The Role of Peripheral Blood Mononuclear Cells (PBMCs) in Drug Discovery and Clinical Trials.
By Dr. Kurt Sales, CSO
In the ever-evolving landscape of drug discovery and development, the ability to accurately assess drug mechanisms, efficacy, and safety is crucial. With more drugs targeting the immune system and circulating immune cells within the blood, whole blood assays or assays using peripheral blood mononuclear cells (PBMCs) are important tools in the pharmacodynamic toolbox.
The pharmacodynamics of a drug refers to its effect on the body and how it interacts with its target. PBMCs are commonly used in clinical trials to evaluate the pharmacodynamics of a new drug, particularly in trials for biologics, immunotherapies, and vaccines. By isolating PBMCs from blood from patients at different time points during the trial, bioanalytical laboratories can assess how a drug affects immune responses.
This article explores the role of PBMCs in drug discovery, their significance in clinical trials, and how bioanalytical laboratories leverage them to assess drug function during clinical studies. Finally using a case study, we present some important stability data for PBMC processing from whole blood and discuss Agilex’s strategy to support PBMC processing and multiparameter flow cytometry analysis in Australia.
Understanding Peripheral Blood Mononuclear Cells (PBMCs)
PBMCs are a diverse group of blood cells that consist of lymphocytes (T cells, B cells, and natural killer cells), monocytes, and dendritic cells. These cells are critical components of the immune system, with each playing a unique role in immune responses. Lymphocytes are responsible for adaptive immunity, while monocytes and dendritic cells are part of the innate immune system and serve as key players in immune regulation and inflammation.
Due to their diverse nature, PBMCs offer a valuable tool for evaluating immune responses, inflammation, and drug effects in both preclinical and clinical settings. They are typically isolated from blood samples using density gradient centrifugation, making them a readily accessible and versatile resource in clinical settings
PBMCs in Drug Discovery
In drug discovery, PBMCs serve as a powerful in vitro model for understanding how a drug interacts with the immune system and modulates immune responses. Drug developers often use PBMCs to investigate various pharmacodynamic endpoints, including immune activation, suppression, cytokine production, cell proliferation, and apoptosis. These assessments provide early insights into the potential efficacy and safety profile of a compound.
PBMCs in Clinical Trials
As drug candidates progress from preclinical to clinical stages, the role of PBMCs expands to help drug developers assess pharmacodynamics in real-world conditions. Clinical trials are designed to evaluate how a drug performs in human subjects, and PBMCs are invaluable in providing data on the functional effects of a drug within the immune system. They are time-critical samples however, with a limited processing window for optimal cell viability. The advantage of PBMCs over whole blood, is the ability to store samples over a long period of time for batch analysis, whereas whole blood typically has a shorter shelf life with the majority of pharmacodynamic assessments needing to be conducted within a short timeframe. To address the need for time-critical PBMC isolation and whole blood processing in Australia, Agilex has established PBMC processing centers in every state. One of the most common endpoints for PBMC analysis is Flow Cytometry. Flow Cytometry is a powerful tool that allows for detailed cell phenotyping, enabling laboratories to assess specific immune cell subsets within whole blood or PBMC populations. This technology is crucial in evaluating how a drug affects different types of immune cells (e.g., T-cells, B-cells, NK cells) in clinical trial participants and can be used to assess markers of activation, proliferation, or immune suppression. At Agilex we have two BD FACSymphony Flow Cytometers with a 20 colour capability offering up to 22 unique parameters per cell. More recently we have expanded our Flow Cytometry capability to include three Cytex Aurora spectral Flow Cytometry instruments to support broader phenotyping of up to 40 colours. Moreover, to address the time-critical nature of sample analysis in real-time (either whole blood or PBMCs), Agilex has recently expanded its flow cytometry capabilities to include laboratories in Brisbane as well as Adelaide, enabling our clients to benefit from our deep immunology expertise, done in real time in both Adelaide and Brisbane.
PBMCs are immensely versatile and generally form part of clinical protocols covering many therapeutic areas and modalities, including:
Immuno-Oncology:
PBMCs are particularly valuable in the field of immuno-oncology, where they are used to assess the effect of novel immunotherapies, such as immune checkpoint inhibitors, on immune cell activation and function. By isolating PBMCs from patients or healthy donors and exposing them to a therapeutic candidate, researchers can evaluate the drug’s ability to modulate immune responses, enhance anti-tumor immunity, and evaluate potential side effects such as cytokine release syndrome.
Vaccine Development:
PBMCs are also pivotal in vaccine research, especially when developing vaccines that stimulate a specific immune response. In this context, PBMCs are used to assess how a candidate vaccine primes the immune system, measuring cellular immune responses and memory recall responses. This evaluation is crucial for determining the potential of the vaccine to elicit long-term immunity.
Common endpoints for vaccine studies include Flow Cytometry analysis, for phenotyping of Humoral immune markers, enzyme-linked immunosorbent spot (ELiSpot) analysis to extrapolate single immune cell responses or recall (memory) responses to the vaccine antigen and cell-based immunogenicity assays to determine presence of neutralizing antibodies. Here PBMCs are commonly used over whole blood due to the ability to isolate and freeze samples for later analysis.
Autoimmune and Inflammatory Diseases:
PBMCs provide an excellent model for understanding how drugs may impact immune dysregulation in autoimmune or inflammatory diseases. PBMCs can be used to test the effect of small molecules or biologics on cytokine production, T-cell activation, and other markers of inflammation, giving insights into whether a drug might help modulate overactive immune responses in diseases like rheumatoid arthritis or multiple sclerosis. Here Flow Cytometry or ligand binding analyses provide insight into how specific immune cells are modulated by drug and monitor the presence of a soluble biomarker which might be key to modulating the immune response.
Safety Monitoring and Cytokine Release Syndrome (CRS):
In clinical trials involving immune-based therapies such as CAR T-cell therapies, there is a risk of cytokine release syndrome (CRS), a potentially life-threatening inflammatory response. PBMCs play a key role in monitoring CRS by measuring cytokine levels in patients undergoing treatment. Through the analysis of PBMCs, researchers can identify early signs of CRS, allowing for timely interventions to mitigate the severity of the response. This is particularly important in ensuring patient safety in trials involving powerful immune-modulating drugs. At Agilex we commonly use enzyme-linked immunosorbent assays (ELISAs), multiplex mesoscale discovery (MSD) assays, Flow Cytometry, and quantitative PCR, to measure changes in immune markers and cytokine production in PBMCs or whole blood (either stimulated or unstimulated). For example, if a drug is expected to modulate T-cell function, we will analyze PBMCs or whole blood for changes in cytokine production (such as IL-2 or IFN-γ) or T-cell activation markers (such as CD69 or CD25). This provides critical insight into the drugs affects across different treatment groups or patient populations in the study. For CRS, there are various off-the-shelf options for multiplex cytokine panels readily available from suppliers such as MSD that provide a ready-made solution for measuring these responses.
Challenges of Using PBMCs in a Clinical Study:
Using PBMCs in clinical studies provides numerous benefits for assessing drug efficacy, safety, and pharmacodynamics, but there are several challenges associated with their use. These challenges span from technical to biological limitations, all of which must be addressed to ensure that PBMCs are used effectively and reliably in clinical research. Below are some of the key challenges we face:
1. Variability Between Samples
One of the most significant challenges when using PBMCs in clinical studies is the variability between samples. PBMCs are isolated from peripheral blood, and immune system responses can vary significantly between individuals due to factors such as genetics, age, gender, lifestyle, and underlying health conditions. In clinical trials, patients often have different immune profiles, which can lead to inconsistent or difficult-to-interpret results.
For example, the response of PBMCs to a drug may vary based on a patient’s baseline immune status, which could be influenced by chronic disease, previous treatments, or even environmental factors. This variability can make it challenging to generalize findings across different patient populations or to establish consistent biomarkers that can predict clinical outcomes.
2. Sample Collection and Processing
The process of collecting and processing PBMCs is complex and can introduce variability. PBMCs are isolated from blood through density gradient centrifugation, and the efficiency of this separation can impact the quality and quantity of the cells collected. Furthermore, PBMCs are highly sensitive to time and temperature during collection and transport. If the sample is delayed or improperly handled, it can lead to degradation of cell viability and function, potentially skewing the results of downstream assays.
For clinical trials, obtaining fresh and high-quality PBMC samples from patients can be difficult, especially in remote or under-resourced areas. Additionally, consistency in processing techniques across different clinical sites is crucial to avoid introducing variability that could compromise the reliability of results.
3. Cell Viability and Functional Integrity
Maintaining PBMC viability and functional integrity is critical for meaningful assay results. After isolation, PBMCs need to be maintained in optimal culture conditions, which can be challenging, especially when dealing with large patient cohorts or during long-term storage. PBMCs are highly sensitive to changes in their environment, and any disruptions in temperature, oxygen levels, or media composition can impact their activation, proliferation, or cytokine production.
Preserving PBMC functionality in clinical trials also involves ensuring that cells remain responsive to the drug or treatment under study. For instance, if PBMCs are exposed to a drug in vitro, they should ideally mimic the same pharmacodynamic response as observed in vivo, which is not always guaranteed. The ex vivo environment may not fully replicate the complex interactions and conditions that occur within the human body, leading to discrepancies between preclinical and clinical results.
4. Complexity of Immune System Response
The immune system is highly complex, and PBMCs represent just a small fraction of the immune cells in the body. While PBMCs are a valuable model for studying immune responses, they do not capture the full range of immune interactions that occur in vivo. For example, PBMCs are isolated from blood, and they may not fully represent the immune microenvironments present in tissues such as tumors or inflamed organs.
Additionally, immune responses are often context-dependent, and the influence of other cell types (such as macrophages, dendritic cells, or tissue-resident lymphocytes) may be crucial for understanding the drug’s effect in a clinical setting. PBMCs alone may not provide a complete picture of how a drug influences the immune system across various tissues and organs.
5. Cost and Logistics
The use of PBMCs in clinical studies adds logistical complexity and cost. Isolating and handling PBMCs requires specialized equipment and expertise, particularly when processing large numbers of patient samples. Furthermore, bioanalytical assays to assess immune responses—such as Flow Cytometry, cytokine analysis, and gene expression profiling—require sophisticated technology and resources and expertise.
For multi-center clinical trials, coordinating the collection, processing, and transportation of PBMCs from different sites is challenging, especially when dealing with strict timelines. Maintaining the integrity of samples during shipping and handling adds to the logistical burden, as samples need to be transported under specific conditions to avoid degradation.
Case Study
Agilex has a well-established network and can do a whole lot with your whole blood! Agilex has PBMC processing capabilities in every state within Australia to provide consistent and reliable PBMC processing, where we have standardized the whole blood preparation to PBMC using a single standard operating procedure and can rapidly analyse these samples for pharmacodynamic endpoints. More recently we are expanding our network to offer rapid Flow Cytometry services and other downstream pharmacodynamics assessments in Brisbane. Using a single SOP and multicentre approach with standardized equipment ensures robust and reproducible sample preparation and data.
Nonetheless, whole blood is often transported under controlled ambient conditions to our headquarters in Adelaide for processing. Multicentre clinical trials pose challenges for immunology bioanalysis due to location and distance from the bioanalytical laboratory as well as limitations within the clinical site with regards to processing of whole blood down to PBMCs and their subsequent storage. We therefore conducted a longitudinal study to evaluate the impact of processing time on PBMC stability, viability composition and function for immunology studies in a multicentre clinical trials setting.
The aim of our study was to; a) investigate the effect of processing laboratory temperature and time to processing on PBMC viability and, b) measure the impact of these on PBMC function using a TBNK flow cytometry panel and TNFα ELiSpot assay as a functional readout.
For our study, PBMC’s were isolated from whole blood from N=3 healthy subjects on day 0 (fresh), and days 1-3 after storage on a rocker at room temperature (either 22°C or 25°C). Viability and cell count was determined by trypan blue exclusion using an automated cell counter (Countess II ThermoFisher Scientific). Cells were initially frozen in 15% DMSO/ 5% FBS/RPMI in Mr Frosty overnight at -80°C before moving into liquid N2 storage. For the PBMC composition assay by flow cytometry, 5 x 105 PBMC were stained with TBNK antibody (BD Biosciences) at 1:2 dilution. Cells were analysed on a BD FACSymphony A3 flow cytometer (BD Biosciences) using a gating strategy as per the manufacturers recommendation. Results were expressed as % grandparent (%CD45). For the PBMC functional assay 5 x 104 cells were plated in triplicate on a TNFα ELiSpot plate (Mabtech) in AIM-V media (ThermoFisher Scientific) in the presence (stimulated) or absence (unstimulated) of 1 mg/ml PMA and 1 ug/ml ionomycin and incubated at 37°C in 5% CO2 for 16 hours. After washing, cells were incubated with anti-TNFα biotin detection antibody (0.5 ug/ml) for 2 hours followed by streptavidin-ALP (1:10000) for 1 hour at ambient temperature. BCIP/NBT-plus was used as a substrate. Cell counts were determined on an iSpot reader (AID) and counts were averaged across triplicates.
We initially assessed the impact of processing temperature on viability. Processing temperature (‘Ambient’ temperature) can differ between laboratories and even a few degrees centigrade can impact the viability and functionality of cells. We found that there was no significant difference in viability of functionality in cells that had been processed from whole blood at either 22°C or 25°C. These were the temperature ranges between the different laboratories that would be used to process samples, indicating that the ambient temperature difference should not impact the data from the multicentre trial.
We next investigated the impact of processing time on cell viability and cell counts, since samples from some sites would require overnight shipping to the processing laboratory. We found no significant difference in cell counts in samples up to day 3 post collection, however we observed a marked reduction in cell viability after day 2 (48 hours after collection; 68.7±11% compared with 94±5% at d0 and 84.3±6% at d1), with less that 50% of cells remaining viable at day 3.
Samples isolated at d0, d1, d2 and d3 were frozen and then thawed out for functional assays, either by Flow Cytometry or ELiSpot analysis to determine the impact of the cell viability on cell type and functionality. Cell viability in the thawed cells was similar to post processing, indicating that the freezing process didn’t impact viability. We subjected the cells to Flow Cytometry analysis using a standard TBNK panel. We found that PBMC’s isolated from whole blood after 24 hours had significantly altered populations of CD3, CD4 and CD8 T Cells, CD16/56 NK Cells and CD19 B Cells, with the greatest impact being on the proportion of CD16/56 NK Cells and CD19 B Cells.
We assessed the impact of sample processing time on functionality using a TNFα ELiSpot assay. We found that cells which had been isolated after 24 hours (d2 and d3) had significantly reduced capacity to generate TNFα after PMA/ionomycin stimulation compared to samples processed at d0 or d1.
Taken together, we found that time to processing of greater than 24 hours significantly reduced cell viability prior to freezing and viability and cell count post freezing. The reduction in viability and cell types in d2 and d3 samples, mainly CD16/56 NK Cells and CD19 B Cells had a significant impact on cellular function. These data showed that whole blood samples need to be processed to PBMC and stored appropriately in LN2 in 15% DMSO/ 5% FBS/RPMI, no later than 24 hours after collection in order to provide robust functional data.
Whilst these data indicated that samples would benefit from immediate isolation and storage at the clinical site, with regular flights from all major cities in Australia to our headquarter laboratories in Adelaide, we are routinely able to process samples with the 24 hour window to ensure that sample and data integrity is maintained. Nonetheless the data generated provided insight and vital information to inform limitations in processing time and correlation with decreased cell function.
Conclusion
PBMCs are invaluable tools in the field of drug discovery and clinical trials. Their ability to reflect immune responses and monitor drug effects in real time makes them an essential resource for biotechnology companies developing novel therapies. While PBMCs are invaluable tools in drug discovery and clinical trials, their use comes with several challenges that need to be addressed to ensure reliable and meaningful results. Variability between patient samples, technical complexities in cell isolation and processing, and the need for careful interpretation of immunological data are just a few of the obstacles that must be overcome. Additionally, logistical considerations concerns must be carefully managed to maintain the integrity of clinical trials and protect patient interests.
Partnering with a Bioanalytical provider with deep expertise and experience is therefore crucial for a successful clinical outcome. Agilex Biolabs, operating since 1996, is Australia’s largest Toxicology and Bioanalytical laboratory. Headquartered in Adelaide with custom-built laboratories for small molecule, large molecule and molecular and cellular biology therapeutics by Flow Cytometry and PCR, Agilex also has a satellite laboratory located within CMAX Phase 1 Unit in Adelaide, for rapid PBMC processing. In addition, Agilex has state-of-the-art GLP rodent toxicology facilities in Brisbane and to address the needs of novel therapeutics coming to Australia, Agilex has recently expanded its service offerings to include Flow Cytometry analysis in Brisbane, to ensure complete coverage of both South Australia and Queensland.
By addressing the challenges associated with whole blood processing to PBMCs, biotechnology companies and clinical researchers can continue to leverage Agilex’s in-depth expertise to better understand the pharmacodynamics of novel therapies, optimize drug development processes, and ultimately bring safer, more effective treatments to patients. To learn more about how Agilex can support your next drug development program, contact us at BD@agilexbiolabs.com, for a confidential discussion on our expanded capabilities.
