Multi-Task Learning with Loop Specific Attention for CDR Structure Prediction

The Complementarity Determining Region (CDR) structure prediction of loops in antibody engineering has gained a lot of attraction by researchers. When designing antibodies, a main challenge is to predict the CDR structure of the H3 loop. Compared with the other CDR loops, that is the H1 and H2 loops, the CDR structure of the H3 loop is more challenging due to its varying length and flexible structure. In this paper, we propose a Multi-task learning model with Loop Specific Attention, namely MLSA. In particular, to the best of our knowledge we are the first to jointly learn the three CDR loops, via a novel multi-task learning strategy. In addition, to account for the structural and functional similarities and differences of the three CDR loops, we propose a loop specific attention mechanism to control the influence of each CDR loop on the training of MLSA. Our experimental evaluation on widely used benchmark data shows that the proposed MLSA method significantly reduces the prediction error of the CDR structure of the H3 loop, by at least 19%, when compared with other baseline strategies. Finally, for reproduction purposes we make the implementation of MLSA publicly available at https://anonymous.4open.science/r/MLSA-2442/.Comment: 8 page

Structural aspects of molecular recognition in the immune system. Part I: Acquired immunity (IUPAC Technical Report)

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 literatur

Impact of Tryptophan Oxidation in Complementarity-Determining Regions of Two Monoclonal Antibodies on Structure-Function Characterized by Hydrogen-Deuterium Exchange Mass Spectrometry and Surface Plasmon Resonance

This work is licensed under a Creative Commons Attribution 4.0 International License.Purpose Tryptophan’s (Trp) unique hydrophobic and structural properties make it an important antigen binding motif when positioned in complementarity-determining regions (CDRs) of monoclonal antibodies (mAbs). Oxidation of Trp residues within the CDR can deleteriously impact antigen binding, particularly if the CDR conformation is altered. The goal of this study was to evaluate the conformational and functional impact of Trp oxidation for two mAb subtypes, which is essential in determining the structure-function relationship and establishing appropriate analytical control strategies during protein therapeutics development. Methods Selective Trp oxidation was induced by 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH) treatment in the presence of free methionine (Met). The native and chemically oxidized mAbs were characterized by hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational changes and surface plasmon resonance (SPR) for antigen-antibody binding. Results Treatment of mAbs with AAPH selectively oxidized solvent accessible Trp residues. Oxidation of Trp within or in proximity of CDRs increased conformational flexibility in variable domains and disrupted antigen binding. Conclusions Trp oxidation in CDRs can adversely impact mAbs’ conformation and antigen binding. Trp oxidation should be carefully evaluated as part of critical quality attribute assessments. Oxidation susceptible Trp should be closely monitored during process development for mAbs to establish appropriate analytical control for manufacturing of drug substance and drug product

Molecular insights of nickel binding to therapeutic antibodies as a possible new antibody superantigen

The binding of nickel by immune proteins can manifest as Type IV contact dermatitis (Ni-specific T cells mediated) and less frequently as Type I hypersensitivity with both mechanisms remaining unknown to date. Since there are reports of patients co-manifesting the two hypersensitivities, a common mechanism may underlie both the TCR and IgE nickel binding. Focusing on Trastuzumab and Pertuzumab IgE variants as serendipitous investigation models, we found Ni-NTA interactions independent of Her2 binding to be due to glutamine stretches. These stretches are both Ni-inducible and in fixed pockets at the antibody complementarity-determining regions (CDRs) and framework regions (FWRs) of both the antibody heavy and light chains with influence from the heavy chain constant region. Comparisons with TCRs structures revealed similar interactions, demonstrating the possible underlying mechanism in selecting for Ni-binding IgEs and TCRs respectively. With the elucidation of the interaction, future therapeutic antibodies could also be sagaciously engineered to utilize such nickel binding for biotechnological purposes

Bowties, Barcodes, and DNA Origami; A Novel Approach for Paired-Chain Immune Receptor Repertoire Analysis

abstract: There are many biological questions that require single-cell analysis of gene sequences, including analysis of clonally distributed dimeric immunoreceptors on lymphocytes (T cells and B cells) and/or the accumulation of driver/accessory mutations in polyclonal tumors. Lysis of bulk cell populations results in mixing of gene sequences, making it impossible to know which pairs of gene sequences originated from any particular cell and obfuscating analysis of rare sequences within large populations. Although current single-cell sorting technologies can be used to address some of these questions, such approaches are expensive, require specialized equipment, and lack the necessary high-throughput capacity for comprehensive analysis. Water-in-oil emulsion approaches for single cell sorting have been developed but droplet-based single-cell lysis and analysis have proven inefficient and yield high rates of false pairings. Ideally, molecular approaches for linking gene sequences from individual cells could be coupled with next-generation high-throughput sequencing to overcome these obstacles, but conventional approaches for linking gene sequences, such as by transfection with bridging oligonucleotides, result in activation of cellular nucleases that destroy the template, precluding this strategy. Recent advances in the synthesis and fabrication of modular deoxyribonucleic acid (DNA) origami nanostructures have resulted in new possibilities for addressing many current and long-standing scientific and technical challenges in biology and medicine. One exciting application of DNA nanotechnology is the intracellular capture, barcode linkage, and subsequent sequence analysis of multiple messenger RNA (mRNA) targets from individual cells within heterogeneous cell populations. DNA nanostructures can be transfected into individual cells to capture and protect mRNA for specific expressed genes, and incorporation of origami-specific bowtie-barcodes into the origami nanostructure facilitates pairing and analysis of mRNA from individual cells by high-throughput next-generation sequencing. This approach is highly modular and can be adapted to virtually any two (and possibly more) gene target sequences, and therefore has a wide range of potential applications for analysis of diverse cell populations such as understanding the relationship between different immune cell populations, development of novel immunotherapeutic antibodies, or improving the diagnosis or treatment for a wide variety of cancers.Dissertation/ThesisDoctoral Dissertation Microbiology 201

Clonal Progression during the T Cell-Dependent B Cell Antibody Response Depends on the Immunoglobulin DH Gene Segment Repertoire

The diversity of the third complementarity determining region of the IgH chain is constrained by natural selection of immunoglobulin diversity (D(H)) sequence. To test the functional significance of this constraint in the context of thymus-dependent (TD) immune responses, we immunized BALB/c mice with WT or altered D(H) sequence with 2-phenyloxazolone-coupled chicken serum albumin (phOx-CSA). We chose this antigen because studies of the humoral immune response to the hapten phOx were instrumental in the development of the current theoretical framework on which our understanding of the forces driving TD responses is based. To allow direct comparison, we used the classic approach of generating monoclonal Ab (mAb) from various stages of the immune response to phOx to assess the effect of changing the sequence of the D(H) on clonal expansion, class switching, and affinity maturation, which are hallmarks of TD responses. Compared to WT, TD-induced humoral IgM as well as IgG antibody production in the D-altered ΔD-DμFS and ΔD-iD strains were significantly reduced. An increased prevalence of IgM-producing hybridomas from late primary, secondary, and tertiary memory responses suggested either impaired class switch recombination (CSR) or impaired clonal expansion of class switched B cells with phOx reactivity. Neither of the D-altered strains demonstrated the restriction in the V(H)/V(L) repertoire, the elimination of V(H)1 family-encoded antibodies, the focusing of the distribution of CDR-H3 lengths, or the selection for the normally dominant Ox1 clonotype, which all are hallmarks of the anti-phOx response in WT mice. These changes in clonal selection and expansion, as well as CSR indicate that the genetic constitution of the D(H) locus, which has been selected by evolution, can strongly influence the functional outcome of a TD humoral response

Generation of Antibody Diversity

Because of the huge diversity, the immunoglobulin repertoire cannot be encoded by static genes, which would explode the genomic capacity comprising about 20,000–25,000 human genes. The immunoglobulin repertoire is provided by the process of somatic germ line recombination, which is the only controlled alteration of the genomic DNA after meiosis. It takes place in mammalian B lymphocyte (B cells) precursors in the bone marrow. The genome germ line sequence of undeveloped B cells is organized in gene segments and compromise V (variable), D (diversity), and J (joining) gene segments constituting the variable domain of the heavy chain and only V and J genes for building up the variable domain of the light chain. The rearrangement of the variable region follows a strict order. The following processes that participate in the generation of antibody diversity were summarized—allelic, combinational, and junctional diversity, pairing of IgH and IgL, and receptor editing—which all together produce the primary antigen repertoire (pre-antigen stimulation). When a B cell encounters a foreign antigen, affinity maturation and class switch are induced. Thereby the antibody repertoire increases. The resulting secondary immunoglobulin repertoire reveals in humans at least 1011 specificities for different antigens

Linking TCR sequence, structure, and function in the DO11.10 mouse model

Thymus-maturated lymphocytes (T-cells) provide the immune system with the ability to appropriately detect and respond to diseases through a unique receptor. These T-cell receptors (TCRs) differentiate between self and foreign peptide antigens and form by hypervariable gene rearrangement and imprecise recombination, both of which can potentially generate upwards of 10¹⁸ unique TCRs in humans. Our work focused on characterizing the chicken ovalbumin (OVA) antigen specific parent hybridoma DO11.10 T-cell with (1) high-throughput sequencing, (2) structural analyses, and (3) TCR reconstitution into T-cells to determine the important factors linking TCR sequence, structure, and function. The repertoire of TCR sequences generated in mice immunized with OVA antigen were sequenced using a novel mouse TCR gene primer set. Using high-throughput sequencing, we determined the relative gene and hypervariable sequence frequencies in the repertoire. Based on our results in these experiments, no significant repertoire differences were found between mouse treatment groups. Additionally no sequences with similarity to previously studied OVA-specific T-cell clones were recovered. The antigen binding loops of TCRs have a high degree of structural similarity across diverse receptor sequences. Key residues in the complementarity determining, antigen-binding regions of TCRs form conserved canonical loop structures. Here we evaluated 249 TCRs and TCR-like antibodies to determine the prevalence of these key residues. Expanding on previous work, we found four new canonical class conformations and validated the key residues for these classes. Finally, we report our work to reconstitute engineered TCRs onto the TCR-deficient mouse 58 [superscript -/-] hybridoma line. We modified an existing antibody expression system to display the engineered TCRs in the hybridomas for activation assays. Expression of the TCR was found to be highly sensitive to the N-terminal signal sequence on each receptor chain. Additional work began to examine how these signal sequences alter stable expression and surface display of these TCRs. This work is important for the discovery of new methods and biological agents to target and respond to designed antigens, especially in the context of altering and engineering TCR specificity for therapeutic purposes.Chemical Engineerin

A structural basis for selection and cross-species reactivity of the semi-invariant NKT cell receptor in CD1d/glycolipid recognition

Little is known regarding the basis for selection of the semi-invariant αβ T cell receptor (TCR) expressed by natural killer T (NKT) cells or how this mediates recognition of CD1d–glycolipid complexes. We have determined the structures of two human NKT TCRs that differ in their CDR3β composition and length. Both TCRs contain a conserved, positively charged pocket at the ligand interface that is lined by residues from the invariant TCR α- and semi-invariant β-chains. The cavity is centrally located and ideally suited to interact with the exposed glycosyl head group of glycolipid antigens. Sequences common to mouse and human invariant NKT TCRs reveal a contiguous conserved “hot spot” that provides a basis for the reactivity of NKT cells across species. Structural and functional data suggest that the CDR3β loop provides a plasticity mechanism that accommodates recognition of a variety of glycolipid antigens presented by CD1d. We propose a model of NKT TCR–CD1d–glycolipid interaction in which the invariant CDR3α loop is predicted to play a major role in determining the inherent bias toward CD1d. The findings define a structural basis for the selection of the semi-invariant αβ TCR and the unique antigen specificity of NKT cells