phe Glu Leu Thr
Leu Arg Tyr 385 390 395 400 Pro Pro Glu Val Ser Val Ile Trp Thr Phe
Ile Asn Gly Ser Gly Thr 405 410 415 Leu Leu Cys Ala Ala Ser Gly Tyr
Pro Gln Pro Asn Val Thr Trp Leu 420 425 430 Gln Cys Ser Gly His Thr
Asp Arg Cys Asp Glu Ala Gln Val Leu Gln 435 440 445 Val Trp Asp Asp
Pro Tyr Pro Glu Val Leu Ser Gln Glu Pro Phe His 450 455 460 Lys Val
Thr Val Gln Ser Leu Leu Thr Val Glu Thr Leu Glu His Asn 465 470 475
480 Gln Thr Tyr Glu Cys Arg Ala His Asn Ser Val Gly Ser Gly Ser Trp
485 490 495 Ala Phe Ile Pro Ile Ser Ala Gly Ala His Thr His Pro Pro
Asp Glu 500 505 510 Phe Leu Phe Thr Pro Val Val Val Ala Cys Met Ser
Ile Met Ala Leu 515 520 525 Leu Leu Leu Leu Leu Leu Leu Leu Leu Tyr
Lys Tyr Lys Gln Lys Pro 530 535 540 Lys Tyr Gln Val Arg Trp Lys Ile
Ile Glu Ser Tyr Glu Gly Asn Ser 545 550 555 560 Tyr Thr Phe Ile Asp
Pro Thr Gln Leu Pro Tyr Asn Glu Lys Trp Glu 565 570 575 Phe Pro Arg
Asn Asn Leu Gln Phe Gly Lys Thr Leu Gly Ala Gly Ala 580 585 590 Phe
Gly Lys Val Val Glu Ala Thr Ala Phe Gly Leu Gly Lys Glu Asp 595 600
605 Ala Val Leu Lys Val Ala Val Lys Met Leu Lys Ser Thr Ala His Ala
610 615 620 Asp Glu Lys Glu Ala Leu Met Ser Glu Leu Lys Ile Met Ser
His Leu 625 630 635 640 Gly Gln His Glu Asn Ile Val Asn Leu Leu Gly
Ala Cys Thr His Gly 645 650 655 Gly Pro Val Leu Val Ile Thr Glu Tyr
Cys Cys Tyr Gly Asp Leu Leu 660 665 670 Asn Phe Leu Arg Arg Lys Ala
Glu Ala Met Leu Gly Pro Ser Leu Ser 675 680 685 Pro Gly Gln Asp Pro
Glu Gly Gly Val Asp Tyr Lys Asn Ile His Leu 690 695 700 Glu Lys Lys
Tyr Val Arg Arg Asp Ser Gly Phe Ser Ser Gln Gly Val 705 710 715 720
Asp Thr Tyr Val Glu Met Arg Pro Val Ser Thr Ser Ser Asn Asp Ser 725
730 735 Phe Ser Glu Gln Asp Leu Asp Lys Glu Asp Gly Arg Pro Leu Glu
Leu 740 745 750 Arg Asp Leu Leu His Phe Ser Ser Gln Val Ala Gln Gly
Met Ala Phe 755 760 765 Leu Ala Ser Lys Asn Cys Ile His Arg Asp Val
Ala Ala Arg Asn Val 770 775 780 Leu Leu Thr Asn Gly His Val Ala Lys
Ile Gly Asp Phe Gly Leu Ala 785 790 795 800 Arg Asp Ile Met Asn Asp
Ser Asn Tyr Ile Val Lys Gly Asn Ala Arg 805 810 815 Leu Pro Val Lys
Trp Met Ala Pro Glu Ser Ile Phe Asp Cys Val Tyr 820 825 830 Thr Val
Gln Ser Asp Val Trp Ser Tyr Gly Ile Leu Leu Trp Glu Ile 835 840 845
Phe Ser Leu Gly Leu Asn Pro Tyr Pro Gly Ile Leu Val Asn Ser Lys 850
855 860 Phe Tyr Lys Leu Val Lys Asp Gly Tyr Gln Met Ala Gln Pro Ala
Phe 865 870 875 880 Ala Pro Lys Asn Ile Tyr Ser Ile Met Gln Ala Cys
Trp Ala Leu Glu 885 890 895 Pro Thr His Arg Pro Thr Phe Gln Gln Ile
Cys Ser Phe Leu Gln Glu 900 905 910 Gln Ala Gln Glu Asp Arg Arg Glu
Arg Asp Tyr Thr Asn Leu Pro Ser 915 920 925 Ser Ser Arg Ser Gly Gly
Ser Gly Ser Ser Ser Ser Glu Leu Glu Glu 930 935 940 Glu Ser Ser Ser
Glu His Leu Thr Cys Cys Glu Gln Gly Asp Ile Ala 945 950 955 960 Gln
Pro Leu Leu Gln Pro Asn Asn Phe Gln Phe Cys 965 970
64493PRTArtificialhuman CSF-1R Extracellular Domain 64Ile Pro Val
Ile Glu Pro Ser Val Pro Glu Leu Val Val Lys Pro Gly 1 5 10 15 Ala
Thr Val Thr Leu Arg Cys Val Gly Asn Gly Ser Val Glu Trp Asp 20 25
30 Gly Pro Pro Ser Pro His Trp Thr Leu Tyr Ser Asp Gly Ser Ser Ser
35 40 45 Ile Leu Ser Thr Asn Asn Ala Thr Phe Gln Asn Thr Gly Thr
Tyr Arg 50 55 60 Cys Thr Glu Pro Gly Asp Pro Leu Gly Gly Ser Ala
Ala Ile His Leu 65 70 75 80 Tyr Val Lys Asp Pro Ala Arg Pro Trp Asn
Val Leu Ala Gln Glu Val 85 90 95 Val Val Phe Glu Asp Gln Asp Ala
Leu Leu Pro Cys Leu Leu Thr Asp 100 105 110 Pro Val Leu Glu Ala Gly
Val Ser Leu Val Arg Val Arg Gly Arg Pro 115 120 125 Leu Met Arg His
Thr Asn Tyr Ser Phe Ser Pro Trp His Gly Phe Thr 130 135 140 Ile His
Arg Ala Lys Phe Ile Gln Ser Gln Asp Tyr Gln Cys Ser Ala 145 150 155
160 Leu Met Gly Gly Arg Lys Val Met Ser Ile Ser Ile Arg Leu Lys Val
165 170 175 Gln Lys Val Ile Pro Gly Pro Pro Ala Leu Thr Leu Val Pro
Ala Glu 180 185 190 Leu Val Arg Ile Arg Gly Glu Ala Ala Gln Ile Val
Cys Ser Ala Ser 195 200 205 Ser Val Asp Val Asn Phe Asp Val Phe Leu
Gln His Asn Asn Thr Lys 210 215 220 Leu Ala Ile Pro Gln Gln Ser Asp
Phe His Asn Asn Arg Tyr Gln Lys 225 230 235 240 Val Leu Thr Leu Asn
Leu Asp Gln Val Asp Phe Gln His Ala Gly Asn 245 250 255 Tyr Ser Cys
Val Ala Ser Asn Val Gln Gly Lys His Ser Thr Ser Met 260 265 270 Phe
Phe Arg Val Val Glu Ser Ala Tyr Leu Asn Leu Ser Ser Glu Gln 275 280
285 Asn Leu Ile Gln Glu Val Thr Val Gly Glu Gly Leu Asn Leu Lys Val
290 295 300 Met Val Glu Ala Tyr Pro Gly Leu Gln Gly Phe Asn Trp Thr
Tyr Leu 305 310 315 320 Gly Pro Phe Ser Asp His Gln Pro Glu Pro Lys
Leu Ala Asn Ala Thr 325 330 335 Thr Lys Asp Thr Tyr Arg His Thr Phe
Thr Leu Ser Leu Pro Arg Leu 340 345 350 Lys Pro Ser Glu Ala Gly Arg
Tyr Ser Phe Leu Ala Arg Asn Pro Gly 355 360 365 Gly Trp Arg Ala Leu
Thr Phe Glu Leu Thr Leu Arg Tyr Pro Pro Glu 370 375 380 Val Ser Val
Ile Trp Thr Phe Ile Asn Gly Ser Gly Thr Leu Leu Cys 385 390 395 400
Ala Ala Ser Gly Tyr Pro Gln Pro Asn Val Thr Trp Leu Gln Cys Ser 405
410 415 Gly His Thr Asp Arg Cys Asp Glu Ala Gln Val Leu Gln Val Trp
Asp 420 425 430 Asp Pro Tyr Pro Glu Val Leu Ser Gln Glu Pro Phe His
Lys Val Thr 435 440 445 Val Gln Ser Leu Leu Thr Val Glu Thr Leu Glu
His Asn Gln Thr Tyr 450 455 460 Glu Cys Arg Ala His Asn Ser Val Gly
Ser Gly Ser Trp Ala Phe Ile 465 470 475 480 Pro Ile Ser Ala Gly Ala
His Thr His Pro Pro Asp Glu 485 490 65388PRTArtificialhuman CSF-1R
fragment delD4 65Ile Pro Val Ile Glu Pro Ser Val Pro Glu Leu Val
Val Lys Pro Gly 1 5 10 15 Ala Thr Val Thr Leu Arg Cys Val Gly Asn
Gly Ser Val Glu Trp Asp 20 25 30 Gly Pro Pro Ser Pro His Trp Thr
Leu Tyr Ser Asp Gly Ser Ser Ser 35 40 45 Ile Leu Ser Thr Asn Asn
Ala Thr Phe Gln Asn Thr Gly Thr Tyr Arg 50 55 60 Cys Thr Glu Pro
Gly Asp Pro Leu Gly Gly Ser Ala Ala Ile His Leu 65 70 75 80 Tyr Val
Lys Asp Pro Ala Arg Pro Trp Asn Val Leu Ala Gln Glu Val 85 90 95
Val Val Phe Glu Asp Gln Asp Ala Leu Leu Pro Cys Leu Leu Thr Asp 100
105 110 Pro Val Leu Glu Ala Gly Val Ser Leu Val Arg Val Arg Gly Arg
Pro 115 120 125 Leu Met Arg His Thr Asn Tyr Ser Phe Ser Pro Trp His
Gly Phe Thr 130 135 140 Ile His Arg Ala Lys Phe Ile Gln Ser Gln Asp
Tyr Gln Cys Ser Ala 145 150 155 160 Leu Met Gly Gly Arg Lys Val Met
Ser Ile Ser Ile Arg Leu Lys Val 165 170 175 Gln Lys Val Ile Pro Gly
Pro Pro Ala Leu Thr Leu Val Pro Ala Glu 180 185 190 Leu Val Arg Ile
Arg Gly Glu Ala Ala Gln Ile Val Cys Ser Ala Ser 195 200 205 Ser Val
Asp Val Asn Phe Asp Val Phe Leu Gln His Asn Asn Thr Lys 210 215 220
Leu Ala Ile Pro Gln Gln Ser Asp Phe His Asn Asn Arg Tyr Gln Lys 225
230 235 240 Val Leu Thr Leu Asn Leu Asp Gln Val Asp Phe Gln His Ala
Gly Asn 245 250 255 Tyr Ser Cys Val Ala Ser Asn Val Gln Gly Lys His
Ser Thr Ser Met 260 265 270 Phe Phe Arg Tyr Pro Pro Glu Val Ser Val
Ile Trp Thr Phe Ile Asn 275 280 285 Gly Ser Gly Thr Leu Leu Cys Ala
Ala Ser Gly Tyr Pro Gln Pro Asn 290 295 300 Val Thr Trp Leu Gln Cys
Ser Gly His Thr Asp Arg Cys Asp Glu Ala 305 310 315 320 Gln Val Leu
Gln Val Trp Asp Asp Pro Tyr Pro Glu Val Leu Ser Gln 325 330 335 Glu
Pro Phe His Lys Val Thr Val Gln Ser Leu Leu Thr Val Glu Thr 340 345
350 Leu Glu His Asn Gln Thr Tyr Glu Cys Arg Ala His Asn Ser Val Gly
355 360 365 Ser Gly Ser Trp Ala Phe Ile Pro Ile Ser Ala Gly Ala His
Thr His 370 375 380 Pro Pro Asp Glu 385 66292PRTArtificialhuman
CSF-1R fragment D1-D3 66Ile Pro Val Ile Glu Pro Ser Val Pro Glu Leu
Val Val Lys Pro Gly 1 5 10 15 Ala Thr Val Thr Leu Arg Cys Val Gly
Asn Gly Ser Val Glu Trp Asp 20 25 30 Gly Pro Pro Ser Pro His Trp
Thr Leu Tyr Ser Asp Gly Ser Ser Ser 35 40 45 Ile Leu Ser Thr Asn
Asn Ala Thr Phe Gln Asn Thr Gly Thr Tyr Arg 50 55 60 Cys Thr Glu
Pro Gly Asp Pro Leu Gly Gly Ser Ala Ala Ile His Leu 65 70 75 80 Tyr
Val Lys Asp Pro Ala Arg Pro Trp Asn Val Leu Ala Gln Glu Val 85 90
95 Val Val Phe Glu Asp Gln Asp Ala Leu Leu Pro Cys Leu Leu Thr Asp
100 105 110 Pro Val Leu Glu Ala Gly Val Ser Leu Val Arg Val Arg Gly
Arg Pro 115 120 125 Leu Met Arg His Thr Asn Tyr Ser Phe Ser Pro Trp
His Gly Phe Thr 130 135 140 Ile His Arg Ala Lys Phe Ile Gln Ser Gln
Asp Tyr Gln Cys Ser Ala 145 150 155 160 Leu Met Gly Gly Arg Lys Val
Met Ser Ile Ser Ile Arg Leu Lys Val 165 170 175 Gln Lys Val Ile Pro
Gly Pro Pro Ala Leu Thr Leu Val Pro Ala Glu 180 185 190 Leu Val Arg
Ile Arg Gly Glu Ala Ala Gln Ile Val Cys Ser Ala Ser 195 200 205 Ser
Val Asp Val Asn Phe Asp Val Phe Leu Gln His Asn Asn Thr Lys 210 215
220 Leu Ala Ile Pro Gln Gln Ser Asp Phe His Asn Asn Arg Tyr Gln Lys
225 230 235 240 Val Leu Thr Leu Asn Leu Asp Gln Val Asp Phe Gln His
Ala Gly Asn 245 250 255 Tyr Ser Cys Val Ala Ser Asn Val Gln Gly Lys
His Ser Thr Ser Met 260 265 270 Phe Phe Arg Val Val Glu Ser Ala Tyr
Leu Asn Leu Ser Ser Glu Gln 275 280 285 Asn Leu Ile Gln 290
6721PRTArtificialsignal peptide 67Met Gly Ser Gly Pro Gly Val Leu
Leu Leu Leu Leu Val Ala Thr Ala 1 5 10 15 Trp His Gly Gln Gly 20
6836DNAArtificialPrimer 68cacctccatg ttcttccggt accccccaga ggtaag
36698PRTMus musculus 69Asp Leu Arg Leu Tyr Phe Asp Val 1 5
7016PRTMus musculus
70Val Ile Trp Ser Gly Gly Gly Thr Asn Tyr Asn Ser Pro Phe Met Ser 1
5 10 15 7110PRTMus musculus 71Gly Phe Ser Leu Thr Ser Tyr Asp Ile
Ser 1 5 10 728PRTMus musculus 72Gly Gln Ser Phe Thr Tyr Pro Thr 1 5
737PRTMus musculus 73Gly Ser Ser Asn Arg Tyr Thr 1 5 7411PRTMus
musculus 74Lys Ala Ser Glu Asp Val Gly Thr Tyr Val Ser 1 5 10
75116PRTMus musculus 75Arg Val Gln Leu Lys Glu Ser Gly Pro Gly Leu
Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser
Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Asp Ile Ser Trp Ile Arg Gln
Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser
Gly Gly Gly Thr Asn Tyr Asn Ser Pro Phe Met 50 55 60 Ser Arg Leu
Arg Ile Ser Lys Asp Asp Ser Arg Ser Gln Val Phe Leu 65 70 75 80 Lys
Val Asn Arg Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Val 85 90
95 Arg Asp Leu Arg Leu Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val
100 105 110 Thr Val Ser Ser 115 76106PRTMus musculus 76Lys Ile Val
Met Thr Gln Ser Pro Lys Ser Met Ser Val Ser Val Gly 1 5 10 15 Glu
Arg Val Ser Leu Ser Cys Lys Ala Ser Glu Asp Val Gly Thr Tyr 20 25
30 Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile
35 40 45 Tyr Gly Ser Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe
Thr Gly 50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser
Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Asp Tyr Ser Cys Gly Gln
Ser Phe Thr Tyr Pro Thr 85 90 95 Phe Gly Thr Gly Thr Lys Leu Glu
Ile Lys 100 105 778PRTMus musculus 77Asp Pro Arg Leu Tyr Phe Asp
Val 1 5 7816PRTMus musculus 78Val Ile Trp Thr Gly Gly Gly Thr Asn
Tyr Asn Ser Gly Phe Met Ser 1 5 10 15 7910PRTMus musculus 79Gly Ser
Ser Leu Asp Ser Phe Asp Ile Ser 1 5 10 808PRTMus musculus 80Gly Gln
Thr Phe Ser Tyr Pro Thr 1 5 817PRTMus musculus 81Gly Ala Ser Asn
Arg Tyr Thr 1 5 8211PRTMus musculus 82Lys Ala Ser Glu Asp Val Val
Thr Tyr Val Ser 1 5 10 83116PRTMus musculus 83Gln Val Gln Leu Lys
Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Lys 1 5 10 15 Ser Leu Ser
Ile Thr Cys Thr Val Ser Gly Ser Ser Leu Asp Ser Phe 20 25 30 Asp
Ile Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40
45 Gly Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Gly Phe Met
50 55 60 Ser Arg Leu Arg Ile Ser Lys Asp Asn Ser Lys Ser Gln Val
Phe Leu 65 70 75 80 Lys Met Ser Ser Leu Gln Ser Asp Asp Thr Ala Ile
Tyr Tyr Cys Val 85 90 95 Arg Asp Pro Arg Leu Tyr Phe Asp Val Trp
Gly Ala Gly Thr Thr Val 100 105 110 Thr Val Ser Ser 115 84106PRTMus
musculus 84Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser
Val Gly 1 5 10 15 Glu Arg Val Thr Leu Ser Cys Lys Ala Ser Glu Asp
Val Val Thr Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln
Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr
Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Ala Thr Asp
Phe Thr Leu Thr Ile Ser Ser Ile Gln Ala 65 70 75 80 Glu Asp Leu Ala
Asp Tyr Tyr Cys Gly Gln Thr Phe Ser Tyr Pro Thr 85 90 95 Phe Gly
Thr Gly Thr Lys Leu Glu Ile Lys 100 105 85218PRTArtificialhuman
CSF-1R fragment domains D4-D5 85Val Val Glu Ser Ala Tyr Leu Asn Leu
Ser Ser Glu Gln Asn Leu Ile 1 5 10 15 Gln Glu Val Thr Val Gly Glu
Gly Leu Asn Leu Lys Val Met Val Glu 20 25 30 Ala Tyr Pro Gly Leu
Gln Gly Phe Asn Trp Thr Tyr Leu Gly Pro Phe 35 40 45 Ser Asp His
Gln Pro Glu Pro Lys Leu Ala Asn Ala Thr Thr Lys Asp 50 55 60 Thr
Tyr Arg His Thr Phe Thr Leu Ser Leu Pro Arg Leu Lys Pro Ser 65 70
75 80 Glu Ala Gly Arg Tyr Ser Phe Leu Ala Arg Asn Pro Gly Gly Trp
Arg 85 90 95 Ala Leu Thr Phe Glu Leu Thr Leu Arg Tyr Pro Pro Glu
Val Ser Val 100 105 110 Ile Trp Thr Phe Ile Asn Gly Ser Gly Thr Leu
Leu Cys Ala Ala Ser 115 120 125 Gly Tyr Pro Gln Pro Asn Val Thr Trp
Leu Gln Cys Ser Gly His Thr 130 135 140 Asp Arg Cys Asp Glu Ala Gln
Val Leu Gln Val Trp Asp Asp Pro Tyr 145 150 155 160 Pro Glu Val Leu
Ser Gln Glu Pro Phe His Lys Val Thr Val Gln Ser 165 170 175 Leu Leu
Thr Val Glu Thr Leu Glu His Asn Gln Thr Tyr Glu Cys Arg 180 185 190
Ala His Asn Ser Val Gly Ser Gly Ser Trp Ala Phe Ile Pro Ile Ser 195
200 205 Ala Gly Ala His Thr His Pro Pro Asp Glu 210 215
86554PRThomo sapiens 86Met Thr Ala Pro Gly Ala Ala Gly Arg Cys Pro
Pro Thr Thr Trp Leu 1 5 10 15 Gly Ser Leu Leu Leu Leu Val Cys Leu
Leu Ala Ser Arg Ser Ile Thr 20 25 30 Glu Glu Val Ser Glu Tyr Cys
Ser His Met Ile Gly Ser Gly His Leu 35 40 45 Gln Ser Leu Gln Arg
Leu Ile Asp Ser Gln Met Glu Thr Ser Cys Gln 50 55 60 Ile Thr Phe
Glu Phe Val Asp Gln Glu Gln Leu Lys Asp Pro Val Cys 65 70 75 80 Tyr
Leu Lys Lys Ala Phe Leu Leu Val Gln Asp Ile Met Glu Asp Thr 85 90
95 Met Arg Phe Arg Asp Asn Thr Pro Asn Ala Ile Ala Ile Val Gln Leu
100 105 110 Gln Glu Leu Ser Leu Arg Leu Lys Ser Cys Phe Thr Lys Asp
Tyr Glu 115 120 125 Glu His Asp Lys Ala Cys Val Arg Thr Phe Tyr Glu
Thr Pro Leu Gln 130 135 140 Leu Leu Glu Lys Val Lys Asn Val Phe Asn
Glu Thr Lys Asn Leu Leu 145 150 155 160 Asp Lys Asp Trp Asn Ile Phe
Ser Lys Asn Cys Asn Asn Ser Phe Ala 165 170 175 Glu Cys Ser Ser Gln
Asp Val Val Thr Lys Pro Asp Cys Asn Cys Leu 180 185 190 Tyr Pro Lys
Ala Ile Pro Ser Ser Asp Pro Ala Ser Val Ser Pro His 195 200 205 Gln
Pro Leu Ala Pro Ser Met Ala Pro Val Ala Gly Leu Thr Trp Glu 210 215
220 Asp Ser Glu Gly Thr Glu Gly Ser Ser Leu Leu Pro Gly Glu Gln Pro
225 230 235 240 Leu His Thr Val Asp Pro Gly Ser Ala Lys Gln Arg Pro
Pro Arg Ser 245 250 255 Thr Cys Gln Ser Phe Glu Pro Pro Glu Thr Pro
Val Val Lys Asp Ser 260 265 270 Thr Ile Gly Gly Ser Pro Gln Pro Arg
Pro Ser Val Gly Ala Phe Asn 275 280 285 Pro Gly Met Glu Asp Ile Leu
Asp Ser Ala Met Gly Thr Asn Trp Val 290 295 300 Pro Glu Glu Ala Ser
Gly Glu Ala Ser Glu Ile Pro Val Pro Gln Gly 305 310 315 320 Thr Glu
Leu Ser Pro Ser Arg Pro Gly Gly Gly Ser Met Gln Thr Glu 325 330 335
Pro Ala Arg Pro Ser Asn Phe Leu Ser Ala Ser Ser Pro Leu Pro Ala 340
345 350 Ser Ala Lys Gly Gln Gln Pro Ala Asp Val Thr Gly Thr Ala Leu
Pro 355 360 365 Arg Val Gly Pro Val Arg Pro Thr Gly Gln Asp Trp Asn
His Thr Pro 370 375 380 Gln Lys Thr Asp His Pro Ser Ala Leu Leu Arg
Asp Pro Pro Glu Pro 385 390 395 400 Gly Ser Pro Arg Ile Ser Ser Leu
Arg Pro Gln Gly Leu Ser Asn Pro 405 410 415 Ser Thr Leu Ser Ala Gln
Pro Gln Leu Ser Arg Ser His Ser Ser Gly 420 425 430 Ser Val Leu Pro
Leu Gly Glu Leu Glu Gly Arg Arg Ser Thr Arg Asp 435 440 445 Arg Arg
Ser Pro Ala Glu Pro Glu Gly Gly Pro Ala Ser Glu Gly Ala 450 455 460
Ala Arg Pro Leu Pro Arg Phe Asn Ser Val Pro Leu Thr Asp Thr Gly 465
470 475 480 His Glu Arg Gln Ser Glu Gly Ser Phe Ser Pro Gln Leu Gln
Glu Ser 485 490 495 Val Phe His Leu Leu Val Pro Ser Val Ile Leu Val
Leu Leu Ala Val 500 505 510 Gly Gly Leu Leu Phe Tyr Arg Trp Arg Arg
Arg Ser His Gln Glu Pro 515 520 525 Gln Arg Ala Asp Ser Pro Leu Glu
Gln Pro Glu Gly Ser Pro Leu Thr 530 535 540 Gln Asp Asp Arg Gln Val
Glu Leu Pro Val 545 550 87242PRThomo sapiens 87Met Pro Arg Gly Phe
Thr Trp Leu Arg Tyr Leu Gly Ile Phe Leu Gly 1 5 10 15 Val Ala Leu
Gly Asn Glu Pro Leu Glu Met Trp Pro Leu Thr Gln Asn 20 25 30 Glu
Glu Cys Thr Val Thr Gly Phe Leu Arg Asp Lys Leu Gln Tyr Arg 35 40
45 Ser Arg Leu Gln Tyr Met Lys His Tyr Phe Pro Ile Asn Tyr Lys Ile
50 55 60 Ser Val Pro Tyr Glu Gly Val Phe Arg Ile Ala Asn Val Thr
Arg Leu 65 70 75 80 Gln Arg Ala Gln Val Ser Glu Arg Glu Leu Arg Tyr
Leu Trp Val Leu 85 90 95 Val Ser Leu Ser Ala Thr Glu Ser Val Gln
Asp Val Leu Leu Glu Gly 100 105 110 His Pro Ser Trp Lys Tyr Leu Gln
Glu Val Glu Thr Leu Leu Leu Asn 115 120 125 Val Gln Gln Gly Leu Thr
Asp Val Glu Val Ser Pro Lys Val Glu Ser 130 135 140 Val Leu Ser Leu
Leu Asn Ala Pro Gly Pro Asn Leu Lys Leu Val Arg 145 150 155 160 Pro
Lys Ala Leu Leu Asp Asn Cys Phe Arg Val Met Glu Leu Leu Tyr 165 170
175 Cys Ser Cys Cys Lys Gln Ser Ser Val Leu Asn Trp Gln Asp Cys Glu
180 185 190 Val Pro Ser Pro Gln Ser Cys Ser Pro Glu Pro Ser Leu Gln
Tyr Ala 195 200 205 Ala Thr Gln Leu Tyr Pro Pro Pro Pro Trp Ser Pro
Ser Ser Pro Pro 210 215 220 His Ser Thr Gly Ser Val Arg Pro Val Arg
Ala Gln Gly Glu Gly Leu 225 230 235 240 Leu Pro 88290PRThomo
sapiens 88Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His
Leu Leu 1 5 10 15 Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr
Val Val Glu Tyr 20 25 30 Gly Ser Asn Met Thr Ile Glu Cys Lys Phe
Pro Val Glu Lys Gln Leu 35 40 45 Asp Leu Ala Ala Leu Ile Val Tyr
Trp Glu Met Glu Asp Lys Asn Ile 50 55 60 Ile Gln Phe Val His Gly
Glu Glu Asp Leu Lys Val Gln His Ser Ser 65 70 75 80 Tyr Arg Gln Arg
Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn 85 90 95 Ala Ala
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr 100 105 110
Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val 115
120 125 Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val
Val 130 135 140 Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala
Glu Gly Tyr 145 150 155 160 Pro Lys Ala Glu Val Ile Trp Thr Ser Ser
Asp His Gln Val Leu Ser 165 170 175 Gly Lys Thr Thr Thr Thr Asn Ser
Lys Arg Glu Glu Lys Leu Phe Asn 180 185 190 Val Thr Ser Thr Leu Arg
Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr 195 200 205 Cys Thr Phe Arg
Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu 210 215 220 Val Ile
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His 225 230 235
240 Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr
245 250 255 Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys
Lys Cys 260 265 270 Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp
Thr His Leu Glu 275 280 285 Glu Thr 290 89118PRTArtificial
sequencesequence is synthesized 89Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30 Trp Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Trp
Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp
Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115
90118PRTArtificial sequencesequence is synthesized 90Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25
30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg His Trp Pro Gly Gly
Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala
115 91118PRTArtificial sequencesequence is synthesized 91Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ser 20
25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Trp Ile Leu Pro Tyr Gly Gly Ser Ser Tyr Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser
Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg His Trp Pro Gly
Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser
Ala 115 92108PRTArtificial sequencesequence is synthesized 92Asp
Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu
Tyr His Pro Ala 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg 100 105 93108PRTArtificial sequencesequence is synthesized
93Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr
Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Tyr Tyr Asn Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105 94108PRTArtificial
sequencesequence is synthesized 94Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser
Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ala Pro
Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 95108PRTArtificial sequencesequence is synthesized 95Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala
20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Tyr Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg 100 105 96108PRTArtificial sequencesequence
is synthesized 96Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Val Ile Asn Thr Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Thr Leu
Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Thr Val Pro Arg 85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
97108PRTArtificial sequencesequence is synthesized 97Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Gly Tyr Gly Val Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg 100 105 98108PRTArtificial sequencesequence is
synthesized 98Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Phe Thr Pro Pro 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
99108PRTArtificial sequencesequence is synthesized 99Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Phe Ile Thr Pro Thr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg 100 105 100108PRTArtificial sequencesequence is
synthesized 100Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Tyr Thr Pro Pro 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
101108PRTArtificial sequencesequence is synthesized 101Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Phe Phe Tyr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg 100 105 102108PRTArtificial sequencesequence is
synthesized 102Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Ser Leu Phe Thr Pro Pro 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
103108PRTArtificial sequencesequence is synthesized 103Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Leu Tyr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg 100 105 104108PRTArtificial sequencesequence is
synthesized 104Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Ser Trp Tyr His Pro Pro 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
105108PRTArtificial sequencesequence is synthesized 105Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Phe Tyr Ile Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg 100 105 106108PRTArtificial sequencesequence is
synthesized 106Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Tyr Thr Pro Thr 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
107108PRTArtificial sequencesequence is synthesized 107Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Tyr Phe Ile Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg 100 105
* * * * *