Tumour-dendritic hybrid cell vaccination for the treatment of patients with malignant melanoma: immunological effects and clinical results

Vaccine 23 (2005) 2367–2373

Tumour-dendritic hybrid cell vaccination for the treatment of patients with malignant melanoma: immunological effects and clinical results Uwe Trefzer, Gunda Herberth, Karolina Wohlan, Annett Milling, Max Thiemann, Tumenjargal Sharav, Katrin Sparbier, Wolfram Sterry, Peter Walden∗ Department of Dermatology and Allergy, Charit´e, University Medicine Berlin, Humboldt University, Schumannstr. 20/21, 10117 Berlin, Germany Available online 26 January 2005

Abstract Hybrid cell vaccines of autologous tumour cells fused with allogenic dendritic cells (DC) combine the tumour’s antigenicity with the immune-stimulatory capacity of mature dendritic cells and allogenic MHC class II molecules to activate T cell help and induce tumour-specific cytotoxic T cells. This concept was tested in a clinical trial with melanoma stage III and IV patients. Seventeen patients were evaluated: one experienced complete, one partial response and six stable disease with long survival times. Eleven of fourteen patients, clinical responders and non-responders alike, mounted high-frequency T cell responses to various tumour-associated antigens. Failing clinical responses correlated with loss of antigenicity. © 2005 Elsevier Ltd. All rights reserved. Keywords: Tumour vaccine; Melanoma; T cell; Immune monitoring; Immune escape; Clinical trial

1. Introduction The current developments of therapeutic vaccination as a promising treatment option for cancer are driven by an increasing understanding of tumour immunology and, in particular, of the antigenicity of tumour cells [1–4]. A number of different vaccine designs have been tested in clinical trials already, however, the clinical response rates reported so far are not satisfying and progression during therapy as much as manifest cancer testifies to insufficient immune defences. These shortcomings are attributed, among other causes such as disease-related immune suppression [5] and immune evasion [6], to poor immunogenicity of the tumours [7]. Fusion of tumour cells with dendritic cells (DC) [8] aims at enhancing the immunogenicity of antigenic tumours [9,10]. The hybrid cell vaccination concept had been tested in animal models for prevention as well as therapy [7,9–14], and first clinical studies were reported for renal cell carcinoma [15], melanoma [16] and glioblastoma [17]. Here we summarize ∗ Corresponding author. Tel.: +49 30 450 518031; fax: +49 30 450 518932. E-mail address: [email protected] (P. Walden).

0264-410X/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2005.01.081

the results of a clinical trial with metastatic melanoma patients at advanced stages of disease [18]. The patients were treated with vaccines generated by fusing autologous tumour cells with mature allogeneic DC (Fig. 1). The autologous tumour cells are expected to present a variety of tumourassociated antigens including members of the MAGE family, Melan A/MART-1, tyrosinase and gp100 [3]. Mature allogeneic DC as fusion partner cells provide the co-stimulatory molecules required for T cell activation [19–22] and, through the allogeneic MHC class II molecules, will recruit and activate helper T cells to support the induction of tumour-specific cytotoxic T lymphocytes (CTL) (Table 1) [23–25]. As shown in earlier studies, productive collaboration of precursor CTL and helper T cells, and induction of CTL responses requires that the antigens for both T cell types are presented by the same antigen-presenting cell [23]. Consequently, it was found in animal experiments that only fused tumour and antigenpresenting cells but not mixtures of these cells induce tumourspecific immune responses and rejection of the tumour [9]. The clinical outcome of vaccination therapy depends on induction of effective tumour-specific CTL as well as on the susceptibility of the tumour cells to the effector mechanisms of these T cells. To test these denominators of therapeutic

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Table 1 Principle of hybrid cell vaccination for cancer immune therapy Utilisation of the complete antigenicity of the tumour cells including shared tumour-associated antigens and tumour-specific mutation via the tumour cell fusion partner with its tumour-specifically loaded MHC class I molecules Recruitment and activation of the T cell help required for induction of tumour-specific CTL via the allogeneic MHC class II molecules of the DC fusion partner Provision of the required co-stimulatory signals via the mature DC fusion partner

efficacy we initiated a clinical trial with melanoma stage III and IV patients and monitored the clinical outcome as well as the immunological effects of the vaccination [18].

2. Patients, materials and methods 2.1. Patients Twenty patients with metastatic melanoma (3 stage III, 17 stage IV) had been enrolled, with written informed consent, in the study, which was approved by the institutional ethics committee of the Charit´e, Humboldt University (EKV 970/98). Complete staging was done before the first and 4 weeks after the third vaccination to establish the clinical response status. The cellular immune response capacity of the patients was assessed with delayed-type hypersensitivity (DTH) tests for recall antigens and only patients with at least one response were enrolled. Seventeen patients were treated according to protocol and considered in this report. All patients had received multiple pretreatments including chemotherapy (six patients), chemoimmunotherapy (two pa-

tients), immunotherapy with interferon-␣ and/or interleukin2 (13 patients), and irradiation of cerebral or mediastinal lesions (two patients). Chemotherapies were terminated at least 3 months, immunotherapies at least 4 weeks before vaccination. The patients received no immune-suppressive treatments, e.g., corticosteroids, immediately before or during the study. None of the patients had active brain metastases, autoimmune diseases detectable by anti-nuclear and anti-thyroid autoantibodies or, as by blood diagnostics, impaired organ functions. At least one metastasis was excised for vaccine preparation. The patients usually had multiple metastases left that could not be removed. 2.2. Hybrid cell vaccination Autologous tumour cells and allogeneic dendritic cells from healthy donors were generated and electrofused as described in details elsewhere [18,26–28]. The hybrid cell vaccines were irradiated and injected every 4 weeks intradermally into healthy skin at the upper arms, upper legs or abdomen away from tumours, near unaffected lymph nodes. The patients received between 3 and 25 vaccinations (average 8–9). 2.3. Immunohistochemistry For analysis of antigen expression formaldehyde-fixed, paraffin-embedded sections were stained after deparaffinisation with xylene with anti-Melan A/MART-1 (DAKO, Hamburg, Germany), 77-b for MAGE-1, 57-b for MAGE-3 (both from C. Sch¨afer, Basle, Switzerland), HMB-45 for gp100, T311 for tyrosinase (Novocastra, Newcastle, UK), and anti␤2 microglobulin and anti-TAP1 (both kindly provided by B.

Fig. 1. Design of the hybrid cell vaccines for cancer immune therapy.

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Seliger, Mainz, Germany), and counterstained with Mayer’s hematoxylin.

Table 2 Clinical results of hybrid cell vaccination of 17 melanoma stage III and IV patients [18]

2.4. Immune monitoring

Patients Merieux positive Progressive disease

17 17 9

Clinical responses Complete responses Mixed responses Stable disease Median survival all patients Median survival responders (CR, MR, SD) Median survival non-responders (PD)

8 1 1 6 22.4 months 28.0 months 12.5 months

PBMC were analysed for tumour-specific T cells by intracellular interferon-␥ staining30 after stimulation with specific peptide (10 ␮g/ml) in 500 ␮l culture medium containing 10% heat-inactivated foetal calf serum (FCS) for 2 h at 37 ◦ C plus 4.5 h including Brefeldin A (10 ␮g/ml), or by MHC tetramer staining as described [18,29]. Frequencies below 0.03% of the CD8+ T cells were considered fluctuations in the background. For tetramer-interferon ␥ doublestaining, cells were first stained with the tetramers then fixed and permeabilised for intracellular staining of interferon ␥. The peptides Melan A/MART-127–35 AAGIGILTV, Melan A/MART-126–35(27L) ELAGIGILTV, tyrosinase369–377 YMNGTMSQV, gp100154–162 KTWGQYWQV, gp100209–217 ITDQVPFSV, gp100280–288 YLEPGPVTA, gp100457–466 LLDGTATLRL, gp100476–485 VLYRYGSFSV, MAGE1161–169 EADPTGHSY, MAGE-3168–176 EVDPIGHLY, MAGE-3271–279 FLWGPRALV and CMV pp65495–503 NLVPMVATV [3,29] had been custom-synthesised by EMCmicrocollections, T¨ubingen, Germany.

3. Results 3.1. Clinical results of the hybrid cell vaccination Because of the rapid fatal courses of malignant melanoma in stage III and stage IV patients, mixed responses (disappearance of metastases with appearance and growth of new ones) and stable disease were counted as clinical responses in addition to objective complete and partial responses (Table 2). Patient 1 had a complete remission, i.e., regression of all sub-

cutaneous metastases after the first vaccination, which is still maintained after 58 months. Patient 2 experienced a mixed response with regression of numerous subcutaneous metastases coinciding with the occurrence of new lesions. Patients 3, 7, 8, 10, 12 and 13 responded with stabilisation of previously progressive disease. The stable disease status lasted for 6–15 months. Thus, 8 of the 17 patients responded clinically with complete or partial response, or stable disease with times to progression between 6 and 27 months. The median survival time for all 17 patients from the time of enrolment in the trial was 22.4 months (range 5–58 months), of the 8 responders including the stable disease patients 28 months (range 12–58 months), and of the 9 progressive disease patients 12.5 months (range 5–22 months) which is somewhat longer than the median survival time of 8 months known for melanoma stage IV patients from the time of diagnosis. The side effects were mild (data not shown). Fourteen of the 17 patients developed signs of cellular immune responses at the injection sites such as erythema and induration which was strongest after 24 h and waned after 48 h. Three patients had fever for not more than 1 day, two pruritus and one patient transient arthralgia. Otherwise, physical examinations and blood diag-

Table 3 Immunological effects of hybrid cell vaccination [18] Patient no.

Antigen gp100

1 2 3 5 6 8 9 10 12 13 14 15 16 19

Melan A/MART-1

Tyrosinase

MAGE-1

Expressed

CTL induced

Expressed

CTL induced

Expressed

CTL induced

Expressed

+ + + + + + + + + + + + + +

+ + + + +

+ + + + +

+ + + + +

+ + + + +

+ + + + +

+

+ n.t. + n.t. + + + +

+ + + + + + + +

+ n.t. + n.t. + + + +

+ + + + + + + +

n.t. + n.t. + + +

+ + +

MAGE-3 CTL induced n.t. n.t. n.t. n.t. n.t. n.t.

Expressed

CTL induced

+ + + +

+

+ + +

n.t. + + + + +

+

+

n.t. n.t. n.t.

n.t.: Not tested. Clinical responses were recorded for patient 1 (CR), patient 2 (MR), and patients 8, 10, 12 and 13 (S.D.).

+

+

+

+

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nostics revealed no indication of serious side effects. Also, no signs of autoimmune disease were detected. 3.2. Immunological effects of the hybrid cell vaccination All metastases used for vaccine production were analysed by immunohistochemistry for antigen expression. These metastases expressed at least two of the melanoma-associated antigens gp100, Melan A/MART-1, tyrosinase, MAGE-1 and MAGE-3 (Table 3a). Expression of ␤2 -microglobulin and TAP-1 was confirmed for all. To examine whether and to what degree this antigenicity translates into induction of tumourspecific T cell responses we monitored the frequencies of CD8+ T cells with specificity for nine HLA-A2.1 and two HLA-A1-restricted T cell epitopes of the above antigens [3]. The specific T cells in the peripheral blood of the patients were visualised by intracellular staining for interferon-␥ after stimulation with the respective peptides, and enumerated by flow cytometry [29,30]. These analyses were done before, and 24 and 48 h after vaccination for at least the first three treatment cycles. As only exception, for patient 3 the T cell responses were analysed for cycles 20–23. The T cell responses were monitored for 14 of the 17 patients and 45 of the total of 147 vaccinations resulting in 1125 assays to document the specific responses to 9 HLA-A2.1 and 2 HLAA1-restricted epitopes chosen for these analyses (Table 3). Patients 11 and 20 were negative for HLA-A1 and HLAA2.1, and the cells of patient 7 could not be recovered for the analyses. Eleven of the 14 patients studied mounted tumourspecific CD8+ T cell responses in their peripheral blood. The induction of T cells was, with exception of some of the Melan A/MART 1-specific responses, transient and peaked 24–48 h after vaccination. Of the three patients without detectable T cell responses, patients 10 and 13 expressed only HLA-A1 so that only two epitopes could be tested. Moreover, the vaccine for patient 10 did not express any and that of patient 13 only one of the HLA-A1-restricted antigens. Patients 8 and 13 had received chemotherapy before which can cause elimination of tumour-reactive T cells as reported by Lee et al. [4]. The 11 immune-responsive patients reacted to between three and five different antigens and to a varying number of T cell epitopes. Overall, in 149 of the 375 analysed cases for which the antigenicity of the vaccines could be compared to the induction of T cell responses induction or enhancement of the frequencies of epitope-specific T cells was established. Fiftyfive of these 149 responses were detected after injection of a vaccine composed of autologous tumour cells and dendritic cells that shared the MHC allomorphs HLA-A2.1 or HLAA1 with the patient. For 94 of the 149 responses this was not the case. For all antigens and in all patients responses were induced where no specific T cells had been detectable prior to vaccination (80 of the 149 responses detected). In the 69 other cases already elevated frequencies of tumour-specific T cells were enhanced. Many of the responses, induced in patients for whom no specific T cells could be detected before vaccination, were detectable within 48 h after the first

vaccination. These responses are too fast for primary T cell responses and, therefore, likely indications of already ongoing immune responses in the patients. For six patients and altogether 11 different epitope-specific responses the T cells were not detectable before or after the first vaccination but only after the second or third boost. These cases are likely examples for primary immune responses induced by the vaccination. As control, the T cell responses to the CMV epitope pp65495–503 NLVPMVATV were analysed. In no case did the frequency of T cell with specificity for this epitope change after hybrid cell vaccination (data not shown). In 36 of 56 cases specific CTL induction correlated with the expression of the corresponding antigen by the tumour cells used for vaccine production (Table 3). In eight cases antigen expression failed to induce T cell responses. In three patients T cells responded to an epitope whose antigen, MAGE3, was not detected in the vaccine. These three patients responded well to several other antigens so that the anti-MAGE3 responses could reflect epitope spreading [31], i.e., responses induced via antigens expressed by tumour cells targeted in the patients. The frequencies of the tumour-specific T cells ranged up to 4.69% as in the case of the anti-Melan A/MART-1 response in patient 3. The time course of the T cell responses over five vaccination cycles in patient 5 and the analyses of the responses after more than 20 vaccinations for patient 3 show that even after prolonged treatment T cells can be stimulated in the patients. A comparison of the immunological responses with the clinical responses failed to show any correlation, meaning, there was no difference between clinical responder and nonresponder patients with respect to the frequencies of tumourspecific T cells induced by vaccination or to the number of antigens and T cell epitopes targeted. 3.3. Immune evasion For six patients it was possible to analyse and compare antigen expression in metastases before and in progressing lesions during or after vaccination. These comparisons revealed clear evidence for immune evasion in all cases including one progressive disease patient (patient 5), the mixed response patient 2 and 4 stable disease patients (patients 3, 7, 10 and 13). In five patients tumour-associated antigens, in two ␤2 -microglobulin and TAP-1 were lost in the progressing lesions (Table 4). These defects have profound effects on the antigenicity of the tumours and apparently result from selection by immune responses against several tumour-associated antigens.

4. Discussion The conclusions from the study presented herein are: • Hybrid cell vaccination can induce clinical responses in late stage malignant melanoma patients.

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Table 4 Immune evasion by the tumour cells in patients receiving hybrid cell vaccination [18] Patient no.

Antigen gp100

2 3 5 7 10 13

Lost Lost Lost Lost

Melan A/MART-1

Tyrosinase

Lost Lost

Lost Lost Lost Lost

MAGE-1

MAGE-3

B2m

TAP-1 Lost Lost

Lost

Lost Lost

Lost Lost

Lost: the respective antigen was expressed by the tumour used for production of the hybrid cell vaccine but was lost in metastases emerging and growing under vaccination. Shading indicates clinical response patients with MR (patient 2) and SD (patients 3, 7, 10, 13).

• Although cure is the exception, the patients experienced a remarkable extension of survival time compared to historic controls. • Hybrid cell vaccination induced strong and extensive immunological responses in the majority of the patients. These immunological responses are directed against a variety of tumour-associated antigens. • However, the immunological and clinical responses do not correlate in that clinical non-responders and responders react equally with induction of high frequencies of circulating and functionally active T cell. • This lack of correlation of immunological and clinical responses is likely due to immune evasion. • The pattern of losses in antigenicity of the tumour cells is complex and corresponds to the complex antigenicity of the hybrid cell vaccine used for treatment. Tumour-specific CD8+ T cells are the most important effector cells in anti-tumour immune responses and, therefore, prime targets for the development of new immunotherapies for the treatment of cancer. They have been demonstrated in patients with different cancers and it was shown in a number of cases that the frequencies of these cells change with disease progression but also in response to therapeutic interventions. Lee et al. have published one case where tumour-specific CD8+ T cells disappeared after cytostatic chemotherapy [4]. On the other hand, it has been reported by several investigators that the frequencies of tumour-specific T cells can increase upon vaccination [32]. Therapeutic vaccination for the treatment of cancer aims at inducing new responses to antigen not yet targeted by the immune system of the patient as well as modulating and boosting existing but inefficient T-cellular immune responses. It is believed that, to cope the disease effectively, cancer vaccines should address and activate high frequencies of cytolytic T cells with specificities for a broad spectrum of tumour-associated antigens and T cell epitopes. Moreover, considering the HLA genetics of T cellmediated immunity and the heterogeneity of the tumours, it seems important to develop vaccines that are suited for individualised therapy. The hybrid cell vaccination is designed to meet these criteria. Fusion of the patients tumour cells with mature dendritic cells creates a hybrid that combines the tumour’s antigenicity with the immune-stimulatory capacity of dendritic cells. For the trial reported here we used allogeneic

dendritic cells in order to stimulate strong helper T cell responses to support the induction of tumour-specific CD8+ cytotoxic T cells. To test whether hybrid cell vaccination indeed induces tumour-specific CTL we evaluated, in addition to the clinical responses, the frequencies of tumour-specific T cells in the peripheral blood of the patients and compared the specificities of these T cells with the antigenicity of the tumour cells used for vaccine production. These analyses revealed induction of a broad range of tumour-specific T cells in the peripheral blood of the vaccinees. In most cases the specificity of the T cell responses corresponded to the expression of the respective antigen by the tumour cells used for vaccine production. Many patients had already elevated levels of tumour-specific CTL in their peripheral blood before vaccination, which, however, usually increased strongly upon vaccination. Often the pre-vaccination frequencies were below detection limit but became detectable within 24–48 h after vaccination, which indicates that the responding cells had been primed already in the patients before vaccination. These secondary T cell responses are likely indications of on-going anti tumour immune responses in the patients. In other cases responses were seen only after the second or even the third booster. These may be primary responses induced by the vaccines. While it needs further investigations to clarify the exact mode of antigen presentation and induction of T cell responses in the cancer patients, it appears likely that the hybrid cells themselves are the critical antigen-presenting cells. It was already established before with animal model experiments that only the hybrid cells but not mixtures of tumour and antigen-presenting cells can induce anti-tumour immune responses [9]. In the present study the vaccines were always injected at sites away from the tumour that do not drain into the same lymph nodes. It is, therefore, unlikely that injected dendritic cells migrate to the tumour sites to pick up and cross-present antigens from indigenous tumours. This conclusion is supported by the fact, that the dendritic cells used for the generation of the vaccines often did not express HLA-A2.1 or HLA-A1, the MHC molecules that present the epitopes used for immune monitoring. Yet, induction of specific T cells was seen. It has been insisted that injection of large quantities of allogeneic dendritic cells might cause nonspecific systemic immune stimulation, which could affect the specific anti-tumour immunity. However, extensive analyses in the context of earlier hybrid cell vaccination trials with

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melanoma patients have never produced any sign of vaccineinduced changes in general immune status indicators such as cellular compositions or cytokine profiles of the peripheral blood [16]. As the frequencies of tumour-specific but not of CMV-specific T cells increased upon vaccination we would exclude such non-specific systemic effects of the vaccines also for the present study. A number of other vaccine designs, which often are based on DC have been developed and, in some cases, tested in clinical trials. Vaccines with defined antigens such as synthetic peptides [32] or protein [33], or DNA coding for these antigens [34] had, so far, limited success. One exception is a trial with DC loaded with mixtures of synthetic peptides for melanoma-associated antigens [35]. These vaccines are restricted to known tumour-associated antigens and T cell epitopes. Strategies that aim at utilising the specific antigenicity of the patients’ tumours like hybrid cell vaccination include DC loaded with lysates [35] or apoptotic bodies [36] of the tumour cells. In a direct comparison with hybrid cell vaccines, Galea-Lauri et al. [37] found these approaches inferior or even ineffective. A promising vaccine designed to represent the individualised antigenicity of a patient’s tumour uses DC transfected with tumour RNA [38]. The degree and complexity of T cell responses induced by vaccination is similar in patients who responded clinically and those who did not. This lack of correlation of immunological and clinical responses is best explained by immune evasion or later silencing of the activated T cells in the tumours. In fact, in all investigated cases tumour progression was associated with defects in antigen expression and/or presentation. Basis for such immune evasion is the heterogeneity of tumours with antigen-loss variants already present in antigen-expressing lesions. It has been shown that this heterogeneity increases with disease progression [6], meaning, at late disease stages antigen-loss variants of tumour cells are more likely than in early stages. Cure of late stage cancer might, therefore, remain a challenge. However, at early stages, cure might become possible and future vaccine developments need to make early treatments possible. Besides loss of antigenicity, immune suppression in the tumour microenvironment has been described to interfere with efficient immune responses [39]. Anergy seen for some tumour-specific T cells in this study (see reference [18] for the data) might result from such immune suppression but could also indicate exhaustion of the immune responses by chronic antigen exposure. However, long-term stable disease maintained by regular vaccination for a high proportion of the patients, which correlates with sustained T cell responses implies that T cell activity can be maintained despite the chronicity of the disease. It also suggests that vaccination might be suitable for maintenance therapy and disease management where cure is not possible. The segregation of the induction of tumour-specific immune responses and clinical responses, and the correlation of this discrepancy with immune evasion mechanisms, on the one hand, can be seen to testify to the tumouricidal potency of the T cell responses but,

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