U.S. patent application number 12/410732 was filed with the patent office on 2010-04-08 for anticancer agent comprising anti-pd-1 antibody or anti-pd-l1 antibody.
This patent application is currently assigned to Seoul National University Industry Foundation. Invention is credited to Woo Sung Chang, Chang Yuil Kang, Ji Yeon Kim.
Application Number | 20100086550 12/410732 |
Document ID | / |
Family ID | 41171109 |
Filed Date | 2010-04-08 |
United States Patent
Application |
20100086550 |
Kind Code |
A1 |
Kang; Chang Yuil ; et
al. |
April 8, 2010 |
ANTICANCER AGENT COMPRISING ANTI-PD-1 ANTIBODY OR ANTI-PD-L1
ANTIBODY
Abstract
Provided is an anticancer agent which comprises an anti-PD-1
antibody or an anti-PD-L1 antibody as an active ingredient,
functioning to reverse the unresponsiveness of iNKT cells in which
anergy has been induced by administration with an iNKT cell ligand.
The anti-PD-1 or anti-PD-L1 antibody blocks the PD-1/PD-L1-mediated
signaling pathway not only to prevent the iNKT cell ligand-induced
iNKT cell anergy, but also to reverse the unresponsiveness of
already anergic iNKT cells to produce cytokines. In addition, the
anti-PD1 or anti-PD-L1 antibody ensures the potent anti-tumor
activity of iNKT cells as demonstrated by a significant reduction
in the number of metastatic nodules in B16F10 melanoma metastasis
models in vivo. Collectively, the anticancer agent can be very
useful in the treatment of cancer, particularly metastatic
cancer.
Inventors: |
Kang; Chang Yuil; (Seoul,
KR) ; Chang; Woo Sung; (Gyeonggi-do, KR) ;
Kim; Ji Yeon; (Seoul, KR) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
Seoul National University Industry
Foundation
Seoul
KR
|
Family ID: |
41171109 |
Appl. No.: |
12/410732 |
Filed: |
March 25, 2009 |
Current U.S.
Class: |
424/138.1 |
Current CPC
Class: |
C07K 2317/76 20130101;
A61K 39/39558 20130101; A61K 2039/505 20130101; C07K 16/2818
20130101; A61P 35/00 20180101; A61K 39/39558 20130101; A61P 35/02
20180101; A61K 2300/00 20130101; C07K 16/2827 20130101 |
Class at
Publication: |
424/138.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 2, 2008 |
KR |
10-2008-0097236 |
Claims
1. An anticancer agent, comprising an iNKT cell ligand as a first
anticancer factor and an anti-PD-1 antibody or anti-PD-L1 antibody
as a second anticancer factor, having a function of reversing
unresponsiveness of iNKT cells in which anergy is induced by
administration with the iNKT cell ligand.
2. The anticancer agent as defined in claim 1, wherein the iNKT
cell ligand is selected from a group consisting of alpha-galactosyl
ceramide, alpha-glucuronosyl ceramide,
phosphatidylinositoltetramannoside, isoglobotrihexosylceramide,
ganglioside GD3, phsphatidylcholine, phosphatidylethanolamine,
phosphatidylinositol, sulfatide, beta-galactosylceramide,
lipophosphoglycan, glycoinositol phospholipid,
alpha-galactosylceramide analogs including beta-anomer
galactoceramide and alpha-anomer galactosylceramide, and bacterial
lipid antigens.
3. The anticancer agent as defined in claim 1, wherein the
anti-PD-1 antibody or the anti-PD-L1 antibody is a monoclonal
antibody or a polyclonal antibody.
4. The anticancer agent as defined in claim 1, wherein the cancer
is selected from a group consisting of lung cancer, stomach cancer,
liver cancer, bone cancer, pancreatic cancer, skin cancer, head and
neck cancer, melanoma, uterine cancer, ovarian cancer, rectal
cancer, colorectal cancer, colon cancer, breast cancer, uterine
sarcoma, Fallopian tube carcinoma, endometrial carcinoma, uterine
cervical carcinoma, vaginal carcinoma, vulva carcinoma, esophageal
cancer, larynx cancer, small intestine cancer, thyroid cancer,
parathyroid cancer, soft tissue sarcoma, uterine cancer, penis
cancer, prostate cancer, chronic or acute leukemia, pediatric solid
tumor, differentiated lymphoma, bladder cancer, kidney cancer,
renal cell carcinoma, renal pelvic carcinoma, primary CNS lymphoma,
spinal cord tumor, brainstem glioma, and pituitary adenoma.
5. A method for reversing the unresponsiveness of iNKT cells with
anergy induced therein by iNKT cell ligand treatment, comprising
treating the anergic iNKT cells with an anti-PD-1 antibody or an
anti-PD-L1 antibody.
6. The method as defined in claim 5, wherein the iNKT cell ligand
is selected from a group consisting of alpha-galactosyl ceramide,
alpha-glucuronosyl ceramide, phosphatidylinositoltetramannoside,
isoglobotrihexosylceramide, ganglioside GD3, phsphatidylcholine,
phosphatidylethanolamine, phosphatidylinositol, sulfatide,
beta-galactosylceramide, lipophosphoglycan, glycoinositol
phospholipid, alpha-galactosylceramide analogs including
beta-anomer galactoceramide and alpha-anomer galactosylceramide,
and bacterial lipid antigens.
7. The method as defined in claim 5, wherein the anti-PD-1 antibody
or the anti-PD-L1 antibody is a monoclonal antibody or a polyclonal
antibody.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from Korean Patent
Application No.10-2008-0097236, filed Oct. 2, 2008, the entire
disclosure of which is incorporated herein by reference in its
entirety.
TECHNICAL FIELD
[0002] The present invention relates, in general, to an anticancer
agent and, more particularly, to an anticancer agent comprising an
anti-PD-1 antibody or an anti-PD-L1 antibody as an active
ingredient, functioning to restore the responsiveness of iNKT cells
in which anergy has been induced by administration with an iNKT
cell ligand.
BACKGROUND OF THE INVENTION
[0003] Natural killer T (NKT) cells, co-expressing a T cell
receptor and NK cell markers, are essential for several aspects of
immunity, such as immunomodulation and immunuopotentiation, in
various immune diseases including autoimmune diseases, infectious
diseases, cancer, etc. NKT cells exist at high levels in the
thymus, the liver, and the bone marrow, but at low levels in the
spleen, lymph nodes and blood.
[0004] Unlike conventional T cells that recognize small peptide
antigens presented by major histocompatibility complex MHC class 1
or MHC class 2, NKT cells recognize glycolipid antigens presented
by CD1d, a MHC class 1-like molecule. A major subset of NKT cells,
called type 1 NKT cells or invariant natural T (iNKT) cells,
express an invariant natural T cell receptor (TCR) composed of
V.alpha.14-J.alpha.18 chains in mice (V.alpha.24-J.alpha.18 in
humans). Upon TCR stimulation with a ligand, such as
.alpha.-galactosylceramide (.alpha.-GC), iNKT cells rapidly produce
a wide range of cytokines including IL-4, IFN-.gamma., L-12, and
GM-CSF. This rapid and potent response to a ligand enables iNKT
cells to enhance or regulate the activity of various immune cells
in innate and acquired immunity. Found in diverse diseases and
promoting tumor rejection or regulating autoimmune disorders, these
immunomodulatory roles of iNKT cells are studied for use in
immunotherapy treatments for cancer and autoimmune diseases.
[0005] However, iNKT cells tend to greatly decrease in
responsiveness following repeated stimulation after a first
stimulation with their ligands via the T cell receptor. For
instance, iNKT cells that have been stimulated in vivo with a-GC
have reduced proliferation and cytokine production upon secondary
stimulation with the same ligand. This iNKT cell anergy is a major
obstacle in immunotherapeutic trials targeting iNKT cells.
[0006] Conventional T cells are known to become anergic when they
receive a TCR signal with insufficient co-stimulatory signals.
Co-stimulatory molecules such as CD28, CD40L and ICOS are known to
be involved in the development and activation of iNKT cells.
Recently, it has been reported that 4-1BB contributes to promote
the activation of iNKT cells as a co-stimulatory molecule and can
affect iNKT cell-mediated allergic lung inflammation (Kim, D. H.,
W. S. Chang, Y. S. Lee, K. A. Lee, Y. K. Kim, B. S. Kwon, and C. Y.
Kang. 2008. 4-1BB engagement co-stimulates NKT cell activation and
exacerbates NKT cell ligand-induced airway hyperresponsiveness and
inflammation. J Immunol 180:2062-2068.). On the other hand, it has
recently been suggested that coinhibitory molecules, such as PD-1,
B7H3, and B7H4, may actively anergize or tolerize T cells by
delivering inhibitory signals into TCR-stimulated T cells. In a
lymphocytic choriomeningitis virus (LCMV) infected model, CD8 T
cells are tolerized by LCMV epitope-presenting dendritic cells.
However, the blockade of the PD-1 signal can reverse the anergic
phenotype of CD8 T cells. It has also been reported that the
inhibition of PD-1/PD-L1 restores the function of exhausted CD8 T
cells in a chronic infection model.
[0007] Programmed death-1 (PD-1) is a 55 KDa type 1 transmebrane
protein of the immunoglobulin superfamily, and is known as a
co-inhibitory molecule on T cells. That is, PD-1 is a member of the
co-inhibitory molecules of the CD28 family (e.g., CD28, CTLA4, ICOS
and BTLA) expressed on activated B cells, T cells and bone marrow
cells. Two ligands for PD-1, PD-L1 and PD-L2, have been identified
thus far. The interaction of PD-1 with the PD ligands can transduce
inhibitory or co-stimulatory signals into the T cells. In
conventional T cells, PD-1 is not expressed on naive T cells, but
is inducibly expressed after T cell activation. As for PD-L1, it is
expressed to some degree on naive T cells and its level is
increased on activated T cells. PD-L1 is found at high levels in
various human cancers and interacts with PD-1 to transduce
inhibitory or co-stimulatory signals from entering into the T
cells. For example, the interaction between PD-1 and PD-L1 induces
a decrease in the level of tumor invasive lymphocytes and in T cell
receptor-mediated proliferation and causes the immune evasion of
tumor cells. In PD-1-deficient animals, PD-1 develops various
autoimmune phenotypes, such as autoimmune cardiac infarction and
lupus-like syndromes with arthritis and nephritis, and plays an
important role in the development of autoimmune encephalomyelitis,
systemic lupus erythematosus, graft-versus-host disease (GVHD),
type 1 diabetes and rheumatic arthritis. Aged PD-1-deficient mice
develop autoimmune diseases, indicating that PD-1 plays a critical
role in the regulation of autoimmunity and immune tolerance. In
particular, PD-1 signals are essential for inducing T cell
exhaustion during chronic infection.
[0008] With the ability thereof to stimulate the T cell receptor to
rapidly produce various cytokines of iNKT cells, the NKT cell
ligand .alpha.-GC has conventionally been used as an anticancer
agent. However, since repeated stimulation of iNKT cells with
.alpha.-GC induces anergy leading to a great decrease in
responsiveness, it cannot achieve effective anticancer effects.
Therefore, there is a pressing need for an anticancer agent that
can restore the responsiveness of iNKT cells even in the state of
anergy caused by stimulation with iNKT cell ligands.
[0009] Leading to the present invention, intensive and thorough
research into an anticancer agent taking advantage of the
responsiveness of iNKT cells, conducted by the present inventors,
resulted in the finding that PD-1 expressed on iNKT cells is
upregulated after stimulation and that blocking of the PD-1/PD-L1
signaling pathway by an anti-PD-1 or anti-PD-L1 antibody allows
iNKT cells under an iNKT cell ligand-induced anergy condition to
recover their responsiveness, such as the production of cytokines.
Also, the anti-PD1 or anti-PD-L1 antibody was found to induce
potent anti-tumor activity of iNKT cells as demonstrated by a
significant reduction in the number of metastatic nodules in B16F10
melanoma metastasis models in vivo.
SUMMARY OF THE INVENTION
[0010] It is therefore an object of the present invention to
provide an anticancer agent which is based on the activity of iNKT
cells, featuring the recovery of iNKT cell responsiveness.
[0011] It is another object of the present invention to provide a
method for the treatment of cancer using the same.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The above and other objects, features and advantages of the
present invention will be more clearly understood from the
following detailed description taken in conjunction with the
accompanying drawings, in which:
[0013] FIG. 1 is of flow histograms showing the expression levels
of PD-1, PD-L1 and PD-L2 on iNKT cells of the splenocytes isolated
from .alpha.-GC-treated C57BL/6 mice at indicated time points (0
hr, 6 hrs, 72 hrs, 7 days, 1 month and 2 months) after
treatment.
[0014] FIG. 2 is of histograms showing IFN-.gamma. and IL-4 levels
in the supernatants obtained after incubating splenocytes isolated
from naive C57BL/6 mice with .alpha.-GC in the presence of control
rat IgG, anti-PD-1 monoclonal antibody, anti-PD-L1 monoclonal
antibody or anti-PD-L2 monoclonal antibody as analyzed by ELISA (in
vitro).
[0015] FIG. 3 is of plots showing IFN-.gamma. and IL-4 levels in
the sera obtained from the C57BL/6 mice, which were
intraperitoneally injected with control rat IgG, anti-PD-1
monoclonal antibody, anti-PD-L1 monoclonal antibody or anti-PD-L2
monoclonal antibody 24 hrs before .alpha.-GC treatment, at 0, 2, 6,
12, 24, 48 and 72 hrs after the .alpha.-GC treatment as analyzed by
ELISA (in vivo).
[0016] FIG. 4 shows FACS results of IFN-.gamma..sup.+ or
IL-.sub.4.sup.+ iNKT cells on which intracellular cytokine staining
was performed to examine if the increased production of IFN-.gamma.
and IL-4 in the sera of FIG. 3 comes from iNKT cells.
[0017] FIG. 5 is of histograms showing IFN-.gamma. and IL-4 levels
in the supernatants obtained after the splenocytes of C57BL/6 mice,
in which iNKT cell anergy was induced by .alpha.-GC treatment 7
days and one month before splenocyte isolation therefrom, were
incubated with .alpha.-GC in the presence of control rat IgG,
anti-PD-1 monoclonal antibody, anti-PD-L1 monoclonal antibody or
anti-PD-L2 monoclonal antibody, as analyzed by ELISA (in
vitro).
[0018] FIG. 6 is of histograms showing IFN-.gamma. and L-4 levels
in sera from C57BL/6 mice, as analyzed by ELISA. The mice were
intraperitoneally injected with control rat IgG, anti-PD-1
monoclonal antibody, anti-PD-L1 monoclonal antibody or anti-PD-L2
monoclonal antibody, followed by double injection with .alpha.-GC
24 hrs and 14 days later. The sera were obtained 2 and 12 hrs after
the secondary injection of .alpha.-GC (in vivo).
[0019] FIG. 7 shows FACS results of IFN-.gamma..sup.+ or IL-4.sup.+
iNKT cells after intracellular cytokine staining was performed on
the splenocytes isolated 2 hrs after the secondary injection (in
vivo).
[0020] FIG. 8 shows CD69 expression levels on iNKT and NK cells as
measured by flow cytometry using the splenocytes isolated 12 hrs
after the secondary injection (in vivo).
[0021] FIG. 9 is a histogram showing weights of normal and
metastatic lungs of C57BL/6 mice. The mice were intraperitoneally
injected with control rat IgG, anti-PD-1 monoclonal antibody or
anti-PD-L1 antibody 24 hrs before intravenous injection with skin
tumor cells. On Days 0, 4 and 8, the mice were co-administered with
.alpha.-GC and the control rat IgG, the anti-PD-1 antibody or the
anti-PD-L1 antibody. The lungs were excised 14 days after the
co-administration.
[0022] FIG. 10 shows effects of the blockage of PD-1/PD-L1
interaction on the antitumor activity of iNKT cells in terms of the
number of metastatic pulmonary nodules (A) and optical microscopic
views (B). C57BL/6 mice were intraperitoneally injected with
control rat IgG, anti-PD-1 antibody or anti-PD-L1 antibody 24 hrs
before .alpha.-GC treatment. 7 days later, the mice were
intravenously injected with skin tumor cells and re-injected with
.alpha.-GC on Days 0, 4 and 8. 14 days later, the lungs were
excised.
DETAILED DESCRIPTION OF THE INVENTION
[0023] In accordance with an aspect thereof, the present invention
provides an anticancer agent, comprising an iNKT cell ligand as a
first anticancer factor and an anti-PD-1 antibody or anti-PD-L1
antibody as a second anticancer factor, having a function of
reversing unresponsiveness of iNKT cells in which anergy is induced
by administration with the iNKT cell ligand.
[0024] In accordance with another aspect thereof, the present
invention provides a method for reversing the unresponsiveness of
iNKT cells with anergy induced therein by iNKT cell ligand
treatment, comprising treating the anergic iNKT cells with an
anti-PD-1 antibody or an anti-PD-L1 antibody.
[0025] The iNKT cell ligand may be selected from a group consisting
of alpha-galactosyl ceramide, alpha-glucuronosyl ceramide,
phosphatidylinositoltetramannoside, isoglobotrihexosylceramide,
ganglioside GD3, phsphatidylcholine, phosphatidylethanolamine,
phosphatidylinositol, sulfatide, beta-galactosylceramide,
lipophosphoglycan, glycoinositol phospholipid,
alpha-galactosylceramide analogs including beta-anomer
galactoceramide and alpha-anomer galactosylceramide, and bacterial
lipid antigens.
[0026] The anti-PD-1 antibody or the anti-PD-L1 antibody may be a
monoclonal antibody or a polyclonal antibody.
[0027] The anti-PD-1 antibody or anti-PD-L1 antibody according to
the present invention can block the signaling of PD-1 or PD-L1 to
prevent the iNKT cell ligand-induced anergy of iNKT cells and can
provide cytokine secretion ability for even anergic iNKT cells to
restore their responsiveness. In addition, the anti-PD-1 antibody
or anti-PD-L1 antibody of the present invention inhibits the anergy
induction of iNKT cells to significantly decrease the number of
pulmonary nodules in a lung metastasis model of B16F10 melanoma,
thus effectively eliciting anticancer immune responses of iNKT
cells and showing anti-tumor effects of iNKT cells against cancer
metastasis. Therefore, an anticancer agent comprising the anti-PD-1
antibody or anti-PD-L 1 antibody of the present invention can be
very useful in the treatment of cancer, particularly, metastatic
cancer.
[0028] The cancer to which the anticancer agent according to the
present invention is therapeutically applicable may be gynecologic
tumor, endocrine gland cancer, CNS (central nervous system) tumor
or ureter cancer. Concrete examples of the cancer include lung
cancer, stomach cancer, liver cancer, bone cancer, pancreatic
cancer, skin cancer, head and neck cancer, melanoma, uterine
cancer, ovarian cancer, rectal cancer, colorectal cancer, colon
cancer, breast cancer, uterine sarcoma, Fallopian tube carcinoma,
endometrial carcinoma, uterine cervical carcinoma, vaginal
carcinoma, vulva carcinoma, esophageal cancer, larynx cancer, small
intestine cancer, thyroid cancer, parathyroid cancer, soft tissue
sarcoma, uterine cancer, penis cancer, prostate cancer, chronic or
acute leukemia, pediatric solid tumor, differentiated lymphoma,
bladder cancer, kidney cancer, renal cell carcinoma, renal pelvic
carcinoma, primary CNS lymphoma, spinal cord tumor, brainstem
glioma, and pituitary adenoma.
[0029] The anticancer agent of the present invention may be
formulated into a pharmaceutical composition with at least one
conventional anticancer ingredient.
[0030] In addition to the active ingredients, the pharmaceutical
composition may further comprise a pharmaceutically acceptable
carrier. Examples of the pharmaceutically acceptable carrier
include saline, sterile water, Ringer's solution, buffered saline,
dextrose solution, maltodextrin solution, glycerol, ethanol, etc.
Optionally, conventional additives, such as antioxidants, buffers,
bacteriostatic agents, etc., may be added to the composition. For
the preparation of dosage forms including injections, such as
aqueous solutions, suspensions and emulsions, pills, capsules,
granules and tablets, the active ingredients may be admixed with a
diluent, a dispersant, a surfactant, a binder and/or a lubricant.
Reference may be made to literature (Remington's Pharmaceutical
Science (recent edition), Mack Publishing Company, Easton Pa.) upon
the formulation of the pharmaceutical composition into suitable
dosage forms.
[0031] The composition of the present invention may be administered
via oral routes or parenteral routes (e.g., intravenous,
subcutaneous, intraperitoneal, topical, etc.). The effective dosage
of the anticancer agent in accordance with the present invention
depends on various factors, including the patient's weight, age,
gender, state of health, diet, the time of administration, route of
administration, excretion rate, severity of diseases, etc. In
general, it may be administered in a single dose, and preferably in
multiple doses per day at a daily dose ranging from 0.01 to 1000
mg/day, and preferably from 0.1 to 100 mg/kg of the anti-PD-1
antibody or anti-PD-L1 antibody.
[0032] For the effective prophylaxis and treatment of cancer, the
composition according to the present invention may be used alone or
in combination with surgical operation, hormonal therapy,
chemotherapy, and/or biological response controllers.
[0033] A better understanding of the present invention may be
obtained through the following examples which are set forth to
illustrate, but are not to be construed as limiting the present
invention.
EXAMPLES
Example 1
Expression of PD-1 and PD Ligands in iNKT Cells
[0034] 1. Experimental Animals
[0035] Six- to eight-week-old female C56BL/6 mice were purchased
from Orient Bio. All mice were bred and maintained in specific
pathogen-free conditions.
[0036] 2. Reagents and Antibodies
[0037] .alpha.-GC was dissolved in PBS containing 0.5% Tween 20.
Hybridoma clones producing antibodies to mouse PD-1 (RMP1-14, rat
IgG2a), PD-L1 (MIH-5, rat IgG2a), and PD-L2 (Ty25, rat IgG2a) were
produced according to methods well known in the art [Yamazaki, T.,
H. Akiba, H. Iwai, H. Matsuda, M. Aoki, Y. Tanno, T. Shin, H.
Tsuchiya, D. M. Pardoll, K. Okumura, M. Azuma, and H. Yagita. 2002.
Expression of programmed death 1 ligands by murine T cells and APC.
J Immunol 169:5538-5545.; Tsushima, F., H. Iwai, N. Otsuki, M. Abe,
S. Hirose, T. Yamazaki, H. Akiba, H. Yagita, Y. Takahashi, K.
Omura, K. Okumura, and M. Azuma. 2003. Preferential contribution of
B7-H1 to programmed death-1-mediated regulation of hapten-specific
allergic inflammatory responses. Eur J Immunol 33:2773-2782.;
Yamazaki, T., H. Akiba, A. Koyanagi, M. Azuma, H. Yagita, and K.
Okumura. 2005. Blockade of B7-H1 on macrophages suppresses CD4+ T
cell proliferation by augmenting IFN-gamma-induced nitric oxide
production. J Immunol 175:1586-1592.]. All clones were cultured in
RPMI 1640 (Gibco) with 10% FBS (Gibco) and 1%
penicillin/streptomycin (BioWhittaker). All antibodies were
prepared from the ascites of nude mice using caprylic acid
purification. Control rat IgG was also prepared by caprylic acid
purification from sera of naive rats.
[0038] 3. Expression of PD-1 and PD Ligands on iNKT Cells After
.alpha.-GC Administration
[0039] Splenocytes were isolated from C57BL/6 mice administered
with 2 .mu.g of .alpha.-GC at different time points (0 hr, 6 hrs,
72 hrs, 7 days, one month and two months) after the administration.
The cells were stained with a PE-conjugated anti-PD-1 monoclonal
antibody, a PE-conjugated anti-PD-L1 monoclonal antibody, a
PE-conjugated anti-PD-L2 monoclonal antibody and a PE-conjugated
isotype control monoclonal antibody, respectively. iNKT cells were
gated on B220.sup.- TCR-.beta..sup.int.alpha.-GC/CD1d:Ig.sup.+
population. PD-1, PD-L1 and PD-L2 expression (open histograms) was
analyzed by FACS. The results are given in FIG. 1. Shaded
histograms indicate staining with isotype control niAbs.
[0040] As depicted in FIG. 1, PD-1 was constitutively expressed on
iNKT cells at a low level and its expression was upregulated after
.alpha.-GC stimulation, followed by the persistence of the
upregulated level for two months. On the other hand, PD-L1
expression was temporarily increased on .alpha.-GC-stimulated iNKT
cells but declined toward naive state levels within 72 hrs. As for
PD-L2, its expression was not found on iNKT cells regardless of
their activation status.
Example 2
Effects of PD-1, PD-L1 and PD-L2 on iNKT Cell Activation
[0041] The following in vitro and in vivo experiments were
conducted to examine the effects of PD-1, PD-L1 and PD-L2 on iNKT
cell activation.
[0042] 1. iNKT Cell Activation In Vitro
[0043] After being prepared from naeve C57BL/6 mice,
5.times.10.sup.5 spelenocytes were incubated for 3 days with 100
ng/ml of .alpha.-GC in the presence of 50 .mu.g/ml of the control
rat IgG, the anti-PD-1 mAb, the anti-PD-L1 mAb, or the anti-PD-L2
mAb. Then, the supernatants were obtained and assayed for
IFN-.gamma. and IL-4 levels by ELISA.
[0044] The results are given in FIG. 2.
[0045] Compared with control IgG treatment, as is apparent from
FIG. 2, anti-PD-1 mAb significantly increased the production of
IFN-.gamma. and, to a lesser extent, IL-4. Anti-PD-L1 mAb also
induced greater production of IFN-.gamma., but anti-PD-L2 mAb did
not.
[0046] 2. iNKT Cell Activation in Vivo
[0047] C57BL/6 mice was injected with 200 .mu.g of the control rat
IgG, the anti-PD-1 mAb, the anti-PD-L1 mAb or the anti-PD-L2 mAb 24
hrs before treatment with 2 .mu.g of .alpha.-GC. Sera were obtained
at 0, 2, 6, 12, 24, 48 and 72 hrs after the treatment, followed by
ELISA analysis for IFN-.gamma. and IL-4 levels.
[0048] In order to determine whether the increased production of
IFN-.gamma. was distinctively attributed to iNKT cells,
intracellular cytokine staining was followed by flow cytometry
analysis. In this regard, splenocytes were isolated 2 hrs after
.alpha.-GC treatment and 5.times.10.sup.6 cells were incubated with
Golgi plug for 2 hrs to accumulate cytokines. Intracellular
cytokine staining was performed using BD Cytofix/Cytoperm Plus with
Golgiplug kit according to the manufacturer's protocol (BD
Biosciences). iNKT cells were gated on B220.sup.-TCR-.beta..sup.int
.alpha.-GC/CD1d:Ig.sup.+ population and IFN-.gamma..sup.+ or
IL-4.sup.+ iNKT cells were analyzed by flow cytometry.
[0049] IFN-.gamma. and IL-4 levels in blood are depicted in FIG. 3
and flow histograms of IFN-.gamma..sup.+ and IL-4.sup.+ iNKT cells
are given in FIG. 4.
[0050] As shown in FIG. 3, the blood IFN-.gamma. and IL-4 levels
were observed to peak, respectively, within 12 hrs and 2 hrs after
.alpha.-GC treatment, indicating iNKT cell activation. IFN-.gamma.
levels in the sera of anti-PD-1 or anti-PD-L1 mAb-treated mice were
significantly enhanced compared with levels in the group treated
with control IgG. Slightly different L-4 production was observed
after treatment with the blocking Abs, but with no
significance.
[0051] As shown in FIG. 4, iNKT cells producing IFN-.gamma.
increased in number because they blocked PD-1/PD-L1 interaction
with the anti-PD-1 mAb or anti-PD-L1 mAb in comparison with control
cells. On the other hand, no significant differences were found
between populations of IL-4.sup.+ iNKT cells and control cells
after mAb treatment. These data indicate that the blockage of
PD-1/PD-L1 interaction allows the delivery of the co-inhibitory
signal during iNKT activation, resulting in increased IFN-.gamma.
secretion from iNKT cells.
Example 3
Effects of Blockage of PD-1/PD-L1 Interaction on Responsiveness of
Anergic iNKT Cells
[0052] The following in vitro and in vivo experiments were
performed in order to examine whether the blockage of PD-1/PD-L 1
interaction reverses iNKT cell anergy.
[0053] 1. Recovery of Responsiveness of Anergic iNKT Cells in
Vitro
[0054] The .alpha.-GC-induced unresponsiveness of iNKT cells was
detected as early as 3 days after primary stimulation and observed
to persist until 7.about.30 days after .alpha.-GC stimulation.
Thus, C57BL/6 mice were injected with 2 .mu.g of .alpha.-GC to
induce iNKT cell anergy. One week and one month later, splenocytes
were isolated from the mice and 5.times.10.sup.5 cells were
incubated for 3 days with 100 ng/ml of .alpha.-GC in the presence
of 50 .mu.g/ml of the control rat IgG, the anti-PD-1 mAb, the
anti-PD-L1 mAb, or the anti-PD-L2 mAb. Also, splenocytes isolated
from the mice were incubated for 3 days with 10 ng/ml of .alpha.-GC
without mAb, and these were represented by `activation`. The
supernatants were then assayed for IFN-.gamma. and IL-4 levels by
ELISA [*:p<0.05 and **:p<0.01 (vs. control rat IgG)].
[0055] The results are depicted in FIG. 5.
[0056] As seen in the graphs of FIG. 5, when restimulated with
.alpha.-GC in the presence of the control IgG, the splenocytes of
the mice in which iNKT cell anergy was induced by pre-treatment
with .alpha.-GC 7 days before the restimulation were found to
greatly decrease in the production of IFN-.gamma. and IL-4. In
contrast, the restimulation of iNKT cell anergy-induced splenocytes
with .alpha.-GC in the presence of the anti-PD-1 antibody or
anti-PD-L1 antibody resulted in a great increase in the production
of IFN-.gamma. and L-4 (A). This upregulation was observed to
persist for one to two months after .alpha.-GC treatment (B).
Therefore, the blockage of PD-1/PD-L1 interaction during
restimulation with .alpha.-GC reverses the established anergic
phenotype of iNKT cells.
[0057] 2. Recovery of Responsiveness of Anergic iNKT Cells in
Vivo
[0058] 200 .mu.g of the control rat IgG, the anti-PD-1 antibody or
the anti-PD-L1 antibody was intraperitoneally injected into C57BL/6
mice 24 hrs before treatment with 2 .mu.g of .alpha.-GC. 14 days
later, 2 .mu.g of .alpha.-GC was injected again, followed by the
preparation of sera 2 and 12 hrs after the re-injection for ELISA
assay of IL-4 and IFN-.gamma. levels, respectively.
[0059] Splenocytes (5.times.10.sup.6 cells), prepared two and
twelve hours after the second injection of .alpha.-GC, were
incubated with Golgi plug for 2 hrs to accumulate cytokines.
Intracellular cytokine staining was performed on the splenocytes
prepared 2 hrs after the secondary GC treatment, using BD
Cytofix/Cytoperm Plus with Golgiplug kit according to the
manufacturer's protocol (BD Biosciences). The splenocytes prepared
12 hrs later were used in an assay for CD69 expression in iNKT and
NK cells. iNKT cells were gated on
B220.sup.-TCR-.beta..sup.int.alpha.-GC/CD1d:1g.sup.+ population and
NK cells were gated on B220.sup.- TCR-.beta..sup.-NK1.1.sup.high
population. IFN-.gamma..sup.+ or IL-4.sup.+ iNKT cells were
analyzed by flow cytometry. Also, CD69 expression on iNKT and NK
cells was analyzed by flow cytometry.
[0060] IFN-.gamma. and L-4 levels in blood are graphed in FIG. 6
and FACS results of IFN-.gamma..sup.+ or L-4.sup.+ iNKT cells and
flow histograms of CD69 expression on iNKT and NK cells are given
in FIGS. 7 and 8, respectively.
[0061] Like mice treated with control rat IgG, as seen in FIG. 6,
mice pretreated with .alpha.-GC produced significantly low
IFN-.gamma. and L-4 upon secondary .alpha.-GC injection. In
contrast, mice treated with the anti-PD-1 antibody during the
primary .alpha.-GC injection produced remarkably higher IFN-.gamma.
and L-4 upon the secondary .alpha.-GC injection than did the
control IgG-treated group.
[0062] As seen in FIG. 7, the increased cytokine production was
attributed to iNKT cells.
[0063] It was also found that CD69 expression upon secondary
.alpha.-GC injection was increased on iNKT and NK cells in a
similar manner by the treatment with anti-PD-1 mAbs, as shown in
FIG. 8. Thus, PD-1/PD-L1 interaction is essential for the induction
of iNKT cell anergy in vivo.
Example 4
Effects of Anti-PD-1 mAb or Anti-PD-L1 mAb on Anti-Tumor Activity
of Activated iNKT Cells
[0064] The following experiments were performed to investigate the
effects of the anti-PD-1 antibody or the anti-PD-L1 antibody on the
anticancer activity of activated iNKT cells in a B16F10 melanoma
metastasis model.
[0065] 1. Weight of Lung
[0066] Skin tumor cells (B16F10, ATCC) were cultured in DMEM media
supplemented with 10% FBS and 1% penicillin/streptomycin. C56BL/6
mice were intraperitoneally injected with 200 g of the control rat
IgG, the anti-PD-1 mAb orthe anti-PD-L1 mAb 24 hrs before i.v.
inoculation with 2.times.10.sup.5 tumor cells. On Days zero, 4 and
8, the mice were treated with 500 ng of .alpha.-GC plus 200 .mu.g
of the control rat IgG, the anti-PD-1 mAb or the anti-PD-L1 mAb. On
Day 14, the lungs were weighed and weight differences between
metastatic and normal lungs are graphed in FIG. 9.
[0067] As depicted in FIG. 9, the B16F10 metastatic lungs from the
group treated with .alpha.-GC plus the anti-PD-1 mAb were
significantly reduced in weight compared with those from the group
treated with .alpha.-GC plus the control rat IgG.
[0068] 2. Number of Pulmonary Nodules
[0069] Anergy-induced iNKT cells were assayed for the recovery of
anti-tumor activity by the antibodies of the present invention. In
this regard, naive C57BL/6 mice were intraperitoneally injected
with 200 .mu.g of the control rat IgG, the anti-PD-1 antibody or
the anti-PD-L1 antibody 24 hrs after which injection with 2 .mu.g
of .alpha.-GC induced iNKT cell anergy. 7 days later,
5.times.10.sup.5 B16F10 tumor cells were i.v. injected. On Day 0, 4
and 8, the mice were treated with 500 ng of .alpha.-GC to induce
anti-tumor activity of iNKT cells. 14 days later, the lungs were
excised and the nodules formed by cancer metastasis were counted
(A). The isolated lungs were observed under an optical microscope
(B). The results are given in FIG. 10.
[0070] As seen in FIG. 10, when induced by treatment with the
control IgG and .alpha.-GC, iNKT cell anergy was unchanged with
time to show a metastasis result comparable to that of the normal
metastasis control group, indicating that .alpha.-GC treatment
during tumor inoculation did not suppress tumor growth. In
contrast, when the anergy induction of .alpha.-GC was prevented by
treatment with the anti-PD-1 antibody or the anti-PD-L1, the
numbers of tumor nodules in the B16F10 melanoma metastasis model
were remarkably reduced compared with those upon treatment with the
control IgG, demonstrating the superior anti-tumor activity of the
antibodies of the present invention. Taken together, the data
obtained above indicates that the blockage of PD-1/PD-L1
interaction by treatment with the anti-PD-1 antibody or the
anti-PD-L1 antibody during the induction phase of iNKT cell anergy
leads to the persistence of the anti-tumor effects of iNKT
cells.
[0071] Formulation examples are given to illustrate dosage
preparations containing the anticancer agent of the present
invention.
Formulation Example 1
Preparation of Powder
TABLE-US-00001 [0072] Anti-PD-1 or Anti-PD-L1 Antibody 0.1 g
Lactose 1.5 g Talc 0.5 g
[0073] These ingredients were mixed and loaded into an airtight sac
to give a powder.
Formulation Example 2
Preparation of Tablet
TABLE-US-00002 [0074] Anti-PD-1 or Anti-PD-L1 Antibody 0.1 g
Lactose 7.9 g Crystalline cellulose 1.5 g Magnesium stearate 0.5
g
[0075] These ingredients were mixed and directly compressed into a
tablet.
Formulation Example 3
Preparation of Capsule
TABLE-US-00003 [0076] Anti-PD-1 or Anti-PD-L1 Antibody 0.1 g Corn
starch 5 g Carboxycellulose 4.9 g
[0077] These ingredients were admixed together and the admixture
was loaded into a conventional capsule using a suitable device.
Formulation Example 4
Preparation of Injection
TABLE-US-00004 [0078] Anti-PD-1 or Anti-PD-L1 Antibody 0.1 g
Sterile water for injection proper quantity pH Adjuster proper
quantity
[0079] Using a conventional method, these ingredients were put into
an ampule (2 ml) to give an injection.
Formulation Example 5
Preparation of Liquid Medicine
TABLE-US-00005 [0080] Anti-PD-1 or Anti-PD-L1 Antibody 0.1 g
Isomerized sugar 10 g Mannitol 5 g Purified water proper
quantity
[0081] Each ingredient was dissolved in purified water and flavored
with lemon before admixing together. Purified water was added to
the admixture to form a final volume of 100 ml which was then
loaded into a brown vial and sterilized.
[0082] As described hitherto, the anti-PD-1 or anti-PD-L1 antibody
according to the present invention blocks the PD-1/PD-L1-mediated
signaling pathway not only to prevent the iNKT cell ligand-induced
iNKT cell anergy, but also to reverse the unresponsiveness of
already anergic iNKT cells to produce cytokines. In addition, the
anti-PD1 or anti-PD-L1 antibody ensures the potent anti-tumor
activity of iNKT cells as demonstrated by a significant reduction
in the number of metastatic nodules in B16F10 melanoma metastasis
models in vivo. Collectively, the anticancer agent comprising an
anti-PD-1 or anti-PD-L1 antibody in accordance with the present
invention can be very useful in the treatment of cancer,
particularly metastatic cancer.
[0083] Although the preferred embodiments of the present invention
have been disclosed for illustrative purposes, those skilled in the
art will appreciate that various modifications, additions and
substitutions are possible, without departing from the scope and
spirit of the invention as disclosed in the accompanying
claims.
* * * * *