Binding Subunit Research Articles

P0717 Real-world data of Mirikizumab in the induction and maintenance therapy for ulcerative colitis

Abstract Background Mirikizumab, a humanized IgG4 monoclonal antibody binding subunit p19 of interleukin 23, has been approved for the therapy of ulcerative colitis since May 2023. Phase 3 studies showed significant efficacy in both inducing and sustaining clinical remission in patients with moderate to severe active ulcerative colitis. Methods All patients with active ulcerative colitis who began induction therapy with Mirikizumab at our outpatient clinic between July 2023 and June 2024 were analyzed retrospectively with a follow-up period until October 2024. Patients with prednisolone therapy exceeding 20 mg, loperamide intake or deviation of standard clinical application period were excluded. The primary endpoints were defined as achieving clinical remission following the completion of intravenous induction (at weeks 12, 16 or 24) and the maintenance of clinical remission at any time between week 8 and week 49 following the initiation of subcutaneous application. Secondary endpoints included rates of clinical remission and relapse in patients previously treated with Ustekinumab, a monoclonal antibody targeting the p40 subunit of interleukin-12 and -23. Results A total of 40 patients started Mirikizumab induction therapy, with 23 meeting the inclusion criteria. Seventeen patients were observed in the maintenance period. After completion of induction, 20 out of 23 patients (87.0%) achieved clinical remission. Among these, 12 (60.0%) had previously been treated with Ustekinumab. During the maintenance period, 6 out of 17 patients (35.3%) experienced a relapse, of whom 5 (83.3%) had prior therapy with Ustekinumab. Within the Ustekinumab subgroup, 5 out of 10 patients (50.0%) suffered relapse after initially achieving clinical remission. Conclusion Mirikizumab is an effective therapy in patients with active ulcerative colitis, including those who had a prior loss of response to Ustekinumab therapy. Patients with prior Ustekinumab therapy exhibited a higher rate of relapse during the maintenance phase.

GABAAR-PPT1 palmitoylation homeostasis controls synaptic transmission and circuitry oscillation

The infantile neuronal ceroid lipofuscinosis, also called CLN1 disease, is a fatal neurodegenerative disease caused by mutations in the CLN1 gene encoding palmitoyl protein thioesterase 1 (PPT1). Identifying the depalmitoylation substrates of PPT1 is crucial for understanding CLN1 disease. In this study, we found that GABAAR, the critical synaptic protein essential for inhibitory neurotransmission, is a substrate of PPT1. PPT1 depalmitoylates GABAAR α1 subunit at Cystein-260, while binding to Cystein-165 and -179. Mutations of PPT1 or its GABAAR α1 subunit binding site enhanced inhibitory synaptic transmission and strengthened oscillations powers but disrupted phase coupling in CA1 region and impaired learning and memory in 1- to 2-months-old PPT1-deficient and Gabra1em1 mice. Our study highlights the critical role of PPT1 in maintaining GABAAR palmitoylation homeostasis and reveals a previously unknown molecular pathway in CLN1 diseases induced by PPT1 mutations.

Variable Assembly and Procapsid Binding of Bacteriophage P22 Terminase Subunits in Solution.

Concatemeric viral DNA is packaged into bacteriophage P22 procapsids via a headful packaging mechanism mediated by a molecular machine consisting of small (gp3) and large (gp2) terminase subunits. Although a negative stain reconstruction exists for the terminase holoenzyme, it is not clear how this complex binds the dodecameric portal protein located at a 5-fold mismatch vertex. Herein, we describe new assemblies for the holoenzyme. Both native mass spectrometry and transmission electron microscopy reveal that the P22 terminase complex adopts three main assemblies, which include a nonameric S-terminase bound to two L-terminase 1(gp3)9:2(gp2), two nonameric S-terminase bound to five L-terminase 2(gp3)9:5(gp2), and three nonameric S-terminase bound to seven L-terminase 3(gp3)9:7(gp2). Native agarose gel electrophoresis shows that the terminase complex interacts with procapsids with mild crosslinking. These results herein illustrate the P22 terminase complex can adopt a variety of conformations and assembly states.

ARID1A governs the silencing of sex-linked transcription during male meiosis in the mouse

We present evidence implicating the BAF (BRG1/BRM Associated Factor) chromatin remodeler in meiotic sex chromosome inactivation (MSCI). By immunofluorescence (IF), the putative BAF DNA binding subunit, ARID1A (AT-rich Interaction Domain 1 a), appeared enriched on the male sex chromosomes during diplonema of meiosis I. Germ cells showing a Cre-induced loss of ARID1A arrested in pachynema and failed to repress sex-linked genes, indicating a defective MSCI. Mutant sex chromosomes displayed an abnormal presence of elongating RNA polymerase II coupled with an overall increase in chromatin accessibility detectable by ATAC-seq. We identified a role for ARID1A in promoting the preferential enrichment of the histone variant, H3.3, on the sex chromosomes, a known hallmark of MSCI. Without ARID1A, the sex chromosomes appeared depleted of H3.3 at levels resembling autosomes. Higher resolution analyses by CUT&RUN revealed shifts in sex-linked H3.3 associations from discrete intergenic sites and broader gene-body domains to promoters in response to the loss of ARID1A. Several sex-linked sites displayed ectopic H3.3 occupancy that did not co-localize with DMC1 (DNA meiotic recombinase 1). This observation suggests a requirement for ARID1A in DMC1 localization to the asynapsed sex chromatids. We conclude that ARID1A-directed H3.3 localization influences meiotic sex chromosome gene regulation and DNA repair.

ARID1A governs the silencing of sex-linked transcription during male meiosis in the mouse.

We present evidence implicating the BAF (BRG1/BRM Associated Factor) chromatin remodeler in meiotic sex chromosome inactivation (MSCI). By immunofluorescence (IF), the putative BAF DNA binding subunit, ARID1A (AT-rich Interaction Domain 1 a), appeared enriched on the male sex chromosomes during diplonema of meiosis I. Germ cells showing a Cre-induced loss of ARID1A arrested in pachynema and failed to repress sex-linked genes, indicating a defective MSCI. Mutant sex chromosomes displayed an abnormal presence of elongating RNA polymerase II coupled with an overall increase in chromatin accessibility detectable by ATAC-seq. We identified a role for ARID1A in promoting the preferential enrichment of the histone variant, H3.3, on the sex chromosomes, a known hallmark of MSCI. Without ARID1A, the sex chromosomes appeared depleted of H3.3 at levels resembling autosomes. Higher resolution analyses by CUT&RUN revealed shifts in sex-linked H3.3 associations from discrete intergenic sites and broader gene-body domains to promoters in response to the loss of ARID1A. Several sex-linked sites displayed ectopic H3.3 occupancy that did not co-localize with DMC1 (DNA meiotic recombinase 1). This observation suggests a requirement for ARID1A in DMC1 localization to the asynapsed sex chromatids. We conclude that ARID1A-directed H3.3 localization influences meiotic sex chromosome gene regulation and DNA repair.

Paip2 protein of <i>Drosophila melanogaster</i> Binds ENY2 protein and interacts with TREX-2 complex in histone mRNP particles

ENY2 is an evolutionarily conserved multifunctional protein that is a member of several complexes regulating different stages of gene expression. ENY2 is a subunit of the TREX-2 complex, which is necessary for the export of bulk mRNA from the nucleus to the cytoplasm through the nuclear pores in many eukaryotes. The wide range of ENY2 functions suggests that it may also associate with other protein factors or complexes. Aiming to search for proteins that interact with ENY2, a cDNA library was screened in a yeast two-hybrid system. As a result, ENY2 was found to interact with the RNA-binding protein Paip2. Paip2 directly binds to ENY2 in vitro and interacts with ENY2 in vivo at the molecular and genetic level. Paip2 is capable to associate with the ENY2-containing TREX-2 complex. We found that Paip2 protein is present at the histone gene cluster locus, both Paip2 and ENY2 are detected at the histone locus body (HLB), a nuclear structure where coordinated histone mRNA transcription and processing take place. Paip2 and the subunits of the TREX-2 complex associate with histone mRNP particles. RNA-interference knockdown of Paip2 results in decreased binding of TREX-2 subunits to histone mRNPs. Thus, Paip2 is a new partner protein of ENY2 within the TREX-2 complex and participates in TREX-2 binding to histone mRNPs.

Structural basis for molecular assembly of fucoxanthin chlorophyll a/c-binding proteins in a diatom photosystem I supercomplex

Photosynthetic organisms exhibit remarkable diversity in their light-harvesting complexes (LHCs). LHCs are associated with photosystem I (PSI), forming a PSI-LHCI supercomplex. The number of LHCI subunits, along with their protein sequences and pigment compositions, has been found to differ greatly among the PSI-LHCI structures. However, the mechanisms by which LHCIs recognize their specific binding sites within the PSI core remain unclear. In this study, we determined the cryo-electron microscopy structure of a PSI supercomplex incorporating fucoxanthin chlorophyll a/c-binding proteins (FCPs), designated as PSI-FCPI, isolated from the diatom Thalassiosira pseudonana CCMP1335. Structural analysis of PSI-FCPI revealed five FCPI subunits associated with a PSI monomer; these subunits were identified as RedCAP, Lhcr3, Lhcq10, Lhcf10, and Lhcq8. Through structural and sequence analyses, we identified specific protein–protein interactions at the interfaces between FCPI and PSI subunits, as well as among FCPI subunits themselves. Comparative structural analyses of PSI-FCPI supercomplexes, combined with phylogenetic analysis of FCPs from T. pseudonana and the diatom Chaetoceros gracilis, underscore the evolutionary conservation of protein motifs crucial for the selective binding of individual FCPI subunits. These findings provide significant insights into the molecular mechanisms underlying the assembly and selective binding of FCPIs in diatoms.

Structural basis for molecular assembly of fucoxanthin chlorophyll a/c-binding proteins in a diatom photosystem I supercomplex.

Photosynthetic organisms exhibit remarkable diversity in their light-harvesting complexes (LHCs). LHCs are associated with photosystem I (PSI), forming a PSI-LHCI supercomplex. The number of LHCI subunits, along with their protein sequences and pigment compositions, has been found to differ greatly among the PSI-LHCI structures. However, the mechanisms by which LHCIs recognize their specific binding sites within the PSI core remain unclear. In this study, we determined the cryo-electron microscopy structure of a PSI supercomplex incorporating fucoxanthin chlorophyll a/c-binding proteins (FCPs), designated as PSI-FCPI, isolated from the diatom Thalassiosira pseudonana CCMP1335. Structural analysis of PSI-FCPI revealed five FCPI subunits associated with a PSI monomer; these subunits were identified as RedCAP, Lhcr3, Lhcq10, Lhcf10, and Lhcq8. Through structural and sequence analyses, we identified specific protein-protein interactions at the interfaces between FCPI and PSI subunits, as well as among FCPI subunits themselves. Comparative structural analyses of PSI-FCPI supercomplexes, combined with phylogenetic analysis of FCPs from T. pseudonana and the diatom Chaetoceros gracilis, underscore the evolutionary conservation of protein motifs crucial for the selective binding of individual FCPI subunits. These findings provide significant insights into the molecular mechanisms underlying the assembly and selective binding of FCPIs in diatoms.

Calcineurin-mediated dephosphorylation stabilizes E2F1 protein by suppressing binding of the FBXW7 ubiquitin ligase subunit

The transcription factor E2F1 serves as a regulator of the cell cycle and promotes cell proliferation. It is highly expressed in cancer tissues and contributes to their malignant transformation. Degradation by the ubiquitin-proteasome system may help to prevent such overexpression of E2F1 and thereby to suppress carcinogenesis. A detailed understanding of the mechanisms underlying E2F1 degradation may therefore inform the development of new cancer treatments. We here identified SCFFBXW7 as a ubiquitin ligase for E2F1 by comprehensive analysis. We found that phosphorylation of E2F1 at serine-403 promotes its binding to FBXW7 (F-box/WD repeat-containing protein 7) followed by its ubiquitination and degradation. Furthermore, calcineurin, a Ca2+/calmodulin-dependent serine-threonine phosphatase, was shown to stabilize E2F1 by mediating its dephosphorylation at serine-403 and thereby preventing FBXW7 binding. Treatment of cells with Ca2+ channel blockers resulted in downregulation of both E2F1 protein and the expression of E2F1 target genes, whereas treatment with the Ca2+ ionophore ionomycin induced upregulation of E2F1. Finally, the calcineurin inhibitor FK506 attenuated xenograft tumor growth in mice in association with downregulation of E2F1 in the tumor tissue. Impairment of the balance between the opposing actions of FBXW7 and calcineurin in the regulation of E2F1 abundance may therefore play an important role in carcinogenesis.

Introduction of a Sodium-Binding Motif into Subunits <i>a</i> and <i>c</i> of <i>Bacillus</i> sp. PS3 Proton F-ATPase Does Not Result in Sodium Specificity of the Enzyme

In bacteria F-type ATPase (F-ATPase) plays a key role in bioenergetics and couples ATP synthesis/hydrolysis with the transport of ions (H+ or Na+) across the membrane. The ion specificity of the enzyme is determined by the amino acid sequence of subunits c and а. Here, we introduced several mutations (7 in subunit c and 6 in subunit a) into F-ATPase of thermophilic bacterium Bacillus sp. PS3 in order to change the ion specificity of the enzyme from proton to sodium. The mutations did not affect the ATPase activity of the enzyme, but led to loss of proton conductivity and impaired the binding of subunit a to the c-subunit oligomer, rather than changed the ion specificity.

Newcastle disease virus induces clathrin-mediated endocytosis to establish infection through the activation of PI3K/AKT signaling pathway by VEGFR2.

Newcastle disease virus (NDV) is a threat to the global poultry industry; however, the mechanisms of NDV infection remain unclear. NDV affects the phosphatidyl-inositol 3-kinase/serine-threonine kinase (PI3K/ AKT) signaling pathway, requiring endocytosis for successful infection. Based on previous studies, we identified a close correlation between NDV infection and replication and the PI3K/AKT signaling pathway activity. This study examined the molecular mechanisms through which NDV activates the PI3K/AKT signaling pathway to regulate endocytosis and facilitate infection. This study showed that early-stage in vitro NDV infection activated the PI3K/AKT signaling pathway, enhancing clathrin-mediated endocytosis, crucial for infection onset. Notably, this process involves the interaction between NDV F protein and the vascular endothelial growth factor receptor 2 tyrosine kinase, leading to the subsequent binding and phosphorylation of the PI3K p85α regulatory subunit. This activation primes PI3K, initiating a cascade that promotes clathrin-mediated endocytosis. Our findings elucidate how NDV capitalizes on the PI3K/AKT signaling pathway to establish infection through endocytosis.

Comparison of Escherichia coli Responses to Different Silver Nanoparticles with Different Particle Sizes and Surface Coatings

AbstractAgNPs are widely used for their excellent antimicrobial properties, whereas the cytotoxicity they possess makes them an unignorable environmental problem. Considering the impact of particle size and surface coating on the antibacterial properties, four types of AgNPs are selected: citrate‐coated 20 nm (C20), polyvinylpyrrolidone‐coated 20 nm (P20), citrate‐coated 100 nm (C100), and polyvinylpyrrolidone‐coated 100 nm (P100) AgNPs. All four AgNPs significantly affect the ribosome pathway, with stronger binding of large and small subunits. 20 nm and 100 nm AgNPs both affected the biosynthesis and metabolism of several amino acids (including arginine, glycine, serine, threonine, glyoxylic acid, and dicarboxylic acid). P20 and C100 exposure affected bacterial chemotaxis and flagellar motility. This study preliminarily explained the response mechanism of E. coli to AgNPs with different properties, which provided a theoretical basis for predicting the response mechanism of E. coli to metal nanoparticles with similar properties.

ARID1A governs the silencing of sex-linked transcription during male meiosis in the mouse.

We present evidence implicating the BAF (BRG1/BRM Associated Factor) chromatin remodeler in meiotic sex chromosome inactivation (MSCI). By immunofluorescence (IF), the putative BAF DNA binding subunit, ARID1A (AT-rich Interaction Domain 1a), appeared enriched on the male sex chromosomes during diplonema of meiosis I. Those germ cells showing a Cre-induced loss of ARID1A were arrested in pachynema and failed to repress sex-linked genes, indicating a defective MSCI. Consistent with this defect, mutant sex chromosomes displayed an abnormal presence of elongating RNA polymerase II coupled with an overall increase in chromatin accessibility detectable by ATAC-seq. By investigating potential mechanisms underlying these anomalies, we identified a role for ARID1A in promoting the preferential enrichment of the histone variant, H3.3, on the sex chromosomes, a known hallmark of MSCI. Without ARID1A, the sex chromosomes appeared depleted of H3.3 at levels resembling autosomes. Higher resolution analyses by CUT&RUN revealed shifts in sex-linked H3.3 associations from discrete intergenic sites and broader gene-body domains to promoters in response to the loss of ARID1A. Several sex-linked sites displayed ectopic H3.3 occupancy that did not co-localize with DMC1 (DNA Meiotic Recombinase 1). This observation suggests a requirement for ARID1A in DMC1 localization to the asynapsed sex chromatids. We conclude that ARID1A-directed H3.3 localization influences meiotic sex chromosome gene regulation and DNA repair.

Direct modulation of G protein-gated inwardly rectifying potassium (GIRK) channels.

Ion channels play a pivotal role in regulating cellular excitability and signal transduction processes. Among the various ion channels, G-protein-coupled inwardly rectifying potassium (GIRK) channels serve as key mediators of neurotransmission and cellular responses to extracellular signals. GIRK channels are members of the larger family of inwardly-rectifying potassium (Kir) channels. Typically, GIRK channels are activated via the direct binding of G-protein βγ subunits upon the activation of G-protein-coupled receptors (GPCRs). GIRK channel activation requires the presence of the lipid signaling molecule, phosphatidylinositol 4,5-bisphosphate (PIP2). GIRK channels are also modulated by endogenous proteins and other molecules, including RGS proteins, cholesterol, and SNX27 as well as exogenous compounds, such as alcohol. In the last decade or so, several groups have developed novel drugs and small molecules, such as ML297, GAT1508 and GiGA1, that activate GIRK channels in a G-protein independent manner. Here, we aim to provide a comprehensive overview focusing on the direct modulation of GIRK channels by G-proteins, PIP2, cholesterol, and novel modulatory compounds. These studies offer valuable insights into the underlying molecular mechanisms of channel function, and have potential implications for both basic research and therapeutic development.

The Role of Speedy/RINGO Protein in Breast Cancer as a Future Biomarker.

Cyclin-dependent kinases (CDKs) are proteins that require the binding of regulatory subunits called cyclins and play a key role in cell cycle progression and activation. CDKs play a key role in carcinogenesis of many solid malignancies, and inhibition of these proteins has produced anti-cancer effects demonstrated in preclinical studies. This narrative review was conducted to develop a hypothetical approach to determine whether Speedy/RINGO, a protein associated with CDK2, could be a possible predictive factor in breast cancer patients treated with a CDK4/6 inhibitor. A literature search was conducted in PubMed, Web of Science, Medline, and Google Scholars search engines to match the following words: "Speedy/RINGO" or "Spy1" and "CDKs" or "Cyclin-dependent kinases (CDKs)" and "CDK4/6 inhibitors" and "Regulation" and "Molecular" and "Breast cancer" and "Carcinogenesis". Only articles investigating the relationship between the Speedy/RINGO protein and CDKs at the molecular level were included. Literature information was compiled by trying to establish a relationship with our hypothesis question. Speedy/RINGO is a tightly regulated proto-oncogenic mammalian protein playing important roles in the somatic cell cycle. Studies have emphasized that although it does not have amino acid sequence homology with cyclins, it can activate CDK2. In addition, results showing molecular compensation of CDK4/6 inhibition through CDK2 activation, also showed that CDK2 can predict drug resistance. Another important finding was that overexpressed Speedy/RINGO, during CDK4/6 inhibitor treatment, could strongly activate CDK2, resulting in a negative response to treatment. Although many predictive factors have been investigated to indicate response to CDK4/6 inhibitors or determine drug resistance, a consensus biomarker has yet to be established. In light of the information obtained from our review, it can be concluded that the Speedy/RINGO protein may have an important role as a predictive biomarker in terms of response to treatment, continuity of treatment and drug resistance in patients treated with CDK4/6 inhibitors.

Discovering optimal kinetic pathways for self-assembly using automatic differentiation

Macromolecular complexes are often composed of diverse subunits. The self-assembly of these subunits is inherently nonequilibrium and must avoid kinetic traps to achieve high yield over feasible timescales. We show how the kinetics of self-assembly benefits from diversity in subunits because it generates an expansive parameter space that naturally improves the "expressivity" of self-assembly, much like a deeper neural network. By using automatic differentiation algorithms commonly used in deep learning, we searched the parameter spaces of mass-action kinetic models to identify classes of kinetic protocols that mimic biological solutions for productive self-assembly. Our results reveal how high-yield complexes that easily become kinetically trapped in incomplete intermediates can instead be steered by internal design of rate-constants or external and active control of subunits to efficiently assemble. Internal design of a hierarchy of subunit binding rates generates self-assembly that can robustly avoid kinetic traps for all concentrations and energetics, but it places strict constraints on selection of relative rates. External control via subunit titration is more versatile, avoiding kinetic traps for any system without requiring molecular engineering of binding rates, albeit less efficiently and robustly. We derive theoretical expressions for the timescales of kinetic traps, and we demonstrate our optimization method applies not just for design but inference, extracting intersubunit binding rates from observations of yield-vs.-time for a heterotetramer. Overall, we identify optimal kinetic protocols for self-assembly as a powerful mechanism to achieve efficient and high-yield assembly in synthetic systems whether robustness or ease of "designability" is preferred.

Structural Evolution of Bacterial Polyphosphate Degradation Enzyme for Phosphorus Cycling.

Living organisms ranging from bacteria to animals have developed their own ways to accumulate and store phosphate during evolution, in particular as the polyphosphate (polyP) granules in bacteria. Degradation of polyP into phosphate is involved in phosphorus cycling, and exopolyphosphatase (PPX) is the key enzyme for polyP degradation in bacteria. Thus, understanding the structure basis of PPX is crucial to reveal the polyP degradation mechanism. Here, it is found that PPX structure varies in the length of ɑ-helical interdomain linker (ɑ-linker) across various bacteria, which is negatively correlated with their enzymatic activity and thermostability - those with shorter ɑ-linkers demonstrate higher polyP degradation ability. Moreover, the artificial DrPPX mutants with shorter ɑ-linker tend to have more compact pockets for polyP binding and stronger subunit interactions, as well as higher enzymatic efficiency (kcat/Km) than that of DrPPX wild type. In Deinococcus-Thermus, the PPXs from thermophilic species possess a shorter ɑ-linker and retain their catalytic ability at high temperatures (70°C), which may facilitate the thermophilic species to utilize polyP in high-temperature environments. These findings provide insights into the interdomain linker length-dependent evolution of PPXs, which shed light on enzymatic adaption for phosphorus cycling during natural evolution and rational design of enzyme.

Ebola Virus Glycoprotein Strongly Binds to Membranes in the Absence of Receptor Engagement.

Ebola virus (EBOV) is an enveloped virus that must fuse with the host cell membrane in order to release its genome and initiate infection. This process requires the action of the EBOV envelope glycoprotein (GP), encoded by the virus, which resides in the viral envelope and consists of a receptor binding subunit, GP1, and a membrane fusion subunit, GP2. Despite extensive research, a mechanistic understanding of the viral fusion process is incomplete. To investigate GP-membrane association, a key step in the fusion process, we used two approaches: high-throughput measurements of single-particle diffusion and single-molecule measurements with optical tweezers. Using these methods, we show that the presence of the endosomal Niemann-Pick C1 (NPC1) receptor is not required for primed GP-membrane binding. In addition, we demonstrate this binding is very strong, likely attributed to the interaction between the GP fusion loop and the membrane's hydrophobic core. Our results also align with previously reported findings, emphasizing the significance of acidic pH in the protein-membrane interaction. Beyond Ebola virus research, our approach provides a powerful toolkit for studying other protein-membrane interactions, opening new avenues for a better understanding of protein-mediated membrane fusion events.

Promiscuous G-protein activation by the calcium-sensing receptor.

The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca2+ concentration and maintains Ca2+ homeostasis1,2. It also mediates diverse cellular processes not associated with Ca2+ balance3-5. The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes6. We determined structures of CaSR in complex with G proteins from three different subfamilies: Gq, Gi and Gs. We found that the homodimeric CaSR of each complex couples to a single G protein through a common mode. This involves the C-terminal helix of each Gα subunit binding to a shallow pocket that is formed in one CaSR subunit by all three intracellular loops (ICL1-ICL3), an extended transmembrane helix 3 and an ordered C-terminal region. G-protein binding expands the transmembrane dimer interface, which is further stabilized by phospholipid. The restraint imposed by the receptor dimer, in combination with ICL2, enables G-protein activation by facilitating conformational transition of Gα. We identified a single Gα residue that determines Gq and Gs versus Gi selectivity. The length and flexibility of ICL2 allows CaSR to bind all three Gα subtypes, thereby conferring capacity for promiscuous G-protein coupling.

Abstract 2373: Functional and specific T-cell engagers against a peptide-MHC tumor target

Abstract Accessing peptides from intracellular tumor antigens displayed on MHC class I (pMHC) can expand the T-cell engager (TCE) target pool for solid tumor applications. A challenge limiting TCE development for pMHC targets is the identification of rare, high-affinity, and high-specificity pMHC-binders. In this work, we describe TCEs in a 1 × 1 format with potent tumor-cell killing activity and high specificity for MAGE-A4, a pMHC tumor antigen expressed in multiple cancer types. We identified functional MAGE-A4 x CD3 TCEs with high specificity and affinity to a human MAGE-A4 peptide sequence of 10 amino acids presented on MHC-I (HLA-A:02*01). We strategically selected MAGE-A4-binders with high binding specificity for pMAGE-A4230-239, but not MHC-I or many closely related MHC-restricted peptides. We then paired them with diverse and developable CD3-binders with a range of binding affinities, subunit specificities, and binding kinetic profiles. High-throughput in vitro functional characterization of hundreds of these TCEs enabled identification of molecules with optimal T-cell dependent cellular cytotoxicity in MAGE-A4-expressing tumor cell lines, with comparable tumor-killing activity and cytokine release to a clinical-stage TCE in a 2 × 1 format. Previous studies have highlighted unique challenges associated with pMHC targets, including their potential for cross-reactive binding to pMHCs on healthy tissues, and the absence of pharmacologically relevant species for preclinical toxicity testing. To address these challenges, we have designed and implemented an in vitro and in silico workflow to identify antibody binding to potential off-target peptides. Using this approach, we selected TCEs with several pMHC binding orientations and high specificity and affinity. These TCEs killed tumor cell lines endogenously expressing MAGE-A4 pMHCs and not isogenic cells with MAGE-A4 knocked out. Results demonstrate that our high-resolution, multiparametric antibody discovery capabilities can identify pMHC-binding antibodies with desired functional and specificity attributes. This approach unlocks the discovery and development of optimal TCEs against complex pMHC antigens. Citation Format: Davide Tortora, Peter Bergqvist, Allison Goodman, Ryan Blackler, Nathalie Blamey, Stefania Carrara, Lauren Chong, Gabrielle Conaghan, Cindy-Lee Crichlow, Valentine de Puyraimond, Harveer Dhupar, Patrick Farber, Jessica Fernandes Scortecci, Kate Gibson, Rodrigo Goya, Ahn Lee, Franco Li, Tova Pinsky, Craig Robb, Patrick Rowe, Antonios Samiotakis, Eduardo Solano Salgado, Ping Xiang, Irene Yu, Kelly Bullock, Tara Fernandez, Stephanie K. Masterman, Kush Dalal, Tim Jacobs, Bryan C. Barnhart. Functional and specific T-cell engagers against a peptide-MHC tumor target [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 2373.