1101MO - Development of CAR T-cells for future treatment of NETs

Background

Neuroendocrine tumors (NETs) overexpress somatostatin receptors (SSTRs). We investigated the antitumor activity of chimeric antigen receptor (CAR) T cells directed against SSTRs.

Methods

A second-generation CAR-like construct containing two molecules of octreotide in the extracellular moiety and CD28 as costimulatory module was cloned in a pMSGV1-28Z retroviral vector and then transduced in human T cells. Luciferase⁺ (Luc⁺) BON1, CM, QGP1, CNDT2.5 and H727 NET cell lines were screened for membrane SSTR_2/5 expression by Western blot (WB) and flow cytometry. Co-culture experiments were performed at effector:target (E:T) ratios ranging from 50:1 to 1:50 for up to 72 hrs. Tumor cell cytotoxicity was assessed by bioluminescence imaging. The release of IFN-γ and IL-2 by activated CAR T cells was investigated by ELISA. NSG female mice (n=11/group) were subcutaneously injected with 2x10⁶ Luc⁺ BON1 or CM cells, and were then intravenously treated either with 7x10⁶ anti-SSTR CAR T cells, or untransduced (UT) T cells. Excised tumors were subjected to PCR to assess the infiltration of CAR T cells. Potential on-target off-tumor toxicities of anti-SSTR CAR T cells were investigated by pathological analysis of mouse brain and pancreas.

Results

All NET cell lines expressed SSTR_2/5, although at variable levels. Following WB confirmation of the CAR expression by transduced lymphocytes, anti-SSTR CAR T cells were co-incubated with target cells. Tumor cell death was induced in approximately 40% (±8%) of CM and BON1 cells at E:T ratio of 1:1. The tumoricidal effect of CAR T cells was time-dependent and peaked at 72 hrs. Compared with UT T cells, CAR T cells secreted significantly higher levels (p<0.01) of IFN-γ and IL-2 after co-incubation with NET cells. Anti-SSTR CAR T cells effectively infiltrated tumors and significantly reduced the growth of subcutaneous CM (p=0.01) and BON1 xenografts (p=0.02) in mice by in vivo bioluminescence imaging. No pathological alterations were seen in the brain and pancreas of mice treated with CAR T cells.

Conclusions

Anti-SSTR CAR T cells exert antitumor activity against SSTR⁺ NET cell lines, both in vitro and in vivo.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

ENETS, AIRC.

Disclosure

D. Abate-Daga: Non-Financial Interests, Personal, Advisory Board: Anixa Biosciences; Financial Interests, Personal and Institutional, Funding: Intellia Therapeutics; Financial Interests, Personal and Institutional, Funding: Bluebird bio. All other authors have declared no conflicts of interest.