New MHC Class II tetramer advances field
NEW YORK, NY, NANTES, FRANCE, and EPALINGES, SWITZERLAND – April 7, 2010 – A team of tumor immunologists in France and Switzerland has established an improved, reliable immune monitoring tool that allows for more accurate measurement of the number, subtype, and activity of cancer antigen-specific CD4+ T cells in cancer patients treated with therapeutic cancer vaccines. The new technology may significantly aid tumor immunologists around the world who are working to improve the effectiveness of this emerging and highly promising class of immune system-based cancer therapies.
In an article published this week in the Proceedings of the National Academy of Sciences, Cancer Research Institute investigators Maha Ayyoub, Ph.D., and Danila Valmori, Ph.D., and their team at the French National Institute of Health and Medical Research (INSERM) in Nantes, France, along with Danijel Dojcinovic, Immanuel Luescher, Ph.D., and colleagues at the Lausanne Branch of the Ludwig Institute for Cancer Research Ltd, describe the successful generation of MHC class II tetramers to evaluate CD4+ T-cell responses in patients who received a therapeutic cancer vaccine in a previously reported study. That vaccine included recombinant NY-ESO-1 cancer antigen in Montanide ISA-51, an oil-and-water emulsion, given in combination with CpG 7909, a non-specific immune boosting adjuvant, to patients with cancers that express NY-ESO-1.
Clinical and laboratory studies have confirmed NY-ESO-1 expression in many different cancer types—including melanoma, lung, breast, and ovarian cancers—but not healthy tissues with the exception of testicular germ cells of normal adults, which are protected from recognition and attack by the immune system. Studies have also shown that the immune system is able to recognize and target the antigen naturally (i.e., spontaneous ESO+ immunity) or as a result of therapeutic cancer vaccination. Taken together, the cancer-specific expression of NY-ESO-1 and its strong immunogenicity make it a highly promising target for vaccine therapy.
Critical to the design of effective cancer vaccines is the accurate assessment of a vaccine’s ability to elicit protective immune responses. Clinical studies have shown that therapeutic cancer vaccines are able to induce tumor antigen-specific CD4+ and CD8+ T cells and antibody-producing B cells. Further studies are required, however, to determine whether these immune responses protect cancer patients from recurrent disease.
Over the past decades, scientists have learned that there are several subsets of CD4+ T cells that serve distinct and opposing functions. For example, the “helper” sub-type (TH) produce interleukins that stimulate the proliferation and activity of other immune cells, including CD8+ T cells and antibody-producing B cells, and play a role in establishing immunological memory, or the ability of the immune system to recognize and mount a rapid immune response against a target it has already encountered. On the other hand, the “regulatory” sub-type (Treg) suppresses immune responses and can be detrimental to vaccine-induced anti-cancer immunity.
Scientists working to develop vaccine strategies that enhance the activity of CD4+ helper T-cell subtypes and inhibit suppressive subtypes must have at their disposal immune monitoring tools that distinguish the different types. In their PNAS paper, Ayyoub et al. describe their development of a new approach to this.
In recent years, tetramers—chemically engineered proteins containing four distinct subunits—have come into widespread use for immune monitoring, enabling scientists to quantify, isolate, and characterize the antigen-specific T-cell responses, such as those induced by a vaccine therapy. Conventional tetramer technology, however, is geared to measure primarily a subset of immune cells, the cytotoxic (“killer”) CD8+ T cells, which recognize antigens presented by major histocompatibility complex (MHC) class I molecules. CD4+ T cells recognize antigens presented by MHC class II molecules, and are more difficult to detect by tetramers.
Previous attempts to establish CD4+ T-cell tetramers have proven challenging, due to factors including the greater degree of heterogeneity of MHC class II peptide complexes compared to refolded MHC class I molecules, and the weaker molecular binding (“low affinity”) of MHC class II tetramers, leading to greater difficulties in detection. The difficulty of producing reliable MHC class II tetramers to identify antigen specific CD4+ T cells has been a major challenge for the field.
Prior to the establishment of the new tetramer, in order to gain a more complete picture of immune responses to cancer vaccination, scientists have been required to carry out immune tests that assess CD4+ T-cell activity by measuring levels of certain cellular products associated with their function, such as cytokines or chemokines.
“These existing tests have been helpful but aren’t optimal,” says principal investigator Danila Valmori, director of the Cancer Vaccine Program at the Nantes Cancer Center. “They are time consuming, and they don’t directly measure the cells we’re trying to stimulate. It’s like pointing to smoke and trying to deduce the size and location of a fire without actually being able to see the fire itself.” She and the others on the study concluded that they required a tetramer to measure reliably CD4+ T-cell activity in the same way that CD8+ T cells are measured.
To overcome the difficulties in preparing high quality class II molecule tetramers, the team developed a new method. They first identified a short and immunogenic NY-ESO-1 peptide epitope presented by a conserved MHC class II molecule, HLA-DR52b, expressed in about half of Caucasians. To help them solve the problem of creating a stable tetramer of this class II molecule, the team in Nantes turned to Dr. Immanuel F. Luescher of the Lausanne Branch of the Ludwig Institute for Cancer Research. Dr. Luescher, who supplies tetramers for immune monitoring to the global network of CRI/LICR Cancer Vaccine Collaborative sites, solved the problem by attaching to one end of this peptide a polyhistidine tag (His-tag). This tag enabled the purification of only those HLA-DR52b molecules that were properly folded and loaded with the peptide for subsequent tetramerization. They demonstrated through laboratory analysis that the new tetramers avidly and stably bound to specific CD4+ T cells with negligible background on non-specific cells.
“With the new tetramer,” Valmori reports, “we are now able to visualize and quantify vaccine induced CD4+ T cells directly in a little more than an hour.”
Valmori and Ayyoub used the new tetramer to detect NY-ESO-1 specific CD4+ T cells among HLA-DR52b+ patients vaccinated in the previously reported study as compared to control groups. Whereas the CD4+ T cells were not detectable before vaccination, they were clearly detected post-vaccination at an average frequency of 1:5000 of total CD4+ T cells. Moreover, they discovered that the large majority of ESO-specific CD4+ T cells were “helper” T cells with the ability to support diverse immune responses, as well as the capacity for immunologic memory, suggesting that the vaccine may enhance protection against tumor recurrence. In addition, the cells did not exhibit suppressive or regulatory characteristics, providing further evidence that the vaccine under study was successful in stimulating a robust and precisely targeted CD4+ T-cell response among patients.
In addition to its role in advancing the current study, the novel tetramer technology developed by Valmori, Ayyoub, Djocinovic, and Luescher offers a powerful new means for scientists to monitor vaccine-induced CD4+ T-cell specificity for ESO and other vaccine targets, accelerating the strategic evaluation and refinement of therapeutic vaccines for the control of cancer.
The study received support from the Cancer Vaccine Collaborative (CVC), a joint program of the Cancer Research Institute and the Ludwig Institute for Cancer Research, by the Atlantic Philanthropies, the Conseil Régional des Pays de la Loire, and the European Structural Funds (FEDER) program, by the Institut National du Cancer (INCa) and by INSERM.
Ayyoub et al. Monitoring of NY-ESO-1 specific CD4+ T cells using molecularly defined MHC class II/His-tag-peptide tetramers. PNAS 201001322; published ahead of print April 5, 2010
Bioley et al. Vaccination with recombinant NY-ESO-1 protein elicits immunodominant HLA-DR52b-restricted CD4+ T cell responses with a conserved T cell receptor repertoire. Clinical Cancer Research July 2009 15; 4467.
Brian M. Brewer, Director of Communications, Cancer Research Institute
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About the Ludwig Institute for Cancer Research
The Ludwig Institute for Cancer Research (LICR) is an international non-profit institute dedicated to understanding and controlling cancer. With operations at 73 sites in 17 countries, the LICR network quite literally spans the world. LICR has developed an impressive portfolio of reagents, knowledge, expertise, and intellectual property, and has assembled the personnel, facilities, and practices necessary to patent, clinically evaluate, license, and thus translate, the most promising aspects of its own laboratory research into cancer therapies. Visit www.licr.org for more information.
About the Cancer Vaccine Collaborative
The Cancer Vaccine Collaborative (CVC), a joint program of the Cancer Research Institute (CRI) and the Ludwig Institute for Cancer Research (LICR), is a coordinated global network of clinical trial sites, with special expertise in immunology, conducting parallel early stage clinical trials to identify the optimal composition of successful therapeutic cancer vaccines. CVC investigators are supported by a comprehensive independent trials management infrastructure that provides regulatory, safety, and compliance expertise and oversight, trial management, shared data collection software, intellectual property management, and funding.
CVC trials test a wide variety of new reagents and multi-component vaccine constructs. By designing complementary trials to be run in parallel at multiple sites, and by standardizing the types of immunological measurements taken, the CVC helps to extract rapidly the immunological knowledge necessary to optimize eventually a therapeutic vaccine construct.
Since the program’s inception in 2001, the Collaborative has completed or is currently conducting more than 40 early phase clinical trials of therapeutic cancer vaccines, which together have produced some of the most comprehensive knowledge on the human immune response to the cancer-specific antigen NY-ESO-1, which is expressed in a broad variety of cancers, including melanoma, a deadly form of skin cancer, and breast, colon, lung, ovarian, and prostate cancers. For more information, go to http://www.cancerresearch.org/collaborative.