Immune modulating activity of the CHK1 inhibitor prexasertib
and anti‑PD‑L1 antibody LY3300054 in patients with high‑grade
serous ovarian cancer and other solid tumors
Khanh T. Do1 · Claire Manuszak2
· Emily Thrash2
· Anita Giobbie‑Hurder3
· Jiani Hu3
· Sarah Kelland1
Allison Powers1
· Adrienne de Jonge1
· Geofrey I. Shapiro1
· Mariano Severgnini2
Received: 30 December 2020 / Accepted: 4 March 2021
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021
Abstract
Background Checkpoint kinase 1 (CHK1) has dual roles in both the DNA damage response and in the innate immune
response to genotoxic stress. The combination of CHK1 inhibition and immune checkpoint blockade has the potential to
enhance anti-tumoral T-cell activation.
Methods This was an open-label phase 1 study evaluating the CHK1 inhibitor prexasertib and the anti-PD-L1 antibody
LY3300054. After a lead-in of LY3300054 (Arm A), prexasertib (Arm B) or the combination (Arm C), both agents were
administered intravenously at their respective recommended phase 2 doses (RP2Ds) on days 1 and 15 of a 28-day cycle. Flow
cytometry of peripheral blood was performed before and during treatment to analyze efects on immune cell populations,
with a focus on T cell subsets and activation. Plasma cytokines and chemokines were analyzed using the Luminex platform.
Results Among seventeen patients enrolled, the combination was tolerable at the monotherapy RP2Ds, 105 mg/m2
prexasertib
and 700 mg LY3300054. Dose-limiting toxicities included one episode each of febrile neutropenia (Arm C) and grade 4 neutropenia lasting>5 days (Arm B). One patient had immune-related AST/ALT elevation after 12 cycles. Three patients with
CCNE1-amplifed, high-grade serous ovarian cancer (HGSOC) achieved partial response (PR), 2 lasting>12 months; a fourth
such patient maintained stable disease>12 months. Analysis of peripheral blood demonstrated evidence of CD8+T-cell
activation in response to treatment.
Conclusions Prexasertib in combination with PD-L1 blockade was tolerable and demonstrated preliminary activity in
CCNE1-amplifed HGSOC with evidence of cytotoxic T-cell activation in patient blood samples.
Trial registration ClinicalTrials.gov identifer: NCT03495323. Registered April 12, 2018.
Keywords Ovarian cancer · Immune checkpoint · Checkpoint kinase 1 · Cyclin E1 · PD-L1
Introduction
The DNA damage response and immune response pathways
are evolutionarily conserved and interrelated. The immune
system plays a critical role in eliminating and controlling
early tumor growth. Upregulation of PD-L1 in tumor cells
has been implicated in immune tolerance and immune
escape [1]. PD-L1 expression has been shown to be diferentially regulated at several crucial timepoints during the
DNA damage response. Accumulating evidence shows that
DNA damage induces cell surface expression of PD-L1 and
that PD-L1 upregulation is mediated by activation of the
cell cycle checkpoint kinases ataxia telangiectasia mutated
(ATM), ataxia telangiectasia and RAD3-related (ATR), and
checkpoint kinase 1 (CHK1) [2, 3]. CHK1, in particular,
Khanh T. Do and Claire Manuszak contributed equally to this
manuscript.
The results of this study were presented at the Society for
Immunotherapy of Cancer (SITC) 34th Annual Conference on
November 6th-10th, 2019 at the Walter E. Washington Convention
Center in Washington, D.C.
* Khanh T. Do
[email protected]
1 Department of Medical Oncology, Dana-Farber Cancer
Institute, 450 Brookline Avenue–DA2010, Boston,
MA 02215, USA
2 Center for Immuno-Oncology, Dana-Farber Cancer Institute,
Boston, USA
3 Division of Biostatistics, Department of Data Science,
Dana-Farber Cancer Institute, Boston, USA
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has been shown to be a central relay point for switch from a
DNA damage response to an immune response, promoting
the upregulation of PD-L1 expression in response to DNA
damage through STAT1/3 and interferon receptor factor 1
(IRF1)-mediated transcription of PD-L1 mRNA [3].
Disruption of the DNA damage response can also lead to
upregulation of PD-L1 expression. Collapse of replication
forks occurring during the S-phase checkpoint in response
to CHK1 inhibition results in accumulation of DNA fragments and generation of cytosolic DNA, which activates a
type I interferon response, involving activation of the cGASSTING pathway, nuclear factor-kappa B mediated release of
infammatory cytokines, and upregulation of PD-L1 expression [4–7]. Additionally, dying cancer cells and the production of neoantigens trigger the release of damage-associated
molecular pattern pathways and activation of a type II interferon response, further reinforcing constitutive upregulation
of PD-L1 expression [8].
Based on these emerging data, we conducted a phase
1 trial combining the CHK1 inhibitor prexasertib and the
PD-L1 antibody LY3300054 in patients with advanced
solid tumors, incorporating profling of peripheral blood to
explore the immune response to this DNA damaging agent
alone and in combination with immune checkpoint blockade.
Patients and methods
Study population
Patients with advanced solid tumors without approved curative therapy or efective palliative therapy,≥18 years of age,
with an Eastern Cooperative Oncology Group (ECOG) performance status 0–1 and measurable disease per Response
Evaluation Criteria in Solid Tumors (RECIST) version 1.1
[9] were eligible for the study. All patients were required to
have adequate organ function defned by: absolute neutrophil count≥1.5× 109
/L, platelet count≥100× 109
/L, total
bilirubin≤1.5×institutional upper limit of reference range
(ULRR), aspartate aminotransferase (AST) and alanine aminotransferase (ALT)≤2.5×ULRR, creatinine≤1.5×institutional upper limit of reference range (ULRR) or creatinine clearance≥60 mL/min by Crockcroft-Gault formula,
free thyroxine within institutional normal limits, and
QTc≤470 ms on screening ECG. Patients were required
to have completed previous cancer therapy at least 3 weeks
prior to study entry (6 weeks for nitrosureas or mitomycin
C). Prior treatment with PD-L1 antibody was permitted if
not the most recent therapy prior to enrollment. Subjects
who had received radiation to>25% of the marrow or more
than 4 lines of cytotoxic chemotherapy were excluded. Additional exclusion criteria included any prior Grade 3 immunerelated adverse events, immune-related neurologic or ocular
toxicities, or immune-related toxicities of any grade which
required permanent discontinuation of prior immune therapy. The presence of untreated brain metastases or carcinomatous meningitis, pregnancy, or active infection (including
Hepatitis B, C, or HIV) was excluded.
Study design and treatment administration
The primary objective of this study was to determine the
safe and tolerable dose of the combination of prexasertib and
LY3300054. Secondary objectives included characterization
of changes in immune cell subsets and cytokine profles in
peripheral blood and tumor biopsies in response to treatment, and assessment of diferences in immune phenotype
between responders and non-responders. Starting dose levels for both prexasertib and LY3300054 were at the RP2D
for the individual agents. Based on expected neutropenia
with prexasertib administration, secondary prophylactic
growth factor support was permitted beginning with cycle
1. Patients were randomized to one of three administration
schedules: lead-in (cycle 0) of LY3300054 alone (Cohort A),
prexasertib alone (Cohort B), or the combination (Cohort
C), designed to accommodate pharmacokinetic (PK) and
pharmacodynamic (PD) assessment of single-agent activity
versus the combination. Thereafter, both agents were administered each as a 1 h intravenous infusion on days 1 and 15
of a 28-day cycle. Up to 6 patients could be enrolled to the
respective arm with observance of a dose-limiting toxicity
(DLT). An expansion cohort with paired tumor biopsies was
planned but enrollment was not completed due to internal
re-prioritization by the drug manufacturer to discontinue further development of prexasertib necessitating early closure
of the study due to limited remaining drug availability.
Dose‑limiting toxicity defnitions and study
assessments
Safety was assessed via monitoring of toxicities during
the lead-in cycle 0+cycle 1 according to National Cancer
Institute Common Terminology Criteria for Adverse Events
(NCI-CTCAE) v.4.03. Dose-limiting toxicities (DLT) were
defned as grade 4 neutropenia>5 days despite growth factor support or febrile neutropenia, grade 4 thrombocytopenia, and any grade 3–4 non-hematologic toxicities related to
study drug and occurring during the lead-in and/or cycle 1.
Grade 3≥nausea, vomiting, diarrhea, electrolyte derangements, or rise in creatinine were considered dose-limiting
if refractory to management and not improved to grade≤2
within 48 h. Any grade 3 or 4 colitis or non-infectious pneumonitis, grade 4 immune-related toxicities, and any onset
of Stevens-Johnson Syndrome, toxic epidermal necrolysis, skin necrosis, or bullous or hemorrhagic skin lesions
were considered dose-limiting. Any non-laboratory grade 3
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immune-related adverse event not downgraded to Grade 2
within 3 days despite optimal medical management was considered dose-limiting. Increases in AST or ALT to>3 times
institutional ULN and concurrent increase in total bilirubin
to>2×institutional ULN were considered dose-limiting.
Additionally, inability to tolerate 100% of scheduled prexasertib and LY3300054 during the lead-in and cycle 1 was
considered dose-limiting. After review of tolerability for
each cohort dose schedule by the Safety Review Committee
in conjunction with the sponsor, a single dose administration schedule was selected as the RP2D for further planned
enrollment. An expansion cohort of up to 10 patients was
planned at the RP2D to further evaluate safety, tolerability,
and PD endpoints.
A physical examination, assessment of vital signs, pertinent tumor biomarker assessments, hematology, and chemistry assessments were performed at screening, on day 1 of
the lead-in cycle, and on days 1 and 15 of every subsequent
cycle. Additional safety labs including CBC with diferential
were collected from patients on days 8 and 22 of the frst
two cycles due to anticipated myelosuppression with treatment. Thyroid function tests were performed at screening
and at the start of each cycle. Standard 12-lead ECGs were
obtained at screening, at the start of the frst two cycles, and
as clinically indicated thereafter.
Pharmacokinetic assessments
Abbreviated plasma samples were collected in 4 mL plastic
Vacutainer tubes with spray dried sodium heparin (prexasertib samples) and K3EDTA tubes (LY3300054 samples) on
C1D1 pre-dose, at 1 h at the completion of the prexasertib
infusion, and 1 h after completion of the LY3300054 infusion; C3D1 pre-dose and at 1 h at the completion of the
prexasertib infusion; and pre-dose every three cycles starting
with C6D1 for all three arms. For Arm C, additional plasma
samples were collected during the lead-in cycle pre-dose, at
1 h at the completion of the prexasertib infusion, 1 h and 2 h
after completion of the LY3300054 infusion. Blood collection tubes were centrifuged to harvest the plasma which was
stored in cryovials at −80 °C. Prexasertib plasma concentrations were quantifed using a validated high-pressure liquid chromatography-mass spectrometry/mass spectrometry
method. LY3300054 plasma concentrations were quantifed
using a validated electrochemiluminescent method. Pharmacokinetic data were analyzed using population pharmacokinetic analysis.
Pharmacodynamic assessments
Whole blood samples were collected during the lead-in
period prior to initiation of therapy on C0D1, on C0D2 after
the frst lead-in administration of drug, on C0D8, C1D8,
C1D1, and C2D1, and at the point of progression. C0D1,
C0D2, and C2D1 samples were chosen for analysis based
on published kinetics of T-cell activation [10]. Peripheral
blood mononuclear cells (PBMC) samples were thawed and
surface stained with fuorochrome-conjugated monoclonal
antibodies against lineage markers (CD3, CD4, CD8, CD19,
CD25, CD56, CD127), memory markers (CD45RA, CCR7)
and functional markers (CD69, CD71). Fluorescence minus
one (FMO) controls were used for compensation, as previously described [11, 12]. Two fow cytometry antibody panels were developed. These are described along with gating
strategies in Supplemental Figure 1. Samples were fxed,
then acquired within 24 h of staining on a four laser BD
Fortessa X20. FCS fles were analyzed by manual gating
using FlowJo v10.0.8 for Mac (BD, Franklin Lakes, NJ).
Data are presented as cellular population frequency based
on percentage of viable cells.
Patient plasma samples were thawed and prepared for
soluble analyte assay according to previously published
methods [13, 14]. Analytes were measured on a Luminex
FLEXMAP3D (Luminex, Austin, TX) per the manufacturer’s protocol. Soluble IL6, IL3, IL7, IL10, VEGFα, IL1β,
IL4, TNFβ, FGFβ, IL5, IL12p70, TGFα, TNFα, ENA78,
GCSF, GMCSF, IFNγ, IL1α, IL1β, IL1RA, IL2, IL8, IL17,
MCP1, MIP1α, and MIP1β were tested. Out of all the soluble markers measured, 15 markers were within detectable
range and could be quantifed by extrapolation of MFIs to
the respective standard curve between lower limit of quantitation and upper limit of quantitation. Analyte concentration
for each patient was calculated using standard curves. Fold
changes were calculated as a ratio relative to the patient’s
baseline (C0D1) [15–17].
Statistical analyses
Summaries of patient demographics, disease, and prior treatment characteristics are presented descriptively. Changes in
immune subsets by fow analysis and cytokine expression
levels are expressed using fold changes relative to C0D1.
As a surrogate for response, we associated patients’ time on
study with changes in immune cell subsets. Patients were
classifed as deriving clinical beneft (DCB) if they had best
response of stable disease or partial response and remained
on study for at least 100 days (N=8). Those patients who
did not derive clinical beneft (Non-DCB) remained on study
for less than 100 days due to disease progression (N=7).
The immunomodulatory efects of prexasertib alone versus
in combination were investigated using Arms B and C. All
comparative, correlative statistical analyses of two groups
were based on two-sided Wilcoxon rank-sum tests with
signifcance level of 0.05. There were no adjustments for
multiple comparisons. Heatmaps were based on hierarchical
clustering using R package “heatmap.2” (R version 3.5.3).
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Results
Patient disposition and characteristics
A total of 17 patients were enrolled between June 2018
through January 2020 (Table 1); 15 patients were enrolled
to the dose optimization phase of the study, and two were
enrolled to the expansion phase of the study prior to early
closure by the sponsor. All patients were evaluable for
both response and toxicity. Three patients received at
least 12 cycles of treatment. The majority of patients had
high-grade serous ovarian carcinoma (HGSOC) (14/17);
six of these patients had tumors harboring CCNE1 amplifcation on targeted next-generation sequencing. Three
patients with CCNE1-amplified HGSOC achieved a
partial response (PR), remaining on study for 7 months,
13 months, and 20 months, respectively; a fourth patient
with CCNE1-amplifed HGSOC maintained stable disease
(SD) lasting>12 months. RECIST v1.1 response for the
subset of ovarian cancer patients is shown for each patient
(Fig. 1).
Adverse events
The most common adverse events (AEs) attributed to treatment and occurring in≥10% of patients are summarized in
(Table 2), reported as highest grade observed. A total of 382
adverse events of any grade and any causality were captured
for the trial. Of these adverse events, 170 or 83% were Grade
1/2. Most common adverse events occurring in greater than
50% of patients include anemia (82%), neutropenia (88%),
Table 1 Patient demographics
*One patient enrolled with 5 prior systemic therapies, one line was on an experimental trial
Patient demographics
Patients enrolled/treated 17
Median age in years (range) 57 (25–76)
Median number of prior therapies (range)
Prior immunotherapy
Diagnoses
Ovarian (incl. high-grade serous ovarian cancer—10; low-grade serous ovarian cancer\\
Soft tissue sarcoma (incl. uterine leiomyosarcoma, malignant solitary fbrous tumor) 2
Colorectal cancer 1
RECIST Response
Best Response
Days on Study
Cohort C- Combo Lead-in
Cohort A- LY3300054 Lead-in
Cohort B- prexaserb Lead-in
Fig. 1 Waterfall Plot of Clinical Response in Ovarian Cancer Patients
(n=14) on Study. Best Response by RECIST v1.1 is shown. Histologies included: *clear cell (n=2), ǂlow-grade serous (n=2), and
†high-grade serous ovarian cancer (n=10); pertinent mutations of
interest, where known, are indicated. Days on study and best response
are shown on the x-axis
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Table 2 Summary of adverse events attributed to study treatment
Dose cohort
Adverse events* Cohort A
Gr 1 Gr 2 Gr 3 Gr 4 Gr 1 Gr 2 Gr 3 Gr 4 Gr 1 Gr 2 Gr 3 Gr 4
Blood and lymphatic system disorders
Anemia 1 – 1 – 2 2 2 – – 5 1 – 14 (82%)
Febrile neutropenia – – – – – – – – – – 2 – 2 (12%)
Endocrine disorders
Hypothyroidism – – – – 1 – – – – 1 – – 2 (12%)
Gastrointestinal disorders
Abdominal pain 1 1 – – 2 1 – – 4 – – – 9 (53%)
Ascites – 2 – – – – – – – – – – 2 (12%)
Bloating 1 – – – 1 – – – 1 – – – 3 (18%)
Constipation – – – – 1 2 – – 1 – – – 4 (24%)
Diarrhea 1 – – – 3 – – – 4 – – – 8 (47%)
Dry mouth 1 – – – 1 – – – – – – – 2 (12%)
Dyspepsia 1 – – – – – – – – – – – 1 (6%)
Flatulence 1 – – – 1 – – – 1 – – – 3 (18%)
Gastroesophageal refux – 2 – – – – – – – – – – 2 (12%)
Gastroparesis 1 – – – – – – – – – – – 1 (6%)
Oral mucositis 1 – – – 1 1 – – 2 – – – 5 (29%)
Oral dysesthesia 1 – – – 1 – – – – – – – 2 (12%)
Nausea 1 – – – 1 1 – – 4 – – – 7 (41%)
Vomiting 1 – – – – – – – 2 – – – 3 (18%)
Immune system disorders
Allergic reaction – – – – – – – – 1 1 – – 2 (12%)
Infections/Infestations
Sepsis – – – – – – – – – – – 1 1 (6%)
Metabolism and nutrition
Anorexia 3 – – – 1 – – – 2 – – – 6 (35%)
Hypokalemia – – – – 1 1 – – 1 – – – 3 (18%)
Investigations
Alkaline phosphatase ↑ 1 – – – 1 1 – – 3 – – – 7 (41%)
AST ↑ – – – – 3 – – – – – 1 – 4 (24%)
ALT ↑ – – – – 2 – – – – – 1 – 3 (18%)
Bilirubin ↑ – – – – – – – – 1 1 – – 2 (12%)
Creatinine ↑ 1 – – – 1 – – – – – – – 2 (12%)
Platelet count ↓ – 1 – – – – 3 – 3 1 – 1 9 (53%)
Lymphocyte count ↓ 2 1 – – 1 1 2 – – 2 2 – 11 (65%)
White blood cell count ↓ – – 1 1 – 1 2 3 – – 2 4 14 (82%)
Neutrophil count ↓ 1 – – 2 – – – 6 – – – 6 15 (88%)
General/Administration site
Infusion related reaction – – – – – 1 – – 1 1 – – 3 (18%)
Fatigue 2 – – – 2 1 – – 3 1 – – 9 (53%)
Flu like symptoms – – – – – 1 – – 1 – – – 2 (12%)
Chills 1 – – – 1 – – – 2 – – – 4 (24%)
Fever 1 – – – 1 – – – 2 – – – 4 (24%)
Malaise – – – – 1 1 – – – – – – 2 (12%)
Localized edema – – – – 1 – – – 1 – – – 2 (12%)
Non-cardiac chest pain – – – – 1 – – – – – – – 1 (6%)
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decreased white blood cell count (82%), decreased platelet
count (53%), fatigue (53%), and abdominal pain (53%) consistent with previous reports for prexasertib monotherapy
[18, 19]. Decreases in lymphocyte count were also observed
but the majority were grade 1 or 2. There were 2 DLTs,
including one event of febrile neutropenia on Arm C and
one event of prolonged grade 4 neutropenia lasting>5 days
on Arm B. A third patient also developed fever in the setting of viral illness and neutropenia at C4D15, outside of
the DLT window. One patient developed immune-related
grade 3 elevation in AST and ALT after 12 cycles, requiring
mycophenolate for management, necessitating discontinuation of therapy.
Pharmacokinetic analyses
Prexasertib exposures ranged from 320 to 1230 ng/mL at
60 min after dosing in the majority of patients, within range
of previously published data [18]. LY3300054 exposures
ranged from 125 to 472 µg/mL at 120 min after dosing and
48–133 µg/mL pre-dose with subsequent cycles (Supplementary Figure 2).
Prexasertib treatment causes changes in T‑cell
proliferation patterns and cytokine profle
Immune cell subset analysis and functional status using soluble analyte and fow cytometry assays from PBMC samples of the 15 patients enrolled to the dose optimization are
shown for C0D2 (Fig. 2a) and C2D1 (Fig. 2b). Cohort A
with a lead-in of LY3300054 alone was used as a comparative control to exclude changes due to immune checkpoint
blockade. Decrease in circulating CD4+Tregs at C0D2
compared to baseline were statistically diferent between
cohort B (5/6) versus cohort C (1/6; P=0.04). Changes
in frequency of activated CD8+T cells positive for transferrin receptor (CD71), Natural Killer T (NKT) cells, and
CD8+NKT cells were also seen at C2D1. CD71+CD8+T
cells were increased in 12/15 patients indicating expansion
of an activated CD8 T cell subset in response to combination treatment. By comparison, the frequency of T cell
memory subsets at C2D1 including CD8+T central memory
(TCM), CD8+T efector memory (TEM), CD8+T efector memory CD45RA+(TEMRA), CD4+TEM cells, and
CD4+TCM were decreased in a majority of patients at
C2D1, as expected post-antigen exposure.
Cytokine analysis revealed similar patterns of immune activation with increase in IL-2 and IL-7 (Fig. 3) consistent with
previously published results [20] and supporting the patterns
of T cell subset proliferation seen in this study. IFNγ levels
were below the limits of detection and could not be interpreted.
Profles were compared between patients who derived clinical
beneft and remained on study for at least 100 days (shown in
red), and patients who did not derive clinical beneft remaining on study for less than 100 days due to disease progression (shown in blue). Levels of IL-6, IL-7, and IL-8 tended
to be overall higher in patients who achieved clinical beneft
compared to those who did not, although levels did not reach
statistical signifcance.
*All adverse events represent the number of patients experiencing the adverse event felt to be related to study treatment, reported as worst grade
for each patient
Table 2 (continued)
Dose cohort
Gr 1 Gr 2 Gr 3 Gr 4 Gr 1 Gr 2 Gr 3 Gr 4 Gr 1 Gr 2 Gr 3 Gr 4
Nervous System
Dizziness 1 – – – 1 – – – 1 – – – 3 (18%)
Headache – – – – 1 2 – – – – – – 3 (18%)
Respiratory/Thoracic/Mediastinal
Dyspnea – – – – – – – – – 1 – – 1 (6%)
Cough – – – – 1 – – – 1 – – – 2 (12%)
Musculoskeletal/Connective tissue
Arthralgia – – – – – 1 – – – – – – 1 (6%)
Back pain – – – – – – – – 1 – – – 1 (6%)
Myalgia 1 – – – 2 – – – 2 – – – 5 (29%)
Skin/Subcutaneous
Pruritus 1 – – – 1 – – – 1 – – – 3 (18%)
Rash maculo-papular – 1 – – – 2 – – 1 – – – 4 (24%)
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Peripheral blood samples could not be compared to tumor
biopsies in the two patients enrolled to the dose expansion
phase of the study. For one patient, biopsy was considered
a high-risk, and the procedure was aborted; for the second
patient, the on-treatment biopsy contained no tumor due to
robust response two weeks into treatment and accordingly
could not be compared to the pre-treatment biopsy.
Antitumor activity in CCNE1‑amplifed high‑grade
serous ovarian cancers
Of the 17 patients enrolled, 14 had recurrent ovarian cancer. Histologies included clear cell (n=2), low-grade serous
(n = 2), and high-grade serous (n = 10). Eight patients
remained on study for at least 4 cycles (≥ 100 days), 3
patients remained on study for at least 12 months. Notable
responses occurred in CCNE1-amplifed high-grade serous
ovarian cancers, including the three patients who achieved
a. Change in Immune Subsets at C0D2 compared to Baseline b. Change in Immune Subsets at C2D1 compared to Baseline
Fig. 2 Flow Cytometry of Patient-derived Peripheral Blood Samples.
Panel a: shows C0D2 compared to baseline after a single lead-in dose
of LY3300054 alone (Cohort A), prexasertib alone (Cohort B), or
the combination of LY3300054 and prexasertib (Cohort C). Panel b:
shows data for the corresponding samples from C2D1. Data are
normalized to baseline pre-treatment samples and expressed as fold
changes
Pre C0D2 C2D1 Pre C0D2 C2D1 Pre C0D2 C2D1 Pre C0D2 C2D1 Pre C0D2 C2D1 Pre C0D2 C2D1 Pre C0D2 C2D1
Pre C0D2 C2D1 Pre C0D2 C2D1 Pre C0D2 C2D1 Pre C0D2 C2D1 Pre C0D2 C2D1 Pre C0D2 C2D1
Clinical benefit
No Clinical benefit
Fig. 3 Cytokine analysis of Patient-derived Plasma Samples. Shown
are Luminex soluble analyte fold change of C0D2 and C2D1 samples normalized to each patient baseline pre-treatment sample. Clinical beneft (red) denotes patients who remained on study≥100 days,
and no clinical beneft (blue) denotes patients who remained on
study<100 days
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a PR (Fig. 1). Immune subset analysis for these patients
showed signifcant interpatient variability, though patterns of
T-cell proliferation including increases in CD71+CD8+T
cells and NK cells were seen in the majority of patients
(Fig. 4). Notable T-cell activation response was seen in
Patient #10 who achieved a durable PR on study lasting past
12 cycles. Lymphocyte lineage analysis shows expansion
of naïve CD4+and CD8+memory cells at C2D1 (Fig. 5a
and b). Additionally, this patient had signifcant elevation of
IL-2 on cytokine profling (Fig. 5c). Sample CT images of
ongoing PR after 12 cycles is shown in Fig. 5d.
Discussion
In this study, we report the immune modulating efect of
CHK1 inhibition with prexasertib in combination with antiPDL1 antibody LY3300054. We additionally show prexasertib and LY3300054 can be given safely in combination at
the RP2D of the individual agents and report preliminary
antitumor activity in CCNE1-amplifed HGSOC.
The design of this study, incorporating a lead-in dose,
allowed for pharmacodynamic assessment of peripheral
T-cell activation and cytokine signatures after a single
dose of prexasertib (Cohort B) compared to the combination (Cohort C; Fig. 2). After a single lead-in dose of
prexasertib, circulating CD4+T regulatory cells (Tregs)
Lymphocyte Lineage
Populaons
CD8+ Memory CD4+ Memory
Subject ID 13 10 3 6
Baseline C0D2 C2D1
Baseline C2D1 Baseline C2D1
Fig. 5 Clinical Response and Corresponding Immune Subset and
Cytokine Analysis in Patient #10: CCNE1-amplifed ovarian cancer. Panel a: Lymphocyte lineage population for Patient #10 based
on percentage of parent population (viable cells). Panel b: Shown
are subset of CD8+and CD4+T memory cells as a function of
CD3+CD4+and CD3+CD8+parent population. Panel c: Heatmap
of C2D1 cytokine analytes normalized to baseline samples for this
patient compared to patient 13 who also achieved a PR and remained
on study for 20 cycles. Panel d: Shown are sample CT images demonstrating durable PR at 12 cycles
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and CD8+ NKT cells decreased compared to baseline in
5/6 patients in Cohort B. Although leukopenia has previously been reported with prexasertib [18], decrease in counts
typically occur 5–8 days after drug exposure. As samples
were obtained on C0D2, within 24 h after dosing, decrease
in Tregs and NKT cells are thought to refect direct immune
modulating activity of CHK1 inhibition on T cells rather
than an artifact of lymphodepletion as previously reported
[21]. Additionally, as both Tregs and NKT cell have been
shown to suppress immune surveillance [22], a decrease in
both of these cell lines after a single dose of prexasertib in
the absence of immune checkpoint blocking antibody further supports a regulatory role for CHK1 in immunity. NK
cells were also increased in 3/6 patients in Cohort B, supporting early activation of the innate immune response in
response to CHK1 inhibition. At C2D1, after the combination of prexasertib and LY3300054 had been administered,
an increase in frequency of CD71+CD8+T cells was seen
in 4/6 patients in cohort B and in 3/6 patients in cohort C,
indicating that the majority of patients had expansion of an
activated CD8+T cell subset in response to treatment.
The signifcance of decreases in the frequency of T cell
memory subsets at C2D1 remains unclear as changes neither
correlated with response nor non-response. It is conceivable that this earlier timepoint may fall between post-antigen exposure and pre-proliferation and is in line with published kinetics of T-cell activation [10]. Cytokine analysis
confrmed the changes seen in T-cell activation signatures.
Soluble IL7 increased during treatment in 9/15 patients at
C2D1 (Fig. 3). This cytokine has been shown to induce B
and CD8+T cell proliferation [23]. Levels of IL-17 also
increased compared to baseline, though this is difcult to
interpret in the setting of G-CSF administration which was
required to support neutropenia in 14/17 patients and did not
correlate with response. Subset analyses of the two patients
who achieved a PR lasting>12 cycles (Patient #10 and #13)
compared to two patients who experienced rapid clinical
progression after 1 cycle (Patient #3 and #6) showed no
consistent diferences in T-cell activation signatures but did
trend towards higher expression of CD69+and CD71+in
the two patients who achieved a PR. Additionally, IL-2 was
found to be signifcantly elevated in P#10, and is consistent
with T-cell activation signatures in this patient (Fig. 5).
Interestingly, Patient #10 was the only patient to experience immune-related toxicities on study. This patient experienced grade 2 hypothyroidism after 10 cycles followed by
grade 3 immune-related elevation in AST and ALT after 12
cycles which initially responded and improved to grade 1
with high-dose steroid administration but required initiation
of mycophenolate for complete resolution. IL-2 has been
implicated in the development of severe irAEs [24] and may
be more a predictive biomarker of irAE rather than response
in this setting.
In this study, clinical beneft was seen in 8/14 recurrent
ovarian cancer patients. Three CCNE1-amplifed HGSOC
patients achieved a PR, a fourth CCNE1-amplifed HGSOC
maintained SD for more than 12 months. These responses
are not thought to be attributed to LY3300054 alone as
previous response rates of immune checkpoint blockade
monotherapy in recurrent ovarian cancers have been modest, averaging 8–10% [25, 26]. Responses are also not attributed to prexasertib alone. In clinical studies of prexasertib
monotherapy in recurrent high-grade serous ovarian cancers
(HGSOC), approximately one-third of patients achieved a
partial response and median duration of response was only
7.4 months [27]. Both the depth and duration of response
seen in this study are attributed to additive activity of the
combination of prexasertib and LY3300054.
Our fndings of T-cell activation and cytokine signatures
in response to treatment with prexasertib and LY3300054
support an immunomodulatory role for CHK1 inhibition.
In contrast to previous reports where an increase in immunosuppressive Tregs were seen at C1D15 [21], we saw an
early decrease in CD4+Tregs and CD8+ NKT cells after
a single dose of prexasertib at C0D2 which would suggest
a more proximal immunoregulatory response with CHK1
inhibition. The discrepancy in Treg response may suggest a
dynamic response to CHK1 inhibition that varies with time
from exposure and may require the addition of immune
checkpoint blockade to further reinforce T-cell activation
signatures. Further correlation with patient-derived tumor
biopsies is needed to confrm changes in tumor immunity
in response to treatment with prexasertib with and without
reinforcement with anti-PD-L1 antibody. The decision to
halt further agent development and early study closure precluded our ability to collect additional tumor biopsies in a
planned expansion cohort. Treatment of recurrent ovarian
cancers presents a therapeutic challenge and combination
immune-based therapies remain an unmet need. The encouraging responses seen in this study warrant further investigation for immune-based therapies in CCNE1 amplifed
ovarian cancers.
Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s00262-021-02910-x.
Acknowledgements We thank the participating patients and their families for their invaluable contributions.
Author contributions All authors contributed to the study conception
and design. Material preparation, data collection, and analysis were
performed by KTD, CM, ET, AG-H, and MS. The frst draft of the
manuscript was written by KTD and CM, and all authors commented
on previous versions of the manuscript. All authors read and approved
the fnal manuscript.
Funding Funding for this study was partially supported by institutional
grant from Eli Lilly.
Cancer Immunology, Immunotherapy
1 3
Availability of data and material All data generated or analyzed during
this study are included in this article and supplementary information
fles.
Declarations
Conflict of interest KTD has served as a consultant/advisory board
member Seattle Genetics and QED Therapeutics; consulting fees from
Jackson Laboratories; and has received commercial research grants (to
institution) from Eli Lilly. CM is an employee of Moderna Therapeutics at the time of this submission. ET is an employee of Fluidigm at
the time of this submission. GIS has received research funding from
Eli Lilly, Merck KGaA/EMD-Serono, Merck, and Sierra Oncology. He
has served on advisory boards for Pfzer, Eli Lilly, G1 Therapeutics,
Roche, Merck KGaA/EMD-Serono, Sierra Oncology, Bicycle Therapeutics, Fusion Pharmaceuticals, Cybrexa Therapeutics, Astex, Almac,
Ipsen, Bayer, Angiex, Daiichi Sankyo, Seattle Genetics, Boehringer
Ingelheim, ImmunoMet, Asana, Artios, Atrin, and Concarlo Holdings.
In addition, he holds a patent entitled, “Dosage regimen for sapacitabine and seliciclib,” also issued to Cyclacel Pharmaceuticals, and a
pending patent, entitled, “Compositions and Methods for Predicting
Response and Resistance to CDK4/6 Inhibition,” together with Liam
Cornell. SK, AP, AA, JH, AG-H, and MS declare no relevant fnancial
or non-fnancial interests to disclose.
Ethical approval This study was conducted in accordance with the
International Conference on Harmonization, Good Clinical Practice
guidelines, and the ethical principles outlined in the Declaration of
Helsinki 2008. The study protocol, any amendments, informed consent, and other information that required pre-approval were reviewed
and approved by the Institutional Review Board, in accordance with
the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use Good Clinical Practice and applicable country-specifc requirements, including US
21 Code of Federal Regulations 312.3(b) for constitution of independent ethics committees.
Consent for publication All participants provided written informed
consent prior to study entry.
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