Yu‐Chuan Lin 1,2, Mei‐Chih Chen 1, Shi‐Wei Huang 1,3,4, Yeh Chen 5, Jennifer Hui‐Chun Ho 2,6,7,8, Fang‐Yu Lin 1, Xiao‐Tong Tan 1, Hung‐Che Chiang 2,9, Chiu‐Ching Huang 2,10, Chih‑Yen Tu 11,12, Der‐Yang Cho 1,3,13,14,✉, Shao‐Chih Chiu 1,2,3,13,
Posted: Sep 5, 2024
Abstract
Immunotherapy targeting immune checkpoints (ICPs), such as programmed death‐ligand‐1 (PD‐L1), is used as a treatment option for advanced or metastatic non‐small cell lung cancer (NSCLC). However, overall response rate to anti‐PD‐L1 treatment is limited due to antigen heterogeneity and the immune‐suppressive tumor microenvironment. Human leukocyte antigen‐G (HLA‐G), an ICP as well as a neoexpressed tumor‐associated antigen, is previously demonstrated to be a beneficial target in combination with anti‐PD‐L1. In this study, a nanobody‐based trispecific T cell engager (Nb‐TriTE) is developed, capable of simultaneously binding to T cells, macrophages, and cancer cells while redirecting T cells toward tumor cells expressing PD‐L1‐ and/or HLA‐G. Nb‐TriTE shows broad spectrum anti‐tumor effects in vitro by augmenting cytotoxicity mediated by human peripheral blood mononuclear cells (PBMCs). In a humanized immunodeficient murine NSCLC model, Nb‐TriTE exhibits superior anti‐cancer potency compared to monoclonal antibodies and bispecific T cell engagers. Nb‐TriTE, at the dose with pharmacoactivity, does not induce additional enhancement of circulating cytokines secretion from PMBCs. Nb‐TriTE effectively prolongs the survival of mice without obvious adverse events. In conclusion, this study introduces an innovative therapeutic approach to address the challenges of immunotherapy and the tumor microenvironment in NSCLC through utilizing the dual ICP‐targeting Nb‐TriTE.
NeoBiotechnologies’ products were used in this study:
ImmunoCult human CD3/CD28 T cell activator (STEMCELL) was used to expand T cells from PBMCs. Macrophages were enriched from T cells-depleted PBMCs using CD3 depletion kit (STEMCELL). The saturated immune cell number toward to 6 × 103 of A549 cells was determined by coculturing respectively with premixed 6 × 105 or 6 × 106 cells of T cells + Nb-TriTE, macrophages + Nb-TriTE, or T cells + macrophages + Nb-TriTE, followed by hybridization with primary antibodies, anti-CD3 (Novus Biologicals), anti-CD68 (NeoBiotechnologies), and HRP-conjugated secondary antibodies. After the optical density of each well was measured by a microplate reader set to 450 nm, the saturated immune cell number against A549 cells (1000:1) was determined. ELISA-based functional assay was then utilized to verify the simultaneously binding ability of Nb-TriTE to tumor cells and immune cells. Macrophages, T cells, and tumor cells were coated on the wells of ELISA plates respectively. A series of concentrations of Nb-TriTE were pre-incubated with the cell mixture then loaded into ELISA plates as follows: A549 cells and T cells to macrophage-coated plate, A549 cells and macrophages to T cell-coated plate, and T cells and macrophages to A549 cell-coated plate. Primary antibodies against CD3, CD68, and E-cadherin (GeneTex), which sequentially recognize T cell, macrophage, and A549 cells were added into the plates followed by application of the appropriate HRP-conjugated secondary antibodies. The absorbance value was then measured at OD 450 nm.
Keywords: human leukocyte antigen‐G (HLA‐G), immune checkpoint (ICP), nanobody‐based trispecific T cell engager (Nb‐TriTE), non‐small cell lung cancer (NSCLC), programmed death‐ligand 1 (PD‐L1)
A nanobody‐based trispecific T cell engager (Nb‐TriTE) is developed to redirect T cells to non‐small cell lung cancer (NSCLC) through dual ICP targeting. In vitro and in vivo evidence suggests that Nb‐TriTE can overcome the immune checkpoints (ICP) heterogeneity of NSCLC without obvious toxicity, implying an innovative approach for the treatment of NSCLC.
Publication History:
Adv Sci (Weinh). 2024 Sep 5;11(41):2309697. doi: 10.1002/advs.202309697
