Academic literature on the topic 'FAB-ANTIGENS COMPLEX STRUCTURE'

The vast majority of platforms for the detection of viral or bacterial antigens rely on immunoassays, typically ELISA or sandwich ELISA, that are contingent on the availability of suitable monoclonal antibodies (mAbs). This is a major bottleneck, since the generation and production of mAbs is time-consuming and expensive. Synthetic antibody fragments (sFabs) generated by phage-display selection offer an alternative with many advantages over Fabs obtained from natural antibodies using hybridoma technology. Unlike mAbs, sFabs are generated using phage display, allowing selection for binding to specific strains or for pan-specificity, for identification of structural epitopes or unique protein conformations and even for complexes. Further, they can easily be produced in Escherichia coli in large quantities and engineered for purposes of detection technologies and other applications. Here, the use of phage-display selection to generate a pan-specific Fab (MJ20), based on a Herceptin Fab scaffold, with the ability to bind selectively and with high affinity to the C-terminal domains of the nucleoproteins (NPs) from all five known strains of the Ebola virus is reported. The high-resolution crystal structure of the complex of MJ20 with the antigen from the Bundibugyo strain of the Ebola virus reveals the basis for pan-specificity and illustrates how the phage-display technology can be used to manufacture suitable Fabs for use in diagnostic or therapeutic applications.

ABSTRACT The three-dimensional structures of the Fab fragment of a neutralizing antibody raised against a foot-and-mouth disease virus (FMDV) of serotype C1, alone and complexed to an antigenic peptide representing the major antigenic site A (G-H loop of VP1), have been determined. As previously seen in a complex of the same antigen with another antibody which recognizes a different epitope within antigenic site A, the receptor recognition motif Arg-Gly-Asp and some residues from an adjacent helix participate directly in the interaction with the complementarity-determining regions of the antibody. Remarkably, the structures of the two antibodies become more similar upon binding the peptide, and both undergo considerable induced fit to accommodate the peptide with a similar array of interactions. Furthermore, the pattern of reactivities of five additional antibodies with versions of the antigenic peptide bearing amino acid replacements suggests a similar pattern of interaction of antibodies raised against widely different antigens of serotype C. The results reinforce the occurrence of a defined antigenic structure at this mobile, exposed antigenic site and imply that intratypic antigenic variation of FMDV of serotype C is due to subtle structural differences that affect antibody recognition while preserving a functional structure for the receptor binding site.

Abstract The vast majority of antigen-antibody interactions rely upon binding to conformational epitopes, which are composed of amino acids that are discontinuous in the protein sequence but are brought together upon three-dimensional protein folding. This character has made conformational epitopes particularly difficult to map because they are only formed in the native structure of the antigen protein. X-ray crystallography is a gold-standard method for mapping conformational epitopes at high-resolution, but it only works for highly purified antigens and is time consuming, and is not applicable when crystals of antigen-antibody complex are unavailable. Here we present a novel middle-down HDX-MS technology with subzero temperature separation that effectively tackles these limitations. It successfully mapped the conformational epitopes in a VPF growth factor targeted by a therapeutic antibody, in a complex protein mixture where the antigen content is as low as 10%. It provided single amino acid resolution by online gas-phase fragmentation on the LC-MS time scale. It also uses directly the whole antibody, pre-digestion of the antibody to Fab fragments is no longer needed. In conclusion, the subzero temperature separation based HDX-MS technology represents an alternative high-resolution epitope mapping approach to X-ray crystallography, and is also a fast and cost-effective solution for new antibody and vaccine development. Its potential in paratope mapping, as well as epitope mapping for other difficult targets and bispecific antibodies will be discussed.

Antibodies classically bind antigens via their complementarity-determining regions, but an alternative mode of interaction involving V-domain framework regions has been observed for some B cell “superantigens.” We report the crystal structure of an antibody employing both modes of interaction simultaneously and binding two antigen molecules. This human antibody from an allergic individual binds to the grass pollen allergen Phl p 7. Not only are two allergen molecules bound to each antibody fragment (Fab) but also each allergen molecule is bound by two Fabs: One epitope is recognized classically, the other in a superantigen-like manner. A single allergen molecule thus cross-links two identical Fabs, contrary to the one-antibody–one-epitope dogma, which dictates that a dimeric allergen at least is required for this to occur. Allergens trigger immediate hypersensitivity reactions by cross-linking receptor-bound IgE molecules on effector cells. We found that monomeric Phl p 7 induced degranulation of basophils sensitized solely with this monoclonal antibody expressed as an IgE, demonstrating that the dual specificity has functional consequences. The monomeric state of Phl p 7 and two structurally related allergens was confirmed by size-exclusion chromatography and multiangle laser light scattering, and the results were supported by degranulation studies with the related allergens, a second patient-derived allergen-specific antibody lacking the nonclassical binding site, and mutagenesis of the nonclassically recognized allergen epitope. The antibody dual reactivity and cross-linking mechanism not only have implications for understanding allergenicity and allergen potency but, importantly, also have broader relevance to antigen recognition by membrane Ig and cross-linking of the B cell receptor.

Abstract Introduction Mature T cell lymphomas are aggressive, treatment resistant cancers that are associated with poor prognosis. Clinical application of immunotherapeutic approaches has been limited by a lack of target antigens that discriminate malignant from healthy T cells. Unlike B cell depletion, pan-T cell aplasia is prohibitively toxic. Previously we reported a targeting strategy based on the mutually exclusive expression of T cell receptor beta-chain constant domains 1 and 2 (TRBC1 and TRBC2). We identified an antibody with unique TRBC1 specificity and demonstrated that anti TRBC1 chimeric antigen receptor (CAR) T cells can ablate cells expressing TRBC1 TCRs while sparing those expressing TRBC2 TCRs. A phase I clinical study investigating the efficacy of our TRBC1 CAR is ongoing. T cell malignancies are clonal, and the ratio of TRBC2 to TRBC1 expressing lymphoma cases is predicted to be approximately 2:1. To treat all cases of T cell lymphoma, a CAR that targets TRBC2 is needed. TRBC1 and 2 are highly homologous. Structural studies suggest that amino acid inversions at positions 4 and 5 of the constant beta chain provide an accessible discriminating portion between the two proteins. Given the structural similarities between TRBC1 and TRBC2 and our characterized binder against TRBC1, we explored generating antibodies with specificity towards TRBC2 via a structure guided computational biology approach; engineering the previously identified TRBC1 antibody and reversing its specificity such that it recognised TRBC2. Results The crystal structure of the TRBC1 specific monoclonal antibody was solved in complex with a TRBC1-TCR to 2.4Å, Figure 1. Through computational biology and protein engineering we rationally designed a mutant version of TRBC1 binder that was specific for TRBC2 and had a 1000 fold decreased affinity towards TRBC1. Flow cytometry analysis of the TRBC2 specific antibody demonstrated the ability to bind to T-cells expressing TRBC2 TCRs. We further showed that the engineered antibody retained favourable biophysical characteristics with high stability (Fab Tm > 65oC) and low aggregation propensity (>99% monomer). We used the engineered monoclonal antibody to generate a 2nd generation anti-TRBC2 CAR. We demonstrated that our anti-TRBC2 CAR showed specificity, cytokine release and cytotoxicity in 72hr co-cultures against TRBC2+ cell lines but not TRBC1+ cell lines or cell lines that did not express TCR on the surface. Anti-TRBC2 CAR T-cells also demonstrated proliferative capacity in long-term co-culture assays. Conclusions We have utilised structural biology and rational protein design to generate CAR-T cells capable of specifically targeting TRBC2. The combination of TRBC1 and 2 targeting CAR-T cell products with a patient stratification companion diagnostic assay offers a therapeutic strategy for the treatment of a wide range of, otherwise untreatable, T-cell lymphomas. Figure 1. A. Structural interface between TCR Beta and Fab fragment of TRBC1 specific antibody. B and C. CDR fold of TRBC1 binder and 90o rotation. Disclosures Onuoha: Autolus Ltd: Employment, Equity Ownership, Patents & Royalties. Ferrari:Autolus Ltd: Employment, Equity Ownership. Bulek:Autolus Ltd: Employment, Equity Ownership, Patents & Royalties. Bughda:Autolus Ltd: Employment, Equity Ownership, Patents & Royalties. Manzoor:Autolus Ltd: Employment, Equity Ownership, Patents & Royalties. Srivastava:Autolus Ltd: Employment, Equity Ownership. Ma:Autolus Ltd: Employment. Karattil:Autolus Ltd: Employment, Equity Ownership, Patents & Royalties. Kinna:Autolus Ltd: Employment, Equity Ownership. Thomas:Autolus Ltd: Employment, Equity Ownership, Patents & Royalties. Cordoba:Autolus Ltd: Employment; Autolus Ltd: Patents & Royalties; Autolus Ltd: Equity Ownership. Maciocia:Autolus: Equity Ownership, Patents & Royalties: UCLB. Pule:Autolus Ltd: Employment, Equity Ownership, Other: Salary contribution paid for by Autolus, Research Funding; University College London: Patents & Royalties: Patent with rights to Royalty share through UCL.

The T cell receptor alpha/beta (TCR-alpha/beta) is encoded by variable (V), diversity (D), joining (J), and constant (C) segments assembled by recombination during thymocyte maturation to produce a heterodimer that imparts antigenic specificity to the T cell. Unlike immunoglobulins (Igs), which bind free antigen, the ligands of TCR-alpha/beta are cell surface complexes of intracellularly degraded antigens (i.e., peptides) bound to and presented by polymorphic products of the major histocompatibility complex (MHC). Therefore, antigen recognition by T cells is defined as MHC restricted. A model has been formulated based upon the similarity between TCR-alpha/beta V region and Ig Fab amino acid sequences, and the crystal structure of the MHC class I and Ig molecules. This model predicts that the complementarity determining regions (CDR) 1 and 2, composed of TCR V alpha and V beta segments, primarily contact residues of the MHC alpha helices, whereas V/J alpha and V/D/J beta junctional regions (the CDR3 equivalent) contact the peptide in the MHC binding groove. Because polymorphism in MHC proteins is limited relative to the enormous diversity of antigenic peptides, the TCR may have evolved to position the highly diverse junctional residues (CDR3), where they have maximal contact with antigen bound in the MHC peptide groove. Here, we demonstrate a definitive association between CDR3 sequences in both TCR alpha and beta chains, and differences in recognition of antigen fine specificity using a panel of I-Ed-restricted, myoglobin-reactive T cell clones. Acquisition of these data relied in part upon a modification of the polymerase chain reaction that uses a degenerate, consensus primer to amplify TCR alpha chains without foreknowledge of the V alpha segments they utilize.

Abstract Chimeric antigen receptor (CAR) T cells have shown promising results for the treatment of blood cancers and various CAR-T cell approaches are in development for use in different tumor indications. Universal or modular CARs do not directly recognize the tumor target antigen, but bind via an adaptor molecule to their respective tumor target. We describe the P329G-CAR-T platform as a novel modular CAR-T cell platform that recognizes the P329G mutation in the Fc portion of IgG1 antibodies, a mutation frequently applied to abolish the Fc immune effector function of therapeutic antibodies. In contrast to other adaptor CAR-T cell platforms this approach does not rely on haptens or artificial tags fused to the targeting antibody.The crystal structure analysis of the anti-P329G Fab fragment in complex with a P329G-Fc portion showed that the Fab fragment recognizes the Fc mutation with 1:1 binding stoichiometry. Surface plasmon resonance analysis determined the equilibrium binding affinity of the P329G antibody to the P329G Fc-portion to be 15 nM. Cell assays using Jurkat-NFAT reporter cell lines and primary T cells transduced with the P329G-CAR showed specific recruitment of P329G CAR-T cells by P329G-containing antibodies, and potent and dose dependent tumor cell killing accompanied by IFNg release and subsequent T cell activation for several unrelated tumor antigens including CD20, CD33, HER2, FOLR1 and mesothelin. Notably, P329G-CAR-T cell killing activity was comparable to the activity of the respective direct scFv-based CAR-T cells, both in terms of kinetics and absolute killing potency. Finally, comparable activity was determined in comparison to CD16 extracellular domain (ECD)-based CAR-T cells engaging the CAR-T cells via the Fc-CD16-ECD interaction. In summary, P329G-CAR-T cells mediate potent tumor cell killing in combination with various tumor targeting antibodies as adaptor molecules. The combination with tumor targeting antibodies enables control of CAR-T activity by adjusting the dose and schedule of the respective antibody adaptor molecule. Importantly, different from CD16-ECD-based CAR-T cells, P329G-CAR-T cells cannot be engaged by endogenous immunoglobulins. In vivo studies to investigate efficacy and safety of P329G CAR-T cells are currently being completed and will be reported. Citation Format: Diana Darowski, Mohamed-Reda Benmebarek, Christian Jost, Kay Stubenrauch, Uwe Wessels, Joerg Benz, Anne Freimoser-Grundschober, Ekkehard Moessner, Pablo Umana, Sebastian Kobold, Christian Klein. Developing a novel adaptor CAR-T cell platform based on the recognition of the P329G Fc mutation in therapeutic IgG1 antibodies for adoptive T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 570.

Abstract Humoral immunity allows the body to mount a defense against pathogens and foreign substances, and to respond with memory to subsequent exposures. The molecular participants may also recognize self-structures, leading to attack on the body and autoimmune disease. The main players in humoral immunity are antibody-producing B lymphocytes, and several classes of T lymphocytes. This review deals with the molecular details of recognition of antigens by soluble antibodies, and of substances presented to receptors on the surfaces of T cells (TCRs). The prototype antibody consists of a dimer of dimers, two heavy (H) chains and two light (L) chains, with antigen recognition capacity lying in variable “head” regions of an H-L pair. Most crystallographic studies are done with this substructure, called a Fab fragment, bound in a soluble antigen complex. Homologous to this arrangement, the prototype TCR consists of two chains (α and β) that complex not soluble antigen, but usually a short peptide or other small molecule presented by proteins of the major histocompatibility complex. In each case a general background on the historical development of understanding the molecular recognition interface is given, followed by a number of examples of crystal structures from the recent literature that have allowed us to refine our understanding of the complex recognition process. Variations on the prototypical structures are also considered. The spectrum of recognition strategies involves interplay of lock-and-key with flexibility, varying degrees of entropic and enthalpic contributions, surface shaping by entrapped water molecules, and combinations of stabilizing hydrogen bonding, electrostatic interactions, salt bridging, and van der Waals forces. Preeminent in the recent literature are details of antibody binding to influenza A and human immunodeficiency viral antigens. Both viral antigens and attempts to understand autoimmunity are prominent in the recent TCR literature.

Complexes formed by binding 125I- or 3H-labeled neuropeptides to one of the two binding sites of their specific antibodies allowed specific and sensitive labeling of various peptidergic neurons, which could be detected by classical autoradiographic methods. To visualize two neuronal antigens on the same material at both light and electron microscopic level, we used a new technique of double immunocytochemical labeling, combining immunoperoxidase and radioimmunocytochemistry. The main steps of the process included: (a) indirect labeling of the first antigen by its specific antibody and by a peroxidase-labeled Fab immunoglobulin fragment directed against the primary antibody; (b) direct labeling of the second antigen by a radiolabeled peptide-antibody complex; (c) revealing of the first label in the presence of peroxidase substrate; and (d) revealing of the second label by autoradiographic treatment of tissue sections. Compared with other known techniques of double immunostaining, this technique offers major advantages for combined visualization of two neuronal antigens at the electron microscopic level: (a) two neuron types can be labeled by a pre-embedding approach, allowing highly sensitive detection of neuronal antigens throughout the 50-microns thickness of vibratome sections; (b) two primary antibodies obtained in the same species can be used to label the two antigens without any risk of crossreactions between the two successive labelings; and (c) the two labels can easily be differentiated, even when they are co-localized within the same neuron structures. Application of this double immunostaining technique is illustrated by data obtained in rat hypothalamus concerning the relationships among a variety of identified neurons and the co-localization of different neuropeptides within the same neuron system.

By using X -ray diffraction and immunochemical techniques, we have exploited the use of monoclonal antibodies raised against hen egg lysozyme (HEL) to study systematically those factors responsible for the high specificity of antigen -antibody interactions. HEL was chosen for our investigations because its three-dimensional structure and immunochemistry have been well characterized and because naturally occurring sequence variants from different avian species are readily available to test the fine specificity of the antibodies. The X-ray crystal structure of a complex formed between HEL and the Fab D1.3 shows a large complementary surface with close interatomic contacts between antigen and antibody. Thus single amino acid sequence changes in heterologous antigens give antigen-antibody association constants that are several orders of magnitude smaller than that of the homologous antigen. For example, a substitution of His for Glu at position 121 in the antigen is sufficient to diminish significantly the binding between D1.3 and the variant lysozyme. The conformation of HEL when complexed to D1.3 shows no significant difference from that seen in the free molecule, and immunobinding studies with other anti-HEL antibodies suggest that this observation may be generally true for the system of monoclonal antibodies that we have studied.

The multi-component vaccine 4CMenB (Bexsero) is the first recombinant vaccine for the prevention of invasive meningococcal disease licensed in over 35 countries. The meningococcal factor H binding protein (fHbp), one of three main protein antigens in 4CMenB, specifically binds human factor H (hfH) and thereby down-regulates the human complement cascade. Over 1000 distinct amino acid sequences of fHbp have been identified and can be classified in three variants which are immunologically distinct. Within the fHbp variant groups the sequence identity is usually above 87%, while between variant groups the sequence identity can be as low as 62%. This high antigenic variability presumably underlies the apparent rarity of cross-reactive antibodies. We cloned and expressed a library of 110 anti-fHbp human monoclonal antibodies as Fabs (huFab), isolated from adults immunized with 4CMenB. We analyzed their antigen binding specificity and affinity, and their ability to inhibit binding of hfH to live meningococci. Interestingly, although 4CMenB vaccine contains only fHbp variant 1.1, thirteen of the 110 huFabs were found to be cross-reactive for fHbp variants 2 and 3. All 13 cross-reactive mAbs showed to be able to kill the bacterium when tested in a serum bactericidal assay using baby rabbit serum as complement source (rSBA); only those 3 mAbs competing with hfH binding showed bactericidial activity also with human complement (hSBA). The crystal structure of the fHbp/huFab 4B3 complex was determined and the identification of key antigen/antibody interactions was performed by structural analyses, obtaining atomic insights into the epitope recognized by this huFab. A detailed analysis of the interface revealed high conservation of key epitope residues some of which lie within the hFH binding site, which explains both the ability of huFab 4B3 to cross-react with different fHbp variants and its ability to inhibit hfH binding, thus explaining its unparalleled potency. In conclusion, we have obtained the first detailed characterization of cross-protective huMabs elicited by 4CMenB and in particular we gained structural data on huFab 4B3, which is able to compete for hFh binding to fHbp. These structural and functional data suggest that the hfH binding site on fHbp can be accessible to the human immune system upon immunization, enabling the generation of a highly potent antibody response