RESUMEN

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20304-y.

RESUMEN

HIV broadly neutralizing antibodies (bnAbs) can suppress viremia and protect against HIV infection. However, their elicitation is made difficult by low frequencies of appropriate precursor B cell receptors and the complex maturation pathways required to generate bnAbs from these precursors. Antibody genes can be engineered into B cells for expression as both a functional antigen receptor on cell surfaces and as secreted antibody. Here, we show that HIV bnAb-engineered primary mouse B cells can be adoptively transferred and vaccinated in immunocompetent mice resulting in the expansion of durable bnAb memory and long-lived plasma cells. Somatic hypermutation after immunization indicates that engineered cells have the capacity to respond to an evolving pathogen. These results encourage further exploration of engineered B cell vaccines as a strategy for durable elicitation of HIV bnAbs to protect against infection and as a contributor to a functional HIV cure.

Asunto(s)

Vacunas contra el SIDA/inmunología , Linfocitos B/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Animales , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Linfocitos B/fisiología , Linfocitos B/trasplante , Anticuerpos ampliamente neutralizantes/sangre , Anticuerpos ampliamente neutralizantes/genética , Femenino , Ingeniería Genética/métodos , Células HEK293 , Anticuerpos Anti-VIH/sangre , Anticuerpos Anti-VIH/genética , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH , Humanos , Inmunización , Memoria Inmunológica/genética , Activación de Linfocitos , Ratones Endogámicos C57BL , Hipermutación Somática de Inmunoglobulina

RESUMEN

We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin (Ig) genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody (bnAb), PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains (LCs) resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses. Peripheral blood derived primary B cells from three different donors were edited using this strategy. Engineered cells could bind the PG9 epitope and sequenced mRNA showed PG9 HC transcribed as several different isotypes after culture with CD40 ligand and IL-4.

Asunto(s)

Reacciones Antígeno-Anticuerpo/genética , Linfocitos B/inmunología , Edición Génica/métodos , Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos , Sistemas CRISPR-Cas , Línea Celular , Citidina Desaminasa/metabolismo , Anticuerpos Anti-VIH/inmunología , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunología

RESUMEN

Recent efforts toward HIV vaccine development include the design of immunogens that can engage B cell receptors with the potential to affinity mature into broadly neutralizing antibodies (bnAbs). V2-apex bnAbs, which bind a protein-glycan region on HIV envelope glycoprotein (Env) trimer, are among the most broad and potent described. We show here that a rare "glycan hole" at the V2 apex is enriched in HIV isolates neutralized by inferred precursors of prototype V2-apex bnAbs. To investigate whether this feature could focus neutralizing responses onto the apex bnAb region, we immunized wild-type rabbits with soluble trimers adapted from these Envs. Potent autologous tier 2 neutralizing responses targeting basic residues in strand C of the V2 region, which forms the core epitope for V2-apex bnAbs, were observed. Neutralizing monoclonal antibodies (mAbs) derived from these animals display features promising for subsequent broadening of the response.

Asunto(s)

Anticuerpos Monoclonales/biosíntesis , Anticuerpos Neutralizantes/biosíntesis , Antígenos Virales/inmunología , Anticuerpos Anti-VIH/biosíntesis , Infecciones por VIH/prevención & control , VIH-1/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/biosíntesis , Vacunas contra el SIDA/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Neutralizantes/química , Antígenos Virales/química , Antígenos Virales/genética , Sitios de Unión , Epítopos/química , Epítopos/inmunología , Femenino , Anticuerpos Anti-VIH/química , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/química , VIH-1/genética , Humanos , Inmunización , Pruebas de Neutralización , Polisacáridos/química , Polisacáridos/inmunología , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Conejos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética

RESUMEN

Vaccination is a reliable method of prophylaxis and a crucial measure for public health. However, the majority of vaccines cannot be administered orally due to their degradation in the harsh gut environment or inability to cross the GI tract. In this study, we report the first proof-of-concept study of orally producible chemically programmed antibodies via specific conjugation of adaptor ligands to endogenous antibodies, in vivo. Pre-immuniztion with 2,4-dinitrophenyl (DNP), or the reactive hapten, 1,3-diketone (DK), or a novel reactive hapten, vinyl sulfone (VS) in mice, followed by oral administration of adaptor ligands composed of the hapten and biotin to the pre-immunized mice resulted in successful in vivo formation of the biotin-hapten-antibody complexes within 2h. Pharmacokinetic evaluations revealed that apparent serum concentrations of programmed antibodies were up to 144nM and that the serum half-lives reached up to 34.4h. These findings show promise for the future development of orally bioavailable drug-hapten-antibody complexes asa strategy to quickly and easily modulate immune targets for aggressive pathogens as well as cancer.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Biotina/inmunología , Haptenos/inmunología , Cetonas/inmunología , Administración Oral , Animales , Anticuerpos Monoclonales/farmacocinética , Reacciones Antígeno-Anticuerpo/efectos de los fármacos , Biotina/administración & dosificación , Haptenos/administración & dosificación , Cetonas/administración & dosificación , Ligandos , Ratones , Ratones Endogámicos BALB C , Estructura Molecular

RESUMEN

Bioorthogonal labeling of antibodies enables the conjugation of compounds, such as small molecules or peptides, which expand targeting capacity or enhance cytotoxicity. Taking advantage of a cyclohexene sulfonamide compound that site-selectively labels Lys64 in human serum albumin (HSA), we demonstrate that domain I of HSA can be used as a fusion protein for the preparation of antibody conjugates. Trastuzumab fusions were expressed at the N-terminus of the light chain or the C-terminus of the heavy chain enabling conjugation to small molecules. Moreover, these conjugates retained HER2 binding and proved to be highly stable in human plasma. Antibody conjugation via HSA domain I fusion should therefore have broad utility for making serum-stable antibody conjugates, particularly for antibody-drug conjugates.

Asunto(s)

Inmunoconjugados/química , Proteínas Recombinantes de Fusión/química , Albúmina Sérica/química , Anticuerpos/química , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Citometría de Flujo , Humanos , Inmunoconjugados/sangre , Inmunoconjugados/metabolismo , Lisina/química , Dominios Proteicos , Ingeniería de Proteínas/métodos , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión/sangre , Proteínas Recombinantes de Fusión/metabolismo , Rodaminas/química , Trastuzumab/química

RESUMEN

Utilization of chemically programmed antibodies (cpAbs) is regarded to be one of the most efficient methods for the development of therapeutic systems. cpAbs can extend the half-life of programming reagents, activate immune systems via the Fc region of antibodies and achieve universal vaccination by attaching varieties of small, programmed molecules. In the current study, we aimed to develop a novel labeling reagent for the preparation of cpAbs and found that N-sulfonyl-ß-lactams (NSBLs) were optimal. NSBL can be synthesized from readily available 4-(bromomethyl)benzenesulfonyl chloride via few simple manipulations and can label the aldolase monoclonal antibody (mAb) 84G3, which could not be labeled effectively by the conventional labeling reagent, N-acyl-ß-lactam (NABL). We also demonstrated that the conjugate, which consists of mAb 84G3 and an NSBL bearing a biotin moiety, maintained strong binding activity to streptavidin. In addition, the stability assay of NSBL revealed that NSBLs can tolerate aqueous media without significant decomposition over 24h.

Asunto(s)

Anticuerpos Monoclonales/química , Fructosa-Bifosfato Aldolasa/química , Haptenos/química , Fragmentos Fab de Inmunoglobulinas/química , beta-Lactamas/química , Anticuerpos Monoclonales/inmunología , Reacciones Antígeno-Anticuerpo , Biotina/química , Biotina/metabolismo , Fructosa-Bifosfato Aldolasa/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Unión Proteica , Estabilidad Proteica , Estreptavidina/química , Estreptavidina/metabolismo

RESUMEN

Site-specific recombinases are powerful tools for genome engineering. Hyperactivated variants of the resolvase/invertase family of serine recombinases function without accessory factors, and thus can be re-targeted to sequences of interest by replacing native DNA-binding domains (DBDs) with engineered zinc-finger proteins (ZFPs). However, imperfect modularity with particular domains, lack of high-affinity binding to all DNA triplets, and difficulty in construction has hindered the widespread adoption of ZFPs in unspecialized laboratories. The discovery of a novel type of DBD in transcription activator-like effector (TALE) proteins from Xanthomonas provides an alternative to ZFPs. Here we describe chimeric TALE recombinases (TALERs): engineered fusions between a hyperactivated catalytic domain from the DNA invertase Gin and an optimized TALE architecture. We use a library of incrementally truncated TALE variants to identify TALER fusions that modify DNA with efficiency and specificity comparable to zinc-finger recombinases in bacterial cells. We also show that TALERs recombine DNA in mammalian cells. The TALER architecture described herein provides a platform for insertion of customized TALE domains, thus significantly expanding the targeting capacity of engineered recombinases and their potential applications in biotechnology and medicine.

Asunto(s)

ADN Nucleotidiltransferasas/química , Proteínas de Unión al ADN/química , Ingeniería de Proteínas , Recombinasas/química , Secuencia de Bases , Dominio Catalítico , ADN/química , ADN/metabolismo , ADN Nucleotidiltransferasas/genética , Proteínas de Unión al ADN/genética , Biblioteca de Genes , Células HEK293 , Humanos , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Recombinasas/metabolismo , Secuencias Repetitivas de Aminoácido

RESUMEN

The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human scFv (single chain antibody fragment) libraries using a short linker (GGSSRSS) or a long linker (GGSSRSSSSGGGGSGGGG). In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final scFv products that are used for cloning.

Asunto(s)

Biblioteca de Genes , Técnicas Inmunológicas/métodos , Anticuerpos de Cadena Única/genética , Tecnología Farmacéutica/métodos , Productos Biológicos , Humanos , Factores Inmunológicos/genética , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/genética

RESUMEN

The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human Fab (fragment antigen binding) antibody libraries. In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final Fab products that are used for cloning.

Asunto(s)

Biblioteca de Genes , Fragmentos Fab de Inmunoglobulinas/genética , Técnicas Inmunológicas/métodos , Tecnología Farmacéutica/métodos , Productos Biológicos , Humanos , Factores Inmunológicos/genética , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/genética

RESUMEN

Engineered zinc-finger transcription factors (ZF-TF) are powerful tools to modulate the expression of specific genes. Complex libraries of ZF-TF can be delivered into cells to scan the genome for genes responsible for a particular phenotype or to select the most effective ZF-TF to regulate an individual gene. In both cases, the construction of highly representative and unbiased libraries is critical. In this protocol, we describe a user-friendly ZF technology suitable for the creation of complex libraries and the construction of customized ZF-TFs. The new technology described here simplifies the building of ZF libraries, avoids PCR-introduced bias and ensures equal representation of every module. We also describe the construction of a customized ZF-TF that can be transferred to a number of expression vectors. This protocol can be completed in 9-11 d.

Asunto(s)

Biblioteca de Péptidos , Ingeniería de Proteínas/métodos , Dedos de Zinc/genética , Animales , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Vectores Genéticos , Humanos , Plásmidos/genética , Proteínas Recombinantes/genética , Factores de Transcripción/genética

RESUMEN

Phage-displayed peptides that selectively bind to aldolase catalytic antibody 93F3 when bound to a particular 1,3-diketone hapten derivative have been developed using designed selection strategies with libraries containing 7-12 randomized amino acid residues. These phage-displayed peptides discriminated the particular 93F3-diketone complex from ligand-free 93F3 and from 93F3 bound to other 1,3-diketone hapten derivatives. By altering the selection procedures, phage-displayed peptides that bind to antibody 93F3 in the absence of 1,3-diketone hapten derivatives have also been developed. With using these phage-displayed peptides, ligand-bound states of the antibody were distinguished from each other. A docking model of one of the peptides bound to the antibody 93F3-diketone complex was created using a sequential divide-and-conquer peptide docking strategy; the model suggests that the peptide interacts with both the antibody and the ligand through a delicate hydrogen bonding network.

Asunto(s)

Anticuerpos Catalíticos/metabolismo , Bacteriófago M13/metabolismo , Sitios de Unión de Anticuerpos , Catalasa/metabolismo , Oligopéptidos/química , Oligopéptidos/metabolismo , Animales , Anticuerpos Catalíticos/química , Anticuerpos Catalíticos/genética , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Bacteriófago M13/química , Catalasa/inmunología , Humanos , Cetonas/química , Cetonas/metabolismo , Ligandos , Ratones , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Oligopéptidos/inmunología , Biblioteca de Péptidos

RESUMEN

A 21-mer peptide that can be used to covalently introduce synthetic molecules into proteins has been developed. Phage-displayed peptide libraries were subjected to reaction-based selection with 1,3-diketones. The peptide was further evolved by addition of a randomized region and reselection for improved binding. The resulting 21-mer peptide had a reactive amino group that formed an enaminone with 1,3-diketone and was used as a tag for labeling of maltose binding protein. Using this peptide tag and 1,3-diketone derivatives, a variety of molecules such as reporter probes and functionalities may be covalently introduced into proteins of interest.

Asunto(s)

Proteínas Portadoras/química , Péptidos/síntesis química , Proteínas Recombinantes de Fusión/química , Cetonas/química , Proteínas de Unión a Maltosa , Biblioteca de Péptidos , Péptidos/química

RESUMEN

A peptide that functions only in the presence of a protein has been developed using reaction-based selection from peptide phage libraries. The peptide was not functional in the absence of the protein, but formed enaminones with 1,3-diketone derivatives when bound to the protein.

Asunto(s)

Péptidos/fisiología , Proteínas/fisiología , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Péptidos/química , Electricidad Estática

RESUMEN

Considerable progress has been made in recent years in the design of transcription factors for the directed regulation of endogenous genes. Although many strategies involve selection methods that must be applied for each new target sequence, we have developed an approach based on linkage of predefined zinc finger domains that each recognize a three-base pair DNA sequence to construct artificial transcription factors that bind to a desired sequence. These domains can be assembled to recognize unique 18-base pair DNA sequences with high specificity. Here we report the development and characterization of zinc finger domains that bind to 15 of the 16 5'-CNN-3' subsites. These domains were created through a combination of phage display selection, site-directed mutagenesis, and de novo design. Furthermore, these domains were used to generate a highly specific six-finger protein targeting the ERBB-2 promoter. When fused to regulatory domains, this protein was capable of up- and down-regulating the expression of the endogenous ERBB-2 gene. With the addition of this collection of predefined zinc finger domains, most 5'-CNN-3'-, 5'-GNN-3'-, and 5'-ANN-3'-containing sequences can now be rapidly targeted for directed gene regulation and nuclease cleavage.

Asunto(s)

Regulación de la Expresión Génica , Ingeniería de Proteínas/métodos , Factores de Transcripción/química , Dedos de Zinc , Secuencia de Aminoácidos , Animales , ADN/química , Desoxirribonucleasa I/metabolismo , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Biblioteca de Genes , Marcación de Gen , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligonucleótidos/química , Biblioteca de Péptidos , Regiones Promotoras Genéticas , Conformación Proteica , Estructura Terciaria de Proteína , Receptor ErbB-2/metabolismo , Retroviridae/genética , Programas Informáticos , Regulación hacia Arriba

RESUMEN

Small (24-35 amino acid residues) peptides that catalyze carbon-carbon bond transformations including aldol, retro-aldol, and Michael reactions in aqueous buffer via an enamine mechanism have been developed. Peptide phage libraries were created by appending six randomized amino acid residues to the C-terminus or to the N-terminus of an 18-mer alpha-helix peptide containing lysine residues. Reaction-based selection with 1,3-diketones was performed to trap the amino groups of reactive lysine residues that were necessary for the catalysis via an enamine mechanism by formation of stable enaminones. The selected 24-mer peptides catalyzed the reactions with improved activities. The improved activities were correlated with improved folded states of the peptides. The catalyst was then improved with respect to substrate specificity by appending a phage display-derived substrate-binding module. The resulting 35-mer peptide functioned with a significant proportion of the catalytic proficiency of larger protein catalysts. These results indicate that small designer enzymes with good rate acceleration and excellent substrate specificity can be created by combination of design and reaction-based selection from libraries.

Asunto(s)

Diseño de Fármacos , Fructosa-Bifosfato Aldolasa/síntesis química , Biblioteca de Péptidos , Ingeniería de Proteínas/métodos , Pliegue de Proteína , Aldehídos/química , Aminas/química , Secuencia de Aminoácidos , Catálisis , Activación Enzimática , Fructosa-Bifosfato Aldolasa/química , Cetonas/química , Cinética , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , Especificidad por Sustrato , Tetrosas/química , beta-Lactamas/química

RESUMEN

Catalytic aldolase antibodies, generated by reactive immunization, catalyze the aldol reaction with the efficiency of natural enzymes, but accept a much broader range of substrates. Two separate groups of aldolase antibodies that catalyze the same aldol reactions with antipodal selectivity were analyzed by comparing their amino acid sequences with their crystal structures, site-directed mutagenesis data, and computational docking of the transition states of the aldol reaction. The crystal structure of aldolase antibody 93F3 Fab' at 2.5A resolution revealed a combining site with two lysine residues, including LysL89 that reacts to form the covalent enamine intermediate. In contrast, antibody 33F12 has one active site lysine, LysH93. The reactive lysine residues in each group of antibodies are differentially located on the heavy and light chain variable regions in pseudo-symmetric opposite orientations, but both within highly hydrophobic environments. Thus, the defining feature for the observed enantioselectivities of these aldolase antibody catalysts is the respective location and relative disposition of the reactive lysine residues within the active sites of these catalysts.

Asunto(s)

Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Fructosa-Bifosfato Aldolasa/inmunología , Secuencia de Aminoácidos , Anticuerpos/química , Anticuerpos/genética , Especificidad de Anticuerpos/genética , Biología Computacional , Cristalografía por Rayos X , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína

RESUMEN

High-throughput screening of cells expressing active catalytic antibody clones by flow cytometry is described. A fluorogenic retro-aldol retro-Michael substrate was designed and synthesized with incorporation of a chloromethyl moiety for intracellular retention. Hybridoma or transfected mammalian cells expressing catalytic antibody molecules could be rapidly isolated.

Asunto(s)

Anticuerpos Catalíticos/farmacología , Fructosa-Bifosfato Aldolasa/metabolismo , Animales , Citometría de Flujo/métodos , Fructosa-Bifosfato Aldolasa/efectos de los fármacos , Hibridomas , Cinética , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Transfección

RESUMEN

Aldolase antibodies that operate via an enamine mechanism were developed by in vitro selection. Antibody Fab phage display libraries were created where the catalytic active site residues of aldolase antibodies 38C2 and 33F12 were combined with a naive human antibody V gene repertoire. Selection from these libraries with 1,3-diketones covalently trapped the amino groups of reactive lysine residues by formation of stable enaminones. The selected aldolase antibodies retained the essential catalytic lysine residue and its function in altered and humanized primary antibody structures. The substrate specificity of the aldolase antibodies was directly related to the structure of the diketone used for selection. The k(cat) values of the antibody-catalyzed retro-aldol reactions were correlated with the K(d) values, i.e. the reactivities of the selected aldolase antibodies for the corresponding diketones. Antibodies that bound to the diketone with a lower K(d) value displayed a higher k(cat) value in the retro-aldol reaction, and a linear relationship was observed in the plots of logk(cat) versus logK(d). These results indicate that selections with diketones directed the evolution of aldolase antibodies in vitro that operate via an enamine mechanism. This strategy provides a route to tailor-made aldol catalysts with different substrate specificities.

Asunto(s)

Fructosa-Bifosfato Aldolasa/metabolismo , Fragmentos Fab de Inmunoglobulinas/metabolismo , Aminas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Sitios de Unión de Anticuerpos , Catálisis , Evolución Molecular Dirigida , Ensayo de Inmunoadsorción Enzimática , Fructosa-Bifosfato Aldolasa/química , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Cetonas/metabolismo , Cinética , Datos de Secuencia Molecular , Pentanonas/metabolismo , Biblioteca de Péptidos , Alineación de Secuencia , Especificidad por Sustrato