Immunogenic Peptides Research Articles

Thirty-seven years of MT1 and MT2 melatonin receptor localization in the brain: Past and future challenges.

Identifying the target cells of a hormone is a key step in understanding its function. Once the molecular nature of the receptors for a hormone has been established, researchers can use several techniques to detect these receptors. Here I will review the different tools used over the years to localize melatonin receptors and the problems associated with each of these techniques. The radioligand 2-[125I] iodomelatonin was the first tool to allow localization of melatonin receptors on tissue sections. Once the MT1 and MT2 receptors were cloned, in situ hybridization could be used to detect the messenger RNA for these receptors. The deduced amino acid sequences for MT1 and MT2 receptors allowed the production of peptide immunogens to generate antibodies against the MT1 and MT2 receptors. Finally, transgenic reporters driven by the promoter elements of the MT1 and MT2 genes have been used to map the expression of MT1 and MT2 in the brain and the retina. Several issues have complicated the localization of melatonin receptors and the characterization of melatonin target cells over the last three decades. Melatonin receptors are expressed at low levels, leading to sensitivity issues for their detection. The second problem are specificity issues with antibodies directed against the MT1 and MT2 melatonin receptors. These receptors are G protein-coupled receptors and many antibodies directed against such receptors have been shown to present similar problems concerning their specificity. Despite these specificity problems which start to be seriously addressed by recent studies, antibodies will be important tools in the future to identify and phenotype melatonin target cells. However, we will have to be more stringent than previously when establishing their specificity. The results obtained by these antibodies will have to be confronted and be coherent with results obtained by other techniques.

Gluten-Free Diet and Other Celiac Disease Therapies: Current Understanding and Emerging Strategies.

A lifelong gluten-free diet (GFD) is the only treatment for celiac disease and other gluten-related disorders. Nevertheless, strict adherence to the GFD is often challenging due to concerns about social isolation, risk of gluten contaminations, high cost, poor quality and the taste of gluten-free products. Moreover, although the GFD is effective in achieving mucosal healing, it may lead to dietary imbalances due to nutrient deficiencies over a long period of time. To overcome these issues, several gluten-free wheat flours have been developed to create products that closely resemble their gluten-containing counterparts. Furthermore, given the critical importance of adhering to the GFD, it becomes essential to promote adherence and monitor possible voluntary or involuntary transgressions. Various methods, including clinical assessment, questionnaires, serology for celiac disease, duodenal biopsies and the detection of Gluten Immunogenic Peptides (GIPs) are employed for this purpose, but none are considered entirely satisfactory. Since adherence to the GFD poses challenges, alternative therapies should be implemented in the coming years to improve treatment efficacy and the quality of life of patients with celiac disease. The aim of this narrative review is to explore current knowledge of the GFD and investigate its future perspectives, focusing on technology advancements, follow-up strategies and insights into a rapidly changing future.

Abstract 24: Utilizing T cell receptor-based therapy to treat anaplastic thyroid cancer

Abstract Anaplastic thyroid cancer (ATC) is one of the most aggressive solid tumor in thyroid tissue, with a 6-month medium overall survival. The effectiveness of conventional treatments in extending the survival of patients is limited. Therefore, there is an urgent need to develop novel systemic therapeutic interventions. Adoptive cell therapy has revolutionized cancer immunotherapy by successfully treating B-cell malignancies through chimeric antigen receptor-based approaches. For solid tumors, there is a need to identify a broader spectrum of tumor target antigens, and the field has now shifted towards leveraging T cell receptors (TCRs) which can recognize both intracellular and extracellular antigen-derived epitopes. Our team has established an immunogenic epitope discovery pipeline, followed by a specialized protocol for empiric validation of candidate epitopes. We characterized immunogenic epitopes by coupling tandem mass spectrometry of MHC-eluted peptides with tumor proteome and MHC-peptide binding prediction databases. By integrating multi-omics data (genomics, transcriptomics, and immunopeptidomics) from both in-house and publicly available ATC databases, we find that ATC exhibits significant upregulation of pathways associated with microtubule-based movement, chromosome segregation, and kinetochore complex components. We have identified several HLA-A2- and A24-restricted immunogenic peptides representing these pathway-associated proteins that covered more than 60% of the patient population. These immunogenic peptides were subsequently utilized to isolate antigen-specific T cells from peripheral blood and identify cognate TCR. These peptide-specific T lymphocytes elicit cytotoxicity toward multiple HLA-matched ATC cell lines and tumor samples in vitro and in vivo. In conclusion, our studies contribute to the development of a new treatment paradigm for treating ATC. Citation Format: Shao-Hsi Hung, Ying C. Henderson, Pan Ke, Yulun Chiu, Shailbala Singh, Yunyun Chen, Stephen Y. Lai, Cassian Yee. Utilizing T cell receptor-based therapy to treat anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 24.

Abstract 6648: Low-intensity pulsed ultrasound enhances the presentation of brain-derived antigens by facilitating the migration of antigen-presenting cells from the brain to the periphery

Abstract Gliomas, the most common primary brain tumors, remain largely incurable despite advancements in surgical and adjuvant therapies. While immunotherapies offer promising avenues, their efficacy has been limited by formidable anatomical barriers, such as the blood-brain barrier (BBB), which restricts the migration of immune cells into the brain and impedes the efficient drainage of brain-derived antigens, establishing an immune-privileged environment. Consequently, neoplastic formations within the brain often escape detection by the peripheral immune system, complicating therapeutic interventions. The application of low-intensity pulsed ultrasound with microbubbles (LIPU/MB) has emerged as a potential solution, enabling the transient opening of the BBB. This facilitates drug transport and immune cell trafficking, including Chimeric Antigen Receptor (CAR) T cells, into brain tumors. However, challenges such as antigen-loss escape persist with CAR T cells. One proposed solution involves promoting antigen/epitope spreading by inducing the presentation of brain (tumor) antigens to the systemic immune system. To investigate this, we conducted intravital imaging using Colony-stimulating factor receptor 1 (CSFR1) and CX3CR1 reporter mice, visualizing antigen-presenting cell locomotion in the brain before and after LIPU/MB treatment using two-photon microscopy. Within one hour after LIPU/MB, we observed the migration of CSFR1+ and CX3CR1+ immune cells from the brain parenchyma and perivascular space into the bloodstream, a phenomenon absent prior to LIPU/MB treatment. Beyond that, flow cytometry identified CD45intermediateCD11b+-CX3CR1+-TMEM119+-P2RY12+microglia-like' cells in the blood. To further assess the immunological effects of LIPU/MB on brain antigen presentation, we utilized transgenic mice expressing a minigene encoding four immunogenic peptides under the glial fibrillary acidic protein (GFAP) promoter. LIPU/MB treatment was associated with increased proliferation and priming of adoptively transferred CNS antigen-specific naïve T cells in cervical lymph nodes, ultimately homing into the CNS, indicating enhanced antigen presentation. Furthermore, in a subset of glioblastoma (GBM) patients undergoing LIPU/MB combined with doxorubicin and anti-PD-1 blocking antibodies, CD11b+CD14+P2YR12+ microglia were detected in blood samples during cycles 3-6 of treatment. This increase was absent in blood samples from healthy donors.Collectively, our findings suggest that LIPU/MB represents a novel approach to augment brain-antigen presentation in the periphery, holding potential implications for improving the immunotherapeutic management of gliomas.[M.G., V.A.A., A.M.S., and H.O. contributed equally to this work.] Citation Format: Marco Gallus, Victor Andres Arrieta, Atsuro Saijo, Pavlina Chuntova, Akane Yamamichi, Jeffrey Haegelin, Su Phyu, Lan Phung, Shayan A. Amir-Kabirian, Senthilnath Lakshmanachetty, Li Chen, Karl Habashy, Andrew Gould, Christina Amidei, Catalina Lee Chang, Roger Stupp, Michael Canney, Hideho Okada. Low-intensity pulsed ultrasound enhances the presentation of brain-derived antigens by facilitating the migration of antigen-presenting cells from the brain to the periphery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6648.

Abstract 4095: Targeting alternative classes of tumor antigens to improve immunotherapy for microsatellite stable, mismatch repair-proficient colorectal cancer

Abstract Background: The clinical outcomes of immunotherapy for microsatellite stable (MSS) colorectal cancer (CRC) remains unsatisfactory. The development of more effective immunotherapies is hampered by limited knowledge of the appropriate target antigens, also considering the low burden of mutations generating neoantigens of these tumors. Alternative classes of MSS CRC antigens may provide actionable epitopes. Methods: We have characterized the immunopeptidome of 2 MSS CRC cell lines derived from the primary tumor (SW480) and a metastatic lymph node (SW620) of the same patient. Given the low mutational burden of MSS, we focused on peptides belonging to alternative sources of HLA-A*02 epitopes, such as proteasomal cis-spliced peptides derived from cancer testis antigens and linear epitopes derived from Human Endogenous Retroviruses (HERVs), which are frequently reactivated in CRC. Peptide immunogenicity was functionally validated based on their ability to expand HLA-A*02+ donor T cells (>3) specifically killing targets cells presenting the relevant peptides or HLA-matched CRC cell lines. In vivo epitope immunogenicity was verified by analyzing polyfunctional responses of human CD8+ T cells obtained from NSG-A2 mice humanized with A*02+ donor PBMCs and immunized with epitope-loaded nano-emulsions selectively targeting human Clec9A+ dendritic cells. Therapeutic exploitation of the novel epitopes was investigated by adoptive transfer of epitope-specific T cells in NSG-A2 mice injected with SW620 cells. Epitope-specific T cell responses were investigated in PBMCs from 10 MSS CRC patients and 7 donors. Results: Among >50,000 HLA-bound peptides identified by in depth immunopeptidomics analysis, 11 cis-spliced peptides and 5 HERV peptides were functionally validated as immunogenic. Epitope-specific T cells showed specific cytotoxic activity against SW480 and SW620 CRC cells. Globally, cis-spliced peptides were more immunogenic in vitro than HERV-derived peptides. The immunogenicity of both groups of peptides was confirmed in vivo in humanized mice. Adoptively transferred HERV or cis-spliced epitope specific T cells significantly inhibited the growth of SW620 cells in vivo, with cis-spliced epitope-specific T cells being more effective. Therapeutic efficacy of Clec9A-targeting vaccines exploiting these epitopes is currently under investigation. T cell responses specific for cis-spliced and HERV epitopes were detected in PBMCs from 3/4 MSS CRC HLA-A*02+ patients but in none of 4 HLA-A*02- MSS CRC patients or 7 healthy donors (4 HLA-A*02+ and 3 HLA-A*02-). Conclusions: We have identified and validated a new class of immunogenic HLA-A*02 epitopes derived from alternative classes of MSS CRC antigens. We also provide the proof of principle supporting their therapeutic exploitation to improve the management of these poorly immunogenic tumors. Citation Format: Riccardo Dolcetti, Bijun Zeng, Jingran Ye, Tima Shamekhi, Madeleine Harpur, Davide Moi, Sara Roth, Rob Ramsay, Divya Duscharla, Antony W. Purcell, Pouya Faridi, Roberta Mazzieri. Targeting alternative classes of tumor antigens to improve immunotherapy for microsatellite stable, mismatch repair-proficient colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4095.

Abstract 3525: Towards the efficient design of shared neoantigen peptide cancer vaccines using artificial intelligence

Abstract Background: The advent of immune checkpoint inhibitors has improved morbidity and mortality for some cancers, and recent breakthroughs in gene & cell therapy have shed light on curing some types of blood cancers. However, many cancers remain intractable and the development of novel, effective and safe therapies continue to be a priority. Cancer vaccines as a cancer immunotherapy approach has seen a resurgence in recent years, due to the success of mRNA vaccines for the COVID-19. However, the accurate prediction of immunogenicity of cancer vaccines remains elusive. Methods: Our models predict the probability of a given peptide derived from the protein of interest to be presented by MHC-I or MHC-II. For MHC-I antigen presentation model development, over 17 million entries in the dataset were collected from published literature and available databases, e.g., IEDB, with peptide lengths ranging from 8 to 11. The peptides were restricted to 150 unique MHC-I alleles. Similarly, ~4 million entries with peptide lengths ranging from 13 to 21 were collected for MHC-II antigen presentation model development, and the peptides were restricted to 19 unique MHC-II alleles. To develop advanced antigen presentation models, a language model was chosen as the backbone network and contrast learning was used to better discriminate the peptide-MHC match versus mismatch. Overall, both MHC-I and MHC-II presentation models were constructed with about 30 million parameters. To validate the model prediction accuracy, automated peptide synthesis and surface plasmon resonance (SPR) technologies were applied. Results: Using open-sourced data, our developed AI models surpassed the performance of state-of-the-art prediction algorithms, the latest versions of NetMHCpan and MixMHCpred, for both MHC-I and MHC-II antigen presentation. Furthermore, to validate the algorithm accuracy and the peptide immunogenicity, 28 predicted patentable peptides derived from mutated TP53 protein were synthesized and their binding to respective common HLA alleles were validated using SPR. We found that greater than 80% of the peptides display binding affinities that are stronger than the positive control, suggesting that AI significantly improves neoantigen peptide vaccine design. Conclusions: We developed advanced AI algorithms to rapidly design shared neoantigen T cell epitopes with predicted strong binding affinity to MHC-I and MHC-II. We envision that the epitopes predicted and designed by our AI algorithms possess great potential in advancing the field of off-the-shelf cancer vaccine development and hold the promise of significantly benefiting patients, once translated into the clinic. Citation Format: Genwei Zhang, Jiewen Du, Xiangrui Gao, Tianyuan Wang, Zhenghui Wang, Qingxia Zhang, Tongren Liu, Dong Chen, Ruohan Zhu, Yalong Zhao, Chi Han Samson Li, Melvin Toh, Lipeng Lai. Towards the efficient design of shared neoantigen peptide cancer vaccines using artificial intelligence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3525.

Abstract 5006: Discovery of immunogenic neo-peptides from actionable RNA fusions for developing cancer vaccines

Abstract The global surge in cancer cases, with a staggering 20 million new instances reported in 2023 alone, underscores an urgent demand for advanced and reliable cancer therapeutics. Immunotherapies centered around neoantigens have emerged as a promising avenue for enhancing treatment efficacy and have become a focal point in cancer research. We hypothesize that the tumor cells present a rich neoantigenic repertoire and the immunogenic neoantigens arising from the junctions of proteins translated from chimeric RNAs present viable targets for peptide and mRNA vaccines, offering a promising approach to impede tumor progression. To validate our hypothesis, we acquired a dataset of RNA fusions detected through RNAseq of tissue extracted from patient-derived xenograft (PDX) models established by XenoSTART, a global non clinical oncology contract research organization. This comprehensive dataset encompassed 985 PDX models across 27 cancer types, revealing three fusions associated with the neuregulin-1 (NRG1) gene. NRG1 gene fusions have been identified as a clinically actionable target for multiple solid tumors as they result in ErbB-mediated pathway activation. CD74-NRG1 has emerged as the most frequently reported NRG1 fusion across many cancers such as pancreatic ductal adenocarcinoma, triple negative breast cancer and ovarian cancer. The CD74-NRG1 fusion was identified in two PDX models harboring lung cancer (ST2891 and ST3204) and in an ovarian cancer model (ST088). The lung cancer PDXs displayed a notable abundance of fusion junction-crossing reads. We generated the CD74 (Exon 1-6) -NRG1 (Exon 6-12) fusion transcript model, where the first 6 exons of CD74 fused to exon 6 of NRG1 leading to an in-frame fusion. Validation of this fusion in PDX tissue was accomplished through PCR and Sanger sequencing of the PCR product. We assessed the affinity of the 9-mer neo peptide sequences formed by the translated junction of CD74-NRG1 to Major Histocompatibility Complex (MHC) Class I molecules using the in-silico prediction pipelines MHCNuggets and MixMHCPred-2 and found HLA-A*68:23 to be the MHC allele with highest binding affinity. HLA-Arena was used to investigate the binding affinity between the peptides and the MHC molecules via molecular docking, which revealed 4 peptides to be strong binders to HLA-A*68:23.The immunogenicity of these neoantigenic peptides to HLA-A*68:23 matched Peripheral Blood Mononuclear Cells (PBMCs) will be assessed by the IFN-γ Enzyme Linked Immunosorbent Spot (ELISpot) assay. To elucidate the specific T-cell clonotypes responding to the neo peptides, the 10X Genomics single-cell 5’ Gene Expression Assay, coupled with T Cell Receptor mapping, will be employed. The objective of the study is to establish a robust combinatorial therapeutic strategy leveraging peptide and mRNA vaccine technology to support an effective framework for personalized cancer treatment. Citation Format: Sakuni Rankothgedera, Micah Castillo, Shiyanth Thevasagayampillai, Cole Woody, Aaranyah Kandasamy, Armando Diaz, Michael Wick, Preethi Gunaratne. Discovery of immunogenic neo-peptides from actionable RNA fusions for developing cancer vaccines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5006.

Abstract 4105: Immune-modulating vaccines targeting CD73

Abstract Background: To realize the generalizable regulation of the tumor microenvironment (TME), immune-modulating vaccines are developed to target shared antigens expressed by immunosuppressive cells and cancer cells. CD73, also known as ecto-5'-nucleotidase, is a pivotal enzyme in the adenosine metabolic pathway and is expressed on the surface of multiple cancer cells and immunosuppressive cells in the TME. In this study, we aim to develop a CD73-targeting immune modulatory peptide vaccine by predicting antigen sequences, screening immunogenic antigen peptides, and assessing their anti-tumor efficacy and capacity to improve the TME. Methods: The expression level of CD73 on various cancer cell lines were evaluated by flow cytometry. Several online software were used to predict CD73-derived peptides that bind to the mouse MHC-I (major histocompatibility complex class I) molecules. Cells from the draining lymph nodes of healthy mice and peptide-immunized mice were harvested and stimulated by the synthetic peptides in vitro. T cell activation level, IFN-γrelease degree and their cytolytic capability towards cancer cells was measured. CD73-derived peptides were mixed with CpG to treat 4T1 tumor-bearing mice and the changes in the TME were investigated by flow cytometry. The online website (www.camoip.net) was used to analyze the correlation between CD73 expression and patient survival in the TCGA Breast invasive carcinoma (BRCA) cohort.Results: 4T1 exhibited a strikingly elevated surface expression of CD73 compared to other mouse cancer cell lines. Three CD73-derived peptides (EP1, EP2, EP3) consisting of 8-10 amino acids were synthesized. In vitro stimulation experiments indicated that EP1 and EP3 significantly elicited T-cell responses in draining lymph nodes. After stimulation, the proportions of CD25+, CD69+, CD107a+, and CD137+ cells markedly increased in CD8+ T cell populations. These cells also displayed enhanced cytotoxicity against 4T1 tumor cells. In vivo experiments demonstrated that EP3 induced a pronounced anti-tumor response in an established 4T1 tumor model without evident toxicity. Moreover, subcutaneous injection of EP3 improves the TME by promoting antigen-specific CD8+ T-cell responses, reducing M2 macrophages, and inducing neutrophil maturation. High tumor CD73 expression was also found to be associated with poor outcomes among BRCA patients, which further indicated clinical relevance. Conclusions: In this study, we successfully identified an immunogenic vaccine targeting CD73. It expressed dual functionality by effectively triggering cytotoxic T-cell responses that directly killing CD73-highexpressing tumor cells and regulating the tumor microenvironment through eliminating immunosuppressive cells with elevated CD73 expression. These findings hold promise for a novel immune-modulating vaccine and suggest a potential therapeutic avenue for the treatment of breast cancer and other types of cancer. Citation Format: Yuting Luo, Tao Shi, Hanbing Wang, Baorui Liu, Jia Wei. Immune-modulating vaccines targeting CD73 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4105.

Abstract 21: Discovery of ALK-specific TCR clonotypes for the development of TCR-T cell therapies against ALK-positive cancers

Abstract Introduction: Anaplastic Lymphoma Kinase (ALK) tyrosine kinase inhibitors (TKIs) have extended the survival of patients with ALK-rearranged cancers, including non-small cell lung cancer (NSCLC). Unfortunately, acquired resistance develops within 2-3 years, highlighting the urgent need for novel and effective therapeutic strategies for these patients. Here, we aimed to identify T cell receptor (TCR) clonotypes against two human ALK immunogenic peptides we previously identified by mass spectrometry in biopsies from patients with ALK-rearranged NSCLC (PMID:37430060) and to develop TCR-T cell therapies for ALK-positive NSCLCs. Methods: BALB/c mice were vaccinated with the murine ALK peptide PGPGRVAKI and transgenic mice expressing human HLA-B*07:02 were vaccinated with human ALK peptides RPRPSQPSSL and IVRCIGVSL. All mice received two priming injections (days 1 and 14) followed by two boosters, and activated CD8+ T cells were sorted and subjected to single-cell sequencing. Results: As proof of concept, we first vaccinated BALB/c mice with the ALK peptide PGPGRVAKI and identified 381 unique ALK-specific clonotypes. Among these, we cloned the most expanded 5 clonotypes and confirmed that these clonotypes were functional as evidenced by the release of significant amounts of IFN-γ and IL2, and potent in vitro killing activity following co-culture with murine ALK-positive lung cancer cells. To identify TCR clonotypes for the development of TCR-T therapy in patients with ALK-positive cancers, we next vaccinated transgenic mice expressing human HLA-B*07:02 with human ALK peptides RPRPSQPSSL and IVRCIGVSL. We again identified multiple unique ALK-specific TCR clonotypes among CD8+/CD137+ cells from these transgenic mice vaccinated with RPRPSQPSSL peptide (N=353 TCR clonotypes) and IVRCIGVSL peptide (N=742 TCR clonotypes). Single-cell RNA sequencing of the expanded clonotypes (TCR clonotype frequency ≥4) versus non-expanded clonotypes (TCR clonotype frequency <4) revealed significant upregulation of Ccl3, Ccl4, Ccl1, Ifng, Xcl1, and Il13 across mice vaccinated with RPRPSQPSSL and IVRCIGVSL (p <0.05). Gene set enrichment analysis (GSEA) confirmed significant upregulation of multiple pathways of adaptive immune response, including T cell activation, IFN-γ signaling and production, cytokine release, and T cell proliferation (adjusted p <0.05), suggesting functional activity of these TCR clonotypes against ALK. Conclusion: Here, we first demonstrated the feasibility of isolating ALK-specific TCR clonotypes by mice vaccination that exerted anti-tumor activity. Additionally, we identified ALK-specific TCR clonotypes from transgenic mice expressing human HLA-B*07:02 vaccinated with two human ALK peptides. This discovery lays the basis for the successful development of TCR-T cell therapies for ALK-positive NSCLCs, and possibly other ALK-positive cancers. Citation Format: Carmen Mecca, Ana Azambuja, Luca Alessandrí, Elisa Bergaggio, Simone Piane, Marcos Simoes-Costa, Ellis L. Reinherz, Rafael Blasco-Patiño, Roberto Chiarle. Discovery of ALK-specific TCR clonotypes for the development of TCR-T cell therapies against ALK-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 21.

Abstract 2340: STRUMP-I: Structure-based binding prediction of MHC class I

Abstract The presentation of short peptides derived from endogenously expressed proteins within the Major Histocompatability Complex-I (MHC-I), together known as the pMHC, allows the adaptive immune system to monitor the internal functioning of a cell and surveil for foreign or mutated self-proteins. With the rising importance of immunotherapies targeting neoantigens in cancers, the ability to accurately predict which peptides will bind to the diverse population of MHC alleles is critically important. Computational methods for predicting MHC/peptide interactions fall into two broad categories: sequence-based methods that utilize machine learning or deep learning to predict binding from information about the MHC and peptide sequence alone, and structure-based methods that leverage the computed structure and energetics of the pMHC to predict binding. While sequence-based methods have outperformed structure-based methods, their performance varies with the quantity and quality of training data for each MHC allele. The pMHC structure also provides additional insights into downstream analyses, such as predicting the immunogenicity of cancer neoantigens, that cannot be determined from the sequence alone. We developed a STRUMP-I (STRUcture-based pMHC Prediction (for class I)), a novel pMHC binding prediction tool that combines structural modelling with a machine learning-based classifier to discriminate binders from non-binders. STRUMP-I has comparable performance to the state-of-the-art sequence-based methods on data taken from IEDB, the HLAthena training set, TESLA, PRIME, and an in-house cancer neoantigen dataset. However, it generalizes better to alleles with low representation or a lower proportion of positive examples in the training data than the sequence-based methods. Additionally, we demonstrate how STRUMP-I can be used in conjunction with PRIME, a sequence-based immunogenicity prediction method, to use structural information about the peptide to filter out false positive predictions and increase the precision of immunogenicity predictions, increasing the precision by up to 2-fold. Further, on a set of 10 experimentally validated peptides from the in-house cancer neoantigen dataset containing only a single immunogenic peptide, STRUMP-I filtered all false positive predictions and retained only the true positive prediction. By leveraging structural information about the pMHC, STRUMP-I provides robust binding predicting performance across MHC alleles and increases the precision of immunogenicity prediction for neoantigen screening. Citation Format: Adam Voshall, Woongyang Park, Eunjung Alice Lee, Yoonjoo Choi. STRUMP-I: Structure-based binding prediction of MHC class I [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2340.

Techniques for Theoretical Prediction of Immunogenic Peptides

Small peptides are an important component of the vertebrate immune system. They are important molecules for distinguishing proteins that originate in the host from proteins derived from a pathogenic organism, such as a virus or bacterium. Consequently, these peptides are central for the vertebrate host response to intracellular and extracellular pathogens. Computational models for prediction of these peptides have been based on a narrow sample of data with an emphasis on the position and chemical properties of the amino acids. In past literature, this approach has resulted in higher predictability than models that rely on the geometrical arrangement of atoms. However, protein structure data from experiment and theory are a source for building models at scale, and, therefore, knowledge on the role of small peptides and their immunogenicity in the vertebrate immune system. The following sections introduce procedures that contribute to theoretical prediction of peptides and their role in immunogenicity. Lastly, deep learning is discussed as it applies to immunogenetics and the acceleration of knowledge by a capability for modeling the complexity of natural phenomena.

Development of celiac-safe foods: prevention of transglutaminase 2 (TG2) deamidation of gluten in healthy non-celiac volunteers.

clinicaltrials.gov, identifier (NCT06005376).

Preclinical Development of a Novel Epitope-based DNA Vaccine Candidate against SARS-CoV-2 and Evaluation of Immunogenicity in BALB/c Mice.

The protective efficacies of current licensed vaccines againstCOVID-19 have significantly reduced as a result of SARS-CoV-2 variants of concern(VOCs) which carried multiple mutations in the Spike (S) protein. Considering thatthese vaccines were developed based on the S protein of the original SARS-CoV-2Wuhan strain, we designed a recombinant plasmid DNA vaccine based on highlyconserved and immunogenic B and T cell epitopes against SARS-CoV-2 Wuhan strainand the Omicron VOC. Literature mining and bioinformatics were used toidentify 6 immunogenic peptides from conserved regions of the SARS-CoV-2 S andmembrane (M) proteins. Nucleotide sequences encoding these peptides representinghighly conserved B and T cell epitopes were cloned into a pVAX1 vector to form thepVAX1/S2-6EHGFP recombinant DNA plasmid vaccine. The DNA vaccine wasintranasally or intramuscularly administered to BALB/c mice and evaluations ofhumoral and cellular immune responses were performed. The intramuscularadministration of pVAX1/S2-6EHGFP was associated with a significantly higherpercentage of CD8+ T cells expressing IFN-γ when compared with the empty vectorand PBS controls. Intramuscular or intranasal administrations of pVAX1/S2-6EHGFPresulted in robust IgG antibody responses. Sera from mice intramuscularly immunizedwith pVAX1/S2-6EHGFP were found to elicit neutralizing antibodies capable ofSARS-CoV-2 Omicron variant with the ACE2 cell surface receptor. This studydemonstrated that the DNA vaccine construct encoding highly conserved immunogenicB and T cell epitopes was capable of eliciting potent humoral and cellular immuneresponses in mice.

Gemcitabine Modulates HLA-I Regulation to Improve Tumor Antigen Presentation by Pancreatic Cancer Cells.

Pancreatic cancer is a lethal disease, harboring a five-year overall survival rate of only 13%. Current treatment approaches thus require modulation, with attention shifting towards liberating the stalled efficacy of immunotherapies. Select chemotherapy drugs which possess inherent immune-modifying behaviors could revitalize immune activity against pancreatic tumors and potentiate immunotherapeutic success. In this study, we characterized the influence of gemcitabine, a chemotherapy drug approved for the treatment of pancreatic cancer, on tumor antigen presentation by human leukocyte antigen class I (HLA-I). Gemcitabine increased pancreatic cancer cells' HLA-I mRNA transcripts, total protein, surface expression, and surface stability. Temperature-dependent assay results indicated that the increased HLA-I stability may be due to reduced binding of low affinity peptides. Mass spectrometry analysis confirmed changes in the HLA-I-presented peptide pool post-treatment, and computational predictions suggested improved affinity and immunogenicity of peptides displayed solely by gemcitabine-treated cells. Most of the gemcitabine-exclusive peptides were derived from unique source proteins, with a notable overrepresentation of translation-related proteins. Gemcitabine also increased expression of select immunoproteasome subunits, providing a plausible mechanism for its modulation of the HLA-I-bound peptidome. Our work supports continued investigation of immunotherapies, including peptide-based vaccines, to be used with gemcitabine as new combination treatment modalities for pancreatic cancer.

Investigating Associations between HLA-DR Genotype, H. pylori Infection, and Anti-CagA IgA Seropositivity in a Turkish Gastritis Cohort.

Helicobacter pylori (H. pylori) is associated with gastric inflammation and mucosal antibodies against its cytotoxin-associated gene A (CagA) are protective. Vaccine-elicited immunity against H. pylori requires MHC class II expression, indicating that CD4+ T cells are protective. We hypothesized that the HLA-DR genotypes in human populations include protective alleles that more effectively bind immunogenic CagA peptide fragments and susceptible alleles with an impaired capacity to present CagA peptides. We recruited patients (n = 170) admitted for gastroendoscopy procedures and performed high-resolution HLA-DRB1 typing. Serum anti-CagA IgA levels were analyzed by ELISA (23.2% positive) and H. pylori classified as positive or negative in gastric mucosal tissue slides (72.9% positive). Pearson Chi-square analysis revealed that H. pylori infection was significantly increased in DRB1*11:04-positive individuals (p = 0.027). Anti-CagA IgA was significantly decreased in DRB1*11:04 positive individuals (p = 0.041). In contrast, anti-CagA IgA was significantly increased in DRB1*03:01 positive individuals (p = 0.030). For these HLA-DRB1 alleles of interest, we utilized two in silico prediction methods to compare their capacity to present CagA peptides. Both methods predicted increased numbers of peptides for DRB1*03:01 than DRB1*11:04. In addition, both alleles preferred distinctively different CagA 15mer peptide sequences for high affinity binding. These observations suggest that DRB1*11:04 is a susceptible genotype with impaired CagA immunity, whereas DRB1*03:01 is a protective genotype that promotes enhanced CagA immunity.

Preparation and Immunochemical Characterization of a Water-Soluble Gluten Peptide Fraction for Improving the Diagnosis of Celiac Disease.

The diagnosis of celiac disease (CD) is complex and requires a multi-step procedure (symptoms, serology, duodenal biopsy, effect of a gluten-free diet, and optional genetic). The aim of the study was to contribute to the improvement of CD diagnosis by preparing a water-soluble gluten peptide fraction (called Solgluten) and by selecting gluten-specific enzyme-linked immunosorbent assays (ELISA) for the detection of gluten immunogenic gluten peptides (GIPs) in urine and blood serum spiked with Solgluten. Food-grade Solgluten was prepared by the extraction of a peptic digest of vital gluten with water, centrifugation, and freeze-drying. The process was relatively easy, repeatable, and cheap. The content of gliadin-derived GIPs was 491 mg/g. Solgluten was used as antigenic material to compare two competitive ELISA kits (R7021 and K3012) and two sandwich ELISA kits (M2114 and R7041) in their quality regarding the quantitation of GIPs in urine and blood serum. The quality parameters were the reactivity, sensitivity, coefficients of variation and determination, and curve shape. The evaluation of the kits showed a number of discrepancies in individual quality parameters measured in urine and serum. Due to the lowest limit of quantitation and the highest coefficient of determination, M2114 may be the first choice, while R7021 appeared to be less suitable because of the high coefficients of variation and unfavorable curve progression. The results set the stage for improving CD diagnosis by supplementing conventional blood tests with oral provocation with Solgluten and subsequent ELISA measurement of GIPs that could support the no-biopsy approach and by better assessing the effect of a gluten-free diet by monitoring adherence to the diet by measuring GIPs in urine and blood.

Reanalysis of Immunopeptidomics Datasets Provides Mechanistic Insight into TAPBPR-Mediated Peptide Editing on HLA-A, -B and -C Molecules.

Major histocompatibility class I (MHC-I, human leukocyte antigen [HLA]-I in humans) molecules present small fragments of the proteome on the cell surface for immunosurveillance, which is pivotal to control infected and malignant cells. Immunogenic peptides are generated and selected in the MHC-I antigen processing and presentation pathway. In this pathway, two homologous molecules, tapasin and TAPBPR, optimise the MHC-I peptide repertoire that is ultimately presented at the plasma membrane. Peptide exchange on HLA-I by human TAPBPR involves the flexible loop region K22-D35, with the leucine at position 30 (L30) involved in mediating peptide dissociation. However, our understanding of the exact molecular mechanisms governing TAPBPR-mediated peptide exchange on HLA-I allotypes remains incomplete. Here, in-depth re-analyses of published immunopeptidomics datasets was used to further examine TAPBPR peptide editing activity and mechanism of action on HLA-I. The role of the TAPBPR editing loop in opening the HLA-I peptide binding groove was assessed using a molecular dynamics simulation. We show that TAPBPR shapes the peptide repertoire on HLA-A, -B and -C allotypes. The TAPBPR editing loop was not essential to allow HLA-I to adopt an open state. L30 in the TAPBPR editing loop was typically sufficient to mediate peptide repertoire restriction on the three HLA-I allotypes expressed by HeLa cells. TAPBPR was also able to load peptides onto HLA-I in a loop-dependent manner. These results unify the previously hypothesised scoop loop and peptide trap mechanisms of TAPBPR-mediated peptide exchange, with the former involved in peptide filtering and the latter in peptide loading.

Unlocking the antibacterial potential of bacteriocin peptides: A subtractive proteomics approach to target food spoiling Serratia marcescens

Food deterioration is a serious environmental issue as well as a major concern of the food industry. Bacteriocins, a natural food preservative, can help resolve these issues. The peptide sequences of 111 bacteriocins were obtained from UniProt. The physicochemical properties and immunogenicity of peptides were identified. Final 55 non immunogenic antimicrobial peptides were selected. Full proteome of Serratia marcescens, containing 4,706 proteins, was downloaded from UniProt. The subtractive proteomic approaches were used to select target pathogenic proteins. Only seventy-two proteins from outer membrane, cytoplasmic membrane, extracellular and periplasm were selected for further analysis. Only two S. marcescens proteins, UPI0002AF278A and UPI0003C9BA85, were docked with 55 bacteriocins. Five protein-bacteriocin complexes were subjected to MD simulation by using Amber tool. Three complexes, UPI0003C9BA85-P36504, UPI0003C9BA85-P80925m and UPI0003C9BA85-P86394m were found stable during 50ns run of MD simulation. These three bacteriocins are suggested for further laboratory testing to be used as food preservatives

Lynch Syndrome: From Multidisciplinary Management to Precision Prevention.

Lynch syndrome (LS) is currently one of the most prevalent hereditary cancer conditions, accounting for 3% of all colorectal cancers and for up to 15% of those with DNA mismatch repair (MMR) deficiency, and it was one of the first historically identified. The understanding of the molecular carcinogenesis of LS tumors has progressed significantly in recent years. We aim to review the most recent advances in LS research and explore genotype-based approaches in surveillance, personalized cancer prevention, and treatment strategies. PubMed was searched to identify relevant studies, conducted up to December 2023, investigating molecular carcinogenesis in LS, surveillance strategies, cancer prevention, and treatment in LS tumors. Multigene panel sequencing is becoming the benchmark in the diagnosis of LS, allowing for the detection of a pathogenic constitutional variant in one of the MMR genes. Emerging data from randomized controlled trials suggest possible preventive roles of resistant starch and/or aspirin in LS. Vaccination with immunogenic frameshift peptides appears to be a promising approach for both the treatment and prevention of LS-associated cancers, as evidenced by pre-clinical and preliminary phase 1/2a studies. Although robust diagnostic algorithms, including prompt testing of tumor tissue for MMR defects and referral for genetic counselling, currently exist for suspected LS in CRC patients, the indications for LS screening in cancer-free individuals still need to be refined and standardized. Investigation into additional genetic and non-genetic factors that may explain residual rates of interval cancers, even in properly screened populations, would allow for more tailored preventive strategies.

Immunoinformatic Identification of Multiple Epitopes of gp120 Protein of HIV-1 to Enhance the Immune Response against HIV-1 Infection.

Acquired Immunodeficiency Syndrome is caused by the Human Immunodeficiency Virus (HIV), and a significant number of fatalities occur annually. There is a dire need to develop an effective vaccine against HIV-1. Understanding the structural proteins of viruses helps in designing a vaccine based on immunogenic peptides. In the current experiment, we identified gp120 epitopes using bioinformatic epitope prediction tools, molecular docking, and MD simulations. The Gb-1 peptide was considered an adjuvant. Consecutive sequences of GTG, GSG, GGTGG, and GGGGS linkers were used to bind the B cell, Cytotoxic T Lymphocytes (CTL), and Helper T Lymphocytes (HTL) epitopes. The final vaccine construct consisted of 315 amino acids and is expected to be a recombinant protein of approximately 35.49 kDa. Based on docking experiments, molecular dynamics simulations, and tertiary structure validation, the analysis of the modeled protein indicates that it possesses a stable structure and can interact with Toll-like receptors. The analysis demonstrates that the proposed vaccine can provoke an immunological response by activating T and B cells, as well as stimulating the release of IgA and IgG antibodies. This vaccine shows potential for HIV-1 prophylaxis. The in-silico design suggests that multiple-epitope constructs can be used as potentially effective immunogens for HIV-1 vaccine development.