Mimotopes of tumor-associated T-cell epitopes for cancer vaccines determined with combinatorial peptide libraries

Cytotoxic T-cells are the most important effector cells in immune responses against tumors. The identification of tumor-associated epitopes for these cells, therefore, has become a key aspect of the development of cancer vaccines. Here, we describe a new approach to the determination of tumor-associated T-cell epitopes which employs combinatorial peptide libraries with singly defined sequence positions in a randomized context. The analysis of the responses of a T-cell clone to these libraries yields the amino acid constituents of the epitope which can be combined to obtain mimotopes that are suitable as vaccine antigens for the induction of tumor-specific responses.     

Identification of a 220-kDa membrane tumor-associated antigen by human anti-UK114 monoclonal antibodies selected from the immunoglobulin repertoire of a cancer patient

Human monoclonal antibodies (HuMAb) specific for a 14-kDa perchloric acid-soluble protein (defined as UK114) were produced by somatic fusion of the human-mouse myeloma K6H6/B5 with Epstein-Barr virus-transformed peripheral B lymphocytes from a cancer patient previously treated with UK101 preparations, containing the UK114 protein. Three IgM-secreting clones were selected on the criteria of specificity for the purified UK114 protein immobilized onto plastic and adapted to grow in a serum-free medium. The reactivity of these antibodies showed a broad distribution pattern restricted to fresh tumor tissues and tumor cell lines, mainly of the adenocarcinoma type. None of the normal cells, nonmalignant cell lines, and normal tissues surrounding the neoplastic lesions were reactive. The immunochemical analysis of the target antigens showed that the HuMAb recognize a molecule of 220 kDa selectively expressed by the surface of tumor cells, as well as a cytoplasmic 14-kDa protein. The 220-kDa antigen was different from other tumor-associated antigens with similar molecular mass and, so far, unique. In the presence of human complement, two of three HuMAb are cytotoxic for tumor cells expressing the 220-kDa surface antigen. The tumor specificity and the lytic ability attributed to these HuMAb are promising features for the exploration of future clinical applications.     

The Tübingen approach: identification,selection, and validation of tumor-associated HLA peptides for cancer therapy

There is substantial need for molecularly defined tumor antigens to prime cytotoxic T cells in vivo for cancer immunotherapy, especially in the case of tumor entities for which only a few tumor antigens have been defined so far. In this review, we present the Tübingen approach to identify, select, and validate large numbers of MHC/HLA class I–associated peptides derived from tumor-associated antigens. Step 1 is the identification of naturally presented HLA-associated peptides directly from primary tumor cells. Step 2 is selection of tumor-associated peptides from step 1 by differential gene expression analysis and data mining. Step 3 is validation of selected candidates by monitoring in vivo T-cell responses in the context of patient-individualized immunizations. Our approach combines methods from genomics, proteomics, bioinformatics, and T-cell immunology. The aim is to develop effective immunotherapeutics consisting of multiple tumor-associated epitopes in order to induce a broad and specific immune response against cancer cells.This work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.     

Identification and characterization of agonist epitopes of the MUC1-C oncoprotein

The MUC1 tumor-associated antigen is overexpressed in the majority of human carcinomas and several hematologic malignancies. Much attention has been paid to the hypoglycosylated variable number of tandem repeats (VNTR) region of the N-terminus of MUC1 as a vaccine target, and recombinant viral vector vaccines are also being evaluated that express the entire MUC1 transgene. While previous studies have described MUC1 as a tumor-associated tissue differentiation antigen, studies have now determined that the C-terminus of MUC1 (MUC1-C) is an oncoprotein, and its expression is an indication of poor prognosis in numerous tumor types. We report here the identification of nine potential CD8⁺ cytotoxic T lymphocyte epitopes of MUC1, seven in the C-terminus and two in the VNTR region, and have identified enhancer agonist peptides for each of these epitopes. These epitopes span HLA-A2, HLA-A3, and HLA-A24 major histocompatibility complex (MHC) class I alleles, which encompass the majority of the population. The agonist peptides, compared to the native peptides, more efficiently (a) generate T-cell lines from the peripheral blood mononuclear cells of cancer patients, (b) enhance the production of IFN-γ by peptide-activated human T cells, and (c) lyse human tumor cell targets in an MHC-restricted manner. The agonist epitopes described here can be incorporated into various vaccine platforms and for the ex vivo generation of human T cells. These studies provide the rationale for the T-cell-mediated targeting of the oncogenic MUC1-C, which has been shown to be an important factor in both drug resistance and poor prognosis for numerous tumor types.     

Identification of tumor antigens and T-cell epitopes,and its clinical application

The capability of antigen-specific CD8⁺ and CD4⁺ T lymphocytes to mediate antitumor immunity has generated remarkable interest in the identification of target antigens and their epitopes. The classical strategy to define tumor antigens is based on the employment of in vivo sensitized tumor-reactive T lymphocytes from cancer patients. These lymphocytes are used to screen an autologous tumor cDNA expression library for the target antigen. Alternatively, antibodies from the serum of cancer patients can be applied to screen a tumor-derived phage expression library for immunogenic cellular structures. In addition, potential target antigens have been selected by gene expression profiling searching for overexpressed gene products in neoplastic cells compared with normal tissues. B-cell target structures and overexpressed gene products have to be verified as T-cell antigens by the strategy of reverse immunology. Therefore, T cells are sensitized in vitro by autologous dendritic cells loaded with predicted antigenic peptide ligands for a given HLA allele or with the global antigen. These different approaches led to the identification of a still growing number of antigenic peptides providing the basis for the development of new active and passive immunotherapies and for the monitoring of spontaneous and vaccine-induced T-cell responses. Some of these antigens and/or their epitopes are now validated in different clinical regimens for their capability to mediate potent T-cell immunity in cancer patients.This work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.     

肿瘤相关巨噬细胞极化的研究进展

肿瘤相关巨噬细胞是肿瘤组织局部浸润的巨噬细胞,在肿瘤组织微环境中,这些巨噬细胞发生M2型极化,从而发挥免疫抑制效应,促进肿瘤增殖。而M2型极化的肿瘤相关巨噬细胞也能够被再次诱导逆向极化形成具有抗肿瘤效应的M1型肿瘤相关巨噬细胞,激发机体产生特异性抗肿瘤免疫应答。促进肿瘤相关巨噬细胞M1型极化由此成为当前抗肿瘤免疫防治研究的热点。将对有关肿瘤相关巨噬细胞极化的新进展进行综述,为抗肿瘤免疫研究提供新的思路。     

Critical testing and parameters for consideration when manufacturing and evaluating tumor–associated antigen-specific T cells

The past year has seen remarkable translation of cellular and gene therapies, with U.S. Food and Drug Administration (FDA) approval of three chimeric antigen receptor (CAR) T-cell products, multiple gene therapy products, and the initiation of countless other pivotal clinical trials. What makes these new drugs most remarkable is their path to commercialization: they have unique requirements compared with traditional pharmaceutical drugs and require different potency assays, critical quality attributes and parameters, pharmacological and toxicological data, and in vivo efficacy testing. What's more, each biologic requires its own unique set of tests and parameters. Here we describe the unique tests associated with ex vivo–expanded tumor-associated antigen T cells (TAA-T). These tests include functional assays to determine potency, specificity, and identity; tests for pathogenic contaminants, such as bacteria and fungus as well as other contaminants such as Mycoplasma and endotoxin; tests for product characterization, tests to evaluate T-cell persistence and product efficacy; and finally, recommendations for critical quality attributes and parameters associated with the expansion of TAA-Ts.     

Isolation of antigen/antibody complexes through organic solvent (ICOS) method

We developed a method termed ICOS (isolation of antigen-antibody complexes through organic solvent) for comprehensive isolation of monoclonal antibodies (mAbs) bound to molecules on the cell surface. By mixing a large number of phage particles of an antibody (Ab) library with living cells, antigen (Ag)-Ab complexes were formed on the cell surface. The mixture was overlaid on organic solution in a tube and subjected to centrifugation. Phages bound to cells were recovered from the precipitate. The phage fraction isolated turned out to contain mAbs that bind to very heterogeneous epitopes and show strong binding activity to Ags. The ICOS method was applied to isolation of human mAbs that may be therapeutic against cancers. Sixty percent of clones isolated by the screening of a phage Ab library against cancer cells turned out to bind to various kinds of tumor-associated Ags. The precise protocol of ICOS method and the rationale of efficient screening were described.     

Emerging roles of the tumor-associated stroma in promoting tumor metastasis

The stroma in human carcinomas consists of extracellular matrix and various types of non-carcinoma cells, mainly leukocytes, endothelial cells, fibroblasts, myofibroblasts and bone marrow-derived progenitors. The tumor-associated stroma actively supports tumor growth by stimulating neo-angiogenesis, as well as proliferation and invasion of apposed carcinoma cells. It has long been accepted that alterations within carcinoma cells mediate metastasis in a cell-autonomous fashion. Recent studies have, however, suggested an additional notion that cancer cells instigate local and systemic changes in the tumor microenvironment and contribute to niche formation for metastasis. Research, aiming to establish the roles of the tumor-associated stroma in facilitating the spread of carcinoma cells into distant organs, has provided an abundance of data and greater knowledge of the biology of metastatic carcinoma cells and associated stromal cells. This has stimulated further advances in the development of novel therapeutic approaches targeting tumor metastasis.     

Tumor-directed targeting of liposomes

Tumor-specific targeting is a critical goal in the research area of liposomal drug delivery. Identification of the specific interactions between ligands and target tumor cells is a principle prerequisite in achieving this goal. Generally, tumor cells aberrantly express tumor-associated antigens that can be utilized as appropriate target molecules. Monoclonal antibodies against tumor-associated antigens have been successfully adopted for targeting to various types of cancer cells. The incorporation of humanized monoclonal antibodies or single chain human antibodies, instead of rodent antibodies into immunoliposomes has resulted in better clinical applicability. Tumor-specific ligands other than monoclonal antibodies have also been investigated as in vivo tumor-directing molecules. However, the number of pre-clinical studies of anticancer treatments using tumor-specific liposomal drugs reporting successful targeting and enhanced therapeutic efficacy has been limited. Further refinement of tumor-specific interactions and liposomal formulations will be necessary for the application of the tumor-specific liposomal drug strategy for anticancer chemotherapy or gene therapy.     

Molecular changes in carbohydrate antigens associated with cancer.

Oncogenic transformation is often associated with aberrant glycosylation in experimental and human tumors. The carbohydrate epitopes, resulting either from incomplete synthesis or neosynthesis, accumulate in high density, possibly in a novel conformation, at the tumor cell surface. A variety of monoclonal antibodies have been developed that recognize tumor-associated carbohydrate antigens and their aberrant organization at the cell surface. These carbohydrate epitopes and the antibodies specific to these structures are being exploited to develop novel diagnostic tools and therapeutic strategies for cancer.     

Three-dimensional microfluidic tumor–macrophage system for breast cancer cell invasion

The recrudescence of breast cancer can partly be attributed to poor understanding of the early steps and the mechanisms involved in breast cancer metastasis, especially how tumor inflammatory cells including tumor-associated macrophages (TAM) affect invasion process. However, invasion-related biological studies in traditional in vitro assays or in vivo models are challenging due to the arduousness in establishing models that precisely reproduce the tumor invasion environment. To this end, we proposed a juxtaposed dual-layer cell-loaded hydrogels biomimetic microfluidic system and formed monolayer size-selective permeable vascular endothelial barriers besides the dual layer to mimic mammalian blood vessels. We clarified that in this system, TAM promoted the invasion of breast cancer cells, whereas breast cancer cells maintained the phenotype of TAM cells and promoted the differentiation of U937 cells into TAM. It formed a tumor–macrophage bidirectional crosstalk system. This system could be used for drug screening. So finally, through the calculation of the survival rate of breast cancer cells when cocultured with different macrophages under paclitaxel treatment, we analyzed the antagonism of tumor–macrophage bidirectional crosstalk on anticancer drugs.     

Autophagy-mediated regulation of macrophages and its applications for cancer

Autophagy is a highly conserved homeostatic pathway that plays an important role in tumor development and progression by acting on cancer cells in a cell-autonomous mechanism. However, the solid tumor is not an island, but rather an ensemble performance that includes nonmalignant stromal cells, such as macrophages. A growing body of evidence indicates that autophagy is a key component of the innate immune response. In this review, we discuss the role of autophagy in the control of macrophage production at different stages (including hematopoietic stem cell maintenance, monocyte/macrophage migration, and monocyte differentiation into macrophages) and polarization and discuss how modulating autophagy in tumor-associated macrophages (TAMs) may represent a promising strategy for limiting cancer growth and progression.     

Animal models of human-derived cancer vaccines

Preclinical cancer vaccine studies must address vaccine safety, immunogenicity, and efficacy, as well as mechanism of vaccine action. Animal models of vaccines employing human tumor-associated antigen or epitopes (TAA, TAE) differ fundamentally from those employing tumor-specific antigens or epitopes (TSA, TSE). TSA and TSE vaccines will most likely demonstrate similar toxicity, immunogenicity, and efficacy in both tumor-bearing animals and patients. In contrast, TAA/TAE immunizations may have to overcome a host’s immunological tolerance to TAA/TAE expressed not only on tumor, but also on normal tissues; immunity to TAA/TAE will potentially target normal tissues and thus may induce autoimmunity. Various experimental models for human-derived TAA/TAE vaccines have been developed. These models include transgenic mice, mice with severe combined immunodeficiency (SCID), and non-human primates. Recently, unique animal models of TAA/TAE cancer vaccines have been developed, taking advantage of the discovery of animal tissue antigens with significant sequence homologies to human TAA/TAE. These models mimic perhaps most closely the situation in cancer patients.     

Activated human γδ T cells induce peptide-specific CD8+ T-cell responses to tumor-associated self-antigens

Specific cellular immunotherapy of cancer requires efficient generation and expansion of cytotoxic T lymphocytes (CTLs) that recognize tumor-associated self-antigens. Here, we investigated the capacity of human γδ T cells to induce expansion of CD8+ T cells specific for peptides derived from the weakly immunogenic tumor-associated self-antigens PRAME and STEAP1. Coincubation of aminobisphosphonate-stimulated human peripheral blood-derived γδ T cells (Vγ9+Vδ2+), loaded with HLA-A*02-restricted epitopes of PRAME, with autologous peripheral blood CD8+ T cells stimulated the expansion of peptide-specific cytolytic effector memory T cells. Moreover, peptide-loaded γδ T cells efficiently primed antigen-naive CD45RA+ CD8+ T cells against PRAME peptides. Direct comparisons with mature DCs revealed equal potency of γδ T cells and DCs in inducing primary T-cell responses and peptide-specific T-cell activation and expansion. Antigen presentation by γδ T-APCs was not able to overcome the limited capacity of peptide-specific T cells to interact with targets expressing full-length antigen. Importantly, T cells with regulatory phenotype (CD4+CD25hiFoxP3+) were lower in cocultures with γδ T cells compared to DCs. In summary, bisphosphonate-activated γδ T cells permit generation of CTLs specific for weakly immunogenic tumor-associated epitopes. Exploiting this strategy for effective immunotherapy of cancer requires strategies that enhance the avidity of CTL responses to allow for efficient targeting of cancer.     

Identification of novel HLA-restricted preferentially expressed antigen in melanoma peptides to facilitate off-the-shelf tumor-associated antigen-specific T-cell therapies

Background aimsPreferentially expressed antigen in melanoma (PRAME) is a cancer/testis antigen that is overexpressed in many human malignancies and poorly expressed or absent in healthy tissues, making it a good target for anti-cancer immunotherapy. Development of an effective off-the-shelf adoptive T-cell therapy for patients with relapsed or refractory solid tumors and hematological malignancies expressing PRAME antigen requires the identification of major histocompatibility complex (MHC) class I and II PRAME antigens recognized by the tumor-associated antigen (TAA) T-cell product. The authors therefore set out to extend the repertoire of HLA-restricted PRAME peptide epitopes beyond the few already characterized.MethodsPeptide libraries of 125 overlapping 15-mer peptides spanning the entire PRAME protein sequence were used to identify HLA class I- and II-restricted epitopes. The authors also determined the HLA restriction of the identified epitopes.ResultsPRAME-specific T-cell products were successfully generated from peripheral blood mononuclear cells of 12 healthy donors. Ex vivo-expanded T cells were polyclonal, consisting of both CD4+ and CD8+ T cells, which elicited anti-tumor activity in vitro. Nine MHC class I-restricted PRAME epitopes were identified (seven novel and two previously described). The authors also characterized 16 individual 15-mer peptide sequences confirmed as CD4-restricted epitopes.ConclusionsTAA T cells derived from healthy donors recognize a broad range of CD4+ and CD8+ HLA-restricted PRAME epitopes, which could be used to select suitable donors for generating off-the-shelf TAA-specific T cells.