Genome edited B cells: a new frontier in immune cell therapies

Abstract

Cell therapies based on reprogrammed adaptive immune cells have great potential as "living drugs." As first demonstrated clinically for engineered chimeric antigen receptor (CAR) T cells, the ability of such cells to undergo clonal expansion in response to an antigen promotes both self-renewal and self-regulation in vivo. B cells also have the potential to be developed as immune cell therapies, but engineering their specificity and functionality is more challenging than for T cells. In part, this is due to the complexity of the immunoglobulin (Ig) locus, as well as the requirement for regulated expression of both cell surface B cell receptor and secreted antibody isoforms, in order to fully recapitulate the features of natural antibody production. Recent advances in genome editing are now allowing reprogramming of B cells by site-specific engineering of the Ig locus with preformed antibodies. In this review, we discuss the potential of engineered B cells as a cell therapy, the challenges involved in editing the Ig locus and the advances that are making this possible, and envision future directions for this emerging field of immune cell engineering.

Keywords: B cells; Cas9; HIV; cell therapy; genome editing; immunotherapy.

Graphical abstract

Figure 1

Potential features of engineered B cells as an immune cell therapy

Figure 2

Simplified life cycle of B cells After immune activation, some B cells can differentiate directly into antibody-secreting plasmablasts and early memory B cells. Others will enter a germinal center (GC) reaction, where the diversification and specialization pathways of somatic hypermutation (SHM) and class-switch recombination (CSR) enhance antibody specificity and functionality. Antibody-secreting plasmablasts and long-lived plasma cells (LLPCs), as well as memory B cells, emerge from the GC. Recall responses from memory B cells (pink arrows) can then re-enter the GC or directly differentiate into both LLPCs and short-lived plasmablasts.

Figure 3

Challenges of engineering the Ig locus (A) Expression of the membrane-bound BCR that coordinates clonal expansion, or the secreted antibody that mediates effector functions, is regulated by alternative splicing as the B cell differentiates. (B) The sequences of the IgH locus are unique in each B cell, with variation generated by three processes: VDJ recombination; CSR, which changes the associated constant region sequences; and SHM. (C) The light chain (LC) can be generated from one of two distinct loci, whose unique sequences are generated by VJ recombination and, for Igλ, choice of constant region. (D) Co-expression of both endogenous and engineered heavy chains (HCs) and LCs in the same cell can lead to HC heterodimers that result in bi-specific antibodies, HC-LC cross-pairings that could generate novel and potentially deleterious antigen-binding domains, or combinations of both.

Figure 4

Strategies for genome editing of the Ig locus (A) A simplified schematic of the IgH and Igκ loci is shown. Arrows indicate gRNA targets used to generate DNA DSBs and are color coded by publication: Voss et al. (teal), Greiner et al. (brown), Moffett et al. (purple), Hartweger et al. (pink), Huang et al. (green), Nahmad et al. (gold), and Rogers et al. (unpublished data) (orange). Homology donor constructs are illustrated and labeled by source publication. The approaches from Voss et al. and Greiner et al. are more specific, either only introducing an engineered HC (Voss et al.) or only able to engineer a subset of cells using common V_H and V_L segments and cannot undergo CSR (Greiner et al.). In contrast, the box highlights homology donors for universal IgH engineering that target the intron upstream of the Eμ enhancer. The approach from Rogers et al. targets immunoglobulin constant regions downstream of the CH1 exon to introduce a promoter-driven HC antibody cassette. (B) Diagram of approaches to ablate cross-pairing between the endogenous LC and engineered HC. (Left) Genetic knockout of the endogenous LC to prevent expression. (Middle) Introduction of a long flexible linker between the CL and VH domains favors the desired HC-LC pairing. (Right) camelid-like HC antibodies are unable to pair with LCs because they lack the CH1 exon necessary for HC-LC heterodimerization. V_H, HC variable region. C_H, Ig HC constant region. V_L, LC variable region. C_L, Ig LC constant region; P, promoter; L, linker; pA, poly(A) signal; 2A, ribosome-skipping 2A peptide motif; V_HH: camelid HC variable region; sd, splice donor.