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.
• 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
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.
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.
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+).
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.
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.
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 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.
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).
Our preclinical program that includes the whole blood loop assay will:
Our clinical safety assessment of biotherapeutics in the whole blood loop system during clinical trials can:
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
Preclinical Evaluation of AdVince, an Oncolytic Adenovirus Adapted for Treatment of Liver Metastases from Neuroendocrine Cancer. Yu D. et al. Neuroendocrinology, 2017
Antibody induced CD4 down-modulation of T cells is site-specifically mediated by CD64+ cells. Vogel S. et al. Scientific Reports, 2015
Complement activation by CpG in a human whole blood loop system: mechanisms and immuno-modulatory effects. Mangsbo S.M. et al. Immunology, 2009
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
|Safety evaluation||Cellular distribution in human blood||Mode-of-action studies|