Extracellular Domain Of Protein Research Articles

S. aureus Eap is a polyvalent inhibitor of neutrophil serine proteases

Staphylococcus aureus expresses three high-affinity neutrophil serine protease (NSP) inhibitors known as the extracellular adherence protein domain (EAPs) proteins. Whereas EapH1 and EapH2 are comprised of a single EAP domain, the modular extracellular adherence protein (Eap) from S. aureus strain Mu50 consists of four EAP domains. We recently reported that EapH2 can simultaneously bind and inhibit cathepsin-G (CG) and neutrophil elastase (NE), which are the two most abundant NSPs. This unusual property of EapH2 arises from independent CG and NE-binding sites that lie on opposing faces of its EAP domain. Here we used X-ray crystallography and enzyme assays to show that all four individual domains of Eap (i.e. Eap1, Eap2, Eap3, and Eap4) exhibit an EapH2-like ability to form ternary complexes with CG and NE that inhibit both enzymes simultaneously. We found that Eap1, Eap2, and Eap3 have similar functional profiles insofar as NSP inhibition is concerned, but that Eap4 displays an unexpected ability to inhibit two NE enzymes simultaneously. Using X-ray crystallography, we determined that this second NE-binding site in Eap4 arises through the same region of its EAP domain that also comprises its CG-binding site. Interestingly, small angle X-ray scattering data showed that stable tail-to-tail dimers of the NE/Eap4/NE ternary complex exist in solution. This arrangement is compatible with NSP-binding at all available sites in a two-domain fragment of Eap. Together, our work implies that Eap is a polyvalent inhibitor of NSPs. It also raises the possibility that higher-order structures of NSP-bound Eap may have unique functional properties.

Clinical and structural insights into the rare but oncogenic HER2-activating missense mutations in non-small cell lung cancer: a retrospective ATLAS cohort study

BackgroundUnlike human epidermal growth factor receptor 2 (HER2) amplification or exon 20 insertions, missense mutations in the extracellular domain (ECD), transmembrane domain (TMD), and intracellular domain (ICD) of the HER2 protein have been implicated as oncogenic in non-small cell lung cancer (NSCLC). However, their molecular subtypes, structural disparities, and clinical responses to current medical treatments, particularly HER2-targeted tyrosine kinase inhibitors (TKIs), remain unclear in NSCLC and warrant investigation.MethodsA real-world observational ATLAS study was conducted to gather and analyze therapeutic outcomes of chemotherapy or TKIs for heterogeneous HER2 missense mutations in NSCLC. Computational models of typical ECD, TMD, and ICD mutations were utilized to explore their structural variances.ResultsWe screened 37 eligible patients with HER2-activating missense mutations, of which 35 patients who had received chemotherapy or HER2-targeted TKIs as first-line therapy were available for response assessment. The median progression-free survival (PFS) for chemotherapy was 4.43 months (95% confidence interval [CI], 3.77–5.10), with an objective response rate (ORR) of 26.1% (6/23) and a disease control rate (DCR) of 17/23 (73.9%). The administration of afatinib, dacomitinib, and pyrotinib, HER2-targeted TKIs, achieved a median PFS of 4.65 months, with an ORR of 33.3% (4/12) and a DCR of 83.3% (10/12). Molecular modeling and computational simulations of ECD, TMD, and ICD mutations revealed their distinct structural characteristics.ConclusionIn comparison to chemotherapy, HER2-targeted TKIs demonstrated similar activity and PFS benefits for HER2-activating missense mutations in NSCLC.

Plant-Produced Chimeric Hepatitis E Virus-like Particles as Carriers for Antigen Presentation.

A wide range of virus-like particles (VLPs) is extensively employed as carriers to display various antigens for vaccine development to fight against different infections. The plant-produced truncated variant of the hepatitis E virus (HEV) coat protein is capable of forming VLPs. In this study, we demonstrated that recombinant fusion proteins comprising truncated HEV coat protein with green fluorescent protein (GFP) or four tandem copies of the extracellular domain of matrix protein 2 (M2e) of influenza A virus inserted at the Tyr485 position could be efficiently expressed in Nicotiana benthamiana plants using self-replicating vector based on the potato virus X genome. The plant-produced fusion proteins in vivo formed VLPs displaying GFP and 4M2e. Therefore, HEV coat protein can be used as a VLP carrier platform for the presentation of relatively large antigens comprising dozens to hundreds of amino acids. Furthermore, plant-produced HEV particles could be useful research tools for the development of recombinant vaccines against influenza.

Utility of protein–protein binding surfaces composed of anti-parallel alpha-helices and beta-sheets selected by phage display

Over the past three decades a diverse collection of small protein domains have been used as scaffolds to generate general-purpose protein-binding reagents using a variety of protein display and enrichment technologies. To expand the repertoire of scaffolds and protein surfaces that might serve this purpose, we have explored the utility of (i) a pair of anti-parallel alpha-helices in a small highly disulfide-bonded 4-helix bundle, the CC4 domain from Reversion-inducing Cysteine-rich Protein with Kazal Motifs (RECK), and (ii) a concave beta-sheet surface and two adjacent loops in the human FN3 domain, the scaffold for the widely used monobody platform. Using M13 phage display and Next Generation Sequencing (NGS), we observe that, in both systems, libraries of ∼30 million variants contain binding proteins with affinities in the low uM range for baits corresponding to the extracellular domains of multiple mammalian proteins. CC4- and FN3-based binding proteins were fused to the N- and/or C-termini of Fc domains and used for immunostaining of transfected cells. Additionally, FN3-based binding proteins were inserted into VP1 of AAV to direct AAV infection to cells expressing a defined surface receptor. Finally, FN3-based binding proteins were insertion into the Pvc13 tail fiber protein of an extracellular contractile injection system particle to direct protein cargo delivery to cells expressing a defined surface receptor. These experiments support the utility of CC4 helices B and C and of FN3 beta-strands C, D, and F together with adjacent loops CD and FG as surfaces for engineering general-purpose protein-binding reagents.

A candidate competitive ELISA based on monoclonal antibody 3A8 for diagnosis of contagious bovine pleuropneumonia

For the development of a competitive ELISA (cELISA) to detect serum antibodies against the Mycoplasma mycoides subsp. Mycoides (Mmm) (strain PG1), the causative agent of contagious bovine pleuropneumonia (CBPP), all the proteins of this pathogen were analyzed. Then, a specific extracellular region of a transmembrane protein with the potential for diagnosis was identified. After that, a monoclonal antibody (Mab) named 3A8 was obtained using this extracellular region as an immunogen. Finally, a cELISA was established with the extracellular domain of this transmembrane protein as the coating antigen, Mab 3A8 as the competitive antibody, and HRP-labeled goat anti-mouse IgG as the enzyme-labeled antibody. This established method was used to detect the antibody dynamic regularity of goats which are artificially immunized Mmm and was also compared with a commercial ELISA kit. Further, the sera of 1011 different cattle from border provinces of China were monitored using a candidate Mab 3A8 cELISA. The detection results of known background sera used in this study indicate that a candidate diagnostic marker was successfully identified by analyzing all the coding proteins of Mmm in this research, and the cELISA established based on the Mab 3A8 against this protein can detect CBPP-positive serum with specificity and has no cross-reaction with other related epidemic disease-positive sera. In addition, we tested the sera collected from the border areas of China using the established ELISA, and no positive sample was detected. The research protocol of the CBPP cELISA established in this study is different from the traditional method, which can greatly reduce the investment of manpower and capital and save development time. We believe that this study’s protocol could serve as a reference for the development of detection methods for mycoplasma and other complex pathogens.Key points• A Mmm-specific diagnostic marker was obtained based on protein characteristics.• A cELISA was established for CBPP serum antibody detection.• The serological investigation was conducted for CBPP in the border areas of China.

Pro-Hemorrhagic Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy Associated with NOTCH3 p.R75P Mutation with Low Vascular NOTCH3 Aggregation Property.

Intracerebral hemorrhage (ICH) and cerebral microbleeds (CMB) in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy are more common in East Asian populations than in people of white European ancestry. We hypothesized that the ethnic difference is explained by the East Asian-specific NOTCH3 p.R75P mutation. This retrospective observational study included 118 patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy in Japanese and Korean cohorts. We investigated whether the p.R75P mutation is associated with symptomatic ICH and multiple CMB (>5) using quasi-Poisson regression models. We predicted the NOTCH3 extracellular domain protein structures in silico and graded NOTCH3 extracellular domain immunostaining in skin vessels of some patients, with subsequent comparisons between p.R75P and other conventional mutations. Among 63 Japanese patients (median age 55 years; 56% men), 15 had a p.R75P mutation, significantly associated with symptomatic ICH (adjusted relative risk 9.56, 95% CI 2.45-37.31), multiple CMB (3.00, 1.34-6.71), and absence of temporopolar lesions (4.91, 2.29-10.52) after adjustment for age, sex, hypertension, and antithrombotics. In the Korean cohort (n = 55; median age 55 years; 51% men), the p.R75P mutation (n = 13) was also associated with symptomatic ICH (8.11, 1.83-35.89), multiple CMB (1.90, 1.01-3.56), and absence of temporopolar lesions (2.32, 1.08-4.97). Structural analysis revealed solvent-exposed free cysteine thiols in conventional mutations, directly causing aggregation, whereas a stereochemically incompatible proline residue structure in p.R75P lowers correct disulfide bond formation probability, indirectly causing aggregation. Pathologically, the p.R75P mutation resulted in less vascular NOTCH3 extracellular domain accumulation than the other conventional mutations. NOTCH3 p.R75P mutation is associated with hemorrhagic presentations, milder temporopolar lesions, and distinct mutant protein structure properties. ANN NEUROL 2024;95:1040-1054.

Abstract 4153: PET/CT imaging of pancreatic ductal adenocarcinoma with a claudin-4 binding peptide in preclinical mouse model

Abstract There is a critical need to develop new imaging and therapeutic strategies for pancreatic cancer. Current positron emission tomography (PET) imaging assessments of pancreatic ductal adenocarcinoma (PDAC) rely on 18F-FDG as a radiopharmaceutical. To increase the sensitivity of PDAC detection, we identified Claudin-4 (CLDN4), a tight junction transmembrane protein, as a PDAC biomarker through spatial transcriptomic and proteomic analyses. CLDN4 expression has previously been reported to be enhanced in 99% of primary and 100% of metastatic pancreatic cancer tissue samples and in 10 of 11 precancerous pancreatic intraepithelial neoplasia (PanIN) specimens. A small fragment of naturally occurring Clostridium perfringens enterotoxin (CPE) was previously reported to bind to the extracellular loop domain of CLDN4 protein (PDB:7kp4) with nanomolar (nM) affinity (Kd). A CLDN4 binding peptide (C4BP) was developed as a radioligand by modifying the n-terminus of C4BP which does not impact receptor binding. Here, we designed and synthesized a DOTA-PEG1-C4BP ligand on a rink amide resin using a microwave-assisted peptide synthesizer. Radiolabeling of DOTA-PEG1-C4BP (2 nmol) with 64CuCl2 (70 MBq) conferred 64Cu-C4BP (28 MBq/nmol) with 80% non-decay corrected yield and >99% radiochemical purity. PET/CT imaging of 64Cu-C4BP (~0.1 μg/mouse) performed in an inducible PDAC (KrasLSL-G12D/+;Rosa26LSL-tdTomato/LSL-tdTomato(KT);Ptf1aCreER;Trp53fl/fl) model at 84 day after tamoxifen injection resulted in ~25% injected activity per cubic centimeter (IA/cc) accumulation in metastases and at ~18% IA/cc retention in pancreatic tumors within 30 minutes of tail vein injection with minimal background accumulation. Ex vivo PET imaging confirmed the localized accumulation of 64Cu-C4BP in transgenic mice within the colon, pancreas, and liver. Blocking of 64Cu-C4BP accumulation with the cold compound in a xenograft model (NOD/SCID) bearing Capan-1 PDAC tumors demonstrated the specificity of 64Cu-C4BP binding. Here we report that CLDN4 is a promising target and 64Cu-C4BP is a promising ligand for the detection of pancreatic cancer. Citation Format: Jai Woong Seo, James Wang, Aris J. Kare, Spencer K. Tumbale, Bo Wu, Marina N. Raie, Mallesh Pandrala, Katherine W. Ferrara. PET/CT imaging of pancreatic ductal adenocarcinoma with a claudin-4 binding peptide in preclinical mouse model [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 4153.

Abstract 1900: TORL-3-600, a novel antibody drug conjugate directed against cadherin 17 (CDH17), has preclinical efficacy in colorectal, gastric, and pancreatic cancer

Abstract Cadherin 17 (CDH17) is a cell-to-cell adhesion protein that is a member of the cadherin superfamily. In normal tissues, expression of this single pass transmembrane protein is restricted to the lateral surfaces of intestinal and pancreatic ductal epithelial cells. However, in cancer, CDH17 is frequently overexpressed in tumors of the colon, stomach, and pancreas. The selective expression of this cell surface protein in these hard-to-treat cancer makes it an attractive target for the development of antibody-based therapeutics. Here, we describe the preclinical development of TORL-3-600, a novel CDH17-targeting antibody drug conjugate (ADC). CDH17 specific monoclonal antibodies were generated using traditional hybridoma technology and splenocytes isolated from mice immunized with cocktails of NIH3T3 cells overexpressing full length hCHD17 plus purified mammalian expressed hCDH17 extracellular domain protein (aa 23-787). Selective mAb binding was confirmed by flow cytometry and mAb internalization rate was assessed by immunofluorescence (IF). TORL-3-600 was generated from a fully humanized CDH17 mAb by MMAE conjugation with a cleavable linker. Cell membrane staining of CDH17 in patient tumor microarrays (TMAs), cell line (CDX) and patient derived xenografts (PDX) was evaluated by IHC assay. Selective binding of the TORL-3-600 to CDH17 was confirmed in human cancer cell lines and cells engineered to overexpress CDH17. Binding of TORL-3-600 to cell surface CDH17 induced Internalization and translocation to the lysosome of the protein-ADC complex for release of the MMAE payload. Treatment with TORL-3-600 induced significant regressions and tumor growth inhibition (TGI) in four CDH17-positive (CDH17+) human colorectal cancer (CRC) CDXs (103.6 - 140.4% TGI) and three CDH17+ CRC PDXs (63.3 - 102.2% TGI). Responses in these models were sustained for up to nine-weeks following cessation of treatment. Sustained inhibition of xenograft tumor progression was also observed in a cell line model of CDH17+ pancreatic cancer (85.8% TGI). In contrast, greatly reduced responses (37.3 - 58.2% TGI) to TORL-3-600 were observed in the CDH17- human colon cancer CDX and PDX models. Each of the doses tested in this study were well tolerated in mice with no dose-limiting toxicities observed. Analyses of large human patient TMAs by CDH17 IHC assay demonstrated detectable expression of CDH17 in 90.1% (173/193) of CRC, 51.8% (86/166) of gastric and 20.4% (69/331) of pancreatic cancers. These numbers suggest that an ADC directed against CDH17 could provide benefit to a significant number of patients diagnosed with these cancers. These data support the clinical development of TORL-3-600 for the treatment of CDH17+ cancers. TORL-3-600 has completed IND enabling toxicity studies with acceptable PK and toxicity profiles and is now in phase 1 clinical testing (NCT05948826). Citation Format: Neil A. O'Brien, Martina SJ McDermott, Jun Zhang, Ming Lu, Ke Wei Gong, Benjamin Hoffstrom, Wei Ping Jia, Tong Luo, Athena M. Madrid, Min Liang, John A. Glaspy, Dennis J. Slamon. TORL-3-600, a novel antibody drug conjugate directed against cadherin 17 (CDH17), has preclinical efficacy in colorectal, gastric, and pancreatic cancer [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 1900.

Role of Mutation of NOTCH1 gene in development of oral squamous cell carcinoma: a narrative review.

Cancer of the oral cavity has numerous types and, among all, oral squamous cell carcinoma represents >90% of all cancers of the oral area. Oral squamous cell carcinoma arises from the squamous lining of the oral cavity. Across the globe, most commonly it develops in the regions of tongue followed by floor of the mouth, and lower lip. Neurogenic locus notch homolog protein 1 gene has its association with oral squamous cell carcinoma and is known to be associated with both oncogenic and tumour suppressor roles. The current narrative review comprised literature published from 2013 to 2023. It was searched on Google Scholar, PubMed and Google databases. Globally, neurogenic locus notch homolog protein 1 mutations are associated with the development of oral squamous cell carcinoma. Most of the mutations are linked to ligand bind epidermal growth factor-like repeat region of extracellular domain of neurogenic locus notch homolog protein 1. Once activated, the pathway is involved in tumour progression and metastasis. The Asians compared to Caucasians are more affected by neurogenic locus notch homolog protein 1 mutations.

Recent Advances, Approaches and Challenges in the Development of Universal Influenza Vaccines.

Every year, influenza virus infections cause significant morbidity and mortality worldwide. They pose a substantial burden of disease, in terms of not only health but also the economy. Owing to the ability of influenza viruses to continuously evolve, annual seasonal influenza vaccines are necessary as a prophylaxis. However, current influenza vaccines against seasonal strains have limited effectiveness and require yearly reformulation due to the virus undergoing antigenic drift or shift. Vaccine mismatches are common, conferring suboptimal protection against seasonal outbreaks, and the threat of the next pandemic continues to loom. Therefore, there is a great need to develop a universal influenza vaccine (UIV) capable of providing broad and durable protection against all influenza virus strains. In the quest to develop a UIV that would obviate the need for annual vaccination and formulation, a multitude of strategies is currently underway. Promising approaches include targeting the highly conserved epitopes of haemagglutinin (HA), neuraminidase (NA), M2 extracellular domain (M2e) and internal proteins of the influenza virus. The identification and characterization of broadly neutralizing antibodies (bnAbs) targeting conserved regions of the viral HA protein, in particular, have provided important insight into novel vaccine designs and platforms. This review discusses universal vaccine approaches presently under development, with an emphasis on those targeting the highly conserved stalk of the HA protein, recent technological advancements used and the future prospects of a UIV in terms of its advantages, developmental obstacles and potential shortcomings.

A transmembrane scaffold from CD20 helps recombinant expression of a chimeric claudin 18.2 in an in vitro coupled transcription and translation system

Cluster of differentiation 20 (CD20) is a nonglycosylated, multispanning transmembrane protein specifically integrated by B lymphocytes. Similar to CD20, another four-pass transmembrane protein, claudin 18.2, has attracted attention as an emerging therapeutic target for cancer. However, their poor solubility and toxic nature often hinder downstream applications, such as antibody drug development. Therefore, developing a cost-effective method for producing drug targets with multiple membrane-spanning domains is crucial. In this study, a high yield of recombinant CD20 was achieved through an E. coli–based in vitro coupled transcription–translation system. Surface plasmon resonance results showed that rituximab (an antileukemia drug) has nanomolar affinity with the CD20 protein, which aligns with published results. Notably, a previously hard-to-express claudin 18.2 recombinant protein was successfully expressed in the same reaction system by replacing its membrane-spanning domains with the transmembrane domains of CD20. The folding of the extracellular domain of the chimeric protein was verified using a commercial anti-claudin 18 antibody. This study provides a novel concept for promoting the expression of four-pass transmembrane proteins and lays the foundation for the large-scale industrial production of membrane-associated drug targets, similar to claudin 18.2.

Nanoparticles Carrying Conserved Regions of Influenza A Hemagglutinin, Nucleoprotein, and M2 Protein Elicit a Strong Humoral and T Cell Immune Response and Protect Animals from Infection.

Current influenza vaccines are mainly strain-specific and have limited efficacy in preventing new influenza A strains. Efficient control of infection can potentially be achieved through the development of broad-spectrum vaccines based on conserved antigens. A combination of several such antigens, including the conserved region of the second subunit of the hemagglutinin (HA2), the extracellular domain of the M2 protein (M2e), and epitopes of nucleoprotein (NP), which together can elicit an antibody- and cell-mediated immune response, would be preferred for vaccine development. In this study, we obtained recombinant virus-like particles formed by an artificial self-assembling peptide (SAP) carrying two epitopes from NP, tandem copies of M2e and HA2 peptides, along with a T helper Pan DR-binding epitope (PADRE). Fusion proteins expressed in Escherichia coli self-assembled in vitro into spherical particles with a size of 15-35 nm. Immunization of mice with these particles induced strong humoral immune response against M2e and the entire virus, and lead to the formation of cytokine-secreting antigen-specific CD4+ and CD8+ effector memory T cells. Immunization provided high protection of mice against the lethal challenge with the influenza A virus. Our results show that SAP-based nanoparticles carrying conserved peptides from M2, HA, and NP proteins of the influenza A virus, as well as T helper epitope PADRE, can be used for the development of universal flu vaccines.

Binding Affinity of Trastuzumab and Pertuzumab Monoclonal Antibodies to Extracellular HER2 Domain.

The binding affinity of trastuzumab and pertuzumab to HER2 has been studied using both experimental and in silico methods. The experiments were conducted using the antibodies in their complete IgG form, as used in clinical therapy, and the extracellular domain of the HER2 protein in solution. This approach provides a precise, reproducible, and reliable view of the interaction between them in physicochemical conditions similar to those found in the tumoral environment. Dynamic light scattering and size exclusion chromatography coupled with tetra detection were utilized to characterize the protein complexes, measure their concentrations, and calculate the equilibrium-free binding energy, ΔGbind. In addition, PRODIGY, a QSAR-like model with excellent predictive ability, was employed to obtain in silico ΔGbind estimations. The results obtained indicate that pertuzumab exhibits a slightly higher binding affinity to HER2 than trastuzumab. The difference in binding affinity was explained based on the contribution of the different interfacial contact (IC) descriptors to the ΔGbind value estimated by the PRODIGY model. Furthermore, experiments revealed that the pertuzumab IgG antibody binds preferentially to two HER2 proteins, one per Fab fragment, while trastuzumab mainly forms a monovalent complex. This finding was interpreted based on a geometrical model that identified steric crowding in the trastuzumab-HER2 complex as compared with the pertuzumab-HER2 complex.

GAS6-based CAR-T cells exhibit potent antitumor activity against pancreatic cancer

BackgroundThe receptor tyrosine kinases TAM family (TYRO3, AXL, and MERTK) are highly expressed in multiple forms of cancer cells and tumor-associated macrophages and promote the development of cancers including pancreatic tumor. Targeting TAM receptors could be a promising therapeutic option.MethodsWe designed a novel CAR based on the extracellular domain of growth arrest-specific protein 6 (GAS6), a natural ligand for all TAM members. The ability of CAR-T to kill pancreatic cancer cells is tested in vitro and in vivo, and the safety is evaluated in mice and nonhuman primate.ResultsGAS6-CAR-T cells efficiently kill TAM-positive pancreatic cancer cell lines, gemcitabine-resistant cancer cells, and cancer stem-like cells in vitro. GAS6-CAR-T cells also significantly suppressed the growth of PANC1 xenografts and patient-derived xenografts in mice. Furthermore, these CAR-T cells did not induce obvious side effects in nonhuman primate or mice although the CAR was demonstrated to recognize mouse TAM.ConclusionsOur findings indicate that GAS6-CAR-T-cell therapy may be effective for pancreatic cancers with low toxicity.

Search of a genomic sequence database for potential novel blood group antigens: Investigation into why some amino acid substitutions are not immunogenic.

Polypeptide blood group antigens are typically identified through investigation of the antibodies they induce. Human genome sequence databases are a new tool to identify AA substitutions that potentially create blood group antigens. The Erythrogene genomic sequence database was searched for missense mutations not known to be blood group antigens in the extracellular domains of selected RBC proteins in European populations. Any mutations found with prevalence of 1%-90% and not known to have induced antibodies in transfusion practice were analyzed using protein structural analysis and epitope prediction programs to determine why they apparently lack immunogenicity. Thirteen missense mutations not known to create blood group antigens were identified in the extracellular domains of Kell, BCAM, and RhD proteins, but not in RhCE, Urea Transporter 1 (Kidd), Atypical Chemokine Receptor 1 (Duffy), glycophorin A or glycophorin B. While 11 of the 13 mutations had low prevalence (<1%), a Kell Ser726Pro substitution and a BCAM Val196Ile substitution had predicted phenotype prevalences of 43.2% and 5.7%, respectively. Ser726Pro had multiple properties of a linear B-cell epitope, but possible suboptimal protein location for B-cell receptor binding and limited T-cell epitope possibilities. Val196Ile was not predicted to be in a linear B-cell epitope. Multiple potential new blood group antigens of low prevalence were identified. Whether they are antigenic remains to be determined. Two higher prevalence variants in Kell and BCAM are unlikely antigens, otherwise their antibodies presumably would already have been identified. Possible reasons for their poor immunogenicity were identified.

Simultaneous inhibition of two neutrophil serine proteases by the S. aureus innate immune evasion protein EapH2

Extracellular adherence protein domain (EAP) proteins are high-affinity, selective inhibitors of neutrophil serine proteases (NSP), including cathepsin-G (CG) and neutrophil elastase (NE). Most Staphylococcus aureus isolates encode for two EAPs, EapH1 and EapH2, that contain a single functional domain and share 43% identity with one another. Although structure/function investigations from our group have shown that EapH1 uses a globally similar binding mode to inhibit CG and NE, NSP inhibition by EapH2 is incompletely understood due to a lack of NSP/EapH2 cocrystal structures. To address this limitation, we further studied NSP inhibition by EapH2 in comparison with EapH1. Like its effects on NE, we found that EapH2 is a reversible, time-dependent, and low nanomolar affinity inhibitor of CG. We characterized an EapH2 mutant which suggested that the CG binding mode of EapH2 is comparable to EapH1. To test this directly, we used NMR chemical shift perturbation to study EapH1 and EapH2 binding to CG and NE in solution. Although we found that overlapping regions of EapH1 and EapH2 were involved in CG binding, we found that altogether distinct regions of EapH1 and EapH2 experienced changes upon binding to NE. An important implication of this observation is that EapH2 might be capable of binding and inhibiting CG and NE simultaneously. We confirmed this unexpected feature by solving crystal structures of the CG/EapH2/NE complex and demonstrating their functional relevance through enzyme inhibition assays. Together, our work defines a new mechanism of simultaneous inhibition of two serine proteases by a single EAP protein.

Multiplex assays to determine correlation of binding and neutralizing antibodies following SARS-CoV-2 infection and vaccination

Abstract The COVID-19 pandemic has led to the rapid development of many different therapeutics and vaccines to reduce the burden of the disease. Most vaccines target the highly immunogenic spike (S) protein for generating functional immune responses to block the entry of the virus into host cells. However, the mutations in the S-protein have led to the emergence of many variants that escape the immune response. We developed two multiplex assays using Luminex® xMAP® technology to assess antibody profiles after infection and vaccination; 1) to quantify the binding antibodies against SARS-CoV2 receptor binding protein (RBD), nucleocapsid protein (NP), S1-protein (S1), and Trimer having S1, S2, and extracellular domain protein; 2) a multiplex surrogate virus neutralization antibody assay targeting CoV2 wild type and various mutant viruses. The quantitative binding assay uses the WHO standard for determining the binding antibody unit (BAU)/mL. Surrogate virus neutralization assay measures the inhibition of ACE2 protein binding to wild-type and S protein variants coupled to beads in the presence of immune or convalescent sera. We determined the negative threshold using 81 pre-pandemic serum samples collected during 2017–18. We used 116 samples collected from 45 vaccinated and 15 infected subjects for antibody quantification. Binding antibodies to RBD, S1, and trimer were higher in vaccinated individuals with minimal reactivity to NP, whereas the NP antibody response was higher in the infected individuals. We observed a direct correlation between spike protein binding and neutralizing antibodies using the two assays. We saw the most elevated neutralization antibodies against variants from individuals with breakthrough infections.

Abstract 5003: Repurposing passenger amplifications for specific therapeutic targeting of solid cancers

Abstract Current cancer therapies focus on targeting driver alterations responsible for tumorigenesis - either oncogenic point mutations or oncogenic driver events within large somatic copy number alterations (SCNAs). However, these alterations are often not actionable or are only present in a small subset of patients. Thus, new therapeutic targets are urgently needed. Apart from oncogenic drivers, SCNAs usually also comprise neutral bystander genes without an active role in neoplastic transformation. We hypothesized that passenger events, specifically in amplified regions, could be therapeutically exploited by providing actionable molecules on the cell surface. Using publicly available multi-omics data, we screened cell surface protein-coding genes for their genomic status and expression levels in liver cancer. This screen identified MPZL1 (Myelin protein zero-like 1), a gene amplified in 75% of hepatocellular carcinomas, accompanied by high mRNA expression in tumors compared to normal livers. MPZL1 is located on chromosome 1q, one of the most commonly amplified regions across several solid cancer types, and codes for a glycosylated cell surface receptor. We further validated MPZL1 protein expression in a wide range of human cancer entities (n=2038 samples) and normal tissues (n=163 samples) by immunohistochemistry and assigned expression scores analogous to the clinically implemented HER2-score. A high percentage of tumors showed scores 2 or 3 (e.g. 48% of HCCs or 89% of TNBCs), whereas healthy tissues were mostly negative or just faintly positive (scores 0 or 1). Moreover, in order to target MPZL1-expressing cells, we generated a highly specific monoclonal antibody directed to the extracellular domain of human MPZL1 protein and thoroughly characterized its binding capacity. Next, the scFv sequence of this antibody was used to generate a CAR (chimeric antigen receptor) construct targeting MPZL1. Luciferase-based cytotoxic assays showed that T cells transduced with this construct can kill various MPZL1-high human cancer cell lines in vitro (liver, breast and lung malignant cell lines, among others), whereas they fail to kill respective isogenic cell lines harboring knockout of MPZL1. Upon antigen-specific exposure, MPZL1-28ζ CAR-T cells underwent antigen-dependent proliferation and showed increased cytokine production (IFNγ, TNFα, IL-2, GZMB), further confirming their specificity. In summary, our findings reveal MPZL1 as a new target for treatment of 1q-amplified cancers and implement a novel immunotherapeutic strategy based on MPZL1-28ζ CAR-T cells. Furthermore, our work provides a proof of concept that passenger events within large chromosomal amplifications can serve as potent therapeutic targets and thus opens a new avenue for innovative approaches in anti-cancer drug development. Citation Format: Sonia Jiménez Vázquez, Christos Patsis, Luise Butthof, Joan Crous Masó, Hendrik Wiethoff, Matthias Gaida, Thomas Longerich, Rosella Pellegrino, Ilse Hofmann, Judith Feucht, Darjus Felix Tschaharganeh. Repurposing passenger amplifications for specific therapeutic targeting of solid cancers. [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 5003.

A lesion-selective albumin-CTLA4Ig as a safe and effective treatment for collagen-induced arthritis

BackgroundCTLA4Ig is a dimeric fusion protein of the extracellular domain of cytotoxic T-lymphocyte protein 4 (CTLA4) and an Fc (Ig) fragment of human IgG1 that is approved for treating rheumatoid arthritis. However, CTLA4Ig may induce adverse effects. Developing a lesion-selective variant of CTLA4Ig may improve safety while maintaining the efficacy of the treatment.MethodsWe linked albumin to the N-terminus of CTLA4Ig (termed Alb-CTLA4Ig) via a substrate sequence of matrix metalloproteinase (MMP). The binding activities and the biological activities of Alb-CTLA4Ig before and after MMP digestion were analyzed by a cell-based ELISA and an in vitro Jurkat T cell activation assay. The efficacy and safety of Alb-CTLA4Ig in treating joint inflammation were tested in mouse collagen-induced arthritis.ResultsAlb-CTLA4Ig is stable and inactive under physiological conditions but can be fully activated by MMPs. The binding activity of nondigested Alb-CTLA4Ig was at least 10,000-fold weaker than that of MMP-digested Alb-CTLA4Ig. Nondigested Alb-CTLA4Ig was unable to inhibit Jurkat T cell activation, whereas MMP-digested Alb-CTLA4Ig was as potent as conventional CTLA4Ig in inhibiting the T cells. Alb-CTLA4Ig was converted to CTLA4Ig in the inflamed joints to treat mouse collagen-induced arthritis, showing similar efficacy to that of conventional CTLA4Ig. In contrast to conventional CTLA4Ig, Alb-CTLA4Ig did not inhibit the antimicrobial responses in the spleens of the treated mice.ConclusionsOur study indicates that Alb-CTLA4Ig can be activated by MMPs to suppress tissue inflammation in situ. Thus, Alb-CTLA4Ig is a safe and effective treatment for collagen-induced arthritis in mice.

Computational and Experimental Evaluation of Linker Peptides and Thioredoxin Fusion Tag in CD20-rituximab Specific Interactions.

Overexpression of CD20 protein on the surface of B cells in lymphoma can be targeted by several anti-CD20 molecules. The development of accessible interactive epitopes is more favorable than the full-length transmembrane CD20 in the affinity assessment of anti-CD20 monoclonal antibodies (mAbs). The sequence of these epitopes was extracted, and the effects of different linker peptides and the location of histidine (His)-tag were computationally analyzed. The impact of thioredoxin (Trx)-tag on the folding of the selected construct and its interaction with rituximab was further investigated. The two final expression cassettes were expressed in Escherichia coli after optimization of culture conditions for incubation temperature, post-induction time, optical density at the induction time, and concentration of the inducer. ELISA evaluated the binding affinity of rituximab towards the recombinant proteins. By homology modeling studies, C-terminal His-tagged structures represented more desirable folded structures. Validation of the models revealed that CD20 extracellular domain linked by the G4S polypeptide had better stereochemical quality and structural compatibility. It was selected due to its more effective interaction with rituximab showing the highest dissociation constant of 5.8E-09M, which improved after the fusion of Trx-tag (7.1E-10M). The most influential parameters in the expression of the two selected proteins were post-induction temperature and optical density at the induction time. Homemade ELISA assays revealed a slightly higher affinity of rituximab towards the Trx-CD20 protein than the CD20/G4S molecule. Experimental in vitro studies confirmed the computationally calculated affinity of rituximab towards the two designed CD20 constructs. Also, the cell-based binding assessment of anti-CD20 mAbs could be substituted by the engineered extracellular domain of human CD20 protein.