How the initial discovery of modified RNA enabled evasion of innate immune responses and facilitated the development of RNA therapeutics.

Besides being the physical link between DNA and proteins, RNAs play several other key roles, including RNA catalysis and gene regulation. Recent advances in the design of lipid nanoparticles have facilitated the development of RNA-based therapeutics. However, chemically and in vitro transcribed RNAs can activate innate immunity, leading to the production of proinflammatory cytokines and interferons, a response similar to the one induced by viral infections. Since these responses are undesirable for certain therapeutic applications, it is important to develop ways to block the sensing of exogenous RNAs by immune cells, such as monocytes, macrophages and dendritic cells. Fortunately, RNA sensing can be blocked by chemical modifications of certain nucleotides, particularly uridine, a finding that has facilitated the development of RNA-based therapeutics such as small interfering RNAs and mRNA vaccines. Here, I provide a backstory on how improved understanding of RNA sensing by innate immunity can be applied to develop more effective RNA therapeutics.

Tumor-Associated Macrophage Subsets: Shaping Polarization and Targeting.

The tumor microenvironment (TME) is a critical regulator of tumor growth, progression, and metastasis. Among the innate immune cells recruited to the tumor site, macrophages are the most abundant cell population and are present at all stages of tumor progression. They undergo M1/M2 polarization in response to signals derived from TME. M1 macrophages suppress tumor growth, while their M2 counterparts exert pro-tumoral effects by promoting tumor growth, angiogenesis, metastasis, and resistance to current therapies. Several subsets of the M2 phenotype have been observed, often denoted as M2a, M2b, M2c, and M2d. These are induced by different stimuli and differ in phenotypes as well as functions. In this review, we discuss the key features of each M2 subset, their implications in cancers, and highlight the strategies that are being developed to harness TAMs for cancer treatment.

Structural Requirements for the Binding of a Peptide to Prohibitins on the Cell Surface of Monocytes/Macrophages.

The screening of phage peptide libraries resulted in the identification of a sequence (named NW peptide, NWYLPWLGTNDW) that specifically binds to human monocytes and macrophages. Although the NW peptide can be used for the targeted delivery of therapeutics without knowledge of its receptor(s), the identification of-its binding partners will support future clinical applications-Here, we used the biotinylated NW peptide for cross-linking cell surface receptor(s) on live cells or as bait in pull-down assays with membrane proteins isolated from monocytes or human THP-1 cells differentiated into macrophages. Proteomic analysis of the captured proteins identified cell surface prohibitins (PHB1 and PHB2) and modified albumin as binding partners. Using flow cytometry and pull-down methods, we demonstrated that PHB1 and PHB2 interact directly with the NW peptide. Confocal imaging showed co-localization of the peptide with PHB1 on the surface of monocytes. Single replacement of either tryptophan or leucine with alanine completely inhibited binding, whereas the replacement of asparagine at position 1 or 10 and aspartic acid at position 11 with alanine did not affect the binding of the peptide variants. Neutral amino acid replacement of tryptophan at positions 2, 6, and 12 with tyrosine or phenylalanine also abolished the binding, implying that the indole ring of tryptophan is indispensable for the NW peptide to bind. Overall, the data suggest that membrane-associated prohibitins might be a useful target for the delivery of therapeutics to monocytes/macrophages and that tryptophan and leucine are key residues for peptide binding.

Microbial sensing by haematopoietic stem and progenitor cells: Vigilance against infections and immune education of myeloid cells.

Bone marrow haematopoietic stem and progenitor cells (HSPCs) express pattern recognition receptors such as Toll-like receptors (TLRs) to sense microbial products and activation of these innate immune receptors induces cytokine expression and redirects bone marrow haematopoiesis towards the increased production of myeloid cells. Secreted cytokines by HSPCs in response to TLR ligands can act in an autocrine or paracrine manner to regulate haematopoiesis. Moreover, tonic activation of HSPCs by microbiota-derived compounds might educate HSPCs to produce superior myeloid cells equipped with innate memory responses to combat pathogens. While haematopoietic stem cell activation through TLRs meets the increased demand for blood leucocytes to protect the host against infection, persistent exposure to inflammatory cytokines or microbial products might impair their function and even induce malignant transformation. This review highlights the potential outcomes of HSPCs in response to TLR ligands.

Development of a new high-affinity human antibody with antitumor activity against solid and blood malignancies.

mAbs have emerged as a promising strategy for the treatment of cancer. However, in several malignancies, no effective antitumor mAbs are yet available. Identifying therapeutic mAbs that recognize common tumor antigens could render the treatment widely applicable. Here, a human single-chain variable fragment (scFv) antibody library was sequentially affinity selected against a panel of human cancer cell lines and an antibody fragment (named MS5) that bound to solid and blood cancer cells was identified. The MS5 scFv was fused to the human IgG1 Fc domain to generate an antibody (MS5-Fc fusion) that induced antibody-dependent cellular cytotoxicity and phagocytosis of cancer cells by macrophages. In addition, the MS5-Fc antibody bound to primary leukemia cells and induced antibody-dependent cellular cytotoxicity. In the majority of analyzed cancer cells, the MS5-Fc antibody induced cell surface redistribution of the receptor complexes, but not internalization, thus maximizing the accessibility of the IgG1 Fc domain to immune effector cells. In vitro stability studies showed that the MS5-Fc antibody was stable after 6 d of incubation in human serum, retaining ∼60% of its initial intact form. After intravenous injections, the antibody localized into tumor tissues and inhibited the growth of 3 different human tumor xenografts (breast, lymphoma, and leukemia). These antitumor effects were associated with tumor infiltration by macrophages and NK cells. In the Ramos B-cell lymphoma xenograft model, the MS5-Fc antibody exhibited a comparable antitumor effect as rituximab, a chimeric anti-CD20 IgG1 mAb. These results indicate that human antibodies with pan-cancer abilities can be generated from phage display libraries, and that the engineered MS5-Fc antibody could be an attractive agent for further clinical investigation.-Sioud, M., Westby, P., Vasovic, V., Fløisand, Y., Peng, Q. Development of a new high-affinity human antibody with antitumor activity against solid and blood malignancies.

T-cell cross-reactivity may explain the large variation in how cancer patients respond to checkpoint inhibitors.

The therapeutic use of the immune system to specifically attack tumours has been a long-standing vision among tumour immunologists. Recently, the use of checkpoint inhibitors to turn-off immunosuppressive signals has proven to be effective in enhancing T-cell reactivity against patient-specific neoantigens, resulting from somatic mutations. Several of the identified T-cell epitopes share similarity with common bacterial and viral antigens, suggesting the involvement of pre-existing microbial cross-reactive T cells in rapid and durable tumour regression seen in some patients. This notion of T-cell cross-reactivity is further supported by the findings that intestinal bacteria can influence checkpoint-blockade therapy. Moreover, early data indicate the presence of such T cells in long-term survival breast cancer patients. This review highlights the main challenges for cancer immunotherapy and discusses the potential contribution of T-cell cross-reactivity in cancer immunotherapy and whether it can be used as a biomarker to predict the responsiveness to checkpoint inhibitors.

Epidermal growth factor receptor (EGFR) density may not be the only determinant for the efficacy of EGFR-targeted photoimmunotherapy in human head and neck cancer cell lines.

OBJECTIVE: The aim of this study was to investigate the effects of targeted photoimmunotherapy (PIT) in vitro on cell lines with various expression levels of epidermal growth factor receptor (EGFR) using an anti-EGFR targeted conjugate composed of Cetuximab and IR700DX, phthalocyanine dye. MATERIALS AND METHODS: Relative EGFR density and cell binding assay was conducted in three human head & neck cancer cell lines (scc-U2, scc-U8, and OSC19) and one reference cell line A431. After incubation with the conjugate for 1 or 24 hours, cellular uptake and localization were investigated by confocal laser scanning microscopy and quantified by image analysis. Cell survival was determined using the MTS assay and alamarBlue assay after PIT with a 690 nm laser to a dose of 7 J.cm-2 (at 5 mW.cm-2 ). The mode of cell death was examined with flow cytometry using apoptosis/necrosis staining by Annexin V/propidium iodide, together with immunoblots of anti-apoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL. RESULTS: A431 cells had the highest EGFR density followed by OSC19, and then scc-U2 and scc-U8. The conjugates were localized both on the surface and in the cytosol of the cells after 1- and 24-hour incubation. After 24-hour incubation the granular pattern was more pronounced and in a similar pattern of a lysosomal probe, suggesting that the uptake of conjugates by cells was via receptor-mediated endocytosis. The results obtained from the quantitative imaging analysis correlate with the level of EGFR expression. Targeted PIT killed scc-U8 and A431 cells efficiently; while scc-U2 and OSC19 were less sensitive to this treatment, despite having similar EGFR density, uptake and localization pattern. Scc-U2 cells showed less apoptotic cell dealth than in A431 after 24-hour targeted PIT. Immunoblots showed significantly higher expression of anti-apoptotic Bcl-2 and Bcl-xL proteins in scc-U2 cell lines compared to scc-U8. CONCLUSION: Our study suggests that the effectiveness of EGFR targeted PIT is not only dependent upon EGFR density. Intrinsic biological properties of tumor cell lines also play a role in determining the efficacy of targeted PIT. We have shown that in scc-U2 cells this difference may be caused by differences in the apoptopic pathway. Lasers Surg. Med. 50:513-522, 2018. © 2018 Wiley Periodicals, Inc.

A novel peptide carrier for efficient targeting of antigens and nucleic acids to dendritic cells.

Dendritic cells (DCs) initiate host immune responses by presenting captured antigens to naive T cells. Hence, DC-binding peptides may be used for antigen targeting to boost naive and memory immune responses. By biopanning peptide phage libraries on human monocyte-derived DCs, we identified novel DC-binding peptides. One of the selected phages, displaying the NW peptide (NWYLPWLGTNDW), bound DCs with high affinity, and its binding was inhibited by the corresponding synthetic peptide. Antigenic peptides or proteins conjugated to the NW peptide bound to DCs and were internalized without negative effects on DC phenotype and function. Ex vivo targeted delivery of CMV-pp65 peptides to DCs via the NW peptide increased T-cell responses in HLA-A2(+)/CMV(+) donors compared to untargeted peptides (P<0.001). Stimulation of CD45RO-depleted peripheral blood mononuclear cells from CMV(-) donors with the NW-pp65 fusion peptides expanded pp65-specific precursor T cells. Moreover, the NW peptide mediated small interfering RNA delivery to DCs, and a significant gene silencing was obtained. Collectively, the data reveal that proteins and nucleic acids can be directed to DCs through the NW peptide, enabling effective uptake and functional effects such as T-cell activation in the context of MHC class I and II molecules.

Exploring the Impact of mRNA Modifications on Translation Efficiency and Immune Tolerance to Self-Antigens.

Therapeutic modified mRNAs are being developed for a broad range of human diseases. However, the impact of potential miscoding of modified mRNAs on self-tolerance remains unknown. Additionally, more studies are needed to explore the effects of nucleoside alkylation on translation. While all six tested modifications are tolerated as substrates by T7 RNA polymerase and inhibited mRNA immunogenicity, the translation efficiency varied significantly depending on the type of modification. In contrast to methylation, ethylation at the N1 position of pseudouridine (Ψ) hindered translation, suggesting that the C5-C1' glycosidic bond alone is not a critical element for high translation. Inhibition of mRNA translation was also observed with 5-methoxyuridine modification. However, this inhibition was partially alleviated through the optimization of mRNA coding sequences. BALB/c mice immunized with syngeneic ψ-modified mRNA encoding for Wilms' tumor antigen-1 (WT1) developed a low but significant level of anti-WT1 IgG antibodies compared to those immunized with either unmodified or N1-methyl ψ-modified mRNA. Overall, the data indicate that adding a simple ethyl group (-CH2CH3) at the N1 position of ψ has a major negative effect on translation despite its reduced immunogenicity. Additionally, mRNA containing Ψ may alter translation fidelity at certain codons, which could lead to a breakdown of immune tolerance to self-antigens. This concern should be taken into account during gene replacement therapies, although it could benefit mRNA-based vaccines by generating a diverse repertoire of antigens.

Inhibition of stromal PlGF suppresses the growth of prostate cancer xenografts.

The growth and vascularization of prostate cancer is dependent on interactions between cancer cells and supporting stromal cells. The primary stromal cell type found in prostate tumors is the carcinoma-associated fibroblast, which produces placental growth factor (PlGF). PlGF is a member of the vascular endothelial growth factor (VEGF) family of angiogenic molecules and PlGF mRNA levels increase after androgen deprivation therapy in prostate cancer. In this study, we show that PlGF has a direct dose-dependent proliferative effect on human PC-3 prostate cancer cells in vitro and fibroblast-derived PlGF increases PC-3 proliferation in co-culture. In xenograft tumor models, intratumoral administration of murine PlGF siRNA reduced stromal-derived PlGF expression, reduced tumor burden and decreased the number of Ki-67 positive proliferating cells associated with reduced vascular density. These data show that targeting stromal PlGF expression may represent a therapeutic target for the treatment of prostate cancer.

Antibody Surface Profiling Identifies Glycoforms in Multiple Myeloma as Targets for Immunotherapy: From Antibody Derivatives to Mimetic Peptides for Killing Tumor Cells.

Despite therapeutic advances in recent years, there are still unmet medical needs for patients with multiple myeloma (MM). Hence, new therapeutic strategies are needed. Using phage display for screening a large repertoire of single chain variable fragments (scFvs), we isolated several candidates that recognize a heavily sulfated MM-specific glycoform of the surface antigen syndecan-1 (CD138). One of the engineered scFv-Fc antibodies, named MM1, activated NK cells and induced antibody-dependent cellular cytotoxicity against MM cells. Analysis of the binding specificity by competitive binding assays with various glycan ligands identified N-sulfation of glucosamine units as essential for binding. Additionally, site-directed mutagenesis revealed that the amino acids arginine and histidine in the complementarily determining regions (CDRs) 2 and 3 of the heavy chain are important for binding. Based on this observation, a heavy-chain antibody, known as a nanobody, and a peptide mimicking the CDR loop sequences were designed. Both variants exhibited high affinity and specificity to MM cells as compared to blood lymphocytes. Specific killing of MM cells was achieved by conjugating the CDR2/3 mimic peptide to a pro-apoptotic peptide (KLAKLAK)2. In a co-culture model, the fusion peptide killed MM cells, while leaving normal peripheral blood mononuclear cells unaffected. Collectively, the development of antibodies and peptides that detect tumor-specific glycoforms of therapeutic targets holds promise for improving targeted therapies and tumor imaging.

Precision Killing of M2 Macrophages with Phage-Displayed Peptide-Photosensitizer Conjugates.

Among the immunosuppressive cells recruited to the tumor microenvironment, macrophages are particularly abundant and involved in angiogenesis, metastasis, and resistance to current cancer therapies. A strategy that simultaneously targets tumor cells and macrophages, particularly pro-tumoral M2 macrophages, would have significant clinical impact for various types of solid malignancies. By the use of phage display technology, we have recently developed a synthetic peptide, named NW, which binds to M1 and M2 macrophages with high affinity. Additional affinity selection on M2 macrophages identified only dominant peptides whose binding motifs are similar to that of the NW peptide. To reduce the frequency of selecting such dominating peptides, the peptide library was affinity selected on M2 macrophages blocked with NW peptide. This approach resulted in the selection of peptides that bind to M2, but not M1 macrophages. To explore the therapeutic potential of the selected peptides, the M13 phage-displayed peptides were conjugated to the photosensitizer IR700, which has been used for cancer photoimmunotherapy. The phage displaying a dominant peptide (SPILWLNAPPWA) killed both M1 and M2 macrophages, while those displaying the M2-specific peptides killed M2 macrophages only upon near-infrared light exposure. A significant fraction of the M2 macrophages were also killed with the untargeted M13 phage-IR700 conjugates. Hence, M2 macrophages can also be selectively targeted by the wild type M13 phage, which displayed a significant tropism to these cells. The benefits of this photoimmunotherapy include an automatic self-targeting ability of the wild type M13 phage, and the option of genetic manipulation of the phage genome to include tumor targeting peptides, allowing the killing of both M2 macrophages and cancer cells.

Efficient siRNA targeted delivery into cancer cells by gastrin-releasing peptides.

Small interfering RNAs (siRNAs) have displayed considerable promise for the treatment of cancer. However, their delivery to the desired cell population remains a challenging task. Here we have covalently conjugated a siRNA against survivin to gastrin-releasing peptides (GRPs) to direct siRNA molecules to cancer cells that express the GRP receptor. The cellular uptake of the peptide-siRNA conjugates was tested in breast MDA-MB 231 cancer cells, which express the GRP receptor. Fluorescein-tagged GRP-siRNA conjugates were taken up by cancer cells but not normal mammary epithelial cells or human blood monocytes. By 120 min of incubation, most of the cells have taken up the conjugates. Excess free peptide inhibited uptake, implying dependence of uptake on GRP receptor. Moreover, bitargeting of siRNA molecules by GR and luteinizing hormone-releasing peptides accelerated the uptake kinetics by MDA-MB 231 cells when compared to monotargeted siRNAs. Peptide-siRNA conjugates, but not free siRNAs, inhibited the expression of survivin, an endogenous gene involved in cancer cell survival. None of the peptide-siRNA conjugates induced the expression of inflammatory cytokines or interferon α in human blood leukocytes. Overall, the data demonstrate the feasibility of GRP receptor-mediated targeted delivery of siRNAs to cancer cells, an important step for RNA interference therapy in humans.

RNA Interference: Story and Mechanisms.

The discovery that gene expression can be silenced by exogenously introduced double-stranded RNAs into cells unveiled a hidden level of gene regulation by a variety of small RNA pathways, which are involved in regulating endogenous gene expression, defending against virus infections, and protecting the genome from invading transposons, both at the posttranscriptional and epigenetic levels. All endogenous RNA interference pathways share a conserved effector complex, which contains at least an argonaute protein and a short single-stranded RNA. Such argonaute-RNA complexes can repress the transcription of genes, target mRNA for site-specific cleavage, or block mRNA translation into proteins. This review outlines the history of RNAi discovery, function, and mechanisms of action. For comparison, it also touches on CRISPR interference.

Evaluation of In Vitro Phototoxicity of a Minibody-IR700 Conjugate Using Cell Monolayer and Multicellular Tumor Spheroid Models.

Photodynamic therapy (PDT) is a treatment strategy that utilizes photosensitizers (PSs) and light of a specific wavelength to kill cancer cells. However, limited tumor specificity is still a drawback for the clinical application of PDT. To increase the therapeutic efficacy and specificity of PDT, a novel human minibody (MS5) that recognizes a cell surface receptor expressed on various cancer cells was labeled with the hydrophilic phthalocyanine PS IR700 to generate an MS5-IR700 conjugate that is activated by near-infrared (NIR) light. The phototoxicity of the conjugate was mainly tested against the PC3 prostate cancer cell line. The MS5-IR700 conjugate killed PC3 cells after NIR light irradiation as compared to untreated cells or cells treated with IR700 alone. Time-course analysis of cell viability revealed a high percentage of cell death during the first hour in PC3 cells exposed to the MS5-IR700 conjugate and NIR light irradiation. After irradiation, the MS5-IR700 conjugate-treated PC3 cells displayed cellular swelling, round shape, and rupture of the cell and nuclear membranes. In a co-culture model, the MS5-IR700 conjugate killed MS5-positive Ramos lymphoma cells specifically, while leaving MS5-negative cells unaffected. In line with the data obtained with the monolayer cultures, the MS5-IR700 conjugate also killed PC3 cancer cell spheroids. The treatment induced relocation of heat shock protein 70 and calreticulin to the cell surface, implying the induction of immunogenic cell death. Overall, the data suggest that the developed MS5-IR700 conjugate is a promising therapeutic agent that warrants further preclinical studies.

Correction: Sioud et al. Evaluation of In Vitro Phototoxicity of a Minibody-IR700 Conjugate Using Monolayer and Multicellular Tumor Spheroid Models. Cancers 2021, 13, 3356.

The authors wish to make the following corrections to this paper [...].

Stromal cell-derived CSF-1 blockade prolongs xenograft survival of CSF-1-negative neuroblastoma.

The molecular mechanisms of tumor-host interactions that render neuroblastoma (NB) cells highly invasive are unclear. Cancer cells upregulate host stromal cell colony-stimulating factor-1 (CSF-1) production to recruit tumor-associated macrophages (TAMs) and accelerate tumor growth by affecting extracellular matrix remodeling and angiogenesis. By coculturing NB with stromal cells in vitro, we showed the importance of host CSF-1 expression for macrophage recruitment to NB cells. To examine this interaction in NB in vivo, mice bearing human CSF-1-expressing SK-N-AS and CSF-1-negative SK-N-DZ NB xenografts were treated with intratumoral injections of small interfering RNAs directed against mouse CSF-1. Significant suppression of both SK-N-AS and SK-N-DZ NB growth by these treatments was associated with decreased TAM infiltration, matrix metalloprotease (MMP)-12 levels and angiogenesis compared to controls, while expression of tissue inhibitors of MMPs increased following mouse CSF-1 blockade. Furthermore, Tie-2-positive and -negative TAMs recruited by host CSF-1 were identified in NB tumor tissue by confocal microscopy and flow cytometry. However, host-CSF-1 blockade prolonged survival only in CSF-1-negative SK-N-DZ NB. These studies demonstrated that increased CSF-1 production by host cells enhances TAM recruitment and NB growth and that the CSF-1 phenotype of NB tumor cells adversely affects survival.

Cell wall anchoring of the 37-kilodalton oncofetal antigen by Lactobacillus plantarum for mucosal cancer vaccine delivery.

The 37-kDa oncofetal antigen (OFA), a tumor immunogen expressed on all mammalian cancers examined to date, was secreted and anchored to the cell wall of Lactobacillus plantarum using homologous signal peptides and LPxTG anchors. Orally administered L. plantarum expressing anchored OFA induced a specific immune response against OFA in mice.

RNA and CRISPR Interferences: Past, Present, and Future Perspectives.

RNA interference (RNAi), a natural gene silencing process, is a widely used technique in basic research, preclinical studies, and drug development strategies. Although the technique has great potential to generate new human therapies and treat undruggable diseases, the clinical application of RNAi is still challenging primarily because of the delivery problem and potential off-target effects. Over the past two decades, great efforts have been undertaken to develop delivery agents and chemical modifications to overcome these challenges. Such advances in RNA delivery and chemical modifications have benefited researchers who are developing gene-editing therapies based on CRISPR-Cas9, an RNA-guided endonuclease, which is already having a major impact on biology and medicine. Here, I review the discovery of these two interference tools, identify the technical challenges yet to be overcome and provide some perspectives on how these two RNA-based technologies can be harnessed to treat human diseases.

Optimized siRNA Delivery into Primary Immune Cells Using Electroporation.

Effective RNA delivery strategies for primary human monocytes and dendritic cells (DCs) are useful tools for both basic research and cancer immunotherapy applications. Compared to viral delivery, electroporation is a relatively safe and simple technique that has been established for most immune cells. This chapter describes the feasibility of introducing small interfering RNAs into human primary monocytes and DCs using either nucleofection or standard electroporation techniques. DC cancer vaccines that integrate siRNA targeting relevant DC-intrinsic immunosuppressive signals induced robust and durable anti-tumor immune responses.