Human Isotype Research Articles

The Crystal Structure of Human IgD-Fc Reveals Unexpected Differences With Other Antibody Isotypes.

Of the five human antibody isotypes, the function of IgD is the least well-understood, although various studies point to a role for IgD in mucosal immunity. IgD is also the least well structurally characterized isotype. Until recently, when crystal structures were reported for the IgD Fab, the only structural information available was a model for intact IgD based on solution scattering data. We now report the crystal structure of human IgD-Fc solved at 3.0 Å resolution. Although similar in overall architecture to other human isotypes, IgD-Fc displays markedly different orientations of the Cδ3 domains in the Cδ3 domain dimer and the lowest interface area of all the human isotypes. The nature of the residues that form the dimer interface also differs from those conserved in the other isotypes. By contrast, the interface between the Cδ2 and Cδ3 domains in each chain is the largest among the human isotypes. This interface is characterized by two binding pockets, not seen in other isotypes, and points to a potential role for the Cδ2/Cδ3 interface in stabilizing the IgD-Fc homodimer. We investigated the thermal stability of IgD-Fc, alone and in the context of an intact IgD antibody, and found that IgD-Fc unfolds in a single transition. Human IgD-Fc clearly has unique structural features not seen in the other human isotypes, and comparison with other mammalian IgD sequences suggests that these unique features might be widely conserved.

Targeting of immune checkpoint regulator V-domain Ig suppressor of T-cell activation (VISTA) with 89Zr-labelled CI-8993

BackgroundCI-8993 is a fully human IgG1κ monoclonal antibody (mAb) that binds specifically to immune checkpoint molecule VISTA (V-domain Ig suppressor of T-cell activation). Phase I safety has been established in patients with advanced cancer (NCT02671955). To determine the pharmacokinetics and biodistribution of CI-8993 in patients, we aimed to develop 89Zr-labelled CI-8993 and validate PET imaging and quantitation in preclinical models prior to a planned human bioimaging trial.MethodsCI-8993 and human isotype IgG1 control were conjugated to the metal ion chelator p-isothiocyanatobenzyl-desferrioxamine (Df). Quality of conjugates were assessed by SE-HPLC, SDS-PAGE, and FACS. After radiolabelling with zirconium-89 (89Zr), radioconjugates were assessed for radiochemical purity, immunoreactivity, antigen binding affinity, and serum stability in vitro. [89Zr]Zr-Df-CI-8993 alone (1 mg/kg, 4.6 MBq) or in combination with 30 mg/kg unlabelled CI-8993, as well as isotype control [89Zr]Zr-Df-IgG1 (1 mg/kg, 4.6 MBq) were assessed in human VISTA knock-in female (C57BL/6 N-Vsirtm1.1(VSIR)Geno, huVISTA KI) or control C57BL/6 mice bearing syngeneic MB49 bladder cancer tumours; and in BALB/c nu/nu mice bearing pancreatic Capan-2 tumours.ResultsStable constructs with an average chelator-to-antibody ratio of 1.81 were achieved. SDS-PAGE and SE-HPLC showed integrity of CI-8993 was maintained after conjugation; and ELISA indicated no impact of conjugation and radiolabelling on binding to human VISTA. PET imaging and biodistribution in MB49 tumour-bearing huVISTA KI female mice showed specific localisation of [89Zr]Zr-Df-CI-8993 to VISTA in spleen and tumour tissues expressing human VISTA. Specific tumour uptake was also demonstrated in Capan-2 xenografted BALB/c nu/nu mice.ConclusionsWe radiolabelled and validated [89Zr]Zr-Df-CI-8993 for specific binding to huVISTA in vivo. Our results demonstrate that 89Zr-labelled CI-8993 is now suitable for targeting and imaging VISTA expression in human trials.Graphical

Impact of isotype on the mechanism of action of agonist anti-OX40 antibodies in cancer: implications for therapeutic combinations

BackgroundOX40 has been widely studied as a target for immunotherapy with agonist antibodies taken forward into clinical trials for cancer where they are yet to show substantial efficacy. Here, we...

Targeting an Old Foe for Cancer: A Molecular Dynamics Perspective to Unravel the Specific Binding Nature of 2-Methoxy Estradiol to Human β-Tubulin Isotypes.

Microtubules, composed of α- and β-tubulin subunits are crucial for cell division with their dynamic tissue-specificity which is dictated by expression of isotypes. These isotypes differ in carboxy-terminal tails (CTTs), rich in negatively charged acidic residues in addition to the differences in the composition of active site residues. 2-Methoxy estradiol (2-ME) is the first antimicrotubule agent that showed less affinity toward hemopoietic-specific β1 isotype consequently preventing myelosuppression toxicity. The present study focuses on the MD-directed conformational analysis of 2-ME and estimation of its binding affinity in the colchicine binding pocket of various β-tubulin isotypes combined with the α-tubulin isotype, α1B. AlphaFold 2.0 was used to predict the 3D structure of phylogenetically divergent human β-tubulin isotypes in dimer form with α1B. The dimeric complexes were subjected to induced-fit docking with 2-ME. The statistical analysis of docking showed differences in the binding characteristics of 2-ME with different isotypes. The replicas of atom-based molecular dynamic simulations of the best conformation of 2-ME provided insights into the molecular-level details of its binding pattern across the isotypes. Furthermore, the MM/GBSA analyses revealed the specific binding energy profile of 2-ME in β-tubulin isotypes. It also highlighed, 2-ME exhibits the lowest binding affinity toward the β1 isotype as supported by experimental study. The present study may offer useful information for designing next-generation antimicrotubule agents that are more specific and less toxic.

Novel Combretastatin A-4 Analogs-Design, Synthesis, and Antiproliferative and Anti-Tubulin Activity.

Combretastatins isolated from the Combretum caffrum tree belong to a group of closely related stilbenes. They are colchicine binding site inhibitors which disrupt the polymerization process of microtubules in tubulins, causing mitotic arrest. In vitro and in vivo studies have proven that some combretastatins exhibit antitumor properties, and among them, combretastatin A-4 is the most active mitotic inhibitor. In this study, a series of novel combretastatin A-4 analogs containing carboxylic acid, ester, and amide moieties were synthesized and their cytotoxic activity against six tumor cell lines was determined using sulforhodamine B assay. For the most cytotoxic compounds (8 and 20), further studies were performed. These compounds were shown to induce G0/G1 cell cycle arrest in MDA and A549 cells, in a concentration-dependent manner. Moreover, in vitro tubulin polymerization assays showed that both compounds are tubulin polymerization enhancers. Additionally, computational analysis of the binding modes and binding energies of the compounds with respect to the key human tubulin isotypes was performed. We have obtained a satisfactory correlation of the binding energies with the IC50 values when weighted averages of the binding energies accounting for the abundance of tubulin isotypes in specific cancer cell lines were computed.

Abstract 6345: In vitro characterization of a dual antagonistic anti-LILRB2/LILRB4 monoclonal antibody

Abstract Introduction: Myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment are a potential therapeutic target in immune checkpoint cancer therapy, but MDSC-targeted therapies have yet been shown to improve survival. The leukocyte immunoglobulin (Ig)-like receptor B2 is a member of the leukocyte Ig-like receptor (LILR) family and is predominantly expressed on the surface of cells of the myelomonocytic lineage (monocytes, macrophages and dendritic cells). LILRB2 is a negative regulator of myeloid cells and is an immune checkpoint receptor. We chose to generate mAbs against LILRB2 as a potential immunotherapeutic for cancer. Methods: PentaMice® strains (5 WT mouse strains comprising 9 MHC class II haplotypes to maximize Ab diversity) were immunized with human LILRB2. Lymphocytes were fused with a myeloma partner to generate lead monoclonal hybridomas. 24 purified anti-LILRB2 mAbs were generated and evaluated by arrayed surface plasma resonance (SPR, Carterra® LSA® instrument) to assign epitope bins. mAb humanization was performed in silico to maximize T20 “humanness” scores (algorithm developed by Curia). Humanized mAbs were transiently expressed using Curia’s TunaCHO™ platform and tested in in vitro MDSC assays. Autologous co-cultures of magnetic bead enriched-human blood-derived MDSC (HLA-DR-CD14+) model cells and T cells (CD3+) were stimulated with plate-bound anti-CD3/CD28 to induce T cell activation. Cytokine secretion was assessed 4 days later. Results: A total of 384 hybridoma supernatants were selected from the primary screen. 245 were confirmed by ELISA and 178 stained HEK293 cells expressing LILRB2 cells by flow cytometry (FACS). 24 hybridomas were further selected, cloned and sequenced. Five epitope bins were identified via Carterra® LSA® instrument. Two clones were chosen for humanization, designated 6-I11A and 4-O19A and were recombinantly expressed with the human IgG4 isotype. FACS analysis on HEK293 cells expressing either huLILRB2 or huLILRB4 revealed that clone hu4-O19A-IgG4 was specific for huLILRB2. Surprisingly, clone hu6-I11A-IgG4 stained both huLILRB2 and huLILRB4. FACS analysis with hu4-O19A-IgG4 on human MDSCs exhibited an EC50 of 11.9 nM and a KD of 1.5 nM in binding to recombinant huLILRB2 antigen, while hu6-I11A-IgG4 had an EC50 of 3.2 nM and a KD of 0.6 nM. In co-cultures with human MDSCs and T cells, both hu6-I11A-IgG4 and hu4-O19A-IgG4 reversed cytokine suppression by the MDSCs by increasing GM-CSF, IL-10, TNFα, and IL-6, while suppressing IL-13, and had little effect on IL-17A, MIP-3α, IL-4, IL-2 and IFNγ compared to treatment with the isotype control, with hu6-I11A-IgG4 having a greater effect as an antagonist than hu4-O19A-IgG4. Conclusions: We have identified a dual anti-LILRB2/LILRB4 antagonist. Targeting MDSCs with this antibody offers great promise for immunotherapy for cancer as well as its immense potential for synergy with PD-1 blockade. Citation Format: Bruce L. Daugherty, Xiaomei Ge, Christine L. Hsieh, Brian A. Zabel, Seth Lederman. In vitro characterization of a dual antagonistic anti-LILRB2/LILRB4 monoclonal antibody [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 6345.

Photoacoustic imaging of the dynamics of a dye-labeled IgG4 monoclonal antibody in subcutaneous tissue reveals a transient decrease in murine blood oxygenation under anesthesia.

Over 100 monoclonal antibodies have been approved by the U.S. Food and Drug Administration (FDA) for clinical use; however, a paucity of knowledge exists regarding the injection site behavior of these formulated therapeutics, particularly the effect of antibody, formulation, and tissue at the injection site. A deeper understanding of antibody behavior at the injection site, especially on blood oxygenation through imaging, will help design improved versions of the therapeutics for a wide range of diseases. The aim of this research is to understand the dynamics of monoclonal antibodies at the injection site as well as how the antibody itself affects the functional characteristics of the injection site [e.g., blood oxygen saturation ()]. We employed triple-wavelength equipped functional photoacoustic imaging to study the dynamics of dye-labeled and unlabeled monoclonal antibodies at the site of injection in a mouse ear. We injected a near-infrared dye-labeled (and unlabeled) human IgG4 isotype control antibody into the subcutaneous space in mouse ears to analyze the injection site dynamics and quantify molecular movement, as well as its effect on local hemodynamics. We performed pharmacokinetic studies of the antibody in different regions of the mouse body to show that dye labeling does not alter the pharmacokinetic characteristics of the antibody and that mouse ear is a viable model for these initial studies. We explored the movement of the antibody in the interstitial space to show that the bolus area grows by over 24h. We discovered that injection of the antibody transiently reduces the local levels in mice after prolonged anesthesia without affecting the total hemoglobin content and oxygen extraction fraction. This finding on local oxygen saturation opens a new avenue of study on the functional effects of monoclonal antibody injections. We also show the suitability of the mouse ear model to study antibody dynamics through high-resolution imaging techniques. We quantified the movement of antibodies at the injection site caused by the interstitial fluid, which could be helpful for designing antibodies with tailored absorption speeds in the future.

Transient Anti-Interferon Auto Antibodies in the Airway Are Associated with Recovery in Mild COVID-19

Pre-existing blood anti-interferon autoantibodies have been associated with susceptibility to severe COVID-19. However, nothing is known about the autoantibody response in the airway mucosa of mild to severe COVID-19 patients. We profiled longitudinal samples collected from the airway and matching blood of more than 100 donors over >18 months, spanning from mild COVID-19 to hospitalized patients in the ICU. We found that transiently induced anti-type I interferon autoantibodies in the nasal mucosa are a common feature and are associated with recovery from mild and moderate COVID-19. To quantify the breadth of antibody response in blood and the airway site of infection, we developed flowBEAT, a novel flow cytometry-based assay capable of simultaneously measuring all 8 human antibody isotypes against 22 SARS-CoV-2 structural and non-structural antigens, including autoantigens. We identified the induction of nasal anti-type I interferon autoantibodies within the first two weeks of infection, waning with disease recovery. Notably, there was an inverse association between nasal and blood anti-interferon responses, while nasal anti-interferon was linked to recovery in cases of mild/moderate disease. In contrast, near-ubiquitous anti-interferon in the endotracheal aspirates of critical cases correlated with heightened airway hyper-inflammation. In both blood and mucosa, anti-interferon positively correlated with a broad anti-viral antibody response, including non-structural proteins. While nasal anti-interferon was universally transient, we identified a group of donors sustaining long-term blood anti-interferon autoantibodies who may be at risk for a more severe subsequent viral infection. Within our longitudinal cohort, we assessed 500 infected-recovered and vaccinated samples and showed that nasal anti-SARS-CoV-2 antibodies are sustained over 18 months by repeated vaccination and boosting. We identified isotype signatures associated with disease severity and an autoantibody signature predictive of vaccine non-response. Thus, our findings emphasize the importance of continued vaccination/boosting to sustain nasal immunity and help prevent ongoing community spread. More importantly, our studies reveal predictive blood markers of protective nasal immunity and highlight a protective role for nasal anti-interferon autoantibodies in the immunopathology of COVID-19.

Expanding the Anti-Phl p 7 Antibody Toolkit: An Anti-Idiotype Nanobody Inhibitor.

We have previously produced a toolkit of antibodies, comprising recombinant human antibodies of all but one of the human isotypes, directed against the polcalcin family antigen Phl p 7. In this work, we complete the toolkit of human antibody isotypes with the IgD version of the anti-Phl p 7 monoclonal antibody. We also raised a set of nanobodies against the IgD anti-Phl p 7 antibody and identify and characterize one paratope-specific nanobody. This nanobody also binds to the IgE isotype of this antibody, which shares the same idiotype, and orthosterically inhibits the interaction with Phl p 7. The 2.1 Å resolution X-ray crystal structure of the nanobody in complex with the IgD Fab is described.

A subset of type-II collagen-binding antibodies prevents experimental arthritis by inhibiting FCGR3 signaling in neutrophils

Rheumatoid arthritis (RA) involves several classes of pathogenic autoantibodies, some of which react with type-II collagen (COL2) in articular cartilage. We previously described a subset of COL2 antibodies targeting the F4 epitope (ERGLKGHRGFT) that could be regulatory. Here, using phage display, we developed recombinant antibodies against this epitope and examined the underlying mechanism of action. One of these antibodies, R69-4, protected against cartilage antibody- and collagen-induced arthritis in mice, but not autoimmune disease models independent of arthritogenic autoantibodies. R69-4 was further shown to cross-react with a large range of proteins within the inflamed synovial fluid, such as the complement protein C1q. Complexed R69-4 inhibited neutrophil FCGR3 signaling, thereby impairing downstream IL-1β secretion and neutrophil self-orchestrated recruitment. Likewise, human isotypes of R69-4 protected against arthritis with comparable efficiency. We conclude that R69-4 abrogates autoantibody-mediated arthritis mainly by hindering FCGR3 signaling, highlighting its potential clinical utility in acute RA.

The influence of variable-heavy chain families on IgG2, 3, 4, FcγRs and B-cell superantigens protein G and L binding using biolayer interferometry.

As the most abundant immunoglobulin in blood and the most common human isotype used for therapeutic monoclonal antibodies, the engagement and activation of its Fc receptors by IgGs are crucial for antibody function. Assumed to be relatively constant within subtypes, recent studies reveal that antibody variable regions exert distal effects of modulating antibody-receptor interactions on antibody isotypes. These variable (V)-region distal effects are also expected for the IgG subtypes. With an in-depth understanding of the V-region effects, researchers can make a more informed antibody engineering approach and antibody purification strategy accounting for the functions of microbial immune evasion . In this study, we created a panel of IgG2/IgG3/IgG4 antibodies by changing the VH family (VH1-7) frameworks while retaining the complementary determining regions of pertumuzab and measured their interactions with FcγRIa, FcγRIIaH167, FcγRIIaR167, FcγRIIb/c, FcγRIIIaF176, FcγRIIIaV176, FcγRIIIbNA1 and FcγRIIIbNA2 receptors alongside B-cell superantigens Protein L and G using biolayer interferometry. The panel of 21 IgGs demonstrated that the VH frameworks influenced receptor binding sites on the constant region in a non-canonical manner. However, there was minimal influence on the binding of bacterial B-cell superantigens Proteins L and Protein G on the IgGs, showing their robustness against V-region effects. These results demonstrate the role of V-regions during the humanization of therapeutic antibodies that can influence FcR-dependent immune responses while retaining binding by bacterial B-cell superantigens for antibody purification. These in vitro measurements provide a clue to detailed antibody engineering and understanding of antibody superantigen functions that would be relevant with in vivo validation.

Integrated regulation of tubulin tyrosination and microtubule stability by human α-tubulin isotypes

Tubulin isotypes are critical for the functions of cellular microtubules, which exhibit different stability and harbor various post-translational modifications. However, how tubulin isotypes determine the activities of regulators for microtubule stability and modifications remains unknown. Here, we show that human α4A-tubulin, a conserved genetically detyrosinated α-tubulin isotype, is a poor substrate for enzymatic tyrosination. To examine the stability of microtubules reconstituted with defined tubulin compositions, we develop a strategy to site-specifically label recombinant human tubulin for single-molecule TIRF microscopy-based invitro assays. The incorporation of α4A-tubulin into the microtubule lattice stabilizes the polymers from passive and MCAK-stimulated depolymerization. Further characterization reveals that the compositions of α-tubulin isotypes and tyrosination/detyrosination states allow graded control for the microtubule binding and the depolymerization activities of MCAK. Together, our results uncover the tubulin isotype-dependent enzyme activity for an integrated regulation of α-tubulin tyrosination/detyrosination states and microtubule stability, two well-correlated features of cellular microtubules.

Generation of recombinant and chickenized scFv versions of an anti-kinesin monoclonal antibody H2.

Kinesin-1, a motor protein composed of the kinesin heavy chain (KHC) and the kinesin light chain (KLC), is essential for proper cellular morphogenesis and function. A monoclonal antibody (mAb) called H2 recognizes the KHC in a broad range of species and is one of the most widely used mAbs in cytoskeletal motor research. Here, we present vectors that express recombinant H2 in mammalian cells. We show the recombinant H2 performs as well as the hybridoma-derived H2 in both western blotting and immunofluorescence assays. Additionally, the recombinant H2 can detect all three human KHC isotypes (KIF5A, KIF5B, and KIF5C) and amyotrophic lateral sclerosis-associated KIF5A aggregates in cells. In addition, we developed a chickenized version of the H2 mAb's single chain variable fragment, which can be used in immunofluorescence microscopy and expands the potential applications of H2. Overall, our results demonstrate that recombinant H2 is a useful tool for studying the functions of KHCs.

Anti-double stranded DNA antibodies: Electrochemical isotyping in autoimmune and neurological diseases

This work reports the first amperometric biosensor for the simultaneous determination of the single or total content of the most relevant human immunoglobulin isotypes (hIgs) of anti-dsDNA antibodies, dsDNA-hIgG, dsDNA-hIgM, dsDNA-hIgA and dsDNA-three hIgs, which are considered relevant biomarkers in prevalent autoimmune diseases such as systemic lupus erythematosus (SLE) as well as of interest in neurodegenerative diseases such as Alzheimer's disease (AD). The bioplatform involves the use of neutravidin-functionalized magnetic microparticles (NA-MBs) modified with a laboratory-prepared biotinylated human double-stranded DNA (b-dsDNA) for the efficient capture of specific autoantibodies that are enzymatically labeled with horseradish peroxidase (HRP) enzyme using specific secondary antibodies for each isotype or a mixture of secondary antibodies for the total content of the three isotypes. Transduction was performed by amperometry (−0.20 V vs. the Ag pseudo-reference electrode) using the H2O2/hydroquinone (HQ) system after trapping the resulting magnetic bioconjugates on each of the four working electrodes of a disposable quadruple transduction platform (SP4CEs). The bioplatform demonstrated attractive operational characteristics for clinical application and was employed to determine the individual or total hIgs classes in serum from healthy individuals and from patients diagnosed with SLE and AD. The target concentrations in AD patients are provided for the first time in this work. In addition, the results for SLE patients and control individuals agree with those obtained by applying ELISA tests as well as with the clinical ranges reported by other authors, using individual detection methodologies restricted to centralized settings or clinical laboratories.

In Silico Exploration of Microtubule Agent Griseofulvin and Its Derivatives Interactions with Different Human β-Tubulin Isotypes

Tubulin isotypes are known to regulate microtubule stability and dynamics, as well as to play a role in the development of resistance to microtubule-targeted cancer drugs. Griseofulvin is known to disrupt cell microtubule dynamics and cause cell death in cancer cells through binding to tubulin protein at the taxol site. However, the detailed binding mode involved molecular interactions, and binding affinities with different human β-tubulin isotypes are not well understood. Here, the binding affinities of human β-tubulin isotypes with griseofulvin and its derivatives were investigated using molecular docking, molecular dynamics simulation, and binding energy calculations. Multiple sequence analysis shows that the amino acid sequences are different in the griseofulvin binding pocket of βI isotypes. However, no differences were observed at the griseofulvin binding pocket of other β-tubulin isotypes. Our molecular docking results show the favorable interaction and significant affinity of griseofulvin and its derivatives toward human β-tubulin isotypes. Further, molecular dynamics simulation results show the structural stability of most β-tubulin isotypes upon binding to the G1 derivative. Taxol is an effective drug in breast cancer, but resistance to it is known. Modern anticancer treatments use a combination of multiple drugs to alleviate the problem of cancer cells resistance to chemotherapy. Our study provides a significant understanding of the involved molecular interactions of griseofulvin and its derivatives with β-tubulin isotypes, which may help to design potent griseofulvin analogues for specific tubulin isotypes in multidrug-resistance cancer cells in future.

A Molecular Docking Study Reveals That Short Peptides Induce Conformational Changes in the Structure of Human Tubulin Isotypes αβI, αβII, αβIII and αβIV.

Microtubules are cylindrical protein polymers assembled in the cytoplasm of all eukaryotic cells by polymerization of aβ tubulin dimers, which are involved in cell division, migration, signaling, and intracellular traffic. These functions make them essential in the proliferation of cancerous cells and metastases. Tubulin has been the molecular target of many anticancer drugs because of its crucial role in the cell proliferation process. By developing drug resistance, tumor cells severely limit the successful outcomes of cancer chemotherapy. Hence, overcoming drug resistance motivates the design of new anticancer therapeutics. Here, we retrieve short peptides obtained from the data repository of antimicrobial peptides (DRAMP) and report on the computational screening of their predicted tertiary structures for the ability to inhibit tubulin polymerization using multiple combinatorial docking programs, namely PATCHDOCK, FIREDOCK, and ClusPro. The interaction visualizations show that all the best peptides from the docking analysis bind to the interface residues of the tubulin isoforms αβl, αβll, αβlll, and αβlV, respectively. The docking studies were further confirmed by a molecular dynamics simulation, in which the computed root-mean-square deviation (RMSD), and root-mean-square fluctuation (RMSF), verified the stable nature of the peptide-tubulin complexes. Physiochemical toxicity and allergenicity studies were also performed. This present study suggests that these identified anticancer peptide molecules might destabilize the tubulin polymerization process and hence can be suitable candidates for novel drug development. It is concluded that wet-lab experiments are needed to validate these findings.

The synergistic effects of the constant region and variable heavy chain families of multimeric immunoglobulin M on its interaction with Fc-mu receptor and antigen

Aim: As the primary response antibody with increasing use as a therapeutic immunoglobulin (Ig) format, IgM is also the largest antibody structure among the five major human isotypes. Spontaneously formed pentamers and hexamers of IgM have avidity effects that could compensate for weaker interactions in monomeric Igs. However, this advantage is counterbalanced by potential steric clashes when binding to multiple large antigens. Recent findings have challenged the expected canonical independence of Fc receptor (FcR) binding at the heavy chain constant (C)-region where the heavy chain C-region isotypes affected antigen binding at the variable (V)-regions, and the variable heavy (VH) families of the V-region affected FcR engagement at the antibody C-regions. With such effects found on other Ig isotypes, IgM candidates need to be investigated with regards to such effects, especially when considering its natural oligomerisation at the C-region that can amplify or modulate such allosteric effects. Methods: Through a panel of 14 recombinant complementarity determining regions (CDRs)-grafted trastuzumab and pertuzumab VH1-7 IgMs subjected to bio-layer interferometry measurements, the interactions with the antigen human epidermal growth factor receptor 2 (Her2), Fc-mu receptor (FcµR), and superantigen Protein L (PpL) were investigated. Results: Significant effects from the V-regions to mitigate FcµR binding and the IgM C-region bidirectional effect modulating Her2 antigen engagements at the V-regions were found. Additional modulatory effects from superantigen PpL binding on the V-region of the kappa chain (Vκ) mitigating antigen binding were also found, revealing possible novel mechanisms of antibody superantigens that can be moderated by the antibody VH frameworks.

Hinge disulfides in human IgG2 CD40 antibodies modulate receptor signaling by regulation of conformation and flexibility.

Antibodies protect from infection, underpin successful vaccines and elicit therapeutic responses in otherwise untreatable cancers and autoimmune conditions. The human IgG2 isotype displays a unique capacity to undergo disulfide shuffling in the hinge region, leading to modulation of its ability to drive target receptor signaling (agonism) in a variety of important immune receptors, through hitherto unexplained molecular mechanisms. To address the underlying process and reveal how hinge disulfide orientation affects agonistic activity, we generated a series of cysteine to serine exchange variants in the hinge region of the clinically relevant monoclonal antibody ChiLob7/4, directed against the key immune receptor CD40. We report how agonistic activity varies with disulfide pattern and is afforded by the presence of a disulfide crossover between F(ab) arms in the agonistic forms, independently of epitope, as observed in the determined crystallographic structures. This structural "switch" affects directly on antibody conformation and flexibility. Small-angle x-ray scattering and ensemble modeling demonstrated that the least flexible variants adopt the fewest conformations and evoke the highest levels of receptor agonism. This covalent change may be amenable for broad implementation to modulate receptor signaling in an epitope-independent manner in future therapeutics.

Abstract 3428: NI-2901, a CD47xPD-L1 bispecific antibody for dual immune checkpoint blockade with fine-tuned affinity to reduce erythrocyte binding and improve biodistribution

Abstract Blocking the CD47/SIRPα checkpoint has recently emerged as an effective approach to mobilize the myeloid cell compartment and to improve antitumor responses in the clinic. Preclinical models have demonstrated the synergistic benefit of combined CD47/SIRPα and PD-1/PD-L1 blockade. Combinations of monoclonal antibodies (mAbs) targeting these two checkpoint pathways are being explored in the clinic. CD47xPD-L1 bispecific antibodies (bsAbs) stand as an attractive alternative to mAb combinations, even more so as they provide a potential solution to improve the pharmacokinetic profile and safety issues faced by CD47 targeted-mAbs and SIRPα-Fc fusion proteins. CD47xPD-L1 bsAbs are expected to preferentially inhibit CD47 on PD-L1 expressing cells, displaying improved safety and pharmacokinetics, but also superior tumor microenvironment targeting capabilities. With the objective of finding the optimal CD47xPD-L1 bsAb, an array of bispecific antibodies (bsAbs) was generated associating a high affinity PD-L1 arm to CD47 arms with varying affinities. The CD47xPD-L1 bsAbs of human IgG4 isotype were generated using our fully human κλ body antibody platform. The candidate molecules were screened for binding and receptor-blocking activity and tested for their capacity to enhance T-cell activation and phagocytosis of tumor cells in the presence of anti-HER-2 mAb, trastuzumab. Selected bsAbs were also evaluated in a xenograft mouse model. The CD47xPD-L1 bsAbs demonstrated an effective blockade of the PD-1/PD-L1 interaction, being able to induce T-cell activation in vitro similar to the anti-PD-L1 clinical benchmark, atezolizumab. Consistent with their CD47 affinities, the bsAbs showed varying levels of CD47 blockade on PD-L1-negative cells and a low binding capacity to red blood cells. Nonetheless, trastuzumab-mediated phagocytosis of tumor cells expressing low levels of PD-L1 could be significantly enhanced by these bsAbs, confirming the PD-L1-independent activity of the CD47 blocking arms. The latter findings were corroborated in vivo using PD-L1-negative Raji cells in a xenograft mouse model. Selected bsAbs will be now tested for tolerability and pharmacokinetic profiles in human-CD47/human-SIRPα transgenic mice. Lead candidate(s) will be evaluated further for PK and safety attributes in non-human primates in early Q1, 2022. Citation Format: Xavier Chauchet, Sébastien Calloud, Margaux Legrand, Laura Cons, Laurence Chatel, Pauline Lloveras, Coline Burnet-Merlin, Louis Hellequin, Nicolas Bosson, Pauline Malinge, Nicolas Pleche, Jérémie Bourguignon, Guillemette Pontini, Christophe Guillamo, Ulla Ravn, Valéry Moine, Bruno Daubeuf, Yves Poitevin, Giovanni Magistrelli, Limin Shang, Walter Ferlin, Krzysztof Masternak. NI-2901, a CD47xPD-L1 bispecific antibody for dual immune checkpoint blockade with fine-tuned affinity to reduce erythrocyte binding and improve biodistribution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3428.

Antibody-Invertase Fusion Protein Enables Quantitative Detection of SARS-CoV-2 Antibodies Using Widely Available Glucometers.

Rapid diagnostics that can accurately inform patients of disease risk and protection are critical to mitigating the spread of the current COVID-19 pandemic and future infectious disease outbreaks. To be effective, such diagnostics must rely on simple, cost-effective, and widely available equipment and should be compatible with existing telehealth infrastructure to facilitate data access and remote care. Commercial glucometers are an established detection technology that can overcome the cost, time, and trained personnel requirements of current benchtop-based antibody serology assays when paired with reporter molecules that catalyze glucose conversion. To this end, we developed an enzymatic reporter that, when bound to disease-specific patient antibodies, produces glucose in proportion to the level of antibodies present in the patient sample. Although a straightforward concept, the coupling of enzymatic reporters to secondary antibodies or antigens often results in low yields, indeterminant stoichiometry, reduced target binding, and poor catalytic efficiency. Our enzymatic reporter is a novel fusion protein that comprises an antihuman immunoglobulin G (IgG) antibody genetically fused to two invertase molecules. The resulting fusion protein retains the binding affinity and catalytic activity of the constituent proteins and serves as an accurate reporter for immunoassays. Using this fusion, we demonstrate quantitative glucometer-based measurement of anti-SARS-CoV-2 spike protein antibodies in blinded clinical sample training sets. Our results demonstrate the ability to detect SARS-CoV-2-specific IgGs in patient serum with precise agreement to benchmark commercial immunoassays. Because our fusion protein binds all human IgG isotypes, it represents a versatile tool for detection of disease-specific antibodies in a broad range of biomedical applications.