Be Biopharma: B cells as protein factories

As a pediatrician at the University of Washington School of Medicine, David Rawlings treats children who have hemophilia and other rare genetic diseases. “They need a therapy from day one of life,” says Rawlings, who also directs the Center for Immunity and Immunotherapies at Seattle Children’s Research Institute.

David Rawlings, co-founder of Be Bio

The standard of care for these disorders is protein replacement therapy, but such treatments require a lifetime of expensive infusions. Gene therapy is one alterative. For people with the bleeding disorder hemophilia B, for example, gene therapy using liver-targeting adeno-associated virus (AAV) vectors can restore long-term expression of factor IX (FIX), a clotting factor deficient in these patients. However, a major problem with AAV gene therapy is immunogenicity: subsequent infusions provoke an immune response to the virus. So “you only get one chance,” Rawlings says. That makes the approach infeasible for children, who need increasing amounts of the therapeutic protein as they grow.

Six years ago, Rawlings and Seattle Children’s colleague Richard James, associate professor in pediatrics and pharmacology at the University of Washington, came up with a possible solution: engineering B cells. During an immune response, exposure to an antigen triggers naive B cells to proliferate and develop into plasma cells or memory cells—each with distinct advantageous features. Plasma cells produce large quantities of disease-fighting antibodies. “They make lots and lots of proteins—comparable to industrial cell line levels,” says James. Memory B cells have longevity. After an infection subsides, memory B cells that have generated specific antibodies can reside in the bone marrow or other lymphoid organs for decades, ready to spring into action if confronted with the same antigen.

Richard James, co-founder of Be Bio

Together, Rawlings and James figured these two features make B cells ideal for treating enzyme deficiencies or other conditions in which patients require long-term protein production. In reality, though, turning B cells into medicines presented technical hurdles. For starters, delivering genes into B cells proved tricky. “It sounds like an obvious thing. Like, of course you could just put a gene in a cell. That’s really easy,” says James. “But for B cells, it wasn’t.” Plasmid delivery is toxic to B cells, and lentiviral vectors had abysmal efficiency or weren’t scalable.

Meanwhile, Rawlings and coworkers had pioneered using designer nucleases, including CRISPR–Cas9, in parallel with AAV vectors to do homology-directed gene editing of primary cells. They showed these techniques could work in human T cells and CD34+ hematopoietic cells. Given the difficulties with other gene delivery methods in B cells, he and James decided to try this AAV technique. “It was so much better than any of the other delivery methods we had tried,” Rawlings says.

But there were more challenges. Not only did the researchers want to engineer new genes into B cells, but they also needed to get naive B cells to differentiate in vitro into protein-producing plasma cells. Rawlings’ lab had previously shown that activating the cell is important for gene editing and homology-directed repair, as terminally differentiated B cells lack endogenous repair systems. “So that was one of the first things we needed to figure out—how to activate the B cells and get them proliferating,” says James.

When the body is responding to an infection, B cells migrate to germinal centers—sites within the spleen or lymph nodes—where activated B cells proliferate and undergo somatic hypermutation, generating a variety of antibodies. From those, higher affinity antibodies are selected for further production. In addition to antigen exposure, B cell proliferation typically requires signals from helper CD4+ T cells and cytokines. To mimic this process in vitro, James and colleagues tested many types of reagents, eventually coming up with a cocktail that included an oligomerized form of CD40 ligand (CD40L; a protein expressed on the surface of activated CD4+ T cells) as well as interleukin (IL)-2, IL-10 and IL-15.

For proof of concept, the researchers used a AAV serotype 2 (AAV-2)-mediated, homology-directed method to engineer plasma B cells to secrete FIX, as reported in Science Translational Medicine. They targeted FIX to the chemokine CC-motif receptor 5 (CCR5) locus—a gene that is not transcriptionally active in human B cells—and used a chromogenic assay (vitamin K–dependent FIX carboxylation) to show that the FIX produced by the engineered cells was active. To promote plasma cell production in culture, the team co-delivered CRISPR–Cas9 to disrupt a developmental gene (PAX5), which normally acts to antagonize plasma cell differentiation.

Separately, the team introduced the B-cell activating factor gene (BAFF) into the CCR5 locus and showed that BAFF-secreting plasma cells survived longer than control cells lacking the Cas9 ribonucleoprotein. Furthermore, when these BAFF-producing cells were transferred into non-obese diabetic–severe combined immunodeficient–IL-2 receptor (IL-2R)-γ κνοχκουτ (NSG) mice, plasma cells engrafted in the bone marrow, produced antibodies and secreted BAFF more than a year later, as reported at the 2019 American Society for Gene and Cell Therapy meeting.

What moved these experiments beyond an academic exercise was the absence of a requirement for cumbersome co-culturing with feeder cells to differentiate the edited B cells into plasma cells. “Now that becomes something that looks a lot more like a product that can go into a patient,” says Lea Hachigian, a principal at the venture capital firm Longwood Fund. She and Longwood colleagues Aleks Radovic-Moreno and David Steinberg worked with Rawlings and James to start a biotech around the B cell engineering technology. Last October, Be Biopharma launched with a $52 million series A financing round led by Atlas Venture and RA Capital Management, joined by Alta Partners and Takeda Ventures.

Coming up with a method to transfect primary B cells with high efficiency was “a major breakthrough,” says James Voss, an immunologist at Scripps Research Institute in San Diego. Furthermore, “it’s very visionary to imagine harnessing a B cell to express a secreted protein. It was exciting to show that these cells could live for so long, even if in an immunocompromised mouse.” Voss wonders, though, if it may be harder for engineered B cells to engraft in a patient with a fully functioning immune system. The absence of other lymphatic cells in an immunocompromised mouse makes it “a bit easier to engraft a foreign component,” he says.

Be Biopharma researchers are doing further experiments to test for engraftment using various proteins with therapeutic potential. The company is focusing on monogenic rare diseases for now. But B cells are versatile. “We think that if you limit their differentiation and engineer B cells to have a regulatory function, you could also use those to modulate autoimmune responses,” says Rawlings, whose poster presented last March at the B Cell Renaissance Keystone Conference describes efforts to test whether antigen-specific B cells expressing IL-10 can tolerize the body to self antigens that cause autoimmune disease. On the flip side, as B cells also work as antigen-presenting cells, they could also be used to drive an anticancer response. “There are multiple ways that you could use B cells,” he says, “and their longevity gives them a lot of leverage as a cell therapeutic.”

Esther Landhuis

San Francisco Bay Area, CA, USA

doi: https://doi.org/10.1038/d41587-021-00007-5

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