Targeting TAG-72 in cutaneous T cell lymphoma.

Purpose: Current monoclonal antibody-based treatment approaches for cutaneous T cell lymphoma (CTCL) rely heavily on the ability to identify a tumor specific target that is essentially absent on normal cells. Herein, we propose tumor associated glycoprotein-72 (TAG-72) as one such target. TAG-72 is a mucin-associated, truncated O-glycan that has been identified as a chimeric antigen receptor (CAR)-T cell target in solid tumor indications. To date, TAG-72 targeting has not been considered in the setting of hematological malignancies. Experimental design: CD3+ cells from patients with CTCL were analyzed for TAG-72 expression by flow cytometry. Immunohistochemistry was used to assess TAG-72 expression in CTCL patient skin lesions and a TAG-72 ELISA was employed to assess soluble TAG-72 (CA 72-4) in patient plasma. TAG-72 CAR transduction was performed on healthy donor (HD) and CTCL T cells and characterized by flow cytometry. In vitro CAR-T cell function was assessed by flow cytometry and xCELLigence® using patient peripheral blood mononuclear cells and proof-of-concept ovarian cancer cell lines. In vivo CAR-T cell function was assessed in a proof-of-concept, TAG-72+ ovarian cancer xenograft mouse model. Results: TAG-72 expression was significantly higher on total CD3+ T cells and CD4+ subsets in CTCL donors across disease stages, compared to that of HDs. TAG-72 was also present in CTCL patient skin lesions, whereas CA 72-4 was detected at low levels in both CTCL patient and HD plasma with no differences between the two groups. In vitro cytotoxicity assays showed that anti-TAG-72 CAR-T cells significantly, and specifically reduced CD3+TAG-72+ expressing CTCL cells, compared to culture with unedited T cells (no CAR). CTCL CAR-T cells had comparable function to HD CAR-T cells in vitro and CAR-T cells derived from CTCL patients eradicated cancer cells in vivo. Conclusion: This study shows the first evidence of TAG-72 as a possible target for the treatment of CTCL.

Engineering T cell receptor fusion proteins using nonviral CRISPR/Cas9 genome editing for cancer immunotherapy.

Manufacture of chimeric antigen receptor (CAR)-T cells usually involves the use of viral delivery systems to achieve high transgene expression. However, it can be costly and may result in random integration of the CAR into the genome, creating several disadvantages including variation in transgene expression, functional gene silencing and potential oncogenic transformation. Here, we optimized the method of nonviral, CRISPR/Cas9 genome editing using large donor DNA delivery, knocked-in an anti-tumor single chain variable fragment (scFv) into the N-terminus of CD3ε and efficiently generated fusion protein (FP) T cells. These cells displayed FP integration within the TCR/CD3 complex, lower variability in gene expression compared to CAR-T cells and good cell expansion after transfection. CD3ε FP T cells were predominantly CD8+ effector memory T cells, and exhibited anti-tumor activity in vitro and in vivo. Dual targeting FP T cells were also generated through the incorporation of scFvs into other CD3 subunits and CD28. Compared to viral-based methods, this method serves as an alternative and versatile way of generating T cells with tumor-targeting receptors for cancer immunotherapy.

CAR-T cell development for Cutaneous T cell Lymphoma: current limitations and potential treatment strategies.

Chimeric antigen receptor (CAR)-T therapy has demonstrated remarkable outcomes for B cell malignancies, however, its application for T cell lymphoma, particularly cutaneous T cell lymphoma (CTCL), has been limited. Barriers to effective CAR-T cell therapy in treating CTCL include T cell aplasia in autologous transplants, CAR-T product contamination with leukemic T cells, CAR-T fratricide (when the target antigen is present on normal T cells), and tumor heterogeneity. To address these critical challenges, innovative CAR engineering by targeting multiple antigens to strike a balance between efficacy and safety of the therapy is necessary. In this review, we discuss the current obstacles to CAR-T cell therapy and highlight potential targets in treating CTCL. Looking forward, we propose strategies to develop more powerful dual CARs that are advancing towards the clinic in CTCL therapy.

Micro-hydrogel injectables that deliver effective CAR-T immunotherapy against 3D solid tumor spheroids.

Chimeric antigen receptor (CAR-) T cells are revolutionizing cancer treatment, as a direct result of their clinical impact on the treatment of hematological malignancies. However for solid tumors, CAR-T cell therapeutic efficacy remains limited, primarily due to the complex immunosuppressive tumor microenvironment, inefficient access to tumor cells and poor persistence of the killer cells. In this in vitro study, an injectable, gelatin-based micro-hydrogel system that can encapsulate and deliver effective CAR-T therapy is investigated. CAR-T cells targeting TAG-72, encapsulated in these microgels possessed high viability (> 87%) after 7 days, equivalent to those grown under normal expansion conditions, with retention of the T cell phenotype and functionality. Microgel recovered CAR-T cells demonstrated potent on-target cytotoxicity against human ovarian cancer in vitro and on three-dimensional tumor spheroids, by completely eliminating tumor cells. The gelatin-based micro-hydrogels have the potential to serve as carrier systems to augment CAR-T immunotherapeutic treatment of solid tumors.

Enhancing a Natural Killer: Modification of NK Cells for Cancer Immunotherapy.

Natural killer (NK) cells are potent innate immune system effector lymphocytes armed with multiple mechanisms for killing cancer cells. Given the dynamic roles of NK cells in tumor surveillance, they are fast becoming a next-generation tool for adoptive immunotherapy. Many strategies are being employed to increase their number and improve their ability to overcome cancer resistance and the immunosuppressive tumor microenvironment. These include the use of cytokines and synthetic compounds to bolster propagation and killing capacity, targeting immune-function checkpoints, addition of chimeric antigen receptors (CARs) to provide cancer specificity and genetic ablation of inhibitory molecules. The next generation of NK cell products will ideally be readily available as an "off-the-shelf" product and stem cell derived to enable potentially unlimited supply. However, several considerations regarding NK cell source, genetic modification and scale up first need addressing. Understanding NK cell biology and interaction within specific tumor contexts will help identify necessary NK cell modifications and relevant choice of NK cell source. Further enhancement of manufacturing processes will allow for off-the-shelf NK cell immunotherapies to become key components of multifaceted therapeutic strategies for cancer.

'Off-the-Shelf' Immunotherapy: Manufacture of CD8+ T Cells Derived from Hematopoietic Stem Cells.

Cellular immunotherapy is revolutionizing cancer treatment. However, autologous transplants are complex, costly, and limited by the number and quality of T cells that can be isolated from and expanded for re-infusion into each patient. This paper demonstrates a stromal support cell-free in vitro method for the differentiation of T cells from umbilical cord blood hematopoietic stem cells (HSCs). For each single HSC cell input, approximately 5 × 104 T cells were created with an initial five days of HSC expansion and subsequent T cell differentiation over 49 days. When the induced in vitro differentiated T cells were activated by cytokines and anti-CD3/CD28 beads, CD8+ T cell receptor (TCR) γδ+ T cells were preferentially generated and elicited cytotoxic function against ovarian cancer cells in vitro. This process of inducing de novo functional T cells offers a possible strategy to increase T cell yields, simplify manufacturing, and reduce costs with application potential for conversion into chimeric antigen receptor (CAR)-T cells for cancer immunotherapy and for allogeneic transplantation to restore immune competence.

Engineered CAR-T cells targeting TAG-72 and CD47 in ovarian cancer.

Chimeric antigen receptor (CAR) T cells have revolutionized blood cancer immunotherapy; however, their efficacy against solid tumors has been limited. A common mechanism of tumor escape from single target therapies is downregulation or mutational loss of the nominal epitope. Targeting multiple antigens may thus improve the effectiveness of CAR immunotherapies. We generated dual CAR-T cells targeting two tumor antigens: TAG-72 (tumor-associated glycoprotein 72) and CD47. TAG-72 is a pan-adenocarcinoma oncofetal antigen, highly expressed in ovarian cancers, with increased expression linked to disease progression. CD47 is ubiquitously overexpressed in multiple tumor types, including ovarian cancer; it is a macrophage "don't eat me" signal. However, CD47 is also expressed on many normal cells. To avoid this component of the dual CAR-T cells killing healthy tissue, we designed a truncated CD47 CAR devoid of intracellular signaling domains. The CD47 CAR facilitates binding to CD47+ cells, increasing the prospect of TAG-72+ cell elimination via the TAG-72 CAR. Furthermore, we could reduce the damage to normal tissue by monomerizing the CD47 CAR. Our results indicate that the co-expression of the TAG-72 CAR and the CD47-truncated monomer CAR on T cells could be an effective, dual CAR-T cell strategy for ovarian cancer, also applicable to other adenocarcinomas.