RESUMEN
B cells and T cells are important components of the adaptive immune system and mediate anticancer immunity. The T cell landscape in cancer is well characterized, but the contribution of B cells to anticancer immunosurveillance is less well explored. Here we show an integrative analysis of the B cell and T cell receptor repertoire from individuals with metastatic breast cancer and individuals with early breast cancer during neoadjuvant therapy. Using immune receptor, RNA and whole-exome sequencing, we show that both B cell and T cell responses seem to coevolve with the metastatic cancer genomes and mirror tumor mutational and neoantigen architecture. B cell clones associated with metastatic immunosurveillance and temporal persistence were more expanded and distinct from site-specific clones. B cell clonal immunosurveillance and temporal persistence are predictable from the clonal structure, with higher-centrality B cell antigen receptors more likely to be detected across multiple metastases or across time. This predictability was generalizable across other immune-mediated disorders. This work lays a foundation for prioritizing antibody sequences for therapeutic targeting in cancer.
Asunto(s)
Linfocitos B , Neoplasias de la Mama , Vigilancia Inmunológica , Humanos , Femenino , Neoplasias de la Mama/inmunología , Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunología , Linfocitos T/inmunología , Monitorización Inmunológica , Secuenciación del Exoma , Antígenos de Neoplasias/inmunología , Metástasis de la Neoplasia , Células ClonalesRESUMEN
Passive immunization using monoclonal antibodies will play a vital role in the fight against COVID-19. The recent emergence of viral variants with reduced sensitivity to some current antibodies and vaccines highlights the importance of broad cross-reactivity. This study describes deep-mining of the antibody repertoires of hospitalized COVID-19 patients using phage display technology and B cell receptor (BCR) repertoire sequencing to isolate neutralizing antibodies and gain insights into the early antibody response. This comprehensive discovery approach has yielded a panel of potent neutralizing antibodies which bind distinct viral epitopes including epitopes conserved in SARS-CoV-1. Structural determination of a non-ACE2 receptor blocking antibody reveals a previously undescribed binding epitope, which is unlikely to be affected by the mutations in any of the recently reported major viral variants including B.1.1.7 (from the UK), B.1.351 (from South Africa) and B.1.1.28 (from Brazil). Finally, by combining sequences of the RBD binding and neutralizing antibodies with the B cell receptor repertoire sequencing, we also describe a highly convergent early antibody response. Similar IgM-derived sequences occur within this study group and also within patient responses described by multiple independent studies published previously.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , COVID-19/prevención & control , COVID-19/terapia , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Técnicas de Visualización de Superficie Celular/métodos , Minería de Datos/métodos , Epítopos/inmunología , Humanos , Inmunización Pasiva/métodos , Sueroterapia para COVID-19RESUMEN
Multiple myeloma is a hematologic cancer that disrupts normal bone marrow function and has multiple lines of therapeutic options, but is incurable as patients ultimately relapse. We developed a novel antibody-drug conjugate (ADC) targeting CS-1, a protein that is highly expressed on multiple myeloma tumor cells. The anti-CS-1 mAb specifically bound to cells expressing CS-1 and, when conjugated to a cytotoxic pyrrolobenzodiazepine payload, reduced the viability of multiple myeloma cell lines in vitro In mouse models of multiple myeloma, a single administration of the CS-1 ADC caused durable regressions in disseminated models and complete regression in a subcutaneous model. In an exploratory study in cynomolgus monkeys, the CS-1 ADC demonstrated a half-life of 3 to 6 days; however, no highest nonseverely toxic dose was achieved, as bone marrow toxicity was dose limiting. Bone marrow from dosed monkeys showed reductions in progenitor cells as compared with normal marrow. In vitro cell killing assays demonstrated that the CS-1 ADC substantially reduced the number of progenitor cells in healthy bone marrow, leading us to identify previously unreported CS-1 expression on a small population of progenitor cells in the myeloid-erythroid lineage. This finding suggests that bone marrow toxicity is the result of both on-target and off-target killing by the ADC.
Asunto(s)
Anticuerpos Monoclonales/química , Antineoplásicos/farmacología , Benzodiazepinas/química , Inmunoconjugados/farmacología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de Microfilamentos/antagonistas & inhibidores , Mieloma Múltiple/tratamiento farmacológico , Pirroles/química , Animales , Antineoplásicos/química , Apoptosis , Proliferación Celular , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Inmunoconjugados/química , Macaca fascicularis , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Microfilamentos/inmunología , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Deep sequencing of B cell receptor (BCR) heavy chains from a cohort of 31 COVID-19 patients from the UK reveals a stereotypical naive immune response to SARS-CoV-2 which is consistent across patients. Clonal expansion of the B cell population is also observed and may be the result of memory bystander effects. There was a strong convergent sequence signature across patients, and we identified 1,254 clonotypes convergent between at least four of the COVID-19 patients, but not present in healthy controls or individuals following seasonal influenza vaccination. A subset of the convergent clonotypes were homologous to known SARS and SARS-CoV-2 spike protein neutralizing antibodies. Convergence was also demonstrated across wide geographies by comparison of data sets between patients from UK, USA, and China, further validating the disease association and consistency of the stereotypical immune response even at the sequence level. These convergent clonotypes provide a resource to identify potential therapeutic and prophylactic antibodies and demonstrate the potential of BCR profiling as a tool to help understand patient responses.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/patología , Receptores de Antígenos de Linfocitos B/genética , SARS-CoV-2/inmunología , Linfocitos B/inmunología , COVID-19/inmunología , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Linfopenia/inmunología , Masculino , Persona de Mediana Edad , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
Background Heart failure is one of the leading causes of death in Western countries, and there is a need for new therapeutic approaches. Relaxin-2 is a peptide hormone that mediates pleiotropic cardiovascular effects, including antifibrotic, angiogenic, vasodilatory, antiapoptotic, and anti-inflammatory effects in vitro and in vivo. Methods and Results We developed RELAX10, a fusion protein composed of human relaxin-2 hormone and the Fc of a human antibody, to test the hypothesis that extended exposure of the relaxin-2 peptide could reduce cardiac hypertrophy and fibrosis. RELAX10 demonstrated the same specificity and similar in vitro activity as the relaxin-2 peptide. The terminal half-life of RELAX10 was 7 days in mouse and 3.75 days in rat after subcutaneous administration. We evaluated whether treatment with RELAX10 could prevent and reverse isoproterenol-induced cardiac hypertrophy and fibrosis in mice. Isoproterenol administration in mice resulted in increased cardiac hypertrophy and fibrosis compared with vehicle. Coadministration with RELAX10 significantly attenuated the cardiac hypertrophy and fibrosis compared with untreated animals. Isoproterenol administration significantly increased transforming growth factor ß1 (TGF-ß1)-induced fibrotic signaling, which was attenuated by RELAX10. We found that RELAX10 also significantly increased protein kinase B/endothelial NO synthase signaling and protein S-nitrosylation. In the reversal study, RELAX10-treated animals showed significantly reduced cardiac hypertrophy and collagen levels. Conclusions These findings support a potential role for RELAX10 in the treatment of heart failure.
Asunto(s)
Cardiomegalia/tratamiento farmacológico , Miocardio/patología , Animales , Cardiomegalia/inducido químicamente , Cardiomegalia/prevención & control , Fibrosis/inducido químicamente , Fibrosis/tratamiento farmacológico , Fibrosis/prevención & control , Isoproterenol/farmacología , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Type 2 diabetes (T2D) is a complex and progressive disease requiring polypharmacy to manage hyperglycaemia and cardiovascular risk factors. However, most patients do not achieve combined treatment goals. To address this therapeutic gap, we have developed MEDI4166, a novel glucagon-like peptide-1 (GLP-1) receptor agonist peptide fused to a proprotein convertase subtilisin/kexin type 9 (PCSK9) neutralising antibody that allows for glycaemic control and low-density lipoprotein cholesterol (LDL-C) lowering in a single molecule. The fusion has been engineered to deliver sustained peptide activity in vivo in combination with reduced potency, to manage GLP-1 driven adverse effects at high dose, and a favourable manufacturability profile. MEDI4166 showed robust and sustained LDL-C lowering in cynomolgus monkeys and exhibited the anticipated GLP-1 effects in T2D mouse models. We believe MEDI4166 is a novel molecule combining long acting agonist peptide and neutralising antibody activities to deliver a unique pharmacology profile for the management of T2D.
Asunto(s)
Anticuerpos Monoclonales , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Péptido 1 Similar al Glucagón , Hipoglucemiantes , Inhibidores de PCSK9 , Proteínas Recombinantes de Fusión , Animales , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/farmacología , Células CHO , Cricetulus , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Células Hep G2 , Humanos , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/farmacología , Macaca fascicularis , Masculino , Ratones , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Humans and higher primates are unique in that they lack uricase, the enzyme capable of oxidizing uric acid. As a consequence of this enzyme deficiency, humans have high serum uric acid levels. In some people, uric acid levels rise above the solubility limit resulting in crystallization in joints, acute inflammation in response to those crystals causes severe pain; a condition known as gout. Treatment for severe gout includes injection of non-human uricase to reduce serum uric acid levels. Krystexxa® is a hyper-PEGylated pig-baboon chimeric uricase indicated for chronic refractory gout that induces an immunogenic response in 91% of treated patients, including infusion reactions (26%) and anaphylaxis (6.5%). These properties limit its use and effectiveness. An innovative approach has been used to develop a therapeutic uricase with improved properties such as: soluble expression, neutral pH solubility, high E. coli expression level, thermal stability, and excellent activity. More than 200 diverse uricase sequences were aligned to guide protein engineering and reduce putative sequence liabilities. A single uricase lead candidate was identified, which showed low potential for immunogenicity in >200 human donor samples selected to represent diverse HLA haplotypes. Cysteines were engineered into the lead sequence for site specific PEGylation and studies demonstrated >95% PEGylation efficiency. PEGylated uricase retains enzymatic activity in vitro at neutral pH, in human serum and in vivo (rats and canines) and has an extended half-life. In canines, an 85% reduction in serum uric acid levels was observed with a single subcutaneous injection. This PEGylated, non-immunogenic uricase has the potential to provide meaningful benefits to patients with gout.
Asunto(s)
Gota/tratamiento farmacológico , Urato Oxidasa/uso terapéutico , Animales , Rastreo Diferencial de Calorimetría , Perros , Escherichia coli/metabolismo , Semivida , Humanos , Concentración de Iones de Hidrógeno , Cinética , Papio , Polietilenglicoles/química , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapéutico , Especificidad por Sustrato , Porcinos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Urato Oxidasa/efectos adversos , Urato Oxidasa/inmunologíaRESUMEN
Antibody-drug conjugate (ADC) which delivers cytotoxic drugs specifically into targeted cells through internalization and lysosomal trafficking has emerged as an effective cancer therapy. We show that a bivalent biparatopic antibody targeting two non-overlapping epitopes on HER2 can induce HER2 receptor clustering, which in turn promotes robust internalization, lysosomal trafficking, and degradation. When conjugated with a tubulysin-based microtubule inhibitor, the biparatopic ADC demonstrates superior anti-tumor activity over ado-trastuzumab emtansine (T-DM1) in tumor models representing various patient subpopulations, including T-DM1 eligible, T-DM1 ineligible, and T-DM1 relapsed/refractory. Our findings indicate that this biparatopic ADC has promising potential as an effective therapy for metastatic breast cancer and a broader patient population may benefit from this unique HER2-targeting ADC.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Inmunotoxinas/uso terapéutico , Maitansina/análogos & derivados , Receptor ErbB-2/inmunología , Trastuzumab/uso terapéutico , Ado-Trastuzumab Emtansina , Animales , Neoplasias de la Mama/inmunología , Femenino , Humanos , Maitansina/uso terapéutico , Ratones , Resultado del TratamientoRESUMEN
The neurotrophin receptor p75(NTR) is utilized by a variety of pathogens to gain entry into the central nervous system (CNS). We tested if this entry portal might be exploited using a phage display library to isolate internalizing antibodies that target the CNS in vivo. By applying a phage library that expressed human single chain variable fragment (scFv) antibodies on their surface to a transected sciatic nerve, we showed that (1) phage conjugated to anti-p75(NTR) antibody or phage scFv library pre-panned against p75(NTR) are internalized by neurons expressing p75(NTR); (2) subsequent retrograde axonal transport separates internalized phage from the applied phage; and, (3) internalized phage can be recovered from a proximal ligature made on a nerve. This approach resulted in 13-fold increase in the number of phage isolated from the injured nerve compared with the starting population, and isolation of 18 unique internalizing p75(NTR) antibodies that were transported from the peripheral nerve into the spinal cord, through the blood-brain barrier. In addition, antibodies recognizing other potentially internalized antigens were identified through in vivo selection using a fully diverse library. Because p75(NTR) expression is upregulated in motor neurons in response to injury and in disease, the p75(NTR) antibodies may have substantial potential for cell-targeted drug/gene delivery. In addition, this novel selection method provides the potential to generate panels of antibodies that could be used to identify further internalization targets, which could aid drug delivery across the blood-brain barrier.
Asunto(s)
Bacteriófagos/genética , Neuronas Motoras/metabolismo , Nervio Ciático/metabolismo , Anticuerpos de Cadena Única/metabolismo , Médula Espinal/metabolismo , Animales , Bacteriófagos/inmunología , Bacteriófagos/metabolismo , Barrera Hematoencefálica , Células Cultivadas , Femenino , Humanos , Terapia Molecular Dirigida , Neuronas Motoras/virología , Biblioteca de Péptidos , Ratas , Ratas Sprague-Dawley , Receptor de Factor de Crecimiento Nervioso/inmunología , Nervio Ciático/virología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/aislamiento & purificación , Médula Espinal/virología , Internalización del VirusRESUMEN
HIV-1 entry into cells is mediated by the envelope glycoprotein receptor-binding (gp120) and membrane fusion-promoting (gp41) subunits. The gp41 heptad repeat 1 (HR1) domain is the molecular target of the fusion-inhibitor drug enfuvirtide (T20). The HR1 sequence is highly conserved and therefore considered an attractive target for vaccine development, but it is unknown whether antibodies can access HR1. Herein, we use gp41-based peptides to select a human antibody, 5H/I1-BMV-D5 (D5), that binds to HR1 and inhibits the assembly of fusion intermediates in vitro. D5 inhibits the replication of diverse HIV-1 clinical isolates and therefore represents a previously unknown example of a crossneutralizing IgG selected by binding to designed antigens. NMR studies and functional analyses map the D5-binding site to a previously identified hydrophobic pocket situated in the HR1 groove. This hydrophobic pocket was proposed as a drug target and subsequently identified as a common binding site for peptide and peptidomimetic fusion inhibitors. The finding that the D5 fusion-inhibitory antibody shares the same binding site suggests that the hydrophobic pocket is a "hot spot" for fusion inhibition and an ideal target on which to focus a vaccine-elicited antibody response. Our data provide a structural framework for the design of new immunogens and therapeutic antibodies with crossneutralizing potential.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Epítopos/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Modelos Moleculares , Sitios de Unión de Anticuerpos/genética , Sitios de Unión de Anticuerpos/inmunología , Epítopos/genética , Proteína gp41 de Envoltorio del VIH/genética , Humanos , Luciferasas , Resonancia Magnética Nuclear Biomolecular , Reacción en Cadena de la Polimerasa , Unión ProteicaRESUMEN
Guided selection is a method of producing a human version of a rodent or any other non-human antibody. The process is a serial transition from rodent to human via rodent-human chimaerics, through to a panel of human antibodies with similar characteristics to those of the starting rodent antibody. The guided selection process can be undertaken using either phage display or ribosome display, and chimaeric antibodies can be made either in series or parallel, with or without the retention of the original rodent CDR3s. Guided selection has successfully been used for the generation of a number of human versions of rodent antibodies, including HUMIRA, an inhibitor of tumour necrosis factor-alpha which is approved for the treatment of moderate to severe rheumatoid arthritis in over 40 countries.
Asunto(s)
Anticuerpos Monoclonales/genética , Ingeniería de Proteínas/métodos , Adalimumab , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Técnicas Químicas Combinatorias , Humanos , Región Variable de Inmunoglobulina/genética , Indicadores y Reactivos , Biblioteca de Péptidos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Ribosomas/inmunología , Ribosomas/metabolismo , Roedores/inmunologíaRESUMEN
Ribosome display is a polymerase chain reaction-based in vitro display technology that is well suited to the selection and evolution of high affinity antibodies. Both eukaryotic and prokaryotic translation systems have been applied to ribosome display, and the technology's utility has been demonstrated in the antibody isolation process. In particular, ribosome display lends itself to the evolution of functional characteristics, such as potency, of lead candidate antibodies to provide therapeutic antibodies. Large libraries (10(12)) can be rapidly constructed, antibodies selected, and sequence space extensively explored by targeted mutagenesis techniques or by random mutagenesis throughout the antibody sequence. Using such approaches in ribosome display systems lead antibodies derived from phage display or from immunised animals have been improved > 1000-fold in potency within 6 months. This review will discuss the technology and give an insight into how ribosome display is being applied to the antibody lead discovery and optimisation processes.
Asunto(s)
Anticuerpos/genética , Ribosomas/genética , Tecnología Farmacéutica/métodos , Animales , Anticuerpos/administración & dosificación , Biblioteca de Genes , HumanosRESUMEN
Autoimmune diseases are a significant area of unmet medical need in the Western World, but human antibodies are an emerging drug class that could address this demand. Some autoimmune diseases, such as rheumatoid arthritis, are currently benefiting from antibody treatment and new and existing technologies for antibody generation could facilitate the production of effective human antibodies as future drug candidates for other autoimmune diseases. Several methods of generating human antibodies for use as therapeutics have been established, the most commonly used being phage display and transgenic mouse technologies and more recently, cell-free display technologies have also emerged. In this review, we explain the principles behind the various methods of antibody generation and highlight some potential benefits of certain approaches in the context of treatment of autoimmune disease.