RESUMO
Many researchers are interested in the possibility of manipulating the targeting specificity of extracellular vesicles (EVs) for their use as physiological delivery vehicles for drugs and bioactive molecules. Our studies demonstrated the possibility of directing EVs toward the desired acceptor cell by coating them with antigen-specific antibody light chains. Here, we describe the methods for detection of the presence of antibody light chains on the EV surface, proving their ability to specifically bind the antigen and for separating the antigen-binding EV subpopulation.
Assuntos
Antígenos , Vesículas Extracelulares , Cadeias Leves de Imunoglobulina , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/química , Humanos , Cadeias Leves de Imunoglobulina/metabolismo , Cadeias Leves de Imunoglobulina/química , Antígenos/imunologia , Citometria de Fluxo/métodosRESUMO
The antigen-binding sites in conventional antibodies are formed by hypervariable complementarity-determining regions (CDRs) from both heavy chains (HCs) and light chains (LCs). A deviation from this paradigm is found in a subset of bovine antibodies that bind antigens via an ultra-long CDR. The HCs bearing ultra-long CDRs pair with a restricted set of highly conserved LCs that convey stability to the antibody. Despite the importance of these LCs, their specific features remained unknown. Here, we show that the conserved bovine LC found in antibodies with ultra-long CDRs exhibits a distinct combination of favorable physicochemical properties such as good secretion from mammalian cells, strong dimerization, high stability, and resistance to aggregation. These physicochemical traits of the LCs arise from a combination of the specific sequences in the germline CDRs and a lambda LC framework. In addition to understanding the molecular architecture of antibodies with ultra-long CDRs, our findings reveal fundamental insights into LC characteristics that can guide the design of antibodies with improved properties.
Assuntos
Regiões Determinantes de Complementaridade , Cadeias Leves de Imunoglobulina , Animais , Bovinos , Cadeias Leves de Imunoglobulina/genética , Anticorpos , Dimerização , Fenótipo , MamíferosRESUMO
Inhibition of amyloid fibril formation could benefit patients with systemic amyloidosis. In this group of diseases, deposition of amyloid fibrils derived from normally soluble proteins leads to progressive tissue damage and organ failure. Amyloid formation is a complex process, where several individual steps could be targeted. Several small molecules have been proposed as inhibitors of amyloid formation. However, the exact mechanism of action for a molecule is often not known, which impedes medicinal chemistry efforts to develop more potent molecules. Furthermore, commonly used assays are prone to artifacts that must be controlled for. Here, potential mechanisms by which small molecules could inhibit aggregation of immunoglobulin light-chain dimers, the precursor proteins for amyloid light-chain (AL) amyloidosis, are studied in assays that recapitulate different aspects of amyloidogenesis in vitro. One molecule reduced unfolding-coupled proteolysis of light chains, but no molecules inhibited aggregation of light chains or disrupted pre-formed amyloid fibrils. This work demonstrates the challenges associated with drug development for amyloidosis, but also highlights the potential to combine therapies that target different aspects of amyloidosis.
Assuntos
Proteínas Amiloidogênicas/metabolismo , Descoberta de Drogas/métodos , Cadeias Leves de Imunoglobulina/química , Amiloidose de Cadeia Leve de Imunoglobulina/tratamento farmacológico , Proteínas Recombinantes/isolamento & purificação , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Amiloidogênicas/química , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina/metabolismo , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Non-native immunoglobulin light chain conformations, including aggregates, appear to cause light chain amyloidosis pathology. Despite significant progress in pharmacological eradication of the neoplastic plasma cells that secrete these light chains, in many patients impaired organ function remains. The impairment is apparently due to a subset of resistant plasma cells that continue to secrete misfolding-prone light chains. These light chains are susceptible to the proteolytic cleavage that may enable light chain aggregation. We propose that small molecules that preferentially bind to the natively folded state of full-length light chains could act as pharmacological kinetic stabilizers, protecting light chains against unfolding, proteolysis and aggregation. Although the sequence of the pathological light chain is unique to each patient, fortunately light chains have highly conserved residues that form binding sites for small molecule kinetic stabilizers. We envision that such stabilizers could complement existing and emerging therapies to benefit light chain amyloidosis patients.
RESUMO
Antibody light chains (LCs), formerly considered a waste product of immunoglobulin synthesis, are currently recognized as important players in the activation of the immune response. However, very little is known about the possible immune regulatory functions of LCs. Recently, we reported that hapten-specific LCs coat miRNA-150-carrying exosomes produced by CD8+ suppressor T cells downregulating the contact hypersensitivity (CHS) reaction in an antigen-specific manner, in mice tolerized by intravenous administration of a high dose of hapten-coupled syngeneic erythrocytes. Thus, the current studies aimed at investigating the role of hapten-specific LCs in antigen-specific, exosome-mediated suppression of CHS effector cells. Suppressor T cell-derived exosomes from tolerized B-cell-deficient µMT-/-, NKT-cell-deficient Jα18-/-, and immunoglobulin-deficient JH-/- mice were nonsuppressive, unless supplemented with LCs of specificity strictly respective to the hapten used for sensitization and CHS elicitation in mice. Thus, these observations demonstrate that B1-cell-derived LCs, coating exosomes in vivo and in vitro, actually ensure the specificity of CHS suppression. Our research findings substantially expand current understanding of the newly discovered, suppressor T cell-dependent tolerance mechanism by uncovering the function of antigen-specific LCs in exosome-mediated, cellâ»cell communication. This express great translational potential in designing nanocarriers for specific targeting of desired cells.
Assuntos
Linfócitos B/imunologia , Dermatite de Contato/imunologia , Exossomos/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Células Matadoras Naturais/imunologia , Linfócitos T/imunologia , Animais , Comunicação Celular , Tolerância Imunológica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
We propose that there is a special B-1a B cell subset ("sB-1a" cells) that mediates linked processes very early after immunization to initiate cutaneous contact sensitivity (CS), delayed-type hypersensitivity (DTH), and immune resistance to pneumococcal pneumonia. Our published data indicate that in CS and DTH, these initiating processes are required for elicitation of the delayed onset and late-occurring classical T cell-mediated responses. sB-1a cells resemble memory B2 cells, as they are stimulated within 1 h of immunization and depend on T helper cytokines-uniquely IL-4 from hepatic iNKT cells--for activation and rapid migration from the peritoneal cavity to the spleen to secrete IgM antibody (Ab) and Ab-derived free light chains (FLCs) by only 1 day after immunization. Unlike conventional B-1a (cB-1a) cell-produced IgM natural Ab, IgM Ab produced by sB-1a cells has high Ag affinity owing to immunoglobulin V-region mutations induced by activation-induced cytidine deaminase (AID). The dominant cB-1a cells are increased in immunized AID-deficient mice but do not mediate initiation, CS, or pneumonia resistance because natural Ab has relatively low Ag affinity because of unmutated germ-line V regions. In CS and DTH, sB-1a IgM Ag affinity is sufficiently high to mediate complement activation for generation of C5a that, together with vasoactive mediators such as TNF-α released by FLC-sensitized mast cells, activate local endothelium for extravascular recruitment of effector T cells. We conclude by discussing the possibility of functional sB-1 cells in humans.