High Thermostability Research Articles

Adjusting photoluminescence of high thermostability Dy3+/Eu3+ co-doped borosilicate glass for white LED device

Due to the adoption of fluorescent powder encapsulated by organic resin, commercial light emitting diodes (LEDs) suffer from low thermal stability and poor light quality under high temperature, and luminescent glass is one of the effective ways to solve this problem. A series of borosilicate (B2O3–SiO2–Al2O3–PbO-Dy2O3–Eu2O3, marked as BSAP: DyEu) was prepared by the gas suspension technique. The basic characterization test indicate that the prepared glass has excellent thermal stability and stable structure. The optical properties of the luminescent glass were investigated in detail. By changing the Eu3+ doping amount, the Dy3+/Eu3+ co-doped glass can realize warm white light emission. The temperature-variable fluorescence spectra of BASP: Dy1.25Eu1.0 were analyzed, and the glass can produce strong warm white light emission in a wide temperature range. At 700 K, the luminous intensity of the glass remains above 38.49 % of the room temperature, and the chroma shift ΔC = 8.57 × 10−3. In addition, the w-LED device based on commercial GaN chip and BSAP: DyEu glass realizes warm white light output with excellent correlated color temperature (3063 K) and high color rendering index (90.3). The analysis shows that Dy3+/Eu3+ co-doped borosilicate glass can be used as a candidate material for w-LED applications.

Kinetics and products of Thermotoga maritima β-glucosidase with lactose and cellobiose

Galacto-oligosaccharides (GOS) are prebiotic compounds that are mainly used in infant formula to mimic bifidogenic effects of mother’s milk. They are synthesized by β-galactosidase enzymes in a trans-glycosylation reaction with lactose. Many β-galactosidase enzymes from different sources have been studied, resulting in varying GOS product compositions and yields. The in vivo role of these enzymes is in lactose hydrolysis. Therefore, the best GOS yields were achieved at high lactose concentrations up to 60%wt, which require a relatively high temperature to dissolve. Some thermostable β-glucosidase enzymes from thermophilic bacteria are also capable of using lactose or para nitrophenyl-galactose as a substrate. Here, we describe the use of the β-glucosidase BglA from Thermotoga maritima for synthesis of oligosaccharides derived from lactose and cellobiose and their detailed structural characterization. Also, the BglA enzyme kinetics and yields were determined, showing highest productivity at higher lactose and cellobiose concentrations. The BglA trans-glycosylation/hydrolysis ratio was higher with 57%wt lactose than with a nearly saturated cellobiose (20%wt) solution. The yield of GOS was very high, reaching 72.1%wt GOS from lactose. Structural elucidation of the products showed mainly β(1 → 3) and β(1 → 6) elongating activity, but also some β(1 → 4) elongation was observed. The β-glucosidase BglA from T. maritima was shown to be a very versatile enzyme, producing high yields of oligosaccharides, particularly GOS from lactose.Key points• β-Glucosidase of Thermotoga maritima synthesizes GOS from lactose at very high yield.• Thermotoga maritima β-glucosidase has high activity and high thermostability.• Thermotoga maritima β-glucosidase GOS contains mainly (β1-3) and (β1-6) linkages.Graphical

Elaborating NH-Bridged Nitrogen-Rich Energetic Materials via Base-Mediated Dimroth Rearrangement: Synthesis, Characterization, and Performance Study.

The pursuit of heat-resistant energetic materials featuring high thermostability and energy has gained keen interest in recent years owing to their use in coal mining and aerospace domains. In this study, we synthesized 4-((4,6-diamino-1,3,5-triazin-2-yl) amino)-1H-1,2,3-triazole-5-carbonitrile (6) and its perchlorate and nitrate energetic salts (6a and 6b) by incorporating amino bridging (-NH-) using the Dimroth rearrangement (DR) from inexpensive starting materials as a heat-resistant energetic materials. All of the compounds were thoroughly characterized by infrared (IR), NMR, elemental analysis (EA), high-resolution mass spectrometry (HRMS), and thermogravimetric analysis-differential scanning calorimetry (TGA-DSC) studies. Compounds 6a and 6b showed good densities (1.81 and 1.80 g cm-3), detonation performance (VOD = 7505 and 8257 m s-1, DP = 23.47 and 24.41 GPa), insensitivity to mechanical stimuli (IS = 40 J and FS = >360 N), and excellent thermal stability (Td = 307 and 334 °C), surpassing presently used heat-resistant explosive HNS (318 °C). The molecular electrostatic potentials and noncovalent interactions were pursued to understand possible interaction sites and structure-directing interactions in these salts. Their facile synthetic approach, good energetic performance, and outstanding thermal stability indicate that they are the ideal combination for replacing current benchmark heat-resistant explosive HNS. Additionally, this study highlights the use of classical DR for making new energetic materials with fine-tuned properties.

Construction and characterization of a novel fusion alginate lyase with endolytic and exolytic cleavage activity for industrial preparation of alginate oligosaccharides

Alginate lyases with high activity and good thermostability are lacking for the preparation of alginate oligosaccharides (AOS) with various biological activities. We constructed a fusion alginate lyase with both endo-and exo-activities. AlyRm6A-Zu7 was successfully constructed by connecting the highly thermostable AlyRm6A to a new exotype lyase, AlyZu7. The fusion enzyme exhibited high catalytic activity and thermostability. It transformed sodium alginate into oligosaccharides with degrees of polymerization (DP) of 2–4 while producing 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). The maximum reducing sugar, AOS, and DP1 + DEH yields were 75 %, 45 %, and 40 %, respectively. Molecular docking confirmed the formation of a stable complex between the substrate and AlyRm6A-Zu7. Protein interactions increased the thermostability of AlyZu7. This work provides new insights into the industrial formation of AOS and monosaccharide DEH using thermally stable fusion enzymes, which has a positive effect in the fields of functional oligosaccharide production and biofuel formation.

Effect of pH and temperature on tropane alkaloids within a processing strategy to provide safe infant cereal-based food

Tropane alkaloids (TAs) are secondary metabolites from weeds that can contaminate cereals and vegetables during harvest. Due to their toxicity, the Regulation (EC) 2023/915 sets maximum levels for atropine and scopolamine in cereal-based foods for infants containing millet, sorghum, buckwheat or their derived products. The aim of this study was to evaluate the effect of pH and temperature on the stability of TAs, as possible parameters in thermal processing to mitigate this chemical hazard in cereal-based infant food. The effect of pH (4 and 7) and temperature (80 °C and 100 °C) was assessed in buffer solutions. Also, treatment at 180 °C was performed in spiked and naturally incurred millet flour to assess the effect of high temperature, simulating cooking or drying, on the stability of TAs in the cereal matrix. The fate of 24 TAs was assessed by UHPLC-MS/MS. TAs showed high thermostability, although it was variable depending on the specific compound, pH, temperature and treatment time. In buffer solutions, higher degradation was found at 100 °C and pH 7. In spiked millet flour at 180 °C for 10 min, scopolamine and atropine contents decreased by 25 % and 22 %, similarly to other TAs which also showed a slow thermal degradation. Atropine, scopolamine, anisodamine, norscopolamine, scopine and scopoline were found in naturally contaminated millet flour. Interestingly, naturally incurred atropine was more thermostable than when spiked, showing a protective effect of the cereal matrix on TAs degradation. The present results highlight the need for an accurate monitorization of TAs in raw materials, as this chemical hazard may remain in infant cereal-based food even after intense thermal processing.

Molecular structure and characterization of the Thermochromatium tepidum light-harvesting 1 photocomplex produced in a foreign host

Purple phototrophic bacteria possess light-harvesting 1 and reaction center (LH1-RC) core complexes that play a key role in converting solar energy to chemical energy. High-resolution structures of LH1-RC and RC complexes have been intensively studied and have yielded critical insight into the architecture and interactions of their proteins, pigments, and cofactors. Nevertheless, a detailed picture of the structure and assembly of LH1-only complexes is lacking due to the intimate association between LH1 and the RC. To study the intrinsic properties and structure of an LH1-only complex, a genetic system was constructed to express the Thermochromatium (Tch.) tepidum LH1 complex heterologously in a modified Rhodospirillum rubrum mutant strain. The heterologously expressed Tch. tepidum LH1 complex was isolated in a pure form free of the RC and exhibited the characteristic absorption properties of Tch. tepidum. Cryo-EM structures of the LH1-only complexes revealed a closed circular ring consisting of either 14 or 15 αβ-subunits, making it the smallest completely closed LH1 complex discovered thus far. Surprisingly, the Tch. tepidum LH1-only complex displayed even higher thermostability than that of the native LH1-RC complex. These results reveal previously unsuspected plasticity of the LH1 complex, provide new insights into the structure and assembly of the LH1-RC complex, and show how molecular genetics can be exploited to study membrane proteins from phototrophic organisms whose genetic manipulation is not yet possible.

Hotspot Wizard-informed engineering of a hyperthermophilic β-glucosidase for enhanced enzyme activity at low temperatures.

Hyperthermophilic enzymes serve as an important source of industrial enzymes due to their high thermostability. Unfortunately, most hyperthermophilic enzymes suffer from reduced activity at low temperatures (e.g., ambient temperature), limiting their applicability. In addition, evolving hyperthermophilic enzymes to increase low temperature activity without compromising other desired properties is generally difficult. In the current study, a variant of β-glucosidase from Pyrococcus furiosus (PfBGL) was engineered to enhance enzyme activity at low temperatures through the construction of a saturation mutagenesis library guided by the HotSpot Wizard analysis, followed by its screening for activity and thermostability. From this library construction and screening, one PfBGL mutant, PfBGL-A4 containing Q214S/A264S/F344I mutations, showed an over twofold increase in β-glucosidase activity at 25 and 50°C compared to the wild type, without compromising high-temperature activity, thermostability and substrate specificity. Our experimental and computational characterizations suggest that the findings with PfBGL-A4 may be due to the elevation of local conformational flexibility around the active site, while slightly compacting the global protein structure. This study showcases the potential of HotSpot Wizard-informed engineering of hyperthermophilic enzymes and underscores the interplays among temperature, enzyme activity, and conformational flexibility in these enzymes.

Effect of processing techniques on functional, antioxidants, and structural properties of finger millet (Eleusine coracana) flour

This study explored the effect of processing methods including milling (control), soaking, parboiling, popping, and germination on functional, antioxidant, structural, and thermal properties of finger millet (Eleusine coracana) flour. These processing techniques increased the water and oil absorption capacity of finger millet flour with an exception for popping method which lacked oil absorption capacity. Additionally, there was also a decrease in bulk density in processed finger millet flour. Antioxidant properties showed a decrease in the anti-nutrient content of millet, and the popping method came out as the best processing method in terms of increase in total phenol content (11.18 mg/g) and antioxidant capacity (40.19%). The tannin's level also declined sharply in all processed millet. The FTIR showed the structural changes by exploiting the changes in bonds and their intensities. Scanning electron microscopy revealed the morphological changes which are attributed to different processing methods. TGA showed that popped finger millet had the highest thermostability. Based on the parameters tested in this research, the popping method came out to show the best results out of all the processing methods in terms of antioxidant and structural properties.

Polyurethane-based thin-film composites with carbon micro- to nanoscale fillers and their microwave properties

Polyurethane-based thin-film composites (PUR-based TFCs) with different carbon fillers, namely, carbon flocs formed by carbon dots (CDs) containing O, N, and F heteroatoms and carbon nanotubes (CNTs), were prepared by hot pressing. The morphology and composition of the carbon fillers were investigated by SEM/EDX. Temperature-programmed desorption mass spectrometry and thermogravimetric analysis revealed that the CNTs have the highest thermostability among the fillers. Microwave studies in the X-band showed that the PUR-based TFCs with the CNTs were characterized by higher reflection losses and lower transmission losses than the PUR-based TFCs with the flocs formed by the studied CDs.

Converting Bulk Sugars into Functional Fibers: Discovery and Application of a Thermostable β-1,3-Oligoglucan Phosphorylase.

Despite their broad application potential, the widespread use of β-1,3-glucans has been hampered by the high cost and heterogeneity associated with current production methods. To address this challenge, scalable and economically viable processes are needed for the production of β-1,3-glucans with tailorable molecular mass distributions. Glycoside phosphorylases have shown to be promising catalysts for the bottom-up synthesis of β-1,3-(oligo)glucans since they combine strict regioselectivity with a cheap donor substrate (i.e., α-glucose 1-phosphate). However, the need for an expensive priming substrate (e.g., laminaribiose) and the tendency to produce shorter oligosaccharides still form major bottlenecks. Here, we report the discovery and application of a thermostable β-1,3-oligoglucan phosphorylase originating from Anaerolinea thermophila (AtβOGP). This enzyme combines a superior catalytic efficiency toward glucose as a priming substrate, high thermostability, and the ability to synthesize high molecular mass β-1,3-glucans up to DP 75. Coupling of AtβOGP with a thermostable variant of Bifidobacterium adolescentis sucrose phosphorylase enabled the efficient production of tailorable β-1,3-(oligo)glucans from sucrose, with a near-complete conversion of >99 mol %. This cost-efficient process for the conversion of renewable bulk sugar into β-1,3-(oligo)glucans should facilitate the widespread application of these versatile functional fibers across various industries.

Expression and biochemical characterization of a novel thermostable alkaline β-1,3–1,4-glucanase (lichenase) from an alkaliphilic Bacillus lehensis G1

New thermostable β-1,3–1,4-glucanase (lichenase) designated as Blg29 was expressed and purified from a locally isolated alkaliphilic bacteria Bacillus lehensis G1. The genome sequence of B. lehensis predicted an open reading frame of Blg29 with a deduced of 249 amino acids and a molecular weight of 28.99 kDa. The gene encoding for Blg29 was successfully amplified via PCR and subsequently expressed as a recombinant protein using the E. coli expression system. Recombinant Blg29 was produced as a soluble form and further purified via immobilized metal ion affinity chromatography (IMAC). Based on biochemical characterization, recombinant Blg29 showed optimal activity at pH9 and temperature 60 °C respectively. This enzyme was stable for more than 2 h, incubated at 50 °C, and could withstand ∼50 % of its activity at 70 °C for an hour and a half. No significant effect on Blg29 was observed when incubated with metal ions except for a small increase with ion Ca2+. Blg29 showed high substrate activity towards lichenan where Vm, Km, Kcat, and kcat/Km values were 2040.82 μmolmin‾1mg‾1, 4.69 mg/mL, and 986.39 s‾1 and 210.32 mLs‾1mg‾1 respectively. The high thermostability and activity make this enzyme useable for a broad prospect in industry applications.

Distinct skyrmion phases at room temperature in two-dimensional ferromagnet Fe3GaTe2

Distinct skyrmion phases at room temperature hosted by one material offer additional degree of freedom for the design of topology-based compact and energetically-efficient spintronic devices. The field has been extended to low-dimensional magnets with the discovery of magnetism in two-dimensional van der Waals magnets. However, creating multiple skyrmion phases in 2D magnets, especially above room temperature, remains a major challenge. Here, we report the experimental observation of mixed-type skyrmions, exhibiting both Bloch and hybrid characteristics, in a room-temperature ferromagnet Fe3GaTe2. Analysis of the magnetic intensities under varied imaging conditions coupled with complementary simulations reveal that spontaneous Bloch skyrmions exist as the magnetic ground state with the coexistence of hybrid stripes domain, on account of the interplay between the dipolar interaction and the Dzyaloshinskii-Moriya interaction. Moreover, hybrid skyrmions are created and their coexisting phases with Bloch skyrmions exhibit considerably high thermostability, enduring up to 328 K. The findings open perspectives for 2D spintronic devices incorporating distinct skyrmion phases at room temperature.

Polarizable H‐Bond Concept in Aromatic Poly(thiourea)s: Unprecedented High Refractive Index, Transmittance, and Degradability at Force to Enhance Lighting Efficiency

AbstractDeveloping transparent and highly refractive environmentally friendly polymers has not been realized yet toward sustainable optoelectronics. This work describes poly(thiourea)s (PTUs) design following a new “polarizable group synergy” concept, combining highly polarizable hydrogen bonding groups and aromatic‐based spacers to form densely packed and high‐refractive‐index polymer networks. Specifically, PTUs containing m‐ and p‐phenylene spacers exhibit an easy synthesis, high thermostability (Tg = 159 °C), visible transparency (>92%T at 1 µm‐film), ultra‐high refractive index (nD = 1.81) based on the random H‐bonding arrays with a high packing constant (Kp = 0.738), and straightforward preparation of flexible films via solvent‐based techniques. Capitalizing on these assets, PTU‐films are integrated into benchmark graphene‐based lighting device architectures based on the light‐emitting electrochemical cells (LECs) concept. A joint optical modeling and experimental validation confirm the increase in external quantum efficiency expected by the enhanced light out‐coupling of PTU‐films. Finally, PTUs are efficiently depolymerized to low molecular weight compounds by simply adding diamines under heating, following the dynamic covalent bond exchange between thiourea moieties. Overall, this work highlights the PTU family as new promising materials with a unique polarizable H‐bond design to meet efficient and sustainable thin‐film lighting devices.

Simultaneous Enhancement of the Thermostability and Catalytic Activity of D-Allulose 3-Epimerase from Clostridium bolteae ATTC BAA-613 Based on the "Back to Consensus Mutations" Hypothesis.

D-Allulose is a high value rare sugar with multiple physiological functions and commercial potential that can be enzymatically synthesized from D-fructose by D-allulose 3-epimerase (DAEase). Poor catalytic activity and thermostability of DAEase prevent the industrial production of D-allulose. In this work, rational design was applied to a previously identified DAEase from Clostridium bolteae ATCC BAA-613 based on the "back to consensus mutations" hypothesis, and the catalytic activity of the Cb-I265 V variant was enhanced 2.5-fold. Furthermore, the Cb-I265 V/E268D double-site variant displayed 2.0-fold higher specific catalytic activity and 1.4-fold higher thermostability than the wild-type enzyme. Molecular docking and kinetic simulation results indicated increased hydrogen bonds between the active pocket and substrate, possibly contributing to the improved thermal stability and catalytic activity of the double-site mutant. The findings outlined a feasible approach for the rational design of multiple preset functions of target enzymes simultaneously.

Bridged Bicyclic Lactam Enables Chemically Recyclable Nylon

AbstractNylon, a widely‐used high‐performance thermoplastic, boasts exceptional durability and resistance to various solvents and weak acids, making it indispensable across diverse applications. However, its nonbiodegradable nature has led to alarming environmental pollution in land and oceans. Chemical recycling to monomers (CRM) stands as a crucial strategy for establishing a circular plastic economy, but the CRM of nylon remains largely unexplored. Herein, we introduce the bridged bicyclic lactam 5‐azabicyclo[2.2.1]octan‐6‐one (5/6‐LM), evolved from δ‐valerolactam and pyrrolidone, to solve the trade‐off in depolymerizability and performance. Notably, 5/6‐LM exhibits nearly 95 % conversion in mild polymerization conditions and efficient depolymerization catalyzed by lewis acids. This compound is synthetically accessible from commercially available chemicals in a single step at room temperature, demonstrating high efficiency and scalability up to 50 g in laboratory. Furthermore, the resulting polyamide displays remarkable attributes including high crystallinity and thermostability up to 283 °C, significantly broadening the scope of chemically recyclable nylons.

Bridged Bicyclic Lactam Enables Chemically Recyclable Nylon.

Nylon, a widely-used high-performance thermoplastic, boasts exceptional durability and resistance to various solvents and weak acids, making it indispensable across diverse applications. However, its nonbiodegradable nature has led to alarming environmental pollution in land and oceans. Chemical recycling to monomers (CRM) stands as a crucial strategy for establishing a circular plastic economy, but the CRM of nylon remains largely unexplored. Herein, we introduce the bridged bicyclic lactam 5-azabicyclo[2.2.1]octan-6-one (5/6-LM), evolved from δ-valerolactam and pyrrolidone, to solve the trade-off in depolymerizability and performance. Notably, 5/6-LM exhibits nearly 95 % conversion in mild polymerization conditions and efficient depolymerization catalyzed by lewis acids. This compound is synthetically accessible from commercially available chemicals in a single step at room temperature, demonstrating high efficiency and scalability up to 50 g in laboratory. Furthermore, the resulting polyamide displays remarkable attributes including high crystallinity and thermostability up to 283 °C, significantly broadening the scope of chemically recyclable nylons.

Production and characterization of recrystallized linear α-glucans at different temperatures for controllable thermostability and digestibility

Molecular structure of linear α-glucans (LAGs) and crystallization temperature have great effects on the thermostability and digestibility of recrystallized LAGs, but the recrystallization behaviors of LAGs in response to temperature remain unclear. Here LAGs with different lengths were prepared from amylopectin via chain elongation and debranching. Recrystallization of LAGs at 4 °C yielded B-type crystalline structure with relative crystallinity ranged from 23.7% to 46.1%. With a chain length of 40.2, an A-type allomorph was observed for a slow recrystallization at 50 °C. Differential scanning calorimetry suggested that A-type crystal had a higher thermostability than the B-type crystal, and increasing LAGs' chain length improved the dimension of double helices, whose assembly produced starch crystallites that enhanced the thermostability and decreased the in vitro digestibility of recrystallized LAGs. An improved thermostability of recrystallized LAGs preserved their ordered structures and kept the resistance to digestive enzymes, with a RS content up to 75.4%.

Towards high conductivity and stability diblock poly(norbornene)s-based anion exchange membranes crosslinked with alkyl quaternary ammonium cationic clusters

Anion exchange membranes (AEMs) with exceptional comprehensive performance, combining high conductivity, alkaline and dimensional stability, have been indispensable for the practical application in fuel cells. Herein, a robust addition poly(norbornene)s-based AEMs with an all-carbon-hydrogen bicyclic skeleton were fabricated by cross-linking of alkyl quaternary ammonium cationic clusters and further quaternization. The partially crosslinked AEMs adopt a synergistic strategy that combines diblock copolymer backbone, cationic cluster crosslinking structure, and long flexible spacer suspended with cationic head groups. Benefiting from these structural design advantages, the prepared AEMs possessed excellent hydroxide conductivity (133.9–146.9 mS cm−1) and low swelling ratio (SR ≦ 20%) at 80 °C. The robust crosslinked AEMs exhibited good mechanical properties and high thermostability, with onset of decomposition temperatures up to 240 °C. Furthermore, after 44 days treatment in 1 M NaOH solution at 80 °C, the hydroxide conductivity of AEMs maintained at 79.4–85.7% of the initial value depending on the crosslinking degree, demonstrating excellent alkaline stability. In addition, the H2/O2 single cell equipped with CL20-aPNB-TAT-QA-1.75 achieved a peak power density of 338.0 mW cm−2 at 80 °C. The study provided a meaningful insight to exploit the practical application of multi-cation clusters crosslinked addition poly(norbornene)s-based AEMs in fuel cells.

Prebiotic levan type fructan from Bacillus subtilis PR-C18 as a potent antibiofilm agent: Structural elucidation and in silico analysis

The global demand for therapeutic prebiotics persuades the quest for novel exopolysaccharides that can retard the growth of pathobionts and healthcare-associated pathogens. In this regard, an exopolysaccharide (3.69 mg/mL) producing strain showing prebiotic and antibiofilm activity was isolated from indigenous pineapple pomace of Tripura and identified as Bacillus subtilis PR-C18. Zymogram analysis revealed EPS PR-C18 was synthesized by levansucrase (∼57 kDa) with a maximal activity of 4.62 U/mg. Chromatography techniques, FTIR, and NMR spectral data revealed the homopolymeric nature of purified EPS with a molecular weight of 3.40 × 104 Da. SEM and rheological study unveiled its microporous structure and shear-thinning effect. Furthermore, EPS PR-C18 showed remarkable emulsification, flocculation, water retention, water solubilization, and antioxidant activity. DSC-TGA data demonstrated its high thermostability and cytotoxicity analysis verified its nontoxic biocompatible nature. In addition, the antibiofilm activity of EPS PR-C18 was validated using molecular docking, molecular simulation, MM-GBSA and PCA studies, which exhibited its strong binding affinity (−20.79 kcal/moL) with PelD, a virulence factor from Pseudomonas aeruginosa. Together, these findings support the future exploitation of EPS PR-C18 as an additive or adjuvant in food and pharmaceutical sectors.

Abstract 6343: AnTenGagerTM, a novel “2+1” T cell engager platform, enables conditional T cell activation with reduced risk of CRS

Abstract Background: CD3+ T cell engaging bispecific antibodies (TCE) redirect T cells to attack targeted tumor cells by simultaneously binding TAA on cancer cells and CD3 complex on a T cell to form a TCR-independent immune synapse. Multiple TCEs have demonstrated promising therapeutic efficacy in the treatment of hematological malignancies. In contrast, suboptimal efficacy in the treatment of solid tumors and the risk of inducing cytokine release syndrome (CRS) continue to be major challenges. Several factors, such as format, affinity, and valency of the antibody, TAA copy number, and antigen size and epitope, can impact the therapeutic efficacy and safety of a TCE. In this study, we developed a novel “2+1” TCE platform, AnTenGagerTM, which enables conditional T cell binding and activation upon TAA-crosslinking, potent anti-tumor activity, and reduced risk of CRS. Methods: Humanized CD3 antibodies were derived from mouse hybridoma, followed by affinity optimization utilizing Computer Aided Drug Design. The Fab-scFv-Fab format was used as the backbone structure of AnTenGagers. The anti-CD3 scFv sequences and the length of peptide linkers between VH/VL, scFv/CH1, and scFv/CH2 were screened and compared in different preclinical assays. LALA mutations (L234A, L235A) were introduced to abolish the Fc receptor binding capability. In a series of in vitro and/or in vivo models, the efficacy, safety and developability of AnTenGagers targeting multiple TAAs, such as Her2, GD2, CDH6, LILRB4, FLT3 and GPRC5D, were evaluated. Results: A library of humanized parental CD3 antibodies with a broad spectrum of affinities were generated, some of which cross react with monkey CD3. AnTenGagers exhibited limited binding to CD3-positive cells before TAA-crosslinking, suggesting a reduced risk of CRS that is caused by systemic CD3+T cell activation. Four CD3 sequences with varying affinities were utilized to construct AnTenGagers with identical TAA-targeting arm. Compared to clinical benchmarks targeting the same TAA, all AnTenGagers demonstrated substantially lower binding capacity to CD3+ cells in the absence of TAA-crosslinking, while inducing increased cytotoxicity against target-positive tumor cells. In addition, they induced significantly less ex-vivo cytokine release by human PBMC than benchmarks. Notably, all AnTenGagers demonstrated enhanced in vivo efficacy compared to the benchmark, exhibiting complete response (CR) in PBMC humanized MM1S mouse CDX model while the serum concentration of proinflammatory cytokines was substantially lower in groups treated with AnTenGagers. AnTenGagers also demonstrated good developability with high expression yield, good thermostability and high stability under different stress conditions. Conclusions: These results suggested that AnTenGagerTM is a promising platform for developing the next generation of TCEs with enhanced efficacy and safety. Citation Format: Bing Hou, Hui Yuwen, Tengteng Li, Yijing Ren, Ao Sun, Zaoshun Hu, Jay Mei, Bo Shan. AnTenGagerTM, a novel “2+1” T cell engager platform, enables conditional T cell activation with reduced risk of CRS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6343.