Flipping out to develop an intracellular protein-targeting CAR T cell therapy to treat AML

From the Meshinchi Lab, Clinical Research and Translational Science and Therapeutics Divisions.

Pediatric acute myeloid leukemia (AML) has pretty dismal outcomes, explained Dr. Danielle Kirkey, a Pediatric Hematology Oncology Fellow conducting research in Dr. Soheil Meshinchi’s lab at Fred Hutch. Dr. Kirkey continued, “AML, which is the most common type of acute leukemia in adults, accounts for only 25% of pediatric leukemias, but is the most deadly form. Even the most intensive chemotherapies don’t work well” for this pediatric cancer. For other blood cancers, chimeric antigen receptor (CAR) T cell therapies have achieved impressive outcomes for improving patient survival. These CAR T cell therapies supercharge a patient’s T cells to fight off cancer, with T cells instructed to attack all cells with a specific protein on their cell surface. This therapy is particularly useful for B-cell malignancies which have specific proteins on their cancer cells that are easy to target. While these markers are also shared with some other healthy non-cancerous B cell populations, depleting those healthy cells fortunately is not deleterious for patients. However, for myeloid blood cancers like AML, developing an effective CAR T cell therapy has been a major hurdle since there is not a good cell surface marker that is specific to the cancer cells. Most AML markers are shared with healthy, essential cells that would have major negative consequences for patients if depleted. Excitingly, recent research has found a candidate AML-specific target, PRAME (Preferentially Expressed Antigen in Melanoma), not shared with non-cancerous cells. There was just one problem- full-length PRAME proteins are only present inside the cell and not on the cell surface. Kirkey stated, “PRAME is a good target, but since it is intracellular, it is not targetable by traditional CAR T cell therapies.” Dr. Kirkey and researchers from the Meshinchi team, got creative and worked to find a way to make this intracellular target work for CAR T cell therapies as a potential treatment for AML. This work led by Kirkey and co-first author, Anisha Loeb in the Meshinchi Lab, was recently published in Blood Advances.

So how does one go about getting a cell surface marker-based therapy to recognize a protein inside the cell? While the researchers could not directly force the full-length protein to be presented on the cell surface, they took advantage of some of the cell’s normal processes. Cells have machinery that chew up proteins into smaller pieces, referred to as peptides. Additionally, the immune system has a class of molecules, called HLA proteins which bind these generated peptides and present them on the cell surface, flipping what’s inside the cell to the outside. This HLA-peptide complex works as part of the immune system’s surveillance system to help distinguish between the body’s own cells and proteins made from foreign invaders like viruses. Recently, it was uncovered that part of the PRAME protein, or a PRAME peptide, gets presented on the cell surface by HLA proteins. Researchers in Dr. David Scheinberg’s lab at Memorial Sloan Kettering were able to develop an antibody that recognizes a specific PRAME peptide-HLA-A2 complex, where HLA-A2 is a specific HLA subtype. Because this antibody recognizes a peptide-HLA complex, like T cell receptors do when they are surveying which cells to attack, this antibody is effectively a mimic T cell receptor (mTCR). Recognizing the potential of this antibody, the Meshinchi team got some help from Dr. Scheinberg to use this antibody to develop a mTCRCAR T cell therapy for AML. Here, the T cells would be instructed to attack cells that express the PRAME peptide-HLA-A2 complex on their cell surface. Since this complex should only be present on AML cells, it would allow specific targeting of these leukemia cells, but not attack any non-cancerous cells that lack this marker.

Overview of PRAME mTCR CAR T cell therapy for AML treatment and its effectiveness in vitro and in vivo.
Overview of PRAME mTCR CAR T cell therapy for AML treatment and its effectiveness in vitro and in vivo. Image provided by Danielle Kirkey.

After engineering the PRAME mTCRCAR T cells, the authors first tested their efficacy in vitro by culturing AML cell lines and primary leukemia patient samples. Kirkey et al. demonstrated that when the mTCRCAR T cells were incubated with AML cell lines expressing HLA-A2 and PRAME, the supercharged T cells were effective at killing off the cancer cells, but not cells that lacked

PRAME or HLA-A2 expression. Moving the work into mouse models, the Meshinchi team generated human leukemia xenograft models by injecting mice with the various AML cells. They then treated these different mouse models with control, unmodified T cells or PRAME mTCRCAR T cells and asked whether their modified CAR T cells were able to clear the leukemia in mice with PRAME-expressing cancer. In mice with PRAME and HLA-A2 expressing leukemia cells, PRAME mTCRCAR T cells were able to kill off the leukemia and the mice remained disease free for the duration of the study. This treatment was also effective at significantly reducing the growth of leukemia cells with lower PRAME expression, however, was unable to completely irradicate it. When the researchers pre-treated these cell lines with IFN-gamma, known to enhance the presentation of tumor associated antigens, this led to increased PRAME expression in both AML cell lines, and increased the efficiency by which the PRAME mTCRCAR T cells were able to kill off the cancer cells. This result demonstrated that certain treatments might be able to increase PRAME expression in patients with low levels of PRAME, potentially overcoming one limitation of this method. Kirkey noted that another limitation of this work is that this treatment approach is “restricted to HLA-A2, which is about 50% of the population, but since you are limited to that HLA status, hopefully novel methods can be developed to overcome that HLA restriction.”

This research developed “a novel immunotherapy for AML where immunotherapies are in their infancy,” Kirkey exclaimed. Traditional CAR T cell therapy relies on cell surface markers. By demonstrating the feasibility of this new approach to targeting non-surface markers, this customizability increases the potential for this immunotherapy to be broadly applicable at treating a new range of cancers that previously could not be targeted. Kirkey emphasized that this important work could not have been done alone, and research technicians in the Meshinchi Lab, Anisha Loeb and Sommer Castro played critical roles in executing this research. Next, Dr. Kirkey is working to push this project forward, with the ultimate goal of moving this research into clinical trials. To do so, she has been testing the PRAME-targeting CAR T cell therapy in patient-derived xenograft mouse models which have shown promising results at clearing the leukemia in these mice and increasing survival. Additionally, she’s exploring ways to manipulate the antibody targeting portion of the CAR T therapy to make this treatment broadly applicable to patients outside of the HLA-A2 status and investigating how a similar approach might extend to the treatment of other pediatric cancers such as neuroblastoma and osteosarcoma.

This work was supported by the Leukemia and Lymphoma Society and St. Baldrick’s Foundation.

UW/Fred Hutch/ Seattle Children’s Cancer Consortium members Soheil Meshinchi and Keith Loeb contributed to this work.

Kirkey DC, Loeb AM, Castro S, McKay CN, Perkins L, Pardo L, Leonti AR, Tang TT, Loken MR, Brodersen LE, Loeb KR, Scheinberg DA, Le Q, Meshinchi S. Therapeutic targeting of PRAME with mTCRCAR T cells in acute myeloid leukemia. Blood Adv. 2023 Apr 11;7(7):1178-1189. doi: 10.1182/bloodadvances.2022008304. PMID: 35984639; PMCID: PMC10111362.