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How Can We Tell When Cancer Vaccines Vaccinate?

Memorial Sloan-Kettering Cancer Center, New York, NY

IN DEVELOPING the first vaccines against infectious diseases, investigators had little to guide them except their clinical observations. When his rabies vaccine made from aged virus was first given to a victim of a rabid dog bite in 1885, all Pasteur could do was to wait and see whether hydrophobia developed. William Park, who developed the first diphtheria vaccine in 1913 made from a mixture of diphtheria toxin bound to antitoxin antibodies, took advantage of the Schick test, an in vivo test of immunity, to determine whether the vaccine preparation was immunogenic. This guided him in determining the optimal mixtures of toxin and antiserum. The absence of reproducible in vitro tests of immunity contributed to the failure of many vaccines developed early in the twentieth century, but as serological techniques became more robust, successful vaccine development—especially more complex vaccines such as the trivalent polio vaccine—depended heavily on the ability to monitor immunologic responses. In our attempts to develop effective cancer vaccines, we too must rely on in vitro immunologic assays to guide us; this is especially true given the low level of immunogenicity that is expected. We now have a variety of methods to measure B-cell and T-cell responses, each with advantages and disadvantages.

Many serological techniques can measure antibody responses against tumor antigens. These techniques, which are well established, extremely sensitive, and reproducible, provide quantitative measurements of antibody concentrations as well as effector functions (ie, complement-mediated lysis). Because they require very little serum, they are repeatable multiple times, which is an important advantage. Measurement of serological responses has fallen somewhat out of favor given current assumptions that T cells are the main mediators of antitumor effects. However, it seems likely that both antibody and T-cell responses will prove to be important, and many protein tumor antigens induce both cellular and humoral immunity. Although minimum antibody titers needed for protection have been defined for some viral infections, we do not yet know whether a specific titer is associated with antitumor effects. However, in some cancer vaccine trials, induction of even low-antibody titers correlated with improved relapse-free survival.¹ In three of the immunized non–small-cell lung cancer patients described in the current report, Salgia et al² detected a modest increase in immunoglobulin G titers against ATP6S1, a component of vacuolar H⁺-ATPase, although the significance of this finding remains unclear.³

Delayed type hypersensitivity (DTH) can be measured by injecting antigenic material intradermally and measuring the inflammatory response several days later, as was done by Salgia et al² in this issue of Journal of Clinical Oncology. The advantages of this monitoring technique are that it is easy to perform and it measures an in vivo reaction in the patient. Several caveats need to be kept in mind in interpreting these results, however. First, it is not clear that this is a quantitative assay and we do not know whether a certain degree of inflammation indicates protective immunity. Second, the DTH reaction involves a choreography of immune function, and it is difficult to tell which cell populations are playing a role. Finally, it is important that the material used for DTH reaction does not contain irrelevant and immunogenic molecules, which could result in a false-positive reaction. In the paper by Salgia et al,² the cells used for the DTH reactions were treated with xenogeneic enzymes that are highly immunogenic, which can lead to false-positive DTH reactions.⁴ There is no way to rule out the possibility that the DTH reactions seen could be against these contaminating proteins rather than against relevant tumor antigens. More intriguing, however, is their observation that immunization induced inflammatory responses at sites of metastatic disease in three of six patients immunized. Although this is not a quantitative measure of immunity, it does imply immune recognition of molecules within the tumor. It is disappointing that these inflammatory responses did not appear to result in tumor regression, which indicates that the antigens recognized were not tumor rejection antigens.

Another way to monitor T-cell responses is by the ELISPOT assay, a quantitative test that can measure antigen-specific T-cell frequencies of approximately 1/10,000. In a typical ELISPOT, purified CD8⁺ T cells are incubated with irradiated antigen-presenting cells (APCs) that have been pulsed with a source of antigen (either whole protein or major histocompatibility complex [MHC] class I restricted peptide). This is done in wells coated with an anti-interferon gamma (IFN) capture antibody, although capture antibodies recognizing other cytokines can also be used. During this incubation period, T cells capable of recognizing antigen will secrete cytokine, in this case IFN, which will be captured by the antibodies coating the well. The cells are washed away and a second antibody to IFN is used to detect the spots where IFN was released and bound to the plate. Each spot corresponds to a T cell secreting IFN, and instruments are available to quantify spots accurately and reproducibly. Ideally, T cells are incubated with antigen-loaded APCs for a minimum amount of time; for example, overnight. In this way, the reactive T cells are not likely to have divided more than once and the T-cell frequency that is measured is likely to represent the frequency in the peripheral blood. The advantage of the ELISPOT is that it provides both quantitative and qualitative data. The disadvantages of this technique are subjectivity of reading plates manually (or the cost of an automated reader) and the need to have a source of antigen and APCs. Many of the antigens used in constructing cancer vaccines are only modestly immunogenic, and the frequency of T cells responding to these antigens are below the level of detection by ELISPOT. To achieve a signal, many investigators expand the T cells in vitro for 10 to 20 days before performing the ELISPOT assay. The T cell frequency results obtained in this way cannot be considered quantitatively valid and have little relationship to the actual frequencies in vivo.

In 1996, the Davis group⁵ described the MHC tetramer assay, a method for quantifying T cells that recognize specific antigenic peptides. This assay is based on the fundamental observation that CD8⁺ T cells recognize, through their T-cell receptors (TcRs), a nine–amino acid peptide derived from the antigen bound to an MHC class I molecule. It is possible to load peptides onto fluorescently labeled tetramers of MHC class I molecules. Recently, it has also become possible to produce MHC class II tetramers. These fluorescent tetramers will bind to T cells that express TcRs against the specific peptide antigen. The advantage of this assay is that once the tetramer is constructed, it is possible to analyze many samples quickly for binding to the specific peptide. A limitation is that a tetramer can only be used to detect T cells that recognize a specific peptide presented by a specific MHC molecule. Without in vitro stimulation, the sensitivity of the tetramer assay is about 1/1,000 T cells, 1 log₁₀ less than the ELISPOT assay. As discussed, expanding antigen-reactive T cells in vitro before performing the assay will increase the signal but invalidates the quantitative results. It is also important to remember that the tetramer assay measures TcR binding to peptide; it provides no functional data, and it is likely that that some of the cells detected by tetramer staining are not functional, either because of low-avidity binding to MHC-peptide complexes or because they are anergic.

The intracellular cytokine (ICC) assay takes advantage of the fact that it is possible to stimulate T cells with antigen leading to cytokine production, but with the addition of specific inhibitors of secretion, the cytokine remains intracellular. After fixation and permeabilization, cells can be stained by fluorescence-activated cell sorting, using antibodies against cytokine as well as cell surface markers. This allows quantification of cells that have produced cytokine in response to the antigen of interest. This technique has several advantages, including the fact that it measures T-cell function. When multilaser machines are used, it is possible to monitor several cytokines at once among several cell types. The main disadvantage appears to be the sensitivity of the technique. It is possible to detect antigen-specific T cells at a frequency as low as 0.05% (1 of 50,000), but whether this is adequate sensitivity for T-cell responses against tumor antigens remains to be seen. If in vitro expansion of T cells is required to obtain an adequate signal in the ICC assay, then the ability to quantify the frequency of T cells against tumor antigens is lost.

Clearly, a variety of options exist for monitoring immune responses against tumor antigens, and each technique has its own set of advantages and disadvantages. The sensitivity of the T-cell assays ranges from 1/1,000 to 1/50,000, but it is unclear if this sensitivity is adequate. The challenge remains to develop an immune monitoring system that not only has adequate sensitivity but that correlates with the relevant clinical effect—tumor shrinkage. If we cannot tell whether the patients have been vaccinated, we are forced to rely on clinical outcomes, as was Pasteur 117 years ago. If, in addition, we do not have convincing evidence of antitumor responses, then we have little to guide our vaccine development.

REFERENCES

1. Kirkwood JM, Ibrahim JG, Sosman JA, et al: High-dose interferon alfa-2b significantly prolongs relapse-free and overall survival compared with the GM2-KLH/QS-21 vaccine in patients with resected stage IIb-III melanoma: Results of intergroup trial E1694/S9512/C509801. J Clin Oncol 19:2370–2380, 2001[Abstract/Free Full Text]

2. Salgia R, Lynch T, Skarin A, et al: Vaccination with irradiated autologous tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor augments antitumor immunity in some patients with metastatic non–small-cell lung carcinoma. J Clin Oncol 21:624–630, 2003[Abstract/Free Full Text]

3. Hodi FS, Schmollinger JC, Soiffer RJ, et al: ATP6S1 elicits potent humoral responses associated with immune-mediated tumor destruction. Proc Natl Acad Sci U S A 99:6919–6924, 2002[Abstract/Free Full Text]

4. Berd D, Maguire HC Jr, Mastrangelo MJ: Induction of cell-mediated immunity to autologous melanoma cells and regression of metastases after treatment with a melanoma cell vaccine preceded by cyclophosphamide. Cancer Res 46:2572–2577, 1986[Abstract/Free Full Text]

5. Altman JD, Moss PA, Goulder PS, et al: Phenotypic analysis of antigen-specific T lymphocytes. Science 274:94–96, 1996[Abstract/Free Full Text]

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