Antibodies — Open Access Journal

Background: Currently, the immunogenicity of influenza vaccines is assessed by detecting an increase of hemagglutination inhibition (HI) antibodies. As neuraminidase (NA)-based immunity may be significant in protecting against influenza infection, detection of neuraminidase inhibiting (NI) antibodies may improve the assessment of the immunogenicity of influenza vaccines. Methods: We investigated the immune response to NA in people after immunization with live influenza vaccines (LAIVs). A number of A/H7NX or A/H6NX viruses were used to detect NI antibodies, using an enzyme-linked lectin assay (ELLA). Results: Seasonal LAIV immunization stimulated an increase in NI antibodies not only to homologous A/H1N1 influenza, but also to A/H1N1pdm09 and A/H5N1 influenza. After A/17/California/09/38 (H1N1) pdm09 LAIV vaccination, there was no statistical relationship between post-vaccinated antibody seroconversion and two surface glycoproteins in serum samples obtained from the same individuals (p = 0.24). Vaccination with LAIV of H5N2, H2N2, H7N3, and H7N9 subtypes led to 7%–29.6% NI antibody seroconversions in the absence of HI antibody conversions. There was relatively low coordination of hemagglutinin (HA) and NA antibody responses (r = 0.24–0.59). Conclusions: The previously noted autonomy for HI and NI immune responses was confirmed when assessing the immunogenicity of LAIVs. Combining the traditional HI test with the detection of NI antibodies can provide a more complete assessment of LAIV immunogenicity. Full article

Anti-idiotypic antibodies (anti-IDs) were discovered at the very beginning of the 20th century and have attracted attention of researchers for many years. Nowadays, there are five known types of anti-IDs: α, β, γ, ε, and δ. Due to the ability of internal-image anti-IDs to compete with an antigen for binding to antibody and to alter the biologic activity of an antigen, anti-IDs have become a target in the search for new treatments of autoimmune illnesses, cancer, and some other diseases. In this review, we summarize the data about anti-IDs that mimic the structural and functional properties of some bioregulators (autacoids, neurotransmitters, hormones, xenobiotics, and drugs) and evaluate their possible medical applications. The immune system is potentially able to reproduce or at least alter the effects of any biologically active endogenous or exogenous immunogenic agent via the anti-idiotypic principle, and probably regulates a broad spectrum of cell functions in the body, being a kind of universal remedy or immunacea, by analogy to the legendary ancient goddess of universal healing Panacea (Πανάκεια, Panakeia in Greek) in the treatment and prevention of diseases, possibly including non-infectious somatic and even hereditary ones. Full article

Aim: Glomerular diseases (GD) associated with malignancies (AM, GDAM) have unique features, which are important to recognize, in the light of the progress made in cancer therapy. We aimed to describe the clinical and histopathological characteristics of patients with GDAM in relation to the presence of circulating autoantibodies, pointing to potential immune pathogenic pathways connecting cancer to GD. Materials and Methods: The included patients were studied retrospectively on the basis of a kidney biopsy proving GD and a related biopsy to establish the diagnosis of AM. We recorded patients’ demographics, serological and laboratory parameters, histopathological findings, and the type of malignancy, GD, and therapy. Results: In total, 41 patients with GDAM, with a mean age of 63.1 (±10.7) years, were studied. In 28 (68.3%) cases, GD was associated with a solid tumor, and in 13 (31.7%) patients with a lymphoid malignancy. The most frequent histopathological pattern was membranous nephropathy (43.9%). Overall, at the time of GD diagnosis, 17% of the patients were positive for antinuclear antibodies (ANA), and 12.2% for antineutrophil cytoplasmic autoantibodies (ANCA), all against myeloperoxidase (MPO). In addition, 93.3% of the patients who had membranous nephropathy were negative for transmembrane glycoprotein M-type phospholipase A₂ receptor (PLA₂R) antibody. Sixteen patients (39.0%) presented with acute nephritic syndrome, of whom five (31.25%) developed rapidly progressive glomerulonephritis. In a mean follow-up time of 36.1 (±28.3) months, nine (21.95%) patients ended up with end-stage kidney disease, and eight (19.5%) died. Conclusion: We found that 3.2% of patients who underwent a native kidney biopsy in our institution during the past decade, for any reason, were identified as having some type of GD associated with a malignancy. Serology indicated a significant presence of ANA or MPO-ANCA antibodies in patients with nephritic syndrome and the absence of PLA₂R antibodies in patients with membranous nephropathy. Full article

Therapeutic monoclonal antibodies for the treatment of cancer came of age in 1997, with the approval of anti-CD20 Rituximab. Since then, a wide variety of antibodies have been developed with many different formats and mechanisms of action. Among these, antibodies blocking immune checkpoint inhibitors (ICI) have revolutionized the field, based on the novelty of their concept and their demonstrated efficacy in several types of cancer otherwise lacking effective immunotherapy approaches. ICI are expressed by tumor, stromal or immune cells infiltrating the tumor microenvironment, and negatively regulate anti-tumor immunity. Antibodies against the first discovered ICI, CTLA-4, PD-1 and PD-L1, have shown significant activity in phase III studies against melanoma and other solid cancers, alone or in combination with chemotherapy or radiotherapy. However, not all cancers and not all patients respond to these drugs. Therefore, novel antibodies targeting additional ICI are currently being developed. In addition, CTLA-4, PD-1 and PD-L1 blocking antibodies are being combined with each other or with other antibodies targeting novel ICI, immunostimulatory molecules, tumor antigens, angiogenic factors, complement receptors, or with T cell engaging bispecific antibodies (BsAb), with the aim of obtaining synergistic effects with minimal toxicity. In this review, we summarize the biological aspects behind such combinations and review some of the most important clinical data on ICI-specific antibodies. Full article

In recent years, site-specific antibody drug conjugates (ADC)s have been in great demand because they have an expanded therapeutic index compared with conventional ADCs. AJICAP™ technology is a chemical conjugation platform to obtain site-specific ADCs through the use of a class of Fc-affinity compounds. Promising results from early technology development studies led to further investigation of AJICAP™ ADC materials to obtain site-specific and homogeneous drug antibody ratio (DAR) ADCs. Here we report site-specific conjugation followed by a preparative hydrophobic interaction chromatography (HIC) purification strategy to obtain purified “DAR = 1.0” and “DAR = 2.0” AJICAP™ ADC materials. Optimization of the mobile phase conditions and resin achieved a high recovery rate. In vitro biological assay demonstrated the target selective activity for purified homogeneous DAR ADCs. These results indicate the ability of a HIC purification strategy to provide “DAR = 1.0” and “DAR = 2.0” AJICAP™ ADCs with considerable potency and target selectivity. Full article

Human cathelicidin LL37 is a cationic antimicrobial peptide active against bacteria and viruses and exerting immune modulatory functions. LL37 can be also a target of autoreactive B- and T-lymphocytes in autoimmune settings. Irreversible post-translational modifications, such as citrullination and carbamylation, mainly occurring at the level of cationic amino acids arginine and lysine, can affect the inflammatory properties and reduce antibacterial effects. Moreover, these modifications could be implicated in the rupture of immune tolerance to LL37 in chronic conditions such as psoriatic disease and cutaneous lupus (LE)/systemic lupus erythematosus (SLE). Here, we describe the generation and fine specificity of six recombinant antibodies (MRB137–MRB142), produced as a monovalent mouse antibody with the antigen-binding scFv portion fused to a mouse IgG2a Fc, and their ability to recognize either native or citrullinated LL37 (cit-LL37) and not cross-react to carbamylated LL37. By using these antibodies, we detected native LL37 or cit-LL37 in SLE and rheumatoid arthritis (RA) sera, and in LE skin, by ELISA and immunohistochemistry, respectively. Such antibodies represent previously unavailable and useful tools to address relationships between the presence of post-translational modified LL37 and the immune system status (in terms of innate/adaptive responses activation) and the clinical characteristics of patients affected by chronic immune-mediated diseases or infectious diseases. Full article

Adaptive immunity in vertebrates is a complex self-organizing network of molecular interactions. While deep sequencing of the immune-receptor repertoire may reveal clonal relationships, functional interpretation of such data is hampered by the inherent limitations of converting sequence to structure to function. In this paper, a novel model of antibody interaction space and network, termed radial adjustment of system resolution, RAdial ADjustment of System Resolution (RADARS), is proposed. The model is based on the radial growth of interaction affinity of antibodies towards an infinity of directions in structure space, each direction corresponding to particular shapes of antigen epitopes. Levels of interaction affinity appear as free energy shells of the system, where hierarchical B-cell development and differentiation takes place. Equilibrium in this immunological thermodynamic system can be described by a power law distribution of antibody-free energies with an ideal network degree exponent of phi square, representing a scale-free fractal network of antibody interactions. Plasma cells are network hubs, memory B cells are nodes with intermediate degrees, and B1 cells function as nodes with minimal degree. Overall, the RADARS model implies that a finite number of antibody structures can interact with an infinite number of antigens by immunologically controlled adjustment of interaction energy distribution. Understanding quantitative network properties of the system should help the organization of sequence-derived predicted structural data. Full article

Driven by its successes across domains such as computer vision and natural language processing, deep learning has recently entered the field of biology by aiding in cellular image classification, finding genomic connections, and advancing drug discovery. In drug discovery and protein engineering, a major goal is to design a molecule that will perform a useful function as a therapeutic drug. Typically, the focus has been on small molecules, but new approaches have been developed to apply these same principles of deep learning to biologics, such as antibodies. Here we give a brief background of deep learning as it applies to antibody drug development, and an in-depth explanation of several deep learning algorithms that have been proposed to solve aspects of both protein design in general, and antibody design in particular. Full article

Abrin, a toxin isolated from the seeds of Abrus precatorius (jequirity pea) is considered a biological threat agent by the Center for Disease Control and Prevention. To date, there is no effective postexposure treatment for abrin poisoning, and efforts are being made to develop an efficient vaccine and measures for postexposure therapy. Epitope mapping is widely applied as an efficient tool for discovering the antigenic moieties of toxins, thus providing invaluable information needed for the development of vaccines and therapies. Aiming to identify the immunodominant epitopes of abrin, several neutralizing antiabrin polyclonal antibodies were screened using a set of 15-mer peptides spanning the amino acid sequence of either the A or B subunits of abrin. Analysis of the antibody-binding pattern revealed 11 linear epitopes for the A subunit and 14 epitopes for the B subunit that are located on the surface of the toxin and thus accessible for antibody interactions. Moreover, the spatial location of several of these epitopes suggests they may block the galactose-binding pockets or the catalytic domain, thus neutralizing the toxin. These findings provide useful information and suggest a possible strategy for the development and design of an improved abrin-based vaccine and therapeutic antibodies. Full article

LT1009 is a humanized version of murine LT1002 IgG1 that employs two bridging Ca²⁺ ions to bind its antigen, the biologically active lipid sphingosine-1-phosphate (S1P). We crystallized and determined the X-ray crystal structure of the LT1009 Fab fragment in 10 mM CaCl₂ and found that it binds two Ca²⁺ in a manner similar to its antigen-bound state. Flame atomic absorption spectroscopy (FAAS) confirmed that murine LT1002 also binds Ca²⁺ in solution and inductively-coupled plasma-mass spectrometry (ICP-MS) revealed that, although Ca²⁺ is preferred, LT1002 can bind Mg²⁺ and, to much lesser extent, Ba²⁺. Isothermal titration calorimetry (ITC) indicated that LT1002 binds two Ca²⁺ ions endothermically with a measured dissociation constant (K_D) of 171 μM. Protein and genome sequence analyses suggested that LT1002 is representative of a small class of confirmed and potential metalloantibodies and that Ca²⁺ binding is likely encoded for in germline variable chain genes. To test this hypothesis, we engineered, expressed, and purified a Fab fragment consisting of naïve murine germline-encoded light and heavy chain genes from which LT1002 is derived and observed that it binds Ca²⁺ in solution. We propose that LT1002 is representative of a class of naturally occurring metalloantibodies that are evolutionarily conserved across diverse mammalian genomes. Full article

In order to increase the successful development of recombinant antibodies and fragments, it seems fundamental to enhance their expression and/or biophysical properties, such as the thermal, chemical, and pH stabilities. In this study, we employed a method bases on replacing the antibody framework region sequences, in order to promote more particularly single-chain Fragment variable (scFv) product quality. We provide evidence that mutations of the VH- C-C′ loop might significantly improve the prokaryote production of well-folded and functional fragments with a production yield multiplied by 27 times. Additional mutations are accountable for an increase in the thermal (+19.6 °C) and chemical (+1.9 M) stabilities have also been identified. Furthermore, the hereby-produced fragments have shown to remain stable at a pH of 2.0, which avoids molecule functional and structural impairments during the purification process. Lastly, this study provides relevant information to the understanding of the relationship between the antibodies amino acid sequences and their respective biophysical properties. Full article

Thousands of antibodies for diagnostic and other analytical purposes are on the market. However, it is often difficult to identify duplicates, reagent changes, and to assign the correct original publications to an antibody. This slows down scientific progress and might even be a cause of irreproducible research and a waste of resources. Recently, activities were started to suggest the sole use of recombinant antibodies in combination with the open communication of their sequence. In this case, such uncertainties should be eliminated. Unfortunately, this approach seems to be rather a long-term vision since the development and manufacturing of recombinant antibodies remain quite expensive in the foreseeable future. Nearly all commercial antibody suppliers also may be reluctant to publish the sequence of their antibodies, since they fear counterfeiting. De novo sequencing of antibodies is also not feasible today for a reagent user without access to the hybridoma clone. Nevertheless, it seems to be crucial for any scientist to have the opportunity to identify an antibody undoubtedly to guarantee the traceability of any research activity using antibodies from a third party as a tool. For this purpose, we developed a method for the identification of antibodies based on a MALDI-TOF MS fingerprint. To circumvent lengthy denaturation, reduction, alkylation, and enzymatic digestion steps, the fragmentation was performed with a simple formic acid hydrolysis step. Eighty-nine unknown monoclonal antibodies were used for this study to examine the feasibility of this approach. Although the molecular assignment of peaks was rarely possible, antibodies could be easily recognized in a blinded test, simply from their mass-spectral fingerprint. A general protocol is given, which could be used without any optimization to generate fingerprints for a database. We want to propose that, in most scientific projects relying critically on antibody reagents, such a fingerprint should be established to prove and document the identity of the used antibodies, as well as to assign a specific reagent to a datasheet of a commercial supplier, public database record, or antibody ID. Full article

The characterization of target binding interactions is critical at each stage of antibody therapeutic development. During early development, it is important to design fit-for-purpose in vitro molecular interaction characterization (MIC) assays that accurately determine the binding kinetics and the affinity of therapeutic antibodies for their targets. Such information enables PK/PD (pharmacokinetics/pharmacodynamics) modeling, estimation of dosing regimens, and assessment of potency. While binding kinetics and affinities seem to be readily obtained, there is little discussion in the literature on how the information should be generated and used in a systematic manner along with other approaches to enable key drug development decisions. The introduction of new antibody modalities poses unique challenges to the development of MIC assays and further increases the need to discuss the impact of developing context-appropriate MIC assays to enable key decision making for these programs. In this paper, we discuss for the first time the challenges encountered when developing MIC assays supporting new antibody modalities. Additionally, through the presentation of several real case studies, we provide strategies to overcome these challenges to enable investigational new drug (IND) filings. Full article

Poliovirus (PV)-specific intestinal IgAs are important for cessation of PV shedding in the gastrointestinal tract following an acute infection with wild type or vaccine-derived PV strains. We sought to produce IgA monoclonal antibodies (mAbs) with PV neutralizing activity. We first performed de novo IgA discovery from primary human B cells using a hybridoma method that allows assessment of mAb binding and expression on the hybridoma surface: On-Cell mAb Screening (OCMS™). Six IgA1 mAbs were cloned by this method; three potently neutralized type 3 Sabin and wt PV strains. The hybridoma mAbs were heterogeneous, expressed in monomeric, dimeric, and aberrant forms. We also used recombinant methods to convert two high-potency anti-PV IgG mAbs into dimeric IgA1 and IgA2 mAbs. Isotype switching did not substantially change their neutralization activities. To purify the recombinant mAbs, Protein L binding was used, and one of the mAbs required a single amino acid substitution in its κ LC in order to enable protein L binding. Lastly, we used OCMS to assess IgA expression on the surface of hybridomas and transiently transfected, adherent cells. These studies have generated potent anti-PV IgA mAbs, for use in animal models, as well as additional tools for the discovery and production of human IgA mAbs. Full article

Abrin and ricin are potent AB toxins, which are considered biological threats. To date, there are no approved treatments against abrin or ricin intoxications. Previously, we showed that the administration of polyclonal anti-abrin antibodies to mice that were intranasally exposed to abrin, even very late post-exposure, conferred an exceedingly high-level of protection, while following ricin intoxication, similar treatment with anti-ricin antibodies resulted in negligible survival rates. To probe this unexpected difference in protection ability, we first examined whether the efficient anti-abrin-induced protection was due to neutralization of the A-subunit responsible for the catalytic effect, or of the B-subunit, which enables binding/internalization, by evaluating the protection conferred by antibodies directed against one of the two subunits. To this end, we generated and immunized rabbits with chimeric toxins containing a single abrin subunit, A_abrinB_ricin in which abrin A-subunit was linked to ricin B-subunit, and A_ricinB_abrin in which ricin A-subunit is linked to abrin B-subunit. Here, we show that antibodies raised against either A_abrinB_ricin or A_ricinB_abrin conferred exceptionally high protection levels to mice following intranasal exposure to a a lethal dose of abrin, suggesting that the high level of protection conferred by anti-abrin antibodies is not related to the neutralization of a particular subunit. Full article

Monoclonal antibodies have evolved from research tools to powerful therapeutics in the past 30 years. Clinical success rates of antibodies have exceeded expectations, resulting in heavy investment in biologics discovery and development in addition to traditional small molecules across the industry. However, protein therapeutics cannot drug targets intracellularly and are limited to soluble and cell-surface antigens. Tremendous strides have been made in antibody discovery, protein engineering, formulation, and delivery devices. These advances continue to push the boundaries of biologics to enable antibody conjugates to take advantage of the target specificity and long half-life from an antibody, while delivering highly potent small molecule drugs. While the “magic bullet” concept produced the first wave of antibody conjugates, these entities were met with limited clinical success. This review summarizes the advances and challenges in the field to date with emphasis on antibody conjugation, linker-payload chemistry, novel payload classes, absorption, distribution, metabolism, and excretion (ADME), and product developability. We discuss lessons learned in the development of oncology antibody conjugates and look towards future innovations enabling other therapeutic indications. Full article