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Cancer Immun. 2008; 8(Suppl 1): 1-20.
Published online 2008 Mar 12.
PMCID: PMC2935788

Cancer Vaccines 2007

Cancer and HIV Vaccines: Shared Lessons
Cancer Immun. 2008; 8(Suppl 1): 1.
Published online 2008 Mar 12.

Cancer vaccines: an overview

Abstract

The long-held hope that vaccination strategies might be effective against cancer has motivated numerous attempts over the past century to put the idea to test in the clinic. Although the generally disappointing results have cast a long shadow over the field, advances in cancer immunology growing on the remarkable insights from basic immunology provide a strong foundation and powerful new tools to guide current attempts to fashion effective cancer vaccines. This review covers the scientific basis and rationale for cancer vaccines, the challenges involved in assembling the many ingredients going into the construction of cancer vaccines, and the daunting obstacles confronting academic investigators wanting to transfer their discoveries into the clinical arena. The Cancer Vaccine Collaborative (CVC), a partnership between the Cancer Research Institute and the Ludwig Institute for Cancer Research, represents a new academic model for developing, coordinating, conducting, and monitoring cancer vaccine trial. NY-ESO-1, a prototype cancer-testis (CT) antigen having strong spontaneous humoral and cellular immunogenicity, has been chosen as the initial CVC vaccine target, and the current status of NY-ESO-1 vaccine trials carried at the multiple CVC sites around the world is discussed.

Keywords: cancer vaccines, cancer immunosurveillance, tumor antigens, immunologic monitoring, clinical trials, NY-ESO-1

It is my pleasure to greet you to this 15th annual symposium organized by the Cancer Research Institute. We inaugurated the series to track the progress of therapeutic approaches to cancer based on immunological principles, with a particular focus on the development of cancer vaccines. Efforts to develop effective cancer and HIV vaccines have much in common, drawing upon the same strategies to vaccinate and tools to monitor, and confronting the same challenges of testing our ideas in the clinic. This meeting of experts from both endeavors provides the opportunity to discuss lessons we have learned and aspirations that we share, find ways to procure needed reagents, and seek solutions to barriers that frustrate our success. The CRI is grateful to Dr. Giuseppe Pantaleo for his critical role in the organization of this meeting and we thank all of you for joining us here today.

I will initiate the meeting now with (i) a brief review of the scientific basis for cancer vaccines, (ii) the personal lamentations of an academic investigator wanting to do clinical trials and the way the Ludwig Institute for Cancer Research that I led for 17 years has attempted to deal with this, (iii) a description of the CVC (Cancer Vaccine Collaborative), a clinical discovery instrument created by a partnership of the Cancer Research Institute and the Ludwig Institute for Cancer Research, and finally (iv) a review of the commercial vaccines that are in advanced clinical testing and a summary of the CVC's experience with vaccines against the highly immunogenic human tumor antigen NY-ESO-1.

Although vaccination against cancer has had a long and controversial history, the field long suffered from the absence of a strong and secure scientific basis. Over the past decade and a half, however, the situation has undergone a remarkable change and this can be directly traced to two sources: (i) the spectacular advances in our understanding of the immune system, and (ii) the validation of widely held but unproven ideas that inspired tumor immunologists.

The first of these, cancer immunosurveillance, the belief that the immune system protects against the development of cancer, a theory put forward by Lewis Thomas and Macfarlane Burnet in the 50s, was broadly accepted then broadly rejected when nu/nu immunodeficient mice did not develop more cancer than their wild-type counterparts. Over the past 5-8 years, the pendulum has swung back and we can now celebrate a formidable body of evidence validating cancer immunosurveillance, from the work of Bob Schreiber, Mark Smyth and their colleagues using mice deficient in individual components of innate and adaptive immunity.

The second deeply held conviction of cancer immunology is that there are specific cancer antigens, the philosopher's stones of the field, that would serve as targets for immune recognition and attack. The search for tumor-specific antigens has been one of the longest uninterrupted lines of inquiry in cancer research, marked by frustration, controversy and disappointment. However, from work carried out over the past two decades by the laboratories of Thierry Boon, Michael Pfreundschuh, Steve Rosenberg, and colleagues of mine, we can now celebrate enormous progress in the definition of cancer antigens with remarkable specificity for cancer and with the capacity to elicit humoral and cellular immunity. The discovery of these antigens is what provides our aspirations for effective cancer vaccines with such a firm theoretical basis.

The third idea that has permeated immunological thinking about cancer comes from the observation that human cancers are frequently infiltrated by cells of the immune system. This was of course postulated to reflect a defensive reaction on the part of the host, but there was little or no proof for this belief. We can now say with confidence from work with several tumor types, including colon cancer, bladder cancer and ovarian cancer, that CD8+ T cells in close contact with tumor cells have highly significant positive prognostic value. Microarray analysis of human tumors has also shown that immunological parameters, such as levels of gamma-interferon, can have a stronger prognostic correlation than the expression of genes generally associated with transformation.

Finally, clinical observations from the time of William B. Coley on the beneficial effect of bacterial infection and bacterial products on the course of human cancer have been interpreted to be due to strengthening immunological reactivity to cancer. This has its counterpart in the current use of BCG (bacillus Calmette-Guerin) as a successful treatment for superficial bladder cancer. The anti-tumor effect of IFN and IL-2, and the capacity of adoptively transferred T cells to cause regressions of large tumors, emphasize the power of the immune system to destroy cancer. Most profound to me, however, is the striking anti-tumor activity of anti-CTLA-4. This antibody to a single molecule on T cells can bring about total regression of tumors in patients with advanced melanoma, a Lazarus effect to be sure. The development of anti-CTLA-4 by Jim Allison is a striking example of how the study of a basic immunological question in mice can be exploited for therapeutic benefit in humans.

Because of these advances in cancer immunology − validation of the theory of cancer immunosurveillance, definition of a large number of tumor antigens as targets for immune recognition, prognostic significance of immunological parameters, such as CD8+ T cells infiltrating human tumors, and therapeutic benefits of immune-related therapies from BCG to anti-CTLA-4 − we now have a firm theoretical basis to test the validity of cancer vaccines as a therapeutic strategy. People who say cancer vaccines have been tested and failed are simply wrong. We only now have the knowledge and tools to put this powerful idea to test.

With our increasingly secure scientific base, how do we go about fashioning effective cancer vaccines? Clearly the model we have for basic research doesn't fit the complex inter-disciplinary, inter-institutional, and regulatory hurdles and requirements involved in an undertaking of this magnitude. The AIDS field confronted this precise challenge some years ago and we can ask at this point how effective the eminently reasonable plan at the time to form a consortium of government, academia, philanthropy and industry as the coordinating and facilitating core of this effort was − how effective was this in achieving the goal of an HIV vaccine? From the onset, the problem with this noble idea was that the component elements had different agendas, different timelines and different cultures − business had intellectual property and bottom lines on their mind, government by its nature is bureaucratic and lacks flexibility, philanthropy tends to be fractionated and uncoordinated, and academics find themselves in a chronic state of underfunding and in an environment that stresses and celebrates individual, not team or programmatic achievements.

The biggest problem, of course, and here I am talking specifically about Phase I clinical entry trials, is the extraordinary challenge confronting any academic wanting to put their ideas to the test in the clinic. Words like "daunting, insurmountable, nightmarish, career-wrecking" are what you hear from the countless investigators who have aspired but failed to scale the barriers separating the laboratory and the clinic. Just how difficult is it? Do a thought experiment with me. Imagine if every experiment we did in our laboratories involved myriad approval steps, was burdened with the extraordinary cost of making and testing the reagents we used, required an IND (Investigational New Drug) submission to the FDA (US Food and Drug Administration) for each experiment, and an additional cost of $500,000 - $1,000,000 to carry out the experiment - just how many discoveries do you think we would have made in our laboratories? The frustration is that never before have we had so much to test in the clinic and never before has it been so difficult or costly to do so.

So industry would seem to be the answer to the academics' prayer - they have the financial resources, the infrastructure to deal with the FDA and other regulatory requirements, and a large staff to initiate and monitor the trials. The problem is that industry, confronting the dictates of the commercial world, follows a different rule book than the academic world. In the real world, industry decides what to test, how to test it, where to test it and when to stop testing. Under these circumstances, the role of the academic becomes essentially a contract researcher, if he or she is lucky enough to be included in conducting the trial. What we need is a new rule book linking academic and commercial aspirations in this early phase of clinical testing.

With D. K. Ludwig's magnificent gift to the world of cancer research and the creation of the Ludwig Institute for Cancer Research, it was possible to construct a new model that allowed the Ludwig Institute to take responsibility for the initial clinical testing of its discoveries. Before describing the Ludwig Model, let me share a principal rule with you from my rule book for the academic clinical investigator. If I wanted to articulate a guiding mantra for individual academics and academic institutions wanting to control the fate and entry of their discoveries into the clinic, it would go something like this overarching statement: "Control the IP (intellectual property), and you control the clinical reagent. Control the reagent, and you control the clinical trial. Control the clinical trial and you control the field".

The Ludwig Institute translated these principles into (i) the development of a strong IP program, (ii) the construction of two facilities - one in Melbourne and one in Ithaca, NY - capable of producing GMP (good manufacturing practice) grade clinical products, (iii) the creation of a clinical trials management team to sponsor and conduct clinical trials but under industry standards, (iv) the assembly of a team of clinical investigators around the world to carry out Phase I/II trials, and (v) linking the licensing of LICR IP to the clinical goals of the LICR. Those of you who have not been involved in the clinical trial arena may wonder why I so stress the issue of IP. I assure you it's not about profit - that's always a toss of the dice. Rather, it has to do with having some control over the fate of your discoveries and having a say in how the validity of your ideas are initially tested in the clinic.

A pivotal event in the development of the cancer vaccine field was the decision of LICR and CRI to form a partnership that created the Cancer Vaccine Collaborative or CVC. This brought together two institutions with different strengths and different histories, but with a common commitment to the exploration of cancer vaccines. The CVC, which I direct, has 24 sites at leading academic institutions in 11 countries around the world and has evolved into a magnificent instrument for clinical discovery. The CVC is centrally managed and coordinated by a CVC Coordinating Committee, and CVC trials are sponsored by the LICR. There is a strong interaction between laboratory and clinical scientists at each site and there is a great deal of inter-site collaboration. Each site has strong expertise in immunological monitoring. Now one of the formidable challenges of creating cancer vaccines is the number of variables that need to be brought together and tested individually in the construction of a successful vaccine. If these are tried serially, one after another, progress would be extremely slow. In contrast, the coordination of CVC activities allows parallel trials that test each variable, and thus a rapid way to decide which is best. Finally, the CVC has established a free online journal − Cancer Immunity − to serve the needs of the cancer vaccine community.

Figure 1 is a global view of the present structure of the CVC and a listing of participating institutions in Australia, Asia, Europe and the USA. There is a strong possibility that the CVC and another organization with the same objective, the Cancer Vaccine Consortium, will join forces and merge. This is an exciting prospect because it will create a single strong voice in the cancer vaccine arena.

Figure 1
Cancer Vaccine Collaborative structure.

Now where are we in the current development of cancer vaccines? As background to our CVC efforts, let me show you a list of the most advanced commercial vaccines in clinical trials (Figure 2). As you can see, they range from whole cell vaccines and heat shock protein vaccines to defined antigen vaccines, including PAP, 5T4, MUC-1 and MAGE-3. The risk, of course, of large scale trials is that they may fail, and if they do, this can have a profound negative impact on the field. The Merck adenovirus HIV vaccine is a good example of this, as was the Therion vaccinia/fowlpox CEA vaccine in pancreatic cancer. There is a strong feeling in the academic community that such large trials should be conducted only if there are compelling indications from early stage trials, a precaution that companies do not always heed.

Figure 2
Commercial cancer vaccines in advanced stage clinical trials.

Now how does an academic enterprise such as CVC contribute to the goal of creating cancer vaccines? At the onset, we had several critical questions to answer. Should we choose a single antigen and use it to compare the multiplicity of different vaccine strategies or choose several antigens and select a single vaccine platform. How critical is immunological monitoring and having a secure base for understanding the immunological response to the vaccine? And when do you have sufficient evidence to justify going after a therapeutic endpoint?

We chose to focus on a single antigen and that antigen was NY-ESO-1, a member of the cancer/testis or CT family of tumor antigens, and one of the most fascinating tumor antigens found to date. There are over 90 CT gene or gene families with an expression pattern that makes them ideal vaccine targets. CT antigens are expressed in spermatogonia, fetal ooblasts and trophoblasts, but with no or highly limited expression in normal adult somatic tissues. In cancer, CT antigens are expressed in a wide range of different tumor types, and their aberrant expression appears to be due to cancer-related hypo-methylation. Over 50% of the CT coding genes are located on the X chromosome and make up over 10% of the coding sequences on that chromosome.

NY-ESO-1 was discovered by Yao Chen and our group in NY, and as a consequence of international collaborative efforts of CVC investigators in Germany, Japan, Australia, the United Kingdom and the USA, we now have a comprehensive view of the immunogenicity of this antigen, with our knowledge of NY-ESO-1 rivaling understanding of HIV and influenza immune responses. NY-ESO-1 ranks as the most immunogenic tumor antigen we know, eliciting a strong integrated humoral and CD4+ and CD8+ T cell immune response in patients with advanced NY-ESO-1 expressing tumors.

Monitoring the immune response to NY-ESO-1, both occurring spontaneously and following vaccination, is central to the CVC mission and, because of its strong immunogenicity, robust and standardized monitoring methodologies have been developed, allowing valid comparisons of immunological monitoring results at different sites.

This centrality of monitoring is exemplified by a maxim I developed for the CVC: "If you want to know how to vaccinate, you need to know how to immunize. And if you want to know how to immunize, you need to know how to monitor". This means that if you haven't defined and maximized the immune response to a vaccine, you probably shouldn't risk going after a therapeutic endpoint. The problem is that we are still learning how to monitor.

Figure 3 is a summary of the CVC sponsored NY-ESO-1 vaccine trials carried out to date. Over 34 trials with different NY-ESO-1 vaccine formulations have been or are being conducted. You will hear from a number of speakers at this meeting about the results of these trials, particularly the detailed immunological studies of these vaccines. The key finding is that NY-ESO-1 peptide, protein and pox-NY-ESO-1 vaccines can all induce strong NY-ESO-1 humoral and cellular immunity in patients with no pre-existing NY-ESO-1 immunity. The NY-ESO-1 Protein/ISCOMATRIX® trial conducted by Jonathan Cebon showed sufficient evidence for possible therapeutic benefit that a Phase II randomized trial is now ongoing, a trial entirely funded and sponsored by the LICR/CRI partnership. The salmonella/NY-ESO-1 vaccine, which shows remarkable therapeutic effects in mice, is now being prepared for the clinic and every effort is going into developing NY-ESO-1 adenovirus constructs for vaccination.

Figure 3
CVC NY-ESO-1 phase I clinical trials in cancer patient.

High priority needs for the CVC are listed in Figure 4 and I would imagine these are also priorities for academic HIV vaccinologists. CVC has a growing emphasis on prime-boost strategies, particularly in view of the success of DNA/vaccinia/adenovirus strategies in the HIV field. Adding additional CT and other antigens to the NY-ESO-1 vaccine constructs to create multivalent vaccines is another priority. But, if I were to say what our highest priority is, it would be to have access to specific reagents that downregulate the suppressive activity of Tregs and the inhibitory activity of CTLA-4 and other restraints on the immune system. Cancer vaccines are only one half of the equation. The other half is finding ways to modulate the regulatory constraints on immunological responses. Of course, no list of vaccine needs would be complete without adding more effective adjuvants and cytokines to maximize the immunogenicity of our vaccines. Finally, the whole point of this effort is to create therapeutic vaccines and, now that we have a good idea of the immunogenicity of NY-ESO-1 vaccines, going after therapeutic endpoints finally becomes justified.

Figure 4
Priorities of the Cancer Vaccine Collaborative.

I'm certain that it doesn't escape anyone's attention that everything on this list is about or depends on reagents, and in my opinion the three major obstacles to our continued success are reagents, reagents, and reagents. We must find ways to combat the chilling territoriality of reagent control, because putting together and testing the necessary ingredients for the construction of effective vaccines requires crossing all sorts of impenetrable IP and commercial boundaries. My hope is that by joining forces, the HIV and cancer vaccine effort will have a much more effective voice in overcoming obstacles to achieving our shared objectives. What we need to say is that our quest to construct effective vaccines against HIV and cancer is only beginning.

Abbreviations

CT
cancer/testis
CVC
Cancer Vaccine Collaborative
IP
intellectual property
LICR
Ludwig Institute for Cancer Research
Cancer Immun. 2008; 8(Suppl 1): 2.
Published online 2008 Mar 12.

Robust T-cell responses and clinical responses following long peptide vaccination against high risk HPV-16

Abstract

A therapeutic vaccine was designed based on long overlapping peptides covering the complete amino acid sequence of the HPV16 E6 and E7 oncogenic proteins, thereby harboring all potential T helper and CTL epitopes. Previously, we demonstrated that HPV16 specific T-cell immunity induced by this vaccine delivered in Montanide ISA 51 adjuvant was able to terminate persistent infections and eradicate established HPV16+ tumors in rabbits.

Currently, 12 patients with histologically proven HPV16+ vulvar intraepithelial neoplasia (VIN) grade III were vaccinated 4 times with a 3-week interval by s.c. injection of the long peptides emulsified in Montanide ISA 51. Immunological monitoring was performed at the systemic level by the analysis of blood samples, drawn before each vaccination and after the last vaccination, and at the local level by the analysis of HPV16-specific T cells in tissue biopsies of the VIN lesion (before and after vaccination) as well as a biopsy from the last vaccination site.

In all 11 patients, already after 2 vaccinations, strong and broad vaccine-induced systemic proliferative responses accompanied with the production of IFNγ and IL-5 were detected. This type of response is similar to the memory T-cell responses observed in healthy individuals with HPV16-specific immunity. Importantly, circulating HPV16 E6 and E7 specific T-cells produced IFNγ upon stimulation with naturally processed and presented antigen. Notably, vaccination resulted in the induction of both CD4+ and CD8+ HPV16-specific T cells. Multiple epitopes were recognized in each patient. Analysis of the local immune response demonstrated the presence of HPV16-specific Th1/Th2 cells infiltrating both the vaccination site and the VIN lesion after vaccination in 6 out of 9 patients analyzed. A complete clinical response was seen in 4 out of 12 patients, as determined by complete clearance of lesions by macroscopy and microscopy. In 3 of these patients HPV 16 was also cleared as determined by PCR.

Further improvement of T-cell responses against the E7 component was achieved by delivering the E6 and E7 peptides into different SC locations, thereby avoiding immunodominance of E6 over E7.

In conclusion, our peptide-based vaccine elicits a strong and broad HPV16-specific T-cell response that displays the capacity to migrate into the persistently HPV16-infected lesion of patients with high grade VIN and causes complete regressions in a substantial proportion of patients.

References

1. Dumortier H, van Mierlo GJ, Egan D, van Ewijk W, Toes RE, Offringa R, Melief CJ. Antigen presentation by an immature myeloid dendritic cell line does not cause CTL deletion in vivo, but generates CD8+ central memory-like T cells that can be rescued for full effector function. J Immunol. 2005;175:855–863. [PubMed]
2. Zwaveling S, Ferreira Mota SC, Nouta J, Johnson M, Lipford GB, Offringa R, van der Burg SH, Melief CJ. Established human papillomavirus type 16-expressing tumors are effectively eradicated following vaccination with long peptides. J Immunol. 2002;169:350–358. [PubMed]
3. Vambutas A, DeVoti J, Nouri M, Drijfhout JW, Lipford GB, Bonagura VR, van der Burg SH, Melief CJ. Therapeutic vaccination with papillomavirus E6 and E7 long peptides results in the control of both established virus-induced lesions and latently infected sites in a pre-clinical cottontail rabbit papillomavirus model. Vaccine. 2005;23:5271–5280. [PubMed]
4. de Jong A, van Poelgeest MI, van der Hulst JM, Drijfhout JW, Fleuren GJ, Melief CJ, Kenter G, Offringa R, van der Burg SH. Human papillomavirus type 16-positive cervical cancer is associated with impaired CD4+ T-cell immunity against early antigens E2 and E6. Cancer Res. 2004;64:5449–5455. [PubMed]
5. van Poelgeest MI, Nijhuis ER, Kwappenberg KM, Hamming IE, Wouter Drijfhout J, Fleuren GJ, van der Zee AG, Melief CJ, Kenter GG, Nijman HW, Offringa R, van der Burg SH. Distinct regulation and impact of type 1 T-cell immunity against HPV16 L1, E2 and E6 antigens during HPV16-induced cervical infection and neoplasia. Int J Cancer. 2006;118:675–683. [PubMed]
6. van Poelgeest MI, van Seters M, van Beurden M, Kwappenberg KM, Heijmans-Antonissen C, Drijfhout JW, Melief CJ, Kenter GG, Helmerhorst TJ, Offringa R, van der Burg SH. Detection of human papillomavirus (HPV) 16-specific CD4+ T-cell immunity in patients with persistent HPV16-induced vulvar intraepithelial neoplasia in relation to clinical impact of imiquimod treatment. Clin Cancer Res. 2005;11:5273–5280. [PubMed]
7. Melief CJ. Cancer immunology: cat and mouse games. Nature. 2005;437:41–42. [PubMed]
Cancer Immun. 2008; 8(Suppl 1): 3.
Published online 2008 Mar 12.

Restoring function in exhausted CD8 T cells during chronic viral infection

Abstract

Functional impairment of antigen-specific T cells is a defining characteristic of many chronic infections, but the underlying mechanisms of T-cell dysfunction are not well understood. To address this question, we analyzed genes expressed in functionally impaired virus-specific CD8 T cells present in mice chronically infected with lymphocytic choriomeningitis virus (LCMV), and compared these with the gene profile of functional memory CD8 T cells. Here we report that PD-1 (programmed death 1; also known as Pdcd1) was selectively upregulated by the exhausted T cells, and that in vivo administration of antibodies that blocked the interaction of this inhibitory receptor with its ligand, PD-L1 (also known as B7-H1), enhanced T-cell responses. Notably, we found that even in persistently infected mice that were lacking CD4 T-cell help, blockade of the PD-1/PD-L1 inhibitory pathway had a beneficial effect on the 'helpless' CD8 T cells, restoring their ability to undergo proliferation, secrete cytokines, kill infected cells and decrease viral load. Blockade of the CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) inhibitory pathway had no effect on either T-cell function or viral control. These studies identify a specific mechanism of T-cell exhaustion and define a potentially effective immunological strategy for the treatment of chronic viral infections.

Cancer Immun. 2008; 8(Suppl 1): 4.
Published online 2008 Mar 12.

Focusing the immune response on protective epitopes: A new approach to vaccine design using classical immunologic principles

Abstract

Protective antibodies (Abs) are needed to reduce the size of a virus inoculum and block infection of target cells. Since sera from some HIV-infected individuals have broad neutralizing activity and several human monoclonal Abs neutralize a broad spectrum of primary isolates, it is clear that the human B-cell repertoire includes genes capable of coding for broadly protective Abs. The epitopes that are known to induce broadly neutralizing Abs include the membrane proximal external region of gp41, the CD4 binding site on gp120, complex glycans on gp120, the CD4-induced epitope in and around the gp120 bridging sheet, and the V3 loop of gp120.

Despite the extensive information on HIV neutralizing Abs, it has proven difficult to induce broadly neutralizing Ab responses against HIV by immunization. This is due to several factors including the predominant induction of non-neutralizing rather than neutralizing Abs, the masking of neutralization-sensitive epitopes, the high mutation rate of HIV leading to antigenic variability, the physicochemical characteristics of the virus membrane, and the variable affinities of the different gp120 proteins for the virus receptors.

While various forms of the HIV envelope proteins have been used as immunogens, the best, albeit modest, results in terms of generating broadly neutralizing Abs have been achieved with strategies utilizing a DNA prime and either a recombinant adenovirus or protein boost. An alternative immunization approach is the construction and use of an immunogen that will focus the immune response on one or a few epitopes that are known to induce neutralizing Abs. An advantage of this approach is the potential to induce an immune response with a larger proportion of neutralizing Abs. To test the concept of "immunofocusing vaccines," we developed an immunization regimen designed to focus the immune response on the V3 loop of the HIV envelope. For this, we used both classical immunologic approaches and more recent data that stress the importance of the conformation of B cell epitopes to prime and selectively stimulate memory B cells.

Here, we report the results of experiments in rabbits in which animals were primed with gp120 DNA and boosted with a V3-fusion protein in order to preferentially induce anti-V3 Abs. The results provide a proof-of-principle that it is possible to focus the immune response on a neutralizing epitope and induce a vigorous neutralizing anti-HIV Ab response. The results suggest that immunofocusing vaccine regimens do indeed, target Abs to particular epitopes. This approach can now be employed to provide a platform for inducing Abs of greater potency and breadth by defining the best combinations of strains from which to build the priming and boosting immunogens and by designing immunogens that will optimally present several neutralizing epitopes.

Cancer Immun. 2008; 8(Suppl 1): 5.
Published online 2008 Mar 12.

New dimensions in serology

Abstract

Immunization of heterologous species with cancer cells or extracts to analyze the resulting antisera for cancer-specific antibodies inaugurated the search for human tumor antigens that could be used in cancer diagnosis and therapy. Technologies for generating monoclonal antibodies revolutionized the discovery process for cell surface and intracellular antigens of human cancer cells, beginning a new era in the clinical application of antibodies. The capacity of the immune system to recognize human cancer antigens was further substantiated with the development of autologous typing−an approach in which tumor cells, lymphocytes, antibody, and control cells are all derived from the same patient, thus eliminating the contribution of alloreactivity in the observed results. This development has led to a growing list of antibody-recognized antigens that are immunogenic in the host of origin, including mutational, overexpressed, viral, and cancer-testis (CT) antigens. Antigens in the latter category, including NY-ESO-1 and MAGE-A3, have been extensively studied and shown to spontaneously elicit antibody responses in a proportion of patients that correlate with T-cell immunity.

With the sequencing of the human genome and rapid and effective protein expression systems, it is now possible to envisage screening the human proteome with the human antibody repertoire, a process we refer to as seromic analysis. To this end, human protein arrays including a subset of antigens identified in the Cancer Immunome: SEREX Database were constructed, in collaboration with ProCognia, using their novel method for attaching proteins to the glass surface. Sera previously shown by ELISA to have reactivity to selected antigens were analyzed for reactivity to the same antigens using the arrays to evaluate their sensitivity and specificity. This technology has provided us with the analytic tools necessary to identify antigens with immunogenicity in cancer patients, as well as to expand the analysis to more antigens in larger cohorts of cancer patients. Preliminary results from array-based serological analyses of NSCLC patients identified a variety of novel antigens that may have utility as targets for cancer vaccine development. These data could also contribute to the elucidation of biomarker signatures for NSCLC based on seroreactivity, and help to define immune responses that can be correlated with clinical benefit for the cancer patient.

Cancer Immun. 2008; 8(Suppl 1): 6.
Published online 2008 Mar 12.

Human monoclonal antibodies and analytic vaccinology

Abstract

Following appropriate priming by infection or vaccination memory B cells and serum antibody levels are sustained for a lifetime conferring immediate protection upon secondary encounter with the pathogen. I will first discuss the differential requirements for activation of human naïve and memory B cells and propose a homeostatic model for the maintenance of the memory B cell pool and of serum antibody levels. I will then describe two methods that can be used to interrogate the human memory B-cell repertoire. The first is based on limiting dilution analysis of polyclonally stimulated mononuclear cells. Using this method we measured the frequency and fine specificity of memory B cells in serial samples under steady state conditions and after vaccination. In particular we found that only a small fraction of virus-specific memory B cells produce neutralizing antibodies, while the majority recognizes internal or denatured antigens. The second method is based on the efficient immortalization and cloning of memory B cells. Using this method we have been able to isolate from the human memory repertoire several potent and broadly neutralizing monoclonal antibodies against viruses such as SARS, Dengue, H5N1, HCMV and HIV-1. I will discuss how such antibodies can be used not only to provide immediate protection, but also as probes for epitope discovery and vaccine design.

Cancer Immun. 2008; 8(Suppl 1): 7.
Published online 2008 Mar 12.

Generating therapeutic/protective T-cell responses to tumors and HIV: "More is better, but will it be enough?"

Abstract

T cells can mediate potent responses to tumors and viruses, providing protection from the development of disease and eradicating existing disease. However, tumors and viruses can evade T-cell responses by multiple mechanisms, and generating effective T-cell responses will require designing strategies that overcome or circumvent these obstacles.

T cells targeting established tumors and chronic infections, if not immediately effective, are confronted by the obstacle of persistent stimulation by antigen, an event that commonly leads to reduction of effector activity and ultimately tolerance or deletion. Insights into the mechanisms by which potentially reactive T cells are rendered unresponsive, and strategies that may overcome such loss of activity, will be discussed.

Therapeutic vaccination in patients with existing tumors or HIV infection provides an opportunity to expand the frequency of reactive T cells in the host, and is based on the presumption that such responses can eliminate the tumor/virus. To better understand the requirements for success and/or reasons for failure, we have examined adoptive T-cell therapy with in vitro expanded tumor- or HIV-specific T-cell clones of defined function and avidity as a model for what can be achieved by vaccination. Preliminary studies in patients with relapsed leukemia and established HIV infection on HAART therapy will be presented.

Many parameters impact the efficacy of a vaccine, such as the ability to induce responses of appropriate magnitude and function that can persist and localize to the required site in vivo where effector activity is required. Developing such vaccines would benefit from the availability of murine models that better mimic human responses and permit efficient analysis of the responses elicited and the limitations and advantages of individual vaccine vectors. Efforts to develop the requisite genetically-modified mice, including the replacement of selected murine genes with human genes, and to establish informative murine models will be discussed, particularly in the context of the current HIV vaccine effort, but the results should also have implications for tumor vaccines.

Cancer Immun. 2008; 8(Suppl 1): 8.
Published online 2008 Mar 12.

The collaboration for AIDS vaccine discovery: A model for accelerating research

Abstract

The Global HIV Vaccine Enterprise was conceived in 2003 to bring new focus, cooperation, and expanded resources to HIV vaccine research. Since its inception, the Enterprise has formed an alliance of independent organizations that includes many of the leading AIDS vaccine research institutions in the world. Together, this group has developed and begun to implement a shared Scientific Strategic Plan, helped mobilize significant new resources, promoted collaborative work among researchers, and sponsored dialogues on major scientific challenges.

The Enterprise concept was first proposed in a June 2003 article in Science signed by 24 leaders in HIV vaccine research. They called for a revitalized, more collaborative research effort that would increase the scale and accelerate the pace of research, establish common standards for comparing products, expand manufacturing capability, and improve the capacity of clinical trials sites. The Enterprise proposal was further developed at a meeting of leading scientists, public health experts and policy makers in August 2003. In 2004, at their annual Summit, the Group of 8 nations endorsed the Enterprise concept and pledged their support. The Enterprise is now engaged in a broad range of activities, but science is its core business. The initial stakeholders in the Enterprise began work on their Scientific Strategic Plan (SSP) in 2004, convening six expert working groups and conducting a comprehensive review of the state of HIV vaccine research. The SSP was published in PLoS Medicine in February 2005.

Enterprise partners are now aligning many of their activities and launching new projects to address the six priority areas identified in the SSP: vaccine discovery, laboratory standardization, product development and manufacturing, clinical trials capacity, regulatory capacity, and intellectual property issues. The first major contribution of the Bill & Melinda Gates Foundation to this initial phase of the Enterprise was the creation of a new initiative called the Collaboration for AIDS Vaccine Discovery (CAVD).

The CAVD was launched in July 2006 and is being developed as a highly collaborative network of 17 research consortia, comprised of almost 200 investigators in over 90 institutions in 22 different countries. Twelve of the consortia are working on different approaches to developing HIV vaccines, six of which focus on vaccine concepts designed to induce cell-mediated immunity, and the other six on vaccine concepts designed to induce protective antibody responses. These 12 Vaccine Discovery Consortia (VDC) are supported by five Central Service Facilities (CSFs), which conduct standardized immunological evaluations and data and statistical analysis for the whole network, allowing for real-time comparison of the results. The CAVD projects are not intended to focus on basic discovery research. Instead, they target a perceived strategic gap in the HIV vaccine research and development continuum, which is the translational or "maturation" phase. This phase is aimed at harnessing the vast amount of knowledge derived from basic research to produce the "proof-of-concept" experiments required to proceed with confidence to the product development phase. To ensure that the whole effort is bigger than the sum of its parts, the 16 CAVD consortia and centers are bound together by a robust communication and alliance management strategy, and by legal agreements to share materials and data among the different CAVD laboratories and, in time, with the rest of the Enterprise alliance and the scientific community at-large. Now at the end of their first year of funding, the CAVD teams have made considerable progress, and a brief description of the main project objectives will be provided in the presentation.

Cancer Immun. 2008; 8(Suppl 1): 9.
Published online 2008 Mar 12.

Immune checkpoint blockade in tumor therapy: new mechanisms and strategies

Abstract

One reason for less than optimal success of clinical strategies to mobilize the immune system to attack cancer cells is that the immune system has multiple cell intrinsic and extrinsic regulatory circuits that serve to limit autoimmunity but can also frustrate anti-tumor responses. The prototype of cell intrinsic circuits is the CD28/CTLA-4 axis, which regulates early stages of the T-cell response. CD28 provides critical costimulatory signals necessary for activation of naïve T cells, while CTLA-4 limits proliferation of the responding T cells. Over the past several years our work has provided some insight into the molecular mechanisms whereby CTLA-4 inhibits T-cell proliferation, and how blockade of this inhibition can enhance anti-tumor responses in mice. As a single agent anti-CTLA-4 can induce the rejection of tumors with inherently high immunogenicity, and in combination with appropriate vaccines can induce rejection of poorly immunogenic tumors. CTLA-4 blockade is being developed as a cancer therapeutic by Medarex and Bristol-Myers Squibb and is currently in a large number of trials in a variety of cancers. To date, objective responses have been observed in many melanoma patients, and anecdotal reports have been obtained in renal, ovarian, and prostate cancer.

FoxP3+ regulatory T cells (Treg) provide a cell extrinsic mechanism for limiting effector T-cell responses, and are associated with poor anti-tumor responses. Data will be presented concerning the interaction of CTLA-4 blockade and Treg cells in tumor rejection in experimental mouse models. We will also present data suggesting that depletion of Treg after establishment of tumors may not be effective in enhancing anti-tumor responses elicited by anti-CTLA-4 and a GM-CSF expressing tumor cell vaccine, as well as a means by which to overcome Treg induced refractoriness.

In the last few years, the number of B7 family members has risen to seven. These fall into four groups, and have distinct expression patterns and immunological functions. The two newest members, B7-H3 and B7x, bind an as yet unidentified receptor and appear to be capable of inhibiting effector T-cell function. It is thus of considerable interest that many mouse and human tumor cells express B7x. We have recently found that high levels of expression of these inhibitory B7 molecules on human prostate cancer cells correlates with a higher rate of clinical failure. These might represent another checkpoint whose blockade would be of value in tumor immunotherapy.

Cancer Immun. 2008; 8(Suppl 1): 10.
Published online 2008 Mar 12.

Clinical and immunologic results of ipilimumab (MDX-010) in patients with metastatic melanoma

Abstract

Checkpoint blockade with anti-CTLA-4 monoclonal antibodies has emerged as an exciting new therapeutic modality for patients with metastatic melanoma. At our center, we have participated in 5 clinical trials using ipilimumab, a fully human IgG1 monoclonal antibody developed by Medarex and Bristol-Myers Squibb. Similar studies are being conducted elsewhere, using tremelimumab, an IgG2 developed by Pfizer. Initial studies of both of these agents have shown clinical activity as monotherapy with a unique pattern of controllable immune related adverse events (irAEs). At MSKCC, we have enrolled a significant number of patients on the phase II ipilimumab trials for patients who have not responded to prior therapy. In these studies, we have made some intriguing observations concerning patterns of response and progression, as well as association with immunologic correlates.

In general, patients demonstrate clinical benefit to ipilimumab at a later time point than would be expected for standard cytotoxic chemotherapy. Pooled data from prior phase I and II trials suggest an objective (partial + complete) response rate of approximately 15% at 12 weeks after initiation of therapy. However, a significant number of patients demonstrate long-term stabilization of disease, which can evolve into an objective response after time periods as long as 5 or 6 months. Even more curious is the observation that patients may have radiologic imaging suggestive of overt progression before eventually showing true clinical response many months later. This suggests that a unique set of criteria need to be developed for accurate classification of response to immunotherapy.

We have also noted that several patients treated with ipilimumab have undergone lymphoid reconstitution during anti-CTLA-4 therapy. Prior lymphopenia, which is frequently induced by temozolomide therapy, was corrected with ipilimumab and this immune reconstitution correlated with clinical response. The underlying biology is currently a topic of investigation and we are actively attempting to model this in mice. One hypothesis is that homeostatic proliferation, present in the context of lymphopenia, is accelerated by checkpoint blockade with anti-CTLA-4. Clearly, this has implications for the design of future clinical trials combining cytotoxic therapies with anti-CTLA-4 antibodies.

As part of the clinical trials, investigators within the Ludwig Center at MSKCC have developed a comprehensive immune monitoring plan for patients receiving anti-CTLA-4 therapy. This includes phenotypic characterization of T-cell subsets pre- and post-treatment, antigen-specific immune responses (both serologic and T-cell) and analyses of T-cell repertoire spectratyping.

Cancer Immun. 2008; 8(Suppl 1): 11.
Published online 2008 Mar 12.

Immunological impact of anti-CTLA4 therapy in a neoadjuvant setting

Abstract

CTLA4 blockade is an active immunotherapeutic strategy that is currently in clinical trials with cancer patients. Anti-CTLA4 therapy has led to measurable decreases in tumor size in patients with metastatic disease, including durable complete responses in some patients. While greater than 1000 cancer patients have been treated with anti-CTLA4 antibody therapy, there has been no hallmark immunological change demonstrated to be associated with drug administration or clinical benefit. Here we present data from a neoadjuvant anti-CTLA4 clinical trial in bladder cancer patients demonstrating immunological changes that are consistent in both the circulating blood and tumor microenvironment. Furthermore, our data demonstrates an increased ratio of effector to regulatory T cells. This is the first report in human patients that anti-CTLA4 therapy favorably shifts the ratio of effector to regulatory T cells, which may be a potential marker for clinical benefit.

Cancer Immun. 2008; 8(Suppl 1): 12.
Published online 2008 Mar 12.

The human HIV vaccine pipeline: an update

Abstract

The number of candidate vaccines that are designed to elicit T cell responses has markedly increased in the last 24 months. In the HVTN system, over 15 different vaccine prototypes have been evaluated. Unfortunately, several prototypes have been less than optimally immunogenic as measured by γIFN producing T cells, while others have elicited high levels of T-cell responses. This disparity has caused the human trialists to look at whether increased standardization of preclinical testing in non-human primates should precede initiation of human clinical trials. Two separate groups (Vaccine Research Center and Eurovac Foundation) have shown immunogenicity of DNA vaccines, especially for priming a subsequent Ad5 or NYVAC vector boost. Overall, replication defective adenoviruses appear to elicit the most consistent immune responses in humans, eliciting γIFN producing T-cell responses in >75% of recipients at levels that range from 200-500 spot forming cells/106 PBMC. Immunogenicity of Ad5 vaccines is influenced by dose, HIV insert and prior immunity of the recipient to Ad5. Efficacy trials are underway to define if the magnitude of current responses with Ad5 or DNA Ad5 vectors is useful for controlling viremia or decreasing acquisition of infection.

Cancer Immun. 2008; 8(Suppl 1): 13.
Published online 2008 Mar 12.

Protein-based vaccination targeting the cancer testis antigen NY-ESO-1

Abstract

The Cancer Vaccine Collaborative (CVC) has pioneered vaccine approaches against Cancer Testis (CT) antigens. These molecules are characterized by expression in a wide variety of cancer types and very limited expression in non-malignant tissues. NY-ESO-1, which has been the focus of particular interest, is highly immunogenic and frequently induces spontaneous immunity, particularly in patients with advanced metastatic disease. It is not clear whether these immune responses modify the natural history of the cancer.

Vaccine trials with NY-ESO-1 have been undertaken using peptides, proteins and genetic vaccines. These trials have been undertaken in patients with fully resected cancer as well as those with advanced metastatic disease. Key observations include: (i) a broad integrated CD4, CD8 T cell and antibody response is induced in the majority of patients vaccinated with full length NY-ESO-1 and an appropriate adjuvant, (ii) the depth and breath of this response is complex and can involve many epitopes in an individual vaccine recipient, (iii) despite only being early phase trials, some strong indications of clinical activity have emerged, particularly in patients with minimal residual disease. We are currently performing a randomized international trial with NY-ESO-1 in such patients with resected melanoma under the aegis of the CVC in Australia and UK. If improved clinical outcomes are observed, this trial will establish the clinical value of targeting such molecules.

In contrast vaccination of patients with advanced metastatic melanoma resulted in attenuated immune responses and this difference appears to be due to tumor-related factors. A variety of regulatory mechanisms appear to be in play. Using immunohistochemistry and fl ow cytometric analysis of melanoma tissue, we detected expression of the transcription factor FoxP3 in the melanoma cells themselves as well as in regulatory T lymphocytes (Treg). Expression of FoxP3 may endow tumor cells with Treg-like activity, as demonstrated by contact-dependent suppression of T cell proliferation and contribute to tumor immune suppression. Although patients with advanced cancer may have NY-ESO-1 specific immune response, the quality of these responses may be critical for determining clinical outcomes and there is emerging evidence that antigen specific Treg may recognize peptides derived from CT antigens, including NY-ESO-1. Additionally, analysis of NY-ESO-1-specific T cells in patients with advanced ovarian cancer before and after depletion of Tregs revealed that Tregs masked spontaneous and vaccine induced effector T cells. The Treg suppressed effectors were generally of higher avidity than vaccine induced effector cells. Together, the CVC trials suggest that vaccine efficacy might be enhanced by targeting regulatory mechanisms via depletion of CD25+FOXP3+ Tregs and treatment with antibody against CTLA4.

References

1. Barrow C, Browning J, MacGregor D, Davis ID, Sturrock S, Jungbluth AA, Cebon J. Tumor Ag expression in melanoma varies according to Ag and stage. Clin Cancer Res. 2006;12:764–771. [PubMed]
2. Jackson H, Dimopoulos N, Mifsud NA, Tai TY, Chen Q, Svobodova S, Browning J, Luescher I, Stockert L, Old LJ, Davis ID, Cebon J, Chen W. Striking immunodominance hierarchy of naturally occurring CD8+ and CD4+ T cell responses to tumor Ag NY-ESO-1. J Immunol. 2006;176:5908–5917. [PubMed]
3. Davis ID, Chen W, Jackson H, Parente P, Shackleton M, Hopkins W, Chen Q, Dimopoulos N, Luke T, Murphy R, Scott AM, Maraskovsky E, McArthur G, MacGregor D, Sturrock S, Tai TY, Green S, Cuthbertson A, Maher D, Miloradovic L, Mitchell SV, Ritter G, Jungbluth AA, Chen YT, Gnjatic S, Hoffman EW, Old LJ, Cebon JS. Recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant induces broad integrated antibody and CD4(+) and CD8(+) T cell responses in humans. Proc Natl Acad Sci U S A. 2004;101:10697–10702. [PMC free article] [PubMed]
4. Chen Q, Jackson H, Parente P, Luke T, Rizkalla M, Tai TY, Zhu HC, Mifsud NA, Dimopoulos N, Masterman KA, Hopkins W, Goldie H, Maraskovsky E, Green S, Miloradovic L, McCluskey J, Old LJ, Davis ID, Cebon J, Chen W. Immunodominant CD4+ responses identified in a patient vaccinated with full-length NY-ESO-1 formulated with ISCOMATRIX adjuvant. Proc Natl Acad Sci U S A. 2004;101:9363–9368. [PMC free article] [PubMed]
5. Cancer-testis antigens are co-ordinately expressed in metastatic melanoma and are associated with poorer clinical outcomes in primary stage II melanoma disease.; Cancer & HIV Vaccines 2007: Shared Lessons; 2007. poster P-63.
6. Antibody responses to the cancer antigen NY-ESO-1 identify highly immunogenic domains in melanoma patients before and after vaccination.; Cancer & HIV Vaccines 2007: Shared Lessons; 2007. poster P-62.
7. The regulatory T cell-associated transcription factor FoxP3 is expressed by tumor cells.; Cancer & HIV Vaccines 2007: Shared Lessons; 2007. poster P-16. [PubMed]
8. T-lymphocytes recognising melanocyte differentiation antigens mediate tumour regression induced by ipilimumab.; Cancer & HIV Vaccines 2007: Shared Lessons; 2007. poster P-34.
Cancer Immun. 2008; 8(Suppl 1): 14.
Published online 2008 Mar 12.

HLA-associated immunodominance and responsiveness to cancer vaccines

Abstract

CTL responses to pathogens are generally limited to a minor fraction of the many potential antigenic determinants encoded by the corresponding genomes, a phenomenon termed immunodominance (1). Both in studies in model systems using inbred mice and in humans, immunodominance has been shown to focus the CTL response to even large viruses to a limited number of determinants often presented in the context of defined HLA class I restricting alleles. The impact of HLA class I allele-associated immunodominance on clinical outcome has been recently documented for HIV, where expression of individual HLA class I alleles restricting immunodominant CTL responses has been clearly associated with slower disease progression (2). However, only few indications of a possible association between certain HLA alleles and clinical course in cancer patients have been reported so far, and the impact of HLA allele-associated immunodominance on the development of CTL responses to full-length recombinant tumor specific antigens has not yet been explored. The non-mutated self-antigen NY-ESO-1 belongs to the group of CTA, developmental antigens frequently expressed in human tumors. Although its function remains unknown, NY-ESO-1 is one of the best characterized human tumor antigens and induces spontaneous integrated antibody and T-cell responses in cancer patients bearing antigen-expressing tumors. For these reasons, NY-ESO-1 is regarded as a model tumor antigen for the development of generic cancer vaccines. In a recent study, we have obtained evidence that immunization with a NY-ESO-1 recombinant protein emulsified with Montanide® ISA-51 and CpG ODN 7909 induces integrated antibody and CD4+ T-cell responses to NY-ESO-1 in all vaccinated patients, at an early phase of the vaccination protocol. However, only half of them also developed specific CTL responses, which often became detectable at a later time point (3). The average level of antibody responses was higher in the group of patients who developed CTL responses as compared to the group that did not, indicating a role of NY-ESO-1 specific antibodies in the cross-priming of CTL. However, some individual patients who failed to develop CTL mounted antibody responses at levels similar to those of some of the responder patients, suggesting that additional factors may impact on the ability of the patients to develop CTL following immunization with the NY-ESO-1 recombinant protein. To unveil these factors, we have assessed epitope recognition and HLA-restriction of vaccine-induced CTL from responder patients in the study. Overall, our data show that recognition of immunodominant NY-ESO-1 epitopes by the large majority of vaccine-induced CTL was restricted by few frequently expressed HLA alleles. All responder patients expressed at least one of these HLA alleles, whereas none of the non-responder patients expressed them. Together, our data show that, in this antigenic system, HLA class I alleles associated immunodominance determines the patient's ability to develop specific CTL responses following vaccination with a full-length recombinant tumor antigen. As recombinant tumor antigens including several CTA but also differentiation and over-expressed antigens are presently among the most promising candidate cancer vaccines under assessment in both academic and industry sponsored clinical trials, we suggest that the immunological monitoring of such trials should systematically include the assessment of HLA association with responsiveness. Finally, our findings emphasize the importance of taking into account human genetic variation in the development of cancer vaccines.

References

1. Yewdell JW. Confronting complexity: real-world immunodominance in antiviral CD8+ T cell responses. Immunity. 2006;25:533–543. [PubMed]
2. Altfeld M, Kalife ET, Qi Y, Streeck H, Lichterfeld M, Johnston MN, Burgett N, Swartz ME, Yang A, Alter G, Yu XG, Meier A, Rockstroh JK, Allen TM, Jessen H, Rosenberg ES, Carrington M, Walker BD. HLA Alleles Associated with Delayed Progression to AIDS Contribute Strongly to the Initial CD8(+) T Cell Response against HIV-1. PLOS Medicine. 2006;3:e403 [PMC free article] [PubMed]
3. Valmori D, Souleimanian NE, Tosello V, Bhardwaj N, Adams S, O'Neill D, Pavlick A, Escalon JB, Cruz CM, Angiulli A, Angiulli F, Mears G, Vogel SM, Pan L, Jungbluth AA, Hoffmann EW, Venhaus R, Ritter G, Old LJ, Ayyoub M. Vaccination with NY-ESO-1 protein and CpG in Montanide induces integrated antibody/Th1 responses and CD8 T cells through cross-priming. Proc Natl Acad Sci U S A. 2007;104:8947–8952. [PMC free article] [PubMed]
Cancer Immun. 2008; 8(Suppl 1): 15.
Published online 2008 Mar 12.

Centralized gene-based HIV-1 vaccine elicits broad cross-clade cellular immune responses in rhesus monkeys

Abstract

One of the major challenges that must be met in developing an HIV-1 vaccine is devising a strategy to generate cellular immunity with sufficient breadth to deal with the extraordinary genetic diversity of the virus. Amino acids in the envelopes of viruses from the same clade can differ by more than 15%, and those from different clades can differ by more than 30%. It has been proposed that creating immunogens using centralized HIV-1 gene sequences might provide a practical solution to this problem. Such centralized genes can be generated employing a number of different strategies: consensus, ancestral, or center of tree sequences. These computer-generated sequences are a shorter genetic distance from any 2 contemporary virus sequences than those contemporary sequences are from each other. The present study was initiated to evaluate the breadth of cellular immunity generated through immunization of rhesus monkeys with vaccine constructs expressing either an HIV-1 global consensus envelope sequence (CON-S) or a single patient isolate clade B envelope sequence (clade B). We show that vaccine immunogens expressing the single centralized gene CON-S generated cellular immune responses with significantly increased breadth compared with immunogens expressing a wildtype virus gene. In fact, CON-S immunogens elicited cellular immune responses to 3-4-fold more discrete epitopes of the envelope proteins from clades A, C and G than did clade B immunogens. These findings suggest that immunization with centralized genes is a promising vaccine strategy for developing a global vaccine for HIV-1 as well as vaccines for other genetically diverse viruses.

Cancer Immun. 2008; 8(Suppl 1): 16.
Published online 2008 Mar 12.

Memory T cells: a matter of life and death

Abstract

Central memory T cells constitute the major correlate of protection in chronic viral diseases including HIV infection. They are essential for the development of protective immune responses in vaccines to viruses. Central memory T cells are endowed with a capacity to persist for up to twenty to thirty years in vivo in an infected or vaccinated individual. The mechanisms that lead to this persistence remain unknown. We have used multiparametric flow cytometry including Phosflow analysis to identify signal transduction pathways involved in central memory T-cell persistence and to validate gene array data. The latter had identified the FOXO3A transcription factor as a critical integrator of survival pathways leading to TCM persistence. FOXO3A is a member of the forkhead box protein family of genes which are essential in regulating survival and differentiation processes. FOXO3A regulates the transcription of several proapoptotic genes such as Bim, Puma and Fas-L and anti-proliferative genes such as Gadd 45 and p130. Upon dephosphorylation of FOXO3A this factor translocates to the nucleus and initiates the transcription of these genes leading to cell cycle arrest and apoptosis. Our results clearly demonstrate that the inhibition of phosphorylation of FOXO3A using several inhibitors leads specifically to TCM cell death. We further show that patients who naturally control HIV infection (Elite Controllers) have significantly higher levels of phosphorylated FOXO3A while HAART treated aviremic patients show mostly non phosphorylated, transcriptionally active FOXO3A; inhibition of FOXO3A using specific siRNAs or dominant negative mutants rescues the cell death of Cell sorted homogenous population of Central memory T cells from ECs allows them to persist in culture for up to 45 days. Our data clearly demonstrate the importance of FOXO3A in long term survival of central memory T cells and have allowed us to correct a major defect which occurs during HIV infection.

Cancer Immun. 2008; 8(Suppl 1): 17.
Published online 2008 Mar 12.

Final results of double-blind, placebo-controlled phase II study to assess the efficacy of MAGE-A3 immunotherapeutic in stage IB/II non-small cell lung cancer (NSCLC)

Abstract

Background: After complete resection, about 50% of patients with stages IB-II NSCLC disease die within 5 years. Adjuvant chemotherapy improves overall survival at the expense of substantial toxicity. Activity of MAGE-A3 immunotherapeutic (i.e. recombinant MAGE-A3 protein and a potent GlaxoSmithKline immunological adjuvant) was previously demonstrated in metastatic melanoma. As about 35% of NSCLCs express MAGE-A3 antigen, post-operative MAGE-treatment may be a tumor-specific, well tolerated, and effective adjuvant therapy.

Methods: Patients with completely resected, MAGE-A3 (+), stage pIB or pII were randomly assigned to postoperative MAGE-A3 or placebo (2:1), with 5 administrations at 3-week intervals, followed by 8 administrations every 3 months. Randomization was stratified for stage (IB vs. II), histology (squamous vs. other), and lymph-node (LN) procedure (sampling vs. dissection). Primary endpoint was disease-free interval (DFI); other endpoints were safety, disease-free survival (DFS), and overall survival (OS). This exploratory Phase II study was designed to detect a clinically relevant HR with a 10% one-sided α.

Results: 182 patients (122 stage IB, 60 stage II) from 59 centers in 14 countries were randomized over 2 years: Median age 63 (45-81); 87% male; 65% squamous cell carcinoma; 65% lymph-node dissection. After a median follow-up of 28 months, 67 recurrences and 45 deaths were recorded. Group comparisons of DFI, DFS and OS gave respectively a hazard ratio (HR) of 0.74 (95% CI 0.44-1.20, P = 0.107), 0.73 (95% CI 0.45-1.16) and 0.66 (95% CI 0.36-1.20) in favor of the MAGE-A3 group. Overall, treatment was well tolerated, with excellent protocol compliance. Subset analysis also suggests that LN dissection may have an effect on survival.

Conclusions: The final analysis of this randomized Phase II study shows a positive trend for activity of MAGE-A3 treatment in NSCLC with a relative improvement of DFI and DFS of 27%. Further Phase III evaluation is planned. This study also suggests that complete lymph-node dissection may have an effect on survival and should be confirmed prospectively.

GSK is now recruiting for a Phase III trial evaluating MAGE-A3 ASCI as adjuvant therapy in MAGE-A3 positive patients with NSCLC. With a target of about 2,270 patients, the randomized, double-blind, and placebo-controlled MAGRIT trial (MAGE-A3 as Adjuvant Non-Small Cell Lung Cancer Immunotherapy) will enroll patients with stage IB, II or IIIA resectable NSCLC. The ASCI administration will be initiated in two groups of patients: after surgery and standard chemotherapy in one group of patients and after surgery in patients who are not eligible for receiving chemotherapy. The primary endpoint of the trial is disease-free survival.

Cancer Immun. 2008; 8(Suppl 1): 18.
Published online 2008 Mar 12.

Immune correlates in HIV infection: application to vaccines

Abstract

Despite evidence that CD8+ T cells play an important role in the control of viral replication and disease progression in HIV-infected individuals, the specific CD8+ T-cell function(s) responsible for this activity remain unclear. We examined simultaneously five separate CD8+ T-cell functional parameters: degranulation ability (CD107a), cytokine expression (IFNγ, TNFα, IL2), and chemokine production (MIP1b) in HIV-specific T cells from HIV progressors (n = 79) and elite long-term non-progressors (n = 9). We found that the functional profile of HIV-specific CD8+ T cells in progressors is limited compared to nonprogressors. While the total magnitude of the CD8+ T-cell response did not correlate inversely with viral load, the magnitude and proportion of the most functional component of the CD8+ T-cell response did. In addition, other virus infections and vaccinations known to provide life-long protection against virus infections were characterized by highly poly-functional CD8+ T-cell responses.

We have also dissected the functions of virus-specific CD4+ T cells, and found that with prolonged maturation, virus-specific CD4+ T cells can express many direct effector functions. Some of these functions could be associated with in vivo protection from HIV infection. These include class II-restricted killer T cell activity and production of MIP-1b. Studies from HIV-infected subjects show that CD4+ T cells that produce MIP-1b are less susceptible to HIV infection in vivo than cells that do not produce MIP-1b. Thus, the T-cell response to HIV and other virus infections is complex, and the quality of the response may be more important to monitor than the quantity.

We have developed flow cytometric panels to monitor 5 separate T-cell functions, and multiple surface phenotypes, which we use to assess the spectrum of T-cell functions induced by our vaccines. These panels will be used to evaluate new vaccine platforms.

Cancer Immun. 2008; 8(Suppl 1): 19.
Published online 2008 Mar 12.

T cells: Functional and molecular analysis of vaccine elicited T-cell responses in melanoma

Abstract

The aim of therapeutic vaccines for cancer or chronic viral infections is to elicit strong antigen-specific T-cell responses able to eliminate transformed or virus infected cells. We have recently identified a strong adjuvant formulation consisting of low doses of a tumor antigenic peptide and of a synthetic TLR-9 agonist, CpG 7909 (PF676), emulsified in Montanide ISA-51 (1). All metastatic melanoma patients (n = 24) immunized with the Melan-A26-35 A27L peptide analogue, which binds strongly to the HLA-A2 molecule, had readily detectable A2/Melan-A tetramer+ CD8 T-cell responses after 2 to 4 subcutaneous injections of the vaccine. In contrast, only half of patients immunized with the same emulsion lacking the TLR-9 agonist had detectable tetramer+ CD8 T-cell responses after repeated vaccination (2). Moreover, the frequencies of specific T cells were approximately 10-fold higher in the group of patients receiving the TLR9 agonist containing vaccine.

Functional assessment of the postvaccination Melan-A tetramer+ CD8 T cells revealed robust differentiation to effector memory type, IFN-γ release and lytic activity comparable to viral antigen specific CD8 T cells present in the autologous peripheral blood lymphocyte pool from the same samples. In contrast, Melan-A tetramer+ CD8 T cells recovered from postvaccination tumor biopsies from two patients displayed reduced function. This was coincident with accumulation of relatively high numbers of regulatory T cells (3). Repeated vaccination led to a progressive increase in the fraction of Melan-A tetramer+ CD8 T cells that downregulated the expression of the CD28 coreceptor present in the circulating lymphocyte pool. These cells displayed an even more marked differentiation towards effector type cells and a reduction in the number of TCR clonotypes. Close to 60% of a series of independent CTL clones isolated from three vaccinated patients efficiently recognized and killed tumor cells in an antigen-specific fashion.

The efficacy of the vaccine formulation described here is additionally demonstrated by its ability to elicit strong CD8 T-cell responses to vaccination with the Melan-A26-35 natural peptide, which forms unstable and low affinity complexes with the HLA-A2 molecule. The weak immunogenicity of such peptide had hindered thus far its use as a vaccine. Functionally, the specific CD8 T cells induced by the non-substituted peptide were also of the effector memory type. Surprisingly, however, they appeared to be superior in terms of both expressed effector molecules and tumor reactivity than those elicited by the substituted peptide analogue. Moreover, close to 100% of a series of independent CTL clones isolated from three melanoma patients immunized with the natural Melan-A peptide were tumor reactive and had a high functional avidity of antigen recognition (4). These results suggest that weak self peptides can be superior to substituted analogues at inducing high avidity T cells provided that appropriately strong adjuvant formulations are used. These results may have wide ranging implications for therapeutic vaccine design.

It has become urgent to identify immune correlates of clinical efficacy of cancer vaccines. This is an important challenge at the present stage of vaccine development because of the low tumor response rates observed in consecutive non-randomized phase I clinical trials including small numbers of patients. In this regard, one approach we favor is to carry the analysis of vaccine-specific T-cell responses at the individual T cell level. We have optimized an experimental strategy based on the labeling of antigen-specific T cells with tetramers combined with cell surface markers for memory and effector subsets and sorting of large collections of single cells from each subset, from each patient and from sequential blood samples taken at different time points before and after vaccination (5). Gene expression analysis and identification of the TCRs is carried out cell by cell using conventional PCR on amplified cDNA. Work in progress shows a very dynamic picture of T-cell responses to peptide vaccines, with the establishment of few dominant clonotypes that are specific to each individual patient. Our results suggest that long term persistence of dominant T-cell clones might correlate with favorable clinical outcomes. In turn, persistence of dominant clones appears to be associated with long telomeres and slow transition to the highly differentiated effector phenotype characterized by high perforin content and loss of the CD28 and CD27 coreceptors.

It is widely acknowledged that poor clinical efficacy of current vaccine approaches may not only be explained by suboptimal vaccine formulation but also by multiple immune regulatory checkpoints active in the advanced metastatic cancer setting. In this regard, regulatory T cells may play a central role. In my talk, I will also discuss recent results on monitoring Melan-A antigen-specific regulatory T cells in vaccinated patients. In summary, functional and molecular monitoring of vaccine induced specific T cells at the single cell level provides invaluable lessons for future therapeutic vaccine development.

References

1. Speiser DE, Liénard D, Rufer N, Rubio-Godoy V, Rimoldi D, Lejeune F, Krieg AM, Cerottini JC, Romero P. Rapid and strong human CD8+ T-cell responses to vaccination with peptide, IFA and CpG oligodeoxynucleotide 7909. J Clin Invest. 2005;115:739–746. [PMC free article] [PubMed]
2. Liénard D, Rimoldi D, Marchand M, Dietrich PY, van Baren N, Geldhof C, Batard P, Guillaume P, Ayyoub M, Pittet MJ, Zippelius A, Fleischhauer K, Lejeune F, Cerottini JC, Romero P, Speiser DE. Ex vivo detectable activation of Melan-A specific T cells correlating with inflammatory skin reactions in melanoma patients vaccinated with peptides in IFA. Cancer Immun. 2004;4:4 http://www.cancerimmunity.org/v4p4/040404.htm [PubMed]
3. Appay V, Jandus C, Voelter V, Reynard S, Coupland SE, Rimoldi D, Lienard D, Guillaume P, Krieg AM, Cerottini JC, Romero P, Leyvraz S, Rufer N, Speiser DE. New generation vaccine in humans induces effective melanoma specific CD8+ T-cells in the circulation but not in the tumor site. J Immunol. 2006;177:1670–1678. [PubMed]
4. Speiser D , Unmodified self antigen triggers human CD8 T cells with stronger tumor reactivity than altered antigen. Manuscript submitted for publication. [PMC free article] [PubMed]
5. Speiser DE, Baumgaertner P, Barbey C, Rubio-Godoy V, Moulin A, Corthesy P, Devevre E, Dietrich PY, Rimoldi D, Liénard D, Cerottini JC, Romero P, Rufer N. A novel approach to characterize clonality and differentiation of human melanoma-specific T-cell response: Spontaneous priming and efficient boosting by vaccination. J Immunol. 2006;177:1338–1348. [PubMed]
Cancer Immun. 2008; 8(Suppl 1): 20.
Published online 2008 Mar 12.

Cell-mediated control of immune mediated inflammation

Abstract

The regulatory T cells (Treg) restrain immune responses through elaboration of suppressor function dependent upon expression of the transcription factor Foxp3. Despite a critical role for Treg cells in maintaining lympho-myeloid homeostasis, it remains unclear whether a single mechanism or multiple mechanisms of Treg-mediated suppression are operating in vivo, and how redundant such mechanisms might be. We address these questions using a genetic approach. Our studies suggest that Treg cells utilize multiple means to limit immune response. Furthermore, these mechanisms are likely non-redundant with a distinct suppressor mechanism playing a prominent and identifiable role at a particular tissue and inflammatory setting.


Articles from Cancer Immunity are provided here courtesy of Academy of Cancer Immunology
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