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The Journal of Immunology, 2008, 180, 8135-8145
Copyright © 2008 by The American Association of Immunologists, Inc.
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CTLs Directed against HER2 Specifically Cross-React with HER3 and HER41

Heinke Conrad*,{ddagger}, Kerstin Gebhard*,{ddagger}, Holger Krönig*, Julia Neudorfer*, Dirk H. Busch{dagger},{ddagger}, Christian Peschel* and Helga Bernhard2,*,{ddagger}

* Department of Hematology/Oncology and {dagger} Department of Microbiology, Immunology and Hygiene, Technical University of Munich, Klinikum Rechts der Isar, Munich, Germany; and {ddagger} Clinical Cooperation Group "Antigen-Specific Immunotherapy," Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, and Technical University Munich, Munich, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The human epidermal growth factor receptor 2 (HER2) has been targeted as a breast cancer-associated Ag by T cell-based immunotherapeutical strategies such as cancer vaccines and adoptive T cell transfer. The prerequisite for a successful T cell-based therapy is the induction of T cells capable of recognizing the HER2-expressing tumor cells. In this study, we generated human cytotoxic T cell clones directed against the HER2369–377 epitope known to be naturally presented with HLA-A*0201. Those HER2-reactive CTLs, which were also tumor lytic, exhibited a similar lysis pattern dividing the targets in lysable and nonlysable tumor cells. Several HER2-expressing tumor cells became susceptible to CTL-mediated lysis after IFN-{gamma} treatment and, in parallel, up-regulated molecules of the Ag-presenting machinery, indicating that the tumor itself also contributes to the success of CTL-mediated killing. Some of the HER2369–377-reactive T cells specifically cross-reacted with the corresponding peptides derived from the family members HER3 and/or HER4 due to a high sequence homology. The epitopes HER3356–364 and HER4361–369 were endogenously processed and contributed to the susceptibility of cell lysis by HER cross-reacting CTLs. The principle of "double" or "triple targeting" the HER Ags by cross-reacting T cells will impact the further development of T cell-based therapies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The human epidermal growth factor receptor (HER)3 2 (HER2; erbB2) is a member of the HER family, which also includes the epidermal growth factor receptor (EGFR; HER1; erbB1), HER3 (erbB3), and HER4 (erbB4). The physiological role of HER2 is to serve as a coreceptor for the other members of the HER family. In particular, the heterodimerization of HER2 with HER3 and HER4 has a major impact on the normal embryonal development (1, 2). In cancer, the HER2 gene can be amplified, which further leads to HER2 protein overexpression and subsequently to the formation of HER2 homodimers and the constitutive activation of the HER2 kinase domain (3). HER2 overexpression is found in 25–30% of breast cancer patients and associated with poor prognosis (4). Only recently has it been shown that the overexpression of both HER2 and HER3 is highly correlated in breast cancer, whereas the combination of HER2 and HER4 overexpression is rare (5). The HER2/HER3 heterodimer functions as an oncogenic unit to drive breast cancer cell proliferation in vitro (6, 7) and in vivo (8, 9). To date, the HER2 oncogenic protein has been the favored target for new therapeutics for breast cancer, including small molecule kinase inhibitors and immunotherapeutical approaches (10, 11).

HER2 has been targeted as a tumor-associated Ag by immunotherapeutical strategies based on HER2-directed mAbs and cancer vaccines, respectively. Objective remissions of HER2-overexpressing breast cancers and prolongation of patient survival can be induced with trastuzumab, an inhibitory mAb against HER2 (12, 13, 14, 15). Following immunization with HER2-directed vaccines, long-lasting HER2-specific T cell responses can be generated in patients with HER2-overexpressing breast cancer (16, 17, 18). However, there is no strong relationship between the level of circulating Ag-specific T cells induced by vaccines and the clinically observed tumor regressions. The paradoxical expansion of noneffective T cells following vaccination can be partially explained by tumor-mediated tolerance mechanisms.

We have been focusing on the generation of HER2-reactive T cells for adoptive therapy for breast cancer patients (19, 20). The rationale for adoptive T cell transfer is based on the attempt to circumvent tolerance by taking out the potentially tumor-reactive T cells from the patient and stimulating the T cells ex vivo. Anergic T cells can be rescued when removed from the tolerizing milieu and stimulated ex vivo (21, 22). Indeed, it has recently been reported by several groups that the transfer of ex vivo activated Ag-specific T cells can promote objective responses and control disease progression in patients with metastatic cancer resistant to conventional treatments (23, 24, 25, 26, 27, 28). Our group has shown that the transfer of HER2-reactive CTLs can lead to the elimination of disseminated HER2-overexpressing tumor cells in vivo (29).

It has been thought that HER2 is an ideal target Ag, because HER2 overexpression is essential for the survival of the tumor cell. However, it has been recently documented that the inhibition of HER2 signaling leads to compensatory changes such as increased membrane HER3 expression and consecutively enhanced transphosphorylation of HER3 (30). This observation is also of particular interest for immunotherapeutical strategies because HER2 can be down-regulated or mutated following HER2-targeted immunotherapies (31, 32, 33), which may further lead to the outgrowth of HER3-overexpressing tumor cells.

In this study, we describe the generation of human CTL clones specific for HER2. Most of these CTL clones displayed a fine cross-specificity to HER2-homologous epitopes derived from HER3 and/or HER4, but not from HER1. The lysis of HER2/HER3-overexpressing tumor cells by HER2/HER3-cross-specific CTLs was dependent on the overexpression of HER2 and HER3. As HER2 and HER3 both contribute to the malignant phenotype of HER2/HER3-overexpressing breast cancer cells, the parallel targeting of HER2 and HER3 by cross-specific CTLs may inhibit the selective outgrowth of escape variants. These results will contribute to the design of T cell-based therapies for the treatment of breast cancer patients.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines

The TAP-defective HLA*A0201+ T2 cell line was provided by P. Cresswell (Yale University School of Medicine, New Haven, CT), the HLA-A02*01-transfected cell line K562tA2 by T. Wölfel (Klinikum der Johannes-Gutenberg-Universität, Mainz, Germany), and the HLA-A*0201+ melanoma cell lines SK-MEL-29 and SK-MEL-37 by L. Old (Memorial Sloan-Kettering Cancer Institute, New York, NY). The HLA-A*0201-transfected ovarian cancer cell line SKOV3tA2 was supplied by M. L. Disis (University of Washington, Seattle, WA) and the HLA-A*0201 breast cancer cell lines KS24.22, GAKL, and HLA-A*0201-transfected SKBR3tA2 by B. Gückel (Eberhard-Karls-Universität, Tübingen, Germany). K562tA2tMIGR1, K562tA2tMIGR1-HER2, and K562tA2tMIGR1tHER2/ECD (where ECD is extracellular domain) were generated by retroviral transduction with MSCV-MIGR1, MSCV-MIGR1-HER2, and MSCV-MIGR1-HER2/ECD into the K562tA2 cell line. K562tA2, K562tA2tMIGR1, K562tA2tMIGR1-HER2, K562tA2tMIGR1tHER2/ECD, SKOV3tA2, and SKBR3tA2 were cultured in RPMI 1640 (Invitrogen). SK-MEL-29, SK-MEL-37, KS24.22, and GAKL were maintained in DMEM (Life Technologies). All media were supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 10% FCS. The HER2 expression of the following cell lines was documented by FACS analyses: K562tA2tMIGR1-HER2 ({Delta}MFI: 569), SKOV3tA2 ({Delta}MFI: 326), KS24.22 ({Delta}MFI: 486). GAKL ({Delta}MFI: 405), SKBR3tA2 ({Delta}MFI: 531), SK-MEL-29 ({Delta}MFI: 240), and SK-MEL-37 ({Delta}MFI: 172) (where {Delta} is difference and MFI is mean fluorescence intensity). The HLA-A2 expression was also confirmed by FACS analyses with a FITC-conjugated anti-HLA-A2 mAb, (BD Pharmingen). For IFN-{gamma} treatment the medium was supplemented with 100 U/ml IFN-{gamma} 48 h before the functional assays.

Generation of CTL clones

The study was in accordance with the precepts established by the Helsinki Declaration and approved by the Ethics Committee of the Technical University of Munich, Munich, Germany. HLA-A*0201-restricted, HER2369–377- reactive CTLs were generated by repetitive stimulation with autologous dendritic cells (DCs) (34) derived from healthy donors. Three different protocols were used for the stimulation of HER2369–377-reactive CTLs. In the first approach, PBMCs were stimulated with HER2369–377-loaded autologous DCs in RPMI 1640 supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 5% autologous serum in 96-well round-bottom plates. The medium was further supplemented with 5 ng/ml human rIL-7 (BD Pharmingen) on day 1 and 20 U/ml human rIL-2 (Chiron Behring) on day 4. The responding T cells were restimulated with HER2369–377-loaded DCs at weekly intervals in the presence of rIL-2 and rIL-7. The stimulator to response ratio was 1:20 for the first stimulation and 1:40 for the restimulations. After three to four stimulations the CTLs were stained using A2/HER2369–377 multimers and sorted with a MoFlo cell sorter (Cytomation) as recently described (35). The sorted CD8+ A2/HER2369–377+ T cells were directly cloned by limiting dilution. Using this technique, seven HER2369–377-specific T cell clones were isolated from the healthy donor NvB.

In the second approach, mature DCs were electroporated with mRNA coding for the full length of HER2 (CureVac). DCs (5 x 106) were suspended in 200 µl of Opti-MEM (Invitrogen) and transferred into a 2-mm cuvette with 25 µl of mRNA (1 µg/µl). The electroporation was conducted using the square wave protocol with 500 volts, 0.25 ms, and 1 pulse. The CD8+ T cells were isolated from PBMCs derived from the same donor using the CD8+ T cell isolation kit II (Miltenyi Biotec) and stimulated with the HER2 mRNA-transfected DCs in 200 µl of AIM-V (Invitrogen) in a stimulator to a response rate of 1:20 in 96-well round-bottom plates. For priming, the T cells and mRNA-transfected DCs were cocultured with 10 ng/ml rhIL-12 and 1000 U/ml rhIL-6 and restimulated in the presence of 5 ng/ml rhIL-7 and 100 U/ml rhIL-2 as previously described (36). HER2369–377-specific minicultures were cloned by limiting dilution. Using this technique, four HER2369–377-specific CTL clones were isolated from the healthy donor KW.

Following the third approach CD8+ T cells from an HLA-A2– donor were stimulated with allogeneic HLA-A2+ DCs loaded with 50 µg peptide using the same medium and cytokines as described for the second protocol. After three stimulations the proliferating T cells were stained with A2/HER2369–377 multimers (35) and the HER2369–377-specific T cells were sorted and cloned. Using this technique 10 HER2369–377-specific CTL clones were isolated from the healthy donor KU.

The expansion of the CTL clones was conducted in the presence of anti-CD3, rhIL-2, rhIL-15, and irradiated lymphoblastoid cell lines (LCL) and PBMCs as feeder cells as previously described (20).

Cytotoxicity assay

Cytolytic activity was analyzed in a standard 4-h chromium release assay as described (19). In brief, the tumor cell lines and the transfectants (5 x 105 cells in 100 µl of FCS) were incubated with 100 µCi of 51Cr (ICN Biochemicals) for 1 h at 37°C, washed, and then used as target cells. Peptide-loaded T2 cells were first labeled with 51Cr for 1.5 h at 37°C and then loaded with the HER2369–377 peptide at 10 µg/ml for an additional 1 h at room temperature. As negative controls, 51Cr-labled T2 cells were loaded with the HLA-A*0201-restricted peptide epitope HIVpol476–484 (ILKEPVHGV), Melan-A26–35A27L (ELAGIGILTV), and NY-ESO- 1157–165C165V (SLLMWITQV), respectively. The 51Cr-labeled targets were cultured with the T cells in RPMI 1640 with 10% FCS at 200 µl/well in V-bottom, 96-well tissue culture plates (Greiner). For evaluating the efficacy of CTL-mediated lysis, the T cells were serially diluted and then cocultured with a fixed amount of target cells, resulting in graded E:T ratios. For testing functional TCR avidity, the T cells were plated at a fixed E:T ratio of 30:1 while the peptide concentration was titrated. After 4 h of coculturing the effector and target cells at 37°C, 100 µl of supernatant was collected and the radioactivity was measured in a gamma counter. The killing was calculated as the percentage of specific 51Cr release using the following equation: percentage of specific lysis = [(sample release – medium release)/(maximal release – medium release)] x 100. Spontaneous release was generally <15%. The data in the figures refer to the mean of two replicates. The SD was below 5% of the mean.

IFN-{gamma} ELISPOT assay

IFN-{gamma} production by CD8+ T cells after two or three stimulations with HER2-expressing DCs was determined in an ELISPOT assay as described (20). The number of spots was counted by an automated ELISPOT reader system (KS ELISPOT; Carl Zeiss).

ELISA

For the detection of the IFN-{gamma} production by the CTL clones, 2 x 104 T cells/well were cocultured with 1 x 104 target cells in 96-well round-bottom plates at 37°C. After 24 h, supernatants were collected and IFN-{gamma} production was determined using a commercially available ELISA kit (BD PharMingen International).

Transfection

Plasmids containing HER1, HER3, and HER4 cDNA were provided by A. Ullrich (Max-Planck-Institut für Biochemie, Martinsried, Germany). K562tA2 cells were electroporated with the vector pCDNA3.1 containing cDNA of HER1, HER2, HER3, and HER4 in a 2-mm cuvette with 200 µl of Opti-MEM using the Xcell system (BioRad).

Small interfering RNA (siRNA) treatment

The siRNA SMARTpool containing four pooled siRNA duplexes directed against ErbB2 (catalog no. M-003126-01), ErbB3 (catalog no. M-003127-02), and a nonspecific siRNA control pool (catalog no. D-001206-13) were purchased from Dharmacon. The transfection of siRNA was performed using Oligofectamine (Invitrogen) according to the manufacturer’s instructions. The knockdown of the specified protein was determined by FACS analyses.

RNA isolation and RT-PCR

Total RNA was isolated using the RNeasy mini kit according to the manufacturer’s instructions (Qiagen). The RNA was reverse transcribed using Moloney murine leukemia virus reverse transcription with oligo(dT) (Invitrogen). PCR was performed with 100 ng of cDNA. The following primers were used: β-actin (sense: 5'-GGCATCGTGATGGACTCC-3': antisense: 5'-GCTGGAAGGTGGACAGCG-3'); tapasin (sense: 5'-CTTGGGATGATGATGAGCCATGG-3'; antisense: 5'-CTGGGCCACCCCGGAGTTCCC-3'), TAP1 (sense: 5'-GGACCGGGACGGCGTCCGAG-3'; antisense: 5'-CTTGGGATGATGATGAGCCATGG-3'); TAP2 (sense: 5'-CACGGCTGAGCTCGGATACCAC-3'; antisense: 5'-CAGCTCAGCATCAGCATCTGC-3'), LMP2 (sense: CAGGAGCGCATCTACTGTGC-3'; antisense: 5'-CAGCTGTAATAGTGACCAGG-3'); and LMP7 (sense: 5'-GAACACTTATGCCTACGGGGT-3'; antisense: 5'-TTTCTACTTTCACCCAACCATC-3') (where LMP is low m.w. protein). The PCR products were electrophoresed on a 2% agarose gel and stained with ethidium bromide.

Flow cytometry

The following mAbs were used for flow cytometry: anti-human mAb c-Erb B2/c-Neu (Ab5; Calbiochem/Merck Biosciences), anti-human mAb c-Erb B3 (Ab4; Calbiochem), and FITC-labeled F(ab')2 anti-mouse IgG (H+L) (Immunotech). Conjugated isotype-matched mAbs (all from BD Biosciences/Pharmingen) were used as negative controls. Fluorescence analyses were performed with the Coulter Epics XL flow cytometer (Coulter Electronics) and documented with FlowJo software (Tree Star).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CTL clones against the HLA-A2-binding peptide HER2369–377 have a different capacity of recognizing the endogenously processed epitope HER2369–377

We sought to isolate CTLs directed against the HER2-derived peptide HER2369–377, which is naturally processed and can serve as an epitope for HLA-A2-restricted, tumor-reactive T cells (37). Given that the natural frequency of HER2-reactive T cells in the blood is often too low to allow direct isolation, we sought to enhance the number of HER2-directed T lymphocytes by in vitro stimulations with HER2-presenting DCs. As a first approach, we stimulated peripheral blood T cells with autologous DCs loaded with the peptide HER2369–377. Following repetitive stimulations, the amount of HER2369–377-specific T cells could be increased to numbers that permitted the visualization and sorting by HLA/peptide multimers. Using this technique, we succeeded in isolating seven HER2369–377- specific T cell clones from the healthy donor NvB. The recognition pattern of CTL NvB2/12, a representative of the seven expanded clones, is shown in Fig. 1 (upper row). CTL NvB2/12 displayed a peptide-specific lytic activity as determined by lysis of T2 cells loaded with the relevant peptide HER2369–377 and by a lack of lysis of T2 cells pulsed with the irrelevant peptides HIVpol476–484 (Fig. 1A, top panel), Melan-A26–35A27L, and NY-ESO-1157–165C165V, respectively (data not shown). We next addressed the question of whether the CTL clone NvB2/12 was also able to recognize the endogenously processed HER2369–377. Therefore, the HLA-A2+ K562tA2 cells were retrovirally transduced with the full length of HER2 and then used as target cells. In addition, K562tA2 cells were transduced with the HER2/ECD, because the amino acid sequence of HER2369–377 is located in this domain. The CTL clone NvB2/12 was unable to lyse the HER2- and HER2/ECD-transduced K562tA2 cells, respectively (Fig. 1B). The failure of CTL NvB2/12 to recognize HER2- as well as HER2/ECD-expressing K562tA2 cells might be due to an insufficient capacity of the K562tA2 cells to process and present HER2369–377 and, therefore, we sought to enhance Ag processing in the presence of IFN-{gamma}. However, the CTL clone NvB2/12, as well as the other peptide-stimulated CTL clones, failed to kill the HER2- and HER2/ECD-transduced K562tA2 cells even after pretreatment with IFN-{gamma} (Fig. 1C). This is in accordance with observations made by several laboratories, including our own group, showing that peptide-stimulated T cells have a deficiency in recognizing the endogenously processed peptide (29, 35, 38, 39).


Figure 1
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  		FIGURE 1. Differential lytic activity of HLA-A2-restricted HER2369–377-directed CTL clones against HLA-A2+ cell lines retrovirally transduced with HER2 or HER2-ECD. The CTL clone NvB2/12 was generated by stimulation with autologous DCs loaded with the peptide HER2369–377, the CTL clone KU1 by activation with allogeneic HLA-A2+ DCs pulsed with peptide HER2369–377, and the CTL clone KW63 by coculture with autologous DCs that had been electroporated with HER2 mRNA. A, The peptide specificity of the three HER2369–377-directed CTL clones was documented by the lysis of T2 cells pulsed with the peptide HER2369–377 (•); T2 cells loaded with peptide HIVpol476–484 ({circ}) were used as negative control. B, CTL recognition of endogenously processed peptide HER2369–377 was evaluated by targeting HLA-A2+ K562tA2 cells that had been retrovirally transduced with MIGR1-HER2 ({diamondsuit}) or MIGR1-HER2/ECD ({blacksquare}); the mock-transduced cell line K562tA2tMIGR1 ({blacktriangleup}) was used as negative control. C, The transduced cell lines K562tA2tMIGR1-HER2 ({diamondsuit}), K562tA2tMIGR1-HER2/ECD ({blacksquare}), and K562tA2tMIGR1 ({blacktriangleup}) were first treated with 100 U/ml IFN-{gamma} for 48 h and then used as target cells for HER2369–377-directed lysis by the CTL clones NvB2/12, KW63, and KU1. Data are representative of two independent experiments.

 
Based on these findings, we were led to question whether HER2-transfected DCs are capable of inducing autologous CTLs recognizing not only target cells exogenously loaded with the peptide but also cells that present the peptide HER2369–377 following processing. Therefore, as a second approach, we stimulated CD8+ T lymphocytes with autologous DCs electroporated with mRNA coding for the full length of HER2. Following repetitive stimulations with mRNA-transfected DCs, the proliferating T cells were screened for peptide specificity, and the HER2369–377-reactive T cell populations were cloned with limiting dilution. We isolated four HER2369–377-specific T cell clones from the healthy donor KW and the reactivity pattern of the CTL clone KW63 is shown in Fig. 1 (middle panel). The peptide specificity of CTL KW63 was confirmed by lysis of T2 cells loaded with HER2369–377 and the lack of lysis of T2 cells loaded with irrelevant peptides including HIVpol476–484 (Fig. 1A, middle panel), Melan-A26–35A27L, and NY-ESO-1157–165C165V, respectively (data not shown). CTL KW63 was able to lyse IFN-{gamma}-treated HER2- and HER2/ECD-expressing K562tA2 cells that had endogenously processed HER2369–377 following transduction with HER2- and HER2/ECD, respectively (Fig. 1C, middle panel). Of note, the CTL recognition of these transfectants was dependent on their pretreatment with IFN-{gamma}, because untreated target cells were not lysed (Fig. 1B, middle panel).

The isolation of HER2-specific T cells with a low recognition efficiency against endogenously processed HER2 may be due to the fact that HER2 is a self-Ag and T cells with high avidity TCRs against HER2 may be partly deleted in the thymus. To generate HER2-specific T cells with increased recognition efficiency, we took advantage of an HLA mismatch between the stimulating DCs and the responding T cells to isolate T cells that recognize the peptide HER2369–377 as foreign. In this way, allo-restricted T cells with peptide-dominant binding and high avidity should be isolated and consecutively show high recognition efficiency. HLA-A2– CD8+ T cells were stimulated with allogeneic HLA-A2+ DCs loaded with the peptide HER2369–377, and A2/HER2369–377 multimer+ T cells were sorted and cloned. Ten HLA-A2-restricted T cell clones with HER2369–377-dominant binding that did not recognize irrelevant peptides such as HIVpol476–484 (Fig. 1A, bottom panel), Melan-A26–35A27L, and NY-ESO-1157–165C165V, respectively (data not shown), could be expanded. As a representative, the allo-restricted CTL clone KU1 is shown in Fig. 1 (bottom row). CTL KU1 was able to lyse the HLA-A2+ HER2-expressing cell lines K562tA2tMIGR1-HER2 and K562tA2tMIGR1-HER2/ECD even without IFN-{gamma} pretreatment. Of note, the lysability of the HER2- and HER2/ECD-expressing K562tA2 cells by CTL KU1 could be significantly increased following incubation with IFN-{gamma}. The recognition patterns of the three different representative CTL clones NvB2/12, KW63, and KU1 were confirmed by cytokine secretion (Table I).


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  		Table I. APM expression of HER2-overexpressing tumor cell lines and recognition by HER2-directed CTL clones

 
Tumor recognition depends on the HER2-specific T lymphocytes and the HER2-expressing tumor cells

To answer the key question of whether the HER2-specific T cells can lyse tumor cells naturally overexpressing HER2, we next investigated various HLA-A2-matched, HER2-overexpressing tumor cell lines as target cells. The HLA-A2+, HER2-overexpressing cell lines were of different tissue origins, such as breast cancer (SK-BR3tA2, KS24.22, GAKL), ovarian cancer (SK-OV3tA2), and melanoma (SK-MEL-29, SK-MEL-37). Before measuring the CTL-mediated tumor reactivity, the tumor cell lines were cultured in the presence or absence of IFN-{gamma}. The pretreatment of tumor cells with IFN-{gamma} did not influence their expression level of HER2 (data not shown). The peptide-stimulated, auto-restricted CTL NvB2/12, which was unable to recognize the HER2-transduced cell lines, was not able to lyse any of the HER2-overexpressing tumor cell lines irrespective of treatment with IFN-{gamma} (Fig. 2, top row). The lack of tumor recognition was observed with an additional HER2369–377-reactive CTL clone, CTL NvB41, which had also been generated by in vitro stimulations with HER2369–377-loaded, autologous DCs. By contrast, the CTL clones KW63 and KU1, both being able to recognize the endogenously processed peptide HER2369–377, exhibited a particular lysis pattern that divided the tumor cell lines into three groups. The first group consisted of the melanoma cell lines SK-MEL-29 and SK-MEL-37, which were lysed by CTLs KW63 and KU1 regardless of whether the tumor cells had been pretreated with IFN-{gamma} or not (Fig. 2A, middle and bottom panels). The lysis of SKOV3tA2 and KS24.22, the representatives of the second group, strongly depended on the pretreatment with IFN-{gamma} (Fig. 2B, middle and bottom panels). The third group was defined by SKBR3tA2 and GAKL, which were not lysable by the CTLs KW63 and KU1 regardless of IFN-{gamma} pretreatment (Fig. 2C, middle and bottom panels); this resistance to lysis was not due to the fact that these tumor cell lines were nonlysable in general, because SKBR3tA2 cells exogenously loaded with HER2369–377 were efficiently killed by the HER2369–377-specific CTLs KU1 (data not shown). A similar lysability pattern was documented with an additional HER2369–377-reactive CTL clone, KU40, which had been generated using HER2369–377-loaded, allogeneic DCs (Table I). The distinct recognizability of tumor cells by HER2369–377-reactive CTLs was verified by measuring CTL-mediated IFN-{gamma} secretion (Table I). In general, there was a correlation between lytic activity and IFN-{gamma} production with some exceptions, which may be due to the divergent functional activity of individual CTL clones.


Figure 2
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  		FIGURE 2. Different lysability of HLA-A2+ HER2-overexpressing tumor cell lines by HER2369–377-directed CTL clones. Six different HER2-overexpressing HLA-A2+ tumor cell lines were used as target cells for HER2369–377-directed lysis by the CTL clones NvB2/12, KW63, and KU1. Before the cytotoxicity assay, the tumor cell lines were either treated with IFN-{gamma} (closed symbols) or not treated (open symbols). Depending on the IFN-{gamma}-induced lysability, three different groups of tumor cell lines were distinguished: SK-MEL-37 with or without IFN-{gamma} (•/{circ}) and SK-MEL-29 with or without IFN-{gamma} ({blacktriangleup}/{triangleup}) (A); KS24.22 with or without IFN-{gamma} (•/{circ}) and SKOV3tA2 with or without IFN-{gamma} ({blacktriangleup}/{triangleup}) (B); and SKBR3tA2 with or without IFN-{gamma} (•/{circ}) and GAKL with or without IFN-{gamma} ({blacktriangleup}/{triangleup}) (C). Data are representative of three independent experiments.

 
The partial dependence of tumor recognition on IFN-{gamma} suggested that deficiencies in the HLA-A2 expression may be involved in this phenomenon. The HLA-A2 surface expression was up-regulated upon treatment with IFN-{gamma}. However, there was a threshold for HLA-A2 expression; if enough HLA-A2/peptide complexes were present on the surface for sufficient CTL recognition, the further increase of HLA-A2 expression did not result in a better CTL recognition. The HLA-A2+ cell line SK-MEL-29 displaying an MFI of 254 was recognized by the HLA-A2-restricted CTL clones KU1 and KW63 without prior treatment with IFN-{gamma}, indicating that an MFI of at least 254 was sufficient for optimal CTL recognition. The KS24.22 cells naturally displayed HLA-A2 with an MFI of 389, the SKOV3tA2 cells with an MFI of 260, and the GAKL cells with an MFI of 265, but were not recognized by the T cells. Hence, the IFN-{gamma}-induced CTL recognition of SKOV3tA2 and KS24.22 was not due to enhanced HLA-A2 expression, because the baseline HLA-A2 expression of these tumor cell lines should have been sufficient for CTL recognition. Therefore, the reason for missing recognition by CTLs was not due to low HLA-A2 expression. Moreover, the SKBR3tA2 cells expressing low levels of HLA-A2 (MFI: 53) up-regulated their HLA-A2 expression upon IFN-{gamma} (MFI: 332) but, nevertheless, were not recognized by the HLA-A2-restricted CTL clones. These data indicated that HLA-A2 expression was not responsible for lack of CTL recognition.

We next addressed the question of whether deficiencies in the HER2 processing pathway were involved in the impaired CTL recognition by investigating the components of the Ag-processing machinery (APM), including LMP2, LMP7, TAP1, TAP2, and tapasin (Table I). The tumor cell lines of group I expressed all of these five APM components in the presence or absence of IFN-{gamma} consistent with their IFN-{gamma}-independent perceptibility. The tumor cell lines of group II, whose lysis was dependent on the presence of IFN-{gamma}, displayed a variable expression of the herein tested APM molecules. Upon IFN-{gamma} treatment, single components were up-regulated in individual cell lines, e.g., tapasin in SKOV3tA2 cells, which may be responsible for IFN-{gamma}-induced, CTL-mediated tumor lysis. However, this was not a consistent finding. For example, the KS24.22 cell line of group II expressed the five tested APM components naturally, but their expression level did not change following IFN-{gamma}-treatment even though the killing of KS24.22 cells was clearly dependent on IFN-{gamma}. Moreover, SKBR3tA2 and GAKL, both members of group III, were not lysed in the presence or absence of IFN-{gamma} despite the fact that they naturally expressed the five APM molecules. In conclusion, there was no consistent correlation between the lysability and the expression of LMP2, LMP7, TAP1, TAP2, or tapasin. Therefore, other effects of IFN-{gamma} may be responsible for the improved tumor recognition by HER2369–377-reactive CTLs.

CTL clones recognizing HER2369–377 have a different ability to cross-react with the homologous peptide epitopes of HER3 and HER4

HER2 shares a high degree of homology with the other members of the HER family. Therefore, we investigated the potential reactivity of the HER2369–377-reactive CTL clones with the corresponding peptides of HER1, HER3, and HER4. The homologous HER peptides, which were identified by amino acid sequence alignment, are located in the same region of the ECD of the respective HER protein (Table II). The selected nonamers HER1364–372, HER3356–364, and HER4361–369 display the same HLA-A2 anchor motifs (position 2: isoleucine; position 9: leucine) as the reference peptide HER2369–377, which turns them into putative epitopes for HLA-A2-restricted CTLs. In particular, the selected peptides HER3356–364 and HER4361–369 display a relatively strong homology with the peptide HER2369–377, differing only in three amino acids.


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  		Table II. HER2369–377 and the homologous peptide epitopes of HER1, 3 and 4

 
The HER2369–377-reactive CTL clones NvB2/12, NvB41, KW63, and KU1 were examined for their cross-reactivity with the corresponding HER peptides. In addition, the functional avidities of these CTL clones toward the peptide HER2369–377 and the other HER peptides were assessed by the recognition of serially diluted amounts of the respective peptide loaded onto T2 cells (Fig. 3). Surprisingly, the nontumor-lytic CTL clones NvB2/12 and NvB41, which had been originally generated with peptide-loaded autologous DCs, displayed a high avidity toward the synthetic peptide HER2369–377 with a half-maximum lysis of ~10–9 and 10–10 M, respectively. In contrast, the half-maximum lysis by the tumor-lytic CTL clones KW63 and KU1 occurred at 10–7 M, indicating low avidity TCRs against HER2369–377. The CTLs NvB41 and KW63 cross-reacted with HER3356–364, the CTL NvB2/12 with HER4361–369, and the CTL KU1 with both HER3356–364 and HER4361–369. None of the CTL clones recognized HER1364–372. The avidities toward HER3356–364 and HER4361–369 varied considerably among the individual CTL clones. The CTLs NvB2/12 and KW63 had lower avidities to HER3356–364 and HER4361–369, respectively, compared with HER2369–377. The CTL NvB displayed an equally high avidity to the HER2- and HER3-derived peptides. In contrast, the CTL clone KU1 expressed a TCR with a higher avidity to HER3356–364 than to HER2369–377, even though the T cells had been originally stimulated with HER2369–377-loaded allogeneic DCs before cloning. Of note, not all CTL clones displayed a cross-reactivity toward HER3356–364 and HER4361–369. For example, CTL clone KU40 (Table I), which had been isolated from the same bulk culture as CTL KU1, did not cross-react with HER3356–364 or HER4361–369 (data not shown). Vice versa, five of five CTL clones induced with HER3356–364 demonstrated cross-specificity with HER2369–377 (data not shown).


Figure 3
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  		FIGURE 3. Specificity and avidity of HER2369–377-directed CTL clones toward homologous peptide epitopes of HER3 and HER4. The CTL clones NvB2/12, NvB41, KW63, and KU1, which had been isolated for specificity to HER2369–377, were tested for their cross-reactivity toward the corresponding peptide epitopes derived from HER1, HER3, and HER4. T2 cells were loaded with HER1364–372 ({blacktriangleup}), HER2369–377 (•), HER3356–364 ({diamondsuit}), and HER4361–369 ({blacksquare}). Functional avidity of CTL clones was determined by the lysis of T2 cells pulsed with graded amounts of peptides at a fixed E:T ratio of 30:1. Data are representative of two independent experiments.

 
We next addressed the question of whether the cross-reactivity of the HER2-directed CTL clones was restricted to target cells exogenously loaded with synthetic HER3 and HER4 peptides or whether the recognition extended to the naturally processed HER3 and HER4 peptides. K562tA2 cells were transfected with cDNA coding for the full length of HER1, HER2, HER3, or HER4 and then cocultured with the CTL KU1, which is known to cross-react with synthetic HER3356–364 and HER4361–369. Indeed, CTL KU1 recognized the naturally processed HER3 and HER4 as determined by IFN-{gamma} secretion (Fig. 4A). Similar data were obtained for CTL KW63 (data not shown).


Figure 4
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  		FIGURE 4. CTL recognition of endogenously processed HER2, HER3, and HER4. A, The CTL clone KU1 was cocultured with K562tA2 transfected with pCDNA3.1-HER1, -HER2, -HER3, or -HER4 and the IFN-{gamma} release was assessed as a parameter for Ag-specific CTL activation. The IFN-{gamma} concentration of the supernatant was determined by ELISA. B, Left panel, The HER2- and HER3-overexpressing melanoma cell line SK-MEL-37 was transfected with siHER2. The down-regulation of HER2 expression was assessed with FACS using a mAb against HER2. As negative control, SK-MEL-37 was transfected with siCo. As specificity control, the HER3 expression of SK-MEL-37 was determined before and after transfection with siHER2. FACS analyses of siHER2-transfected SK-MEL-37 with anti-HER2 mAb (siHER2/anti-HER2; dotted line) demonstrated a down-regulated HER2 expression, whereas the expression of HER3 was unaffected as determined with anti-HER3 mAb (siHER2/anti-HER3; dashed line). Staining of siCo-transfected SK-MEL-37 with mAb against HER2 (siCo/anti-HER2; thick line) was used as control for the natural HER2 expression. Staining of siCo-transfected SK-MEL-37 with anti-IgG1 (siCo/anti-IgG1; thin line) was used as negative control. Right panel, SK-MEL-37 was transfected with siHER3. FACS analyses of siHER3-transfected SK-MEL-37 with mAb anti-HER3 (siHER3/anti-HER3; dotted line) showed a reduced expression of HER3, whereas the expression of HER2 was unchanged (siHER3/anti-HER2; dashed line). Unsilenced HER3 expression of siCo-transfected SK-MEL-37 was determined with mAb against HER3 (siCo/anti-HER3; thick line). As negative control, siCo-transfected SK-MEL-37 cells were stained with anti-IgG1 mAb (siCo/anti-IgG1; thin line). C, HER2+/HER3+ SK-MEL-37 cells were transfected with siHER2 and siHER3, respectively, to determine the influence of HER2 and HER3 regarding the CTL recognition. As negative control, SK-MEL-37 was transfected with siCo. The recognition of HER2+/HER3+ SK-MEL-37 was determined by IFN-{gamma} release with HER2/HER3-directed CTL clone KU1. Data are representative of two (A) and three (B and C) independent experiments.

 
Because the HER2-overexpressing tumor cell lines coexpressed HER3 (data not shown), we investigated whether tumor cell lysis by HER2/3-reactive CTLs depended on HER2 and/or HER3. To clarify the role of a certain HER molecule in tumor cell recognition, the expressions of HER2 and HER3, respectively, were down-regulated by siRNA against HER2 and HER3. The successful down-regulation of the respective HER protein was assessed by flow cytometry (Fig. 4A). The transfection of SK-MEL-37 cells with siRNA against HER2 (siHER2) reduced the HER2 surface expression ({Delta}MFI: 125) compared with the transfection with control siRNA (siCo; {Delta}MFI: 183) (Fig. 4B, left histogram). The down-regulation of HER3 protein expression in SK-MEL-37 cells was almost complete ({Delta}MFI: 0) following transfection with siRNA against HER3 (siHER3) in contrast to siCo ({Delta}MFI: 196) (Fig. 4B, right histogram). The down-regulation of HER2 by siHER2 led to the decreased recognition by HER2/3-reactive CTL KU1 as determined by IFN-{gamma} secretion (Fig. 4C). Furthermore, the silencing of HER3 protein expression was accompanied by diminished cytokine secretion by the CTL KU1.

We finally asked the question of whether additional HER2 epitopes might be shared by the other members of the HER family. T cell clones were isolated from bulk cultures that had been stimulated with autologous DCs loaded with the peptide HER2773–782 known to be naturally processed and presented with HLA-A2 (40). The established HLA-A2-restricted, HER2773–782-specific CTL clones did not cross-react with the corresponding peptides HER1765–774 and HER4771–780 (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The HER2 peptide sequence at position 369–377 was first identified as an immunodominant epitope for HLA-A2-restricted T lymphocytes that had been isolated from malignant ascites of patients with HER2-overexpressing ovarian cancer (37). Since this discovery, the synthetic peptide HER2369–377 has been widely investigated for the ex vivo and in vivo generation of HER2-specific CTLs following stimulation in vitro (39, 41, 42, 43, 44, 45, 46) and vaccination (18, 38, 47, 48, 49), respectively. Results obtained from different research groups led to the conclusion that HER2369–377-specific T cells can potentially lyse HLA-A2-matched HER2-overexpressing tumor cells (39, 41, 42, 43, 44, 45, 46, 48) and may be even able to eliminate tumor cells in vivo (18, 29, 50). However, Sette and colleagues showed for the first time that HER2369–377-specific CTLs generated by in vitro stimulations using the synthetic peptide HER2369–377 do not necessarily recognize the endogenously processed peptide and, therefore, may fail to lyse HLA-A2+ HER2-overexpressing tumor cell lines (45). Moreover, the vaccination of patients using synthetic HER2369–377 in combination with Freund’s incomplete adjuvant led to the induction of peptide-specific CTLs that failed to recognize HER2+ tumors (38).

The herein characterized HER2369–377-specific T cell clones, which had been isolated following repetitive stimulation with autologous DCs loaded with the synthetic peptide HER2369–377, were unable to lyse HLA-A2+ HER2+ tumor cells or transfectants. The inability of the HER2369–377-specific T cells to lyse tumor cells did not correlate with a low TCR avidity toward the synthetic HER2369–377 peptide, as the CTL clones NvB2/12 and NvB41 displayed a half maximum lysis of peptide-loaded T2 cells at a peptide concentration of 5 x 10–10 M (IC50 of 0.5 nM) and 10–11 M (IC50 of 0.01 nM), respectively. These TCR avidities to the HER2369–377 peptide are similar to the high avidity TCRs of virus-specific CTLs, e.g., CMV-specific T cells, that display a half-maximum lysis of peptide-loaded T2 cells at 10–10 M (IC50 of 0.1 nM) (35). Using the herein described culture condition of peptide-stimulation, we predominantly isolated HER2369–377-specific, nontumor-lytic T cell clones (five of five healthy donors). Of note, our clinical study regarding the adoptive transfer of autologous HER2369–377-specific T cells for HLA-A2+ patients with HER2-overexpressing breast cancer was closed prematurely due to the frequent establishment of HER2369–377-specific T cell clones that failed to recognize the endogenously processed peptide HER2369–377 (29). One explanation for the preferential establishment of peptide-specific/nontumor-lytic CTL clones by peptide stimulation may be that their T cell receptors recognize a particular peptide conformation that is present when HLA-A2 molecules are exogenously loaded with peptides but different from the one conferred by endogenous presentation as suggested by Zaks and Rosenberg (38).

Another explanation of this phenomenon may be that the naturally processed HER2369–377 epitope may be glycosylated as it is derived from the ECD known to be heavily glycosylated. It has been described for the glycoprotein MUC1 that complex carbohydrates are not removed during processing (51, 52). Therefore, the majority of CTLs stimulated with the synthetic HER2369–377 peptide may not be able to recognize the naturally processed glycopeptide. A third hypothesis is based on the fact that T cells are known to be cross-reactive. Following stimulation with peptide-loaded autologous as well as allogeneic DCs, certain T cell populations may be enriched that are physiologically directed against a mimotope and, therefore, their TCR may not exactly fit onto the naturally processed A2/HER2 peptide complex even though they recognize the peptide following the exogenous loading of T2 cells.

Alternative Ag delivery modes based on Ag processing, e.g., the viral or nonviral transfer of genes encoding the HER2 Ag, may facilitate the selection of T cells with the ability to recognize HER2-derived peptides that are endogenously processed by HER2+ tumor cells (20, 53, 54). The efficacy of generating tumor-lytic T cells against this or other HER2 epitopes may be also enhanced by using altered or cross-reactive peptides for stimulating T cells in vitro (45, 55, 56, 57).

In this study, we investigated two additional culture conditions for generating HER2369–377-reactive T cells exhibiting tumor-lytic function. The first procedure was based on HLA-A2+ DCs that had been transfected with mRNA encoding the full length of HER2 to stimulate autologous CD8+ T cells toward the endogenously processed HER2369–377. The second method took advantage of the naturally existing pool of allogeneic T cells with peptide-dominant binding (58, 59) by stimulating T cells from an HLA-A2-negative donor with HER2369–377-loaded DCs from an HLA-A2+ donor. With both methods, we succeeded in isolating HER2-reactive CTL clones with the ability to lyse HLA-A2+, HER2-expressing tumor cell lines and transfectants. Surprisingly, the potential of these HER2-reactive CTL clones to lyse HER2-expressing tumor cells did not correlate with their avidity to the synthetic HER2369–377 peptide. Both of the mRNA-stimulated CTL clones, KW63 and the allo-A2-restricted CTL clone KU1, exhibited a half-maximum lysis of peptide-loaded T2 cells at a concentration of 10–7 M (IC50 of 100 nM), which is considerably high compared with the peptide concentration range of 5 x 10–10 to 10–11 M (IC50) that is sufficient for the nontumor-lytic T cell clones generated with HER2369–377-loaded autologous DCs (e.g., NvB2/12 and NvB41). In conclusion, CTLs with a low TCR avidity toward the synthetic HER2369–377 peptide can efficiently lyse HER2-expressing tumor cells, and vice versa, supporting the above-mentioned hypothesis that the conformations of synthetic and endogenously processed peptides may be different. The inverse relationship between TCR avidity and tumor recognition efficiency may also be a consequence of the different cytokine environments during their generation.

The members of the HER family share a high sequence homology. This also holds true for the nonamer HER2369–377 because homologous sequences, including the HLA-A2 binding motif at positions 2 and 9, are found in HER1, HER3, and HER4. Therefore, we investigated the CTL recognition of the peptides corresponding to HER2369–377, which are HER1364–372, HER3356–364, and HER4361–369. Indeed, some of the HER2369–377-stimulated CTL clones cross-reacted with the synthetic peptides HER3356–364 and/or HER4361–369, which both diverge in three of nine amino acids from HER2369–377. Vice versa, HER3356–364-induced CTL clones demonstrated cross-specificity to HER2369–377. In contrast, there were no cross-reactivities of HER2369–377-stimulated CTLs with HER1364–372, which has only four amino acids in common with HER2369–377. Of note, HLA-A2-restricted CTL clones specific for a different HER2 epitope, HER2773–782 (40), did not cross-react with the corresponding peptides HER1765–774 and HER4771–780, differing in four and seven amino acids with HER2773–782, respectively. The recognition of the HER-reactive CTLs is based on the sequence homology of the cognate peptides and, therefore, they can be designated as "cross-specific" rather than "cross-reactive." This is in contrast to CTLs directed against the epitope A2/Melan-A27–35, which are indeed "cross-reactive" due to the degeneracy of Melan-A Ag recognition (60).

The detection of the synthetic peptides HER3356–364 and/or HER4361–369 by HER2369–377-reactive CTLs does not necessarily imply their recognition of the respective epitopes, as the HER3/4 peptides may not be naturally processed or the CTLs may not recognize the processed HER3/4 peptides. Therefore, we selected the HER2/3/4-specific CTL clone KU1 with known tumor-lytic function and tested its ability to recognize the HLA-A2+ K562tA2 cell line transfected with the cDNA encoding the respective HER molecules. Indeed, the HER2/3/4-specific CTLs (e.g., CTL KU1) recognized not only the naturally processed HER2 but also HER3 and HER4 as determined by IFN-{gamma} secretion. This is the first time that cross-specificity of HER2-specific CTLs can be demonstrated toward newly defined epitopes derived from HER3 and HER4.

As most of the HER2+ tumor-cell lines coexpressed HER3, we sought to assess the influence of HER2 and HER3 on the tumor-recognition by the HER2/3-specific, tumor-lytic CTLs. Selective down-regulation of either HER2 or HER3 revealed that both molecules contributed to the Ag-specific recognition by HER2/3-reactive CTLs. Of note, the TCR avidities toward HER3 and/or HER4, as measured by the recognition of titrated synthetic peptides, did not correlate with the capacity of the HER3/4-reactive CTLs to recognize endogenously processed peptides. CTLs with a high avidity toward HER3 (e.g., CTL NvB41) did not recognize the endogenously processed HER3 peptide, whereas CTLs with a low avidity toward HER3 (e.g., CTL KW63) and HER4 (e.g., KU1) recognized the respective epitope presented by HLA-A2+ transfectants or tumor cells. This observation underscores our experiences drawn from the HER2-directed TCR avidity, suggesting that additional factors may play a role in HER-mediated tumor lysis.

The contribution of the three HER-family members, HER2, HER3, and HER4, in the tumor susceptibility toward cross-specific CTLs opens new avenues for the development of HER-directed T cell therapies. As HER2 and HER3 both contribute to the malignant phenotype of the tumor, the "double targeting" of HER2 and HER3 may inhibit the selective outgrowth of immune escape variants (31, 32, 33). This may also be of relevance for cancer types other than breast cancer, e.g., prostate cancer, in which HER2/3 heterodimers also play a decisive role (61). The new principle of "double or triple targeting" two or three HER Ags will broaden the group of cancer patients applicable for HER-directed T cell therapy, because HER2, HER3, and HER4 are differently expressed or coexpressed by individual cancers (5, 62, 63, 64).

The members of the HER family are ubiquitously expressed in normal tissues, including the cardiovascular system and the gastrointestinal, respiratory, and urogenital tracts as well as breast and skin. Until now, HER2-directed immunotherapies, such as vaccines, mAbs, and adoptive T cell transfer have not induced autoimmune disease in patients with HER2-overexpressing tumors. However, it cannot be ruled out that the transfer of HER2-reactive T cells with cross-specificity toward HER3 and/or HER4 may lead to the development of autoimmunity. In particular, normal tissues that are dependent on the heterodimerization of HERs may be at risk, as has been reported for HER2 and HER4 in cardiomyocytes (2, 65, 66).

The efficient elimination of tumor cells by HER-specific T cells is not only dependent on the qualities of the TCR but also on the susceptibility of the tumor cell to CTL killing. For HER2-expressing tumor cells it has been shown that a decreased expression of HLA class I molecules and/or components of the APM may be responsible for the resistance toward CTL-mediated lysis (67, 68). The herein described HER2369–377-reactive, tumor-lytic T cells exhibited a similar lysis pattern by dividing the targeted HLA-A2+ HER2+ cell lines in three groups as follows: 1) cell lines that were lysed independently of IFN-{gamma} pretreatment; 2) cell lines that were only recognized after incubation with IFN-{gamma}; and 3) cell lines that were not lysable regardless of prior IFN-{gamma} treatment. Because HER2-overexpressing tumor cell lines vary tremendously in their expression of APM components (69), we investigated the mRNA expression of LMP2, LMP7, TAP1, TAP2, and tapasin in a panel of cell lines cultured in the presence and absence of IFN-{gamma}. Indeed, the IFN-{gamma} treatment resulted in the up-regulation of certain components in certain cell lines. However, we could not observe a strong correlation between the expression of APM components and the susceptibility to CTL killing. This may partly be due to the fact that, first, not all but only five APM components were examined, and second, that the increased APM component expression was determined on the mRNA level, which does not necessarily translate into a higher density of A2/HER2369–377 complexes. By using a TCR mimic mAb recognizing the A2/HER2369–377 complex, it has been recently demonstrated that the expression level of A2/HER2369–377 complexes predicts tumor cell susceptibility to HER2369–377-specific CTL killing (70). The immunohistochemical analyses of A2/HER2369–377 and other HLA/HER-peptide complexes expressed by cancer samples may have significant implications for the improvement of T cell-based immunotherapies against the HER Ags.

Based on our results, we can conclude that the success of HER2-directed T cell therapies depends on both the characteristics of the HER2-specific T cells and the HER2-expressing tumor cells. As the HER2-specific T lymphocytes vary greatly regarding their cross-specificity and tumor-lytic activity, their broad clinical application is limited due to the laborious procedure of T cell isolation and characterization if tailored for every single patient. For this reason, we will pursue an alternative methodology whereby primary human T cell populations are transduced with the HER-specific TCR of interest. The TCR gene transfer is a convenient method for producing Ag-specific T cells, further allowing individualized therapies to be available for a mass of patients (71, 72, 73, 74, 75, 76).


   Acknowledgments
 
We thank Kathrin Hofer and Kerstin Holtz for excellent technical assistance, Matthias Schiemann for expert cell sorting, Peter Cresswell, Nora Disis, Brigitte Gückel, Thomas Wölfel, and Lloyd Old for providing cell lines, Axel Ullrich for providing plasmids containing HER1, HER3, and HER4, Gert Riethmüller for helpful discussions, and Ekkehard Albert for HLA typing.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Research Council of Germany Grant SFB 456 (to H.B. and D.H.B.) and the Wilhelm Sander foundation Grant 2000.017.3 (to H.B.). Back

2 Address correspondence and reprint requests to Dr. Helga Bernhard, Third Medical Department, Klinikum Rechts der Isar, Technical University of Munich, Ismaninger Strasse 22, D-81675 Munich, Germany. E-mail address: helga.bernhard{at}lrz.tum.de Back

3 Abbreviations used in this paper: HER, human epidermal growth factor receptor; APM, Ag-processing machinery; DC, dendritic cell; ECD, extracellular domain; LMP, low m.w. protein; MFI, mean fluorescence intensity; {Delta}MFI, difference in MFI; siRNA, small interfering RNA; siHER2, siRNA against HER2; siHER3, siRNA against HER3; siCo, control siRNA; LCL, lymphoblastoid cell line. Back

Received for publication June 14, 2007. Accepted for publication April 14, 2008.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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