A bispecific, crosslinking lectibody activates cytotoxic T cells and induces cancer cell death

Abstract

Background Aberrant glycosylation patterns play a crucial role in the development of cancer cells as they promote tumor growth and aggressiveness. Lectins recognize carbohydrate antigens attached to proteins and lipids on cell surfaces and represent potential tools for application in cancer diagnostics and therapy. Among the emerging cancer therapies, immunotherapy has become a promising treatment modality for various hematological and solid malignancies. Here we present an approach to redirect the immune system into fighting cancer by targeting altered glycans at the surface of malignant cells. We developed a so-called “lectibody”, a bispecific construct composed of a lectin linked to an antibody fragment. This lectibody is inspired by bispecific T cell engager (BiTEs) antibodies that recruit cytotoxic T lymphocytes (CTLs) while simultaneously binding to tumor-associated antigens (TAAs) on cancer cells. The tumor-related glycosphingolipid globotriaosylceramide (Gb3) represents the target of this proof-of-concept study. It is recognized with high selectivity by the B-subunit of the pathogen-derived Shiga toxin, presenting opportunities for clinical development. Methods The lectibody was realized by conjugating an anti-CD3 single-chain antibody fragment to the B-subunit of Shiga toxin to target Gb3+ cancer cells. The reactive non-canonical amino acid azidolysine (AzK) was inserted at predefined single positions in both proteins. The azido groups were functionalized by bioorthogonal conjugation with individual linkers that facilitated selective coupling via an alternative bioorthogonal click chemistry reaction. In vitro cell-based assays were conducted to evaluate the antitumoral activity of the lectibody. CTLs, Burkitt´s lymphoma-derived cells and colorectal adenocarcinoma cell lines were screened in flow cytometry and cytotoxicity assays for activation and lysis, respectively. Results This proof-of-concept study demonstrates that the lectibody activates T cells for their cytotoxic signaling, redirecting CTLs´ cytotoxicity in a highly selective manner and resulting in nearly complete tumor cell lysis—up to 93%—of Gb3+ tumor cells in vitro. Conclusions This research highlights the potential of lectins in targeting certain tumors, with an opportunity for new cancer treatments. When considering a combinatorial strategy, lectin-based platforms of this type offer the possibility to target glycan epitopes on tumor cells and boost the efficacy of current therapies, providing an additional strategy for tumor eradication and improving patient outcomes.

Clickable Shiga Toxin B Subunit for Drug Delivery in Cancer Therapy

“…These observations are in line with the studies conducted by Rosato et al The two STxB variantswild-type and AzKwere compared for their binding specificity and affinity on a panel of Burkitt’s lymphoma-derived cells and colon adenocarcinoma cell lines, confirming the specificity of the STxB mutant upon AzK incorporation. The STxB AzK was found to be highly selective for Gb3 + tumor cell lines and successfully targeted them for elimination, while sparing Gb3 – or Gb3-depleted cells.…”

“…BLI-cytotoxicity assays offer a robust and fast evaluation of the cell viability of luciferase-transduced cell lines, in the presence of specific cytotoxic treatments. For this purpose, HT-29 and LS-174 cells were stably transduced to express luciferase . Since BLI is ATP-dependent, dying cells stop emitting BLI once its remaining intracellular ATP has been used up.…”

“…The amino acid sequence of STxB wt protein with a fused C-terminal 6x His-tag was subcloned into pET30a-Cer and 21 and pSCS-T7 × 31 20 , and STxB AzK including a C-terminal 6x His-tag with a mutation on position Lys9 in pSCS_T7 × 31 ( Supporting Information Figure 1 ) was used for expression. 44 The three plasmids were named pET30a<STxB wt >Cer, pSCS-T7 × 31<STxB wt >, and pSCS-T7 × 31<STxB AzK >. All materials used for cloning were purchased from New England Biolabs, Frankfurt, Germany (cloning kits), and IDT, Leuven, Belgium (primers and gBLOCKs).…”

“…For STxB AzK , the full induction condition was supplemented with 5 mM AzK. 44 For all benchtop cultivations, cells were harvested at the end of fermentation and resuspended in 50 mM Na 2 HPO 4 /NaH 2 PO 4 , 500 mM NaCl, and 20 mM imidazole (Sigma-Aldrich, Saint Louis, Missouri, USA) at pH 7.4 to yield a 30 g CD mg/L suspension. The suspension was homogenized at 700/70 (first stage/second stage) bar for 2 passages on a GEA Niro Soavi PANDAPlus 2000 (GEA, Parma, Italy).…”

Dimeric Lectin Chimeras as Novel Candidates for Gb3-Mediated Transcytotic Drug Delivery through Cellular Barriers

“…Remarkably, Gb3-binding lectin-CAR T cells have demonstrated target-specific cytotoxicity against several cancer cell lines in vitro [ 50 ]. In addition, a bispecific construct composed of a Gb3-binding lectin linked to an antibody fragment (anti-CD3), termed ‘lectibody’, has been reported to kill Gb3-positive carcinoma cells in a highly specific and efficient manner by redirecting cytotoxic T lymphocytes in vitro [ 51 ]. Another Gb3-binding lectin recently suggested for therapeutical approaches, including transcytotic drug delivery, is the lectin LecA from Pseudomonas aeruginosa [ 18 ].…”