Detection of soluble isoforms of immuno-oncology checkpoint markers

by S. Oehler, R. Seba, M. Miholits, and I. Rech-Weichselbraun – 04/2017

The ProcartaPlex Immuno-Oncology Checkpoint Panel is the first commercially available multiplex immunoassay system that allows simultaneous detection of the soluble forms of the following analytes: BTLA, GITR, HVEM, IDO, LAG-3, PD-1, PD-L1, PD-L2, Tim-3, CD28, CD80, 4-1BB, CD27, and CTLA-4. Soluble isoforms, or shed variants of these molecules, have been described and can be quantitated by this innovative assay.

Immune checkpoint markers represent potential therapeutic targets

Immune checkpoint markers are recognized as intact transmembrane proteins (for a review, see reference 1) and are known to play a crucial role in the regulation of T cells, leading to either T cell exhaustion or stimulation, thus modifying the antitumor immune response. As such, those molecules have been identified as potential targets for therapeutic intervention by therapeutic antibodies such as the CTLA-4 blockade agent ipilimumab.

In addition to the widely assessed clinical studies of checkpoint blockades such as PD-1 and CTLA-4, a number of additional checkpoint modulators for immunostimulatory therapy are under investigation. Therapeutic antibodies recognizing IDO, GITR, LAG-3, 4-1BB, and TIM-3 are currently being tested in clinical trials.

Quantifying soluble isoforms of checkpoint markers is key for accurate candidate studies

At present only a limited number of studies on soluble immune checkpoint markers can be found in the literature. Correlation between the levels of soluble marker proteins and cancer type and progression is largely unknown, and evidence for the association of the soluble immune checkpoint markers with response to therapy and outcomes is still emerging. There is obviously a tremendous need for further research into the biological functions of these soluble variants.

So far, evidence indicates that soluble isoforms may be generated either by alternative splicing or by shedding of the extracellular portion from the cell surface by matrix metalloproteinases [2]. Once present, however, these molecules have the potential to function as decoy receptors or as immune adjuvants and interfere with the clinical efficacy of the checkpoint modulator drug candidates, making quantification of the soluble form important to enable correlative studies. The relevance of measuring soluble immune checkpoint markers in serum or plasma samples of cancer patients has been shown in several publications [3,4,5,6]. Besides soluble CTLA-4 and its potential as a biomarker enabling the discrimination of responders and nonresponders to ipilimumab, LAG3 is another example of a soluble checkpoint molecule of significant clinical research interest. Soluble LAG3 has been shown to function as an immune adjuvant, but its impact on LAG3 blockade needs to be further investigated (for a review, see reference 7).

Towards a minimally invasive method to identify potential responders

Despite the recent breakthroughs in immuno-oncology and the launch of therapeutic antibodies targeting checkpoint molecules like PD-1 and CTLA-4, many open questions remain. One of the most important questions is why only some patients respond to therapeutic antibodies, creating an intensifying need for tools that may enable the discrimination of responders versus non-responders before applying therapies that may not work and often yield side effects. Ideally, a biomarker assay to identify potential responders prior to treatment should be performed on samples obtained from a minimally invasive procedure (i.e., blood draw). The first evidence supporting the ability to stratify patient groups using soluble immune checkpoint markers was the finding that elevated serum levels of CTLA-4 correlate with clinical outcome benefit in ipilimumab-treated patients [8]. Interestingly, studies show that some patients with histological PD-1– or PD-L1–negative tumors responded to blockade therapy, suggesting that soluble isoforms of checkpoint molecules play an active role in the pathway [9].

Assaying samples with ProcartaPlex Panels

ProcartaPlex Panels are based on spectrally distinct microsphere mixtures

The ProcartaPlex Human Immuno-Oncology Checkpoint Panel’s capture antibodies are covalently bound to the surface of 6.5 µm microspheres that are internally dyed with precise proportions of red and infrared fluorophores. The distinct blends of these two fluorophores (called “bead sets”) result in unique spectral addresses that can be detected when analyzed on Luminex platforms. As with a traditional immunoassay, antigen quantification is enabled by a fluorescently labeled secondary antibody whose signal intensity is proportional to the concentration of protein detected. For multiplexing purposes, a specific bead set is assigned to each analyte, enabling the simultaneous measurement of multiple analytes from a small sample volume (25 µL for serum or plasma samples, 50 µL for cell culture supernatant). The technology allows for the simultaneous detection of multiple soluble immune checkpoint markers using highly specific antibody pairs to create bead-based sandwich immunoassays.

Starting sample material

Soluble isoforms, or shed variants, of the immune checkpoint marker proteins are typically measured using serum, plasma, and cell culture supernatant. Protocols for the use of tissue homogenates and cell lysates are available as well. As with all multiplex assays, ProcartaPlex Panels offer a highly valuable tool for biomarker profiling by delivering maximum information from a limited sample volume.

ProcartaPlex Panel assay results match those obtained with plate-based ELISA

It has been previously suggested in the literature that the anti-cancer immune response is determined by the maintenance of an elegant balance between stimulatory and inhibitory checkpoint molecules [10]. The simultaneous analysis of those factors is expected to yield significant insight and contribute to a better understanding of therapeutic benefit and resistance mechanisms. Figures 1–3 illustrate that equivalent results (R2 of 0.79–0.95) are obtained using ProcartaPlex multiplex immunoassays and traditional plate-based ELISA. The assay sensitivity and specificity enables the detection of soluble biomarkers in serum, plasma, cell culture supernatant, or lysates, with an intra- and inter-CV of ≤15% for all targets (Table 1).


Figure 1. Correlation of PD-L1 sample values determined in PPX and ELISA format.  PD-L1 was measured in cell culture supernatants of hepatocellular carcinoma cells (n = 11). 


Figure 2. Correlation of PD-L2 sample values determined in PPX and ELISA format. PD-L2 was measured in sera (n = 5), EDTA plasma (n = 5), and heparin plasma (n = 5) from healthy donors.



Figure 3. Correlation of PD-1 sample values determined in PPX and ELISA format. PD-1 was measured in sera (n = 8) from healthy donors.


Table 1. Lower limits of quantification (LLOQ) and CVs of ProcartaPlex Human Immuno-Oncology Checkpoint Panel.

Analyte LLOQ* Inter-assay CV* Intra-assay CV*
BTLA 144.65 4.75% 4.89%
CD27 5.76 2.96% 4.20%
CD28 42.02 4.65% 6.70%
CD80 43.58 2.04% 3.92%
CD137/4-1BB 14.21 5.09% 5.68%
CD152/CTLA4 9.52 4.50% 4.87%
GITR 27.2 3.78% 6.11%
HVEM 18.55 4.39% 8.23%
IDO 4.37 5.90% 6.72%
LAG-3 11,21 3.55% 5.24%
PD-1 7.13 3.34% 5.23%
PD-L1 3.64 4.98% 6.13%
PD-L2 48.71 3.71% 6.03%
TIM-3 63.18 3.05% 6.41%

*As determined for Lot 1

Assay flexibility is built into the Immuno-Oncology Checkpoint Panel

The flexibility of the Immuno-Oncology Checkpoint Panel assay format allows customization to individual research needs. The assay can easily be extended by additional analytes, most interestingly chemokines and cytokines. Various cytokines have been demonstrated to be associated with chronic inflammation, which is cancer-promoting, or are associated with acute inflammation, which facilitates cancer rejection [11]. Monitoring serum/plasma cytokine profiles longitudinally can provide information relevant to the function profiles of individual patients or patient groups in the future [12]. 

The multiplex immunoassay for the detection of soluble checkpoint markers enables assessment of multiple protein biomarkers. The analysis of protein from liquid samples (plasma or serum) is easy and convenient. In addition, the ability to monitor soluble checkpoint molecules, correlate their levels with progression of disease in longitudinal studies, and begin to associate biomarker levels with checkpoint blockade therapy response, is a highly promising area of research for the Human Immuno-Oncology Checkpoint Panel. 


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