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The HuMab Chicken : How We Did It

The Cells

In rodents, embryonic stem cells have been and still are the main route to modify the genome. In chickens, embryonic stem cells can be derived but these cells do not become eggs and sperm, which is attributed to the segregation of the germline long before oviposition. To circumvent this problem, cell lines from germ cells have been developed. Both primordial germ cell lines and gonocyte lines have been shown to contribute to eggs and sperm after long-term culture. These cell lines can be genetically modified in vitro using all available modern technologies.

At Crystal Bioscience we have used homologous recombination targeting constructs, as well as CRISPR/Cas9 and Cre recombinase to remove portions of the IgH and IgL loci and simultaneously insert attP sites. This allowed us in subsequent experiments to insert human sequences using phi-C31 integrase in the exact location where the chicken version used to be.

Antibodies of the HuMab chicken only have human variable regions, the constant regions are still chicken. The expressed chicken VH and VL genes are amplified from single B-cells and assembled to generate recombinant antibodies in the scFv-Fc or full IgG formats.

Antibody Diversification

Chickens produce a diverse repertoire of antibodies by gene conversion. In every B cell, the single functional V region at the heavy and light chain immunoglobulin loci is continually altered (i.e. converted) by the donation of sequences from an upstream array of pseudogenes. The continued iterations of gene conversion throughout the life of the bird produce a virtually unlimited antibody repertoire of starting antibodies that can be further improved through antigen-driven selection and affinity maturation, resulting in B cells producing monoclonal antibodies with high affinity and high specificity to a chosen target.

In the case of our HuMab chicken, the functional chicken V genes have been replaced with human sequence V genes, and the upstream pseudogenes have been replaced with human sequence counterparts. We have demonstrated that the human gene array is a functional substrate for the gene conversion machinery through deep sequencing of B cell libraries and through the recovery of affinity matured human-sequence antibodies produced by our engineered birds.

The animation below is a representation of how the gene conversion process generates diversity.