Anti-Human 5T4 Recombinant Antibody Fab Fragment, 5T4 (Naptumomab Estafenatox) (CAT#: TAB-118-F(E))

Figure 1 Superantigen antibody dependent cell-mediated cytotoxicity (SADCC) against Caki-2 cells.

(A) and superantigen dependent cell-mediated cytotoxicity (SDCC) against Raji cells (B) mediated by 4 hours incubation with 5T4Fab-SEA () anatumomab mafenatox (~) or naptumomab estafenatox (') at the indicated concentrations. In brief, the target cell lines Caki-2 or Raji were labeled with (Na)2 51CrO4 and incubated with superantigen-activated and cultured T cells for 4 hours at various concentrations of the fusion proteins, as described in materials and methods. One representative experiment out of 3.

Forsberg, G., Skartved, N. J., Wallén-Öhman, M., Nyhlén, H. C., Behm, K., Hedlund, G., & Nederman, T. (2010). Naptumomab estafenatox, an engineered antibody-superantigen fusion protein with low toxicity and reduced antigenicity. Journal of immunotherapy, 33(5), 492-499.

Figure 2 Calculated bioactivity at different molar ratios of human anti-SEA antibodies and anatumomab mafenatox (x) or naptumomab estafenatox (~).

The anti-SEA antibodies and respective fusion protein were incubated for 20 minutes at ambient temperature, and bioactivity was then measured using SADCC, as described in materials and methods. The bioactivity in the absence of neutralising antibodies was set to 100%.

Forsberg, G., Skartved, N. J., Wallén-Öhman, M., Nyhlén, H. C., Behm, K., Hedlund, G., & Nederman, T. (2010). Naptumomab estafenatox, an engineered antibody-superantigen fusion protein with low toxicity and reduced antigenicity. Journal of immunotherapy, 33(5), 492-499.

Figure 3 ABR-217620 pharmacology.

Geometric mean of (A) interleukin-2 (IL-2) and (B) interferon (IFN)- levels in plasma at before and 3 hours after first infusion of ABR-217620 in the MONO (ABR-217620 dose escalation monotherapy) study. Renal cell carcinoma (RCC; n=9, red lines), non–small-cell lung cancer (NSCLC), and pancreatic cancer (PC; n=8, black lines) patients receiving more than 15 g/kg ABR-217620; patients receiving 2.5 to 15 g/kg (n=9, dashed lines) and less than 2.5 g/kg ABR-217620 (n=12, dotted lines). Plasma samples were taken before and 3 hours after each injection of ABR-217620. (C) Each bar represents an individual subject at the distinct time point. Percentage of T lymphocytes expressing T-cell receptor (TCR)-V 6.4 (percentage TCR-V 6.4 cDNA of total TCR-V cDNA) in peripheral blood from patients before and after treatment with ABR-217620 in the MONO (black) and COMBO (ABR-217620 dose escalation combination with docetaxel; red) studies. (D) Median with first and third quartiles of anti-SAg antibodies before and 28 days after start of treatment with ABR-214936 13 (anti-staphylococcal enterotoxin E [SEA], black line), ABR-217620 MONO (anti-SEA/E-120, red line), and ABR-217620 COMBO (anti-SEA/E-120, blue line).

Borghaei, H., Alpaugh, K., Hedlund, G., Forsberg, G., Langer, C., Rogatko, A., ... & Cohen, R. B. (2009). Phase I dose escalation, pharmacokinetic and pharmacodynamic study of naptumomab estafenatox alone in patients with advanced cancer and with docetaxel in patients with advanced non–small-cell lung cancer. Journal of Clinical Oncology, 27(25), 4116.

Figure 4 Immunohistochemistry for Tlymphocyte (anti-CD3) infiltration in biopsies taken before treatment (archival tissue) and at the third day of the second cycle treatment with ABR-217620 for patient number 2.

The T lymphocytes stain brown and thearrows indicate unstained tumor cells. This patient had a partial response that continues at 30 months.

Borghaei, H., Alpaugh, K., Hedlund, G., Forsberg, G., Langer, C., Rogatko, A., ... & Cohen, R. B. (2009). Phase I dose escalation, pharmacokinetic and pharmacodynamic study of naptumomab estafenatox alone in patients with advanced cancer and with docetaxel in patients with advanced non–small-cell lung cancer. Journal of Clinical Oncology, 27(25), 4116.

Figure 5 Flow cytometry and TCR-mRNA (IMGT TRB variants) analysis.

A) Flow cytometry analysis of [ABR-217620-Biotin/SA-PE]-complex binding to human PBMC from a healthy donor also stained for CD3, CD4, CD8 and HLA-DR. B) Re-analysis of flow cytometry sorted human PBMC from a healthy donor and C) TCR-mRNA analysis (IMGT TRB-variants) of unsorted and sorted cells binding or not binding the [ABR-217620-biotin/SA-PE]-complex. D) TCR-mRNA analysis (IMGT TRB-variants) of T cells from in vitro cultures activated with SEA, SEA/E-120 or SEE. E) TCR-mRNA analysis (IMGT TRB-variants) of T cells from in vitro cultures activated with different concentrations of SEA/E-120.

Hedlund, G., Eriksson, H., Sundstedt, A., Forsberg, G., Jakobsen, B. K., Pumphrey, N., ... & Björk, P. (2013). The tumor targeted superantigen ABR-217620 selectively engages TRBV7-9 and exploits TCR-pMHC affinity mimicry in mediating T cell cytotoxicity. PloS one, 8(10), e79082.

Figure 6 Surface plasmon resonance analysis of ABR-217620 binding to 5T4.

ABR-217620 was injected for 3min at a flow rate of 20uL/min over 5T4FC (density 770RU). Regeneration of the surface was made by injecting 10uL 10mM glycine-HCL, pH 1.5, during 30s. Sensorgrams from bottom to top represent sample buffer and ABR-217620 in the concentration range 1.56-50 nM. An affinity of 2.1*10-10 was calculated after kinetic analysis of binding data by fit of sensorgrams to a 1:1 model with on- and off-rates of 3.6*105(1/Ms) and 7.5*10-5(1/s).

Hedlund, G., Eriksson, H., Sundstedt, A., Forsberg, G., Jakobsen, B. K., Pumphrey, N., ... & Björk, P. (2013). The tumor targeted superantigen ABR-217620 selectively engages TRBV7-9 and exploits TCR-pMHC affinity mimicry in mediating T cell cytotoxicity. PloS one, 8(10), e79082.

Figure 7 Surface plasmon resonance analysis of TRBV7-9 interaction with ABR-217620 or 5T4Fab-SEA.

25 nM ABR-217620 or 5T4Fab-SEA was captured (5 min at 20 µL/min) on immobilized 5T4Fc (density ~ 990 RU) prior to injection (5 min at 20 µL/min) of 0.156-5 µM TRBV7-9. The surface was regenerated with a short 10 µL pulse of 10 mM glycine-HCl, pH 1.5. A) Sensorgrams obtained after injection of 25 nM ABR-217620 (1) followed by 0-5 µM TRBV7-9 (3) with buffer pumped over the surface at (2) and (4). B) Injection of 0.156-5 µM TRBV7-9 over ABR-217620 and 5T4Fab-SEA after subtraction of sensorgram with
sample buffer without TRBV7-9. C) Responses at early association phase (t ~ 530 s) plotted versus TRBV7-9 concentration for binding to captured 5T4Fab-SEA (squares) and ABR-217620 (triangles). Curves were fit to a one-site hyperbola model in GraphPad Prism for calculation of maximal response and apparent affinity.

Hedlund, G., Eriksson, H., Sundstedt, A., Forsberg, G., Jakobsen, B. K., Pumphrey, N., ... & Björk, P. (2013). The tumor targeted superantigen ABR-217620 selectively engages TRBV7-9 and exploits TCR-pMHC affinity mimicry in mediating T cell cytotoxicity. PloS one, 8(10), e79082.