CH2 Domain Research Articles

Nuclear elongation during spermiogenesis depends on physical linkage of nuclear pore complexes to bundled microtubules by Drosophila Mst27D.

Spermatozoa in animal species are usually highly elongated cells with a long motile tail attached to a head that contains the haploid genome in a compact and often elongated nucleus. In Drosophila melanogaster, the nucleus is compacted two hundred-fold in volume during spermiogenesis and re-modeled into a needle that is thirty-fold longer than its diameter. Nuclear elongation is preceded by a striking relocalization of nuclear pore complexes (NPCs). While NPCs are initially located throughout the nuclear envelope (NE) around the spherical nucleus of early round spermatids, they are later confined to one hemisphere. In the cytoplasm adjacent to this NPC-containing NE, the so-called dense complex with a strong bundle of microtubules is assembled. While this conspicuous proximity argued for functional significance of NPC-NE and microtubule bundle, experimental confirmation of their contributions to nuclear elongation has not yet been reported. Our functional characterization of the spermatid specific Mst27D protein now resolves this deficit. We demonstrate that Mst27D establishes physical linkage between NPC-NE and dense complex. The C-terminal region of Mst27D binds to the nuclear pore protein Nup358. The N-terminal CH domain of Mst27D, which is similar to that of EB1 family proteins, binds to microtubules. At high expression levels, Mst27D promotes bundling of microtubules in cultured cells. Microscopic analyses indicated co-localization of Mst27D with Nup358 and with the microtubule bundles of the dense complex. Time-lapse imaging revealed that nuclear elongation is accompanied by a progressive bundling of microtubules into a single elongated bundle. In Mst27D null mutants, this bundling process does not occur and nuclear elongation is abnormal. Thus, we propose that Mst27D permits normal nuclear elongation by promoting the attachment of the NPC-NE to the microtubules of the dense complex, as well as the progressive bundling of these microtubules.

IgA antibody immunotherapy targeting GD2 is effective in preclinical neuroblastoma models

BackgroundImmunotherapy targeting GD2 is very effective against high-risk neuroblastoma, though administration of anti-GD2 antibodies induces severe and dose-limiting neuropathic pain by binding GD2-expressing sensory neurons. Previously, the IgG1 ch14.18 (dinutuximab)...

3D WEBGIS APPLICATIONS FOR DIGITAL HUMANITIES STUDIES: THE TURIN 1911 PROJECT

Abstract. Documentation of CH is paramount to preserve its memory and allow its study, especially when CH is meant to be dismantled, as for the architectures of World’s Fairs. Very few efforts are undertaken to achieve this goal and national and international institutions rarely adopt innovative digital technologies. Digital technologies and products like 3D digital models are successfully and largely applied in CH domains, while webGIS is less explored for CH. Nevertheless, 3D digital models are rarely adopted to accompany digital archives and Digital Humanities studies. Very few cases use 3D models for World’s Fairs’ study and documentation. WebGIS is not used for World’s Fairs, and are scarcely adopted and not fully exploited by Digital Humanities and digital archives for sharing data and information about CH. Turin 1911 is the first digital project aimed to virtually document, recreate, and study an entire World’s Fair. Combining digital technologies (geo-DB, 3D digital reconstruction, and 3D webGIS) with cataloging standards, Turin 1911 is a pioneering initiative that applies these innovations to Digital Humanities in order to share information online. In this paper, dedicated webGIS applications are developed for the Turin 1911 needs, reporting the designed procedure, challenges in the development phase, and potentialities for Digital Humanities research. Finally, BIM models are also integrated in webGIS apps, making visible no more visible architectures. The paper discusses how webGIS apps could become a way for sharing information and data, but also a working environment for Digital Humanities studies where the research takes place in 3D environments.

Crystal structures of the human IgD Fab reveal insights into CH1 domain diversity

Antibodies of the IgD isotype remain the least well characterized of the mammalian immunoglobulin isotypes. Here we report three-dimensional structures for the Fab region of IgD, based on four different crystal structures, at resolutions of 1.45–2.75 Å. These IgD Fab crystals provide the first high-resolution views of the unique Cδ1 domain. Structural comparisons identify regions of conformational diversity within the Cδ1 domain, as well as among the homologous domains of Cα1, Cγ1 and Cμ1. The IgD Fab structure also possesses a unique conformation of the upper hinge region, which may contribute to the overall disposition of the very long linker sequence between the Fab and Fc regions found in human IgD. Structural similarities observed between IgD and IgG, and differences with IgA and IgM, are consistent with predicted evolutionary relationships for the mammalian antibody isotypes.

EB1 decoration of microtubule lattice facilitates spindle-kinetochore lateral attachment in Plasmodium male gametogenesis

Faithful chromosome segregation of 8 duplicated haploid genomes into 8 daughter gametes is essential for male gametogenesis and mosquito transmission of Plasmodium. Plasmodium undergoes endomitosis in this multinucleated cell division, which is highly reliant on proper spindle-kinetochore attachment. However, the mechanisms underlying the spindle-kinetochore attachment remain elusive. End-binding proteins (EBs) are conserved microtubule (MT) plus-end binding proteins and play an important role in regulating MT plus-end dynamics. Here, we report that the Plasmodium EB1 is an orthologue distinct from the canonical eukaryotic EB1. Both in vitro and in vivo assays reveal that the Plasmodium EB1 losses MT plus-end tracking but possesses MT-lattice affinity. This MT-binding feature of Plasmodium EB1 is contributed by both CH domain and linker region. EB1-deficient parasites produce male gametocytes that develop to the anucleated male gametes, leading to defective mosquito transmission. EB1 is localized at the nucleoplasm of male gametocytes. During the gametogenesis, EB1 decorates the full-length of spindle MTs and regulates spindle structure. The kinetochores attach to spindle MTs laterally throughout endomitosis and this attachment is EB1-dependent. Consequently, impaired spindle-kinetochore attachment is observed in EB1-deficient parasites. These results indicate that a parasite-specific EB1 with MT-lattice binding affinity fulfills the spindle-kinetochore lateral attachment in male gametogenesis.

Subclass Effects on Self-Association and Viscosity of Monoclonal Antibodies at High Concentrations.

The effects of a subclass of monoclonal antibodies (mAbs) on protein-protein interactions, formation of reversible oligomers (clusters), and viscosity (η) are not well understood at high concentrations. Herein, we quantify a short-range anisotropic attraction between the complementarity-determining region (CDR) and CH3 domains (KCDR-CH3) for vedolizumab IgG1, IgG2, or IgG4 subclasses by fitting small-angle X-ray scattering (SAXS) structure factor Seff(q) data with an extensive library of 12-bead coarse-grained (CG) molecular dynamics simulations. The KCDR-CH3 bead attraction strength was isolated from the strength of long-range electrostatic repulsion for the full mAb, which was determined from the theoretical net charge and a scaling parameter ψ to account for solvent accessibility and ion pairing. At low ionic strength (IS), the strongest short-range attraction (KCDR-CH3) and consequently the largest clusters and highest η were observed with IgG1, the subclass with the most positively charged CH3 domain. Furthermore, the trend in KCDR-CH3 with the subclass followed the electrostatic interaction energy between the CDR and CH3 regions calculated with the BioLuminate software using the 3D mAb structure and molecular interaction potentials. Whereas the equilibrium cluster size distributions and fractal dimensions were determined from fits of SAXS with the MD simulations, the degree of cluster rigidity under flow was estimated from the experimental η with a phenomenological model. For the systems with the largest clusters, especially IgG1, the inefficient packing of mAbs in the clusters played the largest role in increasing η, whereas for other systems, the relative contribution from stress produced by the clusters was more significant. The ability to relate η to short-range attraction from SAXS measurements at high concentrations and to theoretical characterization of electrostatic patches on the 3D surface is not only of fundamental interest but also of practical value for mAb discovery, processing, formulation, and subcutaneous delivery.

Abstract 214: Sumo2 Regulates Oxidative Function Of Adaptor Protein P66shc

SUMOylation is a highly dynamic post-translational modification which regulates protein function and/or stability. It involves conjugation of SUMOs ( S mall U biquitin-Like Mo difier s ) to the lysine residue/s of target proteins mediated via a highly coordinated enzymatic mechanism. Studies have shown that promoting SUMOylation (SUMO2/3) causes vascular endothelial dysfunction and accelerates atherosclerosis. We reported that SUMO2 overexpression in endothelial cells promotes oxidative stress and impairs endothelial function. However, the mechanism is not well understood. Adaptor protein p66Shc is a master regulator of oxidative stress and endothelial function. Genetic deletion of p66Shc protects mice against endothelial dysfunction due to metabolic dysregulation. Prior studies have shown that lysine modification of p66Shc regulates its oxidative function. As SUMOylation primarily occurs on lysine residue, we examined if p66Shc is a target of SUMO2. In this study, we show that p66Shc is a direct target of SUMO2. Using endothelial cells and HEK-293 cells, we show that overexpression of SUMO2 leads to p66Shc SUMO2ylation which is further increased in presence of E2-ligase Ubc9 and reduced by deSUMOylating enzyme SENP1. SUMO2ylation of p66Shc increases oxidative activation (serine-36 phosphorylation) of p66Shc which is essential for reactive oxygen production by p66Shc. Using mass spectroscopic study, we identified that lysine-81 is the only lysine present in CH2 domain which is getting SUMO2ylated. Rendering p66ShcK81 non-sumoylatable (p66ShcK81R) prevented the SUMO2ylation of p66Shc. In endothelial cells, overexpression of p66ShcK81R prevented the SUMO2-induced increase in serine-36 phosphorylation and mitochondrial translocation of p66Shc, which are essential for oxidative function of p66Shc. Collectively, we present a novel molecular mechanism via which SUMO2 regulates the oxidative function of p66Shc and identify a unique post-translational regulation of p66Shc in endothelial cells.

Abstract 5675: Discovery and development of an asymmetric IgG-like bispecific antibody targeting EGFR and c-MET, engineered through H-H and H-L chain charge-based heterodimerization

Abstract Background: Lung cancer remains the leading cause of cancer mortality and is the most commonly diagnosed cancer with an estimated 2.2 million cases per year. EGFR, a tyrosine kinase receptor, plays a central role in cellular proliferation, survival, differentiation, and migration. Gain-of-function somatic mutations of EGFR significantly drives disease progression. Small molecule tyrosine kinase inhibitors (TKIs), designed to inhibit EGFR signaling specific to these mutations (e.g., L858R and E19del etc.), have achieved excellent clinical outcomes. However, patients carrying TKI-insensitive mutations (e.g., E20ins) and/or those with disease progression after TKI treatment, by the upregulation of complementary signaling pathways (HGF/c-MET, etc.), remain an unmet medical need. Here, we report the discovery of a fully-human afucosylated anti-EGFR x cMET IgG-like bispecific antibody (PM1080). Methods: PM1080 was generated by introducing unique mutations to the CH1-CL domains of each binding arm to promote correct HC-LC pairing. KIH mutations were also introduced to each CH3 domain to support HC-HC heterodimerization and generate an IgG-like bispecific with one arm binding to EGFR and the other to cMET. PM1080 was expressed via a four-chain expression system in CHO cells carrying a fut8−/− knockout to generate afucosylated molecules with enhanced ADCC activity. Results: Through monovalent affinity measurements, PM1080 interacted with EGFR at single digit nanomolar affinity and at sub-nanomolar affinity to c-MET. PM1080 preferentially bound to EGFR/c-MET double-positive cells rather than EGFR single-positive cells. PM1080 blocked both EGF/EGFR and HGF/c-MET signals via physical blockade of the interaction between EGF/HGF to their corresponding receptors and induced EGFR/c-MET internalization and degradation from the cell surface. Through intricate engineering, PM1080 was expressed via a four-chain system and produced as an afucosylated antibody with significantly improved ADCC function. PM1080 induced potent anti-tumor efficacy against EGFR/c-MET-positive tumor cells as a single agent. Importantly, PM1080 induced anti-tumor activity regardless of EGFR mutations at the intracellular signaling domain, and thus may serve as a promising combinational agent to TKI therapies. As expected, synergistic anti-tumor activity was observed when PM1080 was combined with either 1st or 3rd generation TKIs in both in vitro and in vivo models. Conclusion: PM1080, a bispecific antibody targeting EGFR & c-MET was discovered for cancer treatment. PM1080 displayed potent anti-tumor efficacy either as a single agent or in combination with EGFR TKIs. Preclinical PK and toxicity studies have been conducted to support future first-in-human studies. Citation Format: Ping Wang, Weifeng Huang, Zhenting Zhao, Xiaoniu Miao, Shaogang Peng, Chao Wang, Yao Yan, Tiantian Dong, Andy Tsun, Yingda Xu, Xiaolin Liu, Luo Yi. Discovery and development of an asymmetric IgG-like bispecific antibody targeting EGFR and c-MET, engineered through H-H and H-L chain charge-based heterodimerization. [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 5675.

Analysis of the characteristics of SPTB gene variants among 16 children with Hereditary spherocytosis

To analyze the clinical characteristics and spectrum of SPTB gene variants among 16 Chinese children with Hereditary spherocytosis (HS) and explore their genotype-phenotype correlation. Sixteen children who were diagnosed with HS at the Affiliated Hospital of Capital Institute of Pediatrics from November 2018 to July 2022 were selected as the research subjects. Genetic testing was carried out by whole exome sequencing. Candidate variants were verified by Sanger sequencing and subjected to bioinformatic analysis and prediction of 3D structure of the protein. Correlation between the SPTB genotypes and clinical phenotypes was analyzed using Chi-squared test. The male-to-female ratio of the HS patients was 6 : 10, with the median age being 7-year-and-10-month. Clinical features of the patients have included anemia, reticulocytosis and gradual onset of splenomegaly. Mild, moderate and severe anemia have respectively occurred in 56.25% (9/16), 31.25% (5/16) and 12.50% (2/16) of the patients. SPTB gene variants were detected in all patients, among which 10 were unreported previously and 7 were de novo in origin. Loss of function (LOF) variants accounted for 93.75% (15/16). Only one missense variant was detected. Eleven, 4 and 1 of the variants had occurred in the repeat domain, CH1 domain, and dimerization domain, respectively. There was no significant correlation between the type or domain of the SPTB gene variants with the clinical features such as severity of anemia (x² = 3.345, P > 0.05). All of the variants were predicted to be pathogenic or likely pathogenic based on the guidelines from the American College of Medical Genetics and Genomics. Mild to moderate anemia are predominant clinical features of the HS children harboring a SPTB gene variant, for which LOF variants are the main mutational type. The clinical feature of HS is unaffected by the type of the variants.

Tuftsin-tailored fusion protein inhibits the growth of circulating gastric tumor cells associated with macrophage phagocytosis

Circulating tumor cells (CTCs) are a major cause of tumor metastasis and resistance to anticancer therapies. To date, no effective low-toxicity chemotherapeutic agents or antibodies have exhibited significant clinical activity against CTCs. Macrophages are important mediators of antitumor immunity. Tuftsin (TF), a tetrapeptide located at residues 289–292 of the CH2 domain of the Fc region of the IgG heavy chain, binds to Nrp-1, a receptor on the surface of macrophages that promotes phagocytosis and induces nonspecific activation of the immune system against tumors. Lidamycin (LDM) is an antitumor chemotherapy agent that is strongly cytotoxic to tumors and can dissociate into an apoprotein (LDP) and active enediyne (AE) in vitro. We previously constructed the fusion protein LDP-TF through genetic engineering and inserted the chromophore AE to produce LDM-TF, which can target macrophages to promote their phagocytic and cytotoxic activity against tumor cells. Preliminary experiments confirmed the anti-tumor activity of LDM-TFs. In this study, we found that LDM-TF effectively inhibited the growth of CTCs of gastric cancer origin and enhanced macrophage phagocytosis both in vivo and in vitro. Tumor cell expression of CD47, which helps to evade phagocytosis by macrophages, was substantially downregulated by LDM-TF. Notably, our in vitro experiments demonstrated that the combination of LDM-TF and anti-CD47 antibodies promoted phagocytosis more than either component alone. Our findings demonstrate the significant inhibitory effect of LDM-TF on the growth of CTCs of gastric cancer origin and suggest that the combination of LDM-TF and anti-CD47 antibodies may exhibit synergistic effects, thereby providing a new option for the clinical treatment of patients with advanced tumors that have metastasized.

LIMCH1 protein expression associated with lymph node metastasis in breast cancer

Background. Lymph node metastasis in breast cancer is not only a variant of cancer progression, but is also associated with the development of hematogenous metastases. The LIMCH1 protein is involved in the mesenchymal type of migration due to the activation of non-muscle myosin IIA.The objective of the study was to investigate the association of different LIMCH1 protein expression variants with lymph node metastasis.Material and Methods. The retrospective study included 53 patients with invasive breast carcinoma of no special type. The study group included all molecular genetic types (luminal A, luminal B, HER2-positive and triple negative). The median age of the patients was 55.4 ± 14 years. According to the TNM classification, patients had stage T1–3N0–3M0 breast cancer. The patients did not receive preoperative chemotherapy. The expression of the LIMCH1 protein in tumor cells was assessed using immunohistochemistry. Anti-LIMCH1Prestige (HPA004184, Sigma Aldrich, Germany) and anti-LIMCH1-C-term (SAB2700402, Sigma Aldrich, Germany) antibodies were used.Results. Lymph node metastases were more frequent in patients with LIMCH1 protein expression in tumor cells. This is true for cytoplasmic expression of the LIMCH1 protein detected by the CH domain, but not for submembrane expression or expression of the LIMCH1 protein detected by the LIM domain, regardless of localization in the cell. This phenomenon was not associated with the morphological heterogeneity of breast cancer. The most unfavorable factor for worse metastasis-free survival is the combination of the presence of lymph node metastases and cytoplasmic expression of the LIMCH1 protein detected by the CH domain.Conclusion. The development of synchronous lymph node metastases in breast cancer is associated with the presence of cytoplasmic expression of the LIMCH1 protein detected by the CH domain in the primary tumor tissue.

Identification and characterization of immunoglobulin tau (IgT) in Asian Seabass (Lates calcarifer) and mucosal immune response to nervous necrosis virus.

Mucosal immunity plays a critical role in the protection of teleost fish against infection, but mucosal immunoglobulin of important aquaculture species unique to Southeast Asia remained greatly understudied. In this study, the sequence of immunoglobulin T (IgT) from Asian sea bass (ASB) is described for the first time. IgT of ASB possesses the characteristic structure of immunoglobulin with a variable heavy chain and four CH4 domains. The CH2-CH4 domains and full-length IgT were expressed and CH2-CH4 specific antibody was validated against full-length IgT expressed in Sf9 III cells. Subsequent use of the anti-CH2-CH4 antibody in immunofluorescence staining confirmed the presence of IgT-positive cells in the ASB gill and intestine. The constitutive expression of ASB IgT was characterized in different tissues and in response to red-spotted grouper nervous necrosis virus (RGNNV) infection. The highest basal expression of secretory IgT (sIgT) was observed in the mucosal and lymphoid tissues such as the gills, intestine and head kidney. Following NNV infection, IgT expression was upregulated in the head kidney and mucosal tissues. Moreover, a significant increase in localized IgT was found in gills and intestines of infected fish on day 14 post-infection. Interestingly, a significant increase in NNV-specific IgT secretion was only observed in the gills of the infected group. Our results suggest that ASB IgT may play an important role in the adaptive mucosal immune responses against viral infection and could potentially be adapted as a tool for the evaluation of prospective mucosal vaccines and adjuvants for the species.

Molecular and functional characterization of two IgM subclasses in large yellow croaker (Larimichthys crocea)

As the predominant immunoglobulin (Ig) isotype, IgM plays a crucial role in the acquired immunity of vertebrates. There is only one Igμ gene in mammals, except cattle, while the number of Igμ gene varies among teleost fish. In the current study, we found two functional Igμ genes (Igμ1 and Igμ2) and a pseudo Cμ gene (ψIgμ) in large yellow croaker (Larimichthys crocea). Both Igμ1 and Igμ2 genes possessed two transcript variants, which encoded the heavy chains of secreted (sIgM1 and sIgM2) and membrane-bound IgM1 and IgM2 (mIgM1 and mIgM2), respectively. Both the heavy chains of sIgM1 and sIgM2 consisted of a variable Ig domain, four constant Ig domains (CH1, CH2, CH3 and CH4) and a secretory tail, while those of mIgM1 and mIgM2 consisted of a variable Ig domain, three constant Ig domains (CH1, CH2 and CH3), a transmembrane domain and a short cytoplasmic tail. Cysteine residues that are necessary for the formation of intrachain and interchain disulfide bonds and tryptophan residues that are important for the folding of the Ig superfamily domain were well conserved in large yellow croaker IgM1 and IgM2. Interestingly, large yellow croaker IgM2 had an extra cysteine (C94) in the CH1 domain compared with IgM1, which may cause the structural difference between IgM1 and IgM2. A liquid chromatography–tandem mass spectrometry analysis revealed that both IgM1 and IgM2 were present at the protein level in large yellow croaker serum. Both the Igμ1 and Igμ2 genes were mainly expressed in systemic immune tissues, such as head kidney and spleen, but the expression level of Igμ2 was much lower than that of Igμ1. After Pseudomonas plecoglossicida infection, the expression levels of Igμ1 and Igμ2 in both the spleen and head kidney were significantly upregulated, with a higher upregulation of Igμ2 than that of Igμ1. These results suggested that Igμ1 and Igμ2 may play a differential role in the immune response of large yellow croaker against bacterial infection.

Genetic Analysis of a Chinese Pedigree with Hereditary Spherocytosis Caused by Copy Number Variation Deletion of SPTB Gene

To investigate the molecular mechanism of the disease based on the clinical characterization and genetic mutation analysis in a family with hereditary spherocytosis. The proband with jaundice and anemia was referred to Yidu Central Hospital of Weifang in May 2021. Peripheral blood samples were collected from six members of the family. Second-generation sequencing was used to screen the pathological mutations, and the clinically significant variant sites were selected. Then the relevant databases were used to analyze the variant sites, and RT-qPCR was used to detect the relative mRNA levels of candidate gene. The structure and function of SPTB protein were analyzed by UniProt and SMART databases. We infer that the SPTB gene copy number variation (CNV) deletion was co-segregated with the phenotype of the patients in this family based on the results of second-generation sequencing (about 700 target genes). The UCSC Genome Browser demonstrated that the deleted region was mainly located in exon2-3 of SPTB gene. The results of RT-qPCR showed that the relative SPTB mRNA levels of all patients were lower than the healthy control. UniProt and SMART databases analysis showed that SPTB protein without CH1 and CH2 domains could not bind to erythrocyte membrane actin. The CNV deletion of SPTB gene may be the reason for the hereditary spherocytosis in this family.

Ligand-dependent conformational dynamics of MPER-binding HIV-1 antibody Fab235.

The glycoprotein gp160 of HIV-1 is targeted by many antibodies that differ in neutralizing capacity. Due to the high mutation rate of gp160, its relatively conserved membrane-proximal external region (MPER) is an important epitope for eliciting broadly neutralizing antibodies. However, the MPER is located underneath the gp160 ectodomain, making it difficult to access. Thus, the conformational property of the Fab domain of the antibody is likely a key factor in the binding to MPER and HIV-1 neutralization. As a test system, we use vaccine-elicited MPER antibody Fab235, to investigate its ligand-dependent conformational behavior by performing all-atom molecular dynamics simulations. We perform simulations with or without a bound MPER, 1-µs each. Compared to the APO state, the MPER-bound state had increased inter-domain contacts within Fab235 and attendant reduction in conformational fluctuation which propagated down to the constant domains, in particular to CH1. On the other hand, removing MPER from the MPER-bound structure led to reduction of inter-domain contacts and an increase in fluctuation of the CH1 domain, similar to the original APO structure. The compliance of Fab in the APO state may be beneficial for approaching and binding to the MPER, while stiffening upon binding to MPER would support interaction between the antibody and receptors on innate cells or complement proteins interacting via the Fc domain. Our results also indicate that ligand-dependent contacts between the variable and constant domains enable allosteric control of ligand binding.

Rheumatoid factor recognizes specific domains of the IgG heavy chain complexed with HLA class II molecules.

We previously reported that RF recognized the IgG heavy chain (IgGH)/RA-susceptible HLA class II molecule complex. In the present study, we investigated the molecular mechanisms underlying HLA binding to and the RF recognition of IgGH. We synthesized various types of IgGH segments, including VH, CH1, CH2 and CH3, and transfected them with or without HLA class II molecules into the Human Embryonic Kidney 293T cell line. IgGH single domains linked with the HLA-Cw3 peptide, which binds to the binding groove of the HLA class II molecule, were also synthesized. The expression of IgGH domains on the cell surface and their recognition by RF were examined using flow cytometry. Flag-tagged IgGH segments containing CH1 (CH1, VH-CH1, CH1-CH2, VH-CH1-CH2, CH1-CH2-CH3 and VH-CH1-CH2-CH3) were clearly presented on the cell surface by HLA-DR4, while segments without the CH1 domain were expressed at a low level, and the CH3 single domain was only weakly detected on the cell surface, even with HLA-DR4. We then transfected IgGH single domains linked to the Cw3 peptide together with HLA-DR4 and showed that RF-containing sera from RA patients only recognized the CH3 domain and none of the other single domains. When various segments without the Cw3 peptide were transfected with HLA-DR4, only the CH1-CH2-CH3 segment and full-length IgGH were detected by the sera of RA patients. The CH1 domain of IgGH binds to the RA-susceptible HLA-DR molecule and is expressed on the cell surface. RF specifically recognizes the CH3 domain of the IgGH/HLA-DR4 complex.

Novel modular chimeric antigen receptor spacer for T cells derived from signal regulatory protein alpha Ig-like domains.

Background: T cells equipped with chimeric antigen receptors (CAR) have shown remarkable efficacy in targeting B lineage malignancies. Improvement of the CAR structure is needed, however, with a view to developing flexibly modifiable spacers that are inert in interactions with unwanted cells. Specifically, binding to cells carrying receptors for IgG's crystallizable fragment (FcR), that recognize IgG-derived domains in CARs is to be avoided. Methods: Two novel CARs targeting the CD19 antigen where the IgG1-CH2 and -CH3 domains were replaced with Ig-like domains from signal-regulatory protein α (SIRPα) were designed in silico. An IgG1-based CAR and a CAR lacking both SIRPα and IgG1 domains were used as comparators. The phenotype and memory phenotype of the expanded cells were analyzed by flow cytometry, and CAR T cell activation and cytotoxic efficacy were assessed in co-culture experiments in response to CD19+ target cells. Unwanted interactions with FcR-expressing myeloid cells were interrogated in co-culture assays with THP-1 monocytic cells. Results: T cells carrying the novel SIRPα-based CARs enacted potent in vitro cytotoxicity against CD19 positive B-lineage leukemia cells, comparable to traditional IgG1-based CAR T cells. Co-culture of IgG1-based CAR T cells with FcR-expressing THP-1 monocytic cells led to prominent cell surface expression of CD69 on T cells together with production of Interleukin (IL)-2 and Interferon-γ, and production of IL-1β, indicating activation of the T cells and monocytes, respectively. Longer co-culture led to killing of the monocytes. No signs of T cell nor monocyte activation were detected in co-cultures of SIRPα-based CAR T cells with THP-1 cells. Arming T cells with the SIRPα-based CARs favored differentiation towards CD4+ phenotype during expansion, while the effects on memory phenotype of the T cells were equivalent between the SIRPα- and IgG1-based CARs. In a pilot experiment, T cells modified with one of the SIRPα-based CARs showed dose dependent leukemia cell control. Conclusion: The novel SIRPα based spacers offer a suitable backbone for developing chimeric antigen receptors that evade the off-target binding to FcR while the cells retain a favorable memory phenotype and efficient cytotoxicity, establishing a promising candidate for future in vivo and clinical testing.

Efficient Production of Fc Fusion Proteins in the Cytoplasm of Escherichia coli: Dissecting and Mitigating Redox Heterogeneity

Cost-effective production of therapeutic proteins in microbial hosts is an indispensable tool towards accessible healthcare. Many of these heterologously expressed proteins, including all antibody formats, require disulfide bond formation to attain their native and functional state. A system for catalyzed disulfide bond formation (CyDisCo) has been developed allowing efficient production of recombinant proteins in the cytoplasm of one of the most used microbial expression systems, Escherichia coli. Here, we report high-yield production (up to 230 mg/L from 3 mL cultures) of in-demand therapeutics such as IgG1-based Fc fusion proteins in the E. coli cytoplasm. However, the production of this drug class using the CyDisCo system faces bottlenecks related to redox heterogeneity during oxidative folding. Our investigations identified and addressed one of the major causes of redox heterogeneity during CyDisCo-based production of Fc fusion proteins, i.e., disulfide bond formation in the IgG1 CH3 domain. Here, we communicate that mutating the cysteines in the CH3 domain of target Fc fusion proteins allows their production in a homogeneous redox state in the cytoplasm of E. coli without compromising on yields and thermal stability.

Spatially Confined T-Cell Mediated Anti-AML Tumor Immunity By Dual Recognition Antibodies (CD123/CD200)

Definitive cure of acute myeloid leukemia (AML) remains an unmet need. While recombinant antibody-based and CAR-T cell mediated immunotherapies bear great potential in targeting chemotherapy-resistant leukemic stem cells, these approaches are largely limited by the lack of specific targets uniquely expressed by leukemic cells. Here, we propose a combinatorial strategy using a two-target approach which may have improved on-target and reduced off-target effects. The well-known stem cell marker interleukin-3 receptor α (CD123) is expressed on most AML cells. CD200 recently emerged as a cell surface marker mediating immuno-evasion on high-risk AML cells. We engineered the "Prodrug-activating chain Exchange"(PACE) TriFAB system (Dickopf S et al. Biological Chemistry, 2022) to yield a combinatorial anti-AML directed T-cell response. PACE-TriFAB educts are complementary Knob-into-hole IgG derived prodrugs in which one half is exchanged by a dummy containing an interface destabilizing mutation in the CH3 domain. The CH2 domain of the main body is replaced by a CD3 FAB light or heavy chain, respectively. With the hinge region being replaced by a flexible linker and therefore lacking disulfide bonds, a chain exchange between two educts is enabled when in close proximity on the cell surface. This leads to an activated anti CD3 domain and a dummy-dummy by-product. To demonstrate the T-cell activating potential, we co-cultured fresh PBMCs from healthy donors together with the CD200+/CD123+ AML cell line Kasumi-1 and checked T-cell activation by flow cytometry (E:T ratio 5:1, prodrug conc. 20 nM, 24 h stimulation). We observed a marked increase in CD69, CD25 and CD137 expression on CD8+ cells in the case when the CD123 and CD200 prodrugs were applicated simultaneously together with target cells, but not when single prodrugs alone or without target cells were analyzed (Median fluorescence intensity for CD25, CD69 and CD137: No construct: 450, 8.900 & 2.100; prodrug CD200: 460, 8.700 & 2.100; prodrug CD123: 460, 9.600 & 2.000; both prodrugs: 6.500, 344.300 & 43.100; results confirmed in repeat experiments) For the evaluation of the specific cytotoxic effect of the construct we transduced Kasumi-1 cells with an mCherry lentiviral vector and imaged the cells co-cultured with PBMCs every 2 h over a time span of 60 h (E:T ratio 5:1, prodrug conc. 10 nM). Proliferation of the Kasumi-1-mCherry cells was observed when no prodrug or only a single prodrug was added. However, application of both prodrugs together led to a decrease in mCherry positive cell count (log2 fold changes to starting point: No PBMCs 1,2; no construct 0,9; prodrug CD200 0,9; prodrug CD123 0,4; both prodrugs -2,1; n=3). Not only could we show that this elimination of tumor cells was dose dependent but also it was constrained by the E:T ratio (pivot point from killing to proliferation at 0,1 nM and E:T of 1:2 n=3). Similar results were obtained using patient derived xenograft (PDX) AML cells. In contrast to Kasumi-1 AML cells, specific killing of patient-derived PDX cells could be documented with lower E:T ratios and lower prodrug concentrations (pivot point from killing to proliferation at 0,05 nM and E:T of 1:5) We also started to analyze the anti-leukemic effect in vivo. Luciferase-expressing CD200+/CD123+ AML PDX cells were injected into NSG mice. On day +15, PBMC from a healthy individual were transplanted, followed by additional applications of the antibodies on days +16, +18, +21, +23, +25 & +29. Tumor burden was analyzed by bioluminescence imaging (log10 fold change [P/sec] 2 weeks after PBMC injection: Control 1,9; treated -0,4; group size = 3). In conclusion the combination of anti CD200 and anti CD123 as prodrugs of an on-target activating trispecific T-cell engager kills AML cells in vitro and in vivo and has the potential to eliminate AML LSC while minimizing the risk for severe on-target off-leukemia toxicity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Structural Dynamics of a Thermally Stressed Monoclonal Antibody Characterized by Temperature-Dependent H/D Exchange Mass Spectrometry.

Differential scanning calorimetry (DSC) is a standard tool for probing the resilience of monoclonal antibodies (mAbs) and other protein therapeutics against thermal degradation. Unfortunately, DSC usually only provides insights into global unfolding, although sequential steps are sometimes discernible for multidomain proteins. Temperature-dependent hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has the potential to probe heat-induced events at a much greater level of detail. We recently proposed a strategy to deconvolute temperature-dependent HDX data into contributions from local dynamics, global unfolding/refolding, as well as chemical labeling. However, that strategy was validated only for a small protein (Tajoddin, N. N.; Konermann, L. Anal. Chem. 2020, 92, 10058). The current work explores the applicability of this HDX framework to the NIST reference mAb (NISTmAb), a large multidomain protein that is prone to aggregation and has three melting points. Using global fitting, we were able to model HDX profiles across the NISTmAb sequence between zero and 95 °C, and for time points between 15 s and 20 min. We uncovered the enthalpic and entropic contributions of local fluctuations that govern the conformational dynamics at low temperatures. The CH2 and CH3 domains were found to be increasingly affected by global unfolding/refolding in the vicinity of their melting points, although the transiently unfolded protein displayed significant residual protection. Global dynamics were not involved in the deuteration of the Fab domains (which have the highest melting point). Instead, global Fab unfolding was followed immediately by irreversible aggregation. Our results reveal that the thermodynamic HDX-MS strategy applied in this work is well suited for probing spatially resolved dynamics of thermally stressed large proteins such as mAbs, complementing data obtained by DSC.