RESUMO
Lysosomal acid lipase (LAL) deficiency is an autosomal recessive disorder caused by LIPA gene mutations that disrupt LAL activity. We performed in vitro functional testing of 149 LIPA variants to increase the understanding of the variant effects on LAL deficiency and to improve disease prevalence estimates. Chosen variants had been reported in literature or population databases. Functional testing was done by plasmid transient transfection and LAL activity assessment. We assembled a set of 165 published LAL deficient patient genotypes to evaluate this assay's effectiveness to recapitulate genotype/phenotype relationships. Rapidly progressive LAL deficient patients showed negligible enzymatic activity (<1%), whereas patients with childhood/adult LAL deficiency typically have 1-7% average activity. We benchmarked six in silico variant effect prediction algorithms with these functional data. PolyPhen-2 was shown to have a superior area under the receiver operating curve performance. We used functional data along with Genome Aggregation Database (gnomAD) allele frequencies to estimate LAL deficiency birth prevalence, yielding a range of 3.45-5.97 cases per million births in European-ancestry populations. The low estimate only considers functionally assayed variants in gnomAD. The high estimate computes allele frequencies for variants absent in gnomAD, and uses in silico scores for unassayed variants. Prevalence estimates are lower than previously published, underscoring LAL deficiency's rarity.
Assuntos
Predisposição Genética para Doença , Variação Genética , Modelos Genéticos , Esterol Esterase/genética , Doença de Wolman/epidemiologia , Doença de Wolman/genética , Algoritmos , Expressão Gênica , Estudos de Associação Genética , Genótipo , Humanos , Mutação , Fenótipo , Prevalência , Curva ROC , Doença de WolmanRESUMO
We have discovered that a small cationic molecule, GW4869, is cytotoxic to a subset of myeloma cell lines and primary myeloma plasma cells. Biochemical analysis revealed that GW4869 binds to anionic phospholipids such as phosphatidylserine - a lipid normally confined to the intracellular side of the cell membrane. However, interestingly, phosphatidylserine was expressed on the surface of all myeloma cell lines tested (n = 12) and 9/15 primary myeloma samples. Notably, the level of phosphatidylserine expression correlated well with sensitivity to GW4869. Inhibition of cell surface phosphatidylserine exposure with brefeldin A resulted in resistance to GW4869. Finally, GW4869 was shown to delay the growth of phosphatidylserine-high myeloma cells in vivo. To the best of our knowledge, this is the first example of using a small molecule to target phosphatidylserine on malignant cells. This study may provide the rationale for the development of phosphatidylserine-targeting small molecules for the treatment of surface phosphatidylserine-expressing cancers.
Assuntos
Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Compostos de Benzilideno/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Fosfatidilserinas/metabolismo , Compostos de Anilina/administração & dosagem , Compostos de Anilina/uso terapêutico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Compostos de Benzilideno/administração & dosagem , Compostos de Benzilideno/uso terapêutico , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Camundongos SCID , Mieloma Múltiplo/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Helminth parasites secrete molecules that potently modulate the immune responses of their hosts and, therefore, have potential for the treatment of immune-mediated human diseases. FhHDM-1, a 68-mer peptide secreted by the helminth parasite Fasciola hepatica, ameliorated disease in two different murine models of autoimmunity, type 1 diabetes and relapsing-remitting immune-mediated demyelination. Unexpectedly, FhHDM-1 treatment did not affect the proliferation of auto-antigen specific T cells or their production of cytokines. However, in both conditions, the reduction in clinical symptoms was associated with the absence of immune cell infiltrates in the target organ (islets and the brain tissue). Furthermore, after parenteral administration, the FhHDM-1 peptide interacted with macrophages and reduced their capacity to secrete pro-inflammatory cytokines, such as TNF and IL-6. We propose this inhibition of innate pro-inflammatory immune responses, which are central to the initiation of autoimmunity in both diseases, prevented the trafficking of autoreactive lymphocytes from the periphery to the site of autoimmunity (as opposed to directly modulating their function per se), and thus prevented tissue destruction. The ability of FhHDM-1 to modulate macrophage function, combined with its efficacy in disease prevention in multiple models, suggests that FhHDM-1 has considerable potential as a treatment for autoimmune diseases.
Assuntos
Doenças Autoimunes/imunologia , Diabetes Mellitus Tipo 1/imunologia , Esclerose Múltipla/imunologia , Parasitos/imunologia , Peptídeos/imunologia , Animais , Autoimunidade/imunologia , Proliferação de Células/fisiologia , Citocinas/imunologia , Modelos Animais de Doenças , Fasciola hepatica/imunologia , Feminino , Proteínas de Helminto/imunologia , Inflamação/imunologia , Interleucina-6/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/imunologiaRESUMO
We describe a protective early acquired immune response to pneumococcal pneumonia that is mediated by a subset of B1a cells. Mice deficient in B1 cells (xid), or activation-induced cytidine deaminase (AID(-/-) ), or invariant natural killer T (iNKT) cells (Jα18(-/-) ), or interleukin-13 (IL-13(-/-) ) had impaired early clearance of pneumococci in the lung, compared with wild-type mice. In contrast, AID(-/-) mice adoptively transferred with AID(+/+) B1a cells, significantly cleared bacteria from the lungs as early as 3 days post infection. We show that this early bacterial clearance corresponds to an allergic contact sensitivity-like cutaneous response, probably due to a subpopulation of initiating B1a cells. In the pneumonia model, these B1a cells were found to secrete higher affinity antigen-specific IgM. In addition, as in contact sensitivity, iNKT cells were required for the anti-pneumococcal B1a cell initiating response, probably through early production of IL-13, given that IL-13(-/-) mice also failed to clear infection. Our study is the first to demonstrate the importance of AID in generating an appropriate B1a cell response to pathogenic bacteria. Given the antibody affinity and pneumonia resistance data, natural IgM produced by conventional B1a cells are not responsible for pneumonia clearance compared with the AID-dependent subset.
Assuntos
Imunidade Adaptativa , Linfócitos B/enzimologia , Citidina Desaminase/metabolismo , Pulmão/enzimologia , Fagocitose , Pneumonia Pneumocócica/enzimologia , Streptococcus pneumoniae/imunologia , Transferência Adotiva , Tirosina Quinase da Agamaglobulinemia , Animais , Antígenos de Bactérias/imunologia , Linfócitos B/imunologia , Linfócitos B/microbiologia , Linfócitos B/transplante , Ativação do Complemento , Citidina Desaminase/deficiência , Citidina Desaminase/genética , Citidina Desaminase/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Dermatite de Contato/enzimologia , Dermatite de Contato/imunologia , Dermatite de Contato/microbiologia , Modelos Animais de Doenças , Genótipo , Imunoglobulina M/imunologia , Imunoglobulina M/metabolismo , Interleucina-13/deficiência , Interleucina-13/genética , Pulmão/imunologia , Pulmão/microbiologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/microbiologia , Fenótipo , Pneumonia Pneumocócica/imunologia , Pneumonia Pneumocócica/microbiologia , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/metabolismo , Baço/enzimologia , Baço/imunologia , Baço/microbiologia , Streptococcus pneumoniae/patogenicidade , Fatores de TempoRESUMO
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.
Assuntos
Subpopulações de Linfócitos B/imunologia , Citidina Desaminase/imunologia , Dermatite de Contato/imunologia , Pneumonia Pneumocócica/imunologia , Animais , Subpopulações de Linfócitos B/metabolismo , Citidina Desaminase/deficiência , Dermatite de Contato/metabolismo , Humanos , Hipersensibilidade , Imunização , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Pneumonia Pneumocócica/prevenção & controleRESUMO
Monoclonal antibodies (mAb) have had tremendous success in treating a variety of cancers over the past twenty years. Yet despite their widespread clinical use, which includes treatments for haematological malignancies, there are still no approved mAb therapies for multiple myeloma (MM). This is likely to change within the next few years with a number of mAb therapies being assessed in late stage clinical trials, most notably, the anti-CS-1 mAb, elotuzumab, and the anti-CD38 mAb, daratumumab, which are currently being evaluated in Phase III clinical trials for MM. In this review, we will discuss the preclinical and clinical development of MDX-1097, a Phase II candidate which targets cell membrane-associated kappa immunoglobulin free light chains expressed on the surface of MM cells.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Cadeias kappa de Imunoglobulina/imunologia , Imunoterapia/métodos , Mieloma Múltiplo/terapia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Antineoplásicos/imunologia , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Cadeias kappa de Imunoglobulina/química , Cadeias kappa de Imunoglobulina/metabolismo , Modelos Moleculares , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/metabolismo , Ligação Proteica/imunologia , Estrutura Terciária de ProteínaRESUMO
Therapeutic antibodies have come of age with major progress being made in cancer, autoimmunity and chronic inflammation, as well as a wide range of other human diseases. Antibody engineering is further driving development of novel antibody formats and genetically modified cell-based therapies that harness the power of the immune system to progress cures in otherwise intractable human diseases. Nevertheless, there are still significant challenges ahead for basic and applied research relating to therapeutic antibodies. This special issue of the journal provides reviews and opinions that relate to the discovery, design and deployment of antibodies as therapeutics.
Assuntos
Anticorpos/uso terapêutico , Imunoterapia/métodos , Terapia de Alvo Molecular/métodos , Neoplasias/terapia , Anticorpos/imunologia , Antineoplásicos/imunologia , Antineoplásicos/uso terapêutico , Desenho de Fármacos , Descoberta de Drogas , Humanos , Imunoterapia/tendências , Terapia de Alvo Molecular/tendências , Neoplasias/imunologia , Resultado do TratamentoRESUMO
The monoclonal antibody, MDX-1097, is currently progressing through clinical trials as a possible therapy for multiple myeloma. MDX-1097 targets a cell membrane bound form of free immunoglobulin kappa light chain (FκLC), termed kappa myeloma antigen (KMA), which is found on the surface of malignant plasma cells. The clinical potential of MDX-1097 highlights the need to characterise the expression of its cognate antigen, KMA, in normal tissue. In this study, we have analysed the expression of KMA on B cell subsets found in tonsils, peripheral blood and bone marrow. We found KMA expression on a small population of tonsillar and in vitro derived plasmablasts. In contrast, no KMA expression was observed on peripheral blood or bone marrow resident B cell subsets. This study yields important insights into the possible subsets of B cells that might be depleted as a result of an immunotherapy targeting KMA.
Assuntos
Membrana Celular/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Tonsila Palatina/imunologia , Plasmócitos/imunologia , ADP-Ribosil Ciclase 1/imunologia , ADP-Ribosil Ciclase 1/metabolismo , Antígenos CD19/imunologia , Antígenos CD19/metabolismo , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Citometria de Fluxo , Humanos , Cadeias kappa de Imunoglobulina/sangue , Cadeias kappa de Imunoglobulina/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Tonsila Palatina/metabolismo , Plasmócitos/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
Infections with helminth parasites prevent/attenuate auto-inflammatory disease. Here we show that molecules secreted by a helminth parasite could prevent Type 1 Diabetes (T1D) in nonobese diabetic (NOD) mice. When delivered at 4 weeks of age (coincident with the initiation of autoimmunity), the excretory/secretory products of Fasciola hepatica (FhES) prevented the onset of T1D, with 84% of mice remaining normoglycaemic and insulitis-free at 30 weeks of age. Disease protection was associated with suppression of IFN-γ secretion from autoreactive T cells and a switch to the production of a regulatory isotype (from IgG2a to IgG1) of autoantibody. Following FhES injection, peritoneal macrophages converted to a regulatory M2 phenotype, characterised by increased expression levels of Ym1, Arg-1, TGFß and PD-L1. Expression of these M2 genetic markers increased in the pancreatic lymph nodes and the pancreas of FhES-treated mice. In vitro, FhES-stimulated M2 macrophages induced the differentiation of Tregs from splenocytes isolated from naïve NOD mice. Collectively, our data shows that FhES contains immune-modulatory molecules that mediate protection from autoimmune diabetes via the induction and maintenance of a regulatory immune environment.
Assuntos
Autoimunidade/imunologia , Diabetes Mellitus Tipo 1/imunologia , Fasciola hepatica/imunologia , Helmintos/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/parasitologia , Animais , Autoanticorpos/imunologia , Antígeno B7-H1/imunologia , Diferenciação Celular/imunologia , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/parasitologia , Diabetes Mellitus Tipo 1/parasitologia , Feminino , Interferon gama/imunologia , Lectinas/imunologia , Linfonodos/imunologia , Linfonodos/parasitologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos NOD , Pâncreas/imunologia , Fator de Crescimento Transformador beta/imunologia , beta-N-Acetil-Hexosaminidases/imunologiaRESUMO
We have described the presence of cell-membrane-associated κFLCs (free immunoglobulin light chains) on the surface of myeloma cells. Notably, the anti-κFLC mAb (monoclonal antibody) MDX-1097 is being assessed in clinical trials as a therapy for κ light chain isotype multiple myeloma. Despite the clinical potential of anti-FLC mAbs, there have been limited studies on characterizing membrane-associated FLCs at a molecular level. Furthermore, it is not known whether λFLCs can associate with cell membranes of myeloma cells. In the present paper, we describe the presence of λFLCs on the surface of myeloma cells. We found that cell-surface-associated λFLCs are bound directly to the membrane and in an aggregated form. Subsequently, membrane interaction studies revealed that λFLCs interact with saturated zwitterionic lipids such as phosphatidylcholine and phosphatidylethanolamine, and using automated docking, we characterize a potential recognition site for these lipids. Atomic force microscopy confirmed that membrane-associated λFLCs are aggregated. Given the present findings, we propose a model whereby individual FLCs show modest affinity for zwitterionic lipids, with aggregation stabilizing the interaction due to multivalency. Notably, this is the first study to image FLCs bound to phospholipids and provides important insights into the possible mechanisms of membrane association by this unique myeloma surface antigen.
Assuntos
Membrana Celular/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Humanos , Cadeias Leves de Imunoglobulina/química , Microscopia de Força Atômica , Simulação de Acoplamento Molecular , Mieloma Múltiplo , Fosfatidilcolinas/metabolismo , Ligação ProteicaRESUMO
We previously reported the identification of a novel family of immunomodulatory proteins, termed helminth defense molecules (HDMs), that are secreted by medically important trematode parasites. Since HDMs share biochemical, structural, and functional characteristics with mammalian cathelicidin-like host defense peptides (HDPs), we proposed that HDMs modulate the immune response via molecular mimicry of host molecules. In the present study, we report the mechanism by which HDMs influence the function of macrophages. We show that the HDM secreted by Fasciola hepatica (FhHDM-1) binds to macrophage plasma membrane lipid rafts via selective interaction with phospholipids and/or cholesterol before being internalized by endocytosis. Following internalization, FhHDM-1 is rapidly processed by lysosomal cathepsin L to release a short C-terminal peptide (containing a conserved amphipathic helix that is a key to HDM function), which then prevents the acidification of the endolysosomal compartments by inhibiting vacuolar ATPase activity. The resulting endolysosomal alkalization impedes macrophage antigen processing and prevents the transport of peptides to the cell surface in conjunction with MHC class II for presentation to CD4(+) T cells. Thus, we have elucidated a novel mechanism by which helminth pathogens alter innate immune cell function to assist their survival in the host.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/classificação , Antígenos de Helmintos/metabolismo , Fasciola hepatica/metabolismo , Proteínas de Helminto/metabolismo , Macrófagos/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Animais , Catepsina L/genética , Catepsina L/metabolismo , Fasciola hepatica/imunologia , Regulação da Expressão Gênica/fisiologia , Genes MHC da Classe II , Proteínas de Helminto/genética , Humanos , Macrófagos/imunologia , Microdomínios da Membrana/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
During antibody synthesis, immunoglobulin light chains are produced in excess of heavy chains and, as a consequence, can be secreted by plasma cells as free light chains (FLC). Thus, FLC were considered to be a by-product of immunoglobulin synthesis, lacking any biological function or relevance. However, mounting evidence suggests that FLC are bioactive molecules. For example, FLC can induce antigen specific type I hypersensitivity and inhibit viral replication in encephalomyocarditis infected mice. We have recently shown that FLC can associate with the outer membrane of certain plasma cells via interaction with saturated phosphocholine lipids such as sphingomyelin. As these lipids are highly abundant in mammalian cell membranes, we set out to determine whether FLCs can bind to membranes from a variety of cell types. We found that FLCs bind to the plasma membrane of cells from a wide range of lineages. Interestingly, the highest level of binding was to monocytes. As these cells are professional antigen presenting cells, we postulate that membrane-associated FLCs may provide a novel mechanism of antigen uptake by these cells.
Assuntos
Membrana Celular/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Rim/metabolismo , Linfócitos/metabolismo , Monócitos/metabolismo , Animais , Linfócitos B/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/química , Humanos , Cadeias Leves de Imunoglobulina/imunologia , Rim/citologia , Células Matadoras Naturais/metabolismo , Camundongos , Esfingomielinas/metabolismo , Linfócitos T/metabolismoRESUMO
Over the last decade a significant number of studies have highlighted the central role of host antimicrobial (or defence) peptides in modulating the response of innate immune cells to pathogen-associated ligands. In humans, the most widely studied antimicrobial peptide is LL-37, a 37-residue peptide containing an amphipathic helix that is released via proteolytic cleavage of the precursor protein CAP18. Owing to its ability to protect against lethal endotoxaemia and clinically-relevant bacterial infections, LL-37 and its derivatives are seen as attractive candidates for anti-sepsis therapies. We have identified a novel family of molecules secreted by parasitic helminths (helminth defence molecules; HDMs) that exhibit similar biochemical and functional characteristics to human defence peptides, particularly CAP18. The HDM secreted by Fasciola hepatica (FhHDM-1) adopts a predominantly α-helical structure in solution. Processing of FhHDM-1 by F. hepatica cathepsin L1 releases a 34-residue C-terminal fragment containing a conserved amphipathic helix. This is analogous to the proteolytic processing of CAP18 to release LL-37, which modulates innate cell activation by classical toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). We show that full-length recombinant FhHDM-1 and a peptide analogue of the amphipathic C-terminus bind directly to LPS in a concentration-dependent manner, reducing its interaction with both LPS-binding protein (LBP) and the surface of macrophages. Furthermore, FhHDM-1 and the amphipathic C-terminal peptide protect mice against LPS-induced inflammation by significantly reducing the release of inflammatory mediators from macrophages. We propose that HDMs, by mimicking the function of host defence peptides, represent a novel family of innate cell modulators with therapeutic potential in anti-sepsis treatments and prevention of inflammation.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Fasciola hepatica/química , Proteínas de Helminto/farmacologia , Mimetismo Molecular , Proteínas de Fase Aguda/metabolismo , Proteínas de Fase Aguda/farmacologia , Sequência de Aminoácidos , Animais , Anti-Infecciosos/imunologia , Anti-Infecciosos/metabolismo , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , Endotoxemia/imunologia , Endotoxemia/metabolismo , Fasciola hepatica/imunologia , Fasciola hepatica/metabolismo , Fasciolíase/imunologia , Fasciolíase/parasitologia , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Humanos , Imunidade Inata , Inflamação/imunologia , Inflamação/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Sepse/imunologia , Sepse/metabolismo , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismo , CatelicidinasRESUMO
The murine mAb, K-1-21, recognizes a conformational epitope expressed on free Ig kappa light chains (FκLCs) and also on cell membrane-associated FκLCs found on kappa myeloma cells. This has led to the development of a chimeric version of K-1-21, MDX-1097, which is being assessed in a Phase II clinical trial for the treatment of multiple myeloma. The epitope recognized by K-1-21 is of particular interest, especially in the context that it is not expressed on heavy chain-associated light chains such as in an intact Ig molecule. Using epitope excision techniques we have localized the K-1-21 epitope to a region spanning residues 104-110 of FκLC. This short strand of residues links the variable and constant domains, and is a flexible region that adopts different conformations in FκLC and heavy chain-associated light chain. We tested this region using site-directed mutations and found that the reactivity of K-1-21 for FκLC was markedly reduced. Finally, we applied in silico molecular docking to generate a model that satisfied the experimental data. Given the clinical potential of the Ag, this study may aid the development of next generation compounds that target the membrane form of FκLC expressed on the surface of myeloma plasma cells.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Epitopos/química , Epitopos/imunologia , Cadeias kappa de Imunoglobulina/química , Cadeias kappa de Imunoglobulina/imunologia , Sequência de Aminoácidos , Aminoácidos , Animais , Humanos , Região de Troca de Imunoglobulinas/imunologia , Cinética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/imunologia , Mutação/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/imunologia , Homologia de Sequência de Aminoácidos , Tripsina/metabolismoRESUMO
Free κ L chains (FκLCs) are expressed on the surface of myeloma cells and are being assessed as a therapeutic target for the treatment of multiple myeloma. Despite its clinical potential, the mechanism by which FκLCs interact with membranes remains unresolved. In this study, we show that FκLCs associate with sphingomyelin on the plasma membrane of myeloma cells. Moreover, membrane-bound FκLCs are aggregated, suggesting that aggregation is required for intercalation with membranes. Finally, we propose a model where the binding of FκLCs with sphingomyelin on secretory vesicle membranes is stabilized by self-aggregation, with aggregated FκLCs exposed on the plasma membrane after exocytosis. Although it is well known that protein aggregates bind membranes, this is only the second example of an aggregate being found on the surface of cells that also secrete the protein in its native form. We postulate that many other aggregation-prone proteins may associate with cell membranes by similar mechanisms.