Short Peptides Research Articles

Squaric esters as peptide stapling reagents

We report that squaric esters can serve as bifunctional reagents for selective peptide stapling reactions. Formation of the squaric amide staple occurs under mild conditions with amine-containing side chains. We show that short resin-bound peptides are readily stapled on solid phase and that stapling can occur at various relative positions along the peptide and with various amine tether lengths (e.g. Lysine, ornithine, etc). The squaric amide staples are stable to strong acid conditions used to cleave the stapled peptide from the resin and the stapled peptides show an increase in helicity as analyzed through circular dichroism.

Research Strategy for Short-Peptide Fusion Inhibitors Based on 6-HB Core Structure against HIV-1: A Review.

Acquired Immune Deficiency Syndrome (AIDS) is a devastating infectious disease caused by the Human Immunodeficiency Virus type 1 (HIV-1). Enfuvirtide(T20) is the first HIV-1 fusion inhibitor for marketing, which plays an important role in AIDS treatment. However, in the clinical application process, T20 has several drawbacks, such as a high level of development of drug resistance, a short half-life in vivo, and rapid renal clearance, which severely limits the clinical application. Therefore, the development of novel fusion inhibitors to address T20 shortcomings has long been the research hotspot. Short peptides have a long half-life through modification and a high barrier to drug resistance, which is expected to solve the current fusion inhibitors dilemma. In this paper, we summarized six emerging R&D strategies for short peptide-based fusion inhibitors against HIV-1. We hope that this review will provide fresh insights into the development of novel fusion inhibitors, as well as ideas for other viral fusion inhibitor discoveries based on the common membrane fusion 6-HB core structure.

Substrate recruitment via eIF2γ enhances catalytic efficiency of a holophosphatase that terminates the integrated stress response

Dephosphorylation of pSer51 of the α subunit of translation initiation factor 2 (eIF2αP) terminates signaling in the integrated stress response (ISR). A trimeric mammalian holophosphatase comprised of a protein phosphatase 1 (PP1) catalytic subunit, the conserved C-terminally located ~70 amino acid core of a substrate-specific regulatory subunit (PPP1R15A/GADD34 or PPP1R15B/CReP) and G-actin (an essential cofactor) efficiently dephosphorylate eIF2αP in vitro. Unlike their viral or invertebrate counterparts, with whom they share the conserved 70 residue core, the mammalian PPP1R15s are large proteins of more than 600 residues. Genetic and cellular observations point to a functional role for regions outside the conserved core of mammalian PPP1R15A in dephosphorylating its natural substrate, the eIF2 trimer. We have combined deep learning technology, all-atom molecular dynamics simulations, X-ray crystallography, and biochemistry to uncover binding of the γ subunit of eIF2 to a short helical peptide repeated four times in the functionally important N terminus of human PPP1R15A that extends past its conserved core. Binding entails insertion of Phe and Trp residues that project from one face of an α-helix formed by the conserved repeats of PPP1R15A into a hydrophobic groove exposed on the surface of eIF2γ in the eIF2 trimer. Replacing these conserved Phe and Trp residues with Ala compromises PPP1R15A function in cells and in vitro. These findings suggest mechanisms by which contacts between a distant subunit of eIF2 and elements of PPP1R15A distant to the holophosphatase active site contribute to dephosphorylation of eIF2αP by the core PPP1R15 holophosphatase and to efficient termination of the ISR in mammals.

Production and immunological characterization of the novel single-chain variable fragment (scFv) antibodies against the epitopes on Opisthorchis viverrini cathepsin F (OvCatF)

BackgroundOpisthorchis viverrini infection is a significant health problem in several countries, especially Southeast Asia. The infection causes acute gastro-hepatic symptoms and also long-term infection leading to carcinogenesis of an aggressive bile duct cancer (cholangiocarcinoma; CCA). Hence, the early diagnosis of O. viverrini infection could be the way out of this situation. Still, stool examination by microscopic-based methods, the current diagnostic procedure is restricted by low parasite egg numbers in the specimen and unprofessional laboratorians. The immunological procedure provides a better chance for diagnosis of the infection. Hence, this study aims to produce single-chain variable fragment (scFv) antibodies for use as a diagnostic tool for O. viverrini infection. MethodsThis study uses phage display technologies to develop the scFv antibodies against O. viverrini cathepsin F (OvCatF). The OvCatF-deduced amino acid sequence was analyzed and predicted for B-cell epitopes used for short peptide synthesis. The synthetic peptides were used to screen the phage library simultaneously with OvCatF recombinant protein (rOvCatF). The potentiated phages were collected, rescued, and reassembled in XL1-blue Escherichia coli (E. coli) as a propagative host. The positive clones of phagemids were isolated, and the single-chain variable (scFv) fragments were sequenced, computationally predicted, and molecular docked. The complete scFv fragments were digested from the phagemid, subcloned into the pOPE101 expression vector, and expressed in XL1-blue E. coli. Indirect ELISA and Western analysis were used to verify the detection efficiency. ResultsThe scFv phages specific to OvCatF were successfully isolated, subcloned, and produced as a recombinant protein. The recombinant scFv antibodies were purified and refolded to make functional scFv. The evaluation of specific recognition of the particular epitopes and detection limit results by both computational and laboratory performances demonstrated that all three recombinant scFv antibodies against OvCatF could bind specifically to rOvCatF, and the lowest detection concentration in this study was only one hundred nanograms. ConclusionOur produced scFv antibodies will be the potential candidates for developing a practical diagnostic procedure for O. viverrini infection in humans in the future.

RmsdXNA: RMSD prediction of nucleic acid-ligand docking poses using machine-learning method.

Small molecule drugs can be used to target nucleic acids (NA) to regulate biological processes. Computational modeling methods, such as molecular docking or scoring functions, are commonly employed to facilitate drug design. However, the accuracy of the scoring function in predicting the closest-to-native docking pose is often suboptimal. To overcome this problem, a machine learning model, RmsdXNA, was developed to predict the root-mean-square-deviation (RMSD) of ligand docking poses in NA complexes. The versatility of RmsdXNA has been demonstrated by its successful application to various complexes involving different types of NA receptors and ligands, including metal complexes and short peptides. The predicted RMSD by RmsdXNA was strongly correlated with the actual RMSD of the docked poses. RmsdXNA also outperformed the rDock scoring function in ranking and identifying closest-to-native docking poses across different structural groups and on the testing dataset. Using experimental validated results conducted on polyadenylated nuclear element for nuclear expression triplex, RmsdXNA demonstrated better screening power for the RNA-small molecule complex compared to rDock. Molecular dynamics simulations were subsequently employed to validate the binding of top-scoring ligand candidates selected by RmsdXNA and rDock on MALAT1. The results showed that RmsdXNA has a higher success rate in identifying promising ligands that can bind well to the receptor. The development of an accurate docking score for a NA-ligand complex can aid in drug discovery and development advancements. The code to use RmsdXNA is available at the GitHub repository https://github.com/laiheng001/RmsdXNA.

Quantification of serine residue stereoinversion in a short peptide by reversed-phase high-performance liquid chromatography: analysis of mechanisms promoting serine stereoinversion.

Stereoinversion of Ser residues within proteins, which has been identified in long-lived proteins, influences protein function. To quantify the stereoinversion of Ser residues, we investigated the potential adaptation of our direct peptide analytical method originally established for analyzing the isomerization of asparaginyl/aspartyl residues. Peptide pairs containing L-Ser or D-Ser residues with lengths of four or five residues were synthesized. Separation conditions for these peptide pairs were systematically examined by precisely adjusting the pH of the elution solvent using reverse-phase high-performance liquid chromatography (HPLC). Optimal separation conditions were successfully developed for all peptide pairs, enabling the direct quantification of Ser residue stereoinversion through a single HPLC run. Subsequently, the degree of Ser stereoinversion within the model peptide, Gly-Ser-Gly-Tyr, was determined using the method established in this study. Surprisingly, the stereoinversion of Ser residues occurred only when the absolute configurations of Ser and Tyr residues of the peptide differed from each other, whereas no stereoinversion was observed when their absolute configurations were identical. The experiments using peptides similar to the model peptide reveal that both the N-terminal amino group and the hydroxyl group of the C-terminal Tyr residue are involved in the stereoinversion of the Ser residue. By applying a simple method to quantify the stereoinversion of Ser residues, valuable insights into the mechanisms governing these stereoinversions were obtained.

Protein Supplements with Short Peptides Are Better than Complex Protein-Based Supplements on Improving Early Fat-Free Mass Loss Following Bariatric Surgery: A Retrospective Cohort Study.

Bariatric surgery (BS) patients are advised to consume protein supplements to prevent fat-free mass (FFM) loss. However, limited research has explored the efficacy of diverse protein presentations on FFM preservation. This study assesses if short peptide-based (SPB) supplements surpass complex protein-based (CPB) supplements in reducing early FFM loss post-surgery. In this retrospective cohort study, 138 patients who underwent BS other than Roux-en-Y-gastric bypass (RYGB) between January 2021 and March 2021 at the Department of Bariatric Surgery of the Third People's Hospital of Chengdu were included for analysis. Patients were divided into two groups based on their consumption of protein supplements after surgery: SPB group and CPB group. Multiple linear regressions separated by sex were employed to examine the associations between SPB supplements and FFM loss and percentage of FFM (%FFM) loss, respectively. Among participants, 69.6% were female, with a mean age of 33.3years. In multiple linear regression analyses, SPB supplements were significantly and positively associated with a lower FFM loss in both female (ꞵ = - 1.14, P = 0.047) and male (ꞵ = - 2.36, P = 0.024), and were positively associated with a lower %FFM loss in both female (ꞵ = - 1.83) and male (ꞵ = - 2.26) but only significant in male (P = 0.049). SPB supplements may be more effective in preventing early FFM loss after BS, compared to CPB supplements, particularly among male patients. Therefore, SPB supplements may be recommended to patients undergoing BS. Further research is needed to validate these findings.

Exploring a Solvent Dependent Strategy to Control Self-Assembling Behavior and Cellular Interaction in Laminin-Mimetic Short Peptide based Supramolecular Hydrogels.

Self-assembled hydrogels, fabricated through diverse non-covalent interactions, have been extensively studied in regenerative medicines. Inspired from bioactive functional motifs of ECM protein, short peptide sequences have shown remarkable abilities to replicate the intrinsic features of the natural extracellular milieu. In this direction, we have fabricated two short hydrophobic bioactive sequences derived from the laminin protein i. e., IKVAV and YIGSR. Based on the substantial hydrophobicity of these peptides, we selected a co-solvent approach as a suitable gelation technique that included different concentrations of DMSO as an organic phase along with an aqueous solution containing 0.1 % TFA. These hydrophobic laminin-based bioactive peptides with limited solubility in aqueous physiological environment showed significantly enhanced solubility with higher DMSO content in water. The enhanced solubility resulted in extensive intermolecular interactions that led to the formation of hydrogels with a higher-order entangled network along with improved mechanical properties. Interestingly, by simply modulating DMSO content, highly tunable gels were accessed in the same gelator domain that displayed differential physicochemical properties. Further, the cellular studies substantiated the potential of these laminin-derived hydrogels in enhancing cell-matrix interactions, thereby reinforcing their applications in tissue engineering.

Investigation of Rare Earth Element Binding to a Surface-Bound Affinity Peptide Derived from EF-Hand Loop I of Lanmodulin.

Bioinspired strategies have been given extensive attention for the recovery of rare earth elements (REEs) from waste streams because of their high selectivity, regeneration potential, and sustainability as well as low cost. Lanmodulin protein is an emerging biotechnology that is highly selective for REE binding. Mimicking lanmodulin with shorter peptides is advantageous because they are simpler and potentially easier to manipulate and optimize. Lanmodulin-derived peptides have been found to bind REEs, but their properties have not been explored when immobilized on solid substrates, which is required for many advanced separation technologies. Here, two peptides, LanM1 and scrambled LanM1, are designed from the EF-hand loop 1 of lanmodulin and investigated for their binding affinity toward different REEs when surface-bound. First, the ability of LanM1 to bind REEs was confirmed and characterized in solution using circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular dynamics (MD) simulations for Ce(III) ions. Isothermal titration calorimetry (ITC) was used to further analyze the binding of the LanM1 to Ce(III), Nd(III), Eu(III), and Y(III) ions and in low-pH conditions. The performance of the immobilized peptides on a model gold surface was examined using a quartz crystal microbalance with dissipation (QCM-D). The studies show that the LanM1 peptide has a stronger REE binding affinity than that of scrambled LanM1 when in solution and when immobilized on a gold surface. QCM-D data were fit to the Langmuir adsorption model to estimate the surface-bound dissociation constant (Kd) of LanM1 with Ce(III) and Nd(III). The results indicate that LanM1 peptides maintain a high affinity for REEs when immobilized, and surface-bound LanM1 has no affinity for potential competitor calcium and copper ions. The utility of surface-bound LanM1 peptides was further demonstrated by immobilizing them to gold nanoparticles (GNPs) and capturing REEs from solution in experiments utilizing an Arsenazo III-based colorimetric dye displacement assay and ultraviolet-visible (UV-vis) spectrophotometry. The saturated adsorption capacity of GNPs was estimated to be around 3.5 μmol REE/g for Ce(III), Nd(III), Eu(III), and Y(III) ions, with no binding of non-REE Ca(II) ions observed.

SAMP-VGG16: Force-field assisted image-based deep neural network prediction model for short antimicrobial peptides.

During the last three decades, antimicrobial peptides (AMPs) have emerged as a promising therapeutic alternative to antibiotics. The approaches for designing AMPs span from experimental trial-and-error methods to synthetic hybrid peptide libraries. To overcome the exceedingly expensive and time-consuming process of designing effective AMPs, many computational and machine-learning tools for AMP prediction have been recently developed. In general, to encode the peptide sequences, featurization relies on approaches based on (a) amino acid (AA) composition, (b) physicochemical properties, (c) sequence similarity, and (d) structural properties. In this work, we present an image-based deep neural network model to predict AMPs, where we are using feature encoding based on Drude polarizable force-field atom types, which can capture the peptide properties more efficiently compared to conventional feature vectors. The proposed prediction model identifies short AMPs (≤30 AA) with promising accuracy and efficiency and can be used as a next-generation screening method for predicting new AMPs. The source code is publicly available at the Figshare server sAMP-VGG16.

Solid-Phase Synthesis and Screening of Functional Peptides and Their Inhibitory Effects in Cancer Cells

The inhibitory peptide library and functional short peptide RLFGKIW were synthesized by solid-phase synthesis and dichotomous method, and the inhibitory effect and cytocompatibility of the peptide library and peptide chain on breast cancer at the cellular level were evaluated by MTT method.

PEDF-derived peptide protects against Amyloid-β toxicity in vitro and prevents retinal dysfunction in rats

Amyloid-beta (Aβ), a family of aggregation-prone and neurotoxic peptides, has been implicated in the pathophysiology of age-related macular degeneration (AMD). We have previously shown that oligomeric and fibrillar species of Aβ42 exerted retinal toxicity in rats, but while the consequences of exposure to amyloid were related to intracellular effects, the mechanism of Aβ42 internalization in the retina is not well characterized. In the brain, the 67 kDa laminin receptor (67LR) participates in Aβ-related neuronal cell death. A short peptide derived from pigment epithelium-derived factor (PEDF), formerly designated PEDF-335, was found to mitigate experimental models of ischemic retinopathy via targeting of 67LR. In the present study, we hypothesized that 67LR mediates the uptake of pathogenic Aβ42 assemblies in the retina, and that targeting of this receptor by PEDF-335 may limit the internalization of Aβ, thereby ameliorating its retinotoxicity. To test this assumption ARPE-19 cells in culture were incubated with PEDF-335 before treatment with fibrillar or oligomeric structures of Aβ42. Immunostaining confirmed that PEDF-335 treatment substantially prevented amyloid internalization into ARPE-19 cells and maintained their viability in the presence of toxic oligomeric and fibrillar Aβ42 entities in vitro. FRET competition assay was performed and confirmed the binding of PEDF-335 to 67LR in RPE-like cells. Wild-type rats were treated with intravitreal PEDF-335 in the experimental eye 2 days prior to administration of retinotoxic Aβ42 oligomers or fibrils to both eyes. Retinal function was assessed by electroretinography through 6 weeks post injection. The ERG responses in rats treated with oligomeric or fibrillar Aβ42 assemblies were near-normal in eyes previously treated with intravitreal PEDF-335, whereas those measured in the control eyes treated with injection of the Aβ42 assemblies alone showed pathologic attenuation of the retinal function through 6 weeks. The retinal presence of 67LR was determined ex vivo by immunostaining and western blotting. Retinal staining demonstrated the constitutional expression of 67LR mainly in the retinal nuclear layers. In the presence of Aβ42, the levels of 67LR were increased, although its retinal distribution remained largely unaltered. In contrast, no apparent differences in the retinal expression level of 67LR were noted following exposure to PEDF-335 alone, and its pattern of localization in the retina remained similarly concentrated primarily in the inner and outer nuclear layers. In summary, we found that PEDF-335 confers protection against Aβ42-mediated retinal toxicity, with significant effects noted in cells as well as in vivo in rats. The effects of PEDF-335 in the retina are potentially mediated via binding to 67LR and by at least partial inhibition of Aβ42 internalization. These results suggest that PEDF-335 may merit further consideration in the development of targeted inhibition of amyloid-related toxicity in the retina. More broadly, our observations provide evidence on the importance of extracellular versus intracellular Aβ42 in the retina and suggest concepts on the molecular mechanism of Aβ retinal pathogenicity.

Abstract 640: In vivo rescue of p53 tumor suppressor function with ADGN-531 across Pan-p53 alterations

Abstract p53 tumor suppressor mutations result in loss of function of p53 in approximately 50% of all human cancers.TP53 missense, nonsense mutations and p53 deletion are present in about 50%, 12% and 5% of all p53 driven cancers, respectively. To date there is no effective treatment for rescuing p53 function in cancers using a pan-p53 approach. The use of ADGN-531 is a new potent strategy combining mRNA with a tumor selective peptide nanocarrier to rescue of p53 tumor suppressor function. ADGN-531 contains proprietary p53-mRNA complexed with proprietary short amphipathic peptides in the form of nanoparticles. ADGN-531 was evaluated on 20 different cancer cell lines harboring various p53 mutations. In-vivo efficacy of IV-administered ADGN-531 nanoparticles was evaluated on colorectal SW403 (p53 nonsense/KRASG12V), osteosarcoma SaOs2 (p53 null) and NSCLC NCI-H358 (p53 null/KRASG12C) mouse xenografts at a dose of 0.25-1.0 mg/kg weekly. ADGN-531/sotorasib synergy was evaluated in vitro on NCI-H358 (p53 null/KRASG12C) and Mia-PACA (p53R48W/KRASG12C) cells and in vivo on NCI-H358 mouse xenograft. ADGN-531 markedly inhibits the growth of a large panel of cancer cells harboring p53-missense or nonsense mutations or p53-deletion, by rescuing p53 transcriptional activity and subsequent expression of its target proteins. ADGN-531 induces cell cycle arrest in G1 due to p21 upregulation and apoptosis following BAX and PUMA activation. IV-administration of ADGN-531 resulted in dose responsive tumor growth inhibition in SaOs2, SW403 and H358 xenografts independent of KRAS status. We demonstrated a strong synergy in a NSCLC H358 xenograft (tumor regression 80%) when restoring P53 function with ADGN-531 (0.5mg/kg, weekly) along with inhibition of KRASG12C mutant using sotorasib (10mg/kg, daily). ADGN-531 potentiated the effect of sotorasib on sotorasib resistant cells harboring high level of KRASG12C in permanent active state. ADGN-531 treatments are well tolerated, no sign of clinical toxicity was detected after single or repeated administrations. ADGN-531 is effective in rescuing p53 function both in vitro and in vivo across a wide range of p53 alterations. Our study provides a proof-of-concept that restoration of tumor suppressor function by ADGN-531 could be used as a single agent therapy in P53 altered tumors independent of other driver mutations e.g., KRAS, or with other therapies for potent combinatorial cancer treatment. Citation Format: Gilles Divita, Audrey Grunenberger, Elodie Czuba, Veronique Josserand, Melanie Guidetti, Neil Desai. In vivo rescue of p53 tumor suppressor function with ADGN-531 across Pan-p53 alterations [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 640.

Abstract 7237: Peptide-mRNA complex mediated restoration of BRCA-1 tumor suppressor function in BRCA-1 mutated cancers as a new therapeutic strategy

Abstract BRCA1 and BRCA-2 proteins are involved in the repair of DNA damage such as double-strand breaks during S and G2 phases, by intervening in homologous recombination (HR) steps. Breast cancers arising from BRCA-1 mutation carriers (10% of BC) and from epigenetically silenced BRCA-1 (30% of sporadic BC) are associated with a lack of expression and/or function of BRCA-1, which induces genomic instability. Herein, we describe a new potent strategy combining mRNAs with a tumor selective peptide nanocarrier, to selectively rescue BRCA-1 function as potential therapeutic approach in cancers. Proprietary BRCA-1 derived mRNA were complexed with proprietary short amphipathic peptides and the resulting nanoparticles were evaluated on several breast cancer cells, harboring different BRCA-1 alterations. Sensitivity to Veliparib (PARPi) following the nanoparticle treatments were evaluated on PARPI resistant and PARPi sensitive cells. The recruitment of RAD51 to sites of double stranded DNA break was quantified by immunofluorescence. In-vivo efficacy of varying sequences of BRCA-1 mRNA-peptide nanoparticles (2.0 mg/kg, weekly, IV; ADGN-901, -902, -903) and veliparib (50mg/kg, daily, oral) were evaluated on breast cancer (HCC1937-BRCA-1 deficient, PARPi resistant) mouse xenografts. We showed that ADGN-901 inhibits the proliferation of BRCA-1 deficient cell lines from 20 to 60% depending on the type of BRCA-1 alterations. ADGN-902, specifically reduces the proliferation of cells expressing N-terminal truncated BRCA-1, by 50 to 60% and ADGN-903 inhibits proliferation >60% of all cells harboring defects in BRCA-1. Interestingly, ADGN-901 and ADGN-903 were able to resensitize the PARPi resistant cells to veliparib inhibition. We demonstrated that ADGN-903 restores BRCA-1 function in homologous recombination. ADGN-903 prevents RAD51 recruitment to the damage site by reducing RAD51 protein level as well as formation of RAD51 foci following irradiation. IV administration of ADGN-901 and ADGN-903 (2.0 mg/kg) had a strong inhibitory effect on HCC-1937 tumor growth while ADGN-902 and veliparib were inactive. ADGN-901 reduced tumor growth by 95% and ADGN-903 abolished tumor growth with strong tumor regressions. ADGN-901 and ADGN-903, were also evaluated on large tumors of about 500-600 mm3 and were highly effective in preventing further progression and achieving shrinkage of large tumors. ADGN-901 and ADGN-903 treatments are well tolerated, without inducing clinical toxicity. BRCA-1 mRNA-peptide nanoparticles are effective in rescuing BRCA-1 function and inducing tumor regressions in BRCA-1 altered breast cancer cells and tumors. Our study provides a proof-of-concept that restoration of wild type BRCA-1 expression could be a potent therapeutic strategy for BRCA-1 altered tumors whether or not they are PARPi resistant. Citation Format: Gilles Divita, Audrey Grunenberger, Léa Cabrera, Nathalie Durany, Neil Desai. Peptide-mRNA complex mediated restoration of BRCA-1 tumor suppressor function in BRCA-1 mutated cancers as a new therapeutic strategy [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 7237.

Abstract 3761: A modified SELEX enables the efficient identification of DNA aptamers with potential for specific cancer targeting

Abstract Background: Aptamers have sparked significant interest in cell recognition due to their superior binding specificity and biocompatibility. Cell recognition can be mediated by targeting the major histocompatibility complex (MHC) that presents short peptides derived from intracellular antigens. Although numerous antibodies have demonstrated a specific affinity for the peptide-MHC complex, the number of aptamers that exhibit comparable characteristics is limited. However, the success rate of aptamer identification is low. This is possibly due to the presence of complementary sequences or sequences rich in quinine and cytosine that are less accessible for primers. Here, we use ovalbumin model antigen to explore whether aptamers with specificity for binding to a defined peptide-MHC complex can be identified. We developed DNA aptamers with modified SELEX by employing systemic consecutive selections with minimal PCR amplification. Furthermore, the other tumour antigen, claudin-6, was also used as a target to determine whether our modification for SELEX can be used with any selection technology. Methods: To determine whether our modified SELEX has potential for selecting aptamer capable of cancer-specific targeting, we used ovalbumin presented by H-2Kb (to represent the specific antigen presented by MHC) and claudin-6 (a cancer-associated adhesion molecule) as a target for the selection of aptamers. This modified SELEX consisted of consecutive positive and negative selections with minimal PCR amplification. Meanwhile, the selection of candidate aptamers is based on their appearance in multiple rounds rather than enrichment, and is facilitated by several computational tools. The specificity and affinity of the candidate aptamer are validated by flow cytometry, immunohistochemistry, and immunofluorescence analysis with cells with or without these molecules. Results: After high-throughput sequencing, a total of 211 candidate aptamers were selected for further analysis. The quadruplex forming G-rich sequences (QGRS) mapper, mFold modeling, and NUPACK web servers were used to predict the potential of G-quadruplex formation, resulting in only four potential aptamers. Using MTCQ1-OVA cells with FITC-conjugated aptamers, two aptamers showed selective binding to MTCQ1-OVA cells and had no binding specificity to MTCQ1-WT cells. The binding specificity was further validated by immunohistochemistry in tumour derived from mice inoculated with MTCQ1-WT or MTCQ1-OVA cells. In the pull-down assay, the binding affinity of these two candidate aptamers was 136.2 nM and 155.1 nM affinity. Conclusions: This preliminary study demonstrated that our modification for SELEX is effective in the selection of aptamers with promising potential for specific cancer targeting. The identification of aptamers may facilitate further application in diverse biomedical fields. Citation Format: Yang Lin, Cho-Yi Chen, Zhi-Qian Lin, Bing-Hong Chen, Yi-Chen Sun, Kai-Feng Hung. A modified SELEX enables the efficient identification of DNA aptamers with potential for specific cancer targeting [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 3761.

Abstract 3741: Helper/killer hybrid epitope long peptide (H/K-HELP) cancer vaccine augments Th1-dependent CTL induction at tumor-draining lymph node to eradicate solid tumor: its application to develop an innovative combination cancer immunotherapy(iCCI)

Abstract We have initially demonstrated the pivotal role of IFN-γ-producing tumor-antigen-specific Th1 cells for the complete eradication of established tumor mass by CTL. The accumulated evidence has clearly demonstrated that induction of Th1-dependent full activation of CTL at tumor-draining lymph nodes (TDLN) are crucial for inducing complete rejection of solid tumor by CTL. To induce Th1 immunity efficiently at TDLN in tumor-bearing host, we developed an artificially synthesized helper/killer-hybrid epitope long peptide (H/K-HELP), which conjugated killer and helper epitope peptides of cancer antigen by a glycine-linker. In mouse model, OVA-H/K-HELP was selectively presented in vivo by professional DC in vaccinated TDLN and sustained antigen presentation capability of DC to stimulate both CTL and Th1 cells. Thus, in contrast to short peptide, vaccination of OVA-expressing EG7-bearing mice with OVA-H/K-HELP caused a complete eradication of EG7 tumor mass. To apply these basic findings to clinical treatment of cancer patients, we then developed Survivin-H/K-HELP. Treatment of a patient with a triple-negative breast cancer patient with Survivin-H/K-HELP induced a complete response in a triple-negative breast cancer patient concomitantly with the induction of Th1-dependent cellular and humoral immune responses. In this clinical study, the patients showed Th1-dependent C’-fixing IgG antibody production and exhibited IFN-γ-producing Th1 cellular responses. Finally, we also applied H/K-HELP-pulsing DC-vaccine to develop an innovative combination cancer immunotherapy (iCCI). The iCCI consisting of radiation, low dose of immune check point inhibitors (ICIs:Nibolumab, Ipilimumab) and H/K-HELP-pulsed DC, all of which have been shown to initially modulate antitumor immunity at TDLN. Here, we described a successful outcome of a case of the stage IVA inoperable advanced hypopharyngeal squamous cell cancer patient with lymph node-metastasis. The iCCI treatment of the patient caused a complete disappearance of all cancers and the patient has been cancer free for over 15 months so far. Although the destruction mechanism of cancer is not determined, we image this iCCI might improve patient’s antitumor immune capability in immune microenvironment around tumor (IMAT) including TDLN and TME. Our developed iCCI will become a promising strategy to overcome inoperable advanced cancers in future. Citation Format: Takanori Iwasaki, Yuichiro Yazaki, Takashi Masuko, Takashi Nishimura. Helper/killer hybrid epitope long peptide (H/K-HELP) cancer vaccine augments Th1-dependent CTL induction at tumor-draining lymph node to eradicate solid tumor: its application to develop an innovative combination cancer immunotherapy(iCCI) [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 3741.

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.

Specifically targeting antimicrobial peptides for inhibition of Candidatus Liberibacter asiaticus.

Huanglongbing (citrus greening) is a plant disease putatively caused by the unculturable Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas), and it has caused severe damage to citrus plantations worldwide. There are no definitive treatments for this disease, and conventional disease control techniques have shown limited efficacy. This work presents an in silico evaluation of using specifically targeting anti-microbial peptides (STAMPs) consisting of a targeting segment and an antimicrobial segment to inhibit citrus greening by inhibiting the BamA protein of CLas, which is an outer membrane protein crucial for bacterial viability. Initially, a set of peptides with a high affinity toward BamA protein were screened and evaluated via molecular docking and molecular dynamics simulations and were verified in vitro via bio-layer interferometry (BLI). In silico studies and BLI experiments indicated that two peptides, HASP2 and HASP3, showed stable binding to BamA. Protein structures for STAMPs were created by fusing known anti-microbial peptides (AMPs) with the selected short peptides. The binding of STAMPs to BamA was assessed using molecular docking and binding energy calculations. The attachment of high-affinity short peptides significantly reduced the free energy of binding for AMPs, suggesting that it would make it easier for the STAMPs to bind to BamA. Efficacy testing in vitro using a closely related CLas surrogate bacterium showed that STAMPs had greater inhibitory activity than AMP alone. In silico and in vitro results indicate that the STAMPs can inhibit CLas surrogate Rhizobium grahamii more effectively compared to AMPs, suggesting that STAMPs can achieve better inhibition of CLas, potentially via enhancing the site specificity of AMPs.

CLLU1 as an emerging biomarker in chronic lymphoid leukemia.

CLLU1, a disease-specific gene associated with chronic lymphoid leukemia (CLL), is located on chromosome 12q22. Previous studies considered CLLU1 to be a non-coding RNA; however, recent research has discovered that its coding sequence region possesses the potential to encode a short peptide similar to interleukin-4. Remarkably, abnormally elevated expression of CLLU1 has only been detected in chronic lymphoid leukemia among all hematological cancers. High CLLU1 expression often indicates more malignant pathological features and an unfavorable prognosis for patients. Importantly, the expression level of CLLU1 remains unaffected by the passage of time or therapeutic interventions, thus rendering it a novel prognostic marker. This article provides a comprehensive summary of relevant research findings on CLLU1 in the context of CLL prognosis and clinical applications, aiming to guide subsequent theoretical and clinical investigations in this field.

The effect of Lactococcus lactis CICC 23610 fermentation on the physicochemical properties and flavor formation of oat beverage

AbstractBackground and ObjectivesProbiotic fermentation may contribute to enhancing the flavor and nutrients of grain beverages. The aim of this study was to investigate the effect of Lactococcus lactis (L. lactis CICC 23610) fermentation on the physicochemical properties, flavor formation, and metabolomics of oat beverages.FindingsResults showed that L. lactis CICC 23610 grew rapidly in oat substrate and brought a surge in acidity, apparent viscosity, and β‐glucan content. The final amount of lactic acid, sweet amino acids, and umami amino acids metabolites was also increased. Meanwhile, 15 volatile flavor components (VFCs) were identified as key aroma compounds in fermented oat beverages. The levels of 2,3‐butanedione, propionic acid, hexanoic acid, and heptanone rose, giving the beverage a tart, creamy, and sweet flavor, and the undesirable odors of hexanal, n‐octanal, nonanal, and 2‐pentylfuran were reduced. In addition, significant differences in metabolites based on untargeted metabolomics analyses of oat beverages before and after fermentation were mainly focused on organic acids, fatty acids, and short peptides. The sensory analysis showed that the overall acceptability of fermented oat beverages was significantly improved.ConclusionsIt could be concluded that the effects of free amino acids (FAAs), organic acids, and VFCs on flavor were mainly reflected in three indicators of odor, acidity, and umami.Significance and NoveltyThe encouraging results confirmed that probiotic fermentation is a promising method to enhance the overall quality of oat beverages, providing valuable insights into future industrial upgrades of grain beverages.