Innovative Cancer Vaccine Strategies Based on the Identification of Tumour-Associated Antigens

The identification of tumour-associated antigens has opened up new approaches to cancer immunotherapy. While past research focused on CD8+ cytotoxic T-cell responses, accumulating evidence suggests that CD4+ T cells also play an important role in orchestrating the host immune response against cancer. In this article, we summarise new strategies for the identification of major histocompatibility complex (MHC) class II-associated tumour antigens and discuss the importance of engaging both CD4+ and CD8+ T cells in cancer immunotherapy. The cloning of MHC class I- or class II-associated antigens has made it possible to develop synthetic and recombinant cancer vaccines that express specific tumour antigens. There are three major types of synthetic and recombinant cancer vaccines: recombinant viral and bacterial vaccines; naked DNA or RNA vaccines; and recombinant protein and peptide vaccines. In this article, we also discuss a new generation of recombinant cancer vaccines, ‘self-replicating’ DNA and RNA vaccines. Studies on the mechanisms of ‘self-replicating’ nucleic acid vaccines revealed that the enhanced immunogenicity was not due to an enhanced antigen expression, suggesting that the quantitative difference may not be as important as the qualitative difference in antigen presentation. The presence of the RNA replicase in the ‘self-replicating’ nucleic acid vaccines mimics alphavirus infection, which triggers the innate antiviral pathways of the host cells. Studies on how viral and cellular modulators of the innate antiviral pathways affect vaccine function should provide molecular insights crucial to future vaccine design.     

The role of MHC class II-restricted tumor antigens and CD4+ T cells in antitumor immunity

The identification of tumor antigens has generated a resurgence of interest in immunotherapy for cancer. However, both clinical and animal studies suggest that therapeutic strategies that have mainly focused on the use of CD8+ T cells (and MHC class I-restricted tumor antigens) are not effective in eliminating cancer cells. Recent interest has been directed towards the use of CD4+ T cells in generating antitumor immunity. To this end, the identification of MHC class II-restricted tumor antigens that can stimulate CD4+ T cells might provide opportunities for developing effective cancer vaccines.     

In This Issue

The cellular immune responses to ovarian cancer patients treated with viral oncolysates (VO) ovarian tumor vaccines to vaccines are described. CD3+ cells proliferated after stimulation with the tumor vaccines in a dose-dependent manner. The proliferation of CD3+ cells stimulated with the tumor vaccine was blocked by anti-HLA-DR monoclonal antibody and anti-CD4 mAb indicating that CD3+ CD4+ cells from the blood of the patients treated with VO recognize tumor derived determinants in conjunction with MHC class II antigens. The regulatory activity of the T cells collected after VO treatment was assayed by co-cultivation with PBMC collected before VO treatment. These cells demonstrated increased helper activity for immunoglobin production by cells collected before vaccination and secreted IL-2 in response to stimulation by vaccine. Finally, when biochemical fractionation of the components of VO was attempted, PBMC from VO treated patients responded by proliferation to several fractions suggesting that they recognize multiple epitopes in the ovarian tumor vaccine. Therefore, these data provide novel evidence for the involvement of the T cells in response to ovarian tumor vaccines.     

T cell responses to ovarian tumor vaccines: identification and significance for future immunotherapy.

The cellular immune responses to ovarian cancer patients treated with viral oncolysates (VO) ovarian tumor vaccines to vaccines are described. CD3+ cells proliferated after stimulation with the tumor vaccines in a dose-dependent manner. The proliferation of CD3+ cells stimulated with the tumor vaccine was blocked by anti-HLA-DR monoclonal antibody and anti-CD4 mAb indicating that CD3+ CD4+ cells from the blood of the patients treated with VO recognize tumor derived determinants in conjunction with MHC class II antigens. The regulatory activity of the T cells collected after VO treatment was assayed by co-cultivation with PBMC collected before VO treatment. These cells demonstrated increased helper activity for immunoglobin production by cells collected before vaccination and secreted IL-2 in response to stimulation by vaccine. Finally, when biochemical fractionation of the components of VO was attempted, PBMC from VO treated patients responded by proliferation to several fractions suggesting that they recognize multiple epitopes in the ovarian tumor vaccine. Therefore, these data provide novel evidence for the involvement of the T cells in response to ovarian tumor vaccines.     

A phenotype based approach for the immune monitoring of NY-ESO-1-specific CD4+ T cell responses in cancer patients

Because of its frequent expression in tumors and spontaneous immunogenicity in advanced cancer patients, NY-ESO-1 is presently viewed as a prototype tumor antigen for the development of cancer vaccines. A prerequisite for the analysis of NY-ESO-1-specific T cell responses in vaccinated patients is the assessment of the complete T cell repertoire available for the antigen. Here, we have assessed frequency and fine specificity of CD4+ T cells reactive against NY-ESO-1-derived sequences in circulating lymphocytes from cancer patients with spontaneous responses to the antigen. We found that, relative to healthy donors, this frequency was only moderately increased in cancer patients. The reactivity of these cells, however, was directed against the same immunodominant regions previously identified for healthy donors. On account of these data, we developed an approach for the immune monitoring of NY-ESO-1-specific CD4+ T cell responses based on the assessment of CD4+ T cell populations of defined phenotype. Using this approach, a similar frequency of NY-ESO-1-specific CD4+ T cells was found among naive T cells of healthy donors and cancer patients. In contrast, among antigen-experienced T cells, NY-ESO-1-specific CD4+ T cells were exclusively detectable in cancer patients. We anticipate that this phenotype-based approach will be useful for the immune monitoring of vaccine-induced responses in vaccination trials using NY-ESO-1 as well as other tumor antigens.     

B-cell malignancies as a model for cancer vaccines: from prototype protein to next generation genetic chemokine fusions

Summary: B-cell malignancy-derived Ig may be considered a model tumor antigen for vaccine development. However, as a non-immunogenic self antigen, it must also be first rendered immunogenic by chemical or genetic fusion to carriers which enable the induction of protective anti-tumor immunity in murine tumor models. Our group has demonstrated chat active immunizations of human patients with idiotypic vaccines elicited antigen-specific CD8⁺ T-cell responses and antitumor effects. Several alternative preclinical strategies to develop vaccines have been previously reported, including fusion of tumor idiotype-derived single chain Fv with cytokities and immunogenic peptides. On the other hand, we have recently explored a different approach in which the model antigen is rendered immunogenic in mice by genetically fusing it to a chemokine moiety. Administration of these vaccines as fusion proteins or naked DNA vaccines may allow efficient targeting of antigen-presenting cells in vivo. Potent autitumor immunity was dependent on the generation of specific anti-idiotypic antibodies and both CD4⁺ and CD8⁺ effector T cells. We propose that chemokine fusion may represent a novel, general strategy for formulating existing or newly identified tumor and HIV antigens into vaccines for cancer and AIDS, respectively, which elicit potent CD8⁺ T-cell immunity.     

A subpopulation of CD8+ T cells specific for melanocyte differentiation antigens expresses killer inhibitory receptors (KIR) in healthy donors: evidence for a role of KIR in the control of peripheral tolerance

In cancer patients, NK cell inhibitory receptors (IR) are expressed on a fraction of melanoma-specific lymphocytes with a unique reactivity for tumor antigens derived from normal, nonmutated genes (differentiation antigens). It is presently not known whether expression of these receptors is induced during an immune response against melanoma cells or whether these receptors can be found on T cells harboring a self specificity for such differentiation antigens in healthy donors. By analyzing short-term cultures of CD8+ T cells primed in vitro with melanocyte differentiation antigens, we found expression of NK cell receptors on a small but consistent fraction of CD8+ T cells derived from healthy donors. Both long and short forms of NK cell receptors were expressed. Interestingly, only long forms were functional and inhibited effector functions (cytotoxicity and IFN-gamma production) of these CD8+ T cells. Short forms were devoid of any detectable activating function. The presence of T cells specific for differentiation antigens and expressing NK cell receptors, with an exclusive inhibitory function, in healthy donors strengthens the notion that IR may serve to control T cell tolerance to some peripheral antigens.     

Enhancing antitumor immune responses: intracellular peptide delivery and identification of MHC class II-restricted tumor antigens

Summary: The importance of T‐cell‐mediated antitumor immunity has been demonstrated in both animal models and human cancer therapy. The identification of major histocompatibility complex (MHC) class I‐restricted tumor antigens has generated a resurgence of interest in immunotherapy for cancer. However, recent studies suggest that therapeutic strategies that have mainly focused on the use of CD8⁺ T cells (and MHC class I‐restricted tumor antigens) may not be effective in eliminating cancer cells in patients. Novel strategies have been developed for enhancing T‐cell responses against cancer by prolonging antigen presentation of dendritic cells to T cells and the inclusion of MHC class II‐restricted tumor antigens. identification of MHC class II‐restricted tumor antigens, which are capable of stimulating CD4⁺ T cells, not only aids our understanding of the host immune responses against cancer antigens, but also provides opportunities for developing effective cancer vaccines.     

Therapeutic vaccination with tumor cells that engage CD137

Therapeutic cancer vaccination is based on the finding that tumors in both humans and experimental animals, such as mice, express potential immunological targets, some of which have high selectivity for cancer cells. In contrast to the successful vaccination against some infectious diseases, where most vaccines induce neutralizing antibodies that act prophylactically, the aim of therapeutic cancer vaccines is to treat established tumors (primarily micrometastases). Since most tumor-destructive immune responses are cell-mediated, therapeutic cancer vaccination needs to induce and expand such responses and also to overcome "escape" mechanisms that allow tumors to evade immunological destruction. Tumor antigens (as with other antigens) are presented by "professional" antigen-presenting cells, most notably dendritic cells (DC). Therefore DC that have been transfected or "pulsed" to present antigen provide a logical source of tumor vaccines, and some encouraging results have been obtained clinically as well as in preclinical models. An alternative and more physiological approach is to develop vaccines that deliver tumor antigen for in vivo uptake and presentation by the DC. Vaccines of the latter type include tumor cells that have been modified to produce certain lymphokines or express costimulatory molecules, as well as cDNAs, recombinant viruses, proteins, peptides and glycolipids which are often given together with an adjuvant. Several studies over the past 5 years have demonstrated dramatic therapeutic responses against established mouse tumors as a result of repeated injections of agonistic monoclonal antibodies (MAbs) to the costimulatory molecule CD137 (4-1BB). However, the clinical use of such MAbs may be problematic since they depress antibody formation, for example, to infectious agents. The alternative approach to transfect tumor cells to express the CD137 ligand (CD137L) increases their immunogenicity, but vaccination with tumor cells expressing CD137L is ineffective in several systems where injection of anti-CD137 MAb produces tumor regression. Recent findings indicate that a more effective way to engage CD137 towards tumor destruction is to transfect tumor cells to express a cell-bound form of anti-CD137 single-chain Fv fragments (scFv). Notably, tumors from melanoma K1735, growing either subcutaneously or in the lung, could be eradicated following vaccination with K1735 cells that expressed anti-CD137 scFv. This was in spite of the fact that K1735, as with many human neoplasms, expresses very low levels of MHC class I and has low immunogenicity. Similar results were subsequently obtained with other tumors of low immunogenicity, including sarcoma Ag104. We hypothesize that the concomitant expression of tumor antigen and anti-CD137 scFv effectively engages NK cells, monocytes and dendritic cells, as well as activated CD4(+) and CD8(+) T cells (all of which express CD137) so as to induce and expand a tumor-destructive Th1 response. While vaccines in the form of transfected tumor cells can be effective, at least in mouse models, the logical next step is to construct vaccines that combine genes that encode molecularly defined tumor antigens with a gene that encodes anti-CD137 scFv. Before planning any clinical trials, vaccines that engage CD137 via scFv need to be compared in demanding mouse models for efficacy and side effects with vaccines that are already being tested clinically, including transfected DC and tumor cells producing granulocyte-macrophage colony-stimulating factor.     

Cell-based vaccines for the stimulation of immunity to metastatic cancers

Summary: We are developing vaccines for inducing immunity lo metastatic cancers. Although primary tumors are frequently cured by surgery, chemotherapy, or radiation therapy, metastatic lesions often do not respond lo these treatments or proliferate after conventional therapy is terminated. Vaccine therapy for established metastases as well as prophylactic vaccine treatment to prevent outgrowth of latent metastatic tumor cells would therefore be beneficial. Our goal is to activate CD4⁺ and CDS⁺ T lymphocytes; however, we have focused on activating tumor-specific CD4⁺ T-helper lymphocytes because of their pivotal role as regulatory cells and in the generation of long-term immunological memory. The vaccines are based on the premise that tumor cells express potentially immunogenic antigens that could be targeted for T-cell activation, and that if appropriately genetically modified, tumor cells could be antigen presenting cells for these antigens. To facihtate direct antigen presentation to CD4⁺ T cells, tumor cells have been transfected with syngeneic major histocompatibility complex class II, co-stimulatory molecule, and/or superantigen genes. In vivo studies in three mouse tumor models demonstrate that vaccination protects against future challenge with wild-type tumor, cures some solid primary tumors, reduces established metastatic disease, and extends mean survival time. Antigen presentation studies demonstrate that m vivo vaccine efficacy is directly related to in vitro antigen presentation activity. The relevance of antigen presentation activity of the vaccines is further confirmed by in vivo studies demonstrating that during the immunization process, the vaccines directly present tumor-encoded antigens to CD4⁺ T lymphocytes. Adaptation of these vaccines for the treatment of human metastatic cancers is discussed.     

Taming cancer by inducing immunity via dendritic cells

Summary: Immunotherapy seeks to mobilize a patient's immune system for therapeutic benefit. It can be passive, i.e. transfer of immune effector cells (T cells) or proteins (antibodies), or active, i.e. vaccination. In cancer, passive immunotherapy can lead to some objective clinical responses, thus demonstrating that the immune system can reject tumors. However, passive immunotherapy is not expected to yield long-lived memory T cells that might control tumor outgrowth. Active immunotherapy with dendritic cell (DC)-based vaccines has the potential to induce both tumor-specific effector and memory T cells. Early clinical trials testing vaccination with ex vivo -generated DCs pulsed with tumor antigens provide a proof-of-principle that therapeutic immunity can be elicited. Yet, there is a need to improve their efficacy. The next generation of DC vaccines is expected to generate large numbers of high-avidity effector CD8⁺ T cells and to overcome regulatory T cells. Therapeutic vaccination protocols will combine improved ex vivo DC vaccines with therapies that offset the suppressive environment established by tumors.     

Considerations on clinical use of T cell immunotherapy for cancer

The recognition by effector T lymphocytes of novel antigenic targets on tumor cells is the premise of specific, targeted immunotherapy of cancer. With the molecular characterization of peptide epitopes from melanoma antigens and, more recently, broadly expressed tumor antigens, there has been considerable enthusiasm for clinical evaluation of peptide tumor vaccines. Immunologic monitoring of vaccinated patients has demonstrated an expansion of CD8+ T cells that react with the relevant peptide and, more importantly, with native tumor. In most instances, however, vaccine-induced CD8+ T cell responses alone have not been sufficiently robust or sustained to translate into a high percentage of durable clinical responses. Vaccine strategies have also utilized dendritic cells (DCs) that have been modified to present tumor antigens. The superior antigen-processing capacity and co-stimulatory function of DCs convey a powerful stimulatory signal to both CD4+ and CD8+ T cells. Several strategies are attempting to broaden the immune response beyond single antigens by introducing the entire complement of tumor antigens into DCs. Adoptive immunotherapy is a promising strategy to recover tumor-reactive precursor T cells from patients, stimulate them to induce numerical expansion, and then re-infuse them. Ex vivo manipulation of the tumor-reactive T cells also permits cytotoxic therapy to be administered to the patient without damaging the effector cells. Recently, host lymphodepletion prior to adoptive transfer of effector T cells has resulted in an extremely high and sustained frequency of effectors that has achieved therapeutic efficacy against bulky metastatic disease in a substantial fraction of treated patients.     

Integrating immunopeptidome analysis for the design and development of cancer vaccines

The repertoire of naturally presented peptides within the MHC (major histocompatibility complex) or HLA (human leukocyte antigens) system on the cellular surface of every mammalian cell is referred to as ligandome or immunopeptidome. This later gained momentum upon the discovery of CD8 + T cells able to recognize and kill cancer cells in an MHC-I antigen-restricted manner. Indeed, cancer immune surveillance relies on T cell recognition of MHC-I-restricted peptides, making the identification of those peptides the core for designing T cell-based cancer vaccines. Moreover, the breakthrough of antibodies targeting immune checkpoint molecules has led to a new and strong interest in discovering suitable targets for CD8 +T cells. Therapeutic cancer vaccines are designed for the artificial generation and/or stimulation of CD8 +T cells; thus, their combination with ICIs to unleash the breaks of the immune system comes as a natural consequence to enhance anti-tumor efficacy. In this context, the identification and knowledge of peptide candidates take advantage of the fast technology updates in immunopeptidome and mass spectrometric methodologies, paying the way to the rational design of vaccines for immunotherapeutic approaches. In this review, we discuss mainly the role of immunopeptidome analysis and its application for the generation of therapeutic cancer vaccines with main focus on HLA-I peptides. Here, we review cancer vaccine platforms based on two different preparation methods: pathogens (viruses and bacteria) and not (VLPs, nanoparticles, subunits vaccines) that exploit discoveries in the ligandome field to generate and/or enhance anti-tumor specific response. Finally, we discuss possible drawbacks and future challenges in the field that remain still to be addressed.     

Antigen-specific T cell suppression by human CD4+CD25+ regulatory T cells

Anergic/suppressive CD4+CD25+ T cells have been proposed to play an important role in the maintenance of peripheral tolerance. Here we demonstrate that in humans these cells suppress proliferation to self antigens, but also to dietary and foreign antigens. The suppressive CD4+CD25+ T cells display a broad usage of the T cell receptor Vbeta repertoire,suggesting that they recognize a wide variety of antigens. They reside in the primed/memory CD4+CD45RO+CD45RB(low) subset and have short telomeres, indicating that these cells have the phenotype of highly differentiated CD4+ T cells that have experienced repeated episodes of antigen-specific stimulation in vivo. This suggests that anergic/suppressive CD4+CD25+ T cells may be generated in the periphery as a consequence of repeated antigenic encounter. This is supported by the observation that highly differentiated CD4+T cells can be induced to become anergic/suppressive when stimulated by antigen presented by non-professional antigen-presenting cells. We suggest that besides being generated in the thymus, CD4+CD25+ regulatory T cells may also be generated in the periphery. This would provide a mechanism for the generation of regulatory cells that induce tolerance to a wide array of antigens that may not be encountered in the thymus.     

Identification of a promiscuous HLA DR-restricted T-cell epitope derived from the inhibitor of apoptosis protein survivin

The inhibitor of apoptosis protein survivin is a promising tumor-associated antigen specifically recognized by CD8+ cytotoxic effector T-lymphocytes (CTL). To improve current vaccines that aim to induce survivin-specific CTL, it is necessary to study the role of CD4+ T-helper (TH) and CD4+ T-regulatory (Treg) cells. Because both TH and Treg cells recognize antigens in the context of HLA-class II molecules, identification of HLA class II-associated peptide epitopes from survivin is required. Here, we analyzed T-cell responses against survivin using synthetic peptides predicted to serve as HLA-DR-restricted epitopes. Six peptides were shown to induce CD4+ T-cell responses, restricted by HLA-DR molecules. For one peptide epitope, SVN10, T-cell clones were demonstrated to be capable of recognizing naturally processed antigen. SVN10-specific T cells could be stimulated from the blood of healthy individuals and cancer patients with multiple HLA-DR genotypes. Thus the identified SVN10 epitope can be used to study the role of CD4+ TH and Treg cells in immune responses and possibly be included in a multivalent peptide vaccine against survivin.     

Effector-phase tolerance: another mechanism of how cancer escapes antitumor immune response

Growth of cancer in rodent models and in patients elicits immune responses directed toward various antigens expressed by the transformed cell. Clearly though, as most tumors grow, unmanipulated antitumor immune responses are incapable of eliminating cancer. Over the past approximately 15 years, antitumor immunoglobulin and T cells have been used to identify tumor antigens, which in turn, have served as the basis for therapeutic vaccine trials. However, experimental cancer vaccines, although in some patients result in elimination of large tumor burdens, have a low frequency of long-term cancer remission in most patients, ca. <5%. Therefore, as tumors express antigens that distinguish themselves from nontransformed cells in immunological terms (i.e., elicit immune responses to growth of primary tumor and can target tumor cells in vivo), and tumor vaccines prime unsuccessful antitumor immune responses in patients, it is likely that growth of cancer induces immune tolerance to tumor cells. Although there are several types of T cell tolerance, mature, antigen-specific CD8+ T cells isolated from tumors are lytic-defective, implying that the tumor microenvironment inactivates the antitumor effector phase. The nature of the functional local tolerance to antitumor immune response is the subject of this review.     

Origin of CD57+ T cells which increase at tumour sites in patients with colorectal cancer.

Human T cells carrying natural killer (NK) markers, CD57 or CD56 antigens, appear to be distinguishable from other T cell subsets in terms of their granular lymphocyte morphology and their numerical increase in patients with AIDS and in recipients of bone marrow transplantation. At the beginning of this study, we observed that CD57+ T cells as well as CD56+ T cells were abundant at tumour sites in many patients with colorectal cancer. Since all these findings for CD57+ T cells are quite similar to those of extrathymic T cells seen in mice, we investigated how CD57+ T cells are distributed to various immune organs in humans. They were found to be present mainly in the bone marrow and liver, but to be completely absent in the thymus. Similar to the case of extrathymic T cells in mice, they were observed to consist of double-negative CD4-8- subsets as well as single-positive subsets (preponderance of CD8+ cells), and to contain a considerable proportion of gamma delta T cells. These features are striking when compared with those of CD57- T cells, which are characterized by an abundance of CD4+ subsets and alpha beta T cells. Not only at tumour sites but also in the peripheral blood, some patients with colorectal cancer displayed elevated levels of CD57+ cells. These results suggest that CD57+ T cells may be of extrathymic origin, possibly originating in the bone marrow and liver, and may be associated with tumour immunity, similar to another extrathymic population of CD56+ T cells in humans.     

Anti-CD28 has a potent adjuvant effect on the antibody response to soluble antigens mediated through CTLA-4 by-pass

With the surge in potential new vaccines produced as recombinant proteins or synthetic peptides has come a pressing need to identify safe, potent immunological adjuvants to enhance immunogenicity of these antigens. CD28 is an important costimulatory molecule for T cells, and it has been shown that cell surface expression of its ligands, CD80 and CD86, can enhance cellular immune responses against tumor cells, however, these tumor cells do not normally express the ligands. Many new vaccines will be based upon soluble recombinant antigens, and in vaccination with these antigens CD80 and CD86 would normally be expressed on activated antigen-presenting cells and additional stimulation through CD28 would not be predicted to enhance responses further. However, we show here that, surprisingly, CD28 antibody can very strongly enhance immune responses against soluble proteins, but only when directly attached to the antigen. The mode of action of CD28 antibodies appears to be linked to their ability to signal through CD28, but not to bind the negative feedback regulatory antigen, CTLA-4. CD28 stimulants may represent novel, highly effective and safe immunological adjuvants for use with a wide range of prophylactic and therapeutic vaccines.     

Abnormal differentiation of immunoregulatory T-lymphocyte subpopulations in the major histocompatibility complex (MHC) class II antigen deficiency syndrome

The major histocompatibility complex (MHC) class II deficiency syndrome is a rare immunodeficiency disease associated with defective expression of class II MHC antigens. We have examined the consequences of this defect for the differentiation and functional capabilities of immunoregulatory T-cell subpopulations in an affected patient. Although the number of circulating T cells was normal, there was a striking reduction in the number of CD4+ T cells. Furthermore, purified CD4+ cells from the patient were unable to provide help for antibody secretion. This defect in helper function appeared to be due to the abnormal differentiation of the few CD4+ cells present, virtually all of which expressed the CD4 + HB11 + phenotype characteristic of immature virgin T cells. Abnormal development of immunoregulatory CD8+ T cells was also observed. Although increased numbers of CD8+ T cells were present, virtually none had phenotypic properties of suppressor cells (i.e., CD3+/CD8+/9.3-granular lymphocytes that coexpress the Leu-15 or Leu-7 antigens), and purified CD8+ cells from the patient had no suppressor activity. Thus, the absence of class II MHC antigens profoundly disrupts the development of immunoregulatory T cells. We propose that these effects occur by the following mechanisms: (1) the absence of intrathymic class II antigens results in deficient production of CD4+ cells, (2) the CD4+ cells that do emerge from the thymus do not undergo postthymic maturation into CD4+HB11- cells with helper capabilities, and (3) the absence of CD4+HB11- effector cells results in abortive development of suppressor cells involved in feedback suppression.