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Experimental Therapeutics, Preclinical Pharmacology |
Department of Internal Medicine, University of Navarra, 31008 Pamplona, Spain [E. H., P. S., N. C., J. J. L., J. D., M. R., J. P., F. B-C.], and Department of Immunology, Mayo Clinic, 55905 Rochester, Minnesota [J. L., E. C.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design: Substituted peptides from CEA691 were synthesized and tested in HLA-A2-binding assays, and in recognition by CEA691-specific CTL. Selected peptides were used to induce CTL responses in vivo in HLA-A2Kb transgenic mice and in vitro with human cells.
Results: Our experiments afforded several peptides with enhanced binding and/or recognition by CTL specific of CEA691. However, when HLA-A2Kb mice were immunized with these peptides only a few induced a CTL response that cross-reacted with CEA691. Additional replacement of their NH2-terminal amino acid by Y (tyrosine) afforded peptides YMIGMLVGV and YMIGVLLGV with enhanced in vivo and in vitro immunogenicity than CEA691. Indeed, they activated a greater number of precursor cells that recognized CEA691-pulsed cells and tumor cells expressing CEA.
Conclusions: Our results widen the possibility of treating CEA-expressing tumors with enhanced efficacy.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present work we have carried out amino acid changes in the sequence of CTL epitope IMIGVLVGV (from now on CEA691), which encompasses residues 691–699 from CEA. This peptide was selected because it was reported to be presented by HLA-A2+-expressing tumor cells (11) . We decided to replace all of the positions from CEA691 by amino acids representative of different families, with the aim of finding peptides with enhanced binding to HLA-A2 and/or recognition by the TCR. This might afford peptides that would induce CTL recognizing CEA691 with stronger affinity than CTL induced with unmodified CEA691. The results of our findings are discussed below.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines.
The T2 cell line used to measure peptide binding to HLA-A2 was a kind gift of Dr. J. A. Berzofsky (NIH, Bethesda, MD). Jurkat-A2Kb cells, used as target cells in HLA-A2Kb mice CTL assays, were kindly provided by Dr. A. Sette (Epimmune, San Diego, CA). Both cell lines were maintained in CM (RPMI 1640 containing 10% FBS, 4 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-mercaptoethanol). The tumor cell lines SW480 (American Type Culture Collection, Manassas, VA) and SW403 were grown in Leibovitz medium containing 10% FBS, 4 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Tumor line Lyse-4.1 (a kindly gift of Dr. P. Coulie, Ludwig Institute for Cancer Research, Brussels, Belgium) was grown in Iscove’s modified Dulbecco’s medium supplemented with 10% FBS, penicillin/streptomycin, 10 nM hydrocortisone, 5 µg/ml insulin, 100 µg/ml transferrin, 10 nM ß-estradiol, 30 nM sodium selenite, 0.55 mM L-arginine, 0.24 mM L-asparagine, and 1.5 mM L-glutamine.
CEA691-specific CTL clones 1 and 2 were isolated from normal blood donors as described (24) . Briefly, purified CD8+ T cells from HLA-A2-positive normal individuals were stimulated in culture with autologous DCs that were pulsed previously with 40 µM purified (>98%) CEA691 peptide. These DCs were prepared from adherent monocytes that were maintained for one week in culture with granulocyte macrophage colony-stimulating factor (50 ng/ml) and IL-4 (1000 IU/ml). After 7 days, the peptide-activated T cells were restimulated using peptide-pulsed (10 µM) autologous-irradiated monocytes, and IL-2 was added 2 days later at 50 IU/ml. After 2 additional rounds of peptide restimulation, the CTLs were cloned by limiting dilution as described (24) . Both CTL clones were capable of specifically recognizing peptide CEA691 and not other HLA-A2-binding irrelevant (control) peptides (11) . Furthermore, both CTL clones were effective at recognizing CEA-positive tumors expressing the HLA-A2 molecule (11) . CTL clones were maintained in tissue culture by periodic stimulation with anti-CD3 antibodies in the presence of a mixture of feeder cells and IL-2 as described previously (24) .
Transgenic Mice.
HLA-A2Kb transgenic mice were obtained from Dr. L. A. Sherman (The Scripps Research Institute, La Jolla, CA). These mice express 
1 and 2 domains from human HLA-A2.1 molecule, and 3 from mouse H-2Kb molecules (25)
. They were bred and maintained in pathogen-free conditions according to the guidelines for animal care of our institution.
HLA-A2 Binding Assays.
Peptide binding to HLA-A2 molecules was measured using the T2 mutant cell line according to a protocol described previously (26
, 27)
. T2 cells (3 x 105/well) were incubated overnight with different concentrations of peptide in 96-well plates with culture medium (RPMI 1640 containing 2.5% FBS, 4 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin). The next day, cells were washed twice with cold PBS containing 2% FBS and incubated for 30 min at 4°C with anti-HLA-A2 BB7.2 monoclonal AB (1/1000 dilution from ascitis) and 1/100 dilution of FITC-labeled goat antimouse IgG (Sigma Chemical Co, St. Louis, MO). Cells were washed twice after each incubation, and HLA-A2 expression was measured by flow cytometry on a FACScan (Becton Dickinson). HLA-A2 expression (which is a measure of peptide binding to HLA-A2) was quantified as FI according to the formula: FI = (mean fluorescence with peptide - mean fluorescence without peptide)/mean fluorescence without peptide. Background fluorescence without BB7.2 AB was subtracted for each individual value.
In Vivo Induction of CTL Responses by Peptide Immunization of HLA-A2Kb Transgenic Mice.
Peptide mixtures containing 50 nmol of helper peptide PADRE plus 50 nmol of the CTL epitope to be tested (28)
emulsified in incomplete Freund’s adjuvant were injected s.c. to 8–12-week old mice. Two weeks later, a boost was given under the same conditions. Ten to 14 days after the boost, mice were sacrificed and their spleens removed. Spleen cells (4 x 107) from immunized mice were stimulated in 10 ml of CM in T25 flasks with 107 mitomycin C-treated B-cell blasts pulsed with peptides. Blasts were prepared by incubating 4.5 x 107 spleen cells from HLA-A2Kb mice in 30 ml of CM with 1 µg/ml lipopolysaccharide and 7 µg/ml dextrane sulfate for 3 days. Cells were then treated with mitomycin C, washed, incubated with peptide (10 µM) for 2 h at 37°C, and washed. Two days after stimulation of T cells, 2.5 units/ml of IL-2 were added, and cells were grown for an additional 5 days. On day seven, T cells were harvested and stimulated in 24-well plates (106/well) with peptide-pulsed blast cells (3 x 105/well) and C57Bl6 spleen cells (5 x 106/well), and IL-2 was added 1 day after. Five to 6 days after the second stimulation, CTL activity was measured using Jurkat-A2Kb cells as target.
In Vitro Induction of Human CTL Using DCs.
DCs were generated from CD14+ precursor cells as described (24)
and were used as antigen-presenting cells to immunize CTL precursors with the candidate synthetic peptides. Briefly, purified monocytes were cultured for 7 days in the presence of 50 ng/ml granulocyte macrophage colony-stimulating factor and 1000 units/ml of rIL-4 in complete RPMI 1640. The tissue culture-generated DCs were pulsed with 40 µg/ml of synthetic peptides together with 3 µg/ml ß2-microglobulin in PBS containing 1% BSA for 4 h at room temperature. The peptide-pulsed DCs were washed twice and irradiated (4200 rads). The peptide-pulsed DCs were then mixed with autologous purified CD8+ T cells (purified with Miltenyi immunomagnetic beads by positive selection) at 1:20 (DC:T cell) ratio. The CTL immunization cultures were done in 48-well plates, where each well contained 0.25 x 105 DCs and 5 x 105 CD8+ T cells in 0.5 ml complete RPMI 1640 containing 5% human AB serum instead of FBS. This medium was supplemented with 10 ng/ml of recombinant IL-7. One day later, 10 ng/ml of rIL-10 were added to the cultures to increase the efficiency of CTL induction. On days 7 and 14, the T-cell cultures were individually restimulated with peptide-pulsed irradiated autologous antigen-presenting cells (adherent monocytes) as described, adding IL-10 on the following day. Starting on day 9, the T-cell cultures were fed with fresh medium containing 10 units/ml of IL-2 every 2–3 days. The first screening cytotoxicity assay was performed after three rounds of peptide stimulation. And those cultures that exhibited cytotoxic activity toward peptide-pulsed target cells (>20% lysis as compared with the unpulsed target cells) were selected and expanded in tissue culture for additional analysis.
In Vitro Induction of Human CTL Using PBMCs.
This was done according to the protocol described by Plebanski et al. (29)
. Briefly, PBMCs from healthy donors were pulsed with 50 µg/ml of peptide, washed, and cultured in MEM medium supplemented with 5% human AB serum, 4 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. They were plated at 2 x 106 cells/ml in 2-ml wells in the presence of keyhole limpet hemocyanin (5 µg/ml; Sigma) and IL-7 (25 ng/ml; Peprotech). IL-2 (10 units/ml) was added on day 3. On day 7, cells were harvested from bulk cultures, purified by Ficoll gradient, and semicloned at 5000 cells/well in 96-well U-bottomed plates, together with 105 mitomycin C-treated autologous PBMCs pulsed with peptide. IL-2 was added 1 and 4 days later. Stimulation was repeated in the same conditions every 7 days, and 3 weeks after semicloning, cells were tested in CTL assays.
CTL Assays.
One million target cells were incubated with 50 µCi of 51Cr with or without peptide (10 µM) for 2 h at 37°C. Cells were then washed three times and incubated for 4 h with different number of effector cells. After this period, 50 µl of supernatants were harvested and radioactivity counted. The percentage of specific lysis was calculated according to the formula: (cpm experimental-cpm spontaneous)/(cpm maximum-cpm spontaneous) x 100. Spontaneous lysis was measured from wells containing only target cells, whereas maximum lysis was measured from wells containing target cells incubated with 5% Triton X-100.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, peptide CEA691 is a poor binder peptide since, at the highest concentration tested (100 µM) it was unable to enhance HLA-A2 expression by 50%. Most substitutions at positions 4, 8, and 9 were unable to improve binding. By contrast, several substitutions at positions 2 and 6, and most substitutions at 1, 3, 5, and 7 showed enhanced binding to HLA-A2, with a FI0.5 < 20 µM (Fig. 1)
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shows that substitutions 2A and 2T were better recognized by both clones. Also, all of the substitutions at position 1 abolished recognition by clone 1 whereas substitutions 1T, 1L, 1K, and 1A enhanced recognition by clone 2. Moreover, clone 1 had a higher avidity for peptides 7L and 7F, which were not recognized by clone 2. By contrast, clone 2 showed highest avidity for 2L, 2F, 3L, 5T, and 5L that were poorly or not recognized by clone 1. Replacements at positions 6 or 8 afforded peptides, which were recognized by either of the two CTL clones with lower avidity than wild-type epitope CEA691. Substitutions at position 4 abolished recognition by either clone. Changes at position 9 were tested against clone 2 only, and none of these changes improved recognition by this clone.
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shows that some of the new peptide had a high binding affinity. Fig. 4
shows that substitutions 1Q, 2S, and 2V enhanced the reactivity of both clones. Other substitutions enhanced recognition by one of the clones only or abolished recognition by one or both clones.
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Double Substitutions in the Sequence of CEA691.
With the aim of enhancing recognition of peptides by one or preferably both CTL clones, we synthesized a panel of double substituted peptides. The criteria used to select a given combination of changes were enhanced binding to HLA-A2 together with enhanced recognition by one or both CTL clones. In Table 1
we show that most of these peptides had a higher affinity for HLA-A2 than wild-type CEA691, with FI0.5 values usually <30 µM. Six of these peptides, derived from combinations of 2V, 2T, and 2A with 7L and 7F were tested against both CTL clones. As found for single substituted peptides at position 7 (Fig. 2)
, double substituted peptides were recognized by clone 1 but not by clone 2. However, the recognition of these six double substituted peptides from Table 1
by clone 1 was much better than their parent mono substituted peptides at positions 2 or 7. Unfortunately, because clone 1 and clone 2 were lost, the remaining substituted peptides from Table 1
could not be tested against these clones.
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shows that none of the single substituted peptides was more immunogenic than CEA691, but some of them weakly cross-reacted with CEA691. In contrast, double substituted peptides were in general more immunogenic than CEA691, but with the exception of 2V7L, which showed a weak cross-recognition with CEA691, the remaining double substituted peptides were unable to induce CTL responses that recognized CEA691.
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. To determine the effects of these changes in immunogenicity, these peptides were used to immunize HLA-A2Kb transgenic mice. Because we knew that CTLs induced with 2V, 3V, and 7L cross-reacted with CEA691 we decided to stimulate immune spleen cells induced with 1Y, 1Y2V, 1Y3V, 1Y5M, or 1Y7L with CEA691. As shown in Table 3
, with the exception of 1Y3V, all of the peptides tested induced a stronger CTL response against CEA691 than the one induced by CEA691.
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, stimulation with CEA691, 1Y5M, and 1Y7L afforded 3, 3, and 5 wells, respectively, showing specific activity against the peptide used for stimulation. However, in the case of 1Y7L stimulated cells, the levels of specific lysis were higher than in cells stimulated with CEA691. In a second group of experiments, we studied the recognition of CEA691 by the CTL induced after pulsing the PBMCs from a donor with the different peptides. Thus, we stimulated PBMCs with CEA691, 1Y, 1Y5M, or 1Y7L, and after bulk cultures, cells were semicloned seeding 96 wells for each peptide. After several cycles of peptide stimulation, all of the wells were tested in their ability to recognize target cells pulsed with CEA691. As shown in Fig. 5B
, from plates stimulated with CEA691, 1Y or 1Y5M, only one well from CEA691, one from 1Y, and two from 1Y5M of 96 were able to specifically recognize CEA691. By contrast, 7 wells of 96 from the plate stimulated with 1Y7L showed specific activity against CEA691. As for results obtained in Fig. 5A
, peptide 1Y7L not only induced a higher number of positive cultures but also induced higher levels of lysis compared with cultures stimulated with CEA691. We then tested the ability of these CTLs to recognize tumor cells expressing CEA (Fig. 5C)
. These CTL recognized cell line SW480 (CEA+HLA-A2+), but surprisingly they did not recognize cell line SW403 (also described as CEA+HLA-A2+). However, PCR analysis of these cells revealed that they had lost CEA expression, whereas SW480 were clearly CEA+ (data not shown). In another experiment, we induced CTL by pulsing the PBMCs from another donor with CEA691, 1Y, 1Y2V, 1Y5M, and 1Y7L. Cells from those wells showing activity against CEA691 were tested in the recognition of tumor cell lines SW480 (CEA+HLA-A2+) and Lyse-4.1 (CEA-HLA-A2+). As shown in Fig. 5D
, CTLs induced with CEA691 as well as with the substituted peptides were able to lyse SW480 cells but not Lyse-4.1 cells. A greatly enhanced immunogenicity (greater number of active wells) was obtained when using 1Y, 1Y5M, and 1Y7L in regard to using CEA691 or 1Y2V.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and 4
it can be seen that changes at positions 1 and 2, putatively described as pointing to the MHC, not only dramatically affected recognition by the TCR, but also this recognition was different for both CTL clones. Thus, amino acid replacements in a given position may affect the structure of the peptide in other positions and concomitantly the recognition by the MHC or the TCR molecules. For this reason, the assignment of each amino acid position to a particular interaction may in some cases be difficult to conclude.
Double substitutions combining enhanced binding to HLA-A2 together with enhanced recognition by one or both CTL clones did not have an additive effect on binding. In most cases the FI0.5 of the double substituted peptide was similar to the most enhancing substitution of the single modified peptide. As shown in Table 1
, a group of double substituted peptides (the only that could be tested against clone 1 and clone 2 before the loss of these clones) was recognized by clone 1 much better than the parent mono-substituted peptide (compare Table 1
with Figs. 2
and 4
). However, although substitutions at position 2 enhanced recognition by clone 2 in mono-substituted peptides, double substituted peptides were not recognized by clone 2, probably because substitutions 7F and 7L in these peptides disrupted recognition by this clone as found for mono-substituted 7F and 7L (Fig. 2)
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Several single or double substituted peptides that had shown enhanced binding and recognition by one or by both CTL clones were chosen to immunize HLA-A2Kb transgenic mice. It was found that most of the double substituted peptides tested (Table 2)
were more immunogenic than CEA691, but they did not cross-react with CEA691. By contrast, although mono-substituted peptides did not have a higher immunogenicity than CEA691, they retained some cross-reactivity. Because as reported by Tourdot et al. (30)
substitution by Y at position 1 is able to enhance binding to HLA-A2 and concomitantly enhance the immunogenicity of the peptide, we combined this substitution with some of the changes that after immunization of HLA-A2Kb transgenic mice, induced CTL that cross-reacted with wild-type peptide CEA691. Binding experiments revealed that these peptides had a high affinity for HLA-A2. Also, they induced stronger CTL responses against CEA691 than that induced by CEA691. This enhanced CTL activity induced in vivo using transgenic mice was also corroborated using the same peptides for in vitro CTL induction using blood from human donors. Indeed, in this last case, peptide 1Y7L and to a lower extent 1Y5M were much more immunogenic than CEA691, and induced CTL able to recognize CEA691 as well as HLA-A2+ CEA-expressing tumor cells. The low immunogenicity of CEA691 may be related to the poor binding of this epitope to HLA-A2 (see Fig. 1
). It has also been described that CEA691 could not be detected in nine HLA-A2 gastrointestinal tumors (34)
probably because the number of peptides presented per cell is low, and as stated by these authors, the sensitivity of their method after elution of HLA-A2-bound peptides might not be high enough. However, the number of peptides presented by the tumor cells is sufficient to be recognized by CTL.
It is interesting to note that single amino acid replacements 2V, 2S, and 3V that were recognized by both CTL clones, although much more by clone 2, when combined with 7L (which is only recognized by clone 1) induced CTL that recognized CEA691 only marginally (mutation 2V7L) or not at all (mutations 2S7L and 3V7L). This result suggests that changes affecting two regions of the wild-type peptide may alter its structure in such a way that the CTLs induced are no longer able to cross-react with the wild-type peptide. By contrast, double substituted peptide 1Y7L was highly immunogenic and cross-reacted with CEA691. The greater efficacy of 1Y7L may be related to enhancement of binding attributable to 1Y without altering the peptide structure in opposite conflicting directions. From Fig. 5
it is clear that Y at position 1 has a beneficial effect to induce CTLs that cross-react with CEA691 specially when combined with 7L in peptide 1Y7L. However, this beneficial effect is not found when combined with 2V in peptide 1Y2V. We believe that immunization with 1Y7L (and to a lower extent 1Y5M) may have recruited larger fractions of CTL precursors from the TCR repertoire recognizing CEA691 than that induced by CEA691, as reported for other peptide analogues (35)
. Alternatively, immunization with these peptides may have induced CTL with higher affinity.
During the course of our experiments, we became aware that Tangri et al. (20) had reported recently the characterization of other analogues of peptide CEA691. In their case, peptides with substitutions 3M and 5H were much more immunogenic than CEA691, and showed good cross-reactivity against CEA691. At this time, we had found that peptide 3M bound to HLA-A2 with similar affinity than CEA691. Because this peptide was not recognized by clone 2, and we were unable to test it against clone 1, we did not carry out additional experiments with peptide 3M. Regarding peptides with other substitutions, Tangri et al. (20) did not find other analogues clearly improving recognition by their CTL cultures.
We agree with Tangri et al. (20) that replacements giving more immunogenic epitopes are likely conservative changes. Indeed, amino acid replacements in the sequence of CEA691 that enhanced immunogenicity were 3I replaced by M, 5V replaced by H, and in our case 1I replaced by Y, 5V replaced by M, and 7V replaced by L. It is interesting to note that in all of these changes, the volume of the replacing amino acid that confers higher immunogenicity is equal or slightly larger than the original amino acid in the sequence of CEA691. Moreover, when the propensities of interaction of amino acid side chains in proteins reported by Singh and Thornton (36) are compared, it is found that the propensity of the replacing amino acid is higher than that from the replaced amino acid. This volume similarity and enhanced propensity is also observed in the work of Zaremba et al. (19) and that of Hoffmann et al. (21) for peptide analogues with substitutions at TCR positions. Thus, to enhance the immunogenicity of CTL epitopes it would seem advisable, as suggested by Tangri et al. (20) , to make conservative replacements in terms of hydrophilicity/hydrophobicity as well as similarity of volume, but also, and perhaps as important, to carry out replacements that, respecting these rules, have a higher propensity of interaction in proteins. Obviously, the approach suggested above should be taken only as a guide to reduce the number of alternative replacing amino acids to be tested experimentally.
Characterization of peptide analogues derived from the sequence of CEA691 opens the possibility of using these peptides for the treatment of patients with CEA-expressing tumors. These peptides could be used to immunize patients to induce strong CTL responses as reported for analogues of CTL epitopes derived from melanoma antigen gp100 (22) . Moreover, the ability of these peptides to stimulate in vitro antitumor CTL responses might help in the expansion of CTL populations suitable for adoptive immunotherapy. Also, monitoring of CTL responses has been demonstrated in several cases to be more efficient with this kind of peptides (18) . Thus, we believe that our work, in conjunction with that reported by others (19 , 20) , may allow better immunotherapeutic approaches in the treatment of cancer patients with tumors expressing CEA.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant from Comisión Interministerial Ciencia Y Tecnología (SAF2000-0059; to F. B-C.) and NIH Grants R01CA80782, R01CA82677, M01-RR00585, and RR-00585 (to E. C.). 
2 To whom requests for reprints should be addressed, at Department of Internal Medicine, University of Navarra, Irunlarrea 1, 31008 Pamplona, Spain. Phone: 34-948-425600, extension 6366; Fax: 34-948-425700; E-mail: fborras{at}unav.es
3 The abbreviations used are: CEA, carcinoembryonic antigen; TCR, T-cell receptor; AB, antibody; CM, complete medium; FBS, fetal bovine serum; DC, dendritic cell; IL, interleukin; FI, fluorescence index; PBMC, peripheral blood mononuclear cell.
Received 2/14/02; revised 4/10/02; accepted 4/15/02.
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, lysis of peptide-pulsed cells; 
, unpulsed cells. B, peptide-pulsed PBMCs were grown in 24-well plates and semicloned. For each peptide, 96 wells were plated, and after three cycles of stimulation, CTL activity measured in each well. Each bar represents a well with specific activity against peptide CEA691. 
), and SW403 (CEA- HLA-A2+; 
) by CTL induced in vitro by the peptides shown. D, recognition of tumor cell lines SW480 (CEA+ HLA-A2+;