Chimeric antigen receptor (CAR) T-cell (CAR-T) therapies are a potentially curative treatment for patients with chronic lymphocytic leukemia (CLL) who have run out of other options, yet their efficacy is considerably limited. In clinical trials, CD19-targeting CAR-T therapies have proven capable of producing durable responses in relapsed/refractory CLL, but only in 26% of patients.1

Researchers based at the University of Pennsylvania (UPenn) have long been working to understand the factors that underlie such shortcomings of CAR-T therapies in CLL. Some of their recent research, presented at the annual American Society of Gene and Cell Therapy conference in 2020, identified at least one contributing factor: certain characteristics of CLL cells make them less capable of stimulating CAR-T cells sufficiently.2

“I think these are really interesting data that suggest a way forward with trying to modulate the tumor to improve immune responses,” Andrew J. Cowan, MD, of the University of Washington, Seattle, who was not involved in the study, told Cancer Therapy Advisor.

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The findings were a result from a series of in vitro assays performed by the research team (which included a scientist from Yale University). The team repeatedly stimulated CAR-T cells derived from healthy donors with CLL cells from around 20 patients. These CAR-T cells — engineered to target either the CD19 receptor, or ROR1, which is more specific to CLL cells — showed signs of diminished function, characterized by reduced proliferation, the production of cytokines, and their propensity to attach to tumor cells. In contrast, CAR-T cells that were exposed to an antigen-expressing control cell did not exhibit these shortcomings.

The team found, however, that this reduced function was reversible — the activity of the CAR-T cells was restored by subsequently exposing them to the antigen-expression control cell, explained Jan Joseph Melenhorst, PhD, professor of pathology and laboratory medicine and director of the Parker Institute for Cancer Immunotherapy’s biomarker program at UPenn, and a coauthor of the research.

To investigate why the CLL cells had this effect, the researchers took a close look at the proteins they expressed on their cell surfaces. Dr Melenhorst was surprised to see that CLL cells from the majority of patients expressed a receptor for interleukin 2 (IL-2)/CD25, often at high levels. This led the team to hypothesize that CLL cells may be soaking up IL-2 from their environment, effectively removing a cytokine that T cells use to boost proliferation of surrounding cells. 

To test this idea, the team added IL-2 to a mixed culture of CLL and CAR-T cells, and found that this did partially restore cytokine production by CAR-T cells. This lent credence to the idea that the CLL cells were outcompeting T cells for IL-2, Dr Melenhorst explained. “That, in the long run, may have a negative impact on T-cell function.”

The IL-2 decrease explained some, but not all, of the CAR-T cells’ functional defects, leading the team to further hypothesize that maybe the CLL cells weren’t expressing enough surface molecules to stimulate the CAR-T cells, which were specifically engineered to respond to costimulation. And indeed, flow cytometry data demonstrated that CLL cells from most patients were low in — or entirely lacking — CD54, CD58, CD80 and CD86. “Phenotyping actually turned out to demonstrate that costimulation was virtually absent on these tumor cells, and also the adhesion molecules necessary for forming a stable interaction between CAR-T cell and tumor cell were low or virtually absent,” Dr Melenhorst explained.

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When the team used a known method to artificially upregulate these tumor surface molecules — by adding a CD40 ligand and interleukin 4 (IL-4) — and then exposed them to CAR-T cells, this boosted the proliferation of the CAR-T cells, and appeared to trigger activation, as evidenced by the formation of CAR-T–CLL conjugates. “Activation of CLL cells prior to CAR-T interaction caused a partial restoration of CAR-T function,” Dr Melenhorst said.

Noted Dr Cowen: “I think this points to a general idea that optimizing the manufacturing of CAR T cells [as well as optimizing tumor-specific factors] could help improve clinical responses.” Indeed, some other research groups have experimented with incorporating an inverted IL-4 receptor in CAR-T cells, which has been shown to enhance CAR-T potency and promote tumor-targeted cytotoxicity in vitro.3

Another potential way to improve efficacy would be to incorporate a CD40 ligand into the CAR-T construct, with the idea being to stimulate the tumor’s expression of costimulatory molecules, which in turn, then stimulate the T cells and boost their function, Dr Melenhorst explained. “[This] ‘cross-linking’ causes signaling inside a tumor that then triggers the expression of molecules that T cells need for full activation.”

Disclosures: Dr Melenhorst has received research funding from Novartis and Incyte, and currently from IASO Biotherapeutics, has served as a speaker for Novartis and Johnson & Johnson, as a consultant for Simcere of America, Shanghai Unicar Therapy, Johnson & Johnson, Poseida, and IASO Biotherapeutics, and is on the medical and scientific advisory board of IASO Biotherapeutics.


  1. Porter DL, Hwang W, Frey NV, et al. Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. Sci Transl Med. 2015;7(303):303ra139.
  2. Collins MA, Kong W, Jung I, et al. Insufficient activation limits the efficacy of CAR T cell therapy in chronic lymphocytic leukemia. Presentation at: the 2020 American Society of Gene & Cell Therapy; May 12-15, 2020. Abstract 84.
  3. Wang Y, Jiang H, Luo H, et al. An IL-4/21 inverted cytokine receptor improving CAR-T cell potency in immunosuppressive solid-tumor microenvironment. Front Immunol. doi: 10.3389/fimmu.2019.01691