* Genencor International, Palo Alto, California 94304, and
Department of Statistics, Stanford University, Stanford, California 94305
Received July 3, 2003; accepted October 13, 2003
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ABSTRACT |
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Key Words: immunogenicity; cellular activation; human; antigen/peptide/epitopes.
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INTRODUCTION |
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Predictive methods for determining the immunogencity of potential commercial proteins are hampered by the complex nature of the human population. Responsiveness of an individual to a particular protein is controlled by many interacting parameters, including the stability (Hall et al., 2002) and proteolytic activity (Gough et al., 2001
) among other properties (Hall et al., 2002
), and additional extrinsic factors such as the route and dose of exposure (Braun et al., 1997
; Ge et al., 2001
), the presence of adjuvants including the presence or absence of endotoxin contamination (Alving, 2002
; Brimnes et al., 2003
), underlying immune responsivity (for example, tolerance to prevalent self proteins or pre-existing immunity), the presence of protein aggregates (Braun et al., 1997
), and the HLA molecules present. All of these parameters on an individual basis contribute to the population-based response. To analyze the collective response of a representative sample of human donors, a large enough sample set must be tested to ensure the appropriate genetic mix, and both recall and primary responses must be tabulated.
In addition to the ongoing research in the area of food allergens, the immunogenicity of proteins has long been a concern in the enzyme manufacturing industry (Bernstein et al., 1999; Johnsen et al., 1997
; Kimber et al., 1996
; Pepys et al., 1985
; Vanhanen et al., 1997
, 2000
, 2001
). Occupational exposure to proteins has been documented to result in immune responses in industrial and laboratory workers. Conversion to skin prick test positivity (SPT+) can be controlled by reduction of the level of airborne protein (Sarlo and Kirchner, 2002
; Schweigert et al., 2000
). When a new protein is to be manufactured, an occupational exposure guideline must be established. A commonly accepted method to determine these guidelines is the guinea pig intratracheal test (GPIT) (Sarlo et al., 1997
). The GPIT test, while useful, is time consuming and expensive. Recently, a mouse-based test (mouse intranasal test, MINT) has been established that reproduces the results seen in the GPIT (Robinson et al., 1998
).
Animal models have their limitations (Bussiere, 2003). The use of partially outbred guinea pigs in the GPIT necessitates the use of large numbers of animals to achieve statistical significance when comparing responses between groups. Interexperiment variation in control animal responses is very high, which makes potency determinations based on a single set of control responses less convincing. The MINT assay does not suffer from as much variability in antibody responses; the mice used are typically BDF1 mice, a cross between two highly inbred mouse strains. While this additional level of control allows for more robust data analyses, different strains of mice will return very different potency rankings for similar enzymes (Blaikie and Basketter, 1999a
, b
). This is likely due to the specificity of the immune response in a mouse line that has been inbred to express very limited MHC molecules (Sarlo et al., 2000
). Finally, while the data from an individual laboratory using the MINT assay is robust, the MINT assay is also plagued by interlaboratory differences (Blaikie and Basketter, 1999a
).
Finally, any animal test will suffer from the inability to provide a mechanistic description of the immune response to a given protein in humans. First and foremost, inbred strains of mice will present peptide molecules with the specificity conferred by their murine MHC molecules. Human HLA molecules, while highly related to mouse MHC molecules, do not have identical peptide specificities (Bono and Strominger, 1982; Schwaiger et al., 1993
). Human HLA transgenic mice have become available for application to the mechanistic study of human immune responses (Black et al., 2002
; Boyton and Altmann, 2002
; Chen et al., 2002
, 2003
; Das et al., 2000
; Ito et al., 1996
; Raju et al., 2002
; Sonderstrup et al., 1999
). HLA transgenic mice suffer from their species-specific immune system complexities (Farrar et al., 2000
; Kim and Jang, 1992
). HLA transgenic mice are often used for mapping studies when expressing a single HLA molecule, a situation not found in humans. This is especially of note for HLA-DQ transgenic mice where cross-pairing between different HLA-DQ alleles has been shown to create new peptide presentation specificities (Krco et al., 1999
). While the number of available HLA class II transgenic mice expressing common class II alleles is increasing, there are not enough different strains to represent the complexity of human HLA class II frequencies.
To avoid the issues arising from immunogenicity analyses in animals other than humans and to incorporate the complexities of the human target population for commercial proteins, we have developed a method to rank the immunogenicity of proteins using human peripheral blood monocytes (PBMC) as the test subject. The method is based on data gathered using a previously described epitope mapping technique, which relies on the population-based determination of CD4+ T cell responses (Stickler et al., 2000, 2003a
, b
). Because large replicates of human samples are used, the information provided is applicable to general populations. The information is gathered by testing CD4+ T cell responses to peptides presented by dendritic cells and, therefore, includes response data, depending on the protein analyzed, representing both recall and primary immune responses (Harding, 2003
). The data do not suffer from the specificity issues surrounding the use of inbred mice. This method can rank proteins based on their overall immunogenicity but cannot provide relative potency information unless the data are compared to pre-existing animal data. Four well-characterized industrial allergens were placed in the order determined by the GPIT, the BDF1 MINT, and by comparison with human sensitization in occupationally exposed workers (Robinson et al., 1998
). Proteins known to be either immunogenic or presumably tolerizing in humans were tested as positive and negative controls for the method.
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MATERIALS AND METHODS |
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Protein sequences.
Amino acid sequences from the following industrial proteases were tested in the assay: Bacillus lentus subtilisin (Swissprot accession P29600), Bacillus amyloliquifasciens subtilisin (BPN' Y217L, Swissprot accession P00782), Bacillus licheniformis subtilisin (Alcalase®, Swissprot accession P00780), and Bacillus licheniformis -amylase (Swissprot accession P06278). The following other proteins were tested: human interferon-ß (Swissprot accession P01674), human ß2-microglobulin (accession AAH32589), human erythropoietin (Swissprot accession P01588), human thrombopoietin (pir accession G02729), Bertholletia excelsa 2S storage protein (Ber e1, Swissprot accession P04403), and mouse Vh36-60 gene family member (similar to Swissprot accession P01823).
Human donor blood samples.
Buffy coat samples were obtained from two commercial sources (Stanford Blood Center, Palo Alto, CA, and BloodSource, Sacramento, CA). Buffy coat samples were further purified by density separation. Each sample was HLA typed for HLA-DRß and HLA-DQß using a commercial PCR-based kit (Bio-Synthesis, Lewisville, TX).
Preparation of dendritic cells and CD4+ T cells.
The preparation of monocyte-derived dendritic cells and CD4+ T cells has been described previously (Stickler et al., 2000; Zhou and Tedder, 1996
). Briefly, monocytes were purified by adherence to plastic in AIM V medium (Gibco/Life Technologies, Baltimore, MD). Adherent cells were cultured in AIM V media containing 500 units/ml of recombinant human IL-4 (Endogen, Woburn, MA) and 800 units/ml recombinant human GM-CSF (Endogen) for 5 days. On day 5, recombinant human IL-1
(Endogen) and recombinant human TNF-
(Endogen) were added at 50 units/ml and 0.2 units/ml, respectively. On day 7, the fully matured dendritic cells were treated with 50 µg/ml mitomycin c (Sigma Chemical Co., St. Louis, MO) for 1 h at 37°C. Treated dendritic cells were dislodged with 50 mM EDTA in PBS, washed in AIM V media, counted, and resuspended in AIM V media at 2 x 105 cells/ml.
CD4+ T cells were purified by negative selection from frozen aliquots of PBMC using Cellect CD4 columns (Cedarlane, Toronto, Ontario, Canada) or Dynabeads® (Dynal Biotech, Oslo, Norway). CD4+ T cell populations were routinely >80% pure and >95% viable as judged by Trypan blue (Sigma Chemical Co.) exclusion. CD4+ T cells were resuspended in AIM V media at 2 x 106 cells/ml.
Assay conditions.
CD4+ T cells and dendritic cells were plated in round-bottomed 96-well format plates at 100 µl of each cell mix per well. The final cell number per well was 2 x 104 DC and 2 x 105 CD4+ T cells. Peptide was added to a final concentration of ~5 µg/ml in 0.250.5% DMSO. Control wells contained DMSO without added peptide. Each peptide was tested in duplicate. Cultures were incubated at 37°C in 5% CO2 for 5 days. On day 5, 0.5 uCi of tritiated thymidine (NEN/DuPont, Boston, MA) was added to each well. On day 6, the cultures were harvested onto glass fiber mats using a TomTec manual harvester (TomTec, Hamden, CT) and then processed for scintillation counting. Proliferation was assessed by determining the average CPM value for each set of duplicate wells (TriLux Beta, Wallac, Finland).
Data analysis.
For each individual, average CPM values for all the peptides were determined. The average CPM values for each peptide were divided by the average CPM value of the control (DMSO only) wells to calculate a stimulation index (SI). A positive response was recorded if the SI value was equal to or larger than 2.95. The 2.95 value was determined empirically, tested by receiver-operator curve analysis, and shown to result in a highly accurate and efficient value for the analysis of I-mune assay data (Stickler et al., 2003a). For each protein assessed, positive responses to individual peptides by individual donors were compiled. Donor blood samples were tested with each peptide set to yield an average of at least two responses (response = SI of 2.95 or greater) per peptide. For example, since each donor responded to an average of three peptides per 100 peptides tested (average background of ~3.15% for 11 industrial enzymes [Stickler et al., 2003a
]), this would result in the need to test 67 donors to get an expected two responses per peptide for the 100 peptides tested. Data for each protein tested is graphed as the percentage of responders within the population tested to each peptide in the set.
To determine the background response for a given protein, the percentage of responses for each peptide in the set was averaged and a standard deviation was calculated.
Statistical methods.
The total variation distance between the empirical frequencies and the uniform distribution (Kotz and Johnson, 1988), the structure index value, was calculated based on the following equation:
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where is the sum over all peptides in the peptide set of the absolute value of the proportion of responses to each peptide minus the frequency of that peptide in the set; f(i) is defined as the frequency of responses for an individual peptide divided by the total number of responses accumulated; and p is the number of peptides in the peptide set. For more information on this, see Results. For an example of the calculation for BPN' Y217L, see the Supplementary Material online.
Statistical significance of peptide response frequency was calculated based on Poisson statistics (Stickler et al., 2003a). The average frequency of responders to all the peptides was used to calculate a Poisson distribution based on the total number of responses and the number of peptides in the set. A frequency of response to a peptide is considered significant if p < 0.05.
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RESULTS |
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-amylase.
Individuals (n = 82) were tested with peptides derived from the -amylase sequence. The average percentage of response to all peptides in this set was 2.80 ± 3.69% (average ± standard deviation), well within our overall average for 11 industrial enzymes of 3.15 ± 1.57 (Stickler et al., 2003a
). Prominent responses were noted to amino acids 3448 (peptide 12), 160174 (peptide 54), and 442456 (peptide 148; Fig. 2
). All three of these responses were highly significant above the background response (p < 0.0001).
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In theory, if every peptide in the data set had the same number of responses, f(i)-1/p would equal zero. In other words, the proportion of the responses at each peptide would equal the proportion of the data set represented by one peptide, and the difference between these values would equal zero. The absolute value of the sum of the data for all the peptides (zero at each one) would equal zero. On the other hand, if all the accumulated responses were at one peptide, the value would approach 2.0.
To ensure comparability of the structure index values, a stable response pattern must be achieved within the data set. The number of donors necessary was determined empirically for BPN' Y217L, as shown in Figure 6. After about two responses per peptide, the structure index value reaches a plateau level. For the BPN' Y217L peptide set, this occurred after testing approximately 50 donors. For all additional peptide set testing, enough donors to achieve at least two responses per peptide were used.
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Determination of a Cut-off Value for Reduced Immunological Risk
The structure index values for the four respiratory allergens amylase, B. lentus subtilisin, BPN'Y217L, and Alcalase were combined with the values for human IFN-ß, Tpo, and Ber e1 to determine a cut-off value for a putatively immunogenic protein. The average of these seven values was found to be 0.68 with a standard deviation of 0.09 (Table 3). Two standard deviations below the mean is a value of 0.50. To determine the limit using the comparatively less immunogenic proteins, the same analysis was performed using values for ß2-microglobulin, the mouse VH36-60, and Epo. The average structure index value for these three proteins is 0.41 with a standard deviation of 0.04. Two standard deviations above the average are a value of 0.49. Therefore, a protein with a structure index value of less than 0.50 could be categorized by this analysis using these proteins as benchmarks of a comparatively less immunogenic protein.
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DISCUSSION |
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In addition to correctly ranking the allergenicity of four known respiratory allergens, the structure index values for a set of known human immunogens (hTPO [Li et al., 2001], hIFN-ß [PRISMS Study Group, 2001
], and Ber e1 [Nordlee et al., 1996
]) were found to be comparatively high, indicating that these proteins might be capable of inducing immune responses in a significant number of exposed people. The average percentage of response values of the two human-derived protein immunogens were within the range defined by 11 industrial enzymes: 3.15 ± 1.6 (for example, see Table 2
). Background values within this range are assumed to represent responses to novel proteins, as the industrial enzymes tested are not widely encountered by community donors (Pepys et al., 1973
, 1985
; Sarlo et al., 2003
). A high structure value in the context of a low background rate suggests that humans are largely immunologically "naïve" to these human sequencederived proteins, and that the immune system "ignores" the proteins until nonphysiological doses are introduced in the presence of an adjuvant signal. The structure value for Ber e1, the major protein allergen in Brazil nuts, was also found to be high. Our community donor pool is likely exposed to Brazil nuts in food, and, therefore, the value reflects contributions from allergic donors, exposed yet unallergic donors, and donors who are truly unexposed. Conversely, the structure value for a mouse VH 36-60 gene family member was low, commensurate with its predicted immunogenicity (Olsson et al., 1991
). The structure value determined for ß2-microglobulin was also low, as would be expected given that this molecule is presumed to be subject to both peripheral and central tolerance mechanisms (Guery et al., 1995
). Finally, the structure value for human erythropoietin was also found to be low, consistent with its safe use and low frequency of adverse affects (Casadevall et al., 2002
).
The range of structure values was low. A putative nonimmunogenic protein, ß2-microglobulin, had a value of 0.39, while the most immunogenic protein verified by human exposure, amylase, had a value of 0.81. The correlation between these values and potency in animal models is logarithmic. Small differences in the value can indicate large differences in immunogenicity. Therefore, it is critical to test a large enough donor set (greater than two responses per peptide) to make accurate comparisons.
The comparative ranking of proteins tested in this assay assumes that the immunogenicity of whole protein molecules would be compared in vivo at the same dose, in the same formulation, in a matched set of donors, and over the same dose course. This analysis also precludes any processing and/or presentation differences in the proteins, as well as general physical and structural properties (i.e., stability, activity, and multimerization).
The method described also allows for the localization of T cell epitopes in any protein of interest. CD4+ T cell epitopes can be determined in the absence of individuals exposed to the test protein (Stickler et al., 2000, 2003a
, b
). Modification of peptide epitope sequences to select variants less likely to induce immune responses can be performed using unexposed community donors. An analysis of donor responses to the modified peptide variants can be used to calculate structure values for the new protein. Note that testing of protein variants designed to be less immunogenic, by virtue of provoking fewer responses in vitro with large replicates of human donors, cannot be rationally tested in guinea pigs or mice, as rodents often have different CD4+ T cell epitopes than the human population (see [Mucha et al., 2002
] for B. lentus and BPN' Y217L epitopes in guinea pigs). Transgenic mice are limited in their utility, since they typically do not express more than one HLA allele and enough strains to represent the complexity of the human population HLA allele frequencies are not currently available.
Ranking of proteins does not imply any fold potency differences. Potency determination can be extrapolated from an alignment of our data with animal data. However, the values determined would be subject to the same inherent inaccuracies as the animal data used. It follows from the data presented that the selection of a protein with the lowest structure value would minimize the risk of inducing immune responses in human subjects on a population basis. As a general rule for the selection of lead candidates, we calculated that proteins should have structure values less than 0.5. We currently have no human in vivo data to support the use of this method, a criticism that can be levied at almost all predictive and functional methods to modify the immunogenicity of proteins. Full validation of this ranking method will require testing of known immunogenic and allergenic proteins and their modified variants and will not be complete until human donor data become available.
The method described here is an assay to determine the relative immunogenicity of proteins in human subjects that does not expose the donor to the protein of interest. Proteins can be ranked relative to one another. The method encompasses a previously described technique to identify immunodominant peptide epitopes. Taken together, this information may allow for the selection of reduced immunogenicity proteins and can direct the rational modification of proteins to create and test hypo-immunogenic variants appropriate for use in humans.
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NOTES |
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1 To whom correspondence should be addressed at Genencor International, 925 Page Mill Road, Palo Alto, CA 94304. Fax: (650) 845-6509. E-mail: fharding{at}genencor.com.
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