U.S. patent application number 14/715398 was filed with the patent office on 2015-11-19 for depletion of il23r expressing cells in the treatment of various diseases.
The applicant listed for this patent is NUMAB AG. Invention is credited to Tea GUNDE, Sebastian MEYER, David URECH.
Application Number | 20150329637 14/715398 |
Document ID | / |
Family ID | 48463668 |
Filed Date | 2015-11-19 |
United States Patent
Application |
20150329637 |
Kind Code |
A1 |
URECH; David ; et
al. |
November 19, 2015 |
DEPLETION OF IL23R EXPRESSING CELLS IN THE TREATMENT OF VARIOUS
DISEASES
Abstract
The present invention relates, for example, to depletion of
IL23R expressing cells in the treatment of disease and, in an
embodiment thereof, to bispecific constructs that specifically bind
to immune effector cells and, simultaneously, to IL23R-carrying
target cells, as well as nucleic acids, vectors, host cells,
pharmaceutical compositions, and methods of production and use
thereof, including such bispecific constructs for use in, for
example, treating inflammatory and/or autoimmune diseases and/or
cancer.
Inventors: |
URECH; David; (Waedenswil,
CH) ; GUNDE; Tea; (Waedenswil, CH) ; MEYER;
Sebastian; (Waedenswil, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NUMAB AG |
Waedenswil |
|
CH |
|
|
Family ID: |
48463668 |
Appl. No.: |
14/715398 |
Filed: |
May 18, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/EP2014/001282 |
May 12, 2014 |
|
|
|
14715398 |
|
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|
Current U.S.
Class: |
424/135.1 ;
424/136.1 |
Current CPC
Class: |
C07K 2317/73 20130101;
A61P 27/02 20180101; A61P 1/04 20180101; A61P 25/28 20180101; A61P
17/06 20180101; A61P 3/10 20180101; C07K 16/2866 20130101; C07K
2317/622 20130101; C07K 2317/92 20130101; C07K 2317/626 20130101;
A61P 35/00 20180101; A61P 35/02 20180101; C07K 2317/75 20130101;
C07K 2317/569 20130101; A61P 25/16 20180101; C07K 2317/56 20130101;
C07K 2317/55 20130101; A61P 9/10 20180101; A61P 25/00 20180101;
C07K 16/2809 20130101; C07K 2317/33 20130101; A61P 37/06 20180101;
A61P 19/02 20180101; A61P 29/00 20180101; C07K 2317/31
20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
May 10, 2013 |
EP |
13002500.0 |
Claims
1. A method for the treatment of a disease selected from the group
consisting of: an inflammatory disease, an autoimmune disease
and/or cancer, comprising, in a human patient in need thereof,
depleting IL23R expressing cells.
2. The method of claim 1, wherein the patient is refractory to
treatment via inhibition of IL-1.beta., IL-6, TGF-.beta., IL-23,
IL-22, IL-17, IL-8, GM-CSF, G-CSF, IFN-.gamma. and/or TNF.alpha.
signaling.
3. The method of claim 1, wherein said patient has an inflammatory
disease and/or an autoimmune disease and is refractory to treatment
via inhibition of effector cytokine signaling of IL23R expressing
cells and/or cytokine signaling that drives differentiation of
IL23R expressing cells.
4. The method of claim 1, wherein the depleting comprises
administering to the human patient in need thereof a bispecific
construct comprising at least one first binding moiety and at least
one second binding moiety, wherein said first binding moiety
specifically binds to a first antigen present on a cytotoxic
effector cell, and said second binding moiety specifically binds to
an IL-23 receptor specific subunit (IL23R) present on the surface
of a target cell, wherein said bispecific construct is administered
in an effective amount for the treatment of the disease in the
patient.
5. The method of claim 4, wherein the cytotoxic effector cell is
selected from the group consisting of: a cytotoxic effector T (Tc)
cell and a cytotoxic natural killer (NK) cells.
6. The method of claim 4, wherein said first binding moiety
specifically binds to an antigen selected from CD3 and CD28.
7. The method of claim 4, wherein said first binding moiety
specifically binds to an agonistic epitope of CD3.epsilon..
8. The method of claim 4, wherein said first binding moiety and/or
said second binding moiety comprises a heavy chain variable domain
(V.sub.H) or binding fragment thereof and a light chain variable
domain (V.sub.L) or binding fragment thereof.
9. The method of claim 4, wherein the bispecific construct is in a
format selected from the group consisting of a single-chain diabody
(scDb), a tandem scDb (Tandab), a linear dimeric scDb (LD-scDb), a
circular dimeric scDb (CD-scDb), a bispecific T-cell engager (BiTE;
tandem di-scFv), a tandem tri-scFv, a tri(a)body, bispecific
Fab.sub.2, di-miniantibody, tetrabody, scFv-Fc-scFv fusion,
di-diabody, DVD-Ig, IgG-scFab, scFab-dsscFv, Fv2-Fc, IgG-scFv
fusions, including bsAb, Bs1Ab, Bs2Ab, Bs3Ab, Ts1Ab, Ts2Ab, and
Knob-into-Holes (KiHs) and DuoBodies.
10. The method of claim 1, wherein the cancer is cancer
metastasis.
11. The method of claim 1, wherein the cancer is selected from the
group consisting of colorectal cancer, lung cancer, breast cancer,
melanoma, brain cancer, nasopharyngeal cancer, oral cancer,
esophageal cancer, pancreatic cancer, kidney cancer, B-cell
lymphomas, and T-cell lymphomas, including adult T-cell lymphoma
leukemia (ATLL), acute myeloid lymphoma (AML), diffuse large B-cell
lymphoma (DLBCL), follicular lymphoma (FL), pediatric acute
lymphoblastic lymphoma (B-ALL), angioimmunoblastic T-cell lymphoma
(AITL), anaplastic large-cell lymphoma (ALCL), T-/natural
killer-cell lymphomas, and peripheral T-cell lymphoma (PTCL).
12. The method of claim 1, wherein the inflammatory and/or
autoimmune disease is selected from the group consisting of:
rheumatoid arthritis, ankylosing spondylitis, psoriasis, psoriatic
arthritis, ulcerative colitis, irritable bowel disease, Crohn's
disease, systemic lupus erythematosus, juvenile diabetes,
autoimmune uveitis, multiple sclerosis, neuromyelitis
optica/Devic's disease, Parkinson's disease, Alzheimer's disease,
clinically isolated syndrome and ischemia-reperfusion injury.
13. The method of claim 1, wherein the treatment is in an amount to
effective to treat recurrence of the inflammatory and/or autoimmune
disease.
14. The method of claim 3, wherein the effector cytokines are
IL-17, IL-22, GM-CSF, G-CSF, TNF and/or IFN-gamma.
15. The method of claim 3, wherein the cytokines that drive
differentiation are IL-1 beta, TGF-beta, IL23, IFN-gamma and/or
IL-6.
16. The method of claim 7, wherein the IL23R binding moiety VL is:
TABLE-US-00017 (SEQ ID NO: 3)
DIQMTQSPSSLSASVGDRVTITCQASENIYSFLAWYQQKPGKAPKLLIYS
ASKLAAGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTNRYSNPDIY
NVFGQGTKLTVLG;
and the IL23R binding moiety VH is: TABLE-US-00018 (SEQ ID NO: 4)
EVQLVESGGGLVQPGGSLRLSCAASGIDFNSNYYMCWVRQAPGKGLEWIG
CIYVGSHVNTYYANWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAT
SGSSVLYFKFWGQGTLVTVSS.
17. The method of claim 16, wherein the CD3 binding moiety VL is:
TABLE-US-00019 (SEQ ID NO: 5)
DIQMTQSPSSLSASVGDRVTITCQSSESVYNNKRLSWYQQKPGKAPKLLI
YTASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQGEFTCSNAD
CFTFGQGTKLTVLG;
and the CD3 binding moiety VH is: TABLE-US-00020 (SEQ ID NO: 6)
EVQLVESGGGLVQPGGSLRLSCAASGFPLSSYAMIWVRQAPGKGLEWIGM
ILRAGNIYYASWVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARRHY
NREGYPIGIGDLWGQGTLVTVSS.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of
International Application No. PCT/EP2014/001282, filed May 12,
2014, which designated the U.S. and claims the benefit of priority
to European Patent Application No. 13002500.0, filed May 10, 2013,
each of which is hereby incorporated in its entirety including all
tables, figures, and claims.
FIELD OF THE INVENTION
[0002] The present invention relates, for example, to depletion of
IL23R expressing cells in the treatment of disease and, in an
embodiment thereof, to bispecific constructs that specifically bind
to immune effector cells and, simultaneously, to IL23R-carrying
target cells, as well as nucleic acids, vectors, host cells,
pharmaceutical compositions, and methods of production and use
thereof, including such bispecific constructs for use in, for
example, treating inflammatory and/or autoimmune diseases and/or
cancer.
BACKGROUND OF THE INVENTION
[0003] Interleukin (IL)-23 is a heterodimeric cytokine comprised of
two protein subunits, designated p40 and p19 for their approximate
molecular weights. The p40 protein is shared between IL-12 and
IL-23, whereas the p19 protein subunit is unique to IL-23. IL-23
signals through a two-chain receptor complex consisting of the
IL-12 receptor beta-1 (IL-12R.beta.1) chain, which binds to p40,
and a unique IL-23 receptor chain (IL23R), which confers
IL-23-specific intracellular signaling.
[0004] IL-23 induces the differentiation of naive CD4+T cells into
pathogenic IL-17-producing helper T (Th17 or Th.sub.1L-.sub.17)
cells. The IL-17 secreted by this distinct helper T cell subset is
an important effector cytokine during inflammation. Elevated IL-17
levels have been observed in target tissues of various autoimmune
diseases and inflammatory conditions, including rheumatoid
arthritis, inflammatory bowel diseases (i.e., Crohn's disease,
irritable bowel disease and ulcerative colitis), and psoriasis.
Therefore, IL-23 has been implicated as a critical factor in
inflammatory conditions and autoimmune-mediated diseases.
[0005] Several approaches to target IL-23/IL-17 signaling have been
tested so far, including antibodies directed against IL-23 or the
IL-23 receptor, as potential therapeutic approaches for treating
inflammatory and autoimmune diseases. With respect to the use of
IL-23 as a target for potential pharmacological interventions,
various neutralizing antibodies directed to the p40 subunit of
IL-23 have been developed for potential use in the treatment of
autoimmune driven diseases. For example, the monoclonal antibody
known as "ustekinumab" (Janssen-Cilag) is an antibody that targets
the common p40 subunit of IL-12 and IL-23. It has been shown to be
effective in the treatment of psoriasis and psoriatic arthritis.
Another monoclonal antibody that targets the smaller p19 subunit of
IL-23 is described in EP 2 548 577.
[0006] With regard to strategies aimed at blocking the IL-23
receptor function by IL-23 receptor-targeting antibodies, WO
2004/042009 discloses the use of a monoclonal anti-IL-23 receptor
antibody or fragments thereof (Fv, Fab, Fab' and F(ab').sub.2) for
the treatment of an inflammatory disease associated with elevated
expression of IL-17. Moreover, WO 2008/106134 discloses the
generation of humanized antibodies, which recognize the IL23R chain
of the human IL-23 receptor, suitable for use in the treatment of
inflammatory and autoimmune disorders. These anti-IL23R antibodies
include, inter alia, antibodies conjugated to cytotoxic payloads
that can be used in immunotherapy to selectively target and kill
cells expressing IL23R on their surface.
[0007] A promising approach for the antibody-based treatment of
various cancer diseases is the redirection of immune effector cells
to specifically lyse target cells using bispecific antibodies. The
bispecific antibodies recognize a particular antigen on the surface
of a target cell and, simultaneously, an activating surface
molecule of an immune effector cell, such as a natural killer (NK)
cell or a cytotoxic T (Tc) cell, to thereby kill the target
cells.
[0008] The bispecific antibody concept is, for example, used in
cancer therapy where bispecific antibodies are employed that bind
to a cancer antigen on cancer cells and, simultaneously, to the
epsilon chain of CD3 presented on, for example, cytotoxic T cells.
A well-known example of such a bispecific antibody construct is
"blinatumomab", an antibody in the BiTE (bi-specific T cell
engager) format, for the treatment of non-Hodgkin's lymphoma and
acute lymphoblastic leukemia. Blinatumomab was developed by
Micromet and simultaneously binds to the cancer antigen CD19 as
well as to CD3 on the surface of cytotoxic T cells, thereby linking
these two cell types together and activating the cytotoxic T cell
to lyse the target cancer cell.
[0009] The hitherto most successful antibody-based approaches to
treat inflammatory and/or autoimmune diseases exploit mechanisms of
action that interfere with the interaction of cytokines and their
respective receptors in order to prevent signaling through cytokine
receptors. Examples include inhibition of TNF.alpha. (e.g.,
infliximab and adalimumab), inhibition of p40 (e.g., ustekinumab)
or inhibition of IL6R (e.g., tocilizumab). However, these
antibodies often still allow for the signaling through redundant
pathways. As a consequence, a significant number of patients cannot
be effectively treated. For example, up to 40% of the patients are
refractory to treatment with TNF.alpha. inhibiting antibodies.
[0010] Inhibitors of cytokines signaling cascades that are involved
in the differentiation of disease-driving T cells (e.g. IL-1.beta.,
TGF-.beta., IL-6 and IL-23) as well as inhibitors of effector
cytokines that are produced by disease-driving T cells (e.g.
IFN-.gamma., GM-CSF, G-CSF, IL-17, TNF and IL-22) have been
demonstrated to be effective in certain disease conditions.
However, global elimination of individual cytokines is associated
with certain disadvantages: first, it bears the risk of severe side
effects as this may broadly affect immune system's ability to
defend the host against pathogens. Further, redundant signaling
pathways may compensate for each other. The pathogenic cell types
may even become independent of certain upstream cytokines, thus
leaving the patient without effective treatment.
[0011] More recently, antibodies targeting either the downstream
effector cytokine interleukin-17 (IL-17) or the upstream IL-23,
which drives differentiation of pathogenic IL23R expressing T
cells, have proven to be safe and highly efficacious in certain
autoimmune diseases. For example, the anti-IL-23 antibody
ustekinumab/Stelara.RTM. showed excellent safety and efficacy in
psoriasis and PsA, whereas it failed in MS and CD (Longbrake et al
Expert Rev Neurother. 2009;9:319-329; Sandborn et al,
Gastroenterology.2008;135:1130-1141). Similarly, anti-IL-17
therapies were efficacious in psoriasis and uveitis and to some
extent in RA (Jones et al, Nature Immunology.2012;13:1022-1025);
however, studies in CD with secukinumab/AIN457 were recently
terminated due to lack of efficacy (Hueber et al, 2012). These
results suggest that although very similar cell types are
responsible for the inflammatory processes in autoimmune diseases,
the cytokine pattern they produce may differ substantially between
the various diseases.
[0012] By using two different IL-23-blocking antibodies in a murine
experimental autoimmune encephalomyelitis ("EAE") model for
multiple sclerosis it was shown that both antibodies could
completely block development of EAE symptoms in a prophylactic
setting (Chen et al. J.Clin.Invest.2009;116:1317-1325). However,
the blockade of IL-23 signaling appeared to be insufficient for the
therapy of established MS, when the IL-23-blocking antibodies were
administered during an exacerbation episode.
[0013] Instead of targeting a soluble cytokine, it is possible as
well to target the corresponding cytokine receptor. For example, an
antibody against IL23R was employed in a murine EAE model by
administration 2 days after active immunization with an
encephalitogenic PLP, and and a prophylactic activity of the
anti-IL23R could be demonstrated, since the onset of EAE could be
delayed and the EAE score could be reduced (Wojkowska et al.,
Mediators of Inflammation 2014, Article ID 590409, 1-8).
[0014] Instead of systemically blocking selected cytokines or
cytokine-receptor interactions, alternative approaches aim at
specifically eliminating certain immune cells. In MS, the
hypothesis that elimination of immune cells can lead to
long-lasting efficacy has been supported by the recent impressive
clinical results with alemtuzumab/Lemtrada.RTM.. In a follow-up
study to the pivotal phase 3 trial, 80% of patients did not require
any further treatment for up to 24 months after the last treatment
cycle. However, Lemtrada.RTM. unspecifically eliminates the major
components of the adaptive immune system (all B- and T-cells) and
is therefore associated with significant side-effects--such as
opportunistic infections - that would not justify its broad use in
other chronic inflammatory diseases.
[0015] Thus, there is still a need for new and improved treatment
strategies in the treatment of various diseases, such as
inflammatory and/or autoimmune diseases and cancer. In particular,
bispecific molecules for use in such treatments are required that
are stable, easy to produce, highly specific for a given target
antigen, and have a low immunogenicity.
SUMMARY OF THE INVENTION
[0016] The present invention meets the needs presented above by
providing a bispecific construct that specifically binds to an
immune effector cell, i.e. a cytotoxic effector T (Tc) cell and,
simultaneously, to a IL-23 receptor expressing target cell, e.g. a
pathogenic or a non-pathogenic IL-17-producing helper T cell (Th17
cell), in order to kill the IL-23 receptor expressing target cell.
In a further aspect, the binding constructs herein are
multivalent.
[0017] In a first aspect, the present invention provides a
bispecific construct comprising at least one first binding moiety
and at least one second binding moiety for use in the treatment of
cancer comprising IL23R-expressing tumor cells, wherein said first
binding moiety specifically binds to a first antigen present on a
cytotoxic effector T (Tc) cell, and said second binding moiety
specifically binds to the IL-23 receptor specific subunit
(IL23R).
[0018] In a second aspect, the present invention provides a
bispecific construct comprising at least one first binding moiety
and at least one second binding moiety, wherein said first binding
moiety specifically binds to a first antigen present on a cytotoxic
effector cell, e.g., a cytotoxic effector T (Tc) cell or a
cytotoxic natural killer (NK) cell, and said second binding moiety
specifically binds to the IL-23 receptor specific subunit (IL23R)
present on the surface of a target cell.
[0019] The present invention also provides, in further aspects, a
nucleic acid or nucleic acids encoding the bispecific construct of
the present invention, as well as a vector or vectors comprising
said nucleic acid or nucleic acids, and a host cell or host cells
comprising said vector or vectors.
[0020] Another aspect the present invention relates to a method for
producing the bispecific construct of the present invention,
comprising (i) providing a nucleic acid or nucleic acids according
to the present invention, or a vector or vectors according to the
present invention, expressing said nucleic acid or nucleic acids or
said vector or vectors and collecting said bispecific construct
from the expression system, or (ii) providing a host cell or host
cells of the present invention, culturing said host cell or host
cells, and collecting the bispecific construct from the cell
culture.
[0021] Yet another aspect of the present invention relates to a
pharmaceutical composition comprising the bispecific construct of
the present invention and a pharmaceutically acceptable
carrier.
[0022] In still another aspect, the present invention relates to
the use of a bispecific construct of the present invention in the
treatment of an inflammatory and/or autoimmune disease, or in a
method for the treatment of an inflammatory and/or autoimmune
disease, comprising administering to a subject an effective amount
of the bispecific construct of the present invention. Exemplary
inflammatory and/or autoimmune diseases include rheumatoid
arthritis, ankylosing spondylitis, psoriasis, psoriatic arthritis,
ulcerative colitis, Crohn's disease, systemic lupus erythematosus,
juvenile diabetes, autoimmune uveitis, multiple sclerosis,
Parkinson's disease, Alzheimer's disease, and ischemia-reperfusion
injury.
[0023] In still another aspect, the present invention relates to
the use of a bispecific construct of the present invention in the
treatment of multiple sclerosis, in particular after the onset of
an exacerbation episode, comprising administering to a subject an
effective amount of the bispecific construct of the present
invention.
[0024] In still another aspect, the present invention relates to
the use of a bispecific construct of the present invention in the
treatment of tumors whose growth, tissue invasion and/or metastasis
are supported by IL23R expressing cells whereby the tumor may or
may not express IL23R on its surface, comprising administering to a
subject an effective amount of the bispecific construct of the
present invention.
[0025] In a further aspect is a method for the treatment of a
disease selected from the group consisting of: an inflammatory
disease, an autoimmune disease and/or cancer, comprising, in a
human patient in need thereof, depleting IL23R expressing
cells.
[0026] In a further aspect is a method, wherein the patient is
refractory to treatment via inhibition of IL-1.beta., IL-6,
TGF-.beta., IL-23, IL-22, IL-17, IL-8, GM-CSF, G-CSF, IFN-.gamma.
and/or TNF.alpha. signaling.
[0027] In a further aspect is a method, wherein said patient has an
inflammatory disease and/or an autoimmune disease and is refractory
to treatment via inhibition of effector cytokine signaling of IL23R
expressing cells and/or cytokine signaling that drives
differentiation of IL23R expressing cells.
[0028] In a further aspect is a method, wherein the depleting
comprises administering to the human patient in need thereof a
bispecific construct comprising at least one first binding moiety
and at least one second binding moiety, wherein said first binding
moiety specifically binds to a first antigen present on a cytotoxic
effector cell, and said second binding moiety specifically binds to
an IL-23 receptor specific subunit (IL23R) present on the surface
of a target cell, wherein said bispecific construct is administered
in an effective amount for the treatment of the disease in the
patient.
[0029] In a further aspect is a method, wherein the cytotoxic
effector cell is selected from the group consisting of: a cytotoxic
effector T (Tc) cell and a cytotoxic natural killer (NK) cells.
[0030] In a further aspect is a method, wherein said first binding
moiety specifically binds to an antigen selected from CD3 and
CD28.
[0031] In a further aspect is a method, wherein said first binding
moiety specifically binds to an agonistic epitope of
CD3.epsilon..
[0032] In a further aspect is a method, wherein said first binding
moiety and/or said second binding moiety comprises a heavy chain
variable domain (V.sub.H) or binding fragment thereof and a light
chain variable domain (V.sub.L) or binding fragment thereof.
[0033] In a further aspect is a method, wherein the bispecific
construct is in a format selected from the group consisting of a
single-chain diabody (scDb), a tandem scDb (Tandab), a linear
dimeric scDb (LD-scDb), a circular dimeric scDb (CD-scDb), a
bispecific T-cell engager (BiTE; tandem di-scFv), a tandem
tri-scFv, a tri(a)body, bispecific Fab.sub.2, di-miniantibody,
tetrabody, scFv-Fc-scFv fusion, di-diabody, DVD-Ig, IgG-scFab,
scFab-dsscFv, Fv2-Fc, IgG-scFv fusions, including bsAb, Bs1Ab,
Bs2Ab, Bs3Ab, Ts1Ab, Ts2Ab, and Knob-into-Holes (KiHs) and
DuoBodies.
[0034] In a further aspect is a method, wherein the cancer is
cancer metastasis.
[0035] In a further aspect is a method, wherein the cancer is
selected from the group consisting of colorectal cancer, lung
cancer, breast cancer, melanoma, brain cancer, nasopharyngeal
cancer, oral cancer, esophageal cancer, pancreatic cancer, kidney
cancer, B-cell lymphomas, and T-cell lymphomas, including adult
T-cell lymphoma leukemia (ATLL), acute myeloid lymphoma (AML),
diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL),
pediatric acute lymphoblastic lymphoma (B-ALL), angioimmunoblastic
T-cell lymphoma (AITL), anaplastic large-cell lymphoma (ALCL),
T-/natural killer-cell lymphomas, and peripheral T-cell lymphoma
(PTCL).
[0036] In a further aspect is a method, wherein the inflammatory
and/or autoimmune disease is selected from the group consisting of:
rheumatoid arthritis, ankylosing spondylitis, psoriasis, psoriatic
arthritis, ulcerative colitis, irritable bowel disease, Crohn's
disease, systemic lupus erythematosus, juvenile diabetes,
autoimmune uveitis, multiple sclerosis, neuromyelitis
optica/Devic's disease, Parkinson's disease, Alzheimer's disease,
clinically isolated syndrome and ischemia-reperfusion injury.
[0037] In a further aspect is a method, wherein the treatment is in
an amount to effective to treat recurrence of the inflammatory
and/or autoimmune disease.
[0038] In a further aspect is a method, wherein the effector
cytokines are IL-17, IL-22, GM-CSF, G-CSF, TNF and/or
IFN-gamma.
[0039] In a further aspect is a method, wherein the cytokines that
drive differentiation are IL-1 beta, TGF-beta, IL23, IFN-gamma
and/or IL-6.
[0040] In a further aspect is a method, wherein the IL23R binding
moiety VL is:
TABLE-US-00001 (SEQ ID NO: 3)
DIQMTQSPSSLSASVGDRVTITCQASENIYSFLAWYQQKPGKAPKLLIYS
ASKLAAGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTNRYSNPDIY
NVFGQGTKLTVLG;
and the IL23R binding moiety VH is:
TABLE-US-00002 (SEQ ID NO: 4)
EVQLVESGGGLVQPGGSLRLSCAASGIDFNSNYYMCWVRQAPGKGLEWIG
CIYVGSHVNTYYANWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAT
SGSSVLYFKFWGQGTLVTVSS.
[0041] In a further aspect is a method, wherein the CD3 binding
moiety VL is:
TABLE-US-00003 (SEQ ID NO: 5)
DIQMTQSPSSLSASVGDRVTITCQSSESVYNNKRLSWYQQKPGKAPKLLI
YTASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQGEFTCSNAD
CFTFGQGTKLTVLG;
and the CD3 binding moiety VH is:
TABLE-US-00004 (SEQ ID NO: 6)
EVQLVESGGGLVQPGGSLRLSCAASGFPLSSYAMIWVRQAPGKGLEWIGM
ILRAGNIYYASWVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARRHY
NREGYPIGIGDLWGQGTLVTVSS.
[0042] Particular embodiments of the present invention are set
forth in the appended dependent claims and elsewhere herein.
FIGURES
[0043] FIG. 1A describes IL23 signaling and Th1 antitumor response;
FIG. 1B describes proposed mechanisms of bispecific anti-IL23RxCD3c
antibody interactions.
[0044] FIG. 2: Expression of IL23R on colon cancer cell-lines.
IL23R expression was detected by a primary goat polyclonal
anti-IL23R antibody and a PE-labeled anti-goat antibody. Solid
lines, IL23R expression; dashed lines, isotype control. X-axis, PE
intensity; Y-axis, cell counts.
[0045] FIG. 3: IL23R expression on lung adenocarcinoma cell-line.
IL23R expression was detected by a primary goat polyclonal
anti-IL23R antibody and a PE-labeled anti-goat antibody. Solid
lines, IL23R expression; dashed lines, isotype control. X-axis, PE
intensity; Y-axis, cell counts.
[0046] FIG. 4: IL23R expression on lymphoma/leukemia cell-lines.
IL23R expression was detected by a primary goat polyclonal
anti-IL23R antibody and a PE-labeled anti-goat antibody. Solid
lines, IL23R expression; dashed lines, isotype control. X-axis, PE
intensity; Y-axis, cell counts.
[0047] FIG. 5: Phylogenetic tree generated by aligning the unique
CDR sets with the Neighbor Joining algorithm of COBALT.
[0048] FIG. 6: Thermal unfolding of PRO165 by differential scanning
fluorimetry. Duplicate measurement of temperature induced protein
unfolding normalized fluorescence signal.
[0049] FIG. 7. T cell driven lysis of DLD-1 colon carcinoma cells
induced by PRO165. Quantification of early apoptotic and necrotic
DLD-1 cells.
[0050] FIG. 8. Kinetics of skin thickening in the mouse
aldara-induced psoriasis model. The constructs PRO386 and PRO387
were administered by daily intraperitoneal injection of aldara
treated C57B/6 mice, showing, e.g., inhibitiion of skin thickening
in a dose-dependent manner.
[0051] FIG. 9. Flow-cytometry analysis of CD27-negative gamma-delta
T cells from skin lymph nodes in the mouse aldara-induced psoriasis
model showing, e.g., dose-dependent reduction of CD27-negative
gamma-delta T cells.
[0052] FIG. 10. Kinetics of skin thickening in the mouse
aldara-induced psoriasis model. The constructs PRO386 and PRO386
were administered by daily intraperitoneally injecting IL23R-GFP
reporter mice. The anti-CD3 Fab fragment and PBS served as
controls.
[0053] FIG. 11. Flow-cytometry analysis of immune cells in the
thymus of healthy and psoriatic IL23R-GFP reporter mice treated
with PRO386, PRO387, anti-CD3 Fab or PBS.
[0054] FIG. 12. Flow-cytometry analysis of immune cells in skin
draining lymph-nodes (LN) of healthy and psoriatic IL23R-GFP
reporter mice treated with PRO386, PRO387, anti-CD3 Fab or PBS
showing PRO386 and PRO387, showing, e.g., the reduction of counts
of IL23R expressing gamma-delta T cells, IL23R+/CD8+T killer cells
and IL23R+/CD4+T helper cells.
[0055] FIG. 13. Flow-cytometry analysis of immune cells in skin of
healthy and psoriatic IL23R-GFP reporter mice treated with PRO386,
PRO387, anti-CD3 Fab or PBS showing, e.g., the reduction of counts
of IL23R expressing gamma-delta T cells and IL23R expressing T
cells generally.
[0056] FIG. 14. Flow-cytometry analysis of immune cells in skin of
healthy and psoriatic IL23R-GFP reporter mice treated with PRO386,
PRO387, anti-CD3 Fab or PBS.
[0057] FIG. 15. Flow-cytometry analysis of immune cells in spleen
of healthy and psoriatic IL23R-GFP reporter mice treated with
PRO386, PRO387, anti-CD3 Fab or PBS showing, e.g., PRO386 and
PRO387 reduction of IL23R expressing cells.
[0058] FIG. 16. Clinical scores in an active induced mouse EAE
model. SJL mice were treated with PRO386 and PRO387 in a
prophylactic setting, before the onset of disease.
[0059] FIG. 17. Clinical scores in the active induced mouse EAE
model. SJL mice were treated with PRO386, PRO387, anti-CD3 Fab and
PBS in a therapeutic setting after the onset of disease.
[0060] FIG. 18A shows, e.g., lung metastasis counts per animal in
B16F10 melanoma metastasis model. FIG. 18B is photographs from four
representative lungs of mice treated with PBS or PRO386.
DETAILED DESCRIPTION OF THE INVENTION
[0061] In a first aspect, the present invention provides a
bispecific construct comprising at least one first binding moiety
and at least one second binding moiety for use in the treatment of
cancer comprising IL23R-expressing tumor cells, wherein said first
binding moiety specifically binds to a first antigen present on a
cytotoxic effector cell, e.g., a cytotoxic effector T (Tc) cell or
a cytotoxic natural killer (NK) cell, and said second binding
moiety specifically binds to the IL-23 receptor specific subunit
(IL23R).
[0062] Within the meaning of the present invention, the term
"construct" refers to any chemical entity so long as it exhibits
the desired binding activity. Thus, the term "construct" is used in
the broadest sense and specifically covers protein-based molecules,
including recombinant antibodies and fragments thereof comprising
one or more antibody-based domains or binding fragments thereof.
Specific examples include, but are not limited to, monoclonal
chimeric antibodies, humanized antibodies, single-chain diabodies
and the like. Furthermore, the term "comprise" as used within the
present invention, for example in conjunction with the term
"construct", encompasses both "includes" and "consists of".
Moreover, the term "including" used herein means including but not
limited to.
[0063] The term "bispecific", as used herein, is intended to refer
to a construct having two different antigen specificities and,
optionally, other binding moieties that bind to their respective
binding partners, such as a moiety that binds to an Fc receptor or
a tag for detection and/or purification. This means that a
bispecific construct is capable of simultaneously binding to at
least one antigen "A" and at least one antigen "B", wherein A and B
are not the same. Thus, whilst having two different antigen
specificities, a bispecific construct of the present invention does
not necessarily have only two binding moieties, one for each
targeted antigen, but may also include more than two binding
moieties. Furthermore, the term "antigen", as used herein, is to be
interpreted in a broad sense and includes any target moiety that is
bound by the binding moieties of the bispecific construct of the
present invention.
[0064] As used in the present invention, the terms "specific" or
"specifically" are intended to mean that the first and second
binding moieties are able to discriminate between their respective
target molecules (i.e. between the first and second antigen) and/or
one or more reference molecule(s). Thus, in its broadest sense,
"specific binding" or "specifically binding" refers to the first
and second binding moieties' ability to discriminate between the
first antigen on the surface of a Tc cell and the IL23R second
antigen on the surface of a target cell and/or between other target
molecules that are related to or not related to the first antigen
and/or the second antigen (i.e., IL23R).
[0065] The binding specificity of a specific binding moiety can be
determined as known in the art using, for example, surface plasmon
resonance (SPR), western blot, enzyme-linked immunosorbent assay
(ELISA), radioimmunoassay (RIA or IRMA), enhanced chemiluminescence
(ECL), and peptide scan analysis.
[0066] If an ELISA assay is used, the scoring can be carried out by
means of a standard color development reaction, for example by
using horseradish peroxidase (HRP)-conjugated second antibodies in
a HRP, H202, tetramethyl benzidine system. The optical density of
the color development in the reaction vessel (e.g. well) at a given
wavelength is a measure of the binding specificity. A typical
background signal (negative reaction) may be about 0.1 OD, whereas
a typical signal for a positive reaction may be about 1.0 OD or
higher, resulting in a signal to noise ratio of 10:1 or higher.
Typically, the determination of the binding specificity is carried
out using a set of about three to five unrelated biomolecules, such
as milk powder, BSA, transferrin and the like, rather than using
only a single reference biomolecule.
[0067] In particular, the present invention relates to a method for
the treatment of cancer comprising IL23R-expressing tumor cells,
comprising administering to a subject, for example, a mammalian
subject and particularly a human patient, an effective amount of
the bispecific construct of the present invention.
[0068] Further provided herein is a method for the treatment of
tumors whose growth, tissue invasion and/or metastasis are
supported by IL23R expressing cells whereby the tumor may or may
not express IL23R on its surface, comprising administering to a
subject for example a mammalian subject and in particular a human
subject an effective amount of an agent that depletes such IL23
expressing cells, e.g., the bispecific construct described
herein.
[0069] The term "effective amount" is defined below. Typically, an
effective amount of the bispecific construct of the present
invention is administered in form of the above-described
pharmaceutical composition. Suitable administration routes include,
but are not limited to, topical and parenteral administration, in
particular subcutaneous and intravenous injection. The
administration regimen is not particularly limited and includes,
for example, continuous infusion over one week, two weeks or four
weeks.
[0070] The cancer to be treated includes, but is not limited to,
colorectal cancer, lung cancer, breast cancer, melanoma,
nasopharyngeal cancer, oral cancer, esophageal cancer, pancreatic
cancer, kidney cancer, B-cell lymphomas, and T-cell lymphomas,
including adult T-cell lymphoma leukemia (ATLL), acute myeloid
lymphoma (AML), diffuse large B-cell lymphoma (DLBCL), follicular
lymphoma (FL), pediatric acute lymphoblastic lymphoma (B-ALL),
angioimmunoblastic T-cell lymphoma (AITL), anaplastic large-cell
lymphoma (ALCL), T-/natural killer-cell lymphomas, and peripheral
T-cell lymphoma (PTCL). In particular embodiments the cancer is
selected from colorectal cancer, lung cancer, breast cancer,
melanoma, kidney cancer, nasopharyngeal cancer, oral cancer,
esophageal cancer, B-cell lymphomas, and T-cell lymphomas such as
adult T-cell lymphoma leukemia (ATLL), angioimmunoblastic T-cell
lymphoma (AITL), anaplastic large-cell lymphoma (ALCL), T-/natural
killer-cell lymphomas, and peripheral T-cell lymphoma (PTCL).
[0071] The balance between interleukin (IL)-23 and IL-12 plays an
important role in regulating the anti-tumor immune response. IL-23
and IL-12 are heterodimeric cytokines that share one of their
domains (p40). While IL-12--consisting of the two subunits p35 and
p40--has been found to have antitumor effects, IL-23 that is
composed of p40 and p19, seems to promote growth of certain tumors
by direct effects on tumor cells and by indirect mechanisms
creating a tumor supportive inflammatory microenvironement. IL-12
drives the development of IFN-y producing Th1 cells, which are
crucial for the antitumor immunity (Dunn, G. P. et al. 2006. Nat.
rev. Immuno1:6;836-848; Colombo, M. P. and Trinchieri,
G.2002.Cytokine Growth Factor Rev;13:155-168). IL-12 and
IFN-.gamma. are able to inhibit the expansion of intratumoral T
regulatory cells (Tregs) that antagonize the activity of Th1 cells,
as well as angiogenesis in the tumor microenvironment, thus
enhancing tumor control (Cao, X et al.2009.Cancer
Res;69:8700-8709). In line with a converse effect of IL-23 in the
tumor microenvironment, IL-23 suppresses IL-12-dependent
IFN-.gamma. secretion in T cells (Sieve, A. N. et al. 2010.Eur. J.
Immuno1;40;2236-2247). In the gut, IL-23 produced by dendritic
cells (DCs) in response to microbial products, cellular stress and
cell death, may drive the formation of Th17 cells instead of an
antitumor Th1 response. These Th17 cells in turn also produce
IL-23, which may inhibit the immune surveillance activity mediated
by cytotoxic T cells by potentially preventing their ability to
infiltrate into the tumor (Ngiow, S. F.2013.Trend in
Immunology;34:548-555) and to affect antitumor and antimetastatic
functions of NK cells (Teng, M. W. et al. 2010PNAS;107:8328-8333).
In line with the view that IL-23 signaling may have effects
supporting tumor formation and/or growth in certain tumors is the
finding that mice deficient only in IL-12 signaling show increased
tumor growth, while mice deficient for both, IL-12 and IL-23
signaling, show no increased risk of developing tumors (Street, S.
E. et al.2002. J. Exp. Med;196:129-134; Airoldi, I, et al.
2005.Blood;106:3846-3853). Furthermore, mice specifically deficient
for IL-23 signaling--but not for IL-12 signaling--were found to be
almost completely resistant to tumor formation and showed
attenuated tumor growth (Langowski, J. L. eta1.2008.
Nature;442:461-465; Teng, M. W. et al. 2010PNAS;107:8328-8333). In
a recent study it was further found that the coadministration of an
anti-CD40 antibody stimulating T-cells together with an anti-IL-23
antibody attenuating the Th17 response showed better efficacy as
compared to either antibody alone (von Scheidt, B. et al.
2014.Cancer Res;D01:10.1158/0008-5472.CAN-13-1646), possibly by
shifting the balance from Th17 to Th1. In the human situation,
independent clinical studies have reported that serum
concentrations of IL-23 are increased in cancer patients in
comparison with healthy individuals (Li, J. et al.2012. PLoS
ONE;7:e46264; Gangemi, S. et al.2012. J. Cell. Biochem;
113:2122-2125; Ljujic, B. et al.2010. Arch. Med. Res; 41:182-189;
He, S. et al. 2011.Int. J. Mol. Sci;12:7424-7437; Fukuda, M, et
al.2010.1nt. J. Oncol; 36:1355-1365). For example in pancreatic
cancer, elevated IL-23 levels correlated with disease stage and in
breast cancer with poor prognosis (He, S. et al. 2011.Int. J. Mol.
Sci;12:7424-7437). In primary hepatocellular carcinoma (HCC) higher
IL-23 levels in the cancer microenvironment have been associated
with poor prognosis. Also in colorectal cancer serum IL-23 levels
were increased in patients as compared to healthy donors (Stanilov,
N. S.2009.Labmedicine;41:159-163). Further, Th17 gene expression
profiles as well as Th17 cell infiltrations in colorectal cancer
tissue samples were associated with drastically worse prognosis as
opposed to Th1 gene expression or Th1 cell infiltration (Tosolini
et al, Cancer Res 2011;71:1263-1271). Collectively, these data
suggest that IL-23 promotes tumorigenesis by driving protumor
inflammation to suppress antitumor effector cells.
[0072] Human IL23R is predominantly found in activated memory T
cells, natural killer (NK) cells, and innate lymphoid cells (ILCs),
and at lower levels on monocytes, macrophages, and dendritic cells
(DCs). Recently, direct effects of interleukin IL-23 on tumor cells
have been described, suggesting that IL23R is expressed also on
certain tumors. For example IL-23 was shown to have
pro-proliferative effects on human oral squamous cell carcinoma
(SSC) cell lines (Fukuda, M et al.2010. Int. J. Onco1;36:1355-1365;
Fukuda, M et al.2010. Mol. Med. Rep,;3:89-93) and non-small cell
lung cancer (NSCLC) cell lines (Baird, A.M. et al.2013. Lung
Cancer;79:83-90). Using genome-wide association studies, two
sequence variants of IL23R have been associated with the risk of
several solid cancers and the same polymorphisms have been found to
predispose individuals to an increased risk of acute myeloid
leukemia (AML) (Xu, Y, et al.2013.J Gastroentero1;48:125-131; Chu,
H. et al.2012.1nt. J. cancer;130:1093-1097; Chen, J. et al.2010.
mol. Carcinog;49:862-868; Qian, X. et al.2013. PLoS ONE;8:e55473).
IL23R expression was further found to be upregulated in primary
tumor tissue of small cell lung cancer (SCLC), in lung
andenocarcinoma (Li, J. et al.2013. Carcinog;34:658-666), in
follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL)
(Cocco et al.Leukemia.2012:26:1365-1374), in pediatric B-ALL (Cocco
et al.Blood.2010;116:3887-3898) as well as in colorectal carcinoma
(CRC) (Lan et al, Int J Colorectal Dis 2011;26:1511-1518; Suzuki et
al, Oncology Letters 2012;4:199-204). In CRC a correlation between
expression level and severity/metastasis was found (Carlsson et
al,Br J Cancer 2012;106:517-524).
[0073] Taken together, IL-23 seems to create an inflammatory
microenvironment that on one hand favors tumor formation, growth
and metastasis and on the other hand supports tumor immune escape
by attenuating the Th1 antitumor response (see FIG. 1A).
Preclinical studies with co-administration of anti-CD40 and
anti-IL-23 antibodies have demonstrated that this "push" (anti-CD40
driven activation of T cells) and "pull" (anti-IL-23 driven
attenuation of Th17 cells) is effective in certain tumor models.
However, this approach does not specifically target cancer cells.
Thus certain cancer cells are still likely to escape the enhanced
Th1 response. And further, systemic activation of Th1 cells may
lead to adverse effect, such as enhancing inflammatory processes.
Other approaches using bispecific antibodies redirecting T cells to
lyse cancer cells have demonstrated efficacy in different tumor
models, also in solid tumors such as colorectal cancer
(Lutterbuese, R. et al.2010.PNAS;107:12605-12610; Osada T. et al.
2010;British J Cancer;102:124-133).
[0074] Here we present a novel approach aiming at optimally
balancing efficacy and safety, by specifically targeting cancer
cells on one hand and by locally stimulating the anti-tumor
immune-response on the other hand. To achieve this, a bispecific
anti-IL23RxCD3c antibody is employed that acts through the
mechanisms, illustrated in FIG. 1B: [0075] a) Redirecting
CD3+cytotoxic T cells to lyse IL23R expressing tumors; [0076] b)
Stimulating CD3+Th1 and T killer cells specifically in the tumor
microenvironment, as binding to target cells and consequently
cross-linking of CD3c binding domains is required for T cell
stimulation; [0077] c) Redirecting CD3+cytotoxic T cells to lyse
IL23R expressing Th17 cells, thereby eliminating a major source of
Th1 inhibitory cytokines; [0078] d) Redirecting CD3+cytotoxic T
cells to lyse IL23R expressing regulatory T (Treg) cells that
inhibit the Th1 anti-tumor response; and [0079] e) Optionally:
blocking IL-23 signaling by competing with IL-23 binding to its
receptor IL23R.
[0080] In particular embodiments, said human patient is a patient,
which has a high Th17 response.
[0081] As shown in the examples herein, treatment with the
bispecific molecule of this invention indeed drastically reduced
metastasis formation in the B16F10 mouse melanoma metastasis model,
showing that the IL23R expressing cell population is key not only
for tumor growth but surprisingly also for tumor metastasis.
[0082] In the context of the present invention, the term "high Th17
response" refers to a situation, where a patient either shows
increased Th17 gene expression or increased counts of Th17 cells.
An increased Th17 gene expression may be shown by increased levels
of Th17 gene mRNA in the tumor tissue when compared to normal
distant control tissue. In colorectal cancer such increase is at
least 2-fold, particularly 4-fold and most particularly 5- to
6-fold for the genes IL17A and RORC (see Tosolini et al., Cancer
Res 2011;71:1263-1271) between tumor tissue and normal distant
mucosa. An increased count of Th17 cells may be seen in the tumor
tissue or in the tumor adjacent tissue compared to distant control
tissue. In colorectal cancer, such increased counts are observed in
the center or at the invasive margin, particularly in both the
center and the invasive margin of the tumor (see Tosolini et al.,
loc. cit.).
[0083] IL-23R expressing cells include but are not limited to
IL-23R expressing Th17 cells, IL-23R expressing gamma-delta T cells
(for example, CD27-negative gamma-delta T cells), IL-23R expressing
Th22 cells, IL-23R expressing Tc17 cells, IL-23R expressing
leukocytes and other cells expressing IL-23R and/or responding to
IL-23.
[0084] Moreover, the treatment of cancer and the inflammatory
and/or autoimmune diseases described herein further includes
treatment of such diseases by reducing and/or depleting IL-23R
expressing .alpha..beta. T cells that produce effector cytokines,
such as IL-17, GM-CSF, G-CSF, IL-22, IFN-.gamma. and TNF, in
particular IL-17, IFN-.gamma. and GM-CSF. The reduction or
depletion of such cells includes sufficient cell reduction or
depletion to provide the desired level of treatment. As used
herein, to deplete IL-23R expressing cells refers to reducing the
number of such cells in an amount effective to achieve a desired
reduction in the disease, including healing, reduction of disease
symptoms, complete or partial abatement or remission of the
disease, and complete or partial prophylaxis.
[0085] Binding of the constructs, e.g., the bispecific molecules,
described herein to both targets engages cytotoxic effector cell,
e.g., a cytotoxic effector T (Tc) cell or a cytotoxic natural
killer (NK) cells to lyse IL23R expressing cells, thus eliminating
pathogenic disease-causing lymphocytes. This approach results in
better and broader efficacy as compared to blockade of individual
cytokines. Such an anti-IL23RxCD3 bi-specific antibody fragment
very selectively targets a pathogenic subpopulation of activated
leukocytes, the elimination of which is not expected to limit the
protection afforded by the immune system as such, as a broad
repertoire of naive and responsive B- and T-cells will be
maintained. For example in the gamma-delta T cell compartment only
a small subpopulation of CD27 negative/CD3 bright cells is
expressing the typical Th17 cytokines and it is only this
population that is positive for IL23R (Paget et al. Immunology and
Cell Biology.2014:1-15; Chognard et al. PLOS ONE.2014;9:1-15).
Besides the gamma-delta T cells it has been shown in IL23R-GFP
reporter mice that IL23R expression is associated to IL-17
producing CD4+ alpha-beta T cells and certain IL-17 producing
myeloid cells (Awasthi et al. J Immunol.2009;182:1-11). Due to the
restricted expression of IL23R on pathogenic inflammatory cells the
specific elimination of IL23R expressing pathogenic cells will be
even safer than the more traditional blockade of individual
cytokines (e.g. TNF) that are globally involved in immune
processes, particularly because only a very small fraction of T
cells is indeed expressing IL23R.
[0086] Specific elimination of the disease driving Th17 cells in MS
holds the potential to show similar efficacy as
alemtuzumab/Lemtrada.RTM. with better safety though. It has been
demonstrated that expression of IL-23 and IL-17 is upregulated in
lesions and the percentage of IL-17 expressing cells correlates
with disease activity (Tzartos et al, Neurbiology.2008:172:146-155;
Kebir et al, Nature medicine. 2007;13:1173-1175) and further, that
Th17 but not Th1 cells are increased in the cerebrospinal fluid
(CSF) of MS patients (Brucklacher-Waldert et al.
Brain.2009:132;3329-2241). Further, it has been demonstrated that
the Th17 cytokines IL-17 and IL-22 disrupt the blood brain barrier,
which is a prerequisite for the invasion of inflammatory cells into
the CNS (Kebir et al.Nature Medicine.2007;10:1173-1175). In a
monkey EAE model for multiple sclerosis, the IL-23 inhibitory
antibody Stelara, which interferes with differentiation and
activation of Th17 cells, showed good efficacy when tested in a
prophylactic setting in which differentiation of IL23R expressing
cells is blocked (Brok H P M et al. J Immunol 2002;169:6554-6563).
Further, preclinical animal studies suggest that the memory of the
disease (EAE) lies in the Th17 compartment, indicating that
specific elimination of IL23R expressing cells (in particular Th17
memory cells) could result in a long-lasting amelioration of the
disease phenotype (McGeachy et al, Nat Immunol. 2009 March; 10(3):
314-324. doi:10.1038/ni.1698. and Haines et al, Cell Rep. 2013 Apr.
24. pii: S2211-1247(13)00159-9. doi:
10.1016/j.celrep.2013.03.035.). Despite the very strong scientific
support for the role of IL23R expressing cells in MS, the
interleukin-12/23 inhibiting antibody Stelara did not show
significant responses in a phase II clinical study in MS. A
probable explanation for this is the unfortunate patient inclusion
criteria in this phase II study (Longbrake E and Racke M K Expert
Rev Neurother. 9(3),319-321 (2009)). The enrolled patients had
advanced disease, in which the so-called Th17 cells have already
terminally differentiated, and terminally differentiated Th17 cells
may no longer be dependent on IL-23 signaling, do however continue
to express the IL-23 receptor (McGeachy M J Nat
Immuno.2009;10:314-324). In contrast to Stelara, the present
approach targets terminally differentiated IL23R expressing cells.
Recent experiments with a mouse surrogate for the bispecific
constructs in accordance with the present invention have indeed
demonstrated efficacy not only in a prevention setting but
surprisingly also by a therapeutic intervention starting after the
onset of the acute phase of the disease when Th17 cells have
already terminally differentiated. This is in sharp contrast to the
lack of efficacy to block the acute exacerbation in this model of
two different IL-23-blocking antibodies in the same therapeutic
setting (Chen et al. J.Clin.Invest.2009;116:1317-1325). Hence, the
blockade of IL-23 signaling appears to be insufficient for the
therapy of established MS, while depletion of IL23R expressing
cells is showing strong effects.
[0087] Thus, the treatment of the diseases described herein further
includes the treatment of such diseases in a subject refractory or
resistant to treatment with inhibitors of, including blockers of
(e.g., antibodies against) signals leading to the differentiation
of IL23R expressing cells, such as, IL-6, IL-1.beta., TGF-8, and/or
IL-23.
[0088] In particular the treatment of the diseases described herein
further includes the treatment of autoimmune diseases after the
onset of an acute exacerbation. Non-limiting examples for diseases
in which such acute exacerbations may occur, are multiple
sclerosis, clinically isolated syndrome and neuromyelitis
optica.
[0089] Further, the treatment of the diseases described herein
includes the treatment of such diseases in a subject refractory or
resistant to treatment with inhibitors of, including blockers of
(e.g., antibodies against) signals of effector cytokines produced
by IL23R expressing cells, such as IL-17, GM-CSF, G-CSF, IL-22,
IFN-.gamma., and/or TNF.alpha..
[0090] Additionally, the treatment of the diseases described herein
further includes the treatment of such diseases in a subject
refractory or resistant to treatment with conventional tumor
therapies (e.g., chemotherapies), or antibodies against targets
such as HER-2, HER-3, VEGF, CEA, EGFR, IGFR, FASL,
[0091] Moreover, the treatment of cancer and the inflammatory
and/or autoimmune diseases described herein further includes the
treatment of a subject having an advanced stage of such diseases,
wherein Th17 cells and/or other IL-23R expressing cells have
terminally differentiated and therefore are no longer dependent on
IL-23 signaling.
[0092] Moreover, the treatment of cancer and the inflammatory
and/or autoimmune diseases described herein further includes the
treatment for the recurrence of such diseases in a subject. For
example, the treatment steps described herein can be effective in
treating a disease where local memory exists in IL-23R expressing
cells. An example of such a disease is an aspect of psoriasis, in
which, for example, epidermal Th22 and Tc17 cells, which both
express IL23R, form a localized disease memory, even in clinically
healed psoriasis. Accordingly, treatment for disease recurrence can
be achieved, for example, by treatment that reduces or eliminates
this local memory and therefore results in a long lasting treatment
effect.
[0093] In a further aspect, the present invention relates to a
bispecific construct having at least one first binding moiety and
at least one second binding moiety. The first and second binding
moieties specifically bind to a first antigen and a second antigen,
respectively. The first antigen is an antigen present on the
surface of an immune effector cell, namely a cytotoxic effector T
(Tc) cell (also known as cytotoxic T lymphocyte (CTL) or T killer
cell). The second antigen is the IL-23 receptor specific subunit,
IL23R, present on the surface of a target cell.
[0094] Within the present invention, one target cell type is
typically a pathogenic cell, in particular a pathogenic T cell,
more particularly a T cell expressing the transcription factor
ROR.gamma.(t). ROR.gamma.(t) promotes thymocyte differentiation
into pro-inflammatory Th17 cells and also plays a role in
inhibiting apoptosis of undifferentiated T cells and promoting
their differentiation into Th17 cells, possibly by down-regulating
the expression of the Fas ligand and IL-2, respectively. In
particular embodiments, the cells are selected from the group
consisting of IL-17 producing T cells (Th17 cells),
T cells, natural killer T (NKT) cells and invariant natural killer
(iNK) cells. Most particularly, the target cells within the present
invention are selected from Th17 cells and .gamma..delta.
cells.
[0095] Another target cell type within the present invention is a
tumor cell, particularly a tumor cell expressing IL23R. IL23R
expression has for example been shown for epithelial cancers such
as colon carcinoma (CRC), small-cell lung cancer (SCLC),
adenocarcinoma (AC) of the lung, as well as on various B- and
T-cell tumors, exemplified by diffuse large B- cell lymphoma
(DLBCL), follicular lymphoma (FL), pediatric B-ALL and acute
myeloid lymphoma (AML).
[0096] In particular such embodiments, said bispecific construct
has at least one first binding moiety directed against an antigen
present on the surface of an immune effector cell, namely a
cytotoxic effector T (Tc) cell (also known as cytotoxic T
lymphocyte (CTL) or T killer cell), and at least a second binding
moiety directed against the IL-23 receptor specific subunit, IL23R,
present on the surface of a tumor cell, wherein said bispecific
construct is also able to eliminate IL23R expressing cells, such as
Th17 cells and to stimulate an anti-cancer immune response. In
particular such embodiments the anti-cancer immune response is
driven by CD3 expressing cells. In more particular such
embodiments, said first antigen is CD3, particularly
CD3.epsilon..
[0097] The first and second binding moieties are not structurally
limited so long as they specifically bind to the desired first and
second antigens. However, the first and second binding moieties
generally consist of or are formed of one or more oligo- or
polypeptides or parts thereof. Particularly, the first and second
binding moieties are antibody-based binding moieties, which
typically comprise at least one antibody variable domain or binding
fragment thereof.
[0098] In a particular embodiment of the present invention, the
first binding moiety specifically binds to a first antigen selected
from CD3 and CD28. The CD3 protein is associated with the T cell
receptor (TCR) and required for T cell activation. CD3 is a complex
of one CD3y, one CD38 and two CD3s chains which, together with the
TCR and the -chain, form the TCR receptor complex. CD28 is one of
the molecules expressed on T cells that provide co-stimulatory
signals required for T cell activation. Stimulation through CD28
can provide a potent co-stimulatory signal to T cells.
[0099] In a particular embodiment of the present invention, the
first binding moiety binds specifically to CD3, more particularly
to the epsilon chain of CD3 (CD3s), and most particularly to an
agonistic epitope of CD3e. The term "agonistic epitope", as used
herein, means (a) an epitope that, upon binding of the bispecific
construct of the present invention, optionally upon binding of
several bispecific constructs on the same cell, allows said
bispecific constructs to activate TCR signaling and induce T cell
activation, and/or (b) an epitope that is solely composed of amino
acid residues of the epsilon chain of CD3 and is accessible for
binding by the bispecific construct of the present invention, when
presented in its natural context on Tc cells (i.e. surrounded by
the TCR, the CD3.gamma. chain, etc.), and/or (c) an epitope that,
upon binding of the bispecific construct of the present invention,
does not lead to stabilization of the spatial position of
CD3.epsilon. relative to CD3.gamma..
[0100] In another particular embodiment of the present invention,
instead of binding to Tc cells, the first binding moiety
specifically binds to a component of the complement system, such as
C1 q. C1q is a subunit of the C1 enzyme complex that activates the
serum complement system.
[0101] In an alternative embodiment, the present invention also
contemplates the use of a first binding moiety that specifically
binds to an Fc receptor, in particular to an Fc gamma receptor
(Fc.gamma.R). The Fc.gamma.R may be a FcyRIII present on the
surface of natural killer (NK) cells or one of Fc.gamma.RI,
Fc.gamma.RIIA, Fc.gamma.RIIB1, Fc.gamma.RIIB2, and Fc.gamma.RIIIB
present on the surface of macrophages, monocytes, neutrophils
and/or dendritic cells.
[0102] In such embodiment, the first binding moiety particularly is
an Fc region or functional fragment thereof. In the present
context, a "functional fragment" refers to a fragment of an
antibody Fc region that is still capable of binding to an FcR, in
particular to an Fc.gamma.R, with sufficient specificity and
affinity to allow an Fc.gamma.R bearing effector cell, in
particular a macrophage, a monocyte, a neutrophil and/or a
dendritic cell, to kill the target cell by cytotoxic lysis or
phagocytosis. Particularly, a functional Fc fragment is capable of
competitively inhibiting the binding of the original, full-length
Fc portion to an FcR such as the activating FcyRl. Particularly, a
functional Fc fragment retains at least 30%, 40%, 50%, 60%, 70%,
80%, 90% or 95% of its affinity to an activating Fc.gamma.R.
[0103] Within such embodiment of the present invention, the Fc
region or functional fragment thereof is particularly an enhanced
Fc region or functional fragment thereof. The term "enhanced Fc
region", as used herein, refers to an Fc region that is modified to
enhance Fc receptor-mediated effector-functions, in particular
antibody-dependent cell-mediated cytotoxicity (ADCC),
complement-dependent cytotoxicity (CDC), and antibody-mediated
phagocytosis. This can be achieved as known in the art, for example
by altering the Fc region in a way that leads to an increased
affinity for an activating receptor (e.g. Fc.gamma.RIIIA (CD16A)
expressed on natural killer (NK) cells) and/or a decreased binding
to an inhibitory receptor (e.g. Fc.gamma.RIIB1/B2 (CD32B)).
[0104] Suitable alterations within the present invention include
altering glycosylation patterns, in particular afucosylation (also
referred to as "defucosylation"), mutations (point mutations,
deletions, insertions) and fusions with oligo- or polypeptides.
Known techniques for altering glycosylation patterns include
overexpression of heterologous
.beta.1,4-N-acetylglucosaminyltransferase III in the
antibody-producing cell (known as the Glycart-Roche technology) and
knocking out of the gene encoding .alpha.-1,6-fucosyltransferase
(FUT8) in the antibody-producing cell (the Potelligent technology
from Kyowa Hakko Kirin). Specific examples of enhancing mutations
in the Fc part include those described in Shields et al., J. Biol.
Chem. 276:6591-6604 (2001), which is incorporated herein in its
entirety.
[0105] In accordance with the present invention, the bispecific
construct is particularly designed in such a way that the killing
of IL23R-expressing target cells by Tc cells is highly efficient.
Such efficient killing generally involves the ability of the
bispecific construct to effectively redirect Tc cells to lyse
IL23R-expressing target cells. The term "efficient", as used
herein, means that the bispecific construct of the present
invention typically shows an in vitro EC.sub.50 ranging from 10 to
500 ng/ml, and is able to induce redirected lysis of about 50% of
the target cells through Tc cells at a ratio of Tc cells to target
cells of from 1:1 to 50:1, particularly from 1:1 to 15:1, more
particularly from 2:1 to 10:1. As used herein above and below, the
terms "about" and "approximately" refer to .+-.10% of the indicated
value or range.
[0106] Furthermore, the bispecific construct of the present
invention is particularly capable of cross-linking a stimulated or
an (otherwise) unstimulated Tc cell and the target cell in such a
way that the target cell is lysed. This offers the advantage that
no generation of target-specific T cell clones or common antigen
presentation by dendritic cells is required for the bispecific
construct to exert its desired activity. In fact, the bispecific
construct of the present invention is particularly capable of
redirecting Tc cells to lyse the target cells in the absence of
other activating signals. More particularly, if the first binding
moiety of the bispecific construct specifically binds to CD3,
particularly to CD3.epsilon., signaling through CD28 and/or IL-2 is
not required for redirecting Tc cells to lyse the target cells. The
high potential to activate non-target specific and/or unstimulated
Tc cells is considered to be an important feature of the bispecific
construct of the present invention and is believed to contribute to
the efficient killing of target cells.
[0107] The present invention further contemplates that the first
and second binding moieties are particularly arranged relative to
each other in such a manner that the bispecific construct
preferentially binds to the first antigen present on a Tc cell and,
simultaneously, to the second antigen (i.e., IL23R) present on the
target cell, but is essentially not capable of simultaneous binding
to a single cell that co-expresses both the first antigen and IL23R
(e.g. an IL-17 expressing cytotoxic (Tc17) cell).
[0108] Methods for measuring the preference of the bispecific
construct to simultaneously bind to two cells are within the normal
capabilities of a person skilled in the art. For example, the
bispecific construct of the present invention may be contacted with
a mixture of CD3.sup.+/IL23R.sup.- cells and CD3.sup.+/IL23R.sup.+
cells or with a mixture of CD3.sup.+/IL23R.sup.- cells and
CD3.sup.+/IL23R.sup.+ cells. The number of bispecific
construct-positive single cells and the number of cells crosslinked
by bispecific constructs may then be assessed by microscopy or
fluorescence-activated cell sorting (FACS) as known in the art.
About the same number of observed bispecific cross-linked cells in
both set-ups indicate that the bispecific construct of the present
invention does not, or does essentially not, bind to a single
target cell exhibiting both the CD3 and IL23R antigen on its
surface. Alternatively, the apparent binding activity avidity may
be determined. If simultaneous binding to a single target cell is
not, or is essentially not, possible, the apparent binding activity
of the bispecific construct for IL23R.sup.+/CD3.sup.- or
IL23R.sup.+/ CD3.sup.+ cells on one side and IL23R.sup.+/CD3.sup.+
cells on the other side will be about the same. If, however,
simultaneous binding to a single target cell is possible, the
apparent binding activity of the bispecific construct for
IL23R.sup.+/CD3.sup.+cells will be higher than that for
IL23R+/CD3.sup.- or IL23R7CD3.sup.+cells due to avidity
effects.
[0109] In a particular embodiment of the present invention, the
bispecific construct has a structure where the first and second
binding moieties are arranged relative to each other in such a
manner that the part of the first binding moiety recognizing the
first antigen and the part of the second binding moiety recognizing
the second antigen project, relative to the center of the
bispecific construct, outward in essentially opposite directions.
The term "center" particularly relates to the center of geometry
(COG). Without being bound by any theory, it is believed that this
structure of the bispecific construct of the present invention
avoids the undesired double-binding to a single target cell as
outlined above. The term "essentially", as used in the present
context, means that said part of the first binding moiety and said
part of the second moiety are arranged, relative to the center of
the bispecific construct, at an angle of 135.degree. or more up to
180.degree..
[0110] The structure of the bispecific construct is particularly
also characterized in that the angle a between a first vector v1
from the center of geometry (COG) of the entire bispecific
construct to the COG of the first paratope of the first binding
moiety and a second vector v2 from the COG of the entire bispecific
construct to the COG of the second paratope of the second binding
moiety is approximately within the range of
135.degree.<.alpha.<180.degree., more particularly within the
range of 150.degree.<.alpha.<180.degree., and most
particularly within the range of
160.degree.<.alpha.<180.degree.. The term "paratope", as used
herein, refers to the portions of the first and second binding
moieties that directly interact with the first and second antigens,
respectively.
[0111] In the context of the present invention, the first binding
moiety and/or the second binding moiety is an antibody-based
binding moiety, particularly an antibody-based binding moiety
comprising a heavy chain variable domain (VH) or binding fragment
thereof, more particularly an antibody-based binding moiety
comprising a heavy chain variable domain (VH) or binding fragment
thereof and a light chain variable domain (VL) or binding fragment
thereof. The term "binding fragment", as used herein, refers to a
portion of a given domain, region or part, which is (either alone
or in combination with another domain, region or part thereof)
still functional, i.e. capable of binding to the first or second
antigen recognized by the bispecific construct.
[0112] Typically, a binding fragment within the meaning of the
present invention retains at least 10% of its native antigen
binding activity (i.e. of its antigen binding activity as a
monospecific construct) when comprised in a bispecific construct of
the present invention. Particularly, a binding fragment retains at
least 25%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of its native
antigen binding activity, or maintains or even exceeds the full
native antigen binding activity, although any binding fragment with
sufficient affinity to exert the desired biological effect (i.e.
lysing/killing of target cells by Tc cells) will be useful. It is
also intended that a "binding fragment" within the meaning of the
present invention includes variants having conservative amino acid
substitutions that retain their binding activity to the extent
defined above and, particularly, do not substantially alter their
binding or biological activity.
[0113] In another particular embodiment of the present invention,
the bispecific construct is an antibody format selected from the
group consisting of a single-chain diabody (scDb), a tandem scDb
(Tandab), a linear dimeric scDb (LD-scDb), a circular dimeric scDb
(CD-scDb), a bispecific T-cell engager (BiTE; tandem di-scFv), a
tandem tri-scFv, a Tribody, a triabody, bispecific Fab2,
di-miniantibody, tetrabody, scFv-Fc-scFv fusion, di-diabody,
DVD-Ig, IgG-scFab, scFab-dsscFv, Fv2-Fc, IgG-scFv fusions, such as
bsAb (scFv linked to C-terminus of light chain), Bs1Ab (scFv linked
to N-terminus of light chain), Bs2Ab (scFv linked to N-terminus of
heavy chain), Bs3Ab (scFv linked to C-terminus of heavy chain),
Ts1Ab (scFv linked to N-terminus of both heavy chain and light
chain), Ts2Ab (dsscFv linked to C-terminus of heavy chain), and
Knob-into-Holes (KiHs) (bispecific IgGs prepared by the KiH
technology) and DuoBodies (bispecific IgGs prepared by the Duobody
technology). Particularly suitable for use herein is a single-chain
diabody (scDb), in particular a bispecific monomeric scDb.
[0114] The bispecific scDb, in particular the bispecific monomeric
scDb, particularly comprises two variable heavy chain domains (VH)
or fragments thereof and two variable light chain domains (VL) or
fragments thereof connected by linkers L1, L2 and L3 in the order
VHA-L1-VLB-L2-VHB-L3-VLA, VHA-L1-VHB-L2-VLB-L3-VLA,
VLA-L1-VLB-L2-VHB-L3-VHA, VLA-L1-VHB-L2-VLB-L3-VHA,
VHB-L1-VLA-L2-VHA-L3-VLB, VHB-L1-VHA-L2-VLA-L3-VLB,
VLB-L1-VLA-L2-VHA-L3-VHB or VLB-L1-VHA-L2-VLA-L3-VHB, wherein the
VLA and VHA domains jointly form the antigen binding site for the
first antigen, and VLB and VHB jointly form the antigen binding
site for IL23R.
[0115] The linker L1 particularly is a peptide of 2-10 amino acids,
more particularly 3-7 amino acids, and most particularly 5 amino
acids, and linker L3 particularly is a peptide of 1-10 amino acids,
more particularly 2-7 amino acids, and most particularly 5 amino
acids. The middle linker L2 particularly is a peptide of 10-40
amino acids, more particularly 15-30 amino acids, and most
particularly 20-25 amino acids.
[0116] In a particular embodiment of the present invention, the VH
domain of the first and second antibody-based binding moieties of
the bispecific construct comprises rabbit heavy chain
complementarity determining regions (CDRs) grafted onto human heavy
chain framework (FW) regions, and the VL domain of the first and
second antibody-based binding moieties of the bispecific construct
comprises rabbit light chain CDRs grafted onto human light chain FW
regions.
[0117] The heavy chain and light chain CDRs of the first
antibody-based binding moiety are particularly derived from a
rabbit antibody obtained by immunization of a rabbit with the
full-length epsilon chain of human CD3 the full-length, CD28 or the
full-length C1q. The immunization with the full-length chain of
CD3s, CD28 or C1q is suitably conducted by DNA immunization of a
rabbit with a plasmid encoding the full-length chain of human CD3e,
CD28 or C1 q, or, alternatively, with the purified extracellular
domain of the epsilon chain of CD3, or with the purified
extracellular chain of CD28, or with the purified C1 q. Further,
the heavy chain and light chain CDRs of the second antibody-based
binding moiety are particularly derived from a rabbit antibody
obtained by immunization of a rabbit either with the purified
extracellular domain of IL23R or with a plasmid expressing the
full-length IL23R.
[0118] The bispecific constructs of the present invention can be
produced using any convenient antibody manufacturing method known
in the art (see, e.g., Fischer, N. & Leger, O., Pathobiology
74:3-14 (2007) with regard to the production of bispecific
constructs; Hornig, N. & Farber-Schwarz, A., Methods Mol. Biol.
907:713-727, 2012, and WO 99/57150 A2 with regard to bispecific
diabodies and tandem scFvs). In addition, exemplary anti-IL23R and
anti-CD3.epsilon. antibody sequences are disclosed in EP 2 395 025
and EP 1 348 715, respectively. Specific examples of suitable
methods for the preparation of the bispecific construct of the
present invention further include, inter alia, the Genmab (see
Labrijn et al., Proc. Natl. Acad. Sci. USA 110:5145-5150 (2013))
and Merus (see de Kruif et al., Biotechnol. Bioeng. 106:741-750
(2010)) technologies. Methods for production of bispecific
antibodies comprising a functional antibody Fc part are also known
in the art (see, e.g., Zhu et al., Cancer Lett. 86:127-134 (1994));
and Suresh et al., Methods Enzymol. 121:210-228 (1986)).
[0119] These methods typically involve the generation of monoclonal
antibodies, for example by means of fusing myeloma cells with the
spleen cells from a mouse that has been immunized with the desired
antigen using the hybridoma technology (see, e.g., Yokoyama et al.,
Curr. Protoc. Immunol. Chapter 2, Unit 2.5, 2006) or by means of
recombinant antibody engineering (repertoire cloning or phage
display/yeast display) (see, e.g., Chames & Baty, FEMS
Microbiol. Letters 189:1-8 (2000)), and the combination of the
antigen-binding domains or fragments or parts thereof of two
different monoclonal antibodies to give a bispecific construct
using known molecular cloning techniques.
[0120] The bispecific constructs of the present invention are
particularly humanized in order to reduce immunogenicity and/or to
improve stability. Techniques for humanization of antibodies are
well-known in the art. For example, one technique is based on the
grafting of complementarity determining regions (CDRs) of a
xenogeneic antibody onto the variable light chain VL and variable
heavy chain VH of a human acceptor framework (see, e.g., Jones et
al., Nature 321:522-525 (1986); and Verhoeyen et al., Science
239:1534-1536 (1988)). In another technique, the framework of a
xenogeneic antibody is mutated towards a human framework. In both
cases, the retention of the functionality of the antigen-binding
portions is essential (Kabat et al., J. Immunol. 147:1709-1719
(1991)).
[0121] The bispecific constructs of the present invention may
alternatively comprise one or more binding moieties based on
non-antibody based binding domains. Specific examples of suitable
methods for the preparation of the bispecific construct of the
present invention further include, inter alia, the DARPin
technology (Molecular Partners AG), the adnexin technology
(Adnexus), the anticalin technology (Pieris), and the Fynomer
technology (Covagen AG).
[0122] In a particular embodiment of the present invention, the
bispecific construct is PRO165 (SEQ ID NO: 7), or a functionally
active variant of PRO165.
[0123] In the context of the present invention, the term
"functionally active variant of PRO165" refers to a bispecific
construct based on the VL and VH regions of PRO165, wherein (i)
said bispecific construct maintains the functional activity of
PRO165, i.e. wherein a first VH/VL pair specifically binds to CD3c,
and wherein a second VH/VL pair specifically binds to the IL-23
receptor specific subunit (IL23R), and wherein (ii) said VL and VH
regions comprise at least one sequence variation compared the VL
and VH regions of PRO165. In particular embodiments, the CDR
sequences of said functionally active variant are at least 70%,
particularly at least 80%, more particularly at least 90%
homologous to the CDR sequences of the VL and VH regions of PRO165.
Most particularly, the CDR sequences of said functionally active
variant are at least 90% identical and at least 95% homologous to
the CDR sequences of the VL and VH regions of PRO165. In particular
such embodiments, at least the CDR3 regions of the VH regions of
said functional variant are identical to the corresponding CDR3
regions of PRO165. In more particular embodiments, all CDR regions
of said functional variant are identical to the corresponding CDR
regions of PRO165.
[0124] In another aspect, the present invention relates to a
nucleic acid or multiple (i.e. more than one) nucleic acids
encoding the bispecific construct of the present invention. If the
bispecific construct is a single-chain construct, e.g. a
polypeptide or protein, a single nucleic acid codes for the
bispecific construct. However, if the bispecific construct
comprises two or more polypeptides, the bispecific construct of the
present invention may also be encoded by two or more separate
nucleic acids. The nucleic acid molecule(s) according to the
invention can be any nucleic acid molecule, particularly a DNA or
RNA molecule, for example cDNA or mRNA. They can be naturally
occurring molecules or produced through genetic engineering or
chemical synthesis. They may be single-stranded molecules, which
either contain the coding or the non-coding strand, or
double-stranded molecules.
[0125] In a particular embodiment of the present invention, said
nucleic acid or nucleic acids encode the bispecific construct
PRO165 (SEQ ID NO: 7), or a functionally active variant
thereof.
[0126] The nucleic acid(s) of the present invention may be produced
by any suitable method as known to those skilled in the art. The
nucleic acids of the present invention can, for example, be
synthesized by the phosphoramidite method or the like, or can be
produced by polymerase chain reaction (PCR) using specific primers.
Furthermore, methods for introducing a desired mutation into
certain nucleotide sequence, such as site-directed mutagenesis
techniques, are well-known to a person skilled in the art.
[0127] In a further aspect, the present invention relates to a
vector or multiple vectors comprising the nucleic acid(s) of the
present invention. When comprised within a vector, in particular a
plasmid, the nucleic acid(s) particularly is (are) DNA. The types
of vectors used in the present invention are not particularly
limited. For example, the vector may be a vector which replicates
autonomously, such as a plasmid, or may be a vector which is
integrated into the genome of a host cell when introduced into the
host cell and is replicated along with the chromosome.
Particularly, the vector used in the present invention is an
expression vector, in particular an expression plasmid. In an
expression vector, elements necessary for transcription, such as a
promoter, are operatively linked to the DNA nucleic acid(s) of the
present invention.
[0128] Examples of promoters which are operative in bacterial cells
include PR or PL promoters of phage lambda, lac, trp or tac
promoter of Escherichia coli, and the like. Examples of mammalian
promoters include SV40 promoter, MT-1 (metallothionein gene)
promoter, adenovirus 2 major late promoter, and the like.
Furthermore, exemplary promoters for use in insect cells include
polyhedrin promoter, P10 promoter, baculovirus immediate early gene
1 promoter, and the like. Moreover, suitable promoters for yeast
host cells include a promoter derived from yeast glycolysis system
genes, TPI1 promoter and the like. Other promoters suited for
different expression systems are known in the art.
[0129] Further, if necessary, the DNA of the present invention may
be operatively linked to a suitable terminator, such as a human
growth hormone terminator or a TPI1 ADH3 fungal host terminator.
The recombinant vector of the present invention may also have an
element such as a polyadenylation signal (e.g., derived from SV40),
a transcription enhancer sequence (e.g., a SV40 enhancer), or a
translation enhancer sequence (e.g., encoding adenovirus VA
RNA).
[0130] The recombinant vector of the present invention is also
typically provided with a DNA sequence which enables the vector to
replicate inside the host cell, and an example thereof for
mammalian cells is an SV40 origin of replication. Furthermore, the
recombinant vector of the present invention may also contain a
selectable marker. Examples of a selectable marker include, inter
alia, drug resistance genes such as ampicillin, kanamycin,
tetracycline, chloramphenicol, neomycin, and hygromycin. Methods
for connecting the nucleic acid(s) of the present invention with a
promoter and, as desired, other regulatory sequences such as a
terminator and/or a secretion signal sequence, and inserting these
into a suitable vector are known to those skilled in the art.
[0131] In a particular embodiment of the present invention, said
vector or vectors comprise a nucleic acid or nucleic acids, which
encode(s) the bispecific construct PRO165 (SEQ ID NO: 7), or a
functionally active variant thereof.
[0132] In yet another aspect, the present invention relates to a
host cell or multiple host cells that are not identical, comprising
the vector(s) of the present invention. The host cell(s) into which
the recombinant vector of the present invention is (are) introduced
is (are) not particularly limited and include any prokaryotic or
eukaryotic cell which can express the vector of the present
invention. Examples of suitable host cells include bacteria (e.g.,
Bacillus spp., Streptomyces spp., and Escherichia coil), mammalian
cells (e.g., HEK293, HeLa, COS, BHK, CHL, and CHO cells), insect
cells (e.g., baculovirus expression system), yeast cells
(Saccharomyces spp. or Schizosaccharomyces spp., in particular
Saccharomyces cerevisae and Saccharomyces kluyveri), and other
fungal cells (e.g., Aspergillus, Neurospora). Particularly, the
cells are bacterial cells, in particular Escherichia coli
cells.
[0133] A multiple polypeptide chain bispecific construct can be
made in a single host cell expression system wherein the host cell
produces each chain of bispecific construct and assembles the
polypeptide chains into a multimeric structure to form the
bispecific construct, followed by recovery of the bispecific
construct from the host cell. Alternatively, the separate
polypeptide chains of the desired bispecific construct can be made
in separate expression host cells, separately recovered from the
respective host cells, and then mixed in vitro under conditions
permitting the formation of the multi-subunit bispecific constructs
as known in the art.
[0134] Methods for introducing the vector of the present invention
into suitable host cells are known in the art and include the
protoplast method, the competent cell method (for bacterial host
cells), electroporation, the phosphate calcium method, lipofection
(for mammalian cells or for insect cells/baculovirus system),
electroporation, the spheroplast method, and the lithium acetate
method (for yeast and other fungal host cells).
[0135] In yet a further aspect, the present invention relates to a
method for producing the bispecific construct of the present
invention, comprising providing a host cell or host cells of the
present invention, culturing said host cell or said host cells and
collecting the bispecific construct from the cell culture. In the
culturing step, the host cell(s) of the present invention is (are)
cultured in a suitable culture medium under conditions permitting
expression of the bispecific construct of the present invention.
The medium used to culture the cells may be any conventional medium
suitable for growing the host cells, such as minimal or complex
media containing appropriate supplements. Alternatively, the
present invention relates to a method for producing the bispecific
construct of the present invention, comprising providing a nucleic
acid or nucleic acids according to the present invention, or a
vector or vectors according to the present invention, expressing
said nucleic acid or nucleic acids or said vector or vectors,
particularly in an in vitro transcription/translation system (see,
for example, Yin et al., Mabs 2012 Mar. 1;4(2) 217-25), and
collecting said bispecific construct from the expression
system.
[0136] In the step of collecting the bispecific construct from the
cell culture, the produced bispecific construct is recovered by
conventional methods for isolating and purifying a protein,
including separating the host cells from the medium by
centrifugation or filtration, precipitating the proteinaceous
components of the supernatant or filtrate by means of a salt, e.g.
ammonium sulphate, and purification by a variety of chromatographic
procedures, e.g. ion exchange chromatography, gel filtration
chromatography, affinity chromatography or the like. In a case
where the bispecific complex forms insoluble inclusion bodies, for
example when using E. coli as host cell, the inclusion bodies may
be first solubilized in denaturant, followed by a refolding step in
accordance with procedures well known in the art.
[0137] In still another aspect, the present invention relates to a
pharmaceutical composition comprising the bispecific construct of
the present invention and a pharmaceutically acceptable carrier. As
used herein, the term "pharmaceutically acceptable" refers to those
compounds or substances which are, within the scope of sound
medical judgment, suitable for contact with the tissues of mammals,
especially humans, without excessive toxicity, irritation, allergic
response and other problem complications. The term "carrier", as
used herein, relates to a diluent, adjuvant, excipient or vehicle
whereby the active ingredient is administered. Pharmaceutically
acceptable carriers for use herein can be, for example, sterile
liquids or dispersions. Particular carriers are those suited for
intravenous, subcutaneous or topical administration, including
sterile aqueous and non-aqueous solutions or suspensions for
parenteral administration, as discussed in Remington: The Science
and Practice of Pharmacy, 20th Edition (2000).
[0138] The pharmaceutical composition generally includes an
effective amount of the bispecific construct of the present
invention. Within the present invention, the term "effective
amount", with respect to the pharmaceutical composition or
otherwise, refers to the amount of a compound sufficient to effect
beneficial or desired therapeutic or prophylactic results.
Furthermore, as used herein, the term "treatment" includes both
therapeutic and prophylactic treatment and includes both in vivo
and ex vivo treatment. A therapeutically effective or
prophylactically effective treatment amount can be administered in
one or more administrations, applications or dosages and is not
intended to be limited to a particular formulation or
administration route. Also, the pharmaceutical composition may
include one or more additional active substances that are
co-administered with the bispecific construct of the present
invention. In addition, the pharmaceutical composition may contain
additional pharmaceutically acceptable substances, for example
pharmaceutical acceptable excipients such as solubilizing agents,
surfactants, tonicity modifiers and the like. As used herein, the
term "disease" also includes disorders and conditions.
[0139] Additionally, the embodiments herein directed to the
treatment of cancer, include treatment of cancer tumors and the
treatment of cancer metastasis.
[0140] Furthermore, the dosage form of the pharmaceutical
composition of the present invention is not particularly limited
but particularly is a parenteral formulation, such as an aqueous or
non-aqueous solution or dispersion for injection or infusion, or a
formulation suited for topical administration. Another particular
dosage form is a formulation containing the bispecific construct of
the present invention formulated in a controlled or sustained or
delayed release matrix. Further, the pharmaceutical composition may
also be contained in an implantable device that releases the
bispecific construct over time. As used herein, the unit "mcg"
refers to micrograms.
[0141] In still a further aspect, the present invention relates to
the use of the bispecific construct of the present invention in the
treatment of an inflammatory and/or autoimmune disease. In
particular, the present invention relates to a method for the
treatment of an inflammatory and/or autoimmune disease, comprising
administering to a subject, for example, a mammalian subject and
particularly a human patient, an effective amount of the bispecific
construct of the present invention. The meaning of the term
"effective amount" is as defined herein above. Typically, an
effective amount of the bispecific construct of the present
invention is administered in form of the above-described
pharmaceutical composition. Suitable administration routes include,
but are not limited to, topical and parenteral administration, in
particular inhalation, subcutaneous injection, intravenous
injection, and injection into the cerebrospinal fluid. The
administration regimen is not particularly limited and includes,
for example, bi-weekly, monthly, once every other month, once every
third, sixth or ninth month and once-a-year or single application
administration schemes.
[0142] The inflammatory and/or autoimmune disease may be rheumatoid
arthritis, ankylosing spondylitis, psoriasis, psoriatic arthritis,
ulcerative colitis, Crohn's disease, systemic lupus erythematosus,
juvenile diabetes, autoimmune uveitis, and multiple sclerosis,
Parkinson's disease, Alzheimer's disease, and ischemia-reperfusion
injury.
EXAMPLES
Example 1
Cloning of Bispecific Antibody Construct
[0143] A bispecific construct
V.sub.HA-L1-V.sub.LB-L2-V.sub.HB-L3-V.sub.LA (wherein the V.sub.LA
and V.sub.HA domains jointly form the antigen binding site for
CD3e, and V.sub.LB and V.sub.HB jointly form the antigen binding
site for IL23R) can be assembled by cloning V.sub.LA and V.sub.HA
from the humanized anti- CD3s antibody domain comprised in antibody
bscCDl9xCD3 (see EP 1 348 715 A2, FIG. 8, nucleotides 1258-1575,
and nucleotides 847-1203, respectively), and V.sub.LB and V.sub.HB
from the humanized anti-IL23R antibody 20D7 (see EP 2 395 025 A1,
SEQ ID NOs: 9 and 4, respectively) with linkers L1 and L3 each
consisting of the amino acid sequence GGGGS (G.sub.4S) and the
middle linker L2 consisting of the amino acid sequence
GGGGSGGGGSGGGGSGGGGS (G.sub.4S).sub.4 into an expression vector
suitable for expression in E. coli, including an N-terminal signal
sequence for secretion of the bispecific construct into the
periplasm of the E. coli host cells T, using the techniques
described in Holliger et al., Proc. Natl. Acad. Sci USA
90:6444-6448 (1993), Kipriyanov et al., J. Mol. Biol. 293:41-56
(1999) and Brusselbach et al., Tumor Targeting 4:115-123
(1999).
Example 2
Expression Level of IL23R on Cancer Cell-Lines
[0144] The data available in the public domain suggest that IL23R
may be upregulated in certain tumors. However, the available
information is not sufficient to validate IL23R as a suitable
marker to target cancer cells by a bispecific
anti-IL23RxCD3.epsilon. antibody format, as neither the absolute
expression level, nor the specificity of expression nor the
penetrance of IL23R upregulation across different tumor samples are
known. To further study the suitability of IL23R as a label for the
targeted lysis of tumor cells, we assessed the expression level of
IL23R both, on collection of primary cancer tissues, as well as on
a broad spectrum of tumor cell-lines. Surprisingly, IL23R mRNA
expression levels were elevated in most primary tissue samples from
colorectal cancers. Similarly, elevated IL23R mRNA expression was
found in all 129 CRC cell-lines studied. In agreement with these
findings, IL23R was highly upregulated in all six colorectal cancer
cell-lines studied as well as on the A549 lung adenocarcinoma
cell-line also on a protein level as assessed by flow-cytometry
(FIG. 2 and FIG. 3). Quantification of IL23R copies per cell
revealed an expression level of about 10'000-88'000 copies per CRC
cell (Table 1). Remarkably, we found strong expression of IL23R
protein also on cell-lines that were published to be negative for
the validated tumor markers CEA and EGFR, both targets against
which bispecific T cell redirecting antibody therapeutics are
currently in development (Osada, T. et al.2010. British Journal of
Cancer;101:124-133; Lutterbuese, R. et
al.2010.PNAS;107:12605-12610). Our data suggest that IL23R is a
tumor marker that is specifically upregulated in most colorectal
cancers, and that a bispecific anti-IL23RxCD3c antibody for the
targeted lysis of CRC cells has potential also in patients that are
refractory to anti-CEA or anti-EGFR therapies. Interestingly, we
found similar IL23R mRNA expression levels also in a variety of
leukemia and lymphoma cell-lines and confirmed elevated IL23R
protein expression in all cell lines tested (FIG. 4).
TABLE-US-00005 TABLE 1 IL23R molecules expressed on the surface of
cancer cell- lines. .DELTA.MFI, difference in mean fluorescence
intensity, and S/N, Signal to background ratio between anti-IL23R
antibody and isotype control. ABC, antigen binding capacity as
determined by regression to reference standard beads. sample
.DELTA.MFI * S/N * ABC * COLO 678 17476 172.33 88'302 DLD-1 13874
134.40 68'763 HCT 116 1539 9.79 10'553 HT-29 15540 65.21 74'865
LS-174T 8866 51.38 47'011 SW 480 18985 138.57 77'428 A549 1604
15.32 11'864 TF-1 12410 74.43 67'904 697 468 10.64 5'734 KIT225
1694 17.13 15'094 DB 563 3.51 5'677 SU-DHL-4 113 3.4 1'441
Example 3
Identification of Monoclonal Rabbit Antibodies Binding to Human
Interleukin (IL)-23 Receptor
[0145] A total of 475 monoclonal B-cells producing antibodies
specifically binding to interleukin-23 receptor were isolated from
immunized rabbits using flow-cytometry-based single-cell sorting,
the principles of which are well known to the expert and are for
example described by Lalor et al Eur J Immuno1.1992;22.3001-2011.
Monoclonal B cell culture supernatants were first subjected to
ELISA screening for binding to human IL23R ECD. In a second step,
cross-reactivity to IL23R from mouse and cynomolgus monkey was
assessed. Further, the affinity to human and cynomolgus IL23R was
assessed using the surface plasmon resonance (SPR) technology
(Table 2). The median equilibrium dissociation constant (KD) of
those 475 monoclonal antibodies to human IL23R was 6.3E-11M. Five
percent of these antibodies bound with an estimated KD below
4.3E-13M. A total of 92 clones selected either for their high
affinity or their cross-reactivity to cynomolgus and/or mouse, were
subjected to PCR amplification and sequencing of their variable
domains.
[0146] [000141] The sequencing of the obtained rabbit IgG clones
resulted in 68 complete sets of light and heavy chain variable
domains (VL and VH). These sets of rabbit variable domains were
analyzed by sequence alignment to identify unique clones and to
group the sequences into clusters based on sequence homology. This
alignment of the VL and VH domains was performed based on the joint
amino acid sequences of both domains. The analysis led to the
identification of 58 unique clones. In addition to the alignment of
the variable domains, the set of sequences of the six
complementarity determining regions (CDRs) of each rabbit IgG clone
were compared between different clones to identify unique sets of
CDRs. In total 58 unique sets of rabbit CDRs, corresponding to 58
independent clones, were identified. These 58 CDR sets were aligned
using the multiple alignment tool COBALT and a phylogenetic tree
was generated with the Neighbor Joining algorithm as shown in FIG.
5. Twenty three clones from different clusters were selected based
on their affinity for human, cynomolgus and mouse IL23R for
recombinant production and further characterization with the aim to
proceed with high sequence diversity.
Example 4
Heterologous Production of Monoclonal Rabbit Antibodies
[0147] Following the selection of the clones (described above)
rabbit antibodies were expressed and purified for further
characterization. The cloning of the corresponding light and heavy
chain variable domains entailed the in-vitro ligation of the DNA
fragments into a suitable mammalian expression vector. These
expression vectors contained consensus sequences for the constant
domains of the rabbit IgG light and heavy chains to allow for the
assembly and secretion of fully functional rabbit monoclonal IgGs
upon co-expression. Subsequent to the vector construction the
sequence of the resulting constructs was confirmed again and the
plasmid DNA was amplified and purified for mammalian cell
transfections. The expression vectors for the rabbit antibody heavy
and light chains were transfected into a mammalian suspension cell
line for transient heterologous expression by a lipid-based
transfection reagent. The conditions like the ratio of heavy to
light chain vector were optimized for robust expression levels of
secreted monoclonal IgG. The expression culture was cultivated for
7 days in a shaking incubator. At the end of the heterologous
expression period the cell culture supernatant was harvested by
centrifugation and decanting. Subsequently the secreted rabbit IgGs
were affinity purified by Protein A beads. The IgG loaded beads
were washed and the purified antibodies were eluted by a pH shift.
The elution fractions were analyzed by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE), UV absorbance at 280
nm and size-exclusion high performance liquid chromatography
(SE-HPLC) to verify identity, content and purity.
Example 5
Characterization of Purified Monoclonal Rabbit Anti-IL23R
Antibodies
[0148] The affinity of the 23 purified rabbit monoclonal antibodies
towards IL23R from human, cynomolgus monkey and mouse origin was
determined by SPR measurements. The respective results are given in
Table 2 below:
TABLE-US-00006 TABLE 2 Human IL23R Cyno IL23R Mouse IL23R Clone ID
k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d
K.sub.D IgG [M.sup.-1 s.sup.-1] [s.sup.-1] [M] [M.sup.-1 s.sup.-1]
[s.sup.-1] [M] [M.sup.-1 s.sup.-1] [s.sup.-1] [M] NB NB NB ND ND ND
NB NB NB 12-01-F09 1.80E+05 9.12E-05 5.07E-10 1.23E+05 5.37E-03
4.38E-08 NB NB NB 12-02-E05 2.15E+05 <1.0E-06 <4.66E-12
1.03E+05 1.93E-04 1.89E-09 NB NB NB 12-04-H09 1.74E+05 7.59E-05
4.37E-10 4.63E+05 7.71E-03 1.66E-08 NB NB NB 12-06-A05 1.53E+05
1.10E-05 7.17E-11 1.88E+04 8.23E-05 4.39E-09 5.48E+04 4.23E-05
7.72E-10 12-06-E01 1.18E+05 5.66E-05 4.82E-10 7.99E+04 1.14E-03
1.43E-08 1.80E+05 3.64E-03 2.02E-08 12-06-E03 6.58E+04 2.32E-05
3.53E-10 9.57E+04 7.31E-05 7.64E-10 8.91E+04 1.20E-03 1.35E-08
12-06-F06 2.83E+05 1.61E-05 5.70E-11 1.98E+05 5.98E-03 3.02E-08
2.67E+05 2.48E-03 9.31E-09 12-08-E06 9.72E+04 5.44E-05 5.60E-10
6.00E+04 2.49E-05 4.15E-10 NB NB NB 12-09-F05 1.33E+05 4.00E-06
3.00E-11 1.30E+05 4.54E-05 3.49E-10 8.46E+04 1.42E-03 1.68E-08
12-10-A06 NB NB NB ND ND ND NB NB NB 12-19-F08 2.29E+05 2.50E-05
1.10E-10 2.22E+05 1.33E-04 6.00E-10 2.45E+05 3.34E-04 1.36E-09
13-05-H06 NB NB NB NB NB NB NB NB NB 13-10-A03 NB NB NB NB NB NB NB
NB NB 14-03-B01 2.79E+05 <1E-06 <3.58E-12 3.31E+05 4.82E-05
1.46E-10 NB NB NB 14-05-A11 2.26E+05 <1E-06 <4.43E-12
2.52E+05 8.46E-05 3.36E-10 NB NB NB 14-06-H08 3.34E+05 9.92E-06
2.97E-11 4.58E+05 3.17E-06 6.92E-12 NB NB NB 14-07-H07 2.50E+05
1.59E-05 6.33E-11 2.70E+05 3.00E-05 1.11E-10 NB NB NB 14-08-E05
2.17E+05 5.98E-05 2.76E-10 2.45E+05 3.79E-05 1.55E-10 NB NB NB
14-11-D07 4.14E+05 1.98E-07 4.78E-13 4.48E+05 <1E-06
<2.23E-12 2.23E+05 3.34E-04 1.50E-09 14-13-D08 2.06E+05
<1E-06 <4.85E-12 3.07E+05 2.77E-05 9.02E-11 NB NB NB
14-14-E08 5.19E+05 <1E-06 <1.93E-12 5.12E+05 1.58E-05
3.08E-11 NB NB NB 14-17-B09 6.77E+05 7.49E-05 1.11E-10 3.53E+05
1.16E-04 3.30E-10 8.08E+04 <1E-06 <1.24E-11
Example 6
Engineering and Characterization of a Humanized Single-Chain Fv
Fragment
[0149] The humanization of rabbit antibody clone 14-11-D07
comprised the transfer of the rabbit CDRs onto Numab's proprietary
scFv acceptor framework of the VK1/VH3 type. In this process the
amino acid sequence of the six CDR regions was identified on the
rabbit antibody donor sequence as described elsewhere (Borras, L.
et al. 2010. JBC;285:9054-9066) and grafted into the Numab acceptor
scaffold sequence. Two variants were generated, where the variant
sc01 resulted from exclusively engrafting complementarity
determining regions (CDRs) onto the human acceptor variable domain
scaffold, while variant sc02 contains further mutations also in the
human framework sequence. The two resulting humanized scFvs were
characterized for their binding affinity towards IL23R from human,
cynomolgus and mouse origin (see Table 3). As no significant
difference in affinity was detectable for the two variants,
14-11-D07-sc01 was chosen to be engineered in the scDb format
because in this variant no rabbit amino acids were engrafted from
the rabbit donor framework sequences to the human acceptor
sequence, thus minimizing the risk to provoke anti-drug immune
response in humans following application.
TABLE-US-00007 TABLE 3 human IL23R cyno IL23R mouse IL23R Clone ID
k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d
K.sub.D scFv [M.sup.-1 s.sup.-1] [s.sup.-1] [M] [M.sup.-1 s.sup.-1]
[s.sup.-1] [M] [M.sup.-1 s.sup.-1] [s.sup.-1] [M] 14-11-D07-
2.24E+06 2.59E-04 1.16E-10 2.92E+06 3.61E-04 1.24E-10 1.89E+06
4.18E-03 2.21E-09 sc01 14-11-D07- 1.17E+06 1.13E-04 9.60E-11
1.55E+06 2.05E-04 1.33E-10 8.30E+05 1.39E-03 1.68E-09 sc02
Example 7
Engineering and Characterization of a Bispecific Single-Chain
Diabodies (scDb)
[0150] With the aim to redirect CD3E+ T cells to lyse IL23R
expressing target cells, a bispecific antibody fragment of the
so-called single-chain diabody (scDb) format was engineered. This
construct termed PRO165 contains the VH and VL of the anti-IL23R
scFv 14-11-D07-sc01 , as well as the VH and VL of a humanized
rabbit anti-CD3E antibody (clone 6). The anti-CD3E binding domain
was selected a) for its high affinity to human CD3E, b) for its
excellent cross-reactivity to CD3E from cynomolgus origin, c) for
its outstanding stability and resistance towards aggregation, and
d) because this CD3.epsilon. binder activates T cells exclusively
upon cross-linking--as it may for example occur following binding
to target cells--thereby minimizing the risk for potential
side-effects due to unspecific activation of T cells.
[0151] Affinities of the anti-IL23R and anti-CD3.epsilon. binding
moieties towards human, cyno and mouse IL23R, and human and mouse
CD3.epsilon., respectively were measured by SPR (Table 4).
TABLE-US-00008 TABLE 4 human IL23R human CD3.epsilon. k.sub.d
K.sub.D k.sub.d K.sub.D scDb ID [s.sup.-1] K.sub.D [M] [M] k.sub.a
[M.sup.-1 s.sup.-1] [s.sup.-1] [M] PRO165 1.26E-03 1.16E-08
1.82E-10 1.09E+05 1.26E-03 1.16E-08
[0152] The midpoint of transition for the thermal unfolding of the
tested constructs was determined by Differential Scanning
Fluorimetry (DSF), essentially as described by Niesen (Niesen et
al., Nat Protoc. 2 (2007) 2212-21). The DSF assay is performed in a
qPCR machine (e.g. MX3005p, Agilent Technologies). The samples were
diluted in buffer (citrate-phosphate pH 6.4, 0.25 M NaCl)
containing a final concentration of 5.times. SYPRO orange in a
total volume of 25 .mu.L. Samples were measured in duplicate and a
temperature ramp from 25-96.degree. C. programmed. The fluorescence
signal was acquired and the raw data was analyzed with the GraphPad
Prism (GraphPad Software Inc.; results see Table 5).
TABLE-US-00009 TABLE 5 The midpoint of transition for the thermal
unfolding was determined for all constructs by differential
scanning fluorimetry Thermal Unfolding scDb ID anti-IL23R anti-CD3
Tm PRO165 14-11-D07-sc01 clone 6 65.3
Example 8
Targeted Lysis of IL23R Expressing Cells
[0153] To study potency of the scDbs to induce specific lysis of
target cells, IL23R expressing cells were co-cultivated with human
CD8+ T cells in presence of increasing concentrations of the
respective scDb. Lysis of cells in dependence of the scDb
concentration was assessed by measuring fluorescence intensity of
celltox-green intercalated into DNA. The EC50 of PRO165 for the
respective cell-line is given in Table 6.
TABLE-US-00010 TABLE 6 EC.sub.50 of specific IL23R binding target
cell lysis [nM] ID Format moiety DLD-1 SW-480 TF-1 CHO PRO165 scDb
14-11-D07- 45.4 71.7 39.8 no lysis sc01
Methods
1. Assessment of IL23R Expression Levels on Cell Lines
[0154] For the detection of IL23R on the cell membrane, living
cells were stained with 5 .mu.g/mL of a biotinylated polyclonal
goat anti-IL23R antibody (R&D Systems, Cat. No.BAF1400). As
background control, a polyclonal goat IgG isotype was used (R&D
Systems, Cat. No.BAF108). Binding of goat polyclonal antibodies to
cells was in turn detected by a Phycoerythrin-(PE)-labeled
streptavidin (Southern Biotech, Cat. No. 7100-09S). PE fluorescence
intensity of stained cells was measured by flow-cytometry (FACS
aria III, Becton Dickinson). In order to quantify IL23R molecules
per cell anti-human IgG Quantum.TM. Simply Cellular.RTM. (QSC)
microspheres (Bangs Laboratories, Cat. No. 816) loaded with human
IL-23R Fc chimera (R&D Systems, Cat. No.1400-IR-050) were used
as a reference standard. The mean fluorescence intensity (MFI),
reflecting the signal intensity at the geometric mean, was measured
for both, the goat IgG isotype control as well as for the goat
anti-IL23 R antibody. The difference between the MFI of the
specific antibody and isotype control antibody (AMFI) was
calculated. The normalized MFI was calculated by dividing the MFI
obtained with the anti-IL23R antibody by the MFI of the idiotype
control. To quantify IL23R molecules on the cell surface the
specific Antibody Binding Capacity (ABC) of cells was calculated by
regression of the respective MFI to the standard curve generated by
use of Quantum.TM. Simply Cellular.RTM. (QSC) microspheres.
Calculations were performed in the lot-specific QuickCal.RTM.
template. To compare unspecific binding the ABC value of cells
stained with negative control antibody were subtracted from ABC
value of cells stained with the specific antibody.
2. SPR Assay for Determination of Binding Kinetics and Species
Cross-Reactivity of Monoclonal Antibodies in Culture
Supernatant
[0155] Binding affinities of monoclonal rabbit anti-IL23R
antibodies in sort supernatants were measured by surface plasmon
resonance (SPR) using a MASS-1 SPR instrument (Sierra Sensors). For
affinity measurements (run in DPBS with 0.05% Tween) an antibody
specific for the Fc region of rabbit IgGs (Bethyl Laboratories,
Cat. No. A120-111A) was immobilized on a sensor chip (SPR-2
Affinity Sensor, Amine, Sierra Sensors) using a standard
amine-coupling procedure. After immobilization, rabbit monoclonal
antibodies in culture supernatants were captured by the anti-rabbit
IgG antibody while a second immobilized channel served as a control
where the capture was replaced by negative supernatant (cultured
media that does not contain sorted cells). Human IL23R
extracellular domain (ECD) (produced on request by Trenzyme,
Germany) at 90 nM was injected into both flow cells for 3 min and
dissociation of the protein from the captured IgG on the sensor
chip was allowed to proceed for 5 min. In a similar manner the
affinities for cynomolgus and mouse IL23R ECD (both at 90 nM,
produced on request by Trenzyme) were measured. The apparent
dissociation (kd) and association (ka) rate constants and the
apparent dissociation equilibrium constant (KD) were calculated
with the MASS-1 analysis software (Analyzer, Sierra Sensors) using
one-to-one Langmuir binding model.
3. SPR Assay for Determination of Binding Kinetics and Species
Cross-Reactivity of Purified Monoclonal Antibodies
[0156] Binding affinities of purified monoclonal rabbit anti-IL23R
antibodies were measured in a similar setup as the sort
supernatants, except that the sort supernatants were replaced by
purified IgGs. A sensor chip (SPR-2 Affinity Sensor, High Capacity
Amine, Sierra Sensors) was immobilized by the same procedure, and
purified monoclonal antibodies at a concentration of 0.5 .mu.g/ml
(diluted in HEPES running buffer: 0.01 M HEPES, 0.15 M NaCl, 0.05%
Tween) were captured by the anti-rabbit IgG antibody. No capture
was done on the immobilized control channel. Two-fold serial
dilutions of human IL23R extracellular domain ranging from 90 to
2.81 nM were injected into the flow cells for 3 min and
dissociation of the protein from the captured IgG on the sensor
chip was allowed to proceed for 5 min. After each injection cycle,
surfaces were regenerated with a single 1 min injection of 10 mM
glycine-HCl pH 1.5. In a similar manner the affinities for
cynomolgus and mouse IL23R ECD were measured. The apparent
dissociation (kd) and association (ka) rate constants and the
apparent dissociation equilibrium constant (KD) were calculated
with the MASS-1 analysis software (Analyzer, Sierra Sensors) using
one-to-one Langmuir binding model.
4. SPR Assay for Determination of Binding Kinetics and Species
Cross-Reactivity of Anti-IL23R scFvs
[0157] Binding affinities of anti-IL23R scFvs were measured by
surface plasmon resonance (SPR) using a MASS-1 SPR instrument
(Sierra Sensors). For affinity measurements (done in HEPES running
buffer: 0.01 M HEPES, 0.15 M NaCl, 0.05% Tween) an antibody
specific for the Fc region of human IgGs (Bethyl Laboratories, Cat.
No. A80-104A) was immobilized on a sensor chip (SPR-2 Affinity
Sensor, High Capacity Amine, Sierra Sensors) using a standard
amine-coupling procedure. After immobilization, 1 ug/ml recombinant
human IL23R Fc chimera (R&D Systems, Cat. No. 1400-IR-050) was
captured by the anti-human IgG antibody while a second immobilized
channel served as a control where the capture was replaced by
buffer. Two-fold serial dilutions of human scFvs ranging from 180
to 2.81 nM were injected into both flow cells for 3 min and
dissociation of the scFvs from the chimera on the sensor chip was
allowed to proceed for 700 sec. After each injection cycle,
surfaces were regenerated with two 1 min injections of 10 mM
glycine-HCI pH 1.5. In a similar manner the affinities for
cynomolgus and mouse IL23R Fc chimera (R&D Systems, Cat. No.
1686-MR-050) was measured. The apparent dissociation (kd) and
association (ka) rate constants and the apparent dissociation
equilibrium constant (KD) were calculated with the MASS-1 analysis
software (Analyzer, Sierra Sensors) using one-to-one Langmuir
binding model.
5. SPR Assay for Determination of Binding Kinetics and Species
Cross-Reactivity of Anti-CD3.times.IL23R scDbs
[0158] Binding affinities of anti-CD3.times.IL23R scDbs were
measured by surface plasmon resonance (SPR) using a MASS-1 SPR
instrument (Sierra Sensors). For affinity measurements (done in
HEPES running buffer: 0.01 M HEPES, 0.15 M NaCl, 0.05% Tween) to
CD3, human heterodimeric single-chain CD3.gamma..delta.
extracellular domain (produced in-house) was immobilized on a
sensor chip (SPR-2 Affinity Sensor High Capacity Amine, Sierra
Sensors) using a standard amine-coupling procedure. Three-fold
serial dilutions of scDbs ranging from 90 to 0.123 nM were injected
into the flow cells for 3 min and dissociation of the protein from
the immobilized CD3.gamma..delta. on the sensor chip was allowed to
proceed for 700 sec. After each injection cycle, surfaces were
regenerated with a 1 min injection of 10 mM Glycine-HCl pH 2.0. In
a similar manner the binding to cynomolgus CD3.gamma..delta.
(produced in-house) and mouse CD36E (Sino Biologicals Inc., Cat.
No. CT033-M2508H) was measured.
[0159] [000154] For affinity measurements to IL23R, human IL23R
extracellular domain (produced on request by Trenzyme) was
immobilized on a sensor chip (SPR-2 Affinity Sensor High Capacity
Amine, Sierra Sensors) using a standard amine-coupling procedure.
Two-fold serial dilutions of scDbs ranging from 90 to 5.6 nM were
injected into the flow cells for 3 min and dissociation of the
protein from the immobilized IL23R on the sensor chip was allowed
to proceed for 720 sec. After each injection cycle, surfaces were
regenerated with a 1 min injection of 10 mM Glycine-HCl pH 2.0. The
apparent dissociation (kd) and association (ka) rate constants and
the apparent dissociation equilibrium constant (KD) are calculated
with the MASS-1 analysis software (Analyzer, Sierra Sensors) using
one-to-one Langmuir binding model.
6. scDb Mediated Lysis of IL-23R Expressing Cells by Cytotoxic T
Cells
[0160] For the assessment of the potential of bispecific
anti-CD3.times.IL-23R scDbs to induce target cell lysis human
IL-23R expressing cell lines were used. Colon carcinoma DLD-1 or
SW480, and erythroleukemia TF-1 were used as target cell lines
Unstimulated human CD8+ T-cells isolated as described above were
used as effector cells. Target cells were labeled with cell tox
green dye (Promega) according to the manufacturer's instructions.
Cell lysis was monitored by the CellTox.TM. green cytotoxicity
assay (Promega). The assay measures changes in membrane integrity
that occur as a result of cell death. The assay uses an asymmetric
cyanine dye that is excluded from viable cells but preferentially
stains the dead cell DNA. When the dye binds DNA in compromised
cells, its fluorescence properties are substantially enhanced.
Viable cells produce no appreciable increases in fluorescence.
Therefore, the fluorescence signal produced by the binding
interaction with dead cell DNA is proportional to cytotoxicity.
Similarly as described above, labeled IL-23R cells (5'000
cells/well) were incubated with CD8+ cytotoxic T-cells at an
effector:target ratio of 40:1 in presence of 3-fold serially
diluted scDbs (starting concentration 180 nM) in 96 well microtiter
plates. To assess unspecific lysis of cells that do not express the
target, T-cells were co-incubated with labeled wild-type CHO cells.
Fluorescence intensity was analyzed after 48 h of incubation using
a multi-mode microplate reader (FlexStation 3, Molecular Devices).
Data were analyzed using a four-parameter logistic curve fit using
the SoftMax Pro data analysis Software (Molecular Devices), and the
molar concentration of scDb required to induce half maximal target
cell lysis (EC.sub.50) was derived from dose-response curves.
[0161] In order to specifically track the fate of target cells,
target cells were stained with CellVue.RTM. Claret Far Red
Fluorescent (Sigma-Aldrich, Cat.No.MINCLARET-1 KT) in a
concentration of 5nM Dye according to the manufacturer's
instructions. Apoptosis/necrosis was monitored using the Annexin V
Apoptosis Detection Kit FITC (eBioscience, Cat.No.88-8005-74)
according to the manufacturer's instructions (concentration of
dyes: 100 .mu.L/mL Annexin V and 2 .mu.g/mL Propidium iodide).
Stained IL-23R cells (5'000 cells/well) were incubated with CD8+
cytotoxic T-cells at an effector:target ratio of 40:1 in presence
of serially diluted scDbs (starting concentration 180 nM) in 96
well microtiter plates. To assess unspecific lysis of cells that do
not express the target, T-cells were co-incubated with labeled CHO
cells. Fluorescence intensities were analyzed after 48 h of
incubation using a flow cytometer (FACS aria III, Becton
Dickinson). Data were analyzed by discriminate target from effector
cells using the far red fluorescent dye (CeIIVue.RTM. Claret Far
Red Fluorescent). Target cells were gated into live cells and
early-/late-stage apoptotic, necrotic cells by Annexin V and PI
fluorescence. The data obtained were analyzed using a
four-parameter logistic curve fit using the SoftMax.RTM. Pro data
analysis Software (Molecular Devices), and the molar concentration
of scDb required to induce half maximal target cell lysis (EC50)
was derived from dose-response curves.
7. Construct Design and Manufacturing of scDb Constructs
[0162] The single-chain diabody constructs were designed by
arranging the variable domains in a VLA-L1-VHB-L2-VLB-L3-VHA
configuration. In these constructs the VLA and VHA domains jointly
form the binding site for IL23R while the VLB and VHB domains
jointly form the binding site for CD3c. The peptide linkers L1-L3
connecting the variable domains are constructed of the
glycine/serine repeats. The two short linkers L1 and L3 are
composed of a single G4S repeat, whereas the long linker L2 is
composed of the sequence (G4S)4. The nucleotide sequences encoding
the various anti-IL23R.times.CDE3c scDb constructs were de novo
synthesized and cloned into an adapted vector for E.coli expression
that is based on a pET26b(+) backbone (Novagen).
[0163] The expression construct was transformed into the E. coli
strain BL12 (DE3) (Novagen) and the cells were cultivated in 2YT
medium (Sambrook, J., et al., Molecular Cloning: A Laboratory
Manual) as a starting culture. Expression cultures were inoculated
and incubated in shake flasks at 37.degree. C. and 200 rpm. Once an
OD600 nm of 1 was reached protein expression was induced by the
addition of IPTG at a final concentration of 0.5 mM. After
overnight expression the cells were harvested by centrifugation at
4000 g. For the preparation of inclusion bodies the cell pellet was
resuspended in IB Resuspension Buffer (50 mM Tris-HCl pH 7.5, 100
mM NaCl, 5 mM EDTA, 0.5% Triton X-100). The cell slurry was
supplemented with 1 mM DTT, 0.1 mg/mL Lysozyme, 10 mM Leupeptin,
100 .mu.M PMSF and 1 .mu.M Pepstatin. Cells were lysed by 3 cycles
of ultrasonic homogenization while being cooled on ice.
Subsequently 0.01 mg/mL DNAse was added and the homogenate was
incubated at room temperature for 20 min. The inclusion bodies were
sedimented by centrifugation at 15000 g and 4.degree. C. The IBs
were resuspended in IB resuspension Buffer and homogenized by
sonication before another centrifugation. In total a minimum of 3
washing steps with IB Resuspension Buffer were performed and
subsequently 2 washes with IB Wash Buffer (50 mM Tris-HCl pH 7.5,
100 mM NaCl, 5 mM EDTA) were performed to yield the final IBs.
[0164] For protein refolding the isolated lBs were resuspended in
Solubilization Buffer (100 mM Tris/HCl pH 8.0, 6 M Gdn-HCl, 2 mM
EDTA) in a ratio of 5 mL per g of wet IBs. The solubilization
mixture was incubated for 30 min at room temperature until DTT was
added at a final concentration of 20 mM and the incubation was
continued for another 30 min. After the solubilization was
completed the solution was cleared by 10 min centrifugation at
21500 g and 4.degree. C. The refolding was performed by rapid
dilution at a final protein concentration of 0.3 g/L of the
solubilized protein in Refolding Buffer (typically: 100 mM Tris-HCl
pH 8.0, 5.0 M Urea, 5 mM Cysteine,1 mM Cystine). The refolding
reaction was routinely incubated for a minimum of 14 h. The
resulting protein solution was cleared by 10 min centrifugation at
8500 g and 4.degree. C. The refolded protein was purified by
affinity chromatography on Capto L resin (GE Healthcare) followed
by a size-exclusion chromatography on a Superdex 75 column (GE
Healthcare). The proteins were formulated in native buffer (50 mM
Citrate-Phosphate pH 6.4, 150 mM NaCl). The isolated monomer
fraction was analyzed by size-exclusion HPLC, SDS-PAGE for purity
and UV/Vis spectroscopy for protein content.
Example 9
Engineering and Characterization of a Bispecific Single-Chain
Diabody Surrogate (scDb)
[0165] With the aim to redirect murine CD3E+ T cells to lyse IL23R
expressing target cells, a bispecific antibody fragment of the
single-chain diabody (scDb) format was engineered. This construct
termed PRO387 contains the VH and VL of the anti-IL23R scFv
14-11-D07-sc01, as well as the VH and VL of a hamster anti-CD3E
antibody (2C11). The mouse reactive 2C11 has been described in the
literature (Leo O, et al. 1987. P. Natl. Acad. Sci. USA 84:1374)
and has been extensively characterized so that structural and
sequence information is available (Fernandes R A, et al. 2012.
J.Biol.Chem. 287: 13324-13335).
[0166] Affinities of the anti-IL23R and anti-CD3E binding moieties
towards mouse IL23R, and mouse CD3E, respectively were measured by
SPR (Table 8).
TABLE-US-00011 TABLE 7 mouse IL23R mouse CD3 k.sub.d K.sub.D
K.sub.D k.sub.d K.sub.D scDb ID [s.sup.-1] [M] [M] k.sub.a
[M.sup.-1 s.sup.-1] [s.sup.-1] [M] PRO387 7.01E+05 3.26E-03
4.66E-09 2.33E+05 2.13E-03 9.11E-09
Example 10
Engineering and Characterization of a Bispecific, Trivalent
Fab-scFv Fusion Surrogate Molecule and Anti-mCD3 Fab
[0167] With the aim to redirect murine CD3E+ T cells to lyse IL23R
expressing target cells, a bispecific antibody fragment, a Fab-scFv
fusion (EP1049787 B1) format, was engineered. This heterodimeric
construct termed PRO386 contains the Fab fragment of the mouse
reactive 2C11 (VL-CL & VH-CH1), both chains are C-terminally
fused to an anti-IL23R scFv 14-11-D07-sc01 via a flexible
linker.
[0168] In addition to the bispecific molecule a mouse reactive
anti-CD3 Fab (PRO400) was constructed devoid of the fused scFv
modules.
[0169] Affinities of the anti-IL23R and anti-CD3E binding moieties
towards mouse IL23R, and mouse CD3E, respectively were measured by
SPR (Table 8).
TABLE-US-00012 TABLE 8 mouse IL23R mouse CD3 k.sub.d K.sub.D
K.sub.D k.sub.d K.sub.D scDb ID [s.sup.-1] [M] [M] k.sub.a
[M.sup.-1 s.sup.-1] [s.sup.-1] [M] PRO386 9.15E+05 5.33E-04
5.83E-10 1.71E+05 1.96E-03 1.15E-08 PRO400 -- -- -- 2.33E+05
2.66E-03 1.14E-08
Methods
[0170] 1. Construct Design and Manufacturing of Surrogate scDb
Construct
[0171] The single-chain diabody construct was designed by arranging
the variable domains in a VLA-L1-VHB-L2-VLB-L3-VHA configuration.
In these constructs the VLA and VHA domains jointly form the
binding site for IL23R while the VLB and VHB domains jointly form
the binding site for CD3E. The peptide linkers L1-L3 connecting the
variable domains are constructed of the glycine/serine repeats. The
two short linkers L1 and L3 are composed of a single G4S repeat,
whereas the long linker L2 is composed of the sequence (G4S)4. The
nucleotide sequences encoding the anti-IL23R x CDE3E scDb construct
was de novo synthesized and cloned into an adapted vector for
mammalian expression that is based on a pcDNA3.1 backbone
(Invitrogen) with an IL-2 signal sequence preceding the open
reading frame. The transient expression of the functional scDb was
performed with the FreeStyle.TM. MAX system in CHO S cells
(Invitrogen). After cultivation for several days the supernatant of
the expression culture was recovered for purification.
[0172] [000166] The protein was purified by affinity chromatography
on Capto L resin (GE Healthcare) optionally followed by a
size-exclusion chromatography on a Superdex 75 column (GE
Healthcare). The proteins were formulated in PBS buffer (Lonza, REF
BE17-517Q). The isolated monomer fraction was analyzed by
size-exclusion HPLC, SDS-PAGE for purity and UV/Vis spectroscopy
for protein content.
2. Construct Design and Manufacturing of Surrogate Fab-scFv Fusion
Construct and Anti-mCD3 Fab
[0173] The heterodimeric Fab-scFv construct was designed by
preparing to mammalian expression constructs for a co-transfection
in suitable host cells. The configuration of these protein chains
are VLA-CL-L4-VLB-L2-VHB and VHA-CH1-L4-VLB-L2-VHB, respectively.
In these constructs the domains VLA and VHA correspond to the
native amino acid sequence from the 2C11 hamster antibody that have
been combined with human CL and CH1 sequences to form a chimeric
Fab fragment. The scFv modules VLB-L2-VHB from the anti-IL23R
14-11-D07-sc01 have been fused to the C-terminus of the constant
domains via an intermediate linker L4 composed of the sequence
(G4S)2. In addition to the bispecific construct also the chimeric
Fab fragment of the 2C11 was constructed without the fusion of the
scFv modules.
[0174] The nucleotide sequences encoding the constructs were de
novo synthesized and cloned into an adapted vector for mammalian
expression that is based on a pcDNA3.1 backbone (Invitrogen) with
an IL-2 signal sequence preceding the open reading frame. The
transient expression of the functional scDb was performed with the
FreeStyle.TM. MAX system in CHO S cells (Invitrogen). After
cultivation for several days the supernatant of the expression
culture was recovered for purification.
[0175] The protein was purified by affinity chromatography on Kappa
Select resin (GE Healthcare) optionally followed by a
size-exclusion chromatography on a Superdex 75 column (GE
Healthcare). The proteins were formulated in PBS buffer (Lonza, REF
BE17-517Q). The isolated monomer fraction was analyzed by
size-exclusion HPLC, SDS-PAGE for purity and UV/Vis spectroscopy
for protein content.
TABLE-US-00013 TABLE 9 Sequences Sequences (including PRO165:
VLA-L1-VHB-L2-VLB-L3-VHA) SEQ ID Type Sequence 1 Linker L1 GGGGS 2
Linker L2 GGGGS GGGGS GGGGS GGGGS Linker L3 GGGGS 3 VL
DIQMTQSPSSLSASVGDRVTITCQASENIYSFLA anti-IL23R
WYQQKPGKAPKLLIYSASKLAAGVPSRFSGSGS 14-11-D07
GTDFTLTISSLQPEDFATYYCQQTNRYSNPDIYN sc01 VFGQGTKLTVLG 4 VH
EVQLVESGGGLVQPGGSLRLSCAASGIDFNSNY anti-IL23R
YMCWVRQAPGKGLEWIGCIYVGSHVNTYYANW 14-11-D07-
AKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYC sc01 ATSGSSVLYFKFWGQGTLVTVSS 5 VL
DIQMTQSPSSLSASVGDRVTITCQSSESVYNNKR anti-CD3
LSWYQQKPGKAPKLLIYTASSLASGVPSRFSGS clone 6
GSGTDFTLTISSLQPEDFATYYCQGEFTCSNADC FTFGQGTKLTVLG 6 VH
EVQLVESGGGLVQPGGSLRLSCAASGFPLSSYA anti-CD3
MIWVRQAPGKGLEWIGMILRAGNIYYASWVKGR clone 6
FTISRDNSKNTVYLQMNSLRAEDTAVYYCARRH YNREGYPIGIGDLWGQGTLVTVSS 7 PRO165
DIQMTQSPSSLSASVGDRVTITCQASENIYSFLA
WYQQKPGKAPKLLIYSASKLAAGVPSRFSGSGS
GTDFTLTISSLQPEDFATYYCQQTNRYSNPDIYN VFGQGTKLTVLGGGGGSEVQLVESGGGLVQPG
GSLRLSCAASGFPLSSYAMIWVRQAPGKGLEWI
GMILRAGNIYYASWVKGRFTISRDNSKNTVYLQM
NSLRAEDTAVYYCARRHYNREGYPIGIGDLWGQ GTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQ
MTQSPSSLSASVGDRVTITCQSSESVYNNKRLS WYQQKPGKAPKLLIYTASSLASGVPSRFSGSGS
GTDFTLTISSLQPEDFATYYCQGEFTCSNADCFT FGQGTKLTVLGGGGGSEVQLVESGGGLVQPGG
SLRLSCAASGIDFNSNYYMCWVRQAPGKGLEWI
GCIYVGSHVNTYYANWAKGRFTISRDNSKNTVYL
QMNSLRAEDTAVYYCATSGSSVLYFKFWGQGTL VTVSS 8 VL
DIQMTQSPSSLPASLGDRVTINCQASQDISNYLN anti-mCD3
WYQQKPGKAPKLLIYYTNKLADGVPSRFSGSGS 2C11
GRDSSFTISSLESEDIGSYYCQQYYNYPWTFGP GTKLEIKR 9 VH
EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYG anti-D3
MHWVRQAPGRGLESVAYITSSSINIKYADAVKGR 2C11
FTVSRDNAKNLLFLQMNILKSEDTAMYYCARFD WDKNYWGQGTMVTVSS 10 PRO387
DIQMTQSPSSLSASVGDRVTITCQASENIYSFLA
WYQQKPGKAPKLLIYSASKLAAGVPSRFSGSGS
GTDFTLTISSLQPEDFATYYCQQTNRYSNPDIYN VFGQGTKLTVLGGGGGSEVQLVESGGGLVQPG
KSLKLSCEASGFTFSGYGMHWVRQAPGRGLES VAYITSSSINIKYADAVKGRFTVSRDNAKNLLFL
QMNILKSEDTAMYYCARFDWDKNYWGQGTMVTV SSGGGGSGGGGSGGGGSGGGGSDIQMTQSPS
SLPASLGDRVTINCQASQDISNYLNWYQQKPGK
APKLLIYYTNKLADGVPSRFSGSGSGRDSSFTIS
SLESEDIGSYYCQQYYNYPWTFGPGTKLEIKRG GGGSEVQLVESGGGLVQPGGSLRLSCAASGIDF
NSNYYMCWVRQAPGKGLEWIGCIYVGSHVNTY YANWAKGRFTISRDNSKNTVYLQMNSLRAEDTA
VYYCATSGSSVLYFKFWGQGTLVTVSS 11 Linker L4 GGGGS GGGGS 12 PRO386
DIQMTQSPSSLPASLGDRVTINCQASQDISNYLN (CL
WYQQKPGKAPKLLIYYTNKLADGVPSRFSGSGS fusion)
GRDSSFTISSLESEDIGSYYCQQYYNYPWTFGP
GTKLEIKRRTVAAPSVFIFPPSDEQLKSGTASVV
CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECGGGGSGGGGSHMDIQMTQ
SPSSLSASVGDRVTITCQASENIYSFLAWYQQKP
GKAPKLLIYSASKLAAGVPSRFSGSGSGTDFTLT
ISSLQPEDFATYYCQQTNRYSNPDIYNVFGQGTK LTVLGGGGGSGGGGSGGGGSGGGGSEVQLVE
SGGGLVQPGGSLRLSCAASGIDFNSNYYMCWV RQAPGKGLEWIGCIYVGSHVNTYYANWAKGRFT
ISRDNSKNTVYLQMNSLRAEDTAVYYCATSGSS VLYFKFWGQGTLVTVSS 13 PRO386
EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYG (CH1
MHWVRQAPGRGLESVAYITSSSINIKYADAVKGR fusion)
FTVSRDNAKNLLFLQMNILKSEDTAMYYCARFD WDKNYWGQGTMVTVSSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCGGGGSGGGGSH
MDIQMTQSPSSLSASVGDRVTITCQASENIYSFL
AWYQQKPGKAPKLLIYSASKLAAGVPSRFSGSG
SGTDFTLTISSLQPEDFATYYCQQTNRYSNPDIY NVFGQGTKLTVLGGGGGSGGGGSGGGGSGGG
GSEVQLVESGGGLVQPGGSLRLSCAASGIDFNS NYYMCWVRQAPGKGLEWIGCIYVGSHVNTYYA
NWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVY YCATSGSSVLYFKFWGQGTLVTVSS 14
PRO400 DIQMTQSPSSLPASLGDRVTINCQASQDISNYLN (VL-CL)
WYQQKPGKAPKLLIYYTNKLADGVPSRFSGSGS 2C11 Fab
GRDSSFTISSLESEDIGSYYCQQYYNYPWTFGP
GTKLEIKRRTVAAPSVFIFPPSDEQLKSGTASVV
CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 15 PRO400
EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYG (VH-CH1)
MHWVRQAPGRGLESVAYITSSSINIKYADAVKGR 2C11 Fab
FTVSRDNAKNLLFLQMNILKSEDTAMYYCARFD WDKNYWGQGTMVTVSSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSC
Example 11
Mouse Imiquimod (IMQ)-Induced Psoriasis in C57B/6 Mice
Treatment
[0176] This example was performed in wild-type C57B/6 mice in
accordance with an IMQ induced psoriasis disease model. The mouse
back was shaved, and a daily dose of 50 mg of Aldara (5% IMQ cream;
3M Pharmaceuticals) was applied on the back of each mouse for 5
days. Antibody treatment was performed by daily intraperitoneal
injection with 0.4, 2, 10, or 50 mcg/mouse of the bispecific
anti-IL23RxCD3 antibody fragments (PRO386 and PRO387) or PBS as a
control with n=3 per dose group. Injections started two days prior
to first Aldara application on study day -1, and were repeated
daily until day 4. Cell preparation: Skin was cut into small pieces
and digested with 1 mg/ml collagenase type IA and 100 mg/ml DNase
(Sigma-Aldrich) for 60 minutes at 37.degree. C. Isolation of
leukocytes from the lymph nodes (LN) involved teasing the organs
apart. Both were followed by filtering through 70-mcm cell
strainers to obtain single-cell suspensions. Antibodies: Cells were
incubated with antibodies for 20 minutes at 4.degree. C. For
intracellular cytokine staining, cells were stimulated with PMA
(Applichem) and ionomycin (Invitrogen) and treated with GolgiStop
(BD) for 3 hours. After surface staining, cells were permeabilized
according to the manufacturer's (BD) recommendations and stained
intracellularly. Stainings were analyzed with a FACS LSRII Fortessa
(BD). Post-acquisition analysis was performed with FlowJo (Tree
Star) software.
TABLE-US-00014 TABLE 10 Group Test Dose Group Strain size (n)
substance (mcg) A C57B/6 5 PBS 0 B C57B/6 3 PRO387 50 (scDb) C
C57B/6 3 PRO387 10 (scDb) D C57B/6 3 PRO387 2 (scDb) E C57B/6 3
PRO387 0.4 (scDb) F C57B/6 3 PRO386 50 (Tribody) G C57B/6 3 PRO386
10 (Tribody) H C57B/6 3 PRO386 2 (Tribody) I C57B/6 3 PRO386 0.4
(Tribody)
Results
[0177] Intraperitoneal injection of the bispecific anti-IL23RxCD3
antibody constructs PRO386 and PRO387 inhibited skin thickening in
a dose-dependent manner and blocked clinical symptoms nearly
completely at the highest dose of 50 mcg/injection (FIG. 8). The
hypothesis that IL23R expressing T cells are key for the
development of the disease was supported by the dose-dependent
depletion of CD27-negative gamma-delta T cells (FIG. 9). On
gamma-delta T cells, lack of CD27 expression has been demonstrated
to correlate with IL23R expression (Chognard et
al.PLoS.2014;9:e89092).
Example 12
Mouse Imiquimod-Induced Psoriasis in IL23R Reporter Mice
Treatment
[0178] This example was performed in IL23R-GFP reporter mice
(Awasthi et al, J Immuno.2009;182:5904-5908) in accordance with an
IMQ induced psoriasis disease model. The mouse back was shaved, and
a daily dose of 50 mg of Aldara (5% IMQ cream; 3M Pharmaceuticals)
or control vehicle cream (SoftKreme KA, Kantonsapotheke Zurich) was
applied on the back of each mouse for 5 days. Antibody treatment
was performed by daily intraperitoneal injection with 50 mcg/mouse
of the bispecific anti-IL23RxCD3 antibody fragments (PRO386 and
PRO387), the control anti-CD3 Fab fragment, or PBS as a control.
Group sizes were n=5 for the PRO386 and PRO387 groups and n=3 for
the groups treated with PBS or the anti-CD3 Fab. Injections started
two days prior to first Aldara application on study day -1, and
were repeated daily until day 4. Cell preparation: Skin was cut
into small pieces and digested with 1 mg/ml collagenase type IA and
100 mg/ml DNase (Sigma-Aldrich) for 60 minutes at 37.degree. C.
Isolation of leukocytes from the LN involved teasing the organs
apart. Both were followed by filtering through 70-mcm cell
strainers to obtain single-cell suspensions. Antibodies: Cells were
incubated with antibodies for 20 minutes at 4.degree. C. For
intracellular cytokine staining, cells were stimulated with PMA
(Applichem) and ionomycin (Invitrogen) and treated with GolgiStop
(BD) for 3 hours. After surface staining, cells were permeabilized
according to the manufacturer's (BD) recommendations and stained
intracellularly. Stainings were analyzed with a FACS LSRII Fortessa
(BD). Post-acquisition analysis was performed with FlowJo (Tree
Star) software.
TABLE-US-00015 TABLE 11 size Group Mice (n) Inducer Test substance
A C57BL/6 (n = 1); 3 naive PBS IL23R-GFP (n = 2) B IL23R-GFP (n =
5) 5 Aldara PRO386 (Tribody) C IL23R-GFP (n = 5) 5 Aldara PRO387
(scDb) D IL23R-GFP (n = 3) 3 Aldara anti-CD3 Fab E C57BL/6 (n = 1);
3 Aldara PBS IL23R-GFP (n = 2) F IL23R-GFP (n = 5) 5 naive PRO386
(Tribody)
Results
[0179] Intraperitoneal injection of both bispecific anti-IL23RxCD3
antibody constructs PRO386 and PRO387 inhibited skin thickening.
The anti-CD3 Fab fragment had no effect. PRO386 blocked clinical
symptoms nearly completely at 50 mcg per injection (FIG. 10).
Flow-cytometry analysis was performed on cells from thymus, skin
lymph nodes, skin and spleen. In the thymus PRO386 and PRO387
reduced counts of IL23R expressing gamma-delta T cells of both,
psoriatic and healthy control mice, whereas the anti-CD3 Fab
fragment had no effect. No effect was observed generally on CD8+,
CD4+ CD8+/CD4+ double-positive (DPs), CD8-/CD4- double-negative
(DN), CD25+/CD4+and MHCI1+/CD11c positive T cells (FIG. 11). In the
skin lymph nodes (LN) both bispecific IL23RxCD3 constructs PRO386
and PRO387 reduced the counts of IL23R expressing gamma-delta T
cells, IL23R+/CD8+ T killer cells and IL23R+/CD4+ T helper cells
(FIG. 12). Similar to results in the thymus no effects were
observed generally on CD8+, CD4+, CD25+/CD4+ and MHCII+/CD11c+
cells. In the skin, IL23R expressing T cells in general and
gamma-delta T cells in particular were almost completely depleted
with both, PRO386 and PRO387 (FIG. 13). Probably as a secondary
effect of the depletion of IL23R expressing cells, also neutrophil
infiltration was largely reduced in skin (FIG. 14). Interestingly,
no reduction was seen when broadly gating CD3 positive gamma-delta
T cells, suggesting that exclusively IL23R expressing gamma-delta T
cells were depleted. This supports the hypothesis that IL23R
expressing T cells are key for the disease and that specific
depletion of these cells interferes with disease pathogenesis. In
the spleen, only very low numbers of IL23R expressing cells could
be detected in both healthy and psoriatic mice. As in the other
organs, no effects were seen with any treatment neither on CD8+,
CD4+, CD25+/CD4+ T cells, nor on NK1.1+/CD3-, NK1.1/CD3+ or
MHCII+/CD11c+ cells (FIG. 15).
Example 13
Active Induction of Mouse Experimental Autoimmune Encephalomyelitis
in SJL Mice
[0180] An autoimmune encephalomyelitis model (EAE) has been
actively induced by peptide immunization as described elsewhere
(Miller et al.Curr Protoc Immuno1.2007;Chapter:Unit-15.1.doi
:10.1002/0471142735.iml1501s77). Clinical symptoms were assessed on
a daily basis using EAE symptom scoring ranging from 0 (no
detectable signs of EAE) to 5 (dead). As indicated below, the two
bispecific anti-IL23RxCD3 antibody constructs PRO386 and PRO387
were tested in a prophylactic as well a therapeutic setting.
Prevention groups were dosed by daily intraperitoneal injection of
PRO386 and PRO387 from day 1 after induction until day 8. The
therapy groups received daily intraperitoneal injections of 50mcg
of PRO386, PRO387 and an anti-CD3 Fab as a control, for 11 days
after onset of the disease (score of -0.5).
TABLE-US-00016 TABLE 12 Group Test Dose Group Strain size (n)
substance Treatment design (mcg) a SJL 5 PBS prevention/prophylaxis
0 b SJL 5 PRO386 prevention/prophylaxis 50 (Tribody) d SJL 5 PRO387
prevention/prophylaxis 50 (scDb) c SJL 5 PRO386 therapy 50
(Tribody) e SJL 5 PRO387 therapy 50 (scDb) f SJL 3 anti-CD3 Fab
therapy 50
Results
[0181] Disease symptoms, as assessed by EAE scoring peaked at
around day 14 for the PBS control group. Both bispecific constructs
PRO386 and PRO387 blocked the development of clinical symptoms
nearly completely when administered in a prophylactic setting
before disease onset (FIG. 16). This is in line with the findings
by Chen et al.J Clin Invest.2009;116:1317-1325) that antibodies
blocking IL-23 alone (anti-IL-23 p19) or IL-12 and IL-23 together
(anti-IL-12 p40) were able to block disease symptoms in a
prophylactic setting. Surprisingly, and in contrast to anti-IL-12
p40 or anti-IL-23 p19 antibodies, both, PRO386 and PRO387 were able
to ameliorate disease symptoms when administered in a therapeutic
setting, starting after onset of the disease (FIG. 17). A probable
explanation for this is that IL-23 blockade inhibits the de novo
differentiation of pathogenic T cells, such as IL23R expressing
Th17 and gamma-delta T cells. In established disease conditions,
however, these cells are terminally differentiated and may no
longer depend on IL-23 signaling. Thus, IL-23 blockade cannot block
activity of all disease driving cell types. In contrast, depletion
of IL23R expressing cells eliminates the key disease driving cells
independent of their level of differentiation.
Example 14
B16F10 Melanoma Metastasis Model
Treatment
[0182] Mice were injected with B16F10 melanoma cells by the
intravenous route and subsequently treated with 50 mcg PRO386 or
PBS by daily intraperitoneal injection. After 14 days of treatment,
lung metastases were counted for each animal.
Results
[0183] PRO386 significantly reduced the numbers of lung metastasis
(FIG. 18).
Sequence CWU 1
1
1515PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Gly Gly Gly Gly Ser 1 5 220PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10
15 Gly Gly Gly Ser 20 3113PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 3Asp 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 Gln Ala Ser Glu Asn Ile Tyr Ser Phe 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Ser Ala Ser Lys Leu Ala Ala 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 Thr Asn Arg
Tyr Ser Asn 85 90 95 Pro Asp Ile Tyr Asn Val Phe Gly Gln Gly Thr
Lys Leu Thr Val Leu 100 105 110 Gly 4121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
4Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Asp Phe Asn Ser
Asn 20 25 30 Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp 35 40 45 Ile Gly Cys Ile Tyr Val Gly Ser His Val Asn
Thr Tyr Tyr Ala Asn 50 55 60 Trp Ala Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Thr Ser
Gly Ser Ser Val Leu Tyr Phe Lys Phe Trp Gly 100 105 110 Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 5114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
5Asp 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 Gln Ser Ser Glu Ser Val Tyr Asn
Asn 20 25 30 Lys Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu 35 40 45 Leu Ile Tyr Thr Ala Ser Ser Leu Ala Ser Gly
Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gly Glu Phe Thr Cys 85 90 95 Ser Asn Ala Asp Cys
Phe Thr Phe Gly Gln Gly Thr Lys Leu Thr Val 100 105 110 Leu Gly
6123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 6Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Pro Leu Ser Ser Tyr 20 25 30 Ala Met Ile Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Leu Arg
Ala Gly Asn Ile Tyr Tyr Ala Ser Trp Val Lys 50 55 60 Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95 Arg Arg His Tyr Asn Arg Glu Gly Tyr Pro Ile Gly Ile Gly Asp Leu
100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
7501PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 7Asp 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 Gln Ala
Ser Glu Asn Ile Tyr Ser Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Lys
Leu Ala Ala 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 Thr Asn Arg Tyr Ser Asn 85 90
95 Pro Asp Ile Tyr Asn Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu
100 105 110 Gly Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly
Gly Gly 115 120 125 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly 130 135 140 Phe Pro Leu Ser Ser Tyr Ala Met Ile Trp
Val Arg Gln Ala Pro Gly 145 150 155 160 Lys Gly Leu Glu Trp Ile Gly
Met Ile Leu Arg Ala Gly Asn Ile Tyr 165 170 175 Tyr Ala Ser Trp Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 180 185 190 Lys Asn Thr
Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 195 200 205 Ala
Val Tyr Tyr Cys Ala Arg Arg His Tyr Asn Arg Glu Gly Tyr Pro 210 215
220 Ile Gly Ile Gly Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser
225 230 235 240 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser 245 250 255 Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu 260 265 270 Ser Ala Ser Val Gly Asp Arg Val Thr
Ile Thr Cys Gln Ser Ser Glu 275 280 285 Ser Val Tyr Asn Asn Lys Arg
Leu Ser Trp Tyr Gln Gln Lys Pro Gly 290 295 300 Lys Ala Pro Lys Leu
Leu Ile Tyr Thr Ala Ser Ser Leu Ala Ser Gly 305 310 315 320 Val Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 325 330 335
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln 340
345 350 Gly Glu Phe Thr Cys Ser Asn Ala Asp Cys Phe Thr Phe Gly Gln
Gly 355 360 365 Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Gly Ser Glu
Val Gln Leu 370 375 380 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly Ser Leu Arg Leu 385 390 395 400 Ser Cys Ala Ala Ser Gly Ile Asp
Phe Asn Ser Asn Tyr Tyr Met Cys 405 410 415 Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Ile Gly Cys Ile 420 425 430 Tyr Val Gly Ser
His Val Asn Thr Tyr Tyr Ala Asn Trp Ala Lys Gly 435 440 445 Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln 450 455 460
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Thr 465
470 475 480 Ser Gly Ser Ser Val Leu Tyr Phe Lys Phe Trp Gly Gln Gly
Thr Leu 485 490 495 Val Thr Val Ser Ser 500 8108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
8Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Pro Ala Ser Leu Gly 1
5 10 15 Asp Arg Val Thr Ile Asn Cys Gln Ala Ser Gln Asp Ile Ser Asn
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Tyr Thr Asn Lys Leu Ala Asp Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Ser Ser Phe
Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Ile Gly Ser Tyr Tyr
Cys Gln Gln Tyr Tyr Asn Tyr Pro Trp 85 90 95 Thr Phe Gly Pro Gly
Thr Lys Leu Glu Ile Lys Arg 100 105 9116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
9Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Lys 1
5 10 15 Ser Leu Lys Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Gly
Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu
Glu Ser Val 35 40 45 Ala Tyr Ile Thr Ser Ser Ser Ile Asn Ile Lys
Tyr Ala Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp
Asn Ala Lys Asn Leu Leu Phe 65 70 75 80 Leu Gln Met Asn Ile Leu Lys
Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Phe Asp Trp
Asp Lys Asn Tyr Trp Gly Gln Gly Thr Met Val 100 105 110 Thr Val Ser
Ser 115 10488PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 10Asp 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 Gln Ala Ser Glu Asn Ile Tyr Ser Phe 20 25 30 Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser
Ala Ser Lys Leu Ala Ala 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 Thr Asn Arg Tyr
Ser Asn 85 90 95 Pro Asp Ile Tyr Asn Val Phe Gly Gln Gly Thr Lys
Leu Thr Val Leu 100 105 110 Gly Gly Gly Gly Gly Ser Glu Val Gln Leu
Val Glu Ser Gly Gly Gly 115 120 125 Leu Val Gln Pro Gly Lys Ser Leu
Lys Leu Ser Cys Glu Ala Ser Gly 130 135 140 Phe Thr Phe Ser Gly Tyr
Gly Met His Trp Val Arg Gln Ala Pro Gly 145 150 155 160 Arg Gly Leu
Glu Ser Val Ala Tyr Ile Thr Ser Ser Ser Ile Asn Ile 165 170 175 Lys
Tyr Ala Asp Ala Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asn 180 185
190 Ala Lys Asn Leu Leu Phe Leu Gln Met Asn Ile Leu Lys Ser Glu Asp
195 200 205 Thr Ala Met Tyr Tyr Cys Ala Arg Phe Asp Trp Asp Lys Asn
Tyr Trp 210 215 220 Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly 225 230 235 240 Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Asp Ile 245 250 255 Gln Met Thr Gln Ser Pro Ser
Ser Leu Pro Ala Ser Leu Gly Asp Arg 260 265 270 Val Thr Ile Asn Cys
Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 275 280 285 Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr 290 295 300 Thr
Asn Lys Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 305 310
315 320 Ser Gly Arg Asp Ser Ser Phe Thr Ile Ser Ser Leu Glu Ser Glu
Asp 325 330 335 Ile Gly Ser Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro
Trp Thr Phe 340 345 350 Gly Pro Gly Thr Lys Leu Glu Ile Lys Arg Gly
Gly Gly Gly Ser Glu 355 360 365 Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser 370 375 380 Leu Arg Leu Ser Cys Ala Ala
Ser Gly Ile Asp Phe Asn Ser Asn Tyr 385 390 395 400 Tyr Met Cys Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 405 410 415 Gly Cys
Ile Tyr Val Gly Ser His Val Asn Thr Tyr Tyr Ala Asn Trp 420 425 430
Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val 435
440 445 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr 450 455 460 Cys Ala Thr Ser Gly Ser Ser Val Leu Tyr Phe Lys Phe
Trp Gly Gln 465 470 475 480 Gly Thr Leu Val Thr Val Ser Ser 485
1110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 11Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10
12481PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 12Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Pro Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Asn Cys Gln Ala
Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Asn Lys
Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Arg Asp Ser Ser Phe Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu
Asp Ile Gly Ser Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Trp 85 90
95 Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys Arg Arg Thr Val Ala
100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser
Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215
220 Ser His Met Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
225 230 235 240 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser
Glu Asn Ile 245 250 255 Tyr Ser Phe Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys 260 265 270 Leu Leu Ile Tyr Ser Ala Ser Lys Leu
Ala Ala Gly Val Pro Ser Arg 275 280 285 Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser 290 295 300 Leu Gln Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Asn Arg 305 310 315 320 Tyr Ser
Asn Pro Asp Ile Tyr Asn Val Phe Gly Gln Gly Thr Lys Leu 325 330 335
Thr Val Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 340
345 350 Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser
Gly 355 360 365 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
Cys Ala Ala 370 375 380 Ser Gly Ile Asp Phe Asn Ser Asn Tyr Tyr Met
Cys Trp Val Arg Gln 385 390 395 400 Ala Pro Gly Lys Gly Leu Glu Trp
Ile Gly Cys Ile Tyr Val Gly Ser 405 410 415 His Val Asn Thr Tyr Tyr
Ala Asn Trp Ala Lys Gly Arg Phe Thr Ile 420 425 430 Ser Arg Asp Asn
Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu 435 440 445 Arg Ala
Glu Asp Thr Ala
Val Tyr Tyr Cys Ala Thr Ser Gly Ser Ser 450 455 460 Val Leu Tyr Phe
Lys Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser 465 470 475 480 Ser
13485PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 13Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Lys Leu Ser Cys Glu Ala Ser
Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Gly Met His Trp Val Arg Gln
Ala Pro Gly Arg Gly Leu Glu Ser Val 35 40 45 Ala Tyr Ile Thr Ser
Ser Ser Ile Asn Ile Lys Tyr Ala Asp Ala Val 50 55 60 Lys Gly Arg
Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Leu Leu Phe 65 70 75 80 Leu
Gln Met Asn Ile Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90
95 Ala Arg Phe Asp Trp Asp Lys Asn Tyr Trp Gly Gln Gly Thr Met Val
100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly Gly Gly Ser 210 215
220 Gly Gly Gly Gly Ser His Met Asp Ile Gln Met Thr Gln Ser Pro Ser
225 230 235 240 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr
Cys Gln Ala 245 250 255 Ser Glu Asn Ile Tyr Ser Phe Leu Ala Trp Tyr
Gln Gln Lys Pro Gly 260 265 270 Lys Ala Pro Lys Leu Leu Ile Tyr Ser
Ala Ser Lys Leu Ala Ala Gly 275 280 285 Val Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu 290 295 300 Thr Ile Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln 305 310 315 320 Gln Thr
Asn Arg Tyr Ser Asn Pro Asp Ile Tyr Asn Val Phe Gly Gln 325 330 335
Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly 340
345 350 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln
Leu 355 360 365 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
Leu Arg Leu 370 375 380 Ser Cys Ala Ala Ser Gly Ile Asp Phe Asn Ser
Asn Tyr Tyr Met Cys 385 390 395 400 Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Ile Gly Cys Ile 405 410 415 Tyr Val Gly Ser His Val
Asn Thr Tyr Tyr Ala Asn Trp Ala Lys Gly 420 425 430 Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln 435 440 445 Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Thr 450 455 460
Ser Gly Ser Ser Val Leu Tyr Phe Lys Phe Trp Gly Gln Gly Thr Leu 465
470 475 480 Val Thr Val Ser Ser 485 14215PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
14Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Pro Ala Ser Leu Gly 1
5 10 15 Asp Arg Val Thr Ile Asn Cys Gln Ala Ser Gln Asp Ile Ser Asn
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Tyr Thr Asn Lys Leu Ala Asp Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Ser Ser Phe
Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Ile Gly Ser Tyr Tyr
Cys Gln Gln Tyr Tyr Asn Tyr Pro Trp 85 90 95 Thr Phe Gly Pro Gly
Thr Lys Leu Glu Ile Lys Arg Arg Thr Val Ala 100 105 110 Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135
140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys
210 215 15219PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 15Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Lys Leu Ser Cys
Glu Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Gly Met His Trp
Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Ser Val 35 40 45 Ala Tyr
Ile Thr Ser Ser Ser Ile Asn Ile Lys Tyr Ala Asp Ala Val 50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Leu Leu Phe 65
70 75 80 Leu Gln Met Asn Ile Leu Lys Ser Glu Asp Thr Ala Met Tyr
Tyr Cys 85 90 95 Ala Arg Phe Asp Trp Asp Lys Asn Tyr Trp Gly Gln
Gly Thr Met Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185
190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215
* * * * *