First-in-human Phase I dose escalation study assessing safety, tolerability and preliminary efficacy of immunomodulatory nanoparticles

In the rapidly evolving treatment landscape of advanced solid tumors,
immunotherapeutic approaches have revolutionized cancer care for patients.
Despite these advances, there is an undiminished need for the development of
novel immunotherapeutic treatment modalities that can orchestrate an effective
anti-tumor immune response in a highly targeted way, while circumventing
immunosuppressive characteristics of the tumor microenvironment.
In this trial, we investigate a new class of immunomodulatory nanomedicines:
PLGA-nanoparticles loaded with tumor antigen NY- ESO-1 peptides in combination
with the invariant Natural Killer T (iNKT) cell activator IMM60.
PLGA-nanoparticles
Biodegradable PLGA-nanoparticles function as a delivery platform for
immunomodulators and tumor antigens to induce a specific anti-tumor immune
response. PLGA has minimal (systemic) toxicity and is used in various
drug-carrying platforms as encapsulating agent. Uptake of the nanoparticle by
Dendritic Cells (DC) and subsequent prolonged presentation of PLGA-
encapsulated peptides on Major Histocompatibility Complex (MHC) class I and II
molecules are able to generate functional and tumor-antigen-specific CD8+ and
cluster of differentiation (CD)4+ T cell responses in preclinical studies.
NY-ESO-1
NY-ESO-1 is a cancer-testis antigen expressed during embryogenesis and in the
testis, an immune privileged site. Furthermore, NY-ESO-1 expression is observed
in several advanced cancers: lung (2-32%), melanoma (40%), bladder (32-35%),
prostate (38%), ovarian (30%), esophageal (24-33%), and gastric cancers
(8-12%). Clinical trials have shown the safety and tolerability of Good
Manufacturing Practices (GMP)-grade NY-ESO-1 peptides in patients with cancer.
By studying published NY-ESO-1 epitopes and their respective Human Leukocyte
Antigen (HLA) alleles using a cancer antigenic peptide database, we defined two
long peptides of 27 amino acids and one short peptide of 9 amino acids for
nanoparticle encapsulation (85-111, 117-143, and 157- 165). In combination, all
three peptides are able to cover more than 80% of the European population for
both class I and class II HLA alleles. Administration of NY-ESO-1-specific
peptides leads to antigen-specific T-cell responses against NY-ESO-1-positive
tumors. The peptides planned to be investigated in the current trial were
selected based on their previously published immunogenicity (i.e., the capacity
to induce T cell responses).
iNKT cell activator ThrCer6
iNKT cells recognize glycolipid molecules presented with CD1d molecules on DCs
via their invariant T cell receptor (TCR). DCs are considered the most potent
Antigen-Presenting Cells (APCs) of the immune system and induce
antigen-specific cytotoxic (CD8+) T cell responses to tumor antigens. Upon
CD1d-dependent interaction with DCs, iNKT cells will be stimulated to secrete
pro-inflammatory cytokines (e.g., interferon [IFN]-* and interleukin [IL]-12).
Moreover, CD40-CD40 ligand (CD40L) mediated interaction between DCs and iNKT
cells during glycolipid presentation leads to DC maturation demonstrated by the
upregulation of co-stimulatory molecules.
αGalCer consists of a galactose connected via an α-linkage to a ceramide-group.
Once presented with CD1d on antigen presenting cells, it functions as a potent
activator of iNKT cells. ThrCer6 is an alpha-Galactosylceramide (αGalCer)-
derived iNKT cell activator that possesses a superior iNKT cell activation
profile compared to αGalCer in vitro as well as in vivo. Unlike αGalCer, it
does not subject to glycosidase-mediated degradation. Induction of
antigen-specific CD8+ T cell responses at lower concentrations of ThrCer6 than
αGalCer were observed in mice due to a prolonged bioavailability. ThrCer6
maintained the capacity to induce DC maturation in vivo to a similar extent as
αGalCer. Furthermore, iNKT cell-mediated anti-tumor effects include IFN-*-
dependent Natural Killer (NK) cell responses, maturation of DCs, expansion of
antigen-specific CD8+ T cell responses, inhibition of metastatic behavior and
improved survival in cancer mouse models.
Co-encapsulation in nanoparticles: iNKT cell activator and tumor antigen
Preclinical studies in murine cancer models showed αGalCer/CD1d-dependent
superiority of nanoparticles that co-encapsulated αGalCer+Ovalbumin (OVA) in
terms of CD8+-specific T cell induction compared with nanoparticles with OVA
combined with either nanoparticles with αGalCer or soluble αGalCer. These
findings appear independent of CD4+ T cell help. Co-encapsulation of a potent
iNKT cell activator such as αGalCer or ThrCer6, with a tumor antigen, such as
NY-ESO-1, can result in detectable cytotoxic T lymphocyte (CTL) responses
against the respective antigen. With this in mind, in vitro experiments with
peripheral blood mononuclear cells were performed to first assess feasibility
of co-encapsulation. These experiments demonstrated the feasibility of
encapsulating three NY-ESO-1 peptides (the same peptides to be used in this
trial) together with IMM60 in the same particle. Peptides were processed and
presented by multiple HLA types. In mice expressing the human HLA-A2 molecule,
activity of nanoparticle-encapsulated IMM60 was evident by DC maturation and
IFN-γ secretion. Also, CD4+ and CD8+ T cell responses against NY-ESO-1 peptides
were observed with the same nanoparticles co-encapsulating NY-ESO-1 peptides.

The primary objectives of this trial are:
- To determine the safety and tolerability of increasing doses of PRECIOUS-01
after intravenous (i.v.) administration in subjects with solid tumors;
- To assess the effect of increasing doses of PRECIOUS-01 on the composition
and spatial heterogeneity of immune cells directly in the tumor
The secondary objectives of this trial are:
- To determine the Recommended Phase 2 Dose (RP2D) of PRECIOUS-01 for a
subsequent Phase II trial. - To assess immunological responses during and after
therapy with PRECIOUS-01 in blood.
The exploratory objective of this trial is:
- To evaluate the clinical outcome of PRECIOUS-01 using Response Evaluation
Criteria in Solid Tumors (RECIST) v1.1.
Further exploratory objectives will be determined based on results of ongoing
preclinical studies.

This is an open-label, first-in-human Phase I dose escalation trial to
investigate the safety and tolerability of increasing doses of PRECIOUS-01
administered i.v. in subjects with solid tumors.
Eligible subjects will receive three i.v. infusions of PRECIOUS-01 at a
3-weekly interval in three dose-finding cohorts (low: 0.4 mg/kg, intermediate:
0.8 mg/kg, and high: 1.6 mg/kg fixed doses). Subjects will be monitored for
safety and the occurrence of Dose-Limiting Toxicities (DLTs). It is planned to
follow a 3+3 design for the dose escalation steps. Three subjects will be
enrolled sequentially per cohort. If the maximum tolerated dose (MTD) is not
reached in the planned dose escalation cohorts, the RP2D will be based on the
observed safety and immune-modulatory activity as pharmacodynamic parameter
supporting the RP2D. The sample size is based on the determination of the
MTD/RP2D. In order to collect sufficient information regarding changes in
immune-related parameters as readout for pharmacodynamics of the particles
(e.g., NY-ESO-1-specific CD8+ T cells, iNKT cell activation, and NY-ESO-1
antibody responses in serum) it is planned to extend the two highest dosing
cohorts to a total of six subjects.
Based on accumulating data during the trial and no observed toxicity in cohort
2 after three subjects for DLT observation, no further subjects may be enrolled
at that dose level. Instead, cohort 3 would start. If no toxicity is observed
in the six planned subjects in cohort 3 and based on the recommendation of the
Dose Steering Board (DSB), three additional subjects may be enrolled at the
high dose level for a total of nine subjects. If a toxicity is observed in the
first six subjects of cohort 3, three subjects will be enrolled in cohort 2
(for a total of six subjects at that dose level). The total number of subjects
will not change.
The starting dose is 10% of the No Observed Adverse Effect Level (NOAEL) in the
toxicology study. Doses will be escalated after a review of the safety data
from the previous cohort by a DSB upon completion of the DLT observation
period. Pre-defined DLT criteria will be used.

PRECIOUS-01 will be administered i.v. three times at 3-weekly intervals (Day 1,
Day 22 and Day 43).
Doses are specified as follows: low (0.4 mg/kg PRECIOUS-01), intermediate (0.8
mg/kg PRECIOUS-01) and high (1.6 mg/kg PRECIOUS-01). PRECIOUS-01 will be
administered as fixed doses (based on a 70-kg average body weight).

Risks
PRECIOUS-01 has not yet been given to patients, therefore side effects to
PRECIOUS-01 are unknown. The particle has a low potential risk of inducing
allergic reactions. Possible AEs associated with an allergic reaction can
include fever, chills, headache, weakness, nausea, vomiting, diarrhea, low
blood pressure, respiratory symptoms, and rashes. Additionally, since the mode
of action of PRECIOUS-01 includes potential stimulation of the immune response,
there is a likelihood of immune-related AEs. Generally, such AEs have been
observed after specific blocking of inhibitory receptors expressed on T cells
(e.g. Programmed Cell Death Protein 1 [PD-1]) rather than after induction of
tumor-specific immune responses. If PRECIOUS-01 elicits a particularly strong
T-cell immune response in a few subjects predisposed to develop autoimmunity,
immune-related AEs may nonetheless be observed.
Toxicology findings recorded in preclinical studies include hepatocellular
necrosis, vascular/perivascular mononuclear cell infiltration and thrombosis,
all partially recovered. The hepatocellular necrosis correlated with an
increase in alanine aminotransferase (ALT) and aspartate aminotransferase (AST)
levels. To mitigate the thromboembolic risks observed in preclinical studies
with PRECIOUS-01, subjects who have active thromboembolic events that require
anti-coagulation were excluded from the trial. Additionally, all subjects will
be closely monitored for these associated laboratory parameters on Days 1, 7,
and 14 of Cycle 1 and on Days 1 and 7 during Cycle 2 and 3. If needed, the
frequency of monitoring for these changes will be appropriately adapted during
the trail per subject.
The toxicology results also suggest an increase in IFN-γ and TNF-α production,
which can potentially be associated with fever, chills, dizziness and bone
marrow toxicity. The levels of IFN-γ and TNF-α will be carefully monitored as
detailed in the Schedule of Assessments.
Administration of PRECIOUS-01 was associated with lower white blood cell and
platelet count, increased red blood cell mass, reticulocytes and circulating
enzyme levels. These were fully recoverable.
Additional to potential risks related to administration of PRECIOUS-01, there
are also procedural risks related to blood sampling, other possible
subcutaneous injections and diagnostic procedures. Blood sampling and
subcutaneous injections may cause pain, bleeding, bruising, and/or infections
at the site of cannula insertion. Rare complications due to blood sampling are
syncope, thrombophlebitis, as well as accidental punctures of an artery or
nerve.

Benefits
This is the first clinical trial with PRECIOUS-01. The primary aim of this
trial is to obtain safety, and tolerability data when PRECIOUS-01 is
administered to subjects with NY-ESO-1-positive solid tumors. In addition to
the minimal risk using PRECIOUS- 01 (please see IB), preclinical experience
with the compound indicates a potential benefit that might be expected in terms
of an immune response against the tumor that might lead to a beneficial
clinical outcome.
The safety and efficacy data, together with the immunological data obtained
from this trial, will help establish the treatment regimen and a Recommended
Phase II Dose (RP2D) that can form the basis for a recommended dose suitable
for subsequent clinical trials.
Benefit/risk ratio
The anticipated risks, based on the non-clinical experience with PRECIOUS-01,
are expected to be manageable. The importance of the objective of this trial is
considered to outweigh the risks and burdens to the subjects. Measures are
implemented to minimize burdens and risks for subjects. Subjects will be
monitored closely for the occurrence of any significant clinical events and
treatment will only continue if it is considered safe and appropriate to do so.
The benefit/risk assessment is favorable and justifies the planned trial.