15-mer Peptides Research Articles

A magnetic epitope-imprinted microsphere used for selective separation and rapid detection of SHV-type β-lactamases in bacteria: a novel strategy of antimicrobial resistance detection

BackgroundThe production of β-lactamases is the most prevalent resistance mechanism for β-lactam antibiotics in Gram-negative bacteria. Presently, over 4900 β-lactamases have been discovered, and they are categorized into hundreds of families. In each enzyme family, amino acid substitutions result in subtle changes to enzyme hydrolysis profiles; in contrast, certain conserved sequences retained by all of the family members can serve as important markers for enzyme family identification.ResultsThe SHV family was chosen as the study object. First, a unique 10-mer peptide was identified as SHV family's epitope by an approach of protein fingerprint analysis. Then, an SHV-specific magnetic epitope-imprinted gel polymer (MEI-GP) was prepared by an epitope surface imprinting technique, and its sorption behavior and recognition mechanism for template epitope and SHV were both elaborated. Finally, the MEI-GP was successfully applied to selectively extract SHV from bacteria, and the extracted SHV was submitted to MALDI-TOF MS for specific determination. By following this strategy, other β-lactamase families can also be specifically detected. According to the molecular weight displayed in mass spectra, the kind of β-lactamase and its associated hydrolysis profile on β-lactams can be easily identified. Based on this, an initial drug option scheme can be quickly formulated for antimicrobial therapy. From protein extraction to medication guidance reporting, the mean time to detection (MTTD) was less than 2 h, which is much faster than conventional phenotype-based methods (at least 16–20 h) and gene-based techniques (usually about 8 h).ConclusionsThis enzyme-specific detection strategy combined the specificity of epitope imprinting with the sensitivity of mass spectrometry, enabling β-lactamase to be selectively extracted from bacteria and clearly presented in mass spectra. Compared with other drug resistance detection methods, this technique has good specificity, high sensitivity (≤ 15 mg of bacteria), a short MTTD (less than 2 h), and simple operation, and therefore has a broad application prospect in clinical medicine.Graphical

High-Affinity Peptides for Target Protein Screened in Ultralarge Virtual Libraries.

High-throughput virtual screening (HTVS) has emerged as a pivotal strategy for identifying high-affinity peptides targeting functional proteins, which are crucial for diagnostic and therapeutic applications. In the HTVS of peptides, expanding the library capacity to enhance peptide sequence diversity, thereby screening out excellent affinity peptide candidates, remains a significant challenge. This study presents a de novo design strategy that leverages directed mutation driven HTVS to evolve vast virtual libraries and screen peptides with ultrahigh affinities for various target proteins. Utilizing a computer-generated library of 104 random 15-mer peptide scaffolds, we employed a self-developed algorithm for parallelized HTVS with Autodock Vina. The top 1% of designs underwent random mutations at a rate of 20% for six generations, theoretically expanding the library to 1014 members. This approach was applied to various protein targets, including a tumor marker (alpha fetoprotein, AFP) and virus surface proteins (SARS-CoV-2 RBD and norovirus P-domain). Starting from the same 104 random 15-mer peptide library, peptides with high affinities in the nanomolar range for three protein targets were successfully identified. The energy-saving and high-efficient design strategy presents new opportunities for the cost-effective development of more effective high-affinity peptides for various environmental and health applications.

Venomous Peptides: Molecular Origin of the Toxicity of Snake Venom PLA2-like Peptides.

Snakebite envenoming claims 81-138 thousand lives annually, with vipers responsible for many of those. Phospholipase A2 (PLA2) enzymes and PLA2-like proteins are among the most important viper venom toxins. The latter are particularly intriguing, as three decades after their discovery, their molecular mechanism of toxicity is still poorly understood at best. PLA2-like proteins destabilize eukaryotic cell membranes through an unknown mechanism, causing an uncontrolled influx of Ca2+ ions and ultimately triggering cell death. It is now clear that the C-terminal segment is fundamental to the toxicity, as 13-mer peptides with the same sequence exhibit most or all of the activities of the complete PLA2-like proteins. To finally clarify the mechanism of toxicity of these venom peptides, we have simulated their interaction with model cell membranes. Molecular dynamics simulations showed that peptides initially dispersed across the cell membrane quickly and spontaneously migrated, aggregated, induced membrane thinning, and formed clear and transient membrane pores. We calculated the potentials of the mean force for Ca2+ transfer across the cell membranes through the transient pores. The pores significantly lower the free energy barrier for Ca2+ translocation, an effect that grows with the size of the peptide aggregates and, thus, with the pore radius. Ca2+ flowed across the membrane through the largest pores with almost no barrier. The permeability of Ca2+ through the largest pores exceeded the permeability of pharmaceutical drugs by 4 orders of magnitude, revealing the easiness by which Ca2+ overflows the intracellular medium. These results elucidate the illusive molecular origin of the toxicity of this famous class of snake venom-derived peptides.

Discovery of Truncated Cyclic Peptides Targeting an Induced-Fit Pocket on PCSK9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma low-density lipoprotein cholesterol (LDL-C) levels by promoting hepatic LDL receptor (LDL-R) degradation. We previously identified and optimized 13-mer cyclic peptides that bind to a novel, induced-fit pocket adjacent to the binding interface of PCSK9 and LDL-R and effectively disrupted the PCSK9/LDL-R protein-protein interaction (PPI) both in vitro and in vivo. However this series of large cyclic peptides required charged groups for function and lacked oral bioavailability in rodents. We describe herein multiple structure-based modifications to these original peptides to yield truncated, neutral molecules with full PPI function in both biochemical and cellular assays. In parallel, new mRNA-peptide display screens identified non-functional 8- and 9-mer compounds which ligand the induced-fit pocket in a distinct manner. Taken together, these studies indicate multiple directions to reduce the size and complexity of this peptide class toward a true small molecule oral agent.

Fatty Acid Derivatization and Cyclization of the Immunomodulatory Peptide RP-182 Targeting CD206high Macrophages Improve Antitumor Activity.

As tumor-associated macrophages (TAM) exercise a plethora of protumor and immune evasive functions, novel strategies targeting TAMs to inhibit tumor progression have emerged within the current arena of cancer immunotherapy. Activation of the mannose receptor 1 (CD206) is a recent approach that recognizes immunosuppressive CD206high M2-like TAMs as a drug target. Ligation of CD206 both induces reprogramming of CD206high TAMs toward a proinflammatory phenotype and selectively triggers apoptosis in these cells. CD206-activating therapeutics are currently limited to the linear, 10mer peptide RP-182, 1, which is not a drug candidate. In this study, we sought to identify a better suitable candidate for future clinical development by synthesizing and evaluating a series of RP-182 analogs. Surprisingly, fatty acid derivative 1a [RP-182-PEG3-K(palmitic acid)] not only showed improved stability but also increased affinity to the CD206 receptor through enhanced interaction with a hydrophobic binding motif of CD206. Peptide 1a showed superior in vitro activity in cell-based assays of macrophage activation which was restricted to CD206high M2-polarized macrophages. Improvement in responses was disproportionally skewed toward improved induction of phagocytosis including cancer cell phagocytosis. Peptide 1a reprogrammed the immune landscape in genetically engineered murine KPC pancreatic tumors toward increased innate immune surveillance and improved tumor control and effectively suppressed tumor growth of murine B16 melanoma allografts.

Discovery of a proteolysis targeting chimera (PROTAC) as a potent regulator of FOXP3

Regulatory T (Treg) cells play a central role in immune homeostasis. Forkhead box P3 (Foxp3), a hallmark molecule in Treg cells, is a vital transcription factor for their development and function. Studies have shown that degradation of the Foxp3 could provide therapeutic benefits in achieving effective anti-tumor immunity. In this study, we designed three PROTAC molecules, P60-L1-VHL, P60-L2-VHL, and P60-L3-VHL, based on a 15-mer peptide inhibitor of Foxp3 (P60), and explored their potential in regulating Foxp3 expression and function. Our data show that, among these molecules, P60-L3-VHL can inhibit the expression and nuclear localization of Foxp3 in HEK 293 T and HeLa cells, respectively. Meanwhile, use of proteasome inhibitor in P60-L3-VHL treated cells revealed an increased Foxp3 expression, indicating that P60-L3-VHL mediates the inhibition of Foxp3 through its degradation in the proteasome pathway. We further substantiate that P60-L3-VHL reduces the differentiation and Foxp3 expression in the in-vitro activated Treg cells. Overall, our findings suggest that P60-L3-VHL inhibits the differentiation of Treg cells by degrading the Foxp3, which may have potential implications in cancer immunotherapy.

Structural and biochemical analysis of highly similar HLA-B allotypes differentially associated with type 1 diabetes

Type 1 diabetes (T1D) is an autoimmune disease involving T cell-mediated destruction of the insulin-producing beta cells in the pancreatic islets of Langerhans. CD8+ T cells, responding to beta cell peptides presented by class I major histocompatibility complex (MHC) molecules, are important effectors leading to beta cell elimination. Human leukocyte antigen (HLA) B∗39:06, B∗39:01, and B∗38:01 are closely related class I MHC allotypes that nonetheless show differential association with T1D. HLA-B∗39:06 is the most predisposing of all HLA class I molecules and is associated with early age at disease onset. B∗39:01 is also associated with susceptibility to T1D, but to a lesser extent, though differing from B∗39:06 by only two amino acids. HLA-B∗38:01, in contrast, is associated with protection from the disease. Upon identifying a peptide that binds to both HLA-B∗39:06 and B∗39:01, we determined the respective X-ray structures of the two allotypes presenting this peptide to 1.7Å resolution. The peptide residues available for T cell receptor contact and those serving as anchors were identified. Analysis of the F pocket of HLA-B∗39:06 and B∗39:01 provided an explanation for the distinct peptide C terminus preferences of the two allotypes. Structure-based modeling of the protective HLA-B∗38:01 suggested a potential reason for its peptide preferences and its reduced propensity to present 8-mer peptides compared to B∗39:06. Notably, the three allotypes showed differential binding to peptides derived from beta cell autoantigens. Taken together, our findings should facilitate identification of disease-relevant candidate T cell epitopes and structure-guided therapeutics to interfere with peptide binding.

Peptide Blockers of PD-1-PD-L1 Interaction Reinvigorate PD-1-Suppressed T Cells and Curb Tumor Growth in Mice.

The programmed cell death protein 1 (PD-1) plays a critical role in cancer immune evasion. Blocking the PD-1-PD-L1 interaction by monoclonal antibodies has shown remarkable clinical efficacy in treating certain types of cancer. However, antibodies are costly to produce, and antibody-based therapies can cause immune-related adverse events. To address the limitations associated with current PD-1/PD-L1 blockade immunotherapy, we aimed to develop peptide-based inhibitors of the PD-1/PD-L1 interaction as an alternative means to PD-1/PD-L1 blockade antibodies for anti-cancer immunotherapy. Through the functional screening of peptide arrays encompassing the ectodomains of PD-1 and PD-L1, followed by the optimization of the hit peptides for solubility and stability, we have identified a 16-mer peptide, named mL7N, with a remarkable efficacy in blocking the PD-1/PD-L1 interaction both in vitro and in vivo. The mL7N peptide effectively rejuvenated PD-1-suppressed T cells in multiple cellular systems designed to recapitulate the PD-1/PD-L1 interaction in the context of T-cell receptor signaling. Furthermore, PA-mL7N, a chimera of the mL7N peptide coupled to albumin-binding palmitic acid (PA), significantly promoted breast cancer cell killing by peripheral blood mononuclear cells ex vivo and significantly curbed tumor growth in a syngeneic mouse model of breast cancer. Our work raises the prospect that mL7N may serve as a prototype for the development of a new line of peptide-based immunomodulators targeting the PD-1/PD-L1 immune checkpoint with potential applications in cancer treatment.

De novo design of covalent bonding peptides for target protein

To rapidly develop hyper-stable inhibitors that bind specifically and covalently to functional proteins is critical for diagnostics and therapeutics. Taking targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants as an example, we report a fast and low-cost de novo design strategy on covalent bonding peptides toward the SARS-CoV-2 spike protein receptor-binding domain (RBD), hence blocking its interaction with the human angiotensin-converting enzyme 2 (hACE2). As a proof-of-concept, peptide scaffolds built by ligating the hotspot residues at the hACE2-Omicron RBD interface were docked against RBD, and then the chemically modified peptides were designed by predicting their reactivity against RBD using a modified Amber ff14SB force field. Two peptides (15- and 16-mer peptides) were equipped with sulfonyl fluoride warheads bound with the conserved Y449 residue of RBD via the sulfur (VI) fluoride exchange (SuFEx) click chemistry. With permanent bonding and without dissociation, the two peptides blocked Omicron BA.2 pseudovirus infection with IC50 values of 1.07 μM and 1.56 μM, respectively. Our design approach greatly promotes the discovery of hyper-stable inhibitors against SARS-CoV-2 variants and other rapidly evolving viruses, potentially applicable to combat future viral outbreaks efficiently.

Identification and serological responses to a novel Plasmodium vivax merozoite surface protein 1 (PvMSP-1) derived synthetic peptide: a putative biomarker for malaria exposure.

The integration of diagnostic methods holds promise for advancing the surveillance of malaria transmission in both endemic and non-endemic regions. Serological assays emerge as valuable tools to identify and delimit malaria transmission, serving as a complementary method to rapid diagnostic tests (RDT) and thick smear microscopy. Here, we evaluate the potential of antibodies directed against peptides encompassing the entire amino acid sequence of the PvMSP-1 Sal-I strain as viable serological biomarkers for P. vivax exposure. We screened peptides encompassing the complete amino acid sequence of the Plasmodium vivax Merozoite Surface Protein 1 (PvMSP-1) Sal-I strain as potential biomarkers for P. vivax exposure. Here, immunodominant peptides specifically recognized by antibodies from individuals infected with P. vivax were identified using the SPOT-synthesis technique followed by immunoblotting. Two 15-mer peptides were selected based on their higher and specific reactivity in immunoblotting assays. Subsequently, peptides p70 and p314 were synthesized in soluble form using SPPS (Solid Phase Peptide Synthesis) and tested by ELISA (IgG, and subclasses). This study unveils the presence of IgG antibodies against the peptide p314 in most P. vivax-infected individuals from the Brazilian Amazon region. In silico B-cell epitope prediction further supports the utilization of p314 as a potential biomarker for evaluating malaria transmission, strengthened by its amino acid sequence being part of a conserved block of PvMSP-1. Indeed, compared to patients infected with P. falciparum and uninfected individuals never exposed to malaria, P. vivax-infected patients have a notably higher recognition of p314 by IgG1 and IgG3.

T cell independent antibody responses with class switch and memory using peptides anchored on liposomes

Vaccines generally require T lymphocytes for B-cell activation and immunoglobulin class switching in response to peptide or protein antigens. In the absence of T cells, limited IgG class switch takes place, germinal centers are short-lived, and the B cells lack memory. Here, immunization of mice with liposomes containing 15mer peptides and monophosphoryl lipid A (MPLA) as adjuvant, induced T-cell independent (TI) IgG class switch within three days, as well as germinal center formation. The antibody responses were long-lived, strictly dependent on Toll-like receptor 4 (TLR4) signaling, partly dependent on Bruton’s tyrosine kinase (BTK) signal transmission, and independent of signaling through T-cell receptors, MHC class II and inflammasome. The antibody response showed characteristics of both TI type 1 and TI type 2. All IgG subclasses could be boosted months after primary immunization, and the biological function of the secreted antibodies was demonstrated in murine models of allergic anaphylaxis and of bacterial infection. Moreover, antibody responses after immunization with peptide- and MPLA-loaded liposomes could be triggered in neonatal mice and in mice receiving immune-suppressants. This study demonstrates T-cell independent endogenous B-cell memory and recall responses in vivo using a peptide antigen. The stimulation of these antibody responses required a correct and dense assembly and administration of peptide and adjuvant on the surface of liposomes. In the future, TI vaccines may prove beneficial in pathological conditions in which T-cell immunity is compromised through disease or medicines or when rapid, antibody-mediated immune protection is needed.

T-Cell Epitope Mapping of SARS-CoV-2 Reveals Coordinated IFN-γ Production and Clonal Expansion of T Cells Facilitates Recovery from COVID-19.

T-cell responses can be protective or detrimental during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection; however, the underlying mechanism is poorly understood. In this study, we screened 144 15-mer peptides spanning the SARS-CoV-2 spike, nucleocapsid (NP), M, ORF8, ORF10, and ORF3a proteins and 39 reported SARS-CoV-1 peptides in peripheral blood mononuclear cells (PBMCs) from nine laboratory-confirmed coronavirus disease 2019 (COVID-19) patients (five moderate and four severe cases) and nine healthy donors (HDs) collected before the COVID-19 pandemic. T-cell responses were monitored by IFN-γ and IL-17A production using ELISA, and the positive samples were sequenced for the T cell receptor (TCR) β chain. The positive T-cell responses to individual SARS-CoV-2 peptides were validated by flow cytometry. COVID-19 patients with moderate disease produced more IFN-γ than HDs and patients with severe disease (moderate vs. HDs, p < 0.0001; moderate vs. severe, p < 0.0001) but less IL-17A than those with severe disease (p < 0.0001). A positive correlation was observed between IFN-γ production and T-cell clonal expansion in patients with moderate COVID-19 (r = 0.3370, p = 0.0214) but not in those with severe COVID-19 (r = -0.1700, p = 0.2480). Using flow cytometry, we identified that a conserved peptide of the M protein (Peptide-120, P120) was a dominant epitope recognized by CD8+ T cells in patients with moderate disease. Coordinated IFN-γ production and clonal expansion of SARS-CoV-2-specific T cells are associated with disease resolution in COVID-19. Our findings contribute to a better understanding of T-cell-mediated immunity in COVID-19 and may inform future strategies for managing and preventing severe outcomes of SARS-CoV-2 infection.

Anthrax Vaccination, Gulf War Illness, and Human Leukocyte Antigen (HLA).

We report on a highly significant, positive association between anthrax vaccination and occurrence of Gulf War Illness (GWI) in 111 Gulf War veterans (42 with GWI and 69 controls). GWI was diagnosed in 47.1% of vaccinated veterans but only in 17.2% of non-vaccinated veterans (Pearson χ2 = 7.08, p = 0.008; odds ratio = 3.947; relative risk = 2.617), with 1.6x higher GWI symptom severity in vaccinated veterans (p = 0.007, F-test in analysis of covariance). Next, we tested the hypothesis that the susceptibility to GWI following anthrax vaccination could be due to inability to make antibodies against the anthrax protective antigen (PA), the key protein contained in the vaccine. Since the first step in initiating antibody production would be the binding of PA peptide fragments (typically 15-amino acid long [15-mer]) to peptide-binding motifs of human leukocyte antigen (HLA) Class II molecules, we assessed the binding-motif affinities of such HLA specific molecules to all linear 15-mer peptide fragments of the anthrax PA. We identified a total of 58 HLA Class II alleles carried by the veterans in our sample and found that, of those, 18 (31%) were present in the vaccinated group that did not develop GWI but were absent from the vaccinated group who developed GWI. Remarkably, in silico analyses revealed very high binding affinities of peptide-binding motifs of those 18 HLA alleles with fragments of anthrax vaccine PA, leading to the successful production of anti-PA antibodies. Conversely, the absence of these protective HLA alleles points to a reduced ability to develop antibodies against PA, thus resulting in harmful PA persistence and development of GWI.

Plant-Derived, Nodule-Specific Cysteine-Rich Peptides as a Novel Source of Biopesticides for Controlling Citrus Greening Disease.

Nodule-specific cysteine-rich (NCR) peptides, encoded in the genome of the Mediterranean legume Medicago truncatula (barrelclover), are known to regulate plant-microbe interactions. A subset of computationally derived 20-mer peptide fragments from 182 NCR peptides was synthesized to identify those with activity against the unculturable vascular pathogen associated with citrus greening disease, 'Candidatus Liberibacter asiaticus' (CLas). Grounded in a design of experiments framework, we evaluated the peptides in a screening pipeline involving three distinct assays: a bacterial culture assay with Liberibacter crescens, a CLas-infected excised citrus leaf assay, and an assay to evaluate effects on bacterial acquisition by the nymphal stage of hemipteran vector Diaphorina citri. A subset of the 20-mer NCR peptide fragments inhibits both CLas growth in citrus leaves and CLas acquisition by D. citri. Two peptides induced higher levels of D. citri mortality. These findings reveal 20-mer NCR peptides as a new class of plant-derived biopesticide molecules to control citrus greening disease.

Radiolabeled 15-mer peptide internalization is mediated by megalin (LRP2 receptor) in a CRISPR/Cas9-based LRP2 knockout human kidney cell model.

Megalin (LRP2 receptor) mediates the endocytosis of radiolabeled peptides into proximal tubular kidney cells, which may cause nephrotoxicity due to the accumulation of a radioactive tracer. The study aimed to develop a cellular model of human kidney HK2 cells with LRP2 knockout (KO) using CRISPR/Cas9 technique. This model was employed for the determination of the megalin-mediated accumulation of 68Ga- and 99mTc-labeled 15-mer peptide developed to target the vascular endothelial growth factor (VEGF) receptor in oncology radiodiagnostics. The gene editing in the LRP2 KO model was verified by testing two well-known megalin ligands when higher viability of KO cells was observed after gentamicin treatment at cytotoxic concentrations and lower FITC-albumin internalization by the KO cells was detected in accumulation studies. Fluorescent-activated cell sorting was used to separate genetically modified LRP2 KO cell subpopulations. Moreover, flow cytometry with a specific antibody against megalin confirmed LRP2 knockout. The verified KO model identified both 68Ga- and 99mTc-radiolabeled 15-mer peptides as megalin ligands in accumulation studies. We found that both radiolabeled 15-mers enter LRP2 KO HK2 cells to a lesser extent compared to parent cells. Differences in megalin-mediated cellular uptake depending on the radiolabeling were not observed. Using biomolecular docking, the interaction site of the 15-mer with megalin was also described. The CRISPR/Cas9 knockout of LRP2 in human kidney HK2 cells is an effective approach for the determination of radiopeptide internalization mediated by megalin. This in vitro method provided direct molecular evidence for the cellular uptake of radiolabeled anti-VEGFR 15-mer peptides via megalin.

The determinants of the rarity of nucleic and peptide short sequences in nature.

The prevalence of nucleic and peptide short sequences across organismal genomes and proteomes has not been thoroughly investigated. We examined 45785 reference genomes and 21871 reference proteomes, spanning archaea, bacteria, eukaryotes and viruses to calculate the rarity of short sequences in them. To capture this, we developed a metric of the rarity of each sequence in nature, the rarity index. We find that the frequency of certain dipeptides in rare oligopeptide sequences is hundreds of times lower than expected, which is not the case for any dinucleotides. We also generate predictive regression models that infer the rarity of nucleic and proteomic sequences across nature or within each domain of life and viruses separately. When examining each of the three domains of life and viruses separately, the R² performance of the model predicting rarity for 5-mer peptides from mono- and dipeptides ranged between 0.814 and 0.932. A separate model predicting rarity for 10-mer oligonucleotides from mono- and dinucleotides achieved R² performance between 0.408 and 0.606. Our results indicate that the mono- and dinucleotide composition of nucleic sequences and the mono- and dipeptide composition of peptide sequences can explain a significant proportion of the variance in their frequencies in nature.

Epistasis Between HLA-DRB1*16:02:01 and SLC16A11 T-C-G-T-T Reduces Odds for Type 2 Diabetes in Southwest American Indians.

We sought to identify genetic/immunologic contributors of type 2 diabetes in an indigenous American community by genotyping all study participants for both high resolution HLA-DRB1 alleles and SLC16A11 to test their risk and/or protection for T2D. These genes were selected based on independent reports that HLA-DRB1*16:02:01 is protective for T2D and that SLC16A11 associates with T2D in individuals with BMI<35kg/m2, and here test the interaction of the two loci with a more complete dataset and perform a BMI sensitivity test. We define the risk-protection haplotype of SLC16A11, T-C-G-T-T, as allele "2" of a di-allelic genetic model with three genotypes, SLC16A11*11, *12, and *22, where allele "1" is the wildtype. Both earlier findings were confirmed. Together in the same logistic model with BMI≥35, DRB1*16:02:01 remains protective, 0.73, while SLC16A11 switches from risk to protection OR = 0.57 (*22) and 0.78 (*12), respectively; an added interaction term was statistically significant (OR = 0.49 with *12). Bootstrapped (b=10,000) statistical power of interaction, 0.4801, yielded mean OR = 0.43. Sensitivity analysis demonstrated the interaction significant in BMI range 30-41. To investigate the epistasis we used the primary function of the HLA-DRB1 molecule, peptide binding and presentation, to search the entire array of 15mer peptides for both the wildtype and ancient human SLC16A11 molecules for a pattern of strong binding that was associated with risk and protection for T2D. Applying computer binding algorithms suggests the core peptide at SLC16A11 D127G, FSAFASGLL, might be key for moderating risk for T2D with potential implications for T1D.

Abstract 5121: Next-generation immunocompetent and humanized neuroblastoma murine models for the discovery and validation of novel immunotherapies

Abstract The success of CD19-directed chimeric antigen receptor (CAR)-T cell therapy for ALL has yet to be matched in solid pediatric malignancies. Moreover, immune checkpoint blockade has shown limited benefit in pediatric cancer. Despite this unmet need, there is a lack of reliable preclinical models to facilitate novel target discovery, validate therapeutic benefit, anticipate on- or off-target toxicity, and understand mechanisms of resistance. Syngeneic murine model systems are a critical intermediate for preclinical translation, preserving the complex and heterogenous immune landscape and host-immunotherapy interactions. However, model interpretation and validity are limited as expression and function of murine targets and immune effector cells are not always conserved. We have developed a syngeneic immunocompetent C57BL/6 neuroblastoma system to study CAR efficacy, toxicity, and mechanisms of resistance, as well a variety of vaccination strategies. Specifically, we have built two murine CAR T constructs containing a 100% conserved antigen-directed scFv joined to orthologous murine TCR signaling machinery. In parallel, we engineered the murine neuroblastoma cell line 9464D to stably express surface antigen Gpc2 (Bosse, Cancer Cell 2017), or a novel chimeric single chain trimer of a 9mer peptide from intracellular oncoprotein PHOX2B discovered on HLA-A*24:02 (Yarmarkovich, Nature 2023) as Phox2b/A24*02/H2-kb to generate tumor-bearing B6 or B6-A24-Tg mice. We are currently using this system to demonstrate how vaccination approaches enhance the persistence and potency of CART directed to both targets and are extending our syngeneic murine constructs to additional TH-MYCN allograft models. To further study the innate and adaptive immune response to various therapeutics, we have developed peripheral blood mononuclear cell (PBMC) humanized models. Our autologous humanized PDX mouse models of neuroblastoma use immunocompromised NSG mice engrafted with a PDX and humanized with patient-matched PBMCs to evaluate personalized vaccines in vivo. We have shown the ability to stably engraft both lymphoid and myeloid populations in various human cytokine transgenic NSG variants (i.e. SGM3, Flt3L), allowing for T/B cell and APC crosstalk, thus providing a robust means to study targeted interventions. We are currently using this autologously humanized PDX model to test the ability of multivalent, personalized neoantigen vaccines developed specific to the neoantigen landscape of individual PDXs, with a focus on endogenous T cell responses of patient PBMC against their own PDX in vivo. Citation Format: Timothy T. Spear, Elisabeth Posthill, David Groff, Anna M. Giudice, Kristopher R. Bosse, Leyuan Ma, Stephen P. Schoenberger, John M. Maris. Next-generation immunocompetent and humanized neuroblastoma murine models for the discovery and validation of novel immunotherapies [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 5121.

Abstract 3601: Improving killing of renal cell carcinoma through the combined effects of TCR engineered CD4+ and CD8+ T cells

Abstract Patients with late-stage kidney cancer have poor long-term survival rates. Our lab isolated a low affinity CD8 dependent TCR named BZ-4 from CTLs of a patient who had immune-mediated regression of renal cell carcinoma (RCC) following an allogeneic stem cell transplant and found this TCR recognized a human endogenous retrovirus type E (HERV-E) derived 10-mer peptide, CT-RCC-1. The BZ-4 clone belongs to the Vβ7.1 family and binds to CT-RCC-1 in an HLA-A11 dependent manner. Adoptive infusion of CD8+ BZ-4 transduced T cells is currently being explored in an NIH phase I trial (NCT03354390). Because CD4+ T cells have been shown to augment the anti-tumor effects of TCR-modified CD8+ T cells, we hypothesized that CD4+ T cells genetically engineered to co-express BZ-4 and CD8 would recognize and kill RCC tumors expressing the CT-RCC-1 and would improve the in vivo proliferation and tumor cytotoxicity of CD8+ BZ-4 T cells. CD4+ T cells isolated from healthy donor PBMCs were transfected via Sleeping Beauty electroporation with the control MART-1 reactive TCR (DMF5), BZ-4, or BZ-4 alongside the CD8 co-receptor (BZ-4-8). CD8+ T cells were similarly transfected with DMF5 or BZ-4. Following magnetic bead enrichment, 66% to 84% (mean 77%) of transfected T cells stained positive for Vβ7.1. TCR engineered T cells were subsequently tested for their ability to lyse a) luciferase transduced HLA A-11 expressing RCC tumors and b) a T2 cell line modified to express both HLA A-11 and luciferase that was peptide-pulsed with decreasing concentrations (5 × 10−1, 5 × 10−2, 5 × 10−3 &amp; 5 × 10−4 ug/mL) of CT-RCC-1. CD4+ T cells expressing BZ-4 or BZ-4-8 as well as CD8+ T cells expressing BZ-4 or DMF5 were used either alone or in combination in cytotoxicity assays. For combined T cell groups, the total effector/target ratio was maintained at the same ratio as individual T cell groups. For T-2 targets, CD4+ BZ-4 T cells exhibited no cytolytic activity in contrast to both CD8+ BZ-4 T cells and CD4+ BZ-4-8 T cells, which were highly cytolytic to target cells. For RCC tumor targets, CD4+ BZ-4 T cells exhibited no cytolytic activity at 48, 72 and 96 hours in contrast to both CD8+ BZ-4 T cells and CD4+ BZ-4-8 T cells, which exhibited high levels of tumor killing at these timepoints. Remarkably, CD4+ BZ-4-8 T cells alone or in combination with CD8+ BZ-4 T cells demonstrated tumor cytotoxicity that was comparable to that observed with CD8+ BZ-4 T cells alone. In conclusion, these data show that CD4+ BZ-4 T cells transfected with the CD8 co-receptor acquire the ability to lyse RCC tumors at levels comparable to that observed in CD8+ BZ-4 T cells. Additional experiments are planned to evaluate whether mixtures of CD8+ BZ-4 T cells with CD4+ BZ-4-8 T cells will augment the ability of TCR modified T cells to proliferate in vivo and kill human RCC tumors compared to CD8+ BZ-4 T cells alone in tumor bearing NSG mice. Citation Format: Muna Igboko, Long Chen, Stefan Barisic, Elena Cherkasova, David Allan, Mala Chakraborty, Joseph Clara, Angie Parrizzi, Kyra Carney, Rosa Nadal Rios, Robert Reger, Richard Childs. Improving killing of renal cell carcinoma through the combined effects of TCR engineered CD4+ and CD8+ T cells [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 3601.

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

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