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Extended half-life recombinant FIX (rFIX) molecules have been generated to reduce the dosing burden and increase the protection of patients with hemophilia B. Clinical pharmacology studies with recombinant factor IX Fc fusion protein (rFIXFc) report a similar initial peak plasma recovery to that of rFIX, but with a larger volume of distribution. Although the pegylation of N9-GP results in a larger plasma recovery, there is a smaller volume of distribution, suggesting less extravasation of the latter drug. In this study, we set out to compare the biodistribution and tissue localization of rFIX, rFIXFc, and glycoPEGylated rFIX in a hemophilia B mouse model. Radiolabeled rFIX, rFIXFc, and rFIX-GP were employed in in vivo single-photon emission computed tomography imaging (SPECT/CT), microautoradiography (MARG), and histology to assess the distribution of FIX reagents over time. Immediately following injection, vascularized tissues demonstrated intense signal irrespective of FIX reagent. rFIX and rFIXFc were retained in joint and muscle areas through 5 half-lives, unlike rFIX-GP (assessed by SPECT). MARG and immunohistochemistry showed FIX agents localized at blood vessels among tissues, including liver, spleen, and kidney. Microautoradiographs, as well as fluorescent-labeled images of knee joint areas, demonstrated retention over time of FIX signal at the trabecular area of bone. Data indicate that rFIXFc is similar to rFIX in that it distributes outside the plasma compartment and is retained in certain tissues over time, while also retained at higher plasma levels. Overall, data suggest that Fc fusion does not impede the extravascular distribution of FIX., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Introduction: Lipid nanoparticles (LNPs) are one of the most clinically advanced candidates for delivering nucleic acids to target cell populations, such as hepatocytes. Once LNPs are endocytosed, they must release their nucleic acid cargo into the cell cytoplasm. For delivering messenger RNA (mRNA), delivery into the cytosol is sufficient; however, for delivering DNA, there is an added diffusional barrier needed to facilitate nuclear uptake for transcription and therapeutic effect., Method: Here, we use fluorescence microscopy to investigate the intracellular fate of different LNP formulations to determine the kinetics of localization to endosomes and lysosomes. LNPs used in the studies were prepared via self-assembly using a NanoAssemblr for microfluidic mixing. As the content of polyethylene glycol (PEG) within the LNP formulation influences cellular uptake by hepatocyte cells, the content and hydrocarbon chain length within the formulation were assessed for their impact on intracellular trafficking. Standard LNPs were then formed using three commercially available ionizable lipids, Dlin-MC3-DMA (MC3), Dlin-KC2-DMA (KC2), and SS-OP. Plasmid DNA (pDNA) and mRNA were used, more specifically with a mixture of Cyanine 3 (Cy3)-labeled and green fluorescence protein (GFP) producing plasmid DNA (pDNA) as well as Cy5-labeled GFP producing mRNA. After formulation, LNPs were characterized for the encapsulation efficiency of the nucleic acid, hydrodynamic diameter, polydispersity, and zeta potential. All standard LNPs were ~100 nm in diameter and had neutral surface charge. All LNPs resulted in encapsulation efficiency greater than 70%. Confocal fluorescence microscopy was used for the intracellular trafficking studies, where LNPs were incubated with HuH-7 hepatocyte cells at times ranging from 0-48 h. The cells were antibody-stained for subcellular components, including nuclei, endosomes, and lysosomes., Result: Analysis was performed to quantify localization of pDNA to the endosomes and lysosomes. LNPs with 1.5 mol% PEG and a hydrocarbon chain C14 resulted in optimal endosomal escape and GFP production. Results from this study demonstrate that a higher percentage of C14 PEG leads to smaller LNPs with limited available phospholipid binding area for ApoE, resulting in decreased cellular uptake. We observed differences in the localization kinetics depending on the LNP formulation type for SS-OP, KC2, and MC3 ionizable lipids. The results also demonstrate the technique across different nucleic acid types, where mRNA resulted in more rapid and uniform GFP production compared to pDNA delivery., Conclusion: Here, we demonstrated the ability to track uptake and the sub-cellular fate of LNPs containing pDNA and mRNA, enabling improved screening prior to in vivo studies which would aid in formulation optimization., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Effective antitumour immunity depends on the orchestration of potent T cell responses against malignancies 1 . Regression of human cancers has been induced by immune checkpoint inhibitors, T cell engagers or chimeric antigen receptor T cell therapies 2-4 . Although CD8 T cells function as key effectors of these responses, the role of CD4 T cells beyond their helper function has not been defined. Here we demonstrate that a trispecific antibody to HER2, CD3 and CD28 stimulates regression of breast cancers in a humanized mouse model through a mechanism involving CD4-dependent inhibition of tumour cell cycle progression. Although CD8 T cells directly mediated tumour lysis in vitro, CD4 T cells exerted antiproliferative effects by blocking cancer cell cycle progression at G1/S. Furthermore, when T cell subsets were adoptively transferred into a humanized breast cancer tumour mouse model, CD4 T cells alone inhibited HER2 + breast cancer growth in vivo. RNA microarray analysis revealed that CD4 T cells markedly decreased tumour cell cycle progression and proliferation, and also increased pro-inflammatory signalling pathways. Collectively, the trispecific antibody to HER2 induced T cell-dependent tumour regression through direct antitumour and indirect pro-inflammatory/immune effects driven by CD4 T cells., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Recent approval of mRNA vaccines for emergency use against COVID-19 is likely to promote rapid development of mRNA-based vaccines targeting a wide range of infectious diseases. Compared to conventional approaches, this vaccine modality promises comparable potency while substantially accelerating the pace of development and deployment of vaccine doses. Already demonstrated successfully for single antigen vaccines such as for COVID-19, this technology could be optimized for complex multi-antigen vaccines. Herein, utilizing multiple influenza antigens, we demonstrated the suitability of the mRNA therapeutic (MRT) platform for such applications. Seasonal influenza vaccines have three or four hemagglutinin (HA) antigens of different viral subtypes. In addition, influenza neuraminidase (NA), a tetrameric membrane protein, is identified as an antigen that has been linked to protective immunity against severe viral disease. We detail the efforts in optimizing formulations of influenza candidates that use unmodified mRNA encoding full-length HA or full-length NA encapsulated in lipid nanoparticles (LNPs). HA and NA mRNA-LNP formulations, either as monovalent or as multivalent vaccines, induced strong functional antibody and cellular responses in non-human primates and such antigen-specific antibody responses were associated with protective efficacy against viral challenge in mice., (© 2021. The Author(s).)

Emergency use authorization of COVID vaccines has brought hope to mitigate pandemic of coronavirus disease 2019 (COVID-19). However, there remains a need for additional effective vaccines to meet the global demand and address the potential new viral variants. mRNA technologies offer an expeditious path alternative to traditional vaccine approaches. Here we describe the efforts to utilize an mRNA platform for rational design and evaluations of mRNA vaccine candidates based on the spike (S) glycoprotein of SARS-CoV-2. Several mRNA constructs of S-protein, including wild type, a pre-fusion stabilized mutant (2P), a furin cleavage-site mutant (GSAS) and a double mutant form (2P/GSAS), as well as others, were tested in animal models for their capacity to elicit neutralizing antibodies (nAbs). The lead 2P/GSAS candidate was further assessed in dose-ranging studies in mice and Cynomolgus macaques, and for efficacy in a Syrian golden hamster model. The selected 2P/GSAS vaccine formulation, designated MRT5500, elicited potent nAbs as measured in neutralization assays in all three preclinical models and more importantly, protected against SARS-CoV-2-induced weight loss and lung pathology in hamsters. In addition, MRT5500 elicited T H 1-biased responses in both mouse and non-human primate (NHP), thus alleviating a hypothetical concern of potential vaccine-associated enhanced respiratory diseases known associated with T H 2-biased responses. These data position MRT5500 as a viable vaccine candidate for entering clinical development.

Despite the significant therapeutic advances provided by immune-checkpoint blockade and chimeric antigen receptor T cell treatments, many malignancies remain unresponsive to immunotherapy. Bispecific antibodies targeting tumor antigens and activating T cell receptor signaling have shown some clinical efficacy; however, providing co-stimulatory signals may improve T cell responses against tumors. Here, we developed a trispecific antibody that interacts with CD38, CD3 and CD28 to enhance both T cell activation and tumor targeting. The engagement of both CD3 and CD28 affords efficient T cell stimulation, whereas the anti-CD38 domain directs T cells to myeloma cells, as well as to certain lymphomas and leukemias. In vivo administration of this antibody suppressed myeloma growth in a humanized mouse model and also stimulated memory/effector T cell proliferation and reduced regulatory T cells in non-human primates at well-tolerated doses. Collectively, trispecific antibodies represent a promising platform for cancer immunotherapy., (© 2019. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Reproductive hormones are associated with risk for epithelial ovarian cancer. To determine the effect of such hormones on the activation of interleukin 6 (IL-6)/STAT3 (signal transducer and activator of transcription-3) signaling, which may be involved in ovarian cancer, we investigated the status of STAT3, IL-6, and its receptor in immortalized human ovarian surface epithelial (HOSE) and ovarian cancer (OVCA) cell lines., Methods: Two immortalized HOSE cell lines and two OVCA cell lines were cultured with gonadotropins, sex steroid hormones, and/or IL-6, alone or with specific inhibitors or IL-6-neutralizing antibodies. Expression of IL-6, the IL-6 receptor alpha chain (IL-6Ralpha), and phosphorylated and unphosphorylated STAT3 messenger RNAs (mRNAs) and proteins in all cells was determined. Cell proliferation and soft-agar colony formation were assessed. STAT3 activity was investigated in OVCA cells transfected with a dominant negative STAT3 (Dn-STAT3), wild-type STAT3, or an empty control vector. All statistical tests were two-sided., Results: Levels of IL-6 mRNA and protein increased in all cells treated with follicle-stimulating hormone (FSH), luteinizing hormone (LH), 17beta-estradiol, or estrone but increased only in OVCA cells treated with testosterone and 5alpha-dihydrotestosterone. For all lines, IL-6 antibodies partially inhibited hormone-stimulated cell proliferation but completely inhibited IL-6-enhanced cell proliferation. IL-6 induced STAT3 phosphorylation and activation in HOSE cells; STAT3 was constitutively activated in OVCA cells. Higher levels of IL-6Ralpha and STAT3 transcription factors were observed in OVCA cells than in HOSE cells. After transfection, Dn-STAT3 suppressed endogenous STAT3 and inhibited all forms of IL-6-stimulated OVCA cell proliferation (OVCA 429 cells, P<.001; OVCA 432 cells, P<.006), whereas wild-type STAT3 enhanced HOSE cell proliferation (wild-type STAT3 at 0.5 microg/mL in HOSE 306 cells, P<.002; STAT3 at 1.0 microg/mL in HOSE 306 or both concentrations of wild-type STAT3 in HOSE 642 cells, P<.001)., Conclusions: The IL-6/STAT3 signaling pathway may mediate FSH-, LH-, and estrogen-stimulated HOSE cell proliferation. Increased IL-6Ralpha expression and constitutive STAT3 activation may be associated with ovarian cancer.

Epidemiological data have implicated reproductive hormones as probable risk factors for ovarian cancer (OCa) development. Although pituitary and sex hormones have been reported to regulate OCa cell growth, no information is available regarding whether and how they influence normal ovarian surface epithelial (OSE) cell proliferation. To fill this data gap, this study has compared cell growth responses to gonadotropins and sex steroids in primary cultures of human OSE (HOSE) cells with those observed in immortalized, nontumorigenic HOSE cells and in OCa cell lines. Both malignant and normal cell lines/cultures responded equally well to the stimulatory actions of luteinizing hormone and follicle-stimulating hormone and to 17beta-estradiol and estrone, although the latter estrogen has a much lower affinity for estrogen receptor than does the former estrogen. In normal HOSE cell cultures/lines, 5alpha-dihydrotestosterone was found to be more effective than testosterone in stimulating cell growth, but in OCa cell lines, 5alpha-dihydrotestosterone and testosterone are equally potent. One OCa cell line, OVCA 433, was found to be nonresponsive to androgen stimulation. In general, primary cultures of normal HOSE cells exhibited the greatest hormone-stimulated growth responses (>10-fold enhancement), followed by immortalized HOSE cell lines (4-5-fold enhancement) and by OCa cell lines (2-4-fold enhancement). Interestingly, progesterone (P4), at low concentrations (10(-11) to 10(-10) M), was stimulatory to HOSE and OCa cell growth, but at high doses (10(-8) to 10(-6) M), P4 exerted marked inhibitory effects. In all cases, cotreatment of a cell culture/line with a hormone and its specific antagonist blocked the effect of the hormone, confirming specificity of the hormonal action. Taken together, these data support the hypothesis that reproductive states associated with rising levels of gonadotropins, estrogen, and/or androgen promote cell proliferation in the normal OSE, which favors neoplastic transformation. Conversely, those states attended by high levels of circulating P4, such as that seen during pregnancy, induce OSE cell loss and offer protection against ovarian carcinogenesis.