RESUMEN
The Ig fold has had a remarkable success in vertebrate evolution, with a presence in over 2% of human genes. The Ig fold is not just the elementary structural domain of antibodies and TCRs, it is also at the heart of a staggering 30% of immunologic cell surface receptors, making it a major orchestrator of cell-cell interactions. While BCRs, TCRs, and numerous Ig-based cell surface receptors form homo- or heterodimers on the same cell surface (in cis), many of them interface as ligand-receptors (checkpoints) on interacting cells (in trans) through their Ig domains. New Ig-Ig interfaces are still being discovered between Ig-based cell surface receptors, even in well-known families such as B7. What is largely ignored, however, is that the Ig fold itself is pseudosymmetric, a property that makes the Ig domain a versatile self-associative 3D structure and may, in part, explain its success in evolution, especially through its ability to bind in cis or in trans in the context of cell surface receptor-ligand interactions. In this paper, we review the Ig domains' tertiary and quaternary pseudosymmetries, with particular attention to the newly identified double Ig fold in the solved CD19 molecular structure to highlight the underlying fundamental folding elements of Ig domains, i.e., Ig protodomains. This pseudosymmetric property of Ig domains gives us a decoding frame of reference to understand the fold, relate all Ig domain forms, single or double, and suggest new protein engineering avenues.
Asunto(s)
Antígenos CD19/química , Dominios de Inmunoglobulinas , Secuencia de Aminoácidos , Mapas de Interacción de Proteínas , Anticuerpos de Dominio Único/químicaRESUMEN
CD19 is among the most relevant targets in cancer immunotherapy. However, its extracellular domain (ECD) is prone to aggregation and misfolding, representing a major obstacle for the development and analysis of CD19-targeted therapeutics. Here, we engineered stabilized CD19-ECD (termed SuperFolder) variants, which also showed improved expression rates and, in contrast to the wild type protein, they could be efficiently purified in their monomeric forms. Despite being considerably more stable, these engineered mutants largely preserved the wild type sequence (>98.8%). We demonstrate that the variant SF05 enabled the determination of the monovalent affinity between CD19 and a clinically approved FMC63-based CAR, as well as monitoring and phenotypic characterization of CD19-directed CAR-T cells in the blood of lymphoma patients. We anticipate that the SuperFolder mutants generated in this study will be highly valuable tools for a range of applications in basic immunology and CD19-targeted cancer immunotherapy.
Asunto(s)
Sustitución de Aminoácidos , Antígenos CD19/genética , Evolución Molecular Dirigida/métodos , Inmunoterapia Adoptiva/métodos , Linfoma de Células B Grandes Difuso/inmunología , Linfoma de Células B Grandes Difuso/terapia , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/inmunología , Secuencia de Aminoácidos , Aminoácidos/genética , Anticuerpos Monoclonales/inmunología , Antígenos CD19/química , Antígenos CD19/inmunología , Células HEK293 , Humanos , Linfoma de Células B Grandes Difuso/sangre , Proteínas Mutantes , Mutación , Dominios Proteicos/inmunología , Pliegue de Proteína , Estabilidad Proteica , Receptores Quiméricos de Antígenos/genéticaRESUMEN
Signaling through the CD19-CD81 co-receptor complex, in combination with the B cell receptor, is a critical determinant of B cell development and activation. It is unknown how CD81 engages CD19 to enable co-receptor function. Here, we report a 3.8-angstrom structure of the CD19-CD81 complex bound to a therapeutic antigen-binding fragment, determined by cryo-electron microscopy (cryo-EM). The structure includes both the extracellular domains and the transmembrane helices of the complex, revealing a contact interface between the ectodomains that drives complex formation. Upon binding to CD19, CD81 opens its ectodomain to expose a hydrophobic CD19-binding surface and reorganizes its transmembrane helices to occlude a cholesterol binding pocket present in the apoprotein. Our data reveal the structural basis for CD19-CD81 complex assembly, providing a foundation for rational design of therapies for B cell dysfunction.
Asunto(s)
Antígenos CD19/química , Receptores de Antígenos de Linfocitos B/química , Tetraspanina 28/química , Secuencia de Aminoácidos , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/inmunología , Antígenos CD19/inmunología , Linfocitos B/inmunología , Microscopía por Crioelectrón , Humanos , Maitansina/análogos & derivados , Maitansina/química , Maitansina/inmunología , Modelos Moleculares , Mutación , Unión Proteica , Dominios Proteicos , Receptores de Antígenos de Linfocitos B/inmunología , Tetraspanina 28/genética , Tetraspanina 28/inmunologíaRESUMEN
Chimeric antigen receptor (CAR) T-cells targeting CD19 demonstrate remarkable efficacy in treating B-lineage acute lymphoblastic leukemia (BL-ALL), yet up to 39% of treated patients relapse with CD19(-) disease. We report that CD19(-) escape is associated with downregulation, but preservation, of targetable expression of CD20 and CD22. Accordingly, we reasoned that broadening the spectrum of CD19CAR T-cells to include both CD20 and CD22 would enable them to target CD19(-) escape BL-ALL while preserving their upfront efficacy. We created a CD19/20/22-targeting CAR T-cell by coexpressing individual CAR molecules on a single T-cell using one tricistronic transgene. CD19/20/22CAR T-cells killed CD19(-) blasts from patients who relapsed after CD19CAR T-cell therapy and CRISPR/Cas9 CD19 knockout primary BL-ALL both in vitro and in an animal model, while CD19CAR T-cells were ineffective. At the subcellular level, CD19/20/22CAR T-cells formed dense immune synapses with target cells that mediated effective cytolytic complex formation, were efficient serial killers in single-cell tracking studies, and were as efficacious as CD19CAR T-cells against primary CD19(+) disease. In conclusion, independent of CD19 expression, CD19/20/22CAR T-cells could be used as salvage or front-line CAR therapy for patients with recalcitrant disease.
Asunto(s)
Antígenos CD19/inmunología , Inmunoterapia Adoptiva , Leucemia de Células B/inmunología , Leucemia de Células B/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Antígenos CD19/química , Antígenos de Neoplasias , Biomarcadores , Línea Celular Tumoral , Citocinas/metabolismo , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Inmunoterapia Adoptiva/métodos , Leucemia de Células B/genética , Leucemia de Células B/terapia , Ratones Transgénicos , Unión Proteica , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Relación Estructura-Actividad , Transducción Genética , Transgenes , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy is considered as a major scientific breakthrough in cancer immunotherapy. The success of adoptive CAR T-cell therapy for cancer has inspired researchers to expand indications into the area of solid tumors, autoimmune and infectious diseases. The most important factors influencing outcome and durability of the response after infusion of CAR T-cell are proliferation and persistence of this cell subset. It becomes therefore important to detect easily and monitor circulating CAR T-cells into blood samples. Approaches such as quantitative PCR (qPCR) or flow cytometry have been developed. The aim of this study was to set up and optimize a reachable flow cytometry technique using labeled CD19 protein for the measurement of CAR T-cells in infusion bag and patient's blood. METHODS: Patients receiving Yescarta in Cell Therapy Unit (Department of hematology, Lille university hospital, France) between April and October 2019 and healthy volunteers were included to set up the flow cytometry technique. RESULTS AND CONCLUSIONS: We assessed feasibility in clinic and suitability to routine workload of a flow cytometry technique to follow CAR T-cells in infusion bag and patient's blood. With only a few manual steps, the present protocol allows the technician to perform this technique among other routine tasks, meaning a time to results of <2 hr after sample reception. We were also able to assess CAR T-cell heterogenity in terms of CD4+ and CD8+ T lymphocytes within the subset. Moreover, this technique allows monitoring of both authority approved CD19 CAR T-cell.
Asunto(s)
Citometría de Flujo , Inmunoterapia Adoptiva , Receptores Quiméricos de Antígenos/análisis , Linfocitos T/citología , Adulto , Anciano , Antígenos CD19/química , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Tetraspanins, including CD53 and CD81, regulate a multitude of cellular processes through organizing an interaction network on cell membranes. Here, we report the crystal structure of CD53 in an open conformation poised for partner interaction. The large extracellular domain (EC2) of CD53 protrudes away from the membrane surface and exposes a variable region, which is identified by hydrogen-deuterium exchange as the common interface for CD53 and CD81 to bind partners. The EC2 orientation in CD53 is supported by an extracellular loop (EC1). At the closed conformation of CD81, however, EC2 disengages from EC1 and rotates toward the membrane, thereby preventing partner interaction. Structural simulation shows that EC1-EC2 interaction also supports the open conformation of CD81. Disrupting this interaction in CD81 impairs the accurate glycosylation of its CD19 partner, the target for leukemia immunotherapies. Moreover, EC1 mutations in CD53 prevent the chemotaxis of pre-B cells toward a chemokine that supports B-cell trafficking and homing within the bone marrow, a major CD53 function identified here. Overall, an open conformation is required for tetraspanin-partner interactions to support myriad cellular processes.
Asunto(s)
Movimiento Celular , Células Precursoras de Linfocitos B/metabolismo , Tetraspanina 25 , Tetraspanina 28 , Animales , Antígenos CD19/química , Antígenos CD19/genética , Antígenos CD19/metabolismo , Humanos , Ratones , Ratones Noqueados , Dominios Proteicos , Tetraspanina 25/química , Tetraspanina 25/genética , Tetraspanina 25/metabolismo , Tetraspanina 28/química , Tetraspanina 28/genética , Tetraspanina 28/metabolismoRESUMEN
PURPOSE: ADCT-402 (loncastuximab tesirine) is an antibody-drug conjugate comprising a CD19-targeting antibody and pyrrolobenzodiazepine dimers. A first-in-human study evaluated the safety and preliminary clinical activity of loncastuximab tesirine in patients with B-cell non-Hodgkin lymphoma (NHL). PATIENTS AND METHODS: A multicenter, phase I, dose-escalation and dose-expansion study enrolled patients ages ≥18 years with relapsed/refractory (R/R) B-cell NHL. Patients received loncastuximab tesirine every 3 weeks at doses assigned by a 3+3 dose-escalation design. Dose escalation was used to assess the safety and tolerability of loncastuximab tesirine to determine the dose for expansion. Secondary objectives evaluated clinical activity, characterized the pharmacokinetic profile, and evaluated antidrug antibodies. RESULTS: During dose escalation, 88 patients with R/R B-cell NHL were treated with loncastuximab tesirine at doses 15 to 200 µg/kg. Treatment-emergent adverse events (TEAEs) were experienced by 87/88 (98.9%) patients. Most common TEAEs (≥20% of patients) were hematologic abnormalities, fatigue, edema, liver test abnormalities, nausea, rash, and dyspnea. Grade ≥3 TEAEs (≥5% of patients) included hematologic abnormalities, liver test abnormalities, fatigue, and dyspnea. Overall response rate at doses ≥120 µg/kg was 59.4% (41 of 69 patients; 40.6% complete response; 18.8% partial response). Median duration of response, progression-free survival, and overall survival (all doses) were 4.8, 5.5, and 11.6 months, respectively. Drug exposure increased with increasing dose, showing moderate accumulation with multiple doses ≥150 µg/kg. There was no evidence of immunogenicity. CONCLUSIONS: Loncastuximab tesirine had promising activity with acceptable safety in this dose-escalation study. A phase II study with initial dosing at 150 µg/kg has been initiated based on these results.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Antígenos CD19/química , Antineoplásicos/uso terapéutico , Benzodiazepinas/química , Inmunoconjugados/uso terapéutico , Linfoma de Células B/terapia , Recurrencia Local de Neoplasia/terapia , Pirroles/química , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD19/inmunología , Benzodiazepinas/uso terapéutico , Resistencia a Antineoplásicos , Femenino , Estudios de Seguimiento , Humanos , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/patología , Pronóstico , Terapia Recuperativa , Tasa de Supervivencia , Adulto JovenRESUMEN
The B-cell surface protein CD19 is present throughout the cell life cycle and is uniformly expressed in leukemias, making it a target for chimeric antigen receptor engineered immune cell therapy. Identifying the sequence dependence of the binding of CD19 to antibodies empowers fundamental study and more tailored development of CD19-targeted therapeutics. To identify the antibody-binding epitopes on CD19, we screened a comprehensive single-site saturation mutation library of the human CD19 extracellular domain to identify mutations detrimental to binding FMC63-the dominant CD19 antibody used in chimeric antigen receptor development-as well as 4G7-2E3 and 3B10, which have been used in various types of CD19 research and development. All three antibodies had partially overlapping, yet distinct, epitopes near the published epitope of antibody B43. The FMC63 conformational epitope spans spatially adjacent, but genetically distant, loops in exons 3 and 4. The 3B10 epitope is a linear peptide sequence that binds CD19 with 440 pM affinity. Along with their primary goal of epitope mapping, the mutational tolerance data also empowered additional CD19 variant design and analysis. A designed CD19 variant with all N-linked glycosylation sites removed successfully bound antibody in the yeast display context, which provides a lead for aglycosylated applications. Screening for thermally stable variants identified mutations to guide further CD19 stabilization for fusion protein applications and revealed evolutionary affinity-stability trade-offs. These fundamental insights into CD19 sequence-function relationships enhance our understanding of antibody-mediated CD19-targeted therapeutics.
Asunto(s)
Antígenos CD19/química , Antígenos CD19/inmunología , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Antígenos CD19/genética , Mapeo Epitopo , Exones , Humanos , Mutación , Dominios ProteicosAsunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Inmunoconjugados/uso terapéutico , Inmunoterapia Adoptiva/métodos , Inmunoterapia/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Antígenos CD19/química , Niño , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/antagonistas & inhibidores , Resultado del TratamientoRESUMEN
PURPOSE: Anti-CD19 chimeric antigen receptor (CAR) T cells represent a novel immunotherapy and are highly effective in treating relapsed/refractory B-cell non-Hodgkin's lymphoma (B-NHL). How tumor microenvironment influences clinical response to CAR T therapy remains of great interest. PATIENTS AND METHODS: A phase I, first-in-human, dose-escalation study of anti-CD19 JWCAR029 was conducted in refractory B-NHL (NCT03355859) and 10 patients received CAR T cells at an escalating dose of 2.5 × 107(n = 3), 5 × 107(n = 4), and 1 × 108(n = 3) cells. Core needle biopsy was performed on tumor samples collected from diffuse large B-cell lymphoma patients on Day -6 (1 day before lymphodepletion) and on Day 11 after CAR T-cell infusion when adequate CAR T-cell expansion was detected. RESULTS: The overall response rate was 100%, with 6 of 9 (66.7%) evaluable patients achieving complete remission. The most common adverse events of grade 3 or higher were neutropenia (10/10, 100%), anemia (3/10, 30%), thrombocytopenia (3/10, 30%), and hypofibrinogenemia (2/10, 20%). Grade 1 cytokine release syndrome occurred in all patients and grade 3 neurotoxicity in 1 patient. The average peak levels of peripheral blood CAR T cells and cytokines were similar in 3 different dose levels, but CAR T cells were significantly higher in patients achieved complete remission on Day 29. Meanwhile, RNA sequencing identified gene expression signatures differentially enriched in complete and partial remission patients. Increased tumor-associated macrophage infiltration was negatively associated with remission status. CONCLUSIONS: JWCAR029 was effective and safe in treating refractory B-NHL. The composition of the tumor microenvironment has a potential impact in CAR T therapy response.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos CD19/química , Inmunoterapia Adoptiva/métodos , Inmunoterapia/métodos , Linfoma de Células B/terapia , Recurrencia Local de Neoplasia/terapia , Microambiente Tumoral/inmunología , Adulto , Antígenos CD19/inmunología , Terapia Combinada , Resistencia a Antineoplásicos , Femenino , Estudios de Seguimiento , Humanos , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/patología , Pronóstico , Terapia RecuperativaRESUMEN
Chimeric antigen receptor (CAR)-T cells are one of the impressive recent success stories of anti-cancer immunotherapy. Especially in haematological malignancies, this treatment strategy has shown promising results leading to the recent approval of two CAR-T cell constructs targeting CD19 in the United States and the European Union. After the huge success in haematological cancers, the next step will be the evaluation of its efficacy in different solid tumors, which is currently investigated in preclinical as well as clinical settings. A commonly examined tumor model in the context of immunotherapy is melanoma, since it is known for its immunogenic features. However, the first results of CAR-T cell therapy in solid tumors did not reveal the same impressive outcomes that were observed in haematological malignancies, as engineered cells need to cope with several challenges. Obstacles include the lack of migration of CAR-T cells from blood vessels to the tumor site as well as the immunosuppressive tumor microenvironment within solid tumors. Another hurdle is posed by the identification of an ideal target antigen to avoid on-target/off-tumor toxicities. Regarding immune escape mechanisms, which can be developed by tumor cells to bypass immune recognition, the observation of antigen loss should also be considered. This article gives an overview of the challenges displayed in CAR-T cell therapy for the use in solid tumors and discusses different new strategies and approaches that deal with these problems in order to improve CAR-T cell therapy, particularly for its use in melanoma.
Asunto(s)
Inmunoterapia Adoptiva/tendencias , Melanoma/terapia , Receptores Quiméricos de Antígenos/uso terapéutico , Neoplasias Cutáneas/terapia , Antígenos CD19/química , Antígenos de Neoplasias/química , Humanos , Sistema Inmunológico , Terapia de Inmunosupresión , Inmunoterapia Adoptiva/métodos , Receptores de Antígenos de Linfocitos T/inmunología , Resultado del Tratamiento , Microambiente Tumoral/inmunologíaRESUMEN
CD19 is a transmembrane protein expressed on malignant B cells, but not in other lineages or other tissues, which makes it an attractive target for monoclonal antibody-mediated immunotherapy. Anti-CD19 antibody B43 was utilized in a bispecific T-cell engager (BiTE) blinatumomab that demonstrated potency for the treatment of relapsed acute lymphoblastic leukemia. To gain insight into the mechanism of action of the antibody, the crystal structure of B43 Fab was determined in complex with CD19 and in the unbound form. The structure revealed the binding epitope, explained the lack of cross-reactivity toward non-human species, and suggested the key-and-lock mechanism of antigen recognition. Most unexpectedly, the structure revealed a unique molecular topology of CD19. Rather than a tandem of c-type immunoglobulin folds predicted from the amino acid sequence, the extracellular domain of CD19 exhibits an elongated ß-sandwich formed by two immunoglobulin folds by swapping their C-terminal halves. This is the first structure of CD19, which has no sequence homologs.
Asunto(s)
Anticuerpos Monoclonales/química , Antígenos CD19/química , Secuencia de Aminoácidos , Animales , Linfocitos B/citología , Sitios de Unión , Cristalografía por Rayos X , Células HEK293 , Humanos , Ratones , Modelos Moleculares , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de ProteínaRESUMEN
As a 95-kDa member of the immunoglobulin super-family expressed exclusively on B lymphocytes, CD19 is a critical co-receptor for B cell antigen receptor (BCR) signal transduction. Co-ligation of CD19 with the BCR synergistically enhances calcium release, mitogen-activated protein kinase activity and cell proliferation. However, CD19 deficient animals also display hyper-responsiveness under certain circumstances, indicating potential negative regulatory functions in BCR signaling. Thus CD19, like many other signaling molecules, is a double-edged sword and its abnormal expression can result in B cell related diseases. Here in this review, we summarize the latest development on the major functions of CD19 as both positive and negative regulator of BCR signaling in different situations and highlight the correlation and mechanisms of disturbed CD19 expression with autoimmune diseases and B cell lymphomas. Hopefully, the knowledge derived could shed an interesting light on the mechanistic insights of this important B cell surface molecule in both physiological and pathological conditions.
Asunto(s)
Antígenos CD19/genética , Antígenos CD19/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Investigación Biomédica Traslacional , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Antígenos CD19/química , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Regulación de la Expresión Génica , Humanos , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/genética , Linfoma de Células B/inmunología , Linfoma de Células B/metabolismo , Terapia Molecular Dirigida , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Unión Proteica , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Chimeric antigen receptor (CAR) modified T cells targeted CD19 showed promising clinical outcomes in treatment of B cell malignances such as chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL) and other indolent lymphomas. However, the clinical benefit varies tremendously among different trials. This meta-analysis investigated the efficacy (response rates and survival time) of CD19-CAR T cells in refractory B cell malignances in Phase I clinical trials. We searched publications between 1991 and 2014 from PubMed and Web of Science. Pooled response rates were calculated using random-effects models. Heterogeneity was investigated by subgroup analysis and meta-regression. Fourteen clinical trials including 119 patients were eligible for response rate evaluation, 62 patients in 12 clinical trials were eligible for progression-free survival analysis. The overall pooled response rate of CD19-CAR T cells was 73% (95% confidence interval [CI]: 46-94%). Significant heterogeneity across estimates of response rates was observed (p < 0.001, I2=88.3%). ALL patients have higher response rate (93%, 95% CI: 65-100%) than CLL (62%, 95% CI: 27-93%) and lymphoma patients (36%, 95% CI: 1-83%). Meta-regression analysis identified lymphodepletion and no IL-2 administrated T cells as two key factors associated with better clinical response. Lymphodepletion and higher infused CAR T cell number were associated with better prognosis. In conclusion, this meta-analysis showed a high clinical response rate of CD19-CAR T cell-based immunotherapy in treatment of refractory B cell malignancies. Lymphodepletion and increasing number of infused CD19-CAR T cells have positive correlations with the clinical efficiency, on the contrary, IL-2 administration to T cells is not recommended.
Asunto(s)
Antígenos CD19/química , Inmunoterapia Adoptiva/métodos , Leucemia Linfocítica Crónica de Células B/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Ensayos Clínicos Fase I como Asunto , Supervivencia sin Enfermedad , Femenino , Humanos , Interleucina-2/química , Linfoma/terapia , Masculino , Pronóstico , Modelos de Riesgos Proporcionales , Análisis de Regresión , Inducción de Remisión , Resultado del TratamientoRESUMEN
Normal expression of CD19 on the surface of B cells requires the presence of the tetraspanin molecule CD81. Previous studies have shown that surface expression of CD19 is highly reduced in CD81-deficient mouse B cells and that it is completely absent in an antibody deficient human patient with a mutation in the CD81 gene. The current study explored the contribution of an arginine-lysine rich motif, present in the membrane-proximal cytoplasmic domain of CD19, for the maturation and trafficking of this molecule. We demonstrate that this motif plays a role in the maturation and recycling of CD19 but in a CD81-independent manner.
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Antígenos CD19/metabolismo , Arginina/metabolismo , Lisina/metabolismo , Transporte de Proteínas/fisiología , Tetraspanina 28/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Antígenos CD19/química , Arginina/química , Línea Celular Tumoral , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Lisina/química , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Relación Estructura-Actividad , Tetraspanina 28/químicaRESUMEN
Over the last 20-30 years CD19 has gained attention as a potential target in the therapy of B-cell malignancies. In particular, targeting CD19 with the bispecific T-cell engager (BiTE) antibody Blinatumomab and T-cells modified by chimeric antigen receptors (CAR) has shown promising efficacy in early phase clinical trials for adults and children with precursor B-cell ALL (BCP-ALL). This review will discuss the rationale behind targeting CD19 in BCP-ALL and its potential importance in BCP-ALL signaling pathways.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Antígenos CD19/inmunología , Antineoplásicos/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Adulto , Antígenos CD19/química , Ensayos Clínicos como Asunto , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/inmunologíaRESUMEN
Functional studies of cellular immunity in patients with leukemia often require separation of leukemic cells from other peripheral blood mononuclear cells (PBMCs). This can pose a challenge when the number of leukemic cells is very high, such as in untreated patients with chronic lymphocytic leukemia (CLL). We found that when leukemia cell frequency was very high, anti-CD19 coated immunomagnetic beads did not thoroughly deplete B cells when used according to manufacturer's instructions. In this study, we depleted leukemic B cells using a modified protocol comprising serial rounds of depletion using immunomagnetic beads at reduced bead to cell ratios. This resulted in more effective B cell depletion with the use of fewer immunomagnetic beads, and without affecting viability or yield of non-B cells. CD19- PBMC subsets were retained, and serial depletion rounds did not activate T cells and monocytes. The positively isolated CLL cells were of high purity and were available for downstream analysis. This is a convenient and cost-effective method to enable in vitro analysis of immunocompetent cells from patients with leukemia.
Asunto(s)
Antígenos CD19/química , Linfocitos B/inmunología , Separación Inmunomagnética/métodos , Leucemia Linfocítica Crónica de Células B/inmunología , Humanos , Leucocitos Mononucleares/inmunologíaRESUMEN
BACKGROUND: Chimeric Antigen Receptors (CARs) consist of the antigen-recognition portion of a monoclonal antibody fused to an intracellular signaling domain capable of activating T-cells. CARs displayed on the surface of transduced cells perform non-MHC-restricted antigen recognition and activating intracellular signaling pathways for induction of target cytolysis, cytokine secretion and proliferation. Clinical trials are in progress assessing the use of mature T-lymphocytes transduced with CARs targeting CD19 antigen to treat B-lineage malignancies. CD19 is an attractive target for immunotherapy because of its consistent and specific expression in most of the stages of maturation and malignancies of B-lymphocyte origin, but not on hematopoietic stem cells. Antibodies against the extracellular domain of the CAR molecule (anti-Fab, Fc or idiotype) have been used for detection of CAR expression in research and clinical samples by flow cytometry, but may need development for each construct and present significant background in samples from xenograft models. METHODS: A specific reagent for the detection of anti-CD19 CAR expression was developed, a fusion protein consisting of human CD19 extracellular domains and the Fc region of human IgG1 (CD19sIg). Genes encoding CD19sIg fusion proteins were constructed by fusing either exons 1 to 3 (CD19sIg1-3) or exons 1 to 4 (CD19sIg1-4) of the human CD19 cDNA to a human IgG1Fc fragment. These fusion proteins are intended to work in similar fashion as the MHC Tetramers used for identification of antigen-specific T-cells, and may also have other applications in studies of activation of anti-CD19 CAR bearing cells. The CD19sIg proteins were produced from 293 T cells by stable lentiviral vector transduction and purification from culture medium. RESULTS: ELISA assays using several different monoclonal antibodies to CD19 demonstrated dose-related specific binding by the fusion molecule CD19sIg1-4, but no binding by CD19sIg1-3. Conjugation of the CD19sIg1-4 fusion protein to Alexa Fluor 488 allowed specific and sensitive staining of anti-CD19 CAR-bearing cells for flow cytometry assays, detecting as low as 0.5% of CAR-modified primary cells with minimal background staining. CONCLUSIONS: This fusion molecule is a sensitive reagent for detection of anti-CD19 CAR derived from any monoclonal antibody present in CAR-modified T-cells.
Asunto(s)
Antígenos CD19/química , Fragmentos de Inmunoglobulinas/química , Receptores de Antígenos/química , Proteínas Recombinantes de Fusión/metabolismo , Animales , Anticuerpos Monoclonales/química , Proliferación Celular , Trasplante de Células , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Células HEK293 , Humanos , Inmunoglobulina G/química , Leucocitos Mononucleares/citología , Activación de Linfocitos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Linfocitos T/citologíaRESUMEN
BACKGROUND: We developed a single-color multitarget flow cytometry (SM-FC) assay, a single-tube assay with graded mean fluorescence intensities (MFIs). We evaluated the repeatability of SM-FC, and its correlation with multicolor flow cytometry (MFC), to assess its application as a routine FC assay. METHODS: We selected CD19, CD3, CD4, and CD8 as antigen targets to analyze a lymphocyte subset. MFIs were graded by adjusting monoclonal antibody (mAb) volumes to detect several cell populations. Dimly labeled mAb was prepared by decreasing mAb volume and the optimum diluted volume was determined by serial dilution. SM-FC repeatability was analyzed 10 times in 2 normal controls. The correlation between SM-FC and MFC was evaluated in 20 normal and 23 patient samples. RESULTS: CV values (0.8-5.0% and 1.3-4.1% in samples 1 and 2, respectively) acquired by SM-FC with CD3-fluorescein α-isothyocyanate (FITC)(dim)+CD4-FITC(bright) and with CD19-FITC(dim)+CD3-FITC(bright) showed good repeatability, comparable to that acquired by MFC (1.6-3.7% and 1.0-4.8% in samples 1 and 2, respectively). Excellent correlation was observed between the 2 methods in the 20 normal samples (B cells, T cells, non-T(helper) cells, and T(helper) cells; r(2)=0.87, 0.97, 0.97, and 0.98, respectively; P<0.05). There were also linear relationships between SM-FC with CD19-FITC(dim)+CD3-FITC(bright) and CD8-PE(dim)+CD4-PE(bright), and MFC, in the 23 patient samples (B cells, T cells, T(cytotoxic) cells, and T(helper) cells; r(2)≥0.98, 0.99, 0.99, and 0.99, respectively; P<0.05). CONCLUSIONS: The multicolor, single-tube SM-FC technique is a potential alternative tool for identifying a lymphocyte subset.