Constant Domains Research Articles

Undecidability of QLTL and QCTL with two variables and one monadic predicate letter

We study the algorithmic properties of the quantified linear-time temporal logic QLTL in languages with restrictions on the number of individual variables as well as the number and arity of predicate letters. We prove that the satisfiability problem for QLTL in languages with two individual variables and one monadic predicate letter in Σ 11 -hard. Thus, QLTL is Π 11 -hard, and so not recursively enumerable, in such languages. The resultholds both for the increasing domain and the constant domain semantics and is obtained by reduction from a Σ 11 -hard N×N recurrent tiling problem. It follows from the proof for QLTL that similar results hold for the quantified branching-time temporal logic QCTL, and hence for the quantified alternating-time temporal logic QATL. The result presented in this paper strengthens a result by I. Hodkinson, F. Wolter, and M. Zakharyaschev, who have shown that the satisfiability problem for QLTL is Σ 11 -hard in languages with two individual variablesand an unlimited supply of monadic predicate letters.

From IgZ to IgT: A Call for a Common Nomenclature for Immunoglobulin Heavy Chain Genes of Ray-Finned Fish

From IgZ to IgT: A Call for a Common Nomenclature for Immunoglobulin Heavy Chain Genes of Ray-Finned Fish

Design of a Novel Fab-Like Antibody Fragment with Enhanced Stability and Affinity for Clinical use.

With increasing interest in applying recombinant monoclonal antibodies (mAbs) in human medicine, engineered mAb fragments with reduced size and improved stability are in demand to overcome current limitations in clinical use. Herein, a novel Fab-like antibody fragment generated via an in silico-based engineering approach where the CH1 and CL domains of Fab are replaced by the IgG1 CH3 domains is described. This construct, designated as FabCH3, maintains the natural N-terminus and C-terminus of IgG antibody, can be expressed at a high level in bacterial cells and, importantly, exhibits much higher stability and affinity than the parental Fab when tested in a mesothelin-specific Fab m912, as well as a vascular endothelial growth factor A (VEGFA)-specific Fab Ranibizumab (in vivo). The high-resolution crystal structures of m912 FabCH3 and m912 Fab are determined, and the comparative analysis reveals more rigid structures in both constant domains and complementarity-determining regions of FabCH3, explaining its enhanced stability and affinity. Overall, the stabilized FabCH3 described in this report provides a versatile platform for engineering Fab-like antibody fragments with higher stability and antigen-binding affinity that can be used as a distinct class of antibody therapeutics.

Structural study of the recognition mechanism of tau antibody Tau2r3 with the key sequence (VQIINK) in tau aggregation

One of the histopathological features of Alzheimer's disease (AD) is higher order neurofibrillary tangles formed by abnormally aggregated tau protein. The sequence 275VQIINK280 in the microtubule-binding domain of tau plays a key role in tau aggregation. Therefore, an aggregation inhibitor targeting the VQIINK region in tau may be an effective therapeutic agent for AD. We have previously shown that the Fab domain (Fab2r3) of a tau antibody that recognizes the VQIINK sequence can inhibit tau aggregation, and we have determined the tertiary structure of the Fab2r3-VQIINK complex. In this report, we determined the tertiary structure of apo Fab2r3 and analyzed differences in the structures of apo Fab2r3 and Fab2r3-VQIINK to examine the ligand recognition mechanism of Fab2r3. In comparison with the Fab2r3-VQIINK structure, there were large differences in the arrangement of the constant and variable domains in apo Fab2r3. Remarkable structural changes were especially observed in the H3 and L3 loop regions of the complementarity determining regions (CDRs) in apo Fab2r3 and the Fab2r3-VQIINK complex. These structural differences in CDRs suggest that formation of hydrophobic pockets suitable for the antigen is important for antigen recognition by tau antibodies.

The Clonotypic BCR IG of CLL Patients Contain Predicted T-Cell Class I Epitopes with Shared Structural Properties

Targeted therapies have revolutionized the treatment of chronic lymphocytic leukemia (CLL) with remarkable overall response rates. Against that, however, CLL remains incurable, indicating a need for novel strategies towards disease control and eventual eradication, including reinvigoration of anti-tumor immune responses. T cells in CLL display an oligoclonal profile and appear selected by restricted antigens, with recent evidence suggesting that the selecting epitopes may lie within the clonotypic B-cell receptor immunoglobulins (BcR IG). Should this prove to be the case, such neoepitopes could be exploited as idiotypic targets for cellular therapy or for peptide vaccine design, aiming to augment response to current treatments.Here we performed ad hoc prediction of putative T-cell class I neoepitopes contained within the clonotypic BcR IGs of CLL patients, with an intended bias towards major stereotyped CLL subsets. We selected 27 patients to represent the full spectrum of CLL: (i) with mutated IGHV genes (M-CLL, n=5), (ii) with unmutated IGHV genes (U-CLL, n=5), (iii) assigned to major stereotyped subsets (subset #1, n=7; subset #2, n=5; subset #4, n=5). RT-PCR was performed for the heavy (H) and the light (K/L) IG chains using subgroup-specific Leader primers for the IGHV/IGK/LV gene and universal primers annealing to the constant domain (IGHG, IGHM, IGKC, IGLC), in order to produce the full-length V-(D)-J gene rearrangement sequence, plus the start of the constant domain. PCR products were subjected to direct double-strand Sanger sequencing with a quality-optimized protocol. The amino acid sequences were subsequently parsed in peptides and subjected to NetMHCpan. The rank score was calculated, considering the 4-digit HLA-A and -B typing for each individual patient. High- and medium-binding peptides (rank score <2%) were selected. Exact matches to germline and/or proteome databases were excluded.Overall, 1,007 predicted neoepitopes were identified. All patients had predicted CD8 + T-cell epitopes within the clonotypic BcR IG, either in the heavy chain (26/27 pts, n=632 epitopes) or the light chain (26/27 pts, n=375 epitopes). The majority of the peptides resulted from somatic hypermutations (SHMs) across the IGHV gene outside the complementarity-determining region 3 (CDR3; n=538, 53.4%). With the exception of few peptides located within the FR4 region (n=11, 0.1%), the remaining (n=458, 45.5%) involved (at least part of) the CDR3, which is particularly relevant given its small length (9-27 aa) within the full sequence (331-660 aa). There was no statistically significant difference in the rank score of peptides involving the CDR3 vs. all others. Peptide clustering assigned most of the predicted neoepitopes (970/1007, 96.3%) in 54 clusters of similar length and amino acid composition. Also, it revealed similar or identical predicted neoepitopes among different patients (30 clusters of two, 10 clusters of three, 8 clusters of four, 4 clusters of five, 1 cluster of six and 1 cluster of eight). Importantly, these clusters involved: (i) shared CDR3 patterns in patients assigned to the same stereotyped subset, but also (ii) subset-specific recurrent SHMs across the rearranged IGHV gene, e.g. G-to-E SHM at position 28 in the VH CDR1 of subset #4, a recurrent SHM in this subset. Also of note, the two most highly populated clusters involved peptides within the VL CDR3, and were biased towards specific subsets; the cluster of eight patients contained 4 patients assigned to subset #1 and the cluster of six patients included 4 patients assigned to subset #2.In conclusion, in silico prediction identified a significant number of putative T-cell class I neoepitopes contained within the clonotypic BcR IG of CLL patients. The majority of these neoepitopes can be assigned to clusters based on amino acid similarity and are shared among different patients. Many of them culminate from subset-specific (‘stereotyped‘) CDR3 patterns or recurrent SHMs, suggesting that the targeted SHM which shapes the CLL BcR IG repertoire may produce immunogenic CD8 + T-cell epitopes. Their actual immunogenicity has to be tested in ex vivo studies, currently underway by our group. DisclosuresAnagnostopoulos: Abbvie: Other: clinical trials; Sanofi: Other: clinical trials ; Ocopeptides: Other: clinical trials ; GSK: Other: clinical trials; Incyte: Other: clinical trials ; Takeda: Other: clinical trials ; Amgen: Other: clinical trials ; Janssen: Other: clinical trials; novartis: Other: clinical trials; Celgene: Other: clinical trials; Roche: Other: clinical trials; Astellas: Other: clinical trials . Chatzidimitriou: Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

“Snapshotting” Somatic Hypermutation in Single Follicular Lymphoma Cells

IntroductionFollicular Lymphoma (FL) is one of the most prevalent B-cell neoplasms and despite recent advances remains incurable in most cases. FL cells are malignant counterparts of normal germinal center B-cells. At molecular level FL are characterized by the t(14;18) which results in the overexpression of the BCL-2. These apoptosis resistant cells accumulate somatic mutations associated with tumorigenesis and progression. Whereas several mutagenic mechanisms shape the FL genomic landscape, up to 22% of the mutations may be attributed to activation-induced cytidine deaminase (AID) activity.Under physiological conditions AID is responsible for somatic hypermutation (SHM) in immunoglobulin genes (IG) of germinal center B-cells. In fact, it is accepted that ongoing SHM still occurs at relatively high rates in FL cells as compared with other germinal center lymphoid neoplasms. Moreover, we recently demonstrated the direct effect of AID overexpression inducing somatic mutations driving murine and human B-cell neoplasm progression in vivo. Therefore, unveiling the molecular basis of AID activity in lymphoma cells remains essential to understand lymphomagenesis and to develop novel targeted therapies.The advent of single-cell high-throughput sequencing has enabled the analysis of molecular events at an unprecedented resolution. Therefore, we designed a study to capture AID-induced somatic hypermutation at the single cell level in FL.MethodsTumor samples derived from 14 FL patients were analyzed, as controls, we chose a B-cell malignancy with lower SHM rates and included 5 samples derived from chronic lymphocytic leukemia (CLL) and 2 from monoclonal B lymphocytosis (MBL). Single cell whole cDNA libraries were obtained by 10X Genomics. Immunoglobulin gene single cell libraries were prepared by enrichment with seminested amplification using 3 ′ constant domain primers followed by 10X Genomics prep. Both single-cell libraries were sequenced in paired-end mode (2 × 150 bp) on an Illumina Hiseq platform. We developed an immunoglobulin alignment tool to enable the analysis of highly mutated IG sequences derived from FL. A consensus sequence was obtained by transcript associated with a unique molecular identifier (UMI) for every cell. Then, the presence of variants in the IG by position in a particular cell was annotated. These observations were filtered using quality parameters (read depth => 25 , frequency of the event => 20%, UMIs supporting every variant => 5).ResultsWe analyzed an average of 926 cells per case. Our data confirms at the single cell level previous reports on high clonal heterogeneity and hypermutation rate (mean 18.7%) in FL. When analyzing intracellular heterogeneity we observed single FL cells expressing simultaneously transcripts derived from the same immunoglobulin VDJ rearrangement but displaying high confidence single nucleotide variants. After applying strict filtering strategies to account for potential technical issues we defined the occurrence of “SHM snapshot events” when a single cell displayed a set of transcripts from a particular VDJ rearrangement with and without the occurring single nucleotide variant. Such events were detected in 8 of the 14 FL analyzed samples. In those 8 samples, 114 events were observed in which two different transcripts of one specific IG were found within a single cell. AID-related motifs (WRCY, WA and RCG) were found in 45% of the SHM snapshot events. SHM snapshot events were undetectable in CLL (5 samples) and 3 SHM snapshot events were detected in MBL (2 samples). In addition, the occurrence of SHM snapshot events was significantly associated with AID expression (Fisher exact test, p-value < 0.001). On the other hand, AID expression was not detectable on CLL and MBL samples.ConclusionHere we report for the first time the occurrence of ongoing somatic hypermutation at a single cell level in FL. The simultaneous detection of both the pre and post mutation IG mRNA transcripts within a single cell may be indicative of SHM occurring more recently than the lifespan of mRNA transcript. The detection of this phenomenon in 57% of FL samples and its association with AID expression suggests that AID-induced mutagenesis may be acting at a much higher rate than expected. This work highlights the role of AID in shaping the tumor heterogeneity in FL and the need to further understand the role of this enzyme in lymphomagenesis and tumor progression. DisclosuresNo relevant conflicts of interest to declare.

Stress-dependent Flexibility of a Full-length Human Monoclonal Antibody: Insights from Molecular Dynamics to Support Biopharmaceutical Development

After several decades of advancements in drug discovery, product development of biopharmaceuticals remains a time- and resource-consuming endeavor. One of the main reasons is associated to the lack of fundamental understanding of conformational dynamics of such biologic entities, and how they respond to various stresses encountered during manufacturing, storage, and shipping. In this work, we have studied the conformational dynamics of human IgG1κ b12 monoclonal antibody (mAb) using molecular dynamics simulations. The hundreds of nanoseconds long trajectories reveal that b12 mAb is highly flexible. Its variable domains show greater conformational fluctuations than the constant domains. Additionally, it collapses towards a more globular shape in response to thermal stress, leading to decrease in the total solvent exposed surface area and radius of gyration. This behavior is more pronounced for the deglycosylated b12 mAb, and it appears to correlate with increase in inter-domain contacts between specific regions of the antibody. Conformational fluctuations also cause transient formation and disruption of hydrophobic and charged patches on the antibody surface, which is particularly important for the prediction of CMC properties during development phases of antibody-based biotherapeutics. The insights gained through these simulations may help the development of biologic drugs.

Identification and Characterization of a Novel Single Domain Antibody Against Ebola Virus.

Ebola virus (EBOV) belongs to the Filoviridae family and causes severe illnesses such as hemorrhagic fever with a high mortality rate up to 90%. Now two antibody drugs termed Inmazeb and Ebanga have been approved for treating EBOV infection. However, clinical studies have demonstrated that the mortality rate of the patients who received these two antibody drugs remains above 30%. Therefore, novel therapeutics with better efficacy is still desired. The isolated human IgG1 constant domain 2 (CH2 domain) has been proposed as a scaffold for the development of C-based single domain antibodies (C-sdAbs) as therapeutic candidates against viral infections and other diseases. Here, we screened and identified a novel C-sdAb termed M24 that targets EBOV glycoprotein (GP) from a C-sdAb phage display library. M24 neutralizes the pseudotype EBOV with IC50 of 0.8 nmol/L (12 ng/mL) and has modest neutralizing activity against authentic EBOV. Epitope determination, including molecular docking and site mutation analysis, discloses that M24 binds to the internal fusion loop (IFL) within GP2, a transmembrane subunit of GP. Interestingly, we found that the binding of M24 to GP at pH 5.5 has dramatically decreased compared to the binding at pH 7.5, which may lead to weak efficacy in the neutralization of authentic EBOV. Since no sdAb against EBOV infection has been reported to date, our results not only give a proof of concept that sdAbs could be utilized for the development of potential therapeutic candidates against EBOV infection, but also provide useful information for the discovery and improvement of anti-EBOV agents.

A new approach to produce IgG4-like bispecific antibodies

While achieving rapid developments in recent years, bispecific antibodies are still difficult to design and manufacture, due to mispair of both heavy and light chains. Here we report a novel technology to make bispecific molecules. The knob-into-hole method was used to pair two distinct heavy chains as a heterodimer. IgG4 S228P CH1-CL interface was then partially replaced by T-cell receptor α/β constant domain to increase the efficiency of cognate heavy and light chain pairing. Following expression and purification, the bispecific antibody interface exchange was confirmed by Western blotting and LC–MS/MS. To ensure its validity, we combined a monovalent bispecific antibody against PD-1 (sequence from Pembrolizumab) and LAG3 (sequence from Relatlimab). The results showed that the molecule could be assembled correctly at a ratio of 95% in cells. In vitro functional assay demonstrated that the purified bispecific antibody exhibits an enhanced agonist activity compared to that of the parental antibodies. Low immunogenicity was predicted by an open-access software and ADA test.

Protease-sensitive regions in amyloid light chains: what a common pattern of fragmentation across organs suggests about aggregation.

Light‐chain (AL) amyloidosis is characterized by deposition of immunoglobulin light chains (LC) as fibrils in target organs. Alongside the full‐length protein, abundant LC fragments are always present in AL deposits. Herein, by combining gel‐based and mass spectrometry analyses, we identified and compared the fragmentation sites of amyloid LCs from multiple organs of an AL λ amyloidosis patient (AL‐55). The positions pinpointed here in kidney and subcutaneous fat, alongside those previously detected in heart of the same patient, were aligned and mapped on the LC’s dimeric and fibrillar states. All tissues contain fragmented LCs along with the full‐length protein; the fragment pattern is coincident across organs, although microheterogeneity exists. Multiple cleavage positions were detected; some are shared, whereas some are organ‐specific, likely due to a complex of proteases. Cleavage sites are concentrated in ‘proteolysis‐prone’ regions, common to all tissues. Several proteolytic sites are not accessible on native dimers, while they are compatible with fibrils. Overall, data suggest that the heterogeneous ensemble of LC fragments originates in tissues and is consistent with digestion of preformed fibrils, or with the hypothesis that initial proteolytic cleavage of the constant domain triggers the amyloidogenic potential of LCs, followed by subsequent proteolytic degradation. This work provides a unique set of molecular data on proteolysis from ex vivo amyloid, which allows discussing hypotheses on role and timing of proteolytic events occurring along amyloid formation and accumulation in AL patients.

Hinge influences in murine IgG binding to Cryptococcus neoformans capsule.

Decades of studies on antibody structure led to the tenet that the V region binds antigens while the C region interacts with immune effectors. In some antibodies, however, the C region affects affinity and/or specificity for the antigen. One example is the 3E5 monoclonal murine IgG family, in which the mIgG3 isotype has different fine specificity to the Cryptococcus neoformans capsule polysaccharide than the other mIgG isotypes despite their identical variable sequences. Our group serendipitously found another pair of mIgG1/mIgG3 antibodies based on the 2H1 hybridoma to the C.neoformans capsule that recapitulated the differences observed with 3E5. In this work, we report the molecular basis of the constant domain effects on antigen binding using recombinant antibodies. As with 3E5, immunofluorescence experiments show a punctate pattern for 2H1-mIgG3 and an annular pattern for 2H1-mIgG1; these binding patterns have been associated with protective efficacy in murine cryptococcosis. Also as observed with 3E5, 2H1-mIgG3 bound on ELISA to both acetylated and non-acetylated capsular polysaccharide, whereas 2H1-mIgG1 only bound well to the acetylated form, consistent with differences in fine specificity. In engineering hybrid mIgG1/mIgG3 antibodies, we found that switching the 2H1-mIgG3 hinge for its mIgG1 counterpart changed the immunofluorescence pattern to annular, but a 2H1-mIgG1 antibody with an mIgG3 hinge still had an annular pattern. The hinge is thus necessary but not sufficient for these changes in binding to the antigen. This important role for the constant region in antigen binding could affect antibody biology and engineering.

Anti-TRBC1 Antibody-Based Flow Cytometric Detection of T-Cell Clonality: Standardization of Sample Preparation and Diagnostic Implementation.

Simple SummaryThe anti-TRBC1 antibody JOVI-1 has recently been identified as a flow cytometry marker potentially useful for assessment of T-cell clonality. The aim of this study was to optimize a flow cytometric method for routine use of anti-TRBC1 to assess T-cell clonality and validate it in a large series of normal and pathological samples. Our results showed that the best resolution to accurately identify TRBC1+ cells was achieved by adding the CD3 antibody either simultaneously or after TRBC1. In addition, TRBC1+/TRBC1− ratios within different Tαβ-cell subsets are provided as expected reference ranges for polyclonal T-cells. Based on the optimized approach here proposed, we detected monoclonal Tαβ-cell populations with high specificity (96%) and a high analytical sensitivity/level of detection (≤10−4), when clonal T-cells exhibited immunophenotypic aberrancies. These findings further support and extend previous observations about the utility of TRBC1 for the diagnostic screening and monitoring of clonal Tαβ-cell populations.A single antibody (anti-TRBC1; JOVI-1 antibody clone) against one of the two mutually exclusive T-cell receptor β-chain constant domains was identified as a potentially useful flow-cytometry (FCM) marker to assess Tαβ-cell clonality. We optimized the TRBC1-FCM approach for detecting clonal Tαβ-cells and validated the method in 211 normal, reactive and pathological samples. TRBC1 labeling significantly improved in the presence of CD3. Purified TRBC1+ and TRBC1− monoclonal and polyclonal Tαβ-cells rearranged TRBJ1 in 44/47 (94%) and TRBJ1+TRBJ2 in 48 of 48 (100%) populations, respectively, which confirmed the high specificity of this assay. Additionally, TRBC1+/TRBC1− ratios within different Tαβ-cell subsets are provided as reference for polyclonal cells, among which a bimodal pattern of TRBC1-expression profile was found for all TCRVβ families, whereas highly-variable TRBC1+/TRBC1− ratios were observed in more mature vs. naïve Tαβ-cell subsets (vs. total T-cells). In 112/117 (96%) samples containing clonal Tαβ-cells in which the approach was validated, monotypic expression of TRBC1 was confirmed. Dilutional experiments showed a level of detection for detecting clonal Tαβ-cells of ≤10−4 in seven out of eight pathological samples. These results support implementation of the optimized TRBC1-FCM approach as a fast, specific and accurate method for assessing T-cell clonality in diagnostic-FCM panels, and for minimal (residual) disease detection in mature Tαβ+ leukemia/lymphoma patients.

Expression of immunoglobulin constant domain genes in neurons of the mouse central nervous system.

General consensus states that immunoglobulins are exclusively expressed by B lymphocytes to form the first line of defense against common pathogens. Here, we provide compelling evidence for the expression of two heavy chain immunoglobulin genes in subpopulations of neurons in the mouse brain and spinal cord. RNA isolated from excitatory and inhibitory neurons through ribosome affinity purification revealed Ighg3 and Ighm transcripts encoding for the constant (Fc), but not the variable regions of IgG3 and IgM. Because, in the absence of the variable immunoglobulin regions, these transcripts lack the canonical transcription initiation site used in lymphocytes, we screened for alternative 5' transcription start sites and identified a novel 5' exon adjacent to a proposed promoter element. Immunohistochemical, Western blot, and in silico analyses strongly support that these neuronal transcripts are translated into proteins containing four Immunoglobulin domains. Our data thus demonstrate the expression of two Fc-encoding genes Ighg3 and Ighm in spinal and supraspinal neurons of the murine CNS and suggest a hitherto unknown function of the encoded proteins.

Novel oncolytic adenovirus expressing enhanced cross-hybrid IgGA Fc PD-L1 inhibitor activates multiple immune effector populations leading to enhanced tumor killing in vitro, in vivo and with patient-derived tumor organoids

BackgroundDespite the success of immune checkpoint inhibitors against PD-L1 in the clinic, only a fraction of patients benefit from such therapy. A theoretical strategy to increase efficacy would be to...

Host-Pathogen Adhesion as the Basis of Innovative Diagnostics for Emerging Pathogens.

Infectious diseases are an existential health threat, potentiated by emerging and re-emerging viruses and increasing bacterial antibiotic resistance. Targeted treatment of infectious diseases requires precision diagnostics, especially in cases where broad-range therapeutics such as antibiotics fail. There is thus an increasing need for new approaches to develop sensitive and specific in vitro diagnostic (IVD) tests. Basic science and translational research are needed to identify key microbial molecules as diagnostic targets, to identify relevant host counterparts, and to use this knowledge in developing or improving IVD. In this regard, an overlooked feature is the capacity of pathogens to adhere specifically to host cells and tissues. The molecular entities relevant for pathogen–surface interaction are the so-called adhesins. Adhesins vary from protein compounds to (poly-)saccharides or lipid structures that interact with eukaryotic host cell matrix molecules and receptors. Such interactions co-define the specificity and sensitivity of a diagnostic test. Currently, adhesin-receptor binding is typically used in the pre-analytical phase of IVD tests, focusing on pathogen enrichment. Further exploration of adhesin–ligand interaction, supported by present high-throughput “omics” technologies, might stimulate a new generation of broadly applicable pathogen detection and characterization tools. This review describes recent results of novel structure-defining technologies allowing for detailed molecular analysis of adhesins, their receptors and complexes. Since the host ligands evolve slowly, the corresponding adhesin interaction is under selective pressure to maintain a constant receptor binding domain. IVD should exploit such conserved binding sites and, in particular, use the human ligand to enrich the pathogen. We provide an inventory of methods based on adhesion factors and pathogen attachment mechanisms, which can also be of relevance to currently emerging pathogens, including SARS-CoV-2, the causative agent of COVID-19.

Development of a novel anti-B7-H4 antibody enhances anti-tumor immune response of human T cells

BackgroundB7-H4 is a member of the B7 superfamily that is expressed on the surface of tumors and exhibits limited expression on normal tissue. B7-H4 negatively regulates tumor immunity by interacting with the B7-H4 receptor, which is expressed by activated CD8 + T cells. Hence, we sought to generate an immunomodulatory antibody that targets B7-H4 and blocks the immunosuppressive activity of B7-H4. MethodsAnti-B7-H4 antibodies were generated using the hybridoma technique and screened by a binding assay based on B7-H4-expressing tumor cells. The B7-H4 antagonistic antibodies were further screened based on their checkpoint blockade activity using a SEB-stimulated peripheral blood mononuclear cell (PBMC) assay, which comprised B7-H4-expressing antigen presenting cells (APCs) and activated T cells. To assess the immunomodulatory activity of anti-B7-H4 antibodies, activated human CD8+ T cells were cultured in B7-H4 protein-coated plates, and the production of IL-2 and the proliferation rate of CD8+ T cells were measured. In addition, we evaluated the ADCC effect of anti-B7-H4 antibodies against tumor cell lines. The in vivo antitumor efficacy of the anti-B7-H4 antibody was also evaluated in human T cell-engrafted NOG mice. ResultsA panel of anti-B7-H4 antibodies was generated. The top 23 antibodies were screened to identify antibodies that disabled B7-H4-mediated inhibition. Antibody 17 exhibited the greatest induction of the production of IL-2 and IFN-gamma in SEB-stimulated PBMCs. Antibody 17 was constructed as a chimeric antibody (CH17) with a human IgG1 constant domain. CH17 showed high affinity for human B7-H4 and fully cross-reacted with cynomolgus B7-H4. Additionally, CH17 mediated potent antibody-dependent cell cytotoxicity (ADCC) against different B7-H4-positive tumor cell lines. More importantly, CH17 relieved B7-H4-mediated T cell suppression by enhancing IL2 production and promoting T cell proliferation. In an MDA-MB-468-bearing mouse model in which human pan-T cells were engrafted, CH17 delayed tumor growth by engaging T cells and exerted a synergistic effect in combination with an anti-human PD-1 antibody. ConclusionsWe successfully generated an immunomodulatory antibody targeting B7-H4 that possesses both T cell immune checkpoint inhibitory activity and ADCC activity in B7-H4-positive tumors. B7-H4-targeting antibodies might represent a promising immunotherapy for B7-H4-expressing tumors.

Nonlinear dynamic analysis of float-over deck installation for a GBS platform based on a constant parameter time domain model

Float-over deck installation has become a reliable and cost-effective method of installing heavy integrated decks for various types of offshore platforms, such as the jacket and GBS platforms. This operation involves a number of inherent complexities such as the strong nonlinear interactions between the multi-body system and environment. Therefore, dynamic modelling of the float-over system during the deck load transfer stage is a challenging task. Particularly, the wave-induced impacts among the deck, barge and platform substructure may generate strong nonlinear dynamics that cannot be accurately investigated by the current standard simulation packages. This study develops a constant parameter time domain model (CPTDM) based on Cummins equation and state-space model for analysing the nonlinear dynamics arising from the float-over deck installation of a GBS platform. Frequency domain hydrodynamic analyses of the installation barge carrying the deck with and without the presence of the GBS are both carried to identify hydrodynamic interactions. A code-to-code verification is performed to verify the accuracy and efficiency of the developed CPTDM by comparing the present results with ANSYS-AQWA. Based on the verified model, the nonlinear dynamics of the GBS platform at different load transfer stages are investigated by using Poincare maps, bifurcation diagrams and phase trajectories.

Abstract 1514: Engineering off-the-shelf T Cell Receptor Fusion Construct (TRuC) T cells

Abstract The natural T cell receptor (TCR) recognizes its cognate peptide antigen only when presented on human leukocyte antigen (HLA) molecules and requires HLA matching of α/β TCR-engineered T cells for cancer therapy. To bypass the need for HLA matching, we have previously described the generation of autologous T Cell Receptor Fusion Construct (TRuC™) T cells which are engineered to express a fusion protein that comprises an antibody-derived binder tethered to the CD3ϵ signaling subunit. Upon integration of the TRuC into the TCR, it recognizes tumor surface antigens independent of HLA and uses the complete receptor complex to trigger a comprehensive T cell response. Here, we report the engineering of off-the-shelf TRuC-T cells directed against CD19 and mesothelin, respectively. To eliminate the alloreactivity of α/β T cells and reduce the risk of graft-versus-host-disease (GvHD), the TRAC locus is knocked-out. To enable the re-assembly of the TCR, the endogenous TCRα and β subunits are replaced with fusion proteins comprising antibody binders fused to the murine TCRα or β constant domains, or alternatively, to the TCRγ and δ constant domains. Similar to their autologous counterparts, allogeneic CD19 and MSLN-targeting TRuC-T cells upregulate activation markers, secrete cytokines, and lyse tumor cells in an antigen-specific manner. Importantly, TRuC-T cells lack alloreactivity as demonstrated in mixed lymphocyte reactions and clear tumors in NSG xenograft model comparable to autologous anti-CD19 TRuC-T cells, but without signs of GvHD. Likewise, the functional activity and efficacy of allogeneic TRuC-T cells directed against mesothelin is similar to that of autologous anti-MSLN TRuC-T cells. To reduce host rejection and enhance the persistence of the allogeneic TRuC-T cells, MHC class I expression on the surface of the TRuC-T cells was down-regulated by means of RNAi. In summary we have engineered allogeneic TRuC-T cells that maintain the signaling properties of the TCR complex with comparable efficacy as donor-matched autologous TRuC-T cells; moreover, these T cells have the potential to persist in an allogeneic host by diminishing the risk of rejection by the host. Citation Format: Julie Donaghey, Philippe Kieffer-Kwon, Julio Gomez-Rodriguez, Troy Patterson, Jessica Gierut, Tiffany Chan, Ryan Milione, Joshua Daniel, Holly Horton, Robert Tighe, Robert Hofmeister, Dario A. Gutierrez. Engineering off-the-shelf T Cell Receptor Fusion Construct (TRuC) T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1514.

Allosteric activation of Tcell antigen receptor signaling by quaternary structure relaxation.

SummaryThe mechanism of T cell antigen receptor (TCR-CD3) signaling remains elusive. Here, we identify mutations in the transmembrane region of TCRβ or CD3ζ that augment peptide T cell antigen receptor (pMHC)-induced signaling not explicable by enhanced ligand binding, lateral diffusion, clustering, or co-receptor function. Using a biochemical assay and molecular dynamics simulation, we demonstrate that the gain-of-function mutations loosen the interaction between TCRαβ and CD3ζ. Similar to the activating mutations, pMHC binding reduces TCRαβ cohesion with CD3ζ. This event occurs prior to CD3ζ phosphorylation and at 0°C. Moreover, we demonstrate that soluble monovalent pMHC alone induces signaling and reduces TCRαβ cohesion with CD3ζ in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCRαβ to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to license CD3ζ phosphorylation and initiate signal propagation.

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