Vaccination of Stage IV patients with allogeneic IL-4- or IL-2-gene-transduced melanoma cells generates functional antibodies against vaccinating and autologous melanoma cells

The antibody (Ab) response to allogeneic Me14932 and autologous melanoma cells was analyzed in 13 Stage IV (AJCC) melanoma patients immunized with Me14932 cells transduced with the IL-4 (Me14932/IL-4) ( n=10) or IL-2 (Me14932/IL-2) ( n=3) gene. No Ab response was observed before the 4th vaccination. Among 8 patients that received four vaccinations, 3/5 patients vaccinated with Me14932/IL-4 cells developed Ab (IgG and/or IgM) to Me14932 ( n=3) and to autologous ( n=2) melanoma cells, and 2/3 patients vaccinated with Me14932/IL-2 cells developed Ab (IgG) to Me14932, but not to autologous melanoma cells. Further, among these 5 responding patients, circulating Ab against the HLA-A3 allele, expressed only on vaccinating cells, were identified in the immune sera of 4 patients immunized with Me14932/IL-4 ( n=2) or Me14932/IL-2 ( n=2) cells. These sera mediated antibody-dependent cell cytotoxicity (ADCC) of Me14932 cells, and a direct correlation ( r=0.85; P=0.03) between intensity of staining (IgG) and extent of lysis was found. Immune serum of one of these patients also induced ADCC of autologous melanoma cells, and serum from another patient mediated complement cytotoxicity of Me14932, but not of autologous melanoma cells. Thus, Abs against vaccinating and autologous melanoma cells were generated in 62% of patients after four vaccinations with cytokine-transduced melanoma cells. These findings demonstrate that the identification and titration of alloreactive Ab helps to monitor the extent of immunization against cellular vaccines, while the induction of Ab reactive to antigens shared between vaccinating and autologous melanoma cells may contribute to their therapeutic efficacy.     

Pilot study of treatment of biochemotherapy-refractory stage IV melanoma patients with autologous dendritic cells pulsed with a heterologous melanoma cell line lysate

Eleven AJCC stage IV melanoma patients with progressive disease after treatment with biochemotherapy were treated with autologous dendritic cells pulsed with heterologous tumor cell lysates. The vaccine used mature DCs (CD1a⁺⁺⁺, CD40⁺⁺, CD80⁺⁺, CD83⁺, and CD86⁺⁺⁺) generated from peripheral blood monocytes in the presence of GM-CSF and IL-4. After 7 days, DCs were matured with a defined cocktail of cytokines (IL-1+IL-6+TNF-+PGE₂) and simultaneously pulsed with lysates of heterologous melanoma cell lines, for 2 days. A total of 4×10⁶ DCs was injected monthly under ultrasound control in an inguinal lymph node of normal appearance. The study was closed when all patients died as a consequence of tumor progression. No sign of toxicity was observed during the study. One patient experienced a partial response lasting 5 months, and two patients showed a mixed response which lasted 3 months. The median survival of the whole group was 7.3 months (range 3–14 months). This vaccination program had specific antitumoral activity in highly pretreated and large tumor burden stage IV melanoma patients and was well tolerated. The clinical responses and the median survival of the group of patients, together with the low toxicity of our DC vaccine, suggest that this approach could be applied to earlier AJCC stage IV melanoma patients.     

T cell recognition of melanoma antigens in association with HLA-A1 on allogeneic melanoma cells

Previous studies have shown that recognition of melanoma by cytotoxic T lymphocytes may be restricted by HLA-A1, A2 and other HLA antigens. The present study examined the cytotoxic specificity and major histocompatibility complex restriction of cloned cytotoxic T lymphocytes (CTL) isolated from a patient with the HLA phenotype A3,31 who had been immunized with a vaccine prepared from HLA-A1,3 melanoma cells. Cytotoxic assays against HLA-typed allogeneic melanoma cells indicated that cloned CTL from the patient were able to kill allogeneic melanoma cells expressing HLA-A1 but not other HLA-A1-positive cells. Studies on a representative clone indicated that proliferation and cytokine (tumour necrosis factor ) production in response to melanoma cells was also associated with HLA-A1 on melanoma cells. Response to the melanoma cells was associated with interleukin-4 (IL-4) rather than IL-2 production. The antigen recognized in the context of HLA-A1 on allogeneic melanoma cells was detected in cytotoxic assays on cells from 9 of 12 HLA-A1⁺ melanoma cell lines and did not appear to be the product of the MAGE-1 or-3 genes. These findings suggest that T cells can recognize melanoma antigens in the context of alloantigens and that allogeneic vaccines containing immunodominant alloantigens may generate CTL that are ineffective against autologous melanoma. The study does not, however, exclude the possibility that CTL with specificity to the latter may be activated by allogeneic vaccines, and further studies are needed to answer this question.     

Vaccination with autologous dendritic cells pulsed with multiple tumor antigens for treatment of patients with malignant melanoma: results from a phase I/II trial

Background aimsDendritic cells are regarded as the most effective antigen presenting cells and coordinators of the immune response and therefore suitable as vaccine basis. Here we present results from a clinical study in which patients with malignant melanoma (MM) with verified progressive disease received vaccination with autologous monocyte-derived mature dendritic cells (DC) pulsed with p53, survivin and telomerase-derived peptides (HLA-A2⁺ patients) or with autologous/allogeneic tumor lysate (HLA-A2^? patients) in combination with low-dose interleukin (IL)-2 and interferon (IFN)-α2b.ResultsOf 46 patients who initiated treatment, 10 stopped treatment within 1–4 weeks because of rapid disease progression and deterioration. After 8 weeks, 36 patients were evaluable: no patient had an objective response, 11 patients had stable disease (SD); six had continued SD after 4 months, and three patients had prolonged SD for more than 6 months. The mean overall survival time was 9 months, with a significantly longer survival (18.4 months) of patients who attained SD compared with patients with progressive disease (PD) (5 months). Induction of antigen-specific T-cell responses was analyzed by multidimensional encoding of T cells using HLA-A2 major histocompatibility complex (MHC) multimers. Immune responses against five high-affinity vaccine peptides were detectable in the peripheral blood of six out of 10 analyzed HLA-A2⁺ patients. There was no observed correlation between the induction of immune responses and disease stabilization. A significant lower blood level of regulatory T cells (CD25^high CD4 T cells) was demonstrable after six vaccinations in patients with SD compared with PD.ConclusionsVaccination was feasible and safe. Treatment-associated SD was observed in 24% of the patients. SD correlated with prolonged survival suggesting a clinical benefit. Differences in the level of regulatory T cells among SD and PD patients could indicate a significant role of these immune suppressive cells.     

Long-term outcomes in patients with metastatic melanoma vaccinated with melanoma peptide-pulsed CD34+ progenitor-derived dendritic cells

Between March 1999 and May 2000, 18 HLA-A*0201⁺ patients with metastatic melanoma were enrolled in a phase I trial using a dendritic cell (DC) vaccine generated by culturing CD34⁺ hematopoietic progenitors. This vaccine includes Langerhans cells. The DC vaccine was loaded with four melanoma peptides (MART-1/MelanA, tyrosinase, MAGE-3, and gp100), Influenza matrix peptide (Flu-MP), and keyhole limpet hemocyanin (KLH). Ten patients received eight vaccinations, one patient received six vaccinations, one patient received five vaccinations, and six patients received four vaccinations. Peptide-specific immunity was measured by IFN-γ production and tetramer staining in blood mononuclear cells. The estimated median overall survival was 20 months (range: 2–83), and the median event-free survival was 7 months (range: 2–83). As of August 2005, four patients are alive (three patients had M1a disease and one patient had M1c disease). Three of them have had no additional therapy since trial completion; two of them had solitary lymph node metastasis, and one patient had liver metastasis. Patients who survived longer were those who mounted melanoma peptide-specific immunity to at least two melanoma peptides. The present results therefore justify the design of larger follow-up studies to assess the immunological and clinical outcomes in patients with metastatic melanoma vaccinated with peptide-pulsed CD34-derived DCs.Joseph W. Fay and A. Karolina Palucka have equally contributed to this work     

Active specific immunotherapy with hapten-modified autologous melanoma cell vaccine

 We have developed a novel approach to cancer immunotherapy – an autologous whole-cell vaccine modified with the hapten dinitrophenyl (DNP). This approach elicits significant inflammatory responses in metastatic sites and some objective tumor responses. Post-surgical adjuvant immunotherapy with DNP-modified melanoma vaccine in a setting of micrometastatic disease produces significant survival prolongation in stage III melanoma patients. Histologically, the inflammatory responses of the tumor consist of infiltration by lymphocytes, the majority of which are CD8⁺, HLA-DR⁺ T cells. T cells from these lesions tend to have mRNA for interferon γ. T cell receptor analysis suggests that the tumor-infiltrating T cells are clonally expanded. DNP-modified vaccine also induces T cells in the peripheral blood, which respond to DNP-modified autologous cells in a hapten-specific, MHC-restricted manner. Moreover, a T cell line generated from these lymphocytes responded to only a single HPLC fraction of MHC-associated, DNP-modified tumor peptides. Since inflammatory responses in metastases were not consistently associated with dramatic tumor regression, we considered the possibility of immunosuppression at the tumor site. We found that mRNA for the anti-inflammatory cytokine, interleukin-10 (IL-10) is expressed in most metastatic melanoma tissues and subsequently demonstrated that IL-10 protein is produced by melanoma cells. Thus the efficacy of DNP vaccine could be further enhanced by inhibition of IL-10 production or binding. Finally, we expect these results obtained with melanoma to be applicable to other human cancers. Received: 6 August 1996 / Accepted: 20 September 1996     

The prognostic significance of immune changes in patients with renal cancer,melanoma and colorectal cancer,treated with interferon α2b

 In the present study we evaluated the response rate and the immunorestorative properties of interferon α2b (IFnα2b) administered to patients with advanced renal cell carcinoma (RCC), melanoma (MEL) or colorectal cancer (CC). We studied the immune status and correlated it with clinical responses. Thirty-five patients with advanced RCC, and 14 with MEL were treated with recombinant INFα2b. The dose was increased progressively from 5×10⁶ IU/day in the first week (three times every week) to 10×10⁶ IU/day in the second week and thereafter to 15×10⁶ IU/day subcutaneously. In patients with CC INFα2b was given at 5×10⁶ IU/day every other day (three times every week); these patients also received (together with INF) leucovorin 200 mg m^–2 day^–1 in a 1-h i. v. infusion every week, and mid-infusion 400 mg/m² 5-FU was administered as an intravenous bolus every week. The response rate was as follows: for RCC, 6 patients achieved partial response (PR), 10 stable disease (SD), and 21 progressed (PD); for MEL, 5 patients achieved PR and 9 PD; for CC, 6 achieved PR, 5 SD, and 9 PD. In all patients blood was withdrawn prior to INFα2b treatment and then monthly. T lymphocytes, after isolation from peripheral blood, were tested for proliferation in the autologous mixed-lymphocyte reaction and allogeneic mixed-lymphocyte reaction, interleukin-2 (IL-2) production, expression of IL-2 receptors during the allogeneic-mixed-lymphocyte reaction, and the production of IL-1 by peripheral blood monocytes. Striking increases were demonstrated in all parameters 2 months after treatment with INFα2b. In comparison to normal controls, all patients with the malignant neoplasms presented decreased (>45%) mean values of the immunological parameters under investigation (P 0.0001). Responders (patients with RCC, MEL, and PR) presented lower mean values of all the parameters studied than did non-responders (P 0.0001). Patients with CC presented the lowest mean values of the parameters than did the other patients (RCC, MEL) (P 0.0001). After therapy with INFα2b, patients with RCC experiencing PR showed a mean increase of more than 30% (P 0.0001). Patients with SD showed a mean increase of about 20% (P 0.0001), and those with PD showed a 6% increase in the immunological parameters under investigation. Patients with MEL experiencing PR showed a mean increase of more than 30% and patients with PD a decrease of more than 10% (P 0.0001). All patients, regardless of the clinical response, achieved an increase of more than 60% (P 0.0001). Administration of IFNα2b resulted in a marked potentiation of a deficient cellular immune response in vitro in those patients with RCC and MEL who responded to the treatment. On the other hand, non-responders demonstrated a decrease in the examined parameters and, in some, deterioration of the already depressed immunological functions was observed. This observation can have prognostic significance regarding clinical response of INF. In contrast, our findings show that the immune stimulation associated with INFα treatment in all our CC patients did not predict an improved clinical outcome. There are several theoretical explanations for this discrepancy. Received: 30 November 1996 / Accepted: 25 June 1996     

Generation of human-melanoma-specific T lymphocyte clones defining novel cytolytic targets with panels of newly established melanoma cell lines

Melanoma is a cancer where the immune system is believed to play an important role in the control of malignant cell growth. To study the variability of the immune response in melanoma patients, we derived melanoma cell lines from several HLA-A2⁺ and HLA-A2^– patients. The melanoma cell lines studied were designated FM3, FM6, FM9, FM28, FM37, FM45, FM55_P, FM55_M1 and FM55_M2 and were established from eight metastatic tumors as well as from one primary tumor from a total of seven different patients. On the basis of the ability of tumor cells to induce specific cytotoxic T lymphocytes (CTL) from peripheral blood lymphocytes (PBL) in mixed lymphocyte/tumor culture with HLA-A2⁺ melanoma cells, the FM3 cell line was characterized as highly immunogenic. To investigate the expression of different melanoma-associated antigens recognized by CTL on different melanoma cell lines, we selected the cell line FM3 for restimulation and further T cell cloning experiments. The lytic activity of CTL clones with good proliferative activity was examined using a panel of HLA-A2⁺ and HLA-A2^– melanoma cell lines. None of the tested HLA-A2^– melanoma cell lines were susceptible to lysis by the CTL clones, whereas allogeneic HLA-A2⁺ melanoma cell lines were lysed only by a few CTL clones. On the basis of their reactivity with different melanoma cell lines, it was possible to divide the present CTL clones into at least four groups suggesting the recognition of at least four different antigens. Three of these target structures probably are different from already-described HLA-A2-restricted melanoma-associated antigens, because their expression in the different melanoma cell lines do not correlate with the recognition of melanoma cells by these CTL. The results first indicate that poorly immunogenic melanoma cells may express melanoma-associated antigens, and also suggest that, by using CTL clones obtained against different HLA-class-I-matched melanoma cells, it is possible to define such antigens.     

Adjuvant adoptive immunotherapy with tumour-infiltrating lymphocytes and modulated doses of interleukin-2 in 22 patients with melanoma, colorectal and renal cancer, after radical metastasectomy, and in 12 advanced patients

 Adoptive tumour infiltrating lymphocytes (TIL) in combination with a modulated dosage of interleukin-2 (IL-2) can be used with acceptable toxicity in the treatment of immunogenic tumours. Following an experience of reinfusion in advanced melanoma, colorectal and renal cancer patients, treatment was given to disease-free patients after metastasectomy. The high risk of relapse and favourable ratio between reinfused TIL and possible microscopic residual disease determined this choice of adjuvant treatment. A group of 12 patients with advanced disease (7 melanoma, 4 colorectal carcinoma, 1 kidney carcinoma) were treated with TIL (median 5.8×10¹⁰ cells) and IL-2 (West’s schedule) modulated towards a lower dosage (from 12 to 6 MIU/day) in order to maintain an acceptable level of toxicity. As treatment was well tolerated, it was offered to another 22 patients in an adjuvant setting after metastasectomy (11 melanoma, 10 colorectal carcinoma, 1 renal cancer), the median dose of TIL reinfused being 4.95×10¹⁰ cells. No objective response was observed in advanced patients: all patients progressed after a median of 1.5 months (0–8 months) and median survival was 8 months (3–22+ months). Thirteen patients from the second group are still disease-free after a median of 23+ months (9+–47+ months). The remaining 9 patients relapsed after a median of 5 months (3–18 months). Toxicity was moderate as clinical and hepatic/renal function parameters were used to assess the need for dose reductions. Consequently, there was great diversity in IL-2 dosages administered. In particular, there seemed to be a difference in IL-2 doses administered between disease-free cases and those who progressed (17.5 MIU/day versus 7 MIU/day in melanoma patients; 11.2 MIU/day versus 7.1 MIU/day in colorectal cancer patients). By contrast, no differences were observed between number of TIL reinfused and clinical response. Phenotypical characteristics of reinfused TIL were similar to those reported in the literature: 97% were CD3 and 92% were CD8. Aspecific cytolytic activity was evaluated on 12 cases whereas, in 2 melanoma cases, autologous tumour tissue was available for the specific cytotoxicity test. Perforin levels in TIL measured at the end of culture were generally high or very high. Cytokine levels were measured on the supernatant at the end of culture, with an estreme variability in results. Finally, ζ chain and p56^lck were histologically assessed on the resected tissue from which TIL were cultivated. There were virtually none of the former and a complete absence of the latter, which concurs with data reported in the literature. The same immunocytochemical analysis was carried out on TIL at the end of culture. This time an almost complete restoration of both functions was seen, especially in melanoma patients, who are still free from disease. The study is on-going and it has been decided to focus on disease-free patients after metastasectomy in order to increase the number and possibility of clinical and histological correlations.     

Clinical and immunological responses in metastatic melanoma patients vaccinated with a high-dose poly-epitope vaccine

Background

Safety and cellular immunogenicity of rising doses and varying regimens of a poly-epitope vaccine were evaluated in advanced metastatic melanoma. The vaccine comprised plasmid DNA and recombinant modified vaccinia virus Ankara (MVA) both expressing a string (Mel3) of seven HLA.A2/A1 epitopes from five melanoma antigens.

Methods

Forty-one HLA-A2 positive patients with stage III/IV melanoma were enrolled. Patient groups received one or two doses of DNA.Mel3 followed by escalating doses of MVA.Mel3. Immunisations then continued eight weekly in the absence of disease progression. Epitope-specific CD8+ T cell responses were evaluated using ex-vivo tetramer and IFN-γ ELISPOT assays. Safety and clinical responses were monitored.

Results

Prime-boost DNA/MVA induced Melan-A-specific CD8+ T cell responses in 22/31 (71%) patients detected by tetramer assay. ELISPOT detected a response to at least one epitope in 10/31 (32%) patients. T cell responder rates were <50% with low-dose DNA/MVA, or MVA alone, rising to 91% with high-dose DNA/MVA. Among eight patients showing evidence of clinical benefit—one PR (24 months+), five SD (5 months+) and two mixed responses—seven had associated immune responses. Melan-A-tetramer+ immunity was associated with a median 8-week increase in time-to-progression (P = 0.037) and 71 week increase in survival (P = 0.0002) compared to non-immunity. High-dose vaccine was well tolerated. The only significant toxicities were flu-like symptoms and injection-site reactions.

Conclusions

DNA.Mel3 and MVA.Mel3 in a prime-boost protocol generated high rates of immune response to melanoma antigen epitopes. The treatment was well tolerated and the correlation of immune responses with patient outcomes encourages further investigation.     

Phase I trial of combined treatment with ch14.18 and R24 monoclonal antibodies and interleukin-2 for patients with melanoma or sarcoma

Purpose: We conducted a phase I trial of interleukin 2 (IL-2) in combination with chimeric 14.18 (ch14.18) and murine R24 antibodies to determine the maximal tolerated dose (MTD), immunological effects, and toxicity of this treatment combination. Experimental Design: Twenty-seven patients with either melanoma (23 patients) or sarcoma (4 patients) were enrolled to receive a combination therapy with ch14.18 and R24 antibodies together with continuous infusion of Roche IL-2 (1.5×10⁶ U/m²/day, 26 patients) or Chiron IL-2 (4.5×10⁶ U/m²/day, 1 patient) given 4 days/week for 3 weeks. The antibodies ch14.18 (2–7.5 mg/m²/day) and R24 (1–10 mg/m²/day) were scheduled to be administered for 5 days during the second week of IL-2 therapy. Results: When given in combination in this study, the MTD for ch14.18 was 5 mg/m²/day and the MTD for R24 was 5 mg/m²/day. Dose-limiting toxicities were severe allergic reactions to both ch14.18 and R24 as well as pain related to ch14.18. This ch14.18 MTD was lower than the 7.5 mg/m²/day MTD previously determined for ch14.18 given alone with the same dose and schedule of IL-2. Immunological effects included the induction of lymphokine-activated killer (LAK) activity and antibody-dependent cell-mediated cytoxicity (ADCC). Anti-idiotype response to ch14.18 was seen in six patients, including two melanoma patients who had a partial response to treatment. In addition to two partial responses, four patients had a stable disease and one patient remained without any evidence of disease. Conclusions: Immunotherapy with IL-2 in combination with ch14.18 and R24 antibodies augments LAK function and ADCC measured in vitro in all patients. While there exist theoretical advantages of combining these two antibodies, the MTD of ch14.18 and of R24 were lower than the MTD of each antibody in prior studies evaluating single antibody therapy with IL-2. As such, the combination of these two antibodies together with IL-2 therapy appeared to influence the MTD and toxicity of each of the administered antibodies. This work is supported by NIH grants M01-RR03186, R01-CA32685, and P30-CA14520     

Simultaneous transduction of B7-1 and IL-2 genes into human melanoma cells to be used as vaccine: enhancement of stimulatory activity for autologous and allogeneic lymphocytes

 In order to construct an immunogenic cellular vaccine, we transduced three HLA-A*0201 human melanoma lines, selected for expression of classes I and II HLA, adhesion molecules and the T cell-defined melanoma antigens Melan/MART-1, gp100 and tyrosinase, with both interleukin-2 (IL-2) and B7-1 genes by the use of a polycistronic retroviral vector. The lines were selected to share only the HLA-A*0201 allele to avoid generation of strong alloreactivity in case of their multiple in vivo use in HLA-A*0201 + patients. Phenotypic and functional analysis of B7-1-IL2 transduced melanoma lines in comparison with B7-1 transduced and/or parental untransduced counterparts were then carried out. Tumor cells expressing either B7-1 or both genes did not change their original antigenic profile. From a functional point of view, expression of both genes in melanoma lines: (1) improved the response of anti-melanoma cytotoxic T lymphocytes (CTL) over singly transduced or untransduced melanoma cells when subthreshold levels of MHC-peptide complexes were expressed by melanoma cells; (2) conferred a distinct advantage in the ability to stimulate cytotoxicity and interferon-γ release by autologous and/or HLA-A*0201-compatible allogeneic lymphocytes; (3) allowed the generation of a high number of specific CTL by in vitro stimulation of lymphocytes of HLA-A*0201-melanoma patients. Thus, B7-IL2 gene-transduced melanoma lines appear to display a high immunogenicity and could be used as vaccine in melanoma patients. Received: 17 August 2000 / Accepted: 1 February 2001     

Analysis of a rare melanoma patient with a spontaneous CTL response to a MAGE-A3 peptide presented by HLA-A1

We describe an HLA-A1 melanoma patient who has mounted a spontaneous cytolytic T cell (CTL) response against an antigenic peptide encoded by gene MAGE-A3 and presented by HLA-A1. The frequency of anti-MAGE-3.A1 CTLp was 5×10⁻⁷ of the blood CD8 cells, with a dominant clonotype which was present in six out of seven independent anti-MAGE-3.A1 CTL clones. After vaccination with a recombinant poxvirus coding for the MAGE-3.A1 antigen, the blood frequency of anti-MAGE-3.A1 CTLp increased tenfold. Twenty-two independent CTL clones were derived. Surprisingly, only one of them corresponded to the dominant clonotype present before vaccination. Two new clonotypes were repeated 12 and 7 times, respectively. Our interpretation of these results is that the spontaneous anti-MAGE-3.A1 CTL response pre-existing to vaccination was polyclonal, and that the vaccine restimulated only some of these clones. To estimate the incidence of spontaneous anti-MAGE-3.A1 CTL responses in melanoma patients with a tumor expressing gene MAGE-A3, we measured the blood frequency of anti-MAGE-3.A1 T cells in 45 patients, and found only two clear responses.     

Co-expression of immunogenic determinants by the same cellular immunogen is required for the optimum immunotherapeutic benefit in mice with melanoma

 Tumor-associated T cell epitopes are recognized by T cells in the context of determinants specified by class I loci. Since the rejection of foreign histocompatibility antigens is known to enhance tumor immunity, immunization with a cellular vaccine that combined the expression of both syngeneic and allogeneic class I determinants could have important immunological advantages over a vaccine that expressed either syngeneic or allogeneic determinants alone. To investigate this question in a mouse melanoma model system, we tested the immunotherapeutic properties of B16 melanoma × LM fibroblast hybrid cells in C57BL/6J mice with melanoma. Like C57BL/6J mice, B16 cells expressed H-2K^b class I determinants and (antibody-defined) melanoma-associated antigens. LM cells, of C3H mouse origin, formed H-2K^k determinants along with B7.1, a co-stimulatory molecule that can activate T cells. The B16 × LM hybrid cells co-expressed H-2K^b and H-2K^k class I determinants, B7.1 and the melanoma-associated antigens. C57BL/6J mice with melanoma, immunized with the semi-allogeneic hybrid cells, developed CD8-mediated melanoma immunity and survived significantly (P<0.005) longer than mice with melanoma immunized with a mixture of the parental cell types. The failure of melanoma immunity to develop in mice injected with the mixture of parental cells indicated that co-expression of the immunogenic determinants by the same cellular immunogen was necessary for an optimum immunotherapeutic effect. Augmented immunity to melanoma in mice immunized with the semi-allogeneic hybrid cells points toward an analogous form of therapy for patients with melanoma. Received: 19 May 1997 / Accepted: 23 July 1997     

Histamine in immunotherapy of advanced melanoma: a pilot study

Sixteen patients with advanced metastatic malignant melanoma were treated with a high-dose infusion of interleukin-2 (IL-2; 18×10⁶ IU/m^–2 day^–1) together with daily subcutaneous (s.c.) injections of interferon (IFN; 3×10⁶ U/m^–2 day^–1) in 5-day cycles. Nine of these patients were given histamine (1 mg s.c.) twice daily during treatment with IL-2 and IFN. In the seven patients who did not receive histamine, one partial response (that is a reduction of more than 50% in the total tumour burden) was observed in a patient with skin and lymph node melanoma. In the eight histamine-treated patients evaluable for response, four partial responses were observed. Two other patients showed regression at one site of metastasis but tumours remained unchanged at other sites. Two histamine-treated patients showed complete resolution of extensive liver metastasis. Sites of response in histamine-treated patients also included the subcutis, lymph nodes, skeleton, spleen and muscle. Lung melanoma did not respond to histamine/IL-2/IFN. Three patients with lung tumours responded with significant (more than 50%) reduction of the volume of soft-tissue tumours, suggesting that the response to histamine may be organotropic. Survival was significantly prolonged in patients receiving histamine. Our data suggest that treatment with histamine may improve the antitumour efficacy of immunotherapy in metastatic melanoma.     

Spontaneous T cell responses to melanoma differentiation antigens from melanoma patients and healthy subjects

The spontaneous cytotoxic T cell responses to melanoma differentiation antigens and influenza matrix peptide were compared in 20 HLA-A2⁺ melanoma patients and 17 healthy A2⁺ individuals. Cytotoxic T lymphocyte (CTL) responses were determined by mixed lymphocyte peptide culture (MLPC) involving two stimulations of unfractionated peripheral blood lymphocytes (PBLs) with peptide in vitro. CTL responses to Melan-A 9-mer (amino acids 27–35, AAGIGILTV) peptide were detected in 4 out of 16 normal individuals, but in none of the melanoma patients. CTL specific for influenza matrix peptide were frequently found in both normal individuals and melanoma patients, suggesting that generalized immuno-suppression was not responsible for this difference. No significant responses were observed in either normal individuals or melanoma patients to Melan-A 10-mer (26–35, EAAGIGILTV), two gp100 epitopes (280–288, YLEPGPVTA; 457–466, LLDGTATLRL) and two tyrosinase epitopes (1–9, MLLAVLYCL; 368–376, YMDGMSQV). Melan-A (27–35)-specific CTL cells generated by normal individuals and melanoma patients recognized both synthetic peptide-pulsed T2 cells and two HLA-A2⁺, Melan-A⁺ melanoma cell lines (ME272, LAR1) in an antigen-specific, MHC class I restricted manner. T cells generated against Melan-A 9-mer were also able to recognize Melan-A 10-mer-pulsed target cells. Spontaneous CTL responses to Melan-A 9-mer from three known responder normal individuals were further evaluated over a prolonged time course (6–11 months). All 3 subjects demonstrated specific Melan-A 9-mer responses throughout the study period, although lytic activity fluctuated over time for a given individual. We found the MLPC assay to be reliable and easy to perform for monitoring T cell responses, although it may still not be sufficiently sensitive to detect low numbers of precursor T cells. Received: 21 May 1998 / Accepted: 23 July 1998     

Contrasting effects of T cell growth factors on T cell responses to melanoma in vitro

 Determinants of T cell responses to tumor cells remain largely unknown. In the present study we have used long-term cultures of human melanoma cells and autologous peripheral blood lymphocytes to examine the influence of cytokines with T cell growth activity on the phenotype and cytotoxic and proliferative response of T cells to melanoma. It was found that addition of interleukin-4 (IL-4) inhibited the response of CD8⁺ T cells and promoted the response of the CD4 subset. IL-2 or IL-7 was effective in increasing melanoma-specific cytotoxic T lymphocyte (CTL) activity in cultures where CD8 T cells were predominant, whereas IL-4 followed by IL-2 was most effective in cultures where CD4 T cells predominated. IL-10 or IL-12 inhibited proliferation and CTL activity against melanoma in long-term cultures. The effects of IL-12 were reproduced in long-term cultures of T cells stimulated with mAb against CD3 and were shown to depend on prior exposure of T cells to IL-12 before IL-2. As yet unidentified factors, such as co-factor expression on melanoma, appear to be as important as exogenous cytokines in determining the nature of T cell responses to melanoma. These results suggest that analysis of responses in long-term culture may assist in defining the role of key cytokines and other determinants of immune responses to melanoma. Received: 4 June 1996 / Accepted: 12 November 1996     

Low-dose IL-2 induces cytokine cascade, eosinophilia, and a transient Th2 shift in melanoma patients

Purpose: To assess changes in serum cytokine levels in patients treated concomitantly with or without systemic low-dose IL-2. Vaccination targeted CTL responses to peptide antigens, and IL-2 was coadministered to expand activated CTL. Paradoxically, CTL responses were diminished in patients after 2 weeks of IL-2. We hypothesized that changes in the cytokine milieu may have contributed to this result. Experimental design: Serum samples were studied from 37 patients enrolled in two clinical trials of a melanoma peptide vaccine administered with or without low-dose IL-2 therapy. Twenty-two patients enrolled in the MEL36 trial received six weekly vaccinations with the four-peptide mixture and were randomized to receive subcutaneous IL-2 (3×10⁶ IU/m²/day) daily for 6 weeks beginning either at week 1 (upfront group) or at week 4 (delayed group) of vaccine therapy. Fifteen patients on the MEL39 trial were treated with the same vaccine without concurrent IL-2 administration. Results: Circulating levels of IL-5 peaked 1 week after starting IL-2, followed 2 weeks later by a marked eosinophilia, correlating in magnitude with peak IL-5 serum levels. Levels of IFN, GM-CSF, IL-4, IL-10, and IL-12 had no observed relationship to IL-2 administration. At the time of the IL-5 serum peak, PBL responses to mitogen suggested a transient shift to Th2-dominance. Conclusions: Low-dose IL-2 appears to have induced a transient Th2-dominant secondary cytokine cascade at the time of vaccination, for which eosinophilia is a surrogate marker. For future vaccine therapies targeting cytotoxic T-cell responses, delaying IL-2 until after initiation of immune responses may be more effective.William Chad Cragun and Galina V. Yamshchikov contributed equally to this paper.     

A case report: Immune responses and clinical course of the first human use of granulocyte/macrophage-colony-stimulating-factor-transduced autologous melanoma cells for immunotherapy

 The first use of granulocyte/macrophage-colony-stimulating-factor-transduced, lethally irradiated, autologous melanoma cells as a therapeutic vaccine in a patient with rapidly progressive, widely disseminated malignant melanoma resulted in the generation of a novel antitumour immune response associated with partial, albeit temporary, clinical benefit. An initially negative reaction to non-transduced, autologous melanoma cells was converted to a delayed-type hypersensitivity (DTH) reaction of increasing magnitude following successive vaccinations. While intradermal vaccine sites showed prominent dendritic cell accrual, DTH sites revealed a striking influx of eosinophils in addition to activated/memory T lymphocytes and macrophages, recalling the histology of challenge tumour cell rejection in immune mice. Cytotoxic T lymphocytes (CTL) reactive with autologous melanoma cells were detectable at high frequency after vaccination, not only in limiting-dilution analysis, but also in bulk culture without added cytokines. Clonal analysis of CTL showed a conversion from a purely CD8⁺ response to a high proportion of CD4⁺ clones following vaccination. A prominent acute-phase response manifested by a five- to tenfold increase in C-reactive protein was observed, as was a systemic eosinophilia. Vaccination resulted in the regression of axillary lymphatic metastases, stabilisation of pulmonary metastases, and a dramatic, reversible increase in cerebral oedema associated with multiple central nervous system metastases; however, lesions in the adrenal glands, pancreas and spleen proved refractory. The antitumour effects and immune response were not detectable 2 months following the last vaccination. Irradiation of the extensive cerebral metastases resulted in rapid deterioration and death of the patient. Received: 20 September 1996 / Accepted: 5 December 1996     

DNA fusion-gene vaccination in patients with prostate cancer induces high-frequency CD8+ T-cell responses and increases PSA doubling time

We report on the immunogenicity and clinical effects in a phase I/II dose escalation trial of a DNA fusion vaccine in patients with prostate cancer. The vaccine encodes a domain (DOM) from fragment C of tetanus toxin linked to an HLA-A2-binding epitope from prostate-specific membrane antigen (PSMA), PSMA_27–35. We evaluated the effect of intramuscular vaccination without or with electroporation (EP) on vaccine potency. Thirty-two HLA-A2⁺ patients were vaccinated and monitored for immune and clinical responses for a follow-up period of 72 weeks. At week 24, cross-over to the immunologically more effective delivery modality was permitted; this was shown to be with EP based on early antibody data, and subsequently, 13/15 patients crossed to the +EP arm. Thirty-two HLA-A2^? control patients were assessed for time to next treatment and overall survival. Vaccination was safe and well tolerated. The vaccine induced DOM-specific CD4⁺ and PSMA₂₇-specific CD8⁺ T cells, which were detectable at significant levels above baseline at the end of the study (p = 0.0223 and p = 0.00248, respectively). Of 30 patients, 29 had a measurable CD4⁺ T-cell response and PSMA₂₇-specific CD8⁺ T cells were detected in 16/30 patients, with or without EP. At week 24, before cross-over, both delivery methods led to increased CD4⁺ and CD8⁺ vaccine-specific T cells with a trend to a greater effect with EP. PSA doubling time increased significantly from 11.97 months pre-treatment to 16.82 months over the 72-week follow-up (p = 0.0417), with no clear differential effect of EP. The high frequency of immunological responses to DOM-PSMA₂₇ vaccination and the clinical effects are sufficiently promising to warrant further, randomized testing.