Intermediate Affinity Research Articles

Binding characteristics of the major kratom alkaloid, mitragynine, towards serum albumin: Spectroscopic, calorimetric, microscopic, and computational investigations

Mitragynine (MTG) is a prominent indole alkaloid that is present abundantly in Mitragyna speciosa, commonly referred to as kratom. MTG has garnered significant attention due to its selective agonistic characteristics towards opioid receptors and related analgesic effects. In the circulatory system, the in vivo efficacy of MTG is dictated by its interaction with plasma proteins, primarily human serum albumin (HSA). In the present study, we utilized a broad methodology that included spectroscopic, calorimetric, microscopic, and in silico approaches to characterize the interaction between MTG and HSA. Alterations in the UV absorption spectrum of HSA by the presence of MTG demonstrated a ground-state complexation between the protein and the ligand. The Ka values obtained for the MTG–HSA interaction were in the range 103–104 M−1 based on analysis of fluorescence and ITC data, respectively, indicating an intermediate binding affinity. The binding reaction was thermodynamically favorable as revealed by ΔH, ΔS, and ΔG values of −16.42 kJ mol−1, 39.97 J mol−1 K−1, and −28.34 kJ mol−1, respectively. Furthermore, CD spectroscopy results suggested MTG binding induced minimal effects on the structural integrity of HSA, supported by computational methods. Changes in the dimensions of HSA particles due to aggregation, as observed using atomic force microscopy in the presence of MTG. Competitive drug displacement results seemingly suggested site III of HSA located at subdomain IB as the preferred binding site of MTG, but were in inconclusive. However, docking results showed the clear preference of MTG to bind to site III, facilitated by hydrophobic (alkyl and pi-alkyl) and van der Waals forces, together with carbon hydrogen bonds. Additionally, the MTG–HSA complexation was demonstrated to be stable based on molecular dynamics analysis. The outcomes of this study shed light on the therapeutic potential of MTG and can help in the design of more effective derivatives of the compound.

Novel engineered IL-2 Nemvaleukin alfa combined with PD1 checkpoint blockade enhances the systemic anti-tumor responses of radiation therapy

BackgroundCombining interleukin-2 (IL-2) with radiotherapy (RT) and immune checkpoint blockade (ICB) has emerged as a promising approach to address ICB resistance. However, conventional IL-2 cytokine therapy faces constraints owing to its brief half-life and adverse effects. RDB 1462, the mouse ortholog of Nemvaleukin alfa, is an engineered IL-2 with an intermediate affinity that selectively stimulates antitumor CD8 T and NK cells while limiting regulatory T cell expansion. This study aimed to evaluate the antitumor activity and mechanism of action of the combination of RDB 1462, RT, and anti-PD1 in mouse tumor models.MethodsTwo bilateral lung adenocarcinoma murine models were established using 344SQ-Parental and 344SQ anti-PD1-resistant cell lines. Primary tumors were treated with RT, and secondary tumors were observed for evidence of abscopal effects. We performed immune phenotyping by flow cytometry, analyzed 770 immune-related genes using NanoString, and performed T cell receptor (TCR) repertoire analysis. Serum pro-inflammatory cytokine markers were analyzed by 23-plex kit.ResultsCompared to native IL-2 (RDB 1475), RDB 1462 demonstrated superior systemic antitumoral responses, attributable, at least in part, to augmented levels of CD4 and CD8 T cells with the latter. Our findings reveal substantial reductions in primary and secondary tumor volumes compared to monotherapy controls, with some variability observed among different dosing schedules of RDB 1462 combined with RT. Blood and tumor tissue-based flow cytometric phenotyping reveals an increase in effector memory CD8 and CD4 T cells and a decrease in immunosuppressive cells accompanied by a significant increase in IL-2, IFN-γ, and GM-CSF levels in the combination group. Transcriptomic profiling and TCR sequencing reveal favorable gene expression and T cell repertoire patterns with the dual combination. Furthermore, integrating anti-PD1 therapy with RT and RDB 1462 further reduced primary and secondary tumor volumes, prolonged survival, and decreased lung metastasis. Observations of immune cell profiles indicated that RT with escalating doses of RDB 1462 significantly reduced tumor growth and increased tumor-specific immune cell populations.ConclusionThe addition of Nemvaleukin therapy may enhance responses to RT alone and in combination with anti-PD1.Graphical

Understanding the Role of Strain on Electrochemical Reaction Kinetics: Oxygen Evolution on Mechanically Strained Rutile TiO2 As a Model System

Electrocatalysis offers a green alternative (when coupled with renewably-sourced electrons) to conventional carbon-intensive chemical fuel and commodity production processes. Electrochemical device efficiency, however, is limited by the sluggish kinetics of multi-electron transfer reactions, such as the oxygen evolution reaction (OER). Theoretical adsorption free energy scaling relationships predict large oxygen evolution overpotentials (>370 mV) for metal oxides performing the adsorbate evolution mechanism, a series of four proton-concerted electron transfer steps.1 Identifying catalyst design strategies that overcome or “break” scaling relations (i.e. strain,2 high entropy alloys,3 non-concerted proton and electron transfer mechanisms4,5) would provide a means to lower kinetic potential energy penalties and enhance the efficiency and economic incentives for further development of renewably-driven electrochemical reactions.Here we investigate the role of mechanically induced strain on the OER activity of polycrystalline rutile TiO2 thin films, grown on the super elastic alloy nitinol (NiTi) and mechanically strained by tensile tester. Our work characterizes how strain manifests in these complex polycrystalline systems with complementary physical characterization techniques (e.g. X-ray diffraction and Raman spectroscopy) and relates strain to OER performance in alkaline electrolytes. We contextualize our results within the framework of conventional adsorption scaling relationships to hypothesize how tensile strain influences reaction intermediate adsorption affinities, inferring implications for the OER kinetics of benchmark IrO2 and RuO2 catalysts that share the same crystal structure as the rutile TiO2 considered here.(1) Jaramillo, T. F.; Nørskov, J. K.; Rossmeisl, J. Universality in Oxygen Evolution Electrocatalysis on Oxide Surfaces. ChemCatChem 2011, 3 (7), 1159–1165. https://doi.org/10.1002/cctc.201000397.(2) Khorshidi, A.; Violet, J.; Hashemi, J.; Peterson, A. A. How Strain Can Break the Scaling Relations of Catalysis. Nat Catal 2018, 1 (4), 263–268. https://doi.org/10.1038/s41929-018-0054-0.(3) Kante, M. V.; Weber, M. L.; Ni, S.; van den Bosch, I. C. G.; van der Minne, E.; Heymann, L.; Falling, L. J.; Gauquelin, N.; Tsvetanova, M.; Cunha, D. M.; Koster, G.; Gunkel, F.; Nemšák, S.; Hahn, H.; Velasco Estrada, L.; Baeumer, C. A High-Entropy Oxide as High-Activity Electrocatalyst for Water Oxidation. ACS Nano 2023, 17 (6), 5329–5339. https://doi.org/10.1021/acsnano.2c08096.(4) Grimaud, A.; Diaz-Morales, O.; Han, B.; Hong, W. T.; Lee, Y.-L.; Giordano, L.; Stoerzinger, K. A.; Koper, M. T. M.; Shao-Horn, Y. Activating Lattice Oxygen Redox Reactions in Metal Oxides to Catalyse Oxygen Evolution. Nature Chem 2017, 9 (5), 457–465. https://doi.org/10.1038/nchem.2695.(5) Yoo, J. S.; Rong, X.; Liu, Y.; Kolpak, A. M. Role of Lattice Oxygen Participation in Understanding Trends in the Oxygen Evolution Reaction on Perovskites. ACS Catal. 2018, 8 (5), 4628–4636. https://doi.org/10.1021/acscatal.8b00612.

Unraveling the interaction mechanism between orphan drug Nitisinone and bovine serum albumin through spectroscopic and in silico approaches

The interaction between Nitisinone (NTBC) and bovine serum albumin (BSA) as the transport protein in a circulating system was investigated for the first time utilizing various analytical (UV–Vis spectrophotometry, fluorescence spectroscopy, dynamic light scattering, and differential scanning calorimetry) and computational (molecular docking and molecular dynamics simulations) methods. The BSA fluorescence intensity was quenched upon interaction with NTBC, and the quenching mechanism was observed as static. The interaction between NTBC and BSA was examined at 288 K, 298 K, and 308 K where the binding constants were found to be 1.44 × 105 ± 0.22 M−1, 5.18 × 104 ± 0.20 M−1, and 3.02 × 104 ± 0.22 M−1 respectively, suggesting an intermediate binding affinity between NTBC and BSA. Changes in the microenvironment surrounding tryptophan and tyrosine residues of BSA were elucidated using 3-D fluorescence spectroscopy. Thermodynamic studies revealed the calculated values of ΔH = − 54.34 ± 5 kJ/mol and ΔS = − 0.0908 ± 0.24 kJ/mol K−1, indicating the involvement of van der Waals forces and hydrogen bonds in the interaction between NTBC and BSA. Moreover, the interaction’s spontaneous nature was evidenced by negative ΔG values across all temperatures. Using dynamic light scattering, it was observed that higher NTBC concentrations led to a gradual rise in hydrodynamic diameter and notable aggregation of the NTBC-BSA complex. Moreover, changing signal values and shifted peaks of BSA, NTBC, and complex in differential scanning calorimetry curves, meant there were molecular interactions between the NTBC and BSA. In silico approaches also elucidated how NTBC binds to active sites on BSA, further supporting other findings. Moreover, molecular docking studies offer a more profound insight into the changing dynamics of hydrophobic, hydrogen, and halogen bonding involved in stabilizing the NTBC-BSA complex.

Abstract 4083: Pre-clinical development of IAMA-005, a long-acting cytokine fusion of modified IL-2 and IFN-α

Abstract In 1990s, both IL-2 and IFN-α were approved as the first two immunotherapies for metastatic melanoma and renal cell carcinoma; IFN-α was also approved for hairy cell leukemia. Although the response rates are relatively low, along ~25%, those who responded have long term survival benefits. However, the severe toxicity especially in high dose prohibited their broad use in oncology. Recently, there have been tremendous interests in modifying these two cytokines in the efforts to reduce the toxicity and enhance the activity. One such effort is to enhance the binding of IL-2 to the intermediate affinity β/γ receptors, which are expressed and function on CD8 T cells and NK cells. Another effort is to reduce the binding of IL-2 to high affinity α/β/γ receptors which mostly expressed on Treg and stimulate the growth of Treg cells, which suppress immune responses. Recently, whether it’s better to target intermediate affinity or high affinity for cancer therapy has become controversial with some studies showing that high affinity receptor engagements are required for robust anti-tumor activity while others favoring intermediate affinity receptor engagement for better efficacy; However, the clinical comparison is still lacking to be sure which approach is better. Here we have developed a long-acting novel IL-2-Fc-IFN-α fusion molecule termed IAMA-005 that has mutations to reduce the toxicity effects of IL-2 and IFN-α. We have characterized this molecule preclinically both in vitro and in vivo. IAMA-005 has demonstrated a potent tumor cell killing activities in numerous cancer cell lines including BT-474, SK-OV-3, NCI-N87, OE19, HCC-1954, MDA-MB-231, MDA-MB-468. When combined with IAMA-004 (a Her2-CD47-CD16 tri-specific antibody) in vitro, the tumor cell killing activities were dramatically increased. In addition, IAMA-005 was also tested in patient-derived Ex-Vivo fluids (one malignant pleural effusion and one malignant asities) and demonstrated excellent tumor cell killing efficacies. In addition, the killing activities of IAMA-005 could be attributed to the specific part of the fusion molecule because of the TME immune cell compositions. The in vivo assays to demonstrate efficacy and safety are ongoing and will be presented in the poster. These results suggest that this fusion cytokine molecule may be used in monotherapy in certain indications and an excellent complementary therapeutic to antibody targeted therapies or immunotherapies when used in combination. Citation Format: Liming Liu, Zhen Han, Yang Fan, Qi Pan. Pre-clinical development of IAMA-005, a long-acting cytokine fusion of modified IL-2 and IFN-α [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 4083.

Abstract 6318: Development of an anti-PD-L1 CAR T therapeutic treating advanced gastric cancer with enhanced efficacy and lower off-tumor activities

Abstract Purpose: Driven by a relentless unmet need with unsatisfied cancer therapy, we aimed to develop chimeric antigen receptor T-cell (CAR-T) therapy for targeting advanced gastric cancer (AGC) expressing PD-L1. We constructed a 2nd generation CAR lentiviral vector expressing a unique anti-PD-L1 scFv with moderate affinity (VaxCAR001) in order to compare scFvs derived from Avelumab or Atezolizumab. This project focused on the efficacy and safety of PD-L1 CAR-Ts, which are affected by their binding affinity and dissociation with the target protein, PD-L1 during the treatment period. Methods and Results: The findings of this study revealed significant differences among PD-L1-targeting CAR-T cells with different affinities and dissociations. VaxCAR001 cells, showing intermediate affinity, demonstrated a higher CD8 ratio (up to 78% compared to 58% in Avelumab-derived and 45% in Atezolizumab-derived CAR-T), reduced expression of inhibitory markers including Tim-3, LAG-3, and TIGIT, (8%, 39% and 13% in VaxCAR001 vs 35%, 55% and 42 % in Avelumab, 19%, 51% and 34% in Atezolizumab scFv-derived CAR-T, respectively) and more sustained cytotoxicity. Notably, it maintained strong cytotoxicity even one month after production, while the Avelumab or Atezolizumab ScFv-derived CAR-T cells did not. In multicellular spheroid 3D co-culture with CAR T cells, VaxCAR001 exhibited superior infiltration into the core of spheroid and induced higher CAR-T-mediated killing for 3D tumor spheroids as indicated by LDH assay (68%, 800% increased than Avelumab or Atezolizumab scFv-derived CAR-T). Moreover, in an MSI-high AGC mouse model, treatment with VaxCAR001 cells resulted in a significant reduction in tumor size and prolonged survival until the end of the study period. On the other hand, treatment with the Avelumab or Atezolizumab scFv-derived CAR-T cells led to high mortality, with most mice succumbing within three weeks after CAR-T cell injection, suggesting an on-target off-tumor effect. Conclusion: This study demonstrates that VaxCAR001 cells bearing intermediate affinity and high dissociation rate for target PD-L1 showed the superior killing of PD-L expressing tumor cells in vitro and in vivo and manifested better safety profiles, as compared to CAR-T cells bearing scFv derived from commercial anti-PD-L1 targeting monoclonal antibodies such as avelumab and atezolizumab. VaxCAR001 revealed extended cytotoxicity, enhanced infiltration into tumor spheroids, and greater tumor size reduction in in vivo models. Importantly, it also demonstrated superior safety profiles, as evidenced by reduced expression of inhibitory markers and fewer off-tumor adverse effects. These findings would accelerate the clinical stage developments as an effective and safer treatment strategy against AGC. Citation Format: Minwoo Baek, Jae-In Yu, Eun-Ji Choi, Yeojin Lim, Jiwon Yang, Se-Ryeong Choi, Yoonjoo Choi, Mihwa Kim, Joon Haeng Rhee, Je-Jung Lee. Development of an anti-PD-L1 CAR T therapeutic treating advanced gastric cancer with enhanced efficacy and lower off-tumor activities [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 6318.

Abstract 1235: Impact of anti-CD3 and tumor-target binder affinities on in-vitro potency, in-vivo efficacy, and cytokine release

Abstract Background: First generation T cell bispecifics (TCBs) were developed by utilizing high CD3 and tumor target binder affinities to induce a potent response. Instrumentalizing this, TCBs have clinically proven to elicit efficacious target cell killing and anti-tumor efficacy, but they encountered hurdles related to cytokine release. Recent studies suggested that reducing CD3 binder affinity could lead to an efficient tumor killing with lower cytokine levels. However, the answer to whether cytokine release is mainly dependent on CD3 binder affinity or a multifactorial phenomenon remains ambiguous. Here, we aim to characterize the efficacy-safety relationship of different TCBs through investigating the interplay between CD3 binder affinity and the affinity of the tumor target-specific binder. For that purpose, a series of TCBs with varying CD3 affinities, and two target binders of high and low affinities were generated in the 2:1 TCB format, resulting in a bivalent binding to the tumor antigen and a monovalent one to CD3, which offers a unique opportunity to enhance the activity of TCBs. Methods: Firstly, 2:1 TCBs comprising either the high or low affinity target binder and varying CD3 binder affinities were co-cultured in-vitro with human peripheral blood mononuclear cells (PBMCs), and human tumor cell lines, in order to assess T cell mediated cytotoxicity and cytokine release at different time points. Additionally, the impact on in-vivo efficacy, cytokine release, and pharmacokinetics was assessed in humanized mice implanted with a patient-derived xenograft model. Results: Based on the dose-response in the cytotoxicity assay, the CD3 binders were classified into high, intermediate, and low levels of affinity. As expected, we observed that the in-vitro T cell cytotoxicity, in-vivo tumor growth inhibition (TGI), and cytokine release levels were maximal with the highest affine CD3 binder. Simultaneously, we have observed a decrease in cytotoxicity response with decreasing CD3 affinity compared to the high affinity binder. However, this was accompanied by an atypical decrease in cytokine release. Particularly, the TCB comprising the intermediate CD3 affinity binder illustrated a comparable in-vitro cytotoxicity and in-vivo TGI to the high affinity binder with a marked decrease in cytokine release [4x reduction in-vitro and 6x reduction in-vivo]. Upon testing the TCBs comprising the low affinity target binder with the same CD3 binders, we observed an additional decrease in cytokine release with the decreasing CD3 affinity. In conclusion, we designed and generated TCBs with varying affinities in both the CD3 and target binding arms. Reducing cytokine release while maintaining adequate efficacy is feasible through CD3 binder affinity attenuation; however, the target binder affinity should be taken into consideration when interpreting our findings and designing new molecules. Citation Format: Omar Abdelmotaleb, Anneliese Schneider, Thomas Hofer, Johannes Sam, Martin Lechmann, Inja Waldhauer, Ali Bransi, Miro Eigenmann, Anne Freimoser-Grundschober, Christian Gassner, Alex Odermatt, Peter Brünker, Sara Colombetti, Christian Klein. Impact of anti-CD3 and tumor-target binder affinities on in-vitro potency, in-vivo efficacy, and cytokine release [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 1235.

Lanthanide transport in angstrom-scale MoS2-based two-dimensional channels.

Rare earth elements (REEs), critical to modern industry, are difficult to separate and purify, given their similar physicochemical properties originating from the lanthanide contraction. Here, we systematically study the transport of lanthanide ions (Ln3+) in artificially confined angstrom-scale two-dimensional channels using MoS2-based building blocks in an aqueous environment. The results show that the uptake and permeability of Ln3+ assume a well-defined volcano shape peaked at Sm3+. This transport behavior is rooted from the tradeoff between the barrier for dehydration and the strength of interactions of lanthanide ions in the confinement channels, reminiscent of the Sabatier principle. Molecular dynamics simulations reveal that Sm3+, with moderate hydration free energy and intermediate affinity for channel interaction, exhibit the smallest dehydration degree, consequently resulting in the highest permeability. Our work not only highlights the distinct mass transport properties under extreme confinement but also demonstrates the potential of dialing confinement dimension and chemistry for greener REEs separation.

Tracking the role of Aire in immune tolerance to the eye with a TCR transgenic mouse model

Roughly one-half of mice with partial defects in two immune tolerance pathways (AireGW/+Lyn-/- mice) spontaneously develop severe damage to their retinas due to T cell reactivity to Aire-regulated interphotoreceptor retinoid-binding protein (IRBP). Single-cell T cell receptor (TCR) sequencing of CD4+ T cells specific for a predominate epitope of IRBP showed a remarkable diversity of autoantigen-specific TCRs with greater clonal expansions in mice with disease. TCR transgenic mice made with an expanded IRBP-specific TCR (P2.U2) of intermediate affinity exhibited strong but incomplete negative selection of thymocytes. This negative selection was absent in IRBP-/- mice and greatly defective in AireGW/+ mice. Most P2.U2+/- mice and all P2.U.2+/-AireGW/+ mice rapidly developed inflammation of the retina and adjacent uvea (uveitis). Aire-dependent IRBP expression in the thymus also promoted Treg differentiation, but the niche for this fate determination was small, suggesting differences in antigen presentation leading to negative selection vs. thymic Treg differentiation and a stronger role for negative selection in preventing autoimmune disease in the retina.

Controlling gene-expression variability via sequestration-based feedbacks

Expressed Transcription Factors (TFs) not only bind to sites at target promoters but also to decoy sites scattered across the genome. Binding to such “decoys” sequesters TFs critically impacting the response time and stochasticity (noise) in TF and target gene expression level. When the TF is a stable molecule, whose concentration is diluted by cellular growth, our results show that for fixed mean concentration levels, such decoy bindings can both enhance or suppress random fluctuations in TF levels depending on the source of noise (i.e., intrinsic vs. extrinsic noise) and the strength of binding (i.e., weak vs. strong decoys). We implement negative autoregulation where free (unbound) TF molecules inhibit their synthesis. Our analytical results corroborated by numerical simulations reveal that sequestration accentuates the effects of feedback in the sense that noise attenuation by negative feedback is higher with sequestration than in the absence of feedback. We next consider an alternative form of feedback where the TF increases the production of its decoys, and such feedback architectures are frequently seen in endogenous gene regulation involving microRNA-TF circuits and in controlling cellular stress responses. For these circuits where decoy numbers are TF-regulated, we identify limits of noise suppression, and in many cases, these limits occur at intermediate TF-decoy binding affinities.

Impact of SARS-CoV-2 vaccination on FcγRIIIA/CD16 dynamics in Natural Killer cells: relevance for antibody-dependent functions.

Natural Killer (NK) cells contribute to the protective effects of vaccine-induced antibodies thanks to the low affinity receptor for IgG, FcγRIIIA/CD16, whose aggregation leads to the killing of infected cells and IFNγ release, through which they potentiate adaptive immune responses. Forty-seven healthy young individuals undergoing either homologous (ChAdOx1-S/ChAdOx1-S) or heterologous (ChAdOx1-S/BNT162B2) SARS-CoV-2 vaccination settings were recruited. Peripheral blood samples were collected immediately prior to vaccination and 8 weeks after the booster dose. The phenotypic and functional profile of NK cells was evaluated by flow cytometry at both time points. Serum samples were tested to evaluate circulating anti-Spike IgG levels and cytomegalovirus serostatus. CD16 F158V polymorphism was assessed by sequencing analysis. The downregulation of CD16 and the selective impairment of antibody-dependent cytotoxicity and IFNγ production in CD56dim NK population, persisting 8 weeks after boosting, were observed in heterologous, but not in homologous SARS-CoV-2 vaccination scheme. While the magnitude of CD16-dependent functions of the global CD56dim pool correlated with receptor levels before and after vaccination, the responsivity of NKG2C+ subset, that displays amplified size and functionality in HCMV+ individuals, resulted intrinsically insensitive to CD16 levels. Individual CD16 responsiveness was also affected by CD16F158V polymorphism; F/F low affinity individuals, characterized by reduced CD16 levels and functions independently of vaccination, did not show post-vaccinal functional impairment with respect to intermediate and high affinity ones, despite a comparable CD16 downregulation. Further, CD16 high affinity ligation conditions by means of afucosylated mAb overcame vaccine-induced and genotype-dependent functional defects. Finally, the preservation of CD16 expression directly correlated with anti-Spike IgG titer, hinting that the individual magnitude of receptor-dependent functions may contribute to the amplification of the vaccinal response. This study demonstrates a durable downmodulation of CD16 levels and Ab-dependent NK functions after SARS-CoV-2 heterologous vaccination, and highlights the impact of genetic and environmental host-related factors in modulating NK cell susceptibility to post-vaccinal Fc-dependent functional impairment.

Tuning the potency and selectivity of ImmTAC molecules by affinity modulation.

T-cell-engaging bispecifics have great clinical potential for the treatment of cancer and infectious diseases. The binding affinity and kinetics of a bispecific molecule for both target and T-cell CD3 have substantial effects on potency and specificity, but the rules governing these relationships are not fully understood. Using immune mobilizing monoclonal TCRs against cancer (ImmTAC) molecules as a model, we explored the impact of altering affinity for target and CD3 on the potency and specificity of the redirected T-cell response. This class of bispecifics binds specific target peptides presented by human leukocyte antigen on the cell surface via an affinity-enhanced T-cell receptor and can redirect T-cell activation with an anti-CD3 effector moiety. The data reveal that combining a strong affinity TCR with an intermediate affinity anti-CD3 results in optimal T-cell activation, while strong affinity of both targeting and effector domains significantly reduces maximum cytokine release. Moreover, by optimizing the affinity of both parts of the molecule, it is possible to improve the selectivity. These results could be effectively modelled based on kinetic proofreading with limited signalling. This model explained the experimental observation that strong binding at both ends of the molecules leads to reduced activity, through very stable target-bispecific-effector complexes leading to CD3 entering a non-signalling dark state. These findings have important implications for the design of anti-CD3-based bispecifics with optimal biophysical parameters for both activity and specificity.

High-resolution structure of stem-loop 4 from the 5'-UTR of SARS-CoV-2 solved by solution state NMR.

We present the high-resolution structure of stem-loop 4 of the 5'-untranslated region (5_SL4) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) genome solved by solution state nuclear magnetic resonancespectroscopy. 5_SL4 adopts an extended rod-like structure with a single flexible looped-out nucleotide and two mixed tandem mismatches, each composed of a G•U wobble base pair and a pyrimidine•pyrimidine mismatch, which are incorporated into the stem-loop structure. Boththe tandem mismatches and the looped-out residue destabilize the stem-loop structure locally. Their distribution along the 5_SL4 stem-loop suggests a role of these non-canonical elements in retaining functionally important structural plasticity in particular with regard to the accessibility of the start codon of an upstream open reading frame located in the RNA's apical loop. The apical loop-although mostly flexible-harbors residual structural features suggesting an additional role in molecular recognition processes. 5_SL4 is highly conserved among the different variants of SARS-CoV-2 and can be targeted by small molecule ligands, which it binds with intermediate affinity in the vicinity of the non-canonical elements within the stem-loop structure.

An Examination of the Complex Pharmacological Properties of the Non-Selective Opioid Modulator Buprenorphine.

The goal of this review is to provide a recent examination of the pharmacodynamics as well as pharmacokinetics, misuse potential, toxicology, and prenatal consequences of buprenorphine. Buprenorphine is currently a Schedule III opioid in the US used for opioid-use disorder (OUD) and as an analgesic. Buprenorphine has high affinity for the mu-opioid receptor (MOR), delta (DOR), and kappa (KOR) and intermediate affinity for the nociceptin (NOR). Buprenorphine's active metabolite, norbuprenorphine, crosses the blood-brain barrier, is a potent metabolite that attenuates the analgesic effects of buprenorphine due to binding to NOR, and is responsible for the respiratory depressant effects. The area under the concentration curves are very similar for buprenorphine and norbuprenorphine, which indicates that it is important to consider this metabolite. Crowding sourcing has identified a buprenorphine street value (USD 3.95/mg), indicating some non-medical use. There have also been eleven-thousand reports involving buprenorphine and minors (age < 19) at US poison control centers. Prenatal exposure to clinically relevant dosages in rats produces reductions in myelin and increases in depression-like behavior. In conclusion, the pharmacology of this OUD pharmacotherapy including the consequences of prenatal buprenorphine exposure in humans and experimental animals should continue to be carefully evaluated.

Facet-Dependent Selectivity of Rutile IrO2 for Oxygen and Chlorine Evolution Reactions

Water electrolysis is a promising route for sustainable production of hydrogen as an energy storage medium and valuable precursor for industrial chemical syntheses such as ammonia and methanol. Direct electrolysis of seawater circumvents costly desalination and purification steps to reduce the price of renewable hydrogen to achieve cost parity with carbon-intensive steam reforming. However, the significant concentration of chloride salts in seawater poses a challenge to selectivity of seawater electrolysis. In aqueous chloride electrolytes, the chlorine evolution reaction (CER) is kinetically favored over the oxygen evolution reaction (OER). Rutile-type iridium dioxide (IrO2) is a state-of-the-art electrocatalyst for water electrolysis but is also a benchmark chlorine evolution electrocatalyst in the industrial chlor-alkali process. Understanding OER/CER selectivity is needed to make seawater electrolysis a viable energy conversion technology in the future.In this work, we seek to understand the relationship between crystallographic facet and competitive reaction pathway between OER and CER on epitaxial, rutile IrO2 thin films. We investigate facet-dependent OER and CER activity on a series of single-crystalline IrO2 thin films using a rotating disk electrode geometry. The OER/CER selectivity, reaction rate order, and reaction intermediate electroadsorption affinities are explored to add fundamental insight into the reactivity of rutile IrO2 surface. To gain further insight into OER intermediate adsorption affinities, we paired electrochemical measurements with surface-sensitive ambient pressure x-ray photoelectron spectroscopy (AP-XPS). Moving forward, our facet-dependent study of OER/CER selectivity of rutile IrO2 can be used to design selective seawater electrocatalysts for cost-effective and sustainable hydrogen production.

Robust IL-2-dependent antitumor immunotherapy requires targeting the high-affinity IL-2R on tumor-specific CD8+ T cells

BackgroundDevelopment of interleukin (IL)-2-dependent antitumor responses focus on targeting the intermediate affinity IL-2R to stimulate memory-phenotypic CD8+ T and natural killer (NK) cells while minimizing regulatory T cell (Treg) expansion....

Molecular identification and functional analysis of Niemann-Pick type C2 proteins，carriers for semiochemicals and other hydrophobic compounds in the brown dog tick, Rhipicephalus linnaei

Ticks are important vectors of many pathogens with tremendous impact on human and animal health. Studies of semiochemical interactions and mechanisms underlying chemoreception can provide important tools in tick management. Niemann-Pick type C2 (NPC2) proteins have been proposed as one type of chemoreceptor in arthropods. Here, we cloned two NPC2 genes in the brown dog tick, Rhipicephalus linnaei, the tropical lineage previously named R. sanguineus sensu lato and characterized them functionally.R.linNPC2a and R.linNPC2b genes were found to be expressed at each developmental stage with the highest level in adult males. By using quantitative real-time PCR we revealed expression in multiple tissues, including midgut, ovary, salivary glands and legs. Ligand binding analysis revealed that R.linNPC2b bound a wide spectrum of compounds, with β-ionone, α-amylcinnamaldehyde, 2-nitrophenol and benzaldehyde displaying the strongest binding affinity (Ki < 10 μM), whereas R.linNPC2a showed a more narrow ligand binding range, with intermediate binding affinity to α-amylcinnamaldehyde and 2-nitrophenol (Ki < 20 μM). Molecular docking indicated that the amino acid residue Phe89, Leu77 and Val131 of R.linNPC2a and Phe70, Leu132 and Phe73 of R.linNPC2b could bind multiple ligands. These residues might thus play a key role in the identification of the volatiles. Our results contribute to the understanding of olfactory mechanisms of R. linnaei and can offer new pathways towards new management strategies.

Abstract 889: The efficacy and safety of PD-L1-specific CAR-T in advanced gastric cancer

Abstract [Purpose] Heterogeneity and the tumor microenvironment are major obstacles to overcoming incurable advanced gastric cancer (AGC). Although Epstein-Barr virus-positive and microsatellite instability subtypes can be detected early and properly treated, they are rare in AGC. In this regard, recent combinational therapy of immune checkpoint inhibitors (ICIs) such as PD-1/PD-L1 inhibitors and other various therapeutics has been tried, but there are clinically limited doses and drug resistance. We hypothesized that CAR-T targeting PD-L1, the common tumor-associated antigen (TAA), which expresses in most advanced cancer cells, would make a significant breakthrough in curing a solid tumor. This study aimed to evaluate the killing effects of anti-PD-L1 CAR-T against gastric cancer cell lines. Removal of PD-L1-expressing cells in the AGC TME will improve the clinical outcomes of current therapies. [Results] We constructed a second-generation PD-L1 CAR lentiviral vector targeting PD-L1, which has an intermediate affinity with PD-L1 in solid tumor TME. As the secondary signaling motif, CD28 was chosen and tested along with the CD3 zeta chain. The effector CAR-T, named Vax-CAR-T, specifically recognized the PD-L1-expressing AGC cell line, SNU638, and exhibited rapid and robust cytotoxic activity in vitro. When compared to untransduced T cells, the level of the CD25 activation marker was higher in Vax-CAR-T cells. Besides, the inhibitory markers of Tim3 and LAG3 were expressed even higher than the untransduced. Despite high Tim3 and LAG3 expression in the final manufacturing process, Vax-CAR-T cells significantly suppressed tumor growth in both bigger (&amp;gt;95mm3) and smaller (≤50mm3) AGC xenograft NSG mouse models. More importantly, Vax-CAR-T-treated mice have well tolerated the CAR-T therapy with no significant adverse events during the CRS monitoring period, 2 weeks after Vax-CAR-T injection and have gradually recovered weight gains 5 days after CAR-T cell injection. [Conclusion] We believe that a new autologous CAR-T targeting PD-L1 could be a promising new tool for removing heterogeneous solid tumors in TME, which would be a significant step forward in improving current conventional and immunotherapeutic strategies. Citation Format: Hye-Seong Park, Eun-Ji Choi, Jae-In Yu, Kyung-Sik Lee, Yeo-Jin Lim, Se-Ryeong Choi, Yoonjoo Choi, Bum Chan Park, Jae Eun Park, Mihwa Kim, Je-Jung Lee, Joon Haeng Rhee. The efficacy and safety of PD-L1-specific CAR-T in advanced gastric cancer [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 889.

Abstract 4158: Selective targeting of CD122 combined with radiotherapy triggers CD8 and NK mediated immunity, abrogating metastasis in head and neck squamous cell carcinoma

Abstract The implementation of cancer immunotherapies, while revolutionary in certain cancer types, has seen limited clinical success in head and neck squamous cell carcinomas (HNSCC). This revelation has prompted investigation in alternative targets to invigorate anti-tumor immunity. IL-2 signaling governs the survival and functionality of lymphocytes, thus making IL-2 receptor signaling an attractive target for new immunotherapies. However, therapies to enhance IL-2 signaling are often limited in their efficacy by modulating the activation of regulatory T cells (Tregs), which preferentially binds IL-2 via the high affinity receptor IL-2Rα (CD25). Selectively targeting IL2Rβ (CD122), the intermediate affinity receptor, can be leveraged to induce anti-tumor immune responses in effector T cells and natural killer (NK) cells while limiting negative regulation conferred by Tregs. The novel bispecific immunocytokine PD1-IL2v preferentially activates IL-2 signaling through CD122 on PD-1 expressing cells, activating immune effectors, and preventing immune exhaustion by blockade of PD-1. However, PD-1 blockade can amplify immunosuppression by Tregs, necessitating their depletion using αCD25. Here, we observe that radiation therapy and combination of PD1-IL2v with an Fc-optimized αCD25 antibody induces systemic activation of effector CD4 and CD8 T cells, reducing overall tumor burden. Concordant with improved local tumor control, we also observed a decrease in metastatic spread using a newly developed Kras/smad4/p53 mutated metastatic tumor model. NK cells, which are known to play a central role in the control of tumor cell dissemination though direct cytotoxicity, displayed a phenotypic shift towards an activated state upon treatment with PD1-IL2v, highlighted by enhanced cytotoxicity, NK receptor diversity, and metabolic activity. Although relatively uncommon in comparison to other cancer types, those patients who present with metastases display a substantially poorer outcome, demonstrating the need for therapies which target this axis of cancer disease progression. In summary, we find that radiation therapy and PD1-IL2v imparts potent anti-tumor immunity, reducing local tumor growth and distant tumor spread, which taken together contribute to increased overall survival. Citation Format: Jacob Gadwa, Maria Amann, Thomas E. Bickett, Michael W. Knitz, Laurel B. Darragh, Miles Piper, Benjamin Van Court, Sanjana Bukkapatanam, Tiffany T. Pham, Xiao-Jing Wang, Laura Codarri Deak, Christian Klein, Pablo Umana, Angelo D'Alessandro, Sana Karam. Selective targeting of CD122 combined with radiotherapy triggers CD8 and NK mediated immunity, abrogating metastasis in head and neck squamous cell carcinoma. [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 4158.

Formation and characterization of BMP2/GDF5 and BMP4/GDF5 heterodimers

BackgroundProteins of the TGFβ family, which are largely studied as homodimers, are also known to form heterodimers with biological activity distinct from their component homodimers. For instance, heterodimers of bone morphogenetic proteins, including BMP2/BMP7, BMP2/BMP6, and BMP9/BMP10, among others, have illustrated the importance of these heterodimeric proteins within the context of TGFβ signaling.ResultsIn this study, we have determined that mature GDF5 can be combined with mature BMP2 or BMP4 to form BMP2/GDF5 and BMP4/GDF5 heterodimer. Intriguingly, this combination of a BMP2 or BMP4 monomer, which exhibit high affinity to heparan sulfate characteristic to the BMP class, with a GDF5 monomer with low heparan sulfate affinity produces a heterodimer with an intermediate affinity. Using heparin affinity chromatography to purify the heterodimeric proteins, we then determined that both the BMP2/GDF5 and BMP4/GDF5 heterodimers consistently signaled potently across an array of cellular and in vivo systems, while the activities of their homodimeric counterparts were more context dependent. These differences were likely driven by an increase in the combined affinities for the type 1 receptors, Alk3 and Alk6. Furthermore, the X-ray crystal structure of BMP2/GDF5 heterodimer was determined, highlighting the formation of two asymmetric type 1 receptor binding sites that are both unique relative to the homodimers.ConclusionsUltimately, this method of heterodimer production yielded a signaling molecule with unique properties relative to the homodimeric ligands, including high affinity to multiple type 1 and moderate heparan binding affinity.