Applications and Opportunities for Immune Cell CAR Engineering in Comparative Oncology

Abstract

Chimeric antigen receptor (CAR) T-adoptive cell therapy has transformed the treatment of human hematologic malignancies. However, its application for the treatment of solid tumors remains challenging. An exciting avenue for advancing this field lies in the use of pet dogs, in which cancers that recapitulate the biology, immunological features, and clinical course of human malignancies arise spontaneously. Moreover, their large size, outbred genetic background, shared environment with humans, and immunocompetency make dogs ideal for investigating and optimizing CAR therapies before human trials. Here, we will outline how challenges in early clinical trials in patients with canine lymphoma, including issues related to autologous CAR T-cell manufacturing, limited CAR T-cell persistence, and tumor antigen escape, mirrored challenges observed in human CAR T trials. We will then highlight emerging adoptive cell therapy strategies currently under investigation in dogs with hematological and solid cancers, which will provide crucial safety and efficacy data on novel CAR T regimens that can be used to support clinical trials. By drawing from ongoing studies, we will illustrate how canine patients with spontaneous cancer may serve as compelling screening platforms to establish innovative CAR therapy approaches and identify predictive biomarkers of response, with a specific emphasis on solid tumors. With increased funding for canine immunotherapy studies, multi-institutional investigations are poised to generate highly impactful clinical data that should translate into more effective human trials, ultimately benefiting both human and canine cancer patients.

Figure 1.. Canine CAR immunotherapy studies.

A. Translational relevance of canine CAR therapy trials. Pet dogs provide a bridge between in vitro 2D and 3D human systems (1), in vivo murine and non-human primates (NHP) models (–3), and applications in human patients (5). Efficacy and safety data from canine trials can improve the efficiency of CAR immunotherapy development and translation to the human clinics. They can also enable innovative therapeutics along with risk-mitigating strategies that cannot be reliably tested in rodents and NHP, limiting the detrimental impact of failures and unexpected toxicities. (–7) Further optimization of established human CAR therapies may be more readily achieved in canine trials than in human trials, with mutual benefit for canine and human patients. B. Manufacturing and treatment processes for autologous canine CAR-T cells. (1) In most cases, CAR-T products are manufactured from patients’ T cells, although CAR-trials investigating allogeneic and xenogeneic products from canine and human donors have been launched already. (2) T cells are generally isolated following venipuncture or leukapheresis, although TILs can be isolated from patients’ tumors. (3) Subsequent enrichment of specific subsets can be carried out, such as CD1d-restricted T cells for manufacturing of invariant Natural Killer T cell products, or CD5⁺ T cells to minimize the risk of contaminating CAR B cells, MSDCs or other suppressive cells. The isolated T lymphocytes undergo activation either with beads or irradiated artificial Antigen Presenting Cell (APC) in the presence of human cytokines, most of which can also be used to stimulate dog cells. (4) The activated T cells are then engineered to express canine CAR constructs. Both mRNA and virus-based methods have been successfully used, with retroviral systems consistently resulting in the highest percentages of edited canine T cells. (–7) Within 10–23 days, the engineered and expanded CAR cells are infused intravenously or locally into a lymphodepleted canine patient. gen: generation. (B, Created with BioRender.com)