RESUMEN
H chain-only Igs are naturally produced in camelids and sharks. Because these Abs lack the L chain, the Ag-binding domain is half the size of a traditional Ab, allowing this type of Ig to bind to targets in novel ways. Consequently, the H chain-only single-domain Ab (sdAb) structure has the potential to increase the repertoire and functional range of an active humoral immune system. The majority of vertebrates use the standard heterodimeric (both H and L chains) structure and do not produce sdAb format Igs. To investigate if other animals are able to support sdAb development and function, transgenic chickens (Gallus gallus) were designed to produce H chain-only Abs by omitting the L chain V region and maintaining only the LC region to serve as a chaperone for Ab secretion from the cell. These birds produced 30-50% normal B cell populations within PBMCs and readily expressed chicken sequence sdAbs. Interestingly, the H chains contained a spontaneous CH1 deletion. Although no isotype switching to IgY or IgA occurred, the IgM repertoire was diverse, and immunization with a variety of protein immunogens rapidly produced high and specific serum titers. mAbs of high affinity were efficiently recovered by single B cell screening. In in vitro functional assays, the sdAbs produced by birds immunized against SARS-CoV-2 were also able to strongly neutralize and prevent viral replication. These data suggest that the truncated L chain design successfully supported sdAb development and expression in chickens.
Asunto(s)
Animales Modificados Genéticamente , Pollos , Cadenas Pesadas de Inmunoglobulina , Anticuerpos de Dominio Único , Animales , Pollos/inmunología , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , SARS-CoV-2/inmunología , SARS-CoV-2/genética , COVID-19/inmunología , Transgenes/genética , Linfocitos B/inmunología , Anticuerpos Antivirales/inmunología , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , HumanosRESUMEN
Since the discovery of antibody-producing B cells in chickens six decades ago, chickens have been a model for B-cell development in gut-associated lymphoid tissue species. Here we describe targeting of the immunoglobulin light chain locus by homologous recombination in chicken primordial germ cells (PGCs) and generation of VJCL knockout chickens. In contrast to immunoglobulin heavy chain knockout chickens, which completely lack mature B cells, homozygous light chain knockout (IgL(-/-) ) chickens have a small population of B lineage cells that develop in the bursa and migrate to the periphery. This population of B cells expresses the immunoglobulin heavy chain molecule on the cell surface. Soluble heavy-chain-only IgM and IgY proteins of reduced molecular weight were detectable in plasma in 4-week-old IgL(-/-) chickens, and antigen-specific IgM and IgY heavy chain proteins were produced in response to immunization. Circulating heavy-chain-only IgM showed a deletion of the CH1 domain of the constant region enabling the immunoglobulin heavy chain to be secreted in the absence of the light chain. Our data suggest that the heavy chain by itself is enough to support all the important steps in B-cell development in a gut-associated lymphoid tissue species.
Asunto(s)
Anticuerpos/genética , Linfocitos B/inmunología , Linfocitos B/metabolismo , Expresión Génica , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Animales , Animales Modificados Genéticamente , Anticuerpos/inmunología , Formación de Anticuerpos/genética , Formación de Anticuerpos/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Pollos , Eliminación de Gen , Técnicas de Inactivación de Genes , Orden Génico , Marcación de Gen , Vectores Genéticos/genética , Cadenas Ligeras de Inmunoglobulina/química , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Dominios Proteicos/genéticaRESUMEN
Cre recombinase has been extensively used for genome engineering in transgenic mice yet its use in other species has been more limited. Here we describe the generation of transgenic chickens expressing Cre recombinase. Green fluorescent protein (GFP)-positive chicken primordial germ cells were stably transfected with ß-actin-Cre-recombinase using phiC31 integrase and transgenic chickens were generated. Cre recombinase activity was verified by mating Cre birds to birds carrying a floxed transgene. Floxed sequences were only excised in offspring from roosters that inherited the Cre recombinase but were excised in all offspring from hens carrying the Cre recombinase irrespective of the presence of the Cre transgene. The Cre recombinase transgenic birds were healthy and reproductively normal. The Cre and GFP genes in two of the lines were closely linked whereas the genes segregated independently in a third line. These founders allowed development of GFP-expressing and non-GFP-expressing Cre recombinase lines. These lines of birds create a myriad of opportunities to study developmentally-regulated and tissue-specific expression of transgenes in chickens.
Asunto(s)
Pollos/genética , Integrasas/genética , Recombinación Genética , Animales , Animales Modificados Genéticamente , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes , Especificidad de Órganos , Regiones Promotoras Genéticas , TransgenesRESUMEN
Gene targeting by homologous recombination or by sequence-specific nucleases allows the precise modification of genomes and genes to elucidate their functions. Although gene targeting has been used extensively to modify the genomes of mammals, fish, and amphibians, a targeting technology has not been available for the avian genome. Many of the principles of humoral immunity were discovered in chickens, yet the lack of gene targeting technologies in birds has limited biomedical research using this species. Here we describe targeting the joining (J) gene segment of the chicken Ig heavy chain gene by homologous recombination in primordial germ cells to establish fully transgenic chickens carrying the knockout. In homozygous knockouts, Ig heavy chain production is eliminated, and no antibody response is elicited on immunization. Migration of B-lineage precursors into the bursa of Fabricius is unaffected, whereas development into mature B cells and migration from the bursa are blocked in the mutants. Other cell types in the immune system appear normal. Chickens lacking the peripheral B-cell population will provide a unique experimental model to study avian immune responses to infectious disease. More generally, gene targeting in avian primordial germ cells will foster advances in diverse fields of biomedical research such as virology, stem cells, and developmental biology, and provide unique approaches in biotechnology, particularly in the field of antibody discovery.
Asunto(s)
Linfocitos B/citología , Pollos/genética , Técnicas de Inactivación de Genes/métodos , Ingeniería Genética/métodos , Células Germinativas/química , Cadenas Pesadas de Inmunoglobulina/genética , Animales , Linfocitos B/metabolismo , Southern Blotting , Pollos/inmunología , Metilación de ADN , Cartilla de ADN/genética , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Genotipo , Células Germinativas/metabolismo , InmunohistoquímicaRESUMEN
Primordial germ cells (PGCs) are the precursors of sperm and eggs. In most animals, segregation of the germ line from the somatic lineages is one of the earliest events in development; in avian embryos, PGCs are first identified in an extra-embryonic region, the germinal crescent, after approximately 18 h of incubation. After 50-55 h of development, PGCs migrate to the gonad and subsequently produce functional sperm and oocytes. So far, cultures of PGCs that remain restricted to the germ line have not been reported in any species. Here we show that chicken PGCs can be isolated, cultured and genetically modified while maintaining their commitment to the germ line. Furthermore, we show that chicken PGCs can be induced in vitro to differentiate into embryonic germ cells that contribute to somatic tissues. Retention of the commitment of PGCs to the germ line after extended periods in culture and after genetic modification combined with their capacity to acquire somatic competence in vitro provides a new model for developmental biology. The utility of the model is enhanced by the accessibility of the avian embryo, which facilitates access to the earliest stages of development and supplies a facile route for the reintroduction of PGCs into the embryonic vasculature. In addition, these attributes create new opportunities to manipulate the genome of chickens for agricultural and pharmaceutical applications.
Asunto(s)
Linaje de la Célula , Pollos/genética , Células Germinativas/citología , Células Germinativas/metabolismo , Mutación de Línea Germinal/genética , Células Madre/citología , Células Madre/metabolismo , Animales , Línea Celular , Células Cultivadas , Embrión de Pollo , Femenino , Citometría de Flujo , Ingeniería Genética/métodos , Genoma/genética , Células Germinativas/trasplante , Cariotipificación , Masculino , Óvulo/citología , Óvulo/metabolismo , Espermatozoides/citología , Espermatozoides/metabolismo , Trasplante de Células MadreRESUMEN
Bispecific antibodies are an important and growing segment in antibody therapeutics, particularly in the immuno-oncology space. Manufacturing of a bispecific antibody with two different heavy chains is greatly simplified if the light chains can be the same for both arms of the antibody. Here, we introduce a strain of common light chain chickens, called OmniClic®, that produces antibody repertoires largely devoid of light chain diversity. The antibody repertoire in these chickens is composed of diverse human heavy chain variable regions capable of high-affinity antigen-specific binding and broad epitope diversity when paired with the germline human kappa light chain. OmniClic birds can be used in immunization campaigns for discovery of human heavy chains to different targets. Subsequent pairing of the heavy chain with a germline human kappa light chain serves to facilitate bispecific antibody production by increasing the efficiency of correct pairing. Abbreviations: AID: activation-induced cytidine deaminase; bsAb: bispecific antibody; CDR: complementarity-determining region; CL: light chain constant region; CmLC: common light chain; D: diversity region; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; Fc: fragment crystallizable; FcRn: neonatal Fc receptor; FR: framework region; GEM: gel-encapsulated microenvironment; Ig: immunoglobulin; IMGT: the international ImMunoGeneTics information system®; J: joining region; KO: knockout; mAb: monoclonal antibody; NGS: next-generation sequencing; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PGC: primordial germ cell; PGRN: progranulin; TCR: T cell receptor; V: variable region; VK: kappa light chain variable region; VL: light chain variable region; VH: heavy chain variable region.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos/inmunología , Pollos/inmunología , Epítopos/inmunología , Cadenas Ligeras de Inmunoglobulina/inmunología , Animales , Antígenos/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Citometría de Flujo/métodos , Humanos , Inmunización/métodos , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas kappa de Inmunoglobulina/inmunología , Ingeniería de Proteínas/métodosRESUMEN
Most of the approved monoclonal antibodies used in the clinic were initially discovered in mice. However, many targets of therapeutic interest are highly conserved proteins that do not elicit a robust immune response in mice. There is a need for non-mammalian antibody discovery platforms which would allow researchers to access epitopes that are not recognized in mammalian hosts. Recently, we introduced the OmniChicken®, a transgenic animal carrying human VH3-23 and VK3-15 at its immunoglobulin loci. Here, we describe a new version of the OmniChicken which carries VH3-23 and either VL1-44 or VL3-19 at its heavy and light chain loci, respectively. The Vλ-expressing birds showed normal B and T populations in the periphery. A panel of monoclonal antibodies demonstrated comparable epitope coverage of a model antigen compared to both wild-type and Vκ-expressing OmniChickens. Kinetic analysis identified binders in the picomolar range. The Vλ-expressing bird increases the antibody diversity available in the OmniChicken platform, further enabling discovery of therapeutic leads.
Asunto(s)
Animales Modificados Genéticamente/genética , Pollos/genética , Cadenas lambda de Inmunoglobulina/genética , Animales , Animales Modificados Genéticamente/inmunología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Linfocitos B/inmunología , Pollos/inmunología , Humanos , Inmunidad Humoral , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/inmunología , Progranulinas/inmunología , Linfocitos T/inmunología , Transgenes/genéticaRESUMEN
Monoclonal antibodies (mAbs) for therapeutic applications should be as similar to native human antibodies as possible to minimize their immunogenicity in patients. Several transgenic animal platforms are available for the generation of fully human mAbs. Attributes such as specificity, efficacy and Chemistry, Manufacturing and Controls (CMC) developability of antibodies against a specific target are typically established for antibodies obtained from one platform only. In this study, monoclonal antibodies (mAbs) cross-reactive against human and cynomolgus LAMP1 were derived from the human immunoglobulin transgenic TRIANNI mouse and OmniChicken® platforms and assessed for their specificity, sequence diversity, ability to bind to and internalize into tumor cells, expected immunogenicity and CMC developability. Our results show that the two platforms were complementary at providing a large diversity of mAbs with respect to epitope coverage and antibody sequence diversity. Furthermore, most antibodies originating from either platform exhibited good manufacturability characteristics.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Epítopos/inmunología , Proteínas de Membrana de los Lisosomas/inmunología , Animales , Animales Modificados Genéticamente , Anticuerpos Monoclonales/química , Pollos , Células HEK293 , Humanos , Inmunización , Macaca fascicularis , Ratones , Modelos MolecularesRESUMEN
The unique characteristics of the avian embryo, with its large opaque yolk, have necessitated the development of different approaches to transgenesis from those that have been successful in mammalian species. Genetic modification of birds was greatly advanced by the ability to grow long-term cultures of primordial germ cells (PGCs). These cells are obtained from embryos, established in culture, and can be propagated without losing the ability to contribute to the germline when reintroduced into a host animal. PGCs can be genetically modified in culture using traditional transfection and selection techniques, including gene targeting and site-specific nuclease approaches. Here, we describe our methods for deriving cell lines, long-term culture, genetic modification, production of germline chimeras and obtaining fully transgenic birds with the desired genetic modifications.
Asunto(s)
Animales Modificados Genéticamente/crecimiento & desarrollo , Pollos/genética , Quimera/crecimiento & desarrollo , Células Germinativas/citología , Animales , Línea Celular , Células Cultivadas , Pollos/crecimiento & desarrollo , Técnicas de Cocultivo , Femenino , Técnicas de Transferencia de Gen , Células Germinativas/metabolismo , Masculino , RatasRESUMEN
The genome of germline committed cells is thought to be protected by mechanisms of transcriptional silencing, posing a barrier to transgenesis using cultured germline cells. We found that selection for transgene integration into the primordial germ cell genome required that the transgenes be flanked by the chicken beta-globin insulator. However, integration frequency was low, and sequencing of the insertion sites revealed that the transgenes preferentially inserted into active promoter regions, implying that silencing prohibited recovery of insertions in other regions. Much higher frequencies of integration were achieved when the phiC31 integrase was used to insert transgenes into endogenous pseudo attP sites. Despite the evidence for transcriptional silencing in PGCs, gene targeting of a nonexpressed gene was also achieved. The ability to make genetic modifications in PGCs provides unprecedented opportunities to study the biology of PGCs, as well as produce transgenic chickens for applications in biotechnology and developmental biology.
Asunto(s)
Marcación de Gen/métodos , Células Germinativas/fisiología , Integrasas/genética , Animales , Secuencia de Bases , Embrión de Pollo/fisiología , Mapeo Cromosómico , Clonación Molecular , Cartilla de ADN , ADN Circular/genética , Células Germinativas/enzimología , Datos de Secuencia MolecularRESUMEN
The tubular gland of the chicken oviduct is an attractive system for protein expression as large quantities of proteins are deposited in the egg, the production of eggs is easily scalable and good manufacturing practices for therapeutics from eggs have been established. Here we examined the ability of upstream and downstream DNA sequences of ovalbumin, a protein produced exclusively in very high quantities in chicken egg white, to drive tissue-specific expression of human mAb in chicken eggs. To accommodate these large regulatory regions, we established and transfected lines of chicken embryonic stem (cES) cells and formed chimeras that express mAb from cES cell-derived tubular gland cells. Eggs from high-grade chimeras contained up to 3 mg of mAb that possesses enhanced antibody-dependent cellular cytotoxicity (ADCC), nonantigenic glycosylation, acceptable half-life, excellent antigen recognition and good rates of internalization.
Asunto(s)
Anticuerpos Monoclonales/química , Animales , Southern Blotting , Western Blotting , Células CHO , Rastreo Diferencial de Calorimetría , Carbohidratos/química , Pollos , Cricetinae , ADN/metabolismo , Clara de Huevo , Embrión de Mamíferos/citología , Embrión no Mamífero , Ensayo de Inmunoadsorción Enzimática , Femenino , Vectores Genéticos , Genoma , Glicosilación , Humanos , Inmunoglobulina G , Inmunohistoquímica , Focalización Isoeléctrica , Ratones , Ratones Endogámicos BALB C , Modelos Genéticos , Monosacáridos/química , Oligosacáridos/química , Ovalbúmina/genética , Ovalbúmina/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Ionización de Electrospray , Células Madre/citologíaRESUMEN
An important characteristic of chickens is that the antibody repertoire is based on a single framework, with diversity found mainly in the CDRs of the light and heavy chain variable regions. Despite this apparent limitation in the antibody repertoire, high-affinity antibodies can be raised to a wide variety of targets, including those that are highly conserved. Transgenic chickens have previously been generated that express a humanized antibody repertoire, with a single framework that incorporates diversity by the process of gene conversion, as in wild-type chickens. Here, we compare the sequences and antibodies that are generated purely by gene conversion/somatic hypermutation of a pre-rearranged heavy chain, with the diversity obtained by V(D)J rearrangement followed by gene conversion and somatic hypermutation. In a gene converting species, CDR-H3 lengths are more variable with V(D)J rearrangement, but similar levels of amino acid diversity are obtainable with gene conversion/somatic hypermutation alone.
RESUMEN
Transgenic animal platforms for the discovery of human monoclonal antibodies have been developed in mice, rats, rabbits and cows. The immune response to human proteins is limited in these animals by their tolerance to mammalian-conserved epitopes. To expand the range of epitopes that are accessible, we have chosen an animal host that is less phylogenetically related to humans. Specifically, we generated transgenic chickens expressing antibodies from immunoglobulin heavy and light chain loci containing human variable regions and chicken constant regions. From these birds, paired human light and heavy chain variable regions are recovered and cloned as fully human recombinant antibodies. The human antibody-expressing chickens exhibit normal B cell development and raise immune responses to conserved human proteins that are not immunogenic in mice. Fully human monoclonal antibodies can be recovered with sub-nanomolar affinities. Binning data of antibodies to a human protein show epitope coverage similar to wild type chickens, which we previously showed is broader than that produced from rodent immunizations.
Asunto(s)
Anticuerpos Monoclonales Humanizados/biosíntesis , Anticuerpos Monoclonales Humanizados/inmunología , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Antígenos/inmunología , Pollos/inmunología , Epítopos/inmunología , Inmunoglobulinas/inmunología , Animales , Animales Modificados Genéticamente , Antígenos/administración & dosificación , Linfocitos B/inmunología , Pollos/sangre , Pollos/genética , Mapeo Epitopo , Humanos , Inmunización , Inmunoglobulinas/sangre , Inmunoglobulinas/genética , Especificidad de la Especie , Linfocitos T/inmunologíaRESUMEN
The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds.
Asunto(s)
Sistemas CRISPR-Cas , Pollos/genética , Edición Génica/métodos , Genoma , Recombinación Homóloga , Cadenas Pesadas de Inmunoglobulina/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Pollos/crecimiento & desarrollo , Clonación de Organismos , Embrión no Mamífero , Femenino , Técnicas de Inactivación de Genes , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células Germinativas , Proteínas Fluorescentes Verdes/deficiencia , Proteínas Fluorescentes Verdes/genética , Masculino , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismoRESUMEN
Chicken immune responses to human proteins are often more robust than rodent responses because of the phylogenetic relationship between the different species. For discovery of a diverse panel of unique therapeutic antibody candidates, chickens therefore represent an attractive host for human-derived targets. Recent advances in monoclonal antibody technology, specifically new methods for the molecular cloning of antibody genes directly from primary B cells, has ushered in a new era of generating monoclonal antibodies from non-traditional host animals that were previously inaccessible through hybridoma technology. However, such monoclonals still require post-discovery humanization in order to be developed as therapeutics. To obviate the need for humanization, a modified strain of chickens could be engineered to express a human-sequence immunoglobulin variable region repertoire. Here, human variable genes introduced into the chicken immunoglobulin loci through gene targeting were evaluated for their ability to be recognized and diversified by the native chicken recombination machinery that is present in the B-lineage cell line DT40. After expansion in culture the DT40 population accumulated genetic mutants that were detected via deep sequencing. Bioinformatic analysis revealed that the human targeted constructs are performing as expected in the cell culture system, and provide a measure of confidence that they will be functional in transgenic animals.
RESUMEN
Transgenic chickens expressing human sequence antibodies would be a powerful tool to access human targets and epitopes that have been intractable in mammalian hosts because of tolerance to conserved proteins. To foster the development of the chicken platform, it is beneficial to validate transgene constructs using a rapid, cell culture-based method prior to generating fully transgenic birds. We describe a method for the expression of human immunoglobulin variable regions in the chicken DT40 B cell line and the further diversification of these genes by gene conversion. Chicken VL and VH loci were knocked out in DT40 cells and replaced with human VK and VH genes. To achieve gene conversion of human genes in chicken B cells, synthetic human pseudogene arrays were inserted upstream of the functional human VK and VH regions. Proper expression of chimeric IgM comprised of human variable regions and chicken constant regions is shown. Most importantly, sequencing of DT40 genetic variants confirmed that the human pseudogene arrays contributed to the generation of diversity through gene conversion at both the Igl and Igh loci. These data show that engineered pseudogene arrays produce a diverse pool of human antibody sequences in chicken B cells, and suggest that these constructs will express a functional repertoire of chimeric antibodies in transgenic chickens.
Asunto(s)
Anticuerpos/genética , Linfocitos B/metabolismo , Pollos/genética , Conversión Génica , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Anticuerpos/química , Secuencia de Bases , Western Blotting , Línea Celular , Cartilla de ADN , Citometría de Flujo , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
In birds, the primordial germ cell (PGC) lineage separates from the soma within 24 h following fertilization. Here we show that the endogenous population of about 200 PGCs from a single chicken embryo can be expanded one million fold in culture. When cultured PGCs are injected into a xenogeneic embryo at an equivalent stage of development, they colonize the testis. At sexual maturity, these donor PGCs undergo spermatogenesis in the xenogeneic host and become functional sperm. Insemination of semen from the xenogeneic host into females from the donor species produces normal offspring from the donor species. In our model system, the donor species is chicken (Gallus domesticus) and the recipient species is guinea fowl (Numida meleagris), a member of a different avian family, suggesting that the mechanisms controlling proliferation of the germline are highly conserved within birds. From a pragmatic perspective, these data are the basis of a novel strategy to produce endangered species of birds using domesticated hosts that are both tractable and fecund.