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Supplementary Figure Legends 1-4 from Immune Rejection of Mouse Tumors Expressing Mutated Self

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Supplementary Figure 4 from Immune Rejection of Mouse Tumors Expressing Mutated Self

Supplementary Figure 1 from Immune Rejection of Mouse Tumors Expressing Mutated Self

How the immune system recognizes and responds to mutations expressed by cancer cells is a critical issue for cancer immunology. Mutated self-polypeptides are particularly strong tumor-specific rejection antigens for natural tumor immunity, but we know remarkably little about T-cell responses to mutated self during tumor growth in vivo, including levels of response, kinetics, and correlates that predict tumor rejection. To address these questions, a mutated self-antigen, designated tyrosinase-related protein 1 (Tyrp1)-WM, derived from Tyrp1 was expressed in the poorly immunogenic, spontaneously arising B16 melanoma and the immunogenic, chemically induced LiHa fibrosarcoma. Syngeneic mice challenged with LiHa fibrosarcoma cells expressing Tyrp1-WM, but not native Tyrp1, induced specific CD8+ and CD4+ T-cell responses against defined mutated epitopes in tumor-draining lymph nodes and in tumors. Subsequently, specific CD8+ T-cell responses contracted as a minority of tumors progressed. B16 melanomas expressing Tyrp1-WM induced minimal T-cell responses, and no tumor immunity was detected. Treatment with an agonist monoclonal antibody against glucocorticoid-induced tumor necrosis factor receptor family–related gene (GITR) increased the level of CD8+ T cells recognizing a peptide derived from the Tyrp1-WM sequence and the proportion of mice rejecting tumors. These results show that B16 tumors expressing mutations that generate strongly immunogenic epitopes naturally induce T-cell responses, which are insufficient to reject tumors. Immune modulation, such as inducing GITR signaling, is required to enhance CD8+ T-cell responses to specific mutations and to lead to tumor rejection. [Cancer Res 2009;69(8):3545–53]

Supplementary Figure 2 from Immune Rejection of Mouse Tumors Expressing Mutated Self

Granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances immune responses by inducing proliferation, maturation, and migration of dendritic cells (DCs) as well as expansion and differentiation of B and T lymphocytes. The potency of DNA vaccines can be enhanced by the addition of DNA encoding cytokines, acting as molecular adjuvants. We conducted a phase I/II trial of human GM-CSF DNA in conjunction with a multipeptide vaccine (gp100 and tyrosinase) in stage III/IV melanoma patients. Nineteen human leukocyte antigen (HLA)-A*0201+ patients were treated. Three dose levels were studied: 100, 400, and 800 microg DNA/injection, administered subcutaneously every month with 500 microg of each peptide. In the dose-ranging study, three patients were treated at each dose level. The remaining patients were then treated at the highest dose. Most toxicities were grade 1 injection-site reactions. Eight patients (42%) developed CD8+ T-cell responses, defined by aor =3 SD increase in baseline reactivity to tyrosinase or gp100 peptide in tetramer or intracellular cytokine staining (ICS) assays. There was no relationship between dose and T-cell response. Responding T cells had an effector memory cell phenotype. Polyfunctional T cells were also demonstrated. At a median of 31 months follow-up, median survival has not been reached. Human GM-CSF DNA was found to be a safe adjuvant.

Successful approaches to tumor immunotherapy must overcome the physiological state of tolerance of the immune system to self-tumor antigens. Immunization with appropriate variants of syngeneic antigens can achieve this. However, improvements in vaccine design are needed for efficient cancer immunotherapy. Here we explore nine different chimeric vaccine designs, in which the antigen of interest is expressed as an in-frame fusion with polypeptides that impact antigen processing or presentation. In DNA immunization experiments in mice, three of nine fusions elevated relevant CD8(+) T-cell responses and tumor protection relative to an unfused melanoma antigen. These fusions were: Escherichia coli outer membrane protein A (OmpA), Pseudomonas aeruginosa exotoxin A, and VP22 protein of herpes simplex virus-1. The gains of immunogenicity conferred by the latter two are independent of epitope presentation by major histocompatibility complex class II (MHC II). This finding has positive implications for immunotherapy in individuals with CD4(+) T-cell deficiencies. We present evidence that antigen instability is not a sine qua non condition for immunogenicity. Experiments using two additional melanoma antigens identified different optimal fusion partners, thereby indicating that the benefits of fusion vectors remain antigen specific. Therefore large fusion vector panels such as those presented here can provide information to promote the successful advancement of gene-based vaccines.

One strategy to generate T-cell responses to tumors is to alter subdominant epitopes through substitution of amino acids that are optimal anchors for specific MHC molecules, termed heteroclitic epitopes. This approach is manually error-prone and time-consuming. In here, we describe a computer-based algorithm (EpitOptimizer) for the streamlined design of heteroclitic epitopes. Analysis of two cancer-related proteins showed that EpitOptimizer-generated peptides have enhanced MHC-I binding compared with their wild-type counterparts; and were able to induce stronger CD8+ T-cell responses against their native epitope. These data demonstrate that this approach can serve as the basis of epitope-engineering against cancer and intracellular pathogens.

Little is known about the consequences of immune recognition of mutated gene products, despite their potential relevance to autoimmunity and tumor immunity. To identify mutations that induce immunity, here we have developed a systematic approach in which combinatorial DNA libraries encoding large numbers of random mutations in two syngeneic tyrosinase-related proteins are used to immunize black mice. We show that the libraries of mutated DNA induce autoimmune hypopigmentation and tumor immunity through cross-recognition of nonmutated gene products. Truncations are present in all immunogenic clones and are sufficient to elicit immunity to self, triggering recognition of normally silent epitopes. Immunity is further enhanced by specific amino acid substitutions that promote T helper cell responses. Thus, presentation of a vast repertoire of antigen variants to the immune system can enhance the generation of adaptive immune responses to self.

Around 700 BCE, a new military formation called the phalanx was established in ancient Greece: a tight column of heavy infantry carrying long spears, or pikes, used in a single prong of attack. Later, in the battle of Marathon described by Herodotus, the Greeks learned the advantages of multipronged attacks, a strategy still used in modern warfare. Is the immune system similar in its approach to combating pathogens or tumors?

Cancer poses a difficult problem for immunotherapy because it arises from the host's own tissues. Many of the target antigens are tissue-specific molecules shared by cancer cells and normal cells. Thus, these are weak antigens that do not typically elicit immunity. In addition, tumors have several features that make their recognition and destruction by the immune system difficult. Despite these obstacles, several strategies for developing effective tumor immunity have been developed. Crucial to these approaches is the discovery and understanding of the molecular identity of antigens and the mechanisms involved in tumor immunity. In this review, strategies to overcome immune ignorance and tolerance are discussed.

The development of successful cancer vaccines is contingent on the ability to induce effective and persistent anti-tumor immunity against self-antigens that do not typically elicit immune responses. In this study, we examine the effects of a non-myeloablative dose of total body irradiation on the ability of tumor-naïve mice to respond to DNA vaccines against melanoma. We demonstrate that irradiation followed by lymphocyte infusion results in a dramatic increase in responsiveness to tumor vaccination, with augmentation of T cell responses to tumor antigens and tumor eradication. In irradiated mice, infused CD8(+) T cells expand in an environment that is relatively depleted in regulatory T cells, and this correlates with improved CD8(+) T cell functionality. We also observe an increase in the frequency of dendritic cells displaying an activated phenotype within lymphoid organs in the first 24 hours after irradiation. Intriguingly, both the relative decrease in regulatory T cells and increase in activated dendritic cells correspond with a brief window of augmented responsiveness to immunization. After this 24 hour window, the numbers of dendritic cells decline, as does the ability of mice to respond to immunizations. When immunizations are initiated within the period of augmented dendritic cell activation, mice develop anti-tumor responses that show increased durability as well as magnitude, and this approach leads to improved survival in experiments with mice bearing established tumors as well as in a spontaneous melanoma model. We conclude that irradiation can produce potent immune adjuvant effects independent of its ability to induce tumor ablation, and that the timing of immunization and lymphocyte infusion in the irradiated host are crucial for generating optimal anti-tumor immunity. Clinical strategies using these approaches must therefore optimize such parameters, as the correct timing of infusion and vaccination may mean the difference between an ineffective treatment and successful tumor eradication.

How the immune system recognizes and responds to mutations expressed by cancer cells is a critical issue for cancer immunology. Mutated self-polypeptides are particularly strong tumor-specific rejection antigens for natural tumor immunity, but we know remarkably little about T-cell responses to mutated self during tumor growth in vivo, including levels of response, kinetics, and correlates that predict tumor rejection. To address these questions, a mutated self-antigen, designated tyrosinase-related protein 1 (Tyrp1)-WM, derived from Tyrp1 was expressed in the poorly immunogenic, spontaneously arising B16 melanoma and the immunogenic, chemically induced LiHa fibrosarcoma. Syngeneic mice challenged with LiHa fibrosarcoma cells expressing Tyrp1-WM, but not native Tyrp1, induced specific CD8+ and CD4+ T-cell responses against defined mutated epitopes in tumor-draining lymph nodes and in tumors. Subsequently, specific CD8+ T-cell responses contracted as a minority of tumors progressed. B16 melanomas expressing Tyrp1-WM induced minimal T-cell responses, and no tumor immunity was detected. Treatment with an agonist monoclonal antibody against glucocorticoid-induced tumor necrosis factor receptor family–related gene (GITR) increased the level of CD8+ T cells recognizing a peptide derived from the Tyrp1-WM sequence and the proportion of mice rejecting tumors. These results show that B16 tumors expressing mutations that generate strongly immunogenic epitopes naturally induce T-cell responses, which are insufficient to reject tumors. Immune modulation, such as inducing GITR signaling, is required to enhance CD8+ T-cell responses to specific mutations and to lead to tumor rejection. [Cancer Res 2009;69(8):3545–53]

Vaccines are often inefficient in old people and old mice. Few studies have focused on testing vaccines in old populations. Here we used DNA tumor antigen vaccines against melanoma and showed that old mice were not protected. Vaccines incorporating fusions of the tumor antigen with microbial adjuvant proteins OmpA (E. Coli) or Vp22 (Herpes simplex virus-1) dramatically improved protection of old mice. The mechanisms by which these adjuvant proteins act are distinct. TLR2 was not required for either OmpA or Vp22. Antigen processing and presentation were not boosted by these fusion constructs. However, fusion constructs with Vp22 gave a strong CD4 response to B16 melanoma and the OmpA response is MHC-II dependent. Both adjuvant fusion constructs stimulated CD4 and CD8 responses otherwise diminished in old mice.

Malignant relapse remains a major problem for recipients of allogeneic hemopoietic stem cell transplantation (HSCT). We hypothesized that immunization of allogeneic HSCT recipients against tissue-restricted Ags using DNA vaccines would decrease the risk of relapse without enhancing graft-vs-host disease (GVHD). Using the mouse B16 melanoma model, we found that post-HSCT DNA immunization against a single tumor Ag induces tumor rejection that is significantly greater than HSCT alone in a T cell-depleted MHC-matched minor Ag-mismatched allogeneic HSCT model (LP → B6). In treatment models, post-HSCT DNA immunization provides significantly greater overall survival than the vaccine alone. Donor leukocyte infusion further enhances tumor-free survival, including in treatment models. There was no GVHD in HSCT recipients treated with DNA vaccination and donor leukocyte infusion. Further analysis demonstrated that these effects are dependent on CD8+ T cells of donor origin that recognize multiple epitopes. These results demonstrate that DNA immunization against tissue-restricted Ags after allogeneic T cell-depleted HSCT can induce potent antitumor effects without causing GVHD.

A relationship between melanoma and vitiligo, a skin disorder characterized by the loss of melanocytes, has been postulated for many decades. In some cases, vitiligo is almost certainly a manifestation of autoimmune-mediated destruction of melanocytes. Melanocytes and melanoma cells share melanocyte differentiation antigens. Based on a number of observations, de novo vitiligo developing in patients with melanoma has been regarded as a sign of good prognosis. The immune system tolerates or ignores differentiation antigens because these antigens are self-derived. Therefore, immune tolerance or ignorance must be overcome to prime naive T and B cells to induce cancer immunity and autoimmunity against melanocyte differentiation antigens. Mouse models of concurrent melanoma and autoimmune vitiligo have revealed strategies to overcome immune ignorance or tolerance to melanocyte differentiation antigens: immunization with self-antigens as altered self (e.g., orthologues or mutated versions), expression in viral vectors, passive immunization with antibodies or T cells, incorporating potent adjuvants into active immunization, and blockade or removal of a downregulatory mechanism. Extensive investigations into the mechanisms that lead to tumor immunity and autoimmunity elicited by certain differentiation antigens have further revealed a variety of distinct cellular and molecular requirements, which are redundant and alternative.

T cells recognizing self antigens expressed by cancer cells are prevalent in the immune repertoire. However, activation of these autoreactive T cells is limited by weak signals that are incapable of fully priming naive T cells, creating a state of tolerance or ignorance. Even if T cell activation occurs, immunity can be further restricted by a dominant response directed at only a single epitope. Enhanced antigen presentation of multiple epitopes was investigated as a strategy to overcome these barriers. Specific point mutations that create altered peptide ligands were introduced into the gene encoding a nonimmunogenic tissue self antigen expressed by melanoma, tyrosinase-related protein-1 (Tyrp1). Deficient asparagine-linked glycosylation, which was caused by additional mutations, produced altered protein trafficking and fate that increased antigen processing. Immunization of mice with mutated Tyrp1 DNA elicited cross-reactive CD8(+) T cell responses against multiple nonmutated epitopes of syngeneic Tyrp1 and against melanoma cells. These multi-specific anti-Tyrp1 CD8(+) T cell responses led to rejection of poorly immunogenic melanoma and prolonged survival when immunization was started after tumor challenge. These studies demonstrate how rationally designed DNA vaccines directed against self antigens for enhanced antigen processing and presentation reveal novel self epitopes and elicit multi-specific T cell responses to nonimmunogenic, nonmutated self antigens, enhancing immunity against cancer self antigens.

A relationship between melanoma and vitiligo, a skin disorder characterized by the loss of melanocytes, has been postulated for many decades. In some cases, vitiligo is almost certainly a manifestation of autoimmune-mediated destruction of melanocytes. Melanocytes and melanoma cells share melanocyte differentiation antigens. Based on a number of observations, de novo vitiligo developing in patients with melanoma has been regarded as a sign of good prognosis. The immune system tolerates or ignores differentiation antigens because these antigens are self-derived. Therefore, immune tolerance or ignorance must be overcome to prime naive T and B cells to induce cancer immunity and autoimmunity against melanocyte differentiation antigens. Mouse models of concurrent melanoma and autoimmune vitiligo have revealed strategies to overcome immune ignorance or tolerance to melanocyte differentiation antigens: immunization with self-antigens as altered self (e.g., orthologues or mutated versions), expression in viral vectors, passive immunization with antibodies or T cells, incorporating potent adjuvants into active immunization, and blockade or removal of a downregulatory mechanism. Extensive investigations into the mechanisms that lead to tumor immunity and autoimmunity elicited by certain differentiation antigens have further revealed a variety of distinct cellular and molecular requirements, which are redundant and alternative.

Around 700 BCE, a new military formation called the phalanx was established in ancient Greece: a tight column of heavy infantry carrying long spears, or pikes, used in a single prong of attack. Later, in the battle of Marathon described by Herodotus, the Greeks learned the advantages of multipronged attacks, a strategy still used in modern warfare. Is the immune system similar in its approach to combatting pathogens or tumors?

Concomitant tumor immunity describes immune responses in a host with a progressive tumor that rejects the same tumor at a remote site. In this work, concomitant tumor immunity was investigated in mice bearing poorly immunogenic B16 melanoma. Progression of B16 tumors did not spontaneously elicit concomitant immunity. However, depletion of CD4(+) T cells in tumor-bearing mice resulted in CD8(+) T cell-mediated rejection of challenge tumors given on day 6. Concomitant immunity was also elicited by treatment with cyclophosphamide or DTA-1 monoclonal antibody against the glucocorticoid-induced tumor necrosis factor receptor. Immunity elicited by B16 melanoma cross-reacted with a distinct syngeneic melanoma, but not with nonmelanoma tumors. Furthermore, CD8(+) T cells from mice with concomitant immunity specifically responded to major histocompatibility complex class I-restricted epitopes of two melanocyte differentiation antigens. RAG1(-/-) mice adoptively transferred with CD8(+) and CD4(+) T cells lacking the CD4(+)CD25(+) compartment mounted robust concomitant immunity, which was suppressed by readdition of CD4(+)CD25(+) cells. Naturally occurring CD4(+)CD25(+) T cells efficiently suppressed concomitant immunity mediated by previously activated CD8(+) T cells, demonstrating that precursor regulatory T cells in naive hosts give rise to effective suppressors. These results show that regulatory T cells are the major regulators of concomitant tumor immunity against this weakly immunogenic tumor.

Successful active immunization against cancer requires induction of immunity against self or mutated self Ags. However, immunization against self Ags is difficult. Xenogeneic immunization with orthologous Ags induces cancer immunity. The present study evaluated the basis for immunity induced by active immunization against a melanoma differentiation Ag, gp100. Tumor rejection of melanoma was assessed after immunization with human gp100 (hgp100) DNA compared with mouse gp100 (mgp100). C57BL/6 mice immunized with xenogeneic full-length hgp100 DNA were protected against syngeneic melanoma challenge. In contrast, mice immunized with hgp100 DNA and given i.p. tolerizing doses of the hgp100 Db-restricted peptide, hgp10025–33, were incapable of rejecting tumors. Furthermore, mice immunized with DNA constructs of hgp100 in which the hgp10025–27 epitope was substituted with the weaker Db-binding epitope from mgp100 (mgp10025–27) or a mutated epitope unable to bind Db did not reject B16 melanoma. Mice immunized with a minigene construct of hgp10025–33 rejected B16 melanoma, whereas mice immunized with the mgp10025–33 minigene did not develop protective tumor immunity. In this model of xenogeneic DNA immunization, the presence of an hgp100 heteroclitic epitope with a higher affinity for MHC created by three amino acid (25 to 27) substitutions at predicted minor anchor residues was necessary and sufficient to induce protective tumor immunity in H-2b mice with melanoma.

The development of successful cancer vaccines is contingent on the ability to induce effective anti-tumor immunity against self-antigens that do not typically elicit immunity. We are working on strategies to overcome immunologic tolerance/ignorance to cancer through the use of gene products closely related to self-antigens, including xenogeneic DNA and mutated DNA, and have initiated clinical trials of DNA vaccines in patients with advanced melanoma or prostate cancer. Recent studies have demonstrated that homeostasis-driven T-cell proliferation in the reconstituted lymphodepleted host could enhance the efficacy of whole-cell tumor vaccines. We hypothesized that immunization of sub-lethally irradiated and immune reconstituted mice against specific tumor antigens could increase anti-tumor immunity. B6 mice were irradiated with 600 cGy and immediately reconstituted with 30 x 106 syngeneic splenocytes. Weekly DNA immunization using either human tyrosinase-related protein (TRP)-2 DNA (xenogeneic melanoma differentiation antigen (MDA) or Opt-Tyrp1 DNA (a mutated MDA, which has been optimized for CD8 epitopes), was begun on day 1. We have found that: (1) by day 14 after irradiation and reconstitution, recipients have considerable numbers of splenic T cells, including de novo generated donor T cells, which suggests that vaccination aimed at T cells might be feasible; (2) DNA immunization against a single tumor antigen can provide protection from a tumor challenge that is significantly greater than that observed in immunized non-irradiated hosts (Figure); (3) DNA immunization induces higher levels of tumor-specific CD8+ T cells in the irradiated and reconstituted recipients (detected by intracellular cytokine flow cytometry assay); and (4) the effects of DNA immunization after lymphodepletion with immune reconstitution on both tumor-free survival and CD8+ T cell responses have been validated for two different DNA vaccine strategies (TRP-2 DNA and Opt-Tyrp1 DNA). These results demonstrate that DNA immunization following sub-lethal irradiation and immune reconstitution can induce potent anti-tumor effects. Furthermore, they provide a strong rationale for the development of novel therapeutic strategies that combine lymphodepletion with immune reconstitution and DNA immunization in human clinical trials.

Allogeneic HSCT is an important therapy with curative potential for a variety of malignant diseases, including leukemias, lymphomas and some solid tumors. Despite significant progress in reducing treatment-related mortality, malignant relapse remains a major problem. We are developing DNA vaccines that encode gene products closely related to self-antigens, including xenogeneic DNA and mutated DNA, and have initiated clinical trials of DNA vaccines in patients with advanced melanoma or prostate cancer. Using the B16 mouse melanoma model, we have shown that immunization with human TRP-2 DNA (xenogeneic melanoma differentiation antigen - MDA) or Opt-Tyrp1 DNA (a mutated MDA related to TRP-2, which we have optimized for CD8 epitopes), can induce tumor protection, including against established tumors. We hypothesized that immunization of allogeneic HSCT recipients (or their donors) against specific tumor antigens could decrease the risk of relapse without enhancing graft-versus-host disease (GVHD). In an MHC-matched minor antigen-mismatched mouse HSCT model (LP into B6), we found that: (1) by day 28 after transplant, recipients of an allogeneic T-cell depleted (TCD)-HSCT have considerable numbers of splenic T cells, including de novo generated donor T cells, which suggests that vaccination aimed at T cells might be feasible; (2) post-HSCT DNA immunization against a single tumor antigen can provide protection from a tumor challenge that is comparable to that observed with a whole cell vaccine (B16-GM-CSF) and significantly greater than HSCT alone; (3) DNA immunization post-HSCT can induce tumor-specific CD8+ T cells of donor origin (detected by ELISPOT or intracellular cytokine flow cytometry assay); (4) the combination of donor leukocyte infusion (DLI) and post-HSCT DNA immunization further enhances tumor-free survival (Figure); (5) there is no evidence of GVHD in multiple experiments using a clinical GVHD score to monitor recipients; and (6) the effects of post-HSCT DNA immunization on both tumor-free survival and CD8+ T cell responses have been validated for two different DNA vaccine strategies (hTRP-2 + GM-CSF DNA, or Opt-Tyrp1 DNA). These results demonstrate that DNA immunization after allogeneic TCD-HSCT can induce potent anti-tumor effects without the induction of GVHD. This and similar investigations provide a strong rationale for the development of novel therapeutic strategies that combine allogeneic HSCT, post-transplant tumor vaccination and adoptive cell therapy in human clinical trials. Download : Download high-res image (142KB) Download : Download full-size image Figure