γδ T cell antigen receptor polyspecificity enables T cell responses to a broad range of immune challenges.

γδ T cells are essential for immune defense and modulating physiological processes. While they have the potential to recognize large numbers of antigens through somatic gene rearrangement, the antigens which trigger most γδ T cell response remain unidentified, and the role of antigen recognition in γδ T cell function is contentious. Here, we show that some γδ T cell receptors (TCRs) exhibit polyspecificity, recognizing multiple ligands of diverse molecular nature. These ligands include haptens, metabolites, neurotransmitters, posttranslational modifications, as well as peptides and proteins of microbial and host origin. Polyspecific γδ T cells are enriched among activated cells in naive mice and the responding population in infection. They express diverse TCR sequences, have different functional potentials, and include the innate-like γδ T cells, such as the major IL-17 responders in various pathological/physiological conditions. We demonstrate that encountering their antigenic microbiome metabolite maintains their homeostasis and functional response, indicating that their ability to recognize multiple ligands is essential for their function. Human γδ T cells with similar polyspecificity also respond to various immune challenges. This study demonstrates that polyspecificity is a prevalent feature of γδ T cell antigen recognition, which enables rapid and robust T cell responses to a wide range of challenges, highlighting a unique function of γδ T cells.

Comprehensive characterization of the OCT1 phenylalanine-244-alanine substitution reveals highly substrate-dependent effects on transporter function.

Organic cation transporters (OCTs) can transport structurally highly diverse substrates. The molecular basis of this extensive polyspecificity has been further elucidated by cryogenic electron microscopy. Apparently, in addition to negatively charged amino acids, aromatic residues may contribute to substrate binding and substrate selectivity. In this study, we provide a comprehensive characterization of phenylalanine 244 in OCT1 function. We analyzed the uptake of 144 OCT1 substrates for the phenylalanine 244 to alanine substitution compared to wild-type OCT1. This substitution had highly substrate-specific effects ranging from transport reduced to 10% of wild-type activity up to 8-fold increased transport rates. Four percent of substrates showed strongly increased uptake (> 200% of wild type) whereas 39% showed strongly reduced transport (< 50% of wild type). Particularly with larger, more hydrophobic, and more aromatic substrates, the Phe244Ala substitution resulted in higher transport rates and lower inhibition of the transporter. In contrast, substrates with a lower molecular weight and less aromatic rings showed generally decreased uptake rates. A comparison of our data to available transport kinetic data demonstrates that generally, high-affinity low-capacity substrates show increased uptake by the Phe244Ala substitution whereas low-affinity high-capacity substrates are characterized by reduced transport rates. Altogether, our study provides the first comprehensive characterization of the functional role of an aromatic amino acid within the substrate translocation pathway of OCT1. The pleiotropic function further highlights that Phenylalanine 244 interacts in a highly specific manner with OCT1 substrates and inhibitors.

Molecular insights into the determinants of substrate specificity and efflux inhibition of the RND efflux pumps AcrB and AdeB.

Gram-negative bacterial members of the Resistance Nodulation and cell Division (RND) superfamily form tripartite efflux pump systems that span the cell envelope. One of the intriguing features of the multiple drug efflux members of this superfamily is their ability to recognize different classes of antibiotics, dyes, solvents, bile salts, and detergents. This review provides an overview of the molecular mechanisms of multiple drug efflux catalysed by the tripartite RND efflux system AcrAB-TolC from Eschericha coli. The determinants for sequential or simultaneous multiple substrate binding and efflux pump inhibitor binding are discussed. A comparison is made with the determinants for substrate binding of AdeB from Acinetobacter baumannii, which acts within the AdeABC multidrug efflux system. There is an apparent general similarity between the structures of AcrB and AdeB and their substrate specificity. However, the presence of distinct conformational states and different drug efflux capacities as revealed by single-particle cryo-EM and mutational analysis suggest that the drug binding and transport features exhibited by AcrB may not be directly extrapolated to the homolog AdeB efflux pump.

Mobility and disorder in antibody and antigen binding sites do not prevent immunochemical recognition.

The known polyspecificity of antibodies, which is crucial for efficient immune response, is determined by the conformational flexibility and intrinsic disorder encoded in local peculiarities of the amino acid sequence of antibodies within or in the vicinity of their complementarity determining regions. Similarly, epitopes represent fuzzy binding sites, which are also characterized by local structural flexibility. Existing data suggest that the efficient interactions between antigens and antibodies rely on the conformational mobility and some disorder of their binding sites and therefore can be relatively well described by the "flexible lock - adjustable key" model, whereas both, extreme order (rigid lock-and-key) and extreme disorder (viral shape-shifters) are not compatible with the efficient antigen-antibody interactions and are not present in immune interactions.

Antigen binding by conformational selection in near-germline antibodies.

Conformational flexibility in antibody-combining sites has been hypothesized to facilitate polyspecificity toward multiple unique epitopes and enable the limited germline repertoire to match an overwhelming diversity of potential antigens; however, elucidating the mechanisms of antigen recognition by flexible antibodies has been understandably challenging. Here, multiple liganded and unliganded crystal structures of the near-germline anticarbohydrate antibodies S25-2 and S25-39 are reported, which reveal an unprecedented diversity of complementarity-determining region H3 conformations in apparent equilibrium. These structures demonstrate that at least some germline or near-germline antibodies are flexible entities sensitive to their chemical environments, with conformational selection available as an evolved mechanism that preserves the inherited ability to recognize common pathogens while remaining adaptable to new threats.

Induced Polyspecificity of Human Secretory Immunoglobulin A Antibodies: Is It Possible to Improve Their Ability to Bind Pathogens?

INTRODUCTION: As has been shown previously, various protein-modifying agents can change the antigen-binding properties of immunoglobulins. However, induced polyspecificity of human secretory immunoglobulin A (sIgA) has not been previously characterized in detail. METHODS: In the present study, human secretory immunoglobulin A (IgA) was exposed to buffers with acidic pH, to free heme, or to pro-oxidative ferrous ions, and the antigen-binding behavior of the native and modified IgA to viral and bacterial antigens was compared using Western blotting and enzyme-linked immunosorbent assay. The ability of these agents to modulate the antigen-binding properties of human sIgA toward a wide range of pathogen peptides was investigated using an epitope microarray. RESULTS: We have shown that acidic pH, heme, and pro-oxidative ferrous ions influenced the binding of secretory IgA in opposite directions (either increasing or decreasing); however, the strongest effect was observed when using buffers with low pH. This fraction had the highest number of affected reactivities; most of them were increased and most of the new ones were toward common pathogens. CONCLUSIONS: Thus, it was shown that all investigated treatments can alter to some degree the antigen-binding of secretory IgA, but acidic pH has the most potentially beneficial effect by increasing binding to a largest number of common pathogens' antigens.

A New in Silico Antibody Similarity Measure Both Identifies Large Sets of Epitope Binders with Distinct CDRs and Accurately Predicts Off-Target Reactivity.

Developing a therapeutic antibody is a long, tedious, and expensive process. Many obstacles need to be overcome, such as biophysical properties (issues of solubility, stability, weak production yields, etc.), as well as cross-reactivity and subsequent toxicity, which are major issues. No in silico method exists today to solve such issues. We hypothesized that if we were able to properly measure the similarity between the CDRs of antibodies (Ab) by considering not only their evolutionary proximity (sequence identity) but also their structural features, we would be able to identify families of Ab recognizing similar epitopes. As a consequence, Ab within the family would share the property to recognize their targets, which would allow (i) to identify off-targets and forecast the cross-reactions, and (ii) to identify new Ab specific for a given target. Testing our method on 238D2, an antagonistic anti-CXCR4 nanobody, we were able to find new nanobodies against CXCR4 and to identify influenza hemagglutinin as an off-target of 238D2.

Physicochemical Rules for Identifying Monoclonal Antibodies with Drug-like Specificity.

The ability of antibodies to recognize their target antigens with high specificity is fundamental to their natural function. Nevertheless, therapeutic antibodies display variable and difficult-to-predict levels of nonspecific and self-interactions that can lead to various drug development challenges, including antibody aggregation, abnormally high viscosity, and rapid antibody clearance. Here we report a method for predicting the overall specificity of antibodies in terms of their relative risk for displaying high levels of nonspecific or self-interactions at physiological conditions. We find that individual and combined sets of chemical rules that limit the maximum and minimum numbers of certain solvent-exposed amino acids in antibody variable regions are strong predictors of specificity for large panels of preclinical and clinical-stage antibodies. We also demonstrate how the chemical rules can be used to identify sites that mediate nonspecific interactions in suboptimal antibodies and guide the design of targeted sublibraries that yield variants with high antibody specificity. These findings can be readily used to improve the selection and engineering of antibodies with drug-like specificity.

Toward Drug-Like Multispecific Antibodies by Design.

The success of antibody therapeutics is strongly influenced by their multifunctional nature that couples antigen recognition mediated by their variable regions with effector functions and half-life extension mediated by a subset of their constant regions. Nevertheless, the monospecific IgG format is not optimal for many therapeutic applications, and this has led to the design of a vast number of unique multispecific antibody formats that enable targeting of multiple antigens or multiple epitopes on the same antigen. Despite the diversity of these formats, a common challenge in generating multispecific antibodies is that they display suboptimal physical and chemical properties relative to conventional IgGs and are more difficult to develop into therapeutics. Here we review advances in the design and engineering of multispecific antibodies with drug-like properties, including favorable stability, solubility, viscosity, specificity and pharmacokinetic properties. We also highlight emerging experimental and computational methods for improving the next generation of multispecific antibodies, as well as their constituent antibody fragments, with natural IgG-like properties. Finally, we identify several outstanding challenges that need to be addressed to increase the success of multispecific antibodies in the clinic.

Structures of the Multidrug Transporter P-glycoprotein Reveal Asymmetric ATP Binding and the Mechanism of Polyspecificity.

P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancer; it plays important roles in determining the pharmacokinetics of many drugs. Understanding the structural basis of P-gp, substrate polyspecificity has been hampered by its intrinsic flexibility, which is facilitated by a 75-residue linker that connects the two halves of P-gp. Here we constructed a mutant murine P-gp with a shortened linker to facilitate structural determination. Despite dramatic reduction in rhodamine 123 and calcein-AM transport, the linker-shortened mutant P-gp possesses basal ATPase activity and binds ATP only in its N-terminal nucleotide-binding domain. Nine independently determined structures of wild type, the linker mutant, and a methylated P-gp at up to 3.3 Å resolution display significant movements of individual transmembrane domain helices, which correlated with the opening and closing motion of the two halves of P-gp. The open-and-close motion alters the surface topology of P-gp within the drug-binding pocket, providing a mechanistic explanation for the polyspecificity of P-gp in substrate interactions.

Arginine mutations in antibody complementarity-determining regions display context-dependent affinity/specificity trade-offs.

Antibodies commonly accumulate charged mutations in their complementarity-determining regions (CDRs) during affinity maturation to enhance electrostatic interactions. However, charged mutations can mediate non-specific interactions, and it is unclear to what extent CDRs can accumulate charged residues to increase antibody affinity without compromising specificity. This is especially concerning for positively charged CDR mutations that are linked to antibody polyspecificity. To better understand antibody affinity/specificity trade-offs, we have selected single-chain antibody fragments specific for the negatively charged and hydrophobic Alzheimer's amyloid ß peptide using weak and stringent selections for antibody specificity. Antibody variants isolated using weak selections for specificity were enriched in arginine CDR mutations and displayed low specificity. Alanine-scanning mutagenesis revealed that the affinities of these antibodies were strongly dependent on their arginine mutations. Antibody variants isolated using stringent selections for specificity were also enriched in arginine CDR mutations, but these antibodies possessed significant improvements in specificity. Importantly, the affinities of the most specific antibodies were much less dependent on their arginine mutations, suggesting that over-reliance on arginine for affinity leads to reduced specificity. Structural modeling and molecular simulations reveal unique hydrophobic environments near the arginine CDR mutations. The more specific antibodies contained arginine mutations in the most hydrophobic portions of the CDRs, whereas the less specific antibodies contained arginine mutations in more hydrophilic regions. These findings demonstrate that arginine mutations in antibody CDRs display context-dependent impacts on specificity and that affinity/specificity trade-offs are governed by the relative contribution of arginine CDR residues to the overall antibody affinity.

Few Conserved Amino Acids in the Small Multidrug Resistance Transporter EmrE Influence Drug Polyselectivity.

EmrE is the archetypical member of the small multidrug resistance transporter family and confers resistance to a wide range of disinfectants and dyes known as quaternary cation compounds (QCCs). The aim of this study was to examine which conserved amino acids play an important role in substrate selectivity. On the basis of a previous analysis of EmrE homologues, a total of 33 conserved residues were targeted for cysteine or alanine replacement within E. coli EmrE. The antimicrobial resistance of each EmrE variant expressed in Escherichia coli strain JW0451 (lacking dominant pump acrB) to a collection of 16 different QCCs was tested using agar spot dilution plating to determine MIC values. The results determined that only a few conserved residues were drug polyselective, based on ≥4-fold decreases in MIC values: the active-site residue E14 (E14D and E14A) and 4 additional conserved residues (A10C, F44C, L47C, W63A). EmrE variants I11C, V15C, P32C, I62C, L93C, and S105C enhanced resistance to polyaromatic QCCs, while the remaining EmrE variants reduced resistance to one or more QCCs with shared chemical features: acylation, tri- and tetraphenylation, aromaticity, and dicationic charge. Mapping of EmrE variants onto transmembrane helical wheel projections using the highest resolved EmrE structure suggests that polyselective EmrE variants were located closest to the helical faces surrounding the predicted drug binding pocket, while EmrE variants with greater drug specificity mapped onto distal helical faces. This study reveals that few conserved residues are essential for drug polyselectivity and indicates that aromatic QCC selection involves a greater portion of conserved residues than that in other QCCs.

Myelin oligodendrocyte glycoprotein induces incomplete tolerance of CD4(+) T cells specific for both a myelin and a neuronal self-antigen in mice.

T-cell polyspecificity, predicting that individual T cells recognize a continuum of related ligands, implies that multiple antigens can tolerize T cells specific for a given self-antigen. We previously showed in C57BL/6 mice that part of the CD4(+) T-cell repertoire specific for myelin oligodendrocyte glycoprotein (MOG) 35-55 also recognizes the neuronal antigen neurofilament medium (NF-M) 15-35. Such bi-specific CD4(+) T cells are frequent and produce inflammatory cytokines after stimulation. Since T cells recognizing two self-antigens would be expected to be tolerized more efficiently, this finding prompted us to study how polyspecificity impacts tolerance. We found that similar to MOG, NF-M is expressed in the thymus by medullary thymic epithelial cells, a tolerogenic population. Nevertheless, the frequency, phenotype, and capacity to transfer experimental autoimmune encephalomyelitis (EAE) of MOG35-55 -reactive CD4(+) T cells were increased in MOG-deficient but not in NF-M-deficient mice. We found that presentation of NF-M15-35 by I-A(b) on dendritic cells is of short duration, suggesting unstable MHC class II binding. Consistently, introducing an MHC-anchoring residue into NF-M15-35 (NF-M15-35 T20Y) increased its immunogenicity, activating a repertoire able to induce EAE. Our results show that in C57BL/6 mice bi-specific encephalitogenic T cells manage to escape tolerization due to inefficient exposure to two self-antigens.

Polyspecificity of Anti-lipid A Antibodies and Its Relevance to the Development of Autoimmunity.

The process of natural selection favours germ-line gene segments that encode CDRs that have the ability to recognize a range of structurally related antigens. This presents an immunological advantage to the host, as it can confer protection against a common pathogen and still cope with new or changing antigens. Cross-reactive and polyspecific antibodies also play a central role in autoimmune responses, and a link has been shown to exist between auto-reactive B cells and certain bacterial infections. Bacterial DNA, lipids, and carbohydrates have been implicated in the progression of autoimmune diseases such as systemic lupus erythematosus. As well, reports of anti-lipid A antibody polyspecificity towards single-stranded DNA together with the observed sequence homology amongst isolated auto- and anti-lipid A antibodies has prompted further study of this phenomenon. Though the lipid A epitope appears cryptic during Gram-negative bacterial infection, there have been several reported instances of lipid A-specific antibodies isolated from human sera, some of which have exhibited polyspecificity for single stranded DNA. In such cases, the breakdown of negative selection through polyspecificity can have the unfortunate consequence of autoimmune disease. This review summarizes current knowledge regarding such antibodies and emphasizes the features of S1-15, A6, and S55-5, anti-lipid A antibodies whose structures were recently determined by X-ray crystallography.

Structure-Based Reverse Vaccinology Failed in the Case of HIV Because it Disregarded Accepted Immunological Theory.

Two types of reverse vaccinology (RV) should be distinguished: genome-based RV for bacterial vaccines and structure-based RV for viral vaccines. Structure-based RV consists in trying to generate a vaccine by first determining the crystallographic structure of a complex between a viral epitope and a neutralizing monoclonal antibody (nMab) and then reconstructing the epitope by reverse molecular engineering outside the context of the native viral protein. It is based on the unwarranted assumption that the epitope designed to fit the nMab will have acquired the immunogenic capacity to elicit a polyclonal antibody response with the same protective capacity as the nMab. After more than a decade of intensive research using this type of RV, this approach has failed to deliver an effective, preventive HIV-1 vaccine. The structure and dynamics of different types of HIV-1 epitopes and of paratopes are described. The rational design of an anti-HIV-1 vaccine is shown to be a misnomer since investigators who claim that they design a vaccine are actually only improving the antigenic binding capacity of one epitope with respect to only one paratope and not the immunogenic capacity of an epitope to elicit neutralizing antibodies. Because of the degeneracy of the immune system and the polyspecificity of antibodies, each epitope studied by the structure-based RV procedure is only one of the many epitopes that the particular nMab is able to recognize and there is no reason to assume that this nMab must have been elicited by this one epitope of known structure. Recent evidence is presented that the trimeric Env spikes of the virus possess such an enormous plasticity and intrinsic structural flexibility that it is it extremely difficult to determine which Env regions are the best candidate vaccine immunogens most likely to elicit protective antibodies.

Antibody promiscuity: Understanding the paradigm shift in antigen recognition.

Affinity maturation is associated with reduced malleability of the paratope that optimizes an antibody to bind to the bonafide antigen with high specificity and affinity. However, it has been illustrated that mature antibodies tend to exhibit promiscuity despite acquisition of a relatively rigid binding pocket. Such an attribute is contrary to the established paradigm of specificity in antigen recognition. In this review, an explicit dissection of the underlying mechanisms fostering such versatility in mature antibodies has been done. Polyspecificity is essentially achieved by undergoing minimal structural rearrangement at the paratope complemented with plasticity in interaction with antigen. Besides, the structural invariance of the antigen across species could modulate mature antibody specificity. Polyreactivity has been well documented for germline antibodies as broad spectrum antibody repertoire amplification is primarily governed by recombination event of the genetic machinery, which is further expanded at the structural and functional level of interaction. Degenerate specificity in antigen recognition obviates the need to produce distinct antibody for every incoming epitope.

Specificity, polyspecificity, and heterospecificity of antibody-antigen recognition.

The concept of antibody specificity is analyzed and shown to reside in the ability of an antibody to discriminate between two antigens. Initially, antibody specificity was attributed to sequence differences in complementarity determining regions (CDRs), but as increasing numbers of crystallographic antibody-antigen complexes were elucidated, specificity was analyzed in terms of six antigen-binding regions (ABRs) that only roughly correspond to CDRs. It was found that each ABR differs significantly in its amino acid composition and tends to bind different types of amino acids at the surface of proteins. In spite of these differences, the combined preference of the six ABRs does not allow epitopes to be distinguished from the rest of the protein surface. These findings explain the poor success of past and newly proposed methods for predicting protein epitopes. Antibody polyspecificity refers to the ability of one antibody to bind a large variety of epitopes in different antigens, and this property explains how the immune system develops an antibody repertoire that is able to recognize every antigen the system is likely to encounter. Antibody heterospecificity arises when an antibody reacts better with another antigen than with the one used to raise the antibody. As a result, an antibody may sometimes appear to have been elicited by an antigen with which it is unable to react. The implications of antibody polyspecificity and heterospecificity in vaccine development are pointed out.

Polybodies: Next-generation clinical antibodies.

Conventional antibodies [full-length and fragments: F(ab')2, fragment antigen-binding (Fab), single-chain variable fragment (scFv), variable heavy domain of heavy chain (VHH)] are monospecific first-generation antibodies that have dominated the biopharmaceuticals field. However, advanced protein engineering technology has led to the advent of the next-generation polybody, which is a significant improvement over the conventional antibody. Polybodies comprise polyspecific and polyvalent antibodies that enable a single antibody to target multiple specific antigens simultaneously. Polybodies are superior to first-generation antibodies (more efficacious, broad-spectrum, resistance resistant, customizable, etc.) and provide a cost-effective healthcare solution. This review thoroughly addresses developments in polybodies, highlighting their superiority over conventional antibodies and offering future perspectives to encourage the generation of innovative immunotherapies.

Protein language models enable prediction of polyreactivity of monospecific, bispecific, and heavy-chain-only antibodies.

Background: Early assessment of antibody off-target binding is essential for mitigating developability risks such as fast clearance, reduced efficacy, toxicity, and immunogenicity. The baculovirus particle (BVP) binding assay has been widely utilized to evaluate polyreactivity of antibodies. As a complementary approach, computational prediction of polyreactivity is desirable for counter-screening antibodies from in silico discovery campaigns. However, there is a lack of such models. Methods: Herein, we present the development of an ensemble of three deep learning models based on two pan-protein foundational protein language models (ESM2 and ProtT5) and an antibody-specific protein language model (PLM) (Antiberty). These models were trained in a transfer learning network to predict the outcomes in the BVP assay and the bovine serum albumin binding assay, which was developed as a complement to the BVP assay. The training was conducted on a large dataset of antibody sequences augmented with experimental conditions, which were collected through a highly efficient application system. Results: The resulting models demonstrated robust performance on canonical mAbs (monospecific with heavy and light chain), bispecific Abs, and single-domain Fc (VHH-Fc). PLMs outperformed a model built using molecular descriptors calculated from AlphaFold 2 predicted structures. Embeddings from the antibody-specific and foundational PLMs resulted in similar performance. Conclusion: To our knowledge, this represents the first application of PLMs to predict assay data on bispecifics and VHH-Fcs.

Broad-spectrum defenders: γδ T cells take on a multitude of immune challenges.

A recent article in Proceedings of the National Academy of Sciences investigated γδ T cell antigen specificity in mice and humans, in which the authors show that γδ T cell antigen specificity is not constrained to one epitope. Rather, γδ T cells recognize a broad range of diverse antigens containing similar chemical structures or properties. In this News and Views, the importance of γδ T cell antigen polyspecificity during immune responses is highlighted.