U.S. patent application number 15/987996 was filed with the patent office on 2018-11-22 for icos binding proteins.
The applicant listed for this patent is GlaxoSmithKline Intellectual Property Development Limited. Invention is credited to Yao-Bin Liu, Patrick Mayes, Radha Shah Parmar.
Application Number | 20180334503 15/987996 |
Document ID | / |
Family ID | 55262868 |
Filed Date | 2018-11-22 |
United States Patent
Application |
20180334503 |
Kind Code |
A1 |
Liu; Yao-Bin ; et
al. |
November 22, 2018 |
ICOS BINDING PROTEINS
Abstract
The present invention relates to an ICOS binding protein or
antigen binding portion thereof that is an agonist to human ICOS
and does not induce complement, ADCC, or CDC when placed in contact
with a T cell in vivo and methods of treating cancer, infectious
disease and/or sepsis with said ICOS binding protein or antigen
binding portion thereof. Further the ICOS binding proteins or
antigen binding portions thereof of the present invention are
capable of activating a T cell when placed in contact with said T
cell; stimulating T cell proliferation when placed in contact with
said T cell and/or inducing cytokine production when placed in
contact with said T cell. The present invention relates to ICOS
binding proteins or antigen binding portions thereof comprising one
or more of: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ
ID NO:5; and/or SEQ ID NO:6.
Inventors: |
Liu; Yao-Bin; (Collegeville,
PA) ; Mayes; Patrick; (Collegeville, PA) ;
Parmar; Radha Shah; (Stevenage, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GlaxoSmithKline Intellectual Property Development Limited |
Middlesex |
|
GB |
|
|
Family ID: |
55262868 |
Appl. No.: |
15/987996 |
Filed: |
May 24, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15649791 |
Jul 14, 2017 |
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15987996 |
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15387161 |
Dec 21, 2016 |
9738718 |
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15649791 |
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15246662 |
Aug 25, 2016 |
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15387161 |
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15006560 |
Jan 26, 2016 |
9771424 |
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15246662 |
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62247355 |
Oct 28, 2015 |
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62192331 |
Jul 14, 2015 |
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62108605 |
Jan 28, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
C07K 2317/24 20130101; C07K 2317/56 20130101; C07K 16/3023
20130101; C07K 2317/565 20130101; C07K 16/2896 20130101; C07K
16/3038 20130101; C07K 16/2803 20130101; C07K 16/3069 20130101;
C07K 2317/75 20130101; A61K 39/3955 20130101; C07K 16/30 20130101;
A61K 39/39558 20130101; C07K 2317/21 20130101; A61P 35/00 20180101;
C07K 2317/71 20130101; A61K 2039/505 20130101; C07K 16/3015
20130101; C07K 2317/33 20130101; C07K 2317/92 20130101; C07K
16/2818 20130101; A61K 2039/507 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/30 20060101 C07K016/30; A61K 39/395 20060101
A61K039/395; A61K 45/06 20060101 A61K045/06 |
Claims
1. A method of treating cancer in a human in need thereof, the
method comprising administering a pharmaceutical composition
comprising an ICOS binding protein or antigen binding portion
thereof and a pharmaceutically acceptable carrier to said human,
wherein the ICOS binding protein or antigen binding portion thereof
comprises a V.sub.H domain comprising an amino acid sequence at
least 90% identical to the amino acid sequence set forth in SEQ ID
NO:7; and a V.sub.L domain comprising an amino acid sequence at
least 90% identical to the amino acid sequence as set forth in SEQ
ID NO:8 wherein said ICOS binding protein or antigen binding
portion thereof specifically binds to human ICOS, and administering
at least one anti-neoplastic agent selected from the group
consisting of docetaxel, paclitaxel, gemcitabine, and carboplatin
to said human.
2. The method of claim 1, wherein said cancer is selected from
colorectal cancer (CRC), esophageal cancer, cervical cancer,
bladder cancer, breast cancer, head and neck cancer, ovarian
cancer, melanoma, renal cell carcinoma (RCC), EC squamous cell,
non-small cell lung carcinoma, mesothelioma, urothelial cancer, and
prostate cancer.
3. The method of claim 1, wherein the ICOS binding protein or
antigen binding portion thereof comprises one or more of: CDRH1 as
set forth in SEQ ID NO:1; CDRH2 as set forth in SEQ ID NO:2; CDRH3
as set forth in SEQ ID NO:3; CDRL1 as set forth in SEQ ID NO:4;
CDRL2 as set forth in SEQ ID NO:5 and/or CDRL3 as set forth in SEQ
ID NO:6 or a direct equivalent of each CDR wherein a direct
equivalent has no more than two amino acid substitutions in said
CDR.
4. The method of claim 1, wherein the ICOS binding protein or
antigen binding portion thereof is an ICOS agonist.
5. The method of claim 1, wherein the ICOS binding protein or
antigen binding portion comprises a heavy chain variable region
comprising SEQ ID NO:1; SEQ ID NO:2; and SEQ ID NO:3, and a light
chain variable region comprising SEQ ID NO:4; SEQ ID NO:5; and SEQ
ID NO:6.
6. The method of claim 1, wherein the ICOS binding protein or
antigen binding portion thereof comprises a V.sub.H domain
comprising the amino acid sequence set forth in SEQ ID NO:7 and a
V.sub.L domain comprising the amino acid sequence set forth in SEQ
ID NO:8.
7. The method of claim 1, wherein the ICOS binding protein or
antigen binding portion thereof comprises a scaffold selected from
human IgG1 isotype and human IgG4 isotype.
8. The method of claim 1, wherein the ICOS binding protein or
antigen binding portion thereof comprises an hIgG4PE scaffold.
9. The method of claim 1, wherein the ICOS binding protein is a
monoclonal antibody.
10. The method of claim 11, wherein the monoclonal antibody is
humanized.
11. The method of claim 11, wherein the monoclonal antibody
comprises heavy chain CDRs comprising the amino acid sequences set
forth in SEQ ID NO:1; SEQ ID NO:2; and SEQ ID NO:3 and light chain
CDRs comprising the amino acid sequences set forth in SEQ ID NO:4;
SEQ ID NO:5; and SEQ ID NO:6.
12. The method of claim 11, wherein the monoclonal antibody is an
agonist to human ICOS and comprises an IgG4 isotype scaffold.
13. The method of claim 11, wherein the monoclonal antibody
comprises a hIgG4PE scaffold.
14. The method of claim 1 further comprising administering an
anti-PD1 antibody to said human.
15. The method of claim 15, wherein the anti-PD1 antibody is
pembrolizumab.
16. The method of claim 14, wherein the anti-PD1 antibody is
nivolumab.
17. The method of claim 1, wherein the anti-neoplastic agent is
docetaxel.
18. The method of claim 1, wherein the anti-neoplastic agent is
paclitaxel.
19. The method of claim 1, wherein the anti-neoplastic agent is
gemcitabine.
20. The method of claim 1, wherein the anti-neoplastic agent is
carboplatin.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 15/649,791 filed 14 Jul. 2017, which is a continuation of U.S.
application Ser. No. 15/387,161 filed 21 December, 2016, which is a
continuation of U.S. application Ser. No. 15/246,662 filed 25 Aug.
2016, which is a divisional of U.S. application Ser. No. 15/006,560
filed 26 Jan. 2016, which is a 111a application which claims
benefit of U.S. Provisional 62/247,355 filed 28 Oct. 2015, U.S.
Provisional 62/192,331 filed 14 Jul. 2015, and U.S. Provisional
62/108,605 filed 28 Jan. 2015, all of which are incorporated herein
in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to immunotherapy in
the treatment of human disease and reduction of adverse events
related thereto. More specifically, the present invention relates
to the use of ICOS binding proteins including ICOS agonist
antibodies and their use as immunomodulators in the treatment of
cancer, infectious disease and/or sepsis.
BACKGROUND OF THE INVENTION
[0003] Enhancing anti-tumor T cell function and inducing T cell
proliferation is a powerful and new approach for cancer treatment.
Three immune-oncology antibodies (e.g., immuno-modulators) are
presently marketed. Anti-CTLA-4 (YERVOY/ipilimumab) is thought to
augment immune responses at the point of T cell priming and
anti-PD-1 antibodies (OPDIVO/nivolumab and KEYTRUDA/pembrolizumab)
are thought to act in the local tumor microenvironment, by
relieving an inhibitory checkpoint in tumor specific T cells that
have already been primed and activated.
[0004] ICOS is a co-stimulatory T cell receptor with structural and
functional relation to the CD28/CTLA-4-Ig superfamily (Hutloff, et
al., "ICOS is an inducible T-cell co-stimulator structurally and
functionally related to CD28", Nature, 397: 263-266 (1999)).
Activation of ICOS occurs through binding by ICOS-L (B7RP-1/B7-H2).
Neither B7-1 nor B7-2 (ligands for CD28 and CTLA4) bind or activate
ICOS. However, ICOS-L has been shown to bind weakly to both CD28
and CTLA-4 (Yao S et al., "B7-H2 is a costimulatory ligand for CD28
in human", Immunity, 34(5); 729-40 (2011)). Expression of ICOS
appears to be restricted to T cells. ICOS expression levels vary
between different T cell subsets and on T cell activation status.
ICOS expression has been shown on resting TH17, T follicular helper
(TFH) and regulatory T (Treg) cells; however, unlike CD28; it is
not highly expressed on naive T.sub.H1 and T.sub.H2 effector T cell
populations (Paulos C M et al., "The inducible costimulator (ICOS)
is critical for the development of human Th17 cells", Sci Transl
Med, 2(55); 55ra78 (2010)). ICOS expression is highly induced on
CD4+ and CD8+ effector T cells following activation through TCR
engagement (Wakamatsu E, et al., "Convergent and divergent effects
of costimulatory molecules in conventional and regulatory CD4+ T
cells", Proc Natal Acad Sci USA, 110(3); 1023-8 (2013)).
Co-stimulatory signalling through ICOS receptor only occurs in T
cells receiving a concurrent TCR activation signal (Sharpe A H and
Freeman G J. "The B7-CD28 Superfamily", Nat. Rev Immunol, 2(2);
116-26 (2002)). In activated antigen specific T cells, ICOS
regulates the production of both TH1 and TH2 cytokines including
IFN-.gamma., TNF-.alpha., IL-10, IL-4, IL-13 and others. ICOS also
stimulates effector T cell proliferation, albeit to a lesser extent
than CD28 (Sharpe A H and Freeman G J. "The B7-CD28 Superfamily",
Nat. Rev Immunol, 2(2); 116-26 (2002))
[0005] A growing body of literature supports the idea that
activating ICOS on CD4+ and CD8+ effector T cells has anti-tumor
potential. An ICOS-L-Fc fusion protein caused tumor growth delay
and complete tumor eradication in mice with SA-1 (sarcoma), Meth A
(fibrosarcoma), EMT6 (breast) and P815 (mastocytoma) and EL-4
(plasmacytoma) syngeneic tumors, whereas no activity was observed
in the B16-F10 (melanoma) tumor model which is known to be poorly
immunogenic (Ara G et al., "Potent activity of soluble B7RP-1-Fc in
therapy of murine tumors in syngeneic hosts", Int. J Cancer,
103(4); 501-7 (2003)). The anti-tumor activity of ICOS-L-Fc was
dependent upon an intact immune response, as the activity was
completely lost in tumors grown in nude mice. Analysis of tumors
from ICOS-L-Fc treated mice demonstrated a significant increase in
CD4+ and CD8+ T cell infiltration in tumors responsive to
treatment, supporting the immunostimulatory effect of ICOS-L-Fc in
these models.
[0006] Another report using ICOS.sup.-/- and ICOS-L.sup.-/- mice
demonstrated the requirement of ICOS signalling in mediating the
anti-tumor activity of an anti-CTLA4 antibody in the B16/B16
melanoma syngeneic tumor model (Fu T et al., "The ICOS/ICOSL
pathway is required for optimal antitumor responses mediated by
anti-CTLA-4 therapy", Cancer Res, 71(16); 5445-54 (2011)). Mice
lacking ICOS or ICOS-L had significantly decreased survival rates
as compared to wild-type mice after anti-CTLA4 antibody treatment.
In a separate study, B16/B16 tumor cells were transduced to
overexpress recombinant murine ICOS-L. These tumors were found to
be significantly more sensitive to anti-CTLA4 treatment as compared
to a B16/B16 tumor cells transduced with a control protein (Allison
J et al., "Combination immunotherapy for the treatment of cancer",
WO2011/041613 A2 (2009)). These studies provide evidence of the
anti-tumor potential of an ICOS agonist, both alone and in
combination with other immunomodulatory antibodies.
[0007] Emerging data from patients treated with anti-CTLA4
antibodies also point to the positive role of ICOS+ effector T
cells in mediating an anti-tumor immune response. Patients with
metastatic melanoma (Giacomo A M D et al., "Long-term survival and
immunological parameters in metastatic melanoma patients who
respond to ipilimumab 10 mg/kg within an expanded access program",
Cancer Immunol Immunother., 62(6); 1021-8 (2013)); urothelial
(Carthon B C et al., "Preoperative CTLA-4 blockade: Tolerability
and immune monitoring in the setting of a presurgical clinical
trial" Clin Cancer Res., 16(10); 2861-71 (2010)); breast
(Vonderheide R H et al., "Tremelimumab in combination with
exemestane in patients with advanced breast cancer and
treatment-associated modulation of inducible costimulator
expression on patient T cells", Clin Cancer Res., 16(13); 3485-94
(2010)); and prostate cancer which have increased absolute counts
of circulating and tumor infiltrating CD4.sup.+ICOS.sup.+ and
CD8.sup.+ICOS.sup.+ T cells after ipilimumab treatment have
significantly better treatment related outcomes than patients where
little or no increases are observed. Importantly, it was shown that
ipilimumab changes the ICOS.sup.+ T effector:T.sub.reg ratio,
reversing an abundance of T.sub.regs pre-treatment to a significant
abundance of T effectors vs. T.sub.regs following treatment (Liakou
C I et al., "CTLA-4 blockade increases IFN-gamma producing
CD4+ICOShi cells to shift the ratio of effector to regulatory T
cells in cancer patients", Proc Natl Acad Sci USA. 105(39);
14987-92 (2008)) and (Vonderheide R H et al., Clin Cancer Res.,
16(13); 3485-94 (2010)). Therefore, ICOS positive T effector cells
are a positive predictive biomarker of ipilimumab response which
points to the potential advantage of activating this population of
cells with an agonist ICOS antibody.
[0008] Thus, there is a need for additional T cell proliferation
inducing molecules in the treatment of cancer.
SUMMARY OF THE INVENTION
[0009] In one embodiment of the present invention, ICOS binding
proteins or antigen binding portions thereof are provided
comprising one or more of: CDRH1 as set forth in SEQ ID NO:1; CDRH2
as set forth in SEQ ID NO:2; CDRH3 as set forth in SEQ ID NO:3;
CDRL1 as set forth in SEQ ID NO:4; CDRL2 as set forth in SEQ ID
NO:5 and/or CDRL3 as set forth in SEQ ID NO:6 or a direct
equivalent of each CDR wherein a direct equivalent has no more than
two amino acid substitutions in said CDR.
[0010] In one embodiment of the present invention, ICOS binding
proteins or antigen binding portions thereof are provided which
specifically binds to human ICOS wherein said ICOS binding protein
comprises a V.sub.H domain comprising an amino acid sequence at
least 90% identical to the amino acid sequence set forth in SEQ ID
NO:7 and/or a V.sub.L domain comprising an amino acid sequence at
least 90% identical to the amino acid sequence set forth in SEQ ID
NO:8.
[0011] In one embodiment, humanized monoclonal antibodies or
antigen binding portions thereof are provided comprising heavy
chain CDRs having the amino acid sequences set forth in SEQ ID
NO:1; SEQ ID NO:2; and SEQ ID NO:3 and light chain CDRs having the
amino acid sequences set forth in SEQ ID NO:4; SEQ ID NO:5; and SEQ
ID NO:6. In one embodiment, humanized monoclonal antibodies are
provided which comprise a hIgG4PE scaffold; a V.sub.H domain
comprising an amino acid sequence set forth in SEQ ID NO:7; and a
V.sub.L domain comprising an amino acid sequence set forth in SEQ
ID NO:8. The antibodies of the present invention may stimulate
cytokine production when contacted with a T cell.
[0012] In one embodiment, ICOS binding proteins are provided that
compete for binding to human ICOS with any one of the ICOS binding
proteins or antigen binding portions thereof of the invention.
[0013] In one embodiment, methods are provided for treating cancer,
infectious disease and/or sepsis with an ICOS binding protein or a
pharmaceutical composition comprising at least one ICOS binding
protein of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1: IFN-.gamma. production from CD4+CD25- T cells.
[0015] FIG. 2: Proliferation in CD4+CD25- T cells.
[0016] FIG. 3A, FIG. 3B, FIG. 3C: H2L5 humanized variant of
anti-ICOS 422.2 shows better cytokine production in PBMC cells.
[0017] FIG. 4: 422 H2L5 IgG1 induced decreased T cell viability
which was not apparent with Fc-disabled or hIgG4PE isotypes.
[0018] FIG. 5A and FIG. 5B: Dose response of H2L5 hIgG4PE induced
proinflammatory cytokine induction in human CD4+ T cells.
[0019] FIG. 6A, FIG. 6B, FIG. 6C: H2L5 hIgG4PE induces
proliferation, cytokine production and increased cytotoxic
potential in activated PBMCs from healthy human donors.
[0020] FIG. 7: Meso Scale Discovery (MSD) assay showing the
inhibition of ICOS-L binding to ICOS by H2L5 hIgG4PE, indicating
that it binds to the same epitope on ICOS as ICOS-L and competes
for binding.
[0021] FIG. 8: Recovered antibody V.sub.H and V.sub.L genes from
RNA of hybridoma clone 422.2.
[0022] FIG. 9: Protein sequences of heavy and light chains of H2L5
hIgG4PE with signal sequence.
[0023] FIG. 10: DNA sequence of coding region of H2L5 hIgG4PE heavy
chain with signal sequence.
[0024] FIG. 11: DNA sequence of coding region of H2L5 hIgG4PE light
chain with signal sequence.
[0025] FIG. 12A and FIG. 12B: Plasma concentrations of H2L5 hIgG4PE
in cynomolgus monkeys. Concentrations were determined after the (A)
first or (B) second dose (day 15) of H2L5 hIgG4PE. Animals were
sacrificed 48 hours post second dose for tissue sample collection
and histopathology analysis.
[0026] FIG. 13: Detection of H2L5 hIgG4PE binding to CD4+ T cells
from the spleen and axillary lymph nodes of monkeys. Tissue was
collected 48 hours post-second dose (Day 17).
[0027] FIG. 14A and FIG. 14B: Receptor Occupancy of H2L5 hIgG4PE in
blood CD4+ T cells from cynomolgus monkeys.
[0028] FIG. 14A: ICOS "free receptor" as measured by positive
binding of the anti-ICOS fluorescently labelled antibody used for
flow cytometry, which binds only when H2L5 hIgG4PE is not
present.
[0029] FIG. 14B: Receptor bound H2L5 hIgG4PE on peripheral blood
CD4+ cells as measured by fluorescently labelled anti-Human
IgG.
[0030] FIG. 15 (a) Phospho-AKT (T308) expression levels in
Ba/F3-ICOS cells treated with H2L5 hIgG4PE--Intracellular
signalling antibody array; FIG. 15 (b) Phospho-AKT (S473)
expression levels in Ba/F3-ICOS cells treated with H2L5
hIgG4PE--Intracellular signalling antibody array.
[0031] FIG. 16: H2L5 hIgG4PE in combination with ipilimumab results
increased proinflammatory cytokine production as compared to single
antibody treatment in PBMC pre-stimulation assay.
[0032] FIG. 17: H2L5 hIgG4PE in combination with pembrolizumab
results increased proinflammatory cytokine production as compared
to single antibody treatment in PBMC pre-stimulation assay.
[0033] FIG. 18: H2L5 hIgG4PE plus ipilimumab combination induces
increased proinflamatory cytokine production in a modified MLR
assay with CEFT peptide and pre-incubation.
[0034] FIG. 19: H2L5 hIgG4PE plus pembrolizumab combination induces
increased proinflamatory cytokine production in a modified MLR
assay with CEFT peptide and pre-incubation.
[0035] FIG. 20A, FIG. 20B, FIG. 20C: H2L5 hIgG4PE anti-ICOS agonist
mAb alone and in combination with pembrolizumab results in tumor
growth inhibition in a human PBMC A2058 Melanoma mouse tumor
model.
[0036] FIG. 21A and FIG. 21B: anti-ICOS murine surrogate mAb
results in significant tumor growth inhibition and increased
survival in combination with an anti-PD1 murine surrogate mAb in
the CT26 mouse tumor model.
[0037] FIG. 22A, FIG. 22B, FIG. 22C: anti-ICOS murine surrogate mAb
results in significant tumor growth inhibition and increased
survival in combination with an anti-PD1 murine surrogate mAb in
the EMT6 mouse tumor model.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0038] As used herein "ICOS" means any Inducible T-cell
costimulator protein. Pseudonyms for ICOS (Inducible T-cell
COStimulator) include AILIM; CD278; CVID1, JTT-1 or JTT-2,
MGC39850, or 8F4. ICOS is a CD28-superfamily costimulatory molecule
that is expressed on activated T cells. The protein encoded by this
gene belongs to the CD28 and CTLA-4 cell-surface receptor family.
It forms homodimers and plays an important role in cell-cell
signaling, immune responses, and regulation of cell proliferation.
The amino acid sequence of human ICOS is shown below as SEQ ID
NO:10.
MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLK
GGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFD
PPPFKVTLTGGYLHIYESQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKM (SEQ ID
NO:10)
[0039] As used herein "ICOS-L" and "ICOS Ligand" are used
interchangeably and refer to the membrane bound natural ligand of
human ICOS. ICOS ligand is a protein that in humans is encoded by
the ICOSLG gene. ICOSLG has also been designated as CD275 (cluster
of differentiation 275). Pseudonyms for ICOS-L include B7RP-1 and
B7-H2.
[0040] As used herein the term "agonist" refers to an antigen
binding protein, for example an ICOS binding protein, which upon
contact with ICOS causes one or more of the following (1)
stimulates or activates the ICOS receptor, (2) enhances, increases
or promotes, induces or prolongs an activity, function or presence
of ICOS and/or (3) enhances, increases, promotes or induces the
expression of the ICOS. Agonist activity can be measured in vitro
by various assays know in the art such as, but not limited to,
measurement of cell signaling, cell proliferation, immune cell
activation markers, cytokine production. Agonist activity can also
be measured in vivo by various assays that measure surrogate end
points such as, but not limited to the measurement of T cell
proliferation or cytokine production.
[0041] As used herein the term "cross competes for binding" refers
to any ICOS binding protein that will compete for binding to ICOS
with any of the ICOS binding proteins of the present invention.
Competition for binding between two molecules for ICOS can be
tested by various methods known in the art including Flow
cytometry, Meso Scale Discovery and ELISA. Binding can be measured
directly, meaning two or more binding proteins can be put in
contact with ICOS and binding may be measured for one or each.
Alternatively, binding of molecules or interest can be tested
against the binding or natural ligand and quantitatively compared
with each other.
[0042] The term "ICOS binding protein" as used herein refers to
antibodies and other protein constructs, such as domains, which are
capable of binding to ICOS. In some instances, the ICOS is human
ICOS. The term "ICOS binding protein" can be used interchangeably
with "ICOS antigen binding protein." Thus, as is understood in the
art, anti-ICOS antibodies and/or ICOS antigen binding proteins
would be considered ICOS binding proteins. As used herein, "antigen
binding protein" is any protein, including but not limited to
antibodies, domains and other constructs described herein, that
binds to an antigen, such as ICOS. As used herein "antigen binding
portion" of an ICOS binding protein would include any portion of
the ICOS binding protein capable of binding to ICOS, including but
not limited to, an antigen binding antibody fragment.
[0043] The term "antibody" is used herein in the broadest sense to
refer to molecules with an immunoglobulin-like domain (for example
IgG, IgM, IgA, IgD or IgE) and includes monoclonal, recombinant,
polyclonal, chimeric, human, humanized, multispecific antibodies,
including bispecific antibodies, and heteroconjugate antibodies; a
single variable domain (e.g., V.sub.H, V.sub.HH, V.sub.L, domain
antibody (dAb.TM.)), antigen binding antibody fragments, Fab,
F(ab').sub.2, Fv, disulphide linked Fv, single chain Fv,
disulphide-linked scFv, diabodies, TANDABS.TM., etc. and modified
versions of any of the foregoing.
[0044] Alternative antibody formats include alternative scaffolds
in which the one or more CDRs of the antigen binding protein can be
arranged onto a suitable non-immunoglobulin protein scaffold or
skeleton, such as an affibody, a SpA scaffold, an LDL receptor
class A domain, an avimer or an EGF domain.
[0045] The term "domain" refers to a folded protein structure which
retains its tertiary structure independent of the rest of the
protein. Generally domains are responsible for discrete functional
properties of proteins and in many cases may be added, removed or
transferred to other proteins without loss of function of the
remainder of the protein and/or of the domain.
[0046] The term "single variable domain" refers to a folded
polypeptide domain comprising sequences characteristic of antibody
variable domains. It therefore includes complete antibody variable
domains such as V.sub.H, V.sub.HH and V.sub.L and modified antibody
variable domains, for example, in which one or more loops have been
replaced by sequences which are not characteristic of antibody
variable domains, or antibody variable domains which have been
truncated or comprise N- or C-terminal extensions, as well as
folded fragments of variable domains which retain at least the
binding activity and specificity of the full-length domain. A
single variable domain is capable of binding an antigen or epitope
independently of a different variable region or domain. A "domain
antibody" or "dAb.TM." may be considered the same as a "single
variable domain". A single variable domain may be a human single
variable domain, but also includes single variable domains from
other species such as rodent nurse shark and Camelid V.sub.HH
dAbs.TM.. Camelid V.sub.HH are immunoglobulin single variable
domain polypeptides that are derived from species including camel,
llama, alpaca, dromedary, and guanaco, which produce heavy chain
antibodies naturally devoid of light chains. Such V.sub.HH domains
may be humanized according to standard techniques available in the
art, and such domains are considered to be "single variable
domains". As used herein V.sub.H includes camelid V.sub.HH
domains.
[0047] An antigen binding fragment may be provided by means of
arrangement of one or more CDRs on non-antibody protein scaffolds.
"Protein Scaffold" as used herein includes but is not limited to an
immunoglobulin (Ig) scaffold, for example an IgG scaffold, which
may be a four chain or two chain antibody, or which may comprise
only the Fc region of an antibody, or which may comprise one or
more constant regions from an antibody, which constant regions may
be of human or primate origin, or which may be an artificial
chimera of human and primate constant regions.
[0048] The protein scaffold may be an Ig scaffold, for example an
IgG, or IgA scaffold. The IgG scaffold may comprise some or all the
domains of an antibody (i.e. CH1, CH2, CH3, V.sub.H, V.sub.L). The
antigen binding protein may comprise an IgG scaffold selected from
IgG1, IgG2, IgG3, IgG4 or IgG4PE. For example, the scaffold may be
IgG1. The scaffold may consist of, or comprise, the Fc region of an
antibody, or is a part thereof.
[0049] The protein scaffold may be a derivative of a scaffold
selected from the group consisting of CTLA-4, lipocalin, Protein A
derived molecules such as Z-domain of Protein A (Affibody, SpA),
A-domain (Avimer/Maxibody); heat shock proteins such as GroEl and
GroES; transferrin (trans-body); ankyrin repeat protein (DARPin);
peptide aptamer; C-type lectin domain (Tetranectin); human
.gamma.-crystallin and human ubiquitin (affilins); PDZ domains;
scorpion toxin kunitz type domains of human protease inhibitors;
and fibronectin/adnectin; which has been subjected to protein
engineering in order to obtain binding to an antigen, such as ICOS,
other than the natural ligand.
[0050] Antigen binding site refers to a site on an antigen binding
protein which is capable of specifically binding to an antigen,
this may be a single variable domain, or it may be paired
V.sub.H/V.sub.L domains as can be found on a standard antibody.
Single-chain Fv (ScFv) domains can also provide antigen-binding
sites. The term "epitope-binding domain" refers to a domain that
specifically binds to a region of an antigen known as the epitope
independently of a different domain.
[0051] The term multi-specific antigen binding protein refers to
antigen binding proteins which comprise at least two different
antigen binding sites. Each of these antigen-binding sites will be
capable of binding to a different epitope, which may be present on
the same antigen or different antigens. The multi-specific antigen
binding protein will have specificity for more than one antigen,
for example two antigens, or for three antigens, or for four
antigens.
[0052] Examples of multi-specific antigen binding proteins include
those that consist of, or consist essentially of, an Fc region of
an antibody, or a part thereof, linked at each end, directly or
indirectly (for example, via a linker sequence) to a binding
domain. Such an antigen binding protein may comprise two binding
domains separated by an Fc region, or part thereof. By separated is
meant that the binding domains are not directly linked to one
another, and may be located at opposite ends (C and N terminus) of
an Fc region, or any other scaffold region.
[0053] The antigen binding protein may comprise two scaffold
regions each bound to two binding domains, for example at the N and
C termini of each scaffold region, either directly or indirectly
via a linker. Each binding domain may bind to a different
antigen.
[0054] As used herein, the term mAbdAb refers to a monoclonal
antibody linked to a further binding domain, in particular a single
variable domain such as a domain antibody. A mAbdAb has at least
two antigen binding sites, at least one of which is from a domain
antibody, and at least one is from a paired V.sub.H/V.sub.L
domain.
[0055] A "dAb.TM. conjugate" refers to a composition comprising a
dAb to which a drug is chemically conjugated by means of a covalent
or noncovalent linkage. Preferably, the dAb and the drug are
covalently bonded. Such covalent linkage could be through a peptide
bond or other means such as via a modified side chain. The
noncovalent bonding may be direct (e.g., electrostatic interaction,
hydrophobic interaction) or indirect (e.g., through noncovalent
binding of complementary binding partners (e.g., biotin and
avidin), wherein one partner is covalently bonded to drug and the
complementary binding partner is covalently bonded to the dAb.TM.).
When complementary binding partners are employed, one of the
binding partners can be covalently bonded to the drug directly or
through a suitable linker moiety, and the complementary binding
partner can be covalently bonded to the dAb.TM. directly or through
a suitable linker moiety.
[0056] As used herein, "dAb.TM. fusion" refers to a fusion protein
that comprises a dAb.TM. and a polypeptide drug (which could be a
dAb.TM. or mAb). The dAb.TM. and the polypeptide drug are present
as discrete parts (moieties) of a single continuous polypeptide
chain.
[0057] In one embodiment, antigen binding proteins of the present
disclosure show cross-reactivity between human ICOS and ICOS from
another species, such as cynomolgus ICOS. In an embodiment, the
antigen binding proteins of the invention specifically bind human
and cynomolgus ICOS. The provision of a drug that can bind human
and monkey species allows one to test results in these system and
make side-by-side comparisons of data using the same drug. Cross
reactivity between other species used in disease models such as dog
or monkey, in particular monkey, is envisaged.
[0058] Competition between an ICOS binding protein and a reference
ICOS binding protein may be determined by competition MSD, ELISA,
FMAT or BIAcore. In one embodiment, the competition assay is
carried out by comparison of an ICOS binding protein with ICOS
ligand binding. There are several possible reasons for this
competition: the two proteins may bind to the same or overlapping
epitopes, there may be steric inhibition of binding, or binding of
the first protein may induce a conformational change in the antigen
that prevents or reduces binding of the second protein.
[0059] The term "neutralizes" as used throughout the present
specification means that the interaction between ICOS and ICOS-L is
reduced in the presence of an antigen binding protein as described
herein in comparison to the interaction of ICOS and ICOS-L in the
absence of the ICOS binding protein, in vitro or in vivo.
Neutralization may be due to one or more of blocking ICOS binding
to its ligand, preventing ICOS from being activated by its ligand,
down regulating ICOS or its receptor, or affecting effector
functionality. For example, the ligand binding competition
described in Examples 3 and 5 may be used to assess the
neutralizing capability of an ICOS binding protein.
[0060] The effect of an ICOS binding protein on the interaction
between ICOS and ICOS-L may be partial or total. A neutralising
ICOS binding protein may block the interaction of ICOS with ICOS-L
by at least 20%, 30% 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%,
84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
relative to ICOS-ICOS-L interactions in the absence of the ICOS
binding protein.
[0061] Neutralization may be determined or measured using one or
more assays known to the skilled person or as described herein.
[0062] Affinity is the strength of binding of one molecule, e.g. an
antigen binding protein of the invention, to another, e.g. its
target antigen, at a single binding site. The binding affinity of
an antigen binding protein to its target may be determined by
equilibrium methods (e.g. enzyme-linked immunoabsorbent assay
(ELISA) or radioimmunoassay (RIA)), or kinetics (e.g. BIACORE.TM.
analysis). For example, the Biacore.TM. methods described in
Example 5 may be used to measure binding affinity.
[0063] Avidity is the sum total of the strength of binding of two
molecules to one another at multiple sites, e.g. taking into
account the valency of the interaction.
[0064] In an embodiment, the equilibrium dissociation constant (KD)
of the ICOS binding protein-ICOS interaction is 100 nM or less, 10
nM or less, 2 nM or less or 1 nM or less. Alternatively the KD may
be between 5 and 10 nM; or between 1 and 2 nM. The KD may be
between 1 pM and 500 pM; or between 500 pM and 1 nM. A skilled
person will appreciate that the smaller the KD numerical value, the
stronger the binding. The reciprocal of KD (i.e. 1/KD) is the
equilibrium association constant (KA) having units M.sup.-1. A
skilled person will appreciate that the larger the KA numerical
value, the stronger the binding.
[0065] The dissociation rate constant (kd) or "off-rate" describes
the stability of the ICOS binding protein ICOS complex, i.e. the
fraction of complexes that decay per second. For example, a kd of
0.01 s.sup.-1 equates to 1% of the complexes decaying per second.
In an embodiment, the dissociation rate constant (kd) is
1.times.10.sup.-3 s.sup.-1 or less, 1.times.10.sup.-4 s.sup.-1 or
less, 1.times.10.sup.-5 s.sup.-1 or less, or 1.times.10.sup.-6
s.sup.-1 or less. The kd may be between 1.times.10.sup.-5 s.sup.-1
and 1.times.10.sup.-4 s.sup.-1; or between 1.times.10.sup.-4
s.sup.-1 and 1.times.10.sup.-3 s.sup.-1.
[0066] The association rate constant (ka) or "on-rate" describes
the rate of ICOS binding protein-ICOS complex formation. In an
embodiment, the association rate constant (ka) is about
1.0.times.10.sup.5 M.sup.- s.sup.-.
[0067] By "isolated" it is intended that the molecule, such as an
antigen binding protein or nucleic acid, is removed from the
environment in which it may be found in nature. For example, the
molecule may be purified away from substances with which it would
normally exist in nature. For example, the mass of the molecule in
a sample may be 95% of the total mass.
[0068] The term "expression vector" as used herein means an
isolated nucleic acid which can be used to introduce a nucleic acid
of interest into a cell, such as a eukaryotic cell or prokaryotic
cell, or a cell free expression system where the nucleic acid
sequence of interest is expressed as a peptide chain such as a
protein. Such expression vectors may be, for example, cosmids,
plasmids, viral sequences, transposons, and linear nucleic acids
comprising a nucleic acid of interest. Once the expression vector
is introduced into a cell or cell free expression system (e.g.,
reticulocyte lysate) the protein encoded by the nucleic acid of
interest is produced by the transcription/translation machinery.
Expression vectors within the scope of the disclosure may provide
necessary elements for eukaryotic or prokaryotic expression and
include viral promoter driven vectors, such as CMV promoter driven
vectors, e.g., pcDNA3.1, pCEP4, and their derivatives, Baculovirus
expression vectors, Drosophila expression vectors, and expression
vectors that are driven by mammalian gene promoters, such as human
Ig gene promoters. Other examples include prokaryotic expression
vectors, such as T7 promoter driven vectors, e.g., pET41, lactose
promoter driven vectors and arabinose gene promoter driven vectors.
Those of ordinary skill in the art will recognize many other
suitable expression vectors and expression systems.
[0069] The term "recombinant host cell" as used herein means a cell
that comprises a nucleic acid sequence of interest that was
isolated prior to its introduction into the cell. For example, the
nucleic acid sequence of interest may be in an expression vector
while the cell may be prokaryotic or eukaryotic. Exemplary
eukaryotic cells are mammalian cells, such as but not limited to,
COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, HepG2, 653, SP2/0, NS0,
293, HeLa, myeloma, lymphoma cells or any derivative thereof. Most
preferably, the eukaryotic cell is a HEK293, NS0, SP2/0, or CHO
cell. E. coli is an exemplary prokaryotic cell. A recombinant cell
according to the disclosure may be generated by transfection, cell
fusion, immortalization, or other procedures well known in the art.
A nucleic acid sequence of interest, such as an expression vector,
transfected into a cell may be extrachromasomal or stably
integrated into the chromosome of the cell.
[0070] A "chimeric antibody" refers to a type of engineered
antibody which contains a naturally-occurring variable region
(light chain and heavy chains) derived from a donor antibody in
association with light and heavy chain constant regions derived
from an acceptor antibody.
[0071] A "humanized antibody" refers to a type of engineered
antibody having its CDRs derived from a non-human donor
immunoglobulin, the remaining immunoglobulin-derived parts of the
molecule being derived from one or more human immunoglobulin(s). In
addition, framework support residues may be altered to preserve
binding affinity (see, e.g., Queen et al. Proc. Natl Acad Sci USA,
86:10029-10032 (1989), Hodgson, et al., Bio/Technology, 9:421
(1991)). A suitable human acceptor antibody may be one selected
from a conventional database, e.g., the KABAT.TM. database, Los
Alamos database, and Swiss Protein database, by homology to the
nucleotide and amino acid sequences of the donor antibody. A human
antibody characterized by a homology to the framework regions of
the donor antibody (on an amino acid basis) may be suitable to
provide a heavy chain constant region and/or a heavy chain variable
framework region for insertion of the donor CDRs. A suitable
acceptor antibody capable of donating light chain constant or
variable framework regions may be selected in a similar manner. It
should be noted that the acceptor antibody heavy and light chains
are not required to originate from the same acceptor antibody. The
prior art describes several ways of producing such humanized
antibodies--see, for example, EP-A-0239400 and EP-A-054951.
[0072] The term "fully human antibody" includes antibodies having
variable and constant regions (if present) derived from human
germline immunoglobulin sequences. The human sequence antibodies of
the invention may include amino acid residues not encoded by human
germline immunoglobulin sequences (e.g., mutations introduced by
random or site-specific mutagenesis in vitro or by somatic mutation
in vivo). Fully human antibodies comprise amino acid sequences
encoded only by polynucleotides that are ultimately of human origin
or amino acid sequences that are identical to such sequences. As
meant herein, antibodies encoded by human immunoglobulin-encoding
DNA inserted into a mouse genome produced in a transgenic mouse are
fully human antibodies since they are encoded by DNA that is
ultimately of human origin. In this situation, human
immunoglobulin-encoding DNA can be rearranged (to encode an
antibody) within the mouse, and somatic mutations may also occur.
Antibodies encoded by originally human DNA that has undergone such
changes in a mouse are fully human antibodies as meant herein. The
use of such transgenic mice makes it possible to select fully human
antibodies against a human antigen. As is understood in the art,
fully human antibodies can be made using phage display technology
wherein a human DNA library is inserted in phage for generation of
antibodies comprising human germline DNA sequence.
[0073] The term "donor antibody" refers to an antibody that
contributes the amino acid sequences of its variable regions, CDRs,
or other functional fragments or analogs thereof to a first
immunoglobulin partner. The donor, therefore, provides the altered
immunoglobulin coding region and resulting expressed altered
antibody with the antigenic specificity and neutralising activity
characteristic of the donor antibody.
[0074] The term "acceptor antibody" refers to an antibody that is
heterologous to the donor antibody, which contributes all (or any
portion) of the amino acid sequences encoding its heavy and/or
light chain framework regions and/or its heavy and/or light chain
constant regions to the first immunoglobulin partner. A human
antibody may be the acceptor antibody.
[0075] The terms "V.sub.H" and "V.sub.L" are used herein to refer
to the heavy chain variable region and light chain variable region
respectively of an antigen binding protein.
[0076] "CDRs" are defined as the complementarity determining region
amino acid sequences of an antigen binding protein. These are the
hypervariable regions of immunoglobulin heavy and light chains.
There are three heavy chain and three light chain CDRs (or CDR
regions) in the variable portion of an immunoglobulin. Thus, "CDRs"
as used herein refers to all three heavy chain CDRs, all three
light chain CDRs, all heavy and light chain CDRs, or at least two
CDRs.
[0077] Throughout this specification, amino acid residues in
variable domain sequences and full length antibody sequences are
numbered according to the Kabat numbering convention. Similarly,
the terms "CDR", "CDRL1", "CDRL2", "CDRL3", "CDRH1", "CDRH2",
"CDRH3" used in the Examples follow the Kabat numbering convention.
For further information, see Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed., U.S. Department of Health and
Human Services, National Institutes of Health (1991).
[0078] It will be apparent to those skilled in the art that there
are alternative numbering conventions for amino acid residues in
variable domain sequences and full length antibody sequences. There
are also alternative numbering conventions for CDR sequences, for
example those set out in Chothia et al. (1989) Nature 342: 877-883.
The structure and protein folding of the antibody may mean that
other residues are considered part of the CDR sequence and would be
understood to be so by a skilled person.
[0079] Other numbering conventions for CDR sequences available to a
skilled person include "AbM" (University of Bath) and "contact"
(University College London) methods. The minimum overlapping region
using at least two of the Kabat, Chothia, AbM and contact methods
can be determined to provide the "minimum binding unit". The
minimum binding unit may be a sub-portion of a CDR.
[0080] Table 1 below represents one definition using each numbering
convention for each CDR or binding unit. The Kabat numbering scheme
is used in Table 1 to number the variable domain amino acid
sequence. It should be noted that some of the CDR definitions may
vary depending on the individual publication used.
TABLE-US-00001 TABLE 1 Kabat Chothia AbM Contact Minimum CDR CDR
CDR CDR binding unit H1 31-35/ 26-32/ 26-35/ 30-35/ 31-32 35A/35B
33/34 35A/35B 35A/35B H2 50-65 52-56 50-58 47-58 52-56 H3 95-102
95-102 95-102 93-101 95-101 L1 24-34 24-34 24-34 30-36 30-34 L2
50-56 50-56 50-56 46-55 50-55 L3 89-97 89-97 89-97 89-96 89-96
[0081] Accordingly, ICOS binding proteins are provided, which
comprises any one or a combination of the following CDRs:
TABLE-US-00002 (SEQ ID NO: 1) CDRH1: DYAMH (SEQ ID NO: 2) CDRH2:
LISIYSDHTNYNQKFQG (SEQ ID NO: 3) CDRH3: NNYGNYGWYFDV (SEQ ID NO: 4)
CDRL1: SASSSVSYMH (SEQ ID NO: 5) CDRL2: DTSKLAS (SEQ ID NO: 6)
CDRL3: FQGSGYPYT
[0082] In one embodiment of the present invention the ICOS binding
protein comprises CDRH1 (SEQ ID NO:1), CDRH2 (SEQ ID NO:2), and
CDRH3 (SEQ ID NO:3) in the heavy chain variable region having the
amino acid sequence set forth in SEQ ID NO:7. ICOS binding proteins
of the present invention comprising the humanized heavy chain
variable region set forth in SEQ ID NO:7 are designated as "H2." In
some embodiments, the ICOS binding proteins of the present
invention comprise a heavy chain variable region having at least
90% sequence identity to SEQ ID NO:7. Suitably, the ICOS binding
proteins of the present invention may comprise a heavy chain
variable region having about 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity
to SEQ ID NO:7.
Humanized Heavy Chain (V.sub.H) Variable Region (H2):
TABLE-US-00003 [0083] (SEQ ID NO: 7) QVQLVQSGAE VKKPGSSVKV
SCKASGYTFT DYAMHWVRQA PGQGLEWMGL ISIYSDHTNY NQKFQGRVTI TADKSTSTAY
MELSSLRSED TAVYYCGRNN YGNYGWYFDV WGQGTTVTVS S
[0084] In one embodiment of the present invention the ICOS binding
protein comprises CDRL1 (SEQ ID NO:4), CDRL2 (SEQ ID NO:5), and
CDRL3 (SEQ ID NO:6) in the light chain variable region having the
amino acid sequence set forth in SEQ ID NO:8. ICOS binding proteins
of the present invention comprising the humanized light chain
variable region set forth in SEQ ID NO:8 are designated as "L5."
Thus, an ICOS binding protein of the present invention comprising
the heavy chain variable region of SEQ ID NO:7 and the light chain
variable region of SEQ ID NO:8 can be designated as H2L5
herein.
[0085] Suitably a leader sequence for the variable heavy chain and
light chain constructs is show in FIG. 9 and includes, but is not
limited to: MGWSCIILFLVATATGVHS (SEQ ID NO:11)
[0086] In some embodiments, the ICOS binding proteins of the
present invention comprise a light chain variable region having at
least 90% sequence identity to the amino acid sequence set forth in
SEQ ID NO:8. Suitably, the ICOS binding proteins of the present
invention may comprise a light chain variable region having about
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to SEQ ID NO:8.
Humanized Light Chain (V.sub.L) Variable Region (L5)
TABLE-US-00004 [0087] (SEQ ID NO: 8) EIVLTQSPAT LSLSPGERAT
LSCSASSSVS YMHWYQQKPG QAPRLLIYDT SKLASGIPAR FSGSGSGTDY TLTISSLEPE
DFAVYYCFQG SGYPYTFGQG TKLEIK
[0088] CDRs or minimum binding units may be modified by at least
one amino acid substitution, deletion or addition, wherein the
variant antigen binding protein substantially retains the
biological characteristics of the unmodified protein, such as a
murine antibody produced from clone 422.2 or an antibody comprising
SEQ ID NO:7 and SEQ ID NO:8.
[0089] It will be appreciated that each of CDR H1, H2, H3, L1, L2,
L3 may be modified alone or in combination with any other CDR, in
any permutation or combination. In one embodiment, a CDR is
modified by the substitution, deletion or addition of up to 3 amino
acids, for example 1 or 2 amino acids, for example 1 amino acid
acid. Typically, the modification is a substitution, particularly a
conservative substitution, for example as shown in Table 2
below.
TABLE-US-00005 TABLE 2 Side chain Members Hydrophobic Met, Ala,
Val, Leu, Ile Neutral hydrophilic Cys, Ser, Thr Acidic Asp, Glu
Basic Asn, Gln, His, Lys, Arg Residues that influence chain
orientation Gly, Pro Aromatic Trp, Tyr, Phe
[0090] For example, in a variant CDR, the amino acid residues of
the minimum binding unit may remain the same, but the flanking
residues that comprise the CDR as part of the Kabat or Chothia
definition(s) may be substituted with a conservative amino acid
residue.
[0091] Such antigen binding proteins comprising modified CDRs or
minimum binding units as described above may be referred to herein
as "functional CDR variants" or "functional binding unit variants".
Suitably, in one embodiment ICOS binding proteins are provided
comprising one or more CDRs having the amino acid sequences set
forth in SEQ ID NOs:1, 2, 3, 4, 5, and/or 6 and/or a function CDR
variant thereof.
[0092] The term "epitope" as used herein refers to that portion of
the antigen that makes contact with a particular binding domain of
the antigen binding protein. An epitope may be linear or
conformational/discontinuous. A conformational or discontinuous
epitope comprises amino acid residues that are separated by other
sequences, i.e. not in a continuous sequence in the antigen's
primary sequence. Although the residues may be from different
regions of the peptide chain, they are in close proximity in the
three dimensional structure of the antigen. In the case of
multimeric antigens, a conformational or discontinuous epitope may
include residues from different peptide chains. Particular residues
comprised within an epitope can be determined through computer
modelling programs or via three-dimensional structures obtained
through methods known in the art, such as X-ray
crystallography.
[0093] The CDRs L1, L2, L3, H1 and H2 tend to structurally exhibit
one of a finite number of main chain conformations. The particular
canonical structure class of a CDR is defined by both the length of
the CDR and by the loop packing, determined by residues located at
key positions in both the CDRs and the framework regions
(structurally determining residues or SDRs). Martin and Thornton
(1996; J Mol Biol 263:800-815) have generated an automatic method
to define the "key residue" canonical templates. Cluster analysis
is used to define the canonical classes for sets of CDRs, and
canonical templates are then identified by analysing buried
hydrophobics, hydrogen-bonding residues, and conserved glycines and
prolines. The CDRs of antibody sequences can be assigned to
canonical classes by comparing the sequences to the key residue
templates and scoring each template using identity or similarity
matrices.
[0094] There may be multiple variant CDR canonical positions per
CDR, per corresponding CDR, per binding unit, per heavy or light
chain variable region, per heavy or light chain, and per antigen
binding protein, and therefore any combination of substitution may
be present in the antigen binding protein of the invention,
provided that the canonical structure of the CDR is maintained such
that the antigen binding protein is capable of specifically binding
ICOS.
[0095] As discussed above, the particular canonical structure class
of a CDR is defined by both the length of the CDR and by the loop
packing, determined by residues located at key positions in both
the CDRs and the framework regions.
[0096] "Percent identity" between a query nucleic acid sequence and
a subject nucleic acid sequence is the "Identities" value,
expressed as a percentage, that is calculated by the BLASTN
algorithm when a subject nucleic acid sequence has 100% query
coverage with a query nucleic acid sequence after a pair-wise
BLASTN alignment is performed. Such pair-wise BLASTN alignments
between a query nucleic acid sequence and a subject nucleic acid
sequence are performed by using the default settings of the BLASTN
algorithm available on the National Center for Biotechnology
Institute's website with the filter for low complexity regions
turned off. Importantly, a query nucleic acid sequence may be
described by a nucleic acid sequence identified in one or more
claims herein.
[0097] "Percent identity" between a query amino acid sequence and a
subject amino acid sequence is the "Identities" value, expressed as
a percentage, that is calculated by the BLASTP algorithm when a
subject amino acid sequence has 100% query coverage with a query
amino acid sequence after a pair-wise BLASTP alignment is
performed. Such pair-wise BLASTP alignments between a query amino
acid sequence and a subject amino acid sequence are performed by
using the default settings of the BLASTP algorithm available on the
National Center for Biotechnology Institute's website with the
filter for low complexity regions turned off. Importantly, a query
amino acid sequence may be described by an amino acid sequence
identified in one or more claims herein.
[0098] The query sequence may be 100% identical to the subject
sequence, or it may include up to a certain integer number of amino
acid or nucleotide alterations as compared to the subject sequence
such that the % identity is less than 100%. For example, the query
sequence is at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or
99% identical to the subject sequence. Such alterations include at
least one amino acid deletion, substitution (including conservative
and non-conservative substitution), or insertion, and wherein said
alterations may occur at the amino- or carboxy-terminal positions
of the query sequence or anywhere between those terminal positions,
interspersed either individually among the amino acids or
nucleotides in the query sequence or in one or more contiguous
groups within the query sequence.
[0099] The % identity may be determined across the entire length of
the query sequence, including the CDR(s). Alternatively, the %
identity may exclude the CDR(s), for example the CDR(s) is 100%
identical to the subject sequence and the % identity variation is
in the remaining portion of the query sequence, so that the CDR
sequence is fixed/intact.
[0100] The variant sequence substantially retains the biological
characteristics of the unmodified protein, such as SEQ ID NO:7 or
SEQ ID NO:8.
[0101] The V.sub.H or V.sub.L sequence may be a variant sequence
with up to 15 amino acid substitutions, additions or deletions. For
example, the variant sequence may have up to 15, 14, 13, 12, 11,
10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitution(s),
addition(s) or deletion(s).
[0102] The sequence variation may exclude the CDR(s), for example
the CDR(s) is the same as the V.sub.H or V.sub.L (or HC or LC)
sequence and the variation is in the remaining portion of the
V.sub.H or V.sub.L (or HC or LC) sequence, so that the CDR sequence
is fixed/intact.
[0103] The skilled person will appreciate that, upon production of
an antigen binding protein such as an antibody, in particular
depending on the cell line used and particular amino acid sequence
of the antigen binding protein, post-translational modifications
may occur. For example, this may include the cleavage of certain
leader sequences, the addition of various sugar moieties in various
glycosylation and phosphorylation patterns, deamidation, oxidation,
disulfide bond scrambling, isomerisation, C-terminal lysine
clipping, and N-terminal glutamine cyclisation. The present
invention encompasses the use of antigen binding proteins which
have been subjected to, or have undergone, one or more
post-translational modifications. Thus an "antigen binding protein"
or "antibody" of the invention includes an "antigen binding
protein" or "antibody", respectively, as defined earlier which has
undergone a post-translational modification such as described
herein.
[0104] Deamidation is an enzymatic reaction primarily converting
asparagine (N) to isoaspartic acid and aspartic acid (D) at
approximately 3:1 ratio. To a much lesser degree, deamidation can
occur with glutamine residues in a similar manner. Deamidation in a
CDR results in a change in charge of the molecule, but typically
does not result in a change in antigen binding, nor does it impact
on PK/PD.
[0105] Oxidation can occur during production and storage (i.e. in
the presence of oxidizing conditions) and results in a covalent
modification of a protein, induced either directly by reactive
oxygen species or indirectly by reaction with secondary by-products
of oxidative stress. Oxidation happens primarily with methionine
residues, but occasionally can occur at tryptophan and free
cysteine residues.
[0106] Disulfide bond scrambling can occur during production and
basic storage conditions. Under certain circumstances, disulfide
bonds can break or form incorrectly, resulting in unpaired cysteine
residues (--SH). These free (unpaired) sulfhydryls (--SH) can
promote shuffling.
[0107] Isomerization typically occurs during production,
purification, and storage (at acidic pH) and usually occurs when
aspartic acid is converted to isoaspartic acid through a chemical
process.
[0108] N-terminal glutamine in the heavy chain and/or light chain
is likely to form pyroglutamate (pGlu). Most pGlu formation happens
in the production bioreactor, but it can be formed
non-enzymatically, depending on pH and temperature of processing
and storage conditions. pGlu formation is considered as one of the
principal degradation pathways for recombinant mAbs.
[0109] C-terminal lysine clipping is an enzymatic reaction
catalyzed by carboxypeptidases, and is commonly observed in
recombinant mAbs. Variants of this process include removal of
lysine from one or both heavy chains. Lysine clipping does not
appear to impact bioactivity and has no effect on mAb effector
function.
[0110] Naturally occurring autoantibodies exist in humans that can
bind to proteins. Autoantibodies can thus bind to endogenous
proteins (present in naive subjects) as well as to proteins or
peptides which are administered to a subject for treatment.
Therapeutic protein-binding autoantibodies and antibodies that are
newly formed in response to drug treatment are collectively termed
anti-drug antibodies (ADAs). Pre-existing antibodies against
molecules such as therapeutic proteins and peptides, administered
to a subject can affect their efficacy and could result in
administration reactions, hypersensitivity, altered clinical
response in treated patients and altered bioavailability by
sustaining, eliminating or neutralizing the molecule. It could be
advantageous to provide molecules for therapy which comprise human
immunoglobulin (antibody) single variable domains or dAbs' which
have reduced immunogenicity (i.e. reduced ability to bind to
pre-existing ADAs when administered to a subject, in particular a
human subject.
[0111] Thus, in one embodiment of the present invention there is
provided a modified dAb' which has reduced ability to bind to
pre-existing antibodies (ADAs) as compared to the equivalent
unmodified molecule. By reduced ability to bind it is meant that
the modified molecule binds with a reduced affinity or reduced
avidity to a pre-existing ADA. Said modified dAb' comprise one or
more modifications selected from: (a) a C-terminal addition,
extension, deletion or tag, and/or (b) one or more amino acid
framework substitutions.
[0112] Polypeptides and dAbs' of the disclosure and agonists
comprising these can be formatted to have a larger hydrodynamic
size, for example, by attachment of a PEG group, serum albumin,
transferrin, transferrin receptor or at least the
transferrin-binding portion thereof, an antibody Fc region, or by
conjugation to an antibody domain. For example, polypeptides dAbs'
and agonists may be formatted as a larger antigen-binding fragment
of an antibody or as an antibody (e.g., formatted as a Fab, Fab',
F(ab).sub.2, F(ab')2, IgG, scFv).
[0113] As used herein, "hydrodynamic size" refers to the apparent
size of a molecule (e.g., an antigen binding protein) based on the
diffusion of the molecule through an aqueous solution. The
diffusion or motion of a protein through solution can be processed
to derive an apparent size of the protein, where the size is given
by the "Stokes radius" or "hydrodynamic radius" of the protein
particle. The "hydrodynamic size" of a protein depends on both mass
and shape (conformation), such that two proteins having the same
molecular mass may have differing hydrodynamic sizes based on the
overall conformation and charge of the protein. An increase in
hydrodynamic size can give an associated decrease in renal
clearance leading to an observed increase in half life
(t.sub.1/2).
[0114] Hydrodynamic size of the antigen binding proteins (e.g.,
domain antibody monomers and multimers) of the disclosure may be
determined using methods which are well known in the art. For
example, gel filtration chromatography may be used to determine the
hydrodynamic size of an antigen binding protein. Suitable gel
filtration matrices for determining the hydrodynamic sizes of
antigen binding proteins, such as cross-linked agarose matrices,
are well known and readily available.
[0115] The size of an antigen binding protein format (e.g., the
size of a PEG moiety attached to a domain antibody monomer), can be
varied depending on the desired application. For example, where
antigen binding protein is intended to leave the circulation and
enter into peripheral tissues, it is desirable to keep the
hydrodynamic size of the ICOS binding protein low to facilitate
extravazation from the blood stream. Alternatively, where it is
desired to have the antigen binding protein remain in the systemic
circulation for a longer period of time the size of the antigen
binding protein can be increased, for example by formatting as an
Ig like protein.
Pharmaceutical Compositions
[0116] Antigen binding protein as described herein may be
incorporated into pharmaceutical compositions for use in the
treatment of the human diseases described herein. In one
embodiment, the pharmaceutical composition comprises an antigen
binding protein optionally in combination with one or more
pharmaceutically acceptable carriers and/or excipients.
[0117] Such compositions comprise a pharmaceutically acceptable
carrier as known and called for by acceptable pharmaceutical
practice.
[0118] Pharmaceutical compositions may be administered by injection
or continuous infusion (examples include, but are not limited to,
intravenous, intraperitoneal, intradermal, subcutaneous,
intramuscular and intraportal). In one embodiment, the composition
is suitable for intravenous administration. Pharmaceutical
compositions may be suitable for topical administration (which
includes, but is not limited to, epicutaneous, inhaled, intranasal
or ocular administration) or enteral administration (which
includes, but is not limited to, oral or rectal
administration).
[0119] Pharmaceutical compositions may comprise between 0.5 mg to
10 g of ICOS binding protein, for example between 5 mg and 1 g of
antigen binding protein. Alternatively, the composition may
comprise between 5 mg and 500 mg, for example between 5 mg and 50
mg. Methods for the preparation of such pharmaceutical compositions
are well known to those skilled in the art. Other excipients may be
added to the composition as appropriate for the mode of
administration and the particular protein used. Examples of
different excipients and their uses are described in Lowe et al.,
(2011).
[0120] Effective doses and treatment regimes for administering the
antigen binding protein may be dependent on factors such as the
age, weight and health status of the patient and disease to be
treated. Such factors are within the purview of the attending
physician. Guidance in selecting appropriate doses may be found in
e.g Bai et al., (2012).
[0121] The pharmaceutical composition may comprise a kit of parts
of the antigen binding protein together with other medicaments,
optionally with instructions for use. For convenience, the kit may
comprise the reagents in predetermined amounts with instructions
for use.
[0122] The terms "individual", "subject" and "patient" are used
herein interchangeably. In one embodiment, the subject is a mammal,
such as a primate, for example a marmoset or monkey. In another
embodiment, the subject is a human.
[0123] The antigen binding protein described herein may also be
used in methods of treatment. Treatment can be therapeutic,
prophylactic or preventative. Treatment encompasses alleviation,
reduction, or prevention of at least one aspect or symptom of a
disease and encompasses prevention or cure of the diseases
described herein.
[0124] The ICOS binding protein or antigen binding portion thereof
described herein is used in an effective amount for therapeutic,
prophylactic or preventative treatment. A therapeutically effective
amount of the ICOS binding protein or antigen binding portion
thereof described herein is an amount effective to ameliorate or
reduce one or more symptoms of, or to prevent or cure, the
disease.
[0125] Thus, in one embodiment ICOS binding proteins or antigen
binding portions thereof of the present invention are provided for
use in therapy. In one embodiment, ICOS binding proteins or antigen
binding portions thereof of the present invention are provided for
use in the treatment of cancer, infectious disease and/or sepsis.
The present invention also provides the use of an ICOS binding
protein or antigen binding portion thereof of the present invention
in the manufacture of a medicament for the treatment of cancer,
infectious disease and/or sepsis.
[0126] Thus, provided herein are isolated ICOS binding proteins or
antigen binding portions thereof or the pharmaceutical compositions
comprising said isolated ICOS binding proteins or antigen binding
portions thereof for use in the treatment of cancer, infectious
disease and/or sepsis.
Production Methods
[0127] Antigen binding proteins may be prepared by any of a number
of conventional techniques. For example, antigen binding proteins
may purified from cells that naturally express them (e.g., an
antibody can be purified from a hybridoma that produces it), or
produced in recombinant expression systems.
[0128] A number of different expression systems and purification
regimes can be used to generate the antigen binding protein of the
invention. Generally, host cells are transformed with a recombinant
expression vector encoding the desired antigen binding protein. A
wide range of host cells can be employed, including Prokaryotes
(including Gram negative or Gram positive bacteria, for example
Escherichia coli, Bacilli sp., Pseudomonas sp., Corynebacterium
sp.), Eukaryotes including yeast (for example Saccharomyces
cerevisiae, Pichia pastoris), fungi (for example Aspergillus sp.),
or higher Eukaryotes including insect cells and cell lines of
mammalian origin (for example, CHO, Perc6, HEK293, HeLa).
[0129] The host cell may be an isolated host cell. The host cell is
usually not part of a multicellular organism (e.g., plant or
animal). The host cell may be a non-human host cell.
[0130] Appropriate cloning and expression vectors for use with
bacterial, fungal, yeast, and mammalian cellular hosts and methods
of cloning are known in the art.
[0131] The cells can be cultured under conditions that promote
expression of the antigen binding protein, and the polypeptide
recovered by conventional protein purification procedures. The
antigen binding proteins contemplated for use herein include
substantially homogeneous antigen binding proteins substantially
free of contaminating materials.
[0132] The skilled person will appreciate that, upon production of
the antigen binding protein, in particular depending on the cell
line used and particular amino acid sequence of the antigen binding
protein, post-translational modifications may occur. This may
include the cleavage of certain leader sequences, the addition of
various sugar moieties in various glycosylation patterns,
deamidation (for example at an asparagine or glutamine residue),
oxidation (for example at a methionine, tryptophan or free cysteine
residue), disulfide bond scrambling, isomerisation (for example at
an aspartic acid residue), C-terminal lysine clipping (for example
from one or both heavy chains), and N-terminal glutamine
cyclisation (for example in the heavy and/or light chain). The
present invention encompasses the use of antibodies which have been
subjected to, or have undergone, one or more post-translational
modifications. The modification may occur in a CDR, the variable
framework region, or the constant region. The modification may
result in a change in charge of the molecule. The modification
typically does not result in a change in antigen binding, function,
bioactivity, nor does it impact the pharmacokinetic (PK) or
pharmacodynamic (PD) characteristics of the ICOS binding
protein.
[0133] The term "Effector Function" as used herein is meant to
refer to one or more of Antibody dependant cell mediated cytotoxic
activity (ADCC), Complement-dependant cytotoxic activity (CDC)
mediated responses, Fc-mediated phagocytosis or antibody dependant
cellular phagocytosis (ADCP) and antibody recycling via the FcRn
receptor.
[0134] The interaction between the constant region of an antigen
binding protein and various Fc receptors (FcR) including
Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16) is
believed to mediate the effector functions of the antigen binding
protein. Significant biological effects can be a consequence of
effector functionality. Usually, the ability to mediate effector
function requires binding of the antigen binding protein to an
antigen and not all antigen binding proteins will mediate every
effector function.
[0135] Effector function can be measured in a number of ways
including for example via binding of the Fc.gamma.RIII on Natural
Killer cells or via Fc.gamma.RI on monocytes/macrophages to measure
for ADCC/ADCP effector function. For example an antigen binding
protein of the present invention can be assessed for ADCC effector
function in a Natural Killer cell assay.
[0136] Practical approaches to evaluate ADCC and/or CDC function
can be found in (Kellner C et al., "Boosting ADCC and CDC activity
by Fc engineering and evaluation of antibody effector functions",
Methods, 1; 65(1):105-13 (2014))
[0137] Some isotypes of human constant regions, in particular IgG4
and IgG2 isotypes, have reduced function of a) activation of
complement by the classical pathway; and b) antibody-dependent
cellular cytotoxicity. Various modifications to the heavy chain
constant region of antigen binding proteins may be carried out
depending on the desired effector property. IgG1 constant regions
containing specific mutations have separately been described to
reduce binding to Fc receptors and therefore reduce ADCC and CDC.
(Kellner C et al., "Boosting ADCC and CDC activity by Fc
engineering and evaluation of antibody effector functions",
Methods, 1; 65(1):105-13 (2014))
[0138] In one embodiment of the present invention there is provided
an antigen binding protein comprising a constant region such that
the antigen binding protein has reduced ADCC and/or complement
activation or effector functionality. In one such embodiment the
heavy chain constant region may comprise a naturally disabled
constant region of IgG2 or IgG4 isotype or a mutated IgG1 constant
region. One example comprises the substitutions of alanine residues
at positions 235 and 237 (EU index numbering).
[0139] The subclass of an antibody in part determines secondary
effector functions, such as complement activation or Fc receptor
(FcR) binding and antibody dependent cell cytotoxicity (ADCC)
(Huber, et al., Nature 229(5284): 419-20 (1971); Brunhouse, et al.,
Mol Immunol 16(11): 907-17 (1979)). In identifying the optimal type
of antibody for a particular application, the effector functions of
the antibodies can be taken into account. For example, hIgG1
antibodies have a relatively long half life, are very effective at
fixing complement, and they bind to both Fc.gamma.RI and
Fc.gamma.RII. In contrast, human IgG4 antibodies have a shorter
half life, do not fix complement and have a lower affinity for the
FcRs. Replacement of serine 228 with a proline (S228P) in the Fc
region of IgG4 reduces heterogeneity observed with hIgG4 and
extends the serum half life (Kabat, et al., "Sequences of proteins
of immunological interest" 5.sup.th Edition (1991); Angal, et al.,
Mol Immunol 30(1): 105-8 (1993)). A second mutation that replaces
leucine 235 with a glutamic acid (L235E) eliminates the residual
FcR binding and complement binding activities (Alegre, et al., J
Immunol 148(11): 3461-8 (1992)). The resulting antibody with both
mutations is referred to as IgG4PE. The numbering of the hIgG4
amino acids was derived from EU numbering reference: Edelman, G. M.
et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). PMID: 5257969. In
one embodiment of the present invention ICOS antigen binding
proteins comprising an IgG4 Fc region comprising the replacement
S228P and L235E may have the designation IgG4PE. Thus, an ICOS
binding protein having the heavy chain variable region H2 and the
light chain variable region L5 and an IgG4PE Fc region will be
designated as H2L5 IgG4PE or synonymously as H2L5 hIgG4PE.
Enhanced ADCC/CDC
[0140] As is understood in the art various techniques are known
which will increase the ADCC and/or the CDC activity of an
antibody. These include, but are not limited to, various mutation
in the Fc region, Complegent and Potelligent technologies. In one
aspect of the present invention one or more ADCC/CDC enhancing
techniques may be applied to the ICOS binding proteins of the
present invention.
Mutation
[0141] Human IgG1 constant regions containing specific mutations or
altered glycosylation on residue Asn297 have also been described to
enhance binding to Fc receptors. In some cases these mutations have
also been shown to enhance ADCC and CDC, see for example, Kellner
(2013).
[0142] In one embodiment of the present invention, such mutations
are in one or more of positions selected from 239, 332 and 330
(IgG1), or the equivalent positions in other IgG isotypes. Examples
of suitable mutations are S239D and I332E and A330L. In one
embodiment the antigen binding protein of the invention herein
described is mutated at positions 239 and 332, for example S239D
and I332E or in a further embodiment it is mutated at three or more
positions selected from 239 and 332 and 330, for example S239D and
I332E and A330L (EU index numbering).
Complegent
[0143] In one embodiment of the present invention there is provided
an antigen binding protein comprising a chimeric heavy chain
constant region for example an antigen binding protein comprising a
chimeric heavy chain constant region with at least one CH2 domain
from IgG3 such that the antigen binding protein has enhanced
effector function, for example wherein it has enhanced ADCC or
enhanced CDC, or enhanced ADCC and CDC functions. In one such
embodiment, the antigen binding protein may comprise one CH2 domain
from IgG3 or both CH2 domains may be from IgG3.
[0144] Also provided is a method of producing an antigen binding
protein according to the invention comprising the steps of:
a) culturing a recombinant host cell comprising an expression
vector comprising an isolated nucleic acid as described herein
wherein the expression vector comprises a nucleic acid sequence
encoding an Fc domain having both IgG1 and IgG3 Fc domain amino
acid residues; and b) recovering the antigen binding protein.
[0145] Such methods for the production of antigen binding proteins
can be performed, for example, using the COMPLEGENT.TM. technology
system available from BioWa, Inc. (Princeton, N.J.) and Kyowa Hakko
Kogyo (now, Kyowa Hakko Kirin Co., Ltd.) Co., Ltd. In which a
recombinant host cell comprising an expression vector in which a
nucleic acid sequence encoding a chimeric Fc domain having both
IgG1 and IgG3 Fc domain amino acid residues is expressed to produce
an antigen binding protein having enhanced complement dependent
cytotoxicity (CDC) activity that is increased relative to an
otherwise identical antigen binding protein lacking such a chimeric
Fc domain. Aspects of the COMPLEGENT.TM. technology system are
described in WO2007011041 and US20070148165 each of which are
incorporated herein by reference. In an alternative embodiment CDC
activity may be increased by introducing sequence specific
mutations into the Fc region of an IgG chain. Those of ordinary
skill in the art will also recognize other appropriate systems.
Potelligent
[0146] The present invention also provides a method for the
production of an antigen binding protein according to the invention
comprising the steps of:
a) culturing a recombinant host cell comprising an expression
vector comprising the isolated nucleic acid as described herein,
wherein the FUT8 gene encoding alpha-1,6-fucosyltransferase has
been inactivated in the recombinant host cell; and b) recovering
the antigen binding protein.
[0147] Such methods for the production of antigen binding proteins
can be performed, for example, using the POTELLIGENT.TM. technology
system available from BioWa, Inc. (Princeton, N.J.) in which
CHOK1SV cells lacking a functional copy of the FUT8 gene produce
monoclonal antibodies having enhanced antibody dependent cell
mediated cytotoxicity (ADCC) activity that is increased relative to
an identical monoclonal antibody produced in a cell with a
functional FUT8 gene. Aspects of the POTELLIGENT.TM. technology
system are described in U.S. Pat. No. 7,214,775, U.S. Pat. No.
6,946,292, WO0061739 and WO0231240 all of which are incorporated
herein by reference. Those of ordinary skill in the art will also
recognize other appropriate systems.
[0148] It will be apparent to those skilled in the art that such
modifications may not only be used alone but may be used in
combination with each other in order to further enhance effector
function.
[0149] In one such embodiment of the present invention there is
provided an antigen binding protein comprising a heavy chain
constant region which comprises a mutated and chimaeric heavy chain
constant region for example wherein an antigen binding protein
comprising at least one CH2 domain from IgG3 and one CH2 domain
from IgG1, wherein the IgG1 CH2 domain has one or more mutations at
positions selected from 239 and 332 and 330 (for example the
mutations may be selected from S239D and I332E and A330L) such that
the antigen binding protein has enhanced effector function, for
example wherein it has one or more of the following functions,
enhanced ADCC or enhanced CDC, for example wherein it has enhanced
ADCC and enhanced CDC. In one embodiment the IgG1 CH2 domain has
the mutations S239D and I332E.
[0150] In an alternative embodiment of the present invention there
is provided an antigen binding protein comprising a chimaeric heavy
chain constant region and which has an altered glycosylation
profile. In one such embodiment the heavy chain constant region
comprises at least one CH2 domain from IgG3 and one CH2 domain from
IgG1 and has an altered glycosylation profile such that the ratio
of fucose to mannose is 0.8:3 or less, for example wherein the
antigen binding protein is defucosylated so that said antigen
binding protein has an enhanced effector function in comparison
with an equivalent antigen binding protein with an immunoglobulin
heavy chain constant region lacking said mutations and altered
glycosylation profile, for example wherein it has one or more of
the following functions, enhanced ADCC or enhanced CDC, for example
wherein it has enhanced ADCC and enhanced CDC
[0151] In an alternative embodiment the antigen binding protein has
at least one IgG3 CH2 domain and at least one heavy chain constant
domain from IgG1 wherein both IgG CH2 domains are mutated in
accordance with the limitations described herein.
[0152] In one aspect of the invention there is provided a method of
producing an antigen binding protein according to the invention
described herein comprising the steps of:
a) culturing a recombinant host cell containing an expression
vector containing an isolated nucleic acid as described herein,
said expression vector further comprising a Fc nucleic acid
sequence encoding a chimeric Fc domain having both IgG1 and IgG3 Fc
domain amino acid residues, and wherein the FUT8 gene encoding
alpha-1,6-fucosyltransferase has been inactivated in the
recombinant host cell; and b) recovering the antigen binding
protein.
[0153] Such methods for the production of antigen binding proteins
can be performed, for example, using the ACCRETAMAB.TM. technology
system available from BioWa, Inc. (Princeton, N.J.) which combines
the POTELLIGENT.TM. and COMPLEGENT.TM. technology systems to
produce an antigen binding protein having both ADCC and CDC
enhanced activity that is increased relative to an otherwise
identical monoclonal antibody lacking a chimeric Fc domain and
which has fucose on the oligosaccharide
[0154] In yet another embodiment of the present invention there is
provided an antigen binding protein comprising a mutated and
chimeric heavy chain constant region wherein said antigen binding
protein has an altered glycosylation profile such that the antigen
binding protein has enhanced effector function, for example wherein
it has one or more of the following functions, enhanced ADCC or
enhanced CDC. In one embodiment the mutations are selected from
positions 239 and 332 and 330, for example the mutations are
selected from S239D and I332E and A330L. In a further embodiment
the heavy chain constant region comprises at least one CH2 domain
from IgG3 and one Ch2 domain from IgG1. In one embodiment the heavy
chain constant region has an altered glycosylation profile such
that the ratio of fucose to mannose is 0.8:3 or less for example
the antigen binding protein is defucosylated, so that said antigen
binding protein has an enhanced effector function in comparison
with an equivalent non-chimaeric antigen binding protein or with an
immunoglobulin heavy chain constant region lacking said mutations
and altered glycosylation profile.
[0155] The long half-life of IgG antibodies is reported to be
dependent on its binding to FcRn. Therefore, substitutions that
increase the binding affinity of IgG to FcRn at pH 6.0 while
maintaining the pH dependence of the interaction by engineering the
constant region have been extensively studied Kuo and Aveson
(2011).
[0156] Another means of modifying antigen binding proteins of the
present invention involves increasing the in-vivo half life of such
proteins by modification of the immunoglobulin constant domain or
FcRn (Fc receptor neonate) binding domain.
[0157] In adult mammals, FcRn, also known as the neonatal Fc
receptor, plays a key role in maintaining serum antibody levels by
acting as a protective receptor that binds and salvages antibodies
of the IgG isotype from degradation. IgG molecules are endocytosed
by endothelial cells, and if they bind to FcRn, are recycled out
into circulation. In contrast, IgG molecules that do not bind to
FcRn enter the cells and are targeted to the lysosomal pathway
where they are degraded.
[0158] The neonatal FcRn receptor is believed to be involved in
both antibody clearance and the transcytosis across tissues, Kuo
and Aveson, (2011). Human IgG1 residues determined to interact
directly with human FcRn includes Ile253, Ser254, Lys288, Thr307,
Gln311, Asn434 and His435. Switches at any of these positions
described in this section may enable increased serum half-life
and/or altered effector properties of antigen binding proteins of
the invention.
Mutations to Increase Half Life by Increasing Affinity to FcRn
[0159] Antigen binding proteins of the present invention may have
one or more amino acid modifications that increase the affinity of
the constant domain or fragment thereof for FcRn. These may result
in increased half-life of these proteins Kuo and Aveson (2011)
Increasing the half-life of therapeutic and diagnostic IgG's and
other bioactive molecules has many benefits including reducing the
amount and/or frequency of dosing of these molecules. In one
embodiment there is therefore provided an antigen binding according
to the invention provided herein or a fusion protein comprising all
or a portion (an FcRn binding portion) of an IgG constant domain
having one or more of these amino acid modifications and a non-IgG
protein or non-protein molecule conjugated to such a modified IgG
constant domain, wherein the presence of the modified IgG constant
domain increases the in vivo half life of the antigen binding
protein.
[0160] A number of methods are known that can result in increased
half-life (Kuo and Aveson, (2011)), including amino acid
modifications may be generated through techniques including alanine
scanning mutagenesis, random mutagenesis and screening to assess
the binding to FcRn and/or the in vivo behaviour. Computational
strategies followed by mutagenesis may also be used to select one
of amino acid mutations to mutate.
[0161] The present invention therefore provides a variant of an
antigen binding protein with optimized binding to FcRn. In a
preferred embodiment, the said variant of an antigen binding
protein comprises at least one amino acid modification in the Fc
region of said antigen binding protein, wherein said modification
is selected from the group consisting of 226, 227, 228, 230, 231,
233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267,
269, 270, 276, 284, 285, 288, 289, 290, 291, 292, 294, 297, 298,
299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322,
325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350,
352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378,
380, 382, 384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396,
397, 398, 399, 400, 401 403, 404, 408, 411, 412, 414, 415, 416,
418, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439, 440,
443, 444, 445, 446 and 447 of the Fc region as compared to said
parent polypeptide, wherein the numbering of the amino acids in the
Fc region is that of the EU index in Kabat.
[0162] In a further aspect of the invention the modifications are
M252Y/S254T/T256E.
[0163] Additionally, various publications describe methods for
obtaining physiologically active molecules whose half-lives are
modified, see for example Kontermann (2009) either by introducing
an FcRn-binding polypeptide into the molecules or by fusing the
molecules with antibodies whose FcRn-binding affinities are
preserved but affinities for other Fc receptors have been greatly
reduced or fusing with FcRn binding domains of antibodies.
pH Switch Technology to Increase Half Life
[0164] Although substitutions in the constant region are able to
significantly improve the functions of therapeutic IgG antibodies,
substitutions in the strictly conserved constant region have the
risk of immunogenicity in human and substitution in the highly
diverse variable region sequence might be less immunogenic. Reports
concerned with the variable region include engineering the CDR
residues to improve binding affinity to the antigen and
engineering, the CDR and framework residues to improve stability
and decrease immunogenicity risk. As is known, improved affinity to
the antigen can be achieved by affinity maturation using the phage
or ribosome display of a randomized library.
[0165] Improved stability can be rationally obtained from sequence-
and structure-based rational design. Decreased immunogenicity risk
(deimmunization) can be accomplished by various humanization
methodologies and the removal of T-cell epitopes, which can be
predicted using in silico technologies or determined by in vitro
assays. Additionally, variable regions have been engineered to
lower pI. A longer half life was observed for these antibodies as
compared to wild type antibodies despite comparable FcRn binding.
Engineering or selecting antibodies with pH dependent antigen
binding to modify antibody and/or antigen half life eg IgG2
antibody half life can be shortened if antigen-mediated clearance
mechanisms normally degrade the antibody when bound to the antigen.
Similarly, the antigen:antibody complex can impact the half-life of
the antigen, either extending half-life by protecting the antigen
from the typical degradation processes, or shortening the half-life
via antibody-mediated degradation. One embodiment relates to
antibodies with higher affinity for antigen at pH 7.4 as compared
to endosomal pH (i.e., pH 5.5-6.0) such that the KD ratio at
pH5.5/pH 7.4 or at pH 6.0/pH 7.4 is 2 or more. For example to
enhance the pharmacokinetic (PK) and pharmacodynamic (PD)
properties of the antibody, it is possible to engineer pH-sensitive
binding to the antibody by introducing histidines into CDR
residues.
[0166] Additionally, methods of producing an antigen binding
protein with a decreased biological half-life are also provided. A
variant IgG in which His435 is mutated to alanine results in the
selective loss of FcRn binding and a significantly reduced serum
half-life (see for example U.S. Pat. No. 6,165,745 discloses a
method of producing an antigen binding protein with a decreased
biological half-life by introducing a mutation into the DNA segment
encoding the antigen binding protein. The mutation includes an
amino acid substitution at position 253, 310, 311, 433, or 434 of
the Fc-hinge domain.
Linkers
[0167] Protein scaffolds may be the same as naturally occurring
sequences, such as Ig sequences, or be fragments of naturally
occurring sequences, and may contain additional sequences which may
be naturally occurring, from a difference source or synthetic, and
which may be added at the N or C terminus of the scaffold. Such
additional sequences may be considered to be linkers when they link
an epitope binding domain and protein scaffold, such as those
defined herein.
[0168] In another aspect the antigen binding construct consists of,
or consists essentially of, an Fc region of an antibody, or a part
thereof, linked at each end, directly or indirectly (for example,
via a linker sequence) to an epitope binding domain. Such an
antigen binding construct may comprise 2 epitope-binding domains
separated by an Fc region, or part thereof. By separated is meant
that the epitope-binding domains are not directly linked to one
another, and in one aspect are located at opposite ends (C and N
terminus) of an Fc region, or any other scaffold region.
[0169] In one aspect the antigen binding construct comprises 2
scaffold regions each bound to 2 epitope binding domains, for
example at the N and C termini of each scaffold region, either
directly or indirectly via a linker.
[0170] Protein scaffolds of the present invention may be linked to
epitope-binding domains by the use of linkers. Examples of suitable
linkers include amino acid sequences which may be from 1 amino acid
to 150 amino acids in length, or from 1 amino acid to 140 amino
acids, for example, from 1 amino acid to 130 amino acids, or from 1
to 120 amino acids, or from 1 to 80 amino acids, or from 1 to 50
amino acids, or from 1 to 20 amino acids, or from 1 to 10 amino
acids, or from 5 to 18 amino acids. Such sequences may have their
own tertiary structure, for example, a linker of the present
invention may comprise a single variable domain. The size of a
linker in one embodiment is equivalent to a single variable domain.
Suitable linkers may be of a size from 1 to 100 angstroms, for
example may be of a size from 20 to 80 angstroms or for example may
be of a size from 20 to 60 angstroms or for example less than 40
angstroms, or less than 20 angstroms, or less than 5 angstroms in
length.
[0171] In one embodiment of the present invention, ICOS binding
proteins are provided comprising one or more of: CDRH1 as set forth
in SEQ ID NO:1; CDRH2 as set forth in SEQ ID NO:2; CDRH3 as set
forth in SEQ ID NO:3; CDRL1 as set forth in SEQ ID NO:4; CDRL2 as
set forth in SEQ ID NO:5 and/or CDRL3 as set forth in SEQ ID NO:6
or a direct equivalent of each CDR wherein a direct equivalent has
no more than two amino acid substitutions in said CDR.
[0172] In one embodiment of the present invention, ICOS binding
proteins are provided which specifically binds to human ICOS
comprising a V.sub.H domain comprising an amino acid sequence at
least 90% identical to the amino acid sequence set forth in SEQ ID
NO:7 and/or a V.sub.L domain comprising an amino acid sequence at
least 90% identical to the amino acid sequence set forth in SEQ ID
NO:8. In one aspect, the ICOS binding proteins of the present
invention, also bind to cynomolgus ICOS. In one aspect, they do not
bind to murine ICOS.
[0173] In one embodiment, the ICOS binding proteins of the
invention are ICOS agonists. In one aspect, the ICOS binding
proteins increase IFN-gamma production in the presence of Tcells.
In another aspect, the ICOS binding proteins of the present
invention stimulate Tcell proliferation.
[0174] In one embodiment, the ICOS binding protein of the invention
bind to human ICOS with [0175] an association rate constant (kon)
of at least 1.times.10.sup.5 M-1 s-1; and a dissociation rate
constant (koff) of less than 6.times.10-5 s-1; or [0176] a
dissociation constant (Kd) of less than 100 nM
[0177] wherein the high affinity is measured by Biacore.
[0178] In one embodiment the ICOS binding protein comprises CDRH3
(SEQ ID NO:3) or a variant of SEQ ID NO. 3. In another embodiment
the ICOS binding proteins comprise one or more of: CDRH1 (SEQ ID
NO:1); CDRH2 (SEQ ID NO:2); CDRH3 (SEQ ID NO:3); CDRL1 (SEQ ID
NO:4); CDRL2 (SEQ ID NO:5); and/or CDRL3 (SEQ ID NO:6). In one
embodiment, the ICOS binding proteins comprise heavy chain CDRs as
set forth in SEQ ID NO:1; SEQ ID NO:2; and SEQ ID NO:3 and light
chain CDRs as set forth in SEQ ID NO:4; SEQ ID NO:5; and SEQ ID
NO:6.
[0179] In one embodiment, the ICOS binding proteins comprise a
V.sub.H domain having 90% sequence identity to the amino acid
sequence set forth in SEQ ID NO:7; and a V.sub.L domain having 90%
sequence identity to the amino acid sequence shown in the amino
acid sequence set forth in SEQ ID NO:8. In one aspect, the ICOS
binding proteins comprise a V.sub.H domain having the amino acid
sequence set forth in SEQ ID NO.7 and the V.sub.L domain comprising
the amino acid sequence as set forth in SEQ ID NO:8. In one aspect,
the ICOS binding proteins comprise a heavy chain variable domain
consisting of SEQ ID NO:7. In one aspect, the ICOS binding protein
comprises a light chain variable domain consisting of SEQ ID
NO:8.
[0180] In one embodiment, the invention provides an ICOS binding
protein or antigen binding portion thereof comprising a V.sub.H
domain comprising an amino acid sequence at least 90% identical to
the amino acid sequence set forth in SEQ ID NO:7; and a V.sub.L
domain comprising an amino acid sequence at least 90% identical to
the amino acid sequence as set forth in SEQ ID NO:8 wherein said
ICOS binding protein or antigen binding portion thereof
specifically binds to human ICOS. In one embodiment, the ICOS
binding protein or antigen binding portion thereof comprising a
V.sub.H domain comprising an amino acid sequence at least 90%
identical to the amino acid sequence set forth in SEQ ID NO:7; and
a V.sub.L domain comprising an amino acid sequence at least 90%
identical to the amino acid sequence as set forth in SEQ ID NO:8
wherein said ICOS binding protein or antigen binding portion
thereof specifically binds to human ICOS further comprises heavy
chain CDRs having the amino acid sequences set forth in SEQ ID
NO:1; SEQ ID NO:2; and SEQ ID NO:3 and light chain CDRs having the
amino acid sequences set forth in SEQ ID NO:4; SEQ ID NO:5; and SEQ
ID NO:6. In one aspect, the ICOS binding protein or antigen binding
portion thereof comprises a V.sub.H domain comprising an amino acid
sequence set forth in SEQ ID NO:7; and a V.sub.L domain comprises
the amino acid sequence set forth in SEQ ID NO:8. In one
embodiment, the ICOS binding protein or antigen binding portion
thereof is an agonist to human ICOS. In one embodiment the ICOS
binding protein or antigen binding portion thereof further
comprising an IgG4 isotype scaffold or a variant thereof. In one
embodiment, the ICOS binding protein or antigen binding portion
thereof comprises a hIgG4PE scaffold.
[0181] In one embodiment, the ICOS binding protein of the present
invention is a humanized monoclonal antibody comprising a heavy
chain amino acid sequence having at least 90%, 91%, 92,%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid
sequence set forth in SEQ ID NO:23.
TABLE-US-00006 (SEQ ID NO: 23)
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYAMHWVRQAPGQGLEWM
GLISIYSDHTNYNQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC
GRNNYGNYGWYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV
PSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA
KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT
ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA
LHNHYTQKSLSLSLGK
[0182] In one embodiment, the ICOS binding protein of the present
invention is a humanized monoclonal antibody comprising a light
chain amino acid sequence having at least 90%, 91%, 92,%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid
sequence set forth in SEQ ID NO:24.
TABLE-US-00007 (SEQ ID NO: 24)
EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIY
DTSKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCFQGSGYPYT
FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGEC
[0183] In one embodiment, the ICOS binding protein of the present
invention is a humanized monoclonal antibody comprising the heavy
chain amino acid sequence set forth in SEQ ID NO:23 and the light
chain amino acid sequence set forth in SEQ ID NO:24. In one
embodiment, the ICOS binding protein of the present invention
further comprises an hIgGPE scaffold.
[0184] In one embodiment, the ICOS binding protein or antigen
binding portion thereof wherein said ICOS binding protein is a
humanized monoclonal antibody. Also provided by the present
invention are pharmaceutical compositions comprising the ICOS
binding protein or antigen binding portion thereof of claim and a
pharmaceutically acceptable carrier.
[0185] The present invention provides methods of treating a disease
selected from cancer, infectious disease, and/or sepsis in a human
in need thereof which method comprises the step of administering a
pharmaceutical composition of the present invention said human. In
one aspect, the method further comprises administering at least one
anti-neoplastic agent, at least one second immune-modulatory agent,
and/or at least one immunostimulatory adjuvant to said human. In
one aspect, the second immuno-modulatory agent is selected from: an
anti-CTLA4 antibody, and anti-PD-1 antibody, an anti-PDL1 antibody
and an anti OX40 antibody. In one aspect the anti-CTLA4 antibody is
ipilimumab. In one aspect the anti-PD-1 antibody is selected from
pembrolizumab and/or nivolumab.
[0186] In one embodiment of the present invention, methods are
provided for treating cancer in a human comprising administering a
therapeutically acceptable amount of the ICOS binding protein or
antigen binding portion thereof to a human. In some aspects the
cancer is selected from colorectal cancer (CRC), esophageal,
cervical, bladder, breast, head and neck, ovarian, melanoma, renal
cell carcinoma (RCC), EC squamous cell, non-small cell lung
carcinoma, mesothelioma, and prostate cancer.
[0187] In one embodiment, methods are provided for treating
infectious disease in a human comprising administering a
therapeutically acceptable amount of the ICOS binding protein or
antigen binding portion thereof to a human. In one aspect, the
infectious diseases is HIV.
[0188] In one embodiment, methods are provided for treating sepsis
in a human comprising administering a therapeutically acceptable
amount of the ICOS binding protein or antigen binding portion
thereof to a human.
[0189] In one embodiment, methods are provided for stimulating T
cell proliferation, inducing T cell activation and/or inducing
cytokine production in a human comprising administering a
pharmaceutical composition of the invention to said human.
[0190] The present invention also provides polynucleotides encoding
the ICOS binding protein or antigen binding portion thereof of the
present invention. In one embodiment, host cells are provided
comprising polynucleotides encoding the ICOS binding proteins or
antigen binding portions thereof of the present invention. The
present invention also provides methods of making an ICOS binding
protein or antigen binding portion thereof comprising the steps of
a) culturing host cell comprising a polynucleotide encoding an ICOS
binding protein or antigen binding portion thereof of the present
invention under suitable conditions to express said ICOS binding
protein or antigen binding portion thereof and b) isolating said
ICOS binding protein or antigen binding portion thereof.
[0191] The present invention provides an isolated humanized
monoclonal antibody comprising a V.sub.H domain comprising an amino
acid sequence set forth in SEQ ID NO:7; a V.sub.L domain comprising
an amino acid sequence set forth in SEQ ID NO:8; and a hIgG4
scaffold or a variant thereof. In one aspect, the hIgG4 scaffold is
hIgG4PE.
[0192] In one embodiment, ICOS binding proteins or antigen binding
portions thereof are provided, wherein the ICOS binding protein or
antigen binding portion thereof cross-competes for binding for
human ICOS with a reference antibody or antigen binding portion
thereof comprising a V.sub.H domain comprising an amino acid
sequence set forth in SEQ ID NO:7; and a V.sub.L domain comprising
the amino acid sequence set forth in SEQ ID NO:8.
[0193] In one embodiment, the ICOS binding proteins of the
invention stimulate T cell proliferation when placed in contact
with a T cell. In one embodiment, the ICOS binding proteins of the
invention induce T cell activation when placed in contact with a T
cell. T cell activation can be measured by an increase in percent
expression levels of certain activation markers such as, but not
limited to, CD69, CD25, and/or OX40. In one embodiment, the ICOS
binding proteins of the present invention stimulate cytokine
production when placed in contact with a T cell. The ICOS binding
proteins bind to human Fc.gamma.RIIb but do not bind to human
Fc.gamma.RIIa or human Fc.gamma.RIIIa. Additionally, the ICOS
binding proteins suitably do not deplete ICOS expressing T cells
when contacted with ICOS expressing T cells. In some aspects, the
ICOS binding proteins cross-link a T cell with a second cell when
contacted with said T cell in the presence of a second cell. This
cross-linking can occur through engagement of the ICOS binding
protein with a Fc.gamma.R on the second cell. Fc.gamma.R expressing
cells include, but are not limited to monocytes, B lymphocytes,
follicular dendritic cells, natural killer cells, macrophages,
neutrophils, eosinophils, basophils, and mast cells. Thus, in one
embodiment the ICOS binding proteins can be administered to a
mammal wherein the ICOS binding protein will act as an agonist to
ICOS on a T cell and will also engage an Fc.gamma.R on a second
cell.
[0194] In one embodiment, the ICOS binding proteins comprise a
scaffold selected from human IgG1 isotype or variant thereof and
human IgG4 isotype or variant thereof. Suitably, the scaffold
comprises a human IgG4 isotype scaffold or variant thereof. In one
aspect, the scaffold comprises a hIgG4PE scaffold.
[0195] In one embodiment, the ICOS binding protein is a monoclonal
antibody. Suitably the ICOS binding protein is a humanized
monoclonal antibody. In one aspect the monoclonal antibodies of the
present invention can be fully human.
[0196] In another aspect, the ICOS binding protein is a fragment
which is a Fab, Fab', F(ab').sub.2, Fv, diabody, triabody,
tetrabody, miniantibody, minibody, isolated V.sub.H or isolated
V.sub.L. In one embodiment, the ICOS binding protein is an antigen
binding portion thereof.
[0197] In some aspects the ICOS binding protein binds to human ICOS
with an affinity of stronger than 0.6 nM. In one aspect, the
affinity is 100 nM or stronger. In one embodiment the ICOS binding
protein has a KD of 100 nM for ICOS. Suitably, the KD of the ICOS
binding protein for ICOS is 100 nM or less, 50 nM or less, 25 nM or
less, 10 nM or less, 2 nM or less or 1 nM or less.
[0198] In one embodiment, the present invention provides humanized
monoclonal antibodies that are agonists to human ICOS. In one
embodiment, the present invention provides humanized monoclonal
antibodies comprising heavy chain variable region CDRs having the
amino acid sequences set forth in SEQ ID NO:1; SEQ ID NO:2; and SEQ
ID NO:3 and light chain variable region CDRS having the amino acid
sequences set forth in SEQ ID NO:4; SEQ ID NO:5; and SEQ ID NO:6.
In one aspect, the humanized monoclonal antibody is able to
stimulate cytokine production and/or T cell proliferation when
contacted with a T cell while not inducing complement, ADCC or CDC.
In one embodiment, the humanized monoclonal antibody has variant
human IgG1 Fc region. In one embodiment, the humanized monoclonal
antibody has a human IgG4 Fc region or variant thereof. In one
embodiment, the humanized monoclonal antibody has a hIgG4PE Fc
region. In one aspect, the humanized monoclonal antibody comprises
a V.sub.H domain comprising an amino acid sequence at least 90%
identical to the amino acid sequence as set forth in SEQ ID NO:7;
and a V.sub.L domain comprising an amino acid sequence at least 90%
identical to the amino acid sequence as set forth in SEQ ID NO:8.
In one aspect the humanized monoclonal antibody comprises a V.sub.H
domain comprising an amino acid sequence set forth in SEQ ID NO:7;
and a V.sub.L domain comprising the amino acid sequence as set
forth in SEQ ID NO:8. In one aspect the humanized monoclonal
antibody comprises and hIgG4PE scaffold. Furthermore, humanized
monoclonal antibodies of the present invention are shown to
stimulate T cell proliferation when contacted with a CD4+ or a CD8+
T cell. Humanized monoclonal antibodies of the present invention
are shown to induce T cell activation and stimulate cytokine
production.
[0199] In one embodiment, the humanized monoclonal antibody
comprises an hIgG4PE scaffold and comprises a V.sub.H domain
comprising an amino acid sequence set forth in SEQ ID NO:7 and a
V.sub.L domain comprising an amino acid sequence set forth in SEQ
ID NO:8. The antibodies of the present invention may stimulate
cytokine production when contacted with a T cell.
[0200] In one embodiment, an ICOS binding protein is provided that
competes for ICOS binding with any one of the ICOS binding proteins
of the invention. As is understood in the art and described herein,
binding competition can be measured by comparing competition for
ligand binding to ICOS in the presence of one or more ICOS binding
proteins. As is also understood in the art, ICOS is expressed on
CD4+ and CD8+ T cells as well as Treg cells. The ICOS binding
proteins of the present invention act as agonist to ICOS on T
cells. They also act to block the interaction between ICOS-L and
ICOS expressed on both T cells and Treg cells. Thus, in one
embodiment, methods are provided to block the interaction of ICOS-L
with ICOS on Treg cells. ICOS expressing Treg cells can be found in
various types of tumors including liquid tumors such as lymphoma.
Thus, in one aspect of the present invention, methods of treating a
cancer are provided comprising administering an ICOS binding
protein of the invention wherein the ICOS binding protein blocks
the interaction of ICOS-L with ICOS on Treg cells.
[0201] Further to the invention, are pharmaceutical compositions
comprising an ICOS binding protein or a monoclonal antibody
described herein. In one aspect the pharmaceutical composition of
the present invention further comprise at least one anti-neoplastic
agent. In one aspect the pharmaceutical composition of the present
invention further comprise at least one second immunomodulatory
agent. In one aspect, the pharmaceutical composition of the present
invention further comprising at least one immunostimulatory
adjuvant.
[0202] In one embodiment, methods are provided for treating cancer
and/or infectious disease in a human in need thereof wherein said
method comprises the step of administering a pharmaceutical
composition of the invention to said human. In one embodiment the
human has cancer. In one embodiment the human has an infectious
disease. In one embodiment the human has HIV. In one aspect the
method further comprises administering at least one anti-neoplastic
agent to said human. In another aspect the method further comprises
administering at least one second immune-modulatory agent to said
human. In yet another aspect the method further comprises
administering an immunostimulatory adjuvant to said human.
[0203] In one aspect the human has a solid tumor. In one aspect the
tumor is selected from head and neck cancer, gastric cancer,
melanoma, renal cell carcinoma (RCC), esophageal cancer, non-small
cell lung carcinoma, prostate cancer, colorectal cancer, ovarian
cancer and pancreatic cancer. In one aspect the human has one or
more of the following: colorectal cancer (CRC), esophageal,
cervical, bladder, breast, head and neck, ovarian, melanoma, renal
cell carcinoma (RCC), EC squamous cell, non-small cell lung
carcinoma, mesothelioma, and prostate cancer. In another aspect the
human has a liquid tumor such as diffuse large B cell lymphoma
(DLBCL), multiple myeloma, chronic lyphomblastic leukemia (CLL),
follicular lymphoma, acute myeloid leukemia and chronic myelogenous
leukemia.
[0204] The present disclosure also relates to a method for treating
or lessening the severity of a cancer selected from: brain
(gliomas), glioblastomas, Bannayan-Zonana syndrome, Cowden disease,
Lhermitte-Duclos disease, breast, inflammatory breast cancer,
Wilm's tumor, Ewing's sarcoma, Rhabdomyosarcoma, ependymoma,
medulloblastoma, colon, head and neck, kidney, lung, liver,
melanoma, ovarian, pancreatic, prostate, sarcoma, osteosarcoma,
giant cell tumor of bone, thyroid, lymphoblastic T-cell leukemia,
chronic myelogenous leukemia, chronic lymphocytic leukemia,
hairy-cell leukemia, acute lymphoblastic leukemia, acute
myelogenous leukemia, chronic neutrophilic leukemia, acute
lymphoblastic T-cell leukemia, plasmacytoma, immunoblastic large
cell leukemia, mantle cell leukemia, multiple myeloma
megakaryoblastic leukemia, multiple myeloma, acute megakaryocytic
leukemia, promyelocytic leukemia, erythroleukemia, malignant
lymphoma, Hodgkins lymphoma, non-hodgkins lymphoma, lymphoblastic T
cell lymphoma, Burkitt's lymphoma, follicular lymphoma,
neuroblastoma, bladder cancer, urothelial cancer, lung cancer,
vulval cancer, cervical cancer, endometrial cancer, renal cancer,
mesothelioma, esophageal cancer, salivary gland cancer,
hepatocellular cancer, gastric cancer, nasopharangeal cancer,
buccal cancer, cancer of the mouth, GIST (gastrointestinal stromal
tumor) and testicular cancer.
[0205] By the term "treating" and grammatical variations thereof as
used herein, is meant therapeutic therapy. In reference to a
particular condition, treating means: (1) to ameliorate or prevent
the condition of one or more of the biological manifestations of
the condition, (2) to interfere with (a) one or more points in the
biological cascade that leads to or is responsible for the
condition or (b) one or more of the biological manifestations of
the condition, (3) to alleviate one or more of the symptoms,
effects or side effects associated with the condition or treatment
thereof, (4) to slow the progression of the condition or one or
more of the biological manifestations of the condition and/or (5)
to cure said condition or one or more of the biological
manifestations of the condition by eliminating or reducing to
undetectable levels one or more of the biological manifestations of
the condition for a period of time considered to be a state of
remission for that manifestation without additional treatment over
the period of remission. One skilled in the art will understand the
duration of time considered to be remission for a particular
disease or condition. Prophylactic therapy is also contemplated
thereby. The skilled artisan will appreciate that "prevention" is
not an absolute term. In medicine, "prevention" is understood to
refer to the prophylactic administration of a drug to substantially
diminish the likelihood or severity of a condition or biological
manifestation thereof, or to delay the onset of such condition or
biological manifestation thereof. Prophylactic therapy is
appropriate, for example, when a subject is considered at high risk
for developing cancer, such as when a subject has a strong family
history of cancer or when a subject has been exposed to a
carcinogen.
[0206] As used herein, the terms "cancer," "neoplasm," and "tumor"
are used interchangeably and, in either the singular or plural
form, refer to cells that have undergone a malignant transformation
that makes them pathological to the host organism. Primary cancer
cells can be readily distinguished from non-cancerous cells by
well-established techniques, particularly histological examination.
The definition of a cancer cell, as used herein, includes not only
a primary cancer cell, but any cell derived from a cancer cell
ancestor. This includes metastasized cancer cells, and in vitro
cultures and cell lines derived from cancer cells. When referring
to a type of cancer that normally manifests as a solid tumor, a
"clinically detectable" tumor is one that is detectable on the
basis of tumor mass; e.g., by procedures such as computed
tomography (CT) scan, magnetic resonance imaging (MRI), X-ray,
ultrasound or palpation on physical examination, and/or which is
detectable because of the expression of one or more cancer-specific
antigens in a sample obtainable from a patient. Tumors may be a
hematopoietic (or hematologic or hematological or blood-related)
cancer, for example, cancers derived from blood cells or immune
cells, which may be referred to as "liquid tumors." Specific
examples of clinical conditions based on hematologic tumors include
leukemias such as chronic myelocytic leukemia, acute myelocytic
leukemia, chronic lymphocytic leukemia and acute lymphocytic
leukemia; plasma cell malignancies such as multiple myeloma, MGUS
and Waldenstrom's macroglobulinemia; lymphomas such as
non-Hodgkin's lymphoma, Hodgkin's lymphoma; and the like.
[0207] The cancer may be any cancer in which an abnormal number of
blast cells or unwanted cell proliferation is present or that is
diagnosed as a hematological cancer, including both lymphoid and
myeloid malignancies. Myeloid malignancies include, but are not
limited to, acute myeloid (or myelocytic or myelogenous or
myeloblastic) leukemia (undifferentiated or differentiated), acute
promyeloid (or promyelocytic or promyelogenous or promyeloblastic)
leukemia, acute myelomonocytic (or myelomonoblastic) leukemia,
acute monocytic (or monoblastic) leukemia, erythroleukemia and
megakaryocytic (or megakaryoblastic) leukemia. These leukemias may
be referred together as acute myeloid (or myelocytic or
myelogenous) leukemia (AML). Myeloid malignancies also include
myeloproliferative disorders (MPD) which include, but are not
limited to, chronic myelogenous (or myeloid) leukemia (CML),
chronic myelomonocytic leukemia (CMML), essential thrombocythemia
(or thrombocytosis), and polcythemia vera (PCV). Myeloid
malignancies also include myelodysplasia (or myelodysplastic
syndrome or MDS), which may be referred to as refractory anemia
(RA), refractory anemia with excess blasts (RAEB), and refractory
anemia with excess blasts in transformation (RAEBT); as well as
myelofibrosis (MFS) with or without agnogenic myeloid
metaplasia.
[0208] Hematopoietic cancers also include lymphoid malignancies,
which may affect the lymph nodes, spleens, bone marrow, peripheral
blood, and/or extranodal sites. Lymphoid cancers include B-cell
malignancies, which include, but are not limited to, B-cell
non-Hodgkin's lymphomas (B-NHLs). B-NHLs may be indolent (or
low-grade), intermediate-grade (or aggressive) or high-grade (very
aggressive). Indolent Bcell lymphomas include follicular lymphoma
(FL); small lymphocytic lymphoma (SLL); marginal zone lymphoma
(MZL) including nodal MZL, extranodal MZL, splenic MZL and splenic
MZL with villous lymphocytes; lymphoplasmacytic lymphoma (LPL); and
mucosa-associated-lymphoid tissue (MALT or extranodal marginal
zone) lymphoma. Intermediate-grade B-NHLs include mantle cell
lymphoma (MCL) with or without leukemic involvement, diffuse large
cell lymphoma (DLBCL), follicular large cell (or grade 3 or grade
3B) lymphoma, and primary mediastinal lymphoma (PML). High-grade
B-NHLs include Burkitt's lymphoma (BL), Burkitt-like lymphoma,
small non-cleaved cell lymphoma (SNCCL) and lymphoblastic lymphoma.
Other B-NHLs include immunoblastic lymphoma (or immunocytoma),
primary effusion lymphoma, HIV associated (or AIDS related)
lymphomas, and post-transplant lymphoproliferative disorder (PTLD)
or lymphoma. B-cell malignancies also include, but are not limited
to, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia
(PLL), Waldenstrom's macroglobulinemia (WM), hairy cell leukemia
(HCL), large granular lymphocyte (LGL) leukemia, acute lymphoid (or
lymphocytic or lymphoblastic) leukemia, and Castleman's disease.
NHL may also include T-cell non-Hodgkin's lymphoma s(T-NHLs), which
include, but are not limited to T-cell non-Hodgkin's lymphoma not
otherwise specified (NOS), peripheral T-cell lymphoma (PTCL),
anaplastic large cell lymphoma (ALCL), angioimmunoblastic lymphoid
disorder (AILD), nasal natural killer (NK) cell/T-cell lymphoma,
gamma/delta lymphoma, cutaneous T cell lymphoma, mycosis fungoides,
and Sezary syndrome.
[0209] Hematopoietic cancers also include Hodgkin's lymphoma (or
disease) including classical Hodgkin's lymphoma, nodular sclerosing
Hodgkin's lymphoma, mixed cellularity Hodgkin's lymphoma,
lymphocyte predominant (LP) Hodgkin's lymphoma, nodular LP
Hodgkin's lymphoma, and lymphocyte depleted Hodgkin's lymphoma.
Hematopoietic cancers also include plasma cell diseases or cancers
such as multiple myeloma (MM) including smoldering MM, monoclonal
gammopathy of undetermined (or unknown or unclear) significance
(MGUS), plasmacytoma (bone, extramedullary), lymphoplasmacytic
lymphoma (LPL), Waldenstrom's Macroglobulinemia, plasma cell
leukemia, and primary amyloidosis (AL). Hematopoietic cancers may
also include other cancers of additional hematopoietic cells,
including polymorphonuclear leukocytes (or neutrophils), basophils,
eosinophils, dendritic cells, platelets, erythrocytes and natural
killer cells. Tissues which include hematopoietic cells referred
herein to as "hematopoietic cell tissues" include bone marrow;
peripheral blood; thymus; and peripheral lymphoid tissues, such as
spleen, lymph nodes, lymphoid tissues associated with mucosa (such
as the gut-associated lymphoid tissues), tonsils, Peyer's patches
and appendix, and lymphoid tissues associated with other mucosa,
for example, the bronchial linings.
[0210] The ICOS binding proteins, antibodies and antigen binding
fragments of the invention can also be used to cure, prevent or
treat infections and infectious disease. The ICOS binding proteins
can be used alone, or in combination with vaccines, to stimulate
the immune response to pathogens, toxins, and self-antigens. The
ICOS binding proteins of the present invention can be used to
stimulate immune response to viruses infectious to humans, such as,
but not limited to, human immunodeficiency viruses, hepatitis
viruses class A, B and C, Eppstein Barr virus, human
cytomegalovirus, human papilloma viruses, herpes viruses. The ICOS
binding proteins of the present invention can be used to stimulate
immune response to infection with bacterial or fungal parasites,
and other pathogens. Suitably, the present invention provides
methods for treating humans that have been exposed to particular
toxins or pathogens. Accordingly, another aspect of the invention
provides a method of treating an infectious disease in a subject
comprising administering to the subject ICOS binding protein, or
antigen-binding portion thereof.
[0211] Examples of infectious disease for which the ICOS binding
proteins of the present invention may be useful include, but are
not limited to, HIV, Hepatitis (A, B, & C), Influenza, Herpes,
Giardia, Malaria, Leishmania, Staphylococcus aureus, Pseudomonas
Aeruginosa. Some examples of pathogenic viruses causing infections
treatable by methods of the invention include HIV, hepatitis (A, B,
or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV,
Epstein Barr virus), adenovirus, influenza virus, flaviviruses,
echovirus, rhinovirus, coxsackie virus, cornovirus, respiratory
syncytial virus, mumps virus, rotavirus, measles virus, rubella
virus, parvovirus, vaccinia virus, HTLV virus, dengue virus,
papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus
and arboviral encephalitis virus.
[0212] Some examples of pathogenic bacteria causing infections
treatable by methods of the invention include chlamydia,
rickettsial bacteria, mycobacteria, staphylococci, streptococci,
pneumonococci, meningococci and conococci, klebsiella, proteus,
serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli,
cholera, tetanus, botulism, anthrax, plague, leptospirosis, and
Lymes disease bacteria.
[0213] Some examples of pathogenic fungi causing infections
treatable by methods of the invention include Candida (albicans,
krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans,
Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor,
absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis,
Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma
capsulatum.
[0214] Some examples of pathogenic parasites causing infections
treatable by methods of the invention include Entamoeba
histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp.,
Giardia lambia, Cryptosporidium sp., Pneumocystis carinii,
Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma
cruzi, Leishmania donovani, Toxoplasma gondi, and Nippostrongylus
brasiliensis.
[0215] Sepsis is a potentially deadly medical condition that is
characterized by a whole-body inflammatory state (called a systemic
inflammatory response syndrome or SIRS) and the presence of a known
or suspected infection. The body may develop this inflammatory
response by the immune system to microbes in the blood, urine,
lungs, skin, or other tissues. A lay term for sepsis is blood
poisoning, more aptly applied to septicemia, below. Severe sepsis
is the systemic inflammatory response, plus infection, plus the
presence of organ dysfunction.
[0216] Septicemia is a related medical term referring to the
presence of pathogenic organisms in the bloodstream, leading to
sepsis. The term has not been sharply defined.
[0217] Sepsis and cancer share similar immunosuppressive mechanisms
including increased T regulatory cells, increased myeloid derived
suppressor cells, increased expression of negative co-stimulatory
molecules, decreased monocyte/macrophage HLA-DR expression. Sepsis
and cancer are prototypical disorders of chronic inflammation.
Chronic inflammation stimulates potent and sustained
immunoregulatory responses, including expansion of T regulatory
cells and up-regulation of PD-1 and other negative regulators on
effector cells. Barouch D. H. and Deeks S. G.; Immunologic
strategies for HIV-1 remission and eradication. Science 345:169-174
2014. Thus, in one aspect of the present inventions methods are
provided for treating sepsis in a human in need thereof comprising
administering a therapeutically effective amount of an ICOS antigen
binding protein of the present invention to said human. Boomer, et
al. JAMA 306:2594-2605 (2011); Meisel, et al.
Granulocyte-macrophage colony-stimulating factor to reverse
sepsis-associated immunosuppression: a double-blind, randomized,
placebo-controlled multicenter trial. Am J Respir Crit Care Med
180:640-648 (2009); and Hall, et al. Immunoparalysis and nosocomial
infection in children with multiple organ dysfunction syndrome.
Intensive Care Med 37:525-532 (2011).
[0218] The ICOS binding proteins of the invention can be used in
conjunction with other recombinant proteins and/or peptides (such
as tumor antigens or cancer cells) in order to increase an immune
response to these proteins (i.e., in a vaccination protocol).
[0219] For example, ICOS binding proteins thereof may be used to
stimulate antigen-specific immune responses by co-administration of
at least one ICOS binding protein with an antigen of interest
(e.g., a vaccine). Accordingly, in another aspect the invention
provides a method of enhancing an immune response to an antigen in
a subject, comprising administering to the subject: (i) the
antigen; and (ii) an ICOS binding protein of the invention, such
that an immune response to the antigen in the subject is enhanced.
The antigen can be, for example, a tumor antigen, a viral antigen,
a bacterial antigen or an antigen from a pathogen. Non-limiting
examples of such antigens include, without limitation, tumor
antigens, or antigens from the viruses, bacteria or other
pathogens.
[0220] A major hurdle to HIV eradication is the maintenance of
latently infected cells that do not express viral antigens, and
escape immune surveillance. Current strategies to eliminate the
latent viral reservoir referred to as the "kick and kill" strategy,
aims to reactivate HIV gene expression ("kick") as cellular
activation leads to HIV reactivation, and clear reactivated cells
("kill"). Cellular activation is governed by a balance of positive
and negative regulators expressed on the surface of T cells.
Altering this balance by agonising positive regulators and
antagonising negative ones may facilitate HIV reactivation.
[0221] Inducible T cell Co-Stimulator (ICOS) is a positive
regulator whose expression increases on CD4 T cells following
stimulation. ICOS functions to promote T cell proliferation,
cytokine production and differentiation. One important T cell
subset that expresses high levels of PD-1 and ICOS is T follicular
helper cells (Tfh). Tfh cells help B cells undergo differentiation,
class switching, somatic hypermutation and are necessary for
germinal center formation. Tfh cells are significantly expanded
following HIV/SIV infection and their dysregulation during chronic
lentiviral infection contributes to impaired B cell immunity.
Sorted Tfh cells have been shown to contain higher levels of HIV
DNA than other lymphoid CD4 subsets and virus outgrowth is observed
following stimulation. Tfh cells reside in germinal centers and are
exposed to HIV virions trapped on follicular dendritic cells that
may facilitate their infection. In addition, CD8 T cells have
limited access to germinal centers and follicular CD8 cells often
demonstrate reduced cytotoxicity, thus sparing Tfh cells from
antiviral surveillance. Thus, Tfh cells are an important protected
HIV reservoir and strategies that target PD-1 and ICOS may
selectively target Tfh cells and have utility as part of an HIV
cure regimen. Suitably, methods are provided for treating a human
infected with HIV comprising administering an ICOS binding protein
or the antigen binding portion thereof of the present
invention.
[0222] As used herein "tumor antigens" are proteins that are
produced by tumor cells that elicit an immune response,
particularly T-cell mediated immune responses. The term "tumor
antigen" as used herein includes both tumor-specific antigens and
tumor-associated antigens. Tumor-specific antigens are unique to
tumor cells and do not occur on other cells in the body.
Tumor-associated antigens are not unique to a tumor cell and
instead is also expressed on a normal cell under conditions that
fail to induce a state of immunologic tolerance to the antigen. The
expression of the antigen on the tumor may occur under conditions
that enable the immune system to respond to the antigen.
Tumor-associated antigens may be antigens that are expressed on
normal cells during fetal development when the immune system is
immature and unable to respond or they may be antigens that are
normally present at extremely low levels on normal cells but which
are expressed at much higher levels on tumor cells.
[0223] Non-limiting examples of tumor antigens include the
following: differentiation antigens such as MART-1/MelanA (MART-I),
gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific
multilineage antigens such, MAGE family antigens including but not
limited to MAGE1, MAGE3, MAGE10, MAGE11, MAGE12, MAGEA2, MAGEA3,
MAGEA4, MAGEA6, MAGEA8, MAGEA9, MAGEB18, MAGEB6, MABEC1, MAGED2,
MAGEE1, MAGEH1, MAGEL2, BAGE, GAGE-1, GAGE-2, p15; MEL4, melanoma
associated antigen 100+, melanoma gp100, NRIP3, NYS48, OCIAD1,
OFA-iLRP, 01P5, ovarian carcinoma-associated antigen (OV632),
PAGE4, PARP9, PATE, plastin L, PRAME, prostate-specific antigen,
proteinase 3, prostein, Reg3a, RHAMM, ROPN1, SART2, SDCCAG8, SEL1L,
SEPT1, SLC45A2, SPANX, SSX5, STXGALNAC1, STEAP4, survivin, TBC1D2,
TEM1, TRP1, tumor antigens of epithelial origin, XAGE1, XAGE2,
WT-1; overexpressed embryonic antigens such as CEA; overexpressed
oncogenes and mutated tumor-suppressor genes such as p53, Ras,
HER-2/neu; unique tumor antigens resulting from chromosomal
translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR;
and viral antigens, such as the Epstein Barr virus antigens EBVA
and the human papillomavirus (HPV) antigens E6 and E7.
[0224] Other tumor antigens include, but are not limited to,
TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2,
p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM
17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F,
5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125,
CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1,
CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag,
MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding
protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, TPS,
glioma-associated antigen, .beta.-human chorionic gonadotropin,
alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1,
MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS),
intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase,
prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53,
prostein, PSMA, Her2/neu, survivin and telomerase,
prostate-carcinoma tumor antigen-1 (PCTA-1), ELF2M, neutrophil
elastase, ephrinB2, CD19, CD20, CD22, ROR1, CD33/IL3Ra, c-Met,
PSMA, Glycolipid F77, EGFRvIII, GD-2, insulin growth factor
(IGF)-I, IGF-II, IGF-I receptor and mesothelin.
[0225] Typically, any anti-neoplastic agent that has activity
versus a susceptible tumor being treated may be co-administered in
the treatment of cancer in the present invention. Examples of such
agents can be found in Cancer Principles and Practice of Oncology
by V. T. Devita and S. Hellman (editors), 6.sup.th edition (Feb.
15, 2001), Lippincott Williams & Wilkins Publishers. A person
of ordinary skill in the art would be able to discern which
combinations of agents would be useful based on the particular
characteristics of the drugs and the cancer involved. Typical
anti-neoplastic agents useful in the present invention include, but
are not limited to, anti-microtubule agents such as diterpenoids
and vinca alkaloids; platinum coordination complexes; alkylating
agents such as nitrogen mustards, oxazaphosphorines,
alkylsulfonates, nitrosoureas, and triazenes; antibiotic agents
such as anthracyclins, actinomycins and bleomycins; topoisomerase
II inhibitors such as epipodophyllotoxins; antimetabolites such as
purine and pyrimidine analogues and anti-folate compounds;
topoisomerase I inhibitors such as camptothecins; hormones and
hormonal analogues; signal transduction pathway inhibitors;
non-receptor tyrosine kinase angiogenesis inhibitors;
immunotherapeutic agents; proapoptotic agents; and cell cycle
signalling inhibitors.
[0226] Examples of a further active ingredient or ingredients for
use in combination or co-administered with the present ICOS binding
protein are anti-neoplastic agents including any chemotherapeutic
agents, immuno-modulatory agents or immune-modulators and
immunostimulatory adjuvants.
[0227] Anti-microtubule or anti-mitotic agents are phase specific
agents active against the microtubules of tumor cells during M or
the mitosis phase of the cell cycle. Examples of anti-microtubule
agents include, but are not limited to, diterpenoids and vinca
alkaloids.
[0228] Diterpenoids, which are derived from natural sources, are
phase specific anti-cancer agents that operate at the G2/M phases
of the cell cycle. It is believed that the diterpenoids stabilize
the .beta.-tubulin subunit of the microtubules, by binding with
this protein. Disassembly of the protein appears then to be
inhibited with mitosis being arrested and cell death following.
Examples of diterpenoids include, but are not limited to,
paclitaxel and its analog docetaxel.
[0229] Paclitaxel,
5.beta.,20-epoxy-1,2.alpha.,4,7,.beta.,10.beta.,13.alpha.-hexa-hydroxytax-
-11-en-9-one 4,10-diacetate 2-benzoate 13-ester with
(2R,3S)--N-benzoyl-3-phenylisoserine; is a natural diterpene
product isolated from the Pacific yew tree Taxus brevifolia and is
commercially available as an injectable solution TAXOL.RTM.. It is
a member of the taxane family of terpenes. It was first isolated in
1971 by Wani et al. J. Am. Chem, Soc., 93:2325. 1971), who
characterized its structure by chemical and X-ray crystallographic
methods. One mechanism for its activity relates to paclitaxel's
capacity to bind tubulin, thereby inhibiting cancer cell growth.
Schiff et al., Proc. Natl, Acad, Sci. USA, 77:1561-1565 (1980);
Schiff et al., Nature, 277:665-667 (1979); Kumar, J. Biol, Chem,
256: 10435-10441 (1981). For a review of synthesis and anticancer
activity of some paclitaxel derivatives see: D. G. I. Kingston et
al., Studies in Organic Chemistry vol. 26, entitled "New trends in
Natural Products Chemistry 1986", Attaur-Rahman, P. W. Le Quesne,
Eds. (Elsevier, Amsterdam, 1986) pp 219-235.
[0230] Paclitaxel has been approved for clinical use in the
treatment of refractory ovarian cancer in the United States
(Markman et al., Yale Journal of Biology and Medicine, 64:583,
1991; McGuire et al., Ann. lntem, Med., 111:273, 1989) and for the
treatment of breast cancer (Holmes et al., J. Nat. Cancer Inst.,
83:1797, 1991.) It is a potential candidate for treatment of
neoplasms in the skin (Einzig et. al., Proc. Am. Soc. Clin. Oncol.,
20:46) and head and neck carcinomas (Forastire et. al., Sem.
Oncol., 20:56, 1990). The compound also shows potential for the
treatment of polycystic kidney disease (Woo et. al., Nature,
368:750. 1994, lung cancer and malaria. Treatment of patients with
paclitaxel results in bone marrow suppression (multiple cell
lineages, Ignoff, R. J. et. al, Cancer Chemotherapy Pocket Guide,
1998) related to the duration of dosing above a threshold
concentration (50 nM) (Kearns, C. M. et. al., Seminars in Oncology,
3(6) p. 16-23, 1995).
[0231] Docetaxel, (2R,3S)--N-carboxy-3-phenylisoserine,N-tert-butyl
ester, 13-ester with
5.beta.-20-epoxy-1,2.alpha.,4,7.beta.,10.beta.,13.alpha.-hexahydroxytax-1-
1-en-9-one 4-acetate 2-benzoate, trihydrate; is commercially
available as an injectable solution as TAXOTERE.RTM.. Docetaxel is
indicated for the treatment of breast cancer. Docetaxel is a
semisynthetic derivative of paclitaxel q.v., prepared using a
natural precursor, 10-deacetyl-baccatin III, extracted from the
needle of the European Yew tree. The dose limiting toxicity of
docetaxel is neutropenia.
[0232] Vinca alkaloids are phase specific anti-neoplastic agents
derived from the periwinkle plant. Vinca alkaloids act at the M
phase (mitosis) of the cell cycle by binding specifically to
tubulin. Consequently, the bound tubulin molecule is unable to
polymerize into microtubules. Mitosis is believed to be arrested in
metaphase with cell death following. Examples of vinca alkaloids
include, but are not limited to, vinblastine, vincristine, and
vinorelbine.
[0233] Vinblastine, vincaleukoblastine sulfate, is commercially
available as VELBAN.RTM. as an injectable solution. Although, it
has possible indication as a second line therapy of various solid
tumors, it is primarily indicated in the treatment of testicular
cancer and various lymphomas including Hodgkin's Disease; and
lymphocytic and histiocytic lymphomas. Myelosuppression is the dose
limiting side effect of vinblastine.
[0234] Vincristine, vincaleukoblastine, 22-oxo-, sulfate, is
commercially available as ONCOVIN.RTM. as an injectable solution.
Vincristine is indicated for the treatment of acute leukemias and
has also found use in treatment regimens for Hodgkin's and
non-Hodgkin's malignant lymphomas. Alopecia and neurologic effects
are the most common side effect of vincristine and to a lesser
extent myelosupression and gastrointestinal mucositis effects
occur.
[0235] Vinorelbine,
3',4'-didehydro-4'-deoxy-C'-norvincaleukoblastine
[R--(R*,R*)-2,3-dihydroxybutanedioate (1:2)(salt)], commercially
available as an injectable solution of vinorelbine tartrate
(NAVELBINE.RTM.), is a semisynthetic vinca alkaloID Vinorelbine is
indicated as a single agent or in combination with other
chemotherapeutic agents, such as cisplatin, in the treatment of
various solid tumors, particularly non-small cell lung, advanced
breast, and hormone refractory prostate cancers. Myelosuppression
is the most common dose limiting side effect of vinorelbine.
[0236] Platinum coordination complexes are non-phase specific
anti-cancer agents, which are interactive with DNA. The platinum
complexes enter tumor cells, undergo, aquation and form intra- and
interstrand crosslinks with DNA causing adverse biological effects
to the tumor. Examples of platinum coordination complexes include,
but are not limited to, cisplatin and carboplatin.
[0237] Cisplatin, cis-diamminedichloroplatinum, is commercially
available as PLATINOL.RTM. as an injectable solution. Cisplatin is
primarily indicated in the treatment of metastatic testicular and
ovarian cancer and advanced bladder cancer. The primary dose
limiting side effects of cisplatin are nephrotoxicity, which may be
controlled by hydration and diuresis, and ototoxicity.
[0238] Carboplatin, platinum, diammine
[1,1-cyclobutane-dicarboxylate(2-)-O,O'], is commercially available
as PARAPLATIN.RTM. as an injectable solution. Carboplatin is
primarily indicated in the first and second line treatment of
advanced ovarian carcinoma. Bone marrow suppression is the dose
limiting toxicity of carboplatin.
[0239] Alkylating agents are non-phase anti-cancer specific agents
and strong electrophiles. Typically, alkylating agents form
covalent linkages, by alkylation, to DNA through nucleophilic
moieties of the DNA molecule such as phosphate, amino, sulfhydryl,
hydroxyl, carboxyl, and imidazole groups. Such alkylation disrupts
nucleic acid function leading to cell death. Examples of alkylating
agents include, but are not limited to, nitrogen mustards such as
cyclophosphamide, melphalan, and chlorambucil; alkyl sulfonates
such as busulfan; nitrosoureas such as carmustine; and triazenes
such as dacarbazine.
[0240] Cyclophosphamide,
2-[bis(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine
2-oxide monohydrate, is commercially available as an injectable
solution or tablets as CYTOXAN.RTM.. Cyclophosphamide is indicated
as a single agent or in combination with other chemotherapeutic
agents, in the treatment of malignant lymphomas, multiple myeloma,
and leukemias. Alopecia, nausea, vomiting and leukopenia are the
most common dose limiting side effects of cyclophosphamide.
[0241] Melphalan, 4-[bis(2-chloroethyl)amino]-L-phenylalanine, is
commercially available as an injectable solution or tablets as
ALKERAN.RTM.. Melphalan is indicated for the palliative treatment
of multiple myeloma and non-resectable epithelial carcinoma of the
ovary. Bone marrow suppression is the most common dose limiting
side effect of melphalan.
[0242] Chlorambucil, 4-[bis(2-chloroethyl)amino]benzenebutanoic
acid, is commercially available as LEUKERAN.RTM. tablets.
Chlorambucil is indicated for the palliative treatment of chronic
lymphatic leukemia, and malignant lymphomas such as lymphosarcoma,
giant follicular lymphoma, and Hodgkin's disease. Bone marrow
suppression is the most common dose limiting side effect of
chlorambucil.
[0243] Busulfan, 1,4-butanediol dimethanesulfonate, is commercially
available as MYLERAN.RTM. TABLETS. Busulfan is indicated for the
palliative treatment of chronic myelogenous leukemia. Bone marrow
suppression is the most common dose limiting side effects of
busulfan.
[0244] Carmustine, 1,3-[bis(2-chloroethyl)-1-nitrosourea, is
commercially available as single vials of lyophilized material as
BiCNU.RTM.. Carmustine is indicated for the palliative treatment as
a single agent or in combination with other agents for brain
tumors, multiple myeloma, Hodgkin's disease, and non-Hodgkin's
lymphomas. Delayed myelosuppression is the most common dose
limiting side effects of carmustine.
[0245] Dacarbazine,
5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide, is
commercially available as single vials of material as
DTIC-Dome.RTM.. Dacarbazine is indicated for the treatment of
metastatic malignant melanoma and in combination with other agents
for the second line treatment of Hodgkin's Disease. Nausea,
vomiting, and anorexia are the most common dose limiting side
effects of dacarbazine.
[0246] Antibiotic anti-neoplastics are non-phase specific agents,
which bind or intercalate with DNA. Typically, such action results
in stable DNA complexes or strand breakage, which disrupts ordinary
function of the nucleic acids leading to cell death. Examples of
antibiotic anti-neoplastic agents include, but are not limited to,
actinomycins such as dactinomycin, anthrocyclins such as
daunorubicin and doxorubicin; and bleomycins.
[0247] Dactinomycin, also know as Actinomycin D, is commercially
available in injectable form as COSMEGEN.RTM.. Dactinomycin is
indicated for the treatment of Wilm's tumor and rhabdomyosarcoma.
Nausea, vomiting, and anorexia are the most common dose limiting
side effects of dactinomycin.
[0248] Daunorubicin,
(8S-cis-)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyxo-hexopyranos-
yl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12
naphthacenedione hydrochloride, is commercially available as a
liposomal injectable form as DAUNOXOME.RTM. or as an injectable as
CERUBIDINE.RTM.. Daunorubicin is indicated for remission induction
in the treatment of acute nonlymphocytic leukemia and advanced HIV
associated Kaposi's sarcoma. Myelosuppression is the most common
dose limiting side effect of daunorubicin.
[0249] Doxorubicin, (8S,
10S)-10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyxo-hexopyranosyl)oxy]-8-glyc-
oloyl, 7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12
naphthacenedione hydrochloride, is commercially available as an
injectable form as RUBEX.RTM. or ADRIAMYCIN RDF.RTM.. Doxorubicin
is primarily indicated for the treatment of acute lymphoblastic
leukemia and acute myeloblastic leukemia, but is also a useful
component in the treatment of some solid tumors and lymphomas.
Myelosuppression is the most common dose limiting side effect of
doxorubicin.
[0250] Bleomycin, a mixture of cytotoxic glycopeptide antibiotics
isolated from a strain of Streptomyces verticillus, is commercially
available as BLENOXANE.RTM.. Bleomycin is indicated as a palliative
treatment, as a single agent or in combination with other agents,
of squamous cell carcinoma, lymphomas, and testicular carcinomas.
Pulmonary and cutaneous toxicities are the most common dose
limiting side effects of bleomycin.
[0251] Topoisomerase II inhibitors include, but are not limited to,
epipodophyllotoxins.
[0252] Epipodophyllotoxins are phase specific anti-neoplastic
agents derived from the mandrake plant. Epipodophyllotoxins
typically affect cells in the S and G2 phases of the cell cycle by
forming a ternary complex with topoisomerase II and DNA causing DNA
strand breaks. The strand breaks accumulate and cell death follows.
Examples of epipodophyllotoxins include, but are not limited to,
etoposide and teniposide.
[0253] Etoposide, 4'-demethyl-epipodophyllotoxin
9[4,6-0-(R)-ethylidene-.beta.-D-glucopyranoside], is commercially
available as an injectable solution or capsules as VePESID.RTM. and
is commonly known as VP-16. Etoposide is indicated as a single
agent or in combination with other chemotherapy agents in the
treatment of testicular and non-small cell lung cancers.
Myelosuppression is the most common side effect of etoposide. The
incidence of leucopenia tends to be more severe than
thrombocytopenia.
[0254] Teniposide, 4'-demethyl-epipodophyllotoxin
9[4,6-0-(R)-thenylidene-.beta.-D-glucopyranoside], is commercially
available as an injectable solution as VUMON.RTM. and is commonly
known as VM-26. Teniposide is indicated as a single agent or in
combination with other chemotherapy agents in the treatment of
acute leukemia in children. Myelosuppression is the most common
dose limiting side effect of teniposide. Teniposide can induce both
leucopenia and thrombocytopenia.
[0255] Antimetabolite neoplastic agents are phase specific
anti-neoplastic agents that act at S phase (DNA synthesis) of the
cell cycle by inhibiting DNA synthesis or by inhibiting purine or
pyrimidine base synthesis and thereby limiting DNA synthesis.
Consequently, S phase does not proceed and cell death follows.
Examples of antimetabolite anti-neoplastic agents include, but are
not limited to, fluorouracil, methotrexate, cytarabine,
mecaptopurine, thioguanine, and gemcitabine.
[0256] 5-fluorouracil, 5-fluoro-2,4-(1H,3H) pyrimidinedione, is
commercially available as fluorouracil. Administration of
5-fluorouracil leads to inhibition of thymidylate synthesis and is
also incorporated into both RNA and DNA. The result typically is
cell death. 5-fluorouracil is indicated as a single agent or in
combination with other chemotherapy agents in the treatment of
carcinomas of the breast, colon, rectum, stomach and pancreas.
Myelosuppression and mucositis are dose limiting side effects of
5-fluorouracil. Other fluoropyrimidine analogs include 5-fluoro
deoxyuridine (floxuridine) and 5-fluorodeoxyuridine
monophosphate.
[0257] Cytarabine, 4-amino-1-.beta.-D-arabinofuranosyl-2
(1H)-pyrimidinone, is commercially available as CYTOSAR-U.RTM. and
is commonly known as Ara-C. It is believed that cytarabine exhibits
cell phase specificity at S-phase by inhibiting DNA chain
elongation by terminal incorporation of cytarabine into the growing
DNA chain. Cytarabine is indicated as a single agent or in
combination with other chemotherapy agents in the treatment of
acute leukemia. Other cytidine analogs include 5-azacytidine and
2',2'-difluorodeoxycytidine (gemcitabine). Cytarabine induces
leucopenia, thrombocytopenia, and mucositis.
[0258] Mercaptopurine, 1,7-dihydro-6H-purine-6-thione monohydrate,
is commercially available as PURINETHOL.RTM.. Mercaptopurine
exhibits cell phase specificity at S-phase by inhibiting DNA
synthesis by an as of yet unspecified mechanism. Mercaptopurine is
indicated as a single agent or in combination with other
chemotherapy agents in the treatment of acute leukemia.
Myelosuppression and gastrointestinal mucositis are expected side
effects of mercaptopurine at high doses. A useful mercaptopurine
analog is azathioprine.
[0259] Thioguanine, 2-amino-1,7-dihydro-6H-purine-6-thione, is
commercially available as TABLOID.RTM.. Thioguanine exhibits cell
phase specificity at S-phase by inhibiting DNA synthesis by an as
of yet unspecified mechanism. Thioguanine is indicated as a single
agent or in combination with other chemotherapy agents in the
treatment of acute leukemia. Myelosuppression, including
leucopenia, thrombocytopenia, and anemia, is the most common dose
limiting side effect of thioguanine administration. However,
gastrointestinal side effects occur and can be dose limiting. Other
purine analogs include pentostatin, erythrohydroxynonyladenine,
fludarabine phosphate, and cladribine.
[0260] Gemcitabine, 2'-deoxy-2',2'-difluorocytidine
monohydrochloride (.beta.-isomer), is commercially available as
GEMZAR.RTM.. Gemcitabine exhibits cell phase specificity at S-phase
and by blocking progression of cells through the G1/S boundary.
Gemcitabine is indicated in combination with cisplatin in the
treatment of locally advanced non-small cell lung cancer and alone
in the treatment of locally advanced pancreatic cancer.
Myelosuppression, including leucopenia, thrombocytopenia, and
anemia, is the most common dose limiting side effect of gemcitabine
administration.
[0261] Methotrexate, N-[4[[(2,4-diamino-6-pteridinyl)
methyl]methylamino] benzoyl]-L-glutamic acid, is commercially
available as methotrexate sodium. Methotrexate exhibits cell phase
effects specifically at S-phase by inhibiting DNA synthesis, repair
and/or replication through the inhibition of dyhydrofolic acid
reductase which is required for synthesis of purine nucleotides and
thymidylate. Methotrexate is indicated as a single agent or in
combination with other chemotherapy agents in the treatment of
choriocarcinoma, meningeal leukemia, non-Hodgkin's lymphoma, and
carcinomas of the breast, head, neck, ovary and bladder.
Myelosuppression (leucopenia, thrombocytopenia, and anemia) and
mucositis are expected side effect of methotrexate
administration.
[0262] Camptothecins, including, camptothecin and camptothecin
derivatives are available or under development as Topoisomerase I
inhibitors. Camptothecins cytotoxic activity is believed to be
related to its Topoisomerase I inhibitory activity. Examples of
camptothecins include, but are not limited to irinotecan,
topotecan, and the various optical forms of
7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20-camptoth-
ecin described below.
[0263] Irinotecan HCl,
(4S)-4,11-diethyl-4-hydroxy-9-[(4-piperidinopiperidino)
carbonyloxy]-1H-pyrano[3',4',6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)--
dione hydrochloride, is commercially available as the injectable
solution CAMPTOSAR.RTM..
[0264] Irinotecan is a derivative of camptothecin which binds,
along with its active metabolite SN-38, to the topoisomerase I-DNA
complex. It is believed that cytotoxicity occurs as a result of
irreparable double strand breaks caused by interaction of the
topoisomerase I:DNA:irintecan or SN-38 ternary complex with
replication enzymes. Irinotecan is indicated for treatment of
metastatic cancer of the colon or rectum. The dose limiting side
effects of irinotecan HCl are myelosuppression, including
neutropenia, and GI effects, including diarrhea.
[0265] Topotecan HCl,
(S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3',4',6,7]-
indolizino[1,2-b]quinoline-3,14-(4H,12H)-dione monohydrochloride,
is commercially available as the injectable solution HYCAMTIN.RTM..
Topotecan is a derivative of camptothecin which binds to the
topoisomerase I-DNA complex and prevents religation of singles
strand breaks caused by Topoisomerase I in response to torsional
strain of the DNA molecule. Topotecan is indicated for second line
treatment of metastatic carcinoma of the ovary and small cell lung
cancer. The dose limiting side effect of topotecan HCl is
myelosuppression, primarily neutropenia.
[0266] Rituximab is a chimeric monoclonal antibody which is sold as
RITUXAN.RTM. and MABTHERA.RTM.. Rituximab binds to CD20 on B cells
and causes cell apoptosis. Rituximab is administered intravenously
and is approved for treatment of rheumatoid arthritis and B-cell
non-Hodgkin's lymphoma.
[0267] Ofatumumab is a fully human monoclonal antibody which is
sold as ARZERRA.RTM.. Ofatumumab binds to CD20 on B cells and is
used to treat chronic lymphocytic leukemia CLL; a type of cancer of
the white blood cells) in adults who are refractory to treatment
with fludarabine (Fludara) and alemtuzumab Campath).
[0268] Trastuzumab (HEREPTIN.RTM.) is a humanized monoclonal
antibody that binds to the HER2 receptor. It original indication is
HER2 positive breast cancer.
[0269] Cetuximab (ERBITUX.RTM.) is a chimeric mouse human antibody
that inhibits epidermal growth factor receptor (EGFR).
[0270] mTOR inhibitors include but are not limited to rapamycin
(FK506) and rapalogs, RAD001 or everolimus (Afinitor), CCI-779 or
temsirolimus, AP23573, AZD8055, WYE-354, WYE-600, WYE-687 and
Pp121.
[0271] Bexarotene is sold as Targretin.RTM. and is a member of a
subclass of retinoids that selectively activate retinoid X
receptors (RXRs). These retinoid receptors have biologic activity
distinct from that of retinoic acid receptors (RARs). The chemical
name is
4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)
ethenyl] benzoic acID Bexarotene is used to treat cutaneous T-cell
lymphoma CTCL, a type of skin cancer) in people whose disease could
not be treated successfully with at least one other medication.
[0272] Sorafenib marketed as Nexavar.RTM. is in a class of
medications called multikinase inhibitors. Its chemical name is
4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]
phenoxy]-N-methyl-pyridine-2-carboxamide. Sorafenib is used to
treat advanced renal cell carcinoma (a type of cancer that begins
in the kidneys). Sorafenib is also used to treat unresectable
hepatocellular carcinoma (a type of liver cancer that cannot be
treated with surgery).
[0273] Examples of erbB inhibitors include lapatinib, erlotinib,
and gefitinib. Lapatinib,
N-(3-chloro-4-{[(3-fluorophenyl)methyl]oxy}phenyl)-6-[5-({[2-(methylsulfo-
nyl)ethyl]amino}methyl)-2-furanyl]-4-quinazolinamine (represented
by formula II, as illustrated), is a potent, oral, small-molecule,
dual inhibitor of erbB-1 and erbB-2 (EGFR and HER2) tyrosine
kinases that is approved in combination with capecitabine for the
treatment of HER2-positive metastatic breast cancer.
##STR00001##
The free base, HCl salts, and ditosylate salts of the compound of
formula (II) may be prepared according to the procedures disclosed
in WO 99/35146, published Jul. 15, 1999; and WO 02/02552 published
Jan. 10, 2002.
[0274] Erlotinib,
N-(3-ethynylphenyl)-6,7-bis{[2-(methyloxy)ethyl]oxy}-4-quinazolinamine
Commercially available under the tradename Tarceva) is represented
by formula III, as illustrated:
##STR00002##
The free base and HCl salt of erlotinib may be prepared, for
example, according to U.S. Pat. No. 5,747,498, Example 20.
[0275] Gefitinib,
4-quinazolinamine,N-(3-chloro-4-fluorophenyl)-7-methoxy-6-[3-4-morpholin)-
propoxy] is represented by formula IV, as illustrated:
##STR00003##
Gefitinib, which is commercially available under the trade name
IRRESSA.RTM. (Astra-Zenenca) is an erbB-1 inhibitor that is
indicated as monotherapy for the treatment of patients with locally
advanced or metastatic non-small-cell lung cancer after failure of
both platinum-based and docetaxel chemotherapies. The free base,
HCl salts, and diHCl salts of gefitinib may be prepared according
to the procedures of International Patent Application No.
PCT/GB96/00961, filed Apr. 23, 1996, and published as WO 96/33980
on Oct. 31, 1996.
[0276] Also of interest, is the camptothecin derivative of formula
A following, currently under development, including the racemic
mixture (R,S) form as well as the R and S enantiomers:
##STR00004##
known by the chemical name
"7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20(R,S)-camptotheci-
n (racemic mixture) or
"7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20(R)-camptothecin
(R enantiomer) or
"7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20(S)-camptothecin
(S enantiomer). Such compound as well as related compounds are
described, including methods of making, in U.S. Pat. Nos.
6,063,923; 5,342,947; 5,559,235; 5,491,237 and pending U.S. patent
application Ser. No. 08/977,217 filed Nov. 24, 1997.
[0277] Hormones and hormonal analogues are useful compounds for
treating cancers in which there is a relationship between the
hormone(s) and growth and/or lack of growth of the cancer. Examples
of hormones and hormonal analogues useful in cancer treatment
include, but are not limited to, adrenocorticosteroids such as
prednisone and prednisolone which are useful in the treatment of
malignant lymphoma and acute leukemia in children;
aminoglutethimide and other aromatase inhibitors such as
anastrozole, letrazole, vorazole, and exemestane useful in the
treatment of adrenocortical carcinoma and hormone dependent breast
carcinoma containing estrogen receptors; progestrins such as
megestrol acetate useful in the treatment of hormone dependent
breast cancer and endometrial carcinoma; estrogens, androgens, and
anti-androgens such as flutamide, nilutamide, bicalutamide,
cyproterone acetate and 5.alpha.-reductases such as finasteride and
dutasteride, useful in the treatment of prostatic carcinoma and
benign prostatic hypertrophy; anti-estrogens such as tamoxifen,
toremifene, raloxifene, droloxifene, iodoxyfene, as well as
selective estrogen receptor modulators (SERMS) such those described
in U.S. Pat. Nos. 5,681,835, 5,877,219, and 6,207,716, useful in
the treatment of hormone dependent breast carcinoma and other
susceptible cancers; and gonadotropin-releasing hormone (GnRH) and
analogues thereof which stimulate the release of leutinizing
hormone (LH) and/or follicle stimulating hormone (FSH) for the
treatment prostatic carcinoma, for instance, LHRH agonists and
antagagonists such as goserelin acetate and luprolide.
[0278] Signal transduction pathway inhibitors are those inhibitors,
which block or inhibit a chemical process which evokes an
intracellular change. As used herein this change is cell
proliferation or differentiation. Signal tranduction inhibitors
useful in the present invention include inhibitors of receptor
tyrosine kinases, non-receptor tyrosine kinases, SH2/SH3 domain
blockers, serine/threonine kinases, phosphotidyl inositol-3
kinases, myo-inositol signalling, and Ras oncogenes.
[0279] Several protein tyrosine kinases catalyse the
phosphorylation of specific tyrosyl residues in various proteins
involved in the regulation of cell growth. Such protein tyrosine
kinases can be broadly classified as receptor or non-receptor
kinases.
[0280] Receptor tyrosine kinases are transmembrane proteins having
an extracellular ligand binding domain, a transmembrane domain, and
a tyrosine kinase domain. Receptor tyrosine kinases are involved in
the regulation of cell growth and are generally termed growth
factor receptors. Inappropriate or uncontrolled activation of many
of these kinases, i.e. aberrant kinase growth factor receptor
activity, for example by over-expression or mutation, has been
shown to result in uncontrolled cell growth. Accordingly, the
aberrant activity of such kinases has been linked to malignant
tissue growth. Consequently, inhibitors of such kinases could
provide cancer treatment methods. Growth factor receptors include,
for example, epidermal growth factor receptor (EGFr), platelet
derived growth factor receptor (PDGFr), erbB2, erbB4, vascular
endothelial growth factor receptor (VEGFr), tyrosine kinase with
immunoglobulin-like and epidermal growth factor homology domains
(TIE-2), insulin growth factor-I (IGFI) receptor, macrophage colony
stimulating factor Cfms), BTK, ckit, cmet, fibroblast growth factor
(FGF) receptors, Trk receptors (TrkA, TrkB, and TrkC), ephrin (eph)
receptors, and the RET protooncogene. Several inhibitors of growth
receptors are under development and include ligand antagonists,
antibodies, tyrosine kinase inhibitors and anti-sense
oligonucleotides. Growth factor receptors and agents that inhibit
growth factor receptor function are described, for instance, in
Kath, John C., Exp. Opin. Ther. Patents (2000) 10(6):803-818;
Shawver et al DDT Vol 2, No. 2 Feb. 1997; and Lofts, F. J. et al,
"Growth factor receptors as targets", New Molecular Targets for
Cancer Chemotherapy, ed. Workman, Paul and Kerr, David, CRC press
1994, London.
[0281] Tyrosine kinases, which are not growth factor receptor
kinases are termed non-receptor tyrosine kinases. Non-receptor
tyrosine kinases useful in the present invention, which are targets
or potential targets of anti-cancer drugs, include cSrc, Lck, Fyn,
Yes, Jak, cAbl, FAK (Focal adhesion kinase), Brutons tyrosine
kinase, and Bcr-Abl. Such non-receptor kinases and agents which
inhibit non-receptor tyrosine kinase function are described in
Sinh, S. and Corey, S. J., (1999) Journal of Hematotherapy and Stem
Cell Research 8 (5): 465-80; and Bolen, J. B., Brugge, J. S.,
(1997) Annual review of Immunology. 15: 371-404.
[0282] SH2/SH3 domain blockers are agents that disrupt SH2 or SH3
domain binding in a variety of enzymes or adaptor proteins
including, PI3-K p85 subunit, Src family kinases, adaptor molecules
(Shc, Crk, Nck, Grb2) and Ras-GAP. SH2/SH3 domains as targets for
anti-cancer drugs are discussed in Smithgall, T. E. (1995), Journal
of Pharmacological and Toxicological Methods. 34(3) 125-32.
[0283] Inhibitors of Serine/Threonine Kinases including MAP kinase
cascade blockers which include blockers of Raf kinases (rafk),
Mitogen or Extracellular Regulated Kinase (MEKs), and Extracellular
Regulated Kinases (ERKs); and Protein kinase C family member
blockers including blockers of PKCs (alpha, beta, gamma, epsilon,
mu, lambda, iota, zeta). IkB kinase family (IKKa, IKKb), PKB family
kinases, AKT kinase family members, and TGF beta receptor kinases.
Such Serine/Threonine kinases and inhibitors thereof are described
in Yamamoto, T., Taya, S., Kaibuchi, K., (1999), Journal of
Biochemistry. 126 (5) 799-803; Brodt, P, Samani, A., and Navab, R.
(2000), Biochemical Pharmacology, 60. 1101-1107; Massague, J.,
Weis-Garcia, F. (1996) Cancer Surveys. 27:41-64; Philip, P. A., and
Harris, A. L. (1995), Cancer Treatment and Research. 78: 3-27,
Lackey, K. et al Bioorganic and Medicinal Chemistry Letters, (10),
2000, 223-226; U.S. Pat. No. 6,268,391; and Martinez-Iacaci, L., et
al, Int. J. Cancer (2000), 88(1), 44-52.
[0284] Inhibitors of Phosphotidyl inositol-3 Kinase family members
including blockers of PI3-kinase, ATM, DNA-PK, and Ku are also
useful in the present invention. Such kinases are discussed in
Abraham, R. T. (1996), Current Opinion in Immunology. 8 (3) 412-8;
Canman, C. E., Lim, D. S. (1998), Oncogene 17 (25) 3301-3308;
Jackson, S. P. (1997), International Journal of Biochemistry and
Cell Biology. 29 (7):935-8; and Zhong, H. et al, Cancer res, (2000)
60(6), 1541-1545.
[0285] Also useful in the present invention are Myo-inositol
signalling inhibitors such as phospholipase C blockers and
Myoinositol analogues. Such signal inhibitors are described in
Powis, G., and Kozikowski A., (1994 New Molecular Targets for
Cancer Chemotherapy ed., Paul Workman and David Kerr, CRC press
1994, London.
[0286] Another group of signal transduction pathway inhibitors are
inhibitors of Ras Oncogene. Such inhibitors include inhibitors of
farnesyltransferase, geranyl-geranyl transferase, and CAAX
proteases as well as anti-sense oligonucleotides, ribozymes and
immunotherapy. Such inhibitors have been shown to block ras
activation in cells containing wild type mutant ras, thereby acting
as antiproliferation agents. Ras oncogene inhibition is discussed
in Scharovsky, O. G., Rozados, V. R., Gervasoni, S. I. Matar, P.
(2000), Journal of Biomedical Science. 7(4 292-8; Ashby, M. N.
(1998), Current Opinion in Lipidology. 9 (2) 99-102; and Bennett,
C. F. and Cowsert, L. M. BioChim. Biophys. Acta, (1999)
1489(1):19-30.
[0287] As mentioned above, antibody antagonists to receptor kinase
ligand binding may also serve as signal transduction inhibitors.
This group of signal transduction pathway inhibitors includes the
use of humanized antibodies to the extracellular ligand binding
domain of receptor tyrosine kinases. For example Imclone C225 EGFR
specific antibody (see Green, M. C. et al, Monoclonal Antibody
Therapy for Solid Tumors, Cancer Treat. Rev., (2000), 26(4,
269-286); Herceptin.RTM. erbB2 antibody (see Tyrosine Kinase
Signalling in Breast cancer: erbB Family Receptor Tyrosine Kniases,
Breast cancer Res., 2000, 2(3), 176-183); and 2CB VEGFR2 specific
antibody (see Brekken, R. A. et al, Selective Inhibition of VEGFR2
Activity by a monoclonal Anti-VEGF antibody blocks tumor growth in
mice, Cancer Res. (2000) 60, 5117-5124.
[0288] Non-receptor kinase angiogenesis inhibitors may also find
use in the present invention. Inhibitors of angiogenesis related
VEGFR and TIE2 are discussed above in regard to signal transduction
inhibitors (both receptors are receptor tyrosine kinases).
Angiogenesis in general is linked to erbB2/EGFR signaling since
inhibitors of erbB2 and EGFR have been shown to inhibit
angiogenesis, primarily VEGF expression. Thus, the combination of
an erbB2/EGFR inhibitor with an inhibitor of angiogenesis makes
sense. Accordingly, non-receptor tyrosine kinase inhibitors may be
used in combination with the EGFR/erbB2 inhibitors of the present
invention. For example, anti-VEGF antibodies, which do not
recognize VEGFR (the receptor tyrosine kinase), but bind to the
ligand; small molecule inhibitors of integrin (alpha beta.sub.3)
that will inhibit angiogenesis; endostatin and angiostatin
(non-RTK) may also prove useful in combination with the disclosed
erb family inhibitors. (See Bruns C J et al (2000), Cancer Res.,
60: 2926-2935; Schreiber A B, Winkler M E, and Derynck R. (1986),
Science, 232: 1250-1253; Yen L et al. (2000), Oncogene 19:
3460-3469).
[0289] Agents used in immunotherapeutic regimens may also be useful
in combination with the compounds of formula (I). There are a
number of immunologic strategies to generate an immune response
against erbB2 or EGFR. These strategies are generally in the realm
of tumor vaccinations. The efficacy of immunologic approaches may
be greatly enhanced through combined inhibition of erbB2/EGFR
signaling pathways using a small molecule inhibitor. Discussion of
the immunologic/tumor vaccine approach against erbB2/EGFR are found
in Reilly R T et al. (2000), Cancer Res. 60: 3569-3576; and Chen Y,
Hu D, Eling D J, Robbins J, and Kipps T J. (1998), Cancer Res. 58:
1965-1971.
[0290] Agents used in proapoptotic regimens (e.g., bcl-2 antisense
oligonucleotides) may also be used in the combination of the
present invention. Members of the Bcl-2 family of proteins block
apoptosis. Upregulation of bcl-2 has therefore been linked to
chemoresistance. Studies have shown that the epidermal growth
factor (EGF) stimulates anti-apoptotic members of the bcl-2 family
(i.e., mcl-1). Therefore, strategies designed to downregulate the
expression of bcl-2 in tumors have demonstrated clinical benefit
and are now in Phase II/III trials, namely Genta's G3139 bcl-2
antisense oligonucleotide. Such proapoptotic strategies using the
antisense oligonucleotide strategy for bcl-2 are discussed in Water
J S et al. (2000), J. Clin. Oncol. 18: 1812-1823; and Kitada S et
al. (1994, Antisense Res. Dev. 4: 71-79.
[0291] Trastuzumab (HEREPTIN.RTM.) is a humanized monoclonal
antibody that binds to the HER2 receptor. It original indication is
HER2 positive breast cancer.
[0292] Trastuzumab emtansine (trade name Kadcyla) is an
antibody-drug conjugate consisting of the monoclonal antibody
trastuzumab (Herceptin) linked to the cytotoxic agent mertansine
(DM1). Trastuzumab alone stops growth of cancer cells by binding to
the HER2/neu receptor, whereas mertansine enters cells and destroys
them by binding to tubulin. Because the monoclonal antibody targets
HER2, and HER2 is only overexpressed in cancer cells, the conjugate
delivers the toxin specifically to tumor cells. The conjugate is
abbreviated T-DM1.
[0293] Cetuximab (ERBITUX.RTM.) is a chimeric mouse human antibody
that inhibits epidermal growth factor receptor (EGFR).
[0294] Pertuzumab (also called 2C4, trade name Omnitarg) is a
monoclonal antibody. The first of its class in a line of agents
called "HER dimerization inhibitors". By binding to HER2, it
inhibits the dimerization of HER2 with other HER receptors, which
is hypothesized to result in slowed tumor growth. Pertuzumab is
described in WO01/00245 published Jan. 4, 2001.
[0295] Rituximab is a chimeric monoclonal antibody which is sold as
RITUXAN.RTM. and MABTHERA.RTM.. Rituximab binds to CD20 on B cells
and causes cell apoptosis. Rituximab is administered intravenously
and is approved for treatment of rheumatoid arthritis and B-cell
non-Hodgkin's lymphoma.
[0296] Ofatumumab is a fully human monoclonal antibody which is
sold as ARZERRA.RTM.. Ofatumumab binds to CD20 on B cells and is
used to treat chronic lymphocytic leukemia (CLL; a type of cancer
of the white blood cells) in adults who are refractory to treatment
with fludarabine (Fludara) and alemtuzumab (Campath).
[0297] Cell cycle signalling inhibitors inhibit molecules involved
in the control of the cell cycle. A family of protein kinases
called cyclin dependent kinases CDKs) and their interaction with a
family of proteins termed cyclins controls progression through the
eukaryotic cell cycle. The coordinate activation and inactivation
of different cyclin/CDK complexes is necessary for normal
progression through the cell cycle. Several inhibitors of cell
cycle signalling are under development. For instance, examples of
cyclin dependent kinases, including CDK2, CDK4, and CDK6 and
inhibitors for the same are described in, for instance, Rosania et
al, Exp. Opin. Ther. Patents (2000) 10(2):215-230.
[0298] As used herein "immuno-modulators" refer to any substance
including monoclonal antibodies that effects the immune system. The
ICOS binding proteins of the present invention can be considered
immune-modulators. Immuno-modulators can be used as anti-neoplastic
agents for the treatment of cancer. For example, immune-modulators
include, but are not limited to, anti-CTLA-4 antibodies such as
ipilimumab (YERVOY) and anti-PD-1 antibodies (Opdivo/nivolumab and
Keytruda/pembrolizumab). Other immuno-modulators include, but are
not limited to, OX-40 antibodies, PD-L1 antibodies, LAG3
antibodies, TIM-3 antibodies, 41BB antibodies and GITR
antibodies.
[0299] Yervoy (ipilimumab) is a fully human CTLA-4 antibody
marketed by Bristol Myers Squibb. The protein structure of
ipilimumab and methods are using are described in U.S. Pat. Nos.
6,984,720 and 7,605,238.
[0300] Opdivo/nivolumab is a fully human monoclonal antibody
marketed by Bristol Myers Squibb directed against the negative
immunoregulatory human cell surface receptor PD-1 (programmed
death-1 or programmed cell death-1/PCD-1) with immunopotentiation
activity. Nivolumab binds to and blocks the activation of PD-1, an
Ig superfamily transmembrane protein, by its ligands PD-L1 and
PD-L2, resulting in the activation of T-cells and cell-mediated
immune responses against tumor cells or pathogens. Activated PD-1
negatively regulates T-cell activation and effector function
through the suppression of P13k/Akt pathway activation. Other names
for nivolumab include: BMS-936558, MDX-1106, and ONO-4538. The
amino acid sequence for nivolumab and methods of using and making
are disclosed in U.S. Pat. No. 8,008,449.
[0301] KEYTRUDA/pembrolizumab is an anti-PD-1 antibodies marketed
for the treatment of lung cancer by Merck. The amino acid sequence
of pembrolizumab and methods of using are disclosed in U.S. Pat.
No. 8,168,757.
[0302] CD134, also known as OX40, is a member of the
TNFR-superfamily of receptors which is not constitutively expressed
on resting naive T cells, unlike CD28. OX40 is a secondary
costimulatory molecule, expressed after 24 to 72 hours following
activation; its ligand, OX40L, is also not expressed on resting
antigen presenting cells, but is following their activation.
Expression of OX40 is dependent on full activation of the T cell;
without CD28, expression of OX40 is delayed and of fourfold lower
levels. OX-40 antibodies, OX-40 fusion proteins and methods of
using them are disclosed in U.S. patent Nos: U.S. Pat. No.
7,504,101; U.S. Pat. No. 7,758,852; U.S. Pat. No. 7,858,765; U.S.
Pat. No. 7,550,140; U.S. Pat. No. 7,960,515; WO2012027328;
WO2013028231.
[0303] Antibodies to PD-L1 (also referred to as CD274 or B7-H1) and
methods for use are disclosed in U.S. Pat. No. 7,943,743; U.S. Pat.
No. 8,383,796; US20130034559, WO2014055897, U.S. Pat. No.
8,168,179; and U.S. Pat. No. 7,595,048. PD-L1 antibodies are in
development as immuno-modulatory agents for the treatment of
cancer.
[0304] As used herein "immunostimulatory agent" refers to any agent
that can stimulate the immune system. As used herein
immunostimulatory agents include, but are not limited to, vaccine
adjuvants.
[0305] Aminoalkyl glucosaminide phosphates (AGPs) are known to be
useful as vaccine adjuvants and immunostimulatory agents for
stimulating cytokine production, activating macrophages, promoting
innate immune response, and augmenting antibody production in
immunized animals. Aminoalkyl glucosaminide phosphates (AGPs) are
synthetic ligands of the Toll-like Receptor 4 (TLR4). AGPs and
their immunomodulating effects via TLR4 are disclosed in patent
publications such as WO 2006/016997, WO 2001/090129, and/or U.S.
Pat. No. 6,113,918 and have been reported in the literature.
Additional AGP derivatives are disclosed in U.S. Pat. No.
7,129,219, U.S. Pat. No. 6,525,028 and U.S. Pat. No. 6,911,434.
Certain AGPs act as agonists of TLR4, while others are recognized
as TLR4 antagonists.
[0306] Aminoalkyl glucosaminide phosphate compounds employed in the
present invention have the structure set forth in Formula 1 as
follows:
##STR00005## [0307] wherein [0308] m is 0 to 6 [0309] n is 0 to 4;
[0310] X is O or S, preferably O; [0311] Y is O or NH; [0312] Z is
O or H; [0313] each R.sub.1, R.sub.2, R.sub.3 is selected
independently from the group consisting of a C.sub.1-20 acyl and a
C.sub.1-20 alkyl; [0314] R.sub.4 is H or Me; [0315] R.sub.5 is
selected independently from the group consisting of --H, --OH,
--(C.sub.1-C.sub.4) alkoxy, --PO.sub.3R.sub.8R.sub.9,
--OPO.sub.3R.sub.8R.sub.9, --SO.sub.3R.sub.8, --OSO.sub.3R.sub.8,
--NR.sub.8R.sub.9, --SR.sub.8, --CN, --NO.sub.2, --CHO,
--CO.sub.2R.sub.8, and --CONR.sub.8R.sub.9, wherein R.sub.8 and
R.sub.9 are each independently selected from H and
(C.sub.1-C.sub.4) alkyl; and each R.sub.6 and R.sub.7 is
independently H or PO.sub.3H.sub.2.
[0316] In Formula 1 the configuration of the 3' stereogenic centers
to which the normal fatty acyl residues (that is, the secondary
acyloxy or alkoxy residues, e.g., R.sub.1O, R.sub.2O, and R.sub.3O)
are attached is R or S, preferably R (as designated by
Cahn-Ingold-Prelog priority rules). Configuration of aglycon
stereogenic centers to which R.sub.4 and R.sub.5 are attached can
be R or S. All stereoisomers, both enantiomers and diastereomers,
and mixtures thereof, are considered to fall within the scope of
the present invention.
[0317] The number of carbon atoms between heteroatom X and the
aglycon nitrogen atom is determined by the variable "n", which can
be an integer from 0 to 4, preferably an integer from 0 to 2.
[0318] The chain length of normal fatty acids R.sub.1, R.sub.2, and
R.sub.3 can be from about 6 to about 16 carbons, preferably from
about 9 to about 14 carbons. The chain lengths can be the same or
different. Some preferred embodiments include chain lengths where
R.sub.1, R.sub.2 and R.sub.3 are 6 or 10 or 12 or 14.
[0319] Formula 1 encompasses L/D-seryl, -threonyl, -cysteinyl ether
and ester lipid AGPs, both agonists and antagonists and their
homologs (n=1-4), as well as various carboxylic acid bioisosteres
(i.e, R.sub.5 is an acidic group capable of salt formation; the
phosphate can be either on 4- or 6-position of the glucosamine
unit, but preferably is in the 4-position).
[0320] In a preferred embodiment of the invention employing an AGP
compound of Formula 1, n is 0, R.sub.5 is CO.sub.2H, R.sub.6 is
PO.sub.3H.sub.2, and R.sub.7 is H. This preferred AGP compound is
set forth as the structure in Formula 1a as follows:
##STR00006##
[0321] wherein X is O or S; Y is O or NH; Z is O or H; each
R.sub.1, R.sub.2, R.sub.3 is selected independently from the group
consisting of a C.sub.1-20 acyl and a C.sub.1-20 alkyl; and R.sub.4
is H or methyl.
[0322] In Formula 1a the configuration of the 3' stereogenic
centers to which the normal fatty acyl residues (that is, the
secondary acyloxy or alkoxy residues, e.g., R.sub.1O, R.sub.2O, and
R.sub.3O) are attached as R or S, preferably R (as designated by
Cahn-Ingold-Prelog priority rules). Configuration of aglycon
stereogenic centers to which R.sub.4 and CO.sub.2H are attached can
be R or S. All stereoisomers, both enantiomers and diastereomers,
and mixtures thereof, are considered to fall within the scope of
the present invention.
[0323] Formula 1a encompasses L/D-seryl, -threonyl, -cysteinyl
ether or ester lipid AGPs, both agonists and antagonists.
[0324] In both Formula 1 and Formula 1a, Z is O attached by a
double bond or two hydrogen atoms which are each attached by a
single bond. That is, the compound is ester-linked when
Z.dbd.Y.dbd.O; amide-linked when Z.dbd.O and Y.dbd.NH; and
ether-linked when Z.dbd.H/H and Y.dbd.O.
[0325] Especially preferred compounds of Formula 1 are referred to
as CRX-601 and CRX-527. Their structures are set forth as
follows:
##STR00007##
[0326] Additionally, another preferred embodiment employs CRX 547
having the structure shown.
##STR00008##
[0327] Still other embodiments include AGPs such as CRX 602 or CRX
526 providing increased stability to AGPs having shorter secondary
acyl or alkyl chains.
##STR00009##
[0328] In one embodiment methods are provided for treating cancer
in a mammal in need thereof, which comprises: administering to such
mammal a therapeutically effective amount of: [0329] an ICOS
binding protein of the present invention, [0330] and b) at least
one anti-neoplastic agent.
[0331] In one embodiment methods are provided for treating cancer
in a mammal in need thereof, which comprises: administering to such
mammal a therapeutically effective amount of: [0332] an ICOS
binding protein of the present invention, [0333] and b) at least
one second immuno-modulatory agent.
[0334] In one embodiment said second immune-modulatory agent is
selected from the group of: an anti-CTLA4 antibody, an anti-PD-1
antibody, an anti-PD-L1 antibody, an anti-OX40 antibody, an
anti-GITR antibody, and anti-41 BB antibody, an anti-LAG3 antibody
and an anti-TIM3 antibody.
[0335] In one embodiment methods are provided for treating cancer
in a mammal in need thereof, which comprises: administering to such
mammal a therapeutically effective amount of: [0336] an ICOS
binding protein or the present invention, [0337] and b) at least
one immuno-stimulatory agent.
[0338] In one embodiment, the immune-stimulatory agent is a TLR4
agonist. In one embodiment the immune-stimulatory agent is an AGP.
In one aspect, the immune-stimulatory agent is a compound of
Formula I. In one aspect, it is a compound of Formula 1a. In one
aspect, the immune-stimulatory agent is selected from the group
consisting of: CRX-601, CRX-547, CRX-602, CRX-527, and CRX-526.
EXAMPLES
[0339] The following examples illustrate various non-limiting
aspects of this invention.
Example 1: ICOS Expression in Cancer
[0340] In general, solid tumors appear to have low levels of
infiltrating ICOS.sup.+ T cells, consistent with the theory that
ICOS mediates anti-tumor immune responses. ICOS expression analyses
across various tumor histologies was generated using publically
available mRNA expression datasets from The Cancer Genome Atlas
(TCGA) and other databases. Table 3 shows the relative percentage
of tumors from each histology that showed some detectable level of
ICOS mRNA expression. This analysis identifies tumor histologies
known to be sensitive to other cancer immunotherapy approaches
(melanoma, RCC, NSCLC) as having a relatively higher percentage of
tumors (>10%) which have detectable ICOS.sup.+ tumor
infiltrating lymphocytes (TILs), whereas tumor types known to be
poorly immunogenic (prostate, ovarian, and pancreatic) having
relatively lower percentage of tumors which have ICOS.sup.+ TILs
(<10%) (Table 3). Interestingly, tumor types known to be
associated with viral infection and/or chronic inflammation
(H&N, Gastric, Esophageal, and Cervical) were among the tumor
types showing the highest percentage of ICOS.sup.+ TILs. What is
not clear from these mRNA expression analyses is the subpopulation
of TILs which express ICOS in each respective tumor type. In some
instances ICOS may be predominantly expressed on tumor infiltrating
T.sub.regs, while in others it may indicate level of ICOS.sup.+ T
effector cell infiltration.
TABLE-US-00008 TABLE 3 ICOS mRNA expression across different tumor
types Tumor Type Total N ICOS+ (N) ICOS+ (Per.) H&N 426 157
36.9% Gastric 285 75 26.3% Esophageal 70 17 24.3% Melanoma (M) 295
69 23.4% NSCLC(AD) 501 112 22.4% NSCLC(SCC) 489 85 17.4% Cervical
185 32 17.3% Breast 1048 162 15.5% Bladder 244 35 14.3% RCC 522 64
12.3% Melanoma (P) 82 7 8.5% Pancreas 85 7 8.2% Colon 446 34 7.6%
Thyroid 498 34 6.8% HCC 191 11 5.8% Sarcoma 103 4 3.9% Ovarian 412
13 3.2% Prostate 336 10 3.0% Endometrial 532 15 2.8% Rectal 163 4
2.5% GBM 156 0 0.0%
[0341] Analysis of ICOS expression by immunohistochemistry (IHC) in
primary human non-small cell lung carcinoma (NSCLC),
triple-negative breast cancer (TNBC), colo-rectal cancer (CRC),
prostate, pancreatic, ovarian, renal cell cancer (RCC) and melanoma
was performed to better understand which subsets of TILs are
associated with ICOS expression in different tumor types (Table 4).
Similar to what was observed in the mRNA expression analysis, the
abundance of ICOS.sup.+ TILs were relatively low, even in
individual tumors where large numbers of CD4.sup.+, CD8.sup.+
and/or FoxP3.sup.+ TILs were otherwise present. Again, melanoma,
renal cell carcinoma (RCC) and non-small cell lung carcinoma
(NSCLC) histologies had the highest percentage of tumors with some
level of detectable ICOS.sup.+ infiltrate (Table 4, Column 2).
Conversely, prostate, ovarian and pancreatic tumors showed almost
no ICOS.sup.+ TILs (Table 4). These analyses clearly show that
solid tumors have low basal levels of ICOS.sup.+ TILs and may
benefit from the expansion and functional induction of this
population of cells. Future studies using flow cytometry and
dual-color immunohistochemistry to analyze primary human tumors
will help determine which specific T cells subsets express
ICOS.
TABLE-US-00009 TABLE 4 No. of Average number of positive cells
(range) Entity samples ICOS ICOS CD3 CD4 CD8 FOXp3 NSCLC n = 17 - 0
11 (0-74) 2 (0-9) 20 (0-147) 10 (0-28) (squamous) n = 23 + 3 (0-25)
38 (2-143) 8 (0-26) 39 (5-188) 18 (0-75) NSCLC n = 15 - 0 36
(0-157) 4 (0-20) 71 (5-238) 10 (1-41) (adenocarcinoma) n = 25 + 2
(0-7) 56 (0-181) 8 (0-39) 69 (10-201) 19 (0-55) TNBC n = 24 - 0 14
(0-91) 9 (0-132) 17 (0-95) 6 (0-25) n = 11 + 5 (0-20) 85 (3-259) 13
(0-45) 113 (2-393) 30 (2-81) CRC n = 22 - 0 12 (0-47) 14 (0-44) 20
(0-83) 14 (2-52) n = 23 + 2 (0-13) 31 (5-101) 22 (5-48) 53 (4-151)
24 (5-43) Prostate Cancer n = 29 - 0 10 (0-78) 17 (1-96) 23 (1-176)
5 (0-25) n = 1 + 1 30 48 55 11 n = 11 - 0 15 (1-32) 17 (3-71) 20
(3-61) 6 (0-21) Pancreatic Cancer n = 4 + 0 (0-1) 31 (7-85) 17
(6-31) 17 (2-51) 4 (0-9) n = 15 - 0 13 (0-105) 14 (1-78) 13 (0-83)
2 (0-12) Ovarian Cancer n = 5 + 1 (0-3) 19 (6-35) 13 (6-19) 32
(10-76) 7 (4-13) n = 3 - 0 50 (14-104) 58 (36-85) 56 (24-119) 4
(0-9) RCC n = 7 + 5 (1-16) 45 (10-130) 65 (26-164) 71 (12-232) 15
(3-28) n = 7 - 0 42 (1-155) 12 (1-31) 35 (0-156) 5 (0-10) Melanoma
n = 12 + 7 (0-21) 84 (13-222) 32 (13-70) 89 (28-179) 19 (6-40)
Example 2: Screening of ICOS Antibody Agonist
Isolation of Primary Human PBMC
[0342] Fresh blood was obtained from blood donors and was diluted
1:1 with Phenol red free-10% RPMI1640 media. Diluted blood was
layered on top of the density medium in a Uni-Sep Max 50 ml conical
tube and centrifuge at 400.times.g for 20 minutes at room
temperature with BREAK OFF. The resulted white mononuclear layer
(buffy coat) was carefully extracted into a new 50 mL conical tube
through a 100 .mu.M cell strainer. An equal volume of Phenol red
free-10% RPMI1640 media was added to the buffy coat and centrifuged
at 300.times.g for 10 minutes at room temperature. The cell pellet
was resuspended in 10 ml of red blood cell lysis solution (Sigma
Aldrich) and incubated for 5 minutes at room temperature. Cells
were washed once with media and centrifuged as previously
described. Volume was brought to 40 ml with Phenol red free-10%
RPMI1640 media and cells were counted using Vicell cell counter and
viability analyzer (Beckman Coulter).
Isolation of Primary Human CD4+CD25- T Effector Cells
[0343] Human CD4+CD25 cells were further purified from PBMC via
two-step magnetic beads based isolation procedure using human
CD4+CD25+ Regulatory T Cell Isolation kit (Miltenyi Biotec). First,
PBMC cells were incubated with biotin-antibody cocktail at
4.degree. C. for 5 minutes and subsequently incubated anti-Biotin
microbeads for 10 minutes. This step was to label the non-CD4+ T
cells. Cells were then passed through a LD column in the magnetic
field of a MidiMACS separator. The effluent which is the unlabeled
pre-enriched CD4+ cell fraction was collected and incubated with
CD25 MicroBeads at 4.degree. C. for 15 minutes. The labelled cells
were passed through a MS column in the magnetic field of a MiniMACS
Separator. The flow-through containing the unlabeled CD4-CD25- T
effector cells was collected for downstream activation assays.
Isolation of Primary Human CD4+ T Cells Directly from Blood
[0344] Human CD4+ T cells were isolated directly from fresh human
blood using Human CD4+ T cells Enrichment Cocktail (Stem Cell
Technologies). RosetteSep Human CD4+ T Cell Enrichment Cocktail (50
.mu.L/mL) was added to whole blood and mixed well. After 20 minutes
of incubation at room temperature, an equal volume of PBS+2% FBS
was added with gentle mixing. The diluted sample was layered on top
of the density medium and centrifuged for 20 minutes at
1200.times.g at room temperature with the brake off. The enriched
cells from the density medium: plasma interface were carefully
poured into a new conical tube. Next the red blood cells were lysed
with Red Blood Cell Lying Buffer (Sigma Aldrich) and the enriched
cells were washed with PBS+2% FBS twice. The CD4+ T cells were
resuspended in 40 ml of with PBS+2% FBS and counted with Vi-Cell
cell counter.
Experimental Protocols
Human CD4+CD25- T Effector Cell In Vitro Activation Assay--Bound
Assay.
[0345] Non tissue culture treated 96 well flat bottom plates were
coated with 100 .mu.l/well of coating buffer (Biolegend) containing
1 .mu.g/ml anti human CD3 antibody (eBioscience) and various
testing antibodies overnight. On the next day, the pre-coated
plates were washed three times with 10% FBS containing RPMI-1640
medium. Human CD4+CD25- T effectors cells were isolated and
labelled with CFSE as described and seeded onto the plates. After
incubating at 37.degree. C. for 2.5 days, cells were harvested and
analyzed by flow cytometry for proliferation and activation marker
expression. Cell culture supernatants were also collected for
multiplex cytokines measurement by Meso Scale Discovery (MSD).
CFSE Proliferation Assay
[0346] Cells to be labelled were resuspended in 1 ml of pre-warmed
PBS at a final concentration of up to 1E7 cells/mL in a 15 ml of
conical tube. One microliter of 2 mM stock CFSE solution (Life
Technologies) was added directly into the cells followed by
immediately vortexing to ensure uniform labelling. After incubating
at room temperature for 5 minutes, the staining was quenched by
adding 14 ml of ice-cold cell culture media. The labeled cells were
washed three times with ice-cold media. Cells were counted and
adjusted to 1E6 cells/ml in RPMI1640+10% FBS supplemented with 100
IU/ml of IL-2 (PeproTech) and seeded on anti CD3 and testing
antibodies coated plates. After T cell activation, cells were
harvested and washed with PBS+0.5% BSA once before proceeding to
multi-color staining step for flow cytometry analysis.
Multi-Color Flow Cytometry
[0347] Activated T cells were harvested and washed with PBS. Cells
were first stained with LIVE/DEAD Fixable Far Red cell viability
dye (Life Technologies) following vendor's protocol. After washing
the dye off, detection antibodies conjugated with different colors
were incubated with cells at 4.degree. C. for 30 minutes. Stained
cells were washed once with ice cold FACS staining buffer (PBS+0.5%
BSA) before running on FACS Canto or FACS Canto II flow cytometer.
Cytometer performance was checked daily using Cytometer Setup &
Tracking beads (BD Biosciences) and PMT voltages and area scaling
were set based on unstained cells. Compensations were performed
using OneComp or UltraComp beads (eBioscience) that were
individually stained with detection antibodies conjugated with each
fluorochrome.
MSD Cytokine Analysis
[0348] IFN-.gamma., IL-10, IL-2 and TNF-.alpha. cytokine levels in
the tissue culture supernatant were determined using MSD human
V-Plex customized kit. Samples were first diluted 1:200 in
Diluent2. Calibrators were also prepared in Diluent2 following kit
manual. Diluted samples and calibrators were added to black MSD
plate which was subsequently sealed with an adhesive plate seal and
incubated at room temperature with shaking for 2 hours. After
adding 25 .mu.L of detection antibody solution which was freshly
prepared in Diluent2 to each well, the plate was re-sealed and
incubated at room temperature with shaking for another 2 hours. The
plates were washed 3 times with 150 .mu.l/well of PBS plus 0.05%
Tween-20 before adding 150 .mu.l/well of freshly diluted 2.times.
read buffer and immediately read on MESO QuickPlex reader. Data
were analyzed using MSD Workbench software.
Human CD4+ T Cells In Vitro Activation Assay--Bound and Soluble
[0349] Freshly isolated human CD4+ T cells were pre-stimulated on
24 well plates coated with anti-CD3 (1 .mu.g/ml) and anti-CD28 (3
.mu.g/ml) for 48 hours. Cells were harvested, washed and mixed with
anti-CD3 DynaBeads (Life Technologies) at 1:1 ratio in AIM-V medium
supplemented with 100 IU/ml of IL-2 (PeproTech). Cells/beads
mixture were then seeded at 100 k per well onto 96 well flat bottom
plates either non-coated (for soluble format) or coated with H2L5
hIgG4PE (for bound format). For the soluble format, H2L5 hIgG4PE
was added to the wells at the time of cell seeding. After
incubating at 37.degree. C. for 3.5 days, cell culture supernatants
were collected for multiplex cytokines measurement by MSD.
Soluble Human PBMC In Vitro Activation Assay
[0350] Freshly isolated human PBMCs were pre-stimulated on 24 well
plates coated with anti-CD3 (1 .mu.g/ml) and anti-CD28 (5 .mu.g/ml)
for 48 hours. CFSE stained cells were prepared and mixed with
anti-CD3 DynaBeads (Life Technologies) at 1:1 ratio in AIM-V medium
supplemented with 100 IU/ml of IL-2 (PeproTech). Cells/beads
mixture were then seeded at 200 k per well onto 96 well plates that
were pre-coated with 1 .mu.g/ml of anti CD3 antibody. H2L5 hIgG4PE
and control antibody was added directly to the wells in their
soluble form. After incubating at 37.degree. C. for 3.5 days, cell
culture supernatants were collected for multiplex cytokines
measurement by MSD, and cells were harvested for proliferation and
marker expression analysis by flow cytometry.
Data Analysis
Flow Cytometry Data Analysis
[0351] Flow data was analyzed by FlowJo software (FlowJo LLC). Dead
cells were first gated out based on LIVE/DEAD cell viability dye
staining. Doublets were gated out on FSC-H:FSC-W scatter plot. The
resulted live single cells were analyzed for activation marker
expression within different T cell sub-populations such as CD4+ or
CD8+ T cells and reported as percentage of parent population or
Median Fluorescent Intensity (MFI).
CFSE Proliferation Analysis
[0352] CFSE data were also analyzed by Flowjo. After excluding the
dead cells and the doublets, a "proliferated cell" gate was drawn
based on non activated T cells. Any cells fall in this gate in any
given sample were counted as proliferated cells. Data were reported
as percentage of proliferation.
Cell Depletion Analysis by FACS
[0353] Cell depletion was analyzed by FlowJo. First, a live cells
gate was determined based on LIVE/DEAD cell viability dye staining.
Then the doublets were gated out as previously described. The
percentages of live CD4+ or CD8+ T cell sub-population were
calculated as an indicator for cell depletion.
Antibody Dose Response Curve Fitting Analysis
[0354] The dose response data were imported into GraphPad Prism
software and transformed into log scale. Agonist dose response with
various slope model was used to curve fit the data and generate
EC50 values. The fitting formula is listed below:
Y=Bottom+(Top-Bottom)/(1+10 ((Log EC50-X)*HillSlope))
Results
Lead Murine Anti Human ICOS Antibodies Identification
[0355] Fourteen murine mAbs were screened for human and cynomolgus
ICOS binding and agonist activity. Twelve were able to be
re-cloned, sequenced, grown up and purified in sufficient amounts
for functional studies. All were tested for binding characteristics
using BIAcore. Two were found to be very weak/non-binders. Ten
purified mAbs tested by functional "agonism" analysis. The four
best agonist mAbs (designated as 422.2, 279.1, 314.8 and 88.2 in
Table 5 below), based upon their ability to induce T cell
proliferation and IFN-.gamma. cytokine production across multiple
healthy human donors, were selected and made as human IgG1
chimeras. The CDR sequences for 314.8, 88.2, 92.17, 145.1, and 53.3
are shown with other ICOS mAbs in PCT/EP2012/055735 (WO
2012/131004).
The heavy chain variable region for clone 88.2 is presented below
as SEQ ID NO:13:
TABLE-US-00010 (SEQ ID NO: 13)
QVQLQQPGAELVRPGASVKLSCKASGYSFTSYWINWVKQRPGQGLEWIGN
IYPSDSYTNYNQMFKDKATLTVDKSSNTAYMQLTSPTSEDSAVYYCTRWN
LSYYFDNNYYLDYWGQGTTLTVSS
The light chain variable region for clone 88.2 is presented below
as SEQ ID NO:14:
TABLE-US-00011 (SEQ ID NO: 14)
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLI
GGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYNNHLVF GGGTKLTVL
TABLE-US-00012 TABLE 5 ICOS mAb Binding and Competition Compe-
Compe- ICOS Biacore Biacore CD28/ ICOS-L tition tition mAb human
cyno CTLA4 binding against against Clone # (nM) (nM) x-reactive
inhibition 314.8 121.4 53.3 30.4 19.7 - ++ + + 88.2 31 23 - + + +
92.17 27.5 18.8 - ++ + + 145.1 49.5 43.5 - ++ + + 314.8 17 9.3 -
+++ + + 121.4 15 58 - - -/+ -/+ 202.24 46 19 - + + + 279.1 39 33 -
+ + + 293.1 7.6 10 - ++ + + 422.2 5.7 4.46 - ++ ++ +
[0356] Eight of the best ICOS binders were tested for IFN-.gamma.
production and T cell proliferation using a cell based assay in a
dose escalation design against mouse IgG1 and EPR7114 controls.
Based on these assays, clone designated as 422.2 was selected for
humanization. See FIGS. 1 and 2.
Example 3 Antibody Humanization
Experimental Protocol(s)
[0357] Recovery of Antibody Variable Genes from mRNA and Generation
of Chimeric Fc Wild-Type Antibody
[0358] Total RNA was purified from the 422.2 hybridoma cell pellet,
reverse transcribed to generate cDNA from which the variable gene
products, approximately 400 bp, were isolated by PCR and purified
by agarose gel electrophoresis.
[0359] The purified variable region fragments were cloned in to
pTT5 vectors containing either the human IgG1 constant region or
the human Kappa constant region and transformed into DH5a competent
cells. Colonies were picked and used to inoculate L-broth
containing ampicillin. Plasmid DNA was isolated from the cultures
using a QuickLyse mini-prep kit. Variable heavy and light chain
genes were sequenced and sequence data was aligned by informatics
to identify the variable heavy and light chain gene sequences.
Cloning of Codon Optimised Chimeric 422.2 Antibodies
[0360] The mature murine variable region protein sequences were
reverse translated to DNA then codon optimised. The V.sub.H and
V.sub.L sequences were then modified to include the preferred five
prime untranslated region and preferred cloning sites at either
end. The adapted V.sub.H sequence was constructed de novo using a
PCR-based strategy and overlapping oligonucleotides then grafted
onto human IgG1 Fc wild type or Fc disabled hIgG1 (L235A, G237A) or
hinge stabilised hIgG4 (S228P, L235E) (IgG4PE) present in pTT
vectors. The adapted V.sub.L sequence was constructed de novo using
a PCR-based strategy and overlapping oligonucleotides then grafted
onto a kappa constant region present in a pTT5 vector.
The Resulting pTT Plasmids were Used in HEK Transfections to
Produce the Chimeric Antibodies
Humanization of the Variable Domains of 422.2
[0361] Human variable (V) gene templates were chosen for
humanization of 422.2 by searching appropriate in-house human
germline heavy and light chain databases with CDR-masked 422.2 V
regions using BLASTP. IGHV1-69 and IGKV3-11 were chosen from the
top BLASTP hits as the V gene framework templates for 422.2
humanization. IGHJ6 and IGKJ2 human germline Joining (J) genes were
chosen for humanization of 422.2.
[0362] Residue differences between the chosen human germline genes
and the 422.2 sequence were identified to aid in the selection of
back-mutations (mutations made to change the specific human
framework residue to the murine residue). Six humanized VH variants
and six humanized VL variants were designed, codon optimised and
then modified to include preferred 5' and 3' extensions. The
adapted variable region sequences were constructed de novo using a
PCR-based strategy and overlapping oligonucleotides then
respectively cloned into pTT vectors.
[0363] The resulting pTT plasmids were used in HEK transfections to
produce the humanized antibodies. HEK2936E suspension cells were
counted and diluted to 1.5.times.10.sup.6 cells/mL to
2.times.10.sup.6 cells/mL using Freestyle expression medium 293
supplemented with 0.05% Geneticin and for some experiments
supplemented with 1% pluronic F68. DNA and transfection reagent
(Gemini or 293-Fectin) were added to OptiMEM and gently homogenised
prior to incubation for 20 to 30 minutes at room temperature. The
DNA-lipid complexes were then combined with the cell suspensions
and the transfected cells were incubated at 37.degree. C., 5% CO2,
125 rpm. For some transfections, a tryptone feed (Freestyle
expression medium 293 supplemented with 1% pluronic F68 and 20% w/v
casein tryptone) was added to each transfection 24 to 48 hours
after transfection. Transfected cell suspensions were incubated for
5 to 8 days until viable cells dropped below 60% then centrifuged
(construct dependent). Supernatants were harvested and
filtered.
[0364] Antibodies were purified by passing supernatants through a 1
mL HiTrap Protein A HP column to enable antibody capture, washing
the column through with 10 mL of PBS and eluting with 5 mL of IgG
Elute (Pierce, 21009). Purified protein was buffer exchanged into
PBS using the Millipore Centricon.RTM. Centrifugal Filter Units
(30K cut-off) and quantified on the Nandrop spectrophotometer.
Results
Constructed Expression Plasmids
[0365] The murine antibody variable gene sequences of hybridoma
clone 422.2 were successfully recovered and the sequences are shown
below as SEQ ID NOs: 19 and 20, respectively as well as in FIG.
8.
TABLE-US-00013 422 HC (SEQ ID NO: 19)
QVQLQQSGPELVRPGESVKISCMGSGYTFTDYAMHWVKQSHAKSLEWIGL
ISIYSDHTNYNQKFQGKATMTVDKSSSTAYMELARLTSEDSAIYYCGRNN
YGNYGWYFDVWGAGTTVTVSS 422 LC (SEQ ID NO: 20)
ENVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSITSPKLWIYDT
SKLASGVPGRFSGSGSGNSYSLTISSMEAEDVATYYCFQGSGYPYTFGGG TKLEIKR
[0366] Cloning the recovered variable regions into selected pTT5
vectors resulted in generation of plasmids encoding the chimeric
light chain sequence of 422.2 and the chimeric heavy chain
sequences on hIgG1 Fc wild type, hIgG1 Fc disabled (L235A/G237A, EU
numbering) or a hIgG4 PE (S228P/L235E, EU numbering). The chimeric
antibodies were used to confirm functionality of the cloned mouse
V-regions and to identify the most suitable isotype.
[0367] Construction of pTT mammalian expression plasmids encoding
the heavy and light chains of the various humanized variants of
422.2 was carried out successfully.
Expression, Purification and Identification of H2L5 hIgG4PE
[0368] The mature protein sequences of H2L5 hIgG4PE have been
included with additional labeling in FIG. 9. The DNA sequences of
the coding regions of H2L5 hIgG4PE heavy and light chains have been
included in FIGS. 10 and 11.
Example 4: Functional Analysis of H2L5
Fc Receptor Binding
[0369] Humanized antibody H2L5 was modified from a human IgG1
isotype to a modified human IgG4 isotype incorporating mutations
S228P, L235E (EU numbering) to prevent antigen binding fragment
(Fab) arm exchange. Human IgG4PE was selected over human IgG1 as it
diminishes binding of the mAb to activating Fc.gamma. receptors and
C1q, therefore, reducing the potential of the mAb to induce
depletion of ICOS positive cells by antibody-dependent cytotoxicity
(ADCC) or complement dependent cytotoxicity (CDC). In addition,
human IgG4PE (S228P, L235E) retains binding to Fc.gamma.RIIb
(inhibitory Fc.gamma. receptor). Interaction with Fc.gamma.RIIb may
be critical for the agonistic activity of ICOS antibodies by
enabling antibody cross-linking. Interaction with Fc.gamma.RIIb has
been shown to be critical for the agonistic activity of other
immunomodulatory antibodies targeting TNF-.alpha. family receptors
as well as CD28 (Bartholomaeus P et al., "Cell contact-dependent
priming and Fc interaction with CD32+ immune cells contribute to
the TGN1412-triggered cytokine response." J. Immunol., 192(5);
2091-8 (2014)).
[0370] It was further shown that human IgG4PE diminishes binding of
the mAb to activating Fc.gamma. receptors (Fc.gamma.RI,
Fc.gamma.RIIa and Fc.gamma.RIIIa) and C1q, therefore reducing the
potential of the mAb to induce depletion of ICOS positive cells by
antibody-dependent cytotoxicity (ADCC) or complement dependent
cytotoxicity (CDC). In addition, human IgG4PE (S228P, L235E)
retains binding to Fc.gamma.RIIb (inhibitory Fc.gamma. receptor)
(Table 6).
Table 6 below displays representative binding affinities to human
Fc.gamma. receptors of the lead H2L5 as either a hIgG1 or a hIgG4PE
antibody.
TABLE-US-00014 TABLE 6 Representative Affinities of Lead Humanized
ICOS antibody, as either a hIgG1 or a hIgG4PE, to human Fc.gamma.
receptors Fc.gamma.R Fc.gamma.R Fc.gamma.R Fc.gamma.R Fc.gamma.R
IIa IIa Fc.gamma.R IIIa IIIa I H131 R131 IIb V158 F158 KD KD KD KD
KD KD Antibodies (nM) (nM) (nM) (nM) (nM) (nM) 422 H2L5 IgG1 WT
60.8 405 662 1340 281 862 422 H2L5 hIgG4PE 645 NB 2500 1470 NB NB
(H2L5 IgG4PE) NB = no binding.
Experimental Protocol
Functional Evaluation of 422.2 Humanized Variants
[0371] To humanize the four candidate ICOS agonist antibodies,
mouse-human chimeras, which are fusions of mouse V region and human
IgG1 Fc portion, were generated. These four chimera antibodies were
tested in human whole PBMC activation assay as plate bound form.
Anti-ICOS chimera 422.2 showed the best agonistic activity in the
bound PBMC activation assay. Combined with binding data and
biophysical properties, 422.2 clone was chosen for humanization.
Four humanized 422.2 variants were selected based on ICOS binding
and biophysical characteristics (422.2 H2L0, H2L5, H5L0 and H5L5)
and were tested in bound human PBMC activation assays. The H2L5
variant demonstrated comparable or better T cell activation as
measured by cytokine production relative to other variants (FIG.
3).
[0372] The heavy chain (V.sub.H) variable region and mature heavy
chain for the H5 variant are presented below as SEQ ID NOs:15 and
16, respectively.
TABLE-US-00015 H5 VH (SEQ ID NO: 15)
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYAMHWVRQAPGQGLEWIG
LISIYSDHTNYNQKFQGRATMTVDKSTSTAYMELSSLRSEDTAVYYCGR
NNYGNYGWYFDVWGQGTTVTVSS Mature H5 heavy chain (SEQ ID NO: 16)
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYAMHWVRQAPGQGLEWIG
LISIYSDHTNYNQKFQGRATMTVDKSTSTAYMELSSLRSEDTAVYYCGR
NNYGNYGWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQGGSLYSLSSVVTVPSSS
LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK
[0373] The light chain (V.sub.L) variable region and mature light
chain for the L0 variant are presented below as SEQ ID NOs:17 and
18, respectively.
TABLE-US-00016 L0 VL (SEQ ID NO: 17)
EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYD
TSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCFQGSGYPYTFG QGTKLEIK Mature
L0 light chain (SEQ ID NO: 18)
EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYD
TSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCFQGSGYPYTFG
QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
HQGLSSPVTKSFNRGEC
Selection of IgG4[PE] as the Isotype
[0374] The 422 H2L5 IgG1 was subsequently tested in various whole
PBMC activation assays in soluble form. This soluble format is
likely to be more relevant to the in vivo condition as the whole
PBMC assays contain lymphocytes, monocytes and other immune cells
in the same well. However, 422 H2L5 IgG1 mAb consistently showed
decreased viability of T cell populations which is reminiscent of T
cell depletion. This result was observed to different degrees in 11
healthy donors which were tested and was more prominent in CD4+ T
cells than in CD8+ T cells. In contrast, the 422 H2L5 antibody did
not show a significant decrease in T cell viability when expressed
as either an IgG4[PE] or Fc-disabled isotype, suggesting that the
decreased viability may have been due to Fc.gamma.R-mediated
antibody-dependent cellular cytotoxicity (ADCC) (FIG. 4).
Dose Response of H2L5 hIgG4PE in CD4+ T Cell Activation Assay
[0375] To quantify the T cell activation effects of H2L5 hIgG4PE,
human primary CD4+ T cells were first pre-stimulated by plate bound
anti-CD3 (1 .mu.g/ml)/anti-CD28 (3 .mu.g/ml) for 48 hours to induce
levels of ICOS receptor on the surface of T effector cell
populations, followed by re-stimulation with anti CD3 DynaBeads and
H2L5 hIgG4PE. A 10-point dose response curve was generated by
treating the pre-simulated CD4+ T cells with serial concentrations
of bound or soluble H2L5 hIgG4PE in the presence of anti-CD3
DynaBeads. Results showed that both bound and soluble H2L5 hIgG4PE
increased IFN-.gamma. and TNF-.alpha. cytokines productions in a
dose dependent manner in two separate healthy human donors (FIG.
5). A dose-response curve fitting analysis was performed to
generate EC50 values. Interestingly, H2L5 hIgG4PE treatment
resulted in a significantly greater magnitude of cytokine induction
when the antibody was plate-fixed as opposed to being added as a
soluble protein to the supernatant of the T cell cultures.
Functional Testing of H2L5 hIgG4PE in Soluble Human PBMC Activation
Assay
[0376] In order to test the function of H2L5 hIgG4PE in whole human
PBMC ex vivo culture, PBMC from healthy human donors were prepared
with plate bound anti-CD3 and anti-CD28 for 48 hours, followed by
soluble H2L5 hIgG4PE treatment in the presence of anti CD3
dynabeads (bead:cell=1:1 ratio) for 3.5 days. Cytokine production
and T cell Granzyme B expression was examined as functional
readouts for T cell function. The results from 3 donors were
summarized in FIG. 6, which provide evidence to suggest that H2L5
hIgG4PE induces proliferation, cytokine production and increased
cytotoxic potential in activated PBMCs from healthy human donors
(FIG. 6).
ICOS mAb Activity Against Pre-Stimulated Human PBMCs
[0377] The activity of H2L5 hIgG4PE was evaluated in a PBMC
pre-stimulation assay in which the PBMC were pre-stimulated by
plate bound anti-CD3 (clone OKT3, 1 .mu.g/ml) and anti CD28 (clone
CD28.2, 3 .mu.g/ml) for 48 hours. Next, in order to identify the
optimal pre-stimulation conditions, human CD3 Dynabeads and
CD3/CD28 Dynabeads (ThermoFisher) at different bead to cell ratios
were used to pre-stimulate PBMCs. After 48 hr pre-stimulation,
cells were harvested and beads magnetically removed before
re-stimulation with anti CD3 Dynabeads (bead to cell ratio=1:1) in
the presence or absence of soluble ICOS mAb. H2L5 hIgG4PE ICOS
agonist mAb resulted in induction of IFN.gamma. as compared to
isotype control in all pre-stimulation conditions tested; however,
the magnitude of IFN.gamma. production inversely correlated with
strength of the pre-stimulation.
Example 5--T Cell Activation Markers
Methods
[0378] Concentration-dependent changes in functional endpoints were
assessed by treating healthy human PBMCs with immobilized H2L5
hIgG4PE at concentrations ranging from 0.1 .mu.g/mL to 100 .mu.g/mL
concurrently with anti-CD3 antibody treatment at 0.6 .mu.g/mL.
Changes in expression of T cell surface activation markers CD69,
OX40 and CD25 were evaluated by flow cytometry and considered a
measure of T cell activation. Proliferation of T cells was measured
by the changes in Ki67 nuclear staining. Change in levels of
various Th1, Th2 and Th17 cytokines were evaluated on the MSD
platform in response to H2L5 hIgG4PE treatment in the presence of
CD3 engagement. The 24- and 48-hours post treatment timepoints were
selected to ensure the capture of both early cytokine changes as
well as proliferation changes which are predominantly noticed at
later time points.
Experimental Preparation(s)
Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs)
[0379] Whole blood was collected from healthy donors with syringes
coated with liquid sodium heparin (Sagent 10 IU/mL final
concentration) and subsequently diluted 1:1 with phosphate buffered
saline (PBS). Diluted blood (35 mL) was layered on top of 15 mL
Ficoll density gradient medium (GE Healthcare) and centrifuged at
1200.times.g for 20 minutes at room temperature (RT) without
brakes. The white mononucleated cell layer was carefully
transferred into a new 50 ml tube. An equal volume of PBS was added
to the tube and centrifuged at 400.times.g for 5 minutes at RT.
PBMC were washed once with PBS and centrifuged as previously
described. PBMC were resuspended in 50 mL AIM-V media Cells were
counted using a Vi-Cell cell counter and viability analyzer
(BeckmanCoulter).
Antibody Coating
[0380] Anti-human CD3 antibody was diluted in coating buffer to a
final concentration of 0.6 .mu.g/mL. 100 .mu.L diluted antibody was
coated on 96-well, flat-bottom plate overnight at 4.degree. C. Next
day, stock solutions of 10.1 mg H2L5 hIgG4PE and 7.9 mg anti-RSV
IgG4 PE isotype control antibody were 1:2 serially diluted in
coating buffer to give final antibody concentrations ranging from
100 to 0.1 .mu.g/mL. 100 .mu.L diluted antibodies were coated on
anti-CD3 coated plate for 4 hours at room temperature.
Experimental Protocol(s)
Human PBMC Activation Assay
[0381] H2L5 hIgG4PE was tested in a human PBMC activation assay
where TCR engagement via anti-CD3 antibody and ICOS co-stimulation
with H2L5 hIgG4PE occurred concurrently, and the activation effects
were monitored at 24 and 48 hours post activation. This experiment
was repeated four times (n=4) with blood from four different
donors. Two hundred (200) .mu.L PBMCs (1.times.10.sup.6 cells/mL)
in AIM-V medium were added into anti-CD3 antibody-coated wells with
various concentrations of H2L5 hIgG4PE or IgG4 isotype control.
Three technical replicates were included for each assay condition.
PBMCs were cultured at 37.degree. C. and 5% CO.sub.2 for various
times as indicated above. Supernatants were collected at 24- and
48-hour time points and then stored at -80.degree. C. for analysis
of secreted cytokines on the MSD platform. Cells were transferred
into 96-deep well plate at both 24 and 48 hours and washed twice
with 1 mL FACS Staining Buffer stained with fluorophore-conjugated
antibodies or isotype controls.
Cell Surface Staining
[0382] Cells were first stained with 100 .mu.L of fixable viability
dye eFluor 506 pre-diluted 1:1000 in PBS for 30 minutes in the dark
at 4.degree. C. Cells were washed and then incubated with detection
antibodies to cell surface markers conjugated to different
fluorophores on ice for 30 minutes. Post-staining with cell surface
antibodies, samples not to be stained for internalization markers
were washed once with ice cold FACS Staining Buffer before running
on FACS Canto II flow cytometer.
[0383] Cytometer performance was appropriately evaluated daily
using the Cytometer Set-up and Tracking beads. Compensations were
performed using AbC anti-mouse Bead kit that were individually
stained with detection antibodies conjugated with each
fluorochrome. Samples were run and data acquired after proper
compensation set up was ascertained with a mix of beads mentioned
above.
Intracellular Staining for Foxp3 and Ki67
[0384] Following the cell surface staining, the cells were fixed
and permeabilized for staining for intracellular markers. Using the
Transcription Factor Buffer set, the Fixation/Permeabilization
Buffer was prepared by diluting this 1:3 in Diluent Buffer. The
Perm Wash Buffer was prepared by diluting the 5.times. Perm/Wash
Buffer stock in deionized water. One (1) mL of Fix/Perm Buffer was
added to each sample, and the plates were immediately vortexed on
the shaker. The plates were incubated in the dark at 4.degree. C.
for 45 minutes. Following centrifugation, 1 mL of Perm/Wash Buffer
was added, and the plates were mixed and centrifuged for a total of
two washes. The internalization cocktail was prepared with marker
antibodies. 100 .mu.L of the internalization antibodies were added
to the appropriate samples, and the plates were incubated in the
dark at 4.degree. C. for 30 minutes. Samples were washed twice with
1 mL of Perm Wash Buffer, resuspended in 250 .mu.L of flow buffer
and run on FACS Canto II flow cytometer.
Human Special Order 9-Plex Cytokine Assay
[0385] Cytokine levels were measured using the MSD Human Special
Order 9-Plex kit. Samples and calibrators were diluted in Diluent
43. Samples were diluted 1:10 for the 9-plex assay and 1:200 for
the IFN.gamma. assay described in 0.250 .mu.L of the diluent was
added to each of two calibrator panels. After vortexing, the
calibrators were incubated on ice for at least 5 minutes. Two
hundred (200) .mu.L of each calibrator panel was added to 400 .mu.L
of diluent to make the top concentration of calibrator, and a 1:4
serial dilution was used to prepare the 6 additional calibrator
dilutions. Diluent 43 was used as the plate background. 50 .mu.L of
diluted samples (in triplicate) and calibrators (in duplicate) were
added to the MSD plate. Plates were sealed and incubated at RT with
shaking for 2 hours. Plates were washed 3 times with 150 .mu.L of
diluted MSD Wash Buffer from the kit. For each plate, detection
antibody solution was prepared by combining 60 .mu.L of each of the
9 detection antibodies brought up to 3 mL with Diluent 3. Following
the addition of 25 .mu.L of detection antibody solution, the plates
were sealed and incubated at room temperature, in the dark, with
shaking for 2 hours. Plates were washed as above. 150 .mu.L of
2.times. Read Buffer was added to the plates, and they were read on
the QuickPlex.
Human IFN.gamma. Cytokine Assay
[0386] Samples and calibrators were diluted in Diluent 2. 1 mL of
the diluent was added to the calibrator. After vortexing, the
calibrator was incubated on ice for 5 minutes. This is Calibrator
1. A 1:4 serial dilution was used to prepare the 6 additional
calibrator dilutions. Diluent 2 was used as the plate background.
Fifty (50) .mu.L of diluted samples (in tripicate) and calibrators
(in duplicate) were added to the MSD plate. Plates were sealed and
incubated at RT with shaking for 2 hours. Plates were washed 3
times with 150 .mu.L of diluted MSD Wash Buffer from the kit.
Detection antibody solution prepared in Diluent 3. For each plate,
60 .mu.L of each of the detection antibody was added to the diluent
for a total of 3 mL of detection reagent. Following the addition of
25 .mu.L of detection antibodies, the plates were sealed and
incubated at room temperature, in the dark, with shaking for 2
hours. Plates were washed 3 times. Read Buffer was added to the
plates and they were read on the QuickPlex.
Data Analysis
Cytokine and Flow Cytometry Data Analysis
[0387] Results of MSD cytokine assay were analyzed using MSD
Discovery Workbench software, version 4.0 (Meso-Scale), Microsoft
Excel, and Graphpad Prism. Flow cytometry data were analyzed by
DIVA, and numbers were plotted in GraphPad Prism software.
Antibody Dose-Response Curve Fitting Analysis
[0388] The dose response data were imported into GraphPad Prism
software and transformed into log scale. Agonist dose response with
various slope model was used to analyze the data and generate EC50
values. The fitting formula is listed below:
Y=Bottom+(Top-Bottom)/(1+10 ((Log EC50-X)*HillSlope))
Statistical Analyses
[0389] Differences between H2L5 hIgG4PE and isotype antibody
control values in the proliferation study were analyzed for a
statistical significance by two-paired Student's t test.
Results
[0390] Evaluation of Cytokine Changes with H2L5 hIgG4PE
[0391] Treating PBMCs with immobilized H2L5 hIgG4PE in the presence
of anti-CD3 induced the secretion, to different degrees, of Th1
cytokines such as IFN.gamma., TNF.alpha., the Th2 cytokines, IL-6
and IL-10, as well as the Th17 cytokine IL-17a in a
concentration-dependent manner. Other cytokines measured such as
IL-4, IL-5 and IL-13 also showed a lesser extent of
concentration-dependent response to H2L5 hIgG4PE stimulation. The
results from four separate donors are summarized in Table 7.
Functional Evaluation of H2L5 hIgG4PE Activity on Cell Surface
Markers of T Cell Activation by Flow Cytometry
[0392] H2L5 hIgG4PE treatment with concurrent CD3 stimulation in
unactivated human PBMC (n=4 donors) induced significant changes in
T cell activation markers (Table 7 and 8)). Robust increases in
CD25 and OX40 positive CD4 and CD8 T cells were observed in H2L5
hIgG4PE treated samples when compared to the human IgG4 isotope
control samples. The percent of CD69 positive CD4 and CD8 T cells
were also increased at 24- and 48-hour time point in a
concentration-dependent manner.
Characterization of the Effect of H2L5 hIgG4PE on T Cell
Proliferation
[0393] Immobilized H2L5 hIgG4PE treatment with concurrent CD3
activation resulted in a concentration-dependent increase in both
CD4 and CD8 T cell proliferation (n=6 donors) as measured by
intracellular Ki67 staining (Table 8). H2L5 hIgG4PE also increased
CD4+CD25+Foxp3+ regulatory T cell proliferation in a concentration
dependent manner but to a lesser extent than what was observed with
total CD4 and CD8 T cells. The enhancement of T cell proliferation
by H2L5 hIgG4PE was only observed at the 48 hour time point. The
increased proliferation of the regulatory T cells was not
significant whereas the concentration-dependent increase in
proliferating CD4+ cells (p<0.05 for concentrations greater than
0.4 .mu.g/mL H2L5 hIgG4PE) and CD8+ T cells (p<0.05 for
concentrations between 0.2 and 1.6 .mu.g/mL) was significant (see
Table 7).
TABLE-US-00017 TABLE 7 EC50 values (.mu.g/mL) from all functional
endpoints for H2L5 hIgG4PE in human PBMC activation assay.
Donor_1136F50 Donor_185M45 Donor_1124F36 Donor_1149M52 24 hr 48 hr
24 hr 48 hr 24 hr 48 hr 24 hr 48 hr IL-10 1.7 1.3 0.7 0.8 0.7 0.6
0.5 0.6 IFN-.gamma. 0.4 1.5 0.3 0.7 0.3 0.5 0.2 0.6 IL-17a 1.4 1.6
0.8 1.1 0.6 0.8 0.7 1.0 IL-6 0.7 1.1 0.7 0.8 NA NA 0.2 0.3
TNF-.alpha. NA 0.3 NA 0.5 NA 0.3 NA 0.2 CD4+CD69+ 0.5 0.4 0.8 NA
1.1 1.1 0.5 0.4 CD4+CD25+ 0.3 0.6 2.4 0.5 0.6 0.6 0.6 0.4 CD4+OX40+
0.2 0.4 1.6 NA 0.6 1.2 0.6 0.4 CD8+CD69+ 0.5 0.6 1.2 0.5 0.8 1.1
0.6 0.4 CD8+CD25+ 0.4 0.7 2.sup. 0.5 0.6 0.5 1.0 0.4 CD8+OX40+ 0.3
0.5 1.6 NA 2.4 0.5 1.5 0.3 CD4+Ki67+ NT NT NA 0.6 NA 0.8 NA 0.5
CD8+Ki67+ NT NT NA 0.4 NA 0.7 NA 0.3 NA = no analysis (EC50 values
not accurate due to poor curve fitting). NT = no tested.
TABLE-US-00018 TABLE 8 Percent of CD25, Foxp3 and Ki67 positive CD4
or CD8 T cells in human PBMCs after stimulation with H2L5 hIgG4PE
in the presence of anti-CD3 for 48 hours Antibody H2LA hIgG4PE
Isotype Control concen- (% of CD4 or CD8 T cells) (% of CD4 or CD8
T cells) tration Donor Donor Donor Donor Donor Donor TTEST .mu.g/ml
185M45 1124F36 1149M52 185M45 1124F36 1149M52 (p value) % Treg 0
10.2 3.8 6.1 10.2 3.8 6.1 0.415 CD4 T 0.1 12.4 4.2 7.6 8.6 3.6 6.6
0.220 cells 0.2 11.8 4.5 9.8 8.5 3.2 7.1 0.054 0.4 14.3 5.2 11.3
8.6 3.4 7.4 0.076 0.8 18.6 6.9 14.3 8.4 3.4 7.3 0.069 1.6 21.4 7.5
15.9 7.7 3.8 7.2 0.095 3.1 20.8 7.9 15.0 10.4 3.9 7.1 0.058 6.3
18.7 8.3 14.7 10.3 3.6 8.3 0.026 12.5 20.2 7.6 15.1 9.6 4.0 8.1
0.072 25 19.5 7.6 13.9 11.5 5.1 8.3 0.079 50 20.0 7.9 13.3 9.6 5.0
9.7 0.143 100 18.7 7.9 13.3 11.5 5.6 8.8 0.080 % Ki67+ 0 7.2 1.2
2.3 7.2 1.2 2.3 0.693 CD4 T 0.1 9.6 1.5 2.9 6.1 0.9 2.1 0.219 cells
0.2 9.0 2.1 4.4 5.0 0.9 2.4 0.098 0.4 11.9 3.9 7.5 6.8 1.0 2.7
0.025 0.8 18.2 7.1 12.4 7.6 1.3 2.5 0.028 1.6 19.5 8.9 15.9 5.4 0.8
3.2 0.024 3.1 21.1 10.7 14.4 9.1 1.2 2.9 0.005 6.3 18.7 12.6 17.1
8.1 1.1 3.2 0.007 12.5 22.2 12.2 16.5 7.4 1.5 3.4 0.008 25 20.7
11.6 16.1 9.3 1.7 5.2 0.002 50 21.2 12.8 14.3 7.5 2.4 7.3 0.034 100
21.8 12.0 14.0 9.4 2.8 4.7 0.010 % Ki67+ 0 11.0 4.4 2.7 11.0 4.4
2.7 0.841 CD8 T 0.1 15.2 8.3 4.8 9.6 5.3 2.3 0.061 cells 0.2 13.5
10.9 7.8 8.2 3.8 2.9 0.014 0.4 17.4 14.4 11.3 10.1 3.4 4.3 0.023
0.8 20.7 19.6 13.8 14.2 4.5 2.0 0.047 1.6 22.2 22.8 16.1 10.7 4.0
3.7 0.025 3.1 21.8 26.7 13.6 14.5 4.4 3.0 0.099 6.3 20.2 29.8 16.0
10.8 3.3 5.0 0.103 12.5 21.7 29.6 15.6 10.7 4.1 3.4 0.074 25 20.0
28.7 14.6 10.4 6.1 5.0 0.084 50 19.0 31.4 13.5 7.6 5.1 5.5 0.114
100 19.2 27.4 13.4 7.9 7.1 3.5 0.051 % Ki67+ 0 29.7 10.2 11.3 29.7
10.2 11.3 0.920 Treg 0.1 32.8 12.5 14.4 32.4 10.0 12.3 0.127 cells
0.2 33.0 14.8 19.0 30.3 8.3 13.4 0.051 0.4 33.6 22.2 22.2 28.4 10.8
14.1 0.044 0.8 37.0 26.3 26.3 36.7 8.5 14.4 0.191 1.6 35.0 27.7
28.4 26.8 7.7 16.3 0.061 3.1 38.3 30.2 27.2 33.9 9.6 17.4 0.135 6.3
33.9 32.5 26.1 36.7 8.7 16.5 0.315 12.5 37.3 36.8 26.2 31.2 15.1
17.2 0.125 25 36.3 33.0 28.6 33.3 13.7 28.1 0.326 50 37.7 33.6 24.2
32.9 21.1 29.5 0.518 100 40.1 35.8 28.6 36.3 21.5 26.4 0.216
Discussion
[0394] It is well established that ICOS is important for T cell
activation and induction of both Th1 and Th2 cytokines. In this
study, the in vitro activity of H2L5 hIgG4PE (anti-ICOS agonist
antibody), was demonstrated with various measures of T cell
activation and cytokine induction. All measured T cell activation
markers, CD25 (IL-2 receptor alpha-chain), CD69 (early activation
marker) and OX-40 (co-stimulatory marker) were upregulated upon
treatment with H2L5 hIgG4PE in conjunction with CD3 stimulation.
Among the activation markers monitored, the percent of T cells
expressing CD69 and OX40 were strongly increased by H2L5 hIgG4PE
treatment. CD69 is an early activation marker and hence the effects
are predominant in the 24-hour samples. CD25, another important T
cell activation marker, was increased upon treatment with H2L5
hIgG4PE at both time points, suggesting that H2L5 hIgG4PE plays an
important role in maintaining activation of T cells. Ki67 is a
nuclear protein associated with cell proliferation. Flow cytometry
data with Ki67 intracellular staining indicated that immobilized
H2L5 hIgG4PE significantly enhanced both CD4 and CD8 T cell
proliferation in the context of TCR engagement. Though
proliferation of regulatory T cells was also increased by H2L5
hIgG4PE, the changes were not statistically significant.
[0395] Human Th17 cells are a key player in the regulation of
anti-tumor immunity [Nunez, S., et al., T helper type 17 cells
contribute to anti-tumour immunity and promote the recruitment of T
helper type 1 cells to the tumour. Immunology 2013; 139: 61-71].
Studies show ICOS is involved in human Th17 development and
function [Kimura, A., et al., Regulator of Treg/Th17 balance. Eur.
J. Immunol. 2010; 40: 1830-1835; Paulos C M et al. The inducible
costimulator (ICOS) is critical for the development of human Th17
cells. Sci Transl Med. (2010) 2(55); 55ra78; Nelson, M. H., et al.
The inducible costimulator augments Tc17 cell responses to self and
tumor tissue. J Immunol, 2015; 194: 1737-1747]. In the current
functional evaluation of H2L5 hIgG4PE, a majority of cytokines
related to inflammatory and immune responses were measured in the
supernatant of cell cultures after anti-CD3 and H2L5 hIgG4PE
stimulation. H2L5 hIgG4PE strongly induces secretion of Th1
cytokines, IFN-.gamma. and TNF-.alpha. and Th17 cytokine, IL-17a,
in human PBMCs, suggesting that H2L5 hIgG4PE has the potential to
play an important role in anti-tumor responses. IL-6, together with
TGF-.beta., is an important cytokine for induction of the Th17 cell
development from naive T cells. In contrast, IL-6 inhibits Treg
differentiation induced by TGF-.beta. [Kimura, A., 2010; Korn, T.,
et al., IL-6 controls Th17 immunity in vivo by inhibiting the
conversion of conventional T cells into Foxp3.sup.+ regulatory T
cells. PNAS, 2008; 105: 18460-18465]. In this study, H2L5 hIgG4PE
was found to increase the secretion of IL-6 which may further
enhance Th17 cell development. Agonist antibodies of T cell
receptors such as CD28 and TNF receptor family members have been
shown to produce a bell shaped dose response curve [White, A. L.,
et al., Conformation of the Human Immunoglobulin G2 Hinge Imparts
Superagonistic Properties to Immunostimulatory Anticancer
Antibodies. Cancer Cell, 2015, 27: 138-148; Luhder, F., et al,
Topological Requirements and Signaling Properties of T
Cell-activating, Anti-CD28 Antibody Superagonists. J Exp. Med.
2003: 955-966; Stebbings, R., et al., "Cytokine Storm" in the Phase
I Trial of Monoclonal Antibody TGN1412: Better Understanding the
Causes to Improve PreClinical Testing of Immunotherapeutics J.
Immunol., 2007, 179: 3325-3331; Rogers P R and Croft M, CD28,
Ox-40, LFA-1, and CD4 Modulation of Th1/Th2 Differentiation Is
Directly Dependent on the Dose of Antigen. J Immunol 2000 164:
2955-2963; doi: 10.4049/jimmunol.164.6.2955]. H2L5 hIgG4PE also
demonstrates a similar hyperbolic functional response curve. This
information is an important component in ascertaining the best dose
range of the antibody to be used for optimal pharmacodynamic
response.
[0396] Overall, H2L5 hIgG4PE, in conjunction with CD3 stimulation,
was shown to enhance T cell activation, proliferation and
proinflammatory cytokine induction in line with its role as a
potent activator of a T cell co-stimulatory receptor.
Example 6: Binding of Anti-ICOS Antibodies
[0397] The humanization protocol (Example 3) produced 36 heavy and
light chain variants which were screened for binding to human and
cynomolgus ICOS whilst also ensuring that they did not bind to
human CD28 or CTLA-4. The H2L5 variant was identified as the having
high affinity for human and cynomolgus ICOS (1.34 and 0.95 nM
respectively) whilst containing the minimum number of back
mutations.
[0398] Changing the isotype of 422 H2L5 from IgG1 to IgG4PE does
not affect the antigen binding of the antibody as H2L5 hIgG4PE has
an affinity of 1.3 nM to human ICOS. Concentration based inhibition
of ICOS/ICOS-L binding by H2L5 hIgG4PE is shown in FIG. 7.
Experimental Protocols
[0399] Binding of H2L5 hIgG4PE to Human ICOS
[0400] The binding kinetics affinity of the humanized H2L5 hIgG4PE
antibody was determined used the BIAcore T200Anti-human IgG on Fc2
of a CMS chip Anti-ICOS H2L5 hIgG4PE captured on the surface.
Anti-human IgG surface blocked with 0.1 mg/mL hIgG1 control to
prevent non-specific binding of rabbit Fc. Human and cyno ICOS
(rabbit Fc) passed over the captured antibodies at 256 nM, 64 nM,
16 nM, 4 nM and 1 nM. Buffer alone was used to double reference
binding curves. MgCl.sub.2 was used to regenerate surface. Run
carried out at 25.degree. C. Data fitted to 1:1 model using T200
evaluation software. Antibody concentration: 2.5 .mu.g/mL
Results
[0401] Binding of H2L5 hIgG4PE to Human and Cynomolgus ICOS
[0402] The binding kinetics affinity of the humanized H2L5 hIgG4PE
antibody was determined using the BIAcore T200.
[0403] The ICOS binding data was fitted to a 1:1 kinetics model
using the T200 data analysis software.
[0404] The binding affinity of H2L5 hIgG4PE for human ICOS is 1.34
nM and cynomolgus ICOS is 0.95 nM (see Table 9). These values are
comparable and show, as expected, that a change to the Fc region of
the molecule has not affected the binding to the ICOS antigen.
Table 9 shows Ka/Kd/KD for humanized 422 (H2L5) IgG4PE to human and
cynomolgus ICOS.
TABLE-US-00019 TABLE 9 Binding to human ICOS Sample Target Ka(1/Ms)
Kd (1/s) KD (M) 422 H2L5 Human ICOS -Fc 2.97E+05 3.96E-04 1.34E-09
IgG4PE 422 H2L5 Cyno ICOS-Fc 3.91E+05 3.71E-04 9.49E-10 IgG4PE
Discussion
[0405] As shown in Example 1 murine clone 422-2 was identified as
the lead anti-human ICOS murine antibody. Humanization of this
antibody produced 36 heavy and light chain variants which were
screened for binding to human and cynomolgus ICOS whilst also
ensuring that they did not bind to human CD28 or CTLA-4. The H2L5
variant was identified as the having high affinity for human and
cynomolgus ICOS (1.34 and 0.95 nM respectively) whilst containing
the minimum number of back mutations.
[0406] Changing the isotype from IgG1 to IgG4PE does not affect the
binding of the antibody to ICOS.
Example 7: Binding of H2L5 hIgG4PE to Human Activated T Cells
Methods
Experimental Preparation(s)
CD3 Negative Isolation:
[0407] CD3+ T Cells were Negatively Isolated by Stemcell Rosette
Sep Human T Cell Enrichment Kit
Rosette Sep Human T Cell Enrichment:
[0408] 100 mL fresh, whole blood was collected with syringes coated
in liquid sodium heparin (Sagent 10 IU/mL final concentration). The
blood from each collection tube was combined into a flask where 50
.mu.L of Rosette Sep Human T Cell Enrichment cocktail was added per
ml of blood. (5 mL/100 mL Donor Blood). The whole blood/Rosette Sep
antibody cocktail was incubated for 20 minutes at room temperature
(RT). The Blood/Rosette Sep antibody cocktail was then diluted 1:1
with 1.times. phosphate buffered saline (PBS)+2% FBS (fetal bovine
serum). FBS for a final volume of 200 mL. Next, 25 mL of diluted
blood/antibody cocktail was then layered over 15 mL of Ficoll
gradient in Sepmate tubes (8 tubes in total for each Donor). Loaded
Sepmate tubes were then centrifuged at 1200.times.g for 20 minutes
RT with the brakes on. The top layer of plasma down to the
peripheral blood mononuclear cells (PBMC) interface was taken off
with a pipette and discarded. The remaining plasma and buffy coat
interface were decanted from Sepmate tubes into 50 mL conical
centrifuge tubes (4 tubes total). The tubes were topped off to 50
mL with PBS+2% FBS. The cells were centrifuged at 400.times.g for
10 minutes at RT. Supernatants were discarded. The pellets from
each donor were then combined into a single 50 mL conical tube,
re-suspending the pellets in a total volume of 50 mL PBS+2% FBS.
Cells were centrifuged at 400.times.g for 5 minutes at RT.
Supernatants were discarded and the cell pellets were re-suspended
in 2 mLs of RPMI Complete Media (RPMI 1640+10% FCS+1 mM sodium
pyruvate+2 mM L-glutamine+penicillin 100 U/mL+streptomycin 100
.mu.g/mL). Recovered CD3 Cells were counted on the ViCell
instrument and further diluted to 1.2.times.10.sup.6 cells/mL.
1.times.10.sup.6 recovered cells were stained for CD3 PE-Cy7 to
confirm the quality of the T cell isolation.
CD3+ T Cells were Negatively Isolated by Invitrogen Untouched T
Cell Isolation Kit
PBMC Isolation:
[0409] Briefly, 100 mL fresh, whole blood was collected from each
donor with syringes coated in liquid sodium heparin (Sagent 10
IU/mL final concentration). Blood was diluted (1:1) to final volume
of 200 mL with PBS with 2% FBS. Twenty five (25) mL of diluted
blood was layered over 15 mL of Ficoll gradient in Sepmate tubes (8
tubes in total for each donor). Loaded Sepmate tubes were then
centrifuged at 1200.times.g for 20 minutes at RT with the brakes
on. The top layer of plasma down to the PBMC interface was taken
off with a pipette and discarded. The remaining plasma and buffy
coat interface were decanted from Sepmate tubes into 50 mL conical
centrifuge tubes (4 tubes total). The tubes were topped off to 50
mL with PBS+2% FBS. The cells were centrifuged at 400.times.g for
10 minutes at RT. Supernatants were discarded. The pellets from
each donor were then combined into a single 50 ml conical tube and
re-suspended in a total volume of 50 mL PBS+2% FBS. Cells were
centrifuged at 400.times.g for 5 minutes at RT. Supernatants were
discarded, and the cell pellets were re-suspended in an arbitrary
volume of Isolation Buffer (dependent upon the size of the cell
pellet) provided in the Invitrogen Untouched T cell kit. Isolated
PBMC's were then counted on the ViCell instrument and brought to a
final concentration of 1.times.10.sup.8 cells/mL in Isolation
Buffer.
Invitrogen Dynabead Untouched Human T Cell Isolation:
[0410] 2.times.10.sup.8 isolated PBMCs (2 mLs), 400 .mu.l of FBS
and then 400 .mu.l of Antibody Mix from the Invitrogen Untouched T
cell kit were added to each 15 mL tube and incubated for 20 minutes
at 4.degree. C. Cells were washed with 10 mLs of Isolation Buffer
and centrifuged at 350.times.g for 8 minutes at 4.degree. C.
Supernatants were discarded, and the pellets were re-suspended in 2
mLs of Isolation Buffer. Next, 2 mLs of pre-washed Depletion
Dynabeads were added to each tube. Cells were incubated with the
beads for 15 minutes at room temperature with gentle tilting and
rotation. Following the bead incubation, 10 mLs of Isolation Buffer
was added and the cell/bead suspensions were pipeted up and down 10
times. The tubes were placed into a magnet for 2 minutes at room
temperature. Without disturbing the magnetized beads, the
supernatants containing the untouched T Cells were collected. The
beads were washed 1 time with 10 mLs of Isolation Buffer and placed
into the magnet again for 2 minutes at room temperature, and the
bead-cleared buffer collected. The collected cells were centrifuged
at 400.times.g for 5 minutes at RT. Supernatants were discarded,
and the cell pellets were re-suspended in an arbitrary volume of
RPMI Complete Media dependent upon cell pellet size (2 to 35 mLs).
Recovered CD3+ cells were then counted on the ViCell and brought to
a concentration of 1.2.times.10.sup.6 cells/mL in RPMI Complete
Media.
CD3 Confirmation Staining:
[0411] 1.times.10.sup.6 recovered cells were stained with 5 .mu.l
anti-CD3 PE-Cy7 or 5 .mu.l IgG1 Pe-Cy7 Isotype for 40 minutes at
4.degree. C. in the dark. Cells were then washed twice in ice-cold
PBS with 0.1% Tween20, centrifuging at 400.times.g for 5 minutes at
4.degree. C. Stained cells were re-suspended in 1% formaldehyde and
incubated at 4.degree. C. for 20 minutes in the dark. Cells were
then washed twice in ice-cold PBS with 0.1% Tween20, centrifuging
at 400.times.g for 5 minutes at 4.degree. C., and re-suspended in
275 .mu.l of PBS with 0.1% Tween20. Fixed cells were stored at
4.degree. C. in the dark until Flow Cytometry was performed to
confirm the quality of the T cell isolation.
Activation of Isolated Human T Cells:
[0412] T75 flasks were coated with 4 mL of 1 .mu.g/ml CD3/CD28 in
PBS for 2 hours at 37.degree. C. Flasks were washed twice with 12
mL of PBS. 30.times.10.sup.6 cells in 25 mLs of RPMI Complete Media
were added per T75 flask. Cells were incubated for 48 hours at
37.degree. C., 5% CO.sub.2 to allow activation to occur. Binding of
Anti-ICOS (H2L5 hIgG4PE):
[0413] Binding of H2L5 hIgG4PE was assessed in both naive and
activated CD3+ T cells. An 8-point titration from 0.00128 to 100
.mu.g/mL H2L5 hIgG4PE with 5 fold dilutions was employed.
[0414] Either naive and/or activated CD3+ T cells were re-suspended
in PBS with 0.1% BSA (FACS Buffer) containing Human FcR Blocking
Solution at 2.times.10.sup.6 cells/mL (5 .mu.l FcR Blocking
Solution+950 .mu.l FACS Buffer per 1 mL). At a concentration of
2.times.10.sup.5 cells/well, 100 .mu.l cells were placed into 2 mL
96 well Assay Blocks and incubated at room temperature for 15
minutes. During the incubation, a titration of the binding
antibodies, anti-ICOS (H2L5 hIgG4PE) or IgG4 Isotype Control
antibody was prepared as a 2.times. concentration. Following the
incubation of FcR block, 100 .mu.l per well of the 2.times.
concentrated binding antibodies were added to the 100 .mu.l of Fc
blocked T cells per well to achieve a final 1.times. concentration
of anti ICOS (H2L5 hIgG4PE) or IgG4 Isotype Control antibody from
0.00128 to 100 .mu.g/mL final concentration Cells were incubated
with antibodies for 20 minutes at room temperature. Following the
binding incubation, cells were washed twice in 1 mL of FACS buffer,
centrifuging at 400.times.g for 5 minutes at room temperature.
Staining of Naive or Activated T Cells Following Anti-ICOS (H2L5
hIgG4PE) Binding: Cells were Stained for Flow Cytometry with the
Following Cocktails:
Staining Cocktail:
TABLE-US-00020 [0415] Antibody Vol. per well (.mu.l) Vol. for 110
(.mu.l) PE Mouse anti-Human CD4 5 550 APC Mouse anti-Human CD8 5
550 FITC Goat anti-Human IgG Kappa 10 1100 Light Chain
Isotype Cocktail:
TABLE-US-00021 [0416] Antibody Vol. per well (.mu.l) Vol. for 110
(.mu.l) PE Mouse anti-Human CD4 5 550 APC Mouse anti-Human CD8 5
550 FITC IgG1, Kappa Isotype Control 10 1100
Naive or activated, Fc-blocked T cells were re-suspended in 80
.mu.l of FACS Buffer following the binding incubation with H2L5
hIgG4PE or control antibodies. 20 .mu.l per well of either the
Staining or Isotype Cocktail was added per well. Cells were stained
for 20 minutes at room temperature in the dark. Following the
staining incubation, cells were washed twice in 1 mL of FACS
Buffer, centrifuging at 400.times.g for 5 minutes at room
temperature.
Fixation of Stained Cells:
[0417] Cells were re-suspended in 500 .mu.l of 1% formaldehyde (10
mLs of 16.times. concentrated Formaldehyde+150 mLs of 1.times.PBS)
and incubated at room temperature for 20 minutes. Cells were then
washed twice with 1 mL FACS buffer, centrifuging at 400.times.g for
5 minutes at room temperature. Pellets were then re-suspended in
265 .mu.l of FACS Buffer and transferred to a 96 well round bottom
plate. Cells were stored at 4.degree. C. in the dark until analysis
by flow cytometry.
Flow Cytometry:
[0418] Flow cytometry was performed on either the FACS Fortessa X20
or the FACS Canto II using FACSDiva software (Version 8.0).
Compensation was performed at the time of acquisition using single
stained eBioscience Ultracomp beads and the compensation software
in FACSDiva.
Data Analysis
[0419] Data acquisition and compensation were performed on BD FACS
instruments, LSR Fortessa X-20 or FACS Canto II using BD Diva (ver.
8.0) software. Data analysis employed Flow Jo software
(ver.10.0.8r1). Results are reported as both MFI (Median
Fluorescence Intensity) and Percent of cells positive for human IgG
kappa light chain FITC staining out of the total live cells or
appropriate parent population. EC50s were determined using Graphpad
Prism 5 software (ver. 5.04) with non-linear regression of
transformed data (X=(log(X)) using a variable slope with 4
parameters (log(Agonist) vs. response--Variable slope).
Results
[0420] Isolation of T cells from fresh, whole human blood, using
either Rosette Sep CD3 Enrichment kit or Dynabead Untouched T cell
isolation kits was confirmed by staining with anti-CD3 PeCy7.
Donors ranged between 68% and 97% positive for CD3 cells.
Anti-CD.sup.3/anti-CD28 activated CD4+ and CD8+ cell populations
generated H2L5 hIgG4PE-concentration-dependent curves when assessed
for anti-human IgG1 Kappa light chain FITC staining. H2L5 hIgG4PE
binding curves are presented as both percent anti-human IgG1 Kappa
light chain FITC positive and FITC median fluorescence intensity
(MFI). T cells incubated with the IgG4 Isotype Control antibody did
not produce concentration dependent curves when assessed for
anti-human IgG1 Kappa light chain FITC staining. Naive CD4+ or CD8+
cells did not produce full curves; however, there were
concentration-dependent increases observed from 0.1 .mu.g/mL to 100
.mu.g/mL.
[0421] The median (range) EC50 values were 1.04 .mu.g/mL
(0.628-1.31 .mu.g/mL) for CD4+ FITC MFI and 0.652 .mu.g/mL
(0.27-0.74 .mu.g/mL) for CD8+ FITC MFI, respectively. The median
(range) EC50 values were 0.834 .mu.g/mL (0.45-0.965 .mu.g/mL) for
CD4+ Percent IgG Kappa Light Chain FITC Positive and 0.583 .mu.g/mL
(0.371-1.23 .mu.g/mL) for CD8+ Percent IgG Kappa Light Chain FITC.
(Table 10)
TABLE-US-00022 TABLE 10 Summary of 422 H2L5 hIgG4PE binding EC50
values to activated human T cells Activated CD4 T cells Activated
CD8 T cells Percent Percent Donor # MFI Positive MFI Positive
1124F36 0.628 0.45 0.564 0.619 1149M52 1.31 0.882 0.74 0.547
1173F42 0.636 0.612 0.27 0.371 1123F59 1.04 0.853 Not Performed Not
Performed 1141F45 1.27 0.965 Not Performed Not Performed 2100M39 No
Curve Fit 0.814 No Curve Fit 1.23 191F39 No Curve Fit No Curve Fit
No Curve Fit No Curve Fit 1155F49 No Curve Fit No Curve Fit Not
Performed Not Performed 1156F64 No Curve Fit No Curve Fit Not
Performed Not Performed Median 1.04 0.834 0.652 0.583 Mean 0.977
0.763 0.525 0.692 Std. Dev. 0.331 0.193 0.237 0.374
Discussion
[0422] This study demonstrated that H2L5 hIgG4PE (anti-ICOS agonist
antibody) bound to ICOS receptor on activated T cells from healthy
human donors. H2L5 hIgG4PE binding to the cell surface of T cells
was detected using an antibody against human IgG kappa light chain
labelled with FITC.
[0423] The successful isolation of CD3+ T cells was confirmed
through flow cytometry with anti-CD3 Pe-Cy7 staining. Nine of ten
donors resulted in greater than 89% CD3+ T cells after isolation.
However, Donor#2100M39 was only 68.6% CD3+ following isolation. The
cause of this decreased purity in Donor#2100M39's T cell isolation
is unknown. EC50 values generated from gated CD4+ and CD8+
populations from Donor#2100M39 do not appear to be aberrant and
were included in the summarized median values.
[0424] Binding EC50s were determined for H2L5 hIgG4PE in negatively
isolated human T cells. Binding curves were generated when the
isolated T cells were activated by 48 hours exposure to 1 .mu.g/mL
plate bound CD3/CD28 antibodies. Both percent IgG kappa light chain
FITC positive cells and FITC MFI data from CD4+ and CD8+ activated
T cells were considered in the statistical analyses. The median
CD4+EC50 values were similar when calculated as percent FITC
positive cells or FITC MFI, 1.04 and 0.834 .mu.g/mL, respectively.
The median CD8+EC50 values were also similar when calculated as
percent FITC positive cells or FITC MFI, 0.652 and 0.583 .mu.g/mL,
respectively.
[0425] T cells incubated with the IgG4 PE isotype control did not
result in concentration-dependent increases in anti-human IgG kappa
light chain FITC binding regardless of the analysis method
employed, MFI or Percent Positive Cells.
[0426] Full curves could not be obtained from naive or unactivated,
negatively isolated T cells in the 0.00128 to 100 .mu.g/mL H2L5
hIgG4PE range tested. However, a concentration-dependent increase
in binding was observed in donors from 0.1 to 100 .mu.g/mL H2L5
hIgG4PE. EC50s could not be calculated as the curves from naive T
cells were incomplete. The inability of H2L5 hIgG4PE to bind at low
concentrations to naive or unactivated cells was expected since
ICOS is only weakly expressed on resting Th17, T follicular helper
(TFH) and regulatory T (Treg) cells. TCR engagement and activation
are required to induce ICOS expression. Thus, there was likely very
little ICOS receptor expressed on naive or unactivated cells, and
consequently minimal binding of H2L5 hIgG4PE.
Example 8: TK/PD Results from Cyno Dose-Range Finding (DRF)
Study
[0427] To access the in vivo characteristics of H2L5 hIgG4PE in a
target-relevant species a dose-range finding study was performed in
cynomolgus monkeys. The study tested 3 dose levels (0.3, 3 and 30
mg/kg) in addition to a vehicle control cohort. It was a repeat
dose with the second dose administered 14 days after the first. One
male and one female were tested per cohort. H2L5 hIgG4PE exhibited
a dose-dependent increase in C.sub.max (.mu.g/mL) and AUC
(.mu.gh/mL) across the 3 different doses which were tested. At all
three dose levels, antibody was detected in the plasma for two
weeks following the first dose (FIG. 12A). Anti-H2L5 hIgG4PE
antibodies were detected in 3 of the monkeys following a single
dose, both of the 0.3 mg/kg dosed animals as well as the female
dosed at 3 mg/kg. Anti-H2L5 hIgG4PE antibodies correlated with
decreased plasma concentrations following administration of the
second dose in these animals (FIG. 12B). Forty-eight hours after
the second dose all animals were sacrificed to collect tissue for
analysis of pharmacodynamic activity and histopathology
analysis.
[0428] H2L5 hIgG4PE receptor occupancy (RO) was measured in CD4+ T
cells from the spleens and axillary lymph nodes of all animals on
study. A dose-dependent increase in H2L5 hIgG4PE binding was
observed across the dose levels tested in both tissues (FIG.
13).
[0429] Receptor occupancy was also measured on CD4+ T cells from
the peripheral blood of monkeys on study. Blood was drawn at 5 time
points (Day 1 (pre-dose), day 3, day 8, day 15 (pre-second dose)
and day 17). Two different measures were used in this assay to
determine RO. The first was a "free-receptor" assay format in that,
binding of the anti-ICOS mAb used for flow cytometry detection was
determined in the presence or absence H2L5 hIgG4PE which was shown
to compete for ICOS binding. Therefore, the absence of anti-ICOS
signal by FACS was a surrogate for H2L5 hIgG54PE occupied receptor
and conversely, anti-ICOS positivity indicated "free receptor"
which was unbound with H2L5 hIgG4PE. FIG. 14-A shows that ICOS free
receptor decreased in a dose-dependent, and time dependent manner.
Two monkeys (250 and 300) demonstrated "free receptor" signals
which could not be explained, and may be due to the production of
anti-H2L5 hIgG4PE antibodies in these monkeys. In addition, RO was
also measured in peripheral blood CD4+ cells by the same assay used
in spleen and lymph nodes described above. As would be expected the
0 mg/kg dose showed no RO by this read out (FIG. 14-B).
Interestingly, some monkeys at the 3.0 and 30 mg/kg dose levels
showed a time dependent increase in H2L5 hIgG4PE bound CD4+ cell
numbers across the treatment time-course. In particular, animal 350
exhibited a (>5-fold) increase in drug-bound circulating CD4+
cells between days 3 and 17 (FIG. 14-B). It is possible that this
increase in CD4+ICOS+ cell number could be due to H2L5
hIgG4PE-induced proliferation of this population.
Example 9: H2L5 hIgG4PE Induces Intracellular Signalling Changes in
Response to Binding
Experimental Preparation(s)
Cell Lines
[0430] Ba/F3-ICOS cells were obtained from INSERM (Paris, France).
Cells were cultured in the appropriate culture medium supplemented
with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, Mo.),
10 ng/mL recombinant murine IL-3 (R&D Systems, Minneapolis,
Minn.), and 1 mg/mL Geneticin (ThermoFisher, Waltham, Mass.) at
37.degree. C. in humidified incubators under 5% CO.sub.2.
Experimental Protocol(s)
Intracellular Signalling Antibody Array
[0431] Protein lysates were assayed with the PathScan.RTM.
Intracellular Signaling Array Kit (Cell Signaling Technologies)
according to the manufacturer's instructions. Briefly, lysates from
Ba/F3-ICOS cells treated with IgG4-PE (20 .mu.g/mL) or H2L5 hIgG4PE
(0.2, 2, or 20 .mu.g/mL) for 1, 6, 24, and 48 hours were diluted to
1 .mu.g/.mu.L in Array Diluent Buffer and incubated overnight onto
the antibody arrays at 4.degree. C. Images of the arrays were
captured using the Odyssey imaging software (LI-COR Biosciences,
Lincoln, Nebr.).
Phospho-AKT Enzyme-Linked Immunosorbent Assay (ELISA)
[0432] Phosphorylation of AKT was measured using the Meso Scale
Discovery (MSD) Phospho (Ser473)/Total Akt Whole Cell Lysate Kit
and Phospho-Akt (Thr308) Whole Cell Lysate Kit according to the
manufacturer's instructions. Cells were seeded at a cell density of
0.25.times.10.sup.6 cells/well into 96-well U-bottom plates (BD
Falcon) in the appropriate culture media (100 .mu.L/well). Cells
were treated for 1, 2, 4, 6, 24, or 48 hours with either the
control antibody (IgG4 PE), anti-ICOS IgG1 Fc disabled antibody, or
H2L5 hIgG4PE at 7 different concentrations using a 3-fold dilution
scheme (dose range: 20.0-0.03 .mu.g/mL) in duplicate wells. For one
experiment using the Phospho-AKT (Thr308) Whole Cell Lysate Kit,
cells were treated with one concentration of all three antibodies
(10 .mu.g/mL) in triplicate wells. The bottom row of each 96-well
plate contained a no cells control (two blank duplicate wells) and
cells that were left untreated with any antibody. Following
treatment, cells were lysed with 30 .mu.L, of ice cold lysis buffer
containing protease and phosphatase inhibitors, incubated on ice
for 30 minutes, and then 25 .mu.l of lysate was transferred to the
ELISA plate to incubate overnight at 4.degree. C.
Data Analysis
Densitometry Analysis of Intracellular Signalling Antibody
Array
[0433] Densitometry analysis was performed to calculate the
integrated intensity levels of the spots across the antibody array.
The intensities for each spot were normalized to the average of the
positive controls on the array (formula=sample well/average of
positive controls) and graphed using GraphPad Prism 6.0 (La Jolla,
Calif.).
Analysis of MSD ELISA Data
[0434] The percent phosphoprotein was calculated for each well
using the following calculation: %
Phosphoprotein=((2.times.Phospho-signal)/(Phospho-signal+Total
protein signal)).times.100. This value was then normalized to the
untreated cells value at each time point and graphed as "% control"
in Microsoft Excel 2007.
Results
[0435] Prior studies demonstrated that H2L5 hIgG4PE treatment
increased phospho-AKT (S473) levels in Ba/F3-ICOS cells with the
maximum response observed between 30-40 minutes after antibody
exposure. Here, phospho-AKT levels were measured at later time
points to see if increased phosphorylation levels persisted after
several days. Additionally, the regulation of other intracellular
signalling events by ICOS activation was assessed. In Ba/F3-ICOS
cells, phospho-AKT (S473) levels were increased with H2L5 HIGG4PE
treatment compared to IgG4-PE isotype control antibody-treated
cells after one and six hours of treatment, but this effect was
lost after 24 hours (FIG. 15). Interestingly, a similar effect was
observed when cells were treated with an anti-ICOS antibody where
the Fc region of the antibody is disabled. Increased levels of
phospho-AKT (T308) were also observed in H2L5 hIgG4PE and anti-ICOS
IgG1 Fc disabled antibody-treated cells compared to IgG4-PE (FIG.
15) isotype control antibody-treated cells after one hour of
treatment and persisted up to 48 hours which was the last time
point measured. Two other phospho-proteins downstream of AKT,
Glycogen Synthase Kinase 3 Alpha (GSK3.alpha.) and ribosomal
protein S6, were also modestly increased upon ICOS activation, but
the effects were not as robust at those seen with phospho-AKT.
Protein lysates were also analyzed using an antibody array that
measures the phosphorylation or cleavage of 18 proteins involved in
intracellular signalling. Using this approach, only three proteins
showed slight increases in phosphorylation upon ICOS activation:
phospho-AKT (S473), phospho S6 (S235/236), and phospho-SAPK/JNK
(T183/Y185).
[0436] To measure changes to AKT phosphorylation using an assay
format that would allow for direct quantitation, Ba/F3-ICOS cells
were treated with a dose range of control antibody (IgG4 PE),
anti-ICOS IgG1 Fc disabled antibody, or H2L5 hIgG4PE over time and
monitored by ELISA. Increased phospho-AKT (S473) levels were both
dose-dependent and time-dependent in anti-ICOS IgG1 Fc disabled
antibody-treated or H2L5 hIgG4PE-treated cells. As previously
observed, maximal phospho-AKT (S473) activation occurred after 1
hour of treatment. The phospho signal slightly decreased after 2
hours and persisted up to 6 hours but was eventually lost after 24
hours. An ELISA that measures phospho-AKT (T308) levels was also
tested here but no reproducible activation could be observed with
this ELISA kit.
Discussion
[0437] The AKT signalling cascade can be activated by receptor
tyrosine kinases, integrins, B and T cell receptors, cytokine
receptors, G-protein-coupled receptors and other stimuli that
induce production of phosphatidylinositol (3,4,5) trisphosphates
(PIP3) by PI3K [Carnero, 2008]. These lipids serve as plasma
membrane docking sites for Akt and its upstream activator PDK1. At
the membrane, PDK1 phosphorylates AKT at Thr308 leading to partial
activation of Akt [Alessi, 1996]. Phosphorylation of Akt at Ser473
by mTORC2 stimulates full enzymatic activity [Sarbassov, 2005].
[0438] ICOS plays a key role in the function of activated effector
and regulatory CD4+ T cells by promoting T cell survival,
proliferation and memory. Due to its role in sustaining T-cell
activation and effector functions, targeting ICOS with an agonist
antibody could be a plausible approach to enhance antitumor
immunity. In this study, we observed that activation of ICOS by
H2L5 hIgG4PE caused changes to AKT phosphorylation in Ba/F3-ICOS
cells. Subsequently, proteins downstream of AKT, such as
GSK3.alpha. (a direct substrate of AKT) and ribosomal protein S6
were also phosphorylated. This data is consistent with work
performed recently with this model system, and is in-line with data
published externally [Fos, 2008].
Example 10: Functional Effects of Soluble H2L5 hIgG4PE Alone and in
Combination with Anti-PD1 and Anti-CTLA-4 Antibody in Human PBMC
Assay
Experimental Preparation(s)
Isolation of Primary Human PBMC
[0439] Fresh blood was obtained from GSK Health Center blood donors
and was diluted 1:1 with phenol red free-10% RPMI1640 media.
Diluted blood was layered on top of the density medium in a Uni-Sep
Max 50 ml conical tube and centrifuge at 400.times.g for 20 minutes
at room temperature with BREAK OFF. The resulted white mononuclear
layer (buffy coat) was carefully extracted into a new 50 mL conical
tube through a 100 .mu.M cell strainer. An equal volume of Phenol
red free-10% RPMI1640 media was added to the buffy coat and
centrifuged at 300.times.g for 10 minutes at room temperature. The
cell pellet was resuspended in 10 ml of red blood cell lysis
solution (Sigma Aldrich) and incubated for 5 minutes at room
temperature. Cells were washed once with media and centrifuged as
previously described. Volume was brought to 40 ml with Phenol red
free-10% RPMI1640 media and cells were counted using Vicell cell
counter and viability analyzer (Beckman Coulter).
Induction of Monocyte-Derived Immature Dendritic Cells (iDC)
[0440] Human monocytes were isolated using the plastic adherence
method. Briefly, 20 million freshly isolated PBMC were cultured in
a T-75 tissue culture flask in AIM-V media (Thermo Fisher) for 3
hours. Cells that do not bind to plastic were washed off. The
adherent monocytes were cultured in a 37.degree. C. 5% CO.sub.2
incubator in AIM-V media supplemented with 1000 U/ml of human
GM-CSF (Calt#300-03, PeproTech) and 500 U/ml of human IL-4
(cat#200-04). After 7-10 days, the iDC cells were collected for
co-culturing with T cells from a different donor in the allogeneic
Mixed Lymphocyte Reaction assays.
Isolation of Primary Human T Cells Directly from Blood
[0441] Human T cells were isolated directly from fresh human blood
using a human T cell enrichment cocktail (Stem Cell Technologies).
The RosetteSep Human T Cell Enrichment Cocktail (50 .mu.L/mL) was
added to whole blood and mixed well. After 20 minutes of incubation
at room temperature, an equal volume of PBS+2% FBS was added with
gentle mixing. The diluted sample was layered on top of the density
medium and centrifuged for 20 minutes at 1200.times.g at room
temperature with the brake off. The enriched cells from the density
medium: plasma interface were carefully poured into a new conical
tube. Next, the red blood cells were lysed with Red Blood Cell
Lying Buffer (Sigma Aldrich) and the enriched cells were washed
with PBS+2% FBS twice. The T cells were then resuspended in 40 ml
of PBS+2% FBS and counted with a Vi-Cell cell counter.
Experimental Protocols
Human PBMC Pre-Stimulation Assay
[0442] Freshly isolated human PBMCs were pre-stimulated with
CD3/CD28 T cell expander DynaBeads at a bead to cell ratio of 1:20
in a T-75 tissue culture flask in AIM-V medium supplemented with
100 ng/ml of MCSF and 100 IU/ml of IL-2 (PeproTech) at 37.degree.
C. After 48 hours, the pre-stimulation beads were magnetically
removed and cells were washed, counted and re-stimulated with
anti-CD3 DynaBeads and therapeutic antibodies in AIM-V medium
supplemented with 100 IU/ml of IL-2 (PeproTech) in 96-well
non-tissue culture treated round bottom plate. The seeding density
was 100 k cells per 100 .mu.l of medium per well. After incubating
at 37.degree. C. for 3.5 days, cell culture supernatants were
collected for multiplex cytokine measurement by MSD.
Human MLR Activation Assay
[0443] Monocyte-derived iDCs from a healthy human volunteer were
mixed at a 1:10 ratio (iDC:T) with freshly isolated human T cells
from a different donor and pre-incubated at 37.degree. C. in AIM-V
media in the presence of 0.02 .mu.g/ml of a CEFT peptide mixture
for 24 hours. Different groups of treatment antibodies were added
directly to the wells, mixed and further incubated for an
additional 4 days. Cell culture supernatants were collected for
multiplex cytokine measurement by MSD analysis.
MSD Cytokine Analysis
[0444] IFN-.gamma., IL-10, IL-2 and TNF-.alpha. cytokine levels in
the tissue culture supernatant were determined using MSD human
V-Plex customized kits. Samples were first diluted 1:200 in Diluent
2. Calibrators were also prepared in Diluent 2 following the
manufacturer's recommendations. Diluted samples and calibrators
were added to black MSD plates which were subsequently sealed with
an adhesive plate seal and incubated at room temperature with
shaking for 2 hours. After adding 25 pt of the detection antibody
solution, which was freshly prepared in Diluent 2 to each well, the
plate was re-sealed and incubated at room temperature with shaking
for another 2 hours. The plates were washed 3 times with 150
.mu.L/well of PBS plus 0.05% Tween-20 before adding 150 .mu.l/well
of freshly diluted 2.times. read buffer and immediately read on a
MESO QuickPlex reader. Data were analyzed using MSD Workbench
software.
Data Analysis
MSD Data Analysis
[0445] MSD data was analyzed with Discovery Workbench software
(MSD, version 4.0.9). Calibrators in the manufacturer's kit were
included on each MSD plate to generate plate specific standard
curves with R.sup.2 value over 0.99 in all cases. The amounts of
cytokine detected were back calculated based on the standard curve
and the mean and standard deviation from three biological
replicates were used to generate the graphs.
Statistical Analysis
[0446] One-way ANOVA was performed on log-transformed, fold-change
data over each treatment antibody's own isotype control. Dunnett's
Multiple Comparison Test was performed to compare both
mono-therapies vs. combination across different donors. P<0.05
was considered as statistical significant.
Results
[0447] PBMC pre-stimulation assay development and test for
combinatorial activity of H2L5 hIgG4PE with ipilimumab and
pembrolizumab.
[0448] In order to determine the optimal conditions for
pre-stimulation, human anti-CD3 Dynabeads and anti-CD3/CD28
Dynabeads (Thermo Fisher) were tested at different bead to cell
ratios. After 48 hour pre-stimulation, cells were harvested and
beads were magnetically removed prior to stimulation with anti-CD3
Dynabeads (bead to cell ratio=1:1) together with anti-ICOS antibody
alone or in combination with anti-CTLA-4 or anti-PD1. H2L5 hIgG4PE
single agent treatment resulted in induction of IFN-.gamma. as
compared to isotype control in all pre-stimulation conditions
tested. The magnitude of IFN-.gamma. induced by H2L5 hIgG4PE was
inversely correlated with the strength of the pre-stimulation. The
combination of H2L5 hIgG4PE together with ipilimumab demonstrated
enhanced cytokine production as compared to either H2L5 HIGG4PE or
ipilimumab alone in PBMCs that were weakly pre-stimulated. The
combination effect was lost under plate-bound anti-CD3/anti-CD28
pre-stimulation conditions, which is considered a stronger
pre-stimulation condition. Based upon these results, the
pre-stimulation condition using anti-CD3/anti-CD28 beads at a bead
to cell ratio of 1:20 was chosen for all future PBMC assays.
Results from four individual donors are summarized for anti-CTLA-4
combination in FIG. 16 and combination with anti-PD-1 in FIG.
17.
H2L5 hIgG4PE Results in Dose-Dependent Cytokine Induction in a PBMC
Pre-Stimulation Assay
[0449] The dose-dependent activity of H2L5 hIgG4PE was evaluated in
human PBMCs pre-stimulated with anti-CD3/anti-CD28 beads at a
pre-determined bead to cell ratio of 1:20. The anti-RSV IgG4PE and
anti-ICOS 422.2 IgG1 Fc Disabled were included as controls. Eight
concentrations of H2L5 HIGG4PE were tested (100, 30, 10, 3, 1, 0.3,
0.1, and 0.03 .mu.g/ml). IFN-.gamma., IL-10 and TNF-.alpha. were
evaluated by MSD in the tissue culture supernatants of PBMC
samples. H2L5 hIgG4PE, but not isotype control IgG4 or Fc-Disabled
422.2, induced IFN-.gamma., IL-10 and TNF-.alpha. production in a
dose-dependent manner. These results were used to determine the
concentration of H2L5 hIgG4PE to be used in combination
studies.
Human MLR Assay Development
[0450] In an effort to optimize a human MLR assay, in addition to
co-culture of human T cells and monocyte-derived immature DCs from
a different donor, anti-CD3 beads were also added into the wells to
provide a basal TCR stimuli to help prime the cells. Results
demonstrated that anti-CD3 beads greatly increased the range of
IFN-.gamma. induction. Although ipilimumab alone can induce
IFN-.gamma. production in the absence of anti-CD3 beads, H2L5
hIgG4PE alone or the H2L5 HIGG4PE/ipilimumab combination only
showed enhanced IFN-.gamma. production over corresponding controls
in the presence of anti-CD3 beads.
Combinatorial Activity of H2L5 HIGG4PE and Ipilimumab in a Human
MLR Assay
[0451] The immunostimulatory activity of H2L5 hIgG4PE alone or in
combination with ipilimumab was tested in an allogeneic human MLR
assay in which T cells that were pre-incubated with
monocyte-derived immature DCs from an unmatched donor in the
presence of 0.02 .mu.g/ml CEFT peptides for 1 day. The H2L5
hIgG4PE/ipilimumab combination resulted in a significant
enhancement in IFN-.gamma. production as compared to either agent
alone. Results were consistent across three donor pairs tested;
however, modest variability was observed between donors (FIG.
18).
Combinatorial Activity of H2L5 hIgG4PE and Pembrolizumab in a Human
MLR Assay
[0452] The combination of H2L5 hIgG4PE and pembrolizumab was also
tested in the human allogeneic MLR assay described above. H2L5 hIg
G4PE was tested alone and in combination with pembrolizumab at 10
.mu.g/ml. The combination of H2L5 hIg G4PE and pembrolizumab
resulted in increased IFN-.gamma. as compared to either agent
alone. However, statistical significance was not reached due to
high donor variability and significant activity of single agent
anti-PD-1 treatment in some donors (FIG. 19).
Discussion
[0453] ICOS is a costimulatory receptor that is weakly expressed on
naive T cells and quickly upregulated in activated CD4+ and CD8+ T
cells. The ligand for ICOS is ICOS-L (B7h, B7RP-1, CD275), which is
expressed by professional APCs and by peripheral epithelial and
endothelial cells following TNF-.alpha. stimulation. The
ICOS:ICOS-L pathway provides a key costimulatory signal for T-cell
proliferation and function. Due to its role in sustaining T-cell
activation and effector functions, targeting ICOS by agonist
antibodies could be a plausible approach to enhance anti-tumor
immunity.
[0454] Studies have shown an increase the frequency of ICOS.sup.hi
CD4+ effector T cells after CTLA-4 blockade by ipilimumab in
several cancer models. In addition, upon CTLA-4 blockade, this cell
population produced greater levels of INF-.gamma. than ICOS.sup.lo
CD4+ T cells. In fact, the increase in the frequency of ICOS+CD4 T
cells has been identified as a pharmacodynamic biomarker of
ipilimumab treatment in cancer patients. Studies, in wild-type
C57BL/6 mice, demonstrated 80 to 90% tumor rejection follow CTLA-4
blockade therapy; however, in ICOS or ICOSL knockout mice the
efficacy was decreased to less than 50%. The important role played
by ICOS in the effectiveness of CLTA-4 blockade suggests that
stimulating the ICOS pathway during anti-CTLA-4 therapy might
increase therapeutic efficacy. Therefore, we set out to evaluate
the combination activity of H2L5 hIgG4PE and ipilimumab.
[0455] Programmed cell death-1 (PD-1) was reported in 2000 to be
another immune checkpoint molecule. The expression of PD-L1
(B7-H1), which is one of the ligands of PD-1, can be found on many
cell types including T cells, epithelial cells, endothelial cells,
and tumor cells. Antibodies targeting the PD-1/PD-L1 axis have also
shown clinical responses in multiple tumor types. The FDA recently
approved pembrolizumab and nivolumab as second generation of the
immune checkpoint blockers for the treatment of cancer. Merck's
pembrolizumab was shown to lead to response rates of .about.37 to
38% in patients with advanced melanoma, with a subsequent study
reporting an overall response rate of 26% in patients who had
progressive disease after prior ipilimumab treatment. Nivolumab,
the anti-PD-1 antibody from BMS, also showed clinical benefit in
patients with metastatic melanoma with a response rate of 40% and
an overall survival rate of 72.9% at 1 year. In addition, nivolumab
was also FDA-approved for advanced or metastatic non-small cell
lung cancer. As the PD-1 checkpoint blockade antibodies become the
dominant cancer immune therapy in the clinic, it will be important
to evaluate H2L5 hIgG4PE in combination with an anti-PD-1 antibody
for their combined anti-tumor activity.
[0456] Previously, a PBMC activation assay was developed and used
to evaluate the T cell stimulation activity of a panel of anti-ICOS
agonist antibodies. The data generated from those studies supported
the candidate selection of clone 422.2 with an IgG4PE isotype as
H2L5 hIgG4PE. In the previous assay, PBMC cells were pre-stimulated
with plate bound anti-CD3 antibody at 1 .mu.g/ml and anti-CD28
antibody at 3 .mu.g/ml for 48 hours before they were harvested and
re-stimulated with anti-CD3 and soluble ICOS antibodies that were
being investigated. H2L5 hIgG4PE was shown to induce IFN-.gamma.
production in a dose-dependent manner. In order to determine the
optimal conditions for pre-stimulation, human anti-CD3 Dynabeads
and anti-CD3/CD28 Dynabeads (Thermo Fisher) were tested at
different bead to cell ratios. Stimulation by beads is considered
to be more physiological and the strength of the stimulation can be
controlled more easily by constructing different bead to cell
ratios. After 48 hours of pre-stimulation, cells were harvested and
beads were magnetically removed prior to stimulation with anti-CD3
Dynabeads (bead to cell ratio=1:1) together with anti-ICOS antibody
alone or in combination with anti-CTLA-4. The results showed that
H2L5 hIgG4PE single agent treatment resulted in IFN-.gamma.
induction relative to isotype control in all pre-stimulation
conditions tested. The magnitude of IFN-.gamma. induced by H2L5
hIgG4PE was inversely correlated with the strength of the
pre-stimulation. The combination of H2L5 hIgG4PE together with
ipilimumab demonstrated enhanced cytokine production as compared to
either H2L5 hIgG4PE or ipilimumab alone in PBMCs that were weakly
pre-stimulated. The combination effect was lost under plate-bound
anti-CD3/anti-CD28 pre-stimulation conditions, which is considered
a stronger pre-stimulation condition. Based upon these results, the
pre-stimulation condition using anti-CD3/anti-CD28 at a bead to
cell ratio of 1:20 was chosen for all the future PBMC assays. H2L5
hIgG4PE and ipilimumab combination demonstrated a statistically
significant increase in IFN-.gamma. production as compared to
either antibody treatment alone.
[0457] In the assay optimization effort, with an anti-CD3/anti-CD28
stimulation bead to cell ratio fixed at 1:20, the anti-CD3 beads
used during the re-stimulation step were titrated down from bead to
cell ratios of 1:1 to 1:3 and 1:10. The results showed that the
lower the re-stimulation strength yielded lower the IFN-.gamma.
induction by H2L5 hIgG4PE. The combination effect by H2L5 hIgG4PE
and ipilimumab was totally lost under re-stimulation at a bead to
cell ratio of 1:3 and 1:10. Therefore, the re-stimulation anti-CD3
bead to cell ratio of 1:1 was kept for all future experiments.
[0458] With the pre-stimulation and re-stimulation conditions
optimized, this assay was used to evaluate the dose response of
H2L5 hIgG4PE. A total of 8 antibody concentrations were tested,
which were 100, 30, 10, 3, 1, 0.3, 0.1 and 0.03 .mu.g/ml. The
anti-RSV IgG4PE and anti-ICOS 422.2 IgG1 Fc Disabled, the Fc
Disabled version of H2L5 hIgG4PE, were used as controls. Results
showed that H2L5 hIgG4PE, but not isotype control IgG4 or
Fc-Disabled 422.2, induced IFN-.gamma., IL-10 and TNF-.alpha.
production in a dose-dependent manner. It is interesting that the
Fc Disabled version of H2L5 hIg G4PE exhibited a limited cytokine
induction response, indicting the Fc receptor engagement is crucial
for the T cell agonizing function of H2L5 hIg G4PE. These results
were also used to determine the dose of H2L5 hIg G4PE for
combination studies.
[0459] A mixed lymphocytes reaction (MLR) assay was also developed
to evaluate the combination effect of H2L5 hIg G4PE and checkpoint
blocking antibodies. MLR assay is an ex vivo cellular immune assay
in which primary monocyte-derived immature dendritic cells (iDCs)
were mixed with T cells isolated from a different donor. The
mismatch of major histocompatibility complex (MHC) molecules on the
surface of iDC cells can initiate T cell stimulation in an
allogeneic setting. In the clinic, the MLR assay is well-known for
identifying the compatibility of tissue transplants between donors
and recipients.
[0460] In order to develop the MLR assay, fresh human monocytes
were cultured in medium supplemented with human recombinant GM-CSF
and IL-4 for a week to induce an immature DC phenotype. Then fresh
human T cells from a different donor were isolated and mixed with
the iDC cells at a 10:1 ratio (T:iDC). H2L5 hIg G4PE and ipilimumab
mono-therapy or combinational treatments were added to the T
cell/iDC co-culture in the presence or absence of anti-CD3 beads.
The purpose of the anti-CD3 beads was to provide a basal TCR
stimulus to help prime the T cells. Results showed anti-CD3 beads
greatly increased the range of IFN-.gamma. induction in the assay.
Although ipilimumab alone can induce IFN-.gamma. production in the
absence of anti-CD3 beads, H2L5 hIgG4PE alone or the H2L5
hIgG4PE/ipilimumab combination showed enhanced IFN-.gamma.
production over corresponding controls in the presence of anti-CD3
beads. This result suggests that, in this assay, the TCR stimulus
by DC cells alone may not be sufficient to induce ICOS expression
on the surface of resting T cells that were freshly isolated from
PBMCs. In order to improve the situation, a 24 hour iDC and T cells
pre-incubation step was added before the addition of therapeutic
antibodies. The CEFT peptide mix was also added into the assay
procedure to better prime the T cells and to elicit an
antigen-specific response. The CEFT peptide pool consists of 27
peptides selected from defined HLA class I and II-restricted T-cell
epitopes from human Cytomegalovirus (HHV-5; CMV), Epstein-Barr
virus (HHV-4; EBV), Influenza A and Clostridium tetani. Considering
the high vaccination frequency against Influenza and Clostridium
tetani and the high prevalence of CMV and EBV in the general
population, recall antigen responses were expected for a majority
of the human samples. The results showed that increased IFN-.gamma.
production was observed when T cells were pre-incubated with iDC
cells for 24 hours, and the IFN-.gamma. production further
increased when CEFT peptides were added to the co-culture system.
The immunostimulatory activity of H2L5 hIgG4PE alone or in
combination with ipilimumab was tested in the allogeneic human MLR
assay in which T cells that were pre-incubated with
monocyte-derived immature DCs from an unmatched donor in the
presence of 0.02 .mu.g/ml CEFT peptides for 1 day. The H2L5
hIgG4PE/ipilimumab combination resulted in a significant
enhancement in IFN-.gamma. production as compared to either agent
alone. The results were consistent across three donor pairs tested;
however, modest variability was observed between donors.
[0461] Similarly, the combination of H2L5 hIgG4PE and pembrolizumab
was also tested in the human allogeneic MLR assay described above.
H2L5 hIgG4PE was tested alone and in combination with pembrolizumab
at 10 .mu.g/ml. The combination of H2L5 hIgG4PE and pembrolizumab
resulted in increased IFN-.gamma. as compared to either agent
alone. However, statistical significance was not reached due to
high donor variability and significant activity of single agent
anti-PD-1 treatment in some donors.
[0462] In summary, these studies demonstrated the superior
combination activity of H2L5 hIgG4PE with two FDA-approved check
point inhibitors, ipilimumab and pembrolizumab, when compared to
mono-therapies in two human immune cell based assays. In the
studies reported here, H2L5 hIgG4PE was shown to promote T cell
activation and TH1 skewing (e.g. IFN-.gamma. production) that is
characteristic of productive anti-tumor immune responses.
Example 11: Functional Activity of H2L5 hIgG4PE Alone and in
Combination with Anti-PD1 and Anti-CTLA-4 Antibodies In Vivo
Human PBMC Mouse Tumor Model
Methods
Experimental Preparations
[0463] All procedures on animals were reviewed and approved by the
GSK Institutional Animal Care and Use Committee prior to initiation
of the studies protocol.
Preparation of Cell Lines:
[0464] A2058 were propagated according to ATCC protocol.
Materials:
[0465] A2058 human melanoma cell line: ATCC, Cat# CRL-11147,
1.degree. 059349362 [0466] DPBS: ATCC, Cat #30-2200, Lot#63357436
[0467] Dulbecco's Modified Eagle's Medium: ATCC, Cat #30-2002,
Lot#62596471 Expiration: October 2015 [0468] Fetal Bovine Serum:
Sigma-Aldrich, Cat#12176c-1000 ml, lot #13G180R0H1, Expiration:
July-2018 [0469] 0.25% (w/v) Trypsin-0.53 mM EDTA: ATCC, Cat
#30-2102, Lot#62420300 [0470] Antibiotic-Antimycotic (100.times.):
Life Technologies, Cat#15240-062 [0471] T175 cell culture flask:
Greiner bio-one, Cat#661175 [0472] T75 cell culture flask: Greiner
bio-one, Cat#658175
Medium:
[0472] [0473] A2058 complete growth medium: Dulbecco's Modified
Eagle's Medium+10% FBS. Culture conditions: Atmosphere: Air, 95%;
5% carbon dioxide (CO2); Temperature: 37.degree. C. [0474] Upon
receipt of the cells: [0475] Pre-warm complete medium at 37.degree.
C. [0476] Thaw the cells quickly in 37.degree. C. water bath. Wipe
the tube with 70% ethanol and transfer cells to 15 ml tube filled
with prewarmed complete medium. [0477] Centrifuge at 1200 rpm for 5
minutes to collect the cell pellet. [0478] Add the cells back to
T75 flask filled with prewarmed complete medium and incubate at
37.degree. C.
Subculture of the Cells:
[0478] [0479] Volumes are given for a 75 cm.sup.2 flask (For T175
cm.sup.2 flask, adjust the amount of dissociation and culture
medium needed proportionally). [0480] Remove and discard culture
medium. [0481] Briefly rinse the cell layer with DPBS to remove all
traces of serum that contains trypsin inhibitor. [0482] Add 2.0 to
3.0 mL of Trypsin-EDTA solution to flask and observe cells under an
inverted microscope until cell layer is dispersed (2-3 minutes).
[0483] Note: To avoid clumping do not agitate the cells by hitting
or shaking the flask while waiting for the cells to detach. Cells
that are difficult to detach may be placed at 37.degree. C. to
facilitate dispersal. [0484] Add 10 mL of complete growth medium
and aspirate cells by gently pipetting. [0485] Centrifuge at 1200
rpm for 5 minutes to collect the cell pellet, add 10 ml of complete
growth medium [0486] Add appropriate aliquots of the cell
suspension to new culture vessels. Incubate cultures at 37.degree.
C. [0487] Medium Renewal: Every 2 to 3 days
Preparation of Tumor Cells for Mice Inoculation:
[0487] [0488] Wash cells with 1.times.DPBS, add 3 ml 1.times.
Trypsin for 2-3 minutes. [0489] Add complete growth media and
collect the cell suspension in sterile conical centrifuge tube in
the tissue culture hood. [0490] Centrifuge the cells at 1200 rpm
for 5 minutes to obtain cell pellet. [0491] Wash cells with
1.times.DPBS solution, Centrifuge at 1200 rpm for 5 minutes to
obtain cell pellet. Repeat the washing 2 times. [0492] Count the
cells by hemocytometer for cell number and viability. [0493]
Resuspend the cells in ice-cold PBS at concentrations for In Vivo
inoculation (A2058, 2.5e7/ml, 2.5e6/100 .mu.l/mouse).
Tumor Cell Line Inoculation to NSG Mice
Materials:
[0493] [0494] Mouse: NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ. The Jackson
Laboratory Stock: 005557 Female Age: 6 weeks [0495] 1 mL Tuberculin
Syringes with Attached Needle 25 G 5/8: Becton Dickinson,
Cat#305554 [0496] PDI.TM. Alcohol Prep Pads: Professional
Disposables, Cat# B339 [0497] PDI.TM. Povidone-Iodine Prep Pad:
Professional Disposables, [0498] Cat#B40600 [0499] Preparation of
mice [0500] Mice should be 6 weeks old. [0501] Allow 3-5 days
acclimatization period after mice have arrived. [0502] Shave the
mice on right hind flank
Preparation of the Injection
[0502] [0503] Clean and sterilize the inoculation area of the mice
with iodine followed by ethanol pad [0504] Use 1-cc syringe and a
25-gauge needle [0505] Pull out the plunger, mix cells and add 100
.mu.l of cells to the back of syringe, carefully insert the
plunger. [0506] Inject cells subcutaneously (s.c.) into the right
hind flank of the mouse. [0507] Tumor Growth Assessment [0508] To
measure a tumor, wet fur with 70% ethanol to make it easier to find
tumor margins. Measure tumor size and body weight every 2-3 days.
[0509] Tumor size is measured with a digital caliper, and the
volume is determined as follow: Tumor volume
(mm3)=(length).times.(width).sup.2/2 [0510] Human PBMC Intravenous
Administration [0511] Human PBMC administration can start 1 week
after when the tumors have reached an average volume of
approximately 100 mm3.
Materials:
[0511] [0512] Fresh Human PBMC: Allcells, cat#C-PB102-3B2 [0513] 1
mL Tuberculin Syringes with Attached Needle 25 G 5/8: Becton
Dickinson, Cat#305554 [0514] PDI.TM. Alcohol Prep Pads:
Professional Disposables, Cat# B339 [0515] PDI.TM. Povidone-Iodine
Prep Pad: Professional Disposables, Cat# B40600 [0516] Gauze
sponges: Covidien, cat#441211 [0517] Mouse Tail Illuminator
Restrainer: Braintree scientific, cat#MTI STD [0518] PBMC
preparation [0519] Fresh human PBMC are purchased from Allcells by
overnight shipment. [0520] Centrifuge the cells at 1400 rpm for 5
minutes to obtain cell pellet. [0521] Wash cells with 1.times.DPBS
solution, Centrifuge at 1400 rpm for 5 minutes to obtain cell
pellet. [0522] Resuspend the cells in ice-cold PBS at
concentrations for In Vivo injection (20e7/ml). [0523] Use 1-cc
syringe and a 25-gauge needle. [0524] Pull out the plunger, mix
cells and add 100 .mu.l of cells to the back of syringe, carefully
insert the plunger. [0525] Keep the cells on ice. [0526] Tail Vein
injection [0527] Warm the mice with an incandescent lamp for 5
minutes [0528] Restrain the mice with tail illuminator restrainer.
[0529] Rotate the tail slightly to visualize vein. [0530] Clean and
sterilize injection site with iodine followed by ethanol pad [0531]
Insert needle into the vein at a slight angle and inject the cells.
[0532] Remove the needle and apply gentle compression with Gauze
sponges until bleeding has stopped. [0533] Return animals to their
cage and observe for 5-10 minutes to make sure that bleeding has
not resumed.
Therapeutic Antibody Administration
[0533] [0534] 1-3 days post human PBMC injection, mice are
administrated with antibodies by Intraperitoneal injection.
Materials:
[0534] [0535] Fully human IgG1 isotype control: Eureka
therapeutics, cat#ET-901 (preclinical grade) Lot#15-726 Expiration:
February 2017 [0536] Ipilimumab (Yervoy): Bristol-Myers Squibb NDC
0003-2327-11, lot#921873 Expiration: April 2015; lot#4H69490,
Expiration: May 2016 [0537] Fully human IgG4 isotype control:
Eureka therapeutics, cat#ET-904 (preclinical grade) Lot#15-726
Expiration: February 2017 [0538] Anti human ICOS H2L5 hIgG4PE
[0539] Pembrolizumab (Keytruda): Merck, NDC 0006-3026-02, lot#
L010592, Expiration: Apr. 26, 2016 [0540] Intraperitoneal
injection: [0541] Draw up, into the syringe and needle, 100 .mu.l
of to be administered. [0542] Line the bevel of the needle with the
numbers on the syringe. [0543] Sufficiently restrain the animal
with your non-dominant hand. [0544] Entry point for the needle:
Draw an imaginary line across the abdomen just above the knees, the
needle will be inserted along this line on the animal's right side
and close to the midline. As this is a female, you can see that the
point of entry is cranial to and slightly medial of the last
nipple. [0545] Tilt the mouse with its head slightly toward the
ground so that its head is lower than its hind end. [0546] Insert
the needle into the abdomen at about a 30-degree angle. [0547] The
shaft of the needle should enter to a depth of about half a
centimeter. [0548] After injection, withdraw the needle and return
the mouse to its cage. [0549] Blood and tumor sampling [0550]
Materials: [0551] Microvette CB300 (Serum): Braintree Scientific,
Cat# MV-CB300 16440 [0552] Microvette CB300 (Hematology/Potassium
EDTA): Braintree Scientific, Cat# MV-CB300 16444 [0553] Blood:
[0554] Mice were tail vein bled once a week. [0555] 30 .mu.l of
blood was collected in Microvette CB300 (Hematology/Potassium EDTA)
for flow cytometry analysis. [0556] Another 30 .mu.l of blood was
collected in serum Microvette CB300 and incubated for 2 hours at
room temperature to allow clotting, followed by centrifugation at
2000.times.g in order to collect serum. Serum was stored at -20
until further analysis.
Tumor:
[0556] [0557] Mice were euthanized when tumor size reached 2000
mm.sup.3. Tumors were collected and processed in the following
procedure.
Experimental Design
[0558] All studies were prepared according to procedures listed
above. H2L5 hIgG4PE Dose Response
[0559] This study was designed to determine dose-dependent activity
of H2L5 hIgG4PE in human PBMC engrafted NSG mice implanted with
A2058 melanoma tumors. Nine groups with 10 mice per group and 1
control group (Tumor only no PBMC) with 7 mice were assigned into
each study. A summary of the treatment regimen for dose response
using human PBMC from donor #7129 is present in Table 11. H2L5
hIgG4PE was dosed at 0.04, 0.4, 1.2 and 4 mg/kg. Ipilimumab was
dosed at 3 mg/kg and an Fc-Disabled variant of the anti-ICOS
agonist was tested at 1 mg/kg. Test groups were evaluated relative
to the vehicle and matched isotype control groups. Survivability
analysis concluded on day 49 at termination of the study.
TABLE-US-00023 TABLE 11 Summary of Treatment Regimen for H2L5
hIgG4PE Dose Response in Mice # mice/ Groups Treatment 1 Treatment
2 group Dosing 1 Tumor + Vehicle 10 Twice weekly huPBMC for 3 weeks
(donor #7129) 2 Tumor + human IgG1 Isotype 10 Twice weekly huPBMC
(3 mg/kg) for 3 weeks (donor #7129) 3 Tumor + Ipilimumab (3 10
Twice weekly huPBMC mg/kg) for 3 weeks (donor #7129) 4 Tumor +
human IgG4 (4 10 Twice weekly huPBMC mg/kg) for 3 weeks (donor
#7129) 5 Tumor + H2L5 hIgG4PE 10 Twice weekly huPBMC (0.04 mg/kg)
for 3 weeks (donor #7129) 6 Tumor + H2L5 hIgG4PE (0.4 10 Twice
weekly huPBMC mg/kg) for 3 weeks (donor #7129) 7 Tumor + H2L5
hIgG4PE (1.2 10 Twice weekly huPBMC mg/kg) for 3 weeks (donor
#7129) 8 Tumor + H2L5 hIgG4PE (4 10 Twice weekly huPBMC mg/kg) for
3 weeks (donor #7129) 9 Tumor + ICOS-Fc-disabled 10 Twice weekly
huPBMC (1 mg/kg) for 3 weeks (donor #7129) 10 Tumor (no Untreated 7
Twice weekly PBMC) for 3 weeks (donor #7129)
Efficacy and Pharmacodynamic (PD) Activity Study with H2L5 hIgG4PE
in Combination with Ipilimumab and Pembrolizumab
Study Objectives:
[0560] To evaluate the anti-tumor activity of H2L5 hIgG4PE
monotherapy dosed at 0.04 mg/kg and 0.4 mg/kg.
[0561] To evaluate the anti-tumor activity of H2L5 hIgG4PE dosed in
combination with ipilimumab or pembrolizumab with matched isotype
controls.
[0562] Collection of tissue for future pharmacodynamic activity
study of H2L5 hIgG4PE. A total of 22 treatment groups with 10 mice
per group were assigned to this study. Groups 1-16 were the
efficacy cohorts and 17-22 were pharmacodynamic activity
cohorts.
[0563] For combination treatments, H2L5 hIgG4PE (0.04 or 0.4 mg/kg)
and ipilimumab or IgG1 (3 mg/kg) or H2L5 hIgG4PE (0.04 or 0.4
mg/kg) and pembrolizumab or IgG4 (5 mg/kg) were dosed. H2L5 hIgG4PE
and ipilimumab as well as the matched isotype controls were dosed
twice weekly for 6 doses, pembrolizumab and isotype control were
dosed every 5 days until end of the H2L5 hIgG4PE dose. For the
pharmacodynamic tissue collection cohorts, H2L5 hIgG4PE was dosed
at 0.004, 0.04, 0.4 and 1.2 mg/kg. Treatment groups were evaluated
relative to the vehicle and isotype control groups. Treatment
groups for vehicle, isotypes and H2L5 hIgG4PE alone and in
combination with ipilimumab and pembrolizumab using human PBMC from
donor number #6711 are shown in Table 12. Analysis concluded on day
59 at termination of the study.
TABLE-US-00024 TABLE 12 Treatment groups of mice in A2058 melanoma
tumor model # mice/ Group Treatment 1 Treatment 2 group Dosing 1
Tumor + Vehicle 10 Twice a week for 6 doses huPBMC (donor #6711) 2
Tumor + Isotype control (IgG1 10 IgG1 Twice a week for 6 doses
huPBMC 3 mg/kg + IgG4 5 mg/kg) IgG4 every 5 days until the end
(donor #6711) of ICOS dose 3 Tumor + Ipilimumab 3 mg/kg + 10 Twice
a week for 6 doses huPBMC IgG4 5 mg/kg IgG4 every 5 days until the
end (donor #6711) of ICOS dose 4 Tumor + Pembrolizumab 5 mg/kg + 10
IgG1 Twice a week for 6 doses huPBMC IgG1 3 mg/kg Pembrolizumab
every 5 days (donor #6711) until the end of ICOS dose 5 Tumor +
H2L5 hIgG4PE 0.04 mg/kg + 10 IgG1 and ICOS Twice a week huPBMC IgG1
3 mg/kg for 6 doses (donor #6711) 6 Tumor + H2L5 hIgG4PE 0.4 mg/kg
+ 10 IgG1 and ICOS Twice a week huPBMC IgG1 3 mg/kg for 6 doses
(donor #6711) 7 Tumor + Ipilimumab 3 mg/kg + 10 Ipilimumab Twice a
week for 6 huPBMC Pembrolizumab 5 mg/kg doses Pembrolizumab every 5
(donor #6711) days until the end of ICOS dose 8 Tumor + H2L5
hIgG4PE 0.04 mg/kg + 10 Ipilimumab and ICOS Twice a huPBMC
Ipilimumab 3 mg/kg week for 6 doses (donor #6711) 9 Tumor + H2L5
hIgG4PE 0.4 mg/kg + 10 Ipilimumab and ICOS Twice a huPBMC
Ipilimumab 3 mg/kg week for 6 doses (donor #6711) 10 Tumor + H2L5
hIgG4PE 0.04 mg/kg + 10 ICOS Twice a week for 6 doses huPBMC
Pembrolizumab 5 mg/kg Pembrolizumab every 5 days (donor #6711)
until the end of ICOS dose 11 Tumor + H2L5 hIgG4PE 0.4 mg/kg + 10
ICOS Twice a week for 6 doses huPBMC Pembrolizumab 5 mg/kg
Pembrolizumab every 5 days (donor #6711) until the end of ICOS dose
12 Tumor + IgG4 5 mg/kg 10 Twice a week for 6 doses huPBMC (donor
#6711) 13 Tumor + Pembrolizumab 2.5 mg/kg 10 Pembrolizumab every 5
days huPBMC until the end of ICOS dose (donor #6711) 14 Tumor +
Pembrolizumab 5 mg/kg 10 Pembrolizumab every 5 days huPBMC until
the end of ICOS dose (donor #6711) 15 Tumor + H2L5 hIgG4PE 0.4
mg/kg 10 ICOS Twice a week for 6 doses huPBMC (donor #6711) 16
Tumor + H2L5 hIgG4PE 0.4 mg/kg + 10 ICOS Twice a week for 6 doses
huPBMC Pembrolizumab 5 mg/kg + Pembrolizumab every 5 days (donor
#6711) Ipi until the end of ICOS dose 17 Tumor + Vehicle 10 Twice a
week for huPBMC pharmacodynamic activity, 5 (donor #6711) mice
harvested 24 hr post 2nd dose and 5 mice harvested 24 hr post 4nd
dose 18 Tumor + Isotype Control (IgG4) 10 Twice a week for huPBMC
1.2 mg/kg pharmacodynamic activity, 5 (donor #6711) mice harvested
24 hr post 2nd dose and 5 mice harvested 24 hr post 4nd dose 19
Tumor + H2L5 hIgG4PE 0.004 mg/kg 10 Twice a week for huPBMC
pharmacodynamic activity, 5 (donor #6711) mice harvested 24 hr post
2nd dose and 5 mice harvested 24 hr post 4nd dose 20 Tumor + H2L5
hIgG4PE 0.04 mg/kg 10 Twice a week for huPBMC pharmacodynamic
activity, 5 (donor #6711) mice harvested 24 hr post 2nd dose and 5
mice harvested 24 hr post 4nd dose 21 Tumor + H2L5 hIgG4PE 0.4
mg/kg 10 Twice a week for huPBMC pharmacodynamic activity, 5 (donor
#6711) mice harvested 24 hr post 2nd dose and 5 mice harvested 24
hr post 4nd dose 22 Tumor + H2L5 hIgG4PE 1.2 mg/kg 10 Twice a week
for huPBMC pharmacodynamic activity, 5 (donor #6711) mice harvested
24 hr post 2nd dose and 5 mice harvested 24 hr post 4nd dose
Efficacy Study Evaluating H2L5 hIgG4PE Dosed in Combination with
Ipilimumab or Pembrolizumab
[0564] This study was designed to evaluate the anti-tumor efficacy
of H2L5 hIgG4PE (dosed at 0.01 and 0.04 mg/kg) in combination with
ipilimumab or pembrolizumab with matched isotype controls in the
human PBMC engrafted NSG mouse using A2058 melanoma tumor model. A
total of 13 groups with 10 mice per group were assigned into the
study. Group 2 was the combined isotype control of humanized IgG1
and IgG4. H2L5 hIgG4PE was dosed at 0.01 mg/kg (Group 12) and 0.04
mg/kg (Group 13) as single agent. For combination treatments, H2L5
hIgG4PE (0.01 and 0.04 mg/kg) and ipilimumab or IgG1 (3 mg/kg) or
H2L5 hIgG4PE (0.01 and 0.04 mg/kg) and pembrolizumab or IgG4 (5
mg/kg) was dosed. H2L5 hIgG4PE and ipilimumab as well as the
matched isotype controls were dosed twice weekly for 6 doses,
pembrolizumab and isotype control was dosed every 5 days until end
of the H2L5 hIgG4PE dose. A summary of treatment groups, using
human PBMC from donor #4568, is presented in Table 13. Treatment
groups were evaluated relative to the vehicle and isotype control
groups. Survivability analysis was concluded on day 33 at
termination of the study.
TABLE-US-00025 TABLE 13 Treatment groups of mice in A2058 melanoma
tumor model # mice/ Group Treatment 1 Treatment2 group Dosing 1
Tumor + Vehicle 10 Twice a week for 6 doses huPBMC (donor #4568) 2
Tumor + Isotype control (IgG1 10 IgG1 Twice a week for 6 doses
huPBMC 3 mg/kg + IgG4 5 mg/kg) IgG4 every 5 days until the end
(donor #4568) of ICOS dose 3 Tumor + Ipilimumab 3 mg/kg + 10 Twice
a week for 6 doses IgG4 huPBMC IgG4 5 mg/kg every 5 days until the
end of (donor #4568) ICOS dose 4 Tumor + Pembrolizumab 5 mg/kg + 10
IgG1 Twice a week for 6 doses huPBMC IgG1 3 mg/kg Pembrolizumab
every 5 days (donor #4568) until the end of ICOS dose 5 Tumor +
H2L5 hIgG4PE 0.01 mg/kg + 10 IgG1 and ICOS Twice a week huPBMC IgG1
3 mg/kg for 6 doses (donor #4568) 6 Tumor + H2L5 hIgG4PE 0.04 mg/kg
+ 10 IgG1 and ICOS Twice a week huPBMC IgG1 3mg/kg for 6 doses
(donor #4568) 7 Tumor + Ipilimumab 3 mg/kg + 10 Ipilimumab Twice a
week for 6 huPBMC Pembrolizumab 5 mg/kg doses Pembrolizumab every 5
(donor #4568) days until the end of ICOS dose 8 Tumor + H2L5
hIgG4PE 0.01 mg/kg + 10 Ipilimumab and ICOS Twice a huPBMC
Ipilimumab 3 mg/kg week for 6 doses (donor #4568) 9 Tumor + H2L5
hIgG4PE 0.04 10 Ipilimumab and ICOS Twice a huPBMC mg/kg +
Ipilimumab week for 6 doses (donor #4568) 3 mg/kg 10 Tumor + H2L5
hIgG4PE 0.01 mg/kg + 10 ICOS Twice a week for 6 doses huPBMC
Pembrolizumab 5 mg/kg Pembrolizumab every 5 days (donor #4568)
until the end of ICOS dose 11 Tumor + H2L5 hIgG4PE 0.04 mg/kg + 10
ICOS Twice a week for 6 doses huPBMC Pembrolizumab 5 mg/kg
Pembrolizumab every 5 days (donor #4568) until the end of ICOS dose
12 Tumor + H2L5 hIgG4PE 0.01 mg/kg 10 Twice a week for 6 doses
huPBMC (donor #4568) 13 Tumor + H2L5 hIgG4PE 0.04 mg/kg 10 Twice a
week for 6 doses huPBMC (donor #4568)
Statistical Analysis
[0565] The event for survival analysis was tumor volume >2000
mm.sup.3, tumor ulceration, mouse body weight loss >20%,
moribund or found dead, whichever came first. The exact time to
cut-off volume was estimated by fitting a linear line between log
tumor volume and day of two observations, the first observation
that exceed the cut-off volume and the one observation that
immediately preceded the cut-off volume. Kaplan-Meier (KM) method
was carried out to estimate the survival probability of different
treatment groups at a given time. The median time to endpoint and
its corresponding 95% confidence interval was reported. Whether or
not KM survival curves are statistically different between any two
groups was then tested by log-rank test.
[0566] Tumor volume data from the last day in which there were 10
animals per group (i.e. before any animals were euthanized) was
utilized to make tumor volume comparisons between the different
treatment groups. Prior to the analysis, the tumor volume was
natural log transformed due to the inequality of variance in the
different treatment groups. ANOVA followed by pair-wise comparison
were then carried out on the log transformed data. Graphpad Prism
software was used to plot the tumor growth and body weight
data.
Results
[0567] H2L5 hIgG4PE Dose Response (FIG. 20A)
Tumor Growth Inhibition:
Control Group:
[0568] Human PBMC (donor 7129) showed no effect on A2058 tumor
growth in NSG mice. A2058 tumor bearing mice with or without human
PBMC, A2058 tumor bearing mice with human PBMC treated with vehicle
and isotype control antibodies developed tumors that progressed as
expected (Group#1 vs. Group#10, Group#1 vs. Group#2, Group#1 vs.
Group#4, p=1).
Ipilimumab treatment at 3 mg/kg (Group #3) demonstrated significant
tumor growth inhibition (p<0.03) as compared to vehicle control
Group#1, however the statistical significance was lost (p<0.22)
when compared to the isotype control Group #2. This indicated the
isotype antibody may affect tumor growth. H2L5 hIgG4PE treatment at
0.4 mg/kg demonstrated a trend of tumor growth inhibition and
increased survivability of mice compared to other doses, although
the affects were not statistically significant when compared to
either vehicle or isotype control.
Clinical Observations:
[0569] Loss of body weight in mice was observed during the study
which was approximately 20% at the end of study. It has been
reported that both GvHD and tumor burden can result in a drop in
mice body weight, though in this study the body weight loss seemed
to be more related to A2058 tumor since tumor bearing mice without
PBMC engraftment (group #10) showed the same trend. Tumor
ulceration was observed in multiple tumors during the study,
including the isotype control group.
Mouse Fates:
[0570] Most mice were removed upon tumors reaching volumes >2000
mm.sup.3. Three mice were euthanized due to tumor ulceration, and
three mice were euthanized due to body weight loss of >20%. Nine
mice were found dead randomly across the groups, including two in
the vehicle, and three total in the isotype control groups. These
deaths were attributed to the susceptibility of the model for a
Graft-versus-Host Disease state, and not treatment related since no
pattern was observed with treatment groups compared to vehicle or
isotype control groups. Efficacy study with H2L5 hIgG4PE in
combination with ipilimumab and Pembrolizumab (FIG. 20B)
Tumor Growth Inhibition:
[0571] Control group: A2058 tumor bearing mice with human PBMC
treated with vehicle or isotype control antibodies developed tumors
which grew as expected.
Monotherapy:
[0572] Ipilimumab treatment at 3 mg/kg combined with IgG4 (Group#3)
resulted in significant tumor growth inhibition (p<0.04) as
compared to vehicle control Group#1. However, when compared to
isotype control Group#2, the statistical significance was lost
(p<0.23). Pembrolizumab treatment alone at 2.5 or 5 mg/kg
(Group#13, 14) showed observable tumor growth inhibition without
statistical significance when compared to vehicle or isotype
control group#12. Pembrolizumab combined with IgG1 (Group#4),
showed observable tumor growth inhibition without statistical
significance, however a significant increase in survival was
observed (p<0.04) as compared to vehicle control Group#1.
Statistical significance was lost (p<0.4) when compared with
isotype control Group#2. H2L5 hIgG4PE treatment alone at 0.4 mg/kg
(Group#15) showed observable tumor growth inhibition without
statistical significance as compared to vehicle or isotype control
group#12. H2L5 hIgG4PE at 0.04 or 0.4 mg/kg combined with IgG1
(Group#5 and 6) showed observable delay in tumor progression and
mice survival but didn't reach statistical significance.
Combination Treatment:
[0573] Combination of H2L5 hIgG4PE (0.04 or 0.4 mg/kg) with
ipilimumab (3 mg/kg). Groups #8 and #9 showed no additional tumor
growth inhibition as compared to Ipilimumab alone (Group#3).
Combination of H2L5 hIgG4PE (0.04 or 0.4 mg/kg) with pembrolizumab
(5 mg/kg) Groups #10 and #11 demonstrated modest but insignificant
tumor growth inhibition and mice survival compared to pembrolizumab
monotherapy, Group#4, or H2L5 hIgG4PE monotherapy Groups #5 and
#6.
Clinical Observations:
[0574] Mice body weight loss observed during the study was
approximately 20%. Tumor ulceration was apparent in multiple tumors
during the study across the majority of group.
Mouse Fates:
[0575] A total of 100 out of 160 mice were euthanized when tumor
volumes reached >2000 mm.sup.3. 29 mice were euthanized due to
tumor ulceration, 18 mice were found dead, 12 mice were euthanized
due to body weight loss >20%, and one mouse was euthanized as
moribund. Mice were found dead across the groups including the
isotype control group #2. These deaths were attributed to the
susceptibility of the model for a Graft-versus-Host Disease state,
and not treatment related since no pattern was observed with
treatment groups compared to the isotype control group. Efficacy
study evaluating H2L5 hIgG4PE dosed in combination with ipilimumab
or pembrolizumab (FIG. 20C)
Tumor Growth Delay:
Control Group:
[0576] A2058 tumor bearing mice with human PBMC treated with
vehicle or isotype control antibodies developed tumors which grew
as expected.
Monotherapy:
[0577] Ipilimumab treatment at 3 mg/kg combined with IgG4 (Group#3)
demonstrated significant tumor growth inhibition (p<0.02) and
significant increase in survival (p<0.01) as compared to vehicle
control Group#1. Compared to isotype control Group#2 however, the
tumor growth inhibition did not reach significance (p<0.13)
while significant increase in mice survival remained (p<0.04).
Pembrolizumab treatment at 5 mg/kg combined with IgG1 (Group#4)
showed tumor growth inhibition without statistical significance as
compared to vehicle or isotype control Group#2. H2L5 hIgG4PE
treatment alone at 0.01 mg/kg or 0.04 mg/kg (Group#12 and #13)
demonstrated significant tumor growth inhibition (p<0.03)
compared to vehicle control group #1 H2L5 hIgG4PE dosed at 0.04
mg/kg also showed a significant increase in mice survival
(p<0.048) as compared to vehicle control group#1. However, as
compared to isotype control group#2, tumor growth inhibition and
survival did not reach statistical significance for groups #12 and
#13. H2L5 hIgG4PE at 0.01 mg/kg combined with IgG1 (Group#5) showed
significant tumor growth inhibition (p<0.03) and mice survival
(p<0.03) as compared to vehicle control group#1. However, as
compared to isotype control group#2, tumor growth delay and
survival did not reach statistical significance. H2L5 hIgG4PE at
0.04 mg/kg combined with IgG1 (Group#6) showed observable tumor
growth inhibition and mice survival, but did not reach statistical
significance.
Combination Treatment:
[0578] The combination of H2L5 hIgG4PE with ipilimumab (0.01 mg/kg
plus ipilimumab 3 mg/kg; Group#8) showed observable tumor growth
inhibition and mice survival but failed to reach statistical
significance. H2L5 hIgG4PE combination with ipilimumab (0.04 mg/kg
plus ipilimumab 3 mg/kg; Group#9) demonstrated significant tumor
growth inhibition (p<0.00) and a significant increase in mice
survival (p<0.04) as compared to vehicle control group#1 or
isotype control group#2 (p<0.02). However, as compared to
isotype control survival failed to reach statistical significance.
Combination activity did not reach significance as compared to
monotherapy ipilimumab group#3 or H2L5 hIgG4PE monotherapy
groups.
[0579] H2L5 hIgG4PE (0.01 mg/kg or 0.04 mg/kg) combination with
pembrolizumab (5 mg/kg), Groups#10 and #11, showed significant
tumor growth inhibition. (p.ltoreq.0.03) and significant increase
of mice survival observed (p<0.03) when comparing to vehicle
control group#1. When comparing to isotype control group#2, the
tumor growth inhibition significance remained in the 0.04 mg/kg
H2L5 hIgG4PE combination with pembrolizumab (p<0.03). The
survival benefit failed to reach statistical significance however.
The combination failed to reach significance as compared to either
monotherapy treatment group pembrolizumab group#3 or H2L5 hIgG4PE
group#5 or #6. Thus, H2L5 hIgG4PE combined with pembrolizumab (0.01
or 0.04 mg/kg plus pembrolizumab 5 mg/kg) demonstrated an increase
in tumor growth inhibition and mice survival but failed to reach
statistical significance versus isotype control or
monotherapies.
Clinical Observations:
[0580] Mice body weight loss observed during the study was
approximately 20%. Tumor ulceration was observed across the
majority of groups during the study.
Mouse Fates:
[0581] A total of 91 mice were euthanized due to tumor size
>2000 mm.sup.3, 34 mice were euthanized due to tumor
ulcerations, and 5 mice were found dead. These deaths were
attributed to the susceptibility of the model for a
Graft-versus-Host Disease state.
Discussion
[0582] Efficacy of H2L5 hIgG4PE as a monotherapy and in combination
with pembrolizumab as well as ipilimumab was evaluated in the human
PBMC engrafted NSG mouse model with A2058 melanoma tumors. This
model where human PBMC are intravenously injected into adult
immunodeficient NSG (NOD/SCID/IL-2R.gamma.null) mice is known as
the Hu-PBMC NSG model. It induces a Graft-versus-Host Disease
(GvHD) and has been used to study effector and memory T cell
activity. The Hu-PBMC NSG model was implanted with human cancer
cell line A2058 subcutaneously to investigate the effect of human
immunotherapeutic antibodies on tumor growth. The limitations of
this model include onset of GvHD symptoms, loss of body weight, and
frequent tumor ulcerations which prevent survival monitoring for
longer period of time as is possible with syngeneic mouse tumor
models.
[0583] Initial studies evaluating H2L5 hIgG4PE at doses ranging
from 0.04 mg/kg to 4 mg/kg showed that doses in the lower range
demonstrated modest tumor growth inhibition. Delay in tumor
progression and increased survival of mice was observed in dose
groups ranging from 0.04 to 0.4 mg/kg though not statistically
significant when compared to the isotype control groups. Based on
these studies, H2L5 hIgG4PE doses of 0.04 to 0.4 mg/kg were
selected for further evaluation alone and in combination with
pembrolizumab and ipilimumab in two studies with PBMC grafts from
two different donors (donor numbers 4568 and 6711). Modest
responses for H2L5 hIgG4PE monotherapy and combination with
pembrolizumab were observed in one of the two combination studies
performed. The combination study using PBMC donor 4568 (Table 13,
FIG. 20C) demonstrated anti-tumor activity of the monotherapy and
combination while the study using PBMC donor 6711 (Table 12, FIG.
20B) did not show significant anti-tumor effect, which likely was a
result of donor PBMC differences between studies, which reflect the
patient response variability that may be observed in the clinic. In
this second combination study with PBMC donor 4568, enhanced tumor
growth inhibition and increased survivability of mice was observed
in the combination group when compared to either agent alone,
although this difference was not statistically significant.
Combination synergy was observed however, since the H2L5 hIgG4PE
0.04 mg/kg dose in combination with pembrolizumab 5 mg/kg resulted
in a statistically significant decrease in tumor volume ten days
post first dose and increased survivability versus the isotype
control group (p.ltoreq.0.05), while the monotherapies did not. In
fact, 50% of the mice in the H2L5 hIgG4PE and pembrolizumab
combination group remained on study by day 33, but were removed due
to tumor ulcerations. Only four mice were removed from study due to
tumor volume from this combination group, while 8 to 9 mice were
removed from study in the pembrolizumab and isotype groups.
[0584] Anti-PD1 therapy did not demonstrate statistically
significant activity in this model as seen with the limited change
in tumor growth and survival seen with pembrolizumab treated cohort
compared to isotope treated cohort. Ipilimumab monotherapy showed a
trend of tumor growth inhibition modestly better than pembrolizumab
in both studies, and it showed statistically significant increase
in survival versus isotype in the second combination study with the
responsive PBMC donor 4568 (p.ltoreq.0.04). The H2L5 hIgG4PE 0.01
mg/kg dose in combination with ipilimumab 3 mg/kg showed a
significant increase in survival versus ipilimumab (p.ltoreq.0.02),
but not versus H2L5 hIgG4PE monotherapy. There were no additional
significant effects on tumor volume observed with the combination
of H2L5 hIgG4PE and ipilimumab in this model compared to either
agent alone. Mice from across all treatment groups including
vehicle and isotype control groups were found dead as reported in
the Fate Tables. These deaths were attributed to the susceptibility
of the model for a Graft-versus-Host Disease state, and not
treatment related.
Example 12: Functional Activity of Anti-Murine ICOS Agonist
Antibody Alone and in Combination with Anti-PD1 and Anti-CTLA-4
Antibodies In Vivo
CT26 and EMT6 Syngeneic Mouse Tumor Models
[0585] CT26 murine colon carcinoma mouse tumor model
Methods
[0586] This study was conducted under a protocol which was approved
by the GSK Institutional Animal Care and Use Committee prior to
commencement of the study.
Animals
[0587] In this study 164 female BALB/c mice from Harlan Sprague
Dawley. Mice were 6-8 weeks old at the beginning of the study when
they were inoculated.
Cell Culture and Inoculation
[0588] One vial of CT-26 cells (ATCC: CRL-2638) (3.times.10.sup.6
cells; P-11) was thawed from -140.degree. C. and plated in RPMI
with 10% FBS. Cells were subcultured 3 times over 10 days.
Trypsin/EDTA was used to facilitate cell detachment from culture
flask during subculturing. Cells were collected, washed twice, and
re-suspended in RPMI without FBS at 5.times.10.sup.5 cells/ml. Mice
were inoculated subcutaneously with 0.1 ml cells (5.times.10.sup.4
cells/mouse) on the right hind flank. On the day of cell collection
and inoculation, cell counts were done on Beckman Coulter Vi-cell
XR and checked by hemacytometer. Cells were detached from flask
with trypsin/EDTA and washed twice, first with RPMI+10% FBS and
second with RPMI only and resuspended in 10 ml RPMI.
178.times.10.sup.6 cells were collected in 20 ml RPMI with 98.8%
viability. 1.685 ml cell suspension (15.times.10.sup.6 cells total)
was added to 28.315 ml RPMI. 15.times.10.sup.6 cells/30 ml
media=5.times.10.sup.5 cells/ml. This equates to 5.times.10.sup.4
cells/100 .mu.l.
Antibody Formulation and Preparation
[0589] Antibodies were diluted from stock source vials to desired
concentrations in sterile 0.9% saline on the day of dosing.
Anti-ICOS agonist clone C398.4 was tested at 0.05 mg/kg and 0.5
mg/kg. Each dose was also tested with both anti-PD1 10 mg/kg and
anti-CTLA-4 1 mg/kg.
Experimental Protocol(s)
Tumor Monitoring and Dosing
[0590] Mice were inoculated on day 0. On day 11 body weight and
tumor volume were measured. Mice were randomized into the 12 study
groups shown in Table 14 with 10 mice/group based on tumor size.
Randomization was done using Studylog Study Director software. Mice
were dosed based on the study design chart twice weekly starting on
randomization day and continuing for 6 total doses. Dosing was
interperitoneal (IP) in 100 .mu.l volume of 0.9% saline vehicle.
Tumor volume and body weight were measured 3 times per week
throughout the study.
Endpoints
[0591] Mice were removed from the study for tumor burden when tumor
volume was greater than 2000 mm.sup.3. Tumor volume was calculated
by applying length and width caliper measurements to the following
formula: TV=0.52*L*W.sup.2.
[0592] Additionally mice were removed from study when tumors
developed open ulcerations. Ulcerations were observed throughout
the experiment, however scabbed over ulcerations alone were not an
endpoint unless they formed open holes.
[0593] Although it did not apply to any mice in this study a third
endpoint established at the beginning of the study was a decrease
of 20% body weight.
Drugs and Materials
TABLE-US-00026 [0594] Antibody Vendor Catalog # Lot Clone ICOS
Biolegend 93108 B205973 C398.4 PD1 BioXcell BE0146 5792-10/0815B
RMP1-14 CTLA-4 BioXcell BE0164 5632-4/0715 9D9 Mouse IgG2b BioXcell
BE0086 4700/1014 MCP-11 Rat IgG2a BioXcell BE0089 5679-6/0815 2A3
Hamster IgG Biolegend 92257 B205974 HTK888
All antibodies were diluted to desired concentrations in 0.9%
saline and saline was used as a vehicle control.
Data Analysis
[0595] The event for survival analysis is tumor volume of 2000
mm.sup.3 or tumor ulceration, whichever came first. The exact time
to cut-off volume was estimated by fitting a linear line between
log tumor volume and day of two observations, the first observation
that exceed the cut-off volume and the one observation that
immediately preceded the cut-off volume. The Kaplan-Meier (KM)
method was carried out to estimate the survival probability of
different treatment groups at a given time. The median time to
endpoint and its corresponding 95% confidence interval was
reported. Whether or not KM survival curves are statistically
different between any two groups was then tested by the log-rank
test.
[0596] Tumor volumes at 17 days after initial dosing between the
different treatment groups were compared. Prior to the analysis,
the tumor volume was natural log transformed due to the inequality
of variance in the different treatment groups. ANOVA followed by
pair-wise comparison were then carried out on the log transformed
data.
TABLE-US-00027 TABLE 14 Study Groups Group No. Treatment 1 Saline 2
Mouse IgG2b 20 .mu.g + Hamster IgG 10 .mu.g 3 Rat IgG2a 200 .mu.g +
Hamster IgG 10 .mu.g 4 Hamster IgG 10 .mu.g 5 ICOS 1 .mu.g 6 ICOS
10 .mu.g 7 CTLA-4 20 .mu.g 8 PD1 200 .mu.g 9 ICOS 1 .mu.g + CTLA-4
20 .mu.g 10 ICOS 10 .mu.g + CTLA-4 20 .mu.g 11 ICOS 1 .mu.g + PD1
200 .mu.g 12 ICOS 10 .mu.g + PD1 200 .mu.g
[0597] The raw p-value, as well as the false discovery rate (FDR)
adjusted p-values, from the comparisons of days to events by
survival analysis and the comparisons of log transformed tumor
volume at day 10 between treatment groups are shown in the above
table. Comparisons, using FDR adjusted p-values .ltoreq.0.05, are
declared to be statistically significant.
Results
[0598] Mouse fate tracking showed that the number of mice removed
from study for tumor burden and tumor ulceration. All remaining
mice are tumor free at study day 61 except 1 mouse in G7 which has
a tumor volume of 579.16 mm.sup.3.
[0599] For survival (time to endpoints) groups 9 and 12 showed
significant increase in survival compared to the vehicle control
group (p=0.008 and p=0.001 respectively). Group 12 showed
statistically significant extended survival compared to groups 2,
4, and 5 (p=0.006, 0.001, 0.02). However, no combination group
showed statistically significant (p<0.05) increased survival
over either monotherapy. (FIG. 21)
Discussion
[0600] The combination therapy groups, particularly the high dose
anti-ICOS and anti-PD1 combination (Group 12), demonstrated tumor
growth inhibition and increased survival over monotherapy and
isotype control groups, although statistical significance was not
reached at Day 61. The isotype control for group 12 was the Rat
IgG2a+Hamster IgG group 3. The monotherapy groups for comparison
are; ICOS 10 .mu.g (group 6) and PD1 200 .mu.g (group 8). A total
of 5 mice remained as tumor free in group 12 compared to 1 in group
3, 1 in group 6 and 1 in group 8. The survival benefit was
quantified by taking the day each mouse reached any of the
pre-determined study endpoints. A number of mice were removed from
study for open tumor ulcerations and not due to tumor burden.
[0601] In the high dose ICOS+CTLA-4 combination group (group 10) an
increased number of mice were removed due to tumor ulceration by
day 31 which likely masked the survival and anti-tumor benefit that
this combination provided. In this group, 5 mice were removed for
tumor ulcerations and only 2 for tumor burden reaching 2000
mm.sup.3. All tumors removed due to tumor ulceration where still at
modest size when taken off study, and it is expected that tumor
ulceration may have been the result of a therapy-induced anti-tumor
immune response in these mice. Three mice remained tumor free in
this group out to day 61. The 2 mice removed for tumor burden were
the lowest number of mice removed for tumor burden of all
groups.
EMT6 Mammary Carcinoma Mouse Tumor Model
Experimental Protocol(s)
[0602] All procedures and euthanization criteria described in this
document are in accordance with IACUC protocol AUP0606. Animals are
weighed and inoculated on the right hind quarter with 100 .mu.l of
1.times.10.sup.5 EMT6 tumor cells per mouse. The number of mice
inoculated is equal to at least 130% of what was needed for the
study. Assuming 30% failure rate (either too big or too small at
time of start of study), the goal was to have n=10 for each group.
After tumor cell inoculation, tumor growth and total body weight
were measured 3 times a week with a Fowler "ProMax" digital caliper
for 4 weeks or longer. Antibodies were acquired from a commercial
vendor and diluted to desired concentration in 0.9% saline. Dosing
(i.p.) occurred biweekly, for a total of 6 doses and initiated on
the day of randomization, designated as Day 0, when average tumor
volume approximated 100 mm.sup.3, approximately 7 to 8 days after
inoculation. Randomization was performed using the Studylog Study
Director Suite software. Length and width of tumors was measured in
order to determine tumor volume using the formula (tumor
volume=L*W.sup.2*0.52). Tumor measurement of greater than 2,000
mm.sup.3 for an individual animal resulted in removal from study.
Mice may also be removed from the study due to weight loss
(>20%), tumor ulceration, or any other obvious inhibition of
normal mouse activity due to morbidity.
[0603] In this study, a total of 191 animals were inoculated with
EMT6 cells in order to generate enough mice with tumors in the
desired size range for 13 groups of 10 mice each as shown in Table
15. Saline vehicle injected mice and isotype control groups served
as controls for ICOS, PD1 and CTLA-4 mAb treated mice. The isotype
control for ICOS (Hamster IgG) was dosed at 10 .mu.g alone and in
combination with the isotype for CTLA-4 (mouse IgG2b) or PD-1 (rat
IgG2a). Monotherapy treatment groups for anti-CTLA-4 (9D9) and
anti-PD-1 (RMP1-14) were dosed at 20 and 200 .mu.g per mouse,
respectively, and evaluated in combination with the ICOS isotype
control. The C398.4 clone of ICOS agonist was dosed at 10 and 1
.mu.g per mouse. Efficacy of the ICOS agonist was also evaluated at
10 and 1 .mu.g per mouse dosed in combination with anti-CTLA-4 or
anti-PD-1. An additional group of PD-1 and CTLA-4 at predescribed
concentrations was included as a positive control comparator group.
Statistical analysis of tumor volume was performed on day 13 post
randomization. Survivability analysis included mice on study
through day 60.
TABLE-US-00028 TABLE 15 Study Groups Dosing treatment 1 treatment 2
n= Group 1: 1 .times. 10.sup.5 cells per saline 10 Group 2: 1
.times. 10.sup.5 cells per Hamster mIgG2b 20 .mu.g 10 IgG 10 .mu.g
Group 3: 1 .times. 10.sup.5 cells per Hamster rIgG2a 200 .mu.g 10
IgG 10 .mu.g Group 4: 1 .times. 10.sup.5 cells per Hamster 10 IgG
10 .mu.g Group 5: 1 .times. 10.sup.5 cells per ICOS 10 .mu.g 10
Group 6: 1 .times. 10.sup.5 cells per ICOS 1 .mu.g 10 Group 7: 1
.times. 10.sup.5 cells per CTLA4 20 .mu.g Hamster 10 IgG 10 .mu.g
Group 8: 1 .times. 10.sup.5 cells per PD-1 200 .mu.g Hamster 10 IgG
10 .mu.g Group 9: 1 .times. 10.sup.5 cells per ICOS 10 .mu.g CTLA4
20 .mu.g 10 Group 10: 1 .times. 10.sup.5 cells per ICOS 1 .mu.g
CTLA4 20 .mu.g 10 Group 11: 1 .times. 10.sup.5 cells per ICOS 10
.mu.g PD-1 200 .mu.g 10 Group 12: 1 .times. 10.sup.5 cells per ICOS
1 .mu.g PD-1 200 .mu.g 10 Group 13: 1 .times. 10.sup.5 cells per
CTLA4 20 .mu.g PD-1 200 .mu.g 10
Drugs and Materials
Animals
[0604] Female Balb/c mice from 6 to 8 weeks of age were received
from Harlan Sprague Dawley and housed in accordance with IACUC
standards.
EMT6 Cells
[0605] EMT6 cells were thawed and cultured in cell culture flasks
for eight days prior to inoculation. Cells were passed 3 times in
this time. On the day of inoculation, the cells are harvested from
the flask in complete medium. Cells are centrifuged and resuspended
in Weymouth's (with 15% FBS). This step is repeated 3 times in
Weymouth's media without FBS. Cell density and viability are
checked via trypan blue exclusion. Cells are then diluted to
desired density (1.times.10.sup.6 cells per mL).
Immunotherapeutics
[0606] All therapeutics were diluted to desired concentrations in
0.9% sodium chloride on the day of dosing and injected i.p. using a
30 G needle. Therapeutic and control dilutions are presented below
in Table 16.
TABLE-US-00029 TABLE 16 Therapeutic dilutions starting desired
dose/ volume add Total total conc. conc. dilution mouse number
needed stock diluent volume Rx mg/mL mg/mL 1: mg of mice mL mL mL
mL mouse 4.46 0.1 44.6 0.02 10 2 0.10 4.36 4.46 IgG2b rat 6.92 1
6.92 0.2 10 2 0.40 2.37 2.77 IgG2a Hamster 1.47 0.05 29.4 0.01 50
10 0.40 11.36 11.76 IgG CTLA4 6.1 0.1 61 0.02 40 8 0.15 9 9.15 PD-1
7.44 1 7.44 0.2 40 8 1.30 8.372 9.672 ICOS 5 0.05 100 0.01 30 6
0.10 9.9 10 ICOS 0.05 0.005 10 0.001 30 6 1.00 9 10
Data Analysis
Statistical Analysis
[0607] The event for survival analysis was tumor volume of 2000
mm.sup.3 or tumor ulceration, whichever came first. The exact time
to cut-off volume was estimated by fitting a linear line between
log tumor volume and day of two observations, the first observation
that exceed the cut-off volume and the one observation that
immediately preceded the cut-off volume. The Kaplan-Meier (KM)
method was carried out to estimate the survival probability of
different treatment groups at a given time. The median time to
endpoint and its corresponding 95% confidence interval was
reported. Whether or not KM survival curves were statistically
different between any two groups was then tested by the log-rank
test.
[0608] Tumor volumes at 13 days after initial dosing between the
different treatment groups were compared. Prior to the analysis,
the tumor volume was natural log transformed due to the inequality
of variance in the different treatment groups. ANOVA followed by
pair-wise comparison were then carried out on the log transformed
data. SAS 9.3 and R 3.0.2 Analysis Software was utilized.
Results
[0609] Balb/c mice were inoculated and randomized into groups of
ten based on treatment regimen 8 days later. Administration of
therapeutics or controls began on randomization day (Day 0) and
continued twice a week for 3 weeks.
[0610] The saline treated group grew tumors at the expected rate
relative to previous EMT-6 studies. All mice in the saline vehicle
group were euthanized due to tumor size or ulceration by day 30.
Treatment with hamster IgG alone or in combination with rat IgG2a
or mouse IgG2b, resulted in no statistically significant change in
average tumor growth or survival when compared to the saline
vehicle group.
[0611] At 13 days post randomization, the ICOS monotherapy groups
demonstrated little observable change in average tumor growth as
compared to isotype controls. However, the high dose ICOS treatment
group (10 .mu.g) demonstrated an apparent trend towards more tumor
growth inhibition than the low dose group. An effect that was
comparable to the CTLA-4 monotherapy activity was observed.
Monotherapy treatment with PD-1 mAb also resulted in some
observable, but statistically insignificant reduction in average
tumor volume at day 13. However, as with ICOS and CTLA-4
monotherapy, this did not result in increased survival when
compared to that of the appropriate isotype groups. Treatment with
the combination of anti-PD-1 and anti-ICOS antibody clone C398.4 at
the 10 .mu.g dose resulted in considerable tumor growth inhibition
as compared to control and monotherapy treatment groups (FIG. 22).
Three mice in this combination group achieved complete tumor
regression, a considerable improvement over control or monotherapy
treatment groups. However, due to the statistical criteria used,
statistically significant improvement in survival was not reached.
The combination of anti-PD-1 with 1 .mu.g of ICOS agonist antibody
clone C398.4 did result in a statistically significant decrease in
average tumor growth at day 13 as compared to saline vehicle
control (p<0.05) and ICOS monotherapy (p<0.05) groups of 1
and 10 .mu.g. Four mice from this treatment regimen achieved
complete tumor regression resulting in significant trend towards
increased survival that failed to reach statistical
significance.
[0612] The ICOS antibody at both doses in combination with
anti-CTLA-4 demonstrated little observable benefit in tumor growth
inhibition or survival as compared to monotherapy treatment with
either antibody.
Discussion
[0613] While isotype controls resulted in no obvious change in
average tumor volume or overall survival when compared to the
saline vehicle group, there were individual animals in the hamster
IgG group (group 4) and the hamster IgG and rat IgG2a (group 3)
that demonstrated delayed tumor growth. In the hamster IgG &
rat IgG2a isotype group, one mouse survived beyond the last saline
vehicle mouse, being sacrificed on day 36 due to ulceration with a
tumor that measured 1156.56 mm.sup.3 in volume. Two mice in the
hamster IgG group survived longer than the saline group. One animal
was euthanized due to tumor size on day 36, and the second one on
day 41 due to ulceration with a measurement of 1899.28
mm.sup.3.
[0614] The dosing regimen of anti-PD-1 with 10 .mu.g of anti-ICOS
agonist led to an observable inhibition of tumor growth resulting
in a decrease in tumor volume at day 13 when compared to isotype
controls, although this difference was less obvious when compared
to anti-PD-1 monotherapy. However, the combination did result in a
total of five animals surviving beyond any in the anti-PD-1
monotherapy group, with three mice experiencing complete tumor
regression as compared to none in the anti-PD-1 monotherapy
group.
[0615] Pairing anti-PD-1 with a 1 .mu.g dose of ICOS agonist
antibody led to an observable decrease in average tumor size at day
13 when compared to isotype controls and respective monotherapy
groups. This decrease was statistically significant when compared
to saline vehicle control (p<0.05) and the 1 .mu.g ICOS
monotherapy group (p<0.05). Four mice experienced complete tumor
regression and survived beyond any in the PD-1 monotherapy
group
[0616] The survival benefit observed with the ICOS+PD1 combination
group was not found to reach statistical significance relative to
controls by day 60. However, the tumor growth inhibition and
survival benefit of the ICOS+PD1 combination treatment groups was
comparable to the activity observed with the PD1+CTLA-4 combination
group, which was considered a positive control for anti-tumor
activity in this study. This suggests that a combination of ICOS
and PD1 antibodies may have benefit similar to CTLA-4 and PD1
combinations, which have demonstrated significant clinical activity
in some tumor types.
[0617] Of the 130 mice enrolled in this study, 12 remained alive at
day 60 with 11 having achieved complete tumor regression. Of the
118 mice that met endpoints for study removal, 111 were removed due
to reaching a tumor size of 2000 mm.sup.3. The remaining seven mice
were euthanized due to ulceration on the tumor. Occurrences of
ulceration were spread out among the groups. Groups 1 (Saline), 3
(hamster IgG & rat IgG2a), 4 (hamster IgG), 6 (1 .mu.g ICOS),
and 10 (CTLA-4 with 1 .mu.g ICOS) all had one animal removed due to
ulceration. Group 13 (CTLA-4+PD-1) showed two animals sacrificed
due to ulceration. The remaining groups had no animals removed due
to ulceration.
Sequence CWU 1
1
2415PRTMurine 1Asp Tyr Ala Met His 1 5 217PRTMurine 2Leu Ile Ser
Ile Tyr Ser Asp His Thr Asn Tyr Asn Gln Lys Phe Gln 1 5 10 15
Gly312PRTMurine 3Asn Asn Tyr Gly Asn Tyr Gly Trp Tyr Phe Asp Val 1
5 10 410PRTMurine 4Ser Ala Ser Ser Ser Val Ser Tyr Met His 1 5 10
57PRTMurine 5Asp Thr Ser Lys Leu Ala Ser 1 5 69PRTMurine 6Phe Gln
Gly Ser Gly Tyr Pro Tyr Thr 1 5 7121PRTArtificial
SequenceArtificially Synthesized Sequence 7Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Leu Ile Ser Ile Tyr Ser Asp His Thr Asn Tyr Asn Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala
Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Gly Arg Asn Asn Tyr Gly Asn Tyr Gly Trp Tyr
Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 8106PRTArtificial SequenceArtificially Synthesized Sequence
8Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1
5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr
Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr
Ile Ser Ser Leu Glu Pro Glu65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys
Phe Gln Gly Ser Gly Tyr Pro Tyr Thr 85 90 95 Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys 100 105 9467PRTArtificial SequenceArtificially
Synthesized Sequence 9Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val
Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Ala Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met
Gly Leu Ile Ser Ile Tyr Ser Asp His Thr Asn Tyr Asn65 70 75 80 Gln
Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser 85 90
95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
100 105 110 Tyr Tyr Cys Gly Arg Asn Asn Tyr Gly Asn Tyr Gly Trp Tyr
Phe Asp 115 120 125 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser Glu145 150 155 160 Ser Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn 210 215
220 Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu
Ser225 230 235 240 Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
Glu Phe Glu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser Gln 275 280 285 Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr305 310 315 320 Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330
335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu385 390 395 400 Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val 420 425 430 Asp Lys Ser
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455
460 Leu Gly Lys465 10168PRTHomo Sapien 10Met Lys Ser Gly Leu Trp
Tyr Phe Phe Leu Phe Cys Leu Arg Ile Lys 1 5 10 15 Val Leu Thr Gly
Glu Ile Asn Gly Ser Ala Asn Tyr Glu Met Phe Ile 20 25 30 Phe His
Asn Gly Gly Val Gln Ile Leu Cys Lys Tyr Pro Asp Ile Val 35 40 45
Gln Gln Phe Lys Met Gln Leu Leu Lys Gly Gly Gln Ile Leu Cys Asp 50
55 60 Leu Thr Lys Thr Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Ser
Leu65 70 75 80 Lys Phe Cys His Ser Gln Leu Ser Asn Asn Ser Val Ser
Phe Phe Leu 85 90 95 Tyr Asn Leu Asp His Ser His Ala Asn Tyr Tyr
Phe Cys Asn Leu Ser 100 105 110 Ile Phe Asp Pro Pro Pro Phe Lys Val
Thr Leu Thr Gly Gly Tyr Leu 115 120 125 His Ile Tyr Glu Ser Gln Leu
Cys Cys Gln Leu Lys Phe Trp Leu Pro 130 135 140 Ile Gly Cys Ala Ala
Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu145 150 155 160 Ile Cys
Trp Leu Thr Lys Lys Met 165 1119PRTHomo Sapien 11Met Gly Trp Ser
Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His
Ser12232PRTArtificial SequenceArtificially Synthesized Sequence
12Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1
5 10 15 Val His Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser
Leu 20 25 30 Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser
Ser Ser Val 35 40 45 Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu 50 55 60 Leu Ile Tyr Asp Thr Ser Lys Leu Ala
Ser Gly Ile Pro Ala Arg Phe65 70 75 80 Ser Gly Ser Gly Ser Gly Thr
Asp Tyr Thr Leu Thr Ile Ser Ser Leu 85 90 95 Glu Pro Glu Asp Phe
Ala Val Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr 100 105 110 Pro Tyr Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val 115 120 125 Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 130 135
140 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg145 150 155 160 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn 165 170 175 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr Tyr Ser 180 185 190 Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His Lys 195 200 205 Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr 210 215 220 Lys Ser Phe Asn
Arg Gly Glu Cys225 230 13124PRTMurine 13Gln Val Gln Leu Gln Gln Pro
Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser
Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile Asn
Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly
Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Met Phe 50 55
60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala
Tyr65 70 75 80 Met Gln Leu Thr Ser Pro Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95 Thr Arg Trp Asn Leu Ser Tyr Tyr Phe Asp Asn
Asn Tyr Tyr Leu Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Leu Thr
Val Ser Ser 115 120 14109PRTMurine 14Gln Ala Val Val Thr Gln Glu
Ser Ala Leu Thr Thr Ser Pro Gly Glu 1 5 10 15 Thr Val Thr Leu Thr
Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala
Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly 35 40 45 Leu
Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55
60 Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly
Ala65 70 75 80 Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp
Tyr Asn Asn 85 90 95 His Leu Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 15121PRTArtificial SequenceArtificially Synthesized
Sequence 15Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Ser Ile Tyr Ser Asp
His Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Arg Ala Thr Met
Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg
Asn Asn Tyr Gly Asn Tyr Gly Trp Tyr Phe Asp Val Trp Gly 100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 16451PRTArtificial
SequenceArtificially Synthesized Sequence 16Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45
Gly Leu Ile Ser Ile Tyr Ser Asp His Thr Asn Tyr Asn Gln Lys Phe 50
55 60 Gln Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala
Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Gly Arg Asn Asn Tyr Gly Asn Tyr Gly Trp Tyr
Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160 Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180
185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305
310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400 Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425
430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445 Pro Gly Lys 450 17106PRTArtificial SequenceArtificially
Synthesized Sequence 17Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala
Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu
Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu65 70 75 80 Asp
Phe Ala Val Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Tyr Thr 85 90
95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
18213PRTArtificial SequenceArtificially Synthesized Sequence 18Glu
Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg
Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Glu Pro Glu65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Phe
Gln Gly Ser Gly Tyr Pro Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135
140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
Glu145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210
19121PRTMurine 19Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val
Arg Pro Gly Glu 1 5 10 15 Ser Val Lys Ile Ser Cys Met Gly Ser Gly
Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Lys Gln Ser
His Ala Lys Ser Leu Glu Trp Ile 35 40 45 Gly Leu Ile Ser Ile Tyr
Ser Asp His Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Lys Ala
Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80 Met Glu
Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95
Gly Arg Asn Asn Tyr Gly Asn Tyr Gly Trp Tyr Phe Asp Val Trp Gly 100
105 110 Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 20107PRTMurine
20Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1
5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr
Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Ile Thr Ser Pro Lys Leu
Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Asn Ser Tyr Ser Leu Thr
Ile Ser Ser Met Glu Ala Glu65 70 75 80 Asp Val Ala Thr Tyr Tyr Cys
Phe Gln Gly Ser Gly Tyr Pro Tyr Thr 85 90 95 Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys Arg 100 105 211401DNAArtificial
SequenceArtificially Synthesized Sequence 21atgggctggt cctgcatcat
cctgtttctg gtggccaccg ccaccggcgt gcacagccag 60gtgcagctgg tgcagagcgg
agccgaggtg aaaaagcccg gctcaagcgt gaaggtgagc 120tgcaaggcca
gcggctacac cttcaccgac tacgctatgc actgggtgag gcaggccccc
180ggccagggcc tggagtggat gggcctgatc agcatctaca gcgaccacac
caactacaac 240cagaagttcc agggcagggt gaccatcacc gccgataaga
gcaccagcac agcctacatg 300gagctgagca gcctgaggag cgaagacacc
gccgtgtact attgcggcag gaacaactac 360ggcaactacg gctggtactt
cgacgtgtgg ggccagggaa ccactgtcac cgtgagcagc 420gccagcacca
agggccccag cgtgttcccc ctggccccct gcagcagaag caccagcgag
480agcacagccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt
gaccgtgagc 540tggaacagcg gagccctgac cagcggcgtg cacaccttcc
ccgccgtgct gcagagcagc 600ggcctgtaca gcctgagcag cgtggtgacc
gtgcccagca gcagcctggg caccaagacc 660tacacctgca acgtggacca
caagcccagc aacaccaagg tggacaagcg ggtggagagc 720aagtacggcc
ctccctgccc cccctgccct gcccccgagt tcgagggcgg accctccgtg
780ttcctgttcc cccccaagcc caaggacacc ctgatgatca gccggacccc
cgaggtgacc 840tgcgtggtgg tggacgtgag ccaggaagat cccgaggtcc
agttcaattg gtacgtggac 900ggcgtggagg tgcacaacgc caagaccaag
ccccgggagg aacagttcaa cagcacctac 960cgggtggtgt ccgtgctgac
cgtgctgcac caggactggc tgaacggcaa agaatacaag 1020tgcaaggtgt
ccaacaaggg cctgcccagc tccatcgaga aaaccatcag caaggccaag
1080ggccagcctc gggagcccca ggtgtacacc ctgcccccat cccaggaaga
gatgaccaag 1140aaccaggtgt ccctgacctg tctggtgaag ggcttctacc
ccagcgacat cgccgtggag 1200tgggagagca acggccagcc cgagaacaac
tacaagacca ccccccctgt gctggacagc 1260gacggcagct tcttcctgta
cagcaggctg accgtggaca agagccggtg gcaggaaggc 1320aacgtcttta
gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagagc
1380ctgagcctgt ccctgggcaa g 140122696DNAArtificial
SequenceArtificially Synthesized Sequence 22atgggctggt cctgcatcat
cctgtttctg gtggccaccg ccaccggcgt gcacagcgag 60attgtgctga cccagagccc
cgccaccctg agcctgagcc ccggcgaaag ggcaaccctc 120agctgcagcg
ccagcagcag cgtgagctac atgcactggt accagcagaa gcccggccag
180gcccctaggc tgctgatcta cgacacctcc aagctggcca gcggcatccc
agccaggttc 240tcaggcagcg gcagcggcac cgactatact ctgaccatca
gcagcctgga gcccgaggac 300ttcgccgtgt actactgctt ccagggaagc
ggctacccct acaccttcgg ccagggcacc 360aagctggaga tcaagcgtac
ggtggccgcc cccagcgtgt tcatcttccc ccccagcgat 420gagcagctga
agagcggcac cgccagcgtg gtgtgtctgc tgaacaactt ctacccccgg
480gaggccaagg tgcagtggaa ggtggacaat gccctgcaga gcggcaacag
ccaggagagc 540gtgaccgagc aggacagcaa ggactccacc tacagcctga
gcagcaccct gaccctgagc 600aaggccgact acgagaagca caaggtgtac
gcctgtgagg tgacccacca gggcctgtcc 660agccccgtga ccaagagctt
caaccggggc gagtgc 69623448PRTArtificial SequenceArtificially
Synthesized Sequence 23Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Leu Ile Ser Ile
Tyr Ser Asp His Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Gly Arg Asn Asn Tyr Gly Asn Tyr Gly Trp Tyr Phe Asp Val Trp Gly
100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
Glu Ser Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val145 150 155 160 Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser
Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His 195 200 205 Lys
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly 210 215
220 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro
Ser225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser Gln Glu Asp Pro 260 265 270 Glu Val Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320 Lys
Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 325 330
335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp385 390 395 400 Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Arg Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Glu Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445
24213PRTArtificial SequenceArtificially Synthesized Sequence 24Glu
Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg
Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile
Ser Ser Leu Glu Pro Glu65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Phe
Gln Gly Ser Gly Tyr Pro Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu145
150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210
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