The Importance of Quality Immuno-oncology Antibody Reagents In Studying Immune Checkpoint Inhibitors

These days, it’s tough to mention cancer without also mentioning the novel class of cancer-fighting immuno-oncology therapeutics called immune checkpoint inhibitors. These transformative drugs have extended the life of patients with many distinct cancer types, sometimes years longer than they would have lived without receiving treatment. The power of these therapies and the initial discovery of immunotherapeutic reagents were only possible due to the efforts of basic researchers, such as James Allison, whose innovative research led to eventual FDA-approval of Yervoy (ipilimumab).

Basic research is still important for the identification of novel therapeutics and to make currently available therapeutics more efficacious and safe. The ability to do reliable research, however, is dependent on the availability of reliable reagents, specifically antibodies, which are critical to a number of experimental methods. Below, we discuss some best practices for choosing reliable antibodies. Let’s first, review some basics about immune checkpoint inhibitors and the immuno-oncology antibody.

Immune Checkpoint

What is a Checkpoint Inhibitor? The Rise of The Immuno-Oncology Antibody

Checkpoint inhibitors are able to activate the immune system, specifically T-cells, to target and eliminate tumor cells. Typically, cancerous cells are able to keep T-cells in an inhibited state, and checkpoint inhibitors are able to “inhibit the inhibition.” There are lots of checkpoint pathways that are responsible for regulating T-cell activity, however, the FDA-approved checkpoint inhibitors only target one of two pathways: the CTLA-4/CD80 or PD-1/PD-L1 pathway.


CTLA-4 is an inhibitory receptor expressed on T-cells and can bind to proteins named CD80 or CD86 on tumor cells, inhibiting T-cell activity. One FDA-approved treatment, Yervoy, is hypothesized to prevent this interaction.


PD-1 is another inhibitory receptor expressed on T-cells and, like CTLA-4, it can bind to two surface proteins on tumors, PD-L1 or PD-L2. Many human and animal tumors express upregulated PD-L1, suggesting that it is associated with tumor immune evasion. The other five FDA-approved checkpoint inhibitors are thought to prevent the interaction between PD-1 and PD-L1. Therapeutics like Keytruda target PD-1, while others like Tecentriq (atezolizumab), target PD-L1.

Getting Specific With An Immuno-oncology Antibody

These therapeutics are able to specifically target these pathways because they are proteins called antibodies, a class of molecule used by the human immune system to target and destroy “foreign” bacteria or viruses that can potentially cause harm. The specificity of antibodies and their ability to bind to specific proteins, has made them a pillar of basic research for a variety of immuno-oncology applications, and the immune checkpoint inhibitors just discussed act as anti-CTLA-4 antibodies or anti-PD-1/PD-L1 antibodies.

Back to Basics: Using Antibodies For Basic Research Immuno-oncology Applications

There are many outstanding questions in the immuno-oncology field, including those related to:

-the mechanism of action of currently approved checkpoint inhibitors
-whether targeting other checkpoint pathways would be efficacious and safe
-how predictive specific biomarkers are within the course of a patient’s disease or response to a specific therapeutic regimen.

For instance, while many tumors express upregulated PD-L1, there are tumors that are considered to be PD-L1-negative that respond to anti-PD-1 immunotherapy. This not only highlights the necessity for better predictive biomarkers, but also for additional basic research.

So, what is standing in the way of researchers addressing these questions? Well, there are many challenges, but one factor is the availability of reliable reagents such as antibodies.

How to Pick the Right Antibody Reagent

Antibodies are used in basic research for a number of different types of assays, including enzyme-linked immunosorbent assay (ELISA), flow cytometry, western blotting, immunofluorescence, and immunohistochemistry (IHC), as they are the easiest and most efficient methods to track specific proteins, like those involved in regulating immune checkpoints.


While antibodies can be very specific, the reliability of many commercially available antibodies has recently been called into question. Some of the potential problems with these antibodies include:

-the inability to reproduce published results with commercially available antibodies
-batch-to-batch variability
-cross reactivity and lack of specificity for reported target.

These issues can arise due to the production method used for making the antibody or due to incomplete testing. When picking an antibody for any experiment, whether it be for immuno-oncology or another research area, there are some critical questions that you should ask about the reagent.

Here are four questions that you should consider before purchasing an antibody.

Is the Antibody Polyclonal?

Polyclonal antibodies are produced through injection of an antigen, a foreign protein or other molecule that provokes an immune response, into an animal and use of the serum as an antibody source. This presents a significant issue with reproducibility as each animal and batch of antibody generates a different mixture of antibodies. This mixture can range from 0.5% to 5% of the polyclonal antibody actually recognizing the desired antigen.

Is the Antibody Monoclonal?

In contrast to polyclonal antibodies, monoclonal antibodies are produced by immortalizing an antibody-producing cell isolated from an animal that has been injected with an antigen. These cells are called “hybridomas.” In theory, this process creates a cell producing one single antibody and thus 100% of these antibodies should target the desired antigen. In addition, because the antibody-producing cell is immortalized, one could continuously produce this antibody, reducing batch-to-batch variability. In practice, however, monoclonal antibodies can actually target proteins other than the desired antigen or be a mixture of antibodies. Hybridomas can also die during the freeze-thaw process of normal cell handling, leading to the loss of important antibody-producing cell lines. These problems are similar to those with polyclonal antibodies and thus, careful validation that a monoclonal antibody binds to the antigen of interest should be done before using them routinely. 

How Was the Antigen Used For Antibody Generation Produced?

Antigens can be produced in a laboratory using many different expression systems from bacteria to mammalian cells. There are a variety of pros and cons for producing antigens in these systems, but one important downside to producing antigens in bacteria is that they will have very different post-translational modifications than those antigens produced in mammalian cells, and thus can generate very different immune responses during antibody production. This is an important consideration for those looking for an antibody to be used in an immuno-oncology application (or other field that typically uses mammalian model systems), as they will most likely want an antibody that is able to target an antigen most like that in the model system in which you are working. 

What Method Was Used to Validate The Antibody?

The different experimental methods that use antibodies can change how accessible an antigen is to an antibody. For instance, in a western blot, an antigen would be immobilized on a membrane, and in an IHC assay, an antigen would be fixed in an (almost) native cellular environment. It is important to consider whether or not the antibody you are using has been validated for these specific methods, as it may be more ideal for one over the other.

Looking Ahead Towards The Next Generation Of Immune Checkpoint Inhibitors

Answering these questions and making them part of your best practices for reagent choice will help you choose the most reliable antibody reagent and thus generate the most reliable and reproducible data. The more researchers that engage in the use of reliable antibodies, the quicker the basic research questions standing in the way of further development of immune checkpoint inhibitors can be answered.