Engineering Cells for Cancer Therapies

As an emerging cancer treatment, cell-based immunotherapy shows promise by weaponizing a patient’s own immune cells to fight cancer. In adoptive T-cell therapy, T cells are harvested from a patient and engineered to express chimeric antigen receptors (CARs). Thus equipped to target cancer cells, the CAR T cells are then re-infused into the patient. This approach is proving effective against some blood cancers, but solid tumor cancers are proving more recalcitrant. Here’s a look at how next-generation engineering approaches, including genome editing and synthetic biology, are helping to address challenges in cellular immunotherapy.

Improving specificity

New engineering approaches are improving the specificity of CAR T cells, which in turn is improving their safety and efficacy in cancer therapies. “Advances in the design of CAR structure can enable [the development of] synthetic receptors that respond to combinations of cancer-specific antigens, allowing T cells to engage with tumor cells more precisely and effectively,” says Yelena Bronevetsky, Director of Product Management at Xcell Biosciences.

For example, logic-gated CARs may be engineered with an “AND” gate so that the presence of two different antigens are required for receptor activation. “These CARs operate using a gate, activating only when two antigens co-expressed on tumor cells—but absent on healthy cells—are recognized,” says Jon Pileggi, Senior Manager of Process Development at CTMC. “Alternatively, they can employ a “NOT” gate of the T cells, inhibiting activation upon detecting specific antigens that identify healthy cells.” The engineering of logic-gated CARs helps to reduce off-target effects, immune escape, and relapse.

Surviving the tumor microenvironment

Engineering is also helping CAR-T cells survive long enough to be effective in the tumor microenvironment (TME). The TME is an inhospitable landscape in which immune cells are suppressed and even recruited to “the dark side” to serve tumor cells. To help overcome this, researchers are engineering fourth-generation CAR-T cells, also known as T cells Redirected for Universal Cytokine Killing (TRUCKs), that not only express a CAR but also excrete cytokines. “This allows the cells to not only target tumor cells but also to modify the tumor microenvironment to improve tumor infiltration and better support an immune response,” says Pileggi. Bronevetsky agrees that engineering may help CAR-T cells maintain activity in hostile TMEs. “This is something our customers are looking at closely with our AVATAR technology, which enables careful replication of the tumor microenvironment in an incubator,” she says.

James Brady, SVP of Technical Applications and Customer Support at MaxCyte, notes an increase in the sophistication and complexity of the genetic payloads delivered to generate CAR T cells, in which MaxCyte’s electroporation technologies play a central role. "This ‘armored CAR’ approach, which delivers a host of genes to the cell in addition to the CARs, enables the cell to produce various proteins, such as cytokines, that help to neutralize the tumor microenvironment and enhance therapeutic efficacy."

Overcoming autologous limitations

Most CAR-T cell immunotherapy is autologous, in which cells are harvested from the patient, then isolated, engineered to target cancer cells, and re-infused into the patient. The process is cumbersome, time-consuming, and expensive. Ultimately, effective cell therapy will require the development of standardized manufacturing processes for CAR-T cells or other immune cells that can be mobilized upon a patient’s diagnosis, without waiting for the usual autologous procedures. This allogenic approach may use T cells from healthy donors, or possibly T cells produced from emerging stem cell breakthroughs. “iPSC reprogramming technology is improving, introducing the possibility of creating a nearly limitless supply of differentiated T cells that can then be used for off-the-shelf allogeneic cell therapy,” says Brady. “There would be many benefits to doing this, [and] having a ready stock of CAR-T cells against common tumor antigens [may] accelerate the patient’s treatment course.”

No matter where the cells originate, creating successful allogeneic immune cells will depend upon new molecular and cell biology technologies. “By applying synthetic biology principles, modular, “plug-and-play” CAR-T cell vector designs can be developed, enabling efficient production across various tumor targets,” says Pileggi. “Using genome-editing tools, such as CRISPR-Cas9, universal or off-the-shelf CAR-T cells can be generated in bulk from healthy donor cells by knocking out alloreactive markers (such as TCR or HLA markers) that would typically cause rejection or lead to adverse events in a patient following infusion.”

Indeed, such challenges remain an issue facing the development of allogenic approaches to immune cell therapy. But, says Bronevetsky, “CAR engineering is well positioned to address toxicity risks such as cytokine release syndrome and graft-versus-host disease in allogenic treatments.” Using gene editing to remove specific, problematic proteins from T cells “can greatly improve the speed and accessibility of CAR therapies by making allogeneic cell therapy safer,” says Brady. “If we can use donor cells safely, we may begin to build stockpiles of engineered T cells that are then ready at hand the moment they’re needed.”

Another cutting-edge field that bears watching eliminates all the above work described for allogeneic or autologous therapies. Instead, researchers are working on in vivo generation of CAR-T cells by delivering genes using different methods (e.g., viral, lipid nanoparticles, or mRNA). “This emerging approach aims to generate CAR-T cells directly within a patient,” says Pileggi. “It could simplify and accelerate treatments by completely bypassing traditional ex vivo manufacturing, which is almost always complex, time-consuming, and expensive.”

Improving odds against solid tumors

Though CAR-T cell immune therapy is showing success treating blood cancers, it is less effective against solid tumors. One reason is the challenge of delivering CAR-T cells to the tumor sites where they will be effective. The TME is less vascularized and therefore more difficult for immune cells to travel through, and the conditions do not favor their survival. “To reach these tumors, [therapeutic cells] must be capable of exiting the circulatory system (passing beyond endothelial cell barriers), penetrating through multiple layers of tumor tissue; and [then exact] therapeutic effect despite an oppressive environment where tumor cells actively suppress immune cell activation and may be abnormally quiescent,” says Brady.

Immune cell engineering holds promise for increasing our abilities to fight solid tumor cancers, too. “For example, CRISPR/Cas9-mediated knockout of genes that regulate immune checkpoints and cell proliferation can improve CAR-T cell performance in solid tumors,” says Bronevetsky. Brady also notes that researchers are trying to improve CAR-T cells’ ingress into tumors by arming them with chemokine receptors and “allowing them to be drawn more effectively toward the tumor environment.”

A related tool that may improve our odds against solid tumors is CAR-macrophages, which may become complementary to CAR-T cells. Unlike the latter, macrophages are naturally better at infiltrating tumors. “CAR-macrophages are also uniquely capable of phagocytosing tumor cells and presenting tumor antigens to T cells, leading to a more comprehensive anti-tumor immune response,” says Bronevetsky. “These cells can also directly mediate tumor cytotoxicity through the production of proteins like TNF-α and nitric oxide.” Continuing to innovate new attacks on cancer cells with all of the tools at our disposal, including cell engineering, genome editing, and synthetic biology, will soon make more types of cancers treatable and temporary.