March 1, 2017 (Vol. 37, No. 5)

Ramona Seba Product Manager Affymetrix

Understanding Therapeutic Antibody Response in Immuno-Oncology

Immune checkpoint markers have been described as targets for therapeutic intervention because they play a role in the regulation of T cells, leading to either T-cell exhaustion or stimulation, which can modify antitumor immune response. Antibodies such as the CTLA-4 blockade agent ipilimumab have been identified as promising therapeutic agents. However, soluble variants of these checkpoint marker molecules can function as decoy receptors or as immune adjuvants that may interfere with the response to those antibodies.

Despite 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 is why only a portion of patients respond to therapeutic antibodies, while some do not. This question has stimulated demand for tools that can identify responders vs. nonresponders, prior to applying therapies. New information on the activity of soluble immune checkpoint markers may help improve the therapeutic success rate and minimize ineffective therapy along with side effects. The goal is to develop a minimally invasive method, such as biomarker detection, from blood samples for this type of preemptive stratification.

The first evidence of the feasibility of stratifying patient groups using soluble immune checkpoint markers was the discovery that elevated serum levels of CTLA-4 correlate with positive clinical outcomes from ipilimumab treatment. Interestingly, 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.

In addition to the widely assessed clinical activity of checkpoint blockades such as PD-1 or CTLA-4, therapeutic antibodies recognizing IDO, GITR, LAG-3, 4-1BB, and TIM-3 are currently being tested in clinical trials. For example, soluble LAG-3 has been shown to function as an immune adjuvant, but its impact on the LAG-3 blockade needs to be further investigated.


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).

The unresolved questions regarding the biological function of soluble checkpoint markers stand in the way of fully understanding and demonstrating the clinical efficacy of the individual checkpoint modulator drugs. Therefore, the quantitation of the soluble form is important to correlative studies. It has been previously suggested in the literature that the anticancer immune response is determined by the maintenance of an elegant balance between stimulatory and inhibitory checkpoint molecules. The simultaneous analysis of those factors is expected to yield significant insight and contribute to a better understanding of therapeutic benefit and resistance mechanisms.

Evidence of the association of the soluble immune checkpoint markers and response to therapy and outcomes is just emerging, and there is tremendous need for further research into the biological functions of these soluble variants. Furthermore, the precise source of these soluble, circulating checkpoint molecules is still under investigation. Soluble isoforms may be generated by alternative splicing or shedding of the extracellular portion from the cell surface by matrix metalloproteinases.

The multiplex immunoassay is well suited for the detection of soluble checkpoint markers because it provides complementary information to what is obtained by pathologists from tumor tissues. The analysis of protein from liquid biopsies (i.e., plasma or serum) is easy, convenient, minimally invasive, and not biased by the location of the sampling, such as is the case with tissue biopsies. In addition, the ability to monitor soluble checkpoint molecules, correlate these levels with the progression of the disease in longitudinal studies, and begin to associate biomarker levels with checkpoint blockade therapy response is a highly promising area.

The research needs outlined above have been catalysts for the development of the ProcartaPlex Human Immuno-Oncology Checkpoint Panel (Affymetrix), a multiplex immunoassay that can be customized to individual clinical or research needs. The Human Immuno-Oncology Checkpoint Panel can easily be extended for additional analytes, most interestingly chemokines and cytokines, some of which are known to be associated with chronic inflammation, which is cancer-promoting, or are associated with acute inflammation, which facilitates cancer rejection. Monitoring serum/plasma cytokine profiles longitudinally can provide information relevant to the function profile of individual patients or patient groups.

The technology allows for the simultaneous detection of multiple soluble immune checkpoint markers using highly specific antibody pairs to create bead-based sandwich immunoassays. The 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 individual blends of these two fluorophores result in unique spectral addresses that can be detected when run on Luminex instrumentation platforms. As with a traditional immunoassay, antigen quantification is enabled by a fluorescently labeled secondary antibody whose intensity is proportional to the concentration of protein detected.


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.

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). By delivering maximum information from a limited sample volume, the multiplex immunoassay in general is a highly valuable tool for biomarker profiling. The main matrices for samples analyzed with the Human Immuno-Oncology Checkpoint Panel are serum, plasma, and cell culture supernatant—enabling measurement of the soluble isoforms or shed variants of these proteins. Protocols for the usage of tissue homogenates and cell lysates are available as well.

Figures 1–3 illustrate that equivalent results (R2 between 0.79 and 0.95) are obtained using multiplex immunoassays and traditional plate-based ELISAs. The sensitivity and specificity of the immunoassay enable the detection of soluble biomarkers in serum, plasma, CCS, or lysates with an intra- and inter-CV of ≤15% for all targets. The Human Immuno-Oncology Checkpoint Panel is the first commercially available multiplex immunoassay system that allows the 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.

In order to advance the development of immune therapies, it will be important to further study soluble checkpoint molecules. Measurement of these markers is a promising new tool that can also be combined with the measurement of cytokines and chemokines in multiplex immunoassays to support research in the field of immuno-oncology and cancer immunology.


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.

Ramona Seba (ramona_seba@affymetrix .com) is a product manager at Affymetrix.

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