Why are Tissue Cross-Reactivity Studies Needed?
Failure of a therapeutic antibody or antibody-like molecule at the point of clinical trial is phenomenally costly. Pharmaceutical companies, therefore, go to great lengths to try and mitigate any potential issues before “first-in-human” (FIH) studies are initiated. Many pre-clinical investigations undertaken are essentials that form part of a regulatory Investigational New Drug (IND) or Clinical Trial Application (CTA), and are aimed at minimizing the risk of patient harm when a drug is first given to humans. One such pre-clinical safety study is assessment of Tissue Cross-Reactivity (TCR), a series of ex vivo immunohistochemical (IHC) screening assays conducted primarily to identify off-target binding, but also to pick up previously unknown sites of on-target binding. In a nutshell, the presence of staining in frozen tissues ex vivo is used to give an indication of potential organ toxicity in vivo. Whether such staining actually correlates with organ toxicity is still regularly debated, but TCR studies remain a requirement in the data package expected for an IND/CTA submission for most biotherapeutics. TCR studies can also be used to compare patterns of staining between human and animal tissue, providing additional justification for the choice of animal toxicity models used to generate other pre-clinical safety data.
Who conducts a Tissue Cross-Reactivity Study for an IND submission?
TCR studies intended to form part of an IND submission or CTA must be performed in a GLP-compliant manner, so it’s important to select a laboratory whose credentials support this. In the UK this means the test facility must belong to the UK GLP compliance monitoring programme, run by the UK GLP Monitoring Authority (UK GLPMA) which is part of the Medicines and Healthcare products Regulatory Authority (MHRA). Guidelines for conducting TCR studies have been produced by the International Committee for Harmonisation1, but best practice is discussed in more detail by Leach et al. (20102).
One of the most important aspects of a TCR study is the work-up of the IHC protocol, often termed “Phase 1”. Given that the biotherapeutic to be tested (the “Test Item”) has not been designed as an IHC tool, this poses a technical challenge that can only be overcome by rigorous work-up, and sometimes not at all. Since a favorable data set for a TCR study is actually a negative result, it’s very important to know the assay is specific and robust before the test tissues are examined. Many underestimate the length of time needed for the development of a sound IHC method, but cutting corners at this stage can result in misleading TCR results when the “Phase 2” assays are run.
The Test Item
Test Items may come in many forms, some differing substantially from immunoglobulins in structure. Irrespective of format, consideration must be made to how the molecule is to be detected in the context of an IHC assay. Unlabelled human or humanized antibodies can be detected by pre-complexing with an anti-human antibody before applying to the test tissues. However, it is technically easier to work with an antibody that has been labeled with a small molecule such as biotin or FITC, and detect with an antibody specific for the label. Indeed, for many molecules labeling is a necessity to facilitate detection. While biotinylation is a well-established and relatively straightforward technique for labeling biological molecules, FITC may be preferable in a TCR study as it avoids the need for additional avidin-biotin blocking steps in the assay protocol, without which background staining of tissue is likely to prove problematic. In either case, it is important to establish the impact of any labeling on the binding properties of the molecule, and also to ensure Test and Control Items are labeled to the same degree.
The Control Item
Inclusion of a “Control Item” is strongly recommended (Leach et al., 20102). The Control Item, essentially an isotype control, is usually a molecule identical in structure to the Test Item (including labels such as biotin or FITC), but raised against a molecule unlikely to be found in human tissue – e.g., green fluorescent protein, a plant protein or even snake venom. Ideally, such a molecule should be prepared in parallel with the Test Item and is used to ascertain the background level and pattern of binding to tissue that occurs irrespective of the Complementarity-determining region (CDR). In our experience, many clients fail to appreciate the value of including a suitable Control Item and deem it an expensive waste of funds. However, without the Control Item for comparison, binding of the Test Item may be mistakenly interpreted as specific and therefore indicative of potential organ toxicity. There are other ways that specificity of binding can be assessed, for example competing out binding by pre-incubation with a molar excess of soluble antigen, but these methods can be costly, and access to sufficient soluble antigen is not always possible.
Positive control material
As highlighted by Geoly (2015)3, selection of suitable positive control material is crucially important for Phase 1 protocol development, but also for use in Phase 2 to validate the Test Item in each assay run, of which there are likely to be many. The control material needs to be representative of the frozen tissue that the Test Item will encounter in the Phase 2 assays, so overexpressing cell lines or other types of positive control material are inferior to frozen tissue, where the tissue matrix is present. Where no suitable tissue exists that naturally expresses the target of interest, various alternative techniques have been attempted, including subcutaneous injection of antigen-coated beads prior to harvesting skin from mice. To address this where required, BioIVT has developed a proprietary method, based on many years of expertise handling human tissue, which allows incorporation of soluble antigen into a human tissue matrix, resulting in a frozen, sectionable material suitable for use as a positive control material where no other solution exists.
The quality of the frozen tissues used in Phase 2 is of the utmost importance. Whether of post-mortem or surgical origin, human tissues must have good morphological preservation to allow adequate interpretation of staining patterns and crucially must retain antigenicity. BioIVT strongly recommends confirmation of antigenicity of tissues at the time of Phase 2 testing, as testing prior to this doesn’t guarantee antigenicity as a sample is sectioned through.
Interpretation of results
This should be conducted by a qualified pathologist. Staining with the Test Item should be compared to that seen in adjacent sections incubated with the Control Item, and specific staining only considered where Control Item staining is not present. The pathologist may, in addition, make a judgment as to whether smeared or very diffuse staining is specific in nature. The cellular location of specific staining is also important to consider, since staining of cytoplasm, which is unlikely to be accessible to a biotherapeutic in vivo, is less likely to translate into a biological effect than membrane staining, so may be of lower concern from a safety perspective.
The biological relevance of any TCR staining, of course, can never be truly determined until other toxicity data (e.g., from clinical trials or postmarketing surveillance) becomes available, so it’s important to interpret data with caution. Several case studies are presented in an excellent article published in 2010 by authors from a number of pharma giants and CROs, describing both examples of the issues and benefits of TCR studies (Leach et al., 20102). At least for now, TCR studies are here to stay, and it will be interesting to see what regulatory bodies such as the FDA and EMA will require for the assessment of tissue cross-reactivity with newer technologies.
- ICH S6 (R1) Preclinical safety evaluation of biotechnology-derived pharmaceuticals. June 2011 EMA/CHMP/ICH/731268/199
- Leach, M. W., Halpern, W. G., Johnson, C. W., Rojko, J. L., MacLachlan, T. K., Chan, C. M., Galbreath, E. J., Ndifor, A. M., Blanset, D. L., Polack, E., and Cavagnaro, J. A. (2010). Use of tissue cross-reactivity studies in the development of antibody-based biopharmaceuticals: History, experience, methodology, and future directions. Toxicol Pathol 38, 1138–66.
- Geoly, F. J. (2014). Regulatory Forum Opinion Piece*: Tissue Cross-reactivity Studies: What Constitutes an Adequate Positive Control and How Do We Report Positive Staining? Toxicol Pathol, 42: 954-956.