Immune-checkpoint inhibition provides an unmatched level of durable clinical efficacy in various malignancies. Such therapies promote the activation of antigen-specific T cells, although the precise ...targets of these T cells remain unknown. Exploiting these targets holds great potential to amplify responses to treatment, such as by combining immune-checkpoint inhibition with therapeutic vaccination or other antigen-directed treatments. In this scenario, the pivotal hurdle remains the definition of valid HLA-restricted tumour antigens, which requires several levels of evidence before targets can be established with sufficient confidence. Suitable antigens might include tumour-specific antigens with alternative or wild-type sequences, tumour-associated antigens and cryptic antigens that exceed exome boundaries. Comprehensive antigen classification is required to enable future clinical development and the definition of innovative treatment strategies. Furthermore, clinical development remains challenging with regard to drug manufacturing and regulation, as well as treatment feasibility. Despite these challenges, treatments based on diligently curated antigens combined with a suitable therapeutic platform have the potential to enable optimal antitumour efficacy in patients, either as monotherapies or in combination with other established immunotherapies. In this Review, we summarize the current state-of-the-art approaches for the identification of candidate tumour antigens and provide a structured terminology based on their underlying characteristics.
The Peptide Vaccine of the Future Nelde, Annika; Rammensee, Hans-Georg; Walz, Juliane S.
Molecular & cellular proteomics,
01/2021, Letnik:
20
Journal Article
Recenzirano
Odprti dostop
The approach of peptide-based anticancer vaccination has proven the ability to induce cancer-specific immune responses in multiple studies for various cancer entities. However, clinical responses ...remain so far limited to single patients and broad clinical applicability was not achieved. Therefore, further efforts are required to improve peptide vaccination in order to integrate this low-side-effect therapy into the clinical routine of cancer therapy. To design clinically effective peptide vaccines in the future, different issues have to be addressed and optimized comprising antigen target selection as well as choice of optimal adjuvants and vaccination schedules. Furthermore, the combination of peptide-based vaccines with other immuno- and molecular targeted therapies as well as the development of predictive biomarkers could further improve efficacy. In this review, current approaches in the development of peptide-based vaccines and critical implications for optimal vaccine design are discussed.
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•Several prerequisites are mandatory for clinical effective peptide-based vaccines•Overview of current approaches regarding antigen selection and adjuvants•Suitable combinations will enhance the efficacy of peptide vaccines in the future•Biomarkers could be useful to preselect eligible patients for vaccination
Therapeutic peptide-based vaccination approaches for the treatment of cancer patients have shown first glimmers of success. However, to achieve broad clinical efficacy and implement peptide vaccinations in the standard treatment of cancer patients future peptide vaccines need further optimization in terms of target antigen selection, adjuvant choice, vaccination schedules, delivery routes, biomarkers, and combinatorial drugs.
Natural killer (NK) cells are cytotoxic lymphocytes that play an important role in tumor immunosurveillance, preferentially eliminating targets with low or absent expression of MHC class I and ...stress-induced expression of ligands for activating NK receptors. Platelets promote metastasis by protecting disseminating tumor cells from NK cell immunosurveillance, but the underlying mechanisms are not well understood. In this study, we show that tumor cells rapidly get coated in the presence of platelets in vitro, and circulating tumor cells of cancer patients display coexpression of platelet markers. Flow cytometry, immunofluorescent staining, confocal microscopy, and analyses on an ultrastructural level using immunoelectron microscopy revealed that such coating may cause transfer of MHC class I onto the tumor cell surface resulting in high-level expression of platelet-derived normal MHC class I. The resulting "phenotype of false pretenses" disrupts recognition of tumor cell missing self, thereby impairing cytotoxicity and IFN-γ production by NK cells. Thus, our data indicate that platelets, by conferring an unsuspicious "pseudonormal" phenotype, may enable a molecular mimicry that allows metastasizing tumor cells to downregulate MHC class I, to escape T-cell-mediated immunity without inducing susceptibility to NK cell reactivity.
The humoral immune response to SARS-CoV-2 is a benchmark for immunity and detailed analysis is required to understand the manifestation and progression of COVID-19, monitor seroconversion within the ...general population, and support vaccine development. The majority of currently available commercial serological assays only quantify the SARS-CoV-2 antibody response against individual antigens, limiting our understanding of the immune response. To overcome this, we have developed a multiplex immunoassay (MultiCoV-Ab) including spike and nucleocapsid proteins of SARS-CoV-2 and the endemic human coronaviruses. Compared to three broadly used commercial in vitro diagnostic tests, our MultiCoV-Ab achieves a higher sensitivity and specificity when analyzing a well-characterized sample set of SARS-CoV-2 infected and uninfected individuals. We find a high response against endemic coronaviruses in our sample set, but no consistent cross-reactive IgG response patterns against SARS-CoV-2. Here we show a robust, high-content-enabled, antigen-saving multiplex assay suited to both monitoring vaccination studies and facilitating epidemiologic screenings for humoral immunity towards pandemic and endemic coronaviruses.
Immune cell infiltrates have proven highly relevant for colorectal carcinoma prognosis, making colorectal cancer a promising candidate for immunotherapy. Because tumors interact with the immune ...system via HLA-presented peptide ligands, exact knowledge of the peptidome constitution is fundamental for understanding this relationship. Here, we comprehensively describe the naturally presented HLA ligandome of colorectal carcinoma and corresponding nonmalignant colon (NMC) tissue. Mass spectrometry identified 35,367 and 28,132 HLA class I ligands on colorectal carcinoma and NMC, attributable to 7,684 and 6,312 distinct source proteins, respectively. Cancer-exclusive peptides were assessed on source protein level using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein analysis through evolutionary relationships (PANTHER), revealing pathognomonic colorectal carcinoma-associated pathways, including Wnt, TGFβ, PI3K, p53, and RTK-RAS. Relative quantitation of peptide presentation on paired colorectal carcinoma and NMC tissue further identified source proteins from cancer- and infection-associated pathways to be overrepresented merely within the colorectal carcinoma ligandome. From the pool of tumor-exclusive peptides, a selected HLA-ligand subset was assessed for immunogenicity, with the majority exhibiting an existing T-cell repertoire. Overall, these data show that the HLA ligandome reflects cancer-associated pathways implicated in colorectal carcinoma oncogenesis, suggesting that alterations in tumor cell metabolism could result in cancer-specific, albeit not mutation-derived, tumor antigens. Hence, a defined pool of unique tumor peptides, attributable to complex cellular alterations that are exclusive to malignant cells, might comprise promising candidates for immunotherapeutic applications.
Cancer-associated pathways are reflected in the antigenic landscape of colorectal cancer, suggesting that tumor-specific antigens do not necessarily have to be mutation-derived but may also originate from other alterations in cancer cells.
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Therapeutic cancer vaccines have shown limited clinical efficacy so far. Nevertheless, in the meantime, our understanding of immune cell function and the interactions of immune cells with growing ...tumors has advanced considerably. We are now in a position to invest this knowledge into the design of more powerful vaccines and therapy combinations aimed at increasing immunogenicity and decreasing tumor-induced immunosuppression. This review focuses essentially on peptide-based human vaccines. We will discuss two aspects that are critical for increasing their intrinsic immunogenicity: the selection of the antigen(s) to be targeted, and the as yet unmet need for strong adjuvants.
Recombinant bispecific antibodies (bsAbs) are increasingly included in regimens for cancer therapy. Strict good manufacturing practice (GMP) compliant quality control measures are required to ensure ...quality and safety of these innovative biologicals. Gel electrophoresis (sodium dodecyl sulfate‐polyacrylamide gel electrophoresis SDS‐PAGE) and size exclusion chromatography (SEC) are the cornerstones of quality control methods. BsAbs are often prone to aggregation or incomplete synthesis due to their artificial nature. In addition, host cell proteins and host cell DNA as well as impurities from the purification process itself constitute potential contaminants. Such impurities may then appear as additional, unexpected bands or peaks on SDS‐PAGE gels and SEC, respectively. Here we describe a standardized protocol for rapid analysis of recombinant antibodies by mass spectrometry (MS) after tryptic digestion of bands excised from SDS‐PAGE gels. We have used this protocol to characterize unexpected “contaminating bands” that were observed during the clinical development of a novel bsAb with PSMAxCD3 specificity, either during the production of the protein itself or during the development of a surrogate molecule for evaluation in syngeneic mouse models. MS analysis allowed us to precisely determine the origin of these bands, which resulted from artifacts or from incomplete protein synthesis. The combined utilization of SDS‐PAGE und MS can therefore substantially support GMP‐compliant production of recombinant proteins.
Biopharmaceutical products for human use should be as pure as possible and all contaminants should be characterized. In this publication, the authors describe a standardized protocol for the rapid analysis of unknown bands occurring in SDS‐PAGE gels during bispecific antibody production. Using mass spectrometry after tryptic digestion of bands, the origin of unexpected “contaminating bands” can be determined and GMP‐compliant production of such molecules can be supported.
The classical HLA-C and the nonclassical HLA-E and HLA-G molecules play important roles both in the innate and adaptive immune system. Starting already during embryogenesis and continuing throughout ...our lives, these three Ags exert major functions in immune tolerance, defense against infections, and anticancer immune responses. Despite these important roles, identification and characterization of the peptides presented by these molecules has been lacking behind the more abundant HLA-A and HLA-B gene products. In this study, we elucidated the peptide specificities of these HLA molecules using a comprehensive analysis of naturally presented peptides. To that end, the 15 most frequently expressed HLA-C alleles as well as HLA-E*01:01 and HLA-G*01:01 were transfected into lymphoblastoid C1R cells expressing low endogenous HLA. Identification of naturally presented peptides was performed by immunoprecipitation of HLA and subsequent analysis of HLA-bound peptides by liquid chromatographic tandem mass spectrometry. Peptide motifs of HLA-C unveil anchors in position 2 or 3 with high variances between allotypes, and a less variable anchor at the C-terminal end. The previously reported small ligand repertoire of HLA-E was confirmed within our analysis, and we could show that HLA-G combines a large ligand repertoire with distinct features anchoring peptides at positions 3 and 9, supported by an auxiliary anchor in position 1 and preferred residues in positions 2 and 7. The wealth of HLA ligands resulted in prediction matrices for octa-, nona-, and decamers. Matrices were validated in terms of their binding prediction and compared with the latest NetMHC prediction algorithm NetMHCpan-3.0, which demonstrated their predictive power.
The immunopeptidomic landscape of ovarian carcinomas Schuster, Heiko; Peper, Janet K.; Bösmüller, Hans-Christian ...
Proceedings of the National Academy of Sciences,
11/2017, Letnik:
114, Številka:
46
Journal Article
Recenzirano
Odprti dostop
Immunotherapies, particularly checkpoint inhibitors, have set off a revolution in cancer therapy by releasing the power of the immune system. However, only little is known about the antigens that are ...essentially presented on cancer cells, capable of exposing them to immune cells. Large-scale HLA ligandome analysis has enabled us to exhaustively characterize the immunopeptidomic landscape of epithelial ovarian cancers (EOCs). Additional comparative profiling with the immunopeptidome of a variety of benign sources has unveiled a multitude of ovarian cancer antigens (MUC16, MSLN, LGALS1, IDO1, KLK10) to be presented by HLA class I and class II molecules exclusively on ovarian cancer cells. Most strikingly, ligands derived from mucin 16 and mesothelin, a molecular axis of prognostic importance in EOC, are prominent in a majority of patients. Differential gene-expression analysis has allowed us to confirm the relevance of these targets for EOC and further provided important insights into the relationship between gene transcript levels and HLA ligand presentation.
Both B cells and T cells are involved in an effective immune response to SARS‐CoV‐2, the disease‐causing virus of COVID‐19. While B cells—with the indispensable help of CD4+ T cells—are essential to ...generate neutralizing antibodies, T cells on their own have been recognized as another major player in effective anti‐SARS‐CoV‐2 immunity. In this report, we provide insights into the characteristics of individual HLA‐A*02:01‐ and HLA‐A*24:02‐restricted SARS‐CoV‐2‐reactive TCRs, isolated from convalescent COVID‐19 patients. We observed that SARS‐CoV‐2‐reactive T‐cell populations were clearly detectable in convalescent samples and that TCRs isolated from these T cell clones were highly functional upon ectopic re‐expression. The SARS‐CoV‐2‐reactive TCRs described in this report mediated potent TCR signaling in reporter assays with low nanomolar EC50 values. We further demonstrate that these SARS‐CoV‐2‐reactive TCRs conferred powerful T‐cell effector function to primary CD8+ T cells as evident by a robust anti‐SARS‐CoV‐2 IFN‐γ response and in vitro cytotoxicity. We also provide an example of a long‐lasting anti‐SARS‐CoV‐2 memory response by reisolation of one of the retrieved TCRs 5 months after initial sampling. Taken together, these findings contribute to a better understanding of anti‐SARS‐CoV‐2 T‐cell immunity and may contribute to paving the way toward immunotherapeutics approaches targeting SARS‐CoV‐2.
T cell receptors (TCRs) directed against previously identified SARS‐CoV‐2 target epitopes were isolated from convalescent COVID‐19 patients by single‐cell sequencing. Upon ectopic re‐expression, these SARS‐CoV‐2 TCRs conferred potent effector functions such as cytotoxicity and Interferon‐gamma production.
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