Integrin-αvß6 targeted peptide-toxin therapy in a novel αvß6-expressing immunocompetent model of pancreatic cancer.

Previously we reported that a novel αvß6-specific peptide-drug conjugate (SG3299) could eliminate established human pancreatic ductal adenocarcinoma (PDAC) xenografts. However the development of effective therapies for PDAC, which is an essential need, must show efficacy in relevant immunocompetent animals. Previously we reported that the KPC mouse transgenic PDAC model that closely recapitulates most stages of development of human PDAC, unlike in humans, failed to express αvß6 on their tumours or metastases. In this study we have taken the KPC-derived PDAC line TB32043 and engineered a variant line (TB32043mb6S2) that expresses mouse integrin αvß6. We report that orthotopic implantation of the αvß6 over-expressing TB32043mb6S2 cells promotes shorter overall survival and increase in metastases. Moreover, systemic treatment of mice with established TB32043mb6S2 tumours in the pancreas with SG2399 lived significantly longer (p < 0.001; mean OS 48d) compared with PBS or control SG3511 (mean OS 25.5d and 26d, respectively). Thus SG3299 is confirmed as a promising candidate therapeutic for the therapy of PDAC.

Optimization of anti-CD19 CAR T cell production for treatment of patients with chronic lymphocytic leukemia.

T cells expressing anti-CD19 chimeric antigen receptors (CARs) have activity against chronic lymphocytic leukemia (CLL), but complete response rates range from 18% to 29%, so improvement is needed. Peripheral blood mononuclear cells (PBMCs) of CLL patients often contain high levels of CLL cells that can interfere with CAR T cell production, and T cells from CLL patients are prone to exhaustion and other functional defects. We previously developed an anti-CD19 CAR designated Hu19-CD828Z. Hu19-CD828Z has a binding domain derived from a fully human antibody and a CD28 costimulatory domain. We aimed to develop an optimized process for producing Hu19-CD828Z-expressing T cells (Hu19-CAR T) from PBMC of CLL patients. We determined that supplementing Hu19-CAR-T cultures with interleukin (IL)-7 + IL-15 had advantages over using IL-2, including greater accumulation of Hu19-CAR T cells during in vitro proliferation assays. We determined that positive selection with anti-CD4 and anti-CD8 magnetic beads was the optimal method of T cell purification because this method resulted in high T cell purity. We determined that anti-CD3/CD28 paramagnetic beads were the optimal T cell activation reagent. Finally, we developed a current good manufacturing practices-compliant clinical-scale protocol for producing Hu19-CAR T from PBMC of CLL patients. These Hu19-CAR T exhibited a full range of in vitro functions and eliminated leukemia from mice.

Ligand-bound integrin αvß6 internalisation and trafficking.

The integrin αvß6 is expressed at low levels in most normal healthy tissue but is very often upregulated in a disease context including cancer and fibrosis. Integrins use endocytosis and trafficking as a means of regulating their surface expression and thus their functions, however little is known of how this process is regulated in the context of αvß6. As αvß6 is a major target for the development of therapeutics in cancer and fibrosis, understanding these dynamics is critical in the development of αvß6-targeted therapies. Following development of a flow cytometry-based assay to measure ligand (A20FMDV2 or LAP)-bound αvß6 endocytosis, an siRNA screen was performed to identify which genes were responsible for internalising αvß6. These data identified 15 genes (DNM2, CBLB, DNM3, CBL, EEA1, CLTC, ARFGAP3, CAV1, CYTH2, CAV3, CAV2, IQSEC1, AP2M1, TSG101) which significantly decreased endocytosis, predominantly within dynamin-dependent pathways. Inhibition of these dynamin-dependent pathways significantly reduced αvß6-dependent migration (αvß6-specific migration was 547 ± 128 under control conditions, reduced to 225 ± 73 with clathrin inhibition, and 280 ± 51 with caveolin inhibition). Colocalization studies of αvß6 with endosome markers revealed that up to 6 h post-internalisation of ligand, αvß6 remains in Rab11-positive endosomes in a perinuclear location, with no evidence of αvß6 degradation up to 48 h post exposure to A20FMDV2. Additionally, 60% of ligand-bound αvß6 was recycled back to the surface by 6 h. With studies ongoing using conjugated A20FMDV2 to therapeutically target αvß6 in cancer and fibrosis, these data have important implications. Binding of A20FMDV2 seemingly removes much of the αvß6 from the cell membrane, and upon its recycling, a large fraction appears to still be in the ligand-bound state. While these results are observed with A20FMDV2, these data will be of value in the design of αvß6-specific therapeutics and potentially the types of therapeutic load.

Current Perspectives on the Use of off the Shelf CAR-T/NK Cells for the Treatment of Cancer.

CAR T cells have revolutionised the treatment of haematological malignancies. Despite this, several obstacles still prohibit their widespread use and efficacy. One of these barriers is the use of autologous T cells as the carrier of the CAR. The individual production of CAR T cells results in large variation in the product, greater wait times for treatment and higher costs. To overcome this several novel approaches have emerged that utilise allogeneic cells, so called "off the shelf" CAR T cells. In this Review, we describe the different approaches that have been used to produce allogeneic CAR T to date, as well as their current pre-clinical and clinical progress.