Fig 2: Anti-SSTR CAR T cells infiltrate human xenografts and murine SSTR-expressing organs. (A) Visualization of SSTR2 (UMB1 mAb) and SSTR5 (UMB5 mAb) within tumor xenografts by IHC. Magnification: ×20. Scale bar: 100 µm. (B) Explanted CM tumor xenografts as well as SSTR-expressing organs such as murine spleen, pancreas and brain were lysed and subjected to DNA extraction. The infiltration of CAR T cells was demonstrated by PCR using primers specific for the anti-SSTR CAR sequence. The purified CAR construct DNA was used for positive control experiments. Distilled water was used in negative control experiments. (C) FAM-labeled and HEX-labeled probes against the CAR sequence and the MKL2 reference gene, respectively, were employed in ddPCR experiments to quantify the infiltration of CM (blue dots) and BON1 xenografts (red dots) by anti-SSTR CAR T cells. An inverse correlation can be observed between CAR T cell infiltration and tumor bioluminescence increase relative to baseline. (D) Anti-SSTR CAR T cells recognize and kill the MIN6 cells, a murine NET cell line expressing SSTR2/5. By in vitro BLI, CAR T cells induced cell death in the 48% of Luc+ MIN6 cells as compared with UT T cells at an E:T ratio of 1:1 after 72 hours of coculture. (E) IFN-? release on 24 hours coculture of NET cells with CAR T cells or UT T cells at an E:T ratio of 1:1. (F) Histopathological analysis of human NET xenografts and murine pancreas and brain by H&E staining. Extensive necrosis foci could be identified within tumors (arrowhead). Representative microphotographs show the absence of necrosis or other tissue damages in the context of the murine pancreas and brain. Magnification: x20. Scale bar: 100 µm. **P<0.01. CAR, chimeric antigen receptor; ddPCR, droplet digital PCR; E:T, effector:target; IHC, immunohistochemistry; NET, neuroendocrine tumor; SSTR, somatostatin receptor; UT, untransduced.

Fig 3: Effect of SSTR2 expression and activation on CAR T cell activity in vitro and in vivo. (A) The level of antibody binding to SSTR2 was measured to assess SSTR2 internalization induced by SSTR2 agonist (lanreotide). (B) E to T assay of AIC100 cells with or without 1 µM of lanreotide against 293T and 8505C cells. The ratio of T cells to target cells was 2.5:1 (n = 3 each for P2 and P3). (C) A weekly measurement of whole body luminescence to detect tumor growth or killing by AIC100 (P2 and P2b) at MTD doses with or without lanreotide. Lanreotide was administered to mice by intraperitoneal injection at days of 9, 19, 23, 34, and 40 post tumor xenograft. n = 5 for AIC100/Lanreotide; n = 3 for AIC100; n = 4 for No T. Significant difference (p = 0.01) for No T vs. AIC100 or AIC100/Lanreotide; no significance for AIC100 vs. AIC100/Lanreotide by one-way ANOVA with Tukey’s multiple comparisons test.

Fig 4: Comparison of Prodigy manufactured CAR T cells for cell expansion, viability, transduction, VCN, and subsets. (A) A schematic of the insert sequence of AIC100 lentiviral vector. SS = signal sequence; TM = transmembrane; Cyt = cytosolic domain; hSSTR2 = human SSTR2. DNA ruler is shown above features. (B,C) Cell expansion and viability were quantified manually by hemocytometer. CD3 and CD4/CD8 subsets, and CAR positive cells were analyzed by flow cytometer. VCN was determined by ddPCR. (D) CAR expression was determined by dual labeling of cells by anti-Myc and anti-SSTR2 antibodies for CD4/CD8 sorted cells (day 0) and T cells harvested from Prodigy (day 9, 10, or 11).

Fig 5: Anti-SSTR CAR T cells exhibit antigen-specific tumoricidal activity. (A) Anti-SSTR CARs endow human T lymphocytes with reactivity against SSTR-expressing targets. By in vitro BLI assay, CAR T cells induced cell death in up to 58% of Luc+ NET cell lines as compared with UT T cells at an E:T ratio of 1:1. The percentage of specific tumor cell lysis was calculated as the ratio between CAR T cell and UT T cell antitumor activity using the formula: % lysis=1-(mean BLI signal in the presence of anti-SSTR CAR T cells/mean BLI signal in the presence of UT cells) x 100%. (B) In vitro BLI assay to evaluate the cytolytic activity of CAR T cells as compared with UT T cells according to increasing E:T ratios after 24 hours of coculture with NET cells. The degree of cytotoxicity induced by CAR T cells increased when the number of effector cells increased. (C) IFN-? and (D) TNF-a release on 24 hours coculture of NET cells with CAR T cells or UT T cells at an E:T ratio of 1:1. Cytokine production was measured in culture supernatants by ELISA. CD3/CD28 T cell stimulation through TransAct was used for positive control experiments. (E) In vitro BLI assay to investigate the cytolytic activity of CAR T cells as compared with UT T cells against mutant CM cells and parental cells at an E:T ratio of 1:1. (F) Evaluation of the cytotoxic potential of CAR T cells against CM cells harboring wild-type or mutated SSTR2 and/or SSTR5 according to increasing E:T ratios after 24 hours of coculture. (G) IFN-? and (H) TNF-a release on 24 hours coculture of CAR T cells or UT T cells with mutated or parental CM cells at an E:T ratio of 1:1. All experiments were carried out in technical triplicate using lymphocytes from three healthy donors. Mean values and standard errors are represented in figure. *P<0.05, **p<0.01. CAR, chimeric antigen receptor; E:T, effector:target; NET, neuroendocrine tumor; SSTR, somatostatin receptor; UT, untransduced.