Editor’s Choice Articles

Pemphigus vulgaris (PV) is an IgG autoantibody-mediated, potentially fatal mucocutaneous disease manifested by progressive non-healing erosions and blisters. Beyond acting to inhibit adhesion molecules, PVIgGs elicit a unique process of programmed cell death and detachment of epidermal keratinocytes termed apoptolysis. Mitochondrial damage by antimitochondrial antibodies (AMA) has proven to be a critical link in this process. AMA act synergistically with other autoantibodies in the pathogenesis of PV. Importantly, absorption of AMA inhibits the ability of PVIgGs to induce blisters. Pharmacologic agents that protect mitochondrial function offer a new targeted approach to treating this severe immunoblistering disease. Full article

Toxoplasma gondii, an obligate intracellular protozoan parasite, is the causative agent of one of the most prevalent zoonoses worldwide. T. gondii infection is extremely important from a medical point of view, especially for pregnant women, newborns with congenital infections, and immunocompromised individuals. Thus, an accurate and proper diagnosis of this infection is essential. Among the available diagnostic tests, serology is commonly used. However, traditional serological techniques have certain limitations in evaluating the duration of T. gondii infection, which is problematic, especially for pregnant women. Avidity of T. gondii-specific IgG antibodies seems to be a significant tool for discrimination between recent and distant infections. This article describes the problem of diagnosis of T. gondii infection, with regard to IgG avidity tests. The IgG avidity test is a useful serological indicator of toxoplasmosis, which in many cases can confirm or exclude the active form of the disease. IgG antibodies produced in the recent primary T. gondii infection are of low avidity while IgG antibodies with high avidity are detected in the chronic phase of infection. Furthermore, this paper presents important topics of current research that concern the usage of parasite recombinant antigens that may improve the performance of IgG avidity tests. Full article

The accurate and efficient calculation of protein-protein binding affinities is an essential component in antibody and antigen design and optimization, and in computer modeling of antibody affinity maturation. Such calculations remain challenging despite advances in computer hardware and algorithms, primarily because proteins are flexible molecules, and thus, require explicit or implicit incorporation of multiple conformational states into the computational procedure. The astronomical size of the amino acid sequence space further compounds the challenge by requiring predictions to be computed within a short time so that many sequence variants can be tested. In this study, we compare three classes of methods for antibody/antigen (Ab/Ag) binding affinity calculations: (i) a method that relies on the physical separation of the Ab/Ag complex in equilibrium molecular dynamics (MD) simulations, (ii) a collection of 18 scoring functions that act on an ensemble of structures created using homology modeling software, and (iii) methods based on the molecular mechanics-generalized Born surface area (MM-GBSA) energy decomposition, in which the individual contributions of the energy terms are scaled to optimize agreement with the experiment. When applied to a set of 49 antibody mutations in two Ab/HIV gp120 complexes, all of the methods are found to have modest accuracy, with the highest Pearson correlations reaching about 0.6. In particular, the most computationally intensive method, i.e., MD simulation, did not outperform several scoring functions. The optimized energy decomposition methods provided marginally higher accuracy, but at the expense of requiring experimental data for parametrization. Within each method class, we examined the effect of the number of independent computational replicates, i.e., modeled structures or reinitialized MD simulations, on the prediction accuracy. We suggest using about ten modeled structures for scoring methods, and about five simulation replicates for MD simulations as a rule of thumb for obtaining reasonable convergence. We anticipate that our study will be a useful resource for practitioners working to incorporate binding affinity calculations within their protein design and optimization process. Full article

Tumor cells are characterized by overexpressed tumor-associated antigens or mutated neoantigens, which are expressed on the cell surface or intracellularly. One strategy of cancer immunotherapy is to target cell-surface-expressed tumor-associated antigens (TAAs) with therapeutic antibodies. For targeting TAAs or neoantigens, adoptive T-cell therapies with activated autologous T cells from cancer patients transduced with novel recombinant TCRs or chimeric antigen receptors have been successfully applied. Many TAAs and most neoantigens are expressed in the cytoplasm or nucleus of tumor cells. As alternative to adoptive T-cell therapy, the mRNA of intracellular tumor antigens can be depleted by RNAi, the corresponding genes or proteins deleted by CRISPR-Cas or inactivated by kinase inhibitors or by intrabodies, respectively. Intrabodies are suitable to knockdown TAAs and neoantigens without off-target effects. RNA sequencing and proteome analysis of single tumor cells combined with computational methods is bringing forward the identification of new neoantigens for the selection of anti-cancer intrabodies, which can be easily performed using phage display antibody repertoires. For specifically delivering intrabodies into tumor cells, the usage of new capsid-modified adeno-associated viruses and lipid nanoparticles coupled with specific ligands to cell surface receptors can be used and might bring cancer intrabodies into the clinic. Full article

Pancreatic cancer is an aggressive cancer with a dismal prognosis. This is due to the difficulty to detect the disease at an early and curable stage. In addition, only limited treatment options are available, and they are confronted by mechanisms of resistance. Monoclonal antibody (mAb) molecules are highly specific biologics that can be directly used as a blocking agent or modified to deliver a drug payload depending on the desired outcome. They are widely used to target extracellular proteins, but they can also be employed to inhibit intracellular proteins, such as oncoproteins. While mAbs are a class of therapeutics that have been successfully employed to treat many cancers, they have shown only limited efficacy in pancreatic cancer as a monotherapy so far. In this review, we will discuss the challenges, opportunities and hopes to use mAbs for pancreatic cancer treatment, diagnostics and imagery. Full article

Therapeutic monoclonal antibodies (mAbs) are an emerging and very active frontier in clinical oncology, with hundred molecules currently in use or being tested. These treatments have already revolutionized clinical outcomes in both solid and hematological malignancies. However, identifying patients who are most likely to benefit from mAbs treatment is currently challenging and limiting the impact of such therapies. To overcome this issue, and to fulfill the expectations of mAbs therapies, it is urgently required to develop proper culture models capable of faithfully reproducing the interactions between tumor and its surrounding native microenvironment (TME). Three-dimensional (3D) models which allow the assessment of the impact of drugs on tumors within its TME in a patient-specific context are promising avenues to progressively fill the gap between conventional 2D cultures and animal models, substantially contributing to the achievement of personalized medicine. This review aims to give a brief overview of the currently available 3D models, together with their specific exploitation for therapeutic mAbs testing, underlying advantages and current limitations to a broader use in preclinical oncology. Full article

There is increasing interest in the clinical implications and immunology of trogocytosis, a process in which the receptors on acceptor cells remove and internalize cognate ligands from donor cells. We have reported that this phenomenon occurs in cancer immunotherapy, in which cells that express FcγR remove and internalize CD20 and bound mAbs from malignant B cells. This process can be generalized to include other reactions including the immune adherence phenomenon and antibody-induced immunosuppression. We discuss in detail FcγR-mediated trogocytosis and the evidence supporting a proposed predominant role for liver sinusoidal endothelial cells via the action of the inhibitory receptor FcγRIIb2. We describe experiments to test the validity of this hypothesis. The elucidation of the details of FcγR-mediated trogocytosis has the potential to allow for the development of novel therapies that can potentially block or enhance this reaction, depending upon whether the process leads to unfavorable or positive biological effects. Full article

Bullous pemphigoid (BP) is a subepidermal autoimmune blistering disease predominantly affecting elderly patients and carries significant morbidity and mortality. Patients typically suffer from severe itch with eczematous lesions, urticarial plaques, and/or tense blisters. BP is characterized by the presence of circulating autoantibodies against two components of the hemidesmosome, BP180 and BP230. The transmembrane BP180, also known as type XVII collagen or BPAG2, represents the primary pathogenic autoantigen in BP, whereas the intracellular BP230 autoantigen is thought to play a minor role in disease pathogenesis. Although experimental data exist suggesting that anti-BP230 antibodies are secondarily formed following initial tissue damage mediated by antibodies targeting extracellular antigenic regions of BP180, there is emerging evidence that anti-BP230 IgG autoantibodies alone directly contribute to tissue damage. It has been further claimed that a subset of patients has a milder variant of BP driven solely by anti-BP230 autoantibodies. Furthermore, the presence of anti-BP230 autoantibodies might correlate with distinct clinical features. This review summarizes the current understanding of the role of BP230 and anti-BP230 antibodies in BP pathogenesis. Full article

The epithelial cell adhesion molecule (EpCAM) is a cell surface glycoprotein, which is widely expressed on normal and cancer cells. EpCAM is involved in cell adhesion, proliferation, survival, stemness, and tumorigenesis. Therefore, EpCAM is thought to be a promising target for cancer diagnosis and therapy. In this study, we established anti-EpCAM monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening (CBIS) method. We characterized them using flow cytometry, Western blotting, and immunohistochemistry. One of the established recombinant anti-EpCAM mAbs, recEpMab-37 (mouse IgG₁, kappa), reacted with EpCAM-overexpressed Chinese hamster ovary-K1 cells (CHO/EpCAM) or a colorectal carcinoma cell line (Caco-2). In contrast, recEpMab-37 did not react with EpCAM-knocked out Caco-2 cells. The K_D of recEpMab-37 for CHO/EpCAM and Caco-2 was 2.0 × 10⁻⁸ M and 3.2 × 10⁻⁸ M, respectively. We observed that EpCAM amino acids between 144 to 164 are involved in recEpMab-37 binding. In Western blot analysis, recEpMab-37 detected the EpCAM of CHO/EpCAM and Caco-2 cells. Furthermore, recEpMab-37 could stain formalin-fixed paraffin-embedded colorectal carcinoma tissues by immunohistochemistry. Taken together, recEpMab-37, established by the CBIS method, is useful for detecting EpCAM in various applications. Full article

Introduction: We compared the early total spike antibody (S-Ab) and neutralizing antibody (N-Ab) responses to two vaccines. Methods: We studied 96 Pfizer and 34 Sinovac vaccinees over a 14-month period from January 2021 to February 2022. All vaccinees received three doses of one type of vaccine. Antibody levels (Roche Elecsys total S-Ab and the Snibe N-Ab) were tested 10 days after the first dose, 20 days after the second dose, and 20 days after the booster dose. Results: At all time points, the mRNA vaccine generated higher S-Ab and N-Ab responses than the inactivated virus vaccine (S-Ab: first dose 2.48 vs. 0.4 BAU/mL, second dose 2174 vs. 98 BAU/mL, third dose 15,004 vs. 525 BAU/mL; N-Ab: first dose 0.05 vs. 0.02 µg/mL, second dose 3.48 vs. 0.38 µg/mL, third dose 19.8 vs. 0.89 µg/mL). mRNA vaccine recipients had a 6.2/22.2/28.6-fold higher S-Ab and 2.5/9.2/22.2-fold higher N-Ab response than inactivated virus vaccine recipients after the first/second/third inoculations, respectively. Mann–Whitney U analysis confirmed the significant difference in S-Ab and N-Ab titers between vaccination groups at each time point. Conclusions: The mRNA vaccines generated a more robust S-Ab and N-Ab response than the inactivated virus vaccine at all time points after the first, second, and third vaccinations. Full article

In this study, we review the agarose native gel electrophoresis that separates proteins and macromolecular complexes in their native state and transfer of the separated proteins from the agarose gel to membranes by contact blotting which retains the native state of these structures. Green fluorescent protein showed functional state both on agarose gel and blotted membrane. Based on the combined procedures, we discovered conformation-specific monoclonal antibodies against PLXDC2 and SARS-CoV-2 spike protein. Full article

Bispecific T cell receptor (TCR)-based molecules capable of redirecting and activating T cells towards tumor cells represent a novel and promising class of biotherapeutics for the treatment of cancer. Usage of TCRs allows for targeting of intracellularly expressed and highly selective cancer antigens, but also requires a complex maturation process to increase the naturally low affinity and stability of TCRs. Even though TCR domains can be matured via phage and yeast display, these techniques share the disadvantages of non-human glycosylation patterns and the need for a later reformatting into the final bispecific format. Here, we describe the development and application of a Chinese Hamster Ovary (CHO) display for affinity engineering of TCRs in the context of the final bispecific TCR format. The recombinase-mediated cassette exchange (RCME)-based system allows for stable, single-copy integration of bispecific TCR molecules with high efficiency into a defined genetic locus of CHO cells. We used the system to isolate affinity-increased variants of bispecific T cell engaging receptor (TCER) molecules from a library encoding different CDR variants of a model TCR targeting preferentially expressed antigen in melanoma (PRAME). When expressed as a soluble protein, the selected TCER molecules exhibited strong reactivity against PRAME-positive tumor cells associated with a pronounced cytokine release from activated T cells. The obtained data support the usage of the CHO display-based maturation system for TCR affinity maturation in the context of the final bispecific TCER format. Full article

Immunotherapy has revolutionized the care of cancer patients. A diverse set of strategies to overcome cancer immunosuppression and enhance the tumor-directed immune response are in clinical use, but have not achieved transformative benefits for brain tumor patients. Adoptive cell therapies, which employ a patient’s own immune cells to generate directed anti-tumor activity, are emerging technologies that hold promise to improve the treatment of primary brain tumors in children and adults. Here, we review recent advances in chimeric antigen receptor (CAR) T-cell therapies for the treatment of aggressive primary brain tumors, including glioblastoma and diffuse midline glioma, H3 K27M-mutant. We highlight current approaches, discuss encouraging investigational data, and describe key challenges in the development and implementation of these types of therapies in the neuro-oncology setting. Full article

Precision-cut tumor slices (PCTS) have recently emerged as important ex vivo human tumor models, offering the opportunity to study individual patient responses to targeted immunotherapies, including CAR-T cell therapies. In this review, an outline of different human tumor models available in laboratory settings is provided, with a focus on the unique characteristics of PCTS. Standard PCTS generation and maintenance procedures are outlined, followed by an in-depth overview of PCTS utilization in preclinical research aiming to better understand the unique functional characteristics of cytotoxic T cells within human tumors. Furthermore, recent studies using PCTS as an ex vivo model for predicting patient responses to immunotherapies and other targeted therapies against solid tumors are thoroughly presented. Finally, the advantages and limitations of the PCTS models are discussed. PCTS are expected to gain momentum and be fully utilized as a significant tool towards better patient stratification and personalized medicine. Full article

The newly identified strain of the Coronaviridae family called severe acute respiratory syndrome (SARS-CoV-2) recently became the most significant health threat for adults and children. Some main predictors of severe clinical course in patients with SARS-CoV-2 infection are age and concomitant health conditions. Therefore, the proper evaluation of SARS-CoV-2-specific immunity is urgently required to understand and predict the spectrum of possible clinical phenotypes and recommend vaccination options and regimens in children. Furthermore, it is critical to characterize the nature of SARS-CoV-2-specific immune responses in children following asymptomatic infection and COVID-19 and other related conditions such as multisystem inflammatory syndrome (MIS-C), para-infectious and late postinfectious consequences. Recent studies involving children revealed a variety of cytokines, T cells and antibody responses in the pathogenesis of the disease. Moreover, different clinical scenarios in children were observed-asymptomatic seroprevalence, acute SARS-CoV-2 infection, and rarely severe COVID-19 with typical cytokine storm, MIS-C, long COVID-19, etc. Therefore, to gain a better clinical view, adequate diagnostic criteria and treatment algorithms, it is essential to create a realistic picture of the immunological puzzle of SARS-CoV-2 infection in different age groups. Finally, it was demonstrated that children may exert a potent and prolonged adaptive anti-SARS-CoV-2 immune response, with significant cross-reactions against other human Corona Viruses, that might contribute to disease sparing effect in this age range. However, the immunopathology of the virus has to be elucidated first. Full article

In this study, we used sodium chloride (NaCl) to extensively modulate non-specific protein-protein interactions (PPI) of a humanized anti-streptavidin monoclonal antibody class 2 molecule (ASA-IgG2). The changes in PPI with varying NaCl (C_NaCl) and monoclonal antibody (mAb) concentration (C_mAb) were assessed using the diffusion interaction parameter k_D and second virial coefficient B₂₂ measured from solutions with low to moderate C_mAb. The effective structure factor S(q)_eff measured from concentrated mAb solutions using small-angle X-ray and neutron scattering (SAXS/SANS) was also used to characterize the PPI. Our results found that the nature of net PPI changed not only with C_NaCl, but also with increasing C_mAb. As a result, parameters measured from dilute and concentrated mAb samples could lead to different predictions on the stability of mAb formulations. We also compared experimentally determined viscosity results with those predicted from interaction parameters, including k_D and S(q)_eff. The lack of a clear correlation between interaction parameters and measured viscosity values indicates that the relationship between viscosity and PPI is concentration-dependent. Collectively, the behavior of flexible mAb molecules in concentrated solutions may not be correctly predicted using models where proteins are considered to be uniform colloid particles defined by parameters derived from low C_mAb. Full article

Botulinum neurotoxin type E (BoNT/E), the fastest acting toxin of all BoNTs, cleaves the 25 kDa synaptosomal-associated protein (SNAP-25) in motor neurons, leading to flaccid paralysis. The specific detection and quantification of the BoNT/E-cleaved SNAP-25 neoepitope can facilitate the development of cell-based assays for the characterization of anti-BoNT/E antibody preparations. In order to isolate highly specific monoclonal antibodies suitable for the in vitro immuno-detection of the exposed neoepitope, mice and rabbits were immunized with an eight amino acid peptide composed of the C-terminus of the cleaved SNAP-25. The immunized rabbits developed a specific and robust polyclonal antibody response, whereas the immunized mice mostly demonstrated a weak antibody response that could not discriminate between the two forms of SNAP-25. An immune scFv phage-display library was constructed from the immunized rabbits and a panel of antibodies was isolated. The sequence alignment of the isolated clones revealed high similarity between both heavy and light chains with exceptionally short HCDR3 sequences. A chimeric scFv-Fc antibody was further expressed and characterized, exhibiting a selective, ultra-high affinity (pM) towards the SNAP-25 neoepitope. Moreover, this antibody enabled the sensitive detection of cleaved SNAP-25 in BoNT/E treated SiMa cells with no cross reactivity with the intact SNAP-25. Thus, by applying an immunization and selection procedure, we have isolated a novel, specific and high-affinity antibody against the BoNT/E-derived SNAP-25 neoepitope. This novel antibody can be applied in in vitro assays that determine the potency of antitoxin preparations and reduce the use of laboratory animals for these purposes. Full article

Rheumatoid arthritis (RA) is a chronic disease which causes joint inflammation and, ultimately, erosion of the underlying bone. Diagnosis of RA is based on the presence of biomarkers, such as anti-citrullinated protein antibodies (ACPA) and rheumatoid factors, along with clinical symptoms. Much evidence points to a link between the Epstein-Barr virus and RA. In this study, we analyzed ACPA reactivity to citrullinated peptides originating from Epstein-Barr nuclear antigens (EBNA1, EBNA2, and EBNA3) in order to elaborate the diagnostic potential of citrullinated EBNA peptides. Moreover, ACPA cross-reactivity to citrullinated peptides from myelin basic protein (MBP) was analyzed, as citrullinated MBP recently was described to be associated with multiple sclerosis, and some degree of sequence homology between MBP and citrullinated EBNA exists. A peptide from EBNA2, (EBNA2-A, GQGRGRWRG-Cit-GSKGRGRMH) reacted with approximately 70% of all RA sera, whereas only limited reactivity was detected to EBNA1 and EBNA3 peptides. Moreover, screening of ACPA reactivity to hybrid peptides of EBNA3-A (EPDSRDQQS-Cit-GQRRGDENRG) and EBNA2-A and peptides containing citrulline close to the N-terminal confirmed that ACPA sera contain different populations of ACPAs. No notable ACPA reactivity to MBP peptides was found, confirming that ACPAs are specific for RA, and that other factors than the presence of a central Cit-Gly motif are crucial for antibody binding. Collectively, these findings illustrate that citrullinated EBNA2 is an optimal candidate for ACPA detection, supporting current evidence that EBV is linked to RA onset. Full article

Serum anti-HLA-I IgG are present in non-alloimmunized males, cancer patients, and transplant recipients. Anti-HLA-I antibodies are also present in intravenous immunoglobulin (IVIg), prepared from the plasma of thousands of healthy donors. However, the HLA-Ia reactivity of IVIg diminishes markedly after passing through HLA-E HC-affinity columns, suggesting that the HLA-I reactivity is due to antibodies formed against HLA-E. Hence, we examined whether anti-HLA-E antibodies can react to HLA-I alleles. Monoclonal IgG antibodies (mAbs) against HCs of two HLA-E alleles were generated in Balb/C mice. The antibodies were analyzed using multiplex bead assays on a Luminex platform for HLA-I reactivity. Beads coated with an array of HLA heterodimers admixed with HCs (LABScreen) were used to examine the binding of IgG to different HLA-Ia (31-HLA-A, 50-HLA-B, and 16-HLA-C) and Ib (2-HLA-E, one each of HLA-F and HLA-G) alleles. A striking diversity in the HLA-Ia and/or HLA-Ib reactivity of mAbs was observed. The number of the mAbs reactive to (1) only HLA-E (n = 25); (2) all HLA-Ib isomers (n = 8); (3) HLA-E and HLA-B (n = 5); (4) HLA-E, HLA-B, and HLA-C (n = 30); (5) HLA-E, HLA-A*1101, HLA-B, and HLA-C (n = 83); (6) HLA-E, HLA-A, HLA-B, and HLA-C (n = 54); and (7) HLA-Ib and HLA-Ia (n = 8), in addition to four other minor groups. Monospecificity and polyreactivity were corroborated by HLA-E monospecific and HLA-I shared sequences. The diverse HLA-I reactivity of the mAbs are compared with the pattern of HLA-I reactivity of serum-IgG in non-alloimmunized males, cancer patients, and ESKD patients. The findings unravel the diagnostic potential of the HLA-E monospecific-mAbs and immunomodulatory potentials of IVIg highly mimicking HLA-I polyreactive-mAbs. Full article

Identification of new disease-associated biomarkers; specific targeting of such markers by monoclonal antibodies (mAbs); and application of advances in recombinant technology, including the production of humanized and fully human antibodies, has enabled many improved treatment outcomes and successful new biological treatments of some diseases previously neglected or with poor prognoses. Of the 110 mAbs preparations currently approved by the FDA and/or EMA, 46 (including 13 antibody–drug conjugates) recognizing 29 different targets are indicated for the treatment of cancers, and 66, recognizing 48 different targets, are indicated for non-cancer disorders. Despite their specific targeting with the expected accompanying reduced collateral damage for normal healthy non-involved cells, mAbs, may cause types I (anaphylaxis, urticaria), II (e.g., hemolytic anemia, possibly early-onset neutropenia), III (serum sickness, pneumonitis), and IV (Stevens–Johnson syndrome, toxic epidermal necrolysis) hypersensitivities as well as other cutaneous, pulmonary, cardiac, and liver adverse events. MAbs can provoke severe infusion reactions that resemble anaphylaxis and induce a number of systemic, potentially life-threatening syndromes with low frequency. A common feature of most of these syndromes is the release of a cascade of cytokines associated with inflammatory and immunological processes. Epidermal growth factor receptor-targeted antibodies may provoke papulopustular and mucocutaneous eruptions that are not immune-mediated. Full article

Bispecific antibodies (bsAbs) are molecules that simultaneously bind two different antigens (Ags). bsAbs represent a very active field in tumor immunotherapy with more than one hundred molecules currently being tested. More specifically, they have elicited a great interest in the setting of non-Hodgkin’s lymphoma (NHLs), where they could represent a viable option for more fragile patients or those resistant to other conventional therapies. This review aims to give a brief overview of the different available bsAb formats and their mechanisms of action, pinpointing the differences between IgG-like and non-IgG-like classes and will then focus on those in advanced clinical development for NHLs. Full article

This report describes the discovery and characterization of antibodies with potential broad SARS-CoV-2 neutralization profiles. The antibodies were obtained from a phage display library built with the VH repertoire of a convalescent COVID-19 patient who was infected with SARS-CoV-2 B.1.617.2 (Delta). The patient received a single dose of Ad5-nCoV vaccine (Convidecia™, CanSino Biologics Inc.) one month before developing COVID-19 symptoms. Four synthetic VL libraries were used as counterparts of the immune VH repertoire. After three rounds of panning with SARS-CoV-2 receptor-binding domain wildtype (RBD-WT) 34 unique scFvs, were identified, with 27 cross-reactive for the RBD-WT and RBD Delta (RBD-DT), and seven specifics for the RBD-WT. The cross-reactive scFvs were more diverse than the RBD-WT specific ones, being encoded by several IGHV genes from the IGHV1 and IGHV3 families combined with short HCDR3s. Six cross-reactive scFvs and one RBD-WT specific scFv were converted to human IgG1 (hIgG1). Out of the seven antibodies, six blocked the RBD-WT binding to angiotensin converting enzyme 2 (ACE2), suggesting these antibodies may neutralize the SARS-CoV-2 infection. Importantly, one of the antibodies also recognized the RBD from the B.1.1.529 (Omicron) isolate, implying that the VH repertoire of the convalescent patient would protect against SARS-CoV-2 Wildtype, Delta, and Omicron. From a practical viewpoint, the triple cross-reactive antibody provides the substrate for developing therapeutic antibodies with a broad SARS-CoV-2 neutralization profile. Full article

Innate cell engager (ICE^®) constructs are bispecific tetravalent antibodies targeting specific tumor antigens and simultaneously engaging natural killer (NK) cell and macrophage receptors for the destruction of tumor cells. Pre-complexing of ICE^® constructs with adoptive NK cells is a novel approach to enhance NK cell activity. The suitability of such complexes for cryopreservation, whilst retaining the biological activity and specificity, may enable the development of off-the-shelf NK cell products. This study investigates the binding affinity of ICE^® constructs targeting EpCAM and NK cell receptors CD16A, NKG2D, or NKp46 to the corresponding antigens, the ICE^® antitumor activity, and feasibility of cryopreservation. Cell surface retention assays using primary NK cells confirmed a substantially slower ICE^® construct dissociation kinetics compared with control molecules, suggesting the formation of durable complexes independently of the CD16A polymorphism. The high-affinity NK cell and EpCAM/CD16A ICE^® complexes were superior to those engaging NKG2D or NKp46 receptors when tested for the NK-cell-mediated elimination of EpCAM-expressing tumor cells. Moreover, the potency and efficacy of these complexes were unaffected after a single freeze–thaw cycle. CD16A-selective ICE^® drug candidates complexed with NK cells hold promise as novel cryopreserved off-the-shelf NK cell products with chimeric antigen receptor-like NK cell properties, capable of effective depletion of tumor cells. Full article

Therapeutic monoclonal antibodies have exerted a transformative impact on clinical practice in last two decades. However, development of a therapeutic antibody remains a complex process. Various physiochemical and functional liabilities can compromise the production or the therapeutic efficacy of antibodies. One of these liabilities is the susceptibility to oxidation. In the present study, we portrayed an oxidation-dependent vulnerability of immunoglobulins that can be of concern for therapeutic antibodies. By using a library of 119 monoclonal IgG1 molecules, containing variable domain matching clinical-stage antibodies, we demonstrated that a substantial number of these molecules acquired antigen-binding polyreactivity upon exposure to ferrous ions. Statistical analyses revealed that the potential for induction of polyreactivity by the redox-active metal ions correlated with a higher number of somatic mutations in V genes encoding variable domains of heavy and light immunoglobulin chains. Moreover, the sensitive antibodies used with biased frequencies particular V gene families encoding variable domains of their light chains. Besides the exposure to ferrous ions the induction of polyreactivity of therapeutic antibodies occurred after contact with an unrelated pro-oxidative substance—hypochlorite ions. Our data also revealed that induction of polyreactivity by pro-oxidative agents did not impact the binding of antibodies to their cognate antigens. The results from this study may contribute for better selection of antibody therapeutics with suitable developability profiles. Full article

A variable domain of heavy chain antibody (VHH) has different binding properties than conventional antibodies. Conventional antibodies prefer binding to the convex portion of the antigen, whereas VHHs prefer epitopes, such as crevices and clefts on the antigen. Therefore, developing candidates with the binding characteristics of camelid VHHs is important. Thus, To this end, a synthetic VHH library that reproduces the structural properties of camelid VHHs was constructed. First, the characteristics of VHHs were classified according to the paratope formation based on crystal structure analyses of the complex structures of VHHs and antigens. Then, we classified 330 complementarity-determining region 3 (CDR3) structures of VHHs from the Protein Data Bank (PDB) into three loop structures: Upright, Half-Roll, and Roll. Moreover, these structures depended on the number of amino acid residues within CDR3. Furthermore, in the Upright loops, several amino acid residues in the FR2 are involved in the paratope formation, along with CDR3, suggesting that the FR2 design in the synthetic library is important. A humanized synthetic VHH library, comprising two sub-libraries, Upright and Roll, was constructed and named PharmaLogical. A validation study confirmed that our PharmaLogical library reproduces VHHs with the characteristics of the paratope formation of the camelid VHHs, and shows good performance in VHH screening. Full article

Recombinant immunoglobulins, derived from monoclonal antibodies recognizing the defined surface epitopes expressed on dendritic cells, have been employed for the past two decades to deliver antigens to dendritic cells in vivo, serving as critical tools for the investigation of the corresponding T cell responses. These approaches originated with the development of the recombinant chimeric antibody against a multilectin receptor, DEC-205, which is present on subsets of murine and human conventional dendritic cells. Following the widespread application of antigen targeting through DEC-205, similar approaches then utilized other epitopes as entry points for antigens delivered by specific antibodies to multiple types of dendritic cells. Overall, these antigen-delivery methodologies helped to reveal the mechanisms underlying tolerogenic and immunogenic T cell responses orchestrated by dendritic cells. Here, we discuss the relevant experimental strategies as well as their future perspectives, including their translational relevance. Full article

Antibodies against platelet factor 4 (PF4), a protein released from alpha-granules of activated platelets, may cause a number of pathophysiological conditions. The most commonly known is heparin-induced thrombocytopenia (HIT), which develops in a small proportion of people treated with the anticoagulant drug heparin. Notably, PF4 binds with high affinity to heparin, and in HIT, complexes of PF4/H may, in a small proportion of susceptible patients, trigger the development of anti-PF4 antibodies and subsequent platelet activation and aggregation, ultimately leading to the development of pathological thrombosis at sites of vessel occlusion. Of more modern interest, antibodies against PF4 may also arise in patients with COVID-19 (Coronavirus Disease 2019) or in patients who have been vaccinated against COVID-19, especially in recipients of adenovirus-based vaccines. For this latter group of patients, the terms VITT (vaccine-induced [immune] thrombotic thrombocytopenia) and TTS (thrombotic thrombocytopenia syndrome) have been coined. Another category associated with this pathophysiology comprises those in whom a precipitating event is not clear; this category is referred to as ‘spontaneous HIT-like syndrome’. Despite its name, it arises as an HIT-mimicking disorder but without antecedent heparin exposure. In this narrative review, we describe the development of antibodies against PF4, and associated pathophysiology, in such conditions. Full article

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase whose proliferative effects can contribute to the development of many types of solid tumors when overexpressed. For this reason, EGFR inhibitors such as cetuximab can play an important role in treating cancers such as colorectal cancer and head and neck cancer. Cetuximab is a chimeric monoclonal antibody containing mouse variable regions that bind to EGFR and prevent it from signaling. Although cetuximab has been used clinically since 2004 to successfully control solid tumors, advances in protein engineering have created the opportunity to address some of its shortcomings. In particular, the presence of mouse sequences could contribute to immunogenicity in the form of anti-cetuximab antibodies, and an occupied glycosylation site in FR3 can contribute to hypersensitivity reactions and product heterogeneity. Using simple framework graft or sequence-/structure-guided approaches, cetuximab was humanized onto 11 new frameworks. In addition to increasing humanness and removing the VH glycosylation site, dynamic light scattering revealed increases in stability, and bio-layer interferometry confirmed minimal changes in binding affinity, with patterns emerging across the humanization method. This work demonstrates the potential to improve the biophysical and clinical properties of first-generation protein therapeutics and highlights the advantages of computationally guided engineering. Full article

Glycans as sugar polymers are important metabolic, structural, and physiological regulators for cellular and biological functions. They are often classified as critical quality attributes to antibodies and recombinant fusion proteins, given their impacts on the efficacy and safety of biologics drugs. Recent reports on the conjugates of N-acetyl-galactosamine and mannose-6-phosphate for lysosomal degradation, Fab glycans for antibody diversification, as well as sialylation therapeutic modulations and O-linked applications, have been fueling the continued interest in glycoengineering. The current advancements of the human glycome and the development of a comprehensive network in glycosylation pathways have presented new opportunities in designing next-generation therapeutic proteins. Full article