Control Peptide Research Articles

Chaperone-Derived Copper(I)-Binding Peptide Nanofibers Disrupt Copper Homeostasis in Cancer Cells.

Copper (Cu) is a transition metal that plays crucial roles in cellular metabolism. Cu+ homeostasis is upregulated in many cancers and contributes to tumorigenesis. However, therapeutic strategies to target Cu+ homeostasis in cancer cells are rarely explored because small molecule Cu+ chelators have poor binding affinity in comparison to the intracellular Cu+ chaperones, enzymes, or ligands. To address this challenge, we introduce a Cu+ chaperone-inspired supramolecular approach to disrupt Cu+ homeostasis in cancer cells that induces programmed cell death. The Nap-FFMTCGGCR peptide self-assembles into nanofibers inside cancer cells with high binding affinity and selectivity for Cu+ due to the presence of the unique MT/CGGC motif, which is conserved in intracellular Cu+ chaperones. Nap-FFMTCGGCR exhibits cytotoxicity towards triple negative breast cancer cells (MDA-MB-231), impairs the activity of Cu+ dependent co-chaperone super oxide dismutase1 (SOD1), and induces oxidative stress. In contrast, Nap-FFMTCGGCR has minimal impact on normal HEK 293T cells. Control peptides show that the self-assembly and Cu+ binding must work in synergy to successfully disrupt Cu+ homeostasis. We show that assembly-enhanced affinity for metal ions opens new therapeutic strategies to address disease-relevant metal ion homeostasis.

8410 Lipid modification enhances the calcemic and bone building effects of injected PTH(1-14) and PTH(1-11) fragment peptides in mice

Abstract Disclosure: J. Höppner: None. H. Noda: None. A. Khatri: None. H. Jüppner: None. T.J. Gardella: None. PTH analogs with improved actions in vivo are of interest as potential treatment options for bone and mineral ion diseases such as hypoparathyroidism and osteoporosis. The efficacies in vivo of conventional PTH peptides are generally limited by rapid rates of clearance from the circulation (t1/2 = ∼ 5-30 minutes) and relatively short-dwell times on the receptor (PTH1R). This is especially true for small fragment analogs, such as M (modified)-PTH(1-14), and M-PTH(1-11), which have only been shown to be active in vitro. We explored whether PTH fragment peptide efficacy in vivo could be enhanced by addition of a lipid chain (e.g., a C15 palmitoyl chain (palm)) to either the peptide C-terminus or to the side chain of the amino acid residue at position 11 or 13. The C-tail lipid modifications were specifically designed to promote binding to serum albumin, and hence extend circulation half-life, while the lipid chains at sidechains of residue positions 11 and 13 were designed, based on structural models, to anchor the ligand to the receptor in situ -- i.e. by projection of the acyl chain between two adjacent transmembrane helices and into the surrounding plasma membrane -- to thereby extend receptor dwell time. Assessment of cAMP signaling potencies in HEK293/hPTH1R/glosensor cells revealed that both types of lipid modifications can preserve agonist potency, and that the lipid extensions at position 11 or 13 can indeed prolong receptor dwell times, as shown by extended durations of cAMP signaling after initial binding of Palm 11 or 13-modified M-PTH(1-14) and M-PTH(1-11) peptide fragment. Single subcutaneous injection of either Palm-13-M-PTH(1-14) or Palm-11-M-PTH(1-11) into mice resulted in prolonged increases in blood serum calcium levels, while 14 days of daily injection resulted in profound increases in bone mass, as measured by uCT, and marked increases in blood levels of the bone turnover markers CTX1 and P1NP, whereas non-lipidated M-PTH(1-14) control peptides were inactive in vivo. Peptide lipidation thus holds promise as a strategy to employ in the design of new PTH analogs with improved efficacies in vivo. Presentation: 6/1/2024

A Nanobody-Based Proximity Ligation Assay Detects Constitutive and Stimulus-Regulated Native Arc/Arg3.1 Oligomers in Hippocampal Neuronal Dendrites.

Activity-regulated cytoskeleton-associatedprotein (Arc), the product of an immediate early gene, plays critical roles in synaptic plasticity and memory. Evidence suggests that Arc function is determined by its oligomeric state; however, methods for localization of native Arc oligomers are lacking. Here, we developed a nanobody-based proximity ligation assay (PLA) for detection, localization, and quantification of Arc-Arc complexes in primary rat hippocampal neuronal cultures. We used nanobodies with single, structurally defined epitopes in the bilobar Arc capsid domain. Nanobody H11 binds inside the N-lobe ligand pocket, while nanobody C11 binds to the C-lobe surface. For each nanobody, ALFA- and FLAG-epitope tags created a platform for antibody binding and PLA. Surprisingly, PLA puncta in neuronal dendrites revealed widespread constitutive Arc-Arc complexes. Treatment of cultures with tetrodotoxin or cycloheximide had no effect, suggesting stable complexes that are independent of recent neuronal activity and protein synthesis. To assess detection of oligomers, cultures were exposed to a cell-penetrating peptide inhibitor of the Arc oligomerization motif (OligoOFF). Arc-Arc complexes detected by H11 PLA were inhibited by OligoOff but not by control peptide. Notably, Arc complexes detected by C11 were unaffected by OligoOFF. Furthermore, we evaluated Arc complex formation after chemical stimuli that increase Arc synthesis. Brain-derived neurotrophic factor increased Arc-Arc signal detected by C11, but not H11. Conversely, dihydroxyphenylglycine (DHPG) treatment selectively enhanced H11 PLA signals. In sum, nanobody-based PLA reveals constitutive and stimulus-regulated Arc oligomers in hippocampal neuronal dendrites. A model is proposed based on detection of Arc dimer by C11 and higher-order oligomer by H11 nanobody.

Incubation of Amyloidogenic Peptides in Reverse Micelles Allow Active Control of Oligomer Size and Study of Protein-Protein Interactions.

Studies of the structure and dynamics of oligomeric aggregates of amyloidogenic peptides pose challenges due to their transient nature. This concept article provides a brief overview of various nucleation mechanisms with reference to the classical nucleation theory and illustrates the advantages of incubating amyloidogenic peptides in reverse micelles (RMs). The use of RMs not only facilitates size regulation of oligomeric aggregates but also provides an avenue to explore protein-protein interactions among the oligomeric aggregates of various amyloidogenic peptides. Additionally, we envision the feasibility of preparing brain tissue-derived oligomeric aggregates using RMs, potentially advancing the development of monoclonal antibodies with enhanced potency against these pathological species in vivo.

Investigating Cellulose Binding of Peptides Derived from Carbohydrate Binding Module 1.

Carbohydrate-binding modules (CBM) have emerged as useful tools for a wide range of tasks, including the use as purification tags or for cellulose fiber modification. For this purpose, the CBM needs to be attached to a target protein leading to large constructs. We investigated if short peptides from the carbohydrate binding site of CBMs can bind in a similar way as native, full-length CBMs to nanocrystalline cellulose (NCC) or cotton linter paper. We designed our cellulose-binding peptides to be less hydrophobic and shorter than those previously reported. Starting from the binding site of Cel7A-CBM1, we incorporated the essential amino acids involved in cellulose binding into our peptides. These peptides, as well as control peptides with scrambled sequences or a lack of essential amino acids, bound to cellulose with similar affinity as CBM regardless of their secondary structure, sequence, or hydrophobicity. This unspecific mode of cellulose binding displayed by the presented peptides may be exploited to functionalize cellulose-based biomaterials by means of peptide-conjugates.

Engineered extracellular vesicles coated with an antimicrobial peptide for advanced control of bacterial sepsis.

Alarming sepsis-related mortality rates present significant challenges to healthcare services globally. Despite advances made in the field, there is still an urgent need to develop innovative approaches that could improve survival rates and reduce the overall cost of treatment for sepsis patients. Therefore, this study aimed to develop a novel multifunctional therapeutic agent for advanced control of bacterial sepsis. Extracellular vesicles (EVs) isolated from lipopolysaccharide (LPS) induced HepG2 (hepatocellular carcinoma cells) (iEV) displayed an average particle size of 171.63±2.77nm, a poly dispersion index (PDI) of 0.32±0.0, and a zeta potential (ZP) of -11.87±0.18mV. Compared to HepG2 EV, LPS induction significantly increases the EV protein concentration, PDI and ZP, reduces the average size and promotes cell proliferation and cytoprotective effects of the isolated EVs (iEVs) against LPS-induced cytotoxicity. Coating of iEV with a cationic antimicrobial peptide (AMP) to form PC-iEV slightly changed their physical properties and shifted their surface charge toward neutral values. This modification improved the antibacterial activity (2-fold lower minimum bactericidal concentration [MBC] values) and biocompatibility of the conjugated peptide while maintaining iEV cytoprotective and anti-inflammatory activities. Our findings indicate the superior anti-inflammatory and antibacterial dual activity of PC-iEV against pathogens associated with sepsis.

PL3 CendR peptide shows specific uptake in cultured Y79 retinoblastoma cells with nucleolar accumulation

Retinoblastoma is the most common pediatric intraocular malignant tumor affecting 1:15 000–1:20 000 live births. Even though the survival rate in developed countries is over 90 %, more efficient treatment options are needed for better vision salvage and reduction of the adverse effects. Therefore, we investigated fluorescein-labeled PL3 peptide targeting properties towards the Y79 retinoblastoma cell line in vitro. Through the application of cellular imaging and flow cytometry techniques, the PL3 peptide exhibited a rapid and specific internalization within Y79 cells, with subsequent translocation to the cell nuclei, showcasing notable accumulation in the nucleoli. This phenomenon was not present in other investigated cell lines and was not observable with similarly charged and length control peptide. However, the exact mechanism behind this Y79 cell line-specific nuclear and nucleolar targeting pattern remains elusive. In the future, this targeting process could facilitate specific treatment modalities of retinoblastoma with PL3 peptide-coupled drug delivery technologies.

Pathophysiology of hypereosinophilia-associated heart disease.

Cardiac complications in patients with hypereosinophilia cause significant morbidity and mortality. However, mechanisms of how eosinophilic inflammation causes heart damage are poorly understood. We developed a model of hypereosinophilia-associated heart disease by challenging hypereosinophilic mice with peptide from the cardiac myosin heavy chain. Disease outcomes were measured by histology, immunohistochemistry, flow cytometry, and measurement of cells and biomarkers in peripheral blood. Eosinophil dependence was determined by using eosinophil-deficient mice (ΔdblGATA). Single cells from heart were subjected to single cell RNA sequencing to assess cell composition, subtypes and expression profiles. Mice challenged with myocarditic and control peptide had peripheral blood leukocytosis, but only those challenged with myocarditic peptide had heart inflammation. Heart tissue was infiltrated by eosinophil-rich inflammatory infiltrates associated with cardiomyocyte damage. Disease penetrance and severity were dependent on the presence of eosinophils. Single cell RNA sequencing showed enrichment of myeloid cells, T-cells and granulocytes (neutrophils and eosinophils) in the myocarditic mice. Macrophages were M2 skewed, and eosinophils had an activated phenotype. Gene enrichment analysis identified several pathways potentially involved in pathophysiology of disease. Eosinophils are required for heart damage in hypereosinophilia-associated heart disease. Additionally, myeloid cells, granulocytes and T-cell cooperatively or independently participate in the pathogenesis of hypereosinophilia-associated heart disease.

The fermented milk can be a natural ally against obesity? Investigation of bovine milk fermentation by Lacticaseibacillus casei LBC 237, screening, and In silico predictions of bioactive peptides for obesity control

The increasing quest for therapeutic alternatives in treating non-communicable chronic diseases like obesity has propelled research into bioactive peptides, with a particular focus on milk due to its rich protein composition and associated health benefits. Milk fermentation, a traditional process in dairy production, enhances the bioactivity of peptides, broadening their potential therapeutic uses. This study investigated the anti-obesity potential of peptides from bovine milk fermented by Lacticaseibacillus casei LBC 237, identifying 143 peptides, notably LGPV and EVPMP. In silico analyses revealed that LGPV and EVPMP biopeptides exhibited significant interactions with target proteins, employing various molecular interactions such as Van der Waals forces, hydrogen bonds, and electrostatic interactions. These peptides shared common binding sites in some enzymes, suggesting a similar mode of interaction between molecule and target protein, akin to key pharmaceuticals recommended for treating these pathologies. Furthermore, amino acid characteristics present in the peptides, including hydrophobic residues like Leucine, Glutamate, Valine, and Proline, proved essential for their bioactive and inhibitory activities. These findings highlight the potential of LGPV and EVPMP biopeptides as therapeutic agents in managing obesity and metabolic disorders. They provide important insights into their mechanisms of action, paving the way for future research to apply them practically in preventing and treating metabolic conditions.

GluN2A or GluN2B subunits of the NMDA receptor contribute to changes in neuronal excitability and impairments in LTP in the hippocampus of aging mice but do not mediate detrimental effects of oligomeric Aβ (1-42).

Studies in rodent models have revealed that oligomeric beta-amyloid protein [Aβ (1-42)] plays an important role in the pathogenesis of Alzheimer's disease. Early elevations in hippocampal neuronal excitability caused by Aβ (1-42) have been proposed to be mediated via enhanced activation of GluN2B-containing N-methyl-D-aspartate receptors (NMDAR). To what extent GluN2A or GluN2B-containing NMDAR contribute to Aβ (1-42)-mediated impairments of hippocampal function in advanced rodent age is unclear. Here, we assessed hippocampal long-term potentiation (LTP) and neuronal responses 4-5 weeks after bilateral intracerebral inoculation of 8-15 month old GluN2A+/- or GluN2B+/- transgenic mice with oligomeric Aβ (1-42), or control peptide. Whole-cell patch-clamp recordings in CA1 pyramidal neurons revealed a more positive resting membrane potential and increased total spike time in GluN2A+/-, but not GluN2B+/--hippocampi following treatment with Aβ (1-42) compared to controls. Action potential 20%-width was increased, and the descending slope was reduced, in Aβ-treated GluN2A+/-, but not GluN2B+/- hippocampi. Sag ratio was increased in Aβ-treated GluN2B+/--mice. Firing frequency was unchanged in wt, GluN2A+/-, and GluN2B+/-hippocampi after Aβ-treatment. Effects were not significantly different from responses detected under the same conditions in wt littermates, however. LTP that lasted for over 2 h in wt hippocampal slices was significantly reduced in GluN2A+/- and was impaired for 15 min in GluN2B+/--hippocampi compared to wt littermates. Furthermore, LTP (>2 h) was significantly impaired in Aβ-treated hippocampi of wt littermates compared to wt treated with control peptide. LTP induced in Aβ-treated GluN2A+/- and GluN2B+/--hippocampi was equivalent to LTP in control peptide-treated transgenic and Aβ-treated wt animals. Taken together, our data indicate that knockdown of GluN2A subunits subtly alters membrane properties of hippocampal neurons and reduces the magnitude of LTP. GluN2B knockdown reduces the early phase of LTP but leaves later phases intact. Aβ (1-42)-treatment slightly exacerbates changes in action potential properties in GluN2A+/--mice. However, the vulnerability of the aging hippocampus to Aβ-mediated impairments of LTP is not mediated by GluN2A or GluN2B-containing NMDAR.

Enhancing intracellular delivery of protein kinase C beta II peptide inhibitor using myristic acid-trans-activator of transcription via N-terminus conjugation in isolated rat polymorphonuclear leukocytes

Introduction: Protein kinase C beta II (PKCβII) activation promotes polymorphonuclear (PMN) superoxide (SO) production by phosphorylating serine and threonine amino acid residues on NADPH oxidase (NOX-2). Previous studies have shown that cell permeable myristic acid-conjugated PKCβII inhibitor (myr-PKCβII-; SLNPEWNET) (20 μM) remarkably attenuated phorbol 12-myristate 13-acetate (PMA)-induced PMN SO release when compared to scrambled control peptide (myr-PKCβII- scram; WNPESLNTE), unconjugated peptides and nontreated controls by ~35%. This suggests an enhanced intracellular delivery of PKCβII-. Studies have also shown that SO release is attenuated via trans-activator of transcription-conjugated (Tat; YGRKKRRQRRR) NOX-2 inhibitor (Nox2ds-Tat) (80 μM) by ~37%. There have not been evaluations of Tat- or dual myr-Tat-conjugated PKCβII-. We hypothesize that dual myr-Tat-conjugation would enhance the intracellular delivery of PKCβII- and reduce SO release compared to myr-, Tat-, and myr-Tat-PKCβII- scram. Methods: This study focused on the effects of myr-Tat-PKCβII- on intracellular delivery compared to myr-PKCβII-, Tat -PKCβII-, and myr-Tat-PKCβII- scram, with 0.5% dimethyl sulfoxide (DMSO) vehicle as the control. Sprague-Dawley (SD) male rats (~400g) were anesthetized with isoflurane and given a 0.5% glycogen i.p. injection (16ml), and 16-18hrs later, PMNs were harvested from the peritoneal cavity and incubated for 15min at 37oC with 5μM myr-Tat-PKCβII-, myr-PKCβII-, Tat-PKCβII-, or myr-Tat-PKCβII- scram. PMN SO release was calculated spectrophotometrically by the change in absorbance at 550 nm over 390s via ferricytochrome c reduction after PMA stimulation (100nM). Data were analyzed with ANOVA Fisher’s PLSD post-hoc analysis. Results: Myr-Tat-PKCβII- (n=20, 0.338±0.024, p<0.05) and Myr-PKCβII- (n=8, 0.342±0.041, p<0.05) displayed significant attenuation of SO release by 28% and 27%, respectively, compared to 0.5% DMSO control (n=81, 0.470±0.013). Tat-PKCβII- (n=5, 0.404±0.049) showed attenuated SO release by 14% when compared to the vehicle control. Myr-Tat-PKCβII- scram (n=3, 0.542±0.081) showed an increase in SO release by 15% when compared to the vehicle control. Cell viability was > 85% in all groups. Conclusion: The results of the study suggest that intracellular delivery of PKCβII- cargo using myr-Tat dual conjugation is enhanced when compared to myr-, Tat-conjugated PKCβII-, and myr-Tat-PKCβII- scram. Future studies will utilize western blot analysis and immunohistochemistry to assess the translocation and activity of PKCβII- when conjugated with myr-Tat, myr-, or Tat- peptides. Funding: This study was funded by the National Heart, Lung & Blood Institute (1R43HL160338) SBIR phase I grant that was awarded to Young Therapeutics, LLC. Philadelphia College of Osteopathic Medicine, the Department of Biomedical Sciences, the Division of Research and the Center for Chronic Diseases of Aging. and Young Therapeutics, LLC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 123: Hemostatic Fibrin Knob Triggered Microgel To Promote Clotting With Reduced Thrombotic Risks In Neonates

Introduction: Effective neonatal hemostatic therapies are vital for complex surgery-related life-threatening bleeding [1]. Neonatal hemostatic systems differ significantly from adults [2, 3]; however, current treatments for bleeding include adult blood products (ABP). Neonatal ABP use poses an elevated thrombotic risk [4, 5], necessitating alternative approaches. We propose hemostatic therapies leveraging age-dependent clotting differences. In adults, clot formation relies on fibrin(ogen) A:a knob-hole binding, while B:b binding dominates in neonates. Targeting B:b interactions with synthetic Bknob conjugated platelet-like particles (BK-PLP) could crosslink neonatal fibrinogen, catalyze clot formation, and enhance clot strength with lower thrombotic risk than ABP. To test this, we synthesized pNIPAm microgels and conjugated them to B-knob (AHRPYAAK), A-knob (GPRPFPAK), and non-binding control peptides (GPSPFPAK) to prepare BK-PLP, AK-PLP, and NB-PLP. Materials and Methods: We prepared BK-PLP, AK-PLP, and NB-PLP by conjugating microgels with mimetic peptides using EDC-Sulfo-NHS chemistry (Fig. 1A); Fibrin clot polymerization, stability, density, and stiffness, were analyzed via plate-based assays, confocal microscopy, and AFM. Results and Discussion: BK-PLPs significantly increased clot stability compared to AK-PLP, NB-PLP, or fibrin-only clots (Fig. 1B). Fiber density also increased with BK-PLPs, indicating a role in promoting denser fibrin clot structures (Fig. 1C). Clot mechanical properties assessment suggested that AKnob and NB-PLPs may disrupt clot networks, decreasing stiffness (Fig. 1D). Conclusion: Our study explored the effects of peptide conjugate particles on neonatal fibrin clots. BK-PLP incorporation increased clot stability, while AK-PLP and NB-PLP decreased clot stiffness, supporting further BK-PLP development for neonatal hemostasis.

PD-L1 targeted peptide demonstrates potent antitumor and immunomodulatory activity in cancer immunotherapy.

In recent years, immunotherapy has been emerging as a promising alternative therapeutic method for cancer patients, offering potential benefits. The expression of PD-L1 by tumors can inhibit the T-cell response to the tumor and allow the tumor to evade immune surveillance. To address this issue, cancer immunotherapy has shown promise in disrupting the interaction between PD-L1 and its ligand PD-1. We used mirror-image phage display technology in our experiment to screen and determine PD-L1 specific affinity peptides (PPL-C). Using CT26 cells, we established a transplanted mouse tumor model to evaluate the inhibitory effects of PPL-C on tumor growth in vivo. We also demonstrated that PPL-C inhibited the differentiation of T regulatory cells (Tregs) and regulated the production of cytokines. In vitro, PPL-C has a strong affinity for PD-L1, with a binding rate of 0.75 μM. An activation assay using T cells and mixed lymphocytes demonstrated that PPL-C inhibits the interaction between PD-1 and PD-L1. PPL-C or an anti-PD-L1 antibody significantly reduced the rate of tumor mass development in mice compared to those given a control peptide (78% versus 77%, respectively). The results of this study demonstrate that PPL-C prevents or retards tumor growth. Further, immunotherapy with PPL-C enhances lymphocyte cytotoxicity and promotes proliferation in CT26-bearing mice. PPL-C exhibited antitumor and immunoregulatory properties in the colon cancer. Therefore, PPL-C peptides of low molecular weight could serve as effective cancer immunotherapy.

Small animal PET imaging with the 68Ga-labeled pH (low) insertion peptide-like peptide YJL-4 in a triple-negative breast cancer mouse model

BackgroundThe aim of this study was to prepare a novel 68Ga-labeled pH (low) insertion peptide (pHLIP)-like peptide, YJL-4, and determine its value for the early diagnosis of triple-negative breast cancer (TNBC) via in vivo imaging of tumor-bearing nude mice. The novel peptide YJL-4 was designed using a template-assisted method and synthesized by solid-phase peptide synthesis. After modification with the chelator 1,4,7‑triazacyclononane-N,N′,N″-triacetic acid (NOTA), the peptide was labeled with 68Ga. Then, the biodistribution of 68Ga-YJL-4 in tumor-bearing nude mice was investigated, and the mice were imaged by small animal positron emission tomography (PET).ResultsThe radiochemical yield and radiochemical purity of 68Ga-YJL-4 were 89.5 ± 0.16% and 97.95 ± 0.06%, respectively. The biodistribution of 68Ga-YJL-4 in tumors (5.94 ± 1.27% ID/g, 6.72 ± 1.69% ID/g and 4.54 ± 0.58% ID/g at 1, 2 and 4 h after injection, respectively) was significantly greater than that of the control peptide in tumors at the corresponding time points (P < 0.01). Of the measured off-target organs, 68Ga-YJL-4 was highly distributed in the liver and blood. The small animal PET imaging results were consistent with the biodistribution results. The tumors were visualized by PET at 2 and 4 h after the injection of 68Ga-YJL-4. No tumors were observed in the control group.ConclusionsThe novel pHLIP family peptide YJL-4 can adopt an α-helical structure for easy insertion into the cell membrane in an acidic environment. 68Ga-YJL-4 was produced in high radiochemical yield with good stability and can target TNBC tissue. Moreover, the strong concentration of radioactive 68Ga-YJL-4 in the abdomen does not hinder the imaging of early TNBC.

P148 CCN3-derived peptides as anti-fibrotic therapies for systemic sclerosis

Abstract Background/Aims Effective therapy for skin fibrosis in scleroderma (systemic sclerosis: SSc) remains an unmet clinical challenge. Persistently activated M2 macrophages are believed to stimulate myofibroblasts in the disease microenvironment creating a pro-fibrotic niche in the skin. Expression of the anti-fibrotic matricellular protein CCN3 is reduced in animal models of fibrosis. We have identified a small peptide based on an amino acid sequence in CCN3, BLR-200, which mimics the anti-fibrotic activity of CCN3, and propose that BLR-200 could be used to treat patients in whom lack of CCN3 activity is permissive for fibrosis. However, whether there is lack of CCN3 activity in SSc and whether BLR-200 has effective anti-fibrotic activity in SSc are unknown. Methods We used ELISAs (R&amp;D Systems) to detect CCN3 and the M2 macrophage marker CD206 in the serum of scleroderma patients with early stage diffuse subset disease (within two years of disease onset), those with late-stage diffuse subset disease (over five years duration) as well as healthy controls (all n = 20). The bleomycin-induced mouse model of skin scleroderma, in which bleomycin is injected subcutaneously every day for 21 days, was used to assess the antifibrotic ability of BLR200 in vivo. Results In scleroderma serum, CCN3 was significantly reduced, relative to matched healthy controls and patients with inactive disease, in early onset scleroderma patients with active disease that show elevated CD206 levels (p &amp;lt; 0.01). In the mouse model of human SSc, compared to injection with a scrambled control peptide and relative to control mice treated with phosphate buffered saline, injection with BLR-200 prevented bleomycin-induced changes in collagen deposition, myofibroblast differentiation and skin thickness as measured by Trichrome stain, indirect immunofluorescence analysis with an anti-a-smooth muscle actin antibody, and morphometric analysis, respectively (N = 8 mice per group, p &amp;lt; 0.05). Similarly, RNA sequencing and real-time polymerase chain reaction analysis revealed that BLR-200 significantly impaired the ability of bleomycin to induce expression of fibrogenic genes such as: integrin alpha 11, CCN2, CCN1, Smad3, wnt4, YAP1 and tenascin-C (all N = 5, p &amp;lt; 0.05). Spatial transcriptomics analysis revealed that BLR-200 impaired bleomycin-induced alterations in skin cell populations, including the activation and expansion of epithelial and reticular fibroblast niches and the induction of Wnt, hippo, focal adhesion and actin cytoskeleton gene expression cluster, all of which are known to be activators of fibrosis and myofibroblast activation and persistence. Conclusion CCN3 expression is reduced in serum of scleroderma patients with active skin disease that show M2 macrophage activation as visualized by elevated CD206 levels. Since BLR-200 has antifibrotic activity in the bleomycin model of skin scleroderma, our data are consistent with the hypothesis that BLR-200 could be used to block fibrosis progression in early onset diffuse scleroderma patients who possess both low CCN3 and high CD206 in serum. Disclosure J. Nguyen: None. K. Zestranjyan: None. S. Xu: None. B.L. Riser: Corporate appointments; CEO of BLR Bio. R.J. Stratton: None. A. Leask: None.

Merging Modular Molecular Design with High Throughput Screening of Cell Adhesion on Antimicrobial Supramolecular Biomaterials.

A polymer microarray based on the supramolecular ureido-pyrimidinone (UPy) moiety is fabricated to screen antimicrobial materials for their ability to support cell adhesion. UPy-functionalized additives, either cell-adhesive, antimicrobial or control peptides, are used, and investigated in different combinations at different concentrations, resulting in a library of 194 spots. These are characterized on composition and morphology to evaluate the microarray fabrication. Normal human dermal fibroblasts are cultured on the microarrays and cell adhesion to the spots is systematically analyzed. Results demonstrate enhanced cell adhesion on spots with combinations including the antimicrobial peptides. This study clearly proves the power of the high throughput approach in combination with supramolecular molecules, to screen additive libraries for desired biological response.

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 5750: Development of prostate specific membrane antigen-targeted liposomal zinc for the treatment of prostate cancer

Abstract Introduction: Treatment choices for early-stage prostate cancer include mostly watchful waiting, radical prostatectomy, or radiation therapy, whereas for advanced cases hormone therapy followed by chemo/radiotherapy are the preferred options, despite their notable side effects. There is a need for more effective therapy with lower side effects, especially for advanced scenarios. Unlike normal epithelial prostate cells, cancerous ones show reduced zinc (Zn) levels due to ZIP1 zinc transporter downregulation. Zn deficiency allows m-aconitase to remain active, making prostate cancer cells energy efficient and grow. High levels of Zn are needed to inhibit m-aconitase, the enzyme responsible for the first reaction in the Krebs cycle. This study aims to induce energy starvation and cell death of prostate cancer cells by inhibiting m-aconitase using the prostate-specific membrane antigen (PSMA)-targeting liposomal zinc. Methods: Liposomes were prepared by standard reverse phase evaporation method with DSPC, Cholesterol and DSPE-mPEG(2000) lipids. Zinc chloride was used to load Zn+2 into liposome following passive loading method. PSMA-expressing (PC3-PIP) and PSMA-null (PC3-LUC) human prostate cancer cells were used for in vitro studies. In the cell uptake study, cells were incubated with Dil-dye labeled liposomes for 2 hours before analyzing them by microscopy and flow cytometry. For the uptake inhibition study, cells were pre-incubated with PSMA-specific peptide for 2 hours before incubation with liposomes. Cytotoxicity studies were performed with PSMA-targeting Zn-loaded liposomes in PC3-PIP cells. Results: We conjugated the PSMA-specific peptide and control peptide (ASP3) with DSPE-PEG(3400)-NHS, and used them at various amounts (1 to 4 mol% of total lipid) to prepare Zn-loaded PSMA-targeting liposomes (TL) and control liposomes (CL), respectively. The average hydrodynamic diameter of all the liposomes was 120 to 130 nm, and zeta potential ranged from -16 to -12 mV. The encapsulation efficiency and loading capacity of Zn+2 were 0.6% and 8.5%, respectively. Cellular uptake studies showed a higher accumulation of PSMA-targeting liposome (TL) compared to non-targeting liposome (CL) in PSMA-expressing PC3 cells, whereas both liposomes showed low uptake in PSMA non-expressing cell. Furthermore, pre-treatment of PSMA-expressing cells with PSMA-specific peptide reduced the uptake of TL, indicating that the uptake of TL liposome is PSMA dependent. Finally, cytotoxicity studies showed that Zn-loaded TL, induces cell death in the PSMA-expressing cells. Overall, we developed PSMA-targeting Zn-containing liposomes that selectively target and kill PSMA-expressing prostate cancer cells. Future work includes testing the effect of Zn-loaded TL on m-aconitase activity and testing of in vivo efficacy of the formulation in prostate cancer models. Citation Format: Sujan K. Mondal, Alexander L. Klibanov, Anna Moore. Development of prostate specific membrane antigen-targeted liposomal zinc for the treatment of prostate 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 5750.

Abstract 1170: An off-the-shelf vaccine activates mutant KRAS-specific T cells in patients with resected pancreatic cancer

Abstract Background: Oncogenic mutations in KRAS (mKRAS) are expressed in up to 90% of pancreatic ductal adenocarcinomas (PDAC) therefore representing a promising immunotherapeutic target. We developed a pooled mKRAS peptide vaccine targeting the 6 most common mutations in PDAC: G12V, G12A, G12C, G12R, G12D, or G13D (NCT04117087). In a phase I study, we evaluated mKRAS-specific T cell responses raised by the vaccine when given in combination with immune checkpoint inhibitors to PDAC patients following resection and adjuvant chemotherapy. Materials and Methods: Patients received the pooled mKRAS peptide vaccine (0.3mg/peptide and 0.5mg poly-ICLC weekly for 4 doses in combination with ipilimumab and nivolumab followed by boosters every 8 weeks with nivolumab. To detect vaccine-induced mKRAS-specific T cells, pre- and post-vaccine peripheral blood mononuclear cells (PBMCs) were restimulated with control or individual mKRAS peptides and IFNγ release was measured by ELISPOT. mKRAS-specific T cell activation, proliferation, memory, and exhaustion profiles were assessed by CyTOF and cytokine secretion was determined by ELISA. For a subset of patients, mKRAS-specific T cells were expanded in vitro and T cell receptor (TCR) β chain sequencing and scRNA/TCRseq were used to identify the KRAS mutation-specific TCR repertoires and define their phenotypes in circulation. Results: mKRAS-specific T cells were detected by IFNγ ELISPOT post-vaccination for all patients, although magnitude and mutation-specific responses varied between patients. CyTOF analysis identified mKRAS-specific Th1 CD4+ central memory (cm, CCR7+IFNγ+) and effector memory (em, CCR7-IFNγ +IL2+ TNFɑ+) responses as well as CD8+ effector (eff, CD137+GZMBhiKi67hi) and effector memory (CCR7-CD137+GZMB+) responses. mKRAS-specific T cells were also detectable in the extended booster phase of treatment (&amp;gt;1 year post initial vaccination). In vitro expanded mKRAS-specific T cells mapped to CD4+ Tcm/em and CD8+ Tem populations within single cell datasets of unstimulated PBMCs collected post-vaccine. 5-25% of the mKRAS-specific T cells significantly expanded to more than one mKRAS antigen suggesting potential cross-reactive T cell clonotypes. In addition, shared public mKRAS-specific T cell clonotypes were identified across patients. Conclusions: This study demonstrates mKRAS vaccine induction of de novo, high quality mKRAS-specific polyfunctional CD4+ and CD8+ T cells. mKRAS-specific TCR analysis identified shared public TCRs across vaccinated patients that have important implications for future “off the shelf” adoptive therapy approaches. Ongoing studies are aimed to validate antigen specificity and map HLA-restriction of mKRAS-specific T cells. Overall, this study demonstrates a positive immunogenic signal of this pooled mutant KRAS vaccine that may be amenable for interception vaccination strategies against PDAC. Citation Format: Amanda L. Huff, Alex Girgis, Saurav D. Haldar, Emily Davis-Marcisak, Thatcher Heumann, Gabriella Longway, Lalitya Andaloori, Alexei Hernandez, Maximilian F. Konig, Brian Mog, Ludmila Danilova, Luciane T. Kagohara, Julie M. Nauroth, Amy M. Thomas, Elana J. Fertig, Won Jin Ho, Elizabeth M. Jaffee, Nilofer Azad, Neeha Zaidi. An off-the-shelf vaccine activates mutant KRAS-specific T cells in patients with resected pancreatic 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 1170.

Peptide-Drug Conjugate with Statistically Designed Transcellular Peptide for Psoriasis-Like Inflammation.

Peptide-drug conjugates (PDCs) are a promising class of drug delivery systems that utilize covalently conjugated carrier peptides with therapeutic agents. PDCs offer several advantages over traditional drug delivery systems including enhanced target engagement, improved bioavailability, and increased cell permeability. However, the development of efficient transcellular peptides capable of effectively transporting drugs across biological barriers remains an unmet need. In this study, physicochemical criteria based on cell-penetrating peptides are employed to design transcellular peptides derived from an antimicrobial peptides library. Among the statistically designed transcellular peptides (SDTs), SDT7 exhibits higher skin permeability, faster kinetics, and improved cell permeability in human keratinocyte cells compared to the control peptide. Subsequently, it is found that 6-Paradol (PAR) exhibits inhibitory activity against phosphodiesterase 4, which can be utilized for an anti-inflammatory PDC. The transcellular PDC (SDT7-conjugated with PAR, named TM5) is evaluated in mouse models of psoriasis, exhibiting superior therapeutic efficacy compared to PAR alone. These findings highlight the potential of transcellular PDCs (TDCs) as a promising approach for the treatment of inflammatory skin disorders.