Immuno-Services

Unique and comprehensive human whole blood data from one supplier.

The whole blood loop assay

Since 2014, Immuneed has offered a unique, cost-effective, first-in-class extracorporeal whole blood loop assay. The assay measures drug-related rapid infusion reactions mediated by blood components, as well as drug biodistribution and mode-of-action studies. Our advanced characterization services are paired with our immunological know-how and expertise, enabling us to link pre-clinical data to clinical reality.

The whole-blood loop assay is applicable during drug discovery and development – from screening of drug candidates to early clinical development – and is suitable for various drug candidates, including biosimilars, antibodies, peptides and viruses. The assay setup is designed together with you and your team to enhance data output.

Our assay

• Indicates in vivo human effects early in the candidate selection process
• Helps select the right drug candidate and reduce development costs
• Reduces and refines animal studies
• Gives improved understanding of the clinical trial design
• Assays are performed according to FDA and EMA recommended guidelines

BENEFITS OFFERS
Multiple read-outs in one run
Cytokine release assay
Complement activation analysis
Biomarkers evaluation
Cellular drug biodistribution
Mode-of-action studies
Fresh blood
High quality data
Novel technology
Reduced risk
Clinical Relevance
Regulatory bodies
Multiple read-outs in one run
Blood samples can be collected at various time points and each sample can be used for multiple analysis such as cytokine release, complement activation, flow cytometry analysis of cellular distribution etc. Standard time for the whole blood loop assay run is 4 hr time point, but longer incubation times are possible as well (up to 24hr).
Cytokine release assay
Our cytokine release assay (CRA) uniquely presents low donor variability, low background, fast cytokine release read-out (4 hour assay) and intact complement system.
Complement activation analysis
In humans, complement activation can be associated with infusion reactions and therefore it is crucial to study biologicals in system with intact cascade systems. Our loop model, in comparison to other cytokine release methods, is unique as it contains intact complement system.
Biomarkers evaluation
Immuneed can provide biomarker analyses of blood derived cells from individuals in clinical trials as a means to assess pharmacodynamics.
Cellular drug biodistribution
The whole blood loop system allows us to track antibody/drug candidate on different cell populations (surface or intracellular location identified thru quencing of surface staining) as well as cellular activation and viability.
Mode-of-action studies
The whole blood loop assay allows to study immunostimulatory or immunoinhibitory properites of drug candidates. Examples of MOA studies include: antibody-dependent cell-mediated cytotoxicity mechanisms, complement-mediated lysis, signaling on target or non-target cells through antigen-antibody interactions, activation of cells by monomeric or aggregated IgG and circulating immune complexes, IgE-mediated hypersensitivity reaction.
Fresh blood
The whole blood loop model uniquely contains fresh blood in circulation with all blood components present, allowing for proper investigation of potential immunological effects, drug immune cell distribution and adverse events of monoclonal antibodies and other immune targeting therapies ex-vivo.
High quality data
Thanks to our talented team with many years’ experience of laboratory work and immunological methods development, we always ensure our customers the highest quality data.
Novel technology
Listed below are few of the technologies Immuneed has access to:
  • Human whole blood loop model (modified chandler loop model)
  • CytoFLEX Flow cytometer (Beckman Coulter)
  • Meso Scale Discovery system
  • XP-300 Hematology Analyzer (Sysmex)
  • iMark™ Microplate Absorbance Reader (Bio-Rad)
  • iSTAT Blood Analyzer (Abbot)
Reduced risk
The whole blood loop model offers prediction of risk evaluation of first infusion reactions based on cytokine release profile and complement activation.
Clinical relevance
The whole blood loop assay can provide adequate information on acute infusion reactions and immune efficacy of immune modulating drugs, where cellular assays (lacking immunoglobulins and/or complement products) and animal models (cannot show interaction with target molecules and Fc receptors, problems with species differences) fall short.
Regulatory bodies
All assays and methods are performed according to FDA and EMA recommended guidelines. Data is provided in appropriate report format to supplement potential IND or EMA filings, including raw data, LLOD, LLOQ data and statistics.

Cytokine release assay

Our cytokine release assay uniquely presents low donor variability, low background and cytokines measurements performed after only 4 hours (predicting immediate cytokine release).

When a biological drug such as a therapeutic monoclonal antibody is administered, it may react with different components of the human blood. These first infusion reactions are a result of activation of a variety of immune cells. The subsequent release of different cytokines can be severe and life-threatening, but can be managed with administration of steroids prior to drug administration, adjusting the infusion time or by adjusting the dose.

Our cytokine release assay provides a comprehensive tool to measure blood/drug interactions. Table 1 compares our assay with alternative in vitro assays for analysis of cytokine release, showing that our assay is the only available assay with intact cascade systems, short incubation time and it is also highly suitable for investigating drug biodistribution, efficacy and mode-of-action of biological drugs. Figure 1 shows a cytokine release to a panel of monoclonal antibodies used clinically and/or as a standards in cytokine release assays evaluated in our human whole blood loop assay.

As an example of the power of our assay, we have tested a TGN1412-like antibody* and shown that this antibody, but not the isotype-matched control, induce a clear cytokine release. Read more about our study in Fletcher Int Immunopharmacol. 2017 Oct 27;54:1-11.
*TGN1412 was an anti-CD28 monoclonal antibody that caused catastrophic systemic organ failure due to cytokine release in healthy volunteers in a clinical trial some 10 years ago.

Figure 1. Cytokine release in response to monoclonal antibodies in a human whole blood loop assay Freshly acquired whole blood was incubated with the antibodies in a circulating loop assay. The final concentrations of the antibodies in blood are displayed in brackets in the figure [µg/ml]. After 4 hours blood samples were collected and plasma samples were analyzed for cytokines with MSD Discovery® multiplex. Figure includes panel of mAb with low (cetuximab*, tocilizumab, etanercept, natalizumab) and high (CD52-target as alemtuzumab, CD3-target mAb as muromonab-CD3 and CD28-target such as TGN1412) incidence of first infusion reactions. * However a severe hypersensitivity reaction can occur during the initial infusion of cetuximab in individuals with pre-existing IgE antibodies against cetuximab

Figure 2. Frequency responders. The threshold for a positive antibody response was set at above the 95th percentile of the calculated ratio of PBS/baseline value. The ratio value of antibody divided by baseline was set to positive if above the threshold.

Table 1. Characteristics of our whole-blood loop assay compared to available published assays.

Complement activation

The unique whole blood loop system contains intact cascade systems that allow the study of interactions between biologics and intact complement.

In humans, activating the complement system can be associated with infusion reactions. In our assay, complement activation is analyzed by measuring the complement factors anaphylatoxins C3a and C5a at baseline, directly after blood sampling, and at a 15 min time-point (additional time-points are also possible).

Figure 3 gives an example of how complement activation is measured in our whole blood loop assay.

Figure 3. The final concentration of test substances in blood is displayed in brackets in the figure [µg/ml]. After 15 minutes blood samples were collected and cascade systems were inhibited and analyzed for C3a and C5a by ELISA. LPS was used as a positive control and is a potent stimulus that triggers cytokine release and complement activation. Cetuximab was used as a negative comparator as it does not trigger complement activation in blood compartment due to lack of target. Mode of action of alemtuzumab is described as lysis of lymphocytes through complement fixation and antibody-dependent cell-mediated cytotoxicity mechanisms.

Cellular drug biodistribution

The whole blood loop assay is highly suitable for investigating drug biodistribution. It can also be used to track antibodies or drug candidates on different cell populations.

As examples of how our assay can be used to investigate biodistribution, below is shown the cellular distribution of the monoclonal antibody natalizumab (Figure 4).

Figure 4. Freshly acquired whole blood was incubated with the natalizumab (2 µg/ml) or vehicle in a circulating loop assay. Blood was collected at baseline (zero time point) and 4 hour time-point stained with fluorescently labeled monoclonal antibodies to detect: erythrocytes (CD235ab+, CD45-), platelets (CD41+, CD45-), T cells (CD3+), B cells (CD19+), NK cells (CD56+), monocytes (CD14+), granulocytes (CD66b+).

Cell activation patterns

Analysis of cell activation patterns may be included to investigate effects of drug- cell interaction over time.

An example of how cell activation patterns can be analyzed using our whole blood assay is shown in Figure 5.

Figure 5. Freshly acquired whole blood was incubated with different compounds in a circulating loop assay. At baseline (zero time point) and 4 hours blood samples were collected and cellular activation patterns were analyzed by flow cytometry. Expression of activation markers on monocytes (CD86+) and T cells (CD69+) in response to cetuximab, alemtuzumab and vehicle is presented.

Immune recall responses

The whole blood system can be used to assess candidate drug mode-of-action to evaluate immune recall responses.

We use the whole blood system to test the mode-of-action of Immuneed’s therapeutic prostate cancer vaccine. The vaccine incorporates long peptides harboring several T cell epitopes. These long peptides are efficiently delivered to specific immune cells by Immuneed’s TET (tetanus-epitope targeting) system (Read more: Immuno-Oncology TET platform).

As a test for recall responses, Immuneed also makes use of the NLV epitope incorporated into a synthetic long peptide CMV-NLV. By sampling blood from HLA-A201+/CMV+ donors, recall responses to the NLV epitope can be studied using flow cytometry and staining for CD8, tetramer+ cells along with intracellular IFNγ/TNFα production. It is also possible to assess candidate drug mode-of-action to evaluate whether the recall responses can be improved by addition of your candidate drug.

Viability analyses

Determination of cell viability can be performed as part of a safety study to monitor viability of the cells in response to biological drugs.

Our whole blood loop assay does not affect cell viability for individual white blood cell populations and the viability can be evaluated by flow cytometry analysis. Read more about our study in Fletcher Int Immunopharmacol. 2017 Oct 27;54:1-11.

Figure 6. Viability of white blood cells presented by A) white blood cell count using hematology analyzer XP-300 and B) frequency of live cells at 4 hour time-point in response to various substances (measured by flow cytometry).

Blood status

Blood status analysis is a standard read-out performed during the whole blood loop assay with the use of hematology analyzer XP-300 (Sysmex), a proven technology for accuracy of results.

Blood status is an important measurement that can provide valuable information regarding the test substance. Undesired hemolytic or clotting activity can be simply verified and tracked to formulation solution or test substance.

Eight parameters are analyzed: platelet (PLT) count, white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC). WBCs, RBCs and PLTs are counted using the direct current detection method with coincidence correction. Automatic discriminators separate the cell populations based on complex algorithms. The intensity of the electronic pulse from each analyzed cell is proportional to the cell volume. The hematocrit (HCT) is directly determined based on the red cell count and volume detection of each individual RBC. Hemoglobin analysis is conducted using a non-cyanide method.

Mode-of-action studies

The whole blood system can be used to assess candidate drug mode-of-action.

We use the whole blood system to test the mode-of-action of Immuneed’s therapeutic prostate cancer vaccine. The vaccine incorporates long peptides harboring several T cell epitopes. These long peptides are efficiently delivered to specific immune cells by Immuneed’s TET (tetanus-epitope targeting) system that uses MTTE sequences for delivery of the peptides in an antigen-antibody immune complex formula. Since the whole blood loop system has retained intact complement activity, it allows interaction studies of antigen-antibody complexes with human whole blood and complement cascade system. Read more about TET technology under Immuno-Oncology section.

As a test for recall responses, Immuneed also makes use of the immunodominant CMV epitope NLVPMVATV (NLV), incorporated into a synthetic long peptide (SLP). By sampling blood from CMV+/HLA-A*0201 donors, recall responses to the NLV epitope can be studied using flow cytometry and staining for CD8+, tetramer+ cells along with intracellular IFNγ/TNFα production (Figure 7). It is also possible to assess candidate drug mode-of-action to evaluate whether the recall responses can be improved by addition of your candidate drug. Read more about our study in Fletcher J Immunol. 2018 May 11 and about other MOA studies under: Project Examples.

Figure 7. The peptide-conjugates with NLV CD8+ epitope ([MTTE]3-NLV) boost IFNγ and TNFα recall responses of epitope-specific T cells in human whole blood from donors that were both CMV+ and HLA-A*0201, while peptide conjugate with irrelevant peptide ([MTTE]3-SLP] did not. When comparing the naked peptide SLP(NLV) and the conjugate [MTTE]3-NLV, it is possible to achieve similar responses but this requires around 100-fold more peptide than conjugate. (Fletcher J Immunol. 2018 May 11).

Preclinical safety assessment

Our preclinical program that includes the whole blood loop assay will:


  • Support in selecting the right drug candidate

  • Reduce development costs and decrease/complement animal studies

  • Offer preclinical safety evaluation advice

  • Aid risk evaluation of first infusion reactions based on cytokine release profile and complement activation

  • Increase understanding of how to design an initial clinical trial


Clinical safety assessment

Our clinical safety assessment of biotherapeutics in the whole blood loop system during clinical trials can:

  • Aid in safety assessment prior to each dosing to study risk of adverse events related to immunogenicity of the product

  • Perform pre-screening of volunteers prior to or during clinical trial

  • Perform biomarker analyses of blood derived cells from individuals in clinical trials as a means to assess pharmacodynamics

Looking for more info on our assays?

Relevant resources

Demonstrating Immuneed’s unique whole blood loop assay.

Extracorporeal human whole blood assay, as a tool to predict first-infusion reactions and mechanism-of-action of immunotherapeutics. Fletcher E.A.K. et al. International Immunopharmacol, 2017

Read more

The use of whole blood loop assay to investigate biodistribution of human adenovirus.

Preclinical Evaluation of AdVince, an Oncolytic Adenovirus Adapted for Treatment of Liver Metastases from Neuroendocrine Cancer. Yu D. et al. Neuroendocrinology, 2017

Read more

The use of human whole blood loop assay to investigate effector mechanisms of a therapeutic antibody.

Antibody induced CD4 down-modulation of T cells is site-specifically mediated by CD64+ cells. Vogel S. et al. Scientific Reports, 2015

Read more

The use of the human whole blood loop assay to investigate nucleic acid based immunotherapeutic interactions with complement.

Complement activation by CpG in a human whole blood loop system: mechanisms and immuno-modulatory effects. Mangsbo S.M. et al. Immunology, 2009

Read more

The use of whole blood loop assay to study T cell recall responses.

Formation of Immune Complexes with a Tetanus-Derived B Cell Epitope Boosts Human T Cell Responses to Covalently Linked Peptides in an Ex Vivo Blood Loop System. Fletcher E.A.K. et al Journal of Immunology, 2018

Read more

Project examples

Safety evaluationCellular distribution in human bloodMode-of-action studies
  • Evaluation of novel monoclonal antibodies on cytokine release and complement activation
  • Identifying blood cell populations as a source of cytokine release in response to monoclonal antibodies
  • Evaluation of immunoglobulin-mediated cytokine release in response to antigen-antibody complex formation
  • Evaluation of off-target binding of novel drug candidates in blood
  • Biodistribution studies including analysis of activation markers on subsets of blood cells (platelets, granulocytes, monocytes, T cells, B cells, NK cells) of novel monoclonal antibodies drugs and candidate drug impact on blood cell viability and activation
  • Cell surface expression and functional activity of novel candidate drugs
  • Distribution of virus-based drugs in cell and plasma fractions and tracking virus binding to blood cell populations
  • Tracking of drug candidates on different cell populations (surface or intracellular location identified thru quenching of surface staining)
  • Studying antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of monoclonal antibodies in the whole blood and inhibitory effect of complement-blocking agents in LPS-stimulated blood
  • Impact of drug formulation buffers on hemolytic activity and clotting induction
  • Evaluation of immunomodulatory effect of drug candidates on for example lymphocytes
  • Evaluation of immunomodulatory effect of drug candidates on for example lymphocytes
  • Recall T responses to a vaccine candidate in blood from the population intended to treat (patient blood)
  • Immunomodulatory effects on cytokine responses of TNFα inhibitors in LPS-stimulated blood.
  • Impact of small molecule drugs on immune cells
  • Identifying additive/synergistic effects of combinatorial treatments monoclonal antibodies

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