Scientific insights

Bringing you articles full of in-depth scientific insight on subjects ranging from pharmaceutical development through to crop protection registrations.
UK sales@ukorg.huntingdon.com USA sales@priceton.huntingdon.com Japan +81 (0) 3 3238 6381-7

Pharmaceuticals Biologics Crop protection Chemical

Playing with immunological fire predicting potential in vivo cytokine release using an ex vivo assay

You can read more of our articles, posters and case studies on our website

www.huntingdon.com

Immunology

Playing with immunological fire predicting potential in vivo cytokine release using an ex vivo assay

ytokines are soluble signalling molecules which are generated and released by cells of the immune system as well as many others within the body. Within the immune system cytokines act to regulate the magnitude, duration and nature of the immune response to a particular antigen either by acting alone, or more commonly in combination with other cytokines and immune cells. Like many other aspects of human physiology, the immune system uses a series of checks and processes to maintain a constant state of balance between activation and inactivation, and cytokines are key to maintaining this balance. Too little activity will produce an ineffective immune response but too much may produce pathology. Under normal circumstances it is the presence of antigen that tips the balance in favour of the initiation of an immune response; however drugs and, in particular, large molecule therapeutics such as antibodies can also cause this by triggering cytokine release and immune cell activation (Figures 1 and 2). A severe example of this was seen in 2006 during the Phase 1 trial of TGN1412, an antiCD28 IgG4 which activated T-cells, resulting in cytokine generation and release. This initial wave of cytokine release then activated other immune cells in a series of self-perpetuating amplification steps. The resulting massive cytokine storms produced severe pathology which required all six volunteers that received the drug to be placed on life support systems1.

triggering cellular activation. The fourth mechanism is similar to the third but uses the FcR of adjacent cells to cluster antibody in the absence of a cross linking antibody. Whilst it is convenient to describe these mechanisms in isolation, they almost certainly occur simultaneously in vivo, meaning that any response is likely to be a combination of all four mechanisms2. The requirement for an in vitro assay, whilst driven by the TGN14 incident has come about due to the absence of any in vivo models. The most commonly used in vivo model for biologics is the cynomolgous monkey and TGN1412 was indeed tested in this species and was shown to have little ability to generate cytokine release. One of the contributing factors to this is the presence of CD33-related siglec molecules on the T-lymphocytes of cynomolgous monkeys. These transmembrane receptors are coupled to Immonoreceptor inhibitory motifs (ITIM) and act to negatively regulate the activity of the immune system, thereby significantly reducing cytokine mediated activation of the immune system3. Humans have lost siglec expression from the surface of their cells4 which renders them significantly more susceptible to cytokine mediated cellular activation and therefore cytokine storms when compared to the cynomologous monkey. Therefore, whilst for most investigations the cynomolgous monkey is a suitable model, due to these significant differences in responsiveness to cytokines when compared to humans, it is not a relevant species for cytokine release predictions. Given the lack of an in vivo model a number of groups have attempted to produce ex vivo models using human blood. In this situation the challenge is no longer relevance dictated by species differences, but that of cellular distribution and antigen presentation. The immune system uses the lymphatic system to harvest antigen from around the body and concentrates it within the lymph node to increase the chances of its recognition by an effector cell. Therefore, if an antibodydrug is given intravenously, the concentration within the plasma may be substantially lower than that in the lymph node and it is this high density presentation of antibody that in vitro assays often seek to achieve. In addition to this the cellular distribution within the lymph node is significantly different from that of the peripheral blood as a combination of cytokines and adhesion molecules act to exclude granulocytes, red cells and platelets from entering the node. Again, in vitro assay must attempt to

There are four commonly recognised molecular mechanisms by which antibodies may bind to and activate immune cells. The most commonly recognised mechanism is via the interaction of the Fc region of antibodies with the Fc receptors (FcR) of immune cells. The clustering of FcR at the cell surface leads to cellular activation, cytokine release and is a mechanism used by the immune system to target Fc bearing immune cells to pathogens via antibodies. In addition to this FcR based mechanism, immune cells may also be activated by a second mechanism by the cross linking of some (but not all) of their surface antigens. Particular cellsurface markers such as CD3 and CD28 may activate immune cells by clustering at the cell surface and then transferring an activatory signal into the cell. The third mechanism is an elaboration of this second mechanism in which surface receptors are occupied by antibody but not at a sufficient density to trigger a response. In this situation, a second antibody may bind to and cross link the first, thereby increasing antigen density and

Immunology
replicate these conditions if it is to offer a tool that can be used for antibody screening or in vivo predictions. A number of groups, including our own, have attempted to produce in vitro assays to model this process, with the most published in this area being the Biotheraputics group at NIBSC. In early work this group demonstrated that TGN1412 would not activate cells in aqueous phase and that the antibody needed to be immoblilised at a high density surface of a plastic multiwell plate by evaporation (passively or accelerated by hot air)5. In further work the same group were able to show that a number of other clinically used antibody therapeutics would trigger cytokine release in the same assay and that cytokine-induced proliferation of lymphocytes could also be observed6. Although many workers agree on the basic format of these assays there is some controversy over certain aspects. Work performed at HLS suggests that antibody should be immobilised on a protein G surface to present it in a more physiologically relevant confirmation. In addition, the use of freshly isolated PBMC has also been subject to debate. PBMC are routinely rested post isolation before being put into a bioassay; however the work of Huing (unpublished) suggest that extended culture periods may be necessary to transform the cell population to one more representative of the lymph node. Finally, the identification of signature cytokines for potential cytokine storm generation is challenging as the type of cytokine released from stimulated cells may vary according to the cell target of the stimulating antibody. The cytokine release profile of antibodies such as CamPath differs from that of Rituximab, which in turn differs from OKT3 and TGN1412 despite the fact that the latter two target the same cell population. However, some commonality does exist between these. In all cases an initial wave of cytokine is released upon cell stimulation, with a following wave of secondary cytokines released as a result of the first. In most cases the initial wave of release will contain TNF- and IFN- and the second wave IL6, IL8 and IL10. A potential third wave of activity linking IL-2 generation to cellular proliferation may also be considered as a marker. Cytokine release assays have a role in predicting the potential release of cytokines by NBEs and have a place both in lead optimisation work and as tools for setting first in man starting doses. However the current generation of cytokine release assays are basic and only suitable for use as investigative tools or early screening systems. The key to future success in this area will be the understanding of the complex biological processes that dictate it and the evolution of appropriately validated assay systems. Figure 1: Cytokines involved in CRS

TNF- IFN-

IL-6 IL-10 IL-2 IL8

Trigger event

Amplification

Figure 2: CD 3 - Induced release of cytokines from PBMC

Medium alone (-ve control)
IL-2 IL-4 IL-6 IL-10 TNF- IFN-

Anti-human CD3
IL-2 IL-4 IL-10 TNF- IFN- IL-6

Reference

1 Goodyear M Learning from the TGN1412 trial BMJ (2006) 332(7543):677-8 2 Bugelski PJ et al. Monoclonal antibody-induced cytokine-release syndrome Expert Rev Clin Immunol (2009) 5(5):499-521 3 Avril T et al. Negative regulation of leucocyte functions by CD33-related siglecs. Biochem Soc Trans (2006) 34(6):1024-7 4 Nguyen DH et al. Loss of Siglec expression on T lymphocytes during human evolution Proc Natl Acad Sci USA (2006) 103(20):7765-70 5 Stebbings R et al. Cytokine storm in the phase I trial of monoclonal antibody TGN1412: better understanding the causes to improve preclinical testing of immunotherapeutics. J Immunol (2007) 179(5):3325-31 6 Findlay L et al. Improved in vitro methods to predict the in vivo toxicity in man of therapeutic monoclonal antibodies including TGN1412. Journal of Immunological Methods (2010) 352 (1-2):1-12

Key Points

capable of inducing Most therapeutic antibodies aremild and in some it is cytokine release. In many cases this is actually essential differences in the human Due to models are not reliable predictors ofimmune system animal cytokine release this area developed a variety of Investigators inone modelhave yet been demonstratedin vitro models but no has to be an accurate predictor of in vivo CRS beneficial but in a few the reaction can be extreme and life threatening (cytokine release syndrome)

syndrome (CRS) which has necessitated the generation of in vitro models as alternatives