A high affinity monoclonal antibody against HLA-E-VL9 enhances natural killer cell anti-tumor killing.

Natural killer (NK) cells kill target cells following triggering via germline-encoded receptors interacting with target cell-expressed ligands (direct killing), or via antibody-dependent cellular cytotoxicity (ADCC) mediated by FcγRIIIa. NK cytotoxicity is modulated by signaling through activating or inhibitory receptors. A major checkpoint is mediated by the NK inhibitory receptor NKG2A/CD94 and its target cell ligand, HLA-E, which is complexed with HLA signal sequence-derived peptides termed VL9 (HLA-E-VL9). We have previously reported the isolation of a murine HLA-E-VL9-specific IgM antibody 3H4 and the generation of a higher affinity IgG version (3H4v3). Here we have used phage display library selection to generate a high affinity version of 3H4v3, called 3H4v31, with an ∼700 fold increase in binding affinity. We show using an HLA-E-VL9+ K562 tumor model that, in vitro, the addition of 3H4v31 to target cells increased direct killing of targets by CD16-negative NK cell line NK-92 and also mediated ADCC by NK-92 cells transfected with CD16. Moreover, ADCC by primary NK cells was also enhanced in vitro by 3H4v31. 3H4v31 was also able to bind and enhance target cell lysis of endogenously expressed HLA-E-VL9 on human cervical cancer and human pancreatic cancer cell lines. In vivo, 3H4v31 slowed the growth rate of HLA-E-VL9+ K562 tumors implanted into NOD/SCID/IL2rγ null mice compared to isotype control when injected with NK-92 cells intratumorally. Together, these data demonstrate that mAb 3H4v31 can enhance NK cell killing of HLA-E-VL9-expressing tumor cells in vitro by both direct killing activity and by ADCC. Moreover, mAb 3H4v31 can enhance NK cell control of tumor growth in vivo. We thus identify HLA-E-VL9 monoclonal antibodies as a promising novel anti-tumor immunotherapy. One Sentence Summary: A high affinity monoclonal antibody against HLA-E-VL9 enhances natural killer cell anti-tumor killing by checkpoint inhibition and antibody dependent cellular cytotoxicity.

Structural, Biophysical, and Computational Studies of a Murine Light Chain Dimer.

Antibodies are widely used in medicinal and scientific research due to their ability to bind to a specific antigen. Most often, antibodies are composed of heavy and light chain domains. Under physiological conditions, light chains are produced in excess, as compared to the heavy chain. It is now known that light chains are not silent partners of the heavy chain and can modulate the immune response independently. In this work, the first crystal structure of a light chain dimer originating from mice is described. It represents the light chain dimer of 6A8, a monoclonal antibody specific to the allergen Der f 1. Building on the unexpected occurrence of this kind of dimer, we have demonstrated that this light chain is stable in solution alone. Moreover, enzyme-linked immunosorbent assays (ELISA) have revealed that, when the light chain is not partnered to its corresponding heavy chain, it interacts non-specifically with a wide range of proteins. Computational studies were used to provide insight on the role of the 6A8 heavy chain domain in the specific binding to Der f 1. Overall, this work demonstrates and supports the ongoing notion that light chains can function by themselves and are not silent partners of heavy chains.

Engineered immunogens to elicit antibodies against conserved coronavirus epitopes.

Immune responses to SARS-CoV-2 primarily target the receptor binding domain of the spike protein, which continually mutates to escape acquired immunity. Other regions in the spike S2 subunit, such as the stem helix and the segment encompassing residues 815-823 adjacent to the fusion peptide, are highly conserved across sarbecoviruses and are recognized by broadly reactive antibodies, providing hope that vaccines targeting these epitopes could offer protection against both current and emergent viruses. Here we employ computational modeling to design scaffolded immunogens that display the spike 815-823 peptide and the stem helix epitopes without the distracting and immunodominant receptor binding domain. These engineered proteins bind with high affinity and specificity to the mature and germline versions of previously identified broadly protective human antibodies. Epitope scaffolds interact with both sera and isolated monoclonal antibodies with broadly reactivity from individuals with pre-existing SARS-CoV-2 immunity. When used as immunogens, epitope scaffolds elicit sera with broad betacoronavirus reactivity and protect as "boosts" against live virus challenge in mice, illustrating their potential as components of a future pancoronavirus vaccine.

The first reptilian allergen and major allergen for fish-allergic patients: Crocodile ß-parvalbumin.

BACKGROUND: Clinical cross-reactivity between bony fish, cartilaginous fish, frog, and chicken muscle has previously been demonstrated in fish-allergic patients. In indicative studies, two reports of anaphylaxis following the consumption of crocodile meat and IgE-cross-binding were linked to the major fish allergen parvalbumin (PV). This study investigates IgE-binding proteins in crocodile meat with a focus on PV and their clinical relevance. METHODS: Proteins were extracted from muscle tissue of crocodile, three bony fish, and two cartilaginous fish. A cohort of fish-allergic pediatric patients (n = 77) underwent allergen skin prick testing (SPT) to three fish preparations (n = 77) and crocodile (n = 12). IgE-binding proteins were identified and quantified by SDS-PAGE, mass spectrometric analyses, and immunoblotting using commercial and in-house antibodies, as well as individual and pooled patients' serum. PV isoforms were purified or recombinantly expressed before immunological analyses, including human mast cell degranulation assay. RESULTS: Of the tissues analyzed, PV was most abundant in heated crocodile preparation, triggering an SPT of ≥3 mm in 8 of 12 (67%) fish-allergic patients. Seventy percent (31 of 44) of fish PV-sensitized patients demonstrated IgE-binding to crocodile PV. Crocodile ß-PV was the major IgE-binding protein but 20-fold less abundant than α-PV. Cellular reactivity was demonstrated for ß-PV and epitopes predicted, explaining frequent IgE-cross-binding of ß-PVs. Both PV isoforms are now registered as the first reptile allergens with the WHO/IUIS (ß-PV as Cro p 1 and α-PV as Cro p 2). CONCLUSION: Fish-allergic individuals may be at risk of an allergy to crocodile and should seek specialist advice before consuming crocodilian meat.

Crystal structure of timothy grass allergen Phl p 12.0101 reveals an unusual profilin dimer.

Timothy grass pollen is a source of potent allergens. Among them, Phl p 1 and Phl p 5 are thought to be the most important, as a majority of timothy grass-allergic individuals have IgE antibodies directed against these two allergens. The profilin from timothy grass (Phl p 12) has been registered as a minor allergen, with up to 35% of individuals in populations of grass pollen allergic patients showing IgE binding to Phl p 12. Profilins are primarily minor allergens and are known for a high likelihood of co-sensitization as well as cross-reactivity situations caused by their sequence and structure similarity. The crystal structure of Phl p 12.0101 was determined and it revealed that this allergen may form an unusual dimer not previously observed among any profilins. For example, the Phl p 12 dimer has a completely different geometry and interface when compared with the latex profilin (Hev b 8) dimer that has its crystal structure determined. The structure of Phl p 12.0101 is described in the context of allergenic sensitization and allergy diagnostics. Moreover, the structure of the Phl p 12.0101 dimer is discussed, taking into account the production of recombinant allergens and their storage.

Antigen-Antibody Complexes.

In vertebrates, immunoglobulins (Igs), commonly known as antibodies, play an integral role in the armamentarium of immune defense against various pathogens. After an antigenic challenge, antibodies are secreted by differentiated B cells called plasma cells. Antibodies have two predominant roles that involve specific binding to antigens to launch an immune response, along with activation of other components of the immune system to fight pathogens. The ability of immunoglobulins to fight against innumerable and diverse pathogens lies in their intrinsic ability to discriminate between different antigens. Due to this specificity and high affinity for their antigens, antibodies have been a valuable and indispensable tool in research, diagnostics and therapy. Although seemingly a simple maneuver, the association between an antibody and its antigen, to make an antigen-antibody complex, is comprised of myriads of non-covalent interactions. Amino acid residues on the antigen binding site, the epitope, and on the antibody binding site, the paratope, intimately contribute to the energetics needed for the antigen-antibody complex stability. Structural biology methods to study antigen-antibody complexes are extremely valuable tools to visualize antigen-antibody interactions in detail; this helps to elucidate the basis of molecular recognition between an antibody and its specific antigen. The main scope of this chapter is to discuss the structure and function of different classes of antibodies and the various aspects of antigen-antibody interactions including antigen-antibody interfaces-with a special focus on paratopes, complementarity determining regions (CDRs) and other non-CDR residues important for antigen binding and recognition. Herein, we also discuss methods used to study antigen-antibody complexes, antigen recognition by antibodies, types of antigens in complexes, and how antigen-antibody complexes play a role in modern day medicine and human health. Understanding the molecular basis of antigen binding and recognition by antibodies helps to facilitate the production of better and more potent antibodies for immunotherapy, vaccines and various other applications.

Production and Use of Recombinant Profilins Amb a 8, Art v 4, Bet v 2, and Phl p 12 for Allergenic Sensitization Studies.

Four recombinant (r) allergens (rAmb a 8.0101, rArt v 4.0101, rBet v 2.0101, and rPhl p 12.0101) were successfully produced and used for sensitization studies. The allergens belong to the profilin family which is one of the most numerous allergen families. These four proteins represent allergens originating from pollen of weeds (rAmb a 8.0101 and rArt v 4.0101), tree (rBet v 2.0101) and grass (rPhl p 12.0101). The recombinant allergens were characterized using various biochemical and biophysical methods and tested for their ability to bind patient-derived antibodies. One hundred patients aged 2 to 50 years sensitized to pollen and plant-derived food allergens (IgE > 0.35 kU/L) were included. Sensitization to individual allergen sources and components of birch and timothy pollens was evaluated using multiparameter immunoblots. The presence of IgE to pollen-derived recombinant profilins rAmb a 8.0101, rArt v 4.0101, rBet v 2.0101, and rPhl p 12.0101 in serum was evaluated using ELISA method. The presence of IgE against pollen profilins was detected in 20 out of 100 studied patients. High correlation was seen between IgE ELISA results with individual pollen profilins. In summary, it was shown that the recombinant versions of the four allergenic profilins can be used for sensitization studies and for component-resolved allergy diagnostics.

A Human IgE Antibody Binding Site on Der p 2 for the Design of a Recombinant Allergen for Immunotherapy.

Der p 2 is one of the most important allergens from the house dust mite Dermatophagoides pteronyssinus Identification of human IgE Ab binding epitopes can be used for rational design of allergens with reduced IgE reactivity for therapy. Antigenic analysis of Der p 2 was performed by site-directed mutagenesis based on the x-ray crystal structure of the allergen in complex with a Fab from the murine IgG mAb 7A1 that binds an epitope overlapping with human IgE binding sites. Conformational changes upon Ab binding were confirmed by nuclear magnetic resonance using a 7A1-single-chain variable fragment. In addition, a human IgE Ab construct that interferes with mAb 7A1 binding was isolated from a combinatorial phage-display library constructed from a mite-allergic patient and expressed as two recombinant forms (single-chain Fab in Pichia pastoris and Fab in Escherichia coli). These two IgE Ab constructs and the mAb 7A1 failed to recognize two Der p 2 epitope double mutants designed to abolish the allergen-Ab interaction while preserving the fold necessary to bind Abs at other sites of the allergen surface. A 10-100-fold reduction in binding of IgE from allergic subjects to the mutants additionally showed that the residues mutated were involved in IgE Ab binding. In summary, mutagenesis of a Der p 2 epitope defined by x-ray crystallography revealed an IgE Ab binding site that will be considered for the design of hypoallergens for immunotherapy.

Comparative structural and thermal stability studies of Cuc m 2.0101, Art v 4.0101 and other allergenic profilins.

Worldwide, more than one-third of the population suffers from allergies. A significant fraction of officially registered allergens originate from the profilin family of proteins. Profilins are small ubiquitous proteins which are found in plants, viruses and various eukaryotes including mammals. Although they are primarily regarded as minor allergens, profilins are important players in immunoglobulin E (IgE) cross-reactivity. However, in some populations profilins are recognized by IgE from at least 50% of patients allergic to a given allergen source. Cuc m 2.0101 is recognized by IgE in more than 80% of muskmelon-allergic patients. The recombinant isoallergen Cuc m 2.0101 was produced in significant quantities and its X-ray crystal structure was determined. In addition, a new Art v 4.0101 (mugwort profilin) structure was determined. The profilins Cuc m 2.0101 and Art v 4.0101 were compared in terms of their structure and thermal stability. Furthermore, structural similarities and IgE cross-reactivity between profilins from different sources are discussed to explain the molecular basis of various clinical syndromes involving this group of allergens. Special emphasis is placed on discussion of profilins' quaternary structures and their relation to biological function, as well as to protein allergenicity. Moreover, a potential impact of protein purification protocols on the structure of profilins is highlighted.

A robust method for the estimation and visualization of IgE cross-reactivity likelihood between allergens belonging to the same protein family.

Among the vast number of identified protein families, allergens emanate from relatively few families which translates to only a small fraction of identified protein families. In allergy diagnostics and immunotherapy, interactions between immunoglobulin E and allergens are crucial because the formation of an allergen-antibody complex is necessary for triggering an allergic reaction. In allergic diseases, there is a phenomenon known as cross-reactivity. Cross-reactivity describes a situation where an individual has produced antibodies against a particular allergenic protein, but said antibodies fail to discriminate between the original sensitizer and other similar proteins that usually belong to the same family. To expound the concept of cross-reactivity, this study examines ten protein families that include allergens selected specifically for the analysis of cross-reactivity. The selected allergen families had at least 13 representative proteins, overall folds that differ significantly between families, and include relevant allergens with various potencies. The selected allergens were analyzed using information on sequence similarities and identities between members of the families as well as reports on clinically relevant cross-reactivities. Based on our analysis, we propose to introduce a new A-RISC index (Allergens'-Relative Identity, Similarity and Cross-reactivity) which describes homology between two allergens belonging to the same protein family and is used to predict the likelihood of cross-reactivity between them. Information on sequence similarities and identities, as well as on the values of the proposed A-RISC index is used to introduce four categories describing a risk of a cross-reactive reaction, namely: high, medium-high, medium-low and low. The proposed approach can facilitate analysis in component-resolved allergy diagnostics, generation of avoidance guidelines for allergic individuals, and help with the design of immunotherapy.