Optimization of canine CD20 chimeric antigen receptor T cell manufacturing and in vitro cytotoxic activity against B‐cell lymphoma

Abstract

Canine B-cell lymphoma is one of the most common haematopoietic neoplasms in veterinary medicine, and it is considered a relevant model for human diffuse large Bcell lymphoma. Although the standard treatment consisting of multi-drug chemotherapy is effective in most cases, treatment is often challenging because of relapse and drug resistance. The adoptive transfer of autologous T cells genetically modified to express a CD19-specific chimeric antigen receptor (CD19 CART cells) has been shown to be highly effective in human B-cell malignancies. However, there is no clinically available canine CART cell therapy. We generated canine second-generation and third-generation CART cells by retroviral gene transduction. Optimization was performed to investigate effective viral transduction protocols and favourable culture conditions for canine CART cells. The RetroNectin-bound virus infection method resulted in more than 70% transduction efficiency. The effect of culture conditions on the phenotype of CART cells was evaluated by the expression of surface markers. in vitro cytotoxicity assays of target cells cultured with CD20 CAR-transduced cells demonstrated that CD20 CART cells exhibit cytotoxicity against CD20-expressing canine B-cell lymphoma cells and canine CD20-transduced murine cells, whereas no effect was observed against cells that lacked canine CD20 expression. Our study established virus-based canine CART cell generation, providing fundamental data for a better understanding of canine adoptive T-cell therapy.

Genetic re-direction of canine primary T cells for clinical trial use in pet dogs with spontaneous cancer

“…The method below provides step-by-step details to effectively engineer primary canine T cells. Compared to previously published methods ( Panjwani et al., 2020 ; Sakai et al., 2020 ), our implemented protocol based on use of RD114 pseudotyped viral vectors and activation of canine T lymphocytes via anti-canine CD3/CD28-coated beads enables high transduction efficiencies using a single infection. Of note, we have also successfully employed chimeric RD114 and VSVG pseudotyped retrovirus for canine T cell transduction.…”

Spontaneously occurring canine cancer as a relevant animal model for developing novel treatments for human cancers

“…Although several types of CAR-T cell therapies have already been approved in humans [104], there are still no approved CAR-T cell therapies for dogs. However, several groups, including our own, have already reported on how to create canine CAR-T cells with some efficacy in vitro [105] and in vivo [106][107][108]. To maximize the effect of CAR-T cell therapy in patients, CAR-T cells must be administered after myelosuppression using strong anticancer drugs and radiation to eliminate the immune cells.…”

There and back again: Translating adoptive cell therapy to canine cancer and improving human treatment

“…In 2020, a group based out of Yamaguchi, Japan demonstrated that an anti‐CD20 CAR could be stably transduced into canine T cells via a retrovirus vector 48 . Their paper provides data on a variety of optimization experiments they used, comparing how canine CAR‐T cells were differentially transduced, activated, and expanded with a variety of methods, including various agonist antibodies (anti‐CD3, anti‐CD28), cytokines, phytohemagglutinin, aAPCS, and different transduction modes.…”