Gene targeting by homologous recombination or by sequencespecific 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.B-cell development | avian immunology | genome editing T he chicken has historically been an important model vertebrate organism in the fields of developmental biology and immunology and has contributed a number of basic tenets to these fields. For example, B lymphocytes were first recognized in chickens as the antibody-producing cells and are named after the bursa of Fabricius, a gut-associated lymphoid tissue (GALT) that is required for B-cell development in chickens (1). Graft-versushost response was first described in chicken embryos (2), and the first attenuated vaccine was developed by Louis Pasteur against fowl cholera caused by Pasteurella multocida. Nevertheless, the lack of a robust genome editing technology including knockouts has put the chicken at a distinct disadvantage to mammalian species, especially the mouse, as a vertebrate animal model. The discovery of ES cells provided a powerful method to make desired changes to genes of interest in the mouse using homologous recombination, but interestingly, ES cells have not been as easily derived from other species. In the case of chickens, ES cells can contribute to all somatic lineages in high-grade chimeras, but germ-line transmission has not yet been demonstrated, precluding their use in creating fully transgenic chickens (3). Although ES cells are not germ line competent in chickens, embryo-derived primordial germ cells (PGCs) can be cultured indefinitely, transfected, clonally selected, and reintroduced into the embryo where they colonize the gonad and give rise to fully transgenic progeny ...

Native human Abs represent attractive drug candidates; however, the low frequency of B cells expressing high-quality Abs has posed a barrier to discovery. Using a novel single-cell phenotyping technology, we have overcome this barrier to discover human Abs targeting the conserved but poorly immunogenic central motif of respiratory syncytial virus (RSV) G protein. For the entire cohort of 24 subjects with recent RSV infection, B cells producing Abs meeting these stringent specificity criteria were rare, <10 per million. Several of the newly cloned Abs bind to the RSV G protein central conserved motif with very high affinity (Kd 1–24 pM). Two of the Abs were characterized in detail and compared with palivizumab, a humanized mAb against the RSV F protein. Relative to palivizumab, the anti-G Abs showed improved viral neutralization potency in vitro and enhanced reduction of infectious virus in a prophylaxis mouse model. Furthermore, in a mouse model for postinfection treatment, both anti-G Abs were significantly more effective than palivizumab at reducing viral load. The combination of activity in mouse models for both prophylaxis and treatment makes these high-affinity human-derived Abs promising candidates for human clinical testing.

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.

The ability of monoclonal antibodies (mAbs) to target specific antigens with high precision has led to an increasing demand to generate them for therapeutic use in many disease areas. Historically, the discovery of therapeutic mAbs has relied upon the immunization of mammals and various in vitro display technologies. While the routine immunization of rodents yields clones that are stable in serum and have been selected against vast arrays of endogenous, non-target self-antigens, it is often difficult to obtain species cross-reactive mAbs owing to the generally high sequence similarity shared across human antigens and their mammalian orthologs. In vitro display technologies bypass this limitation, but lack an in vivo screening mechanism, and thus may potentially generate mAbs with undesirable binding specificity and stability issues. Chicken immunization is emerging as an attractive mAb discovery method because it combines the benefits of both in vivo and in vitro display methods. Since chickens are phylogenetically separated from mammals, their proteins share less sequence homology with those of humans, so human proteins are often immunogenic and can readily elicit rodent cross-reactive clones, which are necessary for in vivo proof of mechanism studies. Here, we compare the binding characteristics of mAbs isolated from chicken immunization, mouse immunization, and phage display of human antibody libraries. Our results show that chicken-derived mAbs not only recapitulate the kinetic diversity of mAbs sourced from other methods, but appear to offer an expanded repertoire of epitopes. Further, chicken-derived mAbs can bind their native serum antigen with very high affinity, highlighting their therapeutic potential.

Targeting the CD47-signal-regulatory protein α (SIRPα) pathway represents a novel therapeutic approach to enhance anti-cancer immunity by promoting both innate and adaptive immune responses. Unlike CD47, which is expressed ubiquitously, SIRPα expression is mainly restricted to myeloid cells and neurons. Therefore, compared to CD47-targeted therapies, targeting SIRPα may result in differential safety and efficacy profiles, potentially enabling lower effective doses and improved pharmacokinetics and pharmacodynamics. The development of effective SIRPα antagonists is restricted by polymorphisms within the CD47-binding domain of SIRPα, necessitating pan-allele reactive anti-SIRPα antibodies for therapeutic intervention in diverse patient populations. We immunized wild-type and human antibody transgenic chickens with a multi-allele and multi-species SIRPα regimen in order to discover pan-allelic and pan-mammalian reactive anti-SIRPα antibodies suitable for clinical translation. A total of 200 antibodies were isolated and screened for SIRPα reactivity from which approximately 70 antibodies with diverse SIRPα binding profiles, sequence families, and epitopes were selected for further characterization. A subset of anti-SIRPα antibodies bound to both human SIRPα v1 and v2 alleles with high affinity ranging from low nanomolar to picomolar, potently antagonized the CD47/SIRPα interaction, and potentiated macrophage-mediated antibody-dependent cellular phagocytosis in vitro. X-ray crystal structures of five anti-SIRPα antigen-binding fragments, each with unique epitopes, in complex with SIRPα (PDB codes 6NMV, 6NMU, 6NMT, 6NMS, and 6NMR) are reported. Furthermore, some of the anti-SIRPα antibodies cross-react with cynomolgus SIRPα and various mouse SIRPα alleles (BALB/c, NOD, BL/ 6), which can facilitate preclinical to clinical development. These properties provide an attractive rationale to advance the development of these anti-SIRPα antibodies as a novel therapy for advanced malignancies.

The analysis of secreted antibody from large and diverse populations of B cells in parallel at the clonal level can reveal desirable antibodies for diagnostic or therapeutic applications. By immobilizing B cells in microdroplets with particulate reporters, decoding and isolating them in a microscopy environment, we have recovered panels of antibodies with rare attributes to therapeutically relevant targets. The ability to screen up to 100 million cells in a single experiment can be fully leveraged by accessing primary B-cell populations from evolutionarily divergent species such as chickens.

A B lymphocyte that produces the immunoglobulin heavy (H) chain mu may switch to the production of another heavy chain class: gamma, epsilon, or alpha. Since the new heavy chain retains the original variable (V) region, antigenic specificity is maintained. The switch is accompanied by a large deletion of DNA at the heavy chain locus. To explain how this deletion is generated, three models have been proposed: recombination between homologs, unequal sister chromatid exchange, and looping out and deletion. While none of the predicted recombination products of the first two models have been found, both by-products of looping out--inversions and circular DNA--have been isolated. Thus looping out and deletion appears to be the appropriate model to explain the genetic events leading to the immunoglobulin heavy chain class switch. One requirement for switching may be transcription of the constant (C) region to which the cell switches. The switch rearrangement is catalyzed by a switch recombinase, and the isolation of the components of this putative enzyme system is in progress. Although the switch deletion is an accepted fact, the discussion is enlivened by scenarios for switching without DNA rearrangement; such suggestions include processing at the RNA level and trans-splicing.

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.