In vitro re-challenge of CAR T cells.

Chimeric antigen receptor (CAR) T cells (CAR T) have emerged as a potential therapy for cancer patients. CAR T cells are capable of recognizing membrane proteins on cancer cells which initiates a downstream signaling in T cells that ends in cancer cell death. Continuous antigen exposure over time, activation of inhibitory signaling pathways and/or chronic inflammation can lead to CAR T cell exhaustion. In this context, the design of CARs can have a great impact on the functionality of CAR T cells, on their potency and exhaustion. Here, using CD19CAR as model, we provide a re-challenge protocol where CAR T cells are cultured weekly with malignant lymphoid cell lines BL-41 and Nalm-6 to simulate them with continuous antigen pressure over a four-week period. This protocol can be value for assessing CAR T cell functionality and for the comparison of different CAR constructs.

Efficient chimeric antigen receptor targeting of a central epitope of CD22.

Chimeric antigen receptor (CAR) T-cell therapy has had considerable success in the treatment of B-cell malignancies. Targeting the B-lineage marker CD19 has brought great advances to the treatment of acute lymphoblastic leukemia and B-cell lymphomas. However, relapse remains an issue in many cases. Such relapse can result from downregulation or loss of CD19 from the malignant cell population or expression of alternate isoforms. Consequently, there remains a need to target alternative B-cell antigens and diversify the spectrum of epitopes targeted within the same antigen. CD22 has been identified as a substitute target in cases of CD19-negative relapse. One anti-CD22 antibody-clone m971-targets a membrane-proximal epitope of CD22 and has been widely validated and used in the clinic. Here, we have compared m971-CAR with a novel CAR derived from IS7, an antibody that targets a central epitope on CD22. The IS7-CAR has superior avidity and is active and specific against CD22-positive targets, including B-acute lymphoblastic leukemia patient-derived xenograft samples. Side-by-side comparisons indicated that while IS7-CAR killed less rapidly than m971-CAR in vitro, it remains efficient in controlling lymphoma xenograft models in vivo. Thus, IS7-CAR presents a potential alternative candidate for the treatment of refractory B-cell malignancies.

Development of a TGFß-IL-2/15 Switch Receptor for Use in Adoptive Cell Therapy.

Therapy employing T cells modified with chimeric antigen receptors (CARs) is effective in hematological malignancies but not yet in solid cancers. CAR T cell activity in solid tumors is limited by immunosuppressive factors, including transforming growth factor ß (TGFß). Here, we describe the development of a switch receptor (SwR), in which the extracellular domains of the TGFß receptor are fused to the intracellular domains from the IL-2/15 receptor. We evaluated the SwR in tandem with two variants of a CAR that we have developed against STEAP1, a protein highly expressed in prostate cancer. The SwR-CAR T cell activity was assessed against a panel of STEAP1+/- prostate cancer cell lines with or without over-expression of TGFß, or with added TGFß, by use of flow cytometry cytokine and killing assays, Luminex cytokine profiling, cell counts, and flow cytometry phenotyping. The results showed that the SwR-CAR constructs improved the functionality of CAR T cells in TGFß-rich environments, as measured by T cell proliferation and survival, cytokine response, and cytotoxicity. In assays with four repeated target-cell stimulations, the SwR-CAR T cells developed an activated effector memory phenotype with retained STEAP1-specific activity. In conclusion, the SwR confers CAR T cells with potent and durable in vitro functionality in TGFß-rich environments. The SwR may be used as an add-on construct for CAR T cells or other forms of adoptive cell therapy.

How CAR T Cells Breathe.

The manufacture of efficacious CAR T cells represents a major challenge in cellular therapy. An important aspect of their quality concerns energy production and consumption, known as metabolism. T cells tend to adopt diverse metabolic profiles depending on their differentiation state and their stimulation level. It is therefore expected that the introduction of a synthetic molecule such as CAR, activating endogenous signaling pathways, will affect metabolism. In addition, upon patient treatment, the tumor microenvironment might influence the CAR T cell metabolism by compromising the energy resources. The access to novel technology with higher throughput and reduced cost has led to an increased interest in studying metabolism. Indeed, methods to quantify glycolysis and mitochondrial respiration have been available for decades but were rarely applied in the context of CAR T cell therapy before the release of the Seahorse XF apparatus. The present review will focus on the use of this instrument in the context of studies describing the impact of CAR on T cell metabolism and the strategies to render of CAR T cells more metabolically fit.

Use of RNA Interference with TCR Transfer to Enhance Safety and Efficiency.

The gene transfer of T-cell receptors (TCRs) is an attractive strategy for adoptive cell therapy, allowing the transfer of reactivity against antigens that may not otherwise engender an immune response. The TCRs recognize intracellular or extracellular antigens presented in the context of MHC class I or II, respectively. This broadens the range of targets considerably, compared to antibodies and chimeric antigen receptors, that are generally confined to surface antigens. However, TCR transfer must overcome some technical hurdles, relating to interference with endogenous α- and ß-TCR chains and competition with other existing TCR infrastructure of T cells. In this review, we will outline the challenges facing TCR gene transfer and compare several approaches to address them. We will then focus upon one of the most promising amongst these-RNA interference-and detail the methods involved in designing and using this technology.

Novel immunotherapy for adult T-cell leukemia/lymphoma: Targeting aurora kinase A.

Adult T-cell leukemia/lymphoma is caused by infection with HTLV-1, following a long latent period. Immunotherapy targeting Aurora kinase A, a tumor-associated antigen over-expressed in adult T-cell leukemia/lymphoma, holds great therapeutic potential. We review the evidence in favor of a therapeutic strategy combining vaccination and TCR-gene transfer against this target.

A Functionally Superior Second-Generation Vector Expressing an Aurora Kinase-A-Specific T-Cell Receptor for Anti-Leukaemia Adoptive Immunotherapy.

Aurora Kinase A is a cancer-associated protein normally involved in the regulation of mitosis. Being over-expressed in a range of cancers, it is a suitable target for cell-based immunotherapy. Gene transfer of T-cell receptor sequences cognisant of HLA-A*0201-restricted Aurora Kinase A antigen has previously been shown to transfer specific immunoreactivity against the target peptide in a Human Lymphocyte Antigen-restricted manner. While T cell receptor gene-transfer has great potential in overcoming the difficulties of isolating and expanding tumour-reactive lymphocytes from a patient's own cells, one hurdle is potential mispairing and competition between exogenous and endogenous T cell receptor chains. We have used a retroviral vector design bearing a short-interfering RNA that downregulates endogenous T cell receptor chains, without affecting expression of the transgenic T cell receptor sequences. The T cell receptor expression cassette also includes a 2A self-cleaving peptide, resulting in equimolar expression of the T cell receptor alpha and beta chains, further enhancing formation of the desired T cell receptor. Via a simple, modular cloning method, we have cloned the alpha and beta chains of the anti-Aurora Kinase A-reactive T cell receptor into this 'siTCR' vector. We then compared the activity of this vector against the original, 'conventional' vector across a panel of assays. T cell receptors expressed from the siTCR-vector retained the cytotoxic functionality of the original vector, with evidence of reduced off-target reactivity. The rate of expression of correctly-formed T cell receptors was superior using the siTCR design, and this was achieved at lower vector copy numbers. Maintaining T cell receptor efficacy with a reduced vector copy number reduces the risk of genotoxicity. The siTCR design also reduces the risk of mispairing and cross-reactivity, while increasing the functional titre. Such improvements in the safety of T cell receptor gene-transfer will be crucial for clinical applications of this technology.