Applications of Mixed Lymphocyte Reactions

Why use an MLR?

An MLR is an in-vitro method to assess adaptive immune response via cell-mediated immunity. As such, the MLR is one of the single best in-vitro tools available to assess how therapeutics affect our adaptive immune system. It can be used to assess immunogenicity of the therapeutic, test potency and dose-response profiles, investigate mechanism of action, and characterize efficacy and bioactivity: whether it be immunosuppressive properties, checkpoint inhibition, or transplant tolerance. Each of these are critical factors to consider during product development and are often recommended tests for pre-IND applications or during pre-clinical and clinical studies.   

When to use the MLR?

The MLR can be used at any stage of product development where the therapeutic’s effect on cell-mediated immunity needs to assessed. This covers a wide range of applications, from immunogenicity to efficacy and beyond. If the therapeutic drug effects or is expected to effect cell-mediated immunity, then the MLR is an ideal model to characterize those effects. And just as there is not just one MLR format, there are a variety of readouts that can be utilized (e.g. cytokines, cellular proliferation, kinase activity, etc). Any technique that can be used to assess cells, cellular contents, or cellular secretions can be paired with the MLR. And as such, readouts can be tailored to the specific needs of the assay (see MLR readouts), whether that be assessing immunogenicity via pro-inflammatory cytokine production (e.g. multiplexed cytokine assays), or investigating the mechanism of action (e.g. intracellular flow cytometry, western-blot analysis, and RT-qPCR).  

How does the MLR fit into industry guidance?

Assessing a therapeutic’s effect on the adaptive immune system is critically important to pre-clinical safety, and as such, guidance documents often recommend performing an immunogenicity assessment. Beyond immunogenicity, it is important (and recommended) to characterize other effects of the therapeutic on cell-mediated immunity, depending on the therapeutic’s mechanism, intended use, potential side effects, and other factors. Therefore, the MLR is well-suited to address many of these tasks.  

Below we discuss how the MLR can fit into different stages of product development based on recommended regulatory guidance. It should be noted that regulatory guidance for industry rarely recommends one specific assay or technique that should be used to evaluate the immunological properties of a therapeutic; however  the information below is our best interpretation of where the MLR can be best utilized to fulfill recommended guidance criteria associated with the ex vivo evaluation of immunological drug safety or efficacy.

Discovery and Development:

Discovery phase of drug development is primarily concerned with either the de-novo discovery of a therapeutic candidate, or narrowing down a specific candidate from an existing broad class of potential candidates with similar properties. Once ideal candidate(s) have been selected, development continues to further characterize the therapeutic – routes of administration, adsorption, metabolization, side effects, reactivity with other drugs, dosages, and comparisons to current therapeutics/treatments.

The MLR can be valuable tool for assessing in-vitro efficacy, bioactivity, dose response, and immunogenicity. As briefly mentioned above, there are various formats of the MLR and optional readouts that can be tailored to investigate specific aspects of how a therapeutic effects cell-mediated immunity.

Pre-Clinical:

The pre-clinical phase of drug development is primarily concerned with assessing the toxicological potential of the therapeutic using both in-vivo animal models and in-vitro human testing. This is a critical component of an Investigational New Drug (IND) application, that must be submitted prior to human trials. Additionally, depending on the therapeutic, it may be recommended to show Proof of Concept (POC) data and documentation that characterizes therapeutics for factors like efficacy, activity, and mechanism of action, amongst others.

While the MLR is often a good option amongst various other cell-based assays to investigate mechanism of action and efficacy, the MLR is often considered the gold standard for in-vitro immunogenicity assays for adaptive (cell-mediated) immune responses. In some cases these assays are called PBMC assays, or CD8/T-cell depleted PBMC assays, or DC/T-cell assay – all of which refer to the MLR in some format.  

We should note that there are also several other recommended guidances for assessing immunogenicity beyond in-vitro adaptive immunogenicity. Those are:

1. 1. In-vivo adaptive immunogenicity via the detection of anti-drug antibodies (ADA’s) and neutralizing antibodies (NAb’s), which are nearly always required; and

   2. assessing innate immunogenicity via cell-based in-vitro assays like macrophage activation/polarization, dendritic cell (DC) maturation, monocyte/PBMC stimulation, and cytokine release assays.

Clinical:

The clinical phase is the first time the therapeutic is to be tested on human subjects. The clinical phase is divided into 4 smaller phases, starting off with only a handful of subjects for a short period of time and gradually increasing in size to thousands of individuals for up to several years. Phase I is usually conducted to determine appropriate dosage and monitor for safety, after which follow up trials primarily focus on monitoring for safety, efficacy, and side effects in the larger population.

While the MLR has limited uses as an in-vitro method during a clinical trial, it still has its uses. The MLR can be used for cellular therapies or for tissue/organ transplants, where it may be prudent to monitor if the patients’ immune cells have become tolerized or have developed sensitivity to the treatment. In those cases, the patients’ cells (often PBMCs) are co-cultured with the therapeutic. 

Often, the clinical phase will focus on methods aside from cell-based assays to assess safety and efficacy. Among the most common are ADA assays and biomarker analysis, both of which Xeno Dx offers.

CMC Manufacture and Lot Release:

Chemistry, Manufacturing, and Controls (CMC), is the process of producing the therapeutic while ensuring quality and consistency. This is applicable at the pre-clinical phase, clinical phase, and beyond. Recommended guidelines are concerned with a number of factors including: sterility, efficacy/potency, identity, purity, and immunogenicity, among others.

As already discussed, the MLR can be a valuable tool for assessing efficacy and immunogenicity of a therapeutic, and is routinely used to test products prior to lot-release.