U.S. patent application number 16/732764 was filed with the patent office on 2020-07-16 for pharmaceutical composition for cancer treatment.
This patent application is currently assigned to Shionogi & Co., Ltd.. The applicant listed for this patent is Shionogi & Co., Ltd. Osaka University. Invention is credited to Kanji Hojo, Takayuki Kanazawa, Atsunari Kawashima, Yujiro Kidani, Mitsunobu Matsumoto, Norio Nonomura, Naganari Ohkura, Shimon Sakaguchi, Satomi Shinonome, Atsushi Tanaka, Hisashi Wada, Tetsuya Yoshida.
Application Number | 20200222463 16/732764 |
Document ID | / |
Family ID | 63676314 |
Filed Date | 2020-07-16 |
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United States Patent
Application |
20200222463 |
Kind Code |
A1 |
Yoshida; Tetsuya ; et
al. |
July 16, 2020 |
Pharmaceutical Composition for Cancer Treatment
Abstract
The disclosure provides a pharmaceutical composition with a
polypeptide that binds to a peptide produced by cDNA of GenBank ACC
No. NM_005201.3 or NM_007720.2, and methods of use thereof.
Inventors: |
Yoshida; Tetsuya;
(Toyonaka-shi, JP) ; Kidani; Yujiro;
(Toyonaka-shi, JP) ; Matsumoto; Mitsunobu;
(Toyonaka-shi, JP) ; Kanazawa; Takayuki;
(Toyonaka-shi, JP) ; Shinonome; Satomi;
(Toyonaka-shi, JP) ; Hojo; Kanji; (Toyonaka-shi,
JP) ; Ohkura; Naganari; (Suita-shi, JP) ;
Sakaguchi; Shimon; (Suita-shi, JP) ; Tanaka;
Atsushi; (Suita-shi, JP) ; Wada; Hisashi;
(Suita-shi, JP) ; Kawashima; Atsunari; (Suita-shi,
JP) ; Nonomura; Norio; (Suita-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Shionogi & Co., Ltd.
Osaka University |
Osaka
Osaka |
|
JP
JP |
|
|
Assignee: |
Shionogi & Co., Ltd.
Osaka
JP
Osaka University
Osaka
JP
|
Family ID: |
63676314 |
Appl. No.: |
16/732764 |
Filed: |
January 2, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16183216 |
Nov 7, 2018 |
10550191 |
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16732764 |
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PCT/JP2018/012644 |
Mar 28, 2018 |
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16183216 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5011 20130101;
A61K 2039/572 20130101; A61K 9/0019 20130101; A61K 2039/505
20130101; A61P 35/00 20180101; C07K 2317/76 20130101; C07K 16/2827
20130101; A61K 35/17 20130101; A61K 2039/507 20130101; A61K 47/6435
20170801; C07K 2317/732 20130101; C07K 16/2866 20130101; C07K
16/2818 20130101; A61K 47/60 20170801; A61K 47/6883 20170801 |
International
Class: |
A61K 35/17 20060101
A61K035/17; A61K 47/64 20060101 A61K047/64; A61P 35/00 20060101
A61P035/00; A61K 47/60 20060101 A61K047/60; A61K 47/68 20060101
A61K047/68 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 29, 2017 |
JP |
2017-065603 |
Sep 27, 2017 |
JP |
2017-185935 |
Claims
1. A method of treating cancer in a subject in need thereof,
comprising administering to the subject a therapeutically effective
amount of a pharmaceutical composition comprising an antibody
against CCR8.
2. (canceled)
3. The method according to claim 1, wherein the cancer is breast
cancer.
4. The method according to claim 1, wherein the cancer is
colorectal cancer.
5. The method according to claim 1, wherein the cancer is kidney
cancer.
6. The method according claim 1, wherein the cancer is sarcoma.
7. The method according to claim 1, wherein the cancer is lung
cancer.
8-10. (canceled)
11. The method according to claim 1, wherein the antibody is an IgG
antibody.
12. The method according to claim 1, wherein the antibody against
CCR8 is a CCR8-neutralizing antibody.
13. The method according to claim 1, wherein the therapeutically
effective amount is at a dose of 5 mg to 5000 mg.
14. The method according to claim 1, wherein the therapeutically
effective amount is at a dose of 10 mg to 500 mg.
15. The method according to claim 1, wherein the antibody is
administered intravenously.
16. The method according to claim 1, wherein the antibody is
administered intraperitoneally.
17. The method according to claim 1, wherein the antibody has
antibody-dependent cell-mediated cytotoxicity (ADCC) activity
against cells expressing CCR8.
18. The method according to claim 1, wherein in the administering
step, the only antibody administered is the antibody against
CCR8.
19. The method according to claim 1, wherein an Fc region of the
antibody against CCR8 is free from a fucose bond with
N-acetylglucosamine.
20. The method according to claim 1, further comprising
administering an anti-PD-1 antibody or an anti-PD-L1 antibody.
21. The method according to claim 1, wherein the method further
comprises administering at least one antibody selected from the
group consisting of nivolumab, pembrolizumab, atezolizumab,
avelumab, and durvalumab.
Description
TECHNICAL FIELD
[0001] The present invention relates to a pharmaceutical
composition for cancer treatment comprising an antibody against
CCR8.
BACKGROUND ART
[0002] Potent negative regulation mechanisms, including
immunosuppression, mediated by regulatory T cells (Treg cells) in
the tumor microenvironment are major obstacles to the treatment of
tumors (Non Patent Literature 1).
[0003] For example, CD4-positive Treg cells which infiltrate tumors
may be able to strongly inhibit antitumor immune response and may
become a major obstacle to effective cancer treatment.
[0004] Tumor immunosuppression mediated by CD4-positive
FoxP3-positive Treg cells has been sufficiently demonstrated in
animal tumor models. It has been reported that systemic (including
intratumoral) Treg cell removal produces an antitumor effect,
wherein the removal of approximately 50% tumor-infiltrating Treg
cells is not effective (Non Patent Literature 2).
[0005] It has been reported that the increased ratio of
CD4-positive CD25-positive Treg cells (cell population including
Treg cells) to the whole CD4-positive T cell population in humans
is intratumorally detected in patients with various cancers
including lung, breast, and ovary tumors, and the abundance ratio
correlates negatively with the survival probabilities of the
patients (Non Patent Literatures 3 to 8).
[0006] The removal of CD4-positive CD25-positive Treg cells from
tumors using an anti-CD25 antibody has been confirmed to produce an
antitumor effect. However, this removal is not specific for the
Treg cells because CD25 is expressed on the cell surface of the
CD4-positive CD25-positive Treg cells as well as newly activated
effector T cells. Furthermore, the administration of an anti-CD25
antibody to mice brings about a limited antitumor effect. It has
been demonstrated in various tumor models that only the antibody
administration before tumor inoculation exhibits a therapeutic
effect, whereas the administration of the antibody after tumor
engraftment in mice rarely produces a therapeutic effect. The
antitumor effect was attenuated in the case of starting the
administration of an anti-CD25 antibody at post-transplant day 1,
and was rarely observed in the case of starting the administration
of an anti-CD25 antibody at post-transplant day 2 or later (Non
Patent Literature 9).
[0007] Drug efficacy tests have been carried out so far by
administering antibodies to mice for the purpose of removing Treg
cells. Nonetheless, there are few reports showing an antitumor
effect. Thus, it is very difficult to confirm an antitumor
therapeutic effect brought about by Treg cell removal by antibody
administration before inoculation (Non Patent Literature 10).
[0008] CCR8, also previously called CY6, CKR-L1 or TER1, is a G
protein-coupled 7-transmembrane CC chemokine receptor protein
expressed in the thymus, the spleen, etc. A gene encoding this
protein resides on human chromosome 3p21. Human CCR8 consists of
355 amino acids (Non Patent Literature 11). CCL1 is known as an
endogenous ligand for CCR8 (Non Patent Literature 12). Human CCR8
cDNA is constituted by the nucleotide sequence represented by
GenBank ACC No. M_005201.3, and mouse CCR8 cDNA is constituted by
the nucleotide sequence represented by GenBank ACC No.
NM_007720.2.
CITATION LIST
Non Patent Literature
[Non Patent Literature 1]
[0009] Nat. Rev. Immunol., 2006, Vol. 6, No. 4, p. 295-307
[Non Patent Literature 2]
[0009] [0010] Eur. J. Immunol., 2010, Vol. 40, p. 3325-3335
[Non Patent Literature 3]
[0010] [0011] J. Clin. Oncol., 2006, Vol. 24, p. 5373-5380
[Non Patent Literature 4]
[0011] [0012] Nat. Med., 2004, Vol. 10, p. 942-949
[Non Patent Literature 5]
[0012] [0013] J. Clin. Oncol., 2007, Vol. 25, p. 2586-2593
[Non Patent Literature 6]
[0013] [0014] Cancer, 2006, Vol. 107, p. 2866-2872
[Non Patent Literature 7]
[0014] [0015] Eur. J. Cancer, 2008, Vol. 44, p. 1875-1882
[Non Patent Literature 8]
[0015] [0016] Cell. Mol. Immunol. 2011, Vol. 8, p. 59-66
[Non Patent Literature 9]
[0016] [0017] Cancer Res., 1999 Jul. 1; Vol. 59, No. 13, p.
3128-33
[Non Patent Literature 10]
[0017] [0018] Cancer Res., 2010, Vol. 70, No. 7, p. 2665-74
[Non Patent Literature 11]
[0018] [0019] J. Immunol., 1996, Vol. 157, No. 7, p. 2759-63
[Non Patent Literature 12]
[0019] [0020] J. Biol. Chem., 1997, Vol. 272, No. 28, p.
17251-4
SUMMARY OF INVENTION
Technical Problem
[0021] An object of the present invention is to activate the
immunity by inhibiting immunosuppression mediated by Treg cells or
the like and to provide a pharmaceutical composition for cancer
treatment via this mechanism.
Solution to Problem
[0022] The present inventors have conducted diligent studies and
consequently completed the present invention by finding that
tumor-infiltrating Treg cells and tumor-infiltrating macrophage
cells specifically express CCR8, and the administration of an
antibody against CCR8 decreases the cell counts of the
tumor-infiltrating Treg cells and the tumor-infiltrating macrophage
cells and inhibits tumor growth.
[0023] Specifically, the present invention relates to:
(1) a pharmaceutical composition for cancer treatment, comprising
an antibody against CCR8; (2) the pharmaceutical composition
according to (1), wherein the antibody against CCR8 is an antibody
having ADCC activity; (3) the pharmaceutical composition according
to (1) or (2), wherein the antibody against CCR8 is a
CCR8-neutralizing antibody; (4) the pharmaceutical composition
according to any one of (1) to (3), wherein the antibody against
CCR8 has an effect of removing tumor-infiltrating Treg cells; (5)
the pharmaceutical composition according to any one of (1) to (4),
wherein the antibody against CCR8 has an effect of removing
tumor-infiltrating macrophage cells; (6) the pharmaceutical
composition according to any one of (1) to (5), wherein the cancer
is breast cancer, colorectal cancer, kidney cancer or sarcoma; (7)
a medicament for cancer treatment, comprising a combination of an
antibody against CCR8 and an anti-PD-1 antibody or an anti-PD-L1
antibody; (8) a method for treating a cancer, comprising
administering an antibody against CCR8 according to any of one (1)
to (5); (8-1) a method for treating a cancer, comprising
administering an antibody against CCR8; (8-2) the method according
to (8-1), wherein the antibody against CCR8 is an antibody having
ADCC activity; (8-3) the method according to (8-1) or (8-2),
wherein the antibody against CCR8 is a CCR8-neutralizing antibody;
(8-4) the method according to any one of (8-1) to (8-3), wherein
the antibody against CCR8 has an effect of removing
tumor-infiltrating Treg cells; (8-5) the method according to any
one of (8-1) to (8-4), wherein the antibody against CCR8 has an
effect of removing tumor-infiltrating macrophage cells; (8-6) the
method according to any one of (8-1) to (8-5), wherein the cancer
is breast cancer, colorectal cancer, kidney cancer or sarcoma;
(8-7) the method according to any one of (8-1) to (8-6), further
administering an anti-PD-1 antibody or an anti-PD-L1 antibody; (9)
the antibody against CCR8 according to any one of (1) to (5) for
treating a cancer; (9-1) an antibody against CCR8 for treating a
cancer; (9-2) the antibody against CCR8 according to (9-1), wherein
the antibody against CCR8 is an antibody having ADCC activity;
(9-3) the antibody against CCR8 according to (9-1) or (9-2),
wherein the antibody against CCR8 is a CCR8-neutralizing antibody;
(9-4) the antibody against CCR8 according to any one of (9-1) to
(9-3), wherein the antibody against CCR8 has an effect of removing
tumor-infiltrating Treg cells; (9-5) the antibody against CCR8
according to any of one (9-1) to (9-4), wherein the antibody
against CCR8 has an effect of removing tumor-infiltrating
macrophage cells; (9-6) the antibody against CCR8 according to any
of one (9-1) to (9-5), wherein the cancer is breast cancer,
colorectal cancer, kidney cancer or sarcoma; and (9-7) a
combination of an antibody against CCR8 according to any of one
(9-1) to (9-6) and an anti-PD-1 antibody or an anti-PD-L1 antibody
for use in the treatment of a cancer.
Advantageous Effects of Invention
[0024] A pharmaceutical composition comprising the antibody of the
present invention is pharmaceutically very useful for the treatment
of cancers.
BRIEF DESCRIPTION OF DRAWINGS
[0025] FIG. 1 shows results of FACS analysis on kidney cancer
tumor-infiltrating CD3+ CD4+ T cells. A CD25 molecule and a FoxP3
molecule were each stained with an antibody and evaluated for their
expression rates. CD25-expressing cells were found to also express
FoxP3.
[0026] FIG. 2 shows results of flow cytometry analysis on CD45RA
and CD25 expression intensity in peripheral blood mononuclear cells
(hereinafter, referred to as PBMCs) of the same patient. CD3+ CD4+
T cells were fractionated into 6 fractions (Fr1 to Fr6) as shown in
the drawing according to CD45RA and CD25 expression levels, and
cells in each fraction were recovered using a sorter. The numeric
values denote the cell abundance ratio (%) of each fraction. In
this case, Treg fractions are Fr1 and Fr2.
[0027] FIG. 3 shows results of flow cytometry analysis on CD45RA
and CD25 expression intensity in kidney cancer tumor-infiltrating
cells. Tumor-infiltrating CD3+ CD4+ T cells were fractionated into
4 fractions (Fr2 to Fr5) as shown in the drawing according to
CD45RA and CD25 expression levels, and cells in each fraction were
recovered using a sorter. The numeric values denote the cell
abundance ratio (%) of each fraction.
[0028] FIG. 4 shows results of conducting the RNA-Seq analysis of
cells in each of the fractions of FIGS. 2 and 3 and studying
whether any of these fractions would contain Treg cells on the
basis of the mRNA expression levels of Treg-specific expressed
genes FoxP3 and IKZF2. The ordinate depicts a relative mRNA
expression level after normalization. The strong intratumoral
expression of both the genes was observed in Fr2 and Fr3. The
strong expression of IL-2 or IFN.gamma., which is expressed in
effector cells, was observed in Fr4 and Fr5.
[0029] FIG. 5 shows results of analysis on a Treg-specific
demethylation region (chrX, 49118000-49118500, hg19) at a FoxP3
gene locus in each fraction. Most of tumor-infiltrating CD3+ CD4+ T
cells in Fr2 and Fr3 fractions were found to be Treg cells.
[0030] FIG. 6 shows results analyzing the mRNA expression level of
CCR8 in each fraction in the same way as in FIG. 4.
Tumor-infiltrating Treg cell fractions Fr2 and Fr3 exhibited the
strong expression of CCR8, wherein the expression was rarely
observed in Treg cells in peripheral blood mononuclear cells
(PBMCs).
[0031] FIG. 7 shows results of flow cytometry analysis on HEK293
cells expressing mouse CCR8. HEK293 cells were transfected with a
pcDNA3.4 expression vector having an insert of the mouse CCR8 gene
and drug-selected using G418. As for the degree of mouse CCR8
expression, the expression was confirmed with a PE-labeled
anti-mouse CCR8 antibody. HEK293 cells transfected with a pcDNA3.4
vector and drug-selected in the same way as above were used as a
negative control. Almost all the cells were found to express mouse
CCR8.
[0032] FIG. 8 shows that an anti-mouse CCR8 antibody (SA214G2) has
the ability to activate a signaling pathway necessary for
antibody-dependent cell mediated cytotoxicity (ADCC).
[0033] FIG. 9 shows that the anti-mouse CCR8 antibody (SA214G2) has
ADCC activity.
[0034] FIG. 10 shows that the anti-mouse CCR8 antibody (SA214G2)
has activity of inhibiting intracellular calcium influx mediated by
CCR8. An isotype control antibody was used as a negative
control.
[0035] FIG. 11 shows that the anti-mouse CCR8 antibody (SA214G2)
does not recognize CT26 cells. An isotype control antibody was used
as a negative control.
[0036] FIG. 12 shows results of administering a control antibody at
post-transplant day 3 to three BALB/c mice in which mouse
colorectal cancer cell line CT26 cells were transplanted, excising
tumors at post-administration day 4 or 7, and analyzing the
proportion of Treg cells present therein using a flow
cytometer.
[0037] FIG. 13 shows results of analyzing the proportion of CCR8+
Treg cells using a flow cytometer in the same experiment as in FIG.
12.
[0038] FIG. 14 shows results of analyzing the proportion of
CCR8-positive cells in intratumoral CD11b+ Gr1+ CD206+ M2
macrophage cells using a flow cytometer. In both cases, 40 to 50%
cells were found to be CCR8-positive M2 macrophage cells.
[0039] FIG. 15 shows the flow of an experiment of administering the
anti-mouse CCR8 antibody (SA214G2) or an isotype control antibody
at post-transplant day 3 to BALB/c mice in which colorectal cancer
cell line CT26 cells were transplanted, excising tumors at
post-transplant day 7 or 10, and examining the abundance ratios of
T lymphocytes and macrophage cells present therein.
[0040] FIG. 16 shows the ratio of CD25+ FoxP3+ cells to CD45+ CD4+
cells at post-transplant day 7 (d7) or 10 (d10).
[0041] FIG. 17 shows the proportion of CD11b+F4/80+ macrophage
cells at post-transplant day 7 (d7).
[0042] FIG. 18 shows the abundance ratio of IA/IE-positive (IA/IE+)
or IA/IE-negative cells (IA/IE-) at post-transplant day 7 (d7).
[0043] FIG. 19 shows the flow of an experiment of administering the
anti-mouse CCR8 antibody (SA214G2) or an isotype control antibody
(rat anti-KLH) at a single dose of 400 .mu.g/mouse at
post-transplant day 3 (d3) to BALB/c mice in which colorectal
cancer cell line CT26 cells were transplanted, and measuring a
tumor size every 3 to 4 days from post-transplant day 7 (d7) up to
day 21 (d21).
[0044] FIG. 20 shows results of measuring the solid tumor size of
each individual after inoculation and calculating a tumor
volume.
[0045] FIG. 21 shows the mean tumor volume of each mouse group at
each point in time after inoculation. A standard deviation is also
shown. Significance level *** denotes p<0.001, and significance
level ** denotes p<0.01 (t-test).
[0046] FIG. 22 2.times.10.sup.5 colorectal cancer cell line Colon26
cells were intracutaneously transplanted to the back of each BALB/c
mouse. At post-transplant day 3 (d3), the anti-mouse CCR8 antibody
(SA214G2) or an isotype control antibody was administered at a
single dose of 400 .mu.g/mouse. A tumor volume was measured every 3
to 4 days from post-transplant day 3 (d3) up to day 18 (d18). The
mean tumor volume of each group at each point in time after
inoculation is shown.
[0047] FIG. 23 The plot shows the mean fluorescence intensity (MFI)
of each individual in FACS analysis. The central horizontal lines
depict the mean MFI of 14 cases, and the vertical lines depict
standard deviations. Significance level *** denotes P<0.001.
[0048] FIG. 24 shows an individual-based plot of the ratio of cells
that exhibited CCR8-positive signals (percent positivity) equal to
or larger than a background level obtained in an isotype control
antibody, to CD3+ CD4+ FoxP3+ T cells or CD3+ CD4+ FoxP3- T cells
within the human kidney cancer tumors of 14 cases. The central
horizontal lines depict the mean percent positivity of the 14
cases, and the vertical lines depict standard deviations.
[0049] FIG. 25 shows a Kaplan-Meier curve as to the survival
probability of each group obtained by equally dividing clear cell
renal cell carcinoma patients into 2 groups with high expression
(High) and with low expression (Low) on the basis of the CCR8 mRNA
expression levels of intratumoral cells through the use of The
Cancer Genome Atlas (TCGA) database. The ordinate depicts the
survival probability, and the abscissa depicts the number of
months. The numeric values denote the number of individuals in each
group. The P value denotes a log-rank test value.
[0050] FIG. 26 shows results of analyzing prostate cancer patients
in the same way as in FIG. 25.
[0051] FIG. 27 shows results of analyzing bladder cancer patients
in the same way as in FIG. 25.
[0052] FIG. 28 shows that the anti-mouse CCR8 antibody recognizes
neither MethA cells nor LM8 cells, as in FIG. 11. An isotype
control antibody (Isotype) was used as a negative control.
[0053] FIG. 29 3.times.10.sup.5 osteosarcoma cell line LM8 cells
were intracutaneously transplanted to the back of each C3H/He
mouse. At post-transplant day 3 (d3), the anti-mouse CCR8 antibody
(SA214G2) or an isotype control antibody (Control antibody) was
administered at a single dose of 400 .mu.g/mouse. A tumor volume
was measured every 3 to 4 days from 7 days up to 35 days after
tumor inoculation. The mean tumor volume of each group at each
point in time after inoculation is shown. A standard deviation is
also shown. Significance level *** denotes p<0.001, significance
level ** denotes p<0.01, and significance level * denotes
p<0.05 (t-test).
[0054] FIG. 30 1.times.10.sup.5 MethA cells were intracutaneously
transplanted to the back of each Balb/c mouse. At post-transplant
day 3, the anti-mouse CCR8 antibody (SA214G2) or an isotype control
antibody (Control antibody) was administered at a single dose of
400 .mu.g/mouse. A tumor volume was measured every 3 to 4 days from
11 days up to 21 days after tumor inoculation. The mean tumor
volume of each group at each point in time after inoculation is
shown. Significance level * denotes p<0.05 (t-test).
[0055] FIG. 31 1.times.10.sup.5 breast cancer cell line EMT6 cells
were intracutaneously transplanted to the back of each Balb/c
mouse. At 3 and 10 days after tumor inoculation, the anti-mouse
CCR8 antibody (SA214G2) or an isotype control antibody was
administered at 100 .mu.g/mouse. A tumor volume was measured every
3 to 4 days from 4 days up to 22 days after tumor inoculation. The
mean tumor volume of each group at each point in time after
inoculation is shown. Significance level *** denotes p<0.001,
and significance level ** denotes p<0.01 (t-test).
[0056] FIG. 32 2.times.10.sup.5 colorectal cancer cell line Colon26
cells were intracutaneously transplanted to the back of each BALB/c
mouse. At 3 and 10 days after tumor inoculation, an anti-isotype
control antibody (Isotype antibody), the mouse CCR8 antibody
(SA214G2) or an anti-PD-1 antibody (RMP1-14) was administered at
400 .mu.g/mouse. A tumor volume was measured every 3 to 4 days from
3 days up to 24 days after tumor inoculation. The mean tumor volume
of each group at each point in time after inoculation is shown.
[0057] FIG. 33 4.times.10.sup.5 mouse kidney cancer-derived cell
line RAG cells were intracutaneously transplanted to the back of
each BALB/c mouse. 6 days after tumor inoculation, 100 .mu.g (100
.mu.L) of an isotype control antibody, the anti-mouse CCR8 antibody
or an anti-mouse PD-1 antibody (Anti-PD-1 antibody) was
intraperitoneally administered thereto. A tumor volume was measured
every 3 to 4 days from 6 days up to 21 days after tumor
inoculation. The mean tumor volume of each group at each point in
time after inoculation is shown.
[0058] FIG. 34 2.times.10.sup.5 colorectal cancer cell line Colon26
cells were intracutaneously transplanted to the back of each BALB/c
mouse. At 3 and 10 days after tumor inoculation, the anti-mouse
CCR8 antibody (SA214G2) or an isotype control antibody (Control
antibody) was administered at 400 .mu.g/mouse. 24 days after tumor
inoculation, each organ was recovered from the mice, and its weight
was measured. The mean of 10 cases in each group is shown.
[0059] FIG. 35 1.times.10.sup.5 mouse breast cancer cell line EMT6
cells were intracutaneously transplanted to the back of each BALB/c
mouse. The anti-mouse CCR8 antibody was intravenously administered
thereto at 3 and 10 days after tumor inoculation, and an anti-mouse
PD-1 antibody was intravenously administered thereto at 8 and 13
days after tumor inoculation. An isotype control antibody was
intravenously administered to a control group at 3 and 10 days
after tumor inoculation. A tumor volume was measured every 3 to 4
days from 6 days up to 27 days after inoculation. The mean tumor
volume of each group at each point in time after inoculation is
shown.
[0060] FIG. 36 shows the proportion of an individual bearing tumor
larger than 50 mm.sup.3 or smaller at each point in time after
inoculation in each group in the same experiment as in FIG. 35.
[0061] FIG. 37 4.5.times.10.sup.5 mouse kidney cancer-derived cell
line RAG cells were intracutaneously transplanted to the back of
each BALB/c mouse. 8 and 15 days after tumor inoculation, 100 .mu.L
of physiological saline, the anti-mouse CCR8 antibody or an
anti-mouse PD-1 antibody, or the anti-mouse CCR8 antibody and the
anti-mouse PD-1 antibody was intravenously administered thereto. A
tumor volume was measured every 3 to 4 days from 8 days up to 33
days after tumor inoculation. The median tumor volume of each group
at each point in time after inoculation is shown.
[0062] FIG. 38 2.times.10.sup.5 CT26 cells were intracutaneously
transplanted to the back of each wild-type mouse or homozygously
CCR8 gene-deficient mouse of Balb/c lineage (N=5). After
inoculation, an isotype control antibody or the anti-mouse CCR8
antibody was intravenously administered thereto. A tumor volume was
measured every 3 to 4 days after tumor inoculation. The left
diagram shows the mean tumor volume of the wild-type mice in each
group at each point in time after inoculation, and the right
diagram shows the mean tumor volume of the homozygously CCR8
gene-deficient mice in each group at each point in time after
inoculation.
DESCRIPTION OF EMBODIMENTS
[0063] The pharmaceutical composition of the present invention
comprises an antibody against CCR8.
[0064] The CCR8 of the present invention includes those derived
from mice, rats, hamsters, guinea pigs, dogs, pigs, and primate
mammals including monkeys and humans. Human CCR8 is preferred.
[0065] The antibody against CCR8 may be any of a human-derived
antibody, a mouse-derived antibody, a rat-derived antibody, a
rabbit-derived antibody and a goat-derived antibody as long as the
antibody binds to CCR8. The antibody against CCR8 may be a
polyclonal or monoclonal antibody thereof and may be any of a
complete antibody, an antibody fragment (e.g., a F(ab').sub.2,
Fab', Fab or Fv fragment), a chimeric antibody, a humanized
antibody and a complete human antibody. A human-derived antibody, a
humanized antibody or a complete human antibody is preferred.
[0066] The antibody of the present invention can be produced
according to an antibody or antiserum production method known in
the art using a full-length protein or a partial protein of CCR8 as
an antigen. Desirably, the antibody of the present invention binds
to CCR8 expressed on cell surface. Therefore, the partial protein
is desirably an extracellular region of CCR8. These antigens can be
prepared by protein expression and purification methods known in
the art.
[0067] Examples of the antigen, other than those described above,
suitable for the preparation of the antibody against CCR8 include
cells forced to express CCR8 by an expression vector or the like,
CCR8 expression plasmid vectors, and CCR8 expression virus vectors
(adenovirus vectors, etc.).
[0068] The polyclonal antibody can be produced by a method known in
the art. The polyclonal antibody can be produced, for example, by
immunizing an appropriate animal with an antigenic protein or a
mixture thereof with a carrier protein, and harvesting a product
containing an antibody against the antigenic protein from the
immunized animal, followed by the separation and purification of
the antibody. Examples of the animal used generally include mice,
rats, sheep, goats, rabbits, and guinea pigs. In order to enhance
the ability to produce antibodies, a complete Freund's adjuvant or
an incomplete Freund's adjuvant can be administered together with
the antigenic protein. In general, the administration is performed
a total of approximately 3 to 10 times, usually once every
approximately 2 weeks. The polyclonal antibody can be harvested
from the blood, ascitic fluid, or the like of the animal immunized
by the method described above. A polyclonal antibody titer in
antiserum can be measured by ELISA. The separation and purification
of the polyclonal antibody can be performed according to an
immunoglobulin separation and purification method, for example, a
purification method using an antigen binding solid phase or an
active adsorbent such as protein A or protein G, a salting-out
method, an alcohol precipitation method, an isoelectric
precipitation method, electrophoresis, an adsorption and desorption
method using an ion exchanger, an ultracentrifugation method, or a
gel filtration method.
[0069] The monoclonal antibody can be prepared by a known general
production method. Specifically, a mammal, preferably a mouse, a
rat, a hamster, a guinea pig or a rabbit, is immune-sensitized with
the antigen of the present invention, if necessary, together with a
Freund's adjuvant, by subcutaneous, intramuscular, intravenous,
intra-footpad or intraperitoneal injection once to several times.
Usually, immunization was performed once to 4 times every
approximately 1 to 21 days from initial immunization, and
antibody-producing cells can be obtained from the immune-sensitized
mammal approximately 1 to 10 days after the final immunization. The
number of immunizations and the time interval can be appropriately
changed according to the properties, etc. of the immunogen
used.
[0070] Hybridomas secreting the monoclonal antibody can be prepared
according to the method of Kohler and Milstein (Nature, 1975, vol.
256, p. 495-497) and a method equivalent thereto. Specifically, the
hybridomas can be prepared by the cell fusion of antibody-producing
cells contained in the spleen, the lymph node, the bone marrow or
the tonsil, etc., preferably the spleen, obtained from a mammal
immune-sensitized as mentioned above, with preferably mouse-, rat-,
guinea pig-, hamster-, rabbit- or mammal (e.g. human)-derived, more
preferably mouse-, rat- or human-derived myeloma cells lacking the
ability to produce autologous antibodies.
[0071] In general, an established cell line obtained from mice, for
example, P3-U1, NS-1, SP-2, 653, X63, or AP-1, can be used as the
myeloma cells for use in the cell fusion.
[0072] A hybridoma clone producing the monoclonal antibody is
screened for by culturing the hybridomas, for example, in a
microtiter plate, measuring the reactivity of a culture supernatant
in a well where growth is seen, with the antigen of the present
invention used in the mouse immune sensitization mentioned above by
a measurement method such as RIA, ELISA, or FACS, and selecting a
clone producing the monoclonal antibody that exhibits specific
binding to the antigen or hapten. Usually, a method is further used
which involves immobilizing the antigen on a solid phase, and
detecting an antibody in a culture supernatant binding thereto
using a secondary antibody labeled with a radioactive material, a
fluorescent material, an enzyme, or the like. In the case of using
antigen-expressing cells, the hybridoma culture supernatant is
added to the cells, and a fluorescently labeled secondary antibody
can then be reacted therewith, followed by the measurement of
fluorescence intensity of the cells using a fluorescent detection
apparatus such as a flow cytometer to detect a monoclonal antibody
capable of binding to the antigen of the present invention on the
membranes of the cells.
[0073] The monoclonal antibody can be produced from the selected
hybridoma by culturing the hybridoma in vitro or culturing the
hybridoma in the ascitic fluid or the like of a mouse, a rat, a
guinea pig, a hamster or a rabbit, etc., preferably a mouse or a
rat, more preferably a mouse, and isolating the monoclonal antibody
from the obtained culture supernatant or ascetic fluid of the
mammal. For the in vitro culture, the hybridoma is grown,
maintained and preserved according to various conditions such as
the characteristics of the cell type to be cultured, the purpose of
a test and research and a culture method and can be cultured using
a known nutrient medium as used for producing monoclonal antibodies
into a culture supernatant, or every nutrient medium induced and
prepared from a known basal medium.
[0074] Examples of the basal medium include low-calcium media such
as Ham' F12 medium, MCDB153 medium and low-calcium MEM medium, and
high-calcium media such as MCDB104 medium, MEM medium, D-MEM
medium, RPMI1640 medium, ASF104 medium and RD medium. The basal
medium can contain, for example, serum, hormone, cytokine and/or
various inorganic or organic substances, according to a
purpose.
[0075] The monoclonal antibody can be isolated and purified, for
example, by subjecting the culture supernatant or the ascetic fluid
mentioned above to saturated ammonium sulfate, ion-exchange
chromatography (DEAE or DE52, etc.), or affinity column
chromatography using an anti-immunoglobulin column, a protein A
column, or the like.
[0076] A recombinant antibody obtained by cloning an antibody gene
from antibody-producing cells, for example, hybridomas, integrating
the antibody gene into an appropriate vector, and transfecting a
host with this vector, followed by production by use of a gene
recombination technique can be used as the antibody of the present
invention (e.g., Carl et al., THERAPEUTIC MONOCLONAL ANTIBODIES,
published in 1990).
[0077] Specifically, mRNA encoding the variable region (V region)
of the antibody is isolated from hybridomas producing the antibody
of interest or immunocytes producing the antibody, for example,
cells of sensitized lymphocytes immortalized with an oncogene or
the like. For the mRNA isolation, total RNA is prepared by a method
known in the art, for example, a guanidine ultracentrifugation
method (Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299),
and the mRNA is prepared using mRNA Purification Kit (manufactured
by Pharmacia Inc.) or the like.
[0078] cDNA of the antibody V region is synthesized from the
obtained mRNA using reverse transcriptase. The synthesis of the
cDNA can be performed using AMV Reverse Transcriptase First-strand
cDNA Synthesis Kit or the like. 5'-Ampli FINDER RACE Kit
(manufactured by Clontech Laboratories, Inc) and PCR-based 5'-RACE
(Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA, 1988, Vol. 85,
p. 8998, etc.) can be used for cDNA synthesis and amplification.
The DNA fragment of interest is purified from the obtained PCR
product and ligated with vector DNA. A recombinant vector is
further prepared therefrom. E. coli or the like is transfected with
the recombinant vector, and a colony is selected to prepare the
desired recombinant vector. The nucleotide sequence of the DNA of
interest is confirmed by a method known in the art, for example, a
deoxy method.
[0079] Provided that the DNA encoding the V region of the antibody
of interest is successfully obtained, this DNA is linked to DNA
encoding the desired antibody constant region (C region) and the
resultant is integrated into an expression vector. Alternatively,
the DNA encoding the V region of the antibody may be integrated
into an expression vector containing the DNA of the antibody C
region. In order to produce the antibody used in the present
invention, the antibody gene is integrated into an expression
vector such that the antibody gene is expressed under the control
of an expression control region, for example, enhancer/promoter.
Next, host cells can be transformed with this expression vector to
express the antibody.
[0080] For the expression of the antibody gene, DNA encoding the
heavy chain (H chain) and DNA encoding the light chain (L chain) of
the antibody may be separately integrated into expression vectors,
with which a host is co-transformed, or the DNA encoding the H
chain and the DNA encoding the L chain may be integrated into a
single expression vector, with which a host is transformed (see
WO94/11523).
[0081] A so-called phage display technique (Nature Biotechnology
23, 1105 (2005)) can also be used as a method, other than those
described above, for preparing the antibody of the present
invention. Specifically, for example, an antibody gene library
prepared by a method known in the art using human or animal (e.g.,
rabbit, mouse, rat, or hamster) B lymphocytes as a material, or an
antibody gene library completely synthesized by selection and
engineering from a human or animal germ line sequence is displayed
on, for example, bacteriophages, E. coli, yeast or animal cell
surface, or liposomes. In this respect, examples of the form of the
antibody to be displayed on the cell surface include IgG molecules,
IgM molecules, Fab fragments, and single-strand Fv (scFv)
fragments.
[0082] The antibody fragment gene thus obtained can be recombined
with a corresponding region of an IgG antibody gene by a method
known in the art to obtain an antibody gene. Then, the gene thus
obtained can be integrated into an appropriate vector, with which a
host is transfected, followed by the production of the antibody by
use of a gene recombination technique (e.g., Carl et al.,
THERAPEUTIC MONOCLONAL ANTIBODIES, published in 1990).
[0083] The antibody of the present invention includes antibodies
artificially engineered for the purpose of, for example, reducing
xenoantigenicity against humans, for example, chimeric antibodies,
humanized antibodies and complete human antibodies.
[0084] The antibody of the present invention may be a conjugated
antibody in which the antibody is bound with any of various
molecules such as polyethylene glycol (PEG), radioactive
substances, toxins, and sugar chains. Such a conjugated antibody
can be obtained by chemically modifying the obtained antibody. The
method for modifying the antibody has already been established in
the art. The antibody according to the present invention also
encompasses these conjugated antibodies.
[0085] The antibody of the present invention encompasses an
antibody having a Fc region bound with N-glycoside-linked sugar
chains which are free from a fucose bound with N-acetylglucosamine
at their reducing termini. Examples of the antibody having a Fc
region bound with N-glycoside-linked sugar chains which are free
from a fucose bound with N-acetylglucosamine at their reducing
termini include antibodies prepared using
.alpha.1,6-fucosyltransferase gene-deficient CHO cells
(International Publication Nos. WO 2005/035586 and WO 02/31140).
The antibody of the present invention having a Fc region bound with
N-glycoside-linked sugar chains which are free from a fucose bound
with N-acetylglucosamine at their reducing termini has high ADCC
activity.
[0086] The antibody of the present invention may be fused at its N
terminus or C terminus with an additional protein (Clinical Cancer
Research, 2004, 10, 1274-1281). The protein to be fused can be
appropriately selected by those skilled in the art.
[0087] The antibody fragment is a portion of the antibody of the
present invention mentioned above and means a fragment having
CCR8-specific binding activity as in the antibody. Examples of the
antibody fragment can specifically include Fab, F(ab').sub.2, Fab',
single-strand antibody (scFv), disulfide-stabilized antibody
(dsFv), dimerized V region fragment (diabody), and CDR-containing
peptides (Expert Opinion on Therapeutic Patents, Vol. 6, No. 5, p.
441-456, 1996).
[0088] Alternatively, the antibody of the present invention may be
a bispecific antibody which has two different antigenic
determinants and binds to different antigens.
[0089] The ADCC (antibody-dependent cell mediated cytotoxicity)
activity means in vivo activity of damaging tumor cells or the like
by activating effector cells via the binding of the Fc region of
the antibody bound with a cell surface antigen or the like on the
tumor cells or the like to a Fc receptor present on the effector
cell surface. Examples of the effector cells include natural killer
cells and activated macrophages.
[0090] The antibody of the present invention is preferably an
antibody having ADCC activity against cells expressing CCR8 because
this antibody can remove Treg cells or macrophage cells. Whether or
not the antibody of the present invention has such ADCC activity
can be measured by, for example, a method described in Examples
mentioned later.
[0091] The antibody against CCR8 contained in the pharmaceutical
composition of the present invention is preferably a
CCR8-neutralizing antibody from the viewpoint of suppressing the
intratumoral accumulation of Treg cells or macrophage cells. The
CCR8-neutralizing antibody means an antibody having neutralizing
activity against CCR8. Whether or not the antibody of the present
invention has neutralizing activity against CCR8 can be determined
by measuring the presence or absence of suppression of the
physiological effect of CCL1 on CCR8. Examples thereof include, but
are not limited to, the measurement of the binding of CCL1 to CCR8,
the migration of CCR8-expressing cells by CCL1, increase in
intracellular Ca.sup.++ level by CCL1, and variation in the
expression of a gene sensitive to CCL1 stimulation. This can also
be determined by a method described in Examples mentioned
later.
[0092] The antibody against CCR8 of the present invention
preferably has an effect of removing tumor-infiltrating Treg cells.
Whether or not the antibody of the present invention has the effect
of removing tumor-infiltrating Treg cells can be determined by, for
example, a method described in Examples mentioned later.
[0093] The antibody against CCR8 of the present invention
preferably has an effect of removing tumor-infiltrating macrophage
cells. Whether or not the antibody of the present invention has the
effect of removing tumor-infiltrating macrophage cells can be
determined by, for example, a method described in Examples
mentioned later.
[0094] The antibody of the present invention is useful as a
pharmaceutical composition. Thus, the pharmaceutical composition
comprising the antibody of the present invention can be
administered orally or parenterally and systemically or locally.
For example, intravenous injection such as infusion, intramuscular
injection, intraperitoneal injection, subcutaneous injection,
transnasal administration, or inhalation can be selected as
parenteral administration.
[0095] The "cancer" for the "pharmaceutical composition for cancer
treatment" of the present invention includes every solid cancer and
blood cancer. Specifically, examples thereof include breast cancer,
uterine corpus cancer, cervical cancer, ovary cancer, prostate
cancer, lung cancer, stomach cancer (gastric adenocarcinoma),
non-small cell lung cancer, spleen cancer, head and neck squamous
cell carcinoma, esophageal cancer, bladder cancer, melanoma,
colorectal cancer, kidney cancer, non-Hodgkin lymphoma, urothelial
cancer, sarcoma, blood cell carcinoma (leukemia, lymphoma etc.),
bile duct carcinoma, gallbladder carcinoma, thyroid carcinoma,
prostate cancer, testicular carcinoma, thymic carcinoma, and
hepatocarcinoma. Preferably, examples thereof include breast
cancer, uterine corpus cancer, ovary cancer, lung cancer,
colorectal cancer, kidney cancer and sarcoma, and more preferably,
examples thereof include breast cancer, colorectal cancer, kidney
cancer, and sarcoma.
[0096] The "cancer" for the "pharmaceutical composition for cancer
treatment" of the present invention is preferably a cancer
expressing a tumor-specific antigen.
[0097] The "cancer" described in the present specification means
not only epithelial malignant tumors such as ovary cancer and
stomach cancer but non-epithelial malignant tumors including
hematopoietic cancers such as chronic lymphocytic leukemia and
Hodgkin lymphoma. In the present specification, terms such as
"cancer", "carcinoma", "tumor", and "neoplasm" can be used
interchangeably with each other without differentiating
thereamong.
[0098] The antibody against CCR8 of the present invention may be
administered as a concomitant drug in combination with an
additional drug in order to
(1) complement and/or potentiate the therapeutic effect of the
pharmaceutical composition of the present invention, (2) improve
the pharmacokinetics and absorption of the pharmaceutical
composition of the present invention, and reduce the dose thereof,
and/or (3) reduce the adverse reaction of the pharmaceutical
composition of the present invention.
[0099] The concomitant drug of the antibody against CCR8 of the
present invention and an additional drug may be administered in the
form of a combination drug containing both the ingredients in one
preparation or may be administered in the form of separate
preparations. This administration as separate preparations includes
concurrent administration and staggered administration. For the
staggered administration, the antibody of the present invention may
be administered first, and the additional drug may be administered
later, or the additional drug may be administered first, and the
compound of the present invention may be administered later. Their
respective administration methods may be the same or different.
[0100] Examples of the additional drug that may be used in
combination with the antibody against CCR8 of the present invention
include anti-PD-1 antibodies, anti-PD-L1 antibodies and anti-CTLA-4
antibodies. An anti-PD-1 antibody or an anti-PD-L1 antibody is
preferred, and an anti-PD-1 antibody is more preferred.
[0101] In the present invention, examples of the anti-PD-1 antibody
include nivolumab and pembrolizumab.
[0102] In the present invention, examples of the anti-PD-L1
antibody include atezolizumab, avelumab, and durvalumab.
[0103] In the present invention, examples of the anti-CTLA-4
antibody include ipilimumab.
[0104] The patient intended by the pharmaceutical composition of
the present invention is expected to be a cancer patient or a
patient suspected of having a cancer. The effective dose is
selected from the range of 0.01 mg to 100 mg per kg of body weight
per dose. Alternatively, the dose can be selected from 5 to 5000
mg, preferably 10 to 500 mg, per patient. However, the
pharmaceutical composition comprising the antibody of the present
invention or an antibody fragment thereof is not limited by these
doses. Also, the dosing period can be appropriately selected
according to the age and symptoms of the patient. The
pharmaceutical composition of the present invention may further
contain a pharmaceutically acceptable carrier or additive depending
on an administration route. Examples of such a carrier and additive
include water, pharmaceutically acceptable organic solvents,
collagen, polyvinyl alcohol, polyvinylpyrrolidone, sodium alginate,
water-soluble dextran, pectin, methylcellulose, ethylcellulose,
casein, diglycerin, propylene glycol, polyethylene glycol,
Vaseline, human serum albumin (HSA), mannitol, sorbitol, lactose,
and surfactants acceptable as pharmaceutical additives. The
additive used is selected appropriately or in combination from
among those described above according to a dosage form, though the
additive is not limited thereto.
[0105] Hereinafter, the present invention will be specifically
described with reference to Examples. However, the present
invention is not limited by Examples given below. Methods described
in Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring
Harbor Laboratory) were used as gene manipulation approaches unless
otherwise specified.
Example 1
Extraction and Analysis of Kidney Cancer Tumor-Infiltrating Cells
and PBMCs
[0106] The following analysis was conducted using a portion of
primary tumor tissues excised by surgical treatment from clear cell
renal cell carcinoma (ccRCC) patients (3 cases) who were not
preoperatively treated with an anticancer agent, radiation, or the
like. After tumor weight measurement, tumor masses were cut into 2
mm square with scissors, and tumor tissue homogenates were prepared
using Tumor Dissociation Kit, human (130-095-929, Miltenyi Biotec)
and gentleMACS.TM. Dissociator (Miltenyi Biotec, 130-093-235)
according to the protocol attached to the kit. The homogenates were
passed through a 70 um cell strainer and subjected to hemolysis
treatment, followed by the removal of debris and dead cells in a
solution of 30% Percoll in PBS to obtain tumor tissue single
cells.
[0107] Peripheral blood mononuclear cells (PBMCs) of the same
patient were separated from peripheral blood by the density
gradient centrifugation method using Ficoll-Paque PLUS (GE
Healthcare Japan Corp.). After cell count measurement, the
separated intratumoral cells and PBMCs were treated with Human
TruStain FcX.TM. (BioLegend, Inc., 422-301) and Zombie NIR.TM.
Fixable Viability kit (BioLegend, Inc., 423105) according to the
attached protocols and stained 30 minutes in ice. Then, the cells
were washed once with 2% FCS/HEPES/HBSS and then stained with the
following labeling antibodies according to the protocols attached
to the labeling antibodies.
[0108] The cell surface of tumor-infiltrating cells was stained
through reaction for 30 minutes in ice using an anti-CD3 antibody
(BioLegend, Inc., Clone UCHT1), an anti-CD4 antibody (BioLegend,
Inc., Clone OKT4), and an anti-CD25 antibody (BioLegend, Inc.,
Clone BC96). The cells were washed twice with 2% FCS/HEPES/HBSS and
then fixed and membrane-permeabilized using Foxp3/Transcription
Factor Staining Buffer Set (eBioscience, Inc., 00-5523-00)
according to the protocol attached to the kit. FoxP3 was further
stained using a PE-labeled anti-FoxP3 antibody (eBioscience, Inc.,
Clone PCH010). The cells were washed once with a washing solution
attached to the kit and then analyzed by flow cytometry (BD
Biosciences, BD LSRFortessa). Almost all the CD4+ CD25+ T cells
within the ccRCC tumors were confirmed to express FoxP3, a marker
of Treg cells (FIG. 1).
[0109] Subsequently, the tumor-infiltrating cells and the PBMCs
described above were stained with an anti-CD3 antibody, an anti-CD4
antibody, an anti-CD45RA antibody (BD Biosciences, Clone HI100) and
an anti-CD25 antibody. CD3+ CD4+ T cells were two-dimensionally
developed on the basis of CD45RA and CD25 expression levels. The
results about the PBMCs are shown in FIG. 2, and the results about
the tumor-infiltrating cells are shown in FIG. 3. The
tumor-infiltrating cells were fractionated into 4 fractions of
strongly positive cells (Fr2), weakly positive cells (Fr3), and
negative cells (Fr4 and Fr5) as shown in FIG. 1C with CD3+ CD4+
CD45RA- and CD25 expression intensity as an index using a cell
sorter (FACSAria II), and cells contained in each fraction were
recovered. The PBMCs were also two-dimensionally developed, as in
the tumor-infiltrating cells, and fractionated into Fr1 to Fr6 as
shown in FIG. 2 with CD45RA and CD25 expression intensity as an
index, and cells contained in each fraction were recovered.
Example 2
[0110] Separation of RNA from Fractionated Cells and cDNA Sequence
Analysis
[0111] The cells separated and recovered from each fraction were
lysed in RLT buffer (Qiagen N.V.), and total RNA was extracted
using Agencourt RNAClean XP (Beckman Coulter, Inc.). The recovered
RNA was prepared into cDNA using SMART-Seq v4 Ultra Low Input RNA
kit for Sequencing (Clontech Laboratories, Inc.), and a library was
prepared using KAPA Hyper Prep Kit for illumina (Kapa Biosystems,
Inc.). For the cDNA synthesis and the library preparation, quality
control was constantly performed using Agilent 2100 Bioanalyzer
(Agilent Technologies, Inc.) to confirm that these procedures were
free from problems. The finished cDNA library was titrated using a
KAPA library Quantification kit Illumina Platforms (Kapa
Biosystems, Inc.). Then, DNA sequencing was performed by paired end
reads using Hiseq 4000 (Illumina, Inc.) to obtain 20,000,000 reads
or more of 100-base pair sequence data per sample (Fastq file).
[0112] The raw data (Fastq file) was analyzed by FastQC, and
adaptor sequences and repeat sequences were removed using CutAdapt.
Pairs of each paired end read were matched using cmpfastq_pe
program. hg38 was used as a reference sequence in genome mapping,
and the reads were mapped onto the genome at default setting using
TOPHAT2 program having Bowtie 2. The mapped reads were
sequence-sorted using SAMtools program and counted using HTSEQ
program. The count data was normalized using Deseq 2 program. Among
the obtained fractions, a fraction containing Treg cells was
confirmed by the following method.
[0113] Treg cells are known to constitutively express FoxP3 and
Ikzf2 genes as marker genes and to rarely secrete IFN.gamma. or IL2
even when activated by stimulation. Whether or not to contain Treg
cells may be confirmed to some extent by examining the expression
levels of these genes. As a result of examining the expression
levels of these genes as to each fraction of the tumor-infiltrating
cells and the PBMCs on the basis of the RNA-Seq data described
above, Ikzf2 and FoxP3 were found to be specifically expressed in
Fr2 and Fr3 of the tumor-infiltrating cells and Fr2 of the PBMCs
and rarely expressed in the other fractions (FIG. 4). Also,
IFN.gamma. (IFN-gamma) and IL2 were found to be specifically
expressed in Fr4 and Fr5 of the tumor-infiltrating cells and Fr4
and Fr5 of the PBMC cells and not expressed in the other fractions
(FIG. 4). In conclusion, the Treg cells were found to be contained
in Fr2 and Fr3 of the tumor-infiltrating cells and Fr2 of the PBMCs
and not contained in the other fractions.
Example 31
Measurement of Demethylation Rate of FoxP3 Region
[0114] The demethylation rate of a FoxP3 region serves as an index
for accurately determining the proportion of Treg cells. Therefore,
the cells in Fr2 to Fr5 of the kidney cancer tumor-infiltrating
cells obtained as described above were studied for the
demethylation rate of the FoxP3 region. A region demethylated in a
Treg cell-specific manner resides (chrX, 49118000-49118500, hg19)
in a particular CpG region within the first intron of the FoxP3
gene. The cells contained in each fraction of the
tumor-infiltrating cells may be analyzed for the demethylation of
this region to verify whether the fraction obtained this time
consists of only Treg cells or other cells also coexist
therewith.
[0115] Each fraction (Fr2, Fr3, Fr4, and Fr5) of the
tumor-infiltrating CD4+ T cells was recovered, and genome DNA was
recovered by use of the phenol extraction method. The genome DNA
was treated with bisulfite using MethylEasy Xceed kit (Human
Genetic Signatures), and the FOXP3 intron 1 region (chrX,
49118000-49118500, hg19), a Treg cell-specific demethylation
region, was subjected to amplicon PCR. DNA methylation was detected
using a prepared methylated DNA-specific FAM fluorescent probe and
demethylation-specific VIC fluorescent probe and QuantStudio 3D
digital PCR system (Applied Biosystems, Inc.). After the amplicon
PCR, the numbers of light emissions from the FAM and VIC
fluorescent probes were counted, and the DNA methylation rate was
calculated from the ratio between these numbers of fluorescence
emissions and used as the methylation rate of each fraction (Fr2 to
Fr5).
[0116] As a result, 95% or more CpG sequences within the FOXP3
intron 1 region (chrX, 49118000-49118500) were demethylated in the
cells contained in Fr2 and Fr3 of the tumor-infiltrating cells,
whereas the demethylation rates of Fr4 and Fr5 were 50% or less. In
conclusion, almost all the cells contained in Fr2 and Fr3 were
found to be Treg cells (FIG. 5).
Example 4
Identification of CCR8
[0117] In order to identify a gene of one group specifically
expressed in the Treg cells (Fr2 of the tumor-infiltrating cells),
hierarchical clustering analysis was conducted on the gene
expression data on the PBMC-derived CD4+ T cell fraction of the
same patient as in each tumor-derived CD4+ T cell fraction. CCR8
was identified as a gene that was expressed in Fr2 of the Treg
cells and rarely expressed in tumor-derived Fr5 and Fr4 and Fr5 of
the PBMCs (FIG. 6).
Example 51
Preparation of Cells Forced to Express Mouse CCR8
[0118] Full-length ORF of mouse CCR8 (hereinafter, also referred to
as mCCR8) was inserted to an expression vector (pcDNA3.4) to
construct pcDNA3.4-mCCR8 plasmid. The nucleotide sequence was
changed to have codons with high usage frequency in mammals without
changing the amino acids. HEK293 cells were transfected with
pcDNA3.4 or the pcDNA3.4-mCCR8 expression plasmid using
Lipofectamine 3000 and drug-selected at a geneticin (G418)
concentration of 1 mg/ml for 2 weeks.
[0119] Surviving cells were dissociated with trypsin and washed
with DMEM/10% FCS medium. Then, a PE-labeled anti-mCCR8 antibody
(clone SA214G2) diluted 1/200 was added thereto and reacted on ice
for 30 minutes. Then, the cells were washed once with DMEM/10% FCS
to label mCCR8 expressed on the cell surface. A cell population
expressing mCCR8 was enriched by sorting using a cell sorter
(FACSAria II). The positive cell population was cultured at
37.degree. C. for 2 weeks in a CO2 incubator in the presence of
DMEM/10% FCS (medium containing 1 mg/ml G418). For the cells
transformed with pcDNA3.4, only drug selection was performed, and
sorting was not performed. In order to confirm expression, both the
cells were stained with a commercially available anti-PE-labeled
anti-mouse CCR8 antibody (clone SA214G2) and analyzed using a flow
cytometer (FACSAria II). The results are shown (FIG. 7). The
expression of mCCR8 was observed in 99% or more of the cells
transformed with pcDNA3.4-mCCR8 compared with the cells transformed
with pcDNA3.4.
Example 61
Study on Ability of Anti-Mouse CCR8 Antibody (SA214G2) to Stimulate
Fc.gamma.R
[0120] An anti-mouse CCR8 antibody (clone SA214G2, purchased from
BioLegend, Inc.) was evaluated for the ability to stimulate FcgR,
necessary for its ADCC activity, using mFc.gamma.RIV ADCC Reporter
Bioassays Core kit (Promega Corp.). This kit indicates the
activation of Fc.gamma.R on effector cells by the expression level
of luciferase gene linked downstream of NFAT promoter in the cells.
The activation of Fc.gamma.R signals can be quantified by
quantifying this expression level.
[0121] Hereinafter, the procedures will be briefly described.
1.times.10.sup.5 cells/well of mCCR8-expressing HEK293 target cells
(target cells) dissociated with trypsin were mixed with
Fc.gamma.R-expressing effector cells attached to the kit at a ratio
of 1:1.5 in a 96-well plate. Immediately after the cell mixing, the
antibody against mCCR8 was added thereto. The concentration was set
to 33 ug/ml to 0.033 ug/ml as shown in FIG. 8 (N=2). Only the
effector cells were used as a negative control. 14 hours after the
antibody addition, the cells were recovered, and the luciferase
activity was measured (FIG. 8). A mean of N=2 is shown.
[0122] As a result, the luciferase activity was not observed at any
of the antibody concentrations for the negative control, whereas
antibody concentration-dependent activity was observed in the
target cell addition group. The ordinate depicts a relative value
of luminescence intensity. As seen from FIG. 8, the largest
activity value was approximately 6000 relative light units (R.L.U),
and the EC50 value (approximately 3500 R.L.U) was approximately 0.1
.mu.g/ml (lines in the drawing). These results demonstrated that
the anti-mouse CCR8 antibody (SA214G2) can activate
Fc.gamma.RIV.
Example 71
Measurement of ADCC Activity
[0123] The anti-mCCR8 antibody (SA214G2) was evaluated for its
cytotoxic activity using the stably mCCR8-expressing HEK293 cells
prepared in Example 5.
[0124] The spleen of a C57BL/6 mouse was separated, and spleen
cells were recovered through a cell strainer. The cells were washed
and then reacted with a biotinylated anti-CD49b (clone DX5)
antibody at 4.degree. C. for 30 minutes. After washing, NK cells
were purified using streptavidin microbeads (Miltenyi Biotec) and
used as effector cells. The HEK293 cells expressing mouse CCR8 were
stained with Cell Trace Violet (CTV) (Thermo Fisher Scientific
Inc., C34557) at a final concentration of 2.5 uM and used as target
cells. These cells were mixed at a ratio of effector cells:target
cells=5:1 (effector cell count: 2.5.times.10.sup.5 cells) in a
96-well plate (200 .mu.L/well). The anti-mouse CCR8 antibody or an
isotype control antibody (rat IgG2b, clone RTK4530) was added
thereto at a final concentration of 1 .mu.g/ml, followed by
overnight culture in a CO.sub.2 incubator of 37.degree. C. Then,
PE-labeled annexin V (Annexin V-PE, Medical & Biological
Laboratories Co., Ltd. (MBL), 4696-100) diluted 1/100 was added
according to the attached protocol, and the cells were stained at
37.degree. C. for 30 minutes and then washed once. The proportion
of annexin V-positive apoptotic cells in the CTV-stained target
cells was analyzed using a flow cytometer. The test was carried out
in triplicate (N=3), and a mean and a standard deviation thereof
are shown. A typical example of two similar experiments is shown
(FIG. 9). The addition of the anti-mouse CCR8 antibody compared
with the isotype control antibody significantly increased the
proportion of annexin V-positive cells in the target cells by
approximately 6 times. In conclusion, the anti-mouse CCR8 antibody
(SA214G2) was found to have ADCC activity.
Example 81
Measurement of Neutralizing Activity Against CCR8
[0125] The anti-mouse CCR8 antibody (SA214G2) was evaluated for its
neutralizing activity against CCR8 with intracellular calcium
influx mediated by mouse CCL1 (ligand of mouse CCR8) as an index
using HEK293 cells stably expressing mouse CCR8.
[0126] The following reagents were used in calcium measurement.
HEPES (Wako Pure Chemical Industries, Ltd., CAS. NO. 7365-45-9)
HBSS(+) without Phenol Red (Wako Pure Chemical Industries, Ltd.)
Fluo 3-AM (cat F023, Dojindo Laboratories) Probenecid (CAS-No:
57-66-9, Nacalai Tesque, Inc.) Pluronic F127 (P3000MP; Life
Technologies Corp.) 10 mM HEPES/HBSS/0.1% BSA Buffer (HEPES (final
concentration: 10 mM) and BSA (final concentration: 0.1%) were
added to HBSS)
[0127] Fluo 3-AM and Pluronic F127 were dissolved at final
concentrations of 4 .mu.mol/L and 0.04%, respectively, in 10 mM
HEPES/HBSS Buffer. The cells were suspended in this solution and
incubated at 37.degree. C. for 1 hour so that Fluo 3-AM was taken
up by the cells. Then, the cells were washed three times with 10 mM
HEPES/HBSS/0.1% BSA solution and suspended at a cell concentration
of 2.times.10.sup.5 cells/ml in 10 mM HEPES/HBSS/0.1% BSA solution
containing 1.25 uM probenecid. Then, the cells were incubated at
37.degree. C. for 10 minutes in a CO2 incubator. The anti-mCCR8
antibody (SA214G2) or an isotype control antibody (Clone LTF-2, Bio
X Cell) was further added thereto at a concentration of 5 .mu.g/ml.
The cells were further incubated at 37.degree. C. for 20
minutes.
[0128] 2 mL of the solution of the cells was placed in a quartz
glass cuvette and loaded in a spectrophotometer HITACHI F7000 with
the temperature of a measurement room preset to 35.degree. C. The
measurement conditions were as described below.
Excitation wavelength: 508.0 nm, fluorescence (measurement)
wavelength: 527.0 nm, excitation-side slit: 5 nm, fluorescence-side
slit: 5 nm, photomultiplier voltage: 950 V, response: 0.5 s
[0129] The cells were incubated with stirring using a stirrer for
approximately 30 seconds until the fluorescence wavelength was
stabilized. When the wavelength was stabilized, mouse CCL1 was
added thereto at a final concentration of 50 nM (4 .mu.L) to start
measurement. As a result of the measurement, the administration of
the anti-mCCR8 antibody in advance was found to almost completely
suppress intracellular calcium influx mediated by mCCL1 (FIG. 10).
Such suppression was not observed by the addition of the control
antibody. The gaps in the graphs were derived from the opening and
closing of the cover of the instrument in order to administer the
agonist to the cells. In conclusion, the anti-mCCR8 antibody
(SA214G2) was found to have neutralizing activity against mouse
CCR8.
Example 91
[0130] Confirmation of Expression of mCCR8 in CT26
[0131] CT26 cells were cultured in a 6-well dish, and the culture
solution was removed when the cells became approximately 50%
confluent. 5 ml of 10 mM EDTA/PBS was added thereto, and the cells
were incubated at 37.degree. C. for 5 minutes. As a result, almost
all the cells were dissociated, suspended using a pipette and were
thereby able to be separated into almost single cells. The cells
were washed twice with D-MEM/10% FCS, suspended in D-MEM/10% FCS,
and stained in ice with LIVE/DEAD.RTM. Fixable Near-IR Dead Cell
Stain Kit (Thermo Fisher Scientific Inc., L34975) and an
APC-labeled anti-mCCR8 (SA214G2) or APC-labeled isotype control
antibody. 1 hour later, the cells were washed three times with
D-MEM/10% FCS and analyzed for a mCCR8 expression rate using a flow
cytometer (FACSCanto II). A background was set using the isotype
control antibody, and the proportion of positive cells (P6) equal
to or larger than the background level and median APC fluorescence
were calculated (FIG. 11). As a result, no difference in median APC
fluorescence intensity was observed, and the positive cells were
rarely observed (0.2%). In conclusion, the CT26 cells were not
recognized by the anti-mCCR8 antibody, and the CT26 cells were
confirmed to not express mCCR8.
Example 101
Confirmation of CCR8 Expression in Tumor-Infiltrating Cells Using
Colorectal Cancer Cell Line CT26 Cells
[0132] 3.times.10.sup.5 CT26 cells (50 .mu.L) were intracutaneously
transplanted to the back of each Balb/c mouse (7 w, female) (N=3).
At post-transplant day 3, 400 .mu.g of a rat anti-KLH (keyhole
limpet hemocyanin, Clone LTF-2) antibody (IgG2b) was
intraperitoneally administered thereto. At post-administration days
4 (4 d) and 7 (7 d), tumors were recovered from the 3 individuals
(N=3). The tumor masses of the CT26 cells were chopped with
scissors, and tumor-infiltrating cells were prepared using a
commercially available kits (Tumor Dissociation Kit, mouse,
Miltenyi Biotec and gentleMACS.TM. Dissociator, Miltenyi Biotec,
cat. 130-095-929) according to the protocols attached to the
kits.
[0133] The prepared cells were passed through a 70 um cell strainer
and then washed twice with 10 mM HEPES/HBSS/2% FBS. Then, the cells
were treated with an erythrocyte lysis solution (Miltenyi Biotec)
for 5 minutes for the removal of erythrocytes and further washed
twice with 2% FCS (fetal calf serum)/10 mM HEPES/HBSS buffer. The
tumor-infiltrating cells were divided into two parts, one of which
was used in the identification of Treg cells and the other of which
was used in the identification of myeloid (macrophage) cells. The
cells were stained using the following method and antibodies. The
antibodies, staining reagents, and assay buffers used were as
described below.
[0134] The following antibodies were used.
(Antibody Set for Treg Cell Confirmation)
[0135] PE anti-mouse/rat FoxP3 (clone FJK-16s), eBioscience, Inc.
Anti-mouse CD4 PerCP/Cy5.5 (clone RM4-5), eBioscience, Inc.
Anti-mouse CD8a FITC (clone 5H10-1), BioLegend, Inc. Bv421
anti-mouse CD25 (clone PC61), BioLegend, Inc. Bv510 anti-mouse CD45
(clone 30-F11), BioLegend, Inc. AF647 Anti-mouse CCR8 (clone
SA214G2), BioLegend, Inc. AF647 Isotype Control (clone RTK4530),
BioLegend, Inc. (CCR8-negative control)
(Antibody Set for Myeloid and Macrophage Cell Confirmation)
[0136] AF647 Anti-mouse CCR8 (clone SA214G2), BioLegend, Inc. AF647
Isotype Control (clone RTK4530), BioLegend, Inc. (CCR8-negative
control) Bv510 anti-mouse CD45 (clone 30-F11), BioLegend, Inc. FITC
anti-mouse Gr-1 (clone RB6-8C5), BioLegend, Inc. Bv421 anti-mouse
F4/80 (clone BM8), BioLegend, Inc. PECy7 anti-mouse CD11b (clone
M1/70), BioLegend, Inc. PerCP/Cy5.5 Anti-mouse MHC class II IA/IE
(clone M5/114.15.2), BioLegend, Inc. PE anti-mouse CD206 (clone
C068C2), BioLegend, Inc.
(Other Reagents Used)
Zombie NIR Fixable Viability Kit (cat no. 423106), BioLegend,
Inc.
[0137] BD Pharmingen Transcription Factor buffer Set (cat no.
562574)
BD Pharmingen Lysing Buffer (cat no. 555899)
HBSS(-), Wako Pure Chemical Industries, Ltd., 084-08345
[0138] FCS (HyClone Laboratories Inc., cat no. SH30070.03)
[0139] The staining method was as follows: the infiltrating cells
were stained in ice for 30 minutes using a reagent of Zombie NIR
Fixable Viability Kit. The cells were washed once with 2% FCS/10 mM
HEPES/HBSS. Then, Treg- and CCR8-positive cells were stained with
Bv510-labeled anti-CD45, PerCP/Cy5.5-labeled anti-mouse CD4,
FITC-labeled anti-mouse CD8, Bv421-labeled anti-mouse CD25, and
AF647-labeled anti-mouse CCR8 antibody (or AF647-labeled isotype
control antibody). Monocytic cells were stained with Bv510-labeled
anti-CD45, FITC anti-mouse Gr-1, PECy7 anti-mouse CD11b, Bv421
anti-mouse F4/80, PerCP/Cy5.5-labeled MHC class 2 (IA/IE) antibody,
and PE-labeled anti-mouse CD206 antibody.
[0140] The staining was carried out in ice for 30 minutes. The
cells were washed twice with 2% FCS/HEPES/HBSS and then fixed using
a commercially available kit (FoxP3 staining kit, eBioscience,
Inc.) according to the attached protocol, and intracellular FoxP3
was stained using a PE-labeled anti-FoxP3 antibody. The cells were
washed with a buffer attached to the kit and then analyzed using a
flow cytometer.
[0141] CD45+ CD4+ T cells were analyzed. A negative cell region in
the CD45+ CD4+ T cells was determined by staining with an isotype
control antibody, and cells positive to both anti-mouse CD25 and
anti-mouse FoxP3 antibodies were used as Treg cells to calculate
the frequency of presence 4 days after administration (7 days after
inoculation) and 7 days after administration (10 days after
inoculation). As a result, approximately 23% (4 d) and
approximately 30% (7 d) of the CD45+ CD4+ T cells within the mouse
tumors were CD25+ FoxP3+ cells (FIG. 12).
[0142] Next, CCR8 expression in the CD45+ CD4+ CD25+ FoxP3+ T cells
was analyzed. A negative cell region in the CD45+ CD4+ CD25+ FoxP3+
T cells was determined by staining with an isotype control
antibody, and cells positive to an anti-mouse CCR8 antibody were
used as CCR8+ Treg cells to calculate the frequency of presence 4
days after administration (7 days after inoculation) and 7 days
after administration (10 days after inoculation) (FIG. 13). As a
result, approximately 50% (4 d) and approximately 67% (7 d) of the
CD45+ CD4+ CD25+ FoxP3+ T cells within the mouse tumors were CCR8+
cells (FIG. 13).
[0143] As for myeloid cells, the myeloid population was gated on
CD45+ cells and FSC/SSC using a flow cytometer and analyzed for the
proportion of CCR8+ cells in CD11b+ Gr1+ CD206+ cells. As a result,
40 to 50% cells both 7 days after inoculation (4 days after
administration) and 10 days after inoculation (7 days after
administration) were found to be CCR8-positive (FIG. 14). Also, the
CCR8 expression rate in CD45+ CD11b+F4/80+ cells (N=3) as a
macrophage cell population different therefrom was measured in the
same way as above. As a result, 45.3% (standard deviation:
.+-.8.2%) of the cells were confirmed to express CCR8 at
post-transplant day 10 (7 d). From these results, at least CD4+
CD25+ FoxP3+ T cells and CD11b+ Gr1+ CD206+ macrophages (called M2
macrophages) as tumor-infiltrating cells were found to express
CCR8.
Example 11
Study on Effect of Removing Tumor-Infiltrating Treg Cells or
Tumor-Infiltrating Macrophage Cells by Anti-mCCR8 Antibody
Administration
[0144] 3.times.10.sup.5 CT26 cells (50 uL) were intracutaneously
transplanted to the back of each Balb/c mouse (7 w, female). 3 days
after inoculation, 400 .mu.g (liquid volume: 400 .mu.L) of a rat
anti-mouse CD198 (CCR8) antibody (clone SA214G2, BioLegend, Inc.)
or an isotype control antibody (Clone LTF-2) was administered into
the tail vein (each group N=3). 7 days after tumor inoculation (4
days after antibody administration) and 10 days after tumor
inoculation (7 days after antibody administration), tumors were
recovered, and tumor-infiltrating cells were prepared and analyzed
(FIG. 15).
[0145] Tumor-infiltrating Treg cells were recovered in the same way
as in Example 10. The antibodies used were the same as in Example
10.
[0146] First, the infiltrating cells were stained in ice for 30
minutes using Zombie NIR Fixable Viability Kit. The cells were
washed once with 2% FCS/10 mM HEPES/HBSS and then stained with
Bv510-labeled anti-CD45, PerCP/Cy5.5-labeled anti-mouse CD4,
FITC-labeled anti-mouse CD8 antibody, Bv421-labeled anti-mouse
CD25, and AF647-labeled anti-mouse CCR8 antibody (or AF647-labeled
isotype control antibody). The staining was carried out in ice for
30 minutes. The cells were washed twice with 2% FCS/HEPES/HBSS and
then fixed using a commercially available kit (FoxP3 staining kit,
eBioscience, Inc.) according to the attached protocol, and
intracellular FoxP3 was stained using a PE-labeled anti-FoxP3
antibody. The cells were washed with a buffer attached to the kit
and then analyzed using a flow cytometer.
[0147] CD45+ CD4+ FoxP3+ CD25+ cells were used as mouse Treg cells.
A negative cell region in the Treg cells was determined by staining
with an AF647-labeled isotype control antibody, and cells positive
to an AF647-labeled anti-mouse CCR8 antibody compared with the
control were used as CCR8-positive cells to calculate the frequency
thereof.
[0148] As a result, as shown in FIG. 16, the percent positivity of
CD45+ CD4+ CD25+ FoxP3+ T cells (Treg cells) in the mice given the
anti-mouse CCR8 (SA214G2) antibody was approximately 80% 7 days
after tumor inoculation (4 days after antibody administration) and
approximately 40% 10 days after tumor inoculation (7 days after
antibody administration 7) (FIG. 16) when the proportion of
intratumoral CD45+ CD4+ CD25+ FoxP3+ T cells (Treg cells) in the
mice given the isotype antibody was defined as 100% (10 days after
tumor inoculation). Significance level ** was P<0.01 (t test).
These results showed that approximately 60% of the
tumor-infiltrating Treg cells were removed by the anti-CCR8
antibody 7 days after anti-CCR8 antibody administration.
[0149] In the same way as above, tumor-infiltrating cells were
separated from tumors at post-transplant day 7 (d7), and among
CD45+ cells, a myeloid population was gated on FSC/SSC (referred to
as FSC/SSC+), followed by the analysis of CD11b+F4/80+ cells in the
cells. F4/80 (Ly719) is a marker of mouse mature macrophages and
monocytes. As shown in FIG. 17, the abundance ratio of CD11b+F4/80+
cells was decreased in the anti-mCCR8 antibody administration group
(N=3) compared with the isotype control (N=3) (t test; P=0.062).
The graph shows the abundance ratio of F4/80+ cells in a CD45+
FSC/SSC+ mononuclear cell population.
[0150] The abundance ratio of IA/IE-positive or class 2
(IA/IE)-negative cells in the F4/80+ cells shown in FIG. 17 is
further shown as to MHC (tumor histocompatibility antigen) class 2
molecules. As shown in FIG. 18, in the anti-mCCR8 antibody
administration group (N=3) compared with the isotype control (N=3),
the IA/IE-negative group exhibited a decreasing trend, and the
IA/IE-positive group was significantly decreased (t test;
significance level *; P<0.05). In conclusion, the mouse CT26
intratumoral monocyte/macrophage population or a portion of the
population was found to have a decreased intratumoral cell
count.
Example 12
Evaluation of Antitumor Effect of Anti-mCCR8 Antibody
Administration Using Colorectal Cancer-Derived CT26
[0151] 3.times.10.sup.5 colorectal cancer-derived CT26 cells (50
uL) were intracutaneously transplanted to the back of each Balb/c
mouse (7 weeks old, female). 3 days after tumor inoculation, 400
.mu.g (400 .mu.L) of a rat anti-mouse CD198 (CCR8) antibody (clone
SA214G2, BioLegend, Inc.) was intravenously administered thereto
(N=10). An isotype control antibody was administered to a control
(N=10). Tumor volumes were measured every 3 to 4 days from 8 days
after tumor inoculation (5 days after antibody administration). The
tumor volume (mm.sup.3) was calculated according to major axis
(mm).times.minor axis (mm).times.minor axis (mm)/2 (FIG. 19).
[0152] As a result, no significant difference was observed in the
anti-mCCR8 administration group compared with the isotype control
antibody administration group at post-transplant day 7, whereas the
tumor volume of the anti-mCCR8 antibody administration group was
significantly decreased at 11, 14, 17 and 21 days after tumor
inoculation (significance level: ***; P<0.001 at days 11 and 14,
**; P<0.01 at days 17 and 21). Furthermore, in the anti-mouse
CCR8 antibody administration group, the tumor volume was decreased
at post-transplant day 14 or later, and the tumors disappeared
almost completely at day 17 (individual-based data is shown in FIG.
20, and mean data is shown in FIG. 21). From these results, it was
concluded that the anti-mCCR8 antibody administration suppressed
the functions of mCCR8 expressed on Treg and monocytes/macrophages
pointed out as immunosuppressive cells, or killed (removed) these
expressing cells through the ADCC activity of the antibody so that
tumor immunity was enhanced, leading to the regression and
disappearance of the tumors.
[0153] As already reported by many literatures, etc., in the case
of administering an antibody specific for mouse CD25 (anti-CD25), a
marker of mouse Treg cells, to mice and thereby removing mouse Treg
cells, the administration before tumor inoculation exhibits a weak
antitumor effect and the administration at post-transplant day 2 or
later exhibits no antitumor effect. We also carried out the
administration of the anti-CD25 antibody at post-transplant day 3
using the same CT26 cell system as that used this time, but
observed no antitumor effect. From these results, it was concluded
that the anti-mCCR8 antibody has stronger drug efficacy than that
of the anti-CD25 antibody.
Example 131
[0154] Next, anti-PD-1 (clone RMP1-14, Bio X Cell), an antibody
specific for mouse PD-1, was evaluated for its drug efficacy using
CT26 and comparatively studied with anti-mCCR8. 2.times.10.sup.5
colorectal cancer-derived CT26 cells (50 .mu.L) were
intracutaneously transplanted to the back of each Balb/c mouse (7
weeks old, female). The anti-PD-1 antibody (200 .mu.g/head, i.p.)
was administered a total of three times every 3 to 4 days from
post-transplant day 7.
[0155] As a result, an antitumor effect was observed in the group
given the anti-PD-1 antibody (N=8) compared with a group given an
isotype control antibody (N=8). The mean tumor volume and standard
deviation of the isotype control were 601.7.+-.378.1 mm.sup.3,
956.3.+-.467.7 mm.sup.3 and 1528.4.+-.774.1 mm.sup.3 at 14, 17 and
20 days after tumor inoculation, respectively, while the mean tumor
volume and standard deviation of the anti-PD-1 antibody
administration group were 175.3.+-.42.6 mm.sup.3, 174.7.+-.55.8 mg
and 209.6.+-.99.8 mm.sup.3 at 14, 17 and 20 days after tumor
inoculation, respectively. The anti-PD-1 antibody significantly
suppressed increase in tumor volume as compared with the control at
all of 14, 17 and 20 days after tumor inoculation. However, an
individual whose tumor disappeared completely was 1 out of 8 mice
in the observation period (up to post-transplant day 20). On the
other hand, the complete disappearance of tumors was observed in
all of 10 cases in the same period as above by anti-mCCR8 antibody
administration. From these results, it was concluded that the
anti-mCCR8 antibody has stronger drug efficacy than that of the
anti-PD-1 antibody in the standard administration method.
Example 14
Confirmation of Presence or Absence of Induction of Autoimmune
Disease in Mouse Given Anti-mCCR8 Antibody
[0156] Next, the states of the mice of Example 12 were evaluated up
to post-administration day 18. No significant difference in body
weight in this period was found between the control antibody
administration group and the anti-CCR8 antibody administration
group. Piloerection was not observed in both the groups. These mice
were dissected at post-administration day 18. Although the presence
or absence of enlargement of the lymph node and the intestinal
tract was studied in the anti-CCR8 administration group compared
with the control, the enlargement was not observed without any
difference between the groups. From these findings, it was
concluded that any sign of autoimmune disease was not observed in
the period when an antitumor effect was exerted on the mice given
the anti-CCR8 antibody. Papers have reported that, in general, if
Treg is removed from the whole body of a mouse to the extent that
an antitumor effect is induced, severe autoimmune disease is
induced around 14 days after removal. This is a matter of concern
for tumor immunotherapy including Treg suppression therapy. The
results obtained this time showed that any autoimmune disease was
not induced even at post-antibody administration day 18 in the mice
in which a strong anti-tumor immunity effect was observed by
anti-CCR8 antibody administration. One of the explanations therefor
is the low expression of mouse and human CCR8 in PBMCs, the spleen,
and the lymph node compared with tumor tissues according to
reports. However, none of the previous reports state whether or not
an autoimmune disease is induced by removing or functionally
inhibiting CCR8-expressing Treg cells in these peripheral tissues.
Here, it was found for the first time that these approaches induce
no autoimmune disease. This effect may be unexpectable from the
previous findings.
Example 151
Evaluation of Antitumor Effect of Anti-mCCR8 Antibody
Administration Using Colorectal Cancer-Derived Colon-26
[0157] 2.times.10.sup.5 colorectal cancer-derived Colon-26 cells
(50 .mu.L) were intracutaneously transplanted to the back of each
Balb/c mouse (7 weeks old, female). 3 days after tumor inoculation,
400 .mu.g (400 .mu.L) of a rat anti-mouse CCR8 antibody (clone
SA214G2, BioLegend, Inc.) was intravenously administered thereto
(N=10). An isotype control antibody was administered to a control
(N=10). Tumor volumes were measured every 3 to 4 days from 3 days
after tumor inoculation (5 days after antibody administration). The
tumor volume (mm.sup.3) was calculated according to major axis
(mm).times. minor axis (mm).times. minor axis (mm)/2. The point in
time when the tumor reached an endpoint volume (800 mm.sup.3) was
used as the endpoint of each animal. As a result, increase in tumor
volume was suppressed in the anti-mCCR8 administration group
compared with the isotype control antibody administration group at
14 and 18 days after tumor inoculation. The mean tumor volume at
day 14 was 451.3 mm.sup.3 (standard deviation: .+-.177.5 mm.sup.3)
in the isotype control antibody administration group and 322.6
mm.sup.3 (standard deviation: .+-.146.0 mm.sup.3) in the anti-CCR8
antibody administration group. An individual having a tumor volume
of 350 mm.sup.3 or larger at day 14 was 9 out of 10 cases in the
isotype control group and 4 out of 10 cases in the anti-mCCR8
administration group. There was a significant difference with
P=0.019 in the Pearson's chi-square test as to this segregated
form. Thus, the difference was observed in the number of
individuals whose tumor volume reached 350 mm.sup.3 at day 14.
Also, the mean tumor volume at post-transplant day 18 was 874.7
mm.sup.3 (standard deviation: .+-.269.2 mm.sup.3) in the isotype
control antibody administration group and 585.4 mm.sup.3 (standard
deviation: .+-.401.7 mm.sup.3) in the anti-CCR8 antibody
administration group (FIG. 22). An individual having a tumor volume
of 600 mm.sup.3 or larger at day 18 was 9 out of 10 cases in the
isotype control group and, on the other hand, 4 out of 10 cases in
the anti-mCCR8 administration group. There was a significant
difference with P=0.019 in the Pearson's chi-square test as to this
segregated form. Thus, the difference was observed in the number of
individuals whose tumor volume reached 600 mm.sup.3 at day 18.
Further, the point in time when the tumor volume reached 800
mm.sup.3 was preset as an endpoint. An individual regarded as being
dead with the tumor volume exceeding 800 mm.sup.3 was observed in
neither of the groups up to day 14 and was 7 out of 10 cases in the
isotype control group and 3 out of 10 cases in the anti-CCR8
antibody group at day 18. As a result of studying a difference in
the survival probability at day 18 by the Pearson's chi-square
test, there was a significant difference in the survival
probability with P=0.025.
[0158] No antitumor effect was observed in an anti-PD-1 (clone
RMP1-14, Bio X Cell) administration group compared with a group
given an isotype control antibody in a similar experiment using the
same cell line as above. In conclusion, the anti-mCCR8 antibody
exhibited a higher antitumor effect on anti-PD-1 antibody-resistant
Colon26 cells.
Example 161
Analysis on Expression of CCR8 in Human Kidney Cancer Infiltrating
Cells
[0159] The expression of CCR8 was analyzed in human kidney cancer
tumor-infiltrating cells of 14 cases. The backgrounds of the 14
kidney cancer patients were 11 males and 3 females for sex, a
median age of 68.5 years, and pathological stages of T1A for 6
patients, T1B for 2 patients, T3A for 5 patients, and T3b for 1
patient. Specifically, kidney cancer primary tumor-infiltrating
cells were isolated from the 14 kidney cancer (clear cell renal
cell carcinoma, ccRCC) patients in the same way as in FIG. 1 of
Example 1, stained with anti-CD4 (BioLegend, Inc., Clone OKT4),
anti-CD3 (BioLegend, Inc., Clone UCHT1), anti-CD45RA (BD
Biosciences, Clone HI100), anti-CD8 (BioLegend, Inc., RPA-T8),
anti-CCR8 (BioLegend, Inc., Clone L263G8), and anti-FoxP3
(eBioscience, Inc., Clone 236A/E7) or an anti-FoxP3 isotype control
antibody, and analyzed by flow cytometry (BD Biosciences, BD
LSRFortessa). CD3+ CD8+ T cells and CD3+ CD4+ T cells were
analyzed. The CD3+ CD4+ T cells were further divided into 2 groups
according to the presence or absence of FoxP3 expression and
analyzed. A FoxP3 expression-negative control was prepared by
staining with the isotype control antibody. The mean value of FACS
analysis (MFI) of each patient sample was used as the expression
intensity of CCR8. Table 1 shows mean MFI of staining with the
anti-CCR8 antibody or the isotype control antibody thereof, and a
standard deviation thereof.
TABLE-US-00001 TABLE 1 Cell CD8+T FoxP3-CD4+T FoxP3+CD4+T Antibody
Anti- Anti- Anti- Isotype mCCR8 Isotype mCCR8 Isotype mCCR8 Mean
MFI 84.9 267 56.9 423 131.3 3507.2 Standard 26.8 159 62.1 297.5 59
1466.3 deviation
[0160] The CD8+ T cells were found to rarely express CCR8 (Table
1). The CD4+ FoxP3- T cells slightly expressed CCR8, whereas the
CD4+ FoxP3+ T cells had 8 times or more the mean MFI of the CD4+
FoxP3- T cells, revealing that the CD4+ FoxP3+ T cells
significantly strongly express CCR8 (Table 1). FIG. 23 shows the
results of Table 1 in a graph form. Each plot of the graph shows
the mean CCR8 expression level (MFI) of each patient sample in a
flow cytometer. The horizontal lines of the graph depict the mean
MFI of the samples. The bars depict standard deviations.
Significance level *** denotes P<0.001. From these results, the
CCR8 protein was found to be specifically expressed on the surface
of CD3+ CD4+ FoxP3+ T cells which infiltrate tumors in human kidney
cancer (ccRCC). These results are also consistent with the results
of the mRNA expression analysis by the RNA-Seq analysis.
[0161] Tumor-infiltrating CD4+ T cells in the 14 ccRCC samples
described above were subjected to flow cytometry analysis using
FoxP3 and CCR8. The ratio of CCR8-positive cells to FoxP3-positive
cells and the ratio of CCR8-positive cells to FoxP3-negative cells
were plotted on a sample basis (FIG. 24). Staining with an isotype
control antibody was used as negative standards for both FoxP3 and
CCR8, and cells having a value equal to or more than this threshold
were used as positive cells. As a result, the CCR8 expression rate
of intratumoral CD3+ CD4+ FoxP3+ T cells was approximately 75%, and
the CCR8 expression rate of CD3+ CD4+ FoxP3- T cells was
approximately 10%.
[0162] From these results, CCR8 was found to be expressed in most
of FoxP3-expressing Treg cells among human kidney cancer
tumor-infiltrating cells and expressed in approximately 10% of
CD4-positive T cells other than the Treg cells. From these results,
the CCR8 expression rate of human intratumoral FoxP3-positive Treg
cells was similar to that of mouse intratumoral Treg cells,
indicating the possibility that the anti-human CCR8-specific
antibody can remove most of tumor-infiltrating FoxP3-positive Treg
cells, as in mice.
Example 171
[0163] Correlation of CCR8 Expression Rate of Tumor-Infiltrating
Cells in Various Cancers with Survival Probability
[0164] FoxP3 gene has been identified as a gene that is
specifically expressed in Treg cells and not expressed in tumor
cells or most of normal human cells. For example, FoxP3 gene as a
marker gene of Treg cells, CD3G gene as a marker gene of T cells
and NK cells, and CD8A gene as a marker gene of CD8-positive T
cells are known as so-called marker genes, which are expressed only
in certain specific cells as mentioned above.
[0165] It has also been reported as to the FoxP3 gene, a marker
gene of Treg cells, that the mRNA expression level of the FoxP3
gene within each tumor may be measured and thereby used as an index
for the abundance ratio of Treg cells within the tumor (Cell, 2015,
Vol. 160, p. 48-61).
[0166] As also reported in this paper, whether the intratumoral
abundance ratio of Treg cells is related to a survival probability
may be analyzed by drawing a Kaplan-Meier survival curve as to the
intratumoral expression rate (Treg abundance ratio) of the marker
gene and patients' survival probabilities through the use of a
RNA-Seq database such as TCGA. The RNA-Seq data on tumor masses is
mixed data on mRNA expressed in both tumor cells and infiltrating
cells present therein (lymphocytes, vascular cells, etc.). However,
a gene shown to be not expressed in tumor cells can be regarded as
a gene expressed in tumor-infiltrating cells. Through the use
thereof, the tumor-infiltrating cells can be identified by the
analysis as described above, i.e., analysis on marker gene
expression using the RNA-Seq data on tumor masses. Furthermore, the
expression level of a marker gene in a tumor mass can be regarded
as the product of an expressing cell count of particular cells,
corresponding to the marker gene, infiltrating the tumor mass, and
the expression level of the marker gene in each expressing
cell.
[0167] In this context, if the expression level of the marker gene
in each cell is almost constant among individuals, the expression
level is in direct proportion to an infiltrating cell count. Thus,
an intratumoral expressing cell count can be calculated on an
individual basis by use of this expression level and can be
compared among individuals.
(CCR8 Expression Analysis at Cell Level)
[0168] RNA expression data from 1037 different types of human cell
lines is registered in a public database CCLE (Cancer Cell Line
Encyclopedia). Whether CCR8 or CD3G gene would be expressed in
cancer cells other than T cells or normal cells was analyzed using
the database.
[0169] The mRNA expression of CD3G and CCR8 was analyzed as to
kidney cancer-, prostate cancer- and bladder cancer-derived cell
lines using the CCLE database.
[0170] The cell lines examined were 40 kidney cancer-derived cell
lines: VMRCRCW, SKRC20, SNU34, SKRC31, UOK10, SLR20, OSRC2,
TUHR14TKB, SLR24, HK2, A498, RCC4, KMRC1, RCC10RGB, ACHN, SLR25,
SNU1272, UMRC6, SLR23, 769P, SLR21, HEKTE, CAKI1, TUHR4TKB, KMRC2,
VMRCRCZ, KMRC3, KMRC20, CAKI2, BFTC909, 7860, A704, TUHR10TKB,
SLR26, UMRC2, CAL54, FURPNT1, FURPNT2, HEK293, and G402;
8 prostate cancer-derived cell lines:
VCAP, LNCAPCLONEFGC, DU145, PC3, 22RV1, PRECLH, MDAPCA2B, and
NCIH660; and
[0171] 2 bladder cancer-derived cell lines: TCBC14TK and TCBC2TKB.
In all of these solid cancer cell lines examined, the expression of
CCR8 and CD3G was at the same level as the background level, and no
mRNA expression was observed (even the largest value indicating
expression was 1/500 or less of the expression level of G3PDH, and
all the other values were 1/1000 or less of the expression level of
G3PDH). In short, CCR8 and CD3G were able to be confirmed to be
rarely expressed on solid cancer cells. Primary normal cells
derived from each human tissue were also analyzed in the same way
as above. CCR8 and CD3G were found to be expressed only in some
hematopoietic cells and rarely expressed in the other
tissues-derived primary normal cells.
[0172] These results showed that the cells of these 3 cancers
express neither CCR8 nor CD3G. Thus, it was concluded that TCGA RNA
expression data used for tumor masses of kidney cancer, prostate
cancer and bladder cancer reflects the mRNA expression of CCR8 and
CD3G in infiltrating normal cells, other than cancer cells, present
in the tumor masses.
(Analysis Using Public TCGA Database)
[0173] Next, the ratio of the CCR8 gene to the CD3G gene
(CCR8/CD3G) expressed in the tumor of kidney cancer, prostate
cancer, or bladder cancer, and patients' survival probabilities
were analyzed through the use of the public TCGA database. A gene
that most highly correlated (Pearson's correlation) in terms of
expression with the CCR8 and CD3G genes within these 3 tumors was
found to be various genes specifically expressed in T cells (FoxP3,
CD5, IL7R, etc. with correlation coefficient r of 0.7 or more).
These results indicate that CCR8 or CD3G is not expressed on tumor
cells themselves and is specifically expressed on
tumor-infiltrating expressing cells (particularly, T cells).
However, a CCR8-expressing cell population was used here because
this does not deny that CCR8 is expressed on infiltrating cells
other than T cells. CD3G, as already reported in papers, etc., is
specifically expressed on T cells and NK cells. Also, T cells are
major tumor-infiltrating cells. Therefore, an infiltrating T cell
count can be hypothesized from a CD3G expression level. Thus, the
CCR8/CD3G value can be defined as a CCR8-expressing cell count per
T cell count present within a tumor.
[0174] The CCR8/CD3G ratio and patients' survival probabilities
were analyzed as to these 3 carcinomas using a Kaplan-Meier curve.
For kidney cancer, Kidney Renal Clear Cell Carcinoma (TCGA,
Provisional) data in the TCGA data was used, and 523 cases having
complete RNA expression data and patients' survival probability
data were used. Likewise, for prostate cancer, Prostate
Adenocarcinoma (TCGA, Provisional) data in the TCGA data was used,
and 490 cases having complete RNA expression data and patients'
survival probability data were used.
[0175] Also, for bladder cancer, Bladder Urothelial Carcinoma
(TCGA, Provisional) data in the TCGA data was used, and 392 cases
having complete RNA expression data and patients' survival
probability data were used.
[0176] Patients of each cancer were equally divided into 2 groups
(the kidney cancer patients were odd-numbered and therefore divided
into 261:262) with high CCR8/CD3G expression values and with low
CCR8/CD3G expression values, followed by Kaplan-Meier survival
curve analysis using analytical software R (R-Studio). The log-rank
test was conducted as a significant difference test. The results
about the kidney cancer are shown in FIG. 25, the results about the
prostate cancer are shown in FIG. 26, and the results about the
bladder cancer are shown in FIG. 27. The vertical lines in the
graphs show that the patients survived but were treated as dropouts
(corresponding to so-called censors) at the point in this time
because the evaluation period was terminated at this point in time.
The values on the abscissa depict the number of months in all the
graphs.
[0177] As a result, in all the 3 carcinomas, the groups with high
CCR8/CD3G values had significantly low patients' survival
probabilities. The groups with a high ratio of human
tumor-infiltrating CCR8-expressing cells to T cells were found to
have a reduced survival probability. This suggests that in humans
as well, CCR8-expressing cells have a suppressive effect on tumor
immunity. This suggests the possibility that, as in the antitumor
effect of the anti-mCCR8 antibody administered to mice,
intratumoral CCR8-expressing cells in humans are specifically
removed or killed by some method to thereby enhance tumor immunity
and elevate a survival probability.
Example 181
Confirmation of Expression of Mouse CCR8 in LM8 Cells and MethA
Cells
[0178] Osteosarcoma-derived LM8 cells or skin fibrosarcoma-derived
MethA cells were cultured in a 6-well dish, and the culture
solution was removed when the cells became approximately 50%
confluent. 5 ml of 10 mM EDTA/PBS was added thereto, and the cells
were incubated at 37.degree. C. for 5 minutes. As a result, almost
all the cells were dissociated, suspended using a pipette and were
thereby able to be separated into almost single cells. The cells
were washed twice with D-MEM/10% FCS, suspended in D-MEM/10% FCS,
and stained in ice with LIVE/DEAD.RTM. Fixable Near-IR Dead Cell
Stain Kit (Thermo Fisher Scientific Inc., L34975) and an anti-mouse
CCR8 (SA214G2) or isotype control antibody. 1 hour later, the cells
were washed three times with D-MEM/10% FCS and analyzed for a mouse
CCR8 expression rate using a flow cytometer (FACSCanto II). A
background was set using the isotype control antibody, and the
proportion of positive cells equal to or larger than the background
level and median fluorescence were calculated (FIG. 28). As a
result, no difference in median PE fluorescence intensity was
observed in both the cells, and the positive cells were not
observed. In conclusion, these cells were not recognized by the
anti-mouse CCR8 antibody and were confirmed to neither express
mouse CCR8 nor retain an epitope reactive with the antibody.
Example 191
Evaluation of Antitumor Effect of Anti-Mouse CCR8 Antibody
Administration Using Osteosarcoma-Derived LM8
[0179] 3.times.10.sup.5 mouse osteosarcoma-derived LM8 cells (50
uL) were intracutaneously transplanted to the back of each C3H/He
mouse (7 weeks old, male). 3 days after tumor inoculation, 400
.mu.g (400 .mu.L) of a rat anti-mouse CCR8 antibody (clone SA214G2,
BioLegend, Inc.) was intraperitoneally administered thereto (N=11).
An isotype control antibody was administered to a control (N=10).
Tumor volumes were measured every 3 to 4 days from 7 days after
tumor inoculation (4 days after antibody administration). The tumor
volume (mm.sup.3) was calculated according to major axis
(mm).times.minor axis (mm).times.minor axis (mm)/2 (FIG. 29). As a
result, the mean tumor volume of the anti-mCCR8 administration
group compared with the isotype control antibody administration
group was significantly decreased at all the points in time of
measurement at post-transplant day 18 or later (significance level:
*; P<0.05 at day 18, **; P<0.01 at days 21, 24, 27 and 31,
***; P<0.001 at day 35). Furthermore, the tumors disappeared in
6 out of 11 mice in the anti-mouse CCR8 antibody administration
group and 1 out of 10 mice in the isotype control antibody
administration group at post-antibody administration day 31. There
was a significant difference (P=0.031) in the Pearson's chi-square
test conducted as to this segregated form.
Example 201
Evaluation of Antitumor Effect of Anti-Mouse CCR8 Antibody
Administration Using Skin Fibrosarcoma-Derived MethA
[0180] 1.times.10.sup.5 skin fibrosarcoma-derived MethA cells (50
uL) were intracutaneously transplanted to the back of each Balb/c
mouse (7 weeks old, female). 3 days after tumor inoculation, 400
.mu.g (400 .mu.L) of a rat anti-mouse CCR8 antibody (clone SA214G2,
BioLegend, Inc.) was intraperitoneally administered thereto (N=5).
An isotype control antibody was administered to a control (N=5).
Tumor volumes were measured every 3 to 4 days from 11 days after
tumor inoculation (8 days after antibody administration). The tumor
volume (mm.sup.3) was calculated according to major axis
(mm).times.minor axis (mm).times.minor axis (mm)/2 (FIG. 30).
[0181] As a result, the mean tumor volume of the anti-mouse CCR8
administration group compared with the isotype control antibody
administration group was significantly decreased at all the points
in time of measurement at post-transplant day 11 or later
(significance level: *; P<0.05 at all the points in time).
Furthermore, the tumors disappeared in 5 out of 5 mice in the
anti-mouse CCR8 antibody administration group and 0 out of 5 mice
in the isotype control antibody administration group at
post-antibody administration day 21. There was a significant
difference (P=0.0016) in the Pearson's chi-square test conducted as
to this segregated form.
Example 21
Evaluation of Antitumor Effect of Anti-Mouse CCR8 Antibody
Administration Using Breast Cancer-Derived EMT6
[0182] 1.times.10.sup.5 breast cancer-derived EMT6 cells (50 uL)
were intracutaneously transplanted to the back of each Balb/c mouse
(7 weeks old, female). 3 and 10 days after tumor inoculation, 100
.mu.g (100 .mu.L) of a rat anti-mouse CCR8 antibody (clone SA214G2,
BioLegend, Inc.) was intraperitoneally administered thereto (N=20).
An isotype control antibody was administered to a control (N=20).
Tumor volumes were measured every 3 to 4 days from 4 days after
tumor inoculation (1 day after antibody administration). The tumor
volume (mm.sup.3) was calculated according to major axis
(mm).times.minor axis (mm).times.minor axis (mm)/2 (FIG. 31).
[0183] As a result, the mean tumor volume of the anti-mouse CCR8
administration group compared with the isotype control antibody
administration group was significantly decreased at all the points
in time of measurement at post-transplant day 10 or later
(significance level: **; P<0.01 at day 10, ***; P<0.001 at
days 14, 17 and 21). Furthermore, the tumors disappeared in 19 out
of 20 mice in the anti-mouse CCR8 antibody administration group and
2 out of 20 mice in the isotype control antibody administration
group at post-antibody administration day 21. There was a
significant difference (P<0.0001) in the Pearson's chi-square
test conducted as to this segregated form.
Example 22
Confirmation of Superiority of Anti-Mouse CCR8 Antibody Over
Anti-PD-1 Antibody
[0184] 2.times.10.sup.5 colorectal cancer-derived Colon26 cells (50
uL) were intracutaneously transplanted to the back of each Balb/c
mouse (7 weeks old, female). 3 and 10 days after tumor inoculation,
400 .mu.g (400 .mu.L) of an isotype control antibody, a rat
anti-mouse CCR8 antibody (clone SA214G2, BioLegend, Inc.) or an
anti-mouse PD-1 antibody (RMP1-14, Bio X Cell) was intravenously
administered thereto (N=10). Tumor volumes were measured every 3 to
4 days from 3 days after tumor inoculation. The tumor volume
(mm.sup.3) was calculated according to major axis (mm).times.minor
axis (mm).times.minor axis (mm)/2 (FIG. 32). As a result, the tumor
volume of the anti-mouse CCR8 administration group compared with
the isotype antibody administration group was significantly
decreased at days 17, 20, and 24 (Steel's nonparametric test:
significance level P<0.05). No significant difference was
observed in the anti-PD-1 antibody administration group compared
with the isotype antibody administration group at any point in
time.
[0185] A mouse individual bearing a tumor with a volume of 1000
mm.sup.3 or larger at post-antibody administration day 24 was 7 out
of 10 mice in the isotype antibody administration group, 2 out of
10 mice in the anti-mouse CCR8 antibody administration group, and 7
out of 10 mice in the anti-PD-1 administration group. The anti-CCR8
administration group had a significant difference from both the
isotype antibody administration group and the anti-PD-1 antibody
administration group in the Pearson's chi-square test as to the
segregated form (P=0.025 for both). In conclusion, the anti-mouse
CCR8 antibody administration was confirmed to produce an antitumor
therapeutic effect on the colorectal cancer cell line Colon26.
[0186] Furthermore, the tumor volume of the anti-mouse CCR8
administration group compared with the anti-mouse PD-1 antibody
administration group was significantly decreased at 20 and 24 days
after tumor inoculation (Steel-Dwass nonparametric test;
significance level P<0.05). In conclusion, a stronger antitumor
therapeutic effect on the mouse colorectal cell line was observed
in the anti-mouse CCR8 antibody administration group compared with
the anti-PD-1 antibody administration group.
Example 231
Evaluation of Antitumor Effect of Anti-Mouse CCR8 Antibody
Administration Using Kidney Cancer-Derived Cell Line RAG
[0187] A similar study was conducted using a mouse kidney
cancer-derived cell line RAG. 4.times.10.sup.5 kidney
cancer-derived RAG cells (50 uL) were intracutaneously transplanted
to the back of each Balb/c mouse (8 weeks old, female). 6 days
after tumor inoculation, 100 .mu.g (100 .mu.L) of an isotype
control antibody (N=10 except for N=9 at day 21), a rat anti-mouse
CCR8 antibody (N=10) (clone SA214G2, BioLegend, Inc.) or an
anti-mouse PD-1 antibody (N=10) (RMP1-14, Bio X Cell) was
intraperitoneally administered thereto. Tumor volumes were measured
every 3 to 4 days from 6 days after tumor inoculation. The tumor
volume (mm.sup.3) was calculated according to major axis
(mm).times.minor axis (mm).times.minor axis (mm)/2 (FIG. 33). As a
result, the tumor volume of the anti-mouse CCR8 administration
group compared with the isotype antibody administration group was
significantly decreased at 14, 17, and 21 days after tumor
inoculation (Steel's nonparametric test: significance level
P<0.05). No significant difference was observed in the
anti-mouse PD-1 antibody administration group compared with the
isotype antibody administration group. In conclusion, the
anti-mouse CCR8 antibody administration was confirmed to produce an
antitumor therapeutic effect on the kidney cancer cell line.
Furthermore, the tumor volume of the anti-mouse CCR8 administration
group compared with the anti-mouse PD-1 antibody administration
group was significantly decreased at post-transplant day 14
(Steel-Dwass nonparametric test; significance level P<0.05). In
conclusion, a stronger antitumor therapeutic effect on the mouse
kidney cancer cell line was observed in the anti-mouse CCR8
antibody administration group compared with the anti-mouse PD-1
antibody administration group.
Example 24
Analysis on Presence or Absence of Inflammatory Response in Mouse
Given Anti-Mouse CCR8
[0188] 2.times.10.sup.5 colorectal cancer-derived Colon26 cells (50
uL) were intracutaneously transplanted to the back of each Balb/c
mouse (7 weeks old, female). 3 and 10 days after tumor inoculation,
400 .mu.g (400 .mu.L) of a rat anti-mouse CD198 (CCR8) antibody
(clone SA214G2, BioLegend, Inc.) or an isotype control antibody
(LTF-2, Bio X Cell) was intravenously administered thereto (N=10).
The body weight and the weight of each mouse organ (lung, liver,
spleen, small intestine, and inguinal node) were measured at
post-transplant day 24 (FIG. 34). As a result, as shown in FIG. 34,
no significant difference in body weight and each organ weight was
observed between the control administration group (N=10) and the
anti-mouse CCR8 antibody administration group (N=10). From these
results, it was concluded that the anti-mouse CCR8 antibody
administration induced neither inflammatory response nor an
autoimmune disease.
Example 25
Analysis on Expression of CCR8 in Various Clinical
Tumor-Infiltrating Cells
[0189] The expression of CCR8 was analyzed in tumor-infiltrating
cells of human kidney cancer, ovary cancer, uterine corpus cancer,
colorectal cancer, and lung cancer. The numbers of patients with
various clinical tumors used in the expression analysis were 12
kidney cancer patients, 14 ovary cancer patients, 21 uterine corpus
cancer patients, 10 colorectal cancer patients, and 4 lung cancer
patients. Various clinical tumor-infiltrating cells were isolated
in the same way as in FIG. 1 of Example 1 and stained with
anti-CD45 (BioLegend, Inc., Clone H130) and anti-CCR8 (BioLegend,
Inc., Clone L263G8) antibodies, followed by measurement by flow
cytometry (BD Biosciences, BD LSRFortessa). A CCR8-positive cell
count per tumor weight and the ratio of CCR8-positive cells to
CD45-positive leukocytes were analyzed.
[0190] Table 2 shows a mean CCR8-positive cell count per tumor
weight and a standard deviation thereof. Table 3 shows a mean ratio
of CCR8-positive cells to CD45-positive leukocytes and a standard
deviation thereof.
TABLE-US-00002 TABLE 2 CCR8-positive cell count (.times.10.sup.5)
per tumor weight (g) Cancer type Mean Standard deviation Kidney
cancer 8.9 22.7 Ovary cancer 1.7 2.6 Uterine corpus cancer 13.1
28.5 Colorectal cancer 2.9 5.4 Lung cancer 21.8 36.9
TABLE-US-00003 TABLE 3 Ratio (%) of CCR8-positive cells to
CD45-positive leukocytes Cancer type Mean Standard deviation Kidney
cancer 5.6 5.2 Ovary cancer 5.2 6.6 Uterine corpus cancer 9.0 9.2
Colorectal cancer 6.2 6.5 Lung cancer 2.9 2.3
[0191] In the various clinical tumors with kidney cancer as a
reference, as for the CCR8-positive cell count per tumor weight,
ovary cancer and colorectal cancer exhibited a lower mean than that
of kidney cancer, and uterine corpus cancer and lung cancer
exhibited a higher mean than that of kidney cancer. As for the
ratio of CCR8-positive cells to CD45-positive leukocytes, ovary
cancer exhibited a mean equivalent to that of kidney cancer, and
lung cancer exhibited a lower mean than that of kidney cancer.
Also, uterine corpus cancer and colorectal cancer exhibited a
higher mean than that of kidney cancer. The expression of CCR8 was
confirmed in the tumor-infiltrating cells of ovary cancer, uterine
corpus cancer, colorectal cancer and lung cancer, in addition to
the human kidney cancer tumor-infiltrating cells. These results
indicated the possibility that in kidney cancer as well as ovary
cancer, uterine corpus cancer, colorectal cancer and lung cancer,
CCR8-positive tumor-infiltrating cells can be removed using the
anti-human CCR8-specific antibody.
Example 261
Evaluation of Antitumor Effect of Combined Administration of
Anti-Mouse CCR8 Antibody and Anti-PD-1 Antibody Using Breast
Cancer-Derived EMT6
[0192] 1.times.10.sup.5 breast cancer-derived EMT6 cells (50 uL)
were intracutaneously transplanted to the back of each Balb/c mouse
(7 weeks old, female).
[0193] To an anti-mouse CCR8 antibody alone administration group,
15 .mu.g of a rat anti-mouse CCR8 antibody (clone SA214G2,
BioLegend, Inc.) was intravenously administered (100 .mu.L) 3 and
10 days after tumor inoculation, and 200 .mu.g (100 .mu.L) of an
isotype control antibody was administered at 8 and 13 days after
tumor inoculation (N=10). To an anti-PD-1 antibody alone
administration group, 15 .mu.g (100 .mu.L) of an isotype control
antibody was intravenously administered 3 and 10 days after tumor
inoculation, and 200 .mu.g (100 .mu.L) of an anti-mouse PD-1
antibody (RMP1-14, Bio X Cell) was intravenously administered at 8
and 13 days after tumor inoculation (N=10). To an anti-PD-1
antibody and anti-mouse CCR8 antibody combined administration
group, 15 .mu.g (100 .mu.L) of the rat anti-mouse CCR8 antibody was
intravenously administered 3 and 10 days after tumor inoculation,
and 200 .mu.g (100 .mu.L) of the anti-PD-1 antibody was
intravenously administered at 8 and 13 days after tumor inoculation
(N=10). To a control group, 15 .mu.g (100 .mu.L) of an isotype
control antibody was intravenously administered 3 and 10 days after
tumor inoculation, and 100 .mu.L of PBS was intravenously
administered at 8 and 13 days after tumor inoculation (N=10). Tumor
volumes were measured every 3 to 4 days from 3 days after tumor
inoculation (1 day after antibody administration). The tumor volume
(mm.sup.3) was calculated according to major axis (mm).times.minor
axis (mm).times.minor axis (mm)/2 (FIG. 35).
[0194] In the comparison of a mean tumor volume between the alone
administration groups, the mean tumor volume was significantly
small in the anti-mouse CCR8 antibody administration group compared
with the anti-PD-1 antibody administration group at days 10, 14,
17, 20, 23 and 27 (Dunnett method, significance level: P<0.05).
Also, the tumors were small in the combined administration group
compared with each alone administration group.
[0195] A complete remission rate of the tumors at 17 and 27 days
after tumor inoculation was also compared. At post-transplant day
17, the complete remission of the tumors was exhibited in 0 out of
10 mice in the control group and the anti-PD-1 antibody
administration group and 1 out of 10 mice in the anti-mouse CCR8
antibody administration group, whereas the tumors remitted
completely in 6 out of 10 mice in the anti-PD-1 antibody and
anti-mouse CCR8 antibody combined administration group. At
post-transplant day 27, the complete remission of the tumors was
exhibited in 2 and 3 out of 10 mice in the control group and the
anti-PD-1 antibody administration group, respectively, and 7 out of
10 mice in the anti-mouse CCR8 antibody administration group,
whereas the tumors remitted completely in 9 out of 10 mice in the
anti-PD-1 antibody and anti-mouse CCR8 antibody combined
administration group.
[0196] The proportion of an individual bearing tumor larger than 50
mm.sup.3 or smaller was further calculated (FIG. 36). Tue tumors
larger than 50 mm.sup.3 or smaller in all the individuals in the
anti-PD-1 antibody and anti-mouse CCR8 antibody combined
administration group (100%) at post-transplant day 17 and then were
50 mm.sup.3 or smaller up to day 27, whereas the proportion was 10%
and 30% in the anti-PD-1 antibody administration group at days 17
and 27, respectively, and 70% in the anti-mouse CCR8 antibody
administration group at both 17 and 27 days after tumor
inoculation.
[0197] These results demonstrated that the combined administration
group requires a short time to tumor regression and has a strong
regressing effect, as compared with other alone administration
groups.
Example 27
Evaluation of Antitumor Effect of Combined Administration of
Anti-Mouse CCR8 Antibody and Anti-PD-1 Antibody Using Mouse Kidney
Cancer-Derived Cell Line RAG
[0198] 4.5.times.10.sup.5 kidney cancer-derived RAG cells (50 uL)
were intracutaneously transplanted to the back of each Balb/c mouse
(6 weeks old, female). The RAG cells used were RAG cells
(acclimatized cell line) with mouse subcutaneous engraftment
efficiency elevated by transplanting, again to a mouse, a tumor
successfully engrafted in advance by subcutaneous inoculation to a
Balb/c mouse and repeating this operation twice.
[0199] To an anti-PD-1 antibody alone administration group, 50
.mu.g (100 .mu.L) of an anti-PD-1 antibody (RMP1-14, Bio X Cell)
was intravenously administered 8 and 15 days after tumor
inoculation (N=10). To an anti-mouse CCR8 antibody alone
administration group, 25 .mu.g (100 .mu.L) of a rat anti-mouse CCR8
antibody (clone SA214G2, BioLegend, Inc.) was intravenously
administered 8 and 15 days after tumor inoculation (N=10). To an
anti-PD-1 antibody and anti-mouse CCR8 antibody combined
administration group, 50 .mu.g of an anti-PD-1 antibody (RMP1-14,
Bio X Cell) and 25 .mu.g of a rat anti-mouse CCR8 antibody (clone
SA214G2, BioLegend, Inc.) were mixed (100 .mu.L) and intravenously
administered 8 and 15 days after tumor inoculation (N=10). To a
control group, 100 .mu.L of physiological saline was intravenously
administered 8 and 15 days after tumor inoculation (N=10).
[0200] Tumor volumes were measured every 3 to 4 days from 8 days
after tumor inoculation. The tumor volume (mm.sup.3) was calculated
according to major axis (mm).times.minor axis (mm).times.minor axis
(mm)/2 (FIG. 37).
[0201] As a result, the tumors were found to be reduced in size in
the anti-PD-1 antibody and anti-mouse CCR8 antibody combined
administration group compared with the anti-PD-1 antibody or
anti-mouse CCR8 antibody alone administration group.
Example 281
Analysis on Specificity of Anti-Mouse CCR8 Antibody Using
Homozygously CCR8 Gene-Deficient Mouse
[0202] 3.times.10.sup.5 colorectal cancer-derived Colon26 cells (50
uL) were intracutaneously transplanted to the back of each
wild-type mouse (N=10) or homozygously CCR8 gene-deficient mouse
(N=5) of Balb/c lineage. To the wild-type mouse, 100 .mu.g (100
.mu.L) of a rat anti-mouse CCR8 antibody (clone SA214G2, BioLegend,
Inc.) or an isotype control antibody (LTF-2, Bio X Cell) was
intravenously administered 3 and 10 days after tumor inoculation
(N=5). To the homozygously CCR8 gene-deficient mouse, 100 .mu.g
(100 .mu.L) of a rat anti-mouse CCR8 antibody (clone SA214G2,
BioLegend, Inc.) or an isotype control antibody (LTF-2, Bio X Cell)
was also intravenously administered 3 and 10 days after tumor
inoculation (N=5). Tumor sizes were measured from
post-administration day 7.
[0203] As a result, significant tumor regression and final complete
tumor regression were observed in all the wild-type mice by the
anti-mouse CCR8 antibody administration compared with the isotype
control antibody administration. On the other hand, neither change
in tumor volume nor tumor regression was observed in the
homozygously CCR8 gene-deficient mice in the anti-mouse CCR8
antibody administration group compared with the isotype antibody
administration group (FIG. 38).
[0204] The antitumor effect of the anti-mouse CCR8 antibody
disappeared completely in the homozygously CCR8 gene-deficient
mice, demonstrating that the anti-mouse CCR8 antibody (SA214G2)
used exerts an antitumor effect via CCR8.
INDUSTRIAL APPLICABILITY
[0205] The antibody against CCR8 of the present invention has an
effect of activating the immunity by decreasing the number of
tumor-infiltrating Treg cells or the like and is thus
pharmaceutically useful for the treatment of cancers.
* * * * *