Combining antibody therapy, T-cell therapy, transplantation, synthetic biology, and vaccination has given rise to one of the current decade’s greatest hopes for curing cancer. Chimeric antigen receptor T-cell (CAR-T) therapy, using the (MHC-free) targeting ability of a monoclonal antibody to drive a T-cell response, has shown great promise in targeting certain B cell malignancies—with success rates in the case of acute lymphocytic leukemia (ALL) reaching 90%. Yet these miracle cures are less dramatic for other blood cancers, and it is still very early days for CAR-T therapy for solid tumors.

We look here at both why CAR-T therapy for ALL has been so successful and some of the ways it has fallen short. We also examine how the same considerations pose challenges to treating other cancers, and look at strategies being worked on to surmount some of these obstacles.

ALL that Glitters

Generally speaking, CARs are composed of an antibody’s extracellular antigen-recognition domain coupled to the intracellular signaling mechanism of a T-cell receptor (TCR), which are expressed as transgenes in T cells to make CAR-Ts. Because T cells need costimulation to become fully active, second-generation CARs (those that have shown the results most lauded in the media) were designed with an additional intracellular domain (typically from either CD-28 or 4-1BB) that delivers a “second signal” when the receptor is bound.

ALL is a disease in which out-of-control early-stage B cells (blasts) are found mainly in the periphery. The blasts will typically express high levels of CD19—a cell-surface antigen found on nearly all B-cell lineage cells but almost nowhere else. CD19 is “a nearly ideal target” for CAR-T therapy, say Wendell Lim and Carl June in a recent Cell review, because it is required for normal B-cell development, yet loss of B cells can be compensated for by replacement antibody therapy.

The dispersed nature of ALL, and the fact that normal B cells express CD19, means also that virtually all of the CD19 CAR-T cells will be continuously exposed to antigen, driving cytokine production, and thus be maintained in an active state, explains Richard Junghans in a recent Cancer Gene Therapy editorial. The flip side is that in solid tumors—where only a few CAR-Ts will be exposed to tumor at any given time, with most never even encountering their cognate antigen—the bulk of the CAR-T cells will pass to a resting state.

In addition to physical barriers to access, solid tumor microenvironments are generally immunosuppressive, forcing CAR-T cells to “really battle against the forces of evil,” notes David Gilham, vice president research and development at Celyad.

Other blood cancers—even those that express the CD19 antigen, such as chronic lymphocytic leukemia (CLL)—are “more resistant [to CAR-T therapy], mainly because they associate with other cell types that tend to generate a slightly more challenging environment for the T cells to try and target them. And they also might exist elsewhere in the body, more sequestered away,” Gilham says. Non-Hodgkin lymphoma (NHL) cells tends to live in secondary lymphoid organs and have yet more developed structure, “making them a bit more of a halfway house between a liquid and a solid tumor.”

Not All Golden

A 90% cure rate still means that “not everybody gets a complete response; not everybody has a complete response that’s durable” even in ALL, says Lee Greenberger, CSO of the Leukemia and Lymphoma Society. “We’ve also learned that side effects of CAR-T therapy can be life-threatening to manageable.”

Included among these side effects are cerebral edema, central nervous system toxicity, and cytokine release syndrome (CRS, commonly known as cytokine storm). We’re learning how to manage those, but we still need to learn what causes them, how to avoid them, how to shut the T cells down if they become too active, and how to predict which patients will be subject to these bad side effects, he says.

Unfortunately there are no good animal models, so they need to be created, Greenberger adds. For example, “in terms of neurological impairment, it’s pretty difficult to assess in mice unless it’s something very dramatic, [… and] the brain swelling and edema have not been modeled in animals to my knowledge.” Similarly, cytokines and their receptors may be species-specific, and even when they do match up “may have a different response,” notes Gilham. Much of the lab work is by necessity done using cell culture systems.

CAR Polish

Preclinical and translational research are tightly linked in the CAR-T field, with “amazing” movement from preclinical studies into clinical studies. And there are observations in the clinical studies that are then going back for more engineering and evolution in the preclinical setting,” observes Gilham. “This is extremely early technology; there is not any extensive fundamental research going on in the area—it’s just starting, there are some green shoots. But it’s really being driven by translational researchers.”

Much work has been done to address resistance and relapse, an immunosuppressive microenvironment, over- and under-stimulated CAR-T cells, CRS, and a host of other issues. Some of this comes in the form of orthologous or supplemental treatment—such as steroid or antibody therapy, radiation or chemotherapy, or an immune activator or checkpoint inhibitor—to the regimen. Other work has been focused more directly on the CAR-T itself.

Preclinical and translational research are tightly linked in the CAR-T field, with “amazing” movement from preclinical studies into clinical studies.

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For example, “Fourth generation” CARs, in addition to one or more costimulatory domains, also express another protein—for example, the pro-inflammatory cytokine IL-12, which may help make tumor microenvironments less hostile.

Bi-specific CAR-T cells—either with two distinct receptors expressed on a single T cell, or a single CAR with two distinct antigen recognition domains—are being looked at in a variety of ways, using different combinatorial (Boolean logic gate) approaches. In an example of its simplest iteration, T cells may be “programmed” to respond to either CD19 or CD22, allowing escape mutants—known to be a source of resistance—still to be recognized. Perhaps (like most solid tumors) there is no single antigen expressed on all cancer cells that is not expressed on any essential normal cell, but there are combinations of two antigens that together uniquely define the tumor—use an and gate to assure that only cells expressing both antigens will be recognized. Or perhaps instead the tumor cells express a unique combination of one antigen and not another.

In fact, there is only a single protein antigen (variant III of the epidermal growth factor receptor (EGFRvIII)) known to be uniquely expressed on cancerous but not normal tissue. There are, however, instances in which a tumor-associated antigen (TAA) is expressed at significantly higher levels on a tumor. Researchers are investigating ways to tune the affinity of a CAR to take advantage of this.

There is also work being done to generate CARs to TAAs with tumor-specific post-translational modifications such as carbohydrate structures.

Several avenues are being pursued to improve the safety of CAR-T therapy, including engineering in an inducible “suicide switch” that can be triggered upon exposure to a drug that rapidly kills the CAR-Ts in the case of life-threatening reaction.

“Celyad is not using an antibody, we’re using an NK [natural killer] receptor, which gives a different targeting approach. People are using natural receptors, ligands, and are starting to use aptamers,” explains Gilham. “If you can think of it, it’s probably already being expressed in a chimeric receptor in one way or another.”

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