Fibrotic immune microenvironment remodeling mediates superior anti-tumor efficacy of a nano-PD-L1 trap in hepatocellular carcinoma.

The local microenvironment where tumors develop can shape cancer progression and therapeutic outcome. Emerging evidence demonstrate that the efficacy of immune-checkpoint blockade (ICB) is undermined by fibrotic tumor microenvironment (TME). The majority of hepatocellular carcinoma (HCC) develops in liver fibrosis, in which the stromal and immune components may form a barricade against immunotherapy. Here, we report that nanodelivery of a programmed death-ligand 1 (PD-L1) trap gene exerts superior efficacy in treating fibrosis-associated HCC when compared with the conventional monoclonal antibody (mAb). In two fibrosis-associated HCC models induced by carbon tetrachloride and a high-fat, high-carbohydrate diet, the PD-L1 trap induced significantly larger tumor regression than mAb with no evidence of toxicity. Mechanistic studies revealed that PD-L1 trap, but not mAb, consistently reduced the M2 macrophage proportion in the fibrotic liver microenvironment and promoted cytotoxic interferon gamma (IFNγ)+tumor necrosis factor α (TNF-α)+CD8+T cell infiltration to the tumor. Moreover, PD-L1 trap treatment was associated with decreased tumor-infiltrating polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) accumulation, resulting in an inflamed TME with a high cytotoxic CD8+T cell/PMN-MDSC ratio conductive to anti-tumor immune response. Single-cell RNA sequencing analysis of two clinical cohorts demonstrated preferential PD-L1 expression in M2 macrophages in the fibrotic liver, thus supporting the translational potential of nano-PD-L1 trap for fibrotic HCC treatment.

Locally Trapping the C-C Chemokine Receptor Type 7 by Gene Delivery Nanoparticle Inhibits Lymphatic Metastasis Prior to Tumor Resection.

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Currently, no targeted treatment is available for TNBC, and the most common clinical therapy is tumor resection, which often promotes metastasis risks. Strong evidence suggests that the lymphatic metastasis is mediated by the C-C chemokine receptor type 7 (CCR7)/C-C motif chemokine ligand 21 crosstalk between tumor cells and the lymphatic system. It is hypothesized that CCR7 is a key immune modulator in the tumor microenvironment and the local blockade of CCR7 could effectively inhibit TNBC lymphatic metastasis. Accordingly, a plasmid encoding an antagonistic CCR7 affinity protein-CCR7 trap is delivered by tumor targeting nanoparticles in a highly metastatic 4T1 TNBC mouse model. Results show that CCR7 traps are transiently expressed, locally disrupt the signaling pathways in the tumor site, and efficiently inhibit TNBC lymphatic metastasis, without inducing immunosuppression as observed in systemic therapies using CCR7 monoclonal antibody. Significantly, upon applying CCR7 trap therapy prior to tumor resection, a 4T1 TNBC mouse model shows good prognosis without any further metastasis and relapse. In addition, CCR7 trap therapy efficiently inhibits the lymphatic metastasis in a B16F10 melanoma mouse model, indicating its great potential for various metastatic diseases treatment.

LOVTRAP: an optogenetic system for photoinduced protein dissociation.

LOVTRAP is an optogenetic approach for reversible light-induced protein dissociation using protein A fragments that bind to the LOV domain only in the dark, with tunable kinetics and a >150-fold change in the dissociation constant (Kd). By reversibly sequestering proteins at mitochondria, we precisely modulated the proteins' access to the cell edge, demonstrating a naturally occurring 3-mHz cell-edge oscillation driven by interactions of Vav2, Rac1, and PI3K proteins.

Hepatoma-intrinsic CCRK inhibition diminishes myeloid-derived suppressor cell immunosuppression and enhances immune-checkpoint blockade efficacy.

OBJECTIVE: Myeloid-derived suppressor cells (MDSCs) contribute to tumour immunosuppressive microenvironment and immune-checkpoint blockade resistance. Emerging evidence highlights the pivotal functions of cyclin-dependent kinases (CDKs) in tumour immunity. Here we elucidated the role of tumour-intrinsic CDK20, or cell cycle-related kinase (CCRK) on immunosuppression in hepatocellular carcinoma (HCC). DESIGN: Immunosuppression of MDSCs derived from patients with HCC and relationship with CCRK were determined by flow cytometry, expression analyses and co-culture systems. Mechanistic studies were also conducted in liver-specific CCRK-inducible transgenic (TG) mice and Hepa1-6 orthotopic HCC models using CRISPR/Cas9-mediated Ccrk depletion and liver-targeted nanoparticles for interleukin (IL) 6 trapping. Tumorigenicity and immunophenotype were assessed on single or combined antiprogrammed death-1-ligand 1 (PD-L1) therapy. RESULTS: Tumour-infiltrating CD11b+CD33+HLA-DR- MDSCs from patients with HCC potently inhibited autologous CD8+T cell proliferation. Concordant overexpression of CCRK and MDSC markers (CD11b/CD33) positively correlated with poorer survival rates. Hepatocellular CCRK stimulated immunosuppressive CD11b+CD33+HLA-DR- MDSC expansion from human peripheral blood mononuclear cells through upregulating IL-6. Mechanistically, CCRK activated nuclear factor-κB (NF-κB) via enhancer of zeste homolog 2 (EZH2) and facilitated NF-κB-EZH2 co-binding to IL-6 promoter. Hepatic CCRK induction in TG mice activated the EZH2/NF-κB/IL-6 cascade, leading to accumulation of polymorphonuclear (PMN) MDSCs with potent T cell suppressive activity. In contrast, inhibiting tumorous Ccrk or hepatic IL-6 increased interferon γ+tumour necrosis factor-α+CD8+ T cell infiltration and impaired tumorigenicity, which was rescued by restoring PMN-MDSCs. Notably, tumorous Ccrk depletion upregulated PD-L1 expression and increased intratumorous CD8+ T cells, thus enhancing PD-L1 blockade efficacy to eradicate HCC. CONCLUSION: Our results delineate an immunosuppressive mechanism of the hepatoma-intrinsic CCRK signalling and highlight an overexpressed kinase target whose inhibition might empower HCC immunotherapy.

Discovery of small molecule inhibitors for the C. elegans caspase CED-3 by high-throughput screening.

C. elegans has been widely used as a model organism for programmed cell death and apoptosis. Although the CED-3 caspase is the primary effector of cell death in C. elegans, no selective inhibitors have been identified. Utilizing high-throughput screening with recombinant C. elegans CED-3 protein, we have discovered and confirmed 21 novel small molecule inhibitors. Six compounds had IC50 values < 10 µM. From these, four distinct chemotypes were identified. The inhibitor scaffolds described here could lead to the development of selective molecular probes to facilitate our understanding of programmed cell death in this model organism.

Targeted PRINT Hydrogels: The Role of Nanoparticle Size and Ligand Density on Cell Association, Biodistribution, and Tumor Accumulation.

In this Letter, we varied targeting ligand density of an EGFR binding affibody on the surface of two different hydrogel PRINT nanoparticles (80 nm × 320 and 55 nm × 60 nm) and monitored effects on target-cell association, off-target phagocytic uptake, biodistribution, and tumor accumulation. Interestingly, variations in ligand density only significantly altered in vitro internalization rates for the 80 nm × 320 nm particle. However, in vivo, both particle sizes experienced significant changes in biodistribution and pharmacokinetics as a function of ligand density. Overall, nanoparticle size and passive accumulation were the dominant factors eliciting tumor sequestration.

In vitro selection of proteins with desired characteristics using mRNA-display.

mRNA-display is an amplification-based, iterative rounds of in vitro protein selection technique that circumvents a number of difficulties associated with yeast two-hybrid and phage display. Because of the covalent linkage between the genotype and the phenotype, mRNA-display provides a powerful means for reading and amplifying a peptide or protein sequence after it has been selected from a library with very high diversity. The purpose of this article is to provide a summary of the field and practical framework of mRNA-display-based selections. We summarize the advantages and limitations of selections using mRNA-display as well as the recent applications, namely, the identification of novel affinity reagents, target-binding partners, and enzyme substrates from synthetic peptide or natural proteome libraries. Practically, we provide a detailed procedure for performing mRNA-display-based selections with the aim of identifying protease substrates and binding partners of a target protein. Furthermore, we describe how to confirm the function of the selected protein sequences by biochemical assays and bioinformatic tools.

Universal STING mimic boosts antitumour immunity via preferential activation of tumour control signalling pathways.

The efficacy of STING (stimulator of interferon genes) agonists is due to various factors, primarily inefficient intracellular delivery, low/lack of endogenous STING expression in many tumours, and a complex balance between tumour control and progression. Here we report a universal STING mimic (uniSTING) based on a polymeric architecture. UniSTING activates STING signalling in a range of mouse and human cell types, independent of endogenous STING expression, and selectively stimulates tumour control IRF3/IFN-I pathways, but not tumour progression NF-κB pathways. Intratumoural or systemic injection of uniSTING-mRNA via lipid nanoparticles (LNPs) results in potent antitumour efficacy across established and advanced metastatic tumour models, including triple-negative breast cancer, lung cancer, melanoma and orthotopic/metastatic liver malignancies. Furthermore, uniSTING displays an effective antitumour response superior to 2'3'-cGAMP and ADU-S100. By favouring IRF3/IFN-I activity over the proinflammatory NF-κB signalling pathway, uniSTING promotes dendritic cell maturation and antigen-specific CD8+ T-cell responses. Extracellular vesicles released from uniSTING-treated tumour cells further sensitize dendritic cells via exosome-containing miRNAs that reduced the immunosuppressive Wnt2b, and a combination of LNP-uniSTING-mRNA with α-Wnt2b antibodies synergistically inhibits tumour growth and prolongs animal survival. Collectively, these results demonstrate the LNP-mediated delivery of uniSTING-mRNA as a strategy to overcome the current STING therapeutic barriers, particularly for the treatment of multiple cancer types in which STING is downregulated or absent.

Tribody: robust self-assembled trimeric targeting ligands with high stability and significantly improved target-binding strength.

The C-terminal coiled-coil region of mouse and human cartilage matrix protein (CMP) self-assembles into a parallel trimeric complex. Here, we report a general strategy for the development of highly stable trimeric targeting ligands (tribodies), against epidermal growth factor receptor (EGFR) and prostate-specific membrane antigen (PSMA) as examples, by fusing a specific target-binding moiety with a trimerization domain derived from CMP. The resulting fusion proteins can efficiently self-assemble into a well-defined parallel homotrimer with high stability. Surface plasmon resonance (SPR) analysis of the trimeric targeting ligands demonstrated significantly enhanced target-binding strength compared with the corresponding monomers. Cellular-binding studies confirmed that the trimeric targeting ligands have superior binding strength toward their respective receptors. Significantly, the EGFR-binding tribody was considerably accumulated in the tumor of mice bearing xenografted EGFR-positive tumors, indicating its effective cancer-targeting feature under in vivo conditions. Our results demonstrate that CMP-based self-assembly of tribodies can be a general strategy for the facile and robust generation of trivalent targeting ligands for a wide variety of in vitro and in vivo applications.

Ligand-installed polymeric nanocarriers for combination chemotherapy of EGFR-positive ovarian cancer.

Combination chemotherapeutic drugs administered via a single nanocarrier for cancer treatment provides benefits in reducing dose-limiting toxicities, improving the pharmacokinetic properties of the cargo and achieving spatial-temporal synchronization of drug exposure for maximized synergistic therapeutic effects. In an attempt to develop such a multi-drug carrier, our work focuses on functional multimodal polypeptide-based polymeric nanogels (NGs). Diblock copolymers poly (ethylene glycol)-b-poly (glutamic acid) (PEG-b-PGlu) modified with phenylalanine (Phe) were successfully synthesized and characterized. Self-assembly behavior of the resulting polymers was utilized for the synthesis of NGs with hydrophobic domains in cross-linked polyion cores coated with inert PEG chains. The resulting NGs were small (ca. 70 nm in diameter) and were able to encapsulate the combination of drugs with different physicochemical properties such as cisplatin and neratinib. Drug combination-loaded NGs exerted a selective synergistic cytotoxicity towards EGFR overexpressing ovarian cancer cells. Moreover, we developed ligand-installed EGFR-targeted NGs and tested them as an EGFR-overexpressing tumor-specific delivery system. Both in vitro and in vivo, ligand-installed NGs displayed preferential associations with EGFR (+) tumor cells. Ligand-installed NGs carrying cisplatin and neratinib significantly improved the treatment response of ovarian cancer xenografts. We also confirmed the importance of simultaneous administration of the dual drug combination via a single NG system which provides more therapeutic benefit than individual drug-loaded NGs administered at equivalent doses. This work illustrates the potential of our carrier system to mediate efficient delivery of a drug combination to treat EGFR overexpressing cancers.

Development of 18F-Labeled hydrophilic trans-cyclooctene as a bioorthogonal tool for PET probe construction.

We report the development of a hydrophilic 18F-labeled a-TCO derivative [18F]3 (log P = 0.28) through a readily available precursor and a single-step radiofluorination reaction (RCY up to 52%). We demonstrated that [18F]3 can be used to construct not only multiple small molecule/peptide-based PET agents, but protein/diabody-based imaging probes in parallel.

Unexpected multivalent display of proteins by temperature triggered self-assembly of elastin-like polypeptide block copolymers.

We report herein the unexpected temperature triggered self-assembly of proteins fused to thermally responsive elastin-like polypeptides (ELPs) into spherical micelles. A set of six ELP block copolymers (ELP(BC)) differing in hydrophilic and hydrophobic block lengths were genetically fused to two single domain proteins, thioredoxin (Trx) and a fibronectin type III domain (Fn3) that binds the α(v)ß(3) integrin. The self-assembly of these protein-ELP(BC) fusions as a function of temperature was investigated by UV spectroscopy, light scattering, and cryo-TEM. Self-assembly of the ELP(BC) was unexpectedly retained upon fusion to the two proteins, resulting in the formation of spherical micelles with a hydrodynamic radius that ranged from 24 to 37 nm, depending on the protein and ELP(BC). Cryo-TEM images confirmed the formation of spherical particles with a size that was consistent with that measured by light scattering. The bioactivity of Fn3 was retained when presented by the ELP(BC) micelles, as indicated by the enhanced uptake of the Fn3-decorated ELP(BC) micelles in comparison to the unimer by cells that overexpress the α(v)ß(3) integrin. The fusion of single domain proteins to ELP(BC)s may provide a ubiquitous platform for the multivalent presentation of proteins.

Nano-trapping CXCL13 reduces regulatory B cells in tumor microenvironment and inhibits tumor growth.

Interactions between different cell types in the tumor microenvironment (TME) affect tumor growth. Tumor-associated fibroblasts produce C-X-C motif chemokine ligand 13 (CXCL13) which recruits B cells to the TME. B-cells in the TME differentiate into regulatory B cells (Bregs) (IL-10+CD1d+CD5+CD138+CD19+). We highlight these Breg cells as a new important factor in the modulation of the immunosuppressive TME in different desmoplastic murine tumor models. In addition, CXCL13 also stimulates epithelial-mesenchymal transition (EMT) of the tumor cells. The tumorigenic roles of CXCL13 led us to explore an innovative anti-cancer strategy based on delivering plasmid DNA encoding a CXCL13 trap to reduce Bregs differentiation and normalize EMT, thereby suppressing tumor growth. CXCL13 trap suppressed tumor growth in pancreatic cancer, BRAF-mutant melanoma, and triple-negative breast cancer. In this study, following treatment, the affected tumor remained dormant resulting in prolonged progression-free survival of the host.

Identification of a lacosamide binding protein using an affinity bait and chemical reporter strategy: 14-3-3 ζ.

We have advanced a useful strategy to elucidate binding partners of ligands (drugs) with modest binding affinity. Key to this strategy is attaching to the ligand an affinity bait (AB) and a chemical reporter (CR) group, where the AB irreversibly attaches the ligand to the receptor upon binding and the CR group is employed for receptor detection and isolation. We have tested this AB&CR strategy using lacosamide ((R)-1), a low-molecular-weight antiepileptic drug. We demonstrate that using a (R)-lacosamide AB&CR agent ((R)-2) 14-3-3 ζ in rodent brain soluble lysates is preferentially adducted, adduction is stereospecific with respect to the AB&CR agent, and adduction depends upon the presence of endogenous levels of the small molecule metabolite xanthine. Substitution of lacosamide AB agent ((R)-5) for (R)-2 led to the identification of the 14-3-3 ζ adduction site (K120) by mass spectrometry. Competition experiments using increasing amounts of (R)-1 in the presence of (R)-2 demonstrated that (R)-1 binds at or near the (R)-2 modification site on 14-3-3 ζ. Structure-activity studies of xanthine derivatives provided information concerning the likely binding interaction between this metabolite and recombinant 14-3-3 ζ. Documentation of the 14-3-3 ζ-xanthine interaction was obtained with isothermal calorimetry using xanthine and the xanthine analogue 1,7-dimethylxanthine.

Advantages of mRNA display selections over other selection techniques for investigation of protein-protein interactions.

mRNA display is a genotype-phenotype conjugation method that allows for amplification-based, iterative rounds of in vitro selection to be applied to peptides and proteins. mRNA display can be used to display both long natural protein and short synthetic peptide libraries with unusually high diversities for the investigation of protein-protein interactions. Here, we summarize the advantages of mRNA display by comparing it with other widely used peptide or protein-selection techniques, and discuss various applications of this technique in studying protein-protein interactions.

mRNA Delivery of a Bispecific Single-Domain Antibody to Polarize Tumor-Associated Macrophages and Synergize Immunotherapy against Liver Malignancies.

Liver malignancies are among the tumor types that are resistant to immune checkpoint inhibition therapy. Tumor-associated macrophages (TAMs) are highly enriched and play a major role in inducing immunosuppression in liver malignancies. Herein, CCL2 and CCL5 are screened as two major chemokines responsible for attracting TAM infiltration and inducing their polarization toward cancer-promoting M2-phenotype. To reverse this immunosuppressive process, an innovative single-domain antibody that bispecifically binds and neutralizes CCL2 and CCL5 (BisCCL2/5i) with high potency and specificity is directly evolved. mRNA encoding BisCCL2/5i is encapsulated in a clinically approved lipid nanoparticle platform, resulting in a liver-homing biomaterial that allows transient yet efficient expression of BisCCL2/5i in the diseased organ in a multiple dosage manner. This BisCCL2/5i mRNA nanoplatform significantly induces the polarization of TAMs toward the antitumoral M1 phenotype and reduces immunosuppression in the tumor microenvironment. The combination of BisCCL2/5i with PD-1 ligand inhibitor (PD-Li) achieves long-term survival in mouse models of primary liver cancer and liver metastasis of colorectal and pancreatic cancers. The work provides an effective bispecific targeting strategy that could broaden the PD-Li therapy to multiple types of malignancies in the human liver.

Tumor-targeted gene therapy with lipid nanoparticles inhibits tumor-associated adipocytes and remodels the immunosuppressive tumor microenvironment in triple-negative breast cancer.

Adipocytes are the primary cellular components within the tumor microenvironment (TME) of triple-negative breast cancer (TNBC). Increasing evidence suggests that tumor-associated adipocytes (TAAs) can aggravate tumor progression, exacerbate the immunosuppressive TME and compromise therapeutic efficacy. In this study, the biological effect of TAAs within the breast cancer TME is first investigated, and the C-C Motif Chemokine Ligand 2 (CCL2) which is mainly secreted by TAAs in the extracellular environment is identified as the key mediator. CCL2 recruits immune cells such as monocytes and macrophages that further differentiated into immunosuppressive myeloid-derived suppressor cells (MDSCs) and M2 macrophages. To manipulate CCL2-mediated immune response, a protein trap that binds with CCL2 with high affinity and specificity is designed. The plasmid DNA encoding the CCL2 trap (pCCL2) is specifically delivered to the TME by using targeted lipid-protamine-DNA (LPD) nanoparticles to locally express the CCL2 trap and ameliorate the immunosuppressive TME. Significantly, compared with the commercially available CCL2 antibody, this strategy shows enhanced therapeutic efficacy and appreciable tumor growth inhibition. Furthermore, the pCCL2 trap treatment successfully suppresses TAAs, increases T cell infiltration and decreases the population of immunosuppressive M2 macrophages and MDSCs. Further studies show that the pCCL2 trap could facilitate PD-L1 blockade immunotherapy, demonstrating its translation potential.

Proteomic searches comparing two (R)-lacosamide affinity baits: An electrophilic arylisothiocyanate and a photoactivated arylazide group.

We have advanced a novel strategy to search for lacosamide ((R)-1) targets in the brain proteome where protein binding is expected to be modest. Our approach used lacosamide agents containing affinity bait (AB) and chemical reporter (CR) units. The affinity bait moiety is designed to irreversibly react with the target, and the CR group permits protein detection and capture. In this study, we report the preparation and evaluation of (R)-N-(4-azido)benzyl 2-acetamido-3-(prop-2-ynyloxy)propionamide ((R)-3) and show that this compound exhibits potent anticonvulsant activities in the MES seizure model in rodents. We compared the utility of (R)-3 with its isostere, (R)-N-(4-isothiocyanato)benzyl 2-acetamido-3-(prop-2-ynyloxy)propionamide ((R)-2), in proteomic studies designed to identify potential (R)-1 targets. We showed that despite the two-fold improved anticonvulsant activity of (R)-3 compared with (R)-2, (R)-2 was superior in revealing potential binding targets in the mouse brain soluble proteome. The difference in these agents utility has been attributed to the reactivity of the affinity baits (i.e., (R)-2: aryl isothiocyanate moiety; (R)-3: photoactivated aryl azide intermediates) in the irreversible protein modification step, and we conclude that this factor is a critical determinant of successful target detection where ligand (drug) binding is modest. The utility of (R)-2 and (R)-3 in in situ proteome studies is explored.

Tumor neoantigen heterogeneity impacts bystander immune inhibition of pancreatic cancer growth.

The immunogenic clonal-fraction threshold in heterogeneous solid-tumor required to induce effective bystander-killing of non-immunogenic subclones is unknown. Pancreatic cancer poses crucial challenges for immune therapeutic interventions due to low mutational-burden and consequent lack of neoantigens. Here, we designed a model to incorporate artificial-neoantigens into genes-of -interest in cancer-cells and to test their potential to actuate bystander-killing. By precisely controlling a neoantigen's abundance in the tumor, we studied the impact of neoantigen frequency on immune-response and immune-escape. Our results showed single, strong, widely-expressed neoantigen could lead to robust antitumor response when over 80% of cancer cells express the neoantigen. Further, immunological assays demonstrated T-cell responses against non-target self-antigen on KRAS-oncoprotein, when we inoculated animals with a high frequency of tumor-cells expressing test-neoantigen. Using nanoparticle-based gene-therapy, we successfully altered tumor-microenvironment by perturbing interleukin-12 and interleukin-10 gene-expression. The subsequent microenvironment-remodeling reduced the neoantigen frequency threshold at which bioluminescent signal intensity for tumor-burden decreased 1.5-log-fold, marking robust tumor-growth inhibition, from 83% to 29%. Our results thus suggest bystander killing is inefficient in immunologically-cold tumors like pancreatic-cancer and requires high neoantigen abundance. However, bystander killing mediated antitumor response can be rescued by adjuvant-immune therapy.