Optimal Potency Research Articles

Engineering a cleaved, prefusion-stabilized influenza B virus hemagglutinin by identification and locking of all six pH switches.

Vaccine components based on viral fusion proteins require high stability of the native prefusion conformation for optimal potency and manufacturability. In the case of influenza B virus hemagglutinin (HA), the stem's conformation relies on efficient cleavage. In this study, we identified six pH-sensitive regions distributed across the entire ectodomain where protonated histidines assume either a repulsive or an attractive role. Substitutions in these areas enhanced the protein's expression, quality, and stability in its prefusion trimeric state. Importantly, this stabilization enabled the production of a cleavable HA0, which is further processed into HA1 and HA2 by furin during exocytic pathway passage, thereby facilitating correct folding, increased stability, and screening for additional stabilizing substitutions in the core of the metastable fusion domain. Cryo-EM analysis at neutral and low pH revealed a previously unnoticed pH switch involving the C-terminal residues of the natively cleaved HA1. This switch keeps the fusion peptide in a clamped state at neutral pH, averting premature conformational shift. Our findings shed light on new strategies for possible improvements of recombinant or genetic-based influenza B vaccines.

The effects of Fc fusion protein glucagon-like peptide-1 and glucagon dual receptor agonist with different receptor selectivity in vivo studies

BackgroundGlucagon-like peptide-1 (GLP-1)/glucagon (GCG) dual receptor agonists with different receptor selectivity are under investigation and have shown significant improvement in both weight loss and glycemic control, but the optimal potency ratio between the two receptors to balance efficacy and safety remains unclear. Experimental approachWe designed and constructed several dual receptor agonists with different receptor potency ratios using Fc fusion protein technology. The long-term effects of the candidates on body weight and metabolic dysfunction-associated steatotic liver disease (MASLD) were evaluated in diet-induced obese (DIO) model mice, high-fat diet (HFD)-ob/ob mice and AMLN diet-induced MASLD mice. Repeat dose toxicity assays were performed to investigate the safety profile of the candidate (HEC-C070) in Sprague Dawley (SD) rats. Key resultsThe high GCG receptor (GCGR) selectivity of HEC-C046 makes it more prominent than other compounds for weight loss and most MASLD parameters but may lead to safety concerns. The weight change of HEC-C052 with the lowest GCG agonism was inferior to that of selective GLP-1 receptor agonist (GLP-1RA) semaglutide in DIO model mice. The GLP-1R selectivity of HEC-C070 with moderate GCG agonism has a significant effect on weight loss and liver function in obese mice, and its lowest observed adverse effect level (LOAEL) was 30 nmol/kg in the repeat dose toxicity study. ConclusionWe compared the potential of the Fc fusion protein GLP-1/GCG dual receptor agonists with different receptor selectivity to provide the setting for future GLP-1/GCG dual receptor agonists to treat obesity and MASLD.

A Lac Repressor-Inducible Baculovirus Expression Vector for Controlling Adeno-Associated Virus Capsid Ratios.

The baculovirus expression vector (BEV) system is an efficient, cost-effective, and scalable method to produce recombinant adeno-associated virus (rAAV) gene therapy vectors. Most BEV designs emulate the wild-type AAV transcriptome and translate the AAV capsid proteins, VP1, VP2, and VP3, from a single mRNA transcript with three overlapping open reading frames (ORFs). Non-canonical translation initiation codons for VP1 and VP2 reduce their abundances relative to VP3. Changing capsid ratios to improve rAAV vector efficacy requires a theoretical modification of the translational context. We have developed a Lac repressor-inducible system to empirically regulate the expression of VP1 and VP2 proteins relative to VP3 in the context of the BEV. We demonstrate the use of this system to tune the abundance, titer, and potency of a neurospecific rAAV9 serotype derivative. VP1:VP2:VP3 ratios of 1:1:8 gave optimal potency for this rAAV. It was discovered that the ratios of capsid proteins expressed were different than the ratios that ultimately were in purified capsids. Overexpressed VP1 did not become incorporated into capsids, while overexpressed VP2 did. Overabundance of VP2 correlated with reduced rAAV titers. This work demonstrates a novel technology for controlling the production of rAAV in the BEV system and shows a new perspective on the biology of rAAV capsid assembly.

Nonspecificity fingerprints for clinical-stage antibodies in solution.

Monoclonal antibodies (mAbs) have successfully been developed for the treatment of a wide range of diseases. The clinical success of mAbs does not solely rely on optimal potency and safety but also require good biophysical properties to ensure a high developability potential. In particular, nonspecific interactions are a key developability parameter to monitor during discovery and development. Despite an increased focus on the detection of nonspecific interactions, their underlying physicochemical origins remain poorly understood. Here, we employ solution-based microfluidic technologies to characterize a set of clinical-stage mAbs and their interactions with commonly used nonspecificity ligands to generate nonspecificity fingerprints, providing quantitative data on the underlying physical chemistry. Furthermore, the solution-based analysis enables us to measure binding affinities directly, and we evaluate the contribution of avidity in nonspecific binding by mAbs. We find that avidity can increase the apparent affinity by two orders of magnitude. Notably, we find that a subset of these highly developed mAbs show nonspecific electrostatic interactions, even at physiological pH and ionic strength, and that they can form microscale particles with charge-complementary polymers. The group of mAb constructs flagged here for nonspecificity are among the worst performers in independent reports of surface and column-based screens. The solution measurements improve on the state-of-the-art by providing a stand-alone result for individual mAbs without the need to benchmark against cohort data. Based on our findings, we propose a quantitative solution-based nonspecificity score, which can be integrated in the development workflow for biological therapeutics and more widely in protein engineering.

Functional Characterization and Optimization of Switchable Allogeneic Chimeric Antigen Receptor T Cells for Targeting CD19 and CD20 in B Cell Malignancies

Chimeric antigen receptor (CAR) T cell therapy has demonstrated significant efficacy in B cell malignancies. However, despite clinically manageable on-target/off-tumor toxicity when targeting antigens like CD19 and BCMA, severe cytokine release and neurotoxicity remain a clinical challenge. Additionally, patients may require immunoglobulin replacement therapy, and antigen-loss is recognized as a cause of relapse (PMID: 30275569, 31798590, 36263838, 37051246 and 37122700). To overcome these limitations, we developed a novel switchable CAR-T cell therapy that simultaneously targets both CD19 and CD20 in B cell malignancies. This innovative therapy is based on our reverse universal chimeric antigen receptor platform (RevCAR), a 2-component CAR-T technology (Figure 1; PMID: 34638268 and 32923149). The first component is a universal CAR-T cell, incorporating a CAR based on a short, non-immunogenic peptide motif derived from the human nuclear La/SSB autoantigen. The CAR itself does not recognize any human cell surface antigen. However, it is specifically targeted by an antibody-based binder in the second component, a soluble adaptor called targeting module (TM). This TM confers specificity against the desired cancer antigens of choice. Consequently, the availability of the TM effectively controls the activity of RevCAR-T cells. We have recently reported preclinical development of an allogeneic RevCAR-T cell therapy for CD123-positive hematologic malignancies (AVC-201; Ehninger et al. 2023, AACR Abstract 4095; NCT05949125). To target CD123, which is also expressed on normal hematopoietic progenitor cells, a TM with a short half-life was selected (R-TM123), enabling a rapid switch-off of the RevCAR system by TM withdrawal to avoid acute toxicity and continued aplasia. As CD19 and CD20 have a substantially more favorable safety profile and autologous conventional CAR-T cells and bispecific T cell engagers with respective target-specificities are approved in certain B cell malignancies, a half-life extended TM was developed to allow for dosing every 1-2 weeks. After an extensive screen for an optimized TM targeting CD19 and CD20, we screened multiple RevCAR variants differing in hinge, transmembrane and costimulatory domains for optimal anti-tumor potency using both preclinical in vitro and in vivo models. Profound efficacy and complete tumor control could be observed for optimized RevCAR constructs (Figure 2). While short-term in vitro cytotoxicity assays already gave a small hint towards in vivo efficacy, surprisingly, long-term in vitro rechallenge assay results did not correlate with in vivo efficacy. A summary of functional characterization data will be presented at the meeting with a focus on the lack of predictability of certain in vitro models and underlying hypotheses. Insights from these studies are now flowing into preclinical development of a novel switchable allogeneic CAR-T cell product candidate targeting CD19 and CD20 in B cell malignancies engineered to fully overcome graft-versus-host disease as well as graft rejection by host T and NK cells.

A Multiple Stimuli-Responsive NanoCRISPR Overcomes Tumor Redox Heterogeneity to Augment Photodynamic Therapy.

Redox heterogeneity of tumor cells has become one of the key factors leading to the failure of conventional photodynamic therapy (PDT). Exploration of a distinctive therapeutic strategy addressing heterogeneous predicaments is an appealing yet highly challenging task. Herein, a multiple stimuli-responsive nanoCRISPR (Must-nano) with spatial arrangement peculiarities in nanostructure and intracellular delivery is fabricated to overcome redox heterogeneity at both genetic and phenotypic levels for tumor-specific activatable PDT. Must-nano consists of a redox-sensitive core loading CRISPR/Cas9 targeting hypoxia-inducible factors-1α (HIF-1α) and a rationally designed multiple-responsive shell anchored by chlorin e6 (Ce6). Benefiting from the perfect coordination of structure and function, Must-nano avoids enzyme/photodegradation of the CRISPR/Cas9 system and exerts prolonged circulation, precise tumor recognition, and cascade-responsive performances to surmount tumor extra/intracellular barriers. After internalization into tumor cells, Must-nano could undergo hyaluronidase-triggered self-disassembly with charge reversal and rapid endosomal escape, followed by site-specific release and spatially asynchronous delivery of Ce6 and CRISPR/Cas9 under stimulations of redox signals, which not only improves tumor vulnerability to oxidative stress by complete HIF-1α disruption but also destroys the intrinsic antioxidant mechanism through glutathione depletion, thereby homogenizing redox-heterogeneous cells into oxidative stress-sensitive cell subsets. Under laser irradiation, Must-nano eventually exhibits optimal potency to amplify oxidative damage, effectively inhibiting the growth and hypoxia survival of redox-heterogeneous tumor in vitro and in vivo. Overall, our redox homogenization tactic significantly maximizes PDT efficacy and offers a promising strategy to overcome tumor redox heterogeneity in the development of antitumor therapies.

Abstract 1783: High-throughput arrayed screening of logic-gated CARs enables the selection of candidates for ccRCC with optimal potency and fidelity traits

Abstract The development of clinically effective CAR-T cell products for solid tumors will require substantial cell engineering to confer sufficient specificity, potency, and persistence. Advances in genome engineering and synthetic biology have provided an increasingly complex set of features that can be introduced into CAR-T cells to augment their function. However, combining multiple features may result in unpredictable negative interactions between components. Here, we report the use of high-throughput screening to optimize the design of a highly-engineered Integrated Circuit T Cell (ICT) product for the treatment of clear cell renal cell carcinoma (ccRCC). ICT cells are CAR-T cells that contain an AND logic gate requiring two antigens to be present to trigger tumor cell killing together with multiple enhancement modules. First, to create the logic gate we generated hundreds of novel scFv and VH/VHH binders targeting PSMA (as a priming target) and CA9 (as a cytolytic target) via two parallel de novo binder discovery efforts: 1) transgenic mice immunizations and 2) internally-developed phage display panning campaigns. Two independent arrayed screens with 500 PSMA prime receptors (PrimeRTM) and 750 CA9 CARs were conducted to find PrimeRs with high inducibility and CARs with strong on-target potency. From these screens, the top 25 PSMA PrimeRs and 20 CA9 CARs were combined with an shRNA cassette for targeted knockdowns along with two variations of a persistence module. We used a fully-automated workcell to perform end-to-end arrayed screening of the resulting 1,000 member library in T cells engineered from four human donors. Non-viral editing techniques were used to electroporate primary CD4/CD8 cells and robotic handlers were used to set up co-cultures. Circuit specificity and potency were assessed by flow and cytokine secretion and resistance to exhaustion was assessed in a seven day killing assay. Although the library was built from components that functioned well independently, we found that when combined, many of the circuits displayed suboptimal function. Integrated screening identified 20 variants that each far exceeded the performance of a small set of initial prototypes built from “best-guess” selections of individual components. The final candidates are significantly superior to constitutive CAR-T cells in a long term killing assay, show potent cytotoxicity of low expressing antigen lines, and display background levels of cytotoxicity against single antigen targets. Engineering multiple features into T cell products is limited by unpredictable negative interactions between components. We have overcome this limitation by using high-throughput screening which generated development-ready candidates for ccRCC with finely tuned desirability criteria in <18 months. Citation Format: Nishant Mehta, Jamie Thomas, Edward Yashin, Andrea Fua, Jonathan Li, Jonathan Chen, Laura Lim, Je Chua, Andrew Cardozo, Marian Sandoval, Duy P. Nguyen, Ziyan Hong, Jimmy Wu, Catherine Sue, Gustavo Guzman, Li Wang, Sofia K. Panagiotopoulou, Sophie Xu, Angela C. Boroughs, W. Nicholas Haining. High-throughput arrayed screening of logic-gated CARs enables the selection of candidates for ccRCC with optimal potency and fidelity traits [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1783.

Abstract 1886: Identifying T-cell engagers with optimal potency and cytokine-release profiles with a diverse panel of CD3-binding antibodies

Abstract In this study, we describe the characterization and validation of a diverse panel of fully human CD3-binding antibodies, including hundreds of human and cyno cross-reactive binders. We used two proof-of-concept TCE targets to demonstrate that this panel streamlines CD3 T-cell engager (TCE) development, enabling identification of optimal tumor cell-killing and cytokine-release profiles. CD3 TCEs have potential to be powerful cancer treatments, but the small number of available CD3-binding antibodies and limited multispecific engineering technologies have been barriers to development. Identifying TCEs that balance anti-tumor potency with potential toxicities, such as cytokine release syndrome, requires simultaneous tuning of both the CD3- and tumor-binding arms. Pairs of antibodies that achieve this balance are rare, creating a need for diverse panels of developable antibodies that can be combined and tested to identify optimal clinical candidates. To streamline TCE development, we discovered a diverse panel of CD3-binding antibodies. We screened over 5 million single cells from humanized mice and identified 585 unique CD3-specific antibody sequences. Of these, over 170 were identified as cross-reactive to human and cyno CD3 in primary screening. We then used high-throughput characterization to curate a panel of diverse and developable antibodies. We found a wide range of CD3εδ and CD3εγ binding specificities, affinities, and kinetics. Epitope binning analysis revealed multiple bins containing human and cyno cross-reactive binders, some of which are distinct from previously described cross-reactive antibodies, such as SP34-2. We assessed their biophysical properties and identified antibodies with good developability properties, including high thermal stability and low hydrophobicity, self-association, polyspecificity, and aggregation. To validate these antibodies, we used OrthoMab™ to generate proof-of-concept TCE panels with fixed tumor-binding arms. We identified CD3 x EGFR TCEs with high potency, low cytokine release, functional cross-reactivity in a cyno T cell-mediated tumor killing assay, and good pharmacokinetic properties in Tg32 mice. A second proof-of-concept CD3 x PSMA panel further validated our antibodies in bispecific formats. Together, these studies demonstrate that starting with diverse CD3-binding antibodies streamlines identification of developable TCEs with optimal potency and cytokine release. We leveraged data from our extensive characterization of CD3-binding antibodies in mono- and bispecific formats to develop a strategy for down-selection and pairing of CD3- and tumor-binding antibodies, and a high-throughput method for analysis of resulting TCEs. By categorizing antibodies based on functional properties, we are able to rapidly pinpoint optimal potential clinical candidates for specific tumor targets. Citation Format: Juntao (Matt) Mai, Kate Caldwell, Lindsay DeVorkin, Grace P. Leung, Karine Herve, Yuri Hwang, Cristina Faralla, Wei Wei, Emma Lathouwers, Valentine de Puyraimond, Lauren Clifford, Rhys S. Chappell, Stefan Hannie, Katherine J. Lam, Harveer Dhupar, Tran N. Tran, Melissa Cid, Lena M. Bolten, Tova Pinsky, Ping Xiang, Courteney Lai, Ahn Lee, Vivian Z. Li, Patrick Chan, Jasmine Chin, Steve Booth, Amy C. Lee, Stephanie Masterman, Sherie Duncan, Aaron Yamniuk, Kush Dalal, Tim M. Jacobs, Raffi Tonikian, Bryan C. Barnhart. Identifying T-cell engagers with optimal potency and cytokine-release profiles with a diverse panel of CD3-binding antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1886.

Delivery of macromolecules in unstimulated T cells by photoporation with polydopamine nanoparticles.

Ex vivo modification of T cells with exogenous cargo is a common prerequisite for the development of T cell therapies, such as chimeric antigen receptor therapy. Despite the clinical success and FDA approval of several such products, T cell manufacturing presents unique challenges related to therapeutic efficacy after adoptive cell transfer and several drawbacks of viral transduction-based manufacturing, such as high cost and safety concerns. To generate cellular products with optimal potency, engraftment potential and persistence in vivo, recent studies have shown that minimally differentiated T cell phenotypes are preferred. However, genetic engineering of quiescent T cells remains challenging. Photoporation is an upcoming alternative non-viral transfection method which makes use of photothermal nanoparticles, such as polydopamine nanoparticles (PDNPs), to induce transient membrane permeabilization by distinct photothermal effects upon laser irradiation, allowing exogenous molecules to enter cells. In this study, we analyzed the capability of PDNP-photoporation to deliver large model macromolecules (FITC-dextran 500kDa, FD500) in unstimulated and expanded human T cells. We compared different sizes of PDNPs (150, 250 and 400nm), concentrations of PDNPs and laser fluences and found an optimal condition that generated high delivery yields of FD500 in both T cell phenotypes. A multiparametric analysis of cell proliferation, surface activation markers and cytokine production, revealed that unstimulated T cells photoporated with 150nm and 250nm PDNPs retained their propensity to become activated, whereas those photoporated with 400nm PDNPs did less. Our findings show that PDNP-photoporation is a promising strategy for transfection of quiescent T cells, but that PDNPs should be small enough to avoid excessive cell damage.

Prospective approaches to enhancing CAR T cell therapy for glioblastoma.

Glioblastoma (GBM) is the most common malignant brain tumor. The poor clinical outcome and overall ineffectiveness of current standard treatments, including surgery, chemotherapy, and radiation, highlight the urgent need for alternative tumor-specific therapies for GBM. Chimeric antigen receptor (CAR) T cell therapy is a revolutionary therapeutic strategy for hematological malignancies, but the optimal potency of CAR T cell therapy for solid tumors, especially GBM, has not been achieved. Although CAR T cell therapeutic strategies for GBM have been assessed in clinical trials, the current antitumor activity of CAR T cells remains insufficient. In this review, we present our perspective on genetically modifying CAR constructs, overcoming T cell dysfunctions, and developing additional treatments that can improve CAR T cell effectiveness, such as functionality, persistence, and infiltration into tumor sites. Effectively improved CAR T cells may offer patients with GBM new treatment opportunities, and this review is intended to provide a comprehensive overview for researchers to develop potent CAR T cells using genetic engineering or combinatorial preparations.

Abstract 578: A functional approach to identifying and engineering TCRs results in highly-potent and specific TCRs for TCR-T cell therapy

Abstract Peptide-HLA (pHLA)- targeting therapeutics, such as T cell receptor-engineered T cells (TCR-T), have had clinical success in treating solid tumors. However, challenges related to safety exist: major concerns remain surrounding the cross-reactivity of T cell receptors (TCRs) as well as the ability of therapeutics to discriminate between on-target vs off-target pHLAs while maintaining high potency. Therefore, approaches that survey the diversity of the T cell repertoire to discover optimal TCRs, as well as platforms to comprehensively identify potential off-target liabilities, are critical to de-risking and accelerating the development of this promising class of pHLA-targeting therapeutics. We have developed a strategy that (1) queries the TCR repertoire to enrich and identify multiple active, sequence-distinct endogenous TCRs; (2) uses 3T-TRACE, a high-diversity pHLA library, to screen for cross-reactivity; and (3) exploits functional selections to simultaneously optimize for TCR potency and specificity. We applied this approach to identify TCRs of optimal specificity and potency targeting a peptide derived from the cancer-testis antigen NY-ESO-1 (SLLMWITQC) displayed by HLA-A2. We profiled 6 sequence-distinct TCRs using 3T-TRACE and validated potential endogenous off-target cross-reactivities. Many of the identified off-targets exhibited little to no sequence homology to the NY-ESO-1 epitope, highlighting the importance of diverse combinatorial libraries in identifying unexpected cross-reactivities. Leveraging the off-target liabilities identified by 3T-TRACE, we designed a functional library and selection scheme that enabled the identification of TCRs with increased potency and specificity. Optimized TCRs exhibited enhanced killing activity and improved safety against an NY-ESO-1-expressing melanoma cell line compared to benchmark TCRs, indicating that this approach has potential to improve clinical safety and efficacy. Using this multi-faceted and comprehensive approach we rapidly identified highly potent and specific TCRs against NY-ESO-1. Identifying cross-reactivities using 3T-TRACE proved to be critical in selecting TCRs suitable for engineering and functional selections. This approach can be extended to any pHLA target to create safe and effective TCR-Ts for clinical development. Citation Format: John D. Leonard, Alejandro Ramirez, Jason Romero, Jake Kleiner, Joanna Dreux, Bindu Hegde, Bryan Xie, Akshay Sharathchandra, Nathan Katz, Venita I. DeAlmeida, Hans-Peter Gerber, Marvin H. Gee, Leah V. Sibener. A functional approach to identifying and engineering TCRs results in highly-potent and specific TCRs for TCR-T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 578.

Characterizing Lipid-Coated Mesoporous Silica Nanoparticles as CD169-Binding Delivery System for Rilpivirine and Cabotegravir.

Herein, lipid-coated mesoporous silica nanoparticles (LMSN) are investigated as biomimetic delivery vehicle for two antiretroviral compounds (ARVs), rilpivirine (RPV) and cabotegravir (CAB). Monosialodihexosylganglioside (GM3) is incorporated into the membrane to facilitate LMSN binding to CD169 (Siglec-1)-expressing myeloid cells, that are predominantly expressed in secondary lymphoid tissues in vivo. It is demonstrated that in addition to providing CD169-binding functionalities, the lipid membrane around the silica core provides stealth properties that dampen the inflammatory cytokine response to ARVs-loaded LMSN in human monocyte-derived macrophages. Quantification of RPV and CAB releases from nanoparticles, and assessment of antiviral potency to human immunodeficiency virus (HIV-1) infection in vitro reveals that RPV and CAB co-formulated into LMSN retain optimal antiviral potency for 90 days, even upon storage at room temperature, making LMSN an attractive nanoplatform, immune to cold chain requirements. These findings suggest that GM3-LMSN equip the mesoporous silica nanoparticle (MSN) core with lipid-derived properties for surface passivation and lipid-mediated binding that are of high interest for achieving an effective delivery of ARVs to tissue reservoirs of HIV-1 replication.

A novel composite edible film fabricated by incorporating W/O/W emulsion into a chitosan film to improve the protection of fresh fish meat

A novel edible composite film constructed by incorporating W1/O/W2 emulsion (W1: aqueous solution of nisin; W2: water; oil phase: carvacrol) into chitosan film was characterized. Influences of preparing parameters on properties, especially stability, of primary and double emulsions were evaluated, and more persistent antibacterial activity was achieved. The film’s tension strength was increased by incorporating double emulsion at low concentration, but its oxygen permeability increased after this incorporation. The composite film displayed significant inhibitory effects on both Gram-positive and Gram-negative bacteria. SEM showed a sign of aggregation of some emulsion droplets near the surface of the composite film. FTIR found no pronounced interaction between the added active agents and chitosan. TGA proved that the double emulsion helped to increase the thermal stability of the film at high temperature. Coating salmon fillets with the composite film significantly increased the shelf life of fish fillets, demonstrating optimal potency in preserving fish fillets.

Topical corticosteroid therapy for facial acneiform eruption due to EGFR inhibitors in metastatic colorectal cancer patients: a randomized controlled trial comparing starting with a very strong or a weak topical corticosteroid (FAEISS study, NCCH1512, colorectal part).

Although pre-emptive therapy with oral tetracycline, moisturizer, sunscreen, and topical corticosteroid is useful for preventing acneiform eruption (AfE) due to epidermal growth factor receptor (EGFR) inhibitors, no studies have examined the efficacy of topical corticosteroids themselves, or investigated the optimal potency of corticosteroid for treating facial AfE (FAfE). Screened patients with RAS wild-type colorectal cancer started pre-emptive therapy with oral minocycline and moisturizer on initiation of cetuximab or panitumumab therapy. Patients who developed grade 1 or 2 FAfE were randomly allocated to two groups: a ranking-down (RD) group that started with a very strong corticosteroid and serially ranked down every 2weeks unless FAfE exacerbated; and a ranking-up (RU) group that started with a weak corticosteroid and serially ranked up at exacerbation. FAfE grade, patient quality of life, and adverse events (AEs) with topical corticosteroid were evaluated every 2weeks. The primary endpoint was the total number of times grade 2 or higher FAfE was identified in the central review of the 8-week treatment period. No significant differences in total numbers of grade 2 or higher FAfE or in AEs caused by topical corticosteroids were observed between groups during the 8weeks. Incidence of grade 2 or higher FAfE tended to be lower in the RD group during the first 2weeks. Considering the long-term care of FAfE, the RU regimen appears suitable and should be considered the standard treatment for FAfE due to EGFR inhibitor therapy. UMIN Clinical Trials Registry (UMIN000024113).

In vivo evaluation of faloak (Sterculia quadrifida R.Br) stem bark kombucha as hyperglycemia and therapeutic agent

This study aims to investigate the potential differences in the anti-hyperglycemia and antioxidant effects of fermented faloak (Sterculia quadrifida R.Br) stem bark (FSB) kombucha and FSB brew (without fermentation) in alloxan-induced diabetic rats. Black tea (BT) kombucha was used as a kombucha control. FSB kombucha, BT kombucha, and FSB brew were administered orally at a dose of 5 mL/Kg bw/day into the alloxan-induced diabetic rats for 28 days. Fasting blood glucose (FBG), body weight, superoxide dismutase activity, malondialdehyde levels, and pancreatic histopathology of the rats were analyzed. The results of this study showed that FSB kombucha, BT kombucha, and FSB brew were able to effectively reduce FBG, increase superoxide dismutase (SOD) activity, reduce malondialdehyde (MDA) levels, improve lipid profile, and repair pancreatic β-cells in the islets of Langerhans. The administration of FSB kombucha significantly (P<0.05) showed a more optimal potency than the unfermented FSB brew, while the ability was comparable to that of BT kombucha. Thus, faloak (Sterculia quadrifida R.Br) stem bark can be used as an alternative substrate other than black tea in the making of kombucha.

Synthesis and biological evaluation of 2,5-diaryl-1,3,4-oxadiazole derivatives as novel Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2) inhibitors

The Src homology-2 domain containing-protein tyrosine phosphatase-2 (SHP2) is a convergent node for oncogenic cell-signaling cascades including the PD-L1/PD-1 pathway. As an oncoprotein as well as a potential immunomodulator, SHP2 has now emerged as an attractive target for novel anti-cancer agents. Although significant progress has been made in identifying chemotypes of SHP2 inhibitors, these specific compounds might not be clinically useful to inhibit frequently encountered mutated SHP2 variants. Consequently, it is highly desirable to develop chemically different SHP2 inhibitors sensitive to SHP2 mutants. This work developed a new type of SHP2 inhibitors with 2,5-diaryl-1,3,4-oxadiazole scaffold. The representative compound 6l exhibited SHP2 inhibitory activity with IC50 of 2.73 ± 0.20 µM, showed about 1.56-fold, 5.26-fold, and 7.36-fold selectivity for SHP2 over SHP1, PTP1B and TCPTP respectively. Further investigations confirmed that 6l behaved as mixed-type inhibitor sensitive to leukemia cell TF-1 and inhibited SHP2 mediated cell signaling and proliferation. Molecular dynamics simulation provided more detailed information on the binding modes of compounds and SHP2 protein. These preliminary results could provide a possible opportunity for the development of novel SHP2 inhibitors sensitive to SHP2 mutants with optimal potency and improved pharmacological properties.

A proposed method of sample preparation and homogenization of hemp for the molecular analysis of cannabinoids

The use of Cannabis sativa, or Hemp, in commercial, recreational, and pharmacological applications is on the rise in the United States and worldwide. Many of these applications have guidelines associated with them dependent on the concentration of cannabinoid molecules that keep the products classified as hemp versus marijuana or that allow the producer to comment on the purity and potency of their product. Herein, we propose a method for homogenization of hemp that results in small particle sizes, uniform samples, and does not alter the cannabinoid concentrations during processing, allowing for optimal and reproducible potency testing. Using a novel “active grinding media” we homogenized commercially available hemp to analyze approximately 100 mg samples of homogenate via sieve analysis and high-performance liquid chromatography to assess the resulting size and potency of the sample when using this methodology. When processing hemp samples with our proposed methodology, we have demonstrated the ability to produce 60.2% of all particles < 1.25 mm with increased cannabinoid recovery compared to homogenates with larger average particle sizes. Maintaining sample temperatures below 35 °C during processing, we showed that our method does not thermally induce decarboxylation reactions that would result in major cannabinoid profile changes. We have developed a method for hemp processing via homogenization that does not alter the cannabinoid profile during processing, while consistently producing small particle sizes in a uniformly processed sample. This method allows for optimal and reproducible hemp processing when evaluating hemp and hemp-based products being brought to commercial markets.

A Five Immune-Related lncRNA Signature as a Prognostic Target for Glioblastoma.

Background: A variety of regulatory approaches including immune modulation have been explored as approaches to either eradicate antitumor response or induce suppressive mechanism in the glioblastoma microenvironment. Thus, the study of immune-related long noncoding RNA (lncRNA) signature is of great value in the diagnosis, treatment, and prognosis of glioblastoma. Methods: Glioblastoma samples with lncRNA sequencing and corresponding clinical data were acquired from the Cancer Genome Atlas (TCGA) database. Immune-lncRNAs co-expression networks were built to identify immune-related lncRNAs via Pearson correlation. Based on the median risk score acquired in the training set, we divided the samples into high- and low-risk groups and demonstrate the survival prediction ability of the immune-related lncRNA signature. Both principal component analysis (PCA) and gene set enrichment analysis (GSEA) were used for immune state analysis. Results: A cohort of 151 glioblastoma samples and 730 immune-related genes were acquired in this study. A five immune-related lncRNA signature (AC046143.1, AC021054.1, AC080112.1, MIR222HG, and PRKCQ-AS1) was identified. Compared with patients in the high-risk group, patients in the low-risk group showed a longer overall survival (OS) in the training, validation, and entire TCGA set (p = 1.931e-05, p = 1.706e-02, and p = 3.397e-06, respectively). Additionally, the survival prediction ability of this lncRNA signature was independent of known clinical factors and molecular features. The area under the ROC curve (AUC) and stratified analyses were further performed to verify its optimal survival predictive potency. Of note, the high-and low-risk groups exhibited significantly distinct immune state according to the PCA and GSEA analyses. Conclusions: Our study proposes that a five immune-related lncRNA signature can be utilized as a latent indicator of prognosis and potential therapeutic approach for glioblastoma.

Human adipose-derived stem cells enriched with VEGF-modified mRNA promote angiogenesis and long-term graft survival in a fat graft transplantation model

BackgroundFat grafting, as a standard treatment for numerous soft tissue defects, remains unpredictable and technique-dependent. Human adipose-derived stem cells (hADSCs) are promising candidates for cell-assisted therapy to improve graft survival. As free-living fat requires nutritional and respiratory sources to thrive, insufficient and unstable vascularization still impedes hADSC-assisted therapy. Recently, cytotherapy combined with modified mRNA (modRNA) encoding vascular endothelial growth factor (VEGF) has been applied for the treatment of ischemia-related diseases. Herein, we hypothesized that VEGF modRNA (modVEGF)-engineered hADSCs could robustly enhance fat survival in a fat graft transplantation model.MethodshADSCs were acquired from lipoaspiration and transfected with modRNAs. Transfection efficiency and expression kinetics of modRNAs in hADSCs were first evaluated in vitro. Next, we applied an in vivo Matrigel plug assay to assess the viability and angiogenic potential of modVEGF-engineered hADSCs at 1 week post-implantation. Finally, modVEGF-engineered hADSCs were co-transplanted with human fat in a murine model to analyze the survival rate, re-vascularization, proliferation, fibrosis, apoptosis, and necrosis of fat grafts over long-term follow-up.ResultsTransfections of modVEGF in hADSCs were highly tolerable as the modVEGF-engineered hADSCs facilitated burst-like protein production of VEGF in both our in vitro and in vivo models. modVEGF-engineered hADSCs induced increased levels of cellular proliferation and proangiogenesis when compared to untreated hADSCs in both ex vivo and in vivo assays. In a fat graft transplantation model, we provided evidence that modVEGF-engineered hADSCs promote the optimal potency to preserve adipocytes, especially in the long-term post-transplantation phase. Detailed histological analysis of fat grafts harvested at 15, 30, and 90 days following in vivo grafting suggested the release of VEGF protein from modVEGF-engineered hADSCs significantly improved neo-angiogenesis, vascular maturity, and cell proliferation. The modVEGF-engineered hADSCs also significantly mitigated the presence of fibrosis, apoptosis, and necrosis of grafts when compared to the control groups. Moreover, modVEGF-engineered hADSCs promoted graft survival and cell differentiation abilities, which also induced an increase in vessel formation and the number of surviving adipocytes after transplantation.ConclusionThis current study demonstrates the employment of modVEGF-engineered hADSCs as an advanced alternative to the clinical treatment involving soft-tissue reconstruction and rejuvenation.

The pursuit of precision mitochondrial medicine: Harnessing preclinical cellular and animal models to optimize mitochondrial disease therapeutic discovery.

Mitochondria share extensive evolutionary conservation across nearly all living species. This homology allows robust insights to be gained into pathophysiologic mechanisms and therapeutic targets for the heterogeneous class of primary mitochondrial diseases (PMDs) through the study of diverse in vitro cellular and in vivo animal models. Dramatic advances in genetic technologies, ranging from RNA interference to achieve graded knock-down of gene expression to CRISPR/Cas-based gene editing that yields a stable gene knock-out or targeted mutation knock-in, have enabled the ready establishment of mitochondrial disease models for a plethora of individual nuclear gene disorders. These models are complemented and extended by the use of pharmacologic inhibitor-based stressors to characterize variable degrees, onset, duration, and combinations of acute on chronic mitochondrial dysfunction in individual respiratory chain enzyme complexes or distinct biochemical pathways within mitochondria. Herein is described the rationale for, and progress made in, "therapeutic cross-training," a novel approach meant to improve the validity and rigor of experimental conclusions when testing therapies by studying treatment effects in multiple, evolutionarily-distinct species, including Caenorhabditis elegans (invertebrate, worm), Danio rerio (vertebrate, zebrafish), Mus musculus (mammal, mouse), and/or human patient primary fibroblast cell line models of PMD. The goal of these preclinical studies is to identify lead therapies from candidate molecules or library screens that consistently demonstrate efficacy, with minimal toxicity, in specific subtypes of mitochondrial disease. Conservation of in vitro and in vivo therapeutic effects of lead molecules across species has proven extensive, where molar concentrations found to be toxic or efficacious in one species are often consistent with therapeutic effects at similar doses seen in other mitochondrial disease models. Phenotypic outcome studies in all models are prioritized at the level of survival and function, to reflect the ultimate goal of developing highly potent therapies for human mitochondrial disease. Lead compounds that demonstrate significant benefit on gross phenotypes may be further scrutinized in these same models to decipher their cellular targets, mechanism(s), and detailed biochemical effects. High-throughput, automated technologic advances will be discussed that enable efficient, parallel screening in a diverse array of mitochondrial disease disorders and overarching subclasses of compounds, concentrations, libraries, and combinations. Overall, this therapeutic cross-training approach has proven valuable to identify compounds with optimal potency and safety profiles among major biochemical subtypes or specific genetic etiologies of mitochondrial disease. This approach further supports rational prioritization of lead compounds, target concentrations, and specific disease phenotypes, outcomes, and subgroups to optimally inform the design of clinical trials that test their efficacy in human mitochondrial disease subjects.