Mutant Peptides Research Articles

Membrane alteration, anti-virulence properties and metabolomic perturbation of a chionodracine-derived antimicrobial peptide, KHS-Cnd, on two bacteria models

Antarctic fishes, living in an extreme environment and normally exposed to pathogens, are a promising source of antimicrobial peptides (AMPs). These are emerging as next-generation drugs due to their activity against multidrug resistant (MDR) bacteria. To infect hosts, beyond intrinsic/acquired resistance, MDR species also use virulence factors such as protease secretion. Hence, AMPs targeting virulence factors could represent a novel strategy to counteract the antimicrobial resistance (AMR). In this paper, we focused on a mutant peptide, named KHS-Cnd, that was obtained from the scaffold of the chionodracine (Cnd), a natural peptide identified in the icefish Chionodraco hamatus. We studied different effects caused by the peptide interaction with the cell membrane of two model bacteria, E. coli and B. cereus. First, we investigated its membranolytic activity revealing that the peptide action is more evident on E. coli, with a 69 % uptake of the used dye at 3 μM, whereas for B. cereus we found only a 65 % uptake at 6 μM. Successively, we determined the impact of this lysis on total protein concentration in the medium and an increase was estimated for both bacteria (84 % after 1 h for E. coli and 90 % for B. cereus, respectively). Moreover, we evaluated the changes in the proteolytic activity of the supernatant, that is an important aspect of bacterial resistance, showing that there was a significant reduction for both bacteria, although at higher level in the case of E. coli. The membranolytic activity was evidenced also morphologically with TEM analysis and a different alteration was evidenced for the two bacteria. Moreover, NMR metabolomics analysis showed that peptide induces changes in E. coli and B. cereus extracellular metabolites especially at the higher tested concentrations: this metabolic variation could be used as a fingerprinting of the peptide action on bacteria physiology due to its interaction with cell wall. Finally, we determined the KHS-Cnd cytotoxicity on human primary cell lines to verify its selectivity toward bacterial cell membranes and we found low toxicity until a concentration of 5 μM. Considering that the peptide exerts both membranolytic and anti-virulence activity on E. coli at 1.5 μM, we confirmed the interesting potential of this AMP as a new drug to counteract AMR.

NeoaPred: a deep-learning framework for predicting immunogenic neoantigen based on surface and structural features of peptide-human leukocyte antigen complexes.

Neoantigens, derived from somatic mutations in cancer cells, can elicit anti-tumor immune responses when presented to autologous T cells by human leukocyte antigen. Identifying immunogenic neoantigens is crucial for cancer immunotherapy development. However, the accuracy of current bioinformatic methods remains unsatisfactory. Surface and structural features of peptide-HLA class I (pHLA-I) complexes offer valuable insight into the immunogenicity of neoantigens. We present NeoaPred, a deep-learning framework for neoantigen prediction. NeoaPred accurately constructs pHLA-I complex structures, with 82.37% of the predicted structures showing an RMSD of < 1 Å. Using these structures, NeoaPred integrates differences in surface, structural, and atom group features between the mutant peptide and its wild-type counterpart to predict a foreignness score. This foreignness score is an effective factor for neoantigen prediction, achieving an AUROC (Area Under the Receiver Operating Characteristic Curve) of 0.81 and an AUPRC (Area Under the Precision-Recall Curve) of 0.54 in the test set, outperforming existing methods. The source code is released under an Apache v2.0 license and is available at the GitHub repository (https://github.com/Dulab2020/NeoaPred).

Redox-Active Conopeptide Li520 Has Evolved to Catalyze Oxidative Folding of Conotoxins.

The evolution of miniature conopeptide Li520 (COWC*, *: C-terminal amidation) to exhibit the disulfide isomerase activity was probed using structure, function, disulfide conformation, and the precursor gene sequence. The peptides Li520, Li504, [O2A]Li520, [W3A]Li520, and Grx506, homologues active-site motif of glutaredoxin, were chemically synthesized and assessed for their disulfide reduction potential, intrinsic folding of disulfides, and disulfide isomerization activity on α-conotoxin ImI. The reduction potential of the disulfide of peptides varies from -189 to -344 mV, which is within the range observed for the redox family of proteins that modulates the folding of protein disulfides. The oxidative folding studies confirm the significance of the tryptophan residue in engaging Li520 in disulfide-exchange reactions and the role of proline hydroxylation in extending the lifetime of Li520 in a reduced free thiol state. Studies of quenching of tryptophan fluorescence by the disulfide in situ folding reaction in conjunction with the optimized structures by density functional theory (DFT) confirm the difference in conformation of disulfides between the native and mutant peptides. Interestingly, the native peptide Li520/Li504 shares a similar disulfide conformation of (-,-)AntiRHHook with the redox family of proteins known to modulate disulfides, particularly in lieu of the tetrapeptide of glutaredoxin, deviating from its disulfide conformation compared to its naive protein. Analysis of the precursor gene sequences of M-superfamily conotoxins revealed the presence of Li520 in different cone snail species with distinct food habits and possible modes of evolution through the diversification of cysteine motifs. The results of the report suggest that the short redox conopeptide Li520 has evolved to facilitate the oxidative folding of conotoxins and may be useful to develop as reagents for the synthesis of therapeutically important cysteine-rich peptides.

Key charged residues influence the amyloidogenic propensity of the helix-1 region of serum amyloid A

Increased plasma levels of serum amyloid A (SAA), an acute-phase protein that is secreted in response to inflammation, may lead to the accumulation of amyloid in various organs thereby obstructing their functions. Severe cases can lead to a systemic disorder called AA amyloidosis. Previous studies suggest that the N-terminal helix is the most amyloidogenic region of SAA. Moreover, computational studies implicated a significant role for Arg-1 and the residue-specific interactions formed during the fibrillization process. With a focus on the N-terminal region of helix-1, SAA1–13, mutational analysis was employed to interrogate the roles of the amino acid residues, Arg-1, Ser-5, Glu-9, and Asp-12. The truncated SAA1–13 fragment was systematically modified by substituting the key residues with alanine or uncharged but structurally similar amino acids. We monitored the changes in the amyloidogenic propensities, associated conformational markers, and morphology of the amyloids resulting from the mutation of SAA1–13. Mutating out Arg-1 resulted in much reduced aggregation propensity and a lack of detectable β-structures alluding to the importance of salt-bridge interactions involving Arg-1. Our data revealed that by systematically mutating the key amino acid residues, we can modulate the amyloidogenic propensity and alter the time-dependent conformational variation of the peptide. When the behaviors of each mutant peptide were analyzed, they provided evidence consistent with the aggregation pathway predicted by MD simulation studies. Here, we detail the important temporal molecular interactions formed by Arg-1 with Ser-5, Glu-9, and Asp-12 and discuss its mechanistic implications on the self-assembly of the helix-1 region of SAA.

Adoptive transfer of personalized neoantigen-reactive TCR-transduced T cells in metastatic colorectal cancer: phase 2 trial interim results.

Adoptive cell transfer (ACT) with neoantigen-reactive T lymphocytes can mediate cancer regression. Here we isolated unique, personalized, neoantigen-reactive T cell receptors (TCRs) from tumor-infiltrating lymphocytes of patients with metastatic gastrointestinal cancers and incorporated the TCR α and β chains into gamma retroviral vectors. We transduced autologous peripheral blood lymphocytes and adoptively transferred these cells into patients after lymphodepleting chemotherapy. In a phase 2 single-arm study, we treated seven patients with metastatic, mismatch repair-proficient colorectal cancers who had progressive disease following multiple previous therapies. The primary end point of the study was the objective response rate as measured using RECIST 1.1, and the secondary end points were safety and tolerability. There was no prespecified interim analysis defined in this study. Three patients had objective clinical responses by RECIST criteria including regressions of metastases to the liver, lungs and lymph nodes lasting 4 to 7 months. All patients received T cell populations containing ≥50% TCR-transduced cells, and all T cell populations were polyfunctional in that they secreted IFNγ, GM-CSF, IL-2 and granzyme B specifically in response to mutant peptides compared with wild-type counterparts. TCR-transduced cells were detected in the peripheral blood of five patients, including the three responders, at levels ≥10% of CD3+ cells 1 month post-ACT. In one patient who responded to therapy, ~20% of CD3+ peripheral blood lymphocytes expressed transduced TCRs more than 2 years after treatment. This study provides early results suggesting that ACT with T cells genetically modified to express personalized neoantigen-reactive TCRs can be tolerated and can mediate tumor regression in patients with metastatic colorectal cancers. ClinicalTrials.gov registration: NCT03412877 .

Pan-cancer proteogenomics expands the landscape of therapeutic targets

Fewer than 200 proteins are targeted by cancer drugs approved by the Food and Drug Administration (FDA). We integrate Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteogenomics data from 1,043 patients across 10 cancer types with additional public datasets to identify potential therapeutic targets. Pan-cancer analysis of 2,863 druggable proteins reveals a wide abundance range and identifies biological factors that affect mRNA-protein correlation. Integration of proteomic data from tumors and genetic screen data from cell lines identifies protein overexpression- or hyperactivation-driven druggable dependencies, enabling accurate predictions of effective drug targets. Proteogenomic identification of synthetic lethality provides a strategy to target tumor suppressor gene loss. Combining proteogenomic analysis and MHC binding prediction prioritizes mutant KRAS peptides as promising public neoantigens. Computational identification of shared tumor-associated antigens followed by experimental confirmation nominates peptides as immunotherapy targets. These analyses, summarized at https://targets.linkedomics.org, form a comprehensive landscape of protein and peptide targets for companion diagnostics, drug repurposing, and therapy development.

The Characterization of Multifaceted PREP1 Peptides Provides Insights into Correlations between Spectroscopic and Structural Properties of Amyloid-like Assemblies.

The widespread ability of proteins and peptides to self-assemble by forming cross-β structure is one of the most significant discoveries in structural biology. Intriguingly, the cross-β association of proteins/peptides may generate intricate supramolecular architectures with uncommon spectroscopic properties. We have recently characterized self-assembled peptides extracted from the PREP1 protein that are endowed with interesting structural/spectroscopic properties. We here demonstrate that the green fluorescence emission of the peptide PREP1[117-132] (λem ~520 nm), can be induced by excitation with UV radiation. The associated unusually large Stokes shift (Δλ ~150 nm) represents, to the best of our knowledge, the first evidence of an internal resonance energy transfer in amyloid-like structures, where the blue emission of some assemblies becomes the excitation radiation for others. Moreover, the characterization of PREP1[117-132] variants provides insights into the sequence/structure and structure/spectroscopic properties relationships. Our data suggests that the green fluorescence is plausibly associated with antiparallel β-sheet states of the peptide whereas parallel β-sheet assemblies are only endowed with blue fluorescence. Notably, the different PREP1[117-132] variants also form assemblies characterized by distinct morphologies. Indeed, the parent peptide and single mutants form compact but structured aggregates whereas most of the double mutants exhibit elongated and highly extended fibers.

Structure-guided design and cloning of peptide inhibitors targeting CDK9/cyclin T1 protein-protein interaction.

CDK9 (cyclin-dependent kinase 9) plays a significant role in numerous pathological conditions, such as HIV-1 infection and cancer. The interaction between CDK9 and cyclin T1 is crucial for maintaining the kinase's active state. Therefore, targeting this protein-protein interaction offers a promising strategy for inhibiting CDK9. In this study, we aimed to design and characterize a library of mutant peptides based on the binding region of cyclin T1 to CDK9. Using Osprey software, a total of 7,776 mutant peptides were generated. After conducting a comprehensive analysis, three peptides, namely, mp3 (RAADVEGQRKRRE), mp20 (RAATVEGQRKRRE), and mp29 (RAADVEGQDKRRE), were identified as promising inhibitors that possess the ability to bind to CDK9 with high affinity and exhibit low free binding energy. These peptides exhibited favorable safety profiles and displayed promising dynamic behaviors. Notably, our findings revealed that the mp3 and mp29 peptides interacted with a conserved sequence in CDK9 (residues 60-66). In addition, by designing the structure of potential peptides in the plasmid vector pET28a (+), we have been able to pave the way for facilitating the process of their recombinant production in an Escherichia coli expression system in future studies. Predictions indicated good solubility upon overexpression, further supporting their potential for downstream applications. While these results demonstrate the promise of the designed peptides as blockers of CDK9 with high affinity, additional experimental studies are required to validate their biological activity and assess their selectivity. Such investigations will provide valuable insights into their therapeutic potential and pave the way for the future development of peptide-based inhibitors targeting the CDK9-cyclin T1 complex.

Isotope labeled 3D-Raman confocal imaging and atomic force microscopy study on epithelial cells interacting with the fungus Candida albicans

The human pathogenic fungus Candida albicans damages epithelial cells during superficial infections. Here we use three-dimensional-sequential-confocal Raman spectroscopic imaging and atomic force microscopy to investigate the interaction of C. albicans wild type cells, the secreted C. albicans peptide toxin candidalysin and mutant cells lacking candidalysin with epithelial cells. The candidalysin is responsible for epithelial cell damage and exhibits in its deuterated form an identifiable Raman signal in a frequency region distinct from the cellular frequency region. Vibration modes at 2100–2200 cm−1 attributed to carbon‑deuterium bending and at 477 cm−1, attributed to the nitrogen‑deuterium out-of-plane bending, found around the nucleus, can be assigned to deuterated candidalysin. Atomic force microscopy visualized 100 nm deep lesions on the cell and force-distance curves indicate the higher adhesion on pore surrounding after incubation with candidalysin. Candidalysin targets the plasma membrane, but is also found inside of the cytosol of epithelial cells during C. albicans infection.

Cellugyrin (synaptogyrin-2) dependent pathways are used by bacterial cytolethal distending toxin and SARS-CoV-2 virus to gain cell entry.

Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is capable of intoxicating lymphocytes macrophages, mast cells and epithelial cells. Following Cdt binding to cholesterol, in the region of membrane lipid rafts, the CdtB and CdtC subunits are internalized and traffic to intracellular compartments. These events are dependent upon, cellugyrin, a critical component of synaptic like microvesicles (SLMVCg+). Target cells, such as Jurkat cells, rendered unable to express cellugyrin are resistant to Cdt-induced toxicity. Similar to Cdt, SARS-CoV-2 entry into host cells is initiated by binding to cell surface receptors, ACE-2, also associated with cholesterol-rich lipid rafts; this association leads to fusion and/or endocytosis of viral and host cell membranes and intracellular trafficking. The similarity in internalization pathways for both Cdt and SARS-CoV-2 led us to consider the possibility that cellugyrin was a critical component in both processes. Cellugyrin deficient Calu-3 cells (Calu-3Cg-) were prepared using Lentiviral particles containing shRNA; these cells were resistant to infection by VSV/SARS-CoV-2-spike pseudotype virus and partially resistant to VSV/VSV-G pseudotype virus. Synthetic peptides representing various regions of the cellugyrin protein were prepared and assessed for their ability to bind to Cdt subunits using surface plasmon resonance. Cdt was capable of binding to a region designated the middle outer loop (MOL) which corresponds to a region extending into the cytoplasmic surface of the SLMVCg+. SARS-CoV-2 spike proteins were assessed for their ability to bind to cellugyrin peptides; SARS-CoV-2 full length spike protein preferentially binds to a region within the SLMVCg+ lumen, designated intraluminal loop 1A. SARS-CoV-2-spike protein domain S1, which contains the receptor binding domains, binds to cellugyrin N-terminus which extends out from the cytoplasmic surface of SLMV. Binding specificity was further analyzed using cellugyrin scrambled peptide mutants. We propose that SLMVCg+ represent a component of a common pathway that facilitates pathogen and/or pathogen-derived toxins to gain host cell entry.

Abstract 860: Pan-tissue master regulator inference reveals mechanisms of MHC alterations in cancers

Abstract Introduction: Major histocompatibility complex (MHC) genes are an integral part of the adaptive immune system. The T-cells can detect and kill cancer cells through the recognition of mutant peptides on the cell surface via the MHC. As a result, many cancers evade the immune system by altering their MHC regulation which can also lead to resistance to immune checkpoint blockade (ICB). Therefore, understanding MHC regulation in both baseline (normal state) and cancer is critical for uncovering mechanisms behind MHC alterations. Here, we have undertaken a pan-tissue evaluation to comprehensively decode MHC transcriptional regulation in healthy tissues and various cancers using a large compendium of single-cell RNA-seq (scRNA-seq) datasets. Methods: To study MHC class I (MHC-I) and MHC class II (MHC-II) transcriptional regulation at baseline we leveraged a large-scale scRNA-seq dataset encompassing 18 tissues and 124 epithelial cell types. Upon re-clustering and annotation, we investigated MHC expression across cell types and tissues and quantified its heterogeneity using the Gini coefficient. Next, we sought to identify transcription factors (TF) predictive of MHC-I and MHC-II expression, for that, we employed a master regulator inference that utilizes uni- and multi- regression models. Further, to investigate MHC and nominated TFs dysregulation in cancer, we curated a compendium of scRNA-seq datasets comprising 43 datasets, 20 cancer types, across 538 patients. Results: In normal epithelial cell types, we observed a large range (~8 fold) in MHC-I expression. We also detected MHC-II expression in unexpected cell types such as club cells in the prostate. We found that lung epithelial cells showed elevated MHC-II expression. Immunohistochemistry was performed to validate these findings. Using master regulator inference, we nominated 104 and 107 TFs which predict MHC-I and MHC-II expression, respectively. The nominated TFs showed significant enrichment (p &amp;lt; 0.001) for known TFs, alongside putative novel transcriptional regulators such as YBX1 (MHC-I p &amp;lt; 2e-16, MHC-II p = 8e-07) and XBP1 (MHC-I p &amp;lt; 2e-16, MHC-II p = 7e-04). Using our single-cell compendium of 20 cancer types we also observed a pan-cancer repression in MHC genes (p &amp;lt; 0.001). Next, we utilized an ICB cohort to identify specific TFs that could predict therapeutic benefits. Interestingly, XBP1 (p &amp;lt; 1e-16) and SP100 (p &amp;lt; 2.2e-08) were strong predictors of patients' survival. We also observed a large heterogeneity in MHC-II expression among colon cancer patients. Upon investigation, we found that patients with high MHC-II expression were mismatch repair deficient, this implicates MHC-II upregulation as an immune evasion mechanism. Conclusion: In conclusion, we have comprehensively evaluated MHC expression in 124 epithelial cell types and across 20 cancers identifying TFs predictive of MHC-I and MHC-II expression and their effects on patient outcomes. Citation Format: Mahnoor N. Gondal, Rahul Mannan, Yi Bao, Jing Hu, Marcin Cieslik, Arul M. Chinnaiyan. Pan-tissue master regulator inference reveals mechanisms of MHC alterations in 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 860.

Abstract 3855: Kinetics of neoantigen-specific T cells in post-transplant relapse

Abstract Introduction: Graft-versus-leukemia effect (GVL) underpins the curative mechanism of allogeneic stem cell transplantation (alloSCT) for acute leukemias. Donor-derived T cells can recognize various antigens, including human leukocyte antigen (HLA), minor-histocompatibility antigens (miHA), over-expressed antigens, or neoantigens derived from somatic mutations of leukemia. However, the role of neoantigens has not been fully investigated in GVL. Here, we aimed to predict neoantigens and identify neoantigen-reactive T cells using samples after alloSCT. Materials: Leukemia blasts were purified from relapsed samples. Recipient saliva or T cells from pre-transplant peripheral blood (PB) were used as a recipient germline control, while donor monocyte or CD34 cells were used as donor controls. DNA/RNA isolated from these specimens were analyzed for whole exome sequencing (WES) paired with RNAseq. Somatic mutations were called using blasts and recipient germline controls by mutect and mpileup and annotation with SnpEff. Somatic mutations with variant allele frequency &amp;gt;20% were screened for neoantigen prediction bound to recipient HLA (IC50 &amp;lt; 500nM) by pVAC-seq. We also genotyped 73 SNPs associated with publicly known miHAs. Serial PB were stained for HLA-A*02:01 tetramers loaded with neoantigens or CMVpp65. T cells were stimulated with neoantigens for 10 days and tested for cytokine production in responses to target or control peptides. Results: We screened neoantigens in 8 patients relapsing after alloSCT (7 acute myeloid leukemia and 1 acute lymphoblastic leukemia) and their donors (4 matched siblings, 1 matched unrelated, 3 haploidentical). In 7 subjects, WES/RNAseq showed an average of 273 somatic mutations per patient (range 108- 609). In one subject, targeted next-generation sequencing was used to detect 2 somatic mutations. Among these mutations, seven out of eight subjects (87.5%) had potential neoantigen candidates, with a median number of 9 antigens per subject. In contrast, only 2 subjects (25%) had potential miHA. UPN8, a responder to donor lymphocyte infusion (DLI), showed dominant CMVpp65-specific T cells at the time of relapse and emergence of TP53- (neoantigen) or HA-2- (miHA) specific T cells after relapse. HA-2-specific T cells upregulated PD-1 expression at the time of graft-versus-host disease (GVHD), indicating activation of these miHA-specific T cells during GVHD. In two subjects (UPN3 and UPN8) who achieved long-term remission after DLI, CD8 T cells specific to neoantigens were detectable by IFN-γ/CD107a production in reaction to mutant peptides (SDE2.pK123X or TP53.pR248W) from post-relapse samples. Discussion: Our study showed the feasibility of in-silico neoantigens prediction and neoantigen-specific T-cell detection from a subject who developed relapse after alloSCT. Further investigation is needed to test if neoepitope burden and miHA disparity could correlate with transplant outcomes. Citation Format: Biswas Neupane, Jui-En Ray Lee, Kedwin Ventura, Kayla Parr, Kevin Quann, Sawa Ito. Kinetics of neoantigen-specific T cells in post-transplant relapse [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 3855.

Abstract 3986: Exploring nonsense-mediated decay inhibition as a novel approach to improve immunogenicity in small cell lung cancer

Abstract Cancer immunotherapy has revolutionized the field of oncology and is one of the most promising treatments for lung cancer. Small cell lung cancer (SCLC) is highly aggressive and patients often relapse due to chemotherapy resistance. In the recent years, chemotherapy has been combined with immune checkpoint inhibition (ICI) for SCLC treatment, leading to improved responses in some patients, but also leaving a high number of non-responders to address. Paradoxically, SCLC has one of the highest tumor mutational burden, including remarkably abundant frameshifts (FS) which could create highly immunogenic neoantigens (NeoAg). However, FS-harbouring transcripts are often degraded via Nonsense-Mediated Decay (NMD). We therefore aimed to inhibit the NMD pathway to increase the immunogenicity of SCLC. Our lab has so far profiled the mutational landscape of more than 400 SCLC patients and generated over 100 patient-derived xenotransplant models. We analyzed genome and transcriptome sequencing data of multiple tumors to interrogate the NMD target repertoire in SCLC. We determined tumor-specific mutations and predicted NeoAg based on the HLA-I-binding ability of mutant peptides. Upon NMD inhibiton, we validated the expression and presentation of NeoAg via transcriptomics, proteomics and HLA-I-immunopeptidomics. NMD-sensitive mutated transcripts included a wide range of somatic alterations beyond FS-mutations, suggesting an overall increase of potential tumor-specific NeoAg. We furthermore evaluated immunogenicity after NMD perturbation employing in vitro co-cultures as well as immunocompetent in vivo mouse models. Inhibition of NMD in tumor cells resulted in an HLA-I-dependent enhancement of T cell-mediated tumor killing in vitro, and in vivo experiments showed reduced tumor growth accompanied by increased immune infiltration upon controlled induction of NMD inhibition. Altogether, NMD inhibition in SCLC led to an upregulation of NeoAg expression in tumor cells accompanied by enhanced T cell responses in vitro and in vivo. Our strategy provides a novel approach for the treatment of low immunogenic tumors which could be combined with ICI to enhance the efficacy of immunotherapy. Citation Format: Lucia A. Torres Fernández, Volker Böhm, Lukas Maas, Anna Schöllhorn, Joel Kaufmann, Joshua D'Rozario, Laura Kaiser, Niels Gehring, Martin Peifer, Cécile Gouttefangeas, Roman K. Thomas, Julie George. Exploring nonsense-mediated decay inhibition as a novel approach to improve immunogenicity in small cell lung 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 3986.

Binding and Activation of LRP1-Dependent Cell Signaling in Schwann Cells Using a Peptide Derived from the Hemopexin Domain of MMP-9.

Schwann cells (SCs) undergo phenotypic transformation and then orchestrate nerve repair following a peripheral nervous system injury. The low-density lipoprotein receptor-related protein-1 (LRP1) is significantly upregulated in SCs in response to acute injury, activating cJun and promoting SC survival. Matrix-metalloproteinase-9 (MMP-9) is an LRP1 ligand that binds LRP1 through its hemopexin domain (PEX) and activates SC survival signaling and migration. To identify novel peptide mimetics within the hemopexin domain of MMP-9, we examined the crystal structure of PEX, synthesized four peptides, and examined their potential to bind and activate LRP1. We demonstrate that a 22 amino acid peptide, peptide 2, was the only peptide that activated Akt and ERK1/2 signaling in SCs, similar to a glutathione s-transferase (GST)-fused holoprotein, GST-PEX. Intraneural injection of peptide 2, but not vehicle, into crush-injured sciatic nerves activated cJun greater than 2.5-fold in wild-type mice, supporting that peptide 2 can activate the SC repair signaling in vivo. Peptide 2 also bound to Fc-fusion proteins containing the ligand-binding motifs of LRP1, clusters of complement-like repeats (CCRII and CCRIV). Pulldown and computational studies of alanine mutants of peptide 2 showed that positively charged lysine and arginine amino acids within the peptide are critical for stability and binding to CCRII. Collectively, these studies demonstrate that a novel peptide derived from PEX can serve as an LRP1 agonist and possesses qualities previously associated with LRP1 binding and SC signaling in vitro and in vivo.

Molecular Dynamic Study on the Structure and Thermal Stability of Mutant Pediocin PA-1 Peptides Engineered with Cysteine Substitutions.

Pediocin and analogous bacteriocins, valued for thermal stability, serve as versatile antimicrobials in the food sector. Improving their resilience at high temperatures and deriving derivatives not only benefit food production but also offer broad-spectrum antimicrobial potential in pharmaceuticals, spanning treatments for peptic ulcers, women's health, and novel anticancer agents. The study aims to create mutant peptides capable of establishing a third disulfide bond or enhanced through cysteine substitutions. This involves introducing additional Cys residues into the inherent structure of pediocin PA-1 to facilitate disulfide bond formation. Five mutants (Mut 1-5) were systematically generated with double Cys substitutions and assessed for thermal stability through MD simulations across temperatures (298-394K). The most robust mutants (Mut 1, Mut 4-5) underwent extended analysis via MD simulations, comparing their structural stability, secondary structure, and surface accessibility to the reference Pediocin PA-1 molecule. This comprehensive assessment aims to understand how Cys substitutions influence disulfide bonds and the overall thermal stability of the mutant peptides. In silico analysis indicated that Mut 1 and Mut 5, along with the reference structure, lose their helical structure and one natural disulfide bond at high temperatures, and may impacting antimicrobial activity. Conversely, Mut 4 retained its helical structure and exhibited thermal stability similar to Pediocin PA-1. Pending further experimental validation, this study implies Mut 4 may have high stability and exceptional resistance to high temperatures, potentially serving as an effective antimicrobial alternative.

Transmembrane modification of tumor vascular targeting peptide A7R as molecular cargo delivery tool

In recent years, targeting tumor angiogenesis has emerged as a prominent research focus in the treatment and prevention of tumor expansion. A7R (ATWLPPR) exhibits high affinity and specificity for VEGFR-2, which is overexpressed in various tumors. To enhance the tumor tissue and cell penetration capabilities of A7R, we substituted its non-critical amino acid with Arginine (R) and Glutamic acid (E), cyclized the mutant peptide, and linked it to the membrane permeation sequence using coordination principles. We designed and synthesized fifteen novel penetrating peptides that target tumor blood vessels and cells, followed by conducting various biological evaluations and cell imaging experiments. The results demonstrated that Cyclo-A7R-RRR and A7R-RLLRLLR exhibited excellent permeability towards tumor cells, with Cyclo-A7R-RRR showing superior serum stability compared to A7R. Furthermore, the modified peptides showed no toxicity towards HeLa cells, U251 cells, HuH-7 cells, and HEK293 cells under 10 μmol/L. Utilizing Cyclo-A7R-RRR or A7R-RLLRLLR for transmembrane delivery of drug molecules could significantly improve their efficacy. Our findings broaden the potential application scenarios of A7R in targeted tumor angiogenesis.

Dynamics of Molecular Self-Assembly of Short Peptides at Liquid-Solid Interfaces - Effect of Charged Amino Acid Point Mutations.

Self-organizing solid-binding peptides on atomically flat solid surfaces offer a unique bio/nano hybrid platform, useful for understanding the basic nature of biology/solid coupling and their practical applications. The surface behavior of peptides is determined by their molecular folding, which is influenced by various factors and is challenging to study. Here, the effect of charged amino acids is studied on the self-assembly behavior of a directed evolution selected graphite-binding dodecapeptide on graphite surface. Two mutations, M6 and M8, are designed to introduce negatively and positively charged moieties, respectively, at the anchoring domain of the wild-type (WT) peptide, affecting both binding and assembly. The questions addressed here are whether mutant peptides exhibit molecular crystal formation and demonstrate molecular recognition on the solid surface based on the specific mutations. Frequency-modulated atomic force microscopy is used for observations of the surface processes dynamically in water at molecular resolution over several hours at the ambient. The results indicate that while the mutants display distinct folding and surface behavior, each homogeneously nucleates and forms 2D self-organized patterns, akin to the WT peptide. However, their growth dynamics, domain formation, and crystalline lattice structures differ significantly. The results represent a significant step toward the rational design of bio/solid interfaces, potent facilitators of a variety of future implementations.

Stimulating T cell responses against patient-derived breast cancer cells with neoantigen peptide-loaded peripheral blood mononuclear cells

Breast cancer stands as a formidable global health challenge for women. While neoantigens exhibit efficacy in activating T cells specific to cancer and instigating anti-tumor immune responses, the accuracy of neoantigen prediction remains suboptimal. In this study, we identified neoantigens from the patient-derived breast cancer cells, PC-B-142CA and PC-B-148CA cells, utilizing whole-genome and RNA sequencing. The pVAC-Seq pipeline was employed, with minor modification incorporating criteria (1) binding affinity of mutant (MT) peptide with HLA (IC50 MT) ≤ 500 nm in 3 of 5 algorithms and (2) IC50 wild type (WT)/MT > 1. Sequencing results unveiled 2513 and 3490 somatic mutations, and 646 and 652 non-synonymous mutations in PC-B-142CA and PC-B-148CA, respectively. We selected the top 3 neoantigens to perform molecular dynamic simulation and synthesized 9–12 amino acid neoantigen peptides, which were then pulsed onto healthy donor peripheral blood mononuclear cells (PBMCs). Results demonstrated that T cells activated by ADGRL1E274K, PARP1E619K, and SEC14L2R43Q peptides identified from PC-B-142CA exhibited significantly increased production of interferon-gamma (IFN-γ), while PARP1E619K and SEC14L2R43Q peptides induced the expression of CD107a on T cells. The % tumor cell lysis was notably enhanced by T cells activated with MT peptides across all three healthy donors. Moreover, ALKBH6V83M and GAAI823T peptides from PC-B-148CA remarkably stimulated IFN-γ- and CD107a-positive T cells, displaying high cell-killing activity against target cancer cells. In summary, our findings underscore the successful identification of neoantigens with anti-tumor T cell functions and highlight the potential of personalized neoantigens as a promising avenue for breast cancer treatment.

COSMIC-based mutation database enhances identification efficiency of HLA-I immunopeptidome

BackgroundNeoantigens have emerged as a promising area of focus in tumor immunotherapy, with several established strategies aiming to enhance their identification. Human leukocyte antigen class I molecules (HLA-I), which present intracellular immunopeptides to T cells, provide an ideal source for identifying neoantigens. However, solely relying on a mutation database generated through commonly used whole exome sequencing (WES) for the identification of HLA-I immunopeptides, may result in potential neoantigens being missed due to limitations in sequencing depth and sample quality.MethodIn this study, we constructed and evaluated an extended database for neoantigen identification, based on COSMIC mutation database. This study utilized mass spectrometry-based proteogenomic profiling to identify the HLA-I immunopeptidome enriched from HepG2 cell. HepG2 WES-based and the COSMIC-based mutation database were generated and utilized to identify HepG2-specific mutant immunopeptides.ResultThe results demonstrated that COSMIC-based database identified 5 immunopeptides compared to only 1 mutant peptide identified by HepG2 WES-based database, indicating its effectiveness in identifying mutant immunopeptides. Furthermore, HLA-I affinity of the mutant immunopeptides was evaluated through NetMHCpan and peptide-docking modeling to validate their binding to HLA-I molecules, demonstrating the potential of mutant peptides identified by the COSMIC-based database as neoantigens.ConclusionUtilizing the COSMIC-based mutation database is a more efficient strategy for identifying mutant peptides from HLA-I immunopeptidome without significantly increasing the false positive rate. HepG2 specific WES-based database may exclude certain mutant peptides due to WES sequencing depth or sample heterogeneity. The COSMIC-based database can effectively uncover potential neoantigens within the HLA-I immunopeptidomes.

NeoHunter: Flexible software for systematically detecting neoantigens from sequencing data

AbstractComplicated molecular alterations in tumors generate various mutant peptides. Some of these mutant peptides can be presented to the cell surface and then elicit immune responses, and such mutant peptides are called neoantigens. Accurate detection of neoantigens could help to design personalized cancer vaccines. Although some computational frameworks for neoantigen detection have been proposed, most of them can only detect SNV‐ and indel‐derived neoantigens. In addition, current frameworks adopt oversimplified neoantigen prioritization strategies. These factors hinder the comprehensive and effective detection of neoantigens. We developed NeoHunter, flexible software to systematically detect and prioritize neoantigens from sequencing data in different formats. NeoHunter can detect not only SNV‐ and indel‐derived neoantigens but also gene fusion‐ and aberrant splicing‐derived neoantigens. NeoHunter supports both direct and indirect immunogenicity evaluation strategies to prioritize candidate neoantigens. These strategies utilize binding characteristics, existing biological big data, and T‐cell receptor specificity to ensure accurate detection and prioritization. We applied NeoHunter to the TESLA dataset, cohorts of melanoma and non‐small cell lung cancer patients. NeoHunter achieved high performance across the TESLA cancer patients and detected 79% (27 out of 34) of validated neoantigens in total. SNV‐ and indel‐derived neoantigens accounted for 90% of the top 100 candidate neoantigens while neoantigens from aberrant splicing accounted for 9%. Gene fusion‐derived neoantigens were detected in one patient. NeoHunter is a powerful tool to ‘catch all’ neoantigens and is available for free academic use on Github (XuegongLab/NeoHunter).