Class II-restricted Tumor Antigens Research Articles

Abstract 1972: Clinical implications of HLA expression and immunopeptidome-presented tumor antigens in ovarian carcinoma

Abstract Ovarian carcinoma (OvCa) is the seventh most common malignancy in women and the eighth leading cause of cancer-related deaths worldwide. In a previous study, we characterized the antigenic landscape of ovarian carcinoma by mass spectrometry-based immunopeptidomics and identified novel OvCa-associated tumor antigens, including Mucin-16 (MUC-16) and Mesothelin (MSLN) with the aim to develop novel T cell-based immunotherapies (Schuster et al. PNAS 2017). Here, we analyzed the immunopeptidomics data of this OvCa cohort in relation to clinical patient characteristics and disease outcome. Analysis included 43 OvCa patients with respective immunopeptidomics and RNA sequencing data, comprising immunopeptidome diversity, tumor antigen presentation and expression (MUC16, MSLN) as well as HLA mRNA expression.Analyzing HLA class I-restricted tumor antigen presentation in relation to clinical data, we could show that nodal-positive patients presented more frequently HLA-restricted peptides derived from the tumor antigen MUC16 (p = 0.0087) and showed significantly increased numbers of unique MUC16-derived HLA-presented peptides within the total immunopeptidome (p = 0.042) compared to nodal-negative patients. No significant difference in HLA class I immunopeptidome diversity, overall tumor antigen presentation, and expression was observed for histological subtypes, grading, or the prevalence of distant metastases. For HLA class II-restricted tumor antigen presentation and HLA expression in relation to clinical data, we observed a more diverse HLA class II immunopeptidome in terms of different HLA class II-presented peptides (p = 0.011) for patients with high tumor grading (G3) compared to low/intermediate (G1/G2) grading. In line, the tumors of these patients also presented an increased number of different MSLN-derived HLA class II-restricted peptides (p = 0.021). No significant difference in HLA class II immunopeptidome diversity, tumor antigen presentation and expression was seen for the prevalence of distant metastasis, histological subtypes, or nodal positivity.Of note, patients presenting MSLN-derived peptides in their immunopeptidome showed a significantly prolonged recurrence-free survival (RFS, p = 0.011). In addition, patients exhibiting a high expression of HLA-DR showed a significantly increased RFS (p = 0.018 for HLA-DRA, p = 0.0031 for HLA-DRB).In conclusion, this work provides first insights on the relation of immunopeptidomic characteristics, comprising HLA expression and tumor antigen presentation, with clinical characteristics and disease outcome of OvCa patients. The observed correlation of HLA-DR expression and HLA class II tumor antigen presentation with prolonged RFS indicates a central role of CD4+ T cell responses for anti-tumor immune surveillance in ovarian cancer. Citation Format: Christian M. Tegeler, Jonas S. Heitmann, Helmut R. Salih, Juliane S. Walz, Annika Nelde. Clinical implications of HLA expression and immunopeptidome-presented tumor antigens in ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1972.

Unveiling the Hidden Treasury: CIITA-Driven MHC Class II Expression in Tumor Cells to Dig up the Relevant Repertoire of Tumor Antigens for Optimal Stimulation of Tumor Specific CD4+ T Helper Cells.

Simple SummaryIt is becoming clear that combined approaches are required to fight and succeed against cancer. As far as immune-based strategies, new generation anti-tumor vaccines will certainly play a crucial role. A key aspect is to identify tumor antigens which optimally stimulate CD4+ T helper cells, as these cells are crucial to triggering and maintaining all effector mechanisms of the adaptive immune response. Our approach is to render tumor cells surrogate antigen presenting cells for their own tumor antigen by forcing them to express de novo MHC class II molecules after the genetic transfer of CIITA, the major transcriptional controller of MHC class II gene expression. The unprecedented strong stimulation of tumor-specific CD4+ T cells that were obtained with our approach let us hope that this will help in identifying a constellation of MHC class II-restricted tumor antigens for more potent next generation anti-tumor vaccines.Despite the recent enthusiasm generated by novel immunotherapeutic approaches against cancer based on immune checkpoint inhibitors, it becomes increasingly clear that single immune-based strategies are not sufficient to defeat the various forms and types of tumors. Within this frame, novel vaccination strategies that are based on optimal stimulation of the key cell governing adaptive immunity, the CD4+ T helper cell, will certainly help in constructing more efficient treatments. In this review, we will focus on this aspect, mainly describing our past and recent contributions that, starting with a rather unorthodox approach, have ended up with the proposition of a new idea for making available an unprecedented extended repertoire of tumor antigens, both in quantitative and qualitative terms, to tumor-specific CD4+ T helper cells. Our approach is based on rendering the very same tumor cells antigen presenting cells for their own tumor antigens by gene transfer of CIITA, the major transcriptional coordinator of MHC class II expression discovered in our laboratory. CIITA-driven MHC class II-expressing tumor cells optimally stimulate in vivo tumor specific MHC class II-restricted CD4 T cells generating specific and long lasting protective immunity against the tumor. We will discuss the mechanism underlying protection and elaborate not only on the applicability of this approach for novel vaccination strategies amenable to clinical setting, but also on the consequence of our discoveries on sedimented immunological dogmas that are related to antigen presentation.

Abstract B047: Adequate Antigen Availability (AAA) in antitumor immunity: Definition and consequences for novel strategies of tumor prevention and antitumor treatment

Abstract Present immunopreventive and immunotherapeutic approaches against cancer suffer from the limitation of being not “sterilizing” procedures, as very poor protection against the tumor is obtained. Thus, newly conceived anti-tumor strategies of intervention are urgently needed. Here we introduce the concept of Adequate Antigen Availability (AAA) defined as the optimal tumor antigen dose and related antigen processing and MHC class II-restricted presentation necessary to trigger efficient tumor-specific TH cells (1). We will present results on a preventive anti-tumor vaccination system that consists in injecting tumor cells genetically modified to constitutively express MHC class II molecules by transfecting the MHC-II transcriptional activator CIITA. AAA is generated by the function of tumor's newly expressed MHC-II molecules to present tumor-associated antigens to tumor-specific CD4+ TH cells. We will show that tumor rejection against the CIITA-transfected neoplastic cells is brought about by the generation of persistent anti-tumor TH cell memory, responsible of protecting the animals from subsequent challenges with parental, untransfected tumor cells. This approach has been validated in tumor cells of different histotypic origin and of distinct MHC genotype (H-2d and H-2b). Importantly, the anti-tumor TH cell response modifies the tumor microenvironment, favouring the infiltration of CD4+ TH cells, CD8+ cytolytic T cells and granulocytes and dramatically reducing both intratumoral and lymphnode infiltration of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC). Thus the optimal initiation of the adaptive immune response, dictated by AAA, is sufficient to reorient the tumor microenvironment from a pro-tumor to an anti-tumor microenvironment. Our approach is, at present, applied to human clinical setting in hepatocellular carcinomas (2) whereby human tumor cells are rendered HLA class II-positive by CIITA genetic transfer with the aim to purify and sequence immunogenically relevant HLA class II-bound tumor–associated antigenic (TAA) peptides. In conjunction with similarly derived HLA class I-restricted TAA peptides, they will be the basis for the construction of a multi-peptide, multi-epitope vaccine that can target both CD4+ TH and CD8+ CTL anti-tumor responses.

Identification of DRG-1 As a Melanoma-Associated Antigen Recognized by CD4+ Th1 Cells.

Immunotherapy has emerged as a promising strategy for the treatment of metastatic melanoma. Clinical studies have demonstrated the feasibility of cancer immunotherapy using tumor antigens recognized by CD8+ T cells. However, the overall immune responses induced by these antigens are too weak and transient to induce tumor regression in the majority of patients who received immunization. A growing body of evidence suggests that CD4+ T helper (Th) cells play an important role in antitumor immunity. Therefore, the identification of MHC class II-restricted tumor antigens capable of stimulating CD4+ T cells may provide opportunities for developing effective cancer vaccines. To this end, we describe the identification of developmentally regulated GTP-binding protein 1 (DRG-1) as a melanoma-associated antigen recognized by HLA-DR11-restricted CD4+ Th1 cells. Epitope mapping analysis showed that the DRG1248-268 epitope of DRG-1 was required for T cell recognition. Reverse transcription-polymerase chain reaction revealed that DRG-1 was highly expressed in melanoma cell lines but not in normal tissues. DRG-1 knockdown by lentiviral-based shRNA suppressed melanoma cell proliferation and soft agar colony formation. Taken together, these data suggest that DRG-1 plays an important role in melanoma cell growth and transformation, indicating that DRG1 may represent a novel target for CD4+ T cell-mediated immunotherapy in melanoma.

Boosting the MHC Class II-Restricted Tumor Antigen Presentation to CD4+ T Helper Cells: A Critical Issue for Triggering Protective Immunity and Re-Orienting the Tumor Microenvironment Toward an Anti-Tumor State

Although the existence of an immune response against tumor cells is well documented, the fact that tumors take off in cancer patients indicates that neoplastic cells can circumvent this response. Over the years many investigators have described strategies to rescue the anti-tumor immune response with the aim of creating specific and long-lasting protection against the disease. When exported to human clinical settings, these strategies have revealed in most cases a very limited, if any, positive outcome. We believe that the failure is mostly due to the inadequate triggering of the CD4+ T helper (TH) cell arm of the adaptive immunity, as TH cells are necessary to trigger all the immune effector mechanisms required to eliminate tumor cells. In this review, we focus on novel strategies that by stimulating MHC class II-restricted activation of TH cells generate a specific and persistent adaptive immunity against the tumor. This point is of critical importance for both preventive and therapeutic anti-tumor vaccination protocols, because adaptive immunity with its capacity to produce specific, long-lasting protection and memory responses is indeed the final goal of vaccination. We will discuss data from our as well as other laboratories which strongly suggest that triggering a specific and persistent anti-tumor CD4+ TH cell response stably modify not only the tumor microenvironment but also tumor-dependent extratumor microenvironments by eliminating and/or reducing the blood-derived tumor infiltrating cells that may have a pro-tumor growth function such as regulatory CD4+/CD25+ T cells and myeloid-derived-suppressor cells. Within this frame, therefore, we believe that the establishment of a pro-tumor environment is not the cause but simply the consequence of the tumor strategy to primarily counteract components of the adaptive cellular immunity, particularly TH lymphocytes.

Optimal MHC-II-restricted tumor antigen presentation to CD4+ T helper cells: the key issue for development of anti-tumor vaccines

Present immunoprevention and immunotherapeutic approaches against cancer suffer from the limitation of being not “sterilizing” procedures, as very poor protection against the tumor is obtained. Thus newly conceived anti-tumor vaccination strategies are urgently needed. In this review we will focus on ways to provide optimal MHC class II-restricted tumor antigen presentation to CD4+ T helper cells as a crucial parameter to get optimal and protective adaptive immune response against tumor. Through the description of successful preventive or therapeutic experimental approaches to vaccinate the host against the tumor we will show that optimal activation of MHC class II-restricted tumor specific CD4+ T helper cells can be achieved in various ways. Interestingly, the success in tumor eradication and/or growth arrest generated by classical therapies such as radiotherapy and chemotherapy in some instances can be re-interpreted on the basis of an adaptive immune response induced by providing suitable access of tumor-associated antigens to MHC class II molecules. Therefore, focussing on strategies to generate better and suitable MHC class II–restricted activation of tumor specific CD4+ T helper cells may have an important impact on fighting and defeating cancer.

Folding of an MHC class II-restricted tumor antigen controls its antigenicity via MHC-guided processing.

CD4(+) and CD8(+) T cell responses to endogenous retroviral envelope glycoprotein gp90 generate protective immunity to murine colon carcinoma CT26. A panel of I-A(d)-restricted T cell hybridomas recognize gp90 synthesized by CT26 cells but not by other gp90-expressing tumors. Here we report that antigenicity resides in an incompletely folded form of gp90 that is unique to CT26. In contrast to more compact forms of gp90 that are present in other tumors, this open conformer is captured by recycling I-A(d) on antigen-presenting cells and is processed intracellularly. Thus, gp90 acquires immunodominance via MHC-guided processing, and the generation of an MHC class II-restricted response can be controlled by the intracellular folding environment of antigen-expressing cells.

Identification of MHC class II-restricted tumor antigens recognized by CD4+ T cells

CD4+ T cells play a central role in orchestrating host immune responses against cancer as well as autoimmune and infectious diseases. Identification of major histocompatibility complex (MHC) class II-restricted helper T peptides is important for development of effective vaccines. The lack of effective methods to identify such T-cell peptides is a major hurdle in the use of antigen-specific CD4+ T cells in cancer vaccines. Here we describe a genetic targeting expression system for cloning genes encoding for MHC class II-restricted tumor antigens recognized by tumor-reactive CD4+ T cells. Helper T peptides are subsequently identified by using synthetic peptides to test their ability to stimulate CD4+ T cells.

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

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.

Minor Structural Changes in a Mutated Human Melanoma Antigen Correspond to Dramatically Enhanced Stimulation of a CD4 + Tumor-infiltrating Lymphocyte Line

While most immunotherapies for cancer have focused on eliciting specific CD8 + cytotoxic T lymphocyte killing of tumor cells, a mounting body of evidence suggests that stimulation of anti-tumor CD4 + T cell help may be required for highly effective therapy. Several MHC class II-restricted tumor antigens that specifically activate such CD4 + helper T lymphocytes have now been identified, including one from a melanoma tumor that is caused by a single base-pair mutation in the glycolytic enzyme triosephosphate isomerase. This mutation results in the conversion of a threonine residue to isoleucine within the antigenic epitope, concomitant with a greater than five log-fold increase in stimulation of a CD4 + tumor-infiltrating lymphocyte line. Here, we present the crystal structures of HLA-DR1 in complex with both wild-type and mutant TPI peptide antigens, the first structures of tumor peptide antigen/MHC class II complexes recognized by CD4 + T cells to be reported. These structures show that very minor changes in the binding surface for T cell receptor correspond to the dramatic differences in T cell stimulation. Defining the structural basis by which CD4 + T cell help is invoked in an anti-tumor immune response will likely aid the design of more effective cancer immunotherapies.

MHC Class II–Restricted Tumor Antigens Recognized by CD4+ T Cells: New Strategies for Cancer Vaccine Design

The adoptive transfer of tumor-infiltrating lymphocytes (TIL) can mediate tumor regression in patients with melanoma. This finding has led to the identification and characterization of tumor-associated antigens recognized by CD8+ TIL. Several clinical trials based on the genes recognized by these CD8+ T cells have been attempted, but with only limited success. Meanwhile, increasing evidence has demonstrated that CD4+ T cells play important roles in generating and maintaining antitumor immune responses in animal models. These data suggest that it may be necessary to engage both CD4+ and CD8+ T cells for more effective antitumor immunotherapy. In this report, we review emerging molecular approaches in cloning major histocompatibility complex (MHC) class II restricted tumor antigens recognized by CD4+ T cells as well as approaches to identify new MHC class II-restricted epitopes from known tumor antigens recognized by CD8+ cytotoxic T lymphocytes and/or antibodies. Progress made in this field has shed light on the roles of tumor antigen-specific CD4+ T cells in humans; it has also provided new insights into the understanding of tumor genesis and the interaction between tumor and the immune system. More importantly, the discovery of MHC class II-restricted tumor antigens has provided opportunities for developing a new generation of cancer vaccines aimed at eliciting both CD4+ and CD8+ T-cell responses against tumor.

Cross-protective murine graft-versus-leukemia responses to phenotypically distinct myeloid leukemia lines

A c-myc retrovirus-transformed myeloid leukemia line, MMB3.19, of C57BL/6 (B6) origin, was developed to investigate graft-versus-leukemia (GVL) activity in murine bone marrow transplantation (BMT) models. It was previously determined that both naive and leukemia-presensitized CD4+-enriched T cells are capable of mediating GVL activity to MMB3.19 challenge in both syngeneic (B6) and allogeneic (C3H.SW→B6) strain combinations, with the latter coinciding with minimal graft-versus-host disease. In the present study, MMB3.19 and 2 other similarly derived, yet phenotypically diverse, B6 myeloid leukemia lines (MMB1.10 and MMB2.18) were investigated for potential shared tumor antigens in the syngeneic GVL model. Morphologically, all 3 tumor lines are blastic with high cytoplasmic:nuclear ratios, but MMB2.18 displays dendritic processes, whereas MMB1.10 and MMB3.19 have a more rounded appearance. Flow cytometric analysis of the 3 lines revealed constitutive surface molecule expression of Mac-1, Mac-2, F4/80, LFA-1, B7-1, B7-2, H2Kb, H2Db, and macrophage scavenger receptor, consistent with macrophage/monocyte lineages. Furthermore, each of the lines expresses H2I-Ab, but to varying degrees, with MMB2.18 cells having the lowest percentage (31.6%). In vitro 51Cr release assays using MMB3.19-primed T-cell effectors demonstrated equivalent specific lysis of all 3 leukemia-line target cells. In addition, enzyme-linked immunospot analysis of MMB3.19-primed CD4+ T cells revealed significantly increased frequencies of tumor-stimulated interleukin (IL)-2-, IL-4-, and interferon-gamma-secreting cells when restimulated with each of the 3 leukemia lines. Furthermore, when MMB3.19-primed CD4+ T cells were administered in a BMT setting, a protective GVL effect was seen in those mice challenged with MMB1.10, MMB2.18, or MMB3.19. Therefore, in vitro and in vivo experiments indicate that the 3 distinct myeloid leukemia lines share 1 or more common major histocompatibility complex class II-restricted tumor antigens that can elicit a cross-protective in vivo T-cell GVL response. Biol Blood Marrow Transplant 2000;6(5A):537-47.

Human tumor antigens for cancer vaccine development.

The adoptive transfer of tumor-infiltrating lymphocytes (TIL) along with interleukin (IL)-2 into autologous patients with cancer resulted in the objective regression of tumor, indicating that T cells play an important role in tumor regression. In the last few years, efforts have been made towards understanding the molecular basis of T-cell-mediated antitumor immunity and elucidating the molecular nature of tumor antigens recognized by T cells. Tumor antigens identified thus far could be classified into several categories: tissue-specific differentiation antigens, tumor-specific shared antigens and tumor-specific unique antigens. CD4+ T cells play a central role in orchestrating the host immune response against cancer, infectious diseases and autoimmune diseases, and we thus have attempted to identify major histocompatibility complex (MHC) class II-restricted tumor antigens as well. The identification of tumor rejection antigens provides new opportunities for the development of therapeutic strategies against cancer. This review will summarize the current status of MHC class I- and class II-restricted human tumor antigens, and their potential application to cancer treatment.