Description: Development of a novel anti-SSTR bispecific T-cell engager (BiTE)-like molecule for the treatment of neuroendocrine tumors (NETs), focusing  on developing a new type of t-cell engager molecule that may be used in the development of immunotherapies to improve the treatment of SSTR-expressing NETs.

What question will the researchers try to answer?

This research project will test whether the somatostatin receptor (SSTR)- CD3 bispecific T- cell engager (BiTE)-like molecules that we developed, can efficiently activate the tumor infiltrating lymphocytes (TILs) against the SSTR-expressing NET cells.

Why is this important?

Well-differentiated neuroendocrine tumors are resistant to conventional immunotherapy with checkpoint inhibitors.  Other agents are necessary to activate tumor infiltrating lymphocytes (TILs). Our goal is to develop a BiTE-like molecule that targets SSTR-expressing NET cells and is potentially more concordant with the characteristics of the NET immune microenvironment.

What will researchers do?

The project consists of three consecutive parts:

1. Design of the BiTE-like molecule, expression, and purification. In this first part the sequence will be optimized, and the protein produced after transfecting insect cells with Baculovirus containing the DNA sequence coding for the BiTE-like molecule. The appropriate protein folding as well as the absence of aggregation will be investigated using analytical size exclusion chromatography. The protein will be characterized for its purity by SDS-PAGE and mass spectrometry. Flow cytometry will be used to detect the ability of the recombinant protein in binding the respective targets.
2. Assessment of the anti-SSTR BiTE-like molecule antitumor activity in vitro. SSTR-specific tumor toxicity will be assessed by coculturing T cells isolated from peripheral blood of healthy donors with SSTR-expressing NET cells by RTCA.
3. Assessment of the antitumor activity of anti-SSTR BiTE-like molecule against NET murine xenografts. The ability of the BiTE-like molecule to efficiently activate T cells against NETs in vivo will be tested on 6–8-week-old NOD/Shi-scid/IL-2Rγnull mice with SSTR+ NET subcutaneous xenografts. The mice will be treated with T cells alone or in combination with the BiTE-like molecule. A condition with mice treated with phosphate-buffered saline (PBS) solution only will be included as a control.

How might this improve the treatment of NETs?

We will develop a new type of immunotherapy for patients with NETs. This is designed to be in line with the characteristics of the NET immune microenvironment, offering a new treatment option to this group of patients.

What is the next step?

We envision two potential pathways for further drug development. The novel BiTE-like molecule could be further investigated as a therapy for SSTR positive tumors. Alternatively, the BiTE-like molecule could be incorporated into anti-SSTR CAR-T cells already developed by our group and secreted by the same cells, after activation. CAR-T are autologous T cells genetically modified to express a chimeric antigen receptor (CAR) that, elicit a non-MHC restricted immune response against tumor associated antigens (TAA). The CAR developed by our group comprised an octreotide-like extracellular domain with high affinity for SSTR2-5. Its efficacy has been demonstrated in vitro and in vivo against GEP-NET cell lines.

The simultaneous engagement of CAR-T cells and the naïve lymphocytes that spontaneously enrich the neuroendocrine tumor microenvironment (TME) could act in a synergistic way and improve tumor regression in several ways: 1) activate the TILs against NET cells after local release of the BiTE-like molecule 2) boost the activity of anti- SSTR CAR-T cells through the CD3 mediated TCR activation 3) the tumor-neoantigens released after CAR-T cytotoxic activity might stimulate the influx of lymphocytes in the tumor that could be engaged by the BiTE-like molecule resulting in an exponential increase of our CAR-T cells’ cytotoxicity.