Immunization against epitopes in the human melanoma antigen gp100 following patient immunization with synthetic peptides.

gp1OO is a melanocytic lineage-specific antigen recognized by tumor-infiltrating lymphocytes, the adoptive transfer of which is associated with tumor regression in melanoma patients. In this study, peripheral blood mononuclear cells (PBMCs) were harvested from HLA-A2+ melanoma patients before and after immunization with G9-209 (ITDQVPFSY), G9-280 (YLEPGPVTA), or G9-154 (KTWGQYWQV) peptides in Incomplete Freund's Adjuvant and were tested for the ability to be sensitized in vitro using PBMCs pulsed with the native peptides. In addition, PBMCs from patients receiving the G9-209 or G9-280 peptide were stimulated in vitro with peptides modified at anchor residues to enhance binding to HLA-A2: G9-209/2M (IMDQVPFSY) or G9-280-9V (YLEPGPVTV). In patients immunized with G9-209, a single in vitro restimulation with G9-209/2M resulted in the generation of specific antipeptide lymphocytes from seven of seven postimmune PBMCs and only three of seven preimmune PBMCs. In patients immunized with G9-280, a single in vitro restimulation with G9-280/9V resulted in the generation of specific antipeptide lymphocytes from five of six postimmune PBMCs and four of six preimmune PBMCs. In almost all cases, CTLs raised against modified epitopes were capable of recognizing targets displaying the native nonamers. Several anti-G9-209 and anti-G9-209/2M CTLs also demonstrated specific lysis of, and specific IFN-gamma release in response to, gp1OO+-established cell lines. Thus, using peptides modified to enhance immunogenicity for in vitro stimulation improved the sensitivity of immune monitoring of patients immunized with synthetic peptides. These results demonstrate that immunization with a peptide derived from a tumor-associated protein such as gp100 can provoke a measurable antitumor immune response in cancer patients.

Baculovirus recombinants expressing the human carcinoembryonic antigen gene.

Carcinoembryonic antigen (CEA), one of the most extensively studied human tumor-associated antigens, represents a potential target for passive as well as active immunotherapy. We describe here the first baculovirus recombinants expressing the human CEA gene. Eight baculovirus clones were isolated which expressed products of varying molecular weights; one clone, termed BVCEA-140, was shown to contain multiple CEA epitopes by reactivity to a panel of anti-CEA monoclonal antibodies. When purified protein isolated from this clone was deglycosylated, immunoreactive species ranging from M(r) 50,000 to M(r) 110,000 were found. Results of Southern blot analysis carried out on BVCEA-140 DNA were consistent with the hypothesis that these products result from the stable expression of variants which have recombined within the repeated domains of CEA. Other baculovirus recombinants expressing products comprising different portions of the CEA gene were also derived. One, termed BVCEA-35, was shown to be a recombination between the first 87 bases of domains I and III of the CEA gene. A variant, termed BVCEA-16, contained only the NH2-terminal domain of the CEA gene. Moreover, a recombinant expressing the closely related molecule nonspecific cross-reactive antigen was also derived. As shown here, commercially available preparations of CEA, which are derived from tumor biopsies or cell line supernatants, may contain nonspecific cross-reacting antigens and other contaminants. Thus, the recombinant CEA molecules described should have numerous uses including validation of the use of monoclonal antibodies as standards in CEA serum assays, the characterization of immune responses to CEA, the use as immunogen, and the study of structure function relationships.

Recognition of multiple epitopes in the human melanoma antigen gp100 by peripheral blood lymphocytes stimulated in vitro with synthetic peptides.

gp100 is a melanocyte lineage-specific antigen recognized by tumor-infiltrating lymphocytes whose adoptive transfer has been associated with tumor regression in patients with metastatic melanoma. The peripheral blood mononuclear cells of five melanoma patients were sensitized in vitro with synthetic peptides to elicit antigen-specific cytotoxic T lymphocyte (CTL) lines against four gp100 epitopes. These epitope-specific CTL lines were generated following weekly in vitro stimulation with the synthetic decamer G10(476) (V-L-Y-R-Y-G-S-F-S-V) or the nonamers G9(280) (Y-L-E-P-G-P-V-T-A), G9(154) (K-T-W-G-Q-Y-W-Q-V), or G9(209) (I-T-D-Q-V-P-F-S-V) pulsed onto autologous irradiated peripheral blood mononuclear cells. These lines grew as long as 4 months in culture in low-dose interleukin 2 (30 IU/ml) and exhibited antigen-specific, MHC class I-restricted lysis of peptide-pulsed T2 cells and HLA-A2+, gp100+ established melanoma cell lines. G10(476)- and G9(280)-specific CTLs demonstrated specific release of granulocyte-macrophage-colony-stimulating factor and tumor necrosis factor alpha in response to T2 cells pulsed with relevant peptide, as well as to gp100+ melanoma cell lines. These results demonstrate that several peptides derived from the gp100 protein are presented on the surface of melanoma cells and are sufficiently immunogenic to generate, in vitro, potent CTLs capable of cytolysis and the secretion of cytokines. Therefore, for HLA-A2+ melanoma patients, these and possibly other gp100 peptides could represent good candidates for antigen-specific immunotherapy either singly or in a multivalent regimen.

Dendritic cell-based immunotherapy of prostate cancer: immune monitoring of a phase II clinical trial.

We assessed both non- and peptide-specific immune responses in prostate cancer patients before and after immunotherapy with dendritic cells exogenously pulsed with the prostate-specific membrane antigen-derived peptides, PSM-P1 and PSM-P2. For all subjects, we observed that clinical responses were strongly associated with two indicators of immunocompetence: skin test responses to recall antigens and cytokine secretion by T cells after nonspecific stimulation. In a subset of responders, we observed cytokine secretion or cytotoxicity against the immunizing peptides or an immunodominant epitope from an influenza recall antigen. The clinical results support the use of monitoring for overall immunocompetence to help determine why a patient has or has not responded to therapy. Moreover, it could be useful as an inclusion criterion to select those more likely to benefit from treatment.

Dendritic cell-based immunotherapy of prostate cancer.

The immunotherapy of cancer, based on eliciting or enhancing the body's own capacity to mount an effective antitumor response, has produced encouraging early results in the areas of melanoma and renal-cell carcinoma. Such treatments utilizing dendritic cells (DC), immune cells that are excellent antigen presenters, are especially promising. We performed a phase I clinical trial assessing the administration of autologous DC pulsed with HLA-A0201-specific prostate-specific membrane antigen (PSMA) for the treatment of 51 men with hormone-refractory prostate cancer. Participants were divided into five groups receiving four or five infusions of peptides alone (PSM-P1 or PSM-P2; group 1 and 2, respectively), autologous DC (group 3), or DC pulsed with PSM-P1 or P2 (group 4 and 5, respectively). No significant toxicity was observed. Immune reactivity against PSM-P2 was detected in HLA-A2+ patients infused with DC pulsed with PSM-P1 or -P2 (group 4 and 5). An average decrease in PSA was observed only in group 5. Seven partial responders were identified based on NPCP criteria + PSA. The excellent tolerance of this treatment approach, as well as the enhanced cellular responses, decreased PSA levels, and partial clinical responses in some patients suggests that it holds great potential in prostate cancer therapy.

Expression of MAGE genes in ocular melanoma during progression from primary to metastatic disease.

Primary melanomas that form within the eye have a unique pattern of disease progression as compared with melanomas that form within the skin. A high percentage of patients (approximately 50%) develop metastatic tumors that occur predominately in the liver. An unusual characteristic of ocular melanomas is the prolonged disease-free interval that extends for many years between the development of primary and metastatic tumors. It is estimated that the shortest interval between dissemination of tumor cells from the eye and the appearance of clinically detectable metastases is 6 years. A recent report indicated that fresh uveal melanoma tissue and metastatic tumor biopsies failed to express melanoma antigen gene (MAGE)-1, MAGE-2, or MAGE-3. In the present study, we examined the expression of MAGE genes on fresh and cultured tumor cells obtained from an ocular melanoma patient during different stages of progressive disease. MAGE gene expression was determined by reverse transcription-polymerase chain reaction using MAGE-1, MAGE-2 and MAGE-3 specific primers. Our results demonstrate that primary ocular tumor tissue and cultured tumor cells both express significant levels of MAGE-1, 2, and 3 at the time of enucleation. A high percentage of tumor cells within the primary tumor appear to express MAGE as demonstrated by consistent MAGE expression in 16 tumor cell clones. Metastatic liver tumors that developed 3 years after enucleation and 18 years after the initial formation of the primary tumor also expressed high levels of MAGE-1, -2, and -3. MAGE was expressed on fresh tumor tissue from a single biopsy and cultured tumor cells obtained from three of four different metastatic tumor nodules. When the MAGE-negative metastatic tumor cells were treated with the demethylating agent 5-Aza-2-Deoxycytidine (5-Aza-dC), transcription of MAGE-1 was restored, indicating the MAGE genes were not deleted. Our results demonstrate that in some patients, MAGE genes are expressed on primary and metastatic ocular melanomas.

American Association for Cancer Research: dendritic cells: strategies and vaccines. April 1-5, 2000, San Francisco, CA, USA.

Several major sessions on dendritic cells (DCs) reflected the importance of these members of the immune system within cancer research. Technical hurdles to their routine propagation were largely overcome within the past decade, so the study of precursors, as well as the numerous types of dendritic cells, is the focus of intense research. In vitro studies were presented that described further refinements in dendritic cells are collected and culture. Cytokines and other growth factors have been analysed to assess their contribution to the ability of dendritic cells to intake, process and present antigen. In some instances, the dendritic cells themselves are genetically altered to improve their natural biological activity. Many studies have advanced from in vitro work and demonstrated utility in various animal models. Ultimately, there is the hopeful, steady progress into the increasing numbers of experimental immunotherapies against human cancer.

American Association for Cancer Research: Clinical trials of immunotherapeutics and immunologic monitoring. April 1-5, 2000, San Francisco, CA, USA.

A major session at this annual gathering of the cancer researchers from around the globe dealt with the current state of immunotherapy for cancer. Immunotherapy is a form of cancer treatment that enhances its scientific promise and legitimacy with each passing year. As a result, this topic has become one of the most highly attended and anticipated of all the sessions during the AACR. This year's session included further progress from the laboratory to the clinic involving an ever-increasing number of cancers. For example, brain, lung and prostate cancer are now as well-represented as melanoma and lymphoma at such forums. This year's session continued the trend of impressive biosafety of both cell- and antibody-based vaccines. Therefore, these cancer vaccines offer optimism in treatment benefit as well as a minimal impact on quality of life. Lastly, the increasing number of clinical responses allows for true immunological monitoring, as scientists strive to unlock the mysteries behind what makes one patient respond to treatment and another progress. Several groups discussed in vitro immune parameters that were studied in concert with their clinical trials. Others discussed ways in which those in the immunotherapy community can work towards more reliable immune monitoring and ultimately a surrogate marker for response.

Prostate cancer immunotherapy at the dawn of the new millennium.

Standard treatments for adenocarcinoma of the prostate, such as surgery, hormones, radiation and chemotherapy, often achieve a clinical response, but this is usually short-lived. Prostate cancer frequently recurs and second-line therapies have a poor response rate. Many clinicians seem comfortable in limiting their philosophy of treating advanced recurrent disease merely to new regimens of failed therapies, such as combination chemotherapy. However, other medical researchers have chosen to pursue novel approaches, including immunotherapy, several of which are summarised in this review. Although ranging widely in antigen specificity, all attempt to exploit the body's natural antitumour immunity. Furthermore, all aim to stimulate immunity above a threshold level necessary for tumour regression or to induce stability in the face of progression. The goal of in vivo or ex vivo gene therapy is the modification of gene expression within an antigen-presented cell by the introduction of a vector, DNA, or RNA. Within that field, much progress has been made and is ongoing currently concerning gene delivery systems, target identification and characterisation. Comparatively, monoclonal antibodies are an established type of cancer immunotherapy. However, the more recent development of humanized or fully human antibodies, as well as novel moieties they can be coupled to, renews their prospects for clinical impact. Lastly, various cell-based therapies are the focus of several recent clinical studies demonstrating tumour regression or stabilisation. Immune cells, for example, T-lymphocytes and dendritic cells, have already demonstrated treatment benefit, as well as the ability to maintain an excellent quality of life for participants. Overall, there is a multitude of approaches being considered for the treatment of prostate cancer. The following review concentrates on those approaches that are currently in human or animal studies and have a specific emphasis on prostate cancer.

Immunologic approaches to antigen discovery for cancer vaccines.

Since the early 1990s, scientists have identified an ever-expanding number of antigens to serve as targets for experimental cancer treatments, based on the stimulation of a patient's immune system. Using both immune cells and serum to screen potential candidates, several promising antigens are currently components of vaccines directed against a wide range of tumour types. These antigens vary in their tumour- and tissue-specificity. Their utility as a single reagent or as part of a multi-dimensional approach is as varied as the genes themselves. However, there are already reports indicating that the promise of evoking a clinically beneficial immune response, toward human tumours, is being fulfilled. In this review, we provide a summary of the current status of immunologic approaches to antigen discovery. We also discuss the need for additional, supportive data from non-immunologic techniques, as well as the progression of the preclinical process towards target validation.

Progress in active-specific immunotherapy of brain malignancies.

Despite the significant advances in neurosurgical techniques and oncology treatment regimens, the prognosis of patients with brain malignancies remains dismal. Brain tumours remain as lethal in the beginning of this new millennium as they were 30 years ago. Among the promising treatment modalities being tested are various immunotherapeutic approaches. Development of cancer vaccines, also known as active-specific immunotherapy, for malignant brain tumours is summarised in this review. Understanding the mechanisms behind vaccinations and the initiation of immune response have helped the design and improvement of the efficacy of clinical vaccines. The emergence of the antigen-presenting properties of dendritic cells brings the cancer vaccine field into a new generation. Preclinical work on the use of dendritic cell-based vaccine for malignant brain tumours are encouraging. The move from these preliminary studies to the clinic is anticipated with high hope.

Immune monitoring of cancer patients undergoing experimental immunotherapy.

Advancements in the understanding of cellular immunity within the last decade, along with the characterization of tumor antigens, have led to immunotherapeutic approaches for cancer therapy. This mode of treatment is expected to provide more tumor-specific activity, thereby being less toxic to normal cells than standard modalities. Clinical trials are underway throughout the world to determine whether immunotherapy is a practical and viable alternative to conventional cancer therapies. Unlike radiotherapy and chemotherapy, wherein tumor regression is the standard for determining efficacy of the regimens, immunotherapy has to be evaluated by the examination of several immunological parameters within patients. The purpose of this article is to review the methods currently utilized to evaluate the induction, maintenance, and duration of antitumor immune reactivity in cancer patients undergoing immunotherapy.

Viral vectors for gene transfer into antigen presenting cells.

Crucial insights for vaccine development have come from examining how the immune system responds to antimicrobial vaccines, as well as to viral vectors employed for gene therapy. The effectiveness of a vaccine depends upon both the method of antigen delivery and the presentation of antigen to lymphocytes. Much focus has turned to delivering antigens to dendritic cells, to promote clinically beneficial T- and B-cell responses. Recombinant viral vectors represent a powerful vehicle to deliver genes encoding microbial- or tumor-derived antigens to generate clinically beneficial immunity. Dendritic cell-based and viral vector-based vaccines are currently being evaluated in clinical trials as a means of inducing antitumor immunity.

AACR - 91st meeting. Dendritic cell vaccine strategies.

Inevitably, the sessions on dendritic cells at the AACR Annual Meeting were some of the most consistently well attended. Interest has been intense for several years, largely since the technical obstacles to the routine culture of these cells and their precursors, both from animal and human sources, were removed in the early 1990s. Several important advances were presented towards further optimizing dendritic cell-based immunotherapy in general. Groups reported on improved culture conditions, as well as more efficient means of obtaining larger quantities of dendritic cells or their precursors. As expected, there were several strong reports on the beneficial bioactivity of cytokines, such as IL-12, GM-CSF, and IL-2. In addition, enticing work continues with Flt3-ligand and has begun with progenipoietin, a more recently identified hematopoietic growth factor. As shown in this year's sessions, the clinical promise of tumor lysate and apoptotic bodies continues to move steadily from the bench to the clinic. Finally, although dendritic cells are excellent antigen-presenters, work to determine if they could be engineered to be even more effective continues. As a result, several reports were given on gene-modifying this type of immune cell.

Uveal melanomas contain antigenically specific and non-specific infiltrating lymphocytes.

PURPOSE: Tumor-infiltrating lymphocytes (TIL) were recovered from a series of human choroidal melanomas and expanded in cultures containing interleukin-2 (IL-2) to determine whether TIL contained cytotoxic cells that could be activated in vitro. METHODS: TIL were recovered from six ocular melanoma patients and expanded in vitro with IL-2. Cytotoxic activity was tested in a standard 4-hr 51Cr release assay. The HLA class I phenotype of patients was determined, using peripheral blood lymphocytes and the Amos modified-cytotoxicity test. HLA class I expression on tumor cells was determined by flow cytometry. RESULTS: TIL from four patients lysed autologous ocular melanoma cells. Two of these patients possessed TIL that displayed specific cytotoxic activity and failed to lyse tumor cells from other patients (HLA-mismatched, or -matched). TIL from the remaining two patients possessed non-specific cytotoxic cells that lysed ocular melanoma cells from a variety of other patients (HLA-mismatched). TIL from patients that failed to lyse autologous tumor cells possessed cytotoxic activity for ocular melanoma cells from other HLA-mismatched patients. CONCLUSIONS: Ocular melanomas accumulate lymphocytes with the potential to kill tumor cells. Our results imply that elimination of tumor cells may be possible by activation of cytotoxic cells present within progressively growing ocular tumors.

Cancer immunotherapy for prostate cancer.

Our approach to prostate cancer immunotherapy involves two components dendritic cells as antigen-presenting cells; and the antigen used to target T-cell attack, HLA-A0201-associated peptides from prostate specific membrane antigen (PSMA). We have conducted a phase I dose-ranging study in 51 men with advanced prostate cancer, using dendritic cells pulsed with a PSMA peptide. no significant toxicity was observed. In that study, T-cell response was enhanced, with seven men meeting NCPC and PSA criteria for partial response. We are now conducting a phase II study with 67 men, who will receive 6 infusions of dendritic cells that have been pulsed with 2 PSMA peptides, at 6-week intervals. The phase II study design and rationale is described in this paper.