B cells engineered to express an anti-HIV antibody allow memory retention, class switch recombination and clonal selection in mice

Abstract

HIV viremia can be controlled by chronic antiretroviral therapy. As a potentially single-shot alternative, B cells engineered by CRISPR/Cas9 to express anti-HIV broadly neutralizing antibodies (bNAbs) were shown capable of secreting high antibody titers. Here, we demonstrate that, upon immunization of mice, adoptively transferred engineered B cells home to germinal centers (GC) where they 15 predominate over the endogenous response and differentiate into memory and plasma cells while undergoing class switch recombination (CSR). Immunization with a higher affinity antigen increases accumulation in GCs and CSR rates. Boost immunization increases rates of engineered B cells in GCs and antibody secretion, indicating memory retention. Finally, antibody sequences of engineered B cells in the spleen show patterns of clonal selection. B cells may thus be engineered as a living and evolving drug. 20 Main Text:Chronic antiretroviral therapy does not eradicate HIV infection. Broadly neutralizing antibodies (bNAbs) can suppress viremia 1 , but they may have to be chronically administered at a higher cost. Alternatively, bNAbs may be constitutively expressed from muscle 2,3 , but anti-drug antibodies (ADA) are often developed 4 , possibly due to improper glycosylation. In addition, antibodies expressed from muscle 25

Vaccine elicitation of HIV broadly neutralizing antibodies from engineered B cells

“…In the first demonstration of antibody activity, Behring and Kitasato showed in 1890 that serum from immunized mice could protect naive ones from lethal challenge of Diptheria and Tetanus toxins. We show here, and in the complementary study by Nahmed et al 32 , that it is possible to passively transfer genetic information to the adaptive immune system that facilitates a high affinity, highly evolved, yet further evolvable, HIV bnAb response -in effect, demonstrating that one can program immune memory. Thus, we envision that our study represents a new phase in the development of passive immunity, started with the discovery of the antibody itself.…”

Optimization of AAV6 transduction enhances site-specific genome editing of primary human lymphocytes

“…AAV vectors are invaluable reagents for site-specific genome editing of human hematopoietic cells, with AAV6 serotypes in particular being widely used to deliver homology donors to HSPCs, 2,4,11,12 T cells, [13][14][15] and B cells. 16,17,19 The in vitro tropism of AAV6 for human hematopoietic cells, 2,13,16 as well its weak induction of innate immune pathways that could trigger harmful biological consequences in engineered cells, 41,42 may contribute to its success. In addition, the vector's ability to transduce both dividing and quiescent cells 43 may also be beneficial, particularly if the genome is used as a homology template only after second-strand synthesis as some have suggested, 7 though the G2/S phase restriction of cellular factors required for HDR 25 may limit the advantages conferred by this attribute.…”

“…[5][6][7] These procedures can result in high frequency modification of hematopoietic cells, with editing efficiencies ranging from 20-80% across cell types and genomic loci. 2,[8][9][10][11][12][13][14][15][16][17][18][19][20] In developing protocols for genome editing of hematopoietic cells, significant effort has been expended on the targeted nuclease: developing platforms for efficient transient delivery, 2,4,21,22 optimizing protein sequences, 23 and chemically modifying RNA components 24 to maximize on-target nuclease activity while minimizing potentially deleterious off-target DNA break formation. Additional improvements in HSPC genome editing have focused on identifying culture conditions that facilitate HDR through the manipulation of the cell cycle or DNA repair pathways, 21,25,26 as well as to retain optimal stemness and proliferative potential after engraftment.…”

Orthotopic T-Cell Receptor Replacement—An “Enabler” for TCR-Based Therapies

“…Zooming out even further, B and T cells together provide adaptive immunity through humoral and cellular effector function. Analogously to OTR, proof of concept of B-cell receptor (BCR) exchange was recently provided [ 117 , 118 , 119 , 120 ]. The versatility of novel gene editing tools, therefore, creates prospects for the feasibility to engineer complex composite immune responses that reflect natural immunity as much as possible while providing additional or altered function as much as necessary.…”