Vaccination of melanoma patients with an allogeneic, genetically modified interleukin 2-producing melanoma cell line

Thirty-three metastatic melanoma patients were vaccinated according to a phase I-II study with an allogeneic melanoma cell line that was genetically modified by transfection with a plasmid containing the gene encoding human interleukin 2 (IL-2). The cell line expresses the major melanoma-associated antigens and the HLA class I alleles HLA-A1, -A2, -B8, and Cw7. All patients shared one or more HLA class I alleles with this cell line vaccine. Patients were immunized by three vaccinations, each consisting of 60 x 106 irradiated (100 Gy) melanoma cells (secreting 120 ng of IL-2/10(6) cells/24 hr) administered subcutaneously at weekly intervals for 3 consecutive weeks. Side effects of treatment consisted of swelling of locoregional lymph nodes and induration at the site of injection, i.e., a delayed-type hypersensitivity (DTH) reaction. In three patients, vaccination induced inflammatory responses in distant metastases containing necrosis or apoptosis along with T cell infiltration. Apoptosis occurred only in Bcl-2-negative areas, not in Bcl-2-expressing parts of the metastases. Two other patients experienced complete or partial regression of subcutaneous metastases. Seven patients had protracted stabilization (4 to >46 months) of soft tissue metastases, including one patient who developed vitiligo after vaccination. Immune responses to the vaccine could be detected in 67% of the 27 patients measured. Vaccination was shown to induce a variable change in the number of anti-vaccine cytotoxic T lymphocytes (CTLs) in peripheral blood, which did not correlate with response to treatment. However, in two of five patients the frequency of anti-autologous tumor CTLs measured was significantly higher than before vaccination. This study demonstrates the feasibility, safety, and therapeutic potential of vaccination of humans with allogeneic, gene-modified tumor cells, and that frequencies of vaccine-specific CTLs among patient lymphocytes can be determined by using a modified limited dilution analysis (LDA).     

Vaccination of melanoma patients with interleukin 4 gene-transduced allogeneic melanoma cells

A human melanoma line genetically modified to release interleukin 4 (IL-4) was utilized to immunize advanced melanoma patients in order to elicit or increase a specific anti-melanoma immune response, which may affect distant lesions. Twelve metastatic melanoma patients were injected subcutaneously at least three times with 5 x 10(7) IL-4 gene-transduced and irradiated allogeneic melanoma cells per dose. Both systemic and local toxicities were mild, consisting of transient fever and erythema, swelling, and induration at the vaccination site. Two mixed but not complete or partial clinical responses were recorded. To assess the immune response of vaccinated patients, both serological and cell-mediated activities were evaluated. Antibodies to alloantigens could be detected in 2 of 11 patients tested. Mixed tumor-lymphocyte cultures were performed, utilizing autologous and allogeneic HLA-A2-matched melanoma lines as simulators and targets. A significant increase in IFN-gamma release was detected in 7 of 11 cases when postvaccination lymphocytes were stimulated by the untransduced allomelanoma cells. However, induction of a specific recognition of autologous melanoma cells by PBLs was obtained after vaccination in only one of six cases studied. This response involved the melanoma peptide Melan-A/MART-1(27-35) that was recognized in an HLA-A2-restricted fashion. These results indicate that vaccination with allogeneic melanoma cells releasing IL-4 locally can expand a T cell response against antigen(s) of autologous, untransduced tumor, although in a minority of patients.     

Recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) and autologous melanoma vaccine mediate tumor regression in patients with metastatic melanoma

In mice, significant immunoprotection was achieved using B16 melanoma cells transfected with granulocyte-macrophage colony-stimulating factor (GM-CSF) as vaccines (Dranoff G, Jaffee E, Lazenby A, et al. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci USA 1993;90:3539-43). The aim of this study is to test the hypothesis that recombinant human GM-CSF (rhGM-CSF) injected with autologous melanoma vaccine may result in tumor rejection in melanoma patients. Twenty stage IV melanoma patients were treated as outpatients with multiple cycles of autologous melanoma vaccine and bacillus Calmette-Guérin (BCG) plus rhGM-CSF injection in the vaccine sites. Two patients (10%) showed a complete response, with one patient showing resolution of subcutaneous, hepatic, and splenic metastases. In the second patient, buccal, subcutaneous, pulmonary, paraaortic, hepatic, splenic, and retroperitoneal metastases regressed completely. Two patients (10%) showed partial response, with regression of a paraaortic metastasis in one patient. In the second patient, there was shrinkage (> 75%) of a large hepatic lesion. One patient has been rendered free of disease after resection of a single pulmonary metastatic nodule. Three patients (15%) had stable disease during treatment but subsequently developed progression of disease. In 12 patients (60%), the disease progressed. Side effects were minimal. In a separate pilot study, 15 stage IV melanoma patients were also treated with autologous melanoma vaccine with BCG but not with rhGM-CSF; none responded. The fact that four patients showed objective responses to active specific immunotherapy with rhGM-CSF demonstrates that melanoma patients bearing a significant tumor burden may respond specifically to their autologous melanoma.     

Dendritic cells loaded with killed allogeneic melanoma cells can induce objective clinical responses and MART-1 specific CD8+ T-cell immunity

Dendritic cells (DCs) loaded with killed allogeneic tumors can cross-prime tumor-specific naive CD8 T cells in vitro, thereby providing an option to overcome human leukocyte antigen restriction inherent to loading DC vaccines with peptides. We have vaccinated 20 patients with stage IV melanoma with autologous monocyte-derived DCs loaded with killed allogeneic Colo829 melanoma cell line. DCs were generated by culturing monocytes with granulocyte macrophage-colony stimulating factor (granulocyte macrophage-colony stimulating factor) and interleukin (IL-4) and activated by additional culture with tumor necrosis factor and CD40 ligand. A total of 8 vaccines were administered at monthly intervals. The first patient was accrued December 2002 and the last November 2003. Fourteen patients were alive at 12 months, 9 patients were alive at 24 months, and 8 patients are alive as of January 2006. The estimated median overall survival is 22.5 months with a range of 2 to 35.5 months. Vaccinations were safe and tolerable. They induced, in 2 patients who failed previous therapy, durable objective clinical responses, 1 complete regression (CR) and 1 partial regression (PR) lasting 18 and 23 months, respectively. Three out of 13 analyzed patients showed T-cell immunity to melanoma antigen recognized by autologous T cells (MART-1) tissue differentiation antigen. Two of 3 patients showed improved immune function after vaccinations demonstrated by improved secretion of interferon (IFN)-gamma or T-cell proliferation in response to MART-1 derived peptides. In one of these patients, vaccination led to elicitation of CD8 T-cell immunity specific to a novel peptide-derived from MART-1 antigen, suggesting that cross-priming/presentation of melanoma antigens by DC vaccine had occurred. Thus, the present results justify the design of larger follow-up studies to assess the clinical response to DC vaccines loaded with killed allogeneic tumor cells in patients with metastatic melanoma.     

Adoptive cellular therapy with tumor vaccine draining lymph node lymphocytes after vaccination with HLA-B7/beta2-microglobulin gene-modified autologous tumor cells

Adoptive immunotherapy with anti-CD3-expanded lymphocytes from lymph nodes draining alloantigen gene-modified autologous tumor vaccines is an effective treatment of poorly immunogenic murine tumors. This phase I/II study was performed to determine the feasibility and toxicity of combining ex vivo gene transfer of autologous tumor cells and adoptive immunotherapy with anti-CD3-expanded tumor vaccine draining lymph node lymphocytes (TVDLN) in patients with metastatic melanoma and renal cell cancer (RCC). To facilitate the generation of tumor-specific lymphocytes in the TVDLN, autologous tumor cells were modified by gene transfer ex vivo to express the alloantigen HLA-B7, a modification that has the potential to enhance the immunogenicity of the tumor cells. After vaccination with gene-modified tumor cells, patients' lymph nodes were harvested; TVDLN lymphocytes were activated and expanded ex vivo with anti-CD3 and interleukin-2 (IL-2), and adoptively transferred to patients in combination with systemic IL-2. Twenty patients, nine with melanoma and 11 with RCC were treated. Tumor was harvested successfully in all 20 patients. Ex vivo gene transfer was performed using lipofection with a lipid: DNA plasmid complex containing the genes for HLA-B7 and beta2-microglobulin. The mean expression of HLA-B7 by autologous tumor cells after gene transfer was 4.53% (range 0.3%-12.1%). Lymph nodes were harvested from all 20 patients with a mean of 53 x 107 and 60 x 107 cells obtained from the gene-modified and unmodified tumor vaccine sites, respectively. Successful expansion of adequate TVDLN was accomplished in 19 of 20 harvests of unmodified vaccines and in 18 of 20 gene-modified vaccines. No major toxicities were noted after vaccination with autologous tumor cells or adoptive transfer of ex vivo activated TVDLN lymphocytes. Typical IL-2-related toxicities were observed in all patients. No objective tumor regressions were observed. MHC class I restricted, tumor-specific cytokine secretion was observed in lymphocytes from TVDLN and the peripheral blood of vaccinated patients.     

Vaccination of women with metastatic breast cancer,using a costimulatory gene (CD80)-modified,HLA-A2-matched,allogeneic, breast cancer cell line: clinical and immunological results

MDA-MB-231, an HLA-A2(+), HER2/neu(+) allogeneic breast cancer cell line genetically modified to express the costimulatory molecule CD80 (B7-1), was used to vaccinate 30 women with previously treated stage IV breast cancer. Expression of CD80 conferred the ability to deliver a costimulatory signal and thereby improved the antigen presentation capability of the tumor cells to patient T cells in vitro. Patients were vaccinated with 10(7) or 10(8) irradiated gene-modified tumor cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) or BCG, three times at 2-week intervals and then monthly until progressive disease developed. GM-CSF-related flulike symptoms and minor injection site reactions were observed frequently. Prolonged disease stabilization was observed in four patients but no objective tumor regressions were seen. Immune responses were measured in matched peripheral blood samples collected before and after treatment from 9 of 15 patients treated at the 10(8) tumor cell dose. Four patients exhibited MHC class I-restricted cytokine production in response to the parental breast cancer cell line. One patient maintained an increased number of circulating tumor-specific, interferon gamma-secreting CD8(+) T cells for 24 months after the last vaccination. One patient exhibited a tumor-specific interleukin 5 response to an autologous tumor cell line. This immunization strategy proved to be safe and feasible, and induced tumor-specific immune responses in a minority of patients; however, no objective tumor regressions were observed.     

Interferon-gamma or granulocyte-macrophage colony-stimulating factor administered as adjuvants with a vaccine of irradiated autologous tumor cells from short-term cell line cultures: a randomized phase 2 trial of the cancer biotherapy research group

The objective was to study the effects of patient-specific vaccine immunotherapy administered with either interferon-gamma (IFNgamma) or granulocyte-macrophage colony stimulating factor (GM-CSF) in patients with metastatic cancer. Short-term cell lines were established from cancer tissue resected from patients with metastatic cancer for use as autologous tumor cell vaccines. Successful cultures were expanded to 1 to 2 x 108 cells, irradiated, and cryopreserved in aliquots of 106 cells for intradermal testing of delayed tumor hypersensitivity and 107 cells for subcutaneous vaccinations. The study design was that of a randomized phase 2 trial. Patients were stratified by tumor type and by whether they had measurable disease at the time vaccination was to commence, and then randomized to receive either 100 MIU IFNgamma subcutaneously or 500 microg GM-CSF subcutaneously at the time of each tumor cell vaccination. Following a baseline test of delayed-type hypersensitivity (DTH) to an intradermal injection of 106 irradiated autologous tumor cells, patients received 3 weekly subcutaneous injections of 107 cells, had a repeat DTH test at week 4, then received monthly vaccinations for 5 months. A positive DTH test was defined as at least 10 mm of induration; survival was determined from the first DTH test. There were 98 patients enrolled with a median follow-up of over 4 years. The most prevalent diagnoses were melanoma (51), renal cell carcinoma (18), and soft-tissue sarcoma (14). There were 49 patients (26 men, 23 women, average age 50.4 years) randomized to IFNgamma and 49 (28 men, 21 women, average age 54.1 years) to GM-CSF. The average numbers of vaccine and adjuvant injections were 6.3 and 5.9, respectively. For the patients who received IFNgamma, the objective response rate was 0 of 21; for patients who received GM-CSF the response rate was 1 of 26. Only eight patients (four from each arm) had a positive baseline DTH reaction to autologous tumor. The tumor DTH test converted from negative to positive in 13 of 45 of the IFNgamma group and 11 of 43 of the GM-CSF group. With 29 patients deceased in the IFNgamma arm and 31 in the GM-CSF arm, the 2-year and 5-year survival rates were 45% and 29% for the IFNgamma arm and 41% and 23% for the GM-CSF arm (NSD). Both adjuvants were well tolerated and results were similar in both arms of the study. Both adjuvants were associated with a 25% to 30% rate of DTH conversion and a 25% 5-year survival rate. Immune recognition of autologous tumor can be induced with this approach.     

Dendritic cells in vaccination therapies of malignant diseases.

Dendritic cells (DC) are the most potent APC with the unique capacity to initiate primary immune responses. For clinical use DC can be generated in vitro from CD34+ peripheral blood progenitor cells or monocytes. Vaccination of patients with cancer using DC was shown to be effective for B-cell lymphoma, renal cell carcinoma (RCC), prostate cancer and malignant melanoma. We provide evidence that patients with advanced breast and ovarian cancer can be efficiently vaccinated with autologous DC pulsed with HER-2/neu- or MUC1-derived peptides. In 5 of 10 patients, peptide-specific cytotoxic T lymphocytes (CTL) could be detected in the peripheral blood using both intracellular IFN-gamma staining and Cr-release assays. In addition, in one patient vaccinated with the MUC1-derived peptides, CEA- and MAGE-3 peptide-specific T-cell responses were detected after several vaccinations. In a second patient immunized with the HER-2/neu peptides, MUC1-specific T lymphocytes were induced after seven immunizations, suggesting that antigen spreading in vivo might occur after successful immunization with a single tumor antigen. Currently we are analyzing the effect of T-helper epitopes and IL-2 on the CTL induction using peptide pulsed DC. In this ongoing trial one patient with metastatic RCC developed a partial remission of the metastatic sites was induced after the first four vaccinations with MUC1 peptides pulsed DC, that was ongoing after the next cycles containing IL-2. Vaccine-induced peptide-specific T-cell responses in vivo were detected in the PBMNC of this patient and in peptide-specific DTH reactions. This studies demonstrate that peptide pulsed DC can be effective in cancer patients and induce significant clinical and immunological responses.     

Cytokine depot formulations as adjuvants for tumor vaccines. I. Liposome-encapsulated IL-2 as a depot formulation

In an attempt to mimic cytokine gene-transfected tumor cells and to develop an alternative approach to cancer immunotherapy, the authors vaccinated mice with mixtures of inactivated tumor cells and cytokine-containing depots. The RenCa mouse renal carcinoma and the B16 mouse melanoma were used as animal tumor models, with interleukin-2 (IL-2) as a cytokine and liposomes as a depot form. The results obtained show that vaccines consisting of mixtures of irradiated tumor cells and cytokine-containing liposomes can be used as highly effective tumor vaccines. These vaccines are very easy to prepare and, in contrast to vaccines consisting of cytokine gene transfected tumor cells, their composition (cell dosage, cytokine dosage) can be easily varied. Vaccination efficiency depended on (a) on the immunogenicity of the tumor cells: RenCa tumor cells are more immunogenic than B16 melanoma cells; (b) vaccination frequency: a single vaccination with irradiated tumor cells and 10 micrograms of IL-2 in liposome-encapsulated form was sufficient to induce lasting protective immunity against the RenCa tumor, whereas several (four to six) vaccinations in weekly intervals were needed to obtain a similar degree of protective immunity to the B16 melanoma; and (c) the dose of the cytokine encapsulated in the admixed liposome depots: immunity to the tumors could be induced only within a narrow cytokine-dose range ("IL-2-dose window"). The results obtained indicate that, because of the easiness of preparation and handling, vaccine formulations consisting of irradiated tumor cells and IL-2 in depot formulations are candidates for tumor vaccines for the treatment of tumor patients.     

Immunotherapy of metastatic malignant melanoma by a vaccine consisting of autologous interleukin 2-transfected cancer cells: outcome of a phase I study

We performed a phase I trial to evaluate the safety and tolerability of repeated skin injections of IL-2-transfected autologous melanoma cells into patients with advanced disease. Cell suspensions, propagated from excised metastases, were IL-2 gene transfected by adenovirus-enhanced transferrinfection and X-irradiated prior to injection. Vaccine production was successful in 54% of the patients. Fifteen patients (37%) received two to eight skin vaccinations of either 3 x 10(6) (intradermal) or 1 x 10(7) (half intradermal, half subcutaneous) transfected melanoma cells per vaccination (secreting 140-17,060 biological response modifier program units of IL-2/10(6) cells/24 hr). Analyses of safety and efficacy were carried out in 15 and 14 patients, respectively. Overall, the vaccine was well tolerated. All patients displayed modest local reactions (erythema, induration, and pruritus) and some experienced flu-like symptoms. Apart from newly appearing (4 of 14) and increasing (5 of 14) anti-adenovirus and newly detectable anti-nuclear antibody titers (1 of 15), recipients developed de novo or exhibited increased melanoma cell-specific delayed-type hypersensitivity (DTH) reactions (8 of 15) and vitiligo (3 of 15) and showed signs of tumor regression (3 of 15). This supports the idea of a vaccine-induced or -amplified anti-cancer immune response. None of the patients exhibited complete or partial regressions, but five of them experienced periods of disease stabilization. Three of these individuals received more than the four planned vaccinations and their mean survival time was 15.7 +/- 3.5 months as compared to 7.8 +/- 4.6 months for the entire patient cohort. These data indicate that IL-2-producing, autologous cancer cells can be safely administered to stage IV melanoma patients and could conceivably be of benefit to patients with less advanced disease.     

Hybrid cell vaccination in metastatic melanoma: clinical and immunologic results of a phase I/II study

Hybrid cell vaccination with cell fusion products (CFPs) of autologous tumor cells and mature allogenic MHC II bearing dendritic cells has been described to induce cytotoxic T lymphocyte (CTL)-mediated immune responses. The aim of this study was to assess safety, antitumor activity, and immune responses of a CFP-vaccine in patients with disseminated malignant melanoma. In a phase I/II study, we treated 11 patients by monthly intracutaneous or subcutaneous application of a CFP vaccine generated by electrofusion of autologous melanoma cells with mature allogenic dendritic cells. In addition, patients received subcutaneous low-dose interleukin-2 injections for 6 days after each vaccination. No serious adverse effects were observed. Ten patients showed progressive disease and one patient had a short-lasting stable disease. None of the patients developed a positive delayed-type hypersensitivity reaction against irradiated autologous melanoma cells. In 2 patients, who were monitored in more detail, we found no evidence of induction of a specific antimelanoma T-cell response by analyzing the proliferation, cytokine secretion, and cytotoxicity of their T cells toward autologous melanoma cells. No unequivocal beneficial effects of the used CFP vaccine could be demonstrated.     

Therapeutic vaccination with an autologous mRNA electroporated dendritic cell vaccine in patients with advanced melanoma

The immunostimulatory capacity of dendritic cells is improved by co-electroporation with mRNA encoding CD40 ligand, constitutively active toll-like receptor 4, and CD70 (TriMix-DC). This pilot clinical trial evaluated the feasibility, safety, and immunogenicity of a therapeutic vaccination containing autologous TriMix-DC co-electroporated with mRNA encoding a human leukocyte antigen class II-targeting signal linked to 1 of 4 melanoma-associated antigens (MAGE-A3, MAGE-C2, tyrosinase, and gp100) in patients with advanced melanoma. Thirty-five American Joint Committee on Cancer stage III/IV melanoma patients received autologous TriMix-DC (4 administrations 2 weeks apart). Immune monitoring was performed by evaluating skin biopsies of delayed type IV hypersensitivity (DTH) reactions for presence of vaccinal antigen-specific DTH-infiltrating lymphocytes (DIL). Thereafter, patients could receive interferon-alpha-2b (IFN-α-2b) 5 MU subcutaneously 3 times weekly and additional TriMix-DC every 8 weeks. TriMix-DC-related adverse events comprised grade 2 local injection site reactions (all patients), and grade 2 fever and lethargy (2 patients). Vaccinal antigen-specific DIL were found in 0/6 patients tested at vaccine initiation and in 12/21 (57.1%) assessed after the fourth vaccine. A positive postvaccination DTH test correlated with IL-12p70 secretion capacity of TriMix-DC. No objective responses to TriMix-DC alone were seen according to RECIST. Twenty-nine patients received IFN-α-2b after the fourth vaccine without unexpected adverse events. During TriMix-DC/IFN-α-2b combination therapy, 1 partial response and 5 stable disease (disease control of >6 months with regression of metastases) were observed in 17 patients with evaluable disease at baseline. In conclusion, this study demonstrated that therapeutic vaccination with autologous TriMix-DC is feasible, safe, and immunogenic and can be combined with sequential IFN-α-2b.     

Immunization by particle-mediated transfer of the granulocyte-macrophage colony-stimulating factor gene into autologous tumor cells in melanoma or sarcoma patients: report of a phase I/IB study

The primary objective of this phase I study was to determine the safety of an autologous tumor vaccine given by intradermal injection of lethally irradiated granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transfected autologous melanoma and sarcoma cells. Secondary objectives included validation of the gene delivery technology (particle-mediated gene transfer), determining the host immune response to the tumor after vaccination, and monitoring patients for evidence of antitumor response. Sixteen patients were treated with either of two different doses of GM-CSF-treated tumor cells. One patient received treatment with both doses of tumor cells. No treatment-related local or systemic toxicity was noted in any patient. Patients administered 100% treated cells (i.e., with a preparation of tumor cells that had all been exposed to GM-CSF DNA transfection) had a more extensive lymphocytic infiltrate at the vaccine site than did patients given 10% treated cells (a preparation of tumor cells in which 10% had been exposed to GM-CSF transfection) or nontreated tumor. The generation of a systemic immune response to autologous tumor by a delayed-type hypersensitivity response to the intradermal placement of nontransfected tumor cells was noted in one patient. One patient had a transient partial response of metastatic tumor sites. The entire procedure, from tumor removal to vaccine placement, was accomplished in less than 6 hr in all patients. Four of 17 patient tumor preparations produced greater than 3.0 ng of GM-CSF per 10(6) cells per 24 hr in vitro. The one patient with greater than 30 ng of GM-CSF per 10(6) cells per 24 hr in vitro had positive DTH, a significant histologic inflammatory response, and clinically stable disease. This technique of gene transfer was safe and feasible, but resulted in clinically relevant levels of gene expression in only a minority of patients.     

Phase Ib trial assessing autologous, tumor-pulsed dendritic cells as a vaccine administered with or without IL-2 in patients with metastatic melanoma

Twenty-four subjects with metastatic melanoma were treated on a randomized Phase Ib trial evaluating an autologous tumor lysate-pulsed dendritic cell (DC) vaccine with or without interleukin (IL)-2. The vaccine consisted of autologous DCs obtained from peripheral blood mononuclear cells (PBMCs) cultured in granulocyte macrophage-colony stimulating factor and IL-4 then pulsed with autologous tumor cell lysate and keyhole limpet hemocyanin (KLH). The primary end points of the trial were safety and immune response to vaccine. Subjects were randomized to vaccine administered every other week times 3, vaccine x 3 followed by low-dose IL-2, or vaccine x 3 followed by high-dose IL-2. Immune response was monitored pretreatment and at 2 and 4 weeks after the third vaccine administration. Disease evaluation was performed at 4 weeks after the third vaccination. Therapy was well tolerated with no local vaccine toxicity greater than grade 1 in any arm. IL-2 toxicity was as expected without additional toxicity from the addition of IL-2 to vaccine. Immune response defined as delayed-type hypersensitivity, PBMC interferon-gamma enzyme-linked immunosorbent spot, and PBMC proliferation, to both autologous tumor and KLH were detected in all arms. Interferon-gamma enzyme-linked immunosorbent spot response to KLH (7 of 10 patients) and autologous tumor (4 of 10 patients) were also detected in subjects with available vaccine draining lymph node cells. There were no differences in immune response between treatment arms. No clinical responses were seen. Autologous tumor lysate-pulsed DC vaccine with or without IL-2 was well tolerated and immunogenic but failed to induce clinical response in patients with advanced melanoma.     

Assessing the safety of cytotoxic T lymphocytes transduced with a dominant negative transforming growth factor-beta receptor

Transforming growth factor (TGF)-beta, a pleiotropic cytokine that regulates cell growth, is secreted by many human tumors and markedly inhibits tumor-specific cellular immunity. It has previously been shown by our group that transduction of cytotoxic T lymphocytes (CTLs) with a retroviral vector expressing the dominant-negative TGFbeta type II receptor (DNR) overcomes this tumor evasion in a model of Epstein-Barr virus (EBV)-positive Hodgkin disease. TGFbeta is an important physiologic regulator of T-cell growth and survival, however, abrogation of this regulatory signal in genetically modified cells is potentially problematic. To ensure that unresponsiveness to TGFbeta did not lead to the unregulated growth of genetically modified CTLs, the characteristics of DNR-transduced CTLs in vivo were studied. Donor C57BL6 mice were vaccinated with human papillomavirus-E7 plasmid DNA to induce production of E7-specific CTLs. The E7-specific CTLs were genetically modified to express enhanced green fluorescent protein (GFP) or DNR and administered to syngeneic mice. All mice received monthly boosts with E7 DNA for 9 months, and during this time, transduced CTLs were detected in the peripheral blood of most of the mice using a quantitative real-time polymerase chain reaction. By 12 months, 3 months after cessation of vaccination, no DNR-transduced CTLs or GFP-transduced CTLs were detected in the peripheral blood. There were 4 cases of lymphoma (2 DNR-transduced mice and 2 control mice): all tumors were CD3-/CD8- and were also negative for the DNR transgene. Hence, mature antigen-specific cytotoxic T cells can be genetically modified to resist the antiproliferative effects of TGFbeta without undergoing spontaneous lymphoproliferation in vivo. They may be of value for treating human cancers, which use TGFbeta as a powerful immune evasion mechanism.     

High frequency of antitumor T cells in the blood of melanoma patients before and after vaccination with tumor antigens

After vaccination of melanoma patients with MAGE antigens, we observed that even in the few patients showing tumor regression, the frequency of anti-vaccine T cells in the blood was often either undetectable or <10(-5) of CD8 T cells. This frequency being arguably too low for these cells to be sole effectors of rejection, we reexamined the contribution of T cells recognizing other tumor antigens. The presence of such antitumor T cells in melanoma patients has been widely reported. To begin assessing their contribution to vaccine-induced rejection, we evaluated their blood frequency in five vaccinated patients. The antitumor cytotoxic T lymphocyte (CTL) precursors ranged from 10(-4) to 3 x 10(-3), which is 10-10,000 times higher than the anti-vaccine CTL in the same patient. High frequencies were also observed before vaccination. In a patient showing nearly complete regression after vaccination with a MAGE-3 antigen, we observed a remarkably focused antitumoral response. A majority of CTL precursors (CTLp's) recognized antigens encoded by MAGE-C2, another cancer-germline gene. Others recognized gp100 antigens. CTLp's recognizing MAGE-C2 and gp100 antigens were already present before vaccination, but new clonotypes appeared afterwards. These results suggest that a spontaneous antitumor T cell response, which has become ineffective, can be reawakened by vaccination and contribute to tumor rejection. This notion is reinforced by the frequencies of anti-vaccine and antitumor CTLs observed inside metastases, as presented by Lurquin et al. (Lurquin, C., B. Lethe, V. Corbiere, I. Theate, N. van Baren, P.G. Coulie, and T. Boon. 2004. J. Exp. Med. 201:249-257).     

Adoptive cell therapy for patients with melanoma, using tumor-infiltrating lymphocytes genetically engineered to secrete interleukin-2

Adoptive cell transfer of tumor-infiltrating lymphocytes (TILs) after lymphodepletion mediates regression in 50% of patients with metastatic melanoma. In vivo persistence and telomere length of the transferred cells correlate with antitumor response. In an attempt to prolong the in vivo survival of the transferred cells, TILs were genetically engineered to produce interleukin (IL)-2. In vitro, these transduced TILs secreted IL-2 while retaining tumor specificity and exhibited prolonged survival after IL-2 withdrawal. In a phase I/II clinical trial, seven evaluable patients received transduced TILs and one patient experienced a partial response associated with in vivo persistence of IL-2-transduced TILs in circulating lymphocytes. An additional five patients received transduced TILs in conjunction with IL-2 administration. Persistence of IL-2-transduced TILs was observed in three patients, including one partial responder. The transgene DNA as well as vector-derived IL2 mRNA could be detected for 4 months in responding patients. The low response rate in this trial was possibly due to a reduction in telomere length in cells as a result of prolonged in vitro culture. In this study, insertion of the IL-2 gene into antitumor TILs increased their ability to survive after IL-2 withdrawal in vitro but did not increase their in vivo persistence or clinical effectiveness.     

Augmentation of local antitumor immunity in the liver by tumor vaccine modified to secrete murine interleukin 12.

Minimal residual lesions have been a major problem in surgical management of cancer. We transfected M5076 with murine IL-12 gene by a retroviral vector, established a stable transfectant secreting IL-12 and investigated its antitumor effects on a spontaneous liver metastasis murine model of M5076 reticulum cell sarcoma. Subcutaneous vaccination of the irradiated transfectant into the remote skin following the amputation of the tumor-bearing limb improved survival when compared with the vaccination of irradiated parental cells (control). Cytotoxic activities against parental M5076 were significantly stronger in the hepatic lymphocytes from the mice vaccinated with the IL-12 transfectant than those from the control. IFN-gamma production of hepatic lymphocytes when they were cocultured with the parental cells was significantly augmented in mice vaccinated with the IL-12 transfectant compared with the control. On the other hand, both cytotoxic activity and IFN-gamma production of spleen cells in the M5076-vaccinated and transfectant-vaccinated mice were at similar levels. Immunophenotypic analysis revealed the selective increase of CD3+NK1+ population in the liver from the transfectant-vaccinated mice. These results suggest that tumor vaccines genetically modified to secrete IL-12 continuously at a relatively low level preferentially augment local antitumor activity in the liver rather than systemic immune responses. This strategy warrants further investigation as an adjuvant modality in the management of postoperative residual tumors.     

A phase 1/2 study of autologous neuroblastoma tumor cells genetically modified to secrete IL-2 in patients with high-risk neuroblastoma

Autologous neuroblastoma (NB) tumor cells modified to secrete interleukin (IL)-2 (auto-IL-2) can be safely given to patients with advanced neuroblastoma and generate antitumor immune responses. As the benefits of tumor immunization may be greater in patients with minimal residual disease and thus rely on surrogate markers such as immune responses to measure effect, we studied the frequency of immune changes associated with vaccination. Thirteen patients (8 in first remission and 5 after treatment for recurrent NB) received 5 to 8 subcutaneous injections of auto-IL-2 at 0.3 x 10 cells/kg. The vaccine was well tolerated. Injection site biopsies revealed increased cellularity caused by infiltration of CD4 and CD8 lymphocytes, eosinophils, and dendritic cells. Enzyme-linked immunosorbent spot assays for interferon-gamma and IL-5 demonstrated that vaccination produced a rise in circulating CD4 and CD8 T cells responsive to stimulation by autologous tumor cells. Median event-free survival was 22 months for patients in first remission and 3 months for all others. Four patients treated in first remission remain alive and 3 without disease recurrence.     

Semiallogeneic cell hybrids as therapeutic vaccines for cancer

The authors have engineered a cell line that can be used in human studies as a universal donor cell for the formation of semiallogeneic cell hybrids after fusion with patient-derived tumor cells. These hybrids can be irradiated and injected as a patient-tailored therapeutic vaccine in patients affected by virtually any type of cancer. A crucial step in this research effort has been the derivation of an allogeneic cell line (FO1-12) that expresses both a dominant selectable marker (neomycin resistance) and a recessive selectable marker (sensitivity to hypoxanthine, aminopterin, and thymidine), which allows easy selection of semiallogeneic cell hybrids derived from the fusion of FO1-12 cells with patient-derived tumor cells. Tumor-infiltrating lymphocytes derived from select patients with melanoma and exposed to semiallogeneic cell hybrids from the same patient were better able to specifically lyse autologous tumor cells. Furthermore, FO1-12 cells express carcinoembryonic antigen, which is ubiquitous in adenocarcinomas, and fusion of FO1-12 cells with various patient-derived adenocarcinoma cells showed that the hybrid cells also express carcinoembryonic antigen. Because of the results of these preclinical studies, the authors were given permission to use semiallogeneic cell hybrids for immunotherapy of patients with metastatic melanoma or metastatic adenocarcinoma who had not responded to standard treatment regimens. Treatment with semiallogeneic vaccines is associated with minimal or no toxicity and can induce a specific anti-tumor immune response.