A Versatile B Cell Engineering Platform Enables Development of B Cell Medicines for Sustained Delivery of Therapeutic Biologics

The clinical success of T cell therapies has illustrated the power of harnessing or re-directing intrinsic cellular functions for therapeutic benefit. Terminally differentiated B cells (Plasma cells) offer the possibility to expand the tractable pharmacology of the cell therapy toolkit beyond cytotoxicity. Their natural longevity and capacity for high levels of protein secretion make Plasma cells an attractive source for the steady supply of biologics, which require continuous infusion or frequent dosing to achieve a therapeutic benefit in hematological applications and beyond.

We have developed a versatile CRISPR/Cas9-based B cell engineering platform, which allows for the generation of B Cell Medicines (BCMs) that express and secrete therapeutically relevant proteins at high levels. Targeted knock-in is achieved via HDR-mediated insertion of transgene expression cassettes at CRISPR cut sites. Iterative optimizations of culture and engineering conditions enabled efficient DNA editing and gene knockout (>90%), AAV-mediated transgene knock-in (up to 60% without selection), as well as multiplexed gene knockout (>85% at three loci) and multiplexed transgene knock-in (up to 25% dual positive cells). Beyond engineering efficiencies per se, transgene expression levels were found to be tunable via choice of insertion site, regulatory elements, signal peptides, and codon optimizations. To optimize our gene editing methodology for potential clinical applications, we implemented a CRISPR guide screening funnel designed to identify potent guides supportive of HDR and enabled cellular off-target discovery directly in primary human B cells. Candidate off-targets discovered in this assay or via in silico and biochemical tools are followed up in verification studies to nominate guides suitable for clinical use.

The robustness of our BCM platform was demonstrated by the successful expression of diverse therapeutics, including a variety of enzymes, fusion proteins, and full-length antibodies. Herein, we demonstrate the versatility in three areas: enzyme replacement therapy (ERT), immuno-oncology, and coagulopathies. As an alternative to ERT, BCMs were engineered to express tissue nonspecific alkaline phosphatase (ALP) as a potential new treatment option for Hypophosphatasia (HPP), an inherited disorder characterized by defective bone mineralization. Currently there is only one approved ERT for HPP that requires multiple injections per week, which is a significant treatment burden. BCM-produced ALP rescued mineralization in the presence of inorganic pyrophosphate (PPi) in an in vitro osteoblast cell-based model, and active ALP was produced in vivo following BCM administration in NOG-IL6 mice. Beyond genetic disease applications, a BCM prototype engineered to express an anti-CD3:CD19 scFv Bispecific T Cell Engager (BiTE) demonstrated in vitro tumor cytolytic activity when co-cultured with effector cells and CD19-expressing Raji cells. Additionally, a significant reduction in tumor burden was observed in a patient-derived xenograft model treated with BiTE-expressing BCMs demonstrating in vivo efficacy.

Our first clinical program, BE-101, is an investigational autologous BCM therapy engineered to express human factor IX (hFIX) offering a potential new therapeutic option for patients with hemophilia B, an X-linked recessive bleeding disorder. Insertion of an optimized hFIX expression cassette at the CCR5 safe harbor locus enabled hFIX secretion of up to 60ng/1e6 cells/hour with confirmed biological activity as evidenced by reduced clotting times in the activated partial thromboplastin time (aPTT) assay. Following a single IV dose of hFIX-expressing BCMs, NOG-IL6 mice demonstrated sustained hFIX levels in plasma up to 28 weeks, and a second dose administered at 7 weeks post infusion resulted in an additive increase in hFIX levels. A robust nonclinical data package including genome safety assessments as well as short and long-term in vivo studies in NOG-IL6 mice was submitted to the US FDA, who cleared the BeCoMe-9 first-in-human (FIH) Phase 1/2 clinical trial to begin later this year and granted orphan drug designation for BE-101.

In summary, our B cell engineering platform supports the development of novel BCMs which express therapeutically relevant transgenes and have the potential for broad and meaningful therapeutic utility in genetic diseases, cancer, and beyond.