Validation of Pharmacokinetic and Pharmacodynamic Assays for determination of Nivolumab and Pembrolizumab concentrations, and the occupancy of their target receptor Programmed Death-1 (PD-1) on T-cells

Programmed cell death protein 1 (PD-1) is a transmembrane immune checkpoint
molecule, which is expressed on T-cells and pro-B cells. PD-1 fulfils various
anti-inflammatory functions, such as promoting apoptosis of antigen specific
T-cells and preventing activation of T cells. PD-1 binds two ligands: PD-1 and
PD-2. PD-1 in particular is often overexpressed in many tumour types. Anti-PD-1
monoclonal antibodies are able to prevent the binding of PD-1 to its ligands
and sequentially prevent inhibition of the immune system.

Anti PD-1/PD-L1 immunotherapy has quickly risen to be one of the most effective
immunotherapies for various cancer types, such as non-small cell lung cancer
(NSCLC), renal cell carcinoma and melanoma. Investigation on predictive and
prognostic biomarkers is currently ongoing. Biomarkers in the peripheral blood,
which are measured during routine clinical testing, such as lymphocyte numbers,
may predict outcome but are not therapy specific. One biomarker that has
received much attention is the immunologic biomarker PD-L1. PD-L1 expression
has been associated with better response rates to PD-1/PD-L1 treatment1,2.
Predictiveness of clinical outcome based on PD-L1 expression varies between
tumor types. PD-L1 status may have a stronger predictive value in melanoma
patients compared to NSCLC3. Other markers such as T-cell infiltration, T-cell
receptor clonality and somatic mutation burden are currently being
investigated4.

Not one marker will suffice on predicting outcome to PD-1/PD-L1 treatment. For
example, negative PD-L1 staining does not rule out a response to treatment.
Combinations of different markers may be needed in order to select which
patients will respond to treatment. Possible predictive individual markers for
outcome are the serum free drug levels and bound drug levels of the monoclonal
antibody. Upon administration, a large portion of the therapeutic drug
immediately binds to its target on T-cells. The target is mainly located in
peripheral tissues and therefore a large portion of nivolumab may not reach the
tumor infiltrating lymphocytes (TILs). To our knowledge, it has not been
described whether it is binding to the T-cells located in the peripheral blood
or binding to TILs that contribute the most to tumour cell killing. This
knowledge is limited due to lack of well described validated assays to measure
anti-PD1 antibodies and well described pharmacokinetic and PK/PD models.

To better describe the pharmacokinetics of anti PD-1 antibodies, validated
assays to measure serum concentrations of PD-1 antibodies, which can be used
routinely in the clinic are needed. In our lab Enzyme-Linked Immuno Sorbent
Assays are currently developed or in development. These assays use commercially
available reagents to measure pembrolizumab and nivolumab. For these assays,
plates are first coated with PD-1, followed by incubation of serum samples.
During this step the antibody binds to PD-1. During the washing step other
antibodies will be removed from the plate. Next, wells are incubated with
anti-human-IgG4 antibodies, which are horseradish peroxidase conjugated. During
the final step, wells are incubated with enhanced chemiluminescence reagent,
which contains luminol. Horseradish peroxidase catalyses the oxidation of
luminol in a product that emits light. This signal is measured with an ELISA
plate reader. By comparing the sample to a standard curve, the concentration of
the antibody can be calculated.

The aim of this study is to develop and validate the ELISA and potential other
assays (e.g. LC-MS), in order to measure drug concentration levels. For this,
blood and other material will be collected.

For Group A: Patients who receive nivolumab or pembrolizumab treatment will be
asked to participate in this study for blood sampling during treatment. Blood
will be drawn a total of 8 cycles (max 128ml). It will be discussed with the
patient how many times.


For Group B: Patients who will undergo a clinically indicated necessary
procedure, such as ascites/pleural fluid drainage, will be asked to donate
residual material for this study. Additionally, pts will be asked to donate 8ml
of blood.
.

For Group A: Patients who receive nivolumab or pembrolizumab treatment will be
asked to participate in this study for blood sampling during treatment. How
many and when will be discussed with the patient:
-Pre dose will be drawn from drug infusion venflon
-At the end of infusion (extra venipuncture)
-Up to 8 cycles, max 128ml blood will be drawn

Patients do not need to stay longer at the hospital and do not need to make
extra visits. Optionally one extra phlebotomy will be performed at the end of
infusion. The risks for this study are low: there is a chance that a bruise
will be visible at the site of injection.

For group B, no extra burden exists for the donation of residual material, but
patients will be asked to donate 8ml of blood.