Tissue cross-reactivity assay

This chapter will discuss various experimental approaches used to select the relevant species for conduct of toxicology studies for biopharmaceuticals, as well as highlight advances made in scientific approaches and technologies to facilitate this process. Methods discussed include the traditional immunohisto-chemistry and tissue cross-reactivity studies, flow cytometry, protein sequencing, and functional in vitro assays, as well as newer approaches such as utilization of microarray databases for genomic mRNA expression data and use of transcript profiling studies as an adjunct to functional assays, to understand similarity in pharmacological responsiveness between animals and humans. 

Proposed Plasma Level for Clinical Studies The tissue cross-reactivity study is an immunohistochemistry assay, which, because of its nature, does not replicate the conditions obtained in vivo. Moreover, one is looking at tissue sections where the cells are cleaved and all portions of the tissues and cells (membrane, cytoplasm, and nucleus) and surrounding milieu are exposed to the same concentration of the antibody, which is different from the intravascular and perivascular concentrations observed in vivo (see below). Since buffers, protein types, and electrolytes also differ in vivo compared to the immunohistochemistry conditions, one cannot expect to attain high concentra-... 

Tissue cross-reactivity studies, although burdensome, provide a rational in vitro assay to determine the range and intensity of distribution of potential epitopes reactive with a monoclonal antibody test article prior to its administration to humans. In addition, cross-reactivity studies provide a useful tool to identify animal species for safety assessment. The cross-reactivity profiles of different species can be compared to the profiles obtained in human tissues. The predictive value of the assay lies in incorporating the characteristics of the monoclonal antibody (isotype, subtype, and other molecular modifications) with the biological activity of the molecule itself, and the potential in vivo distribution of it. 

The evaluations to determine relevant species for toxicity evaluation include various receptor binding assays and tissue cross-reactivity assessments, the later being routinely performed for monoclonal antibody based products (see Chapters 10 and 26). Species specificity can be determined using properly controlled in vitro cell-based response assays, cloning of receptors and ligands to determine compatibility, receptor-based response assays, immunoassays, genomic-based assays, and traditional biochemical-based assays as well as in vivo assessments with validated endpoints and markers for specificity. 

Fertility and early embryonic development rats Developmental rats, rabbits Prenatal and postnatal development rats Genetic toxicology in vitro bacterial and CFIO cell assays, mouse micronucleus assay Carcinogenicity proliferation in vitro and in vivo Tissue cross-reactivity human tissues Other safety studies in a variety of species Single dose none... 

Several considerations influence the suitability of the immunoassay as a qualitative or quantitative tool for the determination of tissue residues. These include the assay format, the end user (on-farm or laboratory use), effects of sample matrix on the analysis, cross-reactivity considerations, detection levels required of the assay, target tissues to be used in the assay, and the use of incurred or fortified tissues for validation of the immunoassay against accepted instrumental methods. Although these variables are often interrelated, each topic will be discussed in further detail below. 

There are several important advantages RPMAs have over antibody arrays and other proteomic techniques such as immunohis-tochemistry or tissue arrays. Antibody arrays usually require a second specific antibody, made in a different species, for each captured protein to be visualized in a manner analogous to enzyme-linked immunosorbent assays (ELISA). Therefore, it becomes difficult to simultaneously optimize the antibody-antigen hybridization conditions for so many antibodies at once present on antibody arrays while minimizing nonspecific cross-reactivity and ensuring that proteins over a wide range of concentrations can be quantitated in a linear fashion (14). Antibody arrays also consume or require much higher inputs of protein than reverse phase arrays. With antibody arrays. 

Endotoxicity results from the interaction of a bacterial cell envelope component (e.g., LPS or PG with a cell surface receptor constituting part of the nonspecific immune system, (i.e., a toll-like receptor on white blood cells). This results in the production of cytokines [e.g., interleukin 1 (IL-1) or tumor necrosis factor (TNF)] as part of an intracellular enzyme cascade which can cause severe tissue injury. Bioassays or immunoassays can be used to detect such reactions respectively. As noted above the most widely used bioassay is the LAL assay. A lysate of amoebo-cytes of the horseshoe crab (Limulus) contains an enzymatic clotting cascade which is activated by extremely low levels of LPS (nanogram levels or lower). There are variants of this assay that can detect PG, but they are not as widely used. As noted above, other bioassays employ cultured cell lines that respond to LPS or PG, respectively. Unfortunately bioassays are highly amenable to false positives (from the presence of cross-reactive substances) or false negatives from inhibition (by contaminants present in the sample) [10]. A detailed discussion of these assays is beyond the scope of this chapter and has been reviewed elsewhere [1]. 

Antibody arrays immobilized on glass surfaces mimic DNA microarrays in format and spot size. The biggest challenge in protein profiling using antibody microarrays is selection of validated antibodies that are useful in the desired sample environment. Many of the initial reports used antibody arrays assayed for cytokines because serum presents a relatively simple sample assay environment compared to tissue and also because there are numerous validated antibodies available for this clinically important set of proteins. Tissue and cell lysates present more complex assay environments with more opportunities for antibody cross-reactivity and other interferences which erode the biological meaningfulness of the data. 

The observations that fetal tissues in culture produce somatomedins (Section 2.1), and that receptors for somatomedins (especially IGF-II) are abundant in membranes of fetal tissues (Section 4.3), both suggest that somatomedins may play a role in fetal development. Hus is supported by measurements of the peptides in the fetal circulation. While some early studies in this area are inconclusive due to the broad cross-reactivities of the assay methods employed, specific determination of SM-C/ICF-I and IGF-II levels has been possible in recent studies, although much of the fetal data is derived from nonhuman species. 

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