Human population-based identification of CD4+ T-cell peptide epitope determinants

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Abstract

A human cell-based method to identify functional CD4+ T-cell epitopes in any protein has been developed. Proteins are tested as synthetic 15-mer peptides offset by three amino acids. Percent responses within a large donor population are tabulated for each peptide in the set. Peptide epitope regions are designated by difference in response frequency from the overall background response rate for the compiled dataset. Epitope peptide responses are reproducible, with a median coefficient of variance of 21% when tested on multiple random-donor sets. The overall average response rate within the dataset increases with increasing putative human population antigenic exposure to a given protein. The background rate was high for HPV16 E6, and was low for human-derived cytokine proteins. The assay identified recall epitope regions within the donor population for the protein staphylokinase. For an industrial protease with minimal presumed population exposure, immunodominant epitope peptides were identified that were found to bind promiscuously to many HLA class II molecules in vitro. The peptide epitope regions identified in presumably unexposed donors represent a subset of the total recall epitopes. Finally, as a negative control, the assay found no peptide epitope regions in human β2-microglobulin. This method identifies functional CD4+ T-cell epitopes in any protein without pre-selection for HLA class II, suggests whether a donor population is pre-exposed to a protein of interest, and does not require sensitized donors for in vitro testing.

Introduction

The application of proteins to therapeutic, industrial and nutritional uses is limited by the potential for inducing or exacerbating deleterious immune responses. This potential is especially of concern for the use of recombinant human-derived proteins. Recombinant human-derived proteins have been demonstrated to induce immune responses directed at self-proteins, resulting in a state of autoimmunity Li et al., 2001, Casadevall et al., 2002. Subsequent reactivation of the immune system after unintended induction of immune responses to industrial or food proteins can be minimized by avoidance. However, this is not the case with human-derived therapeutic proteins.

The selection or creation of reduced immunogenic protein variants is therefore necessary to improve safety and efficacy of administered proteins. The selection of a naturally occurring hypo-immunogenic protein isomer is an option where several related molecules with similar activities exist. It is not an option for many therapeutic proteins. Therefore, a number of methods for creating reduced immunogenic variants have become available. Where a pre-existing immune response in the target human population is anticipated, modifications to the surface of proteins have been shown to reduce antibody binding. If the target population has not been previously exposed to a potential commercial protein, reducing the risk of de novo sensitization via CD4+ T-cell epitope modification is advised.

For the creation of CD4+ T-cell epitope-modified proteins, the first critical step is the localization of functional epitopes within the protein. There are a number of computer-based methods for predicting the localization of peptide sequences that bind to HLA class II molecules Altuvia et al., 1995, Rammensee et al., 1999, Sturniolo et al., 1999, Yu et al., 2002. Binding to HLA is necessary, but not sufficient, for CD4+ T cell activation. In vitro and in vivo testing must be performed to confirm functionality. Computer-based methods are improving in their ability to correctly identify tight HLA binders, but still suffer from a lack of prediction for binding non-HLA-DR class II molecules, and a significant false negative rate. In addition, functional differences such as the induction of tolerance, and epitopes that induce differential responses by activated T cells cannot be assessed using computer modeling.

In addition to the creation of hypo-allergenic protein variants, T-cell epitope identification is the basis of many vaccine strategies Berzofsky, 1993, Alexander et al., 1998. The identification of T-cell epitopes recognized by individuals who clear pathogens vs. those who do not is of interest to the design of both cancer and viral vaccines Manici et al., 1999, Doolan et al., 2000, Novitsky et al., 2002, Wertheimer et al., 2003.

We present here an in vitro human cell-based method for the localization of immunodominant, promiscuous HLA class II epitopes from any protein of interest. The method relies on CD4+ T cell proliferative responses induced by dendritic cells. The method applies equally well to industrial enzymes, food allergens and human therapeutic proteins as it does to the delineation of population-based epitope responses to pathogen-derived proteins. Large donor sets are tested without pre-selection for HLA type. Epitope determinations are made based on statistical analyses of the response rates by the entire donor set to all the peptides derived from the sequence of the protein, and therefore represent population-based epitopes. We show here the results for proteins that the donor pool was expected to be both unexposed and sensitized to. Both types of analyses were compared to proliferation results from verified antigen-sensitized donors. Finally, human β2-microglobulin was tested as a negative control.

Section snippets

Peptides

All peptides were purchased from Mimotopes (San Diego, CA) as multi-pin syntheses (PepSets) (Maeji et al., 1990). Peptides were resuspended in DMSO (Sigma, St. Louis, MO) at 1 or 2 mg/ml, and stored frozen at −70 until use. Peptides were synthesized as 15-mers that overlapped by 12 amino acids.

Donor pool

All human blood samples were obtained from the Stanford University Blood Center (Palo Alto, CA) or the BloodSource (Sacramento, CA). All samples were handled in accordance with Genencor International's

Background responses to industrial proteases

To establish a background value for proteins not typically encountered by the general donor population, the assay was performed on 11 industrial enzymes including proteases, amylases, laccases, and chitinases (Mathies, 1997). One of the proteases was tested twice using peptides produced in two different formats (PepSet vs. purified peptides from Mimotopes). The number of donors tested per peptide set varied from 19 to 113. The number of peptides in each peptide set varied from 80 to 188. A

Discussion

The i-mune assay is an ex vivo technique for the identification of CD4+ T-cell epitopes on a human population basis. Within a donor population pre-sensitized to the protein of interest, most recall epitopes can be defined. For a donor population defined as un-sensitized to the protein of interest, either primary or cross-reactive epitopes are identified. While the latter cannot be formally ruled out, a number of points support the conclusion that the epitopes found are primary epitopes: First,

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