A Purdue University chemical engineer has improved upon traditional methods to produce off-the-shelf human immune cells that show strong antitumor activity, according to a paper published in the peer-reviewed journal Cell Reports.
Xiaoping Bao, a Purdue University assistant professor from the Davidson School of Chemical Engineering, said CAR-neutrophils, or chimeric antigen receptor neutrophils, and engraftable HSCs, or hematopoietic stem cells, are effective types of therapies for blood diseases and cancer. Neutrophils are the most abundant white cell blood type and effectively cross physiological barriers to infiltrate solid tumors. HSCs are specific progenitor cells that will replenish all blood lineages, including neutrophils, throughout life.
“These cells are not readily available for broad clinical or research use because of the difficulty to expand ex vivo to a sufficient number required for infusion after isolation from donors,” Bao said. “Primary neutrophils especially are resistant to genetic modification and have a short half-life.”
Bao has developed a patent-pending method to mass-produce CAR-neutrophils from human pluripotent stem cells (hPSCs), that is, cells that self-renew and are able to become any type of human cell. The chimeric antigen receptor constructs were engineered to express on the surface of the hPSCs, which were directed into functional CAR-neutrophils through a novel, chemically defined protocol.
The method was created in collaboration with Qing Deng at Purdue’s Department of Biological Sciences, Hal E. Broxmeyer, now deceased, at Indiana University School of Medicine, and Xiaojun Lian at the Pennsylvania State University.
“We developed a robust protocol for massive production of de novo neutrophils from human pluripotent stem cells,” Bao said. “These hPSC-derived neutrophils displayed superior and specific antitumor activities against glioblastoma after engineering with chimeric antigen receptors.”
Bao disclosed the innovation to the Purdue Research Foundation Office of Technology Commercialization, which has applied for an international patent under the Patent Cooperation Treaty system of the World Intellectual Property Organization. The innovation has been optioned to an Indiana-headquartered life sciences company.
“We will also work with Dr. Timothy Bentley, professor of neurology and neurosurgery,and his team at the Purdue College of Veterinary Medicine to run clinical trials in pet dogs with spontaneous glioma,” Bao said.
While immunotherapy has been developed to treat hematologic malignancies, de novo and acquired resistance to targeted cancer therapy is commonly observed in solid tumors due to the complex TME, in which neutrophils are key players (Devlin et al., 2020; Kalafati et al., 2020; Ponzetta et al., 2019).
Improved understanding of neutrophil contributions to the TME has increased our interest in reprograming and/or depleting protumor neutrophils as an alternative approach to treat cancer (Kalafati et al., 2020). Unlike previous neutrophil-depletion approaches, we demonstrate here the feasibility of using CARs to program and maintain neutrophils as antitumor effector cells, representing an advanced neutrophil-based immunotherapy that may complement standard cancer treatments and boost their efficacy.
Due to the short life of primary neutrophils and their resistance to genome editing, engineering hPSCs with synthetic CARs would be an ideal approach to produce off-the-shelf CAR neutrophils. To achieve this goal, we first developed a chemically defined platform for robust production of neutrophils from hPSCs using stage-specific signaling pathway modulators. Based on previous studies, we designed and assessed three different CAR constructs with NK or T cell-specific transmembrane and intracellular activation domains in enhancing neutrophil-mediated tumor killing.
CLTX-T-CAR that contains a GBM-binding peptide CLTX and T cell-specific signaling domains markedly improved tumor antigen-specific cytotoxicity of hPSC neutrophils to a level comparable to CAR T cells with a similar CLTX-T-CAR (Wang et al., 2020a) and superior to CAR macrophages with an anti-CD19 CAR in vitro (Klichinsky et al., 2020), though in vivo quantitative comparison data are unavailable due to the different mouse models used.
Notably, systemically administered CAR neutrophils presented superior anti-GBM activities in mice compared with hPSC-derived CAR NK cells, possibly due to a better ability of neutrophils to cross the BBB and penetrate GBM xenografts. In future studies, it will be interesting to investigate whether neutrophil-specific transmembrane and activation domains can be used to establish neutrophil-specific CAR constructs (Roberts et al., 1998).
Using an inducible gene knockdown system, we identified MMP2 on GBM cells as the target of CLTX binding and recognition that triggers CAR activation in neutrophils. Molecular mechanism investigation revealed that CLTX-T-CAR triggers known downstream intracellular signaling pathways that mediate phagocytosis against tumor cells.
While selected gene-expression profiles, including N1/N2 markers and functional gene-expression patterns, in CAR neutrophils indicated a sustained antitumor phenotype under various TME-like conditions, more authentic markers enabling the tracking of plastic neutrophils are still needed to validate the phenotype and safety of in vivo CAR neutrophils after infusion. Hypoxia reduced ROS generation in wild-type, but not CAR-expressing neutrophils, and thus oxidative stress genes are worthy of further investigation.
In summary, the CAR-neutrophil engineering platform described here may serve as a scalable strategy to make off-the-shelf neutrophils as standardized cellular products for clinical applications in cancer and neutropenia treatment. Given the relative ease of gene editing in hPSCs, other genetic modifications, such as multiple CAR expressions, can also be performed to achieve optimal therapeutic effects in CAR neutrophils.
Due to their native ability to cross the BBB and penetrate brain parenchyma, neutrophil-mediated delivery of therapeutic drugs into brain has improved GBM diagnosis and treatment (Wu et al., 2018; Xue et al., 2017), and such a combination may further enhance antitumor activities of hPSC-derived CAR neutrophils. Importantly, stable CAR-expressing hPSC lines can be also used to produce off-the-shelf CAR T and NK cells.
More information: Yun Chang et al, Engineering chimeric antigen receptor neutrophils from human pluripotent stem cells for targeted cancer immunotherapy, Cell Reports (2022). DOI: 10.1016/j.celrep.2022.111128