The five-year project, titled “Harnessing β-Catenin and NKG2D to Enable HLA-Independent CD8⁺ T-Cell Immunotherapy Against Acute Myeloid Leukemia,” runs through July 2031 and represents one of the most ambitious attempts yet to circumvent a central obstacle in leukemia immunology. At the heart of the problem lies a protein system called HLA class I, a molecular display platform found on the surface of nearly all cells in the body. HLA molecules present fragments of intracellular proteins to circulating CD8⁺ T cells, the cytotoxic “killer” lymphocytes of the adaptive immune system. When a cell becomes cancerous, it displays abnormal peptide fragments on its HLA molecules, effectively raising a flag that invites T-cell destruction. This HLA-dependent recognition is the foundation of most T-cell-based cancer immunotherapies, including engineered CAR T-cell approaches that have produced dramatic remissions in certain blood cancers.

Acute myeloid leukemia, however, has evolved a devastating countermeasure. AML cells can downregulate or completely lose expression of HLA class I molecules, rendering them effectively invisible to the cytotoxic T cells that would otherwise destroy them. This immune-evasion strategy is particularly insidious because it does not merely blunt the effect of natural immunity; it also undermines precisely engineered therapies built around HLA-dependent antigen recognition. For patients with high-risk or relapsed AML, the failure of immune recognition contributes directly to the disease’s grim prognosis, making AML one of the most lethal hematologic malignancies in adults.

Karimi’s laboratory has identified an alternative recognition pathway that may offer a way around this evasion mechanism. “Our research has identified another way that these immune cells may recognize leukemia cells without relying on HLA,” Karimi explained. “In this study, we aim to understand how the alternative pathway is regulated in immune cells from patients with AML and how this pathway could be strengthened to improve immune-based treatments for leukemia.” The pathway centers on NKG2D, an activating receptor expressed on natural killer cells and subsets of T cells, including CD8⁺ T cells. Unlike conventional T-cell receptors, NKG2D does not require HLA-mediated peptide presentation. Instead, it recognizes stress-induced ligands that frequently appear on the surface of transformed or infected cells. By exploiting this HLA-independent axis, Karimi’s team hopes to engineer or enhance CD8⁺ T cells capable of detecting and eliminating AML cells even when those cells have shed their HLA molecules.

The role of β-catenin in this system adds another layer of scientific intrigue. β-catenin is a central signaling molecule in the Wnt pathway, best known for its role in embryonic development and cell proliferation, but increasingly implicated in cancer biology and immune regulation. Aberrant β-catenin activity in tumor cells has been linked to immune suppression in the tumor microenvironment, and signaling events within T cells themselves can shape their function, persistence, and cytotoxic capacity. By mapping how β-catenin influences the NKG2D-dependent recognition pathway, Karimi’s project seeks to uncover the regulatory logic that determines whether this alternative immune recognition can be therapeutically amplified in real patients, whose immune cells may differ substantially from those studied in laboratory models.

The ambition of the project extends beyond simply finding leukemia cells. AML develops primarily in the bone marrow, an anatomical sanctuary where leukemia cells actively sculpt their surroundings into a self-protective niche. “AML develops mainly in the bone marrow, where leukemia cells can create an environment that helps them hide from the immune system and avoid being recognized by cancer-fighting immune cells,” Karimi noted. This immunosuppressive microenvironment can disable infiltrating T cells through metabolic exhaustion, inhibitory checkpoint signaling, and direct suppression by stromal and myeloid cells recruited to the leukemia’s service. Understanding these escape mechanisms in detail, Karimi argues, is the prerequisite for dismantling them. “We want to understand the mechanisms AML cells use to escape immune detection and weaken these modified immune cells,” he said. “By identifying these escape strategies, we hope to find ways to overcome them and improve the ability of immune cells to recognize and destroy AML. Ultimately, this knowledge could help us develop more effective HLA-independent immunotherapies for patients with AML.”

The clinical stakes of this work are illuminated by the current standard of care for patients whose AML returns after initial treatment or who present with high-risk disease features. For these individuals, physicians often turn to allogeneic stem cell transplantation, a procedure in which a patient’s blood-forming system is replaced with that of a healthy donor. The therapeutic power of transplantation comes not merely from the replacement of the marrow itself but from the donor’s immune cells, which can mount what oncologists call a graft-versus-leukemia effect. Donor T cells survey the recipient’s tissues, identify residual leukemia as foreign, and attack it, providing a potent and sometimes curative immunologic assault on the disease. “These donor immune cells can recognize and attack the leukemia, producing what is called a graft-versus-leukemia effect,” Karimi explained.

But this biological double-edged sword cuts in a terrible direction. The same donor immune cells that attack leukemia cannot always distinguish malignant tissue from the patient’s healthy organs, skin, liver, and gastrointestinal tract. The result is graft-versus-host disease, or GVHD, a potentially devastating complication in which the transplanted immune system turns against its new host. “However, the same donor immune cells can also attack the patient’s healthy tissues,” Karimi said. “This serious complication is called graft-versus-host disease, or GVHD.” The problem is compounded by a stark therapeutic vacuum: no approved treatments specifically for GVHD are available, and severe cases can be extraordinarily difficult to control, causing significant illness and death. For many patients and their physicians, the decision to pursue transplantation becomes a calculus of risk, weighing the curative potential of the graft-versus-leukemia effect against the danger of a runaway donor immune response.

This is precisely where Karimi’s HLA-independent strategy could prove transformative. If engineered CD8⁺ T cells can be tuned to recognize AML selectively through NKG2D-dependent mechanisms while remaining inert toward healthy tissue, the therapeutic ideal that allogeneic transplantation only approximates—maximal anti-leukemia activity with minimal collateral damage—could be realized directly. “Therefore, one of our major goals is to develop immune cells that can effectively attack AML while avoiding damage to healthy tissues,” Karimi said. “We will use this knowledge as a foundation to develop new immune-based therapies that can ultimately be translated directly to patients with AML.” The phrase “translated directly to patients” signals the translational orientation of the program: rather than remaining a purely mechanistic inquiry, the project is designed to generate actionable biological knowledge that can inform the design of cellular therapies testable in clinical settings.

The significance of the award extends beyond a single disease. HLA loss and downregulation are not unique to AML; numerous solid tumors and other hematologic malignancies employ similar strategies to escape T-cell surveillance. Immunotherapies that operate independently of HLA presentation could therefore offer a blueprint for treating cancers that have historically eluded the most successful immune-based approaches of the past decade. Moreover, understanding how β-catenin signaling regulates NKG2D-mediated recognition may reveal broader principles governing how innate-like immune recognition can be harnessed in engineered T cells, an area of intense interest across immuno-oncology.

For the roughly twenty thousand Americans diagnosed with AML each year, many of them older adults whose disease carries a five-year survival rate that remains tragically low, the research represents a beacon of rational, mechanistically grounded hope. The five-year funding window through July 2031 gives Karimi’s laboratory the sustained resources needed to move from molecular dissection to therapeutic concept, a timeline that reflects the complexity of the challenge and the National Cancer Institute’s confidence in the scientific foundation underlying it. As the field of cancer immunotherapy enters its second decade of mainstream clinical success, work like this points toward its next frontier: cancers that have learned to hide from the immune system’s primary surveillance system, and the scientists determined to give immune cells a second way to see them.

Standard frontline therapies have improved survival rates; however, patients who experience relapse or are refractory to conventional treatments confront a grim prognosis. A breakthrough in recent years has been the introduction of immunotherapies targeting the CD19 antigen—a surface protein ubiquitously expressed on B-ALL cells. CAR-T (chimeric antigen receptor T-cell) therapies engineered to recognize CD19 have revolutionized treatment paradigms, demonstrating remarkable initial response rates. Yet, clinical outcomes remain unsatisfactory for a substantial subset of patients, with nearly half relapsing primarily due to cancer cells orchestrating immune evasion via downregulation or loss of CD19 expression.

Addressing this mechanism of immune escape, researchers from the Josep Carreras Leukaemia Research Institute and Hospital 12 de Octubre – CNIO undertook pioneering experimental work to develop a dual-targeted CAR-T cell therapy. Led by Dr. Pablo Menéndez, Dr. Clara Bueno, and Dr. Luis Álvarez Vallina, the team designed genetically modified T-cells capable of simultaneously targeting two distinct B-ALL surface antigens: CD19 and CD22. CD22 serves as an alternative, non-overlapping marker consistently present on leukemic blasts, presenting a rational target to complement CD19.

The innovation lies in the sophisticated engineering of the CAR-T cells to combine two complementary strategies within a single therapeutic agent. These CAR-T cells deploy a conventional chimeric antigen receptor construct directed against CD22, facilitating direct cytotoxic engagement of leukemic populations expressing this marker. Simultaneously, these engineered T-cells secrete a bispecific T-cell engager (BiTE) molecule focused on recruiting endogenous T-cells—irrespective of their CAR-T status—towards the CD19 antigen on the tumor cells. This dual mechanism effectively "double-dips" the immune response, enhancing both precision and breadth of leukemic cell targeting.

This approach leverages the unique strengths of both CAR-T technology and bispecific antibody therapy. CAR-T cells offer the advantage of long-lived persistence and sustained cytotoxicity, capable of trafficking throughout the body to eliminate malignancies. In contrast, bispecific antibodies function as bridges that physically tether cytotoxic T-cells to cancer cells, promoting potent immune synapse formation and rapid tumor cell lysis. By integrating BiTE secretion within CAR-T cells, the therapy amplifies immune activation and recruitment, mitigating the risk of tumor cells escaping immune surveillance through antigen loss variants.

Preclinical in vitro investigations revealed that these dual-targeting CAR-T cells markedly improved leukemic control compared to single-antigen targeting counterparts. The bispecific engagement significantly increased the recruitment and activation of bystander T-cells to CD19-expressing leukemia cells, while the direct CD22 CAR-mediated cytotoxicity ensured clearance of leukemic clones that had escaped CD19 targeting. Importantly, this led to prolonged control of leukemic growth and potential reduction in relapse incidence.

Published in the Journal for ImmunoTherapy of Cancer and co-authored by Javier Arroyo Ródenas and Aïda Falgàs, the study marks a pioneering step towards next-generation CAR-T immunotherapies that circumvent antigen escape mechanisms—one of the most formidable barriers to durable remission in relapsed B-ALL. The researchers emphasize that the bifunctional design could redefine treatment paradigms, offering renewed hope for pediatric patients facing limited options after relapse.

The success of this experimental study underscores the importance of multi-antigen targeting within immuno-oncology, particularly in hematological malignancies where antigen heterogeneity and plasticity fuel therapeutic resistance. By harnessing the endogenous immune system’s full cytotoxic potential and imparting long-term surveillance capacity, these "armored" CAR-T cells embody a strategic advance in precision medicine.

Furthermore, the modular construction of these CAR-T cells facilitates adaptability; the BiTE component secreted could theoretically be re-engineered to target alternative tumor-specific antigens or combined with additional immune-stimulatory molecules. This flexibility paves the way for bespoke immunotherapies tailored to diverse malignancies characterized by complex antigenic landscapes.

This research was made possible through funding by several esteemed institutions, including the European Commission’s ERC program, multiple Spanish governmental bodies, regional funds from the Generalitat de Catalunya and Comunidad de Madrid, as well as private entities dedicated to cancer research. Such robust support reflects the growing recognition that innovative immunotherapy solutions are crucial to addressing unmet clinical needs in childhood cancers.

As this CAR-T and bispecific antibody fusion approach progresses towards clinical translation, its implications extend beyond B-ALL. Similar dual-targeting strategies may be exploited for other refractory hematologic cancers and solid tumors notorious for evading mono-targeted immunotherapies. The future of cancer treatment undoubtedly lies in combinatorial immunotherapeutic designs that enhance efficacy while mitigating resistance.

Overall, this study presents a timely, technologically sophisticated immunotherapeutic platform with the potential to transform leukemia care. Its dual targeting mechanism heralds a paradigm shift toward more resilient, lasting remissions for children afflicted with one of the most prevalent and deadly pediatric cancers. Ongoing research and eventual clinical trials will determine its full impact on patient survival and quality of life worldwide.

Subject of Research: Cells

Article Title: CD22 CAR-T cells secreting CD19 T-cell engagers for improved control of B-cell acute lymphoblastic leukemia progression

News Publication Date: 30-Apr-2025

Web References:
https://doi.org/10.1136/jitc-2024-009048

References:
Arroyo-Ródenas J, Falgàs A, Díez-Alonso L, et al. “CD22 CAR-T cells secreting CD19 T-cell engagers for improved control of B-cell acute lymphoblastic leukemia progression”. Journal for ImmunoTherapy of Cancer. 2025;13:e009048. doi:10.1136/jitc-2024-009048

Image Credits: Josep Carreras Leukaemia Research Institute

Keywords: Immunotherapy, Antibody therapy, Cancer immunotherapy, Antibodies, Leukemia