The identification of tumor-associated T cell epitopes has contributed significantly to the understanding of the interrelationship of tumor and immune system and is instrumental in the development of therapeutic vaccines for the treatment of cancer. Most of the known epitopes have been identified with prediction algorithms that compute the potential capacity of a peptide to bind to HLA class I molecules. However, naturally expressed T cell epitopes need not necessarily be strong HLA binders. To overcome this limitation of the available prediction algorithms we established a strategy for the identification of T cell epitopes that include suboptimal HLA binders. To this end, an artificial neural network was developed that predicts HLA-binding peptides in protein sequences by taking the entire sequence context into consideration rather than computing the sum of the contribution of the individual amino acids. Using this algorithm, we predicted seven HLA A*0201-restricted potential T cell epitopes from known melanoma-associated Ags that do not conform to the canonical anchor motif for this HLA molecule. All seven epitopes were validated as T cell epitopes and three as naturally processed by melanoma tumor cells. T cells for four of the new epitopes were found at elevated frequencies in the peripheral blood of melanoma patients. Modification of the peptides to the canonical sequence motifs led to improved HLA binding and to improved capacity to stimulate T cells.
The human X chromosome consists of a high number of large inverted repeat (IR) DNA sequences which fulfill all requirements for formation of cruciform DNA structures. Such alternative DNA structures are suggested to have a great impact in altering the chromatin architecture and function. Our comprehensive analysis of the corresponding orthologous nucleotide sequences of an IR sequence from Homo sapiens and Pan troglodytes revealed that most of the nucleotide differences between the two species are symmetrical to the apex of the IR, and that the spacer region of the orthologous IRs are in reverse orientation. We provide evidence that this IR forms a large non-B DNA structure containing two Holliday junctions, allowing intrastrand nucleotide pairing of the arms and interstrand pairing of the spacer region of the IR. This structure would extrude into a large double-cruciform DNA structure providing the molecular basis of translocation events and regulation of gene expression.
Identification of molecular features that determine peptide interaction with major histocompatibility complex I (MHC I) is essential for vaccine development. We have developed a concept for peptide design by combining an agent-based artificial ant system with artificial neural networks. A jury of feedforward networks classifies octapeptides that are recognized by mouse MHC I protein H-2K(b). Prediction accuracy yielded a correlation coefficient of 0.94. Peptides were designed in machina by the artificial ant system and tested in vitro for their MHC I stabilizing effect. The behavior of the search agents during the design process was controlled by the jury network. The experimentally determined prediction accuracy was 89% for the designed stabilizing and 95% for the non-stabilizing peptides. Novel H-2K(b) stabilizing peptides were conceived that reveal extensions of known residue motifs. The combined network-agent system recognized context dependencies of residue positions. A diverse set of novel sequences exhibiting substantial activity was generated.
Background: FAP is a membrane-bound protease with limited expression in normal tissues but high expression on cancer-associated fibroblasts abundant in the stroma of most tumors. FAP-2286 is a potent and selective FAP-targeted peptide linked to the chelator DOTA that allows for attachment of radionuclides for therapeutic and imaging applications. Assessing patterns of FAP expression in different tumor types and correlating expression with FAP-2286 uptake can help guide tumor selection for FAP-2286 therapy. Methods: FAP immunohistochemistry (IHC) was performed on FFPE tissue specimens from 16 tumor types using the SP325 antibody. Overall and tumor/stroma-specific H-scores were calculated using Visiopharm and HALO analysis, respectively. Autoradiography with 111In-FAP-2286 was performed on a subset of matched frozen tissue sections. Results: Gene expression analysis across The Cancer Genome Atlas data set revealed elevated FAP mRNA expression in multiple tumor types. A pan-tumor IHC screen confirmed that ≥30% of samples in various indications (eg, sarcoma, pancreatic adenocarcinoma, mesothelioma, head and neck squamous cell carcinoma) had high FAP expression (H-score ≥30). While in most tumor types FAP was predominantly localized to the stroma, FAP expression was also observed in tumor cells, especially in tumors of mesenchymal origin, eg, sarcoma and mesothelioma. High FAP expression was independent of tumor stage or grade and detected in both primary and metastatic samples. Multiple sarcoma and mesothelioma subtypes demonstrated high FAP H-scores, suggesting that FAP expression is not limited to a specific subtype. There was significant correlation between FAP expression observed by IHC and FAP-2286 binding as assessed by autoradiography in matched frozen tissues (Pearson r=0.73; p<0.01). Conclusions: Our IHC screen identified high FAP expression in various tumor types that correlated with in vitro FAP-2286 binding, suggesting that FAP is an attractive target across a broad range of tumor types for peptide-targeted radionuclide therapy. Accordingly, the phase 1/2 LuMIERE clinical trial (NCT04939610) will evaluate FAP-2286 as a therapeutic (177Lu-FAP-2286) and imaging (68Ga-FAP-2286) agent in multiple indications. Citation Format: Tanya T. Kwan, Minh Nguyen, Dirk Zboralski, Anne Schumann, Anne Bredenbeck, Matthias Paschke, Christian Haase, Aileen Hoehne, Ulrich Reineke, Christiane Smerling, Frank Osterkamp, Jim Xiao, Andrew D. Simmons, Thomas C. Harding, Kevin L. Lin. Pan-cancer analysis of fibroblast activation protein alpha (FAP) expression to guide tumor selection for the peptide-targeted radionuclide therapy FAP-2286 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA032.
Small peptides bound by Major Histocompatibility Complex (MHC) class I molecules and recognized in this context by the T-cell receptor of CD8 þ T cells are known as T-cell epitopes and are of extraordinary importance for the development of new vaccines against cancer and viral infections. Several algorithms predicting a peptides binding capability to a given MHC class I molecule are currently available and have been successfully applied in the identification of new T-cell epitopes within proteins. Most of these newly identified epitopes obey to the empirically determined anchor residue patterns that are specific for the different MHC I alleles. However, in recent studies an increasing number of weakly binding T-cell epitopes could be identified that do not fit to these canonical amino acid patterns. Therefore there is a need for new prediction algorithms improving the prediction accuracy for weakly binding epitopes that are biologically relevant as they are presented by, e.g. antigen presenting cells. Here we describe the development and application of an Artificial Neural Network (ANN)-based T-cell epitope prediction strategy for the MHC class I allele HLA-A*0201, leading to the identification of seven new melanoma-associated T-cell epitopes, that do not fit to the canonical HLA-A*0201 recognition motif. In this strategy the ANN-based epitope prediction is combined with a filter algorithm that incorporates knowledge of peptide chemistry and a ranking procedure that enables the selection of candidate epitopes according to protein family and phylogenetic conservation.
Background: FAP is a membrane-bound protease under investigation as a pan-cancer target given its limited expression in normal adult tissues but high expression on cancer-associated fibroblasts. FAP-2286 and FAPI-46 are radiotracers in clinical development that target FAP through different modalities: FAP-2286 employs a peptide macrocycle, whereas FAPI-46 is a quinoline-based small molecule. Here, the biochemical and cellular properties of FAP-2286 and FAPI-46 were evaluated, as well as their in vivo biodistribution and efficacy. Methods: FAP-2286 and FAPI-46 were assessed in biochemical and cellular assays for affinity to FAP and cellular uptake. Complexes of the compounds with gallium-68 (68Ga) or lutetium-177 (177Lu) were investigated in imaging and efficacy studies using the HEK-FAP xenograft mouse model. Results: FAP-2286 and FAPI-46 displayed potent affinity to human FAP by surface plasmon resonance with equilibrium dissociation constants of 1.1 and 0.04 nM, respectively. In addition, FAP-2286 and FAPI-46 inhibited human FAP protease activity with IC50 of 3.2 and 1.2 nM, respectively, and competitor binding to FAP-expressing cells with IC50 of 2.7 and 1.3 nM, respectively. In a PET imaging study, 68Ga-FAP-2286 or 68Ga-FAPI-46 resulted in comparable tumor uptake at 1 hour after injection (10 MBq; 10.6 vs 10.1 percent injected dose per gram [%ID/g]). In contrast, 177Lu-FAP-2286 and 177Lu-FAPI-46 had significant differences in tumor uptake at 24 hours (30 MBq; 15.8 vs 3.8 %ID/g, respectively; P=0.001) and 72 hours (16.4 vs 1.6 %ID/g respectively; P=0.002) after dosing as observed by SPECT imaging. Consistent with the SPECT imaging data, Alexa Fluor 488-derivatized FAP-2286 was retained in cells and secluded in endosomes out to 72 hours in vitro, whereas Alexa Fluor 488-derivatized FAPI-46 levels decreased over time starting at 8 hours, despite both showing a similar level of binding and initial internalization to HEK-FAP cells. FAP-2286 and FAPI-46 labeled with 177Lu demonstrated antitumor efficacy with mean tumor volumes (MTV) of 107 and 245 mm3, respectively, on day 9 after dosing compared with 952 mm3 for vehicle control (30 MBq; P<0.001). The suppressed tumor growth was maintained on day 23 for FAP-2286- but not for FAPI-46-treated animals (MTV 12 vs 1210 mm3; P<0.0001). At the end of the study (day 41), all mice treated with 177Lu-FAPI-46 had reached the endpoint MTV of >1500 mm³ with a median survival time (MST) of 27.5 days, whereas the MTV of mice treated with 177Lu-FAP-2286 was 106 mm3 with MST not being reached. Conclusions: In preclinical studies, the radiotherapeutic 177Lu-FAP-2286 showed longer tumor retention, resulting in greater tumor inhibition than 177Lu-FAPI-46. The phase 1/2 LuMIERE clinical trial (NCT04939610) will evaluate FAP-2286 as a therapeutic (177Lu-FAP-2286) and imaging (68Ga-FAP-2286) agent in multiple indications. Citation Format: Dirk Zboralski, Aileen Hoehne, Anne Bredenbeck, Matthias Paschke, Jan Lennart von Hacht, Jim Xiao, Andrew D. Simmons, Frank Osterkamp, Thomas Harding, Minh Nguyen. Comparative biodistribution and radiotherapeutic efficacy of the fibroblast activation protein (FAP)-targeting agents FAP-2286 and FAPI-46 [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 3317.
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