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*
Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, and
Swiss Institute for Cancer Research, Epalinges, Switzerland; and
Centre d’Immunologie Pierre Fabre, Saint Julien-en-Genevois, France
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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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Two options are mainly investigated. The first is based on the use of professional APCs. Dendritic cells transduced with adenovirus vectors expressing melanoma associated Ag or pulsed with whole tumor lysates or synthetic tumor peptides have been used in successful vaccination approaches in experimental models (2). Although the use of dendritic cells allows the targeting of Ag to lymphoid tissue and the induction of a strong and efficient T cell response, it involves steps of cell purification and culture. In this regard, cell-free vaccines would be more suitable for clinical purposes.
In the case of peptide-based vaccines, the way of delivering Ag to the immune system is critical. It has been shown that a complex mixture of tumor peptides conjugated to beads can prime a CTL response in healthy individuals (3), and lipid compounds have been reported to have adjuvant qualities (4). Yet, the most common adjuvant of T cell responses used to date is IFA. IFA protects immunizing peptides from rapid degradation and enhances their immunogenicity by activating inflammatory processes and costimulatory factors and mediating the production of cytokines. It has been successfully used in immunotherapy against melanoma involving gp100 peptide immunization (5). Although IFA is not associated with severe side effects, this adjuvant is not commonly used in human vaccination protocols due to its undesirable effects, such as erythema and induration at the injection site (5). These effects are partly due to the nonmetabolized mineral oil contained in IFA, and efforts have been made to reduce the oil concentration and to use metabolizable mineral oils (6). In addition, specific CTL tolerance rather than immunity against immunizing peptide in IFA has been reported in an experimental model (7). For these reasons, alternative potent and safe adjuvants need to be identified.
Outer membrane proteins
(Omp)2 complexes
extracted from meningococcal membranes have the ability to adsorb to
peptides via hydrophobic bonds (8). Therefore, we asked
whether the 40-kDa major Omp (referred to as P40 hereafter) of
Klebsellia pneumoniae, which contains at least one helper
epitope (9) can be used to target antigenic peptides to
the immune system and elicit a specific CTL response. Peptides from two
melanoma-associated differentiation Ags, Melan-A/MART-1 (Melan-A)
(10, 11) and tyrosinase-related protein 2 (TRP-2)
(12) were used, namely 1) the HLA-A*0201-restricted
decapeptide Melan-A26–35 (EAAGIGILTV)
substituted at position 2 (Melan-A26–35 A27L
peptide analog), which is more immunogenic than the parental peptide
both in vitro in human and in vivo in experimental models (13, 14), and 2) the TRP-2 T cell epitope mapping to residues
181–188 (VYDFFVWL) and presented in association with the mouse
Kb class I molecule (12). To address
the question, we took advantage of transgenic mice expressing a
chimeric MHC class I molecule composed of human HLA-A*0201 
1 and
2 domains and mouse Kb 3, transmembrane and
cytoplasmic domains in the C57BL/6 x DBA/2 background
(15). We found that Melan-A26–35
A27L or TRP-2181–188 peptides are able to
generate a specific CTL response when mixed with P40. This CTL response
is a function of the amount of P40 used for immunization. Moreover, the
CTL response generated against TRP-2181–188
peptide in the presence of P40 is associated with tumor protection in
two different experimental models and is independent of the presence of
CD4+ T lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse EL-4 cells transfected with the HLA-A*0201/Kb gene (EL-4.A2/Kb transfectants) (16) were provided by Dr. Linda Sherman (The Scripps Clinic and Research Foundation, La Jolla, CA) and maintained in DMEM supplemented with 1% HEPES, 1% strepto-penicillin, 10% heat-inactivated FCS, and 0.5 mg/ml G418. The B16F10 melanoma cell line (17) was provided by Dr. Lars French (University Hospital, Geneva, Switzerland) and maintained in DMEM supplemented with 1% HEPES, 1% strepto-penicillin, and 10% FCS.
Synthetic peptides
Peptides were synthesized by standard solid phase chemistry on a multiple peptide synthesizer (Applied Biosystems, Foster City, CA) using standard F-moc for transient NH2-terminal protection and analyzed by mass spectrometry. All peptides were >90% pure as indicated by analytical HPLC. Lyophilized peptides were diluted in DMSO and stored at -20°C.
Biochemical characterization of P40/Melan-A26–35 A27L and P40/TRP-2181–188 mixtures
All chemicals and solvents from commercial sources were of analytical grade. Acetonitrile was of HPLC grade. K. pneumoniae P40 was expressed as a recombinant protein in Escherichia coli, purified as previously described (9, 18), and will be referred to as P40 thereafter. Purification of P40 was designed in apyrogen conditions. The endotoxin level determined by the Limulus assay was <0.25 endotoxin unit/mg of P40. P40 batches used were produced according to pharmaceutical quality standards, intended for clinical trials in humans. Binding of peptides Melan-A26–35 A27L and TRP-2181–188 to P40 was performed by mixing the respective peptide and P40 in a 7/1 (w/w) ratio in PBS. The final concentrations of peptide and P40 were 0.1 and 0.7 mg/ml, respectively. The mixtures were incubated for 2 h at room temperature under gentle agitation. The P40/peptide complexes were purified by gel filtration on a Superose 12 column (Amersham Pharmacia Biotech, Saint-Quentin en Yvelines, France). Fractions containing P40 were pooled and freeze-dried before analysis by HPLC (Hewlett-Packard, Les Ulis, France). For analysis, the samples were dissolved in 50–100 µl of 10% formic acid and further diluted with 1 vol of HPLC equilibration buffer. Samples were injected on a Vydac C18 column (Hesperia, CA; 150 x 2.1 mm, 5 µm). Peptides complexed to P40 were eluted with an acetonitrile/trifluoroacetic acid gradient.
Immunization
Breeding pairs of HLA-A*0201/Kb transgenic mice (line 6) (15) were provided by Harlan Sprague-Dawley (Indianapolis, IN). Different protocols of immunization were used. The protocol of immunization in the presence of IFA has been described previously (19). Briefly, HLA-A*0201/Kb transgenic mice were s.c. preinjected, or not, with an emulsion of IFA at the base of the tail. Three weeks later, these mice were immunized s.c. with 50 µg of Melan-A or TRP-2 peptides emulsified in IFA. In experiments with P40, mice were s.c. immunized with 50 µg of Melan-A or TRP-2 peptides mixed with various doses of P40. Ten days after injection, mice were sacrificed, and lymphocyte suspensions from draining lymph nodes were prepared for in vitro stimulation with the relevant peptides.
Generation of specific mouse CTL by in vitro stimulation
Lymph node cells (4–5 x 106) were
cultured with 2–5 x 105 irradiated (100
Gy) EL-4 A2/Kb cells, prepulsed with 1 µM of
the relevant peptides for 1 h at 37°C, in 24-well cell culture
plates in 2 ml of DMEM supplemented with 10 mM HEPES, 50 µM 
2-ME,
10% FCS, and EL-4 cell culture supernatant containing 30 U/ml of IL-2.
After one or more rounds of weekly stimulation, the cultured cells were
tested for cytolytic activity.
Assessment of in vitro cytolytic activity
The cytolytic activity of specific CTL was determined in a 51Cr release assay. Target cells were labeled with 51Cr for 1 h at 37°C in the presence or the absence of the tested peptides, then washed and coincubated with effector cells at the indicated lymphocyte to target cell ratio in V-bottom 96-well plates in a total volume of 200 µl of DMEM. Chromium release was measured in 100 µl of supernatant harvested after 4–6 h of incubation at 37°C. The percentage of specific lysis was calculated as: % specific lysis = (experimental release - spontaneous release)/(total release - spontaneous release) x 100.
B16F10 tumor challenge
C57BL/6 mice were shaved at the flank and injected s.c. with 2.5 x 103 syngeneic B16F10 melanoma cells. Groups of six mice were immunized s.c. at the base of the tail with 300 µg of P40, 50 µg of TRP-2181–188 peptide, a mixture of both, or 50 µg of TRP-2181–188 peptide emulsified in IFA on the same day or 4 days after tumor injection, then boosted 10 days later. When tumors became apparent, their volumes were measured at regular intervals using an electronic caliper. Mice were generally sacrificed when tumors became necrotic or their volume reached 2500–3000 mm3.
In vivo cell depletion
On days -3, -1, and +1 relative to the day of B16F10 injection and immunization with a mixture of 300 µg of P40 and 50 µg of TRP-2181–188 peptide, C57BL/6 mice (seven mice per group) were injected i.p. with mAb GK1.5 (anti-CD4) or H35 (anti-CD8). Depletion of CD4+ or CD8+ T cells was monitored by flow cytometry. When tumors became apparent, their volumes were measured at regular intervals using an electronic caliper. Mice were generally sacrificed when tumors became necrotic or their volume reached 2500–3000 mm3.
TRP/Tag tumor model
TRP/T Ag of SV40 (Tag) mice (20) were immunized with PBS, 300 µg of P40, 50 µg of TRP-2181–188 peptide, or a mixture of 300 µg of P40 and 50 µg of TRP-2181–188 peptide at 3 wk of age, then boosted at 4 and 5 wk (five mice per group). The appearance of macroscopically detectable ocular pathology was monitored. Mice with severe eye pathology (eye rupture and bleeding) were sacrificed. In untreated mice this generally occurred 10 days from the first appearance of visible eye pathology.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been previously shown that Omp complexes extracted from
meningococcal membranes have the ability to adsorb to peptides via
hydrophobic bonds (8). Because P40 forms hydrophobic
barrels (21), we investigated the possibility of using
mixtures of peptide and P40, rather than chemical conjugate, to prime
CTL responses in vivo, as this would considerably simplify the
preparation of the vaccine. For that purpose we biochemically
characterized the mixture of P40 and
Melan-A26–35 A27L. Fig. 1
A shows the elution pattern
for the P40/Melan-A26–35 A27L mixture as
visualized by gel filtration chromatography with a Superose 12 column.
P40 and Melan-A26–35 A27L were detected only in
the first peak eluting at 40.7 min, suggesting an association of P40
and peptide. Analysis of pooled fractions containing P40 by SDS-PAGE
(data not shown) and reverse phase HPLC on a C18
column (Fig. 1B) confirmed the coelution of P40 and
Melan-A26–35 A27L. Furthermore, the identity of
the C18-purified peptide was confirmed by both
amino acid analysis and coelution with
Melan-A26–35 A27L peptide added to the sample
(data not shown). Similar results have been observed for other
hydrophobic peptides (data not shown), suggesting that P40 and
hydrophobic peptides form relatively stable complexes.
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We then tested the ability of
Melan-A26–35 A27L and
TRP-2181–188 peptides mixed with P40 to
initiate a CTL response after a single immunization of
HLA-A*0201/Kb transgenic mice. As a positive
control of CTL induction, we used the same amount of peptides
emulsified in IFA and coinjected with a Th epitope, P30, derived from
tetanus toxoid 947–967 (22). As a negative control, mice
were injected with the same amount of P40 or P30 Th epitope emulsified
in IFA in the absence of CTL peptide. CTL specific for
Melan-A26–35 A27L peptide generated in vivo
could be detected in vitro after two rounds of stimulation when mice
were immunized with peptide either mixed with P40 or emulsified in IFA
in presence of P30 Th epitope (Fig. 2). A
significant CTL response against TRP-2181–188
peptide was observed after one round of in vitro stimulation in mice
specifically immunized against peptide in the presence of P40 (Fig. 2).
Lymph node cells from mice immunized with the same amount of P40 or IFA
plus P30 in the absence of CTL peptide stimulated in vitro in the same
conditions did not grow, but some viable cells were maintained in
culture for a mean period of 1–2 wk. No CTL activity directed against
Melan-A26–35 A27L or
TRP-2181–188 peptides was found in cells
remaining in these cultures. This suggests that CTLs specific for
Melan-A26–35 A27L and
TRP-2181–188 peptides detected in vitro have
been primed in vivo during immunization with the respective peptides
mixed with P40 and do not result from in vitro primary immunization. No
CTL response was detected when the same peptides were coinjected with
tetanus toxoid alone (not shown), which indicates that the
CTL-eliciting activity of P40 cannot be only ascribed to its properties
as a carrier protein. Taken together, these data indicate that P40 acts
as an adjuvant of the CTL response directed against tumor-associated
CTL epitopes.
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Part of the activity of P40 is as a carrier protein. Given the
epitope suppression observed with certain protein carriers (23, 24), and because humans are often primed to K.
pneumoniae OmpA, presumably following natural exposure
(25), we investigated whether a pre-existing immune
response against P40 could influence its CTL adjuvant potential. For
that purpose, we first preprimed mice with either 50 µg of P40 in PBS
or IFA/PBS emulsion and immunized them 3 wk later with 50 µg of
Melan-A26–35 A27L mixed with 300 µg of P40 or
emulsified in IFA, respectively. Control groups received PBS only for
prepriming. A significant CTL response specific for
Melan-A26–35 A27L was observed after the second
round of in vitro stimulation in each group of mice (Fig. 3). There was no correlation between the
intensity of the CTL response and the protocol of immunization,
indicating that prepriming of mice with P40 does not negatively
influence CTL response induction by peptide mixed with P40. The lack of
epitope suppression with P40 was also confirmed by measuring the IgG
response to a B cell epitope coupled with P40 (not shown).
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To define optimal conditions of immunization with P40, mice were
immunized with 50 µg of CTL peptide mixed with different amounts of
P40. We first measured the number of draining lymph node cells to
evaluate the effect of immunization in vivo. As shown in Table I, the number of draining lymph node
cells recovered was positively correlated with the quantity of P40
injected. A 2.7- to 3.6-fold increase in lymph node cell number was
observed in HLA-A*0201/Kb mice immunized with
Melan-A26–35 A27L peptide plus 30 and 300 µg
of P40, respectively, compared with that in naive mice. No significant
increase in cell yields was observed in mice immunized with
Melan-A26–35 A27L peptide plus 3, 0.3, or 0.03
µg of P40 (Table I). In the same manner, a 3-fold increase in cell
yield was found in the draining lymph nodes derived from C57BL/6 mice
immunized with 300 µg of P40 and 50 µg of
TRP-2181–188 peptide compared with that in naive
mice (Table I). However, the numbers of cells measured in the lymph
nodes derived from mice injected with 300 µg of P40 plus 50 µg of
TRP-2181–188 peptide or with 300 µg of P40
alone were not significantly different. This indicates that P40
contributes to the increase in cell yield found in draining lymph nodes
independently of the presence of CTL peptide. The same results were
obtained in mice injected with IFA in the presence or the absence of
CTL peptide (data not shown). Cytofluorometric analysis of draining
lymph node cells from immunized mice (P40 plus peptide or P40 alone)
did not reveal any significant modification of the percentages of
either CD4+ or CD8+ T cells
and B cells or in the percentage of activated
CD8+ T cells compared with those in naive mice
(data not shown). The second parameter investigated was the specific
CTL activity of lymph node cells after in vitro stimulation. Whereas no
CTL activity was detected after one in vitro stimulation in any
protocol of immunization of HLA-A*0201/Kb
transgenic mice with Melan-A26–35 A27L peptide,
significant levels of peptide-specific lysis were obtained after the
second round of in vitro stimulation of lymph node cells from mice
immunized with peptide plus 300 µg of P40 (Fig. 4). No CTL activity was detectable in any
other group of mice tested. After the third in vitro stimulation, CTL
activity could be detected in all groups of mice. Yet, a lower CTL
activity was measured in lymphocytes from mice immunized with peptide
mixed with 300 µg of P40; this could be attributed to an
overstimulation of T cells during in vivo priming in this group of
mice. These results indicate that generation of CTL specific for the
Melan-A26–35 A27L peptide is strictly dependent
on the dose of P40 used as adjuvant. The first dose of 300 µg is
enough to consistently detect a CTL response after two rounds of
stimulation in vitro, suggesting that the number of CTL precursors
activated in this immunization protocol is higher than the number
activated with the lower doses of P40 tested.
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The B16F10 melanoma cell line is a highly aggressive tumor when
implanted into a syngeneic host. We chose the dose of 2500 B16F10
cells/mouse following injection of different numbers of B16F10 cells
s.c. into C57BL/6 mice (not shown). Of the different doses tested,
injection of 2500 cells yielded nearly 100% tumor take with a
reasonably long kinetics of tumor growth (
3 wk from time of tumor
injection to maximal tumor volume and sacrifice). This situation
(relatively small tumor cell numbers) is an ideal one for testing
immunotherapy protocols, because it presents similarities to that of
potential human immunotherapy recipients. Indeed, in these subjects
tumor mass is generally surgically removed before treatment, so that
only residual numbers of tumor cells remain.
A sharp reduction in the growth kinetics of the tumor was observed in
mice injected with B16F10 and simultaneously immunized with a mixture
of TRP-2181–188 peptide and P40 (Fig. 5B) or with
TRP-2181–188 peptide emulsified in IFA (Fig. 5C) compared with those in mice immunized with
TRP-2181–188 peptide alone (Fig. 5A).
As expected, no effect on tumor growth was observed in mice immunized
with P40 or IFA alone (data not shown). This indicates that P40 in
combination with a tumor-derived peptide could be a good candidate for
therapeutic anti-tumor vaccination.
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To test whether the delay in tumor growth observed following
immunization with a mixture of P40 and
TRP-2181–188 peptide was the result of the CTL
response elicited by this vaccination protocol, and whether this
anti-tumor activity was dependent of CD4+ T
cell help, we depleted the CD4 or CD8 T cell subsets in vivo in mice
that subsequently received both B16F10 cells and a mixture of P40 and
TRP-2181–188 peptide. Subset depletion was
ascertained by flow cytometry (not shown). As shown in Fig. 6, depletion of
CD8+ T cells resulted in complete abrogation of
protection (Fig. 6B) compared with that in nondepleted mice
(Fig. 6A), which, as expected, exhibited very little tumor
growth. Interestingly, CD4+ T cell-depleted mice
remained protected after immunization with P40 and
TRP-2181–188 peptide (Fig. 6C). These
results suggest that the protective effect of immunization with P40 and
TRP-2181–188 peptide is completely mediated by
CD8+ (presumably cytotoxic) T cells and is
independent of the presence of CD4+ (helper) T
cells.
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To confirm the therapeutic potential of P40 and
TRP-2181–188 peptide immunization, we used a
transgenic mouse model of ocular tumor formation resulting from TRP-1
promoter-driven SV40 T Ag expression in the retinal pigmented
epithelium (RPE) (20). Transformation of RPE cells in
these mice starts as early as the fetal stage and continues after birth
until the ocular cavity is completely filled with tumor cells. At this
time point (6 wk of age), the tumor becomes clearly visible
macroscopically. Because TRP-2 is expressed by RPE cells and because
TRP/Tag mice were backcrossed several times to C57BL/6, we asked
whether immunization of these mice with a mixture of P40 and
TRP-2181–188 peptide starting at 3 wk of age
(with boosts at 4 and 5 wk) would influence the kinetics of eye tumor
development. PBS-injected mice or mice inoculated with either
TRP-2181–188 peptide alone or P40 alone rapidly
developed visible eye pathology (Fig. 7).
In contrast, immunization with a mixture of P40 and the peptide
resulted in a significant reduction in the number of eyes presenting
visible pathology. Moreover, most P40- plus
TRP-2181–188 peptide-treated mice that
eventually did develop visible eye symptoms showed relatively mild
pathology (not shown). These data show that mice treated with a mixture
of P40 and peptide several weeks after the onset of the transformation
process are still capable of modulating the course of tumor
progression, again illustrating the potential of P40 as an adjuvant for
immunotherapy of cancer.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Different compounds with potent adjuvant ability on immune responses have been described. However, only a few of them are able to initiate a CTL response when mixed with soluble Ags and are often toxic (26, 27). Compared with these adjuvants, P40 has the advantages of being a potent adjuvant of the CTL response to immunizing peptides and of being safe. No toxic or associated side effects have been observed in our experimental models or in a standard toxicological survey in animals (data not shown).
A simple mixture of P40 with the immunizing peptide allows the
generation of CTL in vivo. P40 is a highly hydrophobic protein in its
native form, reflecting its localization in the outer membrane. We have
demonstrated that hydrophobic peptides, such as
Melan-A26–35 A27L (Fig. 1
) and
TRP-2181–188 (not shown) peptides, are able to
form stable complexes with the protein P40. For several adjuvants, the
Ags are incorporated by means of hydrophobic interactions, and the
physical association has been demonstrated to be essential
for these adjuvants to exert their immunostimulating capacity. As
previously described for proteosomes, which are preparations of Omp of
meningococci (8), we hypothesize that P40/peptide
complexes are also formed by means of hydrophobic interactions of the
peptides with the membrane-spanning domain of the protein P40. Finally,
the fact that an exogenous helper epitope (such as TT) is not required
in the immunizing mixture might be explained by the presence of at
least one CD4 T cell epitope in the amino acid sequence of P40
(9), allowing this protein to act as both an adjuvant and
a carrier protein. In this context, the observation that
preimmunization with P40 does not compromise subsequent immune
responses to P40-associated peptides represents an improvement compared
with classic carrier proteins, such as TT, which have been shown to
elicit epitope suppression (23, 24).
Whereas the main actions of most adjuvants are to form a depot allowing a slow release of Ag in vivo and to induce a strong inflammatory process, their efficiency for CTL induction more likely depends on their capacity to direct soluble Ag to the cytoplasm and class I processing pathway in APCs (28, 29). Different hypotheses have been proposed. For instance, the adjuvant formulation, composed of squalane, Tween 80, and Pluronic L121 (30), when mixed to Ag may result in complexes that are taken up into endosomes of APCs and from there released into the cytoplasm (31). In that case the type of APCs remains unknown. A possible mechanism by which P40 is able to drive a peptide-specific CTL response is suggested by our observation in vitro that P40 selectively binds to professional APC, including dendritic cells, and is internalized by these cells, leading to their activation (32). Experiments are currently in progress in vivo to study the effect of P40 immunization on APC function, and preliminary data indicate the expansion of a subset of CD11c+ cells in the draining lymph node of mice immunized against CTL peptide in the presence of P40. This is of particular interest because dendritic cells provide an excellent costimulatory environment for CTL priming. In this context it is also interesting to note that the protective effect of P40 is independent of CD4+ T cells. Such an independence has been previously reported in immunization protocols, for instance those using Ag-pulsed dendritic cells (33), heat shock proteins (34), or viral pseudoparticles (35), where dendritic cells were efficiently targeted and activated.
The observation that immunization against peptides derived from melanoma Ags in the presence of P40 is associated with tumor protection in vivo reinforces the potential of P40 as an adjuvant in cancer vaccines. Furthermore, we demonstrated the efficiency of TRP-2181–188 immunization in the presence of P40 on tumor regression in a spontaneous tumor model. These data are particularly relevant in cancer treatment in humans, because they show the anti-tumor effect of CTL primed several weeks after the onset of tumor formation. Together, these data indicate that immunization against melanoma Ag-derived peptides in the presence of P40 is efficient enough to prime CTL responses involved in tumor rejection both after tumor challenge and in a spontaneous tumor situation. This is remarkable, especially because the latter is an intraocular tumor, a type particularly resistant to immune-mediated rejection (36).
The demonstration of the adjuvanticity of the protein P40 on the CTL response directed against melanoma Ag-derived peptides both in vitro in a functional assay and in vivo in two different tumor models has important implications for the development of cancer vaccines. Indeed, the facts that no conventional adjuvant is required and that P40/peptide mixtures are effective make the use of P40 a particularly practical immunotherapy approach.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: Omp, outer membrane protein(s); TRP-2, tyrosinase-related protein 2; RPE, retinal pigmented epithelium; Tag, T Ag of SV40.
Received for publication May 22, 2000. Accepted for publication January 22, 2001.
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References |
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3 domain of class I influence the magnitude of the xenogeneic response. J. Exp. Med. 170:1091.This article has been cited by other articles:
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) or the absence (
) of specific peptide in a
chromium release assay. The culture of lymph node cells derived from
mice immunized with 300 µg of P40 in the absence of CTL peptide did
not contain enough viable cells after two rounds of stimulation in
vitro to be tested.
