Send in the CARs: mesothelin-targeted CAR T cells against AML

From the Meshinchi Lab, Clinical Research Division, and Cancer Consortium colleagues

Acute myelogenous leukemia (AML) is a disease for which there is a great unmet clinical need. Whereas immunotherapies, such as engineered adoptive T cell transfer, have offered inroads for other bloodborne cancers, the treatment of AML has proved more difficult. “Chimeric antigen receptor (CAR) T cells have revolutionized cancer treatment for aggressive B-ALL (B cell acute lymphoblastic leukemia), providing a potentially curative treatment option for these high-risk patients,” described Fred Hutch senior scientist Dr. Quy Le. “However, the use of CAR T cell therapy for AML is still in its infancy, with significant barriers such as the inherent heterogeneity in AML and the lack of AML-specific targets.” In recent work, the Meshinchi Laboratory in the Fred Hutch Clinical Research Division identified mesothelin as a potential therapeutic target in AML. Building upon this finding, Dr. Le led an effort to develop mesothelin-specific CAR T cell therapies for the treatment of AML. This work, recently published in Clinical Cancer Research, reveals that CAR T cells directed against mesothelin can safely eliminate AML in preclinical systems, establishing this therapy as a promising strategy for translation into the clinic.

The success of CAR T cell-based therapies hinges on the identification of appropriate target antigens – proteins that are expressed at sufficient levels on cancer cells that can be effectively detected by therapeutic T cells, but not expressed on healthy tissues to avoid toxicities. This is a tricky problem when it comes to AML “due to the substantial overlap of the immunophenotype that is shared between AML and normal hematopoietic cells,” explained Dr. Le. “Current strategies use lineage markers (i.e CD33 and CD123) that may not be ideal targets for CAR T cell therapy in AML as these CAR T cells would not be able to differentiate between normal and malignant cells, which could lead to life-threatening complications such as myeloablation or myelosuppression if the CAR T cells are proven to be effective.” To overcome this problem, the Meshinchi Laboratory and colleagues performed the largest ever multi-omics analysis of AML to identify AML-restricted targets. “By computational analysis of this massive dataset, we identified mesothelin as highly expressed in a significant subset of AML that is entirely absent in normal hematopoiesis, providing a promising AML-restricted target that avoids hematopoietic toxicities,” said Dr. Le.

Engineered T cells expressing chimeric antigen receptors (CARs) against mesothelin detect and destroy AML target cells.
Engineered T cells expressing chimeric antigen receptors (CARs) against mesothelin detect and destroy AML target cells. Image provided by Dr. Le.

Having previously shown that AMLs can successfully be targeted using mesothelin-targeted antibody-drug conjugates (ADCs), the group hypothesized that this antigen might be a good target for CAR T cell therapy. To pursue this, they incorporated the antigen-binding portion of a mesothelin-targeted ADC into a CAR construct, which could in turn be used to direct cytotoxic T cells against cells expressing the mesothelin antigen. The functionality of the CAR was validated using in vitro cytotoxicity assays, in which T cells engineered to express the CAR were cocultured with leukemia cell lines. Encouragingly, mesothelin-targeted CAR T cells selectively reacted to mesothelin-expressing leukemia cells, resulting in their lysis of the target cells, production of inflammatory cytokines, and proliferation.

In order to confirm whether mesothelin-targeted CAR T cells could effectively recognize physiological levels of the target protein, the group tested the functionality of the CAR T cells against primary AML cells isolated from patients. Indeed, these CAR T cells exhibited cytolytic and cytokine responses selectively against AML targets that expressed mesothelin. CAR T cells showed comparable cytolytic activity against bulk AML cells as well as samples enriched with markers of the leukemic stem cell (LSC) compartment, a subset of AML cells that is recalcitrant to traditional chemotherapies and is thought to seed disease relapse after therapy. Importantly, the CAR T cells spared normal hematopoietic cells collected from healthy donors, including hematopoietic stem and progenitor cells (HSPCs), indicating that this therapy can safely eradicate AML cells without compromising healthy blood lineages.

“CAR T cells targeting mesothelin can effectively eliminate both AML blasts and LSCs while sparing normal HSPCs. These results demonstrate that mesothelin-directed CAR T cells may provide an effective approach to eliminate AML without impacting normal hematopoiesis”, said Dr. Le. “Given that the CAR has potency against LSCs, we are optimistic that this CAR may provide a mechanism to prevent relapse as LSCs are thought to resist chemotherapy and can re-initiate the disease.”

Finally, Dr. Le and colleagues assessed preclinical models to test whether the mesothelin-directed CAR T cells could effectively treat AML in vivo. Excitingly, in animals transplanted either with mesothelin-expressing human leukemia cell lines (in the context of immunocompromised mice) or with primary patient AML samples (in ‘humanized’ mice, which have been reconstituted with human hematopoietic lineages), CAR T cell therapy resulted in dramatic disease reduction and extension of survival in the animals.

These findings could have important implications for the treatment of AML in patients. “Our pre-clinical data support advancing the mesothelin-directed CAR into clinical testing for AML,” commented Dr. Le. “We are currently developing the clinical vector with an anticipated clinical trial to open in 3rd-4th quarter of 2022 for relapsed/refractory AML.” Dr. Le credits the Fred Hutch/University of Washington Cancer Consortium for its role in this work and the future translational efforts. “This work was made possible through the collaboration with [Consortium members] Drs. Stan Riddell and Cameron Turtle,” said Dr. Le. Additionally, “the FHCRC TPP (Therapeutic Products Program) core facilitated the process development for the pending clinical trial.”

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

UW/Fred Hutch Cancer Consortium members Shivani Srivastava, Cameron Turtle, Stanley Riddell, Katherine Tarlock, and Soheil Meshinchi contributed to this work.

Le Q, Castro S, Tang T, Loeb AM, Hylkema T, McKay C, Perkins L, Srivastava S, Call L, Smith JL, Leonti A, Ries R, Pardo L, Loken MR, Correnti C, Fiorenza S, Turtle CJ, Riddell S, Tarlock K, Meshinchi S. Therapeutic Targeting of Mesothelin with Chimeric Antigen Receptor T Cells in Acute Myeloid Leukemia. Clin Cancer Res. 2021 Aug 11:clincanres.1546.2021. doi: 10.1158/1078-0432.CCR-21-1546. Epub ahead of print. PMID: 34380639.