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Fetoprotein1
*
Divisions of Surgical Oncology,
Hematology/Oncology,
Pasarow Mass Spectrometry Laboratory, Department of Chemistry and Biochemistry, and Psychiatry and Neuropsychiatric Institute,
Experimental Radiation Oncology,
¶ and the Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles Medical Center, Los Angeles, CA 90095
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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fetoprotein (AFP)-derived peptide epitopes can be recognized by
human T cells in the context of MHC class I. We determined the identity
of AFP-derived peptides, presented in the context of HLA-A*0201, that
could be recognized by the human (h) T cell repertoire. We screened 74
peptides and identified 3 new AFP epitopes, hAFP137–145,
hAFP158–166, and hAFP325–334, in addition to
the previously reported hAFP542–550. Each possesses two
anchor residues and stabilized HLA-A*0201 on T2 cells in a
concentration-dependent class I binding assay. The peptides were stable
for 2–4 h in an off-kinetics assay. Each peptide induced
peptide-specific T cells in vitro from several normal HLA-A*0201
donors. Importantly, these hAFP peptide-specific T cells also were
capable of recognizing HLA-A*0201+/AFP+ tumor
cells in both cytotoxicity assays and IFN-
enzyme-linked immunospot
assays. The immunogenicity of each peptide was tested in vivo with
HLA-A*0201/Kb-transgenic mice. After immunization with each
peptide emulsified in CFA, draining lymph node cells produced IFN-
on recognition of cells stably transfected with hAFP. Furthermore, AFP
peptide-specific T cells could be identified in the spleens of mice
immunized with dendritic cells transduced with an AFP-expressing
adenovirus (AdVhAFP). Three of four AFP peptides could be identified by
mass spectrometric analysis of surface peptides from an HLA-A*0201
human hepatocellular carcinoma (HCC) cell line. Thus, compelling
immunological and physiochemical evidence is presented that at least
four hAFP-derived epitopes are naturally processed and presented in the
context of class I, are immunogenic, and represent potential targets
for hepatocellular carcinoma immunotherapy. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Approximately 80% of HCC reactivate fetoprotein (AFP) expression
(2). The murine and human T cell repertoires can recognize
AFP-derived peptide epitopes in the context of MHC class I (3, 4). Despite being exposed to high plasma levels of this
oncofetal protein during embryonic development, clearly some
AFP-specific T cells have not been deleted during the ontogeny of the
immune system. Activation of these T cell clones can be achieved by
presenting these epitopes in an immunostimulatory context, such as by
professional Ag-presenting dendritic cells (DC). DC, when transduced
with a recombinant adenovirus (AdV) vector encoding AFP, will process
and present peptide epitopes in the context of MHC and will induce
AFP-specific protection in mice (4).
We previously reported that hAFP542–550 (GVALQTMKQ) is naturally processed and presented in the context of HLA-A*0201, is recognized by the human T cell repertoire, and can be used to generate AFP-specific CTL in human T cell cultures and in HLA-A*0201/Kb-transgenic mice (3). Herein, we report a complete analysis of 74 AFP-derived peptides and identify 3 additional HLA-A*0201-restricted immunodominant epitopes as well as 10 potentially subdominant epitopes. These peptides have been characterized by both immunological and physicochemical methods. Our finding suggests that the expression of AFP by HCC cells can be exploited for immunotherapy strategies.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The University of Wisconsin (Madison, WI) Genetics Computer Group program "find patterns" was used to screen the hAFP sequences (GenBank accession numbers: J00077, J00076, and V01514) and identify 9- and 10-mer peptides, as described previously (3). The general HLA-A2.1 motif used was I, L, M, or V in P2 and V, I, or L in P9 (5).
Cells, Abs, cytokines, and viruses
HLA-A*0201 donors and cell lines were screened with the BB7.2
(HLA-A2) Ab and subtyped by PCR analysis by the University of
California Los Angeles (UCLA) Tissue Typing Laboratory. HepG2, Hep3B,
K562, B95-8, BB7.2, and W6/32 cell lines were obtained from American
Type Culture Collection (Manassas, VA). The amount of AFP produced by
HepG2 (606 ng/ml/106 cells/24 h) and Hep3B
(100ng/ml/106 cells/24 h) was determined by the
UCLA Clinical Labs. The M202 human melanoma cell line has been
described previously (6). T2 cells were provided by Peter
Cresswell (Yale University School of Medicine, New Haven, CT). JY cells
(HLA-A*0201 homozygous) were provided by Martin Kast (Loyola University
Cancer Center, Chicago, IL). Jurkat/A2Kb
cells were provided by Linda Sherman (Scripps Research Institute, La
Jolla, CA). EBV-transformed lymphoblastoid cell lines (LCL) were
generated by incubating PBMC with supernatant from B95-8 cells. Cell
lines were cultured in RPMI 1640 (Life Technologies, Gaithersburg, MD)
or IMDM (JY cells; Life Technologies) with 10% FBS (Omega Scientific,
Tarzana, CA) and PSF (penicillin/streptomycin/fungizone; Life
Technologies). Anti-
2-microglobulin and the
anti-CD4, CD14, CD19, and CD56 NA/LE Abs were obtained from BD
PharMingen (San Diego, CA), anti-pan class I Ab was prepared from
concentrated supernatant of W6/32 hybridoma, and CD4-FITC, CD8-PE, and
CD16-PE Abs were obtained from Caltag (Burlingame, CA).
The stable transfectant cell lines LCL/hAFP, M202/hAFP,
JurkatA2/Kb/hAFP (Jurkat/AFP), and
JurkatA2/Kb/MART1 (Jurkat/MART) have been
described previously (3). IL-2 was provided by
Hoffman-LaRoche (Nutley, NJ), IL-7 was obtained from Biosource
(Camarillo, CA), and keyhole limpet hemocyanin (KLH) and
2-microglobulin were obtained from Sigma (St.
Louis, MO).
The AdVhAFP contains the 1.9-kb hAFP cDNA and is driven by the CMV promoter/enhancer in a pAC-CMVpLpA AdV type 5 backbone (3). The empty AdV vector, AdVRR5, has been described previously (7) and served as a control.
Peptide synthesis
Peptides were synthesized by Chiron (Victoria, Australia) and at the UCLA Peptide Synthesis Facility (Dr. Joseph Reeve, Jr., Director) with standard F-moc technology.
T2 binding assay
Each peptide was tested for concentration-dependent binding to T2 cells in an HLA-A*0201 stabilization assay (8, 9). T2 (TAP deficient) cells, that had been incubated at room temperature the previous night to increase cell surface MHC class I molecule expression, were then incubated overnight with each peptide over a range of peptide concentrations from 0.1–100 µM. Stability of HLA-A*0201 was assayed by flow cytometry after staining the cells with anti-HLA-A2 Ab (BB7.2) and goat anti-mouse-FITC. The HLA-A*0201 strongly binding Flu matrix peptide (aa 58–66; Flu) was used as a positive control.
MHC-peptide complex stability
The off-kinetics assay was performed as described (3, 10). HLA-A*0201 LCL were stripped with a mild pH 3.2
citrate-phosphate acid buffer. Each peptide was immediately pulsed onto
cells at 200 µM for 1 h in the presence of
2-microglobulin at 3µg/ml at room temperature. Excess
peptide was washed off and the cells were incubated at 37°C for 0, 2,
4, and 6 h. Cells were washed at the end of each time point and
stained for cell surface HLA-A2 expression and then analyzed by flow
cytometry. The peptide-MHC class I complex was considered stable if the
mean fluorescence intensity increased at least 1.5-fold from cells that
were stripped but not pulsed with peptide.
Human CTL generation from peptide-pulsed PBMC
Peptide-specific CTL were generated as described previously (3, 11, 12) to various peptides. Briefly, normal donor HLA-A*0201 PBMC were pulsed with peptide, washed, and cultured on day 0 with IL-7 (10–25 ng/ml) and KLH (5 µg/ml) in RPMI 1640/10% autologous serum at 3 x 106 cells/1.5 ml/well. Cells were restimulated weekly by removing the nonadherent cells from the culture and adding them to fresh, autologous, peptide-pulsed, washed, and irradiated PBMC at a 1:1 ratio. IL-2 was added twice weekly at 10 U/ml. After 3–4 wk of culture, the cultures were tested for cytotoxicity and/or cytokine release.
Human CTL generation from AdV-transduced DC
DC (prepared as described from PBMC incubated with GM-CSF and IL-4; Refs. 13 and 14) were transduced with AdVhAFP or AdVMART1 at a multiplicity of infection of 1000 for 2 h. Transduced DC were washed, irradiated, and plated at 1–2 x 105 cells/ml to serve as stimulators for CTL generation. Autologous nonadherent cells were depleted of CD4, CD14, CD19, and CD56+ cells by magnetic bead depletion (Dynal, Lake Success, NY) to prepare CD8+-enriched responder cells (population generally 80% CD8+, not shown). The CD8+ cells were plated with the transduced DC at 2 x 106 cells/ml in 5% autologous medium plus IL-7 at 10–25 ng/ml. Cultures were supplemented with IL-2 at 10 U/ml every 3–4 days. The CD8+ CTL were restimulated weekly with fresh, autologous AdV-transduced DC at a ratio of 1 DC to 10–20 CD8+ CTL. Most cultures were phenotyped for CD4+ and CD8+ cells on a weekly basis.
Human and murine cytotoxicity assay
Chromium release assays were performed as described previously
after 6 days of in vitro restimulation of primed splenocytes
(3). Briefly, target cells were chromated (and T2 cells
were peptide pulsed at 50µg/ml) for 1.5–2 h and plated. Target cells
were washed three times, diluted to 5 x 104
(or 1 x 105 for murine assay) cells/ml, and
plated with CTL. To control for nonspecific lysis, a 10–50 fold excess
of unchromated K562 were added to target populations before adding to
CTL (in human assay). After 4–5 h, supernatants were harvested and
counted in a counter. Triplicate wells were averaged and the
percentage of specific lysis was calculated as follows: (sample -
spontaneous release)/(maximum release - spontaneous release).
Human enzyme-linked immunospot (ELISPOT) assay
To determine the frequency of Ag-specific cytokine-producing cells, the ELISPOT technique was used as described previously (3, 15, 16). T cell restimulation was performed with 2–5 x 106 CTL incubated with 1 x 105 autologous LCL or JY cells pulsed with specific or nonspecific peptides or with tumor cell lines. Restimulator cells without CTL served as a negative control. The capture Ab (BD PharMingen)-coated plates (Millipore, Bedford, MA) were incubated with restimulated cells (in duplicate at three dilutions) at 37°C for 24–48 h. The colored spots, representing cytokine-producing cells, were counted under a dissecting microscope.
HLA-A*0201/Kb-transgenic mice
HLA-A*0201/Kb-transgenic female mice (created by Dr. Linda Sherman, Scripps Research Institute) were originally purchased from Harlan Sprague-Dawley (Indianapolis, IN), and currently are bred by the animal facility of the Dept. of Radiation Oncology at UCLA and handled in accordance with the animal care policy of the UCLA. For peptide immunizations, mice received 100 µg of AFP or control peptide emulsified 1:1 in CFA (Difco, Detroit, MI) s.c..
Preparation of murine DC and adenoviral transduction
DC were differentiated from murine bone marrow progenitor cells following the Inaba method (17) with modifications (3, 18). In vitro cultured DC were transduced in RPMI 1640/2% FCS at an multiplicity of infection of 100. Transduction was conducted for 2 h at 37°C, the DC then were washed and resuspended at 5 x 105 DC per 0.2 ml of PBS per animal for injection. In all cases, viability exceeded 95%.
Murine CTL generation
Two weeks after priming (by AdV/DC or peptides), splenocytes (3 x 106/well) were activated ex vivo with irradiated, mitomycin C-treated Jurkat/hAFP or Jurkat/MART (5 x 105/well) in 2 ml RPMI 1640/10% FBS and 50 U/ml IL-2 in 24-well plates for 6 days.
Murine ELISPOT assay
Groups of HLA-A*0201/Kb mice were primed by AdV-transduced or peptide-pulsed DC immunizations, and 2 wk later, splenocytes (4 x 106 cells/ml) were activated ex vivo with an optimal concentration of peptide in complete medium (RPMI/10%FBS) for 48 h, or 5 x 106 cells/ml were cultured in T-25 flasks in an equal volume with irradiated Jurkat-MART or -AFP cells (1 x 105 cells/ml) plus 50 U/ml IL-2. After restimulation for 48 h, the ELISPOT assay was performed as described. The frequency of Ag-specific cells was determined from the difference between the number of spots seen with and without Ag during restimulation.
HPLC separation of naturally processed peptides from viable cells
To elute peptides, HepG2 and Hep3B cells were washed three times with PBS before being incubated with 5 ml of citrate-phosphate buffer at pH 3.2 (19) for 1 min. The suspension was centrifuged (800 x g for 5 min) and a total of 500 ml of cell-free supernatant was collected for each cell line. The materials were lyophilized to dryness and stored at -20°C.
Lyophilized materials were redissolved in 30 ml of water/acetonitrile/triflouroacetic acid (W/A/TFA, 95/5/0.1 all by vol.). This solution was pumped onto an analytical reverse-phase HPLC column (C18 Betasil, 250 mm x 2 mm, 5-µm particle size, 100-Å pore size; Keystone Scientific, Bellefonte, PA) equilibrated in W/A/TFA, at a flow rate of 0.2 ml/min. The column was eluted by using an increasing linear gradient of 0.1% TFA in acetonitrile (time/% acetonitrile = 0/5, 5/5, 55/100, 60/100). Column eluate absorbance was monitored at 215 and 280 nm, and 1-min fractions were collected. The retention times of the synthetic peptides with amino acid sequences corresponding to the immunostimulatory peptides were obtained by using the same separation gradient on a separate column.
Mass analysis
A Voyager-RP (PerSeptive Biosystems, Framingham, MA)
matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF)
instrument was used to acquire the mass spectra. The instrument uses a
stainless-steel target on which the samples are deposited and dried.
All spectra were externally calibrated with insulin, resulting in mass
accuracy typically within ± 0.1%. Lyophilized HPLC fractions
were resuspended in 10 µl of 70% acetonitrile with 0.1% TFA. A
total of 1µl of this material was spotted along with 1 µl of the
matrix -cyano-4-hydroxycinnamic acid (Sigma, 10 mg/ml in 70%
ACN/0.1% TFA). Spectra were obtained by scanning from m/z
500-7000.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We identified potential HLA-A*0201-restricted hAFP peptide
epitopes by using a computer-based analysis of the human AFP amino acid
sequence. A total of 74 9- and 10-mer peptides, the sequences of which
conformed to the well characterized HLA-A*0201 binding motif (3, 20, 21, 22), were synthesized. Several immunological strategies were
used to determine whether any of these peptides were immunogenic and
naturally processed and presented in the context of HLA-A*0201.
Fourteen of these AFP peptides were reproducibly positive in one or
more of these assays and this subset of candidate epitopes is presented
in Table I
(and for comparison, 10
consistently negative hAFP peptides, and positive controls Flu
M158–66 and MART-127–35).
Their structural characteristics also are shown in this table and
include length, number of anchor residues in P2 and P9/10 positions,
concentration required for detectable binding to TAP-deficient T2
cells, and duration of binding to acid-stripped JY cells in an
off-kinetics assay.
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ELISPOT and cytotoxicity
assays (Fig. 1, A and
B, and data not shown). Over a dozen of the 74 AFP peptides
also could be recognized by these primed splenocytes. Three peptides
(hAFP137–145 (PLFQVPEPV),
hAFP158–166 (FMNKFIYEI), and
hAFP325–334 (GLSPNLNRFL)) and the previously
reported hAFP542–550 (GVALQTMKQ; Ref.
3) consistently elicited cytokine production and
cytotoxicity, and therefore are "immunodominant" (23).
The remaining peptides had either weak or less reproducible responses
but were positive in more than one type of assay and therefore are
potential "subdominant" epitopes. These experiments indicated that
the DC from these HLA-A*0201/Kb mice were capable
of processing and presenting peptide epitopes in the context of
HLA-A*0201, which elicited responses from the available murine T cell
repertoire.
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, are formally presented for the four
immunodominant epitopes we have identified. Screening for AFP peptide-specific responses in HLA-A*0201/Kb-transgenic mice immunized with AdVhAFP/DC
HLA-A*0201/Kb mice were immunized
with AdVhAFP/DC, and their splenocytes were restimulated in vitro with
each AFP peptide separately and assayed for both cytotoxicity and
IFN- production by ELISPOT assays. AdVhAFP/DC immunization
induced AFP peptide-specific responses compared with a control
A*0201-restricted melanoma Ag MART-127–35
peptide (Fig. 1C). Conversely, AdVMART1/DC-immunized
mice specifically recognized the immunodominant
MART-127–35 peptide but not AFP-pulsed targets
(not shown). Naive splenocytes showed no cytotoxicity and had few
IFN--producing cells (not shown). Responses to the previously
identified peptide hAFP542–550 were
reproducibly stronger than responses to the three new epitopes.
hAFP peptide-specific human T cell cultures
Synthetic peptides then were used to stimulate human T cells
in vitro. Bulk T cell cultures were generated from PBMC pulsed with
each AFP-derived peptide (supplemented with KLH, IL-7, and IL-2) and
were tested between weeks 3 and 7 of expansion for the ability to
recognize both peptide-pulsed and AFP-expressing targets (Fig. 2). These cultures expanded
peptide-specific T cells (Fig. 2A), as evidenced by the
ability to secrete IFN- on recognition of specific peptide-pulsed JY
cells and not control MART-127–35-pulsed JY in
the ELISPOT assay. The AFP peptide-specific bulk T cells also
recognized both AFP-stably transfected and AdVhAFP-transduced
HLA-A*0201 melanoma cells (M202) compared with unmodified or empty
AdVRR5-transduced parental cells (Fig. 2B) as shown by an
increased frequency of IFN--producing AFP-specific T cells. In
cytotoxicity assays, these bulk AFP peptide-stimulated T cells were
able to lyse the AdVhAFP-transduced melanoma cells (Fig. 2C). To assess the ability to recognize the
HLA-A*0201+, naturally AFP-expressing HCC cell
line HepG2 (compared with the HLA-A2-/AFP+ HCC
line Hep3B), both cytotoxicity (Fig. 2C) and ELISPOT
assays (Fig. 2D) were preformed. Levels of cytotoxicity
were lower than for AFP-transduced melanoma cells, but in general,
frequencies of IFN--producing T cells were similar for the HCC and
transfected/transduced melanoma cells.
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Because AdVhAFP/DC in vitro stimulated human T cells specifically
recognized hAFP-transfected targets in both CTL and ELISPOT assays
(3), we determined whether these four AFP peptides were
specifically recognized by the AdVhAFP/DC stimulated T cells. After
7–21 days of culture, CD8-enriched T cells stimulated weekly with
AdVhAFP/DC were tested for both cytotoxicity (Fig. 3A) and the frequency of hAFP
peptide-specific IFN- cytokine-producing cells (Fig. 3B).
AdVhAFP/DC T cell cultures were cytotoxic for JY cells pulsed with each
of the four AFP peptides, indicating that CTL to these peptides could
be expanded from peripheral blood of normal donors. After restimulation
with autologous peptide-pulsed LCL or JY cells, these bulk cultures
also contained a much higher frequency of IFN--secreting cells
specific for AFP peptides compared with
MART-127–35, indicating that, in addition to
hAFP542–550, these three peptides also are
naturally processed and presented by AdVhAFP-transduced DC.
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C. T
lymphocytes synthesizing the Th1 cytokines IFN- and TNF- were
detected, whereas the Th2 cytokine IL-4 was not detected. IL-10 also
was detected when the HCC lines were plated without T cells, indicating
that production of this cytokine was tumor cell derived.
Cytotoxicity against hAFP137–145- and
hAFP325–334-pulsed targets was generally
more difficult to detect than against
hAFP158–166- and
hAFP542–550-pulsed targets by chromium release
assay. Similarly, a lower frequency of IFN--producing cells were
obtained for these two peptides.
AFP-specific responses in HLA-A*0201/Kb mice immunized with peptide in CFA
To confirm the in vivo immunogenicity of these four hAFP peptides,
HLA-A*0201/Kb mice were immunized with each AFP
peptide pulsed onto syngeneic DC. IFN--specific ELISPOT assays were
performed with splenocytes restimulated in vitro with either the
immunizing AFP peptide (or MART-1 peptide, Fig. 4A) or with Jurkat/AFP or
Jurkat/MART transfected cell lines (Fig. 4B). Immunization
with each hAFP peptide and subsequent restimulation with either peptide
or Jurkat/AFP induced large numbers of AFP-specific IFN--producing
cells. Lymphocytes from PBS-injected mice showed neither cytotoxicity
nor IFN- production regardless of restimulation (Fig. 4 and data not
shown). Mice immunized with MART-127–35 peptide
produced MART-1-specific responses but no AFP peptide responses.
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We then sought physicochemical evidence for their presence on the surface of a human HCC line. We performed MALDI-TOF mass spectrometry to analyze HPLC-fractionated peptides acid-eluted from the AFP-producing HCC cell lines HepG2 (HLA-A2+) and Hep3B (HLA-A2-). We identified potential mass candidates for three of the four peptides in the HPLC-fractionated peptide pool eluted from the HLA-A*0201+ HepG2 cells but not from the HLA-A*0201- Hep3B cells.
The MALDI-TOF analysis of the HPLC fractions established that
almost all fractions contained up to 20 different peptides in the mass
range from 700 to 1500 Da, although frequently with a few dominating
signals. Out of this complex mixture, we identified peaks with
m/z values corresponding to the calculated monoisotropic
protonated molecules ((M+H)+) of
hAFP542–550, hAFP158–166,
and hAFP325–334 in the peptide pool eluted from
HepG2 cells (Table II and Fig. 5). A peptide of m/z 975.6 was
identified in HPLC fraction 21 from the HepG2 peptide pool (Fig. 5A). The calculated (M+H)+ of
hAFP542–550 was 975.5, and the retention time of
the synthetic peptide with amino acids corresponding to
hAFP542–550 was 21.2 min. Furthermore, no signal
at m/z of 975.5 ± 1 was observed in samples with
matrix alone (data not shown) and in HPLC fractions 18–22 from the
Hep3B elution (fraction 21 shown in Fig. 5A). Similarly,
peaks with m/z corresponding to the calculated
(M+H)+ of hAFP158–166 and
hAFP325–334 also were found in the appropriate
fraction (28) derived from HepG2 predicted from the behavior of the
standard peptides (Table II and Fig. 5, B and D).
These peaks were absent in fractions 26–32 in the peptide pool eluted
from Hep3B (fraction 28 shown in Fig. 5). A peak at 1152.2
m/z was observed in fraction 28, suggesting the presence of
the sodium adduct of hAFP325–334 (Fig. 5D).
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C). Because this peak was beyond the
margin of error tolerated by this physicochemical analysis, we
concluded that we could not document the presence of
hAFP137–145 on the surface of HepG2 cells.
To confirm the presence of AFP peptides in these fractions
immunologically, 1 µl of each HPLC fraction from either HepG2 or
Hep3B cells was used to restimulate AdVhAFP/DC-immunized murine
splenocytes in an ELISPOT assay (Table II). A total of 200–250
spots/106 cells were observed from fractions
containing AFP immunodominant peptides, whereas 100–130
spots/106 cells were observed from the other
fractions, and a maximum of 50 spots/106
cells were observed from Hep3B fractions. This further supports the
mass spectrometry identification of these AFP peptides.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human AFP protein has 65% amino acid homology to murine AFP. Therefore, the T cells in these transgenic mice recognize hAFP as a xenoantigen in the context of the human HLA-A*0201. For the four hAFP immunodominant peptides, there are 3, 2, 4, and 1 amino acid differences, respectively, between the human and murine 137, 158, 325, and 542 peptide sequences. Although studies by others have demonstrated that A*0201 mice respond to largely the same set of HLA-A2.1-binding peptides as humans (24, 25), we performed the human T cell culture assays to support the human immunogenicity of each peptide. Moreover, we have demonstrated previously that mAFP is immunogenic in normal inbred mice if presented in the immunogenic context of engineered DC (3).
Each of these three newly reported peptides has two anchor residues and strong binding affinity to HLA-A*0201. Molecular modeling of hAFP137–145 (PLFQVPEPV), hAFP158–166 (FMNKFIYEI), and hAFP325–334 (GLSPNLNRFL) in the HLA-A2 binding groove show that the peptides adopt the general backbone conformation seen in the x-ray structures of HLA-A2 (26, 27): the NH2 and COOH termini of peptides are accommodated by conserved pockets at both ends of the binding groove, whereas the backbone in the central portion of the peptides bulges upward toward the TCR. It should be noted that the proline residues at positions 6 and 8 in hAFP137–145 (PLFQVPEPV) impose a conformation of the peptide that is more rigid than the other peptides in this study. As expected, the side chains of the anchor residues at positions 2 and 9 (10 in hAFP325–334) of the peptides are buried in the deep polymorphic pockets of HLA-A2 (pockets B and F, respectively). Side chains in the central part of the peptide, both hydrophilic and hydrophobic, tend to project upward toward the TCR (K4, F5, Y7, and E8 in hAFP158–166 (FMNKFIYEI); Q4, V5, and E7 in hAFP137–145 (PLFQVPEPV); N5, L6, N7, R8, and F9 in hAFP325–334 (GLSPNLNRFL)). These contrast with hAFP542–550, which lacks a hydrophobic side chain for pocket F.
It proved to be important to screen candidate peptides with fewer than two anchor residues, as 8 of 14 immunogenic peptides had one anchor residue. Although strong binding, or "on-rate" (10 µM or less) was a characteristic of 3 of 4 immunodominant peptides, the fourth immunodominant peptide (hAFP542–550) and 8 of 14 immunogenic peptides overall showed weak (>50 µM) binding. Of the physical characteristics assessed, off-kinetics proved to correlate best with immunogenicity with 9 of 14 immunogenic peptides having off-kinetics greater than 2 h and 5 of 14 greater than 4 h. Of the five peptides with 6 h or longer kinetics, all were immunogenic. A sufficiently long half-life is likely to facilitate the stimulation of many TCRs on a given T cell, which is thought to be important for strong T cell activation (28, 29). Thus, it is possible that additional immunogenic epitopes exist from Ags for which candidate determinants were screened based on only two anchor residues or strong binding affinity (30, 31, 32).
The three new immunodominant and the 10 potentially subdominant peptides described in this report may serve as important tools for direct peptide-based immunotherapy as well as analysis of the diversity of the response to whole AFP Ag-based immunotherapy. We have initiated a phase I/II clinical immunotherapy trial in which A*0201/AFP-positive patients with unresectable HCC are immunized with all four of these AFP immunodominant peptides emulsified in IFA. Our early observations are that these patients are capable of generating peripheral T cell responses to all four peptides as measured by tetramer and ELISPOT assays (data not shown). Together, this data confirms that AFP can serve as a tumor-specific Ag in HCC and may be a suitable target for T cell-mediated immunotherapy strategies.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. James S. Economou, Division of Surgical Oncology, 54-140 Center for Health Sciences, University of California Los Angeles Medical Center, 10833 Le Conte Avenue, Los Angeles, CA 90095-1782.
3 Abbreviations used in this paper: HCC, hepatocellular carcinoma; AFP, fetoprotein; h, human; DC, dendritic cells; AdV, adenovirus; ELISPOT, enzyme-linked immunospot; LCL, lymphoblastoid cell lines; KLH, keyhole limpet hemocyanin; TFA, trifluoroacetic acid; MALDI-TOF, matrix-assisted laser desorption ionization/time-of-flight.
Received for publication June 12, 2000. Accepted for publication February 15, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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fetoprotein. Cancer Res. 59:3134.-fetoprotein-specific genetic immunotherapy for hepatocellular carcinoma. Cancer Res. 59:3064.This article has been cited by other articles:
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