Membrane-anchored Protein Research Articles

Glycosyl Mobile Radical Structures of Folic Acid Receptors Impact the Internalization of Functionalized Folate Amphiphilic Alternating Copolymer in Cancer Cells

Folate receptor alpha (FRα) is a glycosylphosphatidylinositol (GPI) membrane-anchored protein containing three N-glycosylated residues at the N47, N139, and N179 termini. These glycosylation sites have been reported to be crucial for the receptor’s structural integrity and its ability to bind and internalize FA. Here, we investigated the role of FRα glycosylation in the binding and internalization efficacy of FA–DABA–SMA in pancreatic PANC-1 cancer cells. There is a strong association of the FA copolymer with FRα with a Pearson coefficient R-value of 0.7179. PANC-1 cancer cells were pretreated with maackia amurensis lectin II (MAL-2), sambucus Nigra lectin (SNA-1), peanut agglutinin (PNA), and wheat germ agglutinin lectin (WGA) at different doses followed by 20 kDa and 350 kDa FA–DABA–SMA loaded with coumarin 153 (C153). Increasing the dosage of MAL2, SNA-1, PNA, and WGA concomitantly and significantly increased the internalization of C153-loaded FA–DABA–SMA in the cells. The half maximal effective lectin concentrations (EC50) to induce cellular internalization into the cytoplasm of the lectins for MAL-2 were 35.88 µg/mL, 3.051 µg/mL for SNA-1, 7.883 µg/mL for PNA, and 0.898 µg/mL for WGA. Live cell imaging of the internalization of 20 kDa and 350 kDa FA copolymers indicated an aggregation of 350 kDa copolymer with FRα in the cytoplasm. In contrast, the 20 kDa FA copolymer remained in the membrane. The data indicate for the first time that the mobile positions of the glycosyl radical groups and the receptor tilt in generating steric hindrance impacted the individual FRα receptors in the binding and internalization of 350 kDa FA–DABA–SMA in cancer cells.

Optogenetic Control of the Mitochondrial Protein Import in Mammalian Cells.

Mitochondria provide cells with energy and regulate the cellular metabolism. Almost all mitochondrial proteins are nuclear-encoded, translated on ribosomes in the cytoplasm, and subsequently transferred to the different subcellular compartments of mitochondria. Here, we developed OptoMitoImport, an optogenetic tool to control the import of proteins into the mitochondrial matrix via the presequence pathway on demand. OptoMitoImport is based on a two-step process: first, light-induced cleavage by a TEV protease cuts off a plasma membrane-anchored fusion construct in close proximity to a mitochondrial targeting sequence; second, the mitochondrial targeting sequence preceding the protein of interest recruits to the outer mitochondrial membrane and imports the protein fused to it into mitochondria. Upon reaching the mitochondrial matrix, the matrix processing peptidase cuts off the mitochondrial targeting sequence and releases the protein of interest. OptoMitoImport is available as a two-plasmid system as well as a P2A peptide or IRES sequence-based bicistronic system. Fluorescence studies demonstrate the release of the plasma membrane-anchored protein of interest through light-induced TEV protease cleavage and its localization to mitochondria. Cell fractionation experiments confirm the presence of the peptidase-cleaved protein of interest in the mitochondrial fraction. The processed product is protected from proteinase K treatment. Depletion of the membrane potential across the inner mitochondria membrane prevents the mitochondrial protein import, indicating an import of the protein of interest by the presequence pathway. These data demonstrate the functionality of OptoMitoImport as a generic system with which to control the post-translational mitochondrial import of proteins via the presequence pathway.

Ribosome-inactivation by a class of widely distributed C-tail anchored membrane proteins.

Ribosome hibernation is a commonly used strategy that protects ribosomes under unfavorable conditions and regulates developmental processes. Multiple ribosome-hibernation factors have been identified in all domains of life, but due to their structural diversity and the lack of a common inactivation mechanism, it is currently unknown how many different hibernation factors exist. Here, we show that the YqjD/ElaB/YgaM paralogs, initially discovered as membrane-bound ribosome binding proteins in E.coli, constitute an abundant class of ribosome-hibernating proteins, which are conserved across all proteobacteria and some other bacterial phyla. Our data demonstrate that they inhibit invitro protein synthesis by interacting with the 50S ribosomal subunit. Invivo cross-linking combined with mass spectrometry revealed their specific interactions with proteins surrounding the ribosomal tunnel exit and even their penetration into the ribosomal tunnel. Thus, YqjD/ElaB/YgaM inhibit translation by blocking the ribosomal tunnel and thus mimic the activity of antimicrobial peptides and macrolide antibiotics.

Cell-Specific Optical Control of AMPA Glutamate Receptors with a Photoswitchable Tethered Antagonist.

AMPA receptors (AMPARs) are the main drivers of excitatory glutamatergic transmission in the brain, central to synaptic plasticity, and are key drug targets. However, AMPARs are expressed in virtually every neuron in the central nervous system and are activated with complex temporal dynamics, making it difficult to determine their functional roles with sufficient precision.Here we describe a cell specific, light-controllable competitive antagonist for the AMPA receptor called MP-GluAblockthat combines the temporal precision of a photo-switchable ligand with the spatial and cellular specificity of a genetically-encoded membrane-anchor protein. This tool could pave the way for controlling endogenous AMPARs in neural circuits with cellular, spatial, and temporal specificity.

An Aptamer Glue Enables Hyperefficient Targeted Membrane Protein Degradation.

Targeted membrane protein degradation (TMPD) offers significant therapeutic potential by enabling the removal of harmful membrane-anchored proteins and facilitating detailed studies of complex biological pathways. However, existing TMPD methodologies face challenges such as complex molecular architectures, scarce availability, and cumbersome construction requirements. To address these issues, this study presents a highly efficient TMPD system (TMPDS) that integrates an optimized bivalent aptamer glue with a potent protein transport shuttle. Utilizing this approach, we successfully degraded both the highly expressed protein tyrosine kinase 7 in CCRF-CEM cells and the poorly expressed PTK7 in MV-411 cells. This system represents significant advancement in the field of molecular medicine, offering a new avenue for targeted therapeutic interventions and the exploration of cellular mechanisms.

Compound heterozygous mutations of NTNG2 cause intellectual disability via inhibition of the CaMKII signaling

Netrin-G2 is a membrane-anchored protein and is known to play critical roles in neuronal circuit development and synaptic organization. In this study, we identify compound heterozygous mutations of c.547delC, p.(Arg183Alafs∗186) and c.605G > A, p.(Trp202∗) in NTNG2 causing a syndrome exhibiting developmental delay, intellectual disability, hypotonia, and facial dysmorphism. To elucidate the underlying cellular and molecular mechanisms, CRISPR-Cas9 technology is employed to generate a knock-in mouse model expressing the R183Afs and W202X mutations. We report that the Ntng2R183Afs/W202X mice exhibit hypotonia and impaired learning and memory. We find that the levels of CaMKII and p-GluA1Ser831 are decreased and excitatory postsynaptic transmission and long-term potentiation are impaired. To increase the activity of CaMKII, the mutant mice have received intraperitoneal injections of DCP-LA, a CaMKII agonist, and show improved cognitive function. Together, our findings reveal molecular mechanisms of how NTNG2 deficiency leads to impairments of cognitive ability and synaptic plasticity.

The cytoskeleton controls the dynamics of plasma membrane proteins and facilitates their endocytosis in plants.

Plasma membranes (PMs) are highly dynamic structures where lipids and proteins can theoretically diffuse freely. However, reports indicate that PM proteins do not freely diffuse within their planes but are constrained by cytoskeleton networks, though the mechanisms for how the cytoskeleton restricts lateral diffusion of plant PM proteins are unclear. Through single-molecule tracking, we investigated the dynamics of six Arabidopsis (Arabidopsis thaliana) PM proteins with diverse structures and found distinctions in sizes and dynamics among these proteins. Moreover, we showed that the cytoskeleton, particularly microtubules, limits the diffusion of PM proteins, including transmembrane and membrane-anchoring proteins. Interestingly, the microfilament skeleton regulates intracellular transport of endocytic cargo. Therefore, these findings indicate that the cytoskeleton controls signal transduction by limiting diffusion of PM proteins in specific membrane compartments and participating in transport of internalized cargo vesicles, thus actively regulating plant signal transduction.

Biosilica-coated carbonic anhydrase displayed on Escherichia coli: A novel design approach for efficient and stable biocatalyst for CO2 sequestration

A robust and stable carbonic anhydrase (CA) system is indispensable for effectively sequestering carbon dioxide to mitigate climate change. While microbial surface display technology has been employed to construct an economically promising cell-displayed CO2-capturing biocatalyst, the displayed CA enzymes were prone to inactivation due to their low stability in harsh conditions. Herein, drawing inspiration from biomineralized diatom frustules, we artificially introduced biosilica shell materials to the CA macromolecules displayed on Escherichia coli surfaces. Specifically, we displayed a fusion of CA and the diatom-derived silica-forming Sil3K peptide (CA-Sil3K) on the E. coli surface using the membrane anchor protein Lpp-OmpA linker. The displayed CA-Sil3K (dCA-Sil3K) fusion protein underwent a biosilicification reaction under mild conditions, resulting in nanoscale self-encapsulation of the displayed enzyme in biosilica. The biosilicified dCA-Sil3K (BS-dCA-Sil3K) exhibited improved thermal, pH, and protease stability and retained 63 % of its initial activity after ten reuses. Additionally, the BS-dCA-Sil3K biocatalyst significantly accelerated the CaCO3 precipitation rate, reducing the time required for the onset of CaCO3 formation by 92 % compared to an uncatalyzed reaction. Sedimentation of BS-dCA-Sil3K on a membrane filter demonstrated a reliable CO2 hydration application with superior long-term stability under desiccation conditions. This study may open new avenues for the nanoscale-encapsulation of enzymes with biosilica, offering effective strategies to provide efficient, stable, and economic cell-displayed biocatalysts for practical applications.

Virus mimicking liposomes incorporated microneedles delivery platform for efficient skin penetration and enhanced cellular uptake

Transdermal delivery is an attractive route for the treatment of skin disease. However, its therapeutic effectiveness is significantly hampered by the stratum corneum and cell membrane barriers. Herein, we reported a novel liposomes incorporated microneedles (MNs) delivery platform to address these two key challenges. Conventional liposomes were surface modified with membrane-anchoring fusogenic proteins mimicking pH-responsive, comb-like pseudopeptides containing relatively long alkyl side chains. The liposomes-containing MNs were fabricated with biodegradable and biocompatible silk fibroin with sufficient mechanical strength using micromolding technology. The degradation and release profile of liposomes from the MNs delivery platform can be manipulated by controlling the liposomes to silk fibroin ratio, and the released liposomes well-maintained their original spherical shape and pH-responsive functionality. Remarkably enhanced in vitro skin permeation was observed in the polymer modified liposomes incorporating MNs delivery platform compared to the ones without MNs facilitation. Enhanced cellular uptake and potency against cancer cells of the pH-responsive, comb-like pseudopeptides modified liposomes incorporating MNs delivery platform were demonstrated. These results suggest its potential applications for efficient skin penetration and enhanced cellular uptake, which is of crucial importance for skin disease treatment.

Alpha-Synuclein Interaction with UBL3 Is Upregulated by Microsomal Glutathione S-Transferase 3, Leading to Increased Extracellular Transport of the Alpha-Synuclein under Oxidative Stress.

Aberrant aggregation of misfolded alpha-synuclein (α-syn), a major pathological hallmark of related neurodegenerative diseases such as Parkinson's disease (PD), can translocate between cells. Ubiquitin-like 3 (UBL3) is a membrane-anchored ubiquitin-fold protein and post-translational modifier. UBL3 promotes protein sorting into small extracellular vesicles (sEVs) and thereby mediates intercellular communication. Our recent studies have shown that α-syn interacts with UBL3 and that this interaction is downregulated after silencing microsomal glutathione S-transferase 3 (MGST3). However, how MGST3 regulates the interaction of α-syn and UBL3 remains unclear. In the present study, we further explored this by overexpressing MGST3. In the split Gaussia luciferase complementation assay, we found that the interaction between α-syn and UBL3 was upregulated by MGST3. While Western blot and RT-qPCR analyses showed that silencing or overexpression of MGST3 did not significantly alter the expression of α-syn and UBL3, the immunocytochemical staining analysis indicated that MGST3 increased the co-localization of α-syn and UBL3. We suggested roles for the anti-oxidative stress function of MGST3 and found that the effect of MGST3 overexpression on the interaction between α-syn with UBL3 was significantly rescued under excess oxidative stress and promoted intracellular α-syn to extracellular transport. In conclusion, our results demonstrate that MGST3 upregulates the interaction between α-syn with UBL3 and promotes the interaction to translocate intracellular α-syn to the extracellular. Overall, our findings provide new insights and ideas for promoting the modulation of UBL3 as a therapeutic agent for the treatment of synucleinopathy-associated neurodegenerative diseases.

LINC00525 enhances ZNF460-regulated CD24 expression through the sponge miR-125a-5p to promote malignant progression of breast cancer

IntroductionCD24 is a highly glycosylated glycosylphosphatidylinositol anchored membrane protein that plays an important role in tumor progression. The aim of this study was to investigate the effect of abnormal expression of CD24 on the proliferation, migration and invasion of breast cancer (BC) cells, and the molecular mechanism of regulating CD24 expression in breast cancer.MethodologyThe bioinformatics method was used to predict the expression level of CD24 in BC and its relationship with the occurrence and development of BC. IHC, RT-qPCR and WB were used to detect the expression of CD24 in BC tissues and cells. The proliferation of CD24 was evaluated by CCK-8 and colony formation assay, and the migration and invasion of CD24 were evaluated by wound healing and transwell. In addition, the effect of CD24 on the malignancy of BC in vivo was further evaluated by subcutaneous tumorigenesis assay. Molecular mechanisms were measured by luciferase reporter assays, biotin-labeled miRNA pull-down assay, RIP, and western blotting.ResultsThe results show that CD24 is highly expressed in breast cancer tissues and cell lines, and knockdown of CD24 in vivo and in vitro can inhibit the proliferation, migration and invasion of BC cells. Mechanistically, the transcription factor ZNF460 promotes its expression by binding to the CD24 promoter, and the expression of ZNF460 is regulated by miR-125a-5p, which inhibits its expression by targeting the 3’UTR of ZNF460. In addition, LINC00525 acts as a ceRNA sponge to adsorb miR-125a-5p and regulate its expression.ConclusionsOverexpression of CD24 is involved in the development and poor prognosis of BC, which can be used as a potential target for the treatment of BC and provide a theoretical basis for the treatment of BC.

SzM protein of Streptococcus equi ssp. zooepidemicus triggers the release of neutrophil extracellular traps depending on GSDMD

Streptococcus equissp.zooepidemicus (SEZ) is a crucial pathogen and contributes to various infections in numerous animal species. Swine streptococcicosis outbreak caused by SEZ has been reported in several countries in recent years. SzM protein is a cell membrane-anchored protein, which exhibits as an important virulence factor of SEZ. Effects of SzM protein on host innate immune need further study. Here, recombinant SzM (rSzM) protein of the SEZ was obtained, and mice were intraperitoneally injected with rSzM protein. We discovered that rSzM protein can recruit neutrophils into the injected site. In further study, neutrophils were isolated and treated with rSzM protein, NETs release were triggered by rSzM protein independently, and GSDMD protein was promoted-expressed and activated. In order to investigate the role of GSDMD in NETs formation, neutrophils isolated from WT mice and GSDMD−/− mice were treated with rSzM protein. The results showed that GSDMD deficiency suppressed the NETs release. In conclusion, SzM protein of SEZ can trigger the NETs release in a GSDMD-depending manner.

Safety and Immunogenicity of an mRNA-Based hMPV/PIV3 Combination Vaccine in Seropositive Children.

Human metapneumovirus (hMPV) and parainfluenza virus type 3 (PIV3) are common respiratory illnesses in children. The safety and immunogenicity of an investigational mRNA-based vaccine, mRNA-1653, encoding membrane-anchored fusion proteins of hMPV and PIV3, was evaluated in hMPV/PIV3-seropositive children. In this phase 1b randomized, observer-blind, placebo-controlled, dose-ranging study, hMPV/PIV3-seropositive children were enrolled sequentially into 2 dose levels of mRNA-1653 administered 2 months apart; children aged 12 to 36 months were randomized (1:1) to receive 10-μg of mRNA-1653 or placebo and children aged 12 to 59 months were randomized (3:1) to receive 30-μg of mRNA-1653 or placebo. Overall, 27 participants aged 18 to 55 months were randomized; 15 participants received 10-μg of mRNA-1653 (n = 8) or placebo (n = 7), whereas 12 participants received 30-μg of mRNA-1653 (n = 9) or placebo (n = 3). mRNA-1653 was well-tolerated at both dose levels. The only reported solicited local adverse reaction was tenderness at injection site; solicited systemic adverse reactions included grade 1 or 2 chills, irritability, loss of appetite, and sleepiness. A single 10-μg or 30-μg mRNA-1653 injection increased hMPV and PIV3 neutralizing antibody titers (geometric mean fold-rise ratio over baseline: hMPV-A = 2.9-6.1; hMPV-B = 6.2-13.2; PIV3 = 2.8-3.0) and preF and postF binding antibody concentrations (geometric mean fold-rise ratio: hMPV preF = 5.3-6.1; postF = 4.6-6.5 and PIV3 preF = 13.9-14.2; postF = 11.0-12.1); a second injection did not further increase antibody levels in these seropositive children. Binding antibody responses were generally preF biased. mRNA-1653 was well-tolerated and boosted hMPV and PIV3 antibody levels in seropositive children aged 12 to 59 months, supporting the continued development of mRNA-1653 or its components for the prevention of hMPV and PIV3.

The CELL NUMBER REGULATOR FW2.2 protein regulates cell-to-cell communication in tomato by modulating callose deposition at plasmodesmata.

FW2.2 (standing for FRUIT WEIGHT 2.2), the founding member of the CELL NUMBER REGULATOR (CNR) gene family, was the first cloned gene underlying a quantitative trait locus (QTL) governing fruit size and weight in tomato (Solanum lycopersicum). However, despite this discovery over 20 yr ago, the molecular mechanisms by which FW2.2 negatively regulates cell division during fruit growth remain undeciphered. In the present study, we confirmed that FW2.2 is a membrane-anchored protein whose N- and C-terminal ends face the apoplast. We unexpectedly found that FW2.2 is located at plasmodesmata (PD). FW2.2 participates in the spatiotemporal regulation of callose deposition at PD and belongs to a protein complex which encompasses callose synthases. These results suggest that FW2.2 has a regulatory role in cell-to-cell communication by modulating PD transport capacity and trafficking of signaling molecules during fruit development.

Identification of a novel Golgi-localized putative glycosyltransferase protein in Arabidopsis thaliana.

SNAREs play an important role in the process of membrane trafficking. In the present research, we investigated subcellular localization of an uncharacterized Arabidopsis thaliana protein reported to interact with a trans-Golgi network-localized Qa-SNARE, SYNTAXIN OF PLANTS 43. Based on the similarity of its amino acid sequence to metazoan fucosyltransferases, we have named this novel protein AtGTLP (Arabidopsis thaliana GlycosylTransferase-Like Protein) and predicted that it should be a member of yet uncharacterized family of Arabidopsis fucosyltransferases, as it shows no significant sequence similarity to fucosyltransferases previously identified in Arabidopsis. AtGTLP is a membrane-anchored protein, which exhibits a type II-like topology, with a single transmembrane helix and a globular domain in the C-terminal part of its amino acid sequence. Colocalization data we collected suggest that AtGTLP should localize mainly to Golgi apparatus, especially to certain zones of trans-Golgi. As single atgtlp-/- mutants showed no obvious difference in phenotype (primary root length and fresh mass), AtGTLP and proteins related to AtGTLP with high similarity in amino acid sequences may have redundant functions.

Abstract 2585: First in human study with a novel peptide binder to glypican-3, demonstrates high specificity as a PET imaging agent in patients with hepatocellular carcinoma

Abstract Introduction: Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. The American Association for the Study of Liver Disease recommends surveillance for high-risk individuals with abdominal ultrasound, triple phase contrast CT or MRI. However, these imaging techniques have limited sensitivity in classifying lesions smaller than 2 centimeters. The use of [18F]FDG Positron Emission Tomography (PET) is constrained due to insufficient uptake by HCC lesions over background liver uptake. Hence, there is an urgent need for more precise radiotracers for HCC. This study introduces the development of a novel radiotracer that binds to glypican-3 (GPC3), a cell membrane-anchored oncofetal protein overexpressed in HCC with minimal normal tissue expression. Methods: [68Ga]Ga-RYZ-GPC3 is composed of a small macrocyclic peptide binder with high affinity to GPC3 linked with a tetraxetan moiety capable of chelating a variety of radioisotopes including positron-emitting 68Ga and other radiometals. The unlabeled binder to GPC3 was synthesized by the sponsor and provided to three imaging centers in India as part of investigator-initiated trials reviewed and approved by each site’s institutional review board and ethics committee. Labeling with 68Ga and quality checks of the product was performed locally at each center. Patients with high clinical suspicion or with confirmed HCC who gave informed consent received injections of 1.5-5.5 mCi of [68Ga]Ga-RYZ-GPC3 and underwent PET-CT scanning at predetermined intervals. Some patients underwent companion [18F]FDG PET-CT scans in accordance with local guidelines. Results: A total of 64 patients underwent [68Ga]Ga-RYZ-GPC3 PET-CT scans. The median age within the cohort was 61 (41-83), 22% were female. Liver cirrhosis was present in 72% of patients, attributed to hepatitis B, hepatitis C or NASH in 17%, 13% and 20% of cases. Notably, 92% of patients exhibited at least one positive lesion on [68Ga]Ga-RYZ-GPC3 scans. The median SUVmax of GPC3-avid liver lesions was 15.5 (range 1.1 to 137.0) while the median liver SUVmean was 1.6 (0.3-7.9). Physiologic uptake was predominantly observed in the kidneys, the primary route of clearance, with a median SUVmean of 10.9 (2.2-20.7). Additionally, 20 patients underwent [18F]FDG PET-CT scan for comparison. The median SUVmax of lesions on 68Ga-RYZ-GPC3 vs [18F]FDG PET was 11.0 (1.1-137.0) vs 3.8 (1.2-12.2). Conclusion: Early human imaging results in patients with HCC indicate that [68Ga]Ga-RYZ-GPC3 has high tumor lesion specificity compared to normal liverand demonstrates the potential of [68Ga]Ga-RYZ-GPC3 to improve detection of HCC over conventional [18F]FDG PET-CT. In addition, the binder could be complexed with a therapeutic radioisotope as a novel targeted therapeutic option for HCC. Further investigation of this first-in-class binder to HCC is warranted. Citation Format: Chandresakhar Bal, Sanjana Ballal, Kumar Kallur, Anna Karmann, Ye Yuan, Jessica Rearden, Kathryn Shah, Charlotte Lorenz, Susan Moran, Ken Song, Ishita Sen. First in human study with a novel peptide binder to glypican-3, demonstrates high specificity as a PET imaging agent in patients with hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2585.

Abstract 6019: Biodistribution, molecular imaging and efficacy evaluation of a novel GPC3-targeted radiopharmaceutical therapy for hepatocellular carcinoma

Abstract Introduction: Glypican-3 (GPC3) is a membrane-anchored oncofetal protein with minimal expression in normal tissues. Significant upregulation of GPC3 protein has been observed in the majority of hepatocellular carcinomas (HCC), and is associated with poor prognosis. The differential expression of GPC3 between tumor and normal tissues provides an opportunity for targeted radiopharmaceutical therapy (RPT) to treat HCC, a leading cause for cancer-related deaths worldwide. Methods: The novel RPT agent RYZ-GPC3 comprises a small macrocyclic peptide binder with high affinity to GPC3 linked with a tetraxetan moiety capable of chelating a variety of radioisotopes. The affinity of peptide binders to GPC3 was determined by surface plasma resonance (SPR) and radioligand binding assays. Cellular internalization was radiometrically measured at multiple time points. Non-human primate (NHP) PET imaging was performed with 64Cu. In vivo biodistribution, monotherapy and combination treatments with 177Lu or 225Ac were performed in HCC xenografts. Human imaging was conducted in HCC patients with 68Ga PET/CT. Results: The novel agent is a highly potent (KD=0.7 nM) and selective peptide binder to GPC3 of human, mouse, canine and NHP origins, and is compatible with multiple radiometal isotopes. In vivo biodistribution study of [177Lu]Lu-RYZ-GPC3 in HCC xenografts showed sustained tumor uptake of 16.6, 16.4, and 8.8 %ID/g at 24, 48, and 96 hours with fast renal clearance, resulting in favorable tumor/kidney ratios of 3.6, 10.2, and 13.0, respectively. Minimal or no uptake was observed in other normal tissues. PET imaging in NHP confirmed absence of on-target uptake by liver or other organs, and fast renal clearance. As monotherapy, durable (>60 days) tumor regression (TGI>100%) was achieved with 177Lu (111 MBq)- and 225Ac (0.111 MBq)-labeled RYZ-GPC3. At 10x lower injected activity, 177Lu- and 225Ac-labeled RYZ-GPC3 significantly improved anti-tumor effect of lenvatinib in multiple HCC models. Furthermore, PET/CT imaging in a 70-year-old patient with histologically proven HCC demonstrated distinct and avid [68Ga]Ga-RYZ-GPC3 tumor uptake. Conclusion: Preclinical data and preliminary human imaging demonstrate the potential of the novel binder as a theranostic agent for the treatment of patients with GPC3+ HCC. Citation Format: Fanching Lin, Renee Clift, Steven Horton, Matt Guest, Alain Noncovich, Abhijit Bhat, Sanjana Ballal, Chandrasekhar Bal, Kathryn Shah, Anna Karmann, Gary Li. Biodistribution, molecular imaging and efficacy evaluation of a novel GPC3-targeted radiopharmaceutical therapy for hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6019.

Abstract 6020: GPC3-targeted radiopharmaceutical therapy for neuroendocrine prostate cancer

Abstract Introduction: Glypican-3 (GPC3) is a membrane-anchored oncofetal protein with minimal expression in normal tissues. Besides hepatocellular carcinoma, upregulation of GPC3 protein has been observed in a significant subset of treatment-emergent neuroendocrine prostate cancer (NEPC), which accounts for to 30% of prostate cancer cases refractory to anti-androgen and other therapies. The differential expression of GPC3 between NEPC cells and normal tissues provides a theranostic opportunity for targeted radiopharmaceutical therapy (RPT). Methods: The novel RPT agent RYZ-GPC3 comprises a small macrocyclic peptide binder with high affinity to GPC3 linked with a tetraxetan moiety capable of chelating a variety of radioisotopes. The expression of GPC3 in NEPC cells was assessed by flow cytometry and immunohistochemistry (IHC). The affinity of peptide binders to GPC3 was determined by surface plasma resonance (SPR) and radioligand binding assays. In vivo biodistribution of [177Lu]Lu-RYZ-GPC3 and anti-tumor therapy with [177Lu]Lu-RYZ-GPC3 or [225Ac]Ac-RYZ-GPC3 were performed in NEPC xenografts. Results: The novel agent is a highly potent and selective peptide binder to GPC3 of human, mouse and NHP origins, and is compatible with multiple radiometal isotopes. In vivo biodistribution study of [177Lu]Lu-RYZ-GPC3 in GPC3-positive NEPC xenografts showed sustained tumor uptake of 10.8, 12.6, 5.1, and 2.2 %ID/g at 2, 24, 72 and 168 hours post dose, respectively, with fast renal clearance. Minimal or no uptake was observed in other normal tissues. A single injection of [225Ac]Ac-RYZ-GPC3 (11.1 kBq) resulted in significant tumor growth inhibition (TGI 109%) with durable regression and prolonged survival. All treatments were well tolerated. Conclusion: Preclinical in vitro and in vivo data demonstrated the potential of the novel binder as a theranostic agent for the treatment of patients with GPC3+ NEPC. Citation Format: Renee Clift, Fanching Lin, Katrina Salvador, Matt Guest, Daniel Kim, Guangzhou Han, Gary Li. GPC3-targeted radiopharmaceutical therapy for neuroendocrine 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 6020.

Novel GPC3-targeting radiopharmaceutical therapy for hepatocellular carcinoma.

525 Background: Glypican-3 (GPC3) is a membrane-anchored oncofetal protein whose expression is largely absent in normal tissues. Significant upregulation of GPC3 protein has been observed in approximately 75% of hepatocellular carcinomas (HCC), and is associated with poor prognosis. The differential expression of GPC3 between tumor and normal tissues provides an opportunity for targeted radiopharmaceutical therapy (RPT) to treat HCC, a leading cause for cancer-related deaths worldwide. Methods: RAYZ-8009 comprises a novel macrocyclic peptide binder to GPC3, a linker, and a chelator that can be complexed with different radioisotopes. The affinity of peptide binders to GPC3 was determined by surface plasma resonance (SPR) and radioligand binding assays. Target-mediated cellular internalization was radiometrically measured at multiple time points. In vivo biodistribution, monotherapy and combination treatments with 177Lu or 225Ac were performed in HCC xenografts. Results: RAYZ-8009 showed high binding affinity (KD=0.7 nM) to human GPC3, with comparable affinity to GPC3 of human, mouse, canine and monkey origins, and no binding to other GPC family members. Potent cellular binding was confirmed in GPC3+ HepG2 cells, and was not affected by isotope switching. RAYZ-8009 achieved efficient internalization upon binding, with 42% internalized by 20 minutes in HepG2 cells. Biodistribution study of 177Lu-RAYZ-8009 showed sustained tumor uptake and fast renal clearance, with minimal or no uptake in other normal tissues. Exquisite tumor-specific uptake was also demonstrated in orthotopic HCC tumors with no uptake in surrounding normal liver tissue. Therapeutically, significant and durable anti-tumor effect and survival benefit were achieved with 177Lu- and 225Ac-labeled RAYZ-8009, as monotherapy or in combination with lenvatinib, in GPC3+ HCC xenografts. Conclusions: Preclinical in vitro and in vivo data demonstrate the potential of RAYZ-8009 as a theranostic agent for the treatment of patients with GPC3+ HCC.

Cell-membrane engineering strategies for clinic-guided design of nanomedicine.

Cells are the fundamental units of life. The cell membrane primarily composed of two layers of phospholipids (a bilayer) structurally defines the boundary of a cell, which can protect its interior from external disturbances and also selectively exchange substances and conduct signals from the extracellular environment. The complexity and particularity of transmembrane proteins provide the foundation for versatile cellular functions. Nanomedicine as an emerging therapeutic strategy holds tremendous potential in the healthcare field. However, it is susceptible to recognition and clearance by the immune system. To overcome this bottleneck, the technology of cell membrane coating has been extensively used in nanomedicines for their enhanced therapeutic efficacy, attributed to the favorable fluidity and biocompatibility of cell membranes with various membrane-anchored proteins. Meanwhile, some engineering strategies of cell membranes through various chemical, physical and biological ways have been progressively developed to enable their versatile therapeutic functions against complex diseases. In this review, we summarized the potential clinical applications of four typical cell membranes, elucidated their underlying therapeutic mechanisms, and outlined their current engineering approaches. In addition, we further discussed the limitation of this technology of cell membrane coating in clinical applications, and possible solutions to address these challenges.