NANETS2022 15th Annual Multidisciplinary NET Medical Symposium NANETS 2022 Basic Science (16 abstracts)
Development of a Novel Anti-SSTR Bispecific T-Cell Engager (BiTE)-like Molecule for the Treatment of Neuroendocrine Tumors
Eleonora Pelle 1 , Mauro Cives 2 , Elliot Medina 3 , Charlotte C. Mason 3 , Sebastian A. Snedal 4 , Xiomar E. Bustos-Perez 4 , Leticia Tordesillas 4 , Miguel Gomez Fontela 4 , Renata A. Marques Rossetti 4 , Gabriele Maiorano 5 , Vince Luca 3 , Patrick Hwu 6 , Daniel Abate-Daga 7 & Jonathan Strosberg 1
1Department of GI Oncology, Moffitt Cancer Center, Tampa, Florida, USA; 2Department of Interdisciplinary Medicine, University of Bari Aldo Moro, Bari, Italy; 3Department of Drug Discovery, Moffitt Cancer Center, Tampa, Florida, USA; 4Departments of Immunology, Moffitt Cancer Center, Tampa, Florida, USA; 5Institute of Nanotechnology NANOTEC, National Research Council, Lecce, Italy; 6Moffitt Cancer Center, Tampa, Florida, USA; 7Departments of Immunology and Cutaneous Oncology, Moffitt Cancer Center, Tampa, Florida, USA.
Background: Well-differentiated neuroendocrine tumors (NETs) are characterized by the overexpression of somatostatin receptors (SSTRs). To efficiently engage and activate tumor infiltrating lymphocytes against NET cells, we designed a novel bispecific T-cells engager (BiTE) composed of 2 molecules of somatostatin-14 (SST14), the hormone that physiologically binds SSTRs, linked with a single chain variable fragment (scFV)-based anti-CD3.
Methods: The optimized sequence of the BiTE was subcloned into a vector (pAcGP67a) designed for protein expression in insect cells using Baculovirus. Trichoplusia-ni (High Five) cells were used to express the recombinant protein, which was isolated from the supernatant using nickel affinity chromatography. Flow cytometry and confocal microscopy were used to determine the binding potential of the BiTE towards CD3 and SSTR2. CD3+ T cells isolated from the peripheral blood of healthy donors were co-incubated with 293T cells stably transduced to concurrently express SSTR2 and green fluorescent protein (GFP) in the absence or presence of the BiTE. The SSTR2- parental 293T cell line was used as negative control, while anti-CD3/CD28 beads were added as a positive control. The BiTE-induced T cell activation was evaluated measuring the secretion of IFNγ and granzyme B by ELISA and OX40, 41BB and CD69 by flow cytometry.
Results: At a concentration of 100 nM, the BiTE bound the CD3 receptor of approximately 85% of T cells. By confocal microscopy, the BiTE was found to coat SSTR2+ 293T cells. IFN-γ secretion was significantly higher when the T cells were co-cultured with SSTR+ 293T cells in the presence of the BiTE as compared with parallel preparations with SSTR- 293T cells or without the BiTE, suggesting that the BiTE-induced T cell activation is specific. At high concentration of BiTE, OX40, CD69 and 41BB on T cells were upregulated regardless of the presence of target cells. However, at the same concentration, the granzyme B concentration increased only in presence of SSTR+ target cells.
Conclusions: To our knowledge, this is the first BiTE to incorporate a hormone in one binding site. Its non-antibody-like structure efficiently engaged SSTR2 and T cells enabling the formation of immune synapsis.
Abstract ID 21446
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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