Note: Descriptions are shown in the official language in which they were submitted.
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Combination Therapy
FIELD OF THE INVENTION
The present invention relates generally to immunotherapy in the treatment of
human
disease. More specifically, the present invention relates to the use of
sequenced dosing of
immunomodulators such as anti-ICOS antibodies, anti-PD1 antibodies, and anti-
PDL1
antibodies in the treatment of cancer.
BACKGROUND OF THE INVENTION
Cancer immunity is a multistep process that is tightly regulated by a series
of negative
immune checkpoint and positive co-stimulatory receptors that when effectively
triggered can
achieve antitumor response (Mellman, I., et al. (2011) Cancer Immunotherapy
Comes of Age.
Nature 480(7378), 480-489). However, tumors have established various
mechanisms to
circumvent immune clearance by altering the responsiveness of the immune
infiltrate. In
some instances, tumors will be highly dependent on a single mechanism, and in
these cases,
there is the potential to achieve significant clinical activity with single
agent
immunomodulatory therapy (Hoos, A. (2016). Development of immuno-oncology
drugs -
from CTLA4 to PD1 to the next generations. Nat Rev Drug Discov. 15(4), 235-
47).
However, as tumors often utilize multiple, overlapping and redundant
mechanisms to block
antitumor immune response, combination therapy will likely be required for
durable efficacy
across a wide range of tumor types. Therefore, new immune targeted therapies
are needed to
improve the treatment of all cancers.
Thus, there is a need for combination treatments and strategies for dosing of
immunomodulators for the treatment of disease, in particular cancer.
Brief Description of the Drawings
FIG. 1 is a table showing the study design of anti-ICOS antibody / anti-PD1
antibody
concurrent and phased dosing study described herein.
FIG. 2 is a schematic showing the study procedure of anti-ICOS antibody / anti-
PD1 antibody
concurrent and phased dosing study described herein. Shown at the bottom of
FIG. 2 is a table
listing antibodies used in the study.
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FIG. 3 is a plot showing average tumor volume of mice groups treated with anti-
ICOS antibody
and anti-PD1 antibody concurrently or in sequential phases (e.g., lead-in
dose/follow-up dose)
and group(s) treated with control(s), as indicated in the figure legend.
FIG. 4 is a plot showing average tumor volume of mice groups treated with
concurrent dosing
of anti-ICOS antibody and anti-PD1 antibody, and group(s) treated with
control(s), as indicated
in the figure legend.
FIG. 5 is a plot showing average tumor volume of mice groups treated with
phased dosing of
anti-ICOS antibody and anti-PD1 antibody, and group(s) treated with
control(s), as indicated
in the figure legend.
FIG. 6 is a plot showing average tumor volume of mice groups treated with
phased dosing of
anti-PD1 antibody and anti-ICOS antibody, with anti-PD1 antibody as lead-in
dose and anti-
ICOS antibody as follow-up dose, and group(s) treated with control(s), as
indicated in the figure
legend.
FIG. 7 is a plot showing average tumor volume of mice groups treated with
phased dosing of
anti-ICOS antibody and anti-PD1 antibody, with anti-ICOS antibody as lead-in
dose and anti-
PD1 antibody as follow-up dose, and group(s) treated with control(s), as
indicated in the figure
legend.
FIGS. 8A-8C are sets of plots showing individual tumor volumes of mice treated
with
concurrent dosing of anti-ICOS antibody and anti-PD1 antibody, and group(s)
treated with
control(s), as indicated in the corresponding figure legend(s). FIG. 8A shows
individual tumor
volumes of mice in Group 1 (left) and Group 2 (right). FIG. 8B shows
individual tumor
volumes of mice in Group 3 (top left), Group 4 (top right), and Group 5
(bottom). FIG. 8C
shows individual tumor volumes of mice in Group 6 (left) and Group 7 (right).
FIGS. 9A-9C are sets of plots showing individual tumor volumes of mice treated
with
phased dosing of anti-ICOS antibody and anti-PD1 antibody, and group(s)
treated with
control(s), as indicated in the corresponding figure legend(s). FIG. 9A shows
individual
tumor volumes of mice in Group 1 (left) and Group 2 (right). FIG. 9B shows
individual
tumor volumes of mice in Group 8 (top left), Group 9 (top right), and Group 10
(bottom).
FIG. 9C shows individual tumor volumes of mice in Group 11 (top left), Group
12 (top
right), and Group 13 (bottom).
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FIG. 10 is a plot showing survival of mice in all groups (Groups 1-13). Mice
in the groups
were treated with concurrent or phased dosing of anti-ICOS antibody and anti-
PD1 antibody
or treated with control(s), as indicated in the figure legend.
FIG. 11 is a plot showing survival of mice groups treated with concurrent
dosing of anti-
ICOS antibody and anti-PD1 antibody, and group(s) treated with control(s), as
indicated in
the figure legend.
FIG. 12 is a plot showing survival of mice groups treated with phased dosing
of anti-ICOS
antibody and anti-PD1 antibody, and group(s) treated with control(s), as
indicated in the
figure legend.
FIG. 13 is a plot showing survival of mice groups treated with phased dosing
of anti-PD1
antibody and anti-ICOS antibody, with anti-PD1 antibody as lead-in dose and
anti-ICOS
antibody as follow-up dose, and group(s) treated with control(s), as indicated
in the figure
legend.
FIG. 14 is a plot showing survival of mice groups treated with phased dosing
of anti-ICOS
antibody and anti-PD1 antibody, with anti-ICOS antibody as lead-in dose and
anti-PD1
antibody as follow-up dose, and group(s) treated with control(s), as indicated
in the figure
legend.
FIG 15: Development of an anti-human ICOS agonist monoclonal antibody
(A) H2L5 binding to dimeric human ICOS (B) human ICOS-L binding to dimeric
human ICOS
(C) Binding of H2L5 (20 pg/mL) to CD4 (**P=0.0011, t=4.183, df=13) and CD8
(**P=
0.0078, t=3.686, df=7) T cells from healthy donors. Each symbol represents a
separate human
donor, horizontal lines indicate median, and bars are interquartile range (D)
Representative
Western Blot demonstrating induction of AKT signaling in purified activated T
cells after
treatment with H2L5 (E) Quantification of CD69+ CD4 (*P=0.0142, t=3.416 df=6)
or CD8
(**P=0.0012, t=5.734 df=6) T cells and (F) quantification of Ki67+ CD4
(*P=0.0190, t=3.809
df=4) or CD8 (*P=0.0255, t=3.474 df=4) T cells from healthy donor PBMC treated
with (12.5
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ug/mL) of bound H2L5 and anti-CD3 for 48 hours. (G, H) Quantification of
soluble IFN-y
from (G) the culture supernatant of PBMC from healthy subjects treated with
(12.5 ug/mL) of
bound H2L5 and anti-CD3 for 24 hours **P=0.0041, t=4.510 df=6 or 48 hrs
*P=0.0375,
t=2.661 df=6 (H) the supernatant of NSCLC cancer patient PBMC treated with (10
ug/mL)
.. bound H2L5 and anti-CD3 for 72 hours. (I, K) Quantification of RNA
expression of (I) T-Bet
(TBX21) (*P=0.0156, t=2.974 df=9) and (J) Granzyme B (GZMB) (**P=0.0020,
t=4.292
df=9) (K) L-Selectin (SELL) (*P=0.0161, t=2.955 df=9) from healthy donor CD3+
T cells
following indicated treatments analyzed by a two-tailed, unpaired t-test. H2L5
induces
concentration dependent increases in cytokine production and T-cell activation
from
.. disaggregated tumor cell suspensions. Plates were coated with H2L5 +/- anti-
CD3 or isotype
control. (L) IFN-y, (M) CD8+ 0X40+, (N) CD8+ CD25+ following 6 days of
culture. Bars =
Group Medians p < 0.05 by One Way Anova, ** P < 0.05 *** P < 0.0005, **** P <
0.000 by
One Way Anova. Dashed line = CD3 + isotype IgG4 lOug/mL. See Fig. S7 for tumor
types.
FIG 16: Antibody isotype and FcyR-engagement is critical for H2L5 function
.. (A) PBMC from healthy subjects treated with soluble H2L5 of varying
isotypes at 5 ug/m1 for
6 days. Proliferation as measured by CFSE dilution relative to isotype control
(fold change)
(B, C) PBMCs from healthy subjects, with or without depletion of NK cells;
treated with (B)
soluble H2L5 of varying isotypes at (5 ug/mL) for 6 days. (C) Soluble H2L5 of
varying
isotypes (10 ug/mL) for 24 hours and percentage of dead cells determined by
flow cytometry
.. using NIR Live/Dead dye. An anti-CD52 antibody known to induce ADCC-
mediated T-cell
killing was included as a positive control. (D) ICOS Expression on freshly
dissociated patient
TIL. The median fluorescent intensity of ICOS from CD4, CD8, Treg, and Teff
cell populations.
(Tumor types Solid Triangle = NSCLC (6) Solid Circle = CRC (4) Solid Diamond =
Bladder
(2) Solid Square = Head/Neck (1) Open Triangle = RCC (4) Open Circle =
Endometrial (2)
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Open Diamond = Prostate (1) Open Square = Thyroid (1); p < 0.05 by One Way
Anova). Insert
shows histogram of ICOS expression on CD4 (Red), CD8 (orange) and Treg (Blue)
from a
patient with endometrial cancer.
(E) Spearman correlation between total ICOS receptor numbers (calculated by
multiplying the
percent ICOS positive for each cell type by the ICOS receptor number per
positive cell) and
FcyRIIIA reporter assay fold induction in target cells isolated from PBMCs and
patient tumors
in presence of H2L5 IgG1 isotype relative to isotype control for all samples
where both data
points were available (r2=0.681, p< 0.001). (F) Fold induction observed in an
FcyRIIIA reporter
assay using target cells isolated from NSCLC patient tumor 5001003 incubated
with anti-ICOS
antibodies. CD4 Teff, CD8 T cells and Treg were isolated from a dissociated
patient tumor and
utilized as target cells in the FcyRIIIA assay.
FIG. 17: H2L5 exhibits FcR dependent agonism to induce T-cell activation
(A) Isolated CD4 T cells from healthy subjects treated with indicated
concentrations of H2L5
for 60 hours (bound isotype vs. bound H2L5 ***P=0.0006, t=9.777 df=4, soluble
isotype vs.
soluble H2L5 ***P=0.0003, t=11.50 df=4 and (#) bound H2L5 vs. soluble H2L5
**P=0.0017,
t=7.530 df=4) (B) PBMC from a healthy subject treated with soluble H2L5 (ICOS
IgG4PE) or
H2L5 Fc-disabled at (10 [tg/mL) for 3.5 days (isotype control vs. H2L5
**P=0.0056, t=5.426
df=4), (H2L5 vs. H2L5 Fc-disabled **P=0.0012, t=8.297 df=4) (C) MLR with anti-
CD3
antibody followed by treatment with soluble H2L5 or H2L5 Fc-disabled antibody
at (10
[tg/mL) (isotype control vs. H2L5 *P=0.0166, t=3.966 df=4), (H2L5 vs. H2L5 Fc-
disabled
*P=0.0158, t=4.022 df=4) (D) Isolated T cells cultured with and without
monocytes from the
same donor followed by treatment with soluble H2L5 or H2L5 Fc-disabled at (10
[tg/mL) +1-
anti-CD32 or Fc-blocking antibody for 4 days. (#) ***P=0.0009, t=8.734 df=4,
($)
**P=0.0031, t=6.405 df=4, (&) *P=0.0389, t=3.026 df=4, (k) isotype control vs.
H2L5
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"P=0.0027, t=6.612 df=4, H2L5 vs. H2L5 Fc-disabled *P=0.0239, t=3.544 df=4,
H2L5
(control) vs. H2L5 (anti-CD32) *43=0.0066, t=5.184 df=4, H2L5 (anti-CD32) vs.
H2L5 (Fc
block) **P=0.0013, t=8.047 df=4 and H2L5 (control) vs. H2L5 (Fc block)
*P=0.0446, t=2.889
df=4. (E, F) Human T cells pre-stimulated with anti-CD3 for 48 hours and added
to a co-culture
with human DC. AlexaFlour488-labeled H2L5 IgG4PE added at 3p.g/mL to co-
cultures on ice
then moved to 37 C for indicated timepoints. Arrows indicate T cells activated
in response to
H2L5 treatment, polarization and mobilization towards neighboring dendritic
cell. Data
representative of three separate experiments performed using different donor
cells.
FIG. 18: H2L5 induces an EM phenotype and anti-tumor activity in humanized
mouse
model.
(A) Quantification of human CD45 CD3+ cells in the blood of mice H2L5
treatments as
compared to isotype control IgG4PE (****P=<0.0001, F=33.57, df=24) (B)
Quantification of
human CD45 CD3 CD69+ cells from the blood of mice H2L5 (1.2mg/kg ) vs. isotype
control
IgG4PE (*P=0.0119, F=4.179, df=24) (C) Percentage of CD4+ Tcm (0.04mg/kg
*43=0.0038,
0.4mg/kg ***P=0.0002, 1.2mg/kg ***P=0.0005, F=8.172, df=20. This is equivalent
to 0.8, 8
and 24 pg per mouse respectively. (D) CD8+ Tnaive terminally differentiated
effector memory
TTENIRA (0.004mg/kg **P=0.0036, 0.04mg/kg and 0.4mg/kg ****P=<0.0001, 1.2mg/kg
*43=0.0072, F=13.78, df=20) in the spleen of mice (E) The percentage of ICOS+
or PD-1+ T
cells in mice implanted subcutaneously with A549 tumor and identified by using
PE conjugated
.. mouse anti human IgG4 by flow cytometry. (F) The ratio of CD8/Treg cells in
whole tumor
tissues (G) HCT116 tumor volumes on day 13 (0.04mg/kg) *P=0.0273, (0.4mg/kg)
*P=0.0432,
F=2.788, df=36 (H) A549 tumor volumes on day 21 (0.4mg/kg) *P=0.0056, F=3.906,
df=36
(i) Kaplan-Meier survival curve of human PBMC engrafted NSG mice with A549
tumors (A-
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I) horizontal lines represent median values, error bars represent
interquartile range. All
statistical tests were one-way ANOVA.
FIG. 19: The isotype of the murine ICOS mAb influences efficacy in syngeneic
tumors.
(A) Kaplan-Meier plots of mice with murine (A) EMT6 murine (B) CT26 syngeneic
tumors
treated with indicated doses (5, 100 or 200 pg corresponding to 0.5, 5 and
10mg/Kg
respectively of murine IgG1 or IgG2a versions of 7E.17G9 antibody twice weekly
for 3 weeks
or isotype control (200 lig or 10mg/kg).. Results are representative of two
repeat experiments.
Each symbol represents an individual mouse. Horizontal lines represent median
values, error
bars represent interquartile range. All statistical tests were one-way ANOVA,
followed by
specific treatment comparators. (C) The ratio of CD8/Treg in EMT6 or CT26
tumors
determined at tumor size 100 mm3; (D) The percentage of ICOS+ CD4, CD8 and
Treg cells in
tumors (closed circles) or spleens (open circles) of mice implanted with EMT6
tumors; MFI of
ICOS on CD8, CD4 and Treg in tumors (closed circles) or spleen (open circles)
in mice
implanted with (E) CT26 or (F) EMT6 tumors at tumor sizes of 100 mm3. (G)
Histogram of
representative flow plot comparing MFI of ICOS expression on CD4, CD8 and Treg
isolated
from EMT6 and CT26 tumors; (H) Absolute number of TCR clones expanded in post-
treatment
with anti-ICOS 7E.17G9 blood that were also found in EMT6 tumor (10 Lug
*P=0.0173 and
100 jig *P=0.0483; F=3.269 df=28).
FIG. 20: Evaluation of ICOS expression on different cell types in human
cancers. (A)
Expression of ICOS, ICOS-L and PD-Li in different tumor types ranked by
expression of
ICOS from TCGA database. (B) The expression of ICOS+ cells by single plex IHC
and
correlation with expression of PD-L1, PD-1, CD4, CD8, FOXP3 and CD3 in NSCLC.
(C) %
CD45+ cells that are CD3+, B cells, monocytes, NK cells, macrophages,
dendritic cells in
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disaggregated tumors from different solid tumor types. Solid Triangle = NSCLC
(6) Solid
Circle = CRC (4) Solid Diamond = Bladder (2) Solid Square= Head/Neck (1) Open
Triangle =
RCC (4) Open Circle = Endometrial (2) Open Diamond =Prostate (1) Open Square =
Thyroid
(1). (D) The percentage CD3+CD8+, CD3+CD4+Foxp3+ (Treg) and the ratio of CD3+,
CD8+:
.. CD3+CD4+Foxp3 in different tumor types. Horizontal line shows median. (E)
Quantification
of the co-expression of CD3+PD-1+ICOS+ cells in tumor biopsies obtained from
different
tumour types by multiplex IHC. (F) Multiplex IHC of a Head and neck FFPE tumor
sample
co-stained for CD3, PD-1 and ICOS (G) Heatmap summarizing the differentially
expressed
genes in purified human T cells treated with H2L5 plus anti-CD3 mAb compared
to anti-CD3
alone as determined by NanoString nCounter analysis System using Human
PanCancer-
Immune profiling panel (N=6 donors). (H) Gene expression changes (fold
increase) common
between anti-CD3 (0.6 Kg/mL) plus H2L5 (10 Kg/mL) activated human T cells (n=6
donors)
and murine EMT6 transplantable tumors after surrogate anti-ICOS (7E.17G9 rat
IgG2b)
treatment.
.. FIG. 21: ICOS agonist mAb induce PD-1/PD-L1 expression and enhances
activity of anti-
PD-1
(A) Quantification of RNA expression of PD-Li (CD274) (10 lag *P=0.0137 and
100[1g
*P=0.0374; F=5.175 df=10) and (B) PD-1 (Pdcdl) (10 lag *P=0.0194 and 100 lag
P=0.1626;
F=3.911 df=10) in EMT6 following indicated treatments. Each symbol represents
an
individual mouse sample, horizontal lines represent median values, error bars
represent
interquartile range. All statistical tests were one-way ANOVA with square root
transformed
data to stabilize variances (C) Percentage of CD4+113-1 and CD8+113-1 T
cells following
treatment with isotype control or H2L5 at 10 lag/mL for 72 hours in PBMC from
cancer
patients CD4+ *P=0.0128, t=3.026 df=10; CD8+ *43=0.005, t=3.548, df=10. two-
tailed,
unpaired t-tests (D). Percentage of CD4+ ICOS+ in NSCLC or melanoma patients
pre- and
post-PD-1 therapy (either pembrolizumab or nivolumab) compared with healthy
subjects. (E)
Mice with EMT6 tumors treated with 7E.17G9 IgG1 (10 lag equivalent to
0.5mg/kg), anti-
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PD-1 (200 lag equivalent to 10mg/kg) or the combination of 7E.17G9 and anti-PD-
1 dosed
concomitantly, twice weekly for 3 weeks. (N=10 per treatment group) (F) A549
tumor
volume in NSG mice reconstituted with human PBMC and treated with H2L5 at
0.8Kg mouse
equivalent to 0.04mg/kg, isotype 0.8Kg equivalent to 0.04mg/kg or anti-PD-1
.. (pembrolizumab / Keytruda) 100 pg equivalent to 5mg/kg or the combination
of both
antibodies. (G) Quantification of IFN-y from disseminated NSCLC patient tumors
treated
with anti-CD3 and H2L5 (10 ug/mL) for 24 hours. (#) **P=0.0100 ($)
****P=<0.0001 (&)
***P=0.002, F= 15.8, df=20. Horizontal lines represent median values, error
bars represent
interquartile range (H) MLR assay evaluating ICOS + pembrolizumab vs. ICOS
*43=0.0036, IgG4PE ICOS + pembrolizumab vs. pembrolizumab *43=0.0090, ICOS +
pembrolizumab vs. 2x IgG4PE ***P=0.0009, F=7.324, df=10. Bars represent mean
of
triplicate measurement and error bars represent standard deviation (C-E) All
statistical tests
were one-way ANOVA)
FIG. 22: H2L5 IgG4PE epitope binding (A) An ICOS-L competition assay by MSD
demonstrates that H2L5 IgG4PE partially competes with ICOS-L for binding to
human ICOS
receptor. (B) Activated T cells were incubated with different concentrations
of recombinant
ICOS-L (R&D systems) and then incubated with H2L5 and MFI of ICOS CD4+ and
CD8+
cells determined.by flow cytometry.
FIG. 23: H2L5IgG4PE causes dose dependent increases in (A) cytokine production
IFNy, IL-17, IL-10, IL-4, IL-13, IL-5, IL-2, IL-6, TNFa measured by MSD (B)
activation marker 0X40, CD25 and CD69 on CD4 and CD8 T cells. PBMC were
cultured
for 48h with anti-CD3 (0.6ug/m1) and different concentrations of H2L5IgG4PE or
isotype
control and supernatants harvested for cytokine analysis and cells for flow
cytometry.
FIG. 24: H2L5 induces concentration dependent increases in cytokine production
from
disaggregated tumor cell suspensions from different cancer patients.
Disaggregated
tumor cells suspensions were cultured with plate bound H2L5IgG4PE or isotype
control in
.. the presence or absence of anti-CD3 following 6 day in vitro stimulation
with plate bound
anti-CD3 (0.6 g/mL) and IL2 (10Ong/mL) followed by analysis of (A) IL17,(B)
IL10, (C)
IL5, (D) IL13 cytokines in the supernatants by MSD.
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FIG. 25: H2L5 induces concentration dependent increases on percentage of (A)
CD8+LAG3 +, p <0.005 by One Way Anova (B) CD8+ PD-1+, (C) ICOS L + cells and
(D) (CD4+, CD25+ Foxp3+) p <0.05 by One Way Anova from disaggregated tumor
cell
suspensions from different cancer patients. Disaggregated tumor cells
suspensions were
cultured with plate bound H2L5 (ICOS) IgG4PE or isotype control in the
presence or absence
of anti-CD3 following 6 day in vitro stimulation with plate bound anti-CD3
(0.6 ug/mL) and
IL-2(100 ng/mL) followed by flow cytometry . Dashed line = CD3 + IgG4 isotype
lOug/mL
Horizontal bars represent median.
FIG. 26: H2L5 IgG1 induces signaling via the major activating FcyR (FcyRIIIa)
responsible for ADCC in humans. (A) Treatment of Jurkat-FcyRIIIA-NFAT-
luciferase
effector cells and primary human CD4+ T cells at a ratio of 6:1 with soluble
H2L5 of varying
isotypes for 6 hrs. An anti-CD52 antibody known to induce ADCC-mediated T cell
killing
was included as a positive control (B) Treatment of Jurkat-FcyRIIIA-NFAT-
luciferase
effector cells and purified primary human ex vivo tumor derived CD4, CD8 and
Tregs at a
ratio of 6:1 with soluble H2L5 IgG1 for 6 hrs Fold change in luciferase
induction produced
by Jurkat-FcyRIIIA-NFAT-luciferase effector cells relative to isotype control.
FIG. 27: H2L5 causes dose dependent binding to ICOS expressing T cells in
blood and
tumor. The percentage of ICOS+ or PD-1+ T cells in whole blood (A) and tumor
tissues (B)
within each group, 48 hours post 4th dose identified using PE conjugated mouse
anti human
IgG4 by flow cytometry. Bars represent the median values for each group.
FIG. 28: Characterization of an anti-murine ICOS agonist antibody. Anti-mouse
ICOS
agonist antibody (7E.17G9) induces IFNy production in disseminated mouse
splenocytes
cultured ex vivo for 60 hours.
FIG. 29: Tumor growth for (A) EMT6 or (B) CT26 murine syngeneic tumors treated
with 10 (0.5mg/kg) ,100 (5mg/kg) or 200ug (10mg/kg) doses of murine IgG1 or
Ig2a
variants of 7E.17G9 antibody or isotype control (200 jig (10mg/kg) twice
weekly for 3 weeks.
*(numbers) indicate the number of mice with minimally detectable or non-
detectable tumors
at study endpoint.
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FIG. 30: % ICOS+ cells within CD4, CD8 and Treg populations in tumors (closed
circles)
and spleens (open circles) of mice bearing ¨100 mm3 CT26 tumors.
FIG. 31: (A) absolute number of TCR clones contracted in post-treatment with
Anti-ICOS
7E17G9 antibody blood relative to pre-treatment blood (10[Ig *P=0.0327 and
100[Ig
*P=0.0497; F=3.033 df=28) (B) absolute number of TCR clones expanded in post-
treatment
blood relative to pre-treatment blood (10[Ig P=0.0975 and 100 jig P=0.1915;
F=1.958 df=28)
(C) Mean T cell fraction estimate vs Mean productive clonality
FIG. 32: Expression of ICOS positive cells in NSCLC, Breast cancer and CRC by
IHC
singleplex Immunohistochemical detection of ICOS in non-small cell lung cancer
(NSCLC),
breast cancer (BrCA) TNBrCa, and colorectal cancer (CRC), using a rabbit anti-
human
CD278 Monoclonal antibody clone SP98 (Spring Biosciences). Assay was carried
out on the
Leica Bond RX with associated platform reagents. DAB (3, 3'-diaminobenzidine)
was used
for target detection. Sections were counter stained with Hematoxylin (All
scale bars = 20um).
FIG. 33: Changes on cytokine levels from healthy human donor PBMC in response
to
treatment with anti-CD3 plus isotype control or H2L5 IgG4PE antibody at 12.5
ii.tg/mL
FIG. 34: Cytokine induction of PBMC from NSCLC patients following treatment
with
isotype control or H2L5 IgG4PE antibody at 10 ii.tg/mL for 72 hrs.
FIG. 35: Binding affinity of different isotype variants of humanized H2L5
antibody to
human FcgR.
FIG. 36: Binding affinity of different isotype variants 7E-17G9 to murine FcR
FIG. 37: mRNA Expression of ICOS positive cells in different tumor pathologies
from
TCGA
FIG. 38: Gene expression changes with anti CD3 + H2L5 treatment compared to
CD3
alone in human T cells as measured by Nanostring
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SUMMARY OF THE INVENTION
In one aspect, the present invention provides methods of treating cancer in a
patient in
need thereof comprising administering to the patient an effective amount of an
agent directed
to human ICOS and an effective amount of an agent directed to human PD1 or
human PD-Li
sequentially, wherein administration of the agent directed to human ICOS is
followed by
administration of the agent directed to human PD1 or human PD-Li. In one
embodiment, the
agent directed to human ICOS is an ICOS agonist. In one embodiment, the agent
directed to
human PD1 or human PD-Li is a PD1 antagonist.
In one aspect, the present invention provides an anti-ICOS antibody or antigen
binding fragment thereof and an anti-PD1 antibody or antigen binding fragment
thereof for
sequential use in treating cancer in a human in need thereof, wherein
administration of the
anti-ICOS antibody or antigen binding fragment thereof is followed by
administration of the
anti-PD1 antibody or antigen binding fragment thereof In one embodiment, the
anti-PD1
antibody or antigen binding fragment thereof is a PD1 antagonist. In one
embodiment, the
anti-ICOS antibody or antigen binding fragment thereof is an ICOS agonist.
In one aspect, the present invention provides an anti-ICOS antibody or antigen
binding fragment thereof and an anti-PD-Li antibody or antigen binding
fragment thereof for
sequential use in treating cancer in a human in need thereof, wherein
administration of the
anti-ICOS antibody or antigen binding fragment thereof is followed
administration of the
anti-PD-Li antibody or antigen binding fragment thereof In one embodiment, the
anti-PDL1
antibody or antigen binding fragment thereof is a PD1 antagonist. In one
embodiment, the
anti-ICOS antibody or antigen binding fragment thereof is an ICOS agonist.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
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 (isoform 2) (Accession No.: UniProtKB - Q9Y6W8-2) is shown below
as
SEQ ID NO:9.
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MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLK
GGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFD
PPPFKVTLTGGYLHIYESQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKM (SEQ
ID NO:9)
The amino acid sequence of human ICOS (isoform 1) (Accession No.: UniProtKB -
Q9Y6W8-1) is shown below as SEQ ID NO:10.
MKSGLWYFFL FCLRIKVLTG EINGSANYEM FIFHNGGVQI LCKYPDIVQQ
FKMQLLKGGQ ILCDLTKTKG SGNTVSIKSL KFCHSQLSNN SVSFFLYNLD
HSHANYYFCN LSIFDPPPFK VTLTGGYLHI YESQLCCQLK FWLPIGCAAF
VVVCILGCIL ICWLTKKKYS SSVHDPNGEY MFMRAVNTAK KSRLTDVTL
(SEQ ID NO: 10)
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 naïve TH1 and TH2 effector T cell populations (Paulos
CM 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 AH and Freeman GJ. "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-y, TNF-a, IL-10, IL-4, IL-13 and others. ICOS also
stimulates
effector T cell proliferation, albeit to a lesser extent than CD28 (Sharpe AH
and Freeman GJ.
"The B7-CD28 Superfamily", Nat. Rev Immunol, 2(2); 116-26 (2002)). Antibodies
to ICOS
.. and methods of using in the treatment of disease are described, for
instance, in
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W02012/131004, US20110243929, and US20160215059. US20160215059 is incorporated
by reference herein. CDRs for murine antibodies to human ICOS having agonist
activity are
shown in PCT/EP2012/055735 (WO 2012/131004). Antibodies to ICOS are also
disclosed in
WO 2008/137915, WO 2010/056804, EP 1374902, EP1374901, and EP1125585. Agonist
antibodies to ICOS or ICOS binding proteins are disclosed in W02012/13004,
W02014/033327, W02016/120789, US20160215059, and US20160304610. Exemplary
antibodies in US2016/0304610 include 37A10S713. Sequences of 37A105713 are
reproduced below as SEQ ID NOS: 14-21.
37A105713 heavy chain variable region:
EVQLVESGG LVQPGGSLRL SCAASGFTFS DYWMDWVRQA PGKGLVWVSN
IDEDGSITEY SPFVKGRFTI SRDNAKNTLY LQMNSLRAED TAVYYCTRWG
RFGFDSWGQG TLVTVSS (SEQ. ID NO:14)
37A105713 light chain variable region:
DIVMTQSPDS LAVSLGERAT INCKSSQSLL SGSFNYLTWY QQKPGQPPKL
LIFYASTRHT GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCHHHYNAPP
TFGPGTKVDI K (SEQ. ID NO:15)
37A105713 VH CDR1: GFTFSDYWMD (SEQ.ID NO:16)
37A105713 VH CDR2: NIDEDGSITEYSPFVKG (SEQ. ID NO: 17)
37A105713 VH CDR3: WGRFGFDS (SEQ. ID. NO: 18)
37A105713 VL CDR1: KSSQSLLSGSFNYLT (SEQ. ID NO: 19)
37A105713 VL CDR2: YASTRHT (SEQ. ID NO: 20)
37A105713 VL CDR3: HHHYNAPPT (SEQ. ID NO: 21)
By "agent directed to ICOS" is meant any chemical compound or biological
molecule
capable of binding to ICOS. In some embodiments, the agent directed to ICOS is
an ICOS
binding protein. In some other embodiments, the agent directed to ICOS is an
ICOS agonist.
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
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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.
In one embodiment, the ICOS antibodies of the present invention comprise any
one or
a combination of the following CDRs:
CDRH1: DYAMH (SEQ ID NO:1)
CDRH2: LISIYSDHTNYNQKFQG (SEQ ID NO:2)
CDRH3: NNYGNYGWYFDV (SEQ ID NO:3)
CDRL1: SASSSVSYMH (SEQ ID NO:4)
CDRL2: DTSKLAS (SEQ ID NO:5)
CDRL3: FQGSGYPYT (SEQ ID NO:6)
In some embodiments, the anti-ICOS antibodies 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 (VH) Variable Region (H2):
QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DYAMHWVRQA PGQGLEWMGL
ISIYSDHTNY NQKFQGRVTI TADKSTSTAY MELSSLRSED TAVYYCGRNN
YGNYGWYFDV WGQGTTVTVS S
(SEQ ID NO:7)
In one embodiment of the present invention the ICOS antibody 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
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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.
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 (VI) Variable Region (L5)
EIVLTQSPAT LSLSPGERAT LSCSASSSVS YMHWYQQKPG QAPRLLIYDT
SKLASGIPAR FSGSGSGTDY TLTISSLEPE DFAVYYCFQG SGYPYTFGQG TKLEIK
(SEQ ID NO:8)
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 an
antibody
comprising SEQ ID NO:7 and SEQ ID NO:8.
It will be appreciated that each of CDR H1, H2, H3, Li, 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. Typically,
the modification
is a substitution, particularly a conservative substitution, for example as
shown in Table 1
below.
Table 1
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
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Aromatic Trp, Tyr, Phe
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 FcyRI and FcyRII. 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" 5th 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 the ICOS antibody is an IgG4
isotype. In one
embodiment, the ICOS antibody comprises an IgG4 Fc region comprising the
replacement
5228P and L235E may have the designation IgG4PE.
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.
As used herein, an "agent directed to PD-1" or "agent directed to PD1" means
any
chemical compound or biological molecule capable of binding to PD1. In some
embodiments, the agent directed to PD1 is a PD1 antagonist.
The term "PD1 binding protein" or "PD-1 binding protein" as used herein refers
to
antibodies and other protein constructs, such as domains, which are capable of
binding to
PD1. In some instances, the PD1 is human PD1. The term "PD1 binding protein"
can be
used interchangeably with "PD1 antigen binding protein." Thus, as is
understood in the art,
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anti-PD1 antibodies and/or PD1 antigen binding proteins would be considered
PD1 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
PD1. As used herein "antigen binding portion" of a PD1 binding protein would
include any
portion of the PD1 binding protein capable of binding to PD1, including but
not limited to, an
antigen binding antibody fragment.
The protein Programmed Death 1 (PD-1) is an inhibitory member of the CD28
family
of receptors, that also includes CD28, CTLA-4, ICOS and BTLA. PD-1 is
expressed on
activated B cells, T cells, and myeloid cells (Agata et al., supra; Okazaki et
al. (2002) Curr.
Opin. Immunol 14:391779-82; Bennett et al. (2003) J Immunol 170:711-8) The
initial
members of the family, CD28 and ICOS, were discovered by functional effects on
augmenting T cell proliferation following the addition of monoclonal
antibodies (Hutloff et
al. (1999) Nature 397:263-266; Hansen et al. (1980) Immunogenics 10:247-260).
PD-1 was
discovered through screening for differential expression in apototic cells
(Ishida et al. (1992)
EMBO J 11:3887-95) The other members of the family, CTLA-4, and BTLA were
discovered through screening for differential expression in cytotoxic T
lymphocytes and TH1
cells, respectively. CD28, ICOS and CTLA-4 all have an unpaired cysteine
residue allowing
for homodimerization. In contrast, PD-1 is suggested to exist as a monomer,
lacking the
unpaired cysteine residue characteristic in other CD28 family members. PD-1
antibodies
and methods of using in treatment of disease are described in US Patent Nos.:
US
7,595,048; US 8,168,179; US 8,728,474; US 7,722,868; US 8,008,449; US
7,488,802; US
7,521,051; US 8,088,905; US 8,168,757; US 8,354,509; and US Publication Nos.
U520110171220; U520110171215; and U520110271358. Combinations of CTLA-4 and
PD-1 antibodies are described in US Patent No. 9,084,776.
In some embodiments, the agent directed to PD1 is a PD1 antagonist and blocks
binding of PD-Li expressed on a cancer cell to PD-1 expressed on an immune
cell (T cell, B
cell or NKT cell) and may also block binding of PD-L2 expressed on a cancer
cell to the
immune-cell expressed PD-1. Alternative names or synonyms for PD-1 and its
ligands
include: PDCD1, PD1, CD279 and SLEB2 for PD-1; PDCD1L1, PDL1, B7H1, B7-4,
CD274
and B7-H for PD-Li; and PDCD1L2, PDL2, B7-DC, Btdc and CD273 for PD-L2. Human
PD-1 amino acid sequences can be found in NCBI Locus No.: NP_005009. The amino
acid
sequence in NCBI Locus No.: NP_005009 is reproduced below:
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mqipqapwpv vwavlqlgwr pgwfldspdr pwnpptfspa llvvtegdna tftcsfsnts
esfvinwyrm spsnqtdkla afpedrsqpg qdcrfrvtql pngrdfhmsv vrarrndsgt
ylcgaislap kaqikeslra elrvterrae vptahpspsp rpagqfqtiv vgvvggllgs
lvllvwvlav icsraargti garrtgqplk edpsavpvfs vdygeldfqw rektpeppvp
cvpeqteyat ivfpsgmgts sparrgsadg prsaqp1rpe dghcswpl (SEQ ID NO: 11)
Human PD-Li and PD-L2 amino acid sequences can be found in NCBI Locus No.:
NP 054862 and NP 079515, respectively.
The amino acid sequence in NCBI Locus No.: NP_054862 is reproduced below:
mrifavfifin tywhllnaft vtvpkdlyvv eygsnmtiec kfpvekqldl aalivyweme
dkniiqfvhg eedlkvqhss yrqrarllkd qlslgnaalq itdvklqdag vyrcmisygg
adykritvkv napynkinqr ilvvdpvtse heltcqaegy pkaeviwtss dhqvlsgktt
ttnskreekl fnvtstlrin tttneifyct frrldpeenh taelvipelp lahppnerth
lvilgaillc lgvaltfifr lrkgrmmdvk kcgiqdtnsk kqsdthleet (SEQ ID NO: 12)
The amino acid sequence in NCBI Locus No.: NP_079515 is reproduced below:
mifll1m1s1 elqlhqiaal ftvtvpkely iiehgsnvtl ecnfdtgshv nlgaitaslq
kvendtsphr eratlleeql plgkasfhip qvqvrdegqy qciiiygvaw dykyltlkvk
asyrkinthi lkvpetdeve ltcqatgypl aevswpnvsv pantshsrtp eglyqvtsvl
rlkpppgrnf scvfwnthvr eltlasidlq sqmeprthpt wllhifipfc iiafifiatv
ialrkqlcqk lysskdttkr pvtftkrevn sai (SEQ ID NO: 13)
Agents directed to PD-1 in any of the aspects or embodiments of the present
invention
include a monoclonal antibody (mAb), or antigen binding fragment thereof,
which
specifically binds to PD-1. In some embodiments, the mAb to PD-1 specifically
binds to
human PD-1. The mAb may be a human antibody, a humanized antibody or a
chimeric
antibody, and may include a human constant region. In some embodiments, the
human
constant region is selected from the group consisting of IgGl, IgG2, IgG3 and
IgG4 constant
regions, and in preferred embodiments, the human constant region is an IgG1 or
IgG4
constant region. In some embodiments, the antigen binding fragment is selected
from the
group consisting of Fab, Fab'-SH, F(ab')2, scFv and Fv fragments.
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Examples of mAbs that bind to human PD-1, and useful in the various aspects
and
embodiments of the present invention, are described in US Patent No.
8,552,154; US Patent
No. 8,354,509; US Patent No. 8,168,757; US Patent No. 8,008,449; US Patent No.
7,521,051;
US Patent No. 7,488,802; W02004072286; W02004056875; and W02004004771.
Other PD-1 binding proteins useful in any of the aspects and embodiments of
the
present invention include an immunoadhesin that specifically binds to PD-1,
and preferably
specifically binds to human PD-1, e.g., a fusion protein containing the
extracellular or PD-1
binding portion of PD-Li or PD-L2 fused to a constant region such as an Fc
region of an
immunoglobulin molecule. Examples of immunoadhesin molecules that specifically
bind to
PD-1 are described in W02010027827 and W02011066342. Specific fusion proteins
useful
as the PD-1 antagonist in the treatment method, medicaments and uses of the
present
invention include AMP-224 (also known as B7-DCIg), which is a PD-L2-FC fusion
protein
and binds to human PD-1.
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-l/PCD-1) with
immunopotentiation
activity. Nivolumab binds to and blocks the activation of PD-1, an Ig
superfamily
transmembrane protein, by its ligands PD-Li 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
Pl3k/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 US Patent No. US 8,008,449.
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 US Patent No. 8,168,757.
By "agent directed to PD-Li" is meant any chemical compound or biological
molecule capable of binding to PD-Li. In some embodiments, the agent directed
to PD-Li is
a PD-Li binding protein.
The term "PDL1 binding protein" or "PD-Li binding protein" as used herein
refers to
antibodies and other protein constructs, such as domains, which are capable of
binding to PD-
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Ll. In some instances, the PD-Li is human PD1. The term "PD-Li binding
protein" can be
used interchangeably with "PD-Li antigen binding protein." Thus, as is
understood in the
art, anti-PD-Li antibodies and/or PD-Li antigen binding proteins would be
considered PD-
Li 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 PD-Li. As used herein "antigen binding portion" of a PD-Li
binding
protein would include any portion of the PD-Li binding protein capable of
binding to PD-L1,
including but not limited to, an antigen binding antibody fragment.
In some embodiments, the agent directed to PD-Li is a PD1 antagonist and
blocks
binding of PD-Li expressed on a cancer cell to PD-1 expressed on an immune
cell (T cell, B
cell or NKT cell) and may also block binding of PD-L2 expressed on a cancer
cell to the
immune-cell expressed PD-1.
PD-Li is a B7 family member that is expressed on many cell types, including
APCs
and activated T cells (Yamazaki et al. (2002) J. Immunol. 169:5538). PD-Li
binds to both
PD-1 and B7-1. Both binding of T-cell-expressed B7-1 by PD-Li and binding of T-
cell-
expressed PD-Li by B7-1 result in T cell inhibition (Butte et al. (2007)
Immunity 27:111).
There is also evidence that, like other B7 family members, PD-Li can also
provide
costimulatory signals to T cells (Subudhi et al. (2004) J. Clin. Invest.
113:694; Tamura et al.
(2001) Blood 97:1809). PD-Li (human PD-Li cDNA is composed of the base
sequence
shown by EMBL/GenBank Acc. No. AF233516 and mouse PD-Li cDNA is composed of
the
base sequence shown by NM--021893) that is a ligand of PD-1 is expressed
in so-called
antigen-presenting cells (APCs) such as activated monocytes and dendritic
cells (Journal of
Experimental Medicine (2000), vol. 19, issue 7, p 1027-1034). These cells
present interaction
molecules that induce a variety of immuno-inductive signals to T lymphocytes,
and PD-Li is
one of these molecules that induce the inhibitory signal by PD-1. It has been
revealed that
PD-Li ligand stimulation suppressed the activation (cellular proliferation and
induction of
various cytokine production) of PD-1 expressing T lymphocytes. PD-Li
expression has been
confirmed in not only immunocompetent cells but also a certain kind of tumor
cell lines (cell
lines derived from monocytic leukemia, cell lines derived from mast cells,
cell lines derived
from hepatic carcinomas, cell lines derived from neuroblasts, and cell lines
derived from
breast carcinomas) (Nature Immunology (2001), vol. 2, issue 3, p. 261-267).
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Anti-PD-Li antibodies and methods of making the same are known in the art.
Such
antibodies to PD-Li may be polyclonal or monoclonal, and/or recombinant,
and/or
humanized, and/or fully human. PD-Li antibodies are in development as immuno-
modulatory agents for the treatment of cancer.
Exemplary PD-Li antibodies are disclosed in US Patent No. 9,212,224; US Patent
No. 8,779,108; US Patent No 8,552,154; US Patent No. 8,383,796; US Patent No.
8,217,149;
US Patent Publication No. 20110280877; W02013079174; and W02013019906.
Additional
exemplary antibodies to PD-Li (also referred to as CD274 or B7-H1) and methods
for use
are disclosed in US Patent No. 8,168,179; US Patent No. 7,943,743; US Patent
No.
7,595,048; W02014055897; W02013019906; and W02010077634. Specific anti-human
PD-Li monoclonal antibodies useful as a PD-1 antagonist in the treatment
method,
medicaments and uses of the present invention include MPDL3280A, BMS-936559,
MEDI4736, MSB0010718C.
Atezolizumab is a fully humanized monoclonal anti-PD-Li antibody commercially
available as TECENTRIQ. Atezolizumab is indictated for the treatment of some
locally
advanced or metastatic urothelial carcinomas. Atezolizumab blocks the
interaction of PD-Li
with PD-1 and CD80.
Durvalumab (previously known as MEDI4736) is a human monoclonal antibody
directed against PD-Li. Durvalumab blocks the interaction of PD-Li with PD-1
and CD80.
Durvalumab is commercially available as IMFINZITm.
Antibodies to PD-Li (also referred to as CD274 or B7-H1) and methods for use
are
disclosed in US Patent No. 7,943,743; US Patent No. 8,383,796; US20130034559,
W02014055897, US Patent No. 8,168,179; and US Patent No. 7,595,048. PD-Li
antibodies
are in development as immuno-modulatory agents for the treatment of cancer.
As used herein the term "agonist" refers to an antigen binding protein
including but
not limited to an antibody, which upon contact with a co-signalling receptor
causes one or
more of the following (1) stimulates or activates the receptor, (2) enhances,
increases or
promotes, induces or prolongs an activity, function or presence of the
receptor and/or (3)
enhances, increases, promotes or induces the expression of the receptor.
Agonist activity can
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be measured in vitro by various assays know in the art such as, but not
limited to,
measurement of cell signalling, 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.
As used herein the term "antagonist" refers to an antigen binding protein
including
but not limited to an antibody, which upon contact with a co-signalling
receptor causes one or
more of the following (1) attenuates, blocks or inactivates the receptor
and/or blocks
activation of a receptor by its natural ligand, (2) reduces, decreases or
shortens the activity,
function or presence of the receptor and/or (3) reduces, descrease, abrogates
the expression of
the receptor. Antagonist activity can be measured in vitro by various assays
know in the art
such as, but not limited to, measurement of an increase or decrease in cell
signalling, cell
proliferation, immune cell activation markers, cytokine production. Antagonist
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.
As used herein the term "cross competes for binding" refers to any agent such
as an
antibody that will compete for binding to a target with any of the agents of
the present
invention. Competition for binding between two antibodies 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 a co-
signalling receptor and bind 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.
The term "binding protein" as used herein refers to antibodies and other
protein
constructs, such as domains, which are capable of binding to an antigen.
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., VII, Vtni, VL, domain antibody (dAbTm)), antigen binding antibody
fragments, Fab,
F(ab')2, Fv, disulphide linked Fv, single chain Fv, disulphide-linked scFv,
diabodies,
TANDABSTm, etc. and modified versions of any of the foregoing.
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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.
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.
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 VII, VIlli and VL 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 Vtni dAbsTm. Camelid Vtni 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 Vtni 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 VII
includes camelid Vtni domains.
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
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regions may be of human or primate origin, or which may be an artificial
chimera of human
and primate constant regions.
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. CHL
CH2, CH3, VH,
VI). The antigen binding protein may comprise an IgG scaffold selected from
IgGl, IgG2,
IgG3, IgG4 or IgG4PE. For example, the scaffold may be IgGl. The scaffold may
consist of,
or comprise, the Fc region of an antibody, or is a part thereof.
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. BIACORETm analysis). For example, the BIACORETM methods described in
Example
5 may be used to measure binding affinity.
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.
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.
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
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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.
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,
NSO, 293,
HeLa, myeloma, lymphoma cells or any derivative thereof Most preferably, the
eukaryotic
cell is a HEK293, NSO, SP2/0, or CHO cell. E. coil 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.
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.
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 KABATTm 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
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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.
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.
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.
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.
The terms "VH" and "Vi." are used herein to refer to the heavy chain variable
region
and light chain variable region respectively of an antigen binding protein.
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"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.
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).
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.
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.
"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
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"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
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.
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.
In one aspect, methods of treating cancer in a patient in need thereof,
comprising
administering to the patient an effective amount of an agent directed to human
ICOS and an
effective amount of an agent directed to human PD1 or human PD-Li sequentially
are
provided. In one embodiment, administration of the agent directed to human
ICOS is
followed by administration of the agent directed to human PD1 or human PD-Li.
In one
embodiment, the agent directed to human PD1 or human PD-Li is administered
concurrently
with an agent directed to human ICOS in the phase following administration of
the agent
directed to human ICOS.
In another aspect, administration of the agent directed to human PD1 or human
PD-Li
is followed by administration of the agent directed to human ICOS. In one
embodiment, the
agent directed to human ICOS is an anti-ICOS antibody or antigen binding
portion thereof
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In one embodiment, the agent directed to human ICOS is administered
concurrently with an
agent directed to human PD1 or human PD-Li in the phase following
administration of the
agent directed to human PD1 or human PD-Li.
In one aspect, an anti-ICOS antibody or antigen binding fragment thereof and
an anti-
PD1 antibody or antigen binding fragment thereof for sequential use in
treating cancer in a
human in need thereof are provided. In one embodiment, administration of the
anti-ICOS
antibody or antigen binding fragment thereof is followed by administration of
the anti-PD1
antibody or antigen binding fragment thereof In another embodiment,
administration of the
anti-PD1 antibody or antigen binding fragment thereof is followed by
administration of the
anti-ICOS antibody or antigen binding fragment thereof.
In one aspect, an anti-ICOS antibody or antigen binding fragment thereof and
an anti-
PD-Li antibody or antigen binding fragment thereof for sequential use in
treating cancer in a
human in need thereof are provided. In one embodiment, administration of the
anti-ICOS
antibody or antigen binding fragment thereof is followed by administration of
the anti-PD-Li
antibody or antigen binding fragment thereof In another embodiment,
administration of the
anti-PD-Li antibody or antigen binding fragment thereof is followed by
administration of the
anti-ICOS antibody or antigen binding fragment thereof.
In another aspect, use of an anti-ICOS antibody or antigen binding portion
thereof and
an anti-PD1 antibody or antigen binding portion thereof in the manufacture of
a medicament
for the treatment of cancer is provided, wherein the anti-ICOS antibody or
antigen binding
portion thereof and an anti-PD1 antibody or antigen binding portion thereof
are sequentially
administered, and wherein administration of the anti-ICOS antibody or antigen
binding
portion thereof is followed by administration of the anti-PD1 antibody or
antigen binding
portion thereof
In another aspect, use of an anti-ICOS antibody or antigen binding portion
thereof and
an anti-PDL1 antibody or antigen binding portion thereof in the manufacture of
a
medicament for the treatment of cancer is provided, wherein the anti-ICOS
antibody or
antigen binding portion thereof and an anti-PDL1 antibody or antigen binding
portion thereof
are sequentially administered, and wherein administration of the anti-ICOS
antibody or
antigen binding portion thereof is followed by administration of the anti-PDL1
antibody or
antigen binding portion thereof.
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The present invention also provides polynucleotides encoding anti-ICOS
antibodies,
anti-PD1 antibodies, anti-PDL1 antibodies, or antigen binding portion of any
one of said
antibodies, of the present invention. In one embodiment, host cells are
provided comprising
polynucleotides encoding anti-ICOS antibodies, anti-PD1 antibodies, or anti-
PDL1
antibodies, or antigen binding portions of any one of said antibodies, of the
present invention.
The present invention also provides methods of making an anti-ICOS antibody,
anti-PD1
antibody, anti-PDL1 antibody, or an antigen binding portion of said antibody,
comprising the
steps of a) culturing host cell comprising a polynucleotide encoding an anti-
ICOS antibody,
anti-PD1 antibody, or anti-PDL1 antibody or an antigen binding portion of said
antibody of
the present invention under suitable conditions to express said anti-ICOS
antibody, anti-PD1
antibody, or anti-PDL1 antibody or antigen binding portion of said antibody;
and b) isolating
said anti-ICOS, anti-PD1, or anti-PDL1 antibody or antigen binding portion of
said antibody.
In another aspect, a polynucleotide encoding an anti-ICOS antibody or antigen
binding portion thereof is provided, wherein the anti-ICOS antibody or antigen
binding
portion thereof is sequentially administered to a cancer patient with an anti-
PD1 antibody or
antigen binding portion thereof, and wherein administration of the anti-ICOS
antibody or
antigen binding portion thereof is followed by administration of the anti-PD1
antibody or
antigen binding portion thereof.
In another aspect, a polynucleotide encoding an anti-ICOS antibody or antigen
binding portion thereof is provided, wherein the anti-ICOS antibody or antigen
binding
portion thereof is sequentially administered to a cancer patient with an anti-
PDL1 antibody or
antigen binding portion thereof, and wherein administration of the anti-ICOS
antibody or
antigen binding portion thereof is followed by administration of the anti-PDL1
antibody or
antigen binding portion thereof.
In yet another aspect, a polynucleotide encoding an anti-PD1 antibody or
antigen
binding portion thereof is provided, wherein the anti-PD1 antibody or antigen
binding portion
thereof is sequentially administered to a cancer patient with an anti-ICOS
antibody or antigen
binding portion thereof, and wherein administration of the anti-ICOS antibody
or antigen
binding portion thereof is followed by administration of the anti-PD1 antibody
or antigen
binding portion thereof
In still another aspect, a polynucleotide encoding an anti-PDL1 antibody or
antigen
binding portion thereof is provided, wherein the anti-PDL1 antibody or antigen
binding
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portion thereof is sequentially administered to a cancer patient with an anti-
ICOS antibody or
antigen binding portion thereof, and wherein administration of the anti-ICOS
antibody or
antigen binding portion thereof is followed by administration of the anti-PDL1
antibody or
antigen binding portion thereof.
In another aspect, a vector comprising the polynucleotide of any one of the
aspects
herein is provided. In another aspect, a host cell comprising the vector of
any one of the
aspects herein is provided.
In yet another aspect, a method of making an anti-ICOS antibody or antigen
binding
portion thereof is provided, the method comprising a) culturing a host cell
comprising the
.. polynucleotide of any one of the aspects herein under suitable conditions
to express the anti-
ICOS antibody or antigen binding portion thereof; and b) isolating said anti-
ICOS antibody
or antigen binding portion thereof
In another aspect, a method of making an anti-PD1 antibody or antigen binding
portion thereof is provided, the method comprising a) culturing a host cell
comprising the
polynucleotide of any one of the aspects herein under suitable conditions to
express the anti-
PD1 antibody or antigen binding portion thereof; and b) isolating said anti-
PD1 antibody or
antigen binding portion thereof.
In still another aspect, a method of making an anti-PDL1 antibody or antigen
binding
portion thereof is provided, the method comprising a) culturing a host cell
comprising the
polynucleotide of any one of the aspects herein under suitable conditions to
express the anti-
PDL1 antibody or antigen binding portion thereof; and b) isolating said anti-
PDL1 antibody
or antigen binding portion thereof
In one embodiment of any one of the aspects herein, the anti-ICOS antibody is
an
ICOS agonist. In one embodiment, the anti-ICOS antibody comprises a VII domain
comprising an amino acid sequence at least 90% identical to the amino acid
sequence set
forth in SEQ ID NO:7; and a VL domain comprising an amino acid sequence at
least 90%
identical to the amino acid sequence as set forth in SEQ ID NO:8. In another
embodiment,
the the anti-ICOS antibody comprises a Vu domain comprising the amino acid
sequence set
forth in SEQ ID NO:7 and a VL domain comprising the amino acid sequence as set
forth in
SEQ ID NO:8. In one embodiment, the anti-ICOS antibody 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
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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.
In one embodiment of any one of the aspects herein, the agent directed to
human PD1
is an anti-PD1 antibody or antigen binding portion thereof In one embodiment,
the anti-PD1
antibody is a PD1 antagonist. In one embodiment, the anti-PD1 antibody is
pembrolizumab.
In another embodiment, the anti-PD1 antibody is nivolumab. In one embodiment
of any one
of the aspects herein, the agent directed to human PD-Li is an anti-PD-Li
antibody or
antigen binding portion thereof In one embodiment, the anti-PD-Li antibody is
a PD1
antagonist. In one embodiment, the anti-PD-Li antibody is durvalumab.
In one embodiment of any one of the aspects herein, the agent directed to
human
ICOS is administered for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, or 30 consecutive days. In one embodiment of any
one of the
aspects herein, the agent directed to human PD1 or human PD-Li is administered
for 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, or 30
consecutive days.
In one aspect, the cancer is selected from the group consisting of colorectal
cancer
(CRC), gastric, esophageal, cervical, bladder, breast, head and neck, ovarian,
melanoma,
renal cell carcinoma (RCC), EC squamous cell, non-small cell lung carcinoma,
mesothelioma, pancreatic, and prostate cancer.
In one aspect, the present invention provides a method of treating cancer in a
human
in need thereof, the method comprising administering to said human an anti-
ICOS antibody
or antigen binding fragment thereof and/or administering to said human an anti-
PD1 antibody
or antigen binding fragment thereof In one embodiment, the anti-ICOS antibody
or antigen
binding fragment thereof induces T-cell proliferation, expansion, and tumor
infiltration. In
another embodiment, the anti-ICOS antibody or antigen binding fragment thereof
increases
PD-1 expression on a T-cell. In one embodiment, the anti-PD1 antibody or
antigen binding
fragment thereof increases ICOS expression on a T-cell. In one embodiment, the
anti-ICOS
antibody or antigen binding fragment thereof is an IgG4 isotype and reduces
depletion of
ICOS-positive T-cells. In another embodiment, the anti-ICOS antibody or
antigen binding
fragment thereof is an IgG4 isotype and results in increased anti-cancer
efficacy when
compared to an IgG1 isotype anti-ICOS antibody.
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In another embodiment the cancer is selected from head and neck cancer, breast
cancer, lung cancer, colon cancer, ovarian cancer, prostate cancer, gliomas,
glioblastoma,
astrocytomas, glioblastoma multiforme, Bannayan-Zonana syndrome, Cowden
disease,
Lhermitte-Duclos disease, inflammatory breast cancer, Wilm's tumor, Ewing's
sarcoma,
Rhabdomyosarcoma, ependymoma, medulloblastoma, kidney cancer, liver cancer,
melanoma, pancreatic cancer, sarcoma, osteosarcoma, giant cell tumor of bone,
thyroid
cancer, lymphoblastic T cell leukemia, Chronic myelogenous leukemia, Chronic
lymphocytic leukemia, Hairy-cell leukemia, acute lymphoblastic leukemia, acute
myelogenous leukemia, AML, 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, 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.
Some embodiments of the present invention further comprise administering at
least
one neo-plastic agent and/or at least one immunostimulatory agent to said
human.
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 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.
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,
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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.
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
the condition or 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, or (4) to slow the progression of the condition or one or
more of the
biological manifestations of the condition. Prophylactic therapy using the
methods and/or
compositions of the invention is also contemplated. 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.
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
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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.
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.
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
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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.
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
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tissues), tonsils, Peyer's patches and appendix, and lymphoid tissues
associated with other
mucosa, for example, the bronchial linings.
As used herein the term "Compound A2" means an agent directed to human ICOS.
In
some embodiments, Compound A2 is an antibody to human ICOS or the antigen
binding
portion thereof In some embodiments, Compound A2 is an ICOS agonist. Suitably
Compound A2 means a humanized monoclonal antibody having a heavy chain
variable region
as set forth in SEQ ID NO:7 and a light chain variable region as set forth in
SEQ ID NO:8.
As used herein the term "Compound B2" means an agent directed to human PD1 or
an
agent to directed to human PD-Li. In some embodiments, Compound B2 is a PD1
antagonist. In some embodiments, Compound B2 is an antibody to human PD1 or
the antigen
binding portion thereof In some embodiments, Compound B2 is an antibody to
human PD-
Li or the antigen binding portion thereof Suitably, Compound B2 is nivolumab.
Suitably,
Compound B2 is pembrolizumab.
Suitably, the combinations of this invention are administered within a
"specified
period".
The term "specified period" and grammatical variations thereof, as used
herein,
means the interval of time between the administration of one of Compound A2
and
Compound B2 and the other of Compound A2 and Compound B2.
Suitably, if the compounds are administered within a "specified period" and
not
administered simultaneously, they are both administered within about 24 hours
of each other
¨ in this case, the specified period will be about 24 hours; suitably they
will both be
administered within about 12 hours of each other ¨ in this case, the specified
period will be
about 12 hours; suitably they will both be administered within about 11 hours
of each other ¨
in this case, the specified period will be about 11 hours; suitably they will
both be
administered within about 10 hours of each other ¨ in this case, the specified
period will be
about 10 hours; suitably they will both be administered within about 9 hours
of each other ¨
in this case, the specified period will be about 9 hours; suitably they will
both be
administered within about 8 hours of each other ¨ in this case, the specified
period will be
about 8 hours; suitably they will both be administered within about 7 hours of
each other ¨ in
this case, the specified period will be about 7 hours; suitably they will both
be administered
within about 6 hours of each other ¨ in this case, the specified period will
be about 6 hours;
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suitably they will both be administered within about 5 hours of each other ¨
in this case, the
specified period will be about 5 hours; suitably they will both be
administered within about 4
hours of each other ¨ in this case, the specified period will be about 4
hours; suitably they
will both be administered within about 3 hours of each other ¨ in this case,
the specified
period will be about 3 hours; suitably they will be administered within about
2 hours of each
other ¨ in this case, the specified period will be about 2 hours; suitably
they will both be
administered within about 1 hour of each other ¨ in this case, the specified
period will be
about 1 hour. As used herein, the administration of Compound A2 and Compound
B2 in less
than about 45 minutes apart is considered simultaneous administration.
Suitably, when the combination of the invention is administered for a
"specified
period", the compounds will be co-administered for a "duration of time".
The term "duration of time" and grammatical variations thereof, as used herein
means
that both compounds of the invention are administered for an indicated number
of
consecutive days. Unless otherwise defined, the number of consecutive days
does not have to
commence with the start of treatment or terminate with the end of treatment,
it is only
required that the number of consecutive days occur at some point during the
course of
treatment.
Regarding "specified period" administration:
Suitably, both compounds will be administered within a specified period for at
least
one day ¨ in this case, the duration of time will be at least one day;
suitably, during the course
to treatment, both compounds will be administered within a specified period
for at least 3
consecutive days ¨ in this case, the duration of time will be at least 3 days;
suitably, during
the course to treatment, both compounds will be administered within a
specified period for at
least 5 consecutive days ¨ in this case, the duration of time will be at least
5 days; suitably,
during the course to treatment, both compounds will be administered within a
specified
period for at least 7 consecutive days ¨ in this case, the duration of time
will be at least 7
days; suitably, during the course to treatment, both compounds will be
administered within a
specified period for at least 14 consecutive days ¨ in this case, the duration
of time will be at
least 14 days; suitably, during the course to treatment, both compounds will
be administered
within a specified period for at least 30 consecutive days ¨ in this case, the
duration of time
will be at least 30 days.
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Suitably, if the compounds are not administered during a "specified period",
they are
administered sequentially. By the term "sequential administration", and
grammatical
derivates thereof, as used herein is meant that one of Compound A2 and
Compound B2 is
administered for two or more consecutive days and the other of Compound A2 and
.. Compound B2 is subsequently administered for two or more consecutive days.
During the
period of consecutive days in which Compound A2 is administered, at least 1
dose, at least 2
doses, at least 3 doses, at least 4 doses, at least 5 doses, at least 6 doses,
at least 7 doses, at
least 8 doses, at least 9 doses, or at least 10 doses of Compound A2 is
administered. During
the period of consecutive days in which Compound B2 is administered, at least
1 dose, at least
2 doses, at least 3 doses, at least 4 doses, at least 5 doses, at least 6
doses, at least 7 doses, at
least 8 doses, at least 9 doses, or at least 10 doses Compound B2 is
administered. During the
period of consecutive days in which Compound A2 is administered, Compound A2
can be
administered at least three times a day, at least twice a day, at least once a
day, or less than
once a day, e.g., once every 2 days, once every 3 days, once every week, once
every 2 weeks,
once every 3 weeks, or once every 4 weeks. During the period of consecutive
days in which
Compound B2 is administered, Compound B2 can be administered at least three
times a day,
at least twice a day, at least once a day, or less than once a day, e.g., once
every 2 days, once
every 3 days, once every week, once every 2 weeks, once every 3 weeks, or once
every 4
weeks.
Also, contemplated herein is a drug holiday utilized between the sequential
administration of one of Compound A2 and Compound B2 and the other of Compound
A2 and
Compound B2. As used herein, a drug holiday is a period of days after the
sequential
administration of one of Compound A2 and Compound B2 and before the
administration of
the other of Compound A2 and Compound B2 where neither Compound A2 nor
Compound B2
is administered. Suitably the drug holiday will be a period of days selected
from: 1 day, 2
days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11
days, 12 days, 13
days and 14 days.
Sequential administration can also include one of Compound A2 and Compound B2
is
administered for two or more consecutive days and then both of Compound A2 and
Compound B2 is subsequently administered for two or more consecutive days.
Sequential
administration can include both of Compound A2 and Compound B2 being
administered for
two or more consecutive days and then one of Compound A2 and Compound B2 being
subsequently administered for two or more consecutive days
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Regarding sequential administration:
Suitably, one of Compound A2 and Compound B2 is administered for from 1 to 30
consecutive days, followed by an optional drug holiday, followed by
administration of the
other of Compound A2 and Compound B2 for from 1 to 30 consecutive days.
Suitably, one of
.. Compound A2 and Compound B2 is administered for from 1 to 21 consecutive
days, followed
by an optional drug holiday, followed by administration of the other of
Compound A2 and
Compound B2 for from 1 to 21 consecutive days. Suitably, one of Compound A2
and
Compound B2 is administered for from 1 to 14 consecutive days, followed by a
drug holiday
of from 1 to 14 days, followed by administration of the other of Compound A2
and
.. Compound B2 for from 1 to 14 consecutive days. Suitably, one of Compound A2
and
Compound B2 is administered for from 1 to 7 consecutive days, followed by a
drug holiday
of from 1 to 10 days, followed by administration of the other of Compound A2
and
Compound B2 for from 1 to 7 consecutive days.
Suitably, Compound B2 will be administered first in the sequence, followed by
an
optional drug holiday, followed by administration of Compound A2. Suitably,
Compound B2
is administered for from 3 to 21 consecutive days, followed by an optional
drug holiday,
followed by administration of Compound A2 for from 3 to 21 consecutive days.
Suitably,
Compound B2 is administered for from 3 to 21 consecutive days, followed by a
drug holiday
of from 1 to 14 days, followed by administration of Compound A2 for from 3 to
21
consecutive days. Suitably, Compound B2 is administered for from 3 to 21
consecutive days,
followed by a drug holiday of from 3 to 14 days, followed by administration of
Compound
A2 for from 3 to 21 consecutive days. Suitably, Compound B2 is administered
for 21
consecutive days, followed by an optional drug holiday, followed by
administration of
Compound A2 for 14 consecutive days. Suitably, Compound B2 is administered for
14
consecutive days, followed by a drug holiday of from 1 to 14 days, followed by
administration of Compound A2 for 14 consecutive days. Suitably, Compound B2
is
administered for 7 consecutive days, followed by a drug holiday of from 3 to
10 days,
followed by administration of Compound A2 for 7 consecutive days. Suitably,
Compound B2
is administered for 3 consecutive days, followed by a drug holiday of from 3
to 14 days,
followed by administration of Compound A2 for 7 consecutive days. Suitably,
Compound B2
is administered for 3 consecutive days, followed by a drug holiday of from 3
to 10 days,
followed by administration of Compound A2 for 3 consecutive days.
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It is understood that a "specified period" administration and a "sequential"
administration can be followed by repeat dosing or can be followed by an
alternate dosing
protocol, and a drug holiday may precede the repeat dosing or alternate dosing
protocol.
The methods of the present invention may also be employed with other
therapeutic
methods of cancer treatment.
Compound A2 and Compound B2 may be administered by any appropriate
route. Suitable routes include oral, rectal, nasal, topical (including buccal
and sublingual),
intratumorally, vaginal, and parenteral (including subcutaneous,
intramuscular, intravenous,
intradermal, intrathecal, and epidural). It will be appreciated that the
preferred route may
vary with, for example, the condition of the recipient of the combination and
the cancer to be
treated. It will also be appreciated that each of the agents administered may
be administered
by the same or different routes and that Compound A2 and Compound B2 may be
compounded together in a pharmaceutical composition/formulation.
In one embodiment, one or more components of a combination of the invention
are
administered intravenously. In one embodiment, one or more components of a
combination
of the invention are administered orally. In another embodiment, one or more
components of
a combination of the invention are administered intratumorally. In another
embodiment, one
or more components of a combination of the invention are administered
systemically, e.g.,
intravenously, and one or more other components of a combination of the
invention are
administered intratumorally. In any of the embodiments, e.g., in this
paragraph, the
components of the invention are administered as one or more pharmaceutical
compositions.
In one aspect methods are provided for the treatment of cancer, comprising
administering to a human in need thereof a therapeutically effective amount of
(i) an anti-
ICOS antibody or the antigen binding portion thereof, in addition to one of
more diluents,
vehicles, excipients and/or inactive ingredients, and (ii) an anti-PD1
antibody or the antigen
binding portion thereof or an anti-PDL1 antibody or the antigen binding
portion thereof, in
addition to one of more diluents, vehicles, excipients and/or inactive
ingredients. In one
embodiment sequential administration of an anti-ICOS antibody or the antigen
binding
portion thereof and an anti-PD1 antibody or antigen binding portion thereof
provides a
synergistic effect compared to administration of either agent as monotherapy
or concurrently.
In one embodiment, sequential administration of an anti-ICOS antibody or the
antigen
binding portion thereof and an anti-PDL1 antibody or antigen binding portion
thereof
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provides a synergistic effect compared to administration of either agent as
monotherapy or
concurrently.
In one embodiment, the anti-ICOS antibody or antigen binding portion thereof
comprises a VH domain comprising an amino acid sequence at least 90% identical
to the
amino acid sequence set forth in SEQ ID NO:7; and a VL 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 embodiment, methods of treating cancer are provided wherein the anti-
ICOS
antibody or antigen binding portion thereof is administered at a time interval
selected from
once every week, once every two weeks, once every three weeks, and once every
four weeks.
In another embodiment, the anti-PD1 antibody or antigen binding portion
thereof or the anti-
PDL1 antibody or antigen binding portion thereof is administered at a time
interval selected
from once every week, once every two weeks, once every three weeks, and once
every four
weeks. As is understood in the art the start of administration of either agent
can be separated
by an interstitial period. The interstitial period may be 12 hours, one to six
days, one week,
two weeks, three weeks, four weeks, five weeks, or six weeks. By way of
example, an anti-
ICOS antibody could be administered on Day 1 of treatment with an interstitial
period of two
weeks before the start of anti-PD1 antibody therapy which would start on Day
14. In one
aspect, treatment with said anti-ICOS antibody could continue with
administration of a single
IV infusion at a time interval of, for example, every one, two, three or four
weeks. Similarly,
treatment with said anti-PD1 antibody could continue with administration of a
single IV
infusion at a time interval of, for example, every one, two, three or four
weeks.
In one embodiment, the anti-ICOS antibody or antigen binding portion thereof
is
administered as an IV infusion. In one emboditment, the anti-PD1 antibody or
antigen
binding portion thereof is administered as an IV infusion. In one emboditment,
the anti-
PDL1 antibody or antigen binding portion thereof is administered as an IV
infusion. In one
aspect, the anti-ICOS antibody or antigen binding portion thereof is
administered prior to the
anti-PD1 antibody or the antigen binding portion thereof or the anti-PD1
antibody or the
antigen binding portion thereof In one embodiment, administration of the anti-
PD1 antibody
or antigen binding portion thereof or the anti-PDL1 antibody or antigen
binding portion
thereof is initiated at a time point selected from 1 week, 2 weeks, 3 weeks,
and 4 weeks after
the start of the administration of said anti-ICOS antibody or antigen binding
portion thereof
In one aspect, the anti-PD1 antibody or antigen binding portion thereof or the
anti-PDL1
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antibody or antigen binding portion thereof is administered prior to the anti-
ICOS antibody or
the antigen binding portion thereof In one embodiment, the interstitial period
between the
start of the anti-PD1 antibody or anti-PDL1 therapy and the start of the anti-
ICOS antibody
therapy is selected from 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks,
and 6 weeks.
In one embodiment, the anti-ICOS antibody or antigen binding portion thereof
and
said anti-PD1 antibody or antigen binding portion thereof or anti-PDL1
antibody or antigen
binding portion thereof are administered to said human until said human shows
disease
progression or unacceptable toxicity. In one embodiment, methods are provided
for the
treatment of cancer further comprising administering at least one anti
neoplastic agent and/or
at least one immuno- modulatory agent to said human.
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,
T.S. Lawrence, and S.A. Rosenberg (editors), 10th edition (December 5, 2014),
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 or anti-mitotic 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 actinomycins, anthracyclins, and bleomycins; topoisomerase I
inhibitors such as
camptothecins; topoisomerase II inhibitors such as epipodophyllotoxins;
antimetabolites such
as purine and pyrimidine analogues and anti-folate compounds; hormones and
hormonal
analogues; signal transduction pathway inhibitors; non-receptor tyrosine
kinase angiogenesis
inhibitors; immunotherapeutic agents; proapoptotic agents; cell cycle
signalling inhibitors;
proteasome inhibitors; heat shock protein inhibitors; inhibitors of cancer
metabolism; and
cancer gene therapy agents such as genetically modified T cells.
Examples of a further active ingredient or ingredients for use in combination
or co-
administered with the present methods or combinations are anti-neoplastic
agents. Examples
of anti-neoplastic agents include, but are not limited to, chemotherapeutic
agents; immuno-
modulatory agents; immuno-modulators; and immunostimulatory adjuvants.
EXAMPLES
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The following examples illustrate various non-limiting aspects of this
invention.
Example 1
The study design of the anti-ICOS antibody / anti-PD1 antibody concurrent and
phased
dosing study conducted is shown in FIG. 1. FIG. 2 is a schematic showing the
study procedure
.. of anti-ICOS antibody / anti-PD1 antibody concurrent and phased dosing
study. Shown at the
bottom of FIG. 2 is a table listing antibodies used in the study. In FIGS. 3-
7, FIGS. 8A-8C,
FIGS. 9A-9C, and FIGS. 10-14, "Rt ICOS" refers to "rat anti-ICOS antibody;"
"Rt PD1" refers
to "rat anti-PD1 antibody." "Rt IgG2A" refers to "rat IgG2A;" "Rt IgG2B"
refers to "rat
IgG2B."
Monotherapy:
As shown in FIGS. 3, 4, 8B, 10 and 11, rat anti-mouse ICOS antibody (17G9) 100
lag
or 10 lag showed similar tumor growth rate (FIG. 3, FIG. 4, FIG. 8B) and
overall survival
(40%) (FIG. 10, FIG. 11).
Rat anti-mouse anti-PD1 antibody (200 lag) had no effect on tumor growth rate
(FIG.
3, FIG. 4, FIGS. 8A-8B). Overall survival was 10% (FIG. 10, FIG. 11).
Combination:
At day 10, concurrent dosing of anti-ICOS antibody (100 lag or 10 lag)
combined with
anti-PD1 antibody showed synergistic anti-tumor efficacy compared to mono or
phased
dosing regimen (FIGS. 3-7, 8A-8C, 9A-9C).
Mice in Group 12 treated with anti-ICOS lead-in/ anti-PD1 follow up dosing
showed
surprising and unexpected increase in long term survival. Regarding mouse long
term
survival (day 67 post 1st dose), 60% of mice from Group 12 (anti-ICOS lead in
followed by 6
doses of anti-PD1) showed complete response (6 mice were tumor free, 1 mouse
found dead
due to anti-drug antibodies (ADA)) (FIG. 10, FIG. 12, FIG. 14). Twenty percent
(20%) of
mice from Group 11 (anti-ICOS lead in followed by 6 doses of rat IgG2A) showed
complete
response (3 mice were tumor free, 3 mice were found dead due to ADA) (FIG. 10,
FIG. 12,
FIG. 14); the data is comparable to the anti-ICOS monotherapy data. Thirty
percent (30%) of
mice from Group 8 (anti-PD1 lead in followed by 3 doses of anti-PD1+ rat
IgG2b) showed
complete response (3 mice are tumor free) (FIG. 10, FIG. 12, FIG. 13); this
showed better
overall survival than 3 doses of anti-PD1 (10%, 1 tumor free mouse). Twenty
percent (20%)
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of mice from Group 9 (anti-PD1 lead in followed by 3 doses of anti-PD1+ anti-
ICOS)
showed complete response (3 mice were tumor free, 3 mice were found dead due
to ADA)
(FIG. 10, FIG. 12, FIG. 13). ADA occurred at the 4th and 5th doses.
The results described herein in Example 1 were obtained with the following
materials and
methods.
Mice, tumor challenge and treatment
All studies were conducted in accordance with the GSK Policy on the Care,
Welfare and Treatment of Laboratory Animals and were reviewed by the
Institutional
Animal Care and Use Committee (IACUC) either at GSK or by the ethical review
process at
the institution where the work was performed. 6-8 week old female BALB/c mice
(Envigo)
were utilized for in vivo studies in a fully accredited AAALAC facility.
5.0 x 104 cells/mouse of CT26 mouse colon carcinoma (ATCC CRL-2638) tumor
cells were inoculated subcutaneously into the right flank. Tumor volume and
body weight
data were collected using the Study Director software package (Studylog
Systems, South
San Francisco, CA, USA). Tumor volume was calculated using the formula: Tumor
Volume
(mm)3 =0.52* 1 *w2 where w = width and 1 = length, in mm, of the tumor. When
tumors
reached approximately 50-100 mm3, mice were randomized into various groups
(n=10/treatment group) based on tumor volume using stratified sampling method
in the Study
Log" software prior to initiation of treatment. Tumor measurement of greater
than 2,000
mm3 for an individual mouse and/or development of open ulcerations in tumor
and/or body
weight loss greater than 20% resulted in mice being removed from study. Dosing
started on
randomization day. Mice received the mouse anti-ICOS (clone 7E.17G9) and/or
mouse anti-
PD1 (clone RMP1-14) antibodies or saline via intraperitoneal injection twice
weekly starting
on randomization day for a total of 3 doses of anti-ICOS and 3 or 6 doses of
anti-PD1 for
concurrent and sequential dosing respectively. In order to evaluate anti-tumor
activity of
combining the anti-ICOS and anti-PD-1 monoclonal antibodies, mice were dosed
twice a
week with either anti-ICOS (clone 7E.17G9, rat IgG2b 100 lag) or its isotype
control (rat
IgG2b 100 lag) along with anti-PD-1 (clone RMP1-14, rat IgG2a 200 lag) or its
isotype
control (rat IgG2a 200 lag ) concurrently. For the experiments involving
sequential dosing,
either dosing with anti-ICOS antibody started after 3 doses of anti-PD1 which
meant that the
the last 3 of the 6 anti-PD1 doses were given in combination with anti-ICOS or
anti-PD1
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dosing started after all 3 doses of the anti-ICOS antibody were completed.
Apropriate
isotype controls were also employed in a similar dosing regimen.
Data are plotted using Graphpad software and Statistical analysis was
performed by
Statistician.
Example 2
Characterization of an IgG4 anti-ICOS agonist antibody that elicits T-cell
activation and
antitumor responses alone and with PD-1 blockade
Described in Example 2 is the characterization of the immune-stimulatory and
anti-tumor
activity of a humanized non-depleting anti-ICOS agonist antibody, with an
emphasis on the
importance of isotype choice for optimal efficacy and provides strong
rationale for exploring
this in cancer patients as a single agent and in combination with PD-1
checkpoint blockade.
Inducible T-cell Co-Stimulator (ICOS) is a T-cell-restricted co-stimulatory
receptor whose
expression is induced on activated T cells upon T-cell receptor engagement. We
demonstrate
that antibody-mediated ICOS agonism elicits potent T-cell activation,
mobilization of T cells
to the tumor site, and antitumor responses in syngeneic mouse models. Our data
indicate that
the isotype choice for the agonist antibody is crucial to avoid Fc-dependent
cytotoxicity and
depletion of effector T cells (Teff), as observed with an IgG1 version of the
antibody tested.
Furthermore, our data suggest that ICOS expression level on regulatory T cells
(Treg), albeit
high, offers a narrow window for selective depletion of Tregs in most tumors,
due to overlapping
ICOS levels on Teff and the upregulation of ICOS in the presence of checkpoint
blockade.
Exploration of isotypes led to the selection of a humanized IgG4 anti-ICOS
agonist antibody
(H2L5 IgG4PE) for clinical development. We present the characterization of the
immunological activity and therapeutic potential of this ICOS agonist
antibody, currently being
investigated alone and in combination with pembrolizumab in a first-in-human
clinical study.
Introduction
Inducible T-cell co-stimulator (ICOS) is a co-stimulatory receptor with
structural and
functional homology to the CD28/CTLA-4-Ig superfamily (Hutloff, A. Nature
397:263-266
(1999)). ICOS expression is upregulated by antigen stimulation and ICOS
signaling induces
production of both TH1 and TH2 cytokines and effector T-cell (Teff)
proliferation. ICOS
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expression has been observed on resting TH17, T follicular helper (TrH) and
regulatory T (Treg)
cells; however, unlike CD28, it is not highly expressed on most resting naïve
and memory T-
cell populations (Fazilleau, N. etal. Nat Immunol. 8(7):753-61. (2007),
Paulos, C.M. etal. Sci
Transl Med. 2(55): 55-78. (2010)). ICOS plays a crucial role in the survival
and expansion of
Teff and Treg during an immune response (Burmeister, Y. et al. J Immunol.
180(2): 774-82.
(2008)) and has been shown to be critical for the development and function of
TH17 (Paulos,
C.M. et al. Sci Transl Med. 2(55): 55-78. (2010), Guedan, S. et al. Blood
124(7): 1070-80.
(2014)).
Emerging data from patients treated with anti-CTLA-4 antibodies suggest ICOS-
expressing
memory T cells may help mediate antitumor immune responses and long-term
survival
(Liakou, C.I. etal. Proc Natl Acad Sci USA. 105(39): 14987-92. (2008); Di
Giacomo, A.M. et
al. Cancer Immunol Immunother. 62(6): 1021-8. (2013); Carthon, B.C. etal. Clin
Cancer Res.
16(10): 2861-71. (2010); Vonderheide, R.H. etal. Clin Cancer Res. 16(13): 3485-
94. (2010)).
ICOS has been shown to be critical for anti-CTLA-4 antitumor activity in mice
(Fu, T. He, Q.,
Sharma, P. Cancer Res. 71(16): 5445-54. (2011); Fan, X, etal. J Exp Med.
211(4):715-25.
(2014)) and prior reports support the concept that activating ICOS on CD4 and
CD8 T cells
using recombinant murine ICOS ligand has antitumor potential (Ara, G. et al.
Int. J Cancer.
103(4): 501-7 (2003)). Human ICOS ligand (ICOS-L) has been shown to bind both
CTLA-4
and CD28 in addition to ICOS, which limits the potential use of recombinant
ICOS-L as a
therapeutic in humans (Yao, S. et al. Immunity 34(5), 729-40. (2011));
necessitating an
alternative therapeutic approach to activate ICOS in patients with cancer.
Here, we describe the immunologic and antitumor characterization of a first-in-
class
humanized IgG4 anti-ICOS agonist monoclonal antibody (mAb) H2L5 IgG4PE,
designed to
deliver optimal ICOS agonism via Fc gamma receptor (FcyR) cross-linking, with
minimal
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antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis activity;
thereby reducing
the risk of Teff depletion. The comprehensive preclinical data described
herein, support clinical
testing of H2L5 IgG4PE, currently being investigated alone and in combination
with
pembrolizumab in a first-in-human clinical study.
Results
Development of a potent and selective anti-human ICOS agonist monoclonal
antibody
(mAb)
We undertook the generation of an agonistic anti-human ICOS mAb by immunizing
mice with
ICOS extracellular domain. One of these mAb was humanized and expressed as a
human IgG4
with 2 Fc mutations (glutamic acid for leucine at residue 235) (Kabat, E. A.,
et al. Sequences
of Proteins of Immunological Interest, 5th Ed. U.S. Dept. of Health and Human
Services,
Bethesda, MD, NIH Publication no. 91-3242. (1991)) and substitution of proline
for serine at
residue 228 (EU numbering) to reduce antigen binding fragment (Fab) arm
exchange with
native IgG4 (Manjula, P. etal. The Journal of Immunology. 164:1925-1933.
(2000), Rispens,
T. et al. J. Am. Chem. Soc. 133 (26):10302-10311. (2011)). The resulting H2L5
IgG4PE
hereafter referred to as "H2L5".
H2L5 bound to human ICOS with an affinity of 1.34 nM (FIG. 15A), which is
approximately
17-fold higher than the native ICOS-L/CD275 interaction (FIG. 15B). H2L5 did
not bind to
murine ICOS or to human CD28 or CTLA-4, the two nearest structurally related
protein. This
contrasts with the native human ICOS-L, which binds both CTLA-4 and CD28 (Yao,
S. etal.
Immunity 34(5), 729-40. (2011)). H2L5 blocked ICOS/ICOS-L binding by flow
cytometry
and competed partially (<50%) with ICOS-L in binding to ICOS at concentrations
above 1
ug/mL in MSD immunoassays (FIGS. 22A-22B). H2L5 also bound to both CD4 and CD8
T-
cells in activated PBMC samples from healthy human donors (FIG. 15C). ICOS has
previously been shown to activate AKT in response to ICOS-L binding in human
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T cells (Okamoto, N. etal. Biochem Biophys Res Commun. 310(3): 691-702.
(2003)); pre-
activated primary human CD4 T cells demonstrated an increase in pAKT and
pGSK3I3 in
response to treatment with H2L5 (FIG. 15D). H2L5 significantly increased CD4
and CD8 T-
cell activation, when used in a plate-bound format together with anti-CD3, as
measured by
CD69 expression (FIG. 15E) and proliferation (FIG. 15F). Minimal activation
was observed
with H2L5 in the absence of anti-CD3 treatment, indicating that it does not
have superagonist
activity under these assay conditions.
The plate-bound H2L5 antibody induced a dose-dependent increase in TH1, TH2
and TH17
cytokines, IFN-y, TNF-a, IL-17a, IL-10, IL-6 and to a lesser extent IL-2, IL-5
and IL-13 in
PBMC from healthy donors (HD) (FIG. 15G, FIG. 23A, FIG. 33). A similar profile
of cytokine
induction was observed in PBMC from NSCLC patients, with strong induction of
IFN-y, and
lower levels of other cytokines including TNF-a, IL-10 and IL-2 (FIG. 15H,
FIG. 34). A dose-
dependent increase in T-cell activation markers: CD25, 0X40 and CD69 on both
CD4 and
CD8 T cells was also observed with HD following stimulation with plate-bound
anti-CD3 and
H2L5 (FIG. 23B). With isolated human CD3+ T cells, treatment with H2L5 led to
a significant
increase in the mRNA expression of the TH1 transcription factor T-Bet (FIG.
151) as well as
the cytotoxic molecule Granzyme-B (FIG. 15J). A significant decrease in L-
Selectin expression
was observed, indicating a transition towards an activated effector phenotype
(FIG. 15K). The
ability of the plate-bound H2L5 antibody to costimulate T cells isolated ex
vivo from
disaggregated tumors in the presence of anti-CD3 after 6 days of culture, was
also assessed. A
concentration dependent and robust increase in IFN-y was seen in 9/10 donors
(FIG. 15L),
along with less robust induction of IL-17 and IL-10 compared with healthy
PBMC, as well as
low-undetectable levels of TH2 cytokines (IL-5 and IL-13) (FIG. 24A-D).
Significant increases
in activation markers 0X40 (FIG. 15M), CD25 (FIG. 15N) and LAG3 (FIG. 25A)
were
observed on CD8 T cells, in addition to a modest increase in CD8+PD-1+ cells
and
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CD4+CD25+Foxp3+(Treg) cells (FIGS. 25B-25D). Only low percentage of ICOS-L
expressing
cells were observed with most donors (FIG. 25C)
Altogether, these data show that H2L5 is a potent ICOS agonist, capable of
driving T-cell
activation and proliferation, but is not a superagonist capable of driving T-
cell activation in the
absence of TCR stimulation.
Antibody isotype and FcyR-engagement is critical for H2L5 function
FcyR-mediated crosslinking is critical for agonist antibody function (Dahal,
L.N. etal..
Immunol Rev. 268(1): 104-22. (2015); Furness A.J. etal. Trends in Immunology
35(7): 290-
298 (2014)). The results described in FIG. 15 utilized plate-bound antibody,
which
overcomes the need for FcyR cross-linking and suggests that an antibody
isotype capable of
engaging FcyR and mediating crosslinking is key to achieving optimal ICOS
agonism. To
formally assess this, we cloned the heavy and light chain variable regions of
H2L5 and
expressed them as different human IgG isotypes (IgGl, IgG2, IgG4PE and IgG1 Fc-
disabled
[amino acid (AA) substitutions L235A and G237A) (Bartholomew, M. etal.
Immunology
85(1): 41-8 (1995)). The binding of the different H2L5 isotype variants was
determined
against human FcyRI, FcyRIIa (H131), FcyRIIa (R131), FcyRIIb, FcyRIIIa (V158)
and
FcyRIIIa (F158) and demonstrated expected patterns of binding (FIG. 35). The
IgG4PE
contained two AA substitutions from native human IgG4; glutamic acid for
leucine at residue
235 (Kabat, E. A., etal. Sequences of Proteins of Immunological Interest, 5th
Ed. U.S. Dept.
of Health and Human Services, Bethesda, MD, NIH Publication no. 91-3242.
(1991)) and
substitution of proline for serine at residue 228 (EU numbering) to reduce
antigen binding
fragment (Fab) arm exchange with native IgG4 (Manjula, P. etal. The Journal of
Immunology. 164:1925-1933. (2000), Rispens, T. etal. J. Am. Chem. Soc. 133
(26):10302-
10311. (2011)) and decrease binding to activating FcyR and Clq, while
retaining binding to
the inhibitory FcyRIIb. In PBMC assays the H2L5 IgG1 antibody decreased both
CD4 and
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CD8 T-cell proliferation when added in solution in greater than 50% of donors
tested (FIG.
16A). In contrast, IgG2, IgG4PE and Fc-disabled isotype variants of H2L5 did
not result in
substantial inhibition of either CD4 or CD8 T-cell proliferation in any donors
tested, while
the H2L5 IgG4PE format resulted in increased proliferation in a subset of the
donors (FIG.
16A). We next tested whether the inhibitory effect of H2L5 IgG1 was due to
ADCC via NK
cells in the PBMC mixture. In PBMCs from 10 healthy donors (HD), the
inhibitory effect of
H2L5 IgG1 on both CD4 and CD8 populations was lost when NK cells were removed
from
the PBMC pool (FIG. 16B). The H2L5 isotype variants were also tested in a
reporter assay
that detects engagement of FcyRIIIa, the primary activating FcyR responsible
for NK-
mediated ADCC in humans. While the H2L5 IgG1 induced a significant increase in
luciferase signaling, when incubated with activated T cells, neither the H2L5
IgG4PE nor
H2L5 Fc-disabled antibodies induced FcyRIIIa-mediated signaling (FIG. 26A).
Additionally,
H2L5 IgG1 induced T-cell death in an NK-dependent manner whilst neither IgG4PE
nor Fc-
disabled H2L5 resulted in any significant increase in cell death as compared
to isotype
controls (FIG. 16C).
Previous studies have reported that receptor density may influence the
susceptibility of T
cells to killing by ADCC, leading to potential preferential depletion of
different T-cell
subsets, which may differ in the tumor microenvironment compared to the
lymphoid tissue
(Furness A.J. etal. Trends in Immunology 35(7): 290-298 (2014)). The level of
expression of
ICOS on CD4, CD8 and Treg freshly isolated from different tumors was
determined by flow
cytometry. Although there was higher expression observed on Treg vs CD4 and
CD8 T cells,
this was heterogeneous with some tumors showing overlapping levels between
these
populations; consequently, ICOS high expression was not a distinct feature of
Treg (FIG.
16D). To further evaluate the relative contribution of Fc isotype on potential
depletion by
ADCC, the CD4, CD8 and Treg cells were purified directly from different cancer
patients and
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the level of ICOS receptor density was correlated with ability of the IgG1 or
IgG4PE isotype
of H2L5 to stimulate FcyRIIIA in an ex vivo reporter assay (FIG. 16E, FIG.
26B). T cells
isolated from tumors did not stimulate FcyRIIIa when incubated with the H2L5
IgG4PE
isotype; whereas incubation with H2L5 IgG1 did lead to some (variable)
stimulation. In some
tumors, FcRyIlla receptor engagement was seen with Treg, CD4 and CD8,
especially at doses
of 1-10 ug/mL supporting the idea that selective ADCC deletion of Treg without
affecting
CD4 and CD8 may not be universally possible in all tumors (e.g. Breast 1001202
patient
sample conventional CD4 T cells induced similar stimulation to the Treg at
doses of 1-
ug/m1; FIG. 16F, FIG. 26B).
10 Based on the above data, the isotype selected for development was the
engineered IgG4PE
antibody, H2L5.
H2L5 induces FcyR-mediated agonism of TCR dependent T-cell activation.
H2L5 was tested with isolated human CD4 T cells in both a plate-bound
(immobilized
antibody) format as well as in solution. H2L5 in the immobilized format, which
simulates
membrane-bound FcyR-dependent crosslinking, induced significantly greater
levels of IFN-y
compared with the soluble antibody (FIG. 17A). The importance of FcyR
engagement for
optimal H2L5 agonist activity was further confirmed in an activated human PBMC
assay,
where H2L5 resulted in >2-fold induction of IFN-y; whereas the Fc-disabled
version of H2L5
had no cytokine induction activity compared with the isotype control (FIG.
17B). The IgG4PE
and Fc-disabled versions of H2L5 were also tested in a modified mixed
lymphocyte reactions
(MLR). The H2L5 IgG4PE mAb provided >2-fold induction of IFN-y whereas the Fc-
disabled
H2L5 mAb had no activity compared with the isotype control (FIG. 17C). Next, a
CD4 T
cell/CD14 monocyte donor-matched co-culture assay was utilized to determine
whether FcyR-
expressing monocytes increased the agonist potential of soluble H2L5. Like the
MLR assay
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format, H2L5 only induced IFN-y when tested as an IgG4PE isotype; the Fe-
disabled antibody
showed no significant cytokine induction compared with the isotype control.
The addition of
monocytes, which are known to express FcyRs including FcyRII isoforms,
resulted in a
significant increase in H2L5 IgG4PE-induced cytokine production compared with
T cells
alone. Interaction with FcyRIIB has been shown to be critical for the
agonistic activity of other
immunomodulatory antibodies targeting TNF-a family receptors as well as CD28
(Bartholomew, M. et al. Immunology 85(1): 41-8 (1995); Bartholomaeus, P. et
al. J
Immunol. 192(5): 2091-8. (2014)). Conversely, the addition of an FcyR-blocking
antibody
completely inhibited the H2L5-induced cytokine induction (FIG. 17D). These
results indicate
.. that H2L5 can achieve FcyR engagement likely via the FcyRIIB as seen with
other IgG4 agonist
antibodies (Bartholomaeus, P. et al. J Immunol. 192(5): 2091-8. (2014),
Hussain, K. et al.
Blood 125 (1): 102-110 (2015)), while avoiding ADCC killing of ICOS T cells,
as seen with
the IgG1 isotype.
To assess its localization and mobilization at the cell surface, H2L5 was
fluorescently labeled,
added to primary activated human CD3+ T cell cultures, alongside DCs and
imaged. Following
binding, H2L5 rapidly polarized on the T cell surface. The mobilized T cells
began scanning
the culture until binding with a dendritic cell (DC) was initiated. In
instances where T cells
were in cellular contact with DCs, H2L5 accumulated at the point of contact
(FIG. 17E).
Additional studies using co-cultures of human DC and T-cells demonstrated that
H2L5 was
.. rapidly co-localized with CD28 and to a lesser extent CD4 at the polarized
caps of activated T
cells as well as the subsequent immune synapses that formed upon T-cell
binding to DC (FIG.
17F). These results indicate that ICOS induces human T-cell mobilization and
is co-located at
the immune synapse following T cell activation.
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H2L5 induces an effector memory phenotype and antitumor activity in vivo
The in vivo functionality of H2L5 was evaluated in a human PBMC engrafted NSG
mouse
model implanted with A2058 tumors. This model induces a Graft-versus-Host
Disease
(GVHD) response and has been used previously to study effector and memory T-
cell activity
(23). In the blood of H2L5-treated mice, the number of human T cells decreased
in a dose-
dependent manner (FIG. 18A), while a corresponding increase in CD69 expression
(representing T-cell activation) was observed (FIG. 18B). The Fc-disabled
version of H2L5
showed similar, albeit weaker, trends than H2L5 IgG4PE suggesting that the
disappearance of
cells was not due to ADCC. H2L5 induced a dose-dependent increase in
CD4+CD45RO+CD62L- effector memory (TEm) cells (FIG. 18C), and CD8 CD45RO-CD62L-
terminally differentiated CD8 effector cells (TEMRA) (FIG. 18D). H2L5 was next
tested in
human PBMC engrafted NSG mice harboring either HCT116 or A549 tumors.
Detection of
H2L5 binding to ICOS+ T cells (CD4, CD8 and Treg), by a human anti-IgG4
fluorescent
labelled antibody, was observed in blood and tumor at doses of 0.4 and to
lesser extent
0.04 mg/kg demonstrating target engagement in mice bearing the A549 tumors
(FIG. 18E,
FIGS. 27A-27B). Mice treated with anti-PD-1 IgG4 antibody (Keytruda) also
showed the
detection of bound antibody using the same detection reagent. Treatment of
mice with H2L5
was associated with an increase in the CD8:Treg ratio in the A549 tumors,
comparable to that
seen in mice treated with anti-PD-1. (FIG. 18F). H2L5 resulted in significant
tumor growth
inhibition in both HCT116 and A549 tumor models (FIGS. 18G-18H). In the A549
model
where the GVHD response was less severe, tumor growth inhibition resulted in
dose-dependent
increase in survival beyond 50 days (FIG. 181). These experiments suggest that
doses of 0.4
mg/kg, which correlate with successful engagement of the ICOS receptor, result
in subsequent
pharmacological effects associated with T-cell activation in blood and tumor
and reduction of
tumor growth.
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The Fc Isotype of murine anti-ICOS antibody influences efficacy in syngeneic
tumors
Studies in the literature using CTLA-4, PD-L1, 0X40 and CD40 have shown that
selection of
the Fc isotype of mAbs can significantly influence efficacy in different tumor
models (Dahal,
L.N. et al. Immunol Rev. 268(1): 104-22. (2015); Furness A.J. et al. Trends in
Immunology
35(7): 290-298 (2014)). To generate a surrogate mouse anti-ICOS antibody
equivalent to a
human IgG4 in terms of FcyR binding, (FIG. 36) the anti-mouse ICOS antibody 7E-
17G9 was
cloned into murine (m) IgG1 and mIgG2a isotypes and tested in 2 different
tumor models. The
7E-17G9 antibody showed agonistic activity in plate-bound format with anti-CD3
(FIG. 28).
In the EMT6 model the mIgG1 antibody showed greater efficacy than the mIgG2a
especially
at higher doses (>5 mg/kg, 100 [tg/mouse) with both survival (FIG. 19A) and
tumor growth
inhibition (FIG. 29A). However, both isotypes showed only modest dose-
dependent efficacy
as monotherapy in the CT26 model (FIG. 19B, FIG. 29B). As described above for
the human
IgGl, the mIgG2a depleting antibody may be less effective than the mIgGl,
since it has the
potential to deplete both Teff and Treg. A significantly higher CD8:Treg ratio
(FIG. 19C) was
.. observed for EMT6 vs CT26, prior to treatment (100 mm3) and both EMT6 and
CT26 models
showed an increase in the percentage of ICOS positive CD4 and CD8 and Treg
cells in tumor
vs spleen (FIG. 19D, FIG. 30) but higher percentage of ICOS CD8 positive cells
were observed
in spleen in EMT6 vs CT26 (80% vs 10%). Although high levels of ICOS
expression on Treg
from tumor-infiltrating lymphocytes (TILs) were observed for both EMT6 and
CT26 tumors,
ICOS levels on CD8 TILs were approximately 10-fold higher in EMT6 than CT26
(30,000 vs
3000 MFI). This suggests that high ICOS expression on CD8 in both periphery
and tumor may
be associated with response with the agonistic activity of the mIgG1 antibody
in the EMT6
model (FIGS. 19E-19G). To further explore mechanisms of the agonist anti-ICOS
mAb in mice
bearing EMT6 breast tumors, effects on TCR diversity were investigated;
significant changes
in the number of unique circulating TCR clones and a corresponding increase in
TCR clonality
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in the blood of ICOS mAb treated mice was noted (FIGS. 31A-31C). Most clones
that
expanded in mouse blood in response to ICOS agonist mAb treatment were also
found in
tumors (FIG. 19H). These findings indicate that a small pool of tumor-reactive
T-cell clones
expand in response to ICOS mAb treatment.
Characterization of the ICOS / ICOS-L pathway in human cancers
To further explore the translation of an ICOS agonist mAb as an anti-tumor
therapeutic
antibody, human solid tumors from the TCGA database were ranked by ICOS mRNA
expression (FIG. 26). Highest expression was observed in head and neck,
gastric, esophageal,
melanoma, NSCLC, cervical and breast cancer. Expression was confirmed in NSCLC
by
singleplex IHC (FIG. 32). As the H2L5 agonist mAb mode of action is designed
to phenocopy
ICOS-L activity, the co-expression of mRNA for ICOS and ICOS-L was analyzed in
these
tumor types (FIG. 20A). ICOS expression was often not co-expressed with ICOS-
L, supporting
the hypothesis that H2L5 may augment the low level ICOS signaling in these
tumors. We also
assessed the relative expression of PD-Li in the same samples. Expression of
PD-Li has been
associated with increased T-cell infiltration and used as a predictive
biomarker to enrich for
patients responding to anti-PD-1/PD-L1 treatment in different indications.
Overall there was a
clear association between PD-L1 and ICOS expression but this was variable
between different
indications (FIG. 20A). These results were confirmed by IHC staining for
expression of CD4,
CD8 and FOXP3 and tended to localize with ICOS in immune infiltrates in NSCLC
(FIG.
20B).
The presence of key cell types in the tumor microenvironment was analyzed by
flow cytometry
in biopsies from different tumor types. Of the CD45+ leukocyte population, CD3
T cells
appeared to be the dominant cell type, ranging from 20-80%; other cell types
such as B cells,
macrophages, monocytes, NK cells and DC were also present (FIG. 20C). These
cells types
express FcyR including FcyRIIb, which may provide the cross-linking required
for agonistic
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activity of H2L5 in the tumor microenvironment (Furness A.J. et al. Trends in
Immunology
35(7): 290-298 (2014)). The composition of the T-cell sub-populations averaged
CD4 (68%),
CD8 (30%) and Treg (2%) although there was considerable heterogeneity between
different
tumors. When tumor types were analyzed separately, the heterogeneity between
CD8 and Treg
was clear with NSCLC and RCC showing a high CD8/Treg ratio (FIG. 20D). Further
analysis
by multiplex IHC was performed to characterize ICOS expression of different T-
cell sub-types.
Co-expression of ICOS was observed on a proportion of CD3+PD-1+ cells,
especially in head
and neck, esophageal, NSCLC and melanoma supporting a rationale for
combination treatment
with anti-PD-1 therapies (FIGS. 20E-20F).
Next, the effects of H2L5 costimulation on gene expression by purified human T
cells was
determined using the Human PanCancer-Immune profiling panel to identify an
ICOS gene
signature. Compared with anti-CD3 alone, 120 genes were differentially induced
with 85 up-
regulated and 35 down-regulated (FIG. 20G). Several immune related genes or
pathways were
induced by H2L5 compared to anti-CD3 alone including TH1 cytokines, and
chemokines, T-
cell function and cytotoxicity, and TNF family members (FIG. 38). The top
genes identified
from EMT6 mouse tumors treated with 7E. 17G9 that overlapped with the human
ICOS-
induced signature are shown in FIG. 20H. This information is being used to
guide development
of an ICOS transcriptional signature to monitor pharmacodynamic effects of
H2L5 in early
clinical studies.
.. ICOS agonist treatment induces PD-1/PD-L1 in tumors and demonstrates
increased
activity in combination with anti-PD-1 blockade
PD-L1, a known IFN-y responsive gene, as well as PD-1, increased significantly
in the tumors
of ICOS mAb treated mice (FIGS. 21A-21B). Human PBMCs were collected from six
cancer
patients and treated with H2L5, which resulted in a significant increase in PD-
1 expression on
both CD4 and CD8 T cells (FIG. 21C). In addition, NSCLC and melanoma patients
treated
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with anti-PD-1 therapies showed an increase in ICOS expression on CD4 T cells
in peripheral
blood compared with pre-treatment (FIG. 21D). Therefore, we tested whether
combination
with a PD-1 blocking antibody could augment the antitumor activity of the ICOS
agonist mAb.
The ICOS agonist mAb (7E17G9 mIgG1 isotype) was dosed alone or in combination
with anti-
PD-1 antibody in mice with established EMT6 tumors (150 mm3). Combination
resulted in an
increased antitumor response and long-term survival (90% of mice) as compared
with
monotherapy treatment with ICOS or PD-1 antibodies alone (FIG. 21E). The
combination of
H2L5 and anti-PD-1 (pembrolizumab) was also assessed in the humanized mouse
model and
resulted in enhanced antitumor response to A549 tumors compared with
monotherapy alone
(FIG. 21F) These data show that the addition of an ICOS agonist antibody
significantly
improved the antitumor activity induced by a PD-1 antibody.
H2L5 was further tested alone or in combination with pembrolizumab in primary
resected
tumors from 6 patients with NSCLC in an ex vivo assay. While treatment with
H2L5 alone
resulted in a significant increase in IFN-y in 4/6 of the NSCLC tumor samples
tested, the
combination of H2L5 and pembrolizumab resulted in a significant increase in
IFN-y as
compared to pembrolizumab alone and an increase in 5/6 samples as compared to
H2L5
treatment alone (FIG. 21G). The H2L5 combination with pembrolizumab was also
tested in a
modified allogeneic human MLR assay where combination treatment resulted in
increased
IFN-y levels as compared to either agent alone in 3/3 different healthy donor
pairs (FIG. 21H).
Discussion
We have presented the first full characterization of the immunological
activity and therapeutic
potential of a first-in-class, humanized IgG4 anti-ICOS agonist mAb, H2L5. We
have
demonstrated that the H2L5 IgG4PE agonist antibody induces significant
activation and clonal
expansion of both CD4 and CD8 T cells in vitro and in vivo. These T cells have
increased
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effector function through increased expression of TH1 cytokines such as IFN-y,
as well as
increased production of cytotoxic factors such as Granzyme B. ICOS antibody-
activated T cells
displayed increased tissue-homing to tumors with significant accumulation and
infiltration
resulting in antitumor responses. Prior reports using ICOS-/- and ICOS-L-/-
mice as well as
blocking antibodies to ICOS-L have demonstrated the importance of ICOS for the
expansion,
survival and function of both CD4 and CD8 TEm cells in mice (4, 24-25).
Additionally, patients
with common variable immune deficiency, which is characterized by a homozygous
loss of
ICOS, have been found to have fewer memory T cells, specifically those which
are CD62L10w
(26). Our studies using a novel human ICOS-specific agonist antibody have
confirmed the role
of ICOS for inducing this population of memory T cells, providing a viable
therapeutic
approach for targeting this important mechanism in humans.
We show that the engineered form of IgG4 that incorporates the mutations 5228P
and L235E
(EU numbering) relative to the native human IgG4 is the preferred antibody
isotype over
IgG1 for achieving agonist function against human ICOS. These AA changes
prevent
heterogeneous exchange with native IgG4 (Rispens, T. etal. J. Am. Chem. Soc.
133
(26):10302-10311. (2011)). The IgG4PE isotype also has reduced binding to
activating FcyR
and Clq compared to human IgGl, thereby diminishing the cytotoxic potential of
H2L5 that
could result in depletion of ICOS-positive T cells through antibody-dependent
or
complement-dependent mechanisms, respectively (Manjula, P. et al. The Journal
of
Immunology. 164:1925-1933. (2000)). Our in vitro studies have shown that the
IgG1 isotype
of H2L5 (the initial isotype of H2L5 planned for development) is able to kill
activated CD4
and CD8 T cells expressing high levels of ICOS, as well as reduce their
proliferation in an
NK-dependent manner; this was not seen not seen with the IgG4PE isotype.
Importantly, the
IgG4PE isotype retains functional binding to FcyRlIb (the inhibitory FcyR),
critical for
enabling agonist activity against several stimulatory immune receptors
(Bartholomaeus, P. et
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al. J Immunol. 192(5): 2091-8. (2014); Hussain, K. etal. Blood 125 (1): 102-
110 (2015),
Aalberse, R.0 and Schuurman, J. Immunology 105(1): 9-19. (2002); Schuurman J.
and
Parren P.W. Curr Opin Immunol. (2016); White A.L. etal. J Immunol. 187(4):1754-
63.
(2011); White A.L. etal. J Immunol 193 (4): 1828-1835 (2014); Dahal R. etal.
Cancer Cell.
29(6):820-31. (2016); Yu X. etal. Cancer Cell 33 (4): 664-675 (2018)), which
may also be
essential for ICOS agonist activity and associated antitumor effects in
humans. The selection
of the IgG4PE isotype was further supported by in vivo studies using the anti-
murine ICOS
7E17G9 surrogate antibody, where the murine IgG1 isotype showed greater
efficacy than the
deleting IgG2a antibody in the EMT6 syngeneic model. Murine IgG1 has a similar
profile to
human IgG4, with low binding to activating FcyR receptors, yet retaining
binding to some
Fcy-receptors including, inhibitory FcyRIIB and inducing Fc-dependent
crosslinking to
improve agonism of the anti-ICOS antibody; whereas the murine IgG2a can bind
the
activating FcyR, like human IgGl, and is able to mediate effective deletion.
Studies
performed with CTLA-4, PD-L1, 0X40 and CD40 have shown that selection of the
Fc
isotype of mAbs can significantly influence efficacy in different tumor
models; however, this
needs to be optimised for each target, depending on relative expression levels
on different
cell types (e.g. CD8 vs Teff VS Treg) and mode of action of the antibody
(agonism/deletion)
and epitope specificity (Dahal, L.N. etal. Immunol Rev. 268(1): 104-22.
(2015), Furness A.J.
etal., Trends in Immunology 35(7): 290-298 (2014), Yu X. etal. Cancer Cell 33
(4): 664-
675 (2018)). Ex vivo human tumors contain varying proportions of B cells,
macrophages and
DCs, known to express FcyRIIB, which is critical to mediate the FcyR
crosslinking required
for H2L5 in the tumor microenvironment (Furness A.J. etal. Trends in
Immunology 35(7):
290-298 (2014), Hussain, K. etal. Blood 125 (1): 102-110 (2015), Aalberse, R.0
and
Schuurman, J. Immunology 105(1): 9-19. (2002); Schuurman J. and Parren P.W.
Curr Opin
Immunol. (2016); White A.L. etal. J Immunol. 187(4):1754-63. (2011); White
A.L. etal. J
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Immunol 193 (4): 1828-1835 (2014); Dahal R. etal. Cancer Cell. 29(6):820-31.
(2016); Yu
X. etal. Cancer Cell 33 (4): 664-675 (2018)). A balance in favour of
inhibitory FcyRIIB vs
activating FcyR is often seen in the immunosuppressive environment of human
tumors, which
may favor the cross-linking of H2L5 and enhance its agonist activity (Furness
A.J. etal.
Trends in Immunology 35(7): 290-298 (2014), Dahal, L. etal. Cancer Research 77
(13)
3619-3631 (2017)).
Given the agonist activity of H2L5 described above, one factor which must be
considered, is
the expression of ICOS on Treg cells in the tumor microenvironment. The
relationship of ICOS
positive T-cell subsets on response to the murine 7E.17E7 IgG1 surrogate
antibody in the EMT-
6 and CT26 murine tumor models was explored. A higher ratio of ICOS+ CD8: Treg
was
observed at baseline in the EMT6 model vs CT26 in tumors, which may be one
factor leading
to greater efficacy in EMT6 model observed with 7E.17G9 IgGl. Similarly, in
the humanized
mouse model tumor reduction by H2L5 was associated with an increased CD8:Treg
ratio. In
this model, the response to treatment with H2L5 monotherapy was similar to
anti-PD-1
treatment. These results suggest that the presence of ICOS-positive Treg does
not preclude the
ability of an ICOS agonist to provide therapeutic benefit.
Human tumors express varying proportions of CD4 and CD8 Teri. and Treg, with
considerable
variability between tumor types. The percentage of ICOS positive cells and the
level of ICOS
expression was found to be heterogeneous between different cell types, with a
trend for higher
ICOS levels on Treg although in many patients there was overlap between
expression of ICOS
on Treg vs CD4 and CD8 T cells. The IgG1 isotype but not the IgG4 isotype of
H2L5 anti-ICOS
antibody was able to bind and induce activation of the FcyRIIIA luciferase
reporter assay with
of ex vivo purified Treg, and to some extent CD8 and CD4 cells. While the IgG1
mediated
activity in this assay system was found to correlate with ICOS receptor
density, as has been
reported with other targets such as CTLA-4, 0X40 and GITR (19), the
differential expression
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of ICOS on Treg vs Taf was less prominent. Furthermore, since ICOS expression
on Teff is
enhanced with anti-PD-1 and anti-CTLA-4 treatment, this suggests that there
may not be a
large therapeutic window for the IgG1 isotypes to mediate selective depletion
of Treg over Teff
in vivo. Based on the data described above, strategies for the development of
H2L5 include
selection of tumor types which have a high CD8:Treg ratio and high ICOS
expression on CD8
T cells, (e.g., NSCLC) and development of rational combinations with agents
that decrease the
abundance of, or limit the function of, Treg.
A rational combination partner supported by our data is a PD-1/PD-L1 blocking
antibody.
ICOS agonist antibody treatment significantly induced PD-1 on human T cells as
well as PD-
1 and PD-Li expression in tumors of treated mice; furthermore, anti-PD-1
treatment was also
shown to induce expression of ICOS on CD4 and CD8 Teri. cells. Like the
combinatorial activity
observed in mice, the human ICOS agonist H2L5 in combination with the PD-1
blocking
antibody, pembrolizumab, demonstrated increased cytokine production relative
to either agent
alone in ex vivo human immune cell assays. This robust induction of IFN-y by
H2L5 IgG4PE
supports the rationale of anti-PD-1 combination as IFN-y is known to act on
negative feedback
by up-regulation of PD-Li (Mandal, M. etal. Clinical cancer Research
22(10):2329-2334).
Single-agent treatment with anti-PD-1 or anti-PD-Li antibodies has
demonstrated response
rates between 15-30% across many solid tumors (e.g. bladder, head and neck,
lung) (Hoos, A.
Nat. Rev. Drug Disc. 15(4):235-47. (2016)). Emerging clinical data using PD-1
or PD-L1
antibodies in combination with other agents has shown signals of increased
activity in some
settings, however with substantial added toxicity in some instances (Larkin,
J. et al. N Engl J
Med. 373:23-34. (2015) ; Forde P.M., etal. New England Journal of Medicine
(2018); Xu,X.
etal. Int. J Cancer. 142: 2344-2354 (2018)). Several predictive biomarkers
have shown
mechanism of response and resistance to anti-PD-1 treatment and support
rationale for
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combinations (Gibney G.T. etal. Lancet Oncology 17(12): 542-551(2016); Tumeh
P.C. etal.
Nature. 515 (7528): 568-71. (2014); Taube, T.M. etal. Clin. Cancer Research.
20 (19):5064-
5074 (2014)). It has been shown the tumor mutational burden, is a correlate of
the generation
of neoantigens which stimulate expansion of the endogenous tumor specific
repertoire and is
associated with response to anti-PD-1 therapy (Schumacher, T.N. and Schreiber,
RD.
Science 348: 69-74 (2015)). The clonality and degree of T-cell infiltration in
tumors has
recently been shown to be an important positive predictor for immunotherapy
outcomes in
cancer (Xu,X. etal. Int. J Cancer. 142: 2344-2354 (2018)). Our findings
demonstrate that
with the murine surrogate antibody, TCR clones were expanded and shared
between both
blood and tumor. Furthermore, H2L5, which induces T-cell proliferation,
expansion, and
tumor infiltration, may be complimentary to other immunotherapy agents with
distinct
mechanisms of action.
Results described in Example 2 were obtained using the following materials and
methods:
Materials and Methods
.. Humanized H2L5 antibody
H2L5 is a humanized variant of the murine mAb clone 422.2 obtained from the
lab of Daniel
Olive, Institut Paoli-Calmettes, INSERM (Marseille, France). The murine
antibody was
generated using standard hybridoma technology by immunizing BALB/c mice
intraperitoneally with recombinant human ICOS-Fc using C057 cells.
Cell lines and primary cell cultures
Murine tumor cell lines EMT6 (ATCC# CRL-2755) and CT26 (ATCC# CRL-2638) and
human cell lines A549 (ATCC# CCL-185) A2058 (ATCC# CRL-11147), HCT116 (ATCC#
CCL-247) were expanded and frozen upon receipt and used at low passage (< 10
passages) for
inoculation to mice. Prior to in vivo use, cell lines were tested by PCR and
confirmed negative
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for pathogens including mycoplasma using the mouse/rat comprehensive CLEAR
panel
(Charles River Research Animal Diagnostic Services).
All patient material was obtained with the appropriate informed written
consent in accordance
with the GSK human biological sample management (HBSM) policy and SOP. Whole
blood
in sodium heparin tubes (BD Biosciences) and surgically resected tumor tissues
from cancer
patients were obtained from Avaden Biosciences (Seattle) shipped overnight by
post. Primary
T cells or PBMC from healthy human donors were purified from whole blood
collected in
sodium heparin tubes at the GSK on-site blood donation units with appropriate
consent and in
accordance with the GSK HBSM policy. PBMC were isolated by density gradient
centrifugation through Histopaque. T-cells were isolated by negative selection
using
DynabeadsTM UntouchedTM Human T-cell kit (Life Technologies) or RosetteSep
human CD4
or CD8 T-cell enrichment kits (StemCell) for binding and functional assays.
Isolated T cells
were pre-activated with plate-bound anti-CD3 (clone OKT3, eBioscience) and
anti-CD28
(clone CD28.2, eBioscience) for 48-96hrs to upregulate ICOS expression.
Mice, tumor challenge and treatment
All studies were conducted in accordance with the GSK Policy on the Care,
Welfare and
Treatment of Laboratory Animals and were reviewed by the Institutional Animal
Care and Use
Committee either at GSK or by the ethical review process at the institution
where the work was
performed. 6-8 week old female BALB/c mice (Harlan/Envigo) were utilized for
in vivo studies
in a fully accredited AAALAC facility. 5 x 104 cells/mouse CT26 mouse colon
carcinoma or 1
x 105 EMT6 mouse mammary carcinoma tumor cells were inoculated subcutaneously
into the
right flank. Prior to initiation of treatment, mice (n=10/treatment group)
were randomized with
the Study Director software package (Studylog Systems) when the tumors reached
100 mm3
unless otherwise specified. ANOVA was used to ensure similarity between groups
(P>0.9).
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The study investigator was blinded during the group allocation and assessed
the final outcome
to ensure that group distributions were acceptable for study initiation
(P>0.9).
Based on inter-individual variability in tumor growth rates from 5 separate
studies in the CT26
syngeneic model, 10 mice per group were justified as the optimal number
necessary to observe
an effect size of approximately 0.8 between control and drug-treated groups
and to generate
statistically significant data.
Tumor bearing mice received the mouse anti-ICOS (clone 7E.17G9) in different
isotype
backgrounds or H2L5 and/or mouse anti-PD-1 (clone RMP1-14) or an isotype
control in saline
via intraperitoneal injection twice weekly starting on randomization day for a
total of 6 doses.
Tumor measurement of greater than 2,000 mm3 for an individual mouse and/or
development
of open ulcerations resulted in mice being removed from study.
Binding studies
The affinity and kinetics of H2L5 binding to rabbit Fc-tagged recombinant
extracellular human
or cynomolgus ICOS (generated in-house) was determined using a BiacoreTM T200
(GE
HealthcareTm). The ICOS binding data was fitted to a 1:1 kinetics model using
the T200 data
analysis software. Cell surface binding of H2L5 to both freshly isolated
unactivated and
CD3/CD28 activated CD4 and CD8 T cells was determined via detection of anti-
human IgG,
kappa light chain FITC (Sigma) binding to H2L5 by flow cytometry.
Antibodies
The following anti-human antibodies were used for flow cytometry analysis, CD4
(RPA-T4,
BD Biosciences), CD8 (RPA-T8, Biolegend), CD69 (FN50, Biolegend), 0X40 (ACT-
35,
eBioscience), Ki67 (B56, BD Biosciences), ICOS (ISA3, eBioscience). The
following anti-
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mouse antibodies were used for flow cytometry analysis: CD3 (145-2C11, BD
Biosciences),
CD4 (RM4-5, BD Biosciences), CD8 (53-6.7, BD Biosciences), CD25 (PC61, BD
Biosciences), CD44 (IM7, Biolegend), CD62L (MEL14, BD Biosciences), FOXP3 (Fjk-
165,
eBioscience), ICOS (C398.4a, Biolegend), Ki67 (16A8, Biolegend). Apoptotis was
measured
using the Annexin V kit with 7-AAD (Biolegend). For flow cytometry analysis of
the human
PBMC mouse model the following antibodies were used: CD45 (HI30, BD
Biosciences), CD3
(UCHT1, Biolegend), CD4 (SK3, BD Biosciences), CD45R0 (UCHL1, Biolegend),
CD62L
(SK11, BD Biosciences). p-AKT (S473, #4060 and T308, #13038), total Akt
(#9272), pGSK3-
a (#5558), total GSK3-a (#12456), pS6 (S235/236, #2211 and S240/244, #5364),
total S6
(#2317), and pERK (#9101) (all from Cell Signaling Technology) were used for
Western Blots.
ADCC assays
Whole PBMC or NK depleted PBMC were activated with plate-bound anti-CD3 and
anti-
CD28 antibodies. Cells were incubated with anti-ICOS antibodies (H2L5 IgGl,
H2L5 IgG4PE
and H2L5 Fc-disabled) or control antibodies at 10 Kg/mL final concentration
for 24 hours.
Cells were stained with anti-CD8 and CD4 antibodies followed by incubation
with NIR
Live/Dead dye (Invitrogen). Stained cells were analyzed by flow cytometry
(FACSCanto, BD
Biosciences) to measure T-cell killing based on NIR Live/Dead cell dye
staining.
In the FcyRIIIa engagement reporter bioassay (Promega), anti-CD3/CD28 pre-
activated CD4
T cells were incubated with the anti-ICOS and control antibodies for 45
minutes prior to the
addition of Jurkat-FcyRIIIA-NFAT-luciferase effector cells at an E:T cell
ratio of 6:1. ONE-
GLO luciferase reagent was added to each well after 6 hrs of treatment and
luminescence
intensity measured to determine engagement between the target T cells and the
effector cells
on a Victor plate reader (Perkin Elmer). CD4, CD8 and Treg populations were
purified from
either donor PBMC pre-activated with anti-CD3/CD28 or disaggregated tumor
cells and tested
directly ex vivo at 6:1 E:T ratio in presence of IgG1 or IgG4PE H2L5
antibodies.
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Functional assays
H2L5 was tested in human PBMC assays either in a plate-bound format with
concurrent CD3
stimulation using freshly isolated PBMC or in a soluble format in CD3/CD28 pre-
stimulated
PBMC as described earlier. For PBMC from cancer patients, an overnight rest
step was
.. included prior to treatment initiation. 10 ug/mL soluble pembrolizumab was
used in in vitro
assays to study effects of combination. Cytokine concentrations in
supernatants from these
assays were measured using bespoke human multiplex meso-scale detection (MSD)
kits (Meso
Scale Diagnostics).
Human monocytes were isolated from whole blood of healthy human donors, using
CD14
.. MicroBeads (Miltenyi Biotec) for the T cell:monocyte mixed culture assays.
T cell and
monocytes were donor matched. CD3/CD28 pre-stimulated T cells and monocytes
were mixed
at 2:1 ratio in AIM-V serum-free media and cultured together with anti-CD3
dynabeads (Life
Technologies), 100 IU of recombinant human IL-2 and 100 ng/ml of M-CSF
(Peprotech) prior
to incubating with soluble H2L5 or other control antibodies at 37 C for 4
days. 20 g/mL human
Fc block (B564220) (BD biosciences) or anti-CD32 mAb (MCA1075EL, Clone AT10)
(AbD
serotec) were used to test the role of FcyR cross linking.
For the MLR assays, monocytes (Lonza, Switzerland) were grown in GM-CSF and IL-
4 (Pepro
Tech) supplemented LGM-3 media (Lonza) for 9 days for differentiating into
mDCs and TNFa
(R&D Systems) for an additional day before use in the MLR assay. The mDC-T
cell (1:10
ratio) mix was treated with 10 ug/mL soluble H2L5 Fc-disabled or the isotype
control
antibodies either in the presence of anti-CD3 beads at a 1:10 bead to cell
ratio (Life
Technologies) or CEFT peptide mix (0.02 ug/mL) (NT Peptide Technologies) for 4
days
before collecting the supernatants for cytokine analysis by MSD.
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Primary patient tumors were dissociated using GentleMACS (Miltenyi Biotec)
tissue
dissociator. TIL were expanded in IL-2 supplemented RPMI media (Baldan et al.,
2015) before
treating with anti-CD3 plus H2L5. Alternatively, tumor dissociated cells were
directly cultured
ex vivo for up to 6 days following stimulation with anti-CD3 plus H2L5 with
100 ng/ml IL-2
added after 24 hours.
For PBMC assays testing different H2L5 isotypes, anonymized leukocyte cones
from healthy
donors were obtained from the National Blood Service at Southampton General
Hospital, UK
and used within 4 hours. Use of human samples was approved by local ethical
committees in
accordance with the Declaration of Helsinki. PBMC were isolated by density
gradient
centrifugation (Lymphoprep) and cultured in RPMI medium 1640 (Life
Technologies)
supplemented with glutamine (2 mM), sodium pyruvate (1 mM), penicillin, and
streptomycin
(100 IU/mL) at 37 C in 5% CO2.
Proliferation assays were performed as detailed previously (35). Briefly,
fresh PBMC were
labelled with 1 jiM carboxyfluorescein succinimidyl ester (CFSE) and cultured
at high density
(lx 107/mL) for 48 hours prior to antibody stimulations. For the PBMC
stimulation, cells were
transferred into round-bottomed 96-well plates at lx 105 per well and
stimulated with 1 pg/m1
OKT3 (plate-bound) and 5 lag/m1 (soluble) H2L5 mAbs. On day 6, cells were
labelled with
anti-CD8-e450 (SK-1, eBioscience) and anti-CD4-APC (RPA-T4, Insight
Biotechnology), and
proliferation assessed by CFSE dilution on a FACSCantoII flow cytometer (BD
Biosciences).
.. Results are expressed as % divided cells compared to the unstimulated
cells. NK depletion was
performed using CD56 micro beads (Miltenyi Biotec) according to the
manufacturer's
instructions post 48 hours high density culture (Hussain et al. Blood 2014).
Immunofluorescence studies
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Unstimulated and CD3/CD28 stimulated T cells were Fe blocked with 20 g/mL
human Fe
block (B564220) (BD biosciences) or anti-CD32 mAb (MCA1075EL, Clone AT10) (AbD
serotec) to test the role of FcyR cross linking and then treated with 6 ug/mL
cold labeled
antibody (anti¨ICOS or IgG4PE isotype control) on ice for lhr. Cells were
washed in cold
buffer and transferred to 37 C for various times (0, 5, 15, 30 minutes and 1
hour) to allow
protein trafficking before fixing with freshly prepared 4% paraformaldehyde
(Sigma). Samples
1 or 2 hours after the initial pulse at 37 C were re-pulsed with Alexa Fluor
647 labeled anti-
ICOS for 30 minutes at 37 C, washed and fixed in paraformaldehyde. The cells
were
transferred to Poly-L-lysine coated coverslips for 15 minutes and then mounted
on slides in
ProLong Gold with DAPI (Invitrogen). Analysis of the samples was performed
using a ZEISS
LSM510 Meta Confocal microscope with a 63X oil immersion lens.
Human T-cell gene expression
Whole blood was obtained from healthy volunteer donors (n = 6) at the GSK on-
site Blood
Donation Unit and T cells were purified using RosetteSepTM Human T-Cell
Enrichment
Cocktail (Stemcell Technologies) as described above. The cells were re-
suspended (5x106
cells/mL) in AIM-V culture media (Gibco) and incubated in 96-well plates
(Falcon) that were
sequentially pre-coated with 0.6 pg/mL of mouse anti-human CD3 mAb
(eBioscience) and 10
g/mL of anti-human ICOS or corresponding isotype control mAbs ¨ mouse IgG2 a
ic
(eBioscience) and IgG4PE. After 24 hours of incubation at 37 C and 5% CO2,
cells were
pelleted, suspended in RLT buffer (Qiagen), and stored at ¨80 C for RNA
isolation. Total RNA
was extracted using the RNeasy Mini QIAcube Kit (Qiagen). RNA expression
levels were
determined by NanoString nCounter Analysis System. 50 ng of RNA was used in
each reaction
for gene signature using NanoString Human PanCancer Immune profiling CodeSet
according
to the manufacturer's instructions. Raw data was normalized using built-in
positive controls
and house-keeping genes (nCounter Expression Data Analysis Guide, NanoString).
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ArrayStudio (OmicSoft) and GraphPad Prism (GraphPad Software) were used for
further
analysis and graphs.
ICOS/ICOS-L competition assay
MSD plates were incubated overnight at 4 C with 10 pg/mL recombinant ICOS
protein (R&D
Systems) diluted in PBS. Plates were washed and blocked before adding isotype
control or
H2L5 in a 7-point dose curve. After overnight incubation and washes, the
plates were incubated
with 1 g/mL human ICOS ligand (B7-H2) (R&D Systems) followed by incubation
with 10
g/mL biotinylated anti-human ICOS ligand (B7-H2) (R&D Systems) antibody. Sulfo-
tagged
streptavidin at 10 pg/mL in Diluent 100 was used for detection of the
biotinylated ligand. The
plates were read immediately following MSD Read buffer addition on a MSD MESO
Quick
Plex SQ 120 and data analyzed on MSD workbench software. Flow cytometry was
also used
to investigate competition between cell surface ICOS expressed by anti-
CD3/CD28 activated
T cells and ICOS-L by H2L5. Activated T cells were incubated with different
concentrations
of recombinant ICOS-L and then incubated with H2L5 and MFI of ICOS CD4+ and
CD8+
ICOS cells determined.
Human PBMC Mouse model
Adult immunodeficient NOD/SCID/IL-2Rynull (NSG) mice (Jackson Labs) were
injected with
human PBMC (20x106 per mouse) by intravenous injection via the tail vein. Mice
were
implanted with human tumor cell lines A2058, A549, HCT116 (1 x 106) 1-3 days
post human
PBMC injection; mice were administrated isotype control or anti-human ICOS
antibodies at
doses ranging from 0.004 mg/kg to 1.2 mg/kg by intraperitoneal injection twice
weekly for 3
weeks. Tumor bearing mice received the mouse anti-ICOS (clone 7E.17G9) in
different isotype
backgrounds or H2L5 and/or Pembrolizumab (Merck; NDC#0006-3026-02) antibodies
or
isotype controls in saline via intraperitoneal injection twice weekly starting
on randomization
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day for a total of 6 doses. Tumor measurement of greater than 2,000 mm3 for an
individual
mouse and/or development of open ulcerations resulted in mice being removed
from study.
Spleens and whole blood were collected post-euthanization at 24hrs post 2' or
4th dose of
antibodies. Splenocytes were isolated by mechanical dissociation followed by
RBC lysis with
LCK lysis buffer (Lonza) and antibody staining whereas whole blood was stained
with the
appropriate antibodies before RBC lysis with FACSlyse (BD Biosciences). All
samples were
evaluated by flow cytometry on FACScanto (BD) as described below.
Western Blotting
Activated T cells were treated with H2L5 or an isotype control for up to 48
hours. CD4+ T
cells were prestimulated with CD3/CD28 Dynabeads (ThermoFisher) at a cell-to-
bead ratio
of 1:20 for 48 hours, allowed to rest in the absence of stimulation for 24
hours, and then treated
with isotype control antibody or H2L5 (10 pg/mL) in the presence of plate-
bound anti-CD3
antibody. Cells were lysed with cell lysis buffer (Cell Signaling
Technologies) containing
protease and phosphatase inhibitors (Roche). 25-30 jig of protein was run on 4-
12% Bis-Tris
gels (Invitrogen) and transferred onto nitrocellulose membranes (Invitrogen).
Membranes were
blocked using LI-COR Odyssey Blocking Buffer and subsequently immunoblotted
using the
primary and secondary antibodies and scanned on a LI-COR Odyssey imaging
system.
FACS analysis
Non-specific binding on activated T-cells was blocked by incubation with human
or mouse Fc
.. block (Miltenyi Biotec) as appropriate prior to the incubation with
detection antibodies to cell
surface markers conjugated to different fluorophores on ice for 30 minutes.
For intracellular
staining, the cells were fixed and permeabilized using the Transcription
Factor Buffer set (BD
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biosciences). After compensation, data were acquired on FACS Canto II or
Fortessa (BD
biosciences) and analyzed with FACSDiva (BD) or Flowjo (Treestar) software.
Immunohistochemistry
Immunohistochemical detection of ICOS in non-small cell lung cancer (NSCLC),
breast cancer
(BrCA) TNBrCa, and colorectal cancer (CRC), was performed using a rabbit anti-
human
CD278 mAb (clone SP98; Spring Biosciences) on a Leica Bond RX with associated
platform
reagents. DAB (3, 3'-diaminobenzidine) was used for target detection. Sections
were counter
stained with Hematoxylin (All scale bars = 20[Im).
Clarient MultiOmyx platform (Neogenomics, California), a multiplexed
immunofluorescence
(IF) assay was used to evaluate expression of ICOS, PD-1, CD3, CD4 and CD8
among other
T-cell markers on FFPE tumor tissues obtained from vendors vetted by GSK HBS
group as
described above. The iterative process included a round of staining with a Cy3
and Cy5
conjugated antibody each and imaging, followed by dye inactivation, background
fluorescence
imaging and subtraction of the background before the repeating this cycle for
all markers in the
panel.
Statistical analysis
One-way ANOVA or Student's t-tests were used as specified in the figure
legend. Data were
analyzed with GraphPad Prism software (GraphPad) and p values of <0.05 were
considered
statistically significant. (* P< 0.05; ** P<0.01; *** P< 0.005; ****P<0.0001).
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