Specific Peptide Research Articles

Advancing Bone-Targeted Drug Delivery: Leveraging Biological Factors and Nanoparticle Designs to Improve Therapeutic Efficacy.

Designing targeted drug delivery systems to effectively treat bone diseases ranging from osteoporosis to nonunion bone defects remains a significant challenge. Previously, nanoparticles (NPs) self-assembled from diblock copolymers of poly(styrene-alt-maleic anhydride)-b-poly(styrene) (PSMA-b-PS) delivering a Wnt agonist were shown to effectively target bone and improve healing via the introduction of a peptide with high affinity to tartrate-resistant acid phosphatase (TRAP), an enzyme deposited by the osteoclasts during bone remodeling. Despite these promising results, the underlying biological factors governing targeting and subsequent drug delivery system (DDS) design parameters have not been examined to enable the rational design to improve bone selectivity. Therefore, this work investigated the effect of target ligand density, the treatment window after injury, specificity of TRAP binding peptide (TBP), the extent of TRAP deposition, and underlying genetic factors (e.g., mouse strain differences) on TBP-NP targeting. Data based on in vitro binding studies and in vivo biodistribution analyses using a murine femoral fracture model suggest that TBP-NP-TRAP interactions and TBP-NP bone accumulation were ligand-density-dependent; in vitro, TRAP affinity was correlated with ligand density up to the maximum of 200,000 TBP ligands/NP, while NPs with 80,000 TBP ligands showed 2-fold increase in fracture accumulation at day 21 post injury compared with that of untargeted or scrambled controls. While fracture accumulation exhibited similar trends when injected at day 3 compared to that at day 21 postfracture, there were no significant differences observed between TBP-functionalized and control NPs, possibly due to saturation of TRAP by NPs at day 3. Leveraging a calcium-depletion diet, TRAP deposition and TBP-NP bone accumulation were positively correlated, confirming that TRAP-TBP binding leads to TBP-NP bone accumulation in vivo. Furthermore, TBP-NP exhibited similar bone accumulation in both C57BL/6 and BALB/c mouse strains versus control NPs, suggesting the broad applicability of TBP-NP regardless of the underlying genetic differences. These studies provide insight into TBP-NP design, mechanism, and therapeutic windows, which inform NP design and treatment strategies for fractures and other bone-associated diseases that leverage TRAP, such as marrow-related hematologic diseases.

Reduction in systemic muscle stress markers after exercise-induced muscle damage following concurrent training and supplementation with specific collagen peptides - a randomized controlled trial.

Collagen peptide supplementation in conjunction with exercise has been shown to improve structural and functional adaptations of both muscles and the extracellular matrix. This study aimed to explore whether specific collagen peptide (SCP) supplementation combined with a concurrent training intervention can improve muscular stress after exercise-induced muscle damage, verified by reliable blood markers. 55 sedentary to moderately active males participating in a concurrent training (CT) intervention (3x/week) for 12 weeks were administered either 15 g of SCP or placebo (PLA) daily. Before (T1) and after the intervention (T2), 150 muscle-damaging drop jumps were performed. Blood samples were collected to measure creatine kinase (CK), lactate dehydrogenase (LDH), myoglobin (MYO) and high-sensitivity C-reactive protein (hsCRP) before, after, and at 2 h, 24 h and 48 h post exercise. A combination of concurrent training and SCP administration showed statistically significant interaction effects, implying a lower increase in the area under the curve (AUC) of MYO (p = 0.004, ηp2 = 0.184), CK (p = 0.01, ηp2 = 0.145) and LDH (p = 0.016, ηp2 = 0.133) in the SCP group. On closer examination, the absolute mean differences (ΔAUCs) showed statistical significance in MYO (p = 0.017, d = 0.771), CK (p = 0.039, d = 0.633) and LDH (p = 0.016, d = 0.764) by SCP supplementation. In conclusion, 12 weeks of 15 g SCP supplementation combined with CT intervention reduced acute markers of exercise-induced muscle damage and improved post-exercise regenerative capacity, as evidenced by the altered post-exercise time course. The current findings indicate that SCP supplementation had a positive effect on the early phase of muscular recovery by either improving the structural integrity of the muscle and extracellular matrix during the training period or by accelerating membrane and cytoskeletal protein repair. https://www.clinicaltrials.gov/study/NCT05220371?cond=NCT05220371&rank=1, NCT05220371.

Abstract 3598: A new strategy for T cell therapy: T cells secreting TCR anti-CD3 bispecific T-cell engager

Abstract TCR-engineered T (TCR-T) cells and TCR-anti-CD3 bispecific T-cell engagers (TCEs) are potent TCR-based therapeutic agents with distinct advantages and limitations in tumor treatment. TCR-T cells offer durable persistence within patients but necessitate personalized manufacturing and lack the capacity to harness bystander T cells. Conversely, TCEs are readily available as "off-the-shelf" products and can recruit bystander T cells, yet they exhibit a shorter lifespan. In our study, we sought to merge the merits of both approaches by engineering T cells to secrete a TCR-anti-CD3 TCE specific for alpha fetoprotein (AFP), a tumor-associated antigen abundantly expressed in hepatocellular carcinoma (HCC). We initially identified a TCR with specificity for the AFP158-166 peptide bound to HLA-A*02:01 and enhanced its affinity to picomolar via phage display. To facilitate efficient secretion by T cells, we adapted the high-affinity TCR to a single-chain format (scTCR) and fused it with a CD3-specific single-chain antibody fragment (scAFP-TCE). Our findings demonstrated that scAFP-TCE effectively redirected bystander T cells to engage in the lysis of HCC cells. Moreover, scAFP-TCE could be secreted by T cells transduced with lentiviral particles encoding the TCE gene. These transduced T cells exhibited potent antitumor activity both independently and by enlisting bystander T cells. This innovative T cell strategy, combining the bystander-recruiting ability of fusion proteins with the durable persistence seen in T cell therapy after a single infusion, presents a promising alternative to conventional TCR-based therapeutic agents. We anticipate that this novel approach may hold substantial potential for enhancing HCC treatment and expanding the scope of TCR-based immunotherapies. Citation Format: Hanli Sun, Jiao Huang, Kai Zhan, Wanli Wu, Xianqing Tang, Min Liu, Shanshan Guo, Hongjun Zheng, Yingjie Huang, Shi Zhong. A new strategy for T cell therapy: T cells secreting TCR anti-CD3 bispecific T-cell engager [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 3598.

Abstract 6025: Discovery of novel macrocyclic peptide radioligands for tumor therapy by mRNA display

Abstract Radioligand therapeutics (RLT) aim to selectively deliver radioisotopes to cancer tissues to eradicate tumor cells while limiting the damage to surrounding tissues. They are composed of a tumor-targeting ligand and a radioactive payload. The therapeutic potential of RLT strongly depends on their biostability and residence time in tumor and healthy tissue after injection. Macrocyclic peptides are effective tumor-targeting ligands because their size and molecular characteristics confer optimal pharmacologic properties that can be fine-tuned during the development process. mRNA display is a powerful platform for macrocyclic peptide drug discovery that identifies high affinity peptide ligands through in vitro selection. Here, we describe the selection and initial characterization of macrocyclic peptides discovered by mRNA display that bind to the extracellular domain of a tumor target. Enrichment of the library for peptides that bound to the recombinant target protein was observed after four rounds of selection with no further enrichment in round five. The library of round 5 was sequenced by Illumina sequencing and the top 1,000 sequences (highest counts) were used to perform cluster analysis. We tested the top sequences of 43 different clusters in in vitro translation binding assays to validate the selection output and identify sequences for hit confirmation. Most of the sequences tested showed clear binding to the target protein and only a few peptides showed binding to a closely related protein from the same protein family. No non-specific binding of the discovered peptides to an irrelevant protein was observed showing the high specificity of the generated library. From this confirmatory selection data, we chose a subset of peptides for chemical synthesis. Binding affinity measurements were determined by surface plasmon resonance (SPR) and cellular binding of selected candidates to target-positive and -negative tumor cells was analyzed by flow cytometry. From this hit confirmation data, in vitro internalization and in vitro anti-tumor efficacy of radiolabeled peptide candidates for RLT are assessed. Our data highlights the potential of mRNA display platforms for fast and efficient discovery of highly specific macrocyclic peptides with optimal binding properties suitable for RLT. Citation Format: Anika Jaekel, Kathryn Sherry, Prachi Desai, David Fox, Tang Guo-Qing, Manuel Sturzbecher-Hoehne, Dennis Mewis, Kaori Noridomi, Liam Rayman, Ping Ye. Discovery of novel macrocyclic peptide radioligands for tumor therapy by mRNA display [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 6025.

Abstract 5889: An immunoproteasome activator that expands the MHC-class I immunopeptidome, unmasks neoantigens and enhances antimyeloma T-cell activity

Abstract A hallmark of cancer is the ability to evade immunosurveillance mechanisms and thwart the efficacy of immunotherapeutic agents. The proteasome functions as an essential component of immunosurveillance by generating peptides from intracellular proteins that are then presented to T-cells. Antigens generated by the proteasome promote the infiltration of immune cells into tumors and improve tumor regression in response to immunotherapy. Cancer cells can evade elimination by the immune system by deregulating the antigen presentation machinery to downregulate the expression of proteins recognized by immune cells as antigens, creating an immunosuppressive microenvironment. In contrast to constitutive proteasomes, immunoproteasomes are a highly specialized proteasome variant that are highly expressed in antigen-presenting cells. Immunoproteasomes contain distinct catalytic subunits with active site substrate specificities distinct from constitutive proteasomes. Immunoproteasome degrade intracellular proteins to generate antigenic peptides that are the presented by MHC-class I (MHC-I) molecules on tumor cells for recognition by cytotoxic T-cells. We hypothesized that immunoproteasome activation could concomitantly increase the relative abundance and diversity of MHC-I antigens presented on multiple myeloma (MM) cells. High-throughput screening identified a novel small molecule (Compound A) that selectively increased immunoproteasome catalytic activity. Global proteomic integral stability assays determined that Compound A binds the proteasome structural subunit PSMA1 and that treatment with Compound A increased association of the proteasome activator PA28α/β (PSME1/2) activator with immunoproteasomes. The effect of Compound A on immunoproteasomes was abolished after the ablation of PSMA1, PSME1, or PSME2 or by the treatment of cells with suicide inhibitors that target immunoproteasome-specific catalytic sites. Treatment of MM cells with Compound A doubled the total number of MHC-I-bound peptides and increased the level of specific MHC-bound peptides up to 200-fold. Interestingly, a number of tumor-associated antigens and neoantigens were dramatically unmasked following treatment of MM cells with the immunoproteasome activator. Importantly, the treatment of patient CD138+ cells with Compound A promoted the antimyeloma activity of allogenic and autologous patient-derived CD8+ T-cells. Taken together, our results demonstrate the paradigm-shifting translational impact of immunoproteasome activators to expand the myeloma immunopeptidome and have revealed novel, actionable antigenic targets to personalize T-cell-directed immunotherapy. Citation Format: Priyanka S. Rana, James J. Ignatz-Hoover, James J. Driscoll. An immunoproteasome activator that expands the MHC-class I immunopeptidome, unmasks neoantigens and enhances antimyeloma T-cell activity [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 5889.

Abstract 4274: Unraveling collagen signatures in cancer-associated fibroblasts: A biomarker-driven approach

Abstract Background: The tumor microenvironment (TME) plays a crucial role in driving tumor development. Among the constituents of the tumor stroma, cancer-associated fibroblasts (CAFs) are a significant component. CAFs are actively involved in tumor progression by modulating the architecture of the TME through increased deposition of various collagens resulting in tumor fibrosis. Several studies have shown that CAFs have heterogeneity within, and between, individual tissues. TGF-β is thought to be the main driver of tumor fibrosis, however, the field lacks a characterization of the specific collagen deposition of CAFs from different tissues. In this study, we investigated the fibrotic activity of CAFs from various tissues by measuring the production of three specific collagen peptides in vitro by use of non-invasive clinically validated biomarkers. Methods: Primary human CAFs from pancreas (pCAF), colon (cCAF), breast (bCAF) and lung (lCAF) were cultured over a 12-day period in ficoll-based media under both unstimulated conditions and TGF-β stimulation. Additionally, cells were subjected to treatment with the ALK5/TGF-β1 receptor kinase inhibitor (ALK5i). The assessment of collagen I (PRO-C1), collagen III (PRO-C3), and collagen VI (PRO-C6) formation was conducted in the cell supernatant at day 3, day 6, day 9, and day 12 using competitive enzyme-linked immunosorbent assay (ELISA). Results: For lCAFs, cCAFs, and pCAFs, the addition of TGF-β led to a notable elevation in the levels of PRO-C1, PRO-C3, and PRO-C6 within the supernatant. Conversely, in the case of bCAFs, stimulation with TGF-β did not elicit remarkable alterations in the levels of PRO-C1 and PRO-C3 compared to unstimulated cells. Notably, bCAFs exhibit elevated intrinsic activity (high PRO-C3) even in the absence of TGF-β stimulation whereas lCAFs and cCAFs exhibited relatively low level of PRO-C3. Additionally, across all different CAFs, the introduction of ALK5 inhibitor (ALK5i) to the culture medium resulted in a considerable reduction in PRO-C1, PRO-C3, and PRO-C6 levels, returning them to the baseline of the unstimulated control or even lower. Distinct collagen formation patterns were observed among the various CAFs. CAFs derived from breast cancer exhibited the highest release of PRO-C1 and PRO-C3, while CAFs isolated from colon cancer displayed the highest levels of PRO-C6. Conclusion: These findings underscore the heterogeneity in collagen production among different CAFs, providing valuable insights into the extracellular matrix dynamics within distinct tumor microenvironments. Moreover, non-invasive biomarkers demonstrate the capability to differentiate between different CAF subtypes. Lastly, our model proves to be a valuable tool for anti-fibrotic drug screening, as evidenced by the consistent suppression of TGF-β1 signaling and reduced levels of PRO-C1, PRO-C3, and PRO-C6 upon treatment with ALK5i across all cases. Citation Format: Annika Hettich, Neel I. Nissen, Morten A. Karsdal, Nicholas Willumsen. Unraveling collagen signatures in cancer-associated fibroblasts: A biomarker-driven approach [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 4274.

Abstract 1032: Development of a novel companion diagnostic immunohistochemistry antibody for claudin 18.2-targeted therapies

Abstract Background: CLDN18.2, the isoform 2 of CLDN18, is physiologically confined to gastric mucosa tight junctions, the epitopes of which would be exposed on the cancer cell surface upon malignant transformation and is highly expressed in a significant proportion of cancers, e.g., gastric and pancreatic adenocarcinomas, which makes it a promising drug target for cancer therapy. Currently, multiple drug modalities, such as monoclonal antibodies, ADCs, and CARTs targeting CLDN18.2 are being evaluated in clinical trials. A substantial correlation exists between the expression of CLDN18.2 and the efficacy of the these therapies. However, to date, no companion diagnostic (CDx) antibodies that are specific to CLDN18.2 have been approved. Therefore, it is important to promptly develop an anti-CLDN18.2 CDx antibody with a high specificity, sensitivity, and suitability for immunohistochemistry (IHC). In this study, we present the discovery and validation of a novel, highly sensitive IHC antibody that selectively identifies CLDN18.2. Methods: 3 CLDN18.2 specific peptide fragments from extracellular domain of human CLDN18.2 were conjugated with KLH. Antibodies were discovered by immunizing BALB/c and SJL mice with these peptides-KLH conjugates. Screening and selection process was relied on positive ELISA results against the immunogen, as well as FACS and Acumen Explorer Microplate staining of CLDN18.2-experssing cells. Hit antibodies were evaluated for IHC on formalin-fixed paraffin-embedded CHOK1 cells expressing full-length CLDN18.2 or CLDN18.1. The antibodies were further validated on human gastric cancer tissue samples to establish the analytical sensitivity and specificity of the IHC assay. Results: The mAb clone 43F11 exhibited binding to a fusion polypeptide comprising the first extracellular loop of human CLDN18.2 (aa32-53) at a half maximal effective concentration (EC50) of 0.212 nM. 43F11 showed positive cell surface IHC staining on CLDN18.2-experssing cells after fixation but demonstrated no staining on CLDN18.1-experssing cells. The IHC test method was established by utilizing clinical tumor tissues and PDX samples that had predetermined expression levels of CLDN18.2. The 43F11 antibody accurately identified the expression level of CLDN18.2 in the IHC assay, with a minimum concentration of 0.25 μg/mL. When compared to the commercial CLDN18.2 IHC antibody EPR19202, 43F11 demonstrated greater sensitivity, allowing for positive staining on cancer tissues that have significantly lower levels of CLDN18.2 expression. Conclusion: 43F11 exhibits specific recognition of human CLDN18.2 in IHC assays, while showing no reactivity towards CLDN18.1. It demonstrated superior sensitivity compared to the benchmark antibody. These data suggest that 43F11 has the capacity to serve as an effective diagnostic tool for identifying patients who positively express CLDN18.2. Citation Format: Jishun Chen, Suya Bai, Yu Bai, Zaoshun Hu, Peng Chen, Jay Mei, Bo Shan, Bing Hou. Development of a novel companion diagnostic immunohistochemistry antibody for claudin 18.2-targeted therapies [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 1032.

Abstract 3543: TWNeoDB: A web-based database for tumor neoantigens in the Taiwanese population

Abstract Tumor neoantigens are highly immunogenic. Two types of neoantigens have been reported for their ability to be shared among patients. One is a mutated tumor-specific antigen (mTSA) derived from somatic mutations in tumor cells. The other is an aberrantly expressed TSA (aeTSA), influenced by epigenetic changes or abnormal RNA splicing. Tumor neoantigens can bind with the major histocompatibility complex (MHC) and be recognized by the T cell receptor (TCR). Consequently, they can trigger the immune system to attack cancer cells. Until now, some neoantigen databases have been available, they mostly focus on the Western population and primarily contain peptides derived from mTSAs. In contrast, our database provides peptides not only from mTSA but also from aeTSA with a strong emphasis on the Taiwanese population. Initially, we obtained public sequencing raw data from the NCBI database and employed a neoantigen pipeline for analysis, identifying potential neoantigens. The data collection criteria included samples from Taiwanese individuals, with paired DNA-seq or RNA-seq from both normal and tumor tissues of the same patients. RNA sequencing datasets were utilized to identify aeTSAs and mTSAs, whereas DNA sequencing datasets served for mTSA identification. Additionally, human leukocyte antigen (HLA) genotyping was performed for every sample. The identified peptides were further compared to previously validated data available on IEDB. This data was used to develop a web-based database with several functionalities. Users can search for specific peptides and download relevant data from the website. The website also included data cross-referenced and validated with IEDB. In addition, we incorporated clinically validated peptides capable of stimulating T cells to release cytotoxins or interferons. A machine-learning-based LightGBM model was trained to predict immunogenicity for these peptides through a series of cross-validations based on random data splitting. Users can access comprehensive information on tumor-specific peptides in the online database. We collected sequencing data from 243 patients, spanning five different types of cancer. The predominant HLA genotype is HLA-A*11:01, a common allele in the Taiwanese population. Peptide characteristics, such as hydrophobicity, binding affinity, and binding stability, have been calculated and stored in the database. Notably, the LightGBM model excelled in predicting immunogenicity, achieving an AUC of 0.95 on the training dataset and 0.8 on the testing dataset. Implemented in the online database, this model allows users to forecast their own candidates. The state-of-the-art database serves as a comprehensive platform for gathering Taiwanese-specific neoantigens, contributing to the advancement of personalized cancer vaccines and immunotherapies. Citation Format: Yu-Hsuan Tseng, Chia-Hsin Wu, Chia-Yu Sung, Huang Kevin Chih Yang, Mong-Hsun Tsai, Liang-Chuan Lai, Tzu-Pin Lu, K.S. Clifford Chao, Eric Y. Chuang, Chien-Yueh Lee. TWNeoDB: A web-based database for tumor neoantigens in the Taiwanese population [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 3543.

Abstract 5246: Therapeutic efficacy of glypican-3 peptide-linked chimeric antigen receptor-macrophages in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the third most lethal cancer and sixth most diagnosed cancer worldwide. Recently, chimeric antigen receptor-T cell (CAR-T) therapy has been demonstrated to be a promising immunotherapy for the treatment of hematologic malignancies such as leukemia. However, its application in solid tumors has proven to be challenging. To overcome this limitation, chimeric antigen receptor-macrophages (CAR-Ms) have emerged as a new immunotherapy against solid cancers, capitalizing on the unique attributes of macrophages in penetrating the tumor microenvironment, promoting antigen presentation, and priming T cells. Traditionally, CAR-M therapy involves the transfer of an edited CAR gene into macrophages via viral infection. However, this method is expensive, involves tedious procedures, and has long-term safety concerns. In the present study, we employed peptides to mimic the genetic approach for producing CAR-Ms to reduce the above uncertainties. To achieve this, we developed a novel bioconjugation method called the phthalaldehydeamine capture (PAC) reaction to produce peptidic CAR-Ms (pCAR-Ms) targeting Glypican-3 (GPC3) in HCC cells. This was achieved by linking GPC3 specific peptides to Raw264.7 murine macrophages. Using an in vitro co-culture system, we demonstrated the specificity and efficacy of pCAR-Ms against GPC3, as evidenced by a significant increase in the number of phagocytosed cells with high GPC3 expression when compared to their low-expression counterparts, by flow cytometry and confocal microscopy. The specificity of pCAR-Ms was further proven in GPC3-knockdown HCC cells, with a significant decrease in the phagocytic rate. Using an HCC xenograft model derived from MHCC97L cells, we confirmed the specific homing of pCAR-Ms to tumor sites via intravenous injection of lipopolysaccharide (LPS)-differentiated pCAR-Ms. Strikingly, pCAR-Ms suppressed HCC tumor growth when compared with untagged macrophages and PBS control. In conclusion, we provided evidence of the potency and specificity of non-viral-based pCAR-Ms in targeting HCC with simple and short production time and increased flexibility. Citation Format: Wing Hei Leung, Oi Ning Leung, Tsun Ting Wong, Kin Wah Lee. Therapeutic efficacy of glypican-3 peptide-linked chimeric antigen receptor-macrophages in hepatocellular carcinoma [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 5246.

Abstract 2470: Harnessing cytomegalovirus immunity against pancreatic tumors for immunotherapy

Abstract Immunotherapy has had very limited success in pancreatic cancer, due to its low mutational burden and immunosuppressive microenvironment. Our approach consists in redirecting pre-existing antiviral immunity against pancreatic tumors by delivering viral antigens using the tumor penetrating peptide iRGD .This peptide, targets the tumor vasculature through αv integrins and neuropilin-1, delivering conjugated or co-administered cargo to tumors. Here we used mouse cytomegalovirus (CMV) as an infection model. CMV is a β-herpesvirus that induces a strong T-cell response in mice and humans, comprising >10% of all circulating CD4 and CD8 T cells mostly with effector-memory phenotype. Importantly, human CMV infects over 60% of the world’s population rendering it a suitable candidate for translation. Mice latently infected with CMV were orthotopically implanted with KPC pancreatic tumor cells and treated with systemic injections of vehicle or iRGD plus CMV class I and II binding peptides. Another cohort of age matched uninfected mice was used as a control. Tumor growth and mouse weight were monitored twice a week and CMV specific immune responses were measured in spleen and tumor by flow cytometry, using tetramer staining and CMV specific peptide recall. CMV infected mice receiving iRGD + CMV peptides responded to treatment as evidenced by delayed tumor growth associated with increased tumor necrosis and T cell infiltration. In addition, flow cytometry analysis of tumor and spleen showed a significant increase in CMV specific T cells that produced significant amounts of IFN-γ and TNF-α upon CMV peptide recall. Furthermore, tumor regulatory T cells (Treg) were significantly reduced and the CD8/T reg ratio was greatly increased. Survival studies showed a 68% increase in median survival in the treated and infected group. Combining this strategy with anti-PD1 and or anti-IL10R did not significantly improve the benefits of iRGD + CMV therapy alone. Other combinations with chemotherapy and TME remodeling agents are being tested.Taken together, this data shows we can deliver antigens to pancreatic tumors via iRGD and elicit an anti-tumor immune response that results in delayed tumor growth and has a survival benefit. This is a mutation agnostic approach with high translational value, since more than 60% of the population has previous CMV immunity and iRGD in combination with chemotherapy is already in phase II clinical trials for advanced pancreatic cancer. Citation Format: Remi Marrocco, Jay Patel, Siming Sun, Rithika Medari, Kevin Gulay, Eduardo Lucero-Meza, Evangeline Mose, Andrew Lowy, Chris Benedict, Tatiana Hurtado de Mendoza. Harnessing cytomegalovirus immunity against pancreatic tumors for immunotherapy [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 2470.

Abstract 2694: The role of innate lymphoid cells and innate like T cells in hepatocellular carcinoma

Abstract The major focus of cancer immunotherapy has been on conventional T cells due to their unique recognition of specific peptide antigens. Despite advances in the cancer immunotherapy field, including the use of atezolizumab and bevacizumab as first-line therapy for unresectable hepatocellular carcinoma (HCC), the overall mortality rate of HCC continues to rise. Innate lymphoid cells (ILC) and innate-like T cells (ILTC) are broadly categorized within the innate lymphoid population as playing an important role in inflammation, tissue repair and immune tolerance. Their ability to react rapidly to stimulants in their local tissue environment as well as to recruit and activate a variety of other immune cells makes these cells attractive targets for anti-tumor therapy. However, previous studies looking at ILCs and ILTCs in cancer have revealed both pro- and anti-tumor functions. Furthermore, the complex interplay between these cell populations in the tumor microenvironment is not well understood, particularly over the natural course of tumor development. In this study, we aim to better elucidate the role of ILC and ILTC populations in HCC progression in terms of both their kinetics and role as potential therapeutic targets. We performed hydrodynamic tail vein injections in mice with two different plasmid combinations to alter expression of commonly mutated oncogenes and tumor suppressors in human HCC (MYC;TP53 and MYC;CTNNB1). Histologic analysis of the livers was performed to determine appropriate time points to assess ILC and ILTC populations. We then designed a comprehensive 29-plex spectral flow panel to assess broad kinetic changes in the frequency of ILC, ILTC, and conventional T cell populations. We show that in early time points (4-7 days post-injection) when neoplastic transformation of hepatocytes has already occurred, mucosal-associated invariant T (MAIT) cells, group 1 ILCs, ILC2s, and ILC3s expand in frequency for both tumor models. Cytokine analysis shows a generalized decreased trend across timepoints in effector cytokine production in MAITs and group 1 ILCs, suggesting gradual dysfunction over time. Taken together, these preliminary findings show that ILC and ILTC populations expand early during tumor progression but gradually decrease in functionality with tumor progression. Citation Format: Patrick Huang, Benjamin Ruf, Rajiv Trehan, Dana Soika, Chi Ma, Tim F. Greten, Firouzeh Korangy. The role of innate lymphoid cells and innate like T cells in hepatocellular carcinoma [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 2694.

A non-targeting magnetic metal-organic framework probe for highly specific peptide-mediated precise disease monitoring

Alzheimer's disease (AD) is a universal neurodegenerative disease in older adults with incurable and progressive properties, urging for precise monitoring to perform timely treatment to delay its progression. Herein, we introduced a non-targeting magnetic metal-organic framework probe coupled with high-throughput mass spectrometry, creating a rapid screening strategy for highly specific peptides associated with AD. Notably, an elution-free extraction process was proposed, significantly reducing sample preprocessing time while simultaneously ensuring the efficient detection of captured peptides. Using this elution-free extraction process, high-quality peptide profiles were rapidly extracted from the hundreds of samples from both diseased and healthy individuals. By integrating machine learning algorithms, LC-MS/MS, and Uniprot database searching, we identified three specific serum endogenous peptides (m/z = 4215.41, 2884.77 and 2704.61) closely associated with AD. Remarkably, with the use of any single specific peptide, the AUC (Area Under the Curve) values can reach approximately 0.9 during monitoring AD. Moreover, integrating three specific biomarkers provides a robust basis for machine learning algorithms to build monitoring models, with AUC value up to 1.000. This work represents a substantial advancement in the development of peptide-specific precise monitoring approaches for complex diseases, serving as a catalyst for increased dedication to the molecular detection field.

Designing and Developing Serological Test for the Diagnosis of Human Fascioliasis Using a New Recombinant Multi-epitope.

Fascioliasis is a common parasitic disease in humans and herbivores which is caused by Fasciola hepatica and Fasciola gigantica and has a worldwide distribution. Serological tests such as the enzyme-linked immunosorbent assay (ELISA) technique play a prominent role in the fast diagnosis of the disease. However, there are diagnostic limitations, including cross-reactivity with other worms, which decline the specificity of the results. This study aimed to evaluate the structure of a recombinant multi-epitope antigen produced from linear and conformational B-cell epitopes of three parasitic proteins with sera of individuals with fasciolosis, healthy controls, and those with other diseases to gain accurate sensitivity and specificity. After designing the multi-epitope structure of cathepsin L1, FhTP16.5, and SAP-2 antigens and then synthesizing, cloning, and expressing, the extracted purified protein was evaluated by indirect ELISA to detect IgG antibodies against Fasciola hepatica parasite among the sera of 39 serum samples of Fasciola hepatica, 35 healthy individual samples, and 20 samples of other types of parasitic diseases. The synthesized multi-epitope produced from cathepsin L1, FhTP16.5, and SAP-2 antigens was evaluated using the indirect ELISA. The analysis of the samples mentioned for IgG antibody diagnosis against Fasciola hepatica showed 97.43% (95% confidence interval, 94.23-100%) sensitivity and 100% (95% confidence interval, 97-100%) specificity. The recombinant B-cell multi-epitope with high antigenic potency may increase the specificity of epitopic peptides and ultimately help improve and develop indirect ELISA commercial kits for the diagnosis of fascioliasis in humans.

Characterization of a secreted aminopeptidase of M28 family from B. fragilis and its possible role in protein metabolism in the gut

Products of microbial protein metabolism in the gut can influence the health of the host in many ways. Members of the Bacteriodales, major commensals of the human colon have been associated with long-term intake of high-protein diets. Undigested proteins or peptides that reach the colon can be hydrolyzed by extra-cellular proteases found in some Bacteroides species into amino acids and peptides which can be further catabolized. In this communication, we have characterized one such secreted aminopeptidase (BfAP) from Bacteroides fragilis belonging to the M28 family which is capable of degrading peptides released from soybean protein after predigestion in the small intestine. The BfAP enzyme was cloned, expressed in E. coli, and purified to homogeneity. It is a metallopeptidase requiring Co2+ ion for optimum activity at 55 °C and pH 8 and preferentially cleaves neutral aliphatic (Met/Leu) and positively charged (Arg/Lys) amino acids from the N-terminus of peptides. It showed high specificity for long peptides as well as proteins like β-casein. Structural analysis of BfAP and its orthologues using AlphaFold2 reveal a shared highly conserved M28 domain, but vary with respect to their N-terminal region with some of them possessing an additional cap domain which may be important for regulation of substrate binding. Although BfAP lacks the typical cap domain, it shows small extensions that can form a loop adjacent to the proposed active site and may affect substrate binding. We suggest that this secreted enzyme may play an important role in protein metabolism in the colon where Bacteroides species are abundant.

Development of New CD38 Targeted Peptides for Cancer Imaging

PurposeMultiple myeloma (MM) affects over 35,000 patients each year in the US. There remains a need for versatile Positron Emission Tomography (PET) tracers for the detection, accurate staging, and monitoring of treatment response of MM that have optimal specificity and translational attributes. CD38 is uniformly overexpressed in MM and thus represents an ideal target to develop CD38-targeted small molecule PET radiopharmaceuticals to address these challenges.ProceduresUsing phage display peptide libraries and pioneering algorithms, we identified novel CD38 specific peptides. Imaging bioconjugates were synthesized using solid phase peptide chemistry, and systematically analyzed in vitro and in vivo in relevant MM systems.ResultsThe CD38-targeted bioconjugates were radiolabeled with copper-64 (64Cu) with100% radiochemical purity and an average specific activity of 3.3 – 6.6 MBq/nmol. The analog NODAGA-PEG4-SL022-GGS (SL022: Thr-His-Tyr-Pro-Ile-Val-Ile) had a Kd of 7.55 ± 0.291 nM and was chosen as the lead candidate. 64Cu-NODAGA-PEG4-SL022-GGS demonstrated high binding affinity to CD38 expressing human myeloma MM.1S-CBR-GFP-WT cells, which was blocked by the non-radiolabeled version of the peptide analog and anti-CD38 clinical antibodies, daratumumab and isatuximab, by 58%, 73%, and 78%, respectively. The CD38 positive MM.1S-CBR-GFP-WT cells had > 68% enhanced cellular binding when compared to MM.1S-CBR-GFP-KO cells devoid of CD38. Furthermore, our new CD38-targeted radiopharmaceutical allowed visualization of tumors located in marrow rich bones, remaining there for up to 4 h. Clearance from non-target organs occurred within 60 min. Quantitative PET data from a murine disseminated tumor model showed significantly higher accumulation in the bones of tumor-bearing animals compared to tumor-naïve animals (SUVmax 2.06 ± 0.4 versus 1.24 ± 0.4, P = 0.02). Independently, tumor uptake of the target compound was significantly higher (P = 0.003) compared to the scrambled peptide, 64Cu-NODAGA-PEG4-SL041-GGS (SL041: Thr-Tyr-His-Ile-Pro-Ile-Val). The subcutaneous MM model demonstrated significantly higher accumulation in tumors compared to muscle at 1 and 4 h after tracer administration (SUVmax 0.8 ± 0.2 and 0.14 ± 0.04, P = 0.04 at 1 h; SUVmax 0.89 ± 0.01 and 0.09 ± 0.01, P = 0.0002 at 4 h).ConclusionsThe novel CD38-targeted, radiolabeled bioconjugates were specific and allowed visualization of MM, providing a starting point for the clinical translation of such tracers for the detection of MM.

Antitumor Strategies Targeting Peptidergic Systems

Peptidergic systems show promise as targets for fighting tumors. While some peptides encourage the growth and spread of tumor cells and angiogenic mechanisms, others display antitumor properties. As such, peptide ligands and receptor antagonists could be used as antitumor agents alone or in conjunction with chemotherapy or radiotherapy. Peptide receptor antagonists can counteract the oncogenic effects of specific peptides by inducing apoptosis in various types of tumor cells, hindering cancer cell migration and inhibiting angiogenesis. Peptides and peptide receptor antagonists are not currently used in clinical practice as antitumor agents. Still, aprepitant, a neurokinin 1 receptor antagonist, is a promising candidate due to its ability to promote apoptosis in many cancer cells. However, to utilize aprepitant as an anticancer agent, the dosage must be increased and administered for a more extended period. Moving beyond current protocols for aprepitant’s use as an antiemetic is essential. Additionally, a common anticancer strategy with aprepitant is possible regardless of cancer cell type. Finally, combining aprepitant with chemotherapy or radiotherapy is encouraged.

Beyond the model organism-Mechanistic analysis of protein post-translational modifications is ready for all challengers.

A historic challenge for shotgun proteomics has been the requirement for high quality, simple, and nonredundant curated protein sequences in small .fasta text files. Due to the intrinsic informatic challenges and time required to assemble these files, proteomics has struggled to expand beyond the confines of a few model organisms. When considering post-translational modifications that may or may not be present on a specific peptide sequence, these factors inevitably compound. A study on how mangos continue to ripen on the shelf may not be the first thing you'd think of as proof of a scientific discipline shedding historic limitations. However, Bautiste-Valle etal., may be just that. These authors present a quantitative comparison of both peptide and glycopeptide alterations through the complexity of the fruit ripening process and in this we see the present state of a field that no longer needs to wait on genomics to obtain deep mechanistic insights.

AMYGNN: A Graph Convolutional Neural Network-Based Approach for Predicting Amyloid Formation from Polypeptides.

There has been an increasing interest in the use of amyloids for constructing various functional materials. The design of amyloid-associated functional materials requires the identification of the core peptide sequences as the fundamental building block. The existing computational methods are limited in terms of delineating polypeptides, the typical non-Euclidean structural data, and they fail to capture the dynamic interactions between amino acids due to ignoring the contextual information from surrounding amino acids. Here, we first propose the use of a state-of-the-art graph convolutional neural network for predicting the trends of amyloid formation from specific peptide sequences (AMYGNN) by abstracting each polypeptide as a graph, in which the constituting amino acids are viewed as nodes and edges characterizing the connections between pairs of amino acids are established when they meet a given distance threshold (Cα-Cα ≤ 5 Å). Our model achieves high performance with accuracy (0.9208), G-mean (0.9203), MCC (0.8417), and F1 (0.9235) in determining the characteristic peptide sequences to form amyloid. 32 of 534 crucial amino acid properties that greatly contribute to the formation of amyloids are ascertained, and the β-folding-like graph structure of a polypeptide is believed to be essential for the formation of amyloid. Our model enables the mapping of polypeptides with underlying interactions between amino acids and provides a quick and precise predictive framework for directing the construction of amyloid-associated functional materials.

Development of an Enzyme-Linked Immunosorbent Spot Assay for the Assessment of Adeno-Associated Virus Peptides to Examine Immune Safety.

Adeno-associated virus (AAV)-based gene therapies have shown promise as novel treatments for rare genetic disorders such as hemophilia A and spinal muscular atrophy. However, cellular immune responses mediated by cytotoxic (CD8+) and helper (CD4+) T cells may target vector-transduced cells as well as healthy immune cells, impacting safety and efficacy. In this study, we describe the optimization and reproducibility of interferon-γ (IFNγ)-based and interleukin-2 (IL-2)-based enzyme-linked immunosorbent spot (ELISpot) assays for measuring T cell responses against AAV peptide antigens. For method optimization, peripheral blood mononuclear cells (PBMCs) were isolated from healthy human donors and stimulated with commercially available major histocompatibility complex (MHC) class I or II-specific peptides as positive controls. Peptide pools were designed from published AAV8 and AAV9 capsid protein sequences and then used to assess the presence of AAV-specific T cell responses. Our results demonstrate a measurable increase in IFNγ and IL-2-producing cells after AAV peptide presentation. Furthermore, there was an observed difference in the magnitude and specificity of response to peptide pools based on AAV serotype and donor. Finally, using individual peptides, we identified a region of the AAV9 capsid protein that can elicit an immunogenic response. This work shows the applicability of ELISpot in assessing anti-AAV immune responses and provides insight into how novel recombinant AAV vectors could be designed to reduce immunogenic potential.

Design of target specific peptide inhibitors using generative deep learning and molecular dynamics simulations

We introduce a computational approach for the design of target-specific peptides. Our method integrates a Gated Recurrent Unit-based Variational Autoencoder with Rosetta FlexPepDock for peptide sequence generation and binding affinity assessment. Subsequently, molecular dynamics simulations are employed to narrow down the selection of peptides for experimental assays. We apply this computational strategy to design peptide inhibitors that specifically target β-catenin and NF-κB essential modulator. Among the twelve β-catenin inhibitors, six exhibit improved binding affinity compared to the parent peptide. Notably, the best C-terminal peptide binds β-catenin with an IC50 of 0.010 ± 0.06 μM, which is 15-fold better than the parent peptide. For NF-κB essential modulator, two of the four tested peptides display substantially enhanced binding compared to the parent peptide. Collectively, this study underscores the successful integration of deep learning and structure-based modeling and simulation for target specific peptide design.