The Angiogenic and Biomarker Midkine Is a Tumor-Shared Antigen

This information is current as Jerome Kerzerho, Olivier Adotevi, Florence A. Castelli, of September 29, 2021. Magalie Dosset, Karine Bernardeau, Natacha Szely, Francois Lang, Eric Tartour and Bernard Maillere J Immunol 2010; 185:418-423; Prepublished online 28 May 2010;

doi: 10.4049/jimmunol.0901014 Downloaded from http://www.jimmunol.org/content/185/1/418

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The Angiogenic Growth Factor and Biomarker Midkine Is a Tumor-Shared Antigen

Jerome Kerzerho,* Olivier Adotevi,†,‡ Florence A. Castelli,* Magalie Dosset,†,‡ Karine Bernardeau,x Natacha Szely,* Francois Lang,x Eric Tartour,†,‡ and Bernard Maillere*

The angiogenic factor Midkine (MDK) is overexpressed in various human malignant tumors, although its expression is low or undetectable in normal adult tissues. Its expression in tumors and its detection in plasma have been associated with poor disease outcome, whereas its blockade was found to contribute to tumor regression. By weekly stimulation of T lymphocytes harvested in HLA-A2 healthy donors, we derived CD8 T cell lines specific for several MDK peptides. The T cell response was mostly dominated by two nonamer peptides localized in the signal peptide and in the C-terminal part of the , as assessed by IFN-g ELISPOT and HLA-A2 tetramer labeling. Peptide-specific T cell lines recognized cells transfected with an MDK-encoded plasmid and tumor Downloaded from cell lines naturally expressing the MDK protein, but not untransfected cells. T cell presentation of the two MDK epitopes was found to be TAP dependent. Experiments performed in HLA-A2 transgenic mice demonstrated the capacity of the two identified CD8 T cell epitopes to elicit a cytotoxic response. Altogether, our data show that the secreted MDK protein is a candidate vaccine for multiple cancers. The Journal of Immunology, 2010, 185: 418–423. http://www.jimmunol.org/ he number of well-characterized tumor Ags has steadily of tumor. As a result, a few identified tumor Ags exhibit a wide ex- increased in recent years. More than 200 class I-restricted cy- pression in multiple cancer patients and contribute to survival and T totoxic T lymphocyte peptide epitopes have been character- growth of cancer cells. These cancer targets mainly comprise telomer- ized from multiple (1). Tumor Ags differ widely in their ase reverse transcriptase (7), which is required for the control of telo- expression pattern in tumor and healthy cells, in their contribution to mere length in the tumor, and survivin (8), and Bcl-2 (9), which tumor survival, and their immunogenic properties. One major group of provide resistance to apoptosis. T cell epitopes of these proteins (8– tumor Ags is represented by cancer-germline Ags encoded by 10) have been already delineated, but their efficiency in eliciting that are totally silent in most normal tissues and are overexpressed in a tumor-eradicating immune response is not formerly demonstrated. a variety of tumor types (2). Members of this group include genes Midkine (MDK) is a -binding growth factor found as the by guest on September 29, 2021 encoding MAGE, BAGE, or LAGE. Another important type of Ag product of a -responsive (11). This secreted protein is composed of differentiation Ags that are found in both the tumors has been found to be overexpressed in various human malignancies, and the normal cells from which they derived. The most studied dif- such as bladder, prostate, breast, lung, liver, and colon tumors, ferentiation Ags are probably the Melan-A/MART-1 and gp100 pro- whereas its expression is typically low or undetectable in normal adult teins, which are present in both melanocytes and melanoma. Although tissues (12–14). Its natural role is mainly to participate in neuronal both types of Ags appeared to be able to elicit a tumor-specific immune development during embryogenesis (15). It has also been implicated response, they are dispensable for the tumor and hence facilitate tumor in ischemic renal injury (16) and autoimmune responses (17). In escape from immune attack through loss of Ags (3, 4). Because of their tumor cells, MDK has not only an angiogenic role (18), but also almost strict tumor-restricted expression, cancer-germline Ags also a mitogenic (19) and antiapoptotic function (20). Its blockade con- suffer from interindividual expression discrepancies in cancer patients tributes to tumor regression in a mouse model (21), demonstrating its (5, 6), whereas differentiation Ags are only found in a defined type importance in tumor expansion. Interestingly, its expression in the tumor (12, 22) and its concentration in plasma (23) were found to be related to poor disease outcome. Its expression in tumors can be *Commissariat a` l’E´nergie Atomique-Saclay, Institut de Biologie et Technologies, Ser- detected early (13), and it was therefore considered as a relevant bio- † vice d’Inge´nierie Mole´culaire des Prote´ines, Gif-sur-Yvette; Centre de Recherche marker of disease progression. Its expression in tumors can be de- Cardiovasculaire, Institut National de la Sante´ et de la Recherche Me´dicale U970, Universite´ Paris Descartes; ‡Hoˆpital Europe´en Georges Pompidou, Unite´ d’Immunologie termined by blood (23, 24) and urinary analysis (25). Because of its Biologique, Assistance Publique-Hopitaux de Paris, Paris; and xInstitut National de la Sante´ functions, its wide expression in tumors, and its biomarker status, we et de la Recherche Me´dicale U601, Institut de Biologie, Nantes, France have investigated whether MDK can induce a cellular cytotoxic re- Received for publication April 1, 2009. Accepted for publication April 27, 2010. sponse in humans. Both in vitro primary stimulation experiments This work was supported by the Commissariat a` l’E´ nergie Atomique and grants from performed with HLA-A2 healthy donors and immunization trials l’Institut National du Cancer, Immucan, and Canceropole d’Ile de France. performed in HLA-A2 transgenic mice revealed the capacity of Address correspondence and reprint requests to Dr. Bernard Maillere, Commissariat a` MDK to elicit tumor-specific CTL response. l’E´ nergie Atomique, Institute of Biology and Technologies, Service d’Inge´nierie Mole´culaire des Prote´ines, Gif-sur-Yvette, F-91191, France. E-mail address: bernard. [email protected] Materials and Methods The online version of this article contains supplemental material. Peptides and proteins Abbreviations used in this paper: DC, dendritic cell; ER, endoplasmic reticulum; HHD, HLA-A2 transgenic mice; MDK, Midkine; pMDK, Midkine plasmid. MDK nonamer peptides were synthesized using standard 9-fluorenyl- methoxycarbonyl chemistry on an Advanced ChemTech Apex synthesizer Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 (Advanced ChemTech, Louisville, KY) and cleaved from the resin by 95% www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901014 The Journal of Immunology 419 trifluoroacetic acid. If necessary, peptides were purified by reversed-phase mAb coupled to magnetic microbeads (Miltenyi Biotec, Paris, France) and HPLC on a C18 Vydac column (Interchim, Montluc¸on Cedex, France). by magnetic cell sorting, as recommended by the manufacturer. Mature DCs 6 Their purity was .90%, as indicated by analytical HPLC. Sequences of (10 cells/ml) were incubated at 37˚C, 5% CO2, for 4 h in complete IMDM the 13–21 and 114–122 peptides were ALLALTSAV and AQCQETIRV, medium containing 5 mg/ml b2-microglobulin and a mixture of MDK pep- respectively. tides, each peptide being at a concentration of 10 mg/ml. Pulsed cells were washed and added at 104 per round-bottom microwell to 2 3 105 autologous Construction of MDK expression plasmid CD8 lymphocytes in 200 ml IMDM supplemented by 1000 U/ml IL-6 (R&D Systems, Lille, France) and 10 ng/ml IL-12 (R&D Systems). The CD8 T Total RNA was extracted from A549 cells and reverse transcribed into lymphocytes were restimulated on days 7, 14, and 21 with autologous DCs cDNA using reverse transcriptase and oligo(dT) (ImProm-II Reverse Tran- freshly loaded with the MDK peptides and were grown in complete IMDM scription System, Promega, Charbonnie`res-les-bains, France). MDK cDNA medium supplemented with 10 U/ml IL-2 (R&D Systems) and 5 ng/ was amplified by PCR using the upstream primer 59-GTGGTGGAATT- ml IL-7 (R&D Systems). Flow cytometry analysis confirmed that growing CACCACCATGCAGCACCGAGGCTTCCTC-39 and the downstream cells were CD8 T lymphocytes. Specificity of the T cell lines was primer 59-AAACTCGAGCCAGGCTTGGCGTCTAGTC-39. MDK cDNAs investigated by IFN-g ELISPOT assays at day 28 as described previously were digested with EcoRI and XhoI, then ligated into pcDNA3.1 expression (10) using HLA-A2–transfected CIR (CIR-A2) as APC. Cytotoxicity of the vector predigestion. The corresponding plasmid (pMDK) was purified on T cell lines was evaluated using perforin ELISPOT assays or calcein- plasmid columns under endotoxin-free conditions (Qiagen EndoFree acetoxymethyl assays (calcein) (26). Briefly, target cells were incubated with Plasmid Maxi Kit, Qiagen, Courtaboeuf, France). calcein for 30 min at 37˚C and, after extensive washings, were added to Cell culture and transfection culture wells (5000 cells per well) with MDK-specific T cell lines (20,000 A549 human lung carcinoma, CIR human B lymphoblast, HepG2 human hepatocellular carcinoma, and DLD-1 human colorectal adenocarcinoma were from the American Type Culture Collection (Manassas, VA). The Downloaded from pcDNA3.1-MDK (pMDK) (20 mg) was introduced into C1R-A2 cells (3 3 106 cells) by electroporation (0.9 kV,250 mF, 0.4-cm gap) with a Gene Pulser II (Bio-Rad, Marnes-la-coquette, France). The treated cells were suspended in 40 ml prewarmed RPMI 1640 medium with 10% fetal serum and incubated under 5% CO2/air at 37˚C. Forty-eight hours later, transfection efficacy was assessed by flow cytometry as endogenous MDK expression in matured den- dritic cells (DCs) and HepG2 and DLD1 cells. Briefly, cells were permeabi- lized with 75 ml fixation/permeabilization solution (BD Biosciences, Le pont http://www.jimmunol.org/ de Claix, France) for 20 min at 4˚C and washed twice with BD Perm/Wash buffer plus 2% FCS (BD Biosciences). They were stained for 30 min on ice with anti–MDK-PE Ab (BD Biosciences) or anti-isotype control. Cell staining was assessed in an FACSCalibur flow cytometer (BD Biosciences). Generation of Ag-specific CD8+ T cell lines from healthy donors Blood cells were collected from anonymous healthy donors at the Eta- blissement Franc¸ais du Sang (Rungis, France) as buffy-coat preparations after informed consent and following Etablissement Franc¸ais du Sang guide- by guest on September 29, 2021 lines. PBMCs were isolated by density centrifugation on Ficoll-Hyperpaque gradients (Sigma-Aldrich, St. Louis, MO). HLA-A2 donors were identified by using extracellular staining with anti–HLA-A2–PE Ab (BD Biosciences). Monocyte-derived DCs were generated from plastic-adherent cells of PBMCs after 5–7 d of culture in AIM V medium (Invitrogen, Cergy Pontoise, France) supplemented with 1000 U/ml recombinant human IL-4 and recombinant human GM-CSF (tebu-bio, Le-Perray-en-Yvelines, France). Immature DCs were collected at day 5 or 6. Mature DCs were generated after addition of 1 mg/ml LPS (Sigma-Aldrich). CD8 T lymphocytes were isolated from the nonadherent phase by positive selection using an anti-CD8

FIGURE 2. Peptide specificity of T cell lines derived from HLA-A2 FIGURE 1. Flow cytometry analysis of MDK expression in normal and healthy donors. CD8 T cell lines were obtained after three weekly stim- tumor cells. Cells were permeabilized and stained intracellularly with an ulations by autologous mature DCs loaded the selected peptides. Lympho- MDK-specific Ab and analyzed by flow cytometry. HepG2 DC (A), DLD1 cytes (104 per well) were incubated in duplicate with 3 3 103 C1R-A2 (B), and C1R-A2 cells (C) and DCs (D). Black, endogenous expression of cells in the presence or absence of peptides (A, C) or a dose range of MDK; gray, expression of MDK posttransfection with the MDK plasmid; appropriate peptides (B). The specificity of the T cell lines was assessed open, isotype control. by IFN-g ELISPOT after 24-h incubation. 420 MIDKINE IS A TUMOUR-SHARED Ag cells per well). After a 4-h incubation at 37˚C, emitted fluorescence was and characterized by IFN-g ELISPOT. Seven peptides, namely 13– measured on a Gemini Spectramax Fluorimeter (Molecular Devices, St. 21, 12–21, 13–22, 14–22, 63–72, 113–122, and 114–122, elicited Gregoire, France) (excitation at 485 nm and emission at 530 nm). peptide-specific T cell lines and were retained for further experi- In vivo immunogenicity studies ments. As shown in Fig. 2A, T cell lines 314.48 and 267.29 were activated by MDK2HLA-A2+ CIR-A2 cells presenting the peptides H-2 class I knockout, HLA-Ap0201-transgenic mice (HHD II) have been previously described (27). Eight- to 10-wk-old female mice were bred and 113–122 and 114–122 or 12–21 and 13–21, respectively. Omission maintained under specific pathogen-free conditions in our animal facility. of the peptides or addition of inappropriate peptides did not activate HHD mice were injected s.c. at day 0 and day 14 with 50 mg MDK them. Dose-range experiments (Fig. 2B) showed that the optimal b peptides 13–21 or 114–122 along with 140 mg I-A MHC class II helper peptides were the 9-mers 114–122 and 13–21. They were recog- peptide TPPAYRPPNAIL (T13L), which corresponds to amino acids 128– 140 of the hepatitis B virus core protein in IFA. For DNA immunizations, nized at low concentration as compared with the 10-mers 113–122 HHD mice were injected with 50 ml cardiotoxin into each regenerating and 12–21, the midactivating dose being ∼0.01 mg/ml (equivalent 2 tibialis anterior muscle 5 to 6 d prior to DNA injection. Fifty microliters of to 10 8 M). The peptide-specific T cells appeared therefore to pMDK (1 mg/ml in PBS) was injected directly into each pretreated muscle possess a good affinity for the corresponding peptide–HLA-A2 at days 0 and 14. Seven to 10 d after the last immunization, spleen cells of complexes. As shown in Fig. 3, the T cell line 314.7, which was immunized mice were harvested, and Ficoll-purified lymphocytes were isolated. Specific T cells were detected ex vivo using ELISPOT assay. specific for the 114–122 peptide in ELISPOT (not shown), was For CTL function analysis, splenocytes were stimulated for 5 to 6 d with nicely labeled by the corresponding HLA-A2 tetramers (Fig. 3A), peptide-pulsed (10 mg/ml) gamma-irradiated (5000 rad) syngenic spleno- although very few cells were labeled by the 13–21 specific tetramer cytes. Specific cytotoxic activity of effector CTL was measured by stan- (Fig. 3B). In contrast, the T cell line 297.58, which was specific for dard [51Cr] release assay. Downloaded from

Results MDK expression in tumor cell lines and healthy cells As MDK expression has been mostly evaluated at the transcriptional level, we evaluated its protein expression by flow cytometry. As

shown in Fig. 1, MDK was easily detected in the hepatocarcinoma http://www.jimmunol.org/ HepG2 cell line (Fig. 1A), whereas its expression was extremely low in DLD1 cells (Fig. 1B). MDK was also weakly expressed in the CIR-A2 cell line, whereas CIR-A2 cells transfected with pMDK exhibited a high expression of MDK (Fig. 1C). In contrast, human DCs did not express MDK (Fig. 1D). We also observed good ex- pression of MDK in A549 and Caski cell lines, whereas it was not expressed in monocytes, lymphocytes, or activated T lymphocytes (data not shown). We thus confirmed that MDK was overexpressed in several tumor cell lines in contrast to normal cells derived from by guest on September 29, 2021 the blood. Induction of MDK peptide-specific T cell lines Based on HLA-A2 scores provided by the prediction programs BIMAS, SYFPEITHI, and PROPRED, 14 peptides of 9 to 10 aas were initially selected for preliminary T cell priming experiments. These experiments included three HLA-A2+ healthy individuals. CD8 T cell lines were obtained after three weekly stimulations by autologous mature DCs loaded with a mixture of selected peptides

FIGURE 4. HLA-A2 restriction of peptide-specific T cell lines and stim- ulation by tumor cells. A, HLA-A2–transfected C1R cells (C1R-A2) and C1R cells were pulsed for 4 h with peptide 13–21 or 114–122. C1R-A2 cells were transfected with pMDK. These lines were incubated (3 3 104 cells per well) with 114–122 (314.48) or 13–21 (297.58) specific T cell lines in IFN-g ELISPOT assays. B, HLA-A2+ CIR-A2 (MDK2), DLD1 (MDK2) CaSki FIGURE 3. Tetramer labeling of peptide-specific T cell lines. T cell lines (MDK+), and HepG2 (MDK+) were incubated (30,000 cells per well) with 314.7 (A, B) and 297.58 (C, D) are specific to the peptides 114–122 and 13– 114–122 (314.48 and 267.21) and 13–21 (297.58, 616.11, 648.24, and 654.7) 21, respectively. Each line was stained with anti–CD8-FITC and PE-labeled specific T cell lines. C, For inhibition experiments, W6/32 Mab and the HLA-A2/114-122 (A, C) or HLA-A2/13–21 (B, D) tetramers and analyzed isotype control were added at the same concentration (10 mg/ml) directly by flow cytometry. The percentage of each cell population is indicated in the to the wells. T cell activation was revealed by IFN-g ELISPOT assay. D, corresponding quadrants. Same conditions as in A using TAP-deficient T2 cell line. The Journal of Immunology 421 the peptide 13–21 in ELISPOT, was labeled by the 13–21 specific transfection of the T2 cells with pMDK did not restore T cell stim- tetramer (Fig. 3C), but not by the 114–122 specific tetramer (Fig. ulation. This plasmid was, however, used with success to activate the 3D). For both T cell lines, ∼7% of the cells were peptide-specific, same T cell lines (Fig. 4A) by transfection of CIR-A2. Therefore, confirming their good peptide specificity. From 12 different HLA- presentation of both 13–21 and 114–122 appeared to be TAP de- A2+ healthy donors, we derived seven T cell lines specific for pendent. Finally, we evaluated the cytotoxicity of T cell lines by peptide 13–21 and eight T cell lines specific for peptide 114–122, their capacity to release perforin (Fig. 5A) or to lyse labeled cells as illustrated by T cell lines presented Fig. 2C. We therefore (Fig. 5B). T cell lines 365.55 and 365.36, which were specific for demonstrated that peptides 13–21 and 114–122 were able to peptide 114–122 and 13–21, respectively, gave rise to a large num- prime a CD8 T cell response in HLA-A2+ healthy donors. ber of perforin spots when they were in contact with CIR-A2 loaded Restriction, function, and tumor specificity of peptide-specific with the appropriate peptide or with HepG2 (Fig. 5A). They T cell lines remained inactivated in contact with unloaded CIR-A2 cell lines. Cold target inhibition experiments performed with the 114–122 HLA-A2 restriction of five T cell lines was confirmed using HLA- specific T cell line 581.5 confirmed the peptide specificity of the A2–transfected and untransfected APCs. Representative experi- tumor recognition (Fig. 5B). CIR-A2 cells loaded with the 114–122 ments with the T cell lines 314.48 and 297.58 are presented in peptides were more efficient than unloaded CIR-A2 to inhibit the + Fig. 4A. In contrast to the HLA-A2 CIR-A2 cells, HLA class I- T cell activation. Altogether, MDK peptides 13–21 and 114–122 deficient CIR cells were unable to present the appropriate peptide to elicit HLA-A2–restricted T cells in different donors. The T cells T cell lines specific for either 13–21 or 114–122. Moreover, trans- + 2 were cytotoxic and specifically recognized MDK tumor cells and fection of pMDK into MDK CIR-A2 cells was as efficient as MDK-transfected cell lines. Downloaded from peptides 13–21 or 114–122 in stimulating the T cells, demonstrating that both epitopes were appropriately processed and presented by the Induction of MDK-specific T cells in HHD mice APCs. Because transfection led to a higher production of endoge- We also investigated the immunogenic properties of pMDK and nous MDK, we also assessed whether HLA-A2 tumor cells (HepG2) the identified CD8 T cell epitopes in HHD mice. Mice were im- naturally expressing MDK could be recognized by the peptide- munized with pMDK (Fig. 6A) or the individual peptides (Fig. specific T cell lines. As shown for four different T cell lines specific 6B), and the ex vivo T cell response specific to the two HLA-A2– http://www.jimmunol.org/ for 114–122 (Fig. 4B, left panels) and 13–21 (Fig. 4B, right panels), restricted peptides was evaluated by IFN-g ELISPOT. All of the very strong stimulation of the T cell lines occurred in the presence of individual mice immunized with pMDK exhibited a T cell re- + + the HLA-A2 MDK HepG2 cell line, whereas CIR-A2 and DLD1 sponse to the peptides 114–122 and 13–21. Immunization with were almost unable to activate them. The 616.11 T cell line was also individual peptides led to good peptide-specific T cell responses tested with success for recognition of other HLA-A2+ MDK+ tumor cells (Caski) (Fig. 4B). For two T cell lines (Fig. 2C), we showed that tumor recognition occurred in an HLA class I-restricted manner. Activation of the 13–21 specific T cell lines 648.24 and 654.7 was inhibited by the HLA class I-specific mAb (W6/32) but not by the by guest on September 29, 2021 isotype control Ab. We investigated the TAP dependence of the MDK presentation using TAP-deficient T2 cells (Fig. 4D). Although the peptides were efficiently presented to peptide-specific T cells,

FIGURE 6. Induction of MDK-specific CTL responses in HHD transgenic mice. Groups of four HHD transgenic mice (M1, M2, M3, and M4) were immunized with MDK DNA (A) or with the individual peptides 114–122 (B, FIGURE 5. Cytotoxic activites of MDK-specific T cell lines. A, HLA-A2– left panel) and 13–21 (B, right panel). At day 10, splenocytes were harvested transfected C1R cells (C1R-A2) were pulsed for 4 h with peptide 13–21 or and incubated (1 3 105 cells per well) with 2.5 mg/ml MDK peptide 13–21 114–122. Target cells (HepG2 or CIR-A2) were incubated (3 3 104 cells per (gray)or 114–122(black) orwith culture medium (white). T cellactivation was well) with 114–122 (365.55) or 13–21 (365.36) specific T cell lines. T cell evaluated by IFN-g ELISPOT assay. Data were reported for each mouse. C, cytotoxicity was revealed by perforin ELISPOT. B, Calcein-loaded HepG2 Splenocytes of four HHD transgenic mice immunized with MDK DNA were cells (5000 cells per well) were incubated with the MDK 114–122 specific stimulated with the individual peptides 114–122 (left panel)and13–21(right T cell line 581.5 (20,000 cells per well) with CIR-A2 loaded with the peptide panel) for 1 wk. A standard 4-h [51Cr] release assay was then performed using (black) or unloaded (blank). Specific cell lysis was calculated using values RMAS-HHD cells pulsed for 4 h with the individual MDK peptides (black obtained with killed calcein-loaded HepG2 as maximum values and unkilled squares) or an irrelevant peptide (gray squares) as targets at the indicated calcein-loaded HepG2 as background. Data were reported as inhibition per- E:T ratios (100 represents 1 3 105 effector cells per well). Results are centage of the recognition in absence of competitive cells. expressed as the mean specific lysis obtained with four mice. 422 MIDKINE IS A TUMOUR-SHARED Ag in all of the mice. Finally, cytotoxic activity of the peptide-specific feature of MDK CD8 T cell epitopes, as presentation of other epit- T cells was demonstrated by the [51Cr] release assay (Fig. 6C). Our opes derived from the signal peptide was also TAP dependent (43) data obtained in vitro on human cells or in vivo in HLA-A2 trans- Finally, MDK has been proposed as a biomarker for diagnosis of genic mice show that MDK constitutes a novel tumor-shared Ag. tumor-bearing patients. MDK was found to be expressed at early stages of of colorectal (13) and prostate (46) can- Discussion cers. As a secreted protein, MDK can also be quantitated in the We provide the first evidence that the protein MDK can be con- serum (47). In healthy donors, the serum level of MK is ∼0.15 sidered as a tumor-shared Ag. Human CD8+ T cell response spe- ng/ml with a cutoff of 0.5 ng/ml, whereas it is significantly higher cific for MDK-expressing tumor cells was induced in vitro using in various types of human carcinomas (48), such as hepatocellular PBMCs from healthy donors, and immunized HHD transgenic (48) esophageal (49), gastric (50), and neuroblastoma cancers (23). mice developed a cytotoxic response specific for MDK. Because MDK was detectable at early stages (48), and the plasma level has of its wide expression in cancer tissues and its contribution to been reported to be correlated with poor outcome (23, 24). Inter- tumorigenesis, MDK appears as an attractive candidate for a can- estingly, MDK was also detectable in urine samples of patients with cer vaccine. cancer (25). Although significant, the increase in serum concentra- MDK was initially isolated as a protein induced in the early stage tion observed between healthy patients and patients with cancer of differentiation of embryonal carcinoma cells (11). Further inves- remains limited (48). This increase is therefore not expected to tigations demonstrated its wide expression in tumors (12–14) and its dramatically alter the CD8 T cell response observed with healthy multiple functions in favor of tumor expansion (28). These studies donors, because this response seems to be affected by high concen- supported therapeutic strategies based on MDK neutralization using trations of soluble Ags only (51). In contrast to many tumor Ags, Downloaded from antisense DNA (21) or Abs (29) to block the growth of tumor cells. MDK expression in tumors could be revealed by its detection in A gene suicide-based strategy using MDK promoter has been also the blood or the urine. MDK-based vaccination using peptides, proposed (30). We therefore considered the opportunity to target DNA, the whole protein, or viral vectors could be applied to patients MDK by the cellular response to obtain a T cell response specific who have a significant level of MDK in their body fluids. for a wide range of tumors. Although a basal level of MDK was seen We showed that human CD8+ T cell response specific for MDK-

in healthy individuals, we did not fear some deleterious effect in expressing tumor cells can be induced in vitro using PBMCs from http://www.jimmunol.org/ eliciting an MDK-specific cytotoxic response. Although required healthy donors and immunized HHD transgenic mice developed for neuronal embryogenesis, its role in adults rarely appears benefi- a cytotoxic response specific for MDK. Preliminary experiments cial, as MDK seems to participate in ischemic renal injury (16) and performed on eight different patients with cancer did not reveal autoimmune responses (17). Accordingly, MDK-deficient mice do any spontaneous T cell response specific for the MDK epitopes not exhibit serious developmental abnormalities (16, 31), and mice (Supplemental Fig. 1), but we did not have access to MDK ex- vaccinated with peptides and DNA in our studies did not show any pression in these donors. We also did not show any effect of signs of autoimmunity. Finally, its crucial role in tumor growth is peptide immunization on the growth of B16 transfected with expected to limit tumor escape from immune attack through its se- HLA-A2 and naturally expressing the murine MDK (data not lective loss. Its angiogenic function is extremely attractive to limit shown). These preliminary experiments were not fully conclusive by guest on September 29, 2021 metastasis, as proposed for the angiogenic factor-1, for as several differences exist between human and murine MDK which a mutated form has been found to be the natural target of sequences especially in the 13–21 epitope. Further studies will CTL T cells in patients with multiple myeloma (32). address the vaccine potential in HHD mice using stable transfec- MDK is a secreted protein like other identified tumor Ags (33– tants of human MDK. In conclusion, because of its wide expres- 36) and is therefore produced in the endoplasmic reticulum (ER) sion in cancer tissues and its contribution to tumorigenesis, MDK after cleavage of the signal peptide. Because presentation of the appears as a novel attractive candidate for a cancer vaccine. MDK peptide 114–122 appeared to be TAP dependent, three al- ternative pathways could be employed to present it to CD8 T cells. First, secreted proteins like calreticulin (37) could be present in Acknowledgments the cytosol due to an aborted translocation mechanism. Alterna- We thank Dr. S. Raffestin for advice. tively, the secreted molecule is either translocated from the ER into the cytosol (38) or captured by the APCs through a receptor- Disclosures mediated mechanism, as shown for matrix metalloproteinase-2 (34). J.K. and B.M. are coauthors of a pending patent. Capture by the LRP receptor has indeed been implicated in the targeting of MDK to proteasome degradation (39). 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