Gene-edited hematopoietic stem cells sustain therapeutic antibody production in mice
Can cell-based supply reduce the limitations of repeated dosing?
Antibody drugs often require repeated administration over long periods to maintain their therapeutic effect. The researchers engineered hematopoietic stem and progenitor cells (HSPCs) so that, after a single transplant, their B-cell descendants would continuously produce therapeutic antibodies. This preclinical study confirmed antibody production in mice through week 16 and was posted as a preprint on Research Square. Whether the approach can replace repeated dosing or provide lifelong effects in patients remains unknown.
Inserting antibody-expression cassettes with CRISPR and confirming production in mice
The researchers used CRISPR/Cas9 and homology-directed repair (HDR) to insert antibody-expression cassettes at specific genomic sites in HSPCs. The targets were CCR5 in human cells and Rosa26 in mouse cells. The design used so-called safe-harbor sites, but that label does not guarantee clinical safety. Antibody expression was driven by a synthetic promoter designed to function in the B-cell lineage.
The researchers tested an anti-PCSK9 antibody for hypercholesterolemia and an anti-tumor necrosis factor alpha (TNFα) antibody for autoimmune disease. The edited human HSPCs engrafted in immunodeficient mice, but antibody production in the blood was minimal. The researchers pointed to a limitation of this model: human B cells have difficulty maturing sufficiently into antibody-secreting cells. Separately, directly edited human B cells secreted antibodies in culture, and culture medium from cells producing anti-TNFα antibody suppressed TNFα activity in a functional assay.
In vivo production was assessed by transplanting mouse HSPCs into immunocompetent mice. Antibodies were detected in serum starting four weeks after transplantation; concentrations rose through week 12 and were maintained through the final observation point at week 16. At week 16, mean concentrations were 206 μg/mL for anti-PCSK9 antibody and 5.5 μg/mL for anti-TNFα antibody. The researchers cited human treatment-related concentration ranges of approximately 10–15 μg/mL and 3–7 μg/mL, respectively. These are concentration comparisons; the results do not demonstrate disease improvement in the transplanted mice or treatment efficacy in patients.
The potential for secretion control and the challenges of transplantation
The researchers also tested whether antibody secretion could be regulated. In culture experiments with human B cells and plasma cells differentiated from them, they attached an FKBP12 protein-based destabilizing domain to the antibody and reversibly adjusted secretion by adding or removing the small molecule Shield-1. However, adding the domain reduced expression compared with antibodies without the domain, even when Shield-1 was present. These results do not establish whether the same control is possible in vivo in animals.
The platform points to the possibility of long-term antibody supply from a single cell transplant. However, the mouse experiments used radiation conditioning, and bone marrow engraftment was lower in the edited group than in controls. Application to patients receiving repeated antibody doses would require reducing the conditioning burden and establishing adequate engraftment and long-term safety. Combining the approach with reduced-intensity conditioning remains a prospect proposed by the researchers.
Details of the experiments and results are available in the full text; preprint bibliographic information is provided in the PubMed record.
Monoclonal antibodies represent half of the top ten selling drugs. Their proven efficacy, however, generally requires repeated administration for prolonged periods of time. In contrast, cell-based therapies offer a different set of pharmacokinetics and pharmacodynamics than traditional medicines, including the potential to have lifetime durability after a single infusion. Here, we describe a genome-engineered stem cell-based platform for continuous antibody production from a single dose. Using CRISPR/Cas9 homology-directed repair mediated editing, we precisely integrated therapeutic antibody expression cassettes into a safe-harbor locus of hematopoietic stem and progenitor cells (HSPCs). Upon differentiation, these gene-targeted HSPCs generate B cells that secrete monoclonal antibodies. We validated this platform using two clinically approved antibodies, achieving efficient targeted integration of the gene-targeted antibodies (GT-Ab) in human HSPCs that successfully engraft in immunodeficient mice. Direct engineering of human B cells demonstrated robust secretion of therapeutic antibodies. To evaluate in vivo antibody production, we transplanted engineered GT-Ab murine HSPCs into immunocompetent mice, achieving durable serum antibody concentrations within the therapeutic range over several months. Lastly, by fusing the antibody to a destabilization domain, we enabled tunable antibody secretion via small molecule regulation. This modular platform establishes a potentially curative approach for chronic diseases currently reliant on repeated antibody administration, offering durable antibody production from a single treatment.
For hypercholesterolemia or autoimmune diseases that require repeated antibody dosing, B-cell descendants of edited hematopoietic stem cells could offer a strategy to reduce the dosing burden if they provide a sustained antibody supply. This study tested that possibility at the preclinical stage using two antibodies. Assessing practical application will require comparing the benefits of existing antibody injections with the burdens of cell collection, editing, transplantation, and conditioning. Because production levels differed between the antibodies, researchers will also need to determine whether the required concentrations can be maintained reliably and excessive production controlled. Efficacy in patients, lifelong persistence, and cost savings have not been demonstrated; secretion control observed in culture also remains to be tested in vivo.