Retrocyte Display® - a mammalian, B-lineage cell based, fully human antibody discovery technology
Retrocyte Display® (Retroviral B lymphocyte Display) is a high throughput cellular antibody expression platform developed by 4-Antibody to allow expression and screening of full-length immunoglobulin antibody libraries in mammalian B-lineage cells for the identification of antigen-specific fully human monoclonal antibodies. The technology is covered by a family of European, U.S. and PCT patent applications.
Libraries of full length immunoglobulin heavy and light chains are encoded by separate retroviral expression vectors within B-lineage cells to yield efficiently and stably expressed fully human monoclonal antibodies on the surface of the B-lineage cells in the form of B-cell receptors. Retrocyte Display can be used to directly screen combinations of antibody heavy and light chain libraries for antigen-specific binders. Retrocyte Display can also be used in a two-step heavy and light chain shuffling process to convert a non-human antibody with desired properties into a fully human antibody with near-identical characteristics. Both approaches can be used to discover fully human antibodies de novo. Screening of complex heavy and light chain libraries against target antigens or screening of non-human monoclonal antibodies to be converted into human constructs against target antigens is performed by iteratively enriching cells for antigen reactivity with fluorescence activated cell sorting or a combination of magnetic activated and fluorescence-based cell sorting.
After isolation of antigen-reactive Retrocyte Display cell clones, the 4-Antibody method allows the conversion of membrane-expressed antibodies to secreted antibodies such that monoclonal antibody hits can be conveniently screened for antigen specificity by, for example, standard high-throughput ELISA or Luminex®-based assays and for biological activity.
Monoclonal antibody heavy and light chain coding sequences from antigen-reactive Retrocyte Display clones with desired properties can be recovered within days by standard genomic PCR cloning and sequencing for future expression in any cell type and expression format of choice.
Retrocyte Display can utilize any retrovirally transducible mammalian cell line that is able to express antibodies on its cell surface. However, this is of limited utility unless the transduced cell line cannot express endogenous antibody polypeptides that would otherwise interfere with the expression and membrane deposition of the antibody libraries to be screened. We have optimized a method for the retroviral expression and screening of antibody libraries in murine B-lineage cells, devoid of endogenous mouse immunoglobulin expression, capable of growing in suspension with high proliferation rates. These cells are amenable to all aspects of standard cell separation techniques.
The entire procedure - generating antibody-expressing cellular libraries, 3 to 5 rounds of Retrocyte Display enrichments, screening for desired properties, and isolation of the specific antibody VH and VL coding sequences - can be performed in weeks.