Despite its success in treating hematological malignancies, CAR T cell therapy still has challenges in overcoming antigen escape and improving T-cell persistence. A recent study from scientists at the University of Colorado published in Cancer Cell, introduces an innovative approach to addressing these challenges with a novel platform dubbed “ALA-CART.” This CAR T system enhances antigen sensitivity to expand the scope and effectiveness of CAR T therapy.
A major hurdle in CAR T cell therapy is the phenomenon of antigen escape, where cancer cells evade immune attack by downregulating the target antigen. This issue became particularly evident in clinical trials involving CD22-directed CAR T cells for leukemia.
“When I was a fellow at the NIH, I saw patients relapsing with CD22-low disease,” said co-author M. Eric Kohler. “It was clear that low antigen levels were a major vulnerability for CAR T cells. Natural T cells can detect minuscule amounts of antigen, but CAR T cells were failing in this regard.”
To address this, Kohler and his colleagues investigated how CAR T cell signaling breaks down at low antigen levels. They found that the molecule LAT (Linker for Activation of T cells) was not sufficiently engaged when antigen levels were low. This led them to develop ALA-CART—a strategy designed to restore effective T-cell signaling under these conditions.
The ALA-CART approach: Engineering a bicistronic CAR
Instead of relying on a single CAR molecule, ALA-CART employs a bicistronic system, meaning each T cell expresses two different CAR molecules. One is a conventional second-generation CAR that includes CD3ζ and a costimulatory domain (like 4-1BB or CD28) to enhance T-cell activation and persistence. The second is engineered to include the intracellular portion of LAT.
“By incorporating LAT directly into our CAR design, we ensured that even when antigen levels were low, the signal remained strong,” said Kohler. “This effectively ‘plugs the hole’ in the signaling pathway, making the CAR T cells more sensitive to antigen and restoring their ability to attack leukemia cells that would otherwise escape.”
Promising results in preclinical models
The efficacy of ALA-CART was tested in cells and xenograft mouse models of acute lymphoblastic leukemia (ALL). The ALA-CART design successfully eradicated CD22-low leukemia cells that had proven resistant to conventional CAR T cells.
“Our ALA-CART design completely eliminated CD22-low leukemia from mice,” Kohler stated. “We also replicated these results with CD19-low models, demonstrating that the approach is not limited to a single antigen.”
In addition to targeting leukemia, Kohler’s team tested ALA-CART against patient-derived cancer samples. Even in cases where the cancer cells expressed as few as 300 CD22 molecules per cell—a level too low for standard CAR T cells—ALA-CART maintained strong activity.
While the current study focused on hematological malignancies, the team has begun exploring applications for solid tumors.
“This wasn’t part of the publication, but we’re seeing very encouraging early results in solid tumor models,” Kohler revealed. “We’re optimistic that ALA-CART could be just as effective in these settings.”
Additional benefits: Enhanced T-cell persistence and reduced toxicity
Beyond improving antigen sensitivity, ALA-CART appears to confer additional advantages. The modified T cells show enhanced persistence in vivo, maintaining a less differentiated, more “youthful” phenotype that may extend their durability in patients.
“One unexpected benefit was that ALA-CART cells didn’t differentiate as quickly into short-lived effector cells,” Kohler explained. “They retained a stem-like quality that allows them to persist longer, which is critical for long-term tumor control.”
Additionally, the team observed a reduction in cytokine release, suggesting ALA-CART could lower the risk of cytokine release syndrome (CRS)—a common and potentially severe side effect of CAR T therapy.
“In our mouse models, we saw less cytokine release compared to standard second-generation CAR T cells,” Kohler noted. “This suggests a reduced risk of CRS, which would be a major advantage in clinical applications.”
A generalizable platform for CAR T therapy
While this study focused on CD22 and ALL, Kohler emphasized that ALA-CART represents a broader platform technology applicable to multiple antigens and cancer types.
“We see this as more than just a CD22 or leukemia-specific innovation,” Kohler concluded. “This is a platform that we believe can improve CAR T therapy across the board.”
