The consensus that cytotoxic T lymphocytes play a major role in tumor control led to efforts to design vaccines that specifically induce or amplify tumor-specific CTL. Key to production of such vaccines is the identification of tumor antigens recognized by CTL. In humans in particular, the vast majority of tumors arise sporadically, and the associated antigens are highly variable and difficult to predict. Early attempts to define tumor antigens based on antibody reactivity to tumor surface structures seemed of limited use, particularly once it was realized that CTL do not recognize native antigen at cell surfaces.
One of the more efficient ways to identify tumor-associated antigens and peptides is by CTL screening (75, 76). CTL (bulk or cloned; from peripheral blood or isolated from the tumor) known to lyse a particular tumor are used as effector cells, and 51Cr-labeled, MHC-compatible normal cells, transfected with pooled cDNA from the cognate tumor, are used as targets. A target cell that is lysed is assumed to express one or more peptides recognized by the CTL, and candidate peptides are eluted from target MHC molecules and analyzed until the triggering peptide (and parent protein) is found. In many instances, the frequency of tumor-peptide-specific CTL is considerably higher in patients bearing the tumor, reinforcing the notion that such peptides were recognized in vivo and served to expand the corresponding T-cell population (77-79).
Using these and other approaches, a large number of tumor-associated peptides have been identified (80-82). Some are derived from proteins normally expressed at low levels in a limited number of cells, but over-expressed in tumor cells. Some represent proteins (differentiation antigens) normally present only at restricted stages of development, but expressed -again, often at very high levels - in tumor cells. So-called "tumor-unique antigens" may be the most promising targets for vaccine development. These are proteins unique to the oncogenic state, such as oncogenes or other mutated cell-cycle proteins, or in fact any mutated protein associated with, even if not causative of, oncogenesis (83-85).
Tumor-unique antigens occur randomly and unpredictably, either in genes or in their associated introns (86) or even in control-region sequences. Thus the number of unique peptides that could be associated with any given cancer is potentially huge. Moreover, the heterogeneity in class I structures within a population makes it difficult, even with non-mutated tumor proteins, to predict which peptides will interact well with individual MHC structures, or to prepare peptide "cocktails" that will cross-react with sufficiently large numbers of alleles to make this approach economically feasible. On top of all that, selected peptides must find CTL with cognate receptors to be effective as vaccines. In spite of these difficulties, some striking results have been obtained with this approach, and are discussed briefly herewith.
In humans, extensive work has been done to define peptides associated with malignant melanoma such as MART-1 and gp100 (reviewed in 82; 87), and clinical trials have been underway for the past several years. Clinical trials using a modified gp100 peptide administered together with IL-2 yielded promising results. Objective tumor regressions were obtained in 42 percent of HLA-A2-positive patients with advanced melanoma (88). IL-2 appeared to promote sequestration of responding CTL at the tumor site (89). In an unrelated trial a selected peptide from MART-1 was shown to induce significant CTL responses in melanoma patients, which correlated with a prolonged time to relapse (90).
Vaccine trials with peptides representing other cancers including breast (91), cervical (92), and pancreatic (93), are underway, and have given broadly similar results where reported. As with the melanoma trials, these are all Phase I/II studies, restricted to patients with advanced cancer who have failed conventional treatments. However it must be admitted that at present, although CTL and NK cell responses can often be elicited by these vaccination procedures, success has been considerably less than with melanoma. As more peptides are identified, and modified where appropriate to broaden class I affinities, or to enhance responses within individuals, and as treatment is extended to patients with less advanced disease, it is reasonable to expect the present encouraging results will improve still further.
Dendritic cells are key to induction of CD8 T-cell responses to tumors. DC are attracted to tumor sites by cytokines released either by the tumor itself, or nearby DC, NK cells or other elements of the innate immune system. After ingestion of potentially antigenic material, the DC migrate to nearby lymph nodes where they in turn activate CD4 and CD8 T cells to peptides displayed on their MHC molecules (94, 95). Peptide-presenting DC can be prepared in the laboratory either by peptide pulsing or by transfection with cDNA encoding key peptides. It is also possible to present larger antigenic protein structures to DC, and allow them to process these naturally into peptides for MHC loading. A promising recent approach involves introducing tumor antigen genes into hematopoietic stem cells, and then infusing these back into mice where they travel to bone marrow and produce antigen-laden DC. The advantage is better homing of such cells to lymph tissues where they can interact with T cells (96). Key to many current DC-based vaccines is inclusion of substances to activate the DC, such as microbial products. The route of administration of peptide-bearing DC may be critical to the effectiveness of this approach (97). And as with viral vaccines, inclusion of activating cytokines such as GM-CSF as part of the vaccine protocol may further enhance the response (98, 99). Indeed, provision of GMCSF alone to tumors greatly increases CTL activation and tumor reduction (100).
These approaches to tumor vaccination have produced encouraging results in animal models (e.g., 101). Clinical trials are now underway with pulsed DC as vaccines to immunize patients against their own tumors. Again, most such trials involve melanoma patients (25, 102-105). Other trials using DC are aimed at prostate and bladder cancer (106-108). The prostate trials have been disappointing, but a trial using DC pulsed with B-cell lymphoma antigen achieved a response rate of 75 percent (109).
Vaccines based on cell-cycle proteins are typified by Her-2, an oncogene expressed in a wide range of human and animal tumors (110). A recent Phase I trial using DC pulsed with Her-2 peptides resulted in production of CTL able to lyse Her-2-transfected cells in vitro, and positive DTH responses in about a third of patients (111). A major drawback with the use of DC for vaccination procedures is the difficulty in harvesting large numbers of these cells from patients to use in their own vaccines. This limitation may be overcome by using non-dendritic cells geneticaly modified to function as DC (112, 113).
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