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Endocrinology, doi:10.1210/en.2008-0631
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*CALCITONIN, SALMON
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*Thyroid Cancer
Endocrinology Vol. 149, No. 11 5627-5634
Copyright © 2008 by The Endocrine Society

Amino Acid-Modified Calcitonin Immunization Induces Tumor Epitope-Specific Immunity in a Transgenic Mouse Model for Medullary Thyroid Carcinoma

Margret Wuttke, Claudia Papewalis, Yvonne Meyer, Caroline Kessler, Benedikt Jacobs, Holger S. Willenberg, Sven Schinner, Christine Kouatchoua, Thomas Baehring, Werner A. Scherbaum and Matthias Schott

Endocrine Cancer Center, Department of Endocrinology, Diabetes and Rheumatology, University Hospital Duesseldorf, 40225 Duesseldorf, Germany

Address all correspondence and requests for reprints to: Matthias Schott, M.D., Endocrine Cancer Center, Department of Endocrinology, Diabetology and Rheumatology, University Hospital Duesseldorf, Moorenstrasse 5, 40225 Duesseldorf, Germany. E-mail: matthias.schott{at}med.uni-duesseldorf.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Up to now, no relevant tumor antigen has been identified in medullary thyroid carcinoma (MTC). The aim of the present study was to prove the concept of an immunization with an amino acid-modified calcitonin (CT) for the treatment of MTC in a transgenic mouse model. Amino acid-modified (human) CT has been chosen for vaccination because of its higher binding affinity to the murine H2-Kb-MHC molecule. Mice were immunized over 6 months with monthly injections of amino acid-modified CT-pulsed dendritic cells. For enumeration of tumor epitope-specific CD8+ cytotoxic T cells, tetramer analyses were performed. CT peptide-treated mice revealed a mean 0.73 ± 0.45 and 0.91 ± 0.59% positive cells, depending on the two tetramers tested, whereas no increase was seen in control protein-immunized mice (0.08–0.12% tetramer-positive cells). Importantly, the subset of CT-specific CD8+ T cells also showed a high expression of interferon-{gamma}. In line with these results, CT-immunized mice also showed an intratumor infiltration with CD8+ T lymphocytes. Importantly, we also found a diminished tumor outgrowth of –57% and a decrease of the serum CT levels (2.0 ± 0.1 pg/ml) compared with control protein-immunized Ret/Cal mice (3.0 ± 0.4 pg/ml). In summary, we show that amino acid-modified CT is recognized from the immune system leading to a specific antitumor immune response and a diminished tumor outgrowth in transgenic MTC mice. The results are of potential importance because they might be applicable to patients with metastatic spread of a MTC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MEDULLARY THYROID CARCINOMA (MTC) is a rare tumor derived from the parafollicular calcitonin (CT)-secreting cells of the thyroid gland, explaining the key role of CT as a specific and sensitive marker of this cancer. MTC occurs in the sporadic form in about 70–80% of cases, whereas the remaining 20–30% are due to inherited forms (1), including either isolated (familial MTC) or part of multiple endocrine neoplasia syndromes. Prognosis of MTC is relatively good with a 10-yr survival rate ranging from 47–78% (2, 3) and even better in patients with a CT doubling time of more than 2 yr (4). Within the past decade, the prognosis has been improved mainly because of earlier diagnosis and improvement in surgical procedures (5, 6). Nevertheless, more than 50% of nonprophylactic thyroidectomized patients are not cured after surgery (5). In these patients, no effective therapy is known thus far. Most recently, vandetanib (ZD6474), an agent that selectively inhibits tyrosine kinases, has been reported to show some clinical responses in a few patients (8). A promising alternative approach may represent an active immunotherapy using autologous dendritic cells (DCs) pulsed with tumor antigen(s) to generate cytotoxic T lymphocytes (CTLs) directed against cancer cells (9, 10, 11).

Previously, our group has proven the concept that vaccination with mature monocyte-derived, peptide-loaded DCs can expand tumor-specific T lymphocytes in patients with MTC as well (12). After treatment with carcinoembryonic antigen peptide-/CT-pulsed DCs, some of these patients developed an antigen-specific T helper 1 (Th1) immune response [demonstrated by an increased interferon (IFN)-{gamma} production] together with a temporary clinical response in one patient (13). Another group reported on an in vitro immune reactivity in MTC patients treated with tumor lysate-pulsed DCs, however, without achieving a long-term clinical response (14, 15). The major disadvantage in therapy of all neuroendocrine malignancies including MTC is the lack of defined tumor antigens. Tumor cell-specific polypeptide hormones such as CT in MTC may, however, represent specific target molecules (4). Tumor-specific, amino acid-modified PTH peptides have already been used for vaccination of two patients with parathyroid cancer (17, 18). Long-term regression of cancer as well as normalization of tumor marker expression in these patients were due to the induction of a polypeptide hormone-specific cytotoxic immunity (18). Modification of the amino acid sequence of potential tumor antigens has already been proven to be successful in other malignancies such as breast cancer (19), melanoma (20, 21, 22, 23), leukemia (24), and colorectal cancer (25).

The aim of the present study was to prove the concept of an amino acid-modified CT vaccination in a transgenic mouse model for MTC (Ret/Cal mice). These mice display the identical mutation (substitution of Cys for Arg) within the RET protooncogene at codon 634 as most patients with multiple endocrine neoplasia type 2A (6, 27). As in patients with hereditary MTC, Ret/Cal mice develop diffuse C cell hyperplasia and MTC with increased serum CT levels (27). Via tetramer analyses, we were able to detect CT epitope-specific CD8+ cytotoxic T cells. Treated mice also revealed a diminished tumor outgrowth.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Transgenic Ret/Cal mice have been kindly provided by Professor Saarma from the Institute of Biotechnology at the University of Helsinki, Finland. These mice have been first described by Michiels et al. (27) displaying the same mutation within the Ret protooncogene at codon 634 as most patients with multiple endocrine neoplasia type 2A (substitution of Cys-634 for arginine; TGC->CGC). Because of that, all mice display overt diffuse C cell hyperplasia as early as 3 wk of age and subsequently develop diffuse MTCs. As a result, these mice also develop increased serum CT levels. The animal experiments were carried out according to German Law on the Protection of Animals and were approved by the Animal Care Committee of North Rhine-Westphalia.

Generation of DCs and CT peptide pulsing
Bone marrow-derived DCs from wild-type C57BL6 mice were generated as described formerly (28). Briefly, bone marrow cells were prepared from femurs and cultured in complete medium (CellGrowth; CellGenix, Freiburg, Germany) with granulocyte-macrophage colony-stimulating factor (R&D Systems, Wiesbaden, Germany; 1000 U/ml). After 3 d, nonadherent cells were harvested, washed in medium, and pulsed with amino acid-modified full-length CT (Cibacalcine; Novartis, Nürnberg, Germany; 100 µg/ml) representing the same amino acid sequence as human CT. Xenogenic peptide revealed differences at four amino acid positions (amino acids 16, 18, 26, and 30) compared with murine CT. Binding affinity was calculated on the basis of the computer-based algorithm epitope prediction software SYFPEITHI (www.syfpeithi.de). Human albumin (Aventis Behring GmbH, Marburg, Germany) has been used as control protein for pulsing of DCs (100 µg/ml). After 2 h, cells were harvested, washed four times with isotonic NaCl, and resuspended in 50 µl 0.9% NaCl. Thereafter, cells were used for vaccinations. In all preparations, cell viability was 95% or greater as evaluated by the trypan blue exclusion method.

Phenotypic analysis of DCs by flow cytometry analysis
The expression of surface markers characteristic for DCs was determined by flow cytometry. Cell staining was performed using fluorescein isothiocyanate (FITC)-, phycoerythrin, or peridinin chlorophyll protein-conjugated monoclonal antibodies (all purchased from BD Bioscience, Heidelberg, Germany, unless indicated otherwise). All used monoclonal antibodies recognized different DC-specific markers of the cluster of differentiation (CD) or major histocompatibility complex (MHC): hamster antimouse CD80 (clone 16-10A1), CD11c (HL3), CD29 (Ha2/5), CD54 (3H2), CD69 (H1.2F3), rat antimouse CD45 (clone 30-F11), CD45-B220 (RA3-6B2), MHC class II (I-Ak, clone 10-3.6), MHC class I (RT-1A, clone Ox6), CD86 (GL1), CD40 (3/23), CD1d (1B1), CD14 (rmC5-3), NK1.1 (PK136), CD49b (HM{alpha}2), CD3 (17-A2), CD4 (L3T4), CD8 (Ly2), Ly6c (AL-21), CD62L (Immunotools, Friesoythe, Germany; clone MEL-14), mNK (Immunotools, clone MEM-188), and CD19 (Immunotools, PeCa1). Appropriate rat or hamster IgG isotype controls were used to determine the levels of background staining. Samples were analyzed using a FACSCalibur device (BD Biosciences) using CellQuestPRO software (BD Biosciences). A minimum of 10,000 events was measured from each DC preparation before administration.

Immunization of Ret/Cal mice with DCs
Altogether, 80 mice were included into the study. Thirty male and female 4-wk-old Ret/Cal mice were treated with CT-pulsed DCs with six ip injections (4–5 x 107 cells per vaccination) over a time period of 6 months with injection intervals of 4 wk. Another 20 male and female Ret/Cal mice were treated with the same protocol with human albumin-pulsed DCs that served as controls. For evaluating tumor sizes of untreated mice, an additional 30 animals were followed for up to 6 months without any vaccination.

Preparation of peripheral blood mononuclear cells from immunized mice
After 6 months, all mice were killed for further in vitro analyses. Single-cell preparations were generated from spleens and lymph notes. Cells (1 x 107) were incubated in complete medium (RPMI 1640; Invitrogen, Karlsruhe, Germany) supplemented with IL-2 (R&D Systems; 25 U/ml) with respective CT peptides (1 µg/ml) over a time period of 4 d. Thereafter, cells were washed with PBS and incubated for an additional day in IL-2 (25 U/ml).

Tetramer analysis
Two CT-specific H2-Kb tetramers covering the amino acid positions GNLSTCML (tetramer T1) and amino acid positions MLGTYTQD (T2) amino acid-modified CT were provided by Orpegen (Heidelberg, Germany). These tetramers were chosen because of the highest probability of antigen binding calculated by the computer-based algorithm epitope prediction software SYFPEITHI (www. syfpeithi.de).

Tetramer staining of CD8+ cells was performed in PBS supplemented with 10% BSA and incubating these cells with phycoerythrin-labeled tetrameric complexes (10 pmol/ml) for 1 h at 4 C. After subsequent incubation with anti-CD8-FITC (BD Bioscience) for 20 min at 4 C, cells were washed and resuspended in 0.5% BSA in PBS for subsequent flow cytometry analyses. Propidium iodide (20 µg/ml) was used to exclude debris. In each tetramer analysis, control T cells of TCR transgenic OT-1 mice were used for control. OT-1 cells, which were purchased from Orpegen, are isolated cells from MHC class I restricted OVA257–264-expressing TCR transgenic mice. These cells are more than 90% positive for ovalbumin SIINFEKL (OVA257–264) demonstrated by tetramer analysis. The binding affinity score of this tetramer is 25 according to the algorithm epitope prediction software SYFPEITHI. A control tetramer specific for amino acid positions 12–19 LSQELHKL of salmon CT was used as internal control and did not reveal any significant binding affinity (0.06–0.3%).

Immunohistochemical analysis and measurement of tumor size
Cryostat sections (7 µm) of thyroid glands from tumor peptide-immunized, human albumin-vaccinated and nonvaccinated control mice were serially cut, blocked 30 min with normal porcine serum (5%; DakoCytomation), and stained with polyclonal rabbit antihuman CT antibodies (DakoCytomation), or monoclonal antibody rat antimouse CD8 (clone 22378; both from Abcam, Cambridge, UK) at 4 C overnight. For visualizing antirat IgG, biotin-labeled antibodies were used, amplified by avidin biotinylated enzyme complex (ABC; Vector Laboratories, Burlingame, CA), and red staining was performed by high sensitive 3-amino-9-ethylcarbazole and substrate chromogen (DakoCytomation). Sections were counterstained by hematoxylin (Merck, Hamburg, Germany).

Tumor outgrowth and serum CT measurement
Tumor outgrowth was measured using a Nikon Eclips TE300 photomicroscope (with a Nikon digital camera DXM1200) that was connected to an analyzing program for calculation of tumor sizes (Leica QWin V3). Tumor sizes were independently measured by two investigators blinded to the treatment groups. Slides were randomly selected. Each time, tumors within both thyroid lobes were measured in parallel to the entire thyroid gland, respectively. Data are expressed in mean values. Tumor sizes are given in square micrometers. Moreover, tumor sizes were also expressed in relative size of the entire thyroid gland. Serum CT was measured as recommended by the manufacturer (Roche, Mannheim, Germany). The lower detection limit of the assay is 2 pg/ml. The intraassay coefficient of variation is 2.8%, and the interassay coefficient of variation is 6.0%. Data are expressed in mean ± SEM.

Statistical analysis
The results were analyzed for statistical significance by unpaired t test using Prism computer software (GraphPad Software Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DC vaccination in Ret/Cal mice
Altogether, 30 Ret/Cal mice were immunized with bone marrow-generated DCs pulsed with nonmurine (amino acid-modified) CT. Untreated transgenic Ret/Cal mice develop MTCs with 100% tumor prevalence (27). Figure 1Go shows one representative staining of a CT-positive MTC. DCs were pulsed with amino acid-modified (human) CT peptide because of its higher binding affinity to the MHC class I molecule (H2-Kb) compared with murine CT due to amino acid differences at four positions. Binding affinity was calculated on the basis of the computer-based algorithm epitope prediction software SYFPEITHI (www. syfpeithi.de). Another 50 Ret/Cal mice were either immunized with human albumin-pulsed DCs or were not vaccinated for control.


Figure 1
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  		FIG. 1. MTC development in transgenic Ret/Cal mice. A, Representative CT staining of nonimmunized Ret/Cal mice at 6 months of age showing MTC cells (magnification, x20 and x40). B, In contrast, in a C57BL/6 wild-type mouse, neither a MTC nor a precancerous C cell hyperplasia is seen. Position of esophagus (E) and trachea (T) is given for orientation.

 
After a follow-up of 6 months, all Ret/Cal mice were killed for further analyses. Control mice (either nonimmunized or control protein immunized) did not show any macroscopic abnormalities. In contrast, however, most Ret/Cal mice that were immunized with human CT-pulsed DCs developed macroscopically enlarged lymph nodes (Fig. 2AGo). Immunohistochemistry of these lymph nodes revealed a strong infiltration by CD3+ T cells (Fig. 2BGo). Further tests including tetramer analyses and analyses of tumor-infiltrating cells were performed.


Figure 2
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  		FIG. 2. Lymph node enlargement in a xenogenic CT-immunized mouse. A, Ret/Cal mice that were immunized with amino-acid-modified CT-pulsed DCs revealed largely increased lymph nodes. B, Immunohistochemical analyses of the lymph nodes showed a strong infiltration with CD3+ T lymphocytes.

 
Detection of CT epitope-specific CD8+ T cells
For counting of epitope-specific CD8+ T cells, splenic T lymphocytes were extracted and tetramer analyses performed (Fig. 3Go). Two different CT-specific tetramers (T1 and T2) with highest binding affinities to the MHC class I molecule H2-Kb were designed and used for measurement. In control mice, only a negligible number of CT-specific T lymphocytes could be detected in spleens (0.098 ± 0.028% for tetramer 1 and 0.099 ± 0.027% for tetramer 2) (Fig. 3AGo). In contrast, tumor peptide-treated mice showed a significant higher number of CTLs recognizing different CT epitopes. In splenic CTLs of tumor-peptide-vaccinated mice, we observed an increase of CT-specific CTLs (in mean 0.73 ± 0.45 and 0.91 ± 0.59%; P = 0.0007 and 0.0016, compared with controls) depending on the tetramer tested (Fig. 3AGo). Figure 3BGo shows a representative FACS analysis of one tumor peptide treated mouse that revealed a significant number of tetramer-positive CD8+ T cells recognizing T cell receptors specific for the amino acid sequences GNLSTCML (tetramer T1) and MLGTYTQD (T2) of the human CT. In contrast, no significant numbers of tetramer-positive CD8+ T cells were detected by using a control tetramer. In this case, a tetramer (T3) specific for T cells sequence 12–19 (LSQELHKL) was used. Additionally, the subset of CT-specific CD8+ T cells also showed a high expression of IFN-{gamma} that has been measured by intracytoplasmic cytokine analyses (Fig. 4Go), which indicates the induction of a Th1 immune response.


Figure 3
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  		FIG. 3. Tetramer analyses of CT-specific CD8+ T cells. A, Tumor peptide-specific tetramer analyses of immunized mice were performed from T lymphocytes extracted from spleens. Thereafter, epitope-specific CTLs were quantified using two different CT-specific tetramers (T1 and T2) that cover two epitopes of amino-acid-modified human CT with high binding affinity to MHC class I molecule H2-Kb. In nonimmunized Ret/Cal mice (as well as in control protein-immunized mice, left panels), almost no CT-specific T cells were detected. In contrast, in tumor peptide-treated mice, much higher numbers of epitope-recognizing CTLs were detected. ***, P = 0.0007; **, P = 0.0016. B, Representative FACS analysis of viable (propidium iodide negative) CD8+ spleen cells. Upper left and middle panels show the isotype and positive control. Overall assay conditions were verified by OT-1 transgenic mouse cells positive for OVA amino acids 257–264 (SIINFEKL; upper right panel). In tumor peptide-immunized mice, significant amounts of CD8+ T cells revealed specificity to the two CT-specific tetramers tested (lower left and middle panels). In contrast, coincubation with a nonspecific salmon-specific tetramer (lower right panel) did not reveal any significant amounts of tetramer-positive cells.

 

Figure 4
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  		FIG. 4. Analysis of IFN-{gamma}-secreting CD8+ spleen cells. A, Expression of IFN-{gamma} in CTLs of CT-immunized mice was monitored after 1 d culture in complete medium enriched with IL-2 [viable cells gated on proliferating (R2) and resting (R1) cells; left panel). IFN-{gamma} was detected in both regions R1 (B) and R2 (C) using FITC-labeled rat antimouse IFN-{gamma} antibodies for intracytoplasmic detection (gray histogram). Negative controls were performed by using FITC-labeled rat isotype control antibodies (IgG1; white histogram). Experiments were performed three times independently.

 
Detection of tumor-infiltrating cytotoxic T cells
Furthermore, we analyzed whether these CD8+ cells might infiltrate growing MTCs. Therefore, cryopreserved slides of the thyroid glands were stained with anti-CD8+ monoclonal antibodies, and immunohistochemistry was performed. Importantly, in some mice, a weak infiltration with CD8+ T cells was detected (Fig. 5Go), indicating that these CD8+ T cells are attracted from tumor cells and may recognize them. In contrast, control mice did not show any tumor-infiltrating CD8+ T cells.


Figure 5
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  		FIG. 5. Immunohistochemical analyses of tumor-infiltrating cytotoxic T cells. Representative immunohistochemical analyses of serial sections of the thyroid gland. Thyroid tumors were visualized by CT staining (left panels). Tumor peptide-treated Ret/Cal mice revealed infiltrations with CD8+ T cells (upper right panel). In contrast, control protein-immunized Ret/Cal mice did not show any CTL tumor infiltration (lower right panel).

 
Tumor outgrowth and serum tumor marker
Furthermore, we determined whether xenogenic CT-based DC immunization would be sufficient to prevent tumor outgrowth in Ret/Cal mice. Serial cryostat sections of the thyroid glands of long-term treated mice (after 6 months follow-up) were stained for CT, and tumor size of both thyroid lobes was determined. Importantly, CT-immunized mice revealed a diminished overall tumor outgrowth of –57% compared with control protein-immunized Ret/Cal mice (Fig. 6Go). The values were also significantly different (P < 0.04). The tumor size in comparison with the entire thyroid size in immunized mice was 21.3 ± 3.1%, whereas control protein-immunized animals revealed a relative tumor size of 63.6 ± 4.4%. This demonstrates a reduction of the tumor size of 66.5% consistently. In accordance with these findings, the serum levels of CT in tumor peptide-vaccinated Ret/Cal mice were lower (2.0 ± 0.1 pg/ml) compared with (3.0 ± 0.4 pg/ml) and therefore almost reached CT values of those from nontransgenic C57BL/6 wild-type mice (below the detection limit of 2 pg/ml). Because of the low serum CT levels and the limited number of tumor peptide-treated mice, these differences are not significant.


Figure 6
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  		FIG. 6. Tumor outgrowth and serum CT levels in Ret/Cal mice. A, Tumors of all mice were independently measured by two investigators. CT-treated mice showed a significantly reduced tumor outgrowth compared with control mice (–57%; P = 0.0389). B, In accordance with these findings, serum CT levels in tumor peptide-vaccinated Ret/Cal mice were much lower (n = 10; 2.0 ± 0.1 pg/ml) compared with control mice (n = 17; 3.0 ± 0.4 pg/ml) and therefore almost reached values of those from nontransgenic C57BL/6 wild-type mice (below the detection limit of 2 pg/ml). For graphical reasons, values below the cutoff limit were estimated as 2 pg/ml.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
So far, no relevant breakthrough has been achieved in the identification of tumor antigens in the majority of neuroendocrine malignancies, e.g. of MTC. Here we demonstrate that an amino-acid-modified (xenogenic) polypeptide hormone as CT can be recognized by the immune system after immunization. Vaccinations with highly potent antigen-presenting DCs led to the induction of tumor epitope-specific CD8+ T cells, leading to a diminished tumor outgrowth after 6 months. These results are of major interest because they demonstrate that amino-acid-modified polypeptide hormones could in general serve as target molecules for anticancer immunotherapies in diverse neuroendocrine malignancies such as metastatic insulinoma, malignant gastrinoma and glucagonoma, vasointestinal polypeptide-secreting cancer (VIPoma), and others known to secrete different polypeptide hormones.

Polypeptide hormones are secreted in large amounts from neuroendocrine cells. The best examples and most intensively investigated are insulin-producing β-cells of the endocrine pancreas. Numerous studies have demonstrated that insulin may represent an important, possibly the most important, autoantigen in human type 1 diabetes mellitus. This has been supported by the fact that many autoreactive T cells invading pancreatic islets are insulin specific (29, 30). In addition, insulin-reactive T cells were also used within an adoptive T cell transfer model leading to the induction of diabetes in nonobese diabetic (NOD) mice (29, 31, 32). In contrast, T cells specific for another major antigen in type 1 diabetes mellitus, namely glutamic acid decarboxylase 65, induced only a delayed and less frequent diabetes incidence compared with the application of insulin-specific T cells (32). Importantly, the use of insulin gene knockout NOD mice led to disease-free survival, which is due to an abrogation of insulin-reactive T cell clones (33). Additionally, insulin-specific immune epitopes recognized from these CTLs have been identified, too (33, 34, 35, 36). These data clearly demonstrate that polypeptide hormones may serve as target molecules for cytotoxic immunity at least in an autoimmune context.

The 32-amino-acid polypeptide hormone CT is secreted in large amounts from thyroidal C cells. It represents a highly sensitive tumor marker for detecting MTC as well as for monitoring metastatic spread (4, 6, 37). In the past, two studies identified major Th2 epitope clusters of CT at the amino acid positions 13–21 (38) and reported on the in vitro inhibition of a MTC cell line after coculturing with a monoclonal antibody directed toward amino acid positions 10–19 (39). On the basis of the preprohormone (prepro-CT), another group reported on an antigen-specific immune response after cDNA vaccination in a murine model (40). Together with our results in a transgenic mouse model, these data prove that polypeptide hormones may serve as target molecules for cytotoxic immunity.

In the past, different studies have already reported on the use of polypeptide hormones as target molecules for anticancer immunizations. Bradwell and Harvey (17) demonstrated a decline of serum PTH levels and serum calcium levels in one patient with a parathyroid carcinoma who was immunized with Freund’s adjuvant together with amino-acid-modified PTH peptides. Interestingly, another case report also demonstrated a shrinkage of lung metastases by using the same approach (18). On this basis, we already used human CT for immunization of some patients with MTC leading to a CT-specific Th1 immunity in some of them (13) and a partial tumor regression in one patient (12). In contrast to this report in humans, we now applied non-species-specific amino-acid-modified (xenogenic) CT for immunization in mice to increase binding affinity to MHC class I molecules by using a nonself peptide (41). Comparable approaches with xenogenic tumor epitopes have already been described to be successful in other nonendocrine malignancies (20, 21, 24). In these reports, a single amino acid substitution of the original epitope was used, resulting in an improved recognition of tumor antigens by CTLs (20, 21, 24). This might be explained by an increased binding affinity to the MHC molecule itself. In addition to that, the MHC-peptide complex might also be stabilized, which can be explained by binding to anchor positions of the MHC molecule.

Notably, in our present study, we report on a tumor size reduction that was paralleled by a CT tumor marker decrease. It is well known that lymphocytes (7) as well as monocytes (16) may express CT receptors. A small decrease in the level of CT might be explained by the increased binding of CT to the receptor given by an increased number of tumor-specific T cells. However, in our opinion, this appears to be negligible because of the short half-life of CT (26) in comparison with the long presence of T cells and monocytes within the blood in circulation.

In summary, we here describe that an amino-acid-modified xenogenic polypeptide hormone may serve as tumor antigen for immunotherapy in MTC. This is of major importance because up to now, no relevant tumor antigens have been identified in the large number of neuroendocrine malignancies. Our data clearly demonstrate that amino-acid-modified polypeptide hormones are recognized from the immune system and may lead to a Th1 immune response. Applied to other endocrine tumors, alternative polypeptide hormones such as insulin in malignant insulinoma and gastrin in malignant gastrinoma, respectively, might be used as tumor antigens as well. In our group, a new vaccination study with a new DC generation protocol is planned in the future.


   Acknowledgments
 
We thank Professor Saarma from the Institute of Biotechnology at the University of Helsinki, Finland, for providing Ret/Cal mice and Roswitha Charko and Annette Tries for excellent technical support. Sincere thanks goes to our veterinarians Dr. Treiber and Dr. Peter for their supportive cooperation and the helpful support by their staffs.


   Footnotes
 
This work was supported by the Deutsche Forschungsgemeinschaft (Scho 781/4-1) and by the American Thyroid Association (ThyCa).

Disclosure Statement: M.W., C.P., Y.M., C.K., B.J., H.S.W., S.S., C.K., T.B. W.A.S., and M.S. have nothing to declare.

First Published Online July 10, 2008

Abbreviations: CD, Cluster of differentiation; CT, calcitonin; CTL, cytotoxic T lymphocyte; DC, dendritic cell; FITC, fluorescein isothiocyanate; IFN, interferon; MHC, major histocompatibility complex; MTC, medullary thyroid carcinoma; Th1, T helper 1.

Received April 30, 2008.

Accepted for publication July 2, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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