PERSPECTIVE
Immune Therapy for Lung Cancer
Are We Getting Closer?

Paul A. Bunn Jr.

University of Colorado Cancer Center, Denver, Colorado

It is well known that lung cancer patients have abnormalities in their immune system and that the extent of these abnormalities may have prognostic relevance (1, 2). The promise of immune therapy for lung cancer has been suggested for many years, but real improvements in survival have remained elusive. An early report suggested that resected lung cancer patients who developed empyema had a superior outcome (3), perhaps on the basis of immune stimulation. This led to a number of trials of nonspecific immune stimulants, such as Calmette-Guerin bacillus (BCG) and nocardia rubra cell wall skeleton (4, 5). Although several small, uncontrolled trials showed promise to these approaches, larger randomized trials failed to show any survival benefit (6). Prior studies also evaluated the use of thymosin factor V, interferon, and other nonspecific immune stimulants. Once again, promising early study results could not be confirmed for thymosin factor V (10, 11), interferon (12), IL2 with or without adopted cells (16), or other means. However, immune approaches remain appealing because initial therapy with surgery, chemotherapy, and radiotherapy alone and in combination produce high response rates in all histologic types of lung cancer, but relapse is nearly inevitable. Over the past decade, a number of tumor antigens, which are expressed on the majority of lung cancer cells, were described. Some of these are expressed in nearly all small-cell lung cancers (SCLC) and are overexpressed on these tumor cells in comparison with normal lung tissues. These tumor antigens provide a new specific means for development of successful vaccine approaches.

Tumor antigens that are being explored for potential vaccines include mutant oncogene proteins such as p53, ras, or erb B2, and also cell surface proteins such as CEA, MUC1, GD2, or HuD. At this point, the ideal antigen for immune therapy is unknown. There are also many ways in which these antigens may be developed as vaccines. One approach is to isolate the tumor protein, such as mutant p53, and administer the protein alone or with nonspecific immune stimulants. A group from the National Cancer Institute (NCI) evaluated the clinical relevance of human antitumor immune responses to tumor antigens, including p53 and HuD (17). They found serum antibodies against autologous tumor proteins in 50% of SCLC and 75% of non-small-cell lung cancer (NSCLC) patients, including two patients who had anti-p53 antibodies and two patients who had anti-HuD antibodies. The presence of antibodies was found to correlate with improved survival and limited stage. In subsequent studies, the NCI showed that missense p53 mutations give rise to new tumor-specific peptide sequences, which are efficiently processed and presented by human lung cancer cells. They then showed that these mutant p53 peptides were effectively targeted by cytotoxic T cells specific for the endogenous mutant epitopes (18). They are currently conducting a clinical trial using the patient-specific p53 peptides as the immunogen. However, this approach is very labor intensive because the mutant p53 protein must be identified and isolated from each patient and then mass produced.

Another approach to using oncogene mutations evaluated activating mutations of the p21 ras proto-oncogene because these activating mutations are limited in number and occur in many common malignancies and in otherwise completely conserved regions. Triozzi and colleagues (19) engineered a chimeric ras immunogen incorporating a promiscuous T-cell epitope to enhance the immunogenicity of an oligopeptide corresponding to a weakly immunogenic substitution, and they are evaluating this immunogene as a vaccine.

Many lung cancers overexpress erb B1 and its protein, epidermal growth factor receptor (EGFR), and/or erb B2 and its protein, Her2/neu. These oncoproteins have also been used in vaccine development. Gonzalez and associates (20) linked the human EGF protein to either tetanus toxoid or Neisseria meningitidis recombinant protein and immunized patients with intradermal injection using aluminum hydroxyde as an adjuvant. Anti-EGF antibodies developed in 60% of the immunized patients without major toxicities. Finally, Amici and coworkers developed a plasmid DNA encoding the rat neu NT oncogene (21). In a transgenic mouse model, intramuscular injections of neu NT plasmids drastically reduced the outgrowth of new and mammory neoplasms.

Common tumor antigens such as CEA, MUC1, GD2, and HuD have been used as the basis for vaccine strategies. Human MUC1 is overexpressed in many human carcinomas but is largely restricted due to the apical surface of secretory cells in normal tissues. Zhang and his fellow investigators developed a vaccine containing MUC1-KLH conjugate prepared with an ester linker plus the adjuvant OSS021 (22). They showed that this approach induced high titer antibody against MUC1-expressing tumor cells. Although T-cell responses, including delayed-type hypersensitivity by lymphocyte proliferation, and cytotoxic T lymphocytes (CTL) were not observed in immunized mice, significant protection from MUC1-expressing tumor cell challenge was observed. This vaccine is in early clinical trials.

Dr. Schlom's laboratory at the NCI developed a recombinant carcinoembryonic antigen (CEA) vaccina virus vaccine, which has been studied in a phase I clinical trial with postvaccination CEA peptide challenge (23). The approach has been safe, and antibodies have developed. Further studies are ongoing.

A unique vaccine strategy based on the widely expressed GD2 antigen developed antiidiotypic antibodies as the immunogen. The antiganglioside GD2 monoclonal antibody 14G2A (Ab1) served as an immunogen to generate the anti-Id, 1A7 (24). Anti-Id 1A7 induced anti-GD2 antibodies in mice, rabbits, and monkeys, which reacted with GD2 positive tumor cells. There were no side effects. The authors then administered the antiidiotypic antibody to patients with GD2 positive melanomas (25) and SCLC. An immune response was documented (25). In SCLC patients long-term survival was superior to that predicted by historic controls. Thus, randomized phase III trials evaluating this promising approach are in progress in the United States.

The scientific basis for vaccines based on the HuD antigen centers around the fact that this antigen is expressed on nearly all SCLC tumors. Some patients (a minority of about 17%) develop detectable serum antibodies reacting with HuD. These patients appear to have a better prognosis than those who did not develop anti-HuD antibodies (26). A group from Memorial Sloan Kettering Cancer Center (MSKCC) in New York developed a plasmid coding for a secreted form of HuD and studied this in an animal model of neuroblastoma, which also expressed HuD (27). They found that mice immunized with the secretory HuD plasmid showed significant tumor growth inhibition and that 14% had complete tumor rejection. Tumors from immunized animals had more T-cell infiltration of the tumor, and there was no evidence of neurologic deficits or central nervous system (CNS) pathologic change.

In this issue, Ohwada and colleagues report on a very similar approach using HuD as a candidate antigen for vaccination (28). The authors created a similar plasmid containing gene sequences, which encode for a portion of the HuD antigen. Their plasmid was constructed to contain a cytomegalovirus (CMV) promoter and the HuD gene sequences. They evaluated this plasmid in an animal model consisting of a mouse colon cancer cell line termed Colon 26 (C26), which they transfected with HuD. This cell line grows in mice injected with the tumor cells. The tumor cells transfected with HuD were shown to produce the HuD protein. They also grew more slowly than nontransfected C26 cells in vitro. This slowed growth is a potential complicating factor in analyzing their results. However, they used appropriate controls, including C26 cells with and without transfected HuD. Mice were transplanted with native tumor cells and with tumor cells containing HuD. The mice with these tumors were then vaccinated with plasmids containing the HuD gene sequences or control plasmids. Mice bearing HuD⁺ tumors immunized with the HuD plasmids were shown to have slower tumor growth than both mice with native (HuD) tumors and mice treated with HuD plasmids.

Ohwada and coworkers also showed that anti-HuD antibody was detected in the mice with HuD⁺ tumors vaccinated with HuD plasmids. The delay in tumor growth was assumed to be due to an immune response mounted against this tumor protein. The authors conclude that these studies show the potential utility of this approach. The authors also point out one potential danger in this approach. The HuD protein is present on normal cells in the cerebellum. It is possible that an immune response to this antigen could inadvertently induce a CNS disease. The authors looked for CNS toxicity in the treated mice but found no evidence of such CNS disease, as did other authors using this approach (27).

What are the prospects for vaccination strategies in humans with SCLC in the near future? The good news is that a randomized phase III trial with the anti-Id to GD2 is in clinical trial. If this study confirms a positive survival advantage, it will provide a huge impetus to additional vaccine strategies.

Even if this trial proves negative, the data from this study by Ohwada and associates and the data from MSKCC suggest that human clinical trials of the HuD DNA plasmid strategy should continue. There are clearly some drawbacks to the studies reported thus far. Neither of these studies employed a typical SCLC tumor model. It is hoped, but not proven, that the results with the neuroblastoma and HuD-transfected colon cancer cells will predict results with SCLC. Concerns about inducing neurologic disease will continue until human clinical trials are initiated. However, if no toxicities are observed in monkeys, human trials should proceed.

	Footnotes

Abbreviations BCG, Calmette-Guerin bacillus; C26, Colon 26 (colon cancer cell line); CNS, central nervous system; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; MSKCC, Memorial Sloan Kettering Cancer Center; NCI, National Cancer Insitute; SCLC, small-cell lung cancer; NSCLC, non-small-cell lung cancer.

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