U.S. patent application number 15/007345 was filed with the patent office on 2016-06-30 for combining cd27 agonists and immune checkpoint inhibition for immune stimulation.
The applicant listed for this patent is ADURO BIOTECH HOLDINGS, EUROPE B.V.. Invention is credited to Hans VAN EENENNAAM, Andrea VAN ELSAS.
Application Number | 20160185870 15/007345 |
Document ID | / |
Family ID | 51494473 |
Filed Date | 2016-06-30 |
United States Patent
Application |
20160185870 |
Kind Code |
A1 |
VAN EENENNAAM; Hans ; et
al. |
June 30, 2016 |
COMBINING CD27 AGONISTS AND IMMUNE CHECKPOINT INHIBITION FOR IMMUNE
STIMULATION
Abstract
The present invention relates to treatments of conditions
ameliorated by stimulation of an immune response, in particular by
the stimulation of antigen-specific T-lymphocytes. Treatment of
such conditions according to the invention is effected by the
combination of an anti-human CD27 agonistic antibody together with
a number of immune checkpoint inhibitors.
Inventors: |
VAN EENENNAAM; Hans;
(Nijmegen, NL) ; VAN ELSAS; Andrea; (Oss,
NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ADURO BIOTECH HOLDINGS, EUROPE B.V. |
OSS |
|
NL |
|
|
Family ID: |
51494473 |
Appl. No.: |
15/007345 |
Filed: |
January 27, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/NL2014/050543 |
Aug 2, 2014 |
|
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15007345 |
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Current U.S.
Class: |
424/133.1 ;
424/142.1; 424/143.1; 424/144.1; 424/172.1; 530/388.22;
530/389.1 |
Current CPC
Class: |
A61K 39/3955 20130101;
C07K 16/2803 20130101; A61K 45/06 20130101; C07K 16/2827 20130101;
A61P 31/00 20180101; C07K 2317/75 20130101; A61P 35/00 20180101;
C07K 16/2818 20130101; C07K 16/2896 20130101; A61K 2039/507
20130101; C07K 16/2878 20130101; A61P 31/04 20180101; A61P 31/12
20180101; A61P 33/00 20180101; A61P 31/10 20180101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 45/06 20060101 A61K045/06; A61K 39/395 20060101
A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 2, 2013 |
NL |
2011262 |
Mar 4, 2014 |
NL |
2012361 |
Claims
1. An anti-human CD27 agonistic antibody, such as hCD27.15 or 1F5,
or an antibody analogue thereof, for use in the treatment of a
condition ameliorated by stimulation of an immune response, in
particular stimulation of antigen-specific T-lymphocytes, wherein
in said treatment a number of immune checkpoint protein inhibitors
is administered.
2. An anti-human CD27 agonistic antibody according to claim 1,
wherein an immune checkpoint protein inhibitor is selected from an
inhibitor of CTLA-4, PD1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4,
TIM3 or KIR.
3. An anti-human CD27 agonistic antibody according to claim 1,
wherein the condition ameliorated by immune stimulation, in
particular stimulation of antigen-specific T-lymphocytes is
selected from infectious diseases, such as bacterial, fungal, viral
and parasitic infectious diseases, immunization against a pathogen,
such as a pathogen selected from bacteria, fungi, viruses or
parasites, or vaccination against toxins, or self-antigens,
including antigens expressed on benign or malignant tumors, such as
cancers, or conditions associated with uncontrolled proliferation
of cells such as cancers.
4. An anti-human CD27 agonistic antibody according to claim 1,
wherein the treatment is vaccination and a vaccine is administered
in the treatment.
5. Immune checkpoint inhibitor for use in the treatment of a
condition ameliorated by stimulation of an immune response, in
particular stimulation of antigen-specific T-lymphocytes, wherein
in said treatment an anti-human CD27 agonistic antibody, such as
hCD27.15 or 1F5, or an antibody analogue thereof, is
administered.
6. Immune checkpoint inhibitor according to claim 5, wherein the
immune checkpoint inhibitor is selected from an inhibitor of
CTLA-4, PD1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3 or
KIR.
7. Immune checkpoint inhibitor according to claim 5, wherein the
condition ameliorated by immune stimulation is selected from
infectious diseases, such as bacterial, fungal, viral and parasitic
infectious diseases, immunization against a pathogen, such as a
pathogen selected from bacteria, fungi, viruses or parasites, or
vaccination against toxins, or self-antigens, including antigens
expressed on benign or malignant tumors, such as cancers, or
conditions associated with uncontrolled proliferation of cells such
as cancers.
8. An immune checkpoint inhibitor according to claim 5, wherein the
treatment is vaccination and wherein a vaccine is administered in
the treatment.
9. Combination of an anti-human CD27 agonistic antibody, such as
hCD27.15 or 1F5, or an antibody analogue thereof, together with a
number of immune checkpoint inhibitors for use in the treatment of
a condition ameliorated by stimulation of an immune response,
particularly stimulation of antigen-specific T-lymphocytes.
10. Combination according to claim 9, wherein an immune checkpoint
inhibitor is selected from an inhibitor of CTLA-4, PD1, PD-L1,
PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3 or KIR.
11. Combination according to claim 9, wherein the condition
ameliorated by immune stimulation is selected from infectious
diseases, such as bacterial, fungal, viral and parasitic infectious
diseases, immunization against a pathogen, such as a pathogen
selected from bacteria, fungi, viruses or parasites, or vaccination
against toxins, or self-antigens, including antigens expressed on
benign or malignant tumors, such as cancers, or conditions
associated with uncontrolled proliferation of cells such as
cancers.
12. Combination according to claim 9, wherein the treatment is
vaccination and wherein a vaccine is administered in the
treatment.
13. A method of treating a condition ameliorated by stimulation of
an immune response, comprising administering to a subject in need
thereof a therapeutically effective amount of an anti-human CD27
agonistic antibody and further an immune checkpoint protein
inhibitor.
14. The method of claim 13, wherein said anti-human CD27 agonistic
antibody is selected from the group consisting of: an anti-human
CD27 agonistic antibody comprising the CDR amino acid sequences of
SEQ ID NO: 1, 2, 3, 4, 5, 6, or a variant sequence; a humanized
analogue of antibody hCD27.15; an analogue of antibody hCD27.15
that binds to the same epitope as hCD27.15; antibody 1F5; an
anti-human CD27 agonistic antibody that does not require
cross-linking.
15. The method of claim 13, wherein said further immune checkpoint
inhibitor protein is selected from the group consisting of: an
CTLA-4 antibody, an anti-PD1 antibody, an anti-PD-L1 antibody, an
anti-PD-L2 antibody, an anti LAG-3 antibody, an anti-BTLA antibody,
an anti-B7H3 antibody, an anti-B7H4 antibody, an anti-TIM3 antibody
and an anti-MR antibody.
16. The method of claim 13, wherein said further immune checkpoint
inhibitor protein is an anti-PD1 antibody.
17. The method of claim 13, wherein said anti-PD-1 antibody is
pembrolizumab.
18. The method of claim 13, wherein said anti-PD-1 antibody is
nivolumab.
19. The method of claim 13, wherein said further immune checkpoint
inhibitor protein is an anti-LAG3 antibody.
20. The method of claim 19, wherein said anti-LAG3 antibody
comprises the heavy chain and light chain amino acid sequences of
SEQ ID NO: 23 and SEQ ID NO: 24, respectively.
21. The method of claim 13, wherein the subject in need of
treatment suffers from cancer.
22. The method of claim 13, wherein the subject in need of
treatment suffers from an infection (such as a bacterial, fungal,
viral and parasitic infectious diseases).
23. A vaccine comprising an anti-human CD27 agonistic antibody and
further comprising an immune checkpoint protein inhibitor.
24. The vaccine of claim 23, wherein said further immune checkpoint
protein inhibitor is selected from the group consisting of: an
CTLA-4 antibody, an anti-PD1 antibody, an anti-PD-L1 antibody, an
anti-PD-L2 antibody, an anti LAG-3 antibody, an anti-BTLA antibody,
an anti-B7H3 antibody, an anti-B7H4 antibody, an anti-TIM3 antibody
and an anti-MR antibody.
25. The vaccine of claim 23, wherein said further immune checkpoint
inhibitor protein is an anti-PD1 antibody.
26. The vaccine of claim 23, wherein said anti-PD-1 antibody is
pembrolizumab.
27. The vaccine of claim 23, wherein said anti-PD-1 antibody is
nivolumab.
28. The vaccine of claim 23, wherein said further immune checkpoint
inhibitor protein is an anti-LAG3 antibody.
29. The vaccine of claim 23, wherein said anti-LAG3 antibody
comprises the heavy chain and light chain amino acid sequences of
SEQ ID NO:23 and SEQ ID NO:24, respectively.
Description
RELATED APPLICATIONS AND INCORPORATION BY REFERENCE
[0001] This application is a continuation-in-part application of
international patent application Serial No. PCT/NL2014/050543 filed
Aug. 2, 2014, which published as PCT Publication No. WO 2015/016718
on Feb. 5, 2015, which claims benefit of Netherlands patent
applications NL 2011262 filed Aug. 2, 2013 and NL 2012361 filed
Mar. 4, 2014.
[0002] The foregoing applications, and all documents cited therein
or during their prosecution ("appln cited documents") and all
documents cited or referenced in the appln cited documents, and all
documents cited or referenced herein ("herein cited documents"),
and all documents cited or referenced in herein cited documents,
together with any manufacturer's instructions, descriptions,
product specifications, and product sheets for any products
mentioned herein or in any document incorporated by reference
herein, are hereby incorporated herein by reference, and may be
employed in the practice of the invention. More specifically, all
referenced documents are incorporated by reference to the same
extent as if each individual document was specifically and
individually indicated to be incorporated by reference.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Dec. 17, 2015, is named 45468_00_2002 SL.txt and is 24,139 bytes
in size.
FIELD OF THE INVENTION
[0004] The present invention relates to the field of human and
veterinary medicine, including medical/veterinary diagnosis and
medical/veterinary research. More specifically the present
invention relates to treatments of conditions ameliorated by
stimulation of an immune response, in particular by the stimulation
of antigen-specific T-lymphocytes. The various aspects of the
present invention are suitable for treatment of any condition known
or expected to be ameliorated by stimulation of CD27+ immune cells
or by inhibition of one or more immune checkpoint protein(s).
Conditions suitably treated by the invention are those ameliorated
by immune stimulation, such as infectious diseases and cancers, by
using a combination of a CD27 agonist and one or more immune
checkpoint blocking agents.
BACKGROUND
[0005] CD27, a TNF receptor family member was identified as a
membrane molecule on human T cells (van Lier et al., 1987, J
Immunol 139:1589-96). According to current evidence, CD27 has a
single ligand, CD70, which is also a TNF family member (Goodwin et
al., 1993, Cell 73:447-56).
[0006] CD27 is exclusively expressed by hematopoietic cells, in
particular those of the lymphocyte lineage, i.e. T-, B- and NK
cells. CD27 was originally defined as a human T-cell co-stimulatory
molecule that increments the proliferative response to TCR
stimulation (van Lier et al., 1987, J Immunol 139:1589-96).
Presence of CD70, the ligand of CD27, dictates the timing and
persistence of CD27-mediated co-stimulation.
[0007] Transgenic expression of CD70 in immature dendritic cells
sufficed to convert immunological tolerance to virus or tumors into
CD8.sup.+ T cell responsiveness. Likewise, agonistic soluble CD70
promoted the CD8.sup.+ T cell response upon such peptide
immunization (Rowley et al., 2004, J Immunol 172:6039-6046) and in
CD70 transgenic mice, CD4.sup.+ and CD8.sup.+ effector cell
formation in response to TCR stimulation was greatly facilitated
(Arens et al. 2001, Immunity 15:801-12; Tesselaar et al., 2003, Nat
Immunol 4:49-54; Keller et al. 2008, Immunity 29: 334-346). In
mouse lymphoma models, tumor rejection was improved upon CD70
transgenesis or injection of an anti-mouse CD27 antibody (Arens et
al., 2003, J Exp Med 199:1595-1605; French et al., 2007, Blood 109:
4810-15; Sakanishi and Yagita, 2010, Biochem. Biophys. Res. Comm.
393: 829-835; WO 2008/051424; WO 2012/004367).
[0008] In WO2012/004367 the first anti-human agonistic antibody
(designated hCD27.15) was described that does not require
crosslinking to activate CD27-mediated co-stimulation of the immune
response. In addition, an anti-human CD27 antibody, designated 1F5
was disclosed that activates CD27 upon crosslinking (WO2011/130434
and Vitale et al., Clin. Cancer Res, 2012, 18(14): 3812-3821).
[0009] Recently, the first clinical successes of agents that
modulate cancer immunity have validated cancer immunotherapy as a
novel path to obtain durable and long-lasting clinical responses in
cancer patients (Mellman et al., Nature, 2011, 480:480-489). The
first such agent, ipilimumab (Yervoy, BMS), that obtained marketing
approval for treatment of metastatic melanoma is an antibody
blocking the CTLA4 receptor, an immune checkpoint protein. Further
immune checkpoint inhibitors under development are antibodies that
block the interaction between the PD-1 receptor and its ligands
PD-L1 and PD-L2 (Mullard, Nat. Rev. Drug Disc., 2013, 12:489-492).
Several antibodies targeting the PD-1 pathway are currently in
clinical development for treatment of melanoma, renal cell cancer,
non-small cell lung cancer, diffuse large B cell lymphoma and other
tumors. Although these agents have not yet been filed for marketing
approval, impressive results have been obtained in early clinical
studies, for example with Lambrolizumab (anti-PD1) in melanoma
(Hamid et al., 2013, New. Eng. J. Med., 369:134-44).
[0010] The current state of the art does not suggest that agonists
of the CD27 receptor would be rationally combined with immune
checkpoint inhibitors, such as anti-PD1, anti-PDL1 or anti-CTLA4
antibodies to improve cancer immunity. In fact, available data
suggest that CD27 acts, at least in part via overruling PD-1 and
CTLA-4 signals or via downregulation of these immune checkpoints.
First, in a T-cell tolerance model based on LCMV-derived epitopes
that was demonstrated to be highly dependent on PD-1 and CTLA4
receptors the forced expression of CD70 ligand was sufficient to
turn T cell tolerance into activation of T-cell immunity.
Apparently these data strongly suggest that CD27 activation
overrules tolerance mediated via PD-1 and CTLA4 (Keller et al.,
Immunity, 2008, 29:934-946. In a second model, activation of CD27
using a rat anti-mouse CD27 agonistic antibody was demonstrated to
support the maintenance of CD8.sup.+ T-cells, to reduce the
frequency of FoxP3-expressing CD4.sup.+ T-cells within tumors and
to potentiate the ability of NK1.1.sup.+ and CD8.sup.+ tumor
infiltrating cells to secrete IFN-.gamma. in coculture with tumor
cells. This enhanced function correlated with decreased levels of
PD-1 expression on CD8.sup.+ T-cells (Roberts et al., J
Immunother., 2010, 33: 769-79).
[0011] Citation or identification of any document in this
application is not an admission that such document is available as
prior art to the present invention.
SUMMARY OF THE INVENTION
[0012] The current state of the art thus is, that stimulation of
CD27 overrules PD-1 and CTLA-4 mediated immune tolerance and that
activation using anti-CD27 antibodies results in down-regulation of
these immune checkpoint receptors. Therefore, according to the
state of the art the combination of an agent that activates the
CD27 receptor together with one or more immune checkpoint
inhibitors would result in either efficacy that is equal or similar
to CD27 activation or immune checkpoint inhibition alone.
[0013] The inventors of the present invention however have
surprisingly found that the combination of a CD27 agonistic
antibody together with an immune checkpoint inhibitor does have
additional effects on T-cell stimulation in comparison to a CD27
agonistic antibody or an immune checkpoint inhibitor alone. In
particular, the additional effects of the combination of a CD27
agonistic antibody together with an immune checkpoint inhibitor has
been tested in established assays that have been clinically
validated to predict anti-cancer immune responses. In these assays
immune checkpoint inhibitory antibodies were demonstrated to induce
elevated levels of the T-cell cytokine IL-2 upon stimulation with
Staphylococcus Enterotoxin B in either human peripheral blood
mononuclear cells or whole human blood (Dulos et al., J.
Immunother, 2012, 35:169-78). The clinical validation and the
predictive value of this assay was subsequently established in
Phase I/II clinical studies (Patnaik et al., ASCO, Chicago, 2012;
Ribas et al., PEGS Summit, Boston, 2013; Hamid et al., N. Engl. J.
Med., 2013, 369: 134-144).
[0014] The present invention is thus based on the surprising
discovery that the combination of an anti-human CD27 agonistic
antibody together with an immune checkpoint inhibitor results in
immune stimulation to an unexpected level. In view of the so far
known relation between CD27 and immune checkpoint inhibitors the
higher level of immune stimulation resulting from the combination
is surprising. In view of its surprising discovery the present
invention relates to treatments of conditions ameliorated by
stimulation or enhancement of the immune response, in particular
the treatments of conditions that result in the stimulation or
enhancement of antigen-specific T-lymphocytes, such as cancer and
infectious disease. More specifically, the present invention is
aimed at treatment of any condition known or expected to be
ameliorated by stimulation of CD27+ immune cells or by inhibition
of one or more immune checkpoint protein(s). Treatment of these
conditions may be further improved by using a combination of a CD27
agonist and one or more immune checkpoint blocking agents.
[0015] According to a first aspect the invention relates to an
anti-human CD27 agonistic antibody, such as hCD27.15 or 1F5, or an
antibody derived therefrom, for use in the treatment of a condition
ameliorated by stimulation of an immune response, in particular the
treatment of a condition ameliorated by stimulation of
antigen-specific T-lymphocytes wherein in said treatment a number
of immune checkpoint protein inhibitors is administered. By this
co-administration of the anti-human CD27 agonistic antibody with a
number of immune checkpoint protein inhibitors, surprising effects
are obtained.
[0016] According to a further aspect the invention relates to an
immune checkpoint protein inhibitor for use in the treatment of a
condition ameliorated by stimulation of an immune response, in
particular the treatment of a condition ameliorated by stimulation
of antigen-specific T-lymphocytes, wherein in said treatment an
anti-human CD27 agonistic antibody, such as hCD27.15 or 1F5, or an
antibody derived therefrom, is administered. By this
co-administration of the immune checkpoint protein inhibitor with
an anti-human CD27 agonistic antibody, surprising effects are
obtained.
[0017] Yet a further aspect of the invention relates to a
combination of an anti-human CD27 agonistic antibody, such as
hCD27.15 or 1F5, or an antibody derived therefrom, together with a
number of immune checkpoint protein inhibitors for use in the
treatment of a condition ameliorated by stimulation of an immune
response, in particular the treatment of a condition ameliorated by
stimulation of antigen-specific T-lymphocytes.
[0018] Still a further aspect of the invention relates to a method
for treating a condition ameliorated by stimulation of an immune
response, in particular the treatment of a condition ameliorated by
stimulation of antigen-specific T-lymphocytes, said method
comprising administering an anti-human CD27 agonistic antibody,
such as hCD27.15 or 1F5, or an antibody derived therefrom, in
combination with a number of immune checkpoint inhibitors.
[0019] An anti-human CD27 antibody of the invention may be selected
from hCD27.15 or analogues thereof, in particular analogues which
may comprise the CDRs of hCD27.15, analogues (cross-)blocking the
binding of hCD27.15 to human CD27, analogues binding to the same
epitope of hCD27.15 or humanized analogues of hCD27.15.
[0020] In another embodiment, the anti-human CD27 antibody is
administered in combination with an anti-PD1 antibody. In one
embodiment, the anti-human CD27 antibody is administered in
combination with nivolumab. In another embodiment, the anti-human
CD27 antibody is administered in combination with pembrolizumab. In
another embodiment, the anti-human CD27 antibody is administered in
combination with an anti-CTLA4 antibody. In another embodiment, the
anti-human CD27 antibody is administered in combination with an
anti-LAG3 antibody. In another embodiment, the anti-human CD27
antibody is administered in combination with an anti-LAG3 antibody
which may comprise the heavy chain amino acid sequence of SEQ ID
NO: 23 and the light chain amino acid sequence of SEQ ID NO:
24.
[0021] As the skilled person will know the published sequences for
the heavy and light chain of nivolumab and pembrolizumab are as
presented in SEQ ID NO: 21, 22, 19 and 20 respectively.
TABLE-US-00001 TABLE 1 Sequence Listing SEQ ID NO: Description 1
hCD27.15 heavy chain CDR1 (AA) 2 hCD27.15 heavy chain CDR2 (AA) 3
hCD27.15 heavy chain CDR3 (AA) 4 hCD27.15 light chain CDR1 (AA) 5
hCD27.15 light chain CDR2 (AA) 6 hCD27.15 light chain CDR3 (AA) 7
hCD27.15 heavy chain variable region (DNA) 8 hCD27.15 heavy chain
variable region (AA) 9 hCD27.15 light chain variable region (DNA)
10 hCD27.15 light chain variable region (AA) 11 1F5 heavy chain
CDR1 (AA) 12 1F5 heavy chain CDR2 (AA) 13 1F5 heavy chain CDR3 (AA)
14 1F5 light chain CDR1 (AA) 15 1F5 light chain CDR2 (AA) 16 1F5
light chain CDR3 (AA) 17 1F5 heavy chain variable region (AA) 18
1F5 light chain variable region (AA) 19 Pembrolizumab heavy chain
(AA) 20 Pembrolizumab light chain (AA) 21 Nivolumab heavy chain
(AA) 22 Nivolumab light chain (AA) 23 Anti-human LAG3 mature heavy
chain (AA) 24 Anti-human LAG3 mature light chain (AA)
[0022] Accordingly, it is an object of the invention not to
encompass within the invention any previously known product,
process of making the product, or method of using the product such
that Applicants reserve the right and hereby disclose a disclaimer
of any previously known product, process, or method. It is further
noted that the invention does not intend to encompass within the
scope of the invention any product, process, or making of the
product or method of using the product, which does not meet the
written description and enablement requirements of the USPTO (35
U.S.C. .sctn.112, first paragraph) or the EPO (Article 83 of the
EPC), such that Applicants reserve the right and hereby disclose a
disclaimer of any previously described product, process of making
the product, or method of using the product. It may be advantageous
in the practice of the invention to be in compliance with Art.
53(c) EPC and Rule 28(b) and (c) EPC. All rights to explicitly
disclaim any embodiments that are the subject of any granted
patent(s) of applicant in the lineage of this application or in any
other lineage or in any prior filed application of any third party
is explicitly reserved Nothing herein is to be construed as a
promise.
[0023] It is noted that in this disclosure and particularly in the
claims and/or paragraphs, terms such as "comprises", "comprised",
"comprising" and the like can have the meaning attributed to it in
U.S. Patent law; e.g., they can mean "includes", "included",
"including", and the like; and that terms such as "consisting
essentially of" and "consists essentially of" have the meaning
ascribed to them in U.S. Patent law, e.g., they allow for elements
not explicitly recited, but exclude elements that are found in the
prior art or that affect a basic or novel characteristic of the
invention.
[0024] These and other embodiments are disclosed or are obvious
from and encompassed by, the following Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The following detailed description, given by way of example,
but not intended to limit the invention solely to the specific
embodiments described, may best be understood in conjunction with
the accompanying drawings.
[0026] FIGS. 1A-B.
[0027] CD27 agonistic antibodies in combination with immune
checkpoint blockers unexpectedly enhance SEB induced T-cell
activation in human PBMCs in comparison to CD27 agonistic
antibodies and immune checkpoint inhibitors alone. FIG. 1A. CD27
agonistic antibodies in combination with anti-PD1 antibodies
enhance SEB induced T-cell activation in human PBMCs. Equal amounts
of the indicated antibodies were added, with a final concentration
as indicated on the X-axis. Human IgG4 was used as an
isotype-matched control. FIG. 1B. CD27 agonistic antibodies in
combination with anti-PDL1 antibodies enhance SEB induced T-cell
activation in human PBMCs. Equal amounts of the indicated
antibodies were added, with a final concentration as indicated on
the X-axis. Human IgG4 and/or mouse IgG1 were used as
isotype-matched controls.
[0028] FIGS. 2A-C.
[0029] CD27 agonistic antibodies in combination with immune
checkpoint blockers unexpectedly enhance SEB induced T-cell
activation in human whole blood in comparison to CD27 agonistic
antibodies and immune checkpoint inhibitors alone. FIG. 2A. CD27
agonistic antibodies in combination with anti-PD1 antibodies
enhance SEB induced T-cell activation in human whole blood. Equal
amounts of the indicated antibodies were added, with a final
concentration as indicated on the X-axis. Human IgG4 was used as
isotype-matched control. FIG. 2B. CD27 agonistic antibodies in
combination with anti-PDL1 antibodies enhance SEB induced T-cell
activation in human whole blood. Equal amounts of the indicated
antibodies were added, with a final concentration as indicated on
the X-axis. Human IgG4 and/or mouse IgG1 were used as
isotype-matched controls. FIG. 2C. CD27 agonistic antibodies in
combination with anti-CTLA-4 antibodies enhance SEB induced T-cell
activation in human whole blood. Equal amounts of the indicated
antibodies were added, with a final concentration as indicated on
the X-axis. Human IgG4 and/or mouse IgG2A were used as
isotype-matched controls.
[0030] FIGS. 3A-D.
[0031] CD27 agonistic antibodies in combination with immune
checkpoint blockers enhance SEB induced T-cell activation in human
whole blood in comparison to CD27 agonistic antibodies and immune
checkpoint inhibitors alone. FIGS. 3A-3D present data for the
effect of a hCD27.15 analogue in combination with anti-LAG3 (left
panel) and anti-PD1 (right panel). Combination of the antibodies
enhances SEB induced T-cell activation in human whole blood of
different donors.
DETAILED DESCRIPTION OF THE INVENTION
[0032] In a first aspect the invention relates to an anti-human
CD27 agonistic antibody for use in the treatment of a condition
ameliorated by stimulation of an immune response, in particular the
treatment of a condition ameliorated by stimulation of
antigen-specific T-lymphocytes, wherein in said treatment a number
of immune checkpoint protein inhibitors is administered.
[0033] Anti-human CD27 agonistic antibodies should be construed as
meaning an antibody demonstrating activation of the CD27 receptor
on CD27.sup.+ immune cells. Agonistic properties of anti-human CD27
antibodies can be assayed by CD27 receptor activation using for
example the NF-.kappa.B luciferase reporter assay as described in
WO2012/004367. Activation of CD27 receptor using activating
anti-human CD27 antibodies has been shown to induce activation,
proliferation and/or survival of human CD27.sup.+ immune cells. By
displaying its CD27 receptor-stimulating effect the CD27 agonist is
capable of inducing and/or enhancing an immune response (e.g. an
antigen-specific T-cell mediated immune response). An anti-human
CD27 antibody used in the present invention may exert its agonistic
activity when in soluble form. Alternatively an anti-human CD27
antibody used in the present invention may exert its agonistic
activity when cross-linked. For cross-linking the anti-human CD27
antibody may be adapted in respect of its Fc function. The use of
anti-human CD27 antibodies exerting agonistic activity when in
soluble form is preferred.
[0034] Anti-human CD27 agonistic antibodies are known in the art.
For example hCD27.15 is disclosed in WO2012/004367 and 1F5 is
disclosed in WO2011/130434 and Vitale et al., Clin. Cancer Res,
2012, 18(14): 3812-3821. The use of 1F5 or hCD27.15 or an antibody
derived from one of these known antibodies is preferred. The use of
hCD27.15 or an antibody analogue therefrom is most preferred in
view of its beneficial binding properties and its ability to
display good agonistic activity when in soluble form (without any
further cross-linking). Within the present invention an antibody
derived from a certain antibody may be considered an analogue. The
skilled person will understand that for a proper functioning of an
antibody analogue within the context of this invention, a derived
antibody (or antibody analogue), according to certain embodiments,
may comprise antigen binding regions of its originating antibody or
will at least bind to the same epitope. Antibody analogues may
(cross-)block binding of the anti-CD27 antibody, for example
hCD27.15 or 1F5, to human CD27. Antibody analogues in particular
may comprise antibody fragments, antibodies having modified
effector function, chimeric antibodies and humanized antibodies as
defined below. The antibody analogue according to the invention
maintains CD27 agonistic functionality.
[0035] The heavy chain CDR1, CDR2 and CDR3 amino acid sequences and
light chain CDR1, CDR2 and CDR3 amino acid sequences of hCD27.15,
identifying the antigen binding region of this antibody, are
already disclosed in WO2012/004367. These sequences have also been
presented in SEQ ID NO: 1-6 of the sequence listing of this
description, together with the amino acid sequences of the heavy
chain variable region and the light chain variable region (SEQ ID
NO: 8 and 10 respectively). Antibody analogues of hCD27.15 which
may comprise these CDR sequences, or sequence variants thereof, are
particularly considered for use in the invention.
[0036] The heavy chain CDR1, CDR2 and CDR3 amino acid sequences and
light chain CDR1, CDR2 and CDR3 amino acid sequences of 1F5,
identifying the antigen binding region of this antibody, are
already disclosed in WO2011/130434. These sequences have also been
presented in SEQ ID NO: 11-16 of the sequence listing of this
description, together with the amino acid sequences of the heavy
chain variable region and the light chain variable region (SEQ ID
NO: 17 and 18). Antibody analogues of 1F5 which may comprise these
CDR sequences, or sequence variants thereof, are particularly
considered for use in the invention.
[0037] Alternatively, analogues of hCD27.15 or 1F5 binding to the
same epitope of these antibodies, but having differing CDRs may
also be selected. To screen for antibodies that bind to the
hCD27.15 or 1F5 epitope on human CD27, a routine cross-blocking
assay such as that described in "Antibodies, A Laboratory Manual,
Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988)",
can be performed. Antibodies that bind to the same epitope are
likely to cross-block in such assays, but not all cross-blocking
antibodies will necessarily bind at precisely the same epitope
since cross-blocking may result from steric hindrance of antibody
binding by antibodies binding at overlapping epitopes, or even
nearby non-overlapping epitopes. Such cross-blocking antibodies
maintaining CD27 agonistic functionality are also within the scope
of the present invention.
[0038] Alternatively, the known technique of epitope mapping, e.g.,
as described in Champe et al., 1995, J. Biol. Chem. 270:1388-1394,
can be performed to determine whether the antibody binds an epitope
of interest. "Alanine scanning mutagenesis," as described by
Cunningham and Wells, 1989, Science 244: 1081-1085, or some other
form of point mutagenesis of amino acid residues in human CD27 may
also be used to determine the functional epitope for anti-CD27
antibodies of the present invention.
[0039] Another known method to map the epitope of an antibody is to
study binding of the antibody to synthetic linear and CLIPS
peptides that can be screened using credit-card format mini PEPSCAN
cards as described by Slootstra et al. (Slootstra et al., 1996,
Mol. Diversity 1: 87-96) and Timmerman et al. (Timmerman et al.,
2007, J. Mol. Recognit. 20: 283-299). The binding of antibodies to
each peptide is determined in a PEPSCAN-based enzyme-linked immuno
assay (ELISA).
[0040] Additional antibodies binding to the same epitope as
hCD27.15 may be obtained with known techniques, for example, by
screening of antibodies raised against CD27 for binding to the
epitope, or by immunization of an animal with a peptide which may
comprise a fragment of human CD27 which may comprise the epitope
sequences. Antibodies that bind to the same functional epitope
might be expected to exhibit similar biological activities, such as
CD27 agonistic activity, and such activities can be confirmed by
functional assays of the antibodies. For analogues of 1F5 binding
to the same epitope of this antibody, these techniques can also be
used in analogy.
[0041] According to an embodiment an analogue of hCD27.15 binding
to the same epitope of this antibody, but having differing CDRs may
block binding of hCD27.15 to human CD27 with an IC.sub.50 of about
50 nM or lower. Alternatively an analogue of hCD27.15 binding to
the same epitope of this antibody, but having differing CDRs may be
blocked in its binding to human CD27 by hCD27.15 with an IC.sub.50
of about 50 nM or lower. Similarly analogues of 1F5 binding to the
same epitope of this antibody, but having differing CDRs may block
binding of 1F5 to human CD27 with an IC.sub.50 of about 50 nM or
lower or alternatively may be blocked in its binding to human CD27
by 1F5 with an IC.sub.50 of about 50 nM or lower. About 50 nM or
lower is to be understood to include 50*10.sup.-9 to 0.1*10.sup.-12
M, such as 20*10.sup.-9 to 1.0*10.sup.-11 M, 10*10.sup.-9 to
1.0*10.sup.-10 M or 10*10.sup.-9 to 1.0*10.sup.-19.
[0042] The differing CDRs may be sequence variants of the known
CDRs of the human CD27 binding antibody used in the invention, for
example hCD27.15 or 1F5. As used herein, a sequence "variant"
refers to a sequence that differs from the disclosed sequence at
one or more amino acid residues but which retains the biological
activity of the resulting molecule. The invention includes the
variants of antibodies explicitly disclosed by the various
sequences, for example hCD27.15 or 1F5. For the V.sub.H domain
CDR1, CDR2 and CDR3 sequences, according to some embodiments,
variant sequences may comprise up to 6 amino acid substitutions,
such as 1, 2, 3, 4, 5 or 6 amino acid substitutions, for the CDR1,
CDR2 and CDR3 sequences taken together. Similarly for the V.sub.L
domain CDR1, CDR2 and CDR3 sequences, according to some
embodiments, variant sequences may comprise up to 6 amino acid
substitutions, such as 1, 2, 3, 4, 5 or 6 amino acid substitutions,
for the CDR1, CDR2 and CDR3 sequences taken together. The skilled
person will understand that in particular conservative amino acid
substitutions may result in maintaining biological activity. For
all amino acid and DNA sequences disclosed, the sequence variants
are also envisaged within this invention.
[0043] "Conservatively modified variants" or "conservative amino
acid substitution" refers to substitutions of amino acids are known
to those of skill in this art and may be made generally without
altering the biological activity of the resulting molecule. Those
of skill in this art recognize that, in general, single amino acid
substitutions in non-essential regions of a polypeptide do not
substantially alter biological activity (see, e.g., Watson, et al.,
Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p.
224 (4th Edition 1987)). Such exemplary substitutions are
preferably made in accordance with those set forth above in Table
2.
TABLE-US-00002 TABLE 2 Exemplary Conservative Amino Acid
Substitutions Original residue Conservative substitution Ala (A)
Gly; Ser Arg (R) Lys, His Asn (N) Gln; His Asp (D) Glu; Asn Cys (C)
Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly (G) Ala His (H) Asn; Gln
Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg; His Met (M) Leu;
Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) Thr Thr (T) Ser
Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu
[0044] The anti-human CD27 agonistic antibody of the invention is
intended for use in the treatment of a condition ameliorated by
stimulation of an immune response, in particular the treatment of
conditions which are ameliorated by stimulation of antigen-specific
T-lymphocytes. Characteristic of this treatment is that a number of
immune checkpoint protein inhibitors is administered. Thus the CD27
agonistic antibody is co-administered with a number of immune
checkpoint protein inhibitors. Within the present invention the
term "number of" should be understood as meaning at least one or
alternatively one or more, such as, 1, 2, 3, 4, 5 or 6.
[0045] The term "immune checkpoint protein" is known in the art.
Within the known meaning of this term it will be clear to the
skilled person that on the level of "immune checkpoint proteins"
the immune system provides inhibitory signals to its components in
order to balance immune reactions. Known immune checkpoint proteins
may comprise CTLA-4, PD1 and its ligands PD-L1 and PD-L2 and in
addition LAG-3, BTLA, B7H3, B7H4, TIM3, MR. The pathways involving
LAG3, BTLA, B7H3, B7H4, TIM3, and MR are recognized in the art to
constitute immune checkpoint pathways similar to the CTLA-4 and
PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer
12:252-264; Mellman et al., 2011. Nature 480:480-489).
[0046] Within the present invention an immune checkpoint protein
inhibitor is any compound inhibiting the function of an immune
checkpoint protein. Inhibition includes reduction of function and
full blockade. In particular the immune checkpoint protein is a
human immune checkpoint protein. Thus the immune checkpoint protein
inhibitor preferably is an inhibitor of a human immune checkpoint
protein. Immune checkpoint proteins are described in the art (see
for instance Pardoll, 2012. Nature Rev. cancer 12: 252-264). The
designation immune checkpoint includes the experimental
demonstration of stimulation of an antigen-receptor triggered T
lymphocyte response by inhibition of the immune checkpoint protein
in vitro or in vivo, e.g. mice deficient in expression of the
immune checkpoint protein demonstrate enhanced antigen-specific T
lymphocyte responses or signs of autoimmunity (such as disclosed in
Waterhouse et al., 1995. Science 270:985-988; Nishimura et al.,
1999. Immunity 11:141-151). It may also include demonstration of
inhibition of antigen-receptor triggered CD4+ or CD8+ T cell
responses due to deliberate stimulation of the immune checkpoint
protein in vitro or in vivo (e.g. Zhu et al., 2005. Nature Immunol.
6:1245-1252).
[0047] Preferred immune checkpoint protein inhibitors are
antibodies that specifically recognize immune checkpoint proteins.
A number of CTLA-4, PD1, PDL-1, PD-L2, LAG-3, BTLA, B7H3, B7H4,
TIM3 and MR inhibitors are known and in analogy of these known
immune checkpoint protein inhibitors, alternative immune checkpoint
inhibitors may be developed in the (near) future. For example
ipilimumab is a fully human CTLA-4 blocking antibody presently
marketed under the name Yervoy (Bristol-Myers Squibb). A second
CTLA-4 inhibitor is tremelimumab (referenced in Ribas et al., 2013,
J Clin. Oncol. 31:616-22). Examples of PD-1 inhibitors include
without limitation humanized antibodies blocking human PD-1 such as
lambrolizumab (e.g. disclosed as hPD109A and its humanized
derivatives h409A11, h409A16 and h409A17 in WO2008/156712; Hamid et
al., N Engl. J. Med. 369: 134-144 2013,), or pidilizumab (disclosed
in Rosenblatt et al., 2011. J Immunother. 34:409-18), as well as
fully human antibodies such as nivolumab (previously known as
MDX-1106 or BMS-936558, Topalian et al., 2012. N Eng. J. Med.
366:2443-2454, disclosed in U.S. Pat. No. 8,008,449 B2). Other PD-1
inhibitors may include presentations of soluble PD-1 ligand
including without limitation PD-L2 Fc fusion protein also known as
B7-DC-Ig or AMP-244 (disclosed in Mkrtichyan M, et al. J Immunol.
189:2338-47 2012) and other PD-1 inhibitors presently under
investigation and/or development for use in therapy. In addition,
immune checkpoint inhibitors may include without limitation
humanized or fully human antibodies blocking PD-L1 such as
MEDI-4736 (disclosed in WO2011066389 A1), MPDL3280A (disclosed in
U.S. Pat. No. 8,217,149 B2) and MIH1 (Affymetrix obtainable via
eBioscience (16.5983.82)) and other PD-L1 inhibitors presently
under investigation. According to this invention an immune
checkpoint inhibitor is preferably selected from a CTLA-4, PD-1 or
PD-L1 inhibitor, such as selected from the known CTLA-4, PD-1 or
PD-L1 inhibitors mentioned above (ipilimumab, tremelimumab,
labrolizumab, nivolumab, pidilizumab, AMP-244, MEDI-4736,
MPDL3280A, MIH1). Known inhibitors of these immune checkpoint
proteins may be used as such or analogues may be used, in
particular chimerized, humanized or human forms of antibodies.
[0048] As the skilled person will know, alternative and/or
equivalent names may be in use for certain antibodies mentioned
above. Such alternative and/or equivalent names are interchangeable
in the context of the present invention. For example it is known
that lambrolizumab is also known under the alternative and
equivalent names MK-3475 and pembrolizumab.
[0049] The selection of an immune checkpoint inhibitor from PD1 and
PD-L1 inhibitors, such as a known PD-1 or PD-L1 inhibitor mentioned
above, is more preferred and most preferably a selection is made
from a PD-1 inhibitor, such as a known PD1 inhibitor mentioned
above. In preferred embodiments, the PD1 inhibitor is nivolumab or
pembrolizumab or another antagonist antibody against human PD1.
[0050] The invention also includes the selection of other immune
checkpoint inhibitors that are known in the art to stimulate immune
responses. This includes inhibitors that directly or indirectly
stimulate or enhance antigen-specific T-lymphocytes. These other
immune checkpoint inhibitors include, without limitation, agents
targeting immune checkpoint proteins and pathways involving PD-L2,
LAG3, BTLA, B7H4 and TIM3. For example, human PD-L2 inhibitors
known in the art include MIH18 (disclosed in (Pfistershammer et
al., 2006. Eur J Immunol. 36:1104-13). Another example, LAG3
inhibitors known in the art include soluble LAG3 (I P321, or
LAG3-Ig disclosed in WO2009044273 A2, and in Brignon et al. 2009.
Clin. Cancer Res. 15:6225-6231) as well as mouse or humanized
antibodies blocking human LAG3 (for instance IMP701 disclosed in
and derived from WO2008132601 A1), or fully human antibodies
blocking human LAG3 (such as disclosed in EP 2320940 A2). Another
example is provided by the use of blocking agents towards BTLA,
including without limitation antibodies blocking human BTLA
interaction with its ligand (such as 4C7 disclosed in
WO2011014438). Yet another example is provided by the use of agents
neutralizing B7H4 including without limitation antibodies to human
B7H4 (disclosed in WO 2013025779 A1, and in WO 2013067492 A1) or
soluble recombinant forms of B7H4 (such as disclosed in
US20120177645 A1 or Anti-human B7H4 clone H74: eBiocience
#14-5948). Yet another example is provided by agents neutralizing
B7-H3, including without limitation antibodies neutralizing human
B7-H3 (e.g. MGA271 disclosed as BRCA84D and derivatives in US
20120294796 A1). Yet another example is provided by agents
targeting TIM3, including without limitation antibodies targeting
human TIM3 (e.g. as disclosed in WO 2013006490 A2 or the anti-human
TIM3, blocking antibody F38-2E2 disclosed by Jones et al., J Exp
Med. 2008 November 24; 205(12):2763-79). Known inhibitors of immune
checkpoint proteins may be used in their known form or analogues
may be used, in particular chimerized forms of antibodies, most
preferably humanized forms.
[0051] The invention also includes the selection of more than one
immune checkpoint inhibitor selected from CTLA-4, PD-1 or PDL1
inhibitors for combination with an anti-human CD27 agonistic
antibody within the various aspects of the invention. For example
concurrent therapy of ipilimumab (anti-CTLA4) with Nivolumab
(anti-PD1) has demonstrated clinical activity that appears to be
distinct from that obtained in monotherapy (Wolchok et al., 2013,
N. Eng. J. Med., 369:122-33). Also included are combinations of
agents that have been shown to improve the efficacy of checkpoint
inhibitors, such as Lirilumab (also known as anti-KIR, BMS-986015
or IPH2102, as disclosed in U.S. Pat. No. 8,119,775 B2 and Benson
et al., Blood 120:4324-4333 (2012)) in combination with ipilimumab
(Rizvi et al., ASCO 2013, and clinicaltrials.gov NCT01750580) or in
combination with nivolumab (Sanborn et al., ASCO 2013, and
clinicaltrials.gov NCT01714739), agents targeting LAG3 combined
with anti-PD-1 (Woo et al., 2012 Cancer Res. 72:917-27) or
anti-PD-L1 (Butler N S et al., Nat Immunol. 2011 13:188-95), agents
targeting ICOS in combination with anti-CTLA-4 (Fu et al., Cancer
Res. 2011 71:5445-54, or agents targeting 4-1BB in combination with
anti-CTLA-4 (Curran et al., PLoS One. 2011 6(4):e19499).
Combinations of anti-human CD27 agonistic antibodies and immune
checkpoint inhibitors envisaged within the various embodiments of
the present invention include those presented in Table 3. In this
table the names of anti-human CD27 agonistic antibodies and immune
checkpoint inhibitors refers to both the known compound and
analogues thereof. For antibodies, analogues include analogues
having modified Fc-function, chimerized antibodies and humanized
antibodies. For antibodies preferably human or humanized forms are
selected. CD27 agonist refers to an anti-human CD27 agonistic
antibody.
TABLE-US-00003 TABLE 3 COMBINATIONS OF ANTI-HUMAN CD27 AGONISTS AND
IMMUNE CHECKPOINT PROTEIN INHIBITORS B7H4 Lambro- LAG-3 inhibitor,
lizimab PD- KIR inhibitor BTLA e.g. B7H3 TIM3 PD-1 (pembro- L1
inhibitor e.g. inhibitor, H74, inhibitor, inhibitor, CTLA-4 Ipili-
tremeli- inhib- lizumab/ Nivo- pidili- inhib- MEDI- PD-L2 e.g.
IMP321, e.g. soluble e.g. e.g. inhibitor mumab mumab itor MK-3475)
lumab zumab AMP244 itor 4736 MPDL3280A MIH1 inhibitor MIH18
lirilumab IMP701 4C7 B7H4 MA271 F38-2E2 hCD27.15 1 hCD27.15 1
hCD27.15 1 hCD27.15 1 hCD27.15 1 hCD27.15 1 hCD27.15 1 hCD27.15 1
hCD27.15 1 hCD27.15 1 hCD27.15 1 hCD27.15 1 hCD27.15 2 2 hCD27.15 2
2 hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2
hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2
hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2
hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2
hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2
hCD27.15 1 hCD27.15 1 hCD27.15 1 hCD27.15 1 hCD27.15 1 hCD27.15 1
hCD27.15 hCD27.15 2 hCD27.15 2 hCD27.15 2 hCD27.15 2 2 hCD27.15 2 2
hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2
hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2
hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2
hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2 hCD27.15 2 2 hCD27.15 1F5 1
1F5 1 1F5 1 1F5 1 1F5 1 1F5 1 1F5 1 1F5 1 1F5 1 1F5 1 1F5 1 1F5 1
1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5
2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2
1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5
2 2 1F5 2 2 1F5 1 1F5 1 1F5 1 1F5 1 1F5 1 1F5 1 1F5 2 1F5 2 1F5 2
1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5
2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2
1F5 2 2 1F5 2 2 1F5 2 2 1F5 2 2 CD27 agonist 1 CD27 agonist 1 CD27
agonist 1 CD27 agonist 1 CD27 agonist 1 CD27 agonist 1 CD27 agonist
1 CD27 agonist 1 CD27 agonist 1 CD27 agonist 1 CD27 agonist 1 CD27
agonist 1 CD27 agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27
agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27
agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27
agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27
agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27
agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27
agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27
agonist 1 CD27 agonist 1 CD27 agonist 1 CD27 agonist 1 CD27 agonist
1 CD27 agonist 1 CD27 agonist CD27 agonist 2 CD27 agonist 2 CD27
agonist 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27
agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27
agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27
agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27
agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27 agonist 2 2 CD27
agonist 2 2 CD27 agonist 2 2 In the table above presenting
combinations of anti-human CD27 antibodies with Immune checkpoint
inhibitors of the invention, numbers indicated (N) refer to the
number of immune checkpoint inhibitors (N = 1 or N = 2) combined
with an anti-human CD27 agonistic antibody. When a number is
presented the combination comprises the immune checkpoint
inhibitor(s) listed in the column at the position of the
number.
[0052] Most of the immune checkpoint protein inhibitors presently
known are antibodies. In view of the advances in antibody
technology the use of an antibody as an immune checkpoint protein
inhibitor is preferred in the present invention. However, the use
of alternative immune checkpoint inhibitors based on other
technologies is also envisaged in alternative embodiments.
[0053] As the skilled person will know other technologies are
available for developing binding compounds interfering with the
function of proteins such as immune checkpoint proteins and thus
acting as an inhibitor. For example as the skilled person will
understand, a library of binding peptides engineered on
non-immunoglobulin protein scaffolds can be used to select binding
peptides that inhibit immune checkpoint proteins. Examples of such
protein scaffolds include, but at not restricted to Adnectins,
Affibodies, Anticalins and DARPins (Gebauer and Skerra, Current
opinion Chem. Biol., 2009, 13:245-255 and Caravella and Lugovskoy,
Current opinion Chem. Biol., 2010, 14:520-528). Selection methods
for example include phage display to identify protein scaffolds
that express peptides binding to immune check point proteins. In
addition, combinatorial peptide libraries which may comprise
peptides potentially presenting immune checkpoint protein
inhibitory functions may be screened for suitable immune checkpoint
protein inhibitors. From such a library For example,
one-bead-one-compound combinatorial libraries expressing a broad
set of peptides on beads, where one bead is binding one peptide,
may be used. After selection procedures, beads are recovered and
the peptide is identified (Lam et al., Methods, 1996, 9:482-93;
Xiao et al., Comb. Chem. High Throughput Screen, 2013 16:441-8).
For example using mass-spectrometry methods.
[0054] In the present invention the term "antibody" is used in the
broadest sense and specifically covers, but is not limited to,
monoclonal antibodies (including full length monoclonal
antibodies), polyclonal antibodies, and multispecific antibodies
(e.g., bispecific antibodies) and binding fragments thereof.
[0055] "Antibody fragment" and "antibody binding fragment" mean
antigen-binding fragments of an antibody, typically including at
least a portion of the antigen binding or variable regions (e.g.
one or more CDRs) of the parental antibody. An antibody fragment
retains at least some of the binding specificity of the parental
antibody. Typically, an antibody fragment retains at least 10% of
the parental binding activity when that activity is expressed on a
molar basis. Preferably, an antibody fragment retains at least 20%,
50%, 70%, 80%, 90%, 95% or 100% or more of the parental antibody's
binding affinity for the target. Therefore, as is clear for the
skilled person, "antibody fragments" in many applications may
substitute antibodies and the term "antibody" should be understood
as including "antibody fragments" when such a substitution is
suitable. Examples of antibody fragments include, but are not
limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear
antibodies; single-chain antibody molecules, e.g., sc-Fv, unibodies
or duobodies (technology from Genmab); nanobodies (technology from
Ablynx); domain antibodies (technology from Domantis); and
multispecific antibodies formed from antibody fragments. Engineered
antibody variants are reviewed in Holliger and Hudson, 2005, Nat.
Biotechnol. 23:1126-1136.
[0056] An "Fab fragment" may be comprised of one light chain and
the CH1 and variable regions of one heavy chain. The heavy chain of
a Fab molecule cannot form a disulfide bond with another heavy
chain molecule.
[0057] An "Fc" region contains two heavy chain fragments which may
comprise the C.sub.H1 and C.sub.H2 domains of an antibody. The two
heavy chain fragments are held together by two or more disulfide
bonds and by hydrophobic interactions of the C.sub.H3 domains.
[0058] An "Fab' fragment" contains one light chain and a portion of
one heavy chain that contains the V.sub.H domain and the C.sub.H1
domain and also the region between the C.sub.H1 and C.sub.H2
domains, such that an interchain disulfide bond can be formed
between the two heavy chains of two Fab' fragments to form a
F(ab').sub.2 molecule.
[0059] An "F(ab').sub.2 fragment" contains two light chains and two
heavy chains containing a portion of the constant region between
the C.sub.H1 and C.sub.H2 domains, such that an interchain
disulfide bond is formed between the two heavy chains. A
F(ab').sub.2 fragment thus is composed of two Fab' fragments that
are held together by a disulfide bond between the two heavy
chains.
[0060] The "Fv region" may comprise the variable regions from both
the heavy and light chains, but lacks the constant regions.
[0061] A "single-chain Fv antibody" (or "scFv antibody") refers to
antibody fragments which may comprise the V.sub.H and V.sub.L
domains of an antibody, wherein these domains are present in a
single polypeptide chain. Generally, the Fv polypeptide may further
comprise a polypeptide linker between the V.sub.H and V.sub.L
domains which enables the scFv to form the desired structure for
antigen binding. For a review of scFv, see Pluckthun, 1994, The
Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds. Springer-Verlag, New York, pp. 269-315. See also,
International Patent Application Publication No. WO 88/01649 and
U.S. Pat. Nos. 4,946,778 and 5,260,203.
[0062] A "diabody" is a small antibody fragment with two
antigen-binding sites. The fragments may comprise a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L or
V.sub.L-V.sub.H). By using a linker that is too short to allow
pairing between the two domains on the same chain, the domains are
forced to pair with the complementary domains of another chain and
create two antigen-binding sites. Diabodies are described more
fully in, e.g., EP 404,097; WO 93/11161; and Holliger et al., 1993,
Proc. Natl. Acad. Sci. USA 90: 6444-6448.
[0063] A "domain antibody fragment" is an immunologically
functional immunoglobulin fragment containing only the variable
region of a heavy chain or the variable region of a light chain. In
some instances, two or more V.sub.H regions are covalently joined
with a peptide linker to create a bivalent domain antibody
fragment. The two V.sub.H regions of a bivalent domain antibody
fragment may target the same or different antigens.
[0064] An antibody fragment of the invention may comprise a
sufficient portion of the constant region to permit dimerization
(or multimerization) of heavy chains that have reduced disulfide
linkage capability, for example where at least one of the hinge
cysteines normally involved in inter-heavy chain disulfide linkage
is altered with known methods available to the skilled person. In
another embodiment, an antibody fragment, for example one that may
comprise the Fc region, retains at least one of the biological
functions normally associated with the Fc region when present in an
intact antibody, such as FcRn binding, antibody half life
modulation, ADCC (antibody dependent cellular cytotoxicity)
function, and/or complement binding (for example, where the
antibody has a glycosylation profile necessary for ADCC function or
complement binding).
[0065] The antibody is directed against human CD27 and thus may
comprise binding domains that bind to and/or interact with human
CD27. The antibody may be raised in an animal from a non-human
species suitable for eliciting antibodies against human antigens.
Alternatively, the antibody may be isolated from antibody phage
libraries generated using the techniques described in McCafferty et
al., 1990, Nature, 348:552-554. Clackson et al., 1991, Nature,
352:624-628, and Marks et al., 1991, J. Mol. Biol. 222:581-597. The
skilled person will be able to select a suitable non-human species
for eliciting antibodies against human antigens. For example a
selection may be made from a non-human mammal, such as a rodent,
including murine (rat or mouse) or hamster species, or
alternatively a camelid species.
[0066] The antibody, when raised in a non-human species, preferably
is chimerized with methods and techniques known in the art to form
a "chimeric antibody". The term "chimeric" antibody refers to
antibodies in which a portion of the heavy and/or light chain is
identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(See, for example, U.S. Pat. No. 4,816,567 and Morrison et al.,
1984, Proc. Natl. Acad. Sci. USA 81:6851-6855). Within the present
invention a "chimeric antibody" preferably is a "humanized
antibody".
[0067] As used herein, the term "humanized antibody" refers to
forms of antibodies that contain sequences from non-human (e.g.,
murine) antibodies as well as human antibodies. Such antibodies
contain minimal sequence derived from non-human immunoglobulin. In
general, the humanized antibody may comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FR
regions are those of a human immunoglobulin sequence. The humanized
antibody optionally also may comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. The humanized forms of rodent antibodies may
essentially comprise the same CDR sequences of the parental rodent
antibodies, although certain amino acid substitutions may be
included to increase affinity, increase stability of the humanized
antibody, or for other reasons However, as CDR loop exchanges do
not uniformly result in an antibody with the same binding
properties as the antibody of origin, changes in framework residues
(FR), residues involved in CDR loop support, might also be
introduced in humanized antibodies to preserve antigen binding
affinity (Kabat et al., 1991, J. Immunol. 147:1709).
[0068] The term "antibody" also includes "fully human" antibodies,
i.e., antibodies that may comprise human immunoglobulin protein
sequences only. A fully human antibody may contain non-human, such
as murine (rat or mouse) carbohydrate chains if produced in a
non-human cell (e.g. mouse or hamster), or in a hybridoma derived
from a murine cell. Similarly, "mouse antibody" or "rat antibody"
refer to an antibody that may comprise only mouse or rat
immunoglobulin sequences, respectively.
[0069] A fully human antibody may be generated in a human being, in
a transgenic non-human animal having human immunoglobulin germline
sequences, by phage display or other molecular biological methods.
Also, recombinant immunoglobulins may also be made in transgenic
mice. See Mendez et al., 1997, Nature Genetics 15:146-156. See also
Abgenix, Medarex, MeMo and Kymab technologies.
[0070] The antibodies of the present invention also include
antibodies with modified (or blocked) Fc regions to provide altered
effector functions. See, e.g. U.S. Pat. No. 5,624,821;
WO2003/086310; WO2005/120571; WO2006/0057702; Presta, 2006, Adv.
Drug Delivery Rev. 58:640-656; Vincent and Zurini, Biotechnol. J.,
2012, 7:1444-50; Kaneko and Niwa, Biodrugs, 2011, 25: 1-11. Such
modification can be used to enhance or suppress various reactions
of the immune system, with possible beneficial effects in diagnosis
and therapy. Alterations of the Fc region include amino acid
changes (substitutions, deletions and insertions), glycosylation or
deglycosylation, and adding multiple Fc. Preferably, Fc regions
displaying reduced Fc effector functions are used. The antibodies
of the present invention also include antibodies that have a human
IgG4 containing Fc regions. And/or Fc regions carrying a N297Q
glycosylation deficient mutant are used.
[0071] Changes to the Fc can also alter the half-life of antibodies
in therapeutic antibodies, and a longer half-life would result in
less frequent dosing, with the concomitant increased convenience
and decreased use of material. See Presta, 2005, J. Allergy Clin.
Immunol. 116:731 at 734-35.
[0072] The antibodies of the present invention, although less
preferred, also include antibodies with intact Fc regions that
provide full effector functions, e.g. antibodies of isotype IgG1,
which induce complement-dependent cytotoxicity (CDC) or antibody
dependent cellular cytotoxicity (ADCC) in the a cell associated
with the target for the antibody.
[0073] The antibodies may also be conjugated (e.g., covalently
linked) to molecules that improve stability of the antibody during
storage or increase the half-life of the antibody in vivo. Examples
of molecules that increase the half-life are albumin (e.g., human
serum albumin) and polyethylene glycol (PEG). Albumin-linked and
PEGylated derivatives of antibodies can be prepared using
techniques well known in the art. See, e.g. Chapman, 2002, Adv.
Drug Deliv. Rev. 54:531-545; Anderson and Tomasi, 1988, J. Immunol.
Methods 109:37-42; Suzuki et al., 1984, Biochim. Biophys. Acta
788:248-255; and Brekke and Sandlie, 2003, Nature Rev. 2:52-62.
[0074] As used herein, the term "about" refers to a value that is
within an acceptable error range for the particular value as
determined by one of ordinary skill in the art, which will depend
in part on how the value is measured or determined, i.e. the
limitations of the measurement system. For example, "about" can
mean within 1 or more than 1 standard deviation per the practice in
the art. Alternatively, "about" or "comprising essentially of" can
mean a range of up to 20%. Furthermore, particularly with respect
to biological systems or processes, the terms can mean up to an
order of magnitude or up to 5-fold of a value. When particular
values are provided in the application and claims, unless otherwise
stated, the meaning of "about" or "comprising essentially of"
should be assumed to be within an acceptable error range for that
particular value.
[0075] The antibody can be selected from any class of
immunoglobulins, including IgM, IgG, IgD, IgA, and IgE. Preferably,
the antibody is an IgG antibody. Any isotype of IgG can be used,
including IgG1, IgG2, IgG3, and IgG4. Variants of the IgG isotypes
are also contemplated. The humanized antibody may comprise
sequences from more than one class or isotype. Optimization of the
necessary constant domain sequences to generate the desired
biologic activity is readily achieved by screening the antibodies
in the biological assays described in the Examples.
[0076] Likewise, either class of light chain can be used in the
compositions and methods herein. Specifically, kappa, lambda, or
variants thereof are useful in the present compositions and
methods.
[0077] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies which may comprise the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations that typically include different
antibodies directed against different determinants (epitopes), each
monoclonal antibody is directed against a single determinant on the
antigen. The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by the hybridoma method first described by
Kohler et al., 1975, Nature 256:495, or may be made by recombinant
DNA methods (see, for example, U.S. Pat. No. 4,816,567). The
"monoclonal antibodies" may also be isolated from phage antibody
libraries using the techniques described in Clackson et al., 1991,
Nature 352: 624-628 and Marks et al., 1991, J. Mol. Biol.
222:581-597, for example. The monoclonal antibodies herein
specifically include "chimeric" antibodies.
[0078] Monoclonal antibodies can be made according to knowledge and
skill in the art of injecting test subjects with human antigen and
then generating hybridomas expressing antibodies having the desired
sequence or functional characteristics. DNA encoding the monoclonal
antibodies is readily isolated and sequenced using conventional
procedures (e.g., by using oligonucleotide probes that are capable
of binding specifically to genes encoding the heavy and light
chains of the monoclonal antibodies). The hybridoma cells serve as
a preferred source of such DNA.
[0079] For therapeutic applications the antibodies may be used as
such or as a treatment conjugate. As used herein, a treatment
"conjugate" refers to an antibody, or a fragment thereof,
conjugated to a therapeutic moiety, such as a bacterial toxin, a
cytotoxic drug or a radiotoxin. Toxic moieties can be conjugated to
antibodies, of the invention using methods available in the
art.
[0080] In view of the fact that the present invention resides in
the surprising immune stimulatory effects of the combination of an
anti-human CD27 agonistic antibody together with an immune
checkpoint inhibitor, the present invention, in its various
embodiments is suitable for treatment of a condition known or
expected to be ameliorated by immune stimulation, in particular
stimulation of antigen-specific T-lymphocytes. In this invention
the term antigen-specific T-lymphocyte in particular includes CD4+
and/or CD8+ T cells.
[0081] The immune stimulation, in particular the stimulation of
antigen-specific T-lymphocytes, may be achieved by stimulation of
CD27.sup.+ immune cells or by inhibition of an immune checkpoint
protein, such as CTLA-4, PD1, PD-L1, PD-L2, LAG-3, BTLA, B7H3,
B7H4, TIM3 and KIR. Thus according to certain embodiments the
present invention, is suitable for treatment of a condition known
or expected to be ameliorated by stimulation of CD27.sup.+ immune
cells. According to certain alternative embodiments the present
invention, is suitable for treatment of a condition known or
expected to be ameliorated by inhibition of an immune checkpoint
protein, such as CTLA-4, PD1, PD-L1, PD-L2, LAG-3, BTLA, B7H3,
B7H4, TIM3 or MR.
[0082] The meaning of the term stimulation in the context of an
immune response will be known to the skilled person. It will be
clear that this term includes enhancement and thus relates to both
elevation of existing immune responses and induction or de novo
generation of an immune response.
[0083] The skilled person will know which cells are associated with
CD27, CTLA-4, PD1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3 and
MR. In particular it is known that CD27 positive cells may or may
not express on their cell surface CTLA-4, PD1, PD-L1, PD-L2, LAG-3,
BTLA, B7H3, B7H4, TIM3 and MR (and vice versa). Thus conditions
treatable within the present invention are most certainly not
restricted to conditions involving cells expressing both CD27 and
an immune checkpoint protein, such as CTLA-4, PD-1 or PD-L1, PD-L2,
LAG-3, BTLA, B7H3, B7H4, TIM3 or MR.
[0084] The various aspects of the invention are aimed at "treatment
of a condition amelioratated by stimulation of an immune response,
in particular stimulation of antigen-specific T-lymphocytes". "The
treatment of a condition amelioratated by stimulation of an immune
response, in particular stimulation of antigen-specific
T-lymphocytes" may alternatively be defined as "a treatment wherein
stimulation of an immune response, in particular stimulation of
antigen-specific T-lymphocytes, is beneficial". The terms
"amelioration" and "beneficial" in the context of treatment both
refer to clinically meaning full improvement as can be predicted
and/or established by a physician.
[0085] Within the present invention the treatment of the
"condition" includes any therapeutic use including prophylactic and
curative uses of the anti-human CD27 agonistic antibody and the
number of immune checkpoint inhibitors. Therefore the term
"condition" may refer to disease states but also to physiological
states in the prophylactic setting where physiology is not altered
to a detrimental state. Conditions in the setting of prophylactic
use of the anti-human CD27 agonistic antibody and the number of
immune checkpoint inhibitors include for example immunization (the
physiological process of inducing and/or generating immune memory
against an antigen) after vaccination. The treatment within the
context of the present invention thus may be aimed at supporting
prophylactically induced physiological processes.
[0086] Conditions ameliorated by immune stimulation, in particular
stimulation of antigen-specific T-lymphocytes include infectious
diseases, such as bacterial, fungal, viral and parasitic infectious
diseases. In addition immunization after vaccination may also be
ameliorated by immune stimulation, in particular stimulation of
antigen-specific T-lymphocytes. The vaccination may be against a
pathogen, such as pathogen selected from bacteria, fungi, viruses
or parasites, or against toxins, or self-antigens, including
antigens expressed on benign or malignant tumors, including
cancers. Also conditions associated with uncontrolled proliferation
of cells such as cancers may be ameliorated by immune stimulation,
in particular stimulation of antigen-specific T-lymphocytes. These
conditions may be ameliorated by stimulation of CD27.sup.+ immune
cells and/or by inhibition of an immune checkpoint protein, such as
CTLA-4, PD1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3 or
KIR.
[0087] Cancers within the present invention include, but are not
limited to, leukemia, acute lymphocytic leukemia, acute myelocytic
leukemia, myeloblasts promyelocyte, myelomonocytic monocytic
erythroleukemia, chronic leukemia, chronic myelocytic
(granulocytic) leukemia, chronic lymphocytic leukemia, mantle cell
lymphoma, primary central nervous system lymphoma, Burkitt's
lymphoma and marginal zone B cell lymphoma, Polycythemia vera
Lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple
myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,
solid tumors, sarcomas, and carcinomas, fibrosarcoma, myxosarcoma,
liposarcoma, chrondrosarcoma, osteogenic sarcoma, osteosarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal
carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical cancer, uterine cancer,
testicular tumor, lung carcinoma, small cell lung carcinoma,
non-small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma,
retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma,
basal cell carcinoma, biliary tract cancer, bladder cancer, bone
cancer, brain and central nervous system (CNS) cancer, cervical
cancer, choriocarcinoma, colorectal cancers, connective tissue
cancer, cancer of the digestive system, endometrial cancer,
esophageal cancer, eye cancer, head and neck cancer, gastric
cancer, intraepithelial neoplasm, kidney cancer, larynx cancer,
liver cancer, lung cancer (small cell, large cell), melanoma,
neuroblastoma; oral cavity cancer (for example lip, tongue, mouth
and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma,
rhabdomyosarcoma, rectal cancer; cancer of the respiratory system,
sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid
cancer, uterine cancer, and cancer of the urinary system.
[0088] Less preferred cancers include CD27-expressing tumors, such
as those selected from the group consisting of chronic lymphocytic
leukemia, mantle cell lymphoma, primary central nervous system
lymphoma, Burkitt's lymphoma and marginal zone B cell lymphoma.
[0089] Some examples of pathogenic viruses causing infections
ameliorated by immune stimulation, in particular stimulation of
antigen-specific T-lymphocytes include HIV, hepatitis (A, B, C, D
or E), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV,
Epstein Barr virus), adenovirus, influenza virus, flaviviruses,
echovirus, rhinovirus, coxsackie virus, cornovirus, respiratory
syncytial virus, mumps virus, rotavirus, measles virus, rubella
virus, parvovirus, vaccinia virus, HTLV virus, dengue virus,
papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus
and arboviral encephalitis virus.
[0090] Some examples of pathogenic bacteria causing infections
ameliorated by immune stimulation, in particular stimulation of
antigen-specific T-lymphocytes, include chlamydia, rickettsial
bacteria, mycobacteria, staphylococci, streptococci, pneumonococci,
meningococci and conococci, klebsiella, proteus, serratia,
pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera,
tetanus, botulism, anthrax, plague, leptospirosis, and Lyme disease
bacteria.
[0091] Some examples of pathogenic fungi causing infections
ameliorated by immune stimulation, in particular stimulation of
antigen-specific T-lymphocytes, include Candida (albicans, krusei,
glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus
(fumigatus, niger, etc.), Genus Mucorales (mucor, absidia,
rhizophus), Sporothrix schenkii, Blastomyces dermatitidis,
Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma
capsulatum.
[0092] Some examples of pathogenic parasites causing infections
ameliorated by immune stimulation, in particular stimulation of
antigen-specific T-lymphocytes, include Entamoeba histolytica,
Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia
lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium
vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi,
Leishmania donovani, Toxoplasma gondi, and Nippostrongylus
brasiliensis.
[0093] If the condition ameliorated by immune stimulation, in
particular stimulation of antigen-specific T-lymphocytes is
immunization against an antigen, the anti-human CD27 agonistic
antibody and the number of immune checkpoint inhibitors are in
general administered in combination with a vaccine. The vaccine may
comprise a number of antigen or antigenic determinants specific
for, a pathogen, such as a pathogen selected from bacteria, fungi,
viruses or parasites, or of a toxin, or a self-antigen, including
antigens expressed on benign or malignant tumors, such as cancers.
The pathogens and cancers against which the vaccination is directed
may be selected as indicated above.
[0094] According to certain embodiments the vaccination is directed
against HIV, Hepatitis (A, B, C, D, E), Influenza, Herpes, Giardia,
Malaria, Leishmania, Staphylococcus aureus, or Pseudomonas
Aeruginosa, preferably HIV. For these pathogens there is currently
no effective vaccine, or existing vaccines are less than completely
effective.
[0095] The inventors of the present invention have found unexpected
immune stimulatory effects when combining an anti-human CD27
agonistic antibody and a number of immune checkpoint protein
inhibitors, in particular an inhibitor of CTLA4 or PD1, PD-L1,
PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3 or MR. Tests indicate
synergistic and/or potentiating effects of the combinations. Thus
according to certain embodiments the anti-human CD27 agonistic
antibody is for synergistic and/or potentiating stimulation of the
immune response, particularly synergistic and/or potentiating
stimulation of antigen-specific T-lymphocytes, together with the
number of immune checkpoint inhibitors.
[0096] The anti-human CD27 agonistic antibody of the present
invention preferably is presented in a composition which may
comprise the antibody. The composition may comprise the antibody or
antibodies together with a carrier. The composition according to
certain embodiments preferably is a pharmaceutical composition.
[0097] To prepare pharmaceutical or sterile compositions, the
antibody, antibodies or fragment thereof, is admixed with a
pharmaceutically acceptable carrier and/or excipient, see, e.g.,
Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National
Formulary, Mack Publishing Company, Easton, Pa. (1984).
Formulations of therapeutic and diagnostic agents may be prepared
by mixing with physiologically acceptable carriers, excipients, or
stabilizers in the form of, e.g., lyophilized powders, slurries,
aqueous solutions or suspensions (see, e.g., Hardman, et al., 2001,
Goodman and Gilman's The Pharmacological Basis of Therapeutics,
McGraw-Hill, New York, N.Y.; Gennaro, 2000, Remington: The Science
and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New
York, N.Y.; Avis, et al. (eds.), 1993, Pharmaceutical Dosage Forms:
Parenteral Medications, Marcel Dekker, NY; Lieberman, et al.
(eds.), 1990, Pharmaceutical Dosage Forms: Tablets, Marcel Dekker,
NY; Lieberman, et al. (eds.), 1990, Pharmaceutical Dosage Forms:
Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie, 2000,
Excipient Toxicity and Safety, Marcel Dekker, Inc., New York,
N.Y.).
[0098] Toxicity and therapeutic efficacy of the binding compound,
in particular antibody, compositions, administered alone or in
combination with another agent, such as the usual anti-cancer
drugs, can be determined by standard pharmaceutical procedures in
cell cultures or experimental animals, e.g., for determining the
LD.sub.50 (the dose lethal to 50% of the population) and the
ED.sub.50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects
is the therapeutic index and it can be expressed as the ratio
between LD.sub.50 and ED.sub.50. The data obtained from these cell
culture assays and animal studies can be used in formulating a
range of dosage for use in humans. The dosage of such compounds
lies preferably within a range of circulating concentrations that
include the ED.sub.50 with little or no toxicity. The dosage may
vary within this range depending upon the dosage form employed and
the route of administration utilized.
[0099] Suitable routes of administration include parenteral
administration, such as intramuscular, intravenous, or subcutaneous
administration and oral administration. Administration of
antibodies, used in the pharmaceutical composition or to practice
the method of the present invention can be carried out in a variety
of conventional ways, such as oral ingestion, inhalation, topical
application or cutaneous, subcutaneous, intraperitoneal,
parenteral, intraarterial or intravenous injection. In one
embodiment, the binding compound of the invention is administered
intravenously. In another embodiment, the binding compound of the
invention is administered subcutaneously.
[0100] Alternatively, one may administer the antibodies in a local
rather than systemic manner, for example, via injection of the
antibody or antibodies directly into the site of action, often in a
depot or sustained release formulation. Furthermore, one may
administer the antibody in a targeted drug delivery system.
[0101] Guidance in selecting appropriate doses of antibodies,
cytokines, and small molecules are available (see, e.g.,
Wawrzynczak, 1996, Antibody Therapy, Bios Scientific Pub. Ltd,
Oxfordshire, UK; Kresina (ed.), 1991, Monoclonal Antibodies,
Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.),
1993, Monoclonal Antibodies and Peptide Therapy in Autoimmune
Diseases, Marcel Dekker, New York, N.Y.; Baert, et al., 2003, New
Engl. J. Med. 348:601-608; Milgrom, et al., 1999, New Engl. J. Med.
341:1966-1973; Slamon, et al., 2001, New Engl. J. Med. 344:783-792;
Beniaminovitz, et al., 2000, New Engl. J. Med. 342:613-619; Ghosh,
et al., 2003, New Engl. J. Med. 348:24-32; Lipsky, et al., 2000,
New Engl. J. Med. 343:1594-1602).
[0102] Determination of the appropriate dose is made by the
clinician, e.g., using parameters or factors known or suspected in
the art to affect treatment or predicted to affect treatment.
Generally, the dose begins with an amount somewhat less than the
optimum dose and it is increased by small increments thereafter
until the desired or optimum effect is achieved relative to any
negative side effects. Important diagnostic measures include those
of symptoms of, e.g., the inflammation or level of inflammatory
cytokines produced.
[0103] A preferred dose protocol is one involving the maximal dose
or dose frequency that avoids significant undesirable side effects.
A total weekly dose is generally at least 0.05 .mu.g/kg body
weight, more generally at least 0.2 .mu.g/kg, most generally at
least 0.5 .mu.g/kg, typically at least 1 .mu.g/kg, more typically
at least 10 .mu.g/kg, most typically at least 100 .mu.g/kg,
preferably at least 0.2 mg/kg, more preferably at least 1.0 mg/kg,
most preferably at least 2.0 mg/kg, optimally at least 10 mg/kg,
more optimally at least 25 mg/kg, and most optimally at least 50
mg/kg (see, e.g., Yang, et al., 2003, New Engl. J. Med.
349:427-434; Herold, et al., 2002, New Engl. J. Med. 346:1692-1698;
Liu, et al., 1999, J. Neurol. Neurosurg. Psych. 67:451-456;
Portielji, et al., 2003, Cancer Immunol. Immunother. 52:133-144).
The desired dose of a small molecule therapeutic, e.g., a peptide
mimetic, natural product, or organic chemical, is about the same as
for an antibody or polypeptide, on a moles/kg basis.
[0104] "Administration", "therapy" and "treatment," as it applies
to an animal, human, experimental subject, cell, tissue, organ, or
biological fluid, refers to contact of an exogenous pharmaceutical,
therapeutic, diagnostic agent, or composition to the animal, human,
subject, cell, tissue, organ, or biological fluid.
"Administration", "therapy" and "treatment" can refer, e.g., to
therapeutic, pharmacokinetic, diagnostic, research, and
experimental methods. Treatment of a cell encompasses contact of a
reagent to the cell, as well as contact of a reagent to a fluid,
where the fluid is in contact with the cell. "Administration",
"therapy" and "treatment" also mean in vitro and ex vivo
treatments, e.g., of a cell, by a reagent, diagnostic, binding
composition, or by another cell.
[0105] As used herein, "inhibit" or "treat" or "treatment" includes
a postponement of development of the symptoms associated with
disease and/or a reduction in the severity of such symptoms that
will or are expected to develop with said disease. The terms
further include ameliorating existing symptoms, preventing
additional symptoms, and ameliorating or preventing the underlying
causes of such symptoms. Thus, the terms denote that a beneficial
result has been conferred on a vertebrate subject with a
disease.
[0106] As used herein, the term "therapeutically effective amount"
or "effective amount" refers to an amount of antibody or
antibodies, that when administered alone or in combination with an
additional therapeutic agent to a cell, tissue, or subject is
effective to prevent or ameliorate the disease or condition to be
treated. A therapeutically effective dose further refers to that
amount of the compound sufficient to result in amelioration of
symptoms, e.g., treatment, healing, prevention or amelioration of
the relevant medical condition, or an increase in rate of
treatment, healing, prevention or amelioration of such conditions.
When applied to an individual active ingredient administered alone,
a therapeutically effective dose refers to that ingredient alone.
When applied to a combination, a therapeutically effective dose
refers to combined amounts of the active ingredients that result in
the therapeutic effect, whether administered in combination,
serially or simultaneously. An effective amount of therapeutic will
decrease the symptoms typically by at least 10%; usually by at
least 20%; preferably at least about 30%; more preferably at least
40%, and most preferably by at least 50%.
[0107] Methods for co-administration or treatment with a second
therapeutic agent are well known in the art, see, e.g., Hardman, et
al. (eds.), 2001, Goodman and Gilman's The Pharmacological Basis of
Therapeutics, 10th ed., McGraw-Hill, New York, N.Y.; Poole and
Peterson (eds.), 2001, Pharmacotherapeutics for Advanced Practice:
A Practical Approach, Lippincott, Williams & Wilkins, Phila.,
Pa.; Chabner and Longo (eds.), 2001, Cancer Chemotherapy and
Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.
[0108] Within the present invention the term `co-administered`
should be understood as meaning that the individual active
components (here the anti-human CD27 agonistic antibody and the
number of immune checkpoint inhibitors) are administered in the
same subject. Such administration may be simultaneously or
alternatively the active components may be administered within a
time frame of up to 3 months, in view of the fact that most
antibody therapeutics, including those targeting immune checkpoint
inhibitors and CD27 display a terminal half-life in human subjects
of 2-4 weeks. Therefore, pharmacodynamic responses to such antibody
may be detectable for months after administration.
[0109] The pharmaceutical composition of the invention may also
contain other agents, including but not limited to a cytotoxic,
chemotherapeutic, cytostatic, anti-angiogenic or antimetabolite
agents, a tumor targeted agent, an immune stimulating or immune
modulating agent or an antibody conjugated to a cytotoxic,
cytostatic, or otherwise toxic agent. The pharmaceutical
composition can also be employed with other therapeutic modalities
such as surgery, chemotherapy and radiation.
[0110] According to a preferred embodiment the anti-human CD27
agonistic antibody is provided, in a pharmaceutical composition
which may comprise the anti-human CD27 agonistic antibody together
with a pharmaceutically acceptable carrier, said pharmaceutical
composition being packed in a container and said container being
associated with an information carrier, wherein said information
carrier may comprise information indicating the combined use of the
anti-human CD27 agonistic antibody, preferably in the
pharmaceutical composition, together with an immune checkpoint
inhibitor for use in the treatment of a condition ameliorated by
stimulation of an immune response, in particular stimulation of
antigen-specific T-lymphocytes. The container may be any container
suitable for holding a pharmaceutical composition, preferably a
sterile pharmaceutical composition, more preferably an injectable
pharmaceutical composition. According to a preferred embodiment the
container may comprise a unit dosage of the anti-human CD27
agonistic antibody.
[0111] The information carrier may be any suitable information
carrier such as an object having a surface suitable for carrying
information, for example paper or cardboard. Alternatively a data
carrier which may comprise machine readable information may be
used. The information may be presented in visual form such as in
the form of written information or in the form of a number of
pictures. Alternatively the information may be presented in a form
"readable" by touch, such as in braille. In case the information is
on a data carrier which may comprise machine readable information,
the information may be stored as machine code or analogous signals
which can be converted to visual and/or audio information.
[0112] The information carrier and the container which may comprise
the pharmaceutical composition are associated such that they can be
presented together in a single product. For example they may be
associated by being enclosed together in an enclosure, such as a
container, including a box.
[0113] A further aspect of the invention relates to an immune
checkpoint inhibitor for use in the treatment of a condition
ameliorated by stimulation of an immune response, in particular the
treatment of a condition that benefits from the stimulation of
antigen-specific T-lymphocytes, wherein in said treatment an
anti-human CD27 agonistic antibody is administered. This immune
checkpoint inhibitor together with the anti-human CD27 agonistic
antibody forms a therapeutic combination resulting in unexpected
immune stimulation. According to certain embodiments the immune
checkpoint inhibitor is for synergistic and/or potentiating
stimulation of the immune response, particularly synergistic and/or
potentiating stimulation of antigen-specific T-lymphocytes,
together with the anti-human CD27 agonistic antibody. The technical
details of the various features and preferred embodiments of the
immune checkpoint inhibitor of the invention is similar to what has
already been discussed in connection to the anti-human CD27
agonistic antibody of the invention.
[0114] Similar to the anti-human CD27 agonistic antibody, the
immune checkpoint inhibitor of the invention may be provided, in a
pharmaceutical composition which may comprise the immune check
point inhibitor together with a pharmaceutically acceptable
carrier, said pharmaceutical composition being packed in a
container and said container being associated with an information
carrier, wherein said information carrier may comprise information
indicating the combined use of the immune checkpoint inhibitor,
preferably in the pharmaceutical composition, together with an
anti-human CD27 agonistic antibody for use in the treatment of a
condition ameliorated by stimulation of an immune response, in
particular the treatment of a condition that benefits from
stimulation of antigen-specific T-lymphocytes. The container may be
any container suitable for holding a pharmaceutical composition,
preferably a sterile pharmaceutical composition more preferably an
injectable pharmaceutical. According to a preferred embodiment the
container may comprise a unit dosage of the anti-human CD27
agonistic antibody. Also for this embodiment of the invention the
details of the various technical features and preferred embodiments
will be clear from the parts of the description relating to the
anti-human CD27 agonistic antibody.
[0115] Yet a further aspect of the invention relates to a
combination of an anti-human CD27 agonistic antibody together with
a number of immune checkpoint inhibitors for use in the treatment
of a condition ameliorated by stimulation of an immune response, in
particular the treatment of a condition that benefits from the
stimulation of antigen-specific T-lymphocytes. This combination
according to the invention is primarily a functional combination
and any physical combination considered suitable by the skilled
person for making this functional combination is to be considered
within the scope of this invention. For example in the combination
the anti-human CD27 agonistic antibody may be co-administered with
the number of immune checkpoint inhibitors to a subject.
Alternatively, the combination may be a kit of parts which may
comprise: [0116] (i) a first container holding a first
pharmaceutical composition which may comprise the anti-human CD27
agonistic antibody together with a pharmaceutically acceptable
carrier; [0117] (ii) a second container holding a second
pharmaceutical composition which may comprise the immune checkpoint
inhibitor together with a pharmaceutically acceptable carrier
[0118] (iii) optionally an information carrier which may comprise
information indicating the combined use of the anti-human CD27
agonistic antibody, preferably in the first pharmaceutical
composition, together with an immune checkpoint inhibitor,
preferably in the second pharmaceutical composition, for use in the
treatment of a condition ameliorated by stimulation of an immune
response, in particular the treatment of conditions that benefit
from the stimulation or enhancement of antigen-specific
T-lymphocytes.
[0119] According to an alternative embodiment the first and second
container and the first and second pharmaceutical composition
coincide thus presenting a kit of parts which may comprise: [0120]
(A) a container holding a pharmaceutical composition which may
comprise the anti-human CD27 agonistic antibody and the immune
checkpoint inhibitor together with a pharmaceutically acceptable
carrier; [0121] (B) optionally an information carrier which may
comprise information indicating the combined use of the anti-human
CD27 agonistic antibody, preferably in the pharmaceutical
composition, together with an immune checkpoint inhibitor,
preferably in the pharmaceutical composition, for use in the
treatment of a condition ameliorated by stimulation of an immune
response, in particular the treatment of a condition that benefits
from the stimulation of antigen-specific T-lymphocytes.
[0122] Also for the combination of the invention the details of the
various technical features and preferred embodiments is similar to
what has been discussed in the parts of the description relating to
the anti-human CD27 agonistic antibody.
[0123] A further aspect of the invention relates to a method for
treating a condition ameliorated by stimulation of an immune
response, in particular the treatments of a condition that result
in the stimulation of antigen-specific T-lymphocytes, said method
which may comprise administering a CD27 agonist, preferably an
anti-human CD27 agonistic antibody, in combination with a number of
immune checkpoint inhibitors.
[0124] Also for the method of treatment of the invention the
details of the various technical features and preferred embodiments
are similar to what has been discussed above in relation to the
anti-human CD27 agonistic antibody.
[0125] Although the present invention and its advantages have been
described in detail, it should be understood that various changes,
substitutions and alterations can be made herein without departing
from the spirit and scope of the invention as defined in the
appended claims.
[0126] The present invention will be further illustrated in the
following Examples which are given for illustration purposes only
and are not intended to limit the invention in any way.
EXAMPLES
Example 1
CD27 Agonism Combined with Checkpoint Protein Blockade Results in
Unexpected Immune Stimulation
[0127] To study the effect of combining CD27 agonists with PD-1 or
PD-L1 blocking antibodies human Peripheral Blood Mononuclear cells
(PBMNCs) were isolated from buffy coat. First, the Buffy coat was
diluted to a total volume of 300 ml with DMEM/F12 medium (Gibco,
11320) supplemented with heparin solution (Leo Pharma, DB6132, 5000
U/ml) at RT. After mixing the cell suspension, aliquotes were
loaded on a Ficoll-Paque Plus gradient in conical tubes and
centrifuged at 450 g for 30 min, at 20.degree. C. without a brake.
Next, plasma was removed by aspiration and PBMCs were recovered
from the plasma/Ficoll interface. PBMCs were washed twice with
DMEM/F12 medium and resuspended in RPMI 1640 medium (Gibco, 52400)
supplemented with 10% Foetal Calf Serum. PBMCs were plated at
2.times.10.sup.5 cells/well in 96-well flat bottom plates (Nuclon).
Antibodies (anti-PD1 (hIgG4 chimera, see below, of hPD109A,
WO/2008/156712), anti-hCD27 (hIgG4 chimera of hCD27.15). See below
for chimerization), anti-PDL1 (MIH1, eBioscience 16.5983.82), human
IgG4 isotype control (Sigma, 14639) and mouse IgG1 isotype control
(eBioscience, 16.4714.85)) and dilutions thereof were diluted in
PBS and added to the PBMCs. Human IgG4 chimeric versions of
hPD1.09A and hCD27.15 were constructed by cloning of the VH and VL
genes upstream of the human IgG4 and human Kappa constant domain
encoding cDNA, respectively. Full cDNAs were subcloned in pcDNA3.1
(Invitrogen) and transiently transfected in 293T/17 cells (ATCC)
using Lipofectamine 2000 (Invitrogen) following the manufacturer's
instructions. After 5-7 days, supernatants were harvested and
antibodies were purified using standard protein A purification.
Finally, Staphylococcus Enterotoxin B (Sigma S4881) diluted in RPMI
1640 medium supplemented with 10% Foetal Calf Serum was added to
the wells in a final concentration of 25 ng/ml. Cells were
incubated for two days at 37.degree. C., 5% CO.sub.2 and 95%
humidity. To assess the level of immune activation, IL-2 secretion
levels were determined in the supernatant in accordance with the
method described in Dulos et al., J. Immunother, 2012, 35:169-78.
To that aim, supernatants were aspirated and cleared from any cell
material by centrifugation. Next, supernatants were added to Nunc
maxisorp ELISA plates that had been coated with 2 .mu.g/ml
anti-hIL-2 antibody (BDPharmingen, 555051) in PBS by overnight
incubation at 4.degree. C. Prior to addition of the supernatants,
wells were emptied and blocked with 200 .mu.l/well PBS/1% BSA for
one hour at Room Temperature (RT). Supernatants were incubated for
one hour at RT, washed three times with PBST (PBS with 0.01% Tween
20). Subsequently, 100 .mu.l of 0.5 .mu.g/ml of anti-hIL2-biotin
(BD Pharmingen 555040) was added in PBS/PBS-1% BSA (1:1) and
incubated for one hour at RT. After three washes with PBST, 1:5000
diluted streptavidin-HRP (BD Pharmingen, 554066) was added in 100
.mu.l PBS/PBS-1% BSA (1:1). After three washes with PBST and one
wash with water, IL-2 was detected by addition of 100 .mu.l/well
TMB stabilized chromogen (Invitrogen, SB02). Reactions were stopped
with 100 .mu.l 0.5 M H.sub.2SO.sub.4 and absorbances were read at
450 and 620 nm. In this assay, recombinant human IL-2 (Sigma,
H7041) was used to quantify IL-2 protein levels in the
supernatants. In FIG. 1A the cooperative action between CD27
agonistic antibody and PD-1 blocking antibody and in FIG. 1B the
cooperative action between CD27 agonistic antibody and PD-L1
blocking antibody is shown.
[0128] To study the effect of combining CD27 agonist with PD-1 and
PD-L1 blocking antibody in whole human blood, blood was diluted 10
times in RPMI 1640 medium (Gibco, 52400) supplemented with 10%
Foetal Bovine Serum (Hyclone). Diluted blood was plated in 96-well
Nunclon delta surface flat bottom plates (100 .mu.l/well).
Antibodies anti-hCD27 (hIgG4 chimera of hCD27.15, see above)),
anti-PD-1 (hIgG4 chimera of hPD1.09A, see above), anti-PDL1 (MIH1,
eBioscience 16.5983.82), human IgG4 isotype control (Sigma, 14639)
and mouse IgG1 isotype control (eBioscience, 16.4714.85) and
dilutions thereof were diluted in PBS and added to the diluted
blood. Finally, Staphylococcus Enterotoxin B (Sigma S4881) diluted
in RPMI 1640 medium supplemented with 10% Foetal Calf Serum was
added to the wells in a final concentration of 25 ng/ml. Cells were
incubated for two days at 37.degree. C., 5% CO.sub.2 and 95%
humidity. To assess the level of immune activation, IL-2 secretion
levels were determined in the supernatant. To that aim,
supernatants were aspirated and cleared from any cell material by
centrifugation. Next, supernatants were added to Nunc maxisorp
ELISA plates that had been coated with 2 .mu.g/ml anti-hIL-2
antibody (BD Pharmingen, 555051) in PBS by overnight incubation at
4.degree. C. Prior to addition of the supernatant, wells were
emptied and blocked with 200 PBS/1% BSA for one hour at Room
Temperature (RT). Supernatants were incubated for one hour at RT,
washed three times with PBST (PBS with 0.01% Tween 20).
Subsequently, 100 .mu.l of 0.5 .mu.g/ml of anti-hIL2-biotin (BD
Pharmingen 555040) was added in PBS/PBS-1% BSA (1:1) and incubated
for one hour at RT. After three washes with PBST, 1:5000 diluted
streptavidin-HRP (BD Pharmingen, 554066) was added in 100 .mu.l
PBS/PBS-1% BSA (1:1). After three washes with PBST and one wash
with water, IL-2 was detected by addition of 100 .mu.l/well TMB
stabilized chromogen (Invitrogen, SB02). Reactions were stopped
with 100 .mu.l 0.5 M H.sub.2SO.sub.4 and absorbances were read at
450 and 620 nm. In this assay, recombinant human IL-2 (Sigma,
H7041) was used to quantify IL-2 protein levels in the
supernatants. Also in this test the IL-2 level was increased to an
unexpected level by the combination of the CD27 agonistic antibody
and the PD-1 or PDL-1 inhibiting antibody in comparison to these
antibodies alone.
[0129] The expectation presented by these results, that
combinations of a CD27 agonistic antibody with other immune check
point inhibitors will give similar effects, was confirmed in
additional experiments performed with the same whole blood test
protocol, which included the combination of a CD27 agonist (hIgG4
chimera of hCD27.15, see above) with anti-CTLA4 (14D3, eBioscience
16.1529.82). The results of these experiments (see FIGS. 2A-2C)
showed a similar cooperative action by the combination of the CD27
agonistic antibody and the CTLA4 inhibiting antibody in comparison
to the individual antibodies alone (IgG4 isotype control as above;
IgG2A isotype control: BD Pharmingen 554126).
[0130] On the basis of these results it may be expected that the
combination of an anti-human CD27 agonistic antibody together with
at least one immune checkpoint inhibitor will have beneficial
effects in conditions ameliorated by stimulation of an immune
response, such as conditions ameliorated by stimulation of
antigen-specific T-lymphocytes.
Example 2
Evaluating the Activity of Anti-CD27, Anti-LAG-3, and Anti-PD1
Antibodies in a Human PBMC SEB Superantigen-Induced IL2
Secretion
[0131] An additional confirmatory experiment was conducted to
further show the effects of the combination of anti-human CD27
agonistic antibodies with immune checkpoint protein inhibitors. In
this experiment a hCD27.15 analogue was used in combination with an
anti-LAG3 antibody and an anti-PD1 antibody. The analogue used
contains functional heavy chain CDR1, CDR2, CDR3 (SEQ ID NO: 1, 2,
3, respectively) and light chain CDR1, CDR2, CDR3 (SEQ ID NO: 4, 5,
6, respectively) of hCD27.15. In addition it contains functionally
linked functional human IgG4 constant domains (CH1-CH3, GenBank
accession #K01316). Functionality of the hCD27.15 analogue was
established in a CD27 binding assay corresponding to the one
disclosed in example 2 (page 49-50) of WO2012/004367 and a CD27
signaling test corresponding to the NF-.kappa.B tests disclosed in
example 3 (page 54-55) of WO2012/004367.
[0132] The human anti-LAG3 antibody used comprised a heavy chain
amino acid sequence according to SEQ ID NO: 23 and a light chain
amino acid sequence according to SEQ ID NO: 24. The human anti-PD1
antibody used comprised a heavy chain amino acid sequence according
to SEQ ID NO: 21 and a light chain amino acid sequence according to
SEQ ID NO: 22. To study the effect of the combination of anti-human
CD27 agonistic antibodies with immune checkpoint protein inhibitors
human PBMCs were isolated from heparinized human blood from healthy
donors using SepMate.RTM. tubes (Stem Cell). 15 ml of Ficoll-1077
was added to SepMate.RTM. tubes, which was carefully overlayed with
25 ml of human blood and centrifuged at 1,250 g for 12 min. at RT
with light brake (5 out of 10--10 being max brake). White blood
cells were isolated at the interphase of the Ficoll and dilute into
40 ml of Hanks Balances Salt Solution (HMSS) at RT. Next, cells
were centrifuged at 300 g for 10 min. at 4.degree. C. and the
pellet was resuspended in 50 ml of HBSS. Finally, the cell
suspension was centrifuged at 250 g for 10 min to remove platelets
and the pellet was resuspended in 12 ml complete media (RPMI 1640
containing HEPES and Penn/Step and 10% human A+B+ serum)). Cells
were quantified by Vi-cell. Next, 50 .mu.l of PBMC cell suspension
(1.times.10.sup.7 cells/ml (final 5.times.10.sup.5 cells/well) were
plated in a 96-well round bottom plate. 50 .mu.l of antibody (per
antibody treatment, so 100 .mu.l total antibody added for
combination conditions) at 10 .mu.g/ml end concentrations for 30
min. at 37.degree. C. Next, a 4.times. concentration of 50 .mu.l
Staphylococcus enterotoxin B (SEB) superantigen (Toxin Technology,
Sarasota, Fla.) at 0.5, 1, 8, 16, 32, 64 and 80 ng/ml end
concentration was added and incubate 72 hr at 37.degree. C.
Supernatants were collected and cleared from any cell material by
centrifugation. IL-2 secretion levels as a measure for immune
activation was detected using Human IL-2 V-PLEX Kit (Catalog No.
K151QQD-4, from Meso Scale Discovery, Rockville, Md.), according to
the manufacturer's instructions. As a negative control,
(sub)-isotype matched antibodies were used.
[0133] FIGS. 3A-3D show the results obtained from four separate
donors using the experimental methods described above. For each
donor the results of the anti-CD27/Anti-LAG3 combination is shown
in the left panel and the results of the anti-CD27/Anti-PD1
combination is shown in the right panel. For each SEB concentration
the bars represent from left to right: isotype control; immune
checkpoint inhibitor (anti-Lag3 or anti-PD1); anti-CD27; immune
checkpoint inhibitor (anti-Lag3 or anti-PD1)+anti-CD27. The
anti-CD27 antibody demonstrates approximately 1.5-2 fold IL-2
increase above isotype controls as a single agent. The anti-CD27
antibody showed combination activity with anti-LAG-3 antibody and
with anti-PD1 antibody. Similar effects may be expected for
combinations of anti-human CD27 agonistic antibody (including
analogues) with other immune checkpoint protein inhibitors.
[0134] The invention is further described by the following numbered
paragraphs:
[0135] 1. An anti-human CD27 agonistic antibody, such as hCD27.15
or 1F5, or an antibody analogue thereof, for use in the treatment
of a condition ameliorated by stimulation of an immune response, in
particular stimulation of antigen-specific T-lymphocytes, wherein
in said treatment a number of immune checkpoint protein inhibitors
is administered.
[0136] 2. An anti-human CD27 agonistic antibody according to
paragraph 1, wherein an immune checkpoint protein inhibitor is
selected from an inhibitor of CTLA-4, PD1, PD-L1, PD-L2, LAG-3,
BTLA, B7H3, B7H4, TIM3 or KIR.
[0137] 3. An anti-human CD27 agonistic antibody according to any of
the paragraphs 1-2, wherein the condition ameliorated by immune
stimulation, in particular stimulation of antigen-specific
T-lymphocytes is selected from infectious diseases, such as
bacterial, fungal, viral and parasitic infectious diseases,
immunization against a pathogen, such as a pathogen selected from
bacteria, fungi, viruses or parasites, or vaccination against
toxins, or self-antigens, including antigens expressed on benign or
malignant tumors, such as cancers, or conditions associated with
uncontrolled proliferation of cells such as cancers.
[0138] 4. An anti-human CD27 agonistic antibody according to any of
the paragraphs 1-3, wherein the treatment is vaccination and a
vaccine is administered in the treatment.
[0139] 5. Immune checkpoint inhibitor for use in the treatment of a
condition ameliorated by stimulation of an immune response, in
particular stimulation of antigen-specific T-lymphocytes, wherein
in said treatment an anti-human CD27 agonistic antibody, such as
hCD27.15 or 1F5, or an antibody analogue thereof, is
administered.
[0140] 6. Immune checkpoint inhibitor according to paragraph 5,
wherein the immune checkpoint inhibitor is selected from an
inhibitor of CTLA-4, PD1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4,
TIM3 or KIR.
[0141] 7. Immune checkpoint inhibitor according to any of the
paragraphs 5-6, wherein the condition ameliorated by immune
stimulation is selected from infectious diseases, such as
bacterial, fungal, viral and parasitic infectious diseases,
immunization against a pathogen, such as a pathogen selected from
bacteria, fungi, viruses or parasites, or vaccination against
toxins, or self-antigens, including antigens expressed on benign or
malignant tumors, such as cancers, or conditions associated with
uncontrolled proliferation of cells such as cancers.
[0142] 8. An immune checkpoint inhibitor according to any of the
paragraphs 5-7, wherein the treatment is vaccination and wherein a
vaccine is administered in the treatment.
[0143] 9. Combination of an anti-human CD27 agonistic antibody,
such as hCD27.15 or 1F5, or an antibody analogue thereof, together
with a number of immune checkpoint inhibitors for use in the
treatment of a condition ameliorated by stimulation of an immune
response, particularly stimulation of antigen-specific
T-lymphocytes.
[0144] 10. Combination according to paragraph 9, wherein an immune
checkpoint inhibitor is selected from an inhibitor of CTLA-4, PD1,
PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3 or MR.
[0145] 11. Combination according to any of the paragraphs 9-10,
wherein the condition ameliorated by immune stimulation is selected
from infectious diseases, such as bacterial, fungal, viral and
parasitic infectious diseases, immunization against a pathogen,
such as a pathogen selected from bacteria, fungi, viruses or
parasites, or vaccination against toxins, or self-antigens,
including antigens expressed on benign or malignant tumors, such as
cancers, or conditions associated with uncontrolled proliferation
of cells such as cancers.
[0146] 12. Combination according to any of the paragraphs 9-11,
wherein the treatment is vaccination and wherein a vaccine is
administered in the treatment.
[0147] 13. A method of treating a condition ameliorated by
stimulation of an immune response, comprising administering to a
subject in need thereof a therapeutically effective amount of an
anti-human CD27 agonistic antibody and further an immune checkpoint
protein inhibitor.
[0148] 14. The method of paragraph 13, wherein said anti-human CD27
agonistic antibody is selected from the group consisting of: an
anti-human CD27 agonistic antibody comprising the CDR amino acid
sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, or a variant sequence; a
humanized analogue of antibody hCD27.15; an analogue of antibody
hCD27.15 that binds to the same epitope as hCD27.15; antibody 1F5;
an anti-human CD27 agonistic antibody that does not require
cross-linking.
[0149] 15. The method of any of the paragraphs 13-14, wherein said
further immune checkpoint inhibitor protein is selected from the
group consisting of: an CTLA-4 antibody, an anti-PD1 antibody, an
anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti LAG-3
antibody, an anti-BTLA antibody, an anti-B7H3 antibody, an
anti-B7H4 antibody, an anti-TIM3 antibody and an anti-MR
antibody.
[0150] 16. The method of any of the paragraphs 13-15, wherein said
further immune checkpoint inhibitor protein is an anti-PD1
antibody.
[0151] 17. The method of any of the paragraphs 13-16, wherein said
anti-PD-1 antibody is pembrolizumab.
[0152] 18. The method of any of the paragraphs 13-17, wherein said
anti-PD-1 antibody is nivolumab.
[0153] 19. The method of any of the paragraphs 13-18, wherein said
further immune checkpoint inhibitor protein is an anti-LAG3
antibody.
[0154] 20. The method of paragraph 19, wherein said anti-LAG3
antibody comprises the heavy chain and light chain amino acid
sequences of SEQ ID NO: 23 and SEQ ID NO: 24, respectively.
[0155] 21. The method of any of the paragraphs 13-20, wherein the
subject in need of treatment suffers from cancer.
[0156] 22. The method of any of the paragraphs 13-20, wherein the
subject in need of treatment suffers from an infection (such as a
bacterial, fungal, viral and parasitic infectious diseases).
[0157] 23. A vaccine comprising an anti-human CD27 agonistic
antibody and further comprising an immune checkpoint protein
inhibitor.
[0158] 24. The vaccine of paragraph 23, wherein said further immune
checkpoint protein inhibitor is selected from the group consisting
of: an CTLA-4 antibody, an anti-PD1 antibody, an anti-PD-L1
antibody, an anti-PD-L2 antibody, an anti LAG-3 antibody, an
anti-BTLA antibody, an anti-B7H3 antibody, an anti-B7H4 antibody,
an anti-TIM3 antibody and an anti-MR antibody.
[0159] 25. The vaccine of any of the paragraphs 23-24, wherein said
further immune checkpoint inhibitor protein is an anti-PD1
antibody.
[0160] 26. The vaccine of any of the paragraphs 23-25, wherein said
anti-PD-1 antibody is pembrolizumab.
[0161] 27. The vaccine of paragraphs 23-26, wherein said anti-PD-1
antibody is nivolumab.
[0162] 28. The vaccine of any of the paragraphs 23-27, wherein said
further immune checkpoint inhibitor protein is an anti-LAG3
antibody.
[0163] 29. The vaccine of any of the paragraphs 23-28, wherein said
anti-LAG3 antibody comprises the heavy chain and light chain amino
acid sequences of SEQ ID NO:23 and SEQ ID NO:24, respectively.
[0164] Having thus described in detail preferred embodiments of the
present invention, it is to be understood that the invention
defined by the above paragraphs is not to be limited to particular
details set forth in the above description as many apparent
variations thereof are possible without departing from the spirit
or scope of the present invention.
Sequence CWU 1
1
24110PRTMus musculus 1Gly Phe Ile Ile Lys Ala Thr Tyr Met His 1 5
10 217PRTMus musculus 2Arg Ile Asp Pro Ala Asn Gly Glu Thr Lys Tyr
Asp Pro Lys Phe Gln 1 5 10 15 Val 37PRTMus musculus 3Tyr Ala Trp
Tyr Phe Asp Val 1 5 411PRTMus musculus 4Arg Ala Ser Glu Asn Ile Tyr
Ser Phe Leu Ala 1 5 10 57PRTMus musculus 5His Ala Lys Thr Leu Ala
Glu 1 5 610PRTMus musculus 6Gln His Tyr Tyr Gly Ser Pro Leu Thr Phe
1 5 10 7348DNAMus musculus 7gaggttcggc tgcagcagtc tggggcagac
cttgtgaagc caggggcctc agtcaagttg 60tcctgcacag cttctggctt catcattaaa
gccacctata tgcactgggt gaggcagagg 120cctgaacagg gcctggagtg
gattggaagg attgatcctg cgaatggtga gactaaatat 180gacccgaagt
tccaggtcaa ggccactata acagcagaca catcctccag cacagcctac
240ctgcagctca acagcctgac atctgacgac actgccgtct attactgtgc
tagatacgcc 300tggtacttcg atgtctgggg cgcagggacc acggtcaccg tctcctca
3488116PRTMus musculus 8Glu Val Arg Leu Gln Gln Ser Gly Ala Asp Leu
Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser
Gly Phe Ile Ile Lys Ala Thr 20 25 30 Tyr Met His Trp Val Arg Gln
Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro
Ala Asn Gly Glu Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Val Lys
Ala Thr Ile Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Leu
Gln Leu Asn Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Tyr Ala Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val
100 105 110 Thr Val Ser Ser 115 9321DNAMus musculus 9gacatccaga
tgactcagtc tccagcctcc ctgtctgcat ctgtgggaga cactgtcact 60atcacatgtc
gggcaagtga gaatatttac agttttttag catggtatca tcagaaacag
120ggaaggtctc cgcaactcct ggtctatcat gcaaaaaccc tagcagaagg
tgtgccatca 180aggttcagtg gcagtggatc aggcacacag ttttctctga
agatcaacag cctgcaggct 240gaagattttg ggagttatta ctgtcaacat
tattatggta gtccgctcac gttcggtgct 300gggaccaagc tggaggtgaa a
32110107PRTMus musculus 10Asp Ile Gln Met Thr Gln Ser Pro Ala Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Thr Val Thr Ile Thr Cys Arg
Ala Ser Glu Asn Ile Tyr Ser Phe 20 25 30 Leu Ala Trp Tyr His Gln
Lys Gln Gly Arg Ser Pro Gln Leu Leu Val 35 40 45 Tyr His Ala Lys
Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Ala 65 70 75 80
Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Tyr Tyr Gly Ser Pro Leu 85
90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Val Lys 100 105
1110PRTMus musculus 11Gly Phe Thr Phe Ser Ser Tyr Asp Met His 1 5
10 1217PRTMus musculus 12Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 1310PRTMus musculus 13Gly Ser
Gly Asn Trp Gly Phe Phe Asp Tyr 1 5 10 1411PRTMus musculus 14Arg
Ala Ser Gln Gly Ile Ser Arg Trp Leu Ala 1 5 10 157PRTMus musculus
15Ala Ala Ser Ser Leu Gln Ser 1 5 1610PRTMus musculus 16Gln Gln Tyr
Asn Thr Tyr Pro Arg Thr Phe 1 5 10 17119PRTMus musculus 17Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20
25 30 Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Gly Asn Trp
Gly Phe Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val
Ser Ser 115 18107PRTMus musculus 18Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly Ile Ser Arg Trp 20 25 30 Leu Ala Trp Tyr
Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Thr Tyr
Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 19447PRTArtificial SequenceDescription of Artificial Sequence
Synthetic humanized polypeptide 19Gln Val Gln Leu Val Gln Ser Gly
Val Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Met Tyr Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly
Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60
Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr 65
70 75 80 Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp
Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185
190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys
195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr
Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly
Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val
Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310
315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440
445 20218PRTArtificial SequenceDescription of Artificial Sequence
Synthetic humanized polypeptide 20Glu Ile Val Leu Thr Gln Ser Pro
Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser
Cys Arg Ala Ser Lys Gly Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr
Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu
Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala 50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65
70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His
Ser Arg 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185
190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
21440PRTHomo sapiens 21Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser
Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr
Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Cys Ser 115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 180 185 190 Thr Tyr Thr
Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 210 215
220 Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
225 230 235 240 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val 245 250 255 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val 260 265 270 Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln 275 280 285 Phe Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln 290 295 300 Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 305 310 315 320 Leu Pro
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 325 330 335
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 340
345 350 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser 355 360 365 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr 370 375 380 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr 385 390 395 400 Ser Arg Leu Thr Val Asp Lys Ser
Arg Trp Gln Glu Gly Asn Val Phe 405 410 415 Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys 420 425 430 Ser Leu Ser Leu
Ser Leu Gly Lys 435 440 22214PRTHomo sapiens 22Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser
Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 23447PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
23Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp
Tyr 20 25 30 Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Arg Gly Ser Thr Asn Ser
Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Leu Ser Leu Asp Thr
Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Arg Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Phe Gly Tyr Ser Asp
Tyr Glu Tyr Asn Trp Phe Asp Pro Trp Gly Gln 100 105 110 Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135
140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr
Thr Cys Asn Val Asp His Lys 195
200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315
320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445
24214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 24Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn
Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu 85 90
95 Thr Phe Gly Gln Gly Thr Asn Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe
Asn Arg Gly Glu Cys 210
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