Hazard Identification

Less refined methods have been developed using the assumption that hydro-philic polypeptide regions contain antigenic sites. Conversely, hydrophobic sites would be buried and inaccessible (Krystek etal., 1985). In a study of 29 epitopes on four model proteins, statistical analyses showed that a segmental mobility scale and a hydrophilicity scale based on chromatographic peptide retention times gave the highest correct predictions (Van Regernmortel and Daney de Marcillac, 1988).

It is also possible to determine whether similar linear epitopes occur in other proteins. Protein data bases have been growing at a rapid rate. Currently, the Swiss-Prot data basecontains 1.5 X 107 residues and the NBRF contains 1.1 X 107 residues. Others such as the National Centerfor Biotechnology Information, the Protein Identification Resource, and the Brookhaven Protein data bases contain smaller numbers of residues. The Brookhaven data base also has three-dimensional structures for a number of proteins. Using these data bases, it is easy to find five, six, or seven amino acid matches between unrelated proteins.

The rate-limiting step in processing of antigen and presentation to the immune system appears to be unfolding of proteins at the cleavage site. Immunodominant epitopes are associated with structurally unstable protein segments associated with highly flexible polypeptide loops (Landry, 1997). These epitopes can be measured by NMR relaxation parameters.

The instability allows structural "breathing" of the protein. In the relaxed or unstable form, the protein undergoes proteolytic cleavage and binding of the proximal C-terminus to the MHC class II marker. Additional modification or trimming of the peptide occurs in the binding cleft (Landry, 1997).

The characteristic of antigenic peptides used for the generation of vaccines has been discerned from Monte Carlo computer experiments. Peptides should have the propensity to form a-helices that do not develop coil conformations. In addition, they should have a lysine at the C-terminus (Spouge et al., 1987). Moreover, the T-cell epitopes should not be segmentally amphipathic (e.g., two disjointed subpeptides one of which is hydrophobic while the other is hydro-philic).

The importance offlanking regions in the generation of antigenic epitopes is controversial. In the generation of CD8 cytotoxic T cells both in vivo and in vitro, recognition is influenced by the C- and N-terminal flanking residues. Amino acids with aromatic (tyrosine), basic (lysine), or aliphatic side chains (alanine) enhance CTL recognition. Acidic and helix-breaking amino acids (glycine and proline) inhibit recognition ofthe N-terminus epitope (Bergmann etal., 1996). In contrast, the generation of CD4 T helper cells to HIV gpl20 in recombinant proteins was dependent on the insertion position in the protein. Moreover, antigenicity was influenced by the protein region flanking the HIV peptide (Manca et al., 1996).

Differences in antigenic potency are related to the size of the epitope. Linear sequences of seven amino acids delineated T-cell epitopes. Increasing the peptide length often results in increased antigenic potency. Increased potency may be related to the ability of longer peptides to adopt the appropriate secondary structure following binding to the MHC molecule.

Although it is possible to predict antigenic fragments within molecules, it is difficult to predict immunogenicity of proteins in humans. Factors such as the presence of inflammation, local protease production, presence of molecular chap-erones, and N-glycosylation influence the immunogenicity of a protein (Landry, 1997).

N-glycosylation is often critical to the expression of antigenicity. Proteins are posttranslationally modified shortly after synthesis at the ribosome and further modified when the protein is translocated through intracellular membranes (Hounsell, 1994). The addition of the oligosaccharides has a large influence on the antigenicity of the molecule. For example, patients receiving recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) develop antibodies to the recombinant protein. The antibodies interact with native protein at sites normally protected by 0-linked glycosylation but are exposed in rhGM-CSF produced in yeast and E. coli (Gribben et al., 1990).

It is likely that products of biotechnology designed for human usage will be immunogenic in most animal species. Moreover, some of the animals will form neutralizing antibodies, causing a loss of efficacy (Galbraith, 1987). Humans often develop antibodies against humanized proteins. But the therapeutic response usually persists in the presence of the antibody. This suggests that human antibodies are necessarily neutralizing.

Proper species selection may negate the production of antibodies. Animals closely related to humans may not respond to the proteins, yet they have pharmacological and metabolic activity similarto ours. Therefore, the primate is the animal of choice for testing human recombinant proteins. Testing in primates is, however, expensive and laborous.

When in vivo or in vitro models or correlates are not available, transgenic models canbe used. One system is the SCID-hu model where human stem cells are placed in the mouse to create afunctional human immune system (Namikawae/a/., 1990). Normal human ratios ofCD4/CD8 are found in the blood and lymphocytes respond to mitogens, alloantigens, and anti-CD3 (Kaneshima etal., 1990).

The immunogenicity of low-molecular-weight compounds requires a different approach to hazard identification. Initially, structure-activity relationships are determined with classes or families of chemicals known to be immunogenic. For example, chemicals with a structure similar to toluene diisocyanates would be flagged as a possible immunogen. Second, the chemical would be reacted with albumin in the test tube to determine whether hapten—protein conjugates are formed. Finally, the complexes would be injected into animals to determine whether hapten-specific antibodies are formed.

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