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Cardinal Bernardin Cancer Center, Loyola University Chicago, Maywood, IL 60153; and
University of Chicago, Chicago IL 60637
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the human setting, peptide-based vaccines have also induced diverse effects. Several clinical trials applying peptide-based vaccines in cancer patients have demonstrated peptide-specific immune responses in the majority of the patients (7, 8, 9, 10). However, many of the clinical trials testing peptide-based vaccines have shown limited or no peptide-specific immune response (11, 12) with undetectable clinical effects (13, 14, 15). Although many explanations for the absence of immune induction have been offered, the deletion of specific CTLs after peptide vaccination has not been studied. Moreover, vaccination-induced T cell tolerance would be deleterious to the desired objective of establishing immunity and to the immediate health of the patients. Therefore, it is important to determine why peptide-based vaccines can give such contrasting results. To our knowledge, there is no published study that has explored a possible difference in pharmacokinetics as an important property that determines immunogenicity of peptides in vivo. Information on peptide pharmacokinetics could greatly influence the design of peptide-based vaccine strategies.
In this study, we compared the pharmacokinetics of a T cell-activating
peptide derived from the HPV16 E7 protein and a T cell-tolerizing
peptide of the Ad5 E1A protein. The results show that the Ad5 E1A
peptide egressed from the vaccine site at a higher rate than the HPV16
E7 peptide. As a consequence, the Ad5 E1A peptide reached its maximum
concentration in all organs much faster than the HPV16 E7 peptide;
maximum concentrations were reached at 
10 and 160 h in all
organs for the Ad5 E1A and the HPV16 E7 peptide, respectively.
Furthermore, the HPV16 E7 peptide remained in all organs significantly
longer than the Ad5 E1A peptide. These differences in pharmacokinetics
could contribute to the differential effects of HPV16 E7 and Ad5 E1A
peptide injection on peptide-specific CTLs in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six-week-old female C57BL/6 mice were purchased from Taconic Farms (Germantown, NY) and were housed in a specified pathogen-free environment. RMA-S cells were cultured in IMDM (BioWhittaker, Walkersville, MD) supplemented with 2 mM L-glutamine (Mediatech, Herndon, VA), 10% FCS (HyClone Laboratories, Logan, UT), and 1 mg/ml kanamycin (Sigma, St. Louis, MO). Peptides were dissolved in PBS and emulsified in IFA (Difco, Detroit, MI) at a ratio of 1:1 using the Polytron homogenizer PT-3100 (Brinkmann Instruments, Westbury, NY). This method of emulsification of peptides is superior to the classical method of emulsification (i.e., vortexing or repeatedly passing through a syringe). This method guaranteed that both peptides were equally well emulsified. A total of 300 µg of 3H-labeled peptide, equivalent to 0.51 µCi 3H, was injected s.c. in a volume of 250 µl on the right flank of the mouse.
Peptide synthesis
Ad5 E1A234–243 (NH2-SGPSNTPPEI-COOH) (16) and HPV16 E749–57 (NH2-RAHYNIVTF-COOH) (3) were synthesized and purified as reported previously for 13C-labeled peptides (17). Briefly, uniformly labeled 3H-L-Ile (New England Nuclear, Billerica, MA) with a specific radioactivity of 108 Ci/mmol were protected by tBOC or F-moc groups as described (18). Peptides were dissolved in 0.1% trifluoroacetic acid (TFA) and purified by preparative reverse phase HPLC using a Rainin Dynamax SD-200 HPLC (Rainin Instruments, Emeryville, CA) and a Zorbax 300-SB C4 column (MAC-MOD Analytical, Chaddsford, PA). Purity was assessed by analytical HPLC using a Rainin 25 x 0.5 cm C18 column (Rainin Instruments) and a linear gradient over 1 h of 0.1% TFA in water to 0.1% TFA in 70/30 acetonitrile/water. The identity was confirmed by matrix-assisted laser desorption ionization/time-of-flight or electron spray mass spectrometry. The peptides were determined to be >99% pure. The specific activity of the Ad5 E1A and HPV16 E7 peptides were 1899 cpm/nmole and 2062 cpm/nmol, respectively.
RMA-S-peptide binding assay
The MHC-peptide binding assay, which measures the ability of the peptide to bind H2-Db, was assessed using the RMA-S cell line as described previously (3). Briefly, RMA-S cells were incubated for 36 h at 26°C followed by a 4-h incubation with 100 µg/ml tritiated and nontritiated HPV16 E7 or Ad5 E1A peptides. Cells were stained with a biotin-conjugated H2-Db-specific Ab (BD PharMingen, San Diego, CA), followed by avidin-FITC (BD Biosciences, San Jose, CA). The level of H2-Db expression, as determined by FACS analysis, correlates with the peptide binding affinity.
Organ collection
At the following time points after injection, mice were sacrificed by cervical dislocation: 0, 0.5, 1, 2, 4, 8, 24, 48, 72, 120, 168, and 336 h. At these times, the following organs were collected: vaccine site, lymph nodes, spleen, thymus, heart, liver, lungs, kidney, intestines, brain, and blood. Each organ was frozen at -20°C until analysis. Whole blood was heparinized and processed as described below.
Organ processing
All organs were homogenized in deionized water at 3 ml/g wet weight using the Polytron PT-3100. A total of 0.3 ml homogenate was digested in 1 ml Soluene-350 (Packard, Meriden, CT). To the vaccine site and organs weighing <100 mg, 0.3 ml deionized water and 1 ml Soluene-350 was added. The mixture was incubated at 50°C overnight, after which 10 ml Hionic Fluor scintillation mixture (Packard) was added. To whole blood, 1 ml Soluene-350:isopropanol (1:1) was added. After a 2-h incubation at 50°C, 0.2 ml of 30% H2O2 was added. Following a 30-min incubation at 50°C, 10 ml Hionic Fluor scintillation mixture was added. 3H content of all samples was measured by a liquid scintillation counter (Beckman Coulter, Fullerton, CA). The cpm were converted to dpm by standard quench curves. Furthermore, the measured dpm were corrected for the percentage of dpm due to chemiluminescence.
HPLC of homogenates
Homogenates were centrifuged at 17,000 x g, filtered using a 0.22-nm filter, and subjected to reverse phase HPLC as described above but with the gradient shortened to 0.5 h. Fractions were collected, and the radioactivity was measured by liquid scintillation counting.
Analysis of kinetic data
The data were analyzed as the mean dpm for each data point (i.e., three mice at each time point and each organ). Multiple kinetic schemes were tested; nonlinear fit of the data to the appropriate equation was performed using the Kaleidagraph program (Abelback software).
Enzyme-linked immunospot (ELISPOT)
Spleens were isolated at defined times after peptide injection.
Splenocytes were frozen as a single-cell suspension. Multiscreen
96-well filtration plates (Millipore, Bedford, MA) were coated with 5
µg/ml anti-IFN-
Ab (BD PharMingen) overnight. Splenocytes were
thawed and plated in triplicate at 5 x 105
and 2 x 105 cells/well in the presence of
30 U/ml IL-2 and 5 µg/ml Ad5 E1A, HPV16 E7 peptide, or no peptide.
After a 24-h incubation, ELISPOT plates were washed with PBS containing
0.1% Tween 20, and 2.5 µg/ml anti-IFN--biotin (BD PharMingen)
was added to each well. Plates were incubated for 2 h at room
temperature, and 1.25 µg/ml avidin-alkaline phosphatase (Sigma) was
added to each well for 2 h. Plates were washed with PBS containing
0.1% Tween 20 followed by PBS. A solution of
5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium substrate
(Promega, Madison, WI) in alkaline phosphatase buffer was added
to each well and, after a 15-min incubation, plates were washed with
deionized water and air-dried. Spots in the lower dilution wells were
counted, averaged, and subtracted from the background spots counted in
the wells stimulated without peptide. This number was converted to the
average number of CTL precursor cells per 1 x
106 cells present in the immunized mice.
Tetramer analysis
C57BL/6 splenocytes isolated for the ELISPOT were used for this assay. CD8+ T cells were positively selected using anti-CD8 conjugated magnetic beads (Miltenyi Biotech, Auburn, CA). These cells were stained for anti-CD8-FITC (BD PharMingen), and the HPV16 E7 (RAHYNIVTF)-H2-Db tetramer or Ad5 E1A (SGPSNTPPEI)-H2-Db tetramer, which were both conjugated with PE (National Institute of Allergy and Infectious Diseases tetramer facility, Atlanta, GA), for 1 h on ice. Expression levels of CD8 and specific TCRs on 4 x 104 gated viable cells were analyzed by FACS.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test whether the HPV16 E7 and Ad5 E1A peptide binding
capacity to H2-Db was altered by tritiation, a
standard RMA-S binding assay was performed. The level of
H2-Db expression on the cell surface when RMA-S
cells were loaded with regular peptide or tritiated peptide was
compared by FACS analysis. No difference in MHC expression could be
detected (Fig. 1
), indicating that the
peptide-MHC binding capacity was not affected by intrinsic tritiation.
Based on these data, the tritiated peptides emulsified in IFA were
compared for in vivo biodistribution after s.c. injection in
mice.
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The tritium content of the vaccine site was assessed at different
times after injection with 300 µg 3H-labeled
Ad5 E1A or HPV16 E7 peptide emulsified in IFA. Fig. 2A shows that, 3 h after
injection, the tolerizing Ad5 E1A peptide started to egress from the
vaccine site, whereas increased egression of the activating HPV16 E7
peptide started 10 h after injection. Furthermore, egression of
the Ad5 E1A peptide halted after 72 h, and egression of the HPV16
E7 peptide stopped after 200 h (Fig. 2B). Comparison of
the egression rate constants (K2) of
the two peptides (Table I) showed that
the Ad5 E1A peptide egressed from the Ag depot 2.7 times faster than
the HPV16 E7 peptide.
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To establish the pharmacokinetic behavior of the two peptides with
respect to organ distribution, several organs were analyzed for their
tritium content at different points in time, and influx and egression
parameters were calculated. In four independent experiments with three
mice per time point, the pharmacokinetic behavior of each individual
peptide in all organs was found to be very consistent (Table I
). To
determine the pharmacokinetics in lymphoid organs, in which induction
of the CTL response should take place, spleen, thymus, and lymph nodes
were analyzed for their tritium content. Fig. 3 shows the pharmacokinetics of both
peptides in the spleen. The maximum Ad5 E1A peptide concentration in
the spleen was similar to that of the HPV16 E7 peptide concentration;
however, the maximum Ad5 E1A peptide concentration was reached after
10 h, and it declined to an equilibrium after 100 h. The HPV16 E7
peptide reached the maximum concentration in the spleen 140 h after
injection, and its concentration had not equilibrated to baseline
levels as late as 350 h (Fig. 3B). Identical peptide
concentration and kinetics were detected in the thymus as indicated in
Table I. Tritium concentration in pooled lymph nodes was also measured
but never attained levels above background. This is most likely due to
the extremely small size of these organs, resulting in an undetectable
tritium concentration.
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. The kinetics of both HPV16 E7 and Ad5
E1A peptides in the lungs are very similar to those detected in the
spleen in that maximum peptide concentrations are reached at 10 and
170 h after Ad5 E1A or HPV16 E7 peptide injection, respectively
(Fig. 4A), due to the large difference (36-fold) in
influx between both peptides into the lungs as represented by the slope
of the curves (Fig. 4B). Moreover, the HPV16 E7 peptide
persists in the lungs at least 100 h longer then the Ad5 E1A
peptide (Fig. 4B). These results indicate that, while both
peptides spread to and egress from all organs with similar kinetics,
each individual peptide does so with distinct kinetics. This difference
in kinetic behavior between these two peptides depends on the nature of
the peptide rather than that of the organ.
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To determine whether the tritium measured in the different organs
originated from intact peptide, free amino acids, or other degradation
products, homogenates of most organs obtained 24 h after injection
were analyzed by reversed phase HPLC analysis and compared with pure
synthetic tritiated peptide (Fig. 5).
With the exception of the intestines, the tritium label eluted from all
organs in the same fraction as intact synthetic peptide (fraction 17
and 18 for the Ad5 E1A peptide and fraction 23 for the HPV16 E7
peptide), demonstrating that the label was still contained within
intact peptide. In the intestines, the label eluted in the void volume,
indicating that the peptide had been degraded to amino acids or other
polar products.
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The previous data indicate a clear difference in the
kinetics of the tolerizing (Ad5 E1A) and the activating peptides (HPV16
E7). However, these differences may not be the cause of the observed
functional differences in the induction of a CTL response. Therefore,
we assessed the kinetics of the Ad5 E1A- and HPV16 E7-specific CTL
responses after peptide injection. Mice were immunized s.c. with 100
µg Ad5 E1A or HPV16 E7 peptide in IFA. At defined times after peptide
injection, splenocytes were isolated and the number of Ad5 E1A- and
HPV16 E7-specific CTLs was measured by ELISPOT. Fig. 6A shows that an increase in
HPV16 E7-specific CTL precursors was observed 72 h after peptide
injection. The CTL response peaked at 120–168 h after peptide
injection and returned to the original CTL level after 2 wk. A similar
pattern was observed in the HPV16 E7-H2-Db
tetramer analysis performed at the same time points after peptide
injection (Fig. 6B). As observed in the ELISPOT assay, the
number of HPV16 E7-specific CTLs increased 120 h after peptide
injection and declined 2 wk after injection. The E7-specific CTL
kinetics corresponds strongly with E7 peptide kinetics, which also
showed a peak in E7 peptide concentration between 120 and 168 h
after injection (Fig. 3). In contrast, a significant Ad5 E1A-specific
CTL response was not detected by ELISPOT (Fig. 6C) or
by Ad5 E1A- H2-Db tetramer staining (Fig. 6D). The positive control (splenocytes isolated from mice
immunized with irradiated tumor cells expressing the Ad5 E1A protein)
showed a significant induction of Ad5 E1A-specific CTLs by tetramer
staining (Fig. 6D). This proves that Ad5 E1A-specific CTLs
can be induced, but Ad5 E1A peptide in IFA did not induce Ad5
E1A-specific CTLs.
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The absence of an Ad5 E1A-specific CTL response after Ad5
E1A peptide injection could be due to a functional or physical deletion
of Ad5 E1A-specific CTLs. To test this, C57BL/6 mice were preimmunized
twice with irradiated Ad5 E1A-positive tumor cells to induce a
significant number of Ad5 E1A-specific CTLs. Two weeks after the last
cellular immunization, mice were injected s.c. with Ad5 E1A peptide in
IFA. At defined times after peptide injection, splenocytes were
isolated and the number of Ad5 E1A-specific CTLs was determined by Ad5
E1A-H2-Db tetramer analysis. As shown in Fig. 7, before peptide injection (0 h after
peptide injection), a significant Ad5 E1A-specific CTL response was
present: 1.58% of the CD8+ splenocytes was Ad5
E1A specific. Within 24 h after Ad5 E1A peptide injection, the
number of Ad5 E1A-specific CTLs gradually reduced to levels detected in
a naive mouse and remained unchanged up to 2 wk after peptide
injection. These data indicate that Ad5 E1A peptide injection results
in physical deletion of Ad5 E1A-specific CTLs. Furthermore, the
observed physical deletion is very rapid, as is the appearance of the
Ad5 E1A peptide in the spleen. The rapid increase of Ad5 E1A peptide
concentration within the first 10 h of peptide injection results
in deletion of Ad5 E1A-specific CTLs in that same time period.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). As a consequence of the rapid diffusion of the Ad5 E1A
peptide from the vaccine site, the Ad5 E1A peptide concentration
reaches its maximum concentration in a relatively short time in all
organs, but it also declines more rapidly than the HPV16 E7 peptide
concentration (Figs. 3 and 4).
The peptide kinetics offers an explanation for the observation that
mice injected with Ad5 E1A-specific CTLs i.v. and the Ad5 E1A peptide
s.c. die of extensive lung destruction within 16 h after injection
(4). When the Ad5 E1A peptide reaches the lungs within
16 h (Fig. 4A), it activates Ad5 E1A-specific CTLs that
are entrapped in the microvascular beds of the lungs after adoptive
transfer. Peptides will bind to the MHC class I molecules of the
endothelial cells, and the activated CTLs will cause destruction of the
endothelial cells presenting the peptide. In contrast, mice injected
simultaneously with HPV16 E7-specific CTLs and HPV16 E7 peptide
remained alive, because the HPV16 E7 peptide slowly egresses and does
not reach its maximum concentration until 160 h after injection
(Fig. 4B). At that time, the HPV16 E7-specific CTLs will
have migrated from the lungs and will not cause the lung destruction as
observed in the Ad5 E1A peptide-injected mice.
Interestingly, we observed that the peptides detected after 24 h
in most organs were intact (Fig. 5). These findings are in contrast to
a report that studied the stability of a MART-1 peptide in human plasma
in vitro in which the peptide half-life was calculated to be 22 s
(23). Our data indicate that both Ad5 E1A and HPV16 E7
peptides remained in circulation for a much longer period. Peptides may
be protected from degradation by binding to MHC class I molecules. This
could explain the difference between these two studies, because
MHC-expressing cells are absent in plasma. One exception to this
generalization is the intestines, in which the radioactivity was
recovered in a different fraction from the intact peptide after HPLC
analysis, indicating peptide degradation. This should not be
surprising, because the intestines contain both proteases and bacteria,
which may have degraded the peptides.
The mechanism by which the peptide-specific CTLs are tolerized is
not fully understood. However, based on the kinetics of the Ad5 E1A
peptide, we infer that tolerance is likely induced by
activation-induced cell death (AICD). AICD occurs as the consequence of
repeated antigenic stimulation of T cells and is mediated by Fas-Fas
ligand interactions in CD4+ T cells
(24) and engagement of TNFRs in CD8+
T cells (25). We show in the spleen that Ad5 E1A
peptide-specific CTLs are confronted with a rapid influx of a high
peptide concentration within 24 h after peptide injection (Fig. 3). The analysis of the Ad5 E1A-specific CTL precursor frequency
obtained from preimmunized mice after Ad5 E1A peptide injection
demonstrated that, within 24 h after peptide injection, the number
of preexisting Ad5 E1A-specific CTL (1.6% of the
CD8+ T cell population) was significantly
reduced. We propose that this is the result of AICD, even though we did
not actually measure apoptosis. This is in agreement with a study
showing that i.p. injection of high peptide doses, resulting in
systemic distribution, readily deleted their specific CTLs in wild-type
mice. However, in TNFR/Fas double-knockout mice, i.p. injection of
peptide did not induce AICD, suggesting that peptide-induced tolerance
is dependent on the presence of these two death receptors
(26). In contrast, a slow buildup of the HPV16 E7 peptide
and persistence of the peptide in lymphoid organs seems optimal for
immunization. This is supported by our finding that the increased HPV16
E7 peptide concentration is closely followed by an increase in HPV16 E7
CTLs (Fig. 6, A and B). Another possible
explanation for the reduction of Ad5 E1A-specific CTLs could be
retrafficking of these CTLs. However, because the Ad5 E1A peptide
spreads to all organs with similar kinetics, this seems less
likely.
It remains to be answered why the Ad5 E1A peptide displays such different kinetics compared with the HPV16 E7 peptide. Differences in kinetics may be the consequence of a combination of intrinsic peptide characteristics, i.e., hydrophobicity, charge, structure, and MHC class I binding affinities of the peptide. Comparison of these two peptide sequences revealed that the Ad5 E1A peptide is more hydrophilic than the HPV16 E7 peptide. However, tolerance could not be prevented by using an Ad5 E1A peptide that was engineered to increase hydrophobicity (27). Furthermore, the Ad5 E1A peptide is negatively charged as compared with a positively charged HPV16 E7. Other structural features, such as the presence of aromatic amino acids in HPV16 E7 and not in Ad5 E1A, may contribute to their functional differences. Another important difference is the fact that the Ad5 E1A peptide has a 100-fold higher binding affinity for the H2-Db molecule than for the HPV16 E7 peptide (28). However, this feature by itself does not explain the functional difference between the two peptides, because MHC-peptide binding analysis of a number of T cell-tolerizing and -activating peptides showed that tolerizing peptides do not have a consistently higher binding affinity than activating peptides (29). The pharmacokinetic properties of other T cell-tolerizing and -activating peptides should be analyzed to determine whether differences in pharmacokinetics could indeed predict the tolerizing or activating property of peptides in general. Ultimately, prediction of the effects that peptides may have on the immune system using pharmacokinetic profiles may increase the safety and efficacy of peptide-based vaccinations in humans.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. W. Martin Kast, Cardinal Bernardin Cancer Center, Loyola University Chicago, 2160 South First Avenue, Maywood, IL 60153. E-mail address: mkast{at}lumc.edu
3 Abbreviations used in this paper: HPV, human papilloma virus; Ad5, Adenovirus type 5; TFA, trifluoroacetic acid; ELISPOT, enzyme-linked immunospot; AICD, activation-induced cell death; DC, dendritic cell.
Received for publication February 5, 2001. Accepted for publication April 5, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-amyloid10–35 is parallel -sheet with residues in exact register. Proc. Natl. Acad. Sci. USA 95:13407.This article has been cited by other articles:
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H.-K. Kang, M. Liu, and S. K. Datta Low-Dose Peptide Tolerance Therapy of Lupus Generates Plasmacytoid Dendritic Cells That Cause Expansion of Autoantigen-Specific Regulatory T Cells and Contraction of Inflammatory Th17 Cells J. Immunol., June 15, 2007; 178(12): 7849 - 7858. [Abstract] [Full Text] [PDF]
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P. Krebs, E. Scandella, B. Odermatt, and B. Ludewig Rapid Functional Exhaustion and Deletion of CTL following Immunization with Recombinant Adenovirus J. Immunol., April 15, 2005; 174(8): 4559 - 4566. [Abstract] [Full Text] [PDF]
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A. Th. den Boer, G. J. D. van Mierlo, M. F. Fransen, C. J. M. Melief, R. Offringa, and R. E. M. Toes The Tumoricidal Activity of Memory CD8+ T Cells Is Hampered by Persistent Systemic Antigen, but Full Functional Capacity Is Regained in an Antigen-Free Environment J. Immunol., May 15, 2004; 172(10): 6074 - 6079. [Abstract] [Full Text] [PDF]
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J.-S. Blanchet, D. Valmori, I. Dufau, M. Ayyoub, C. Nguyen, P. Guillaume, B. Monsarrat, J.-C. Cerottini, P. Romero, and J. E. Gairin A New Generation of Melan-A/MART-1 Peptides That Fulfill Both Increased Immunogenicity and High Resistance to Biodegradation: Implication for Molecular Anti-Melanoma Immunotherapy J. Immunol., November 15, 2001; 167(10): 5852 - 5861. [Abstract] [Full Text] [PDF]
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B
b, and
a, b, and c are aggregated
kinetic parameters including terms for rate constants and apparent pool
sizes. Curve fits are shown on a logaritmic (A) and a
linear (B) scale to enable data analysis at different
times after peptide injection. This is one representative experiment of
four independent experiments performed. The SEM has been included for
each time point. Squares represent the HPV16 E7 peptide, and circles
represent the Ad5 E1A peptide.
,
heart; 
, lungs; 
, kidneys; 
, liver; 
, intestines; and 
,
brain. The arrow indicates the fraction in which the purified synthetic
tritiated peptide eluted.
