by Rapamycin Press

Schematic of experimental procedures for generation of novel anti-Sonic Hedgehog (Shh) candidate therapeutic antibodies directed at the carboxy (C)-terminal. (A) Two synthetic peptide mimics of Shh (AA 247-264 and AA 448-462) were conjugated to carrier protein KLH and used as the immunogen. (B) Dual administration of both peptides was used to elicit an immune response in Sp2/0-Ag1 mice. (C) B cells from immunized murine lymph nodes were isolated and fused to myeloma cells for hybridoma generation. (D) Protein-based screening analyses guided the selection of the best hybridoma clones followed by one round of sub-cloning to isolate monoclonal antibody-producing hybridoma cells (E) Large-scale preparations of purified antibodies from hybdridomas were used for in vitro, in vivo, and ex vivo investigations. Credit: Bhairavi Tolani

Even though the Shh pathway is mainly quiescent in adults, the safety of Shh-targeting with therapeutic antibodies was questioned initially because not only does the N-terminus of the Shh protein play an important role in embryonic development, but Shh also plays a poorly understood role in tissue homeostasis and repair in adults.

Furthermore, the research team previously showed that Shh+ cells expressing the full-length protein appeared to provide a signal for proliferation, migration and chemotherapy-resistance properties, making functional targeting of full-length Shh by the use of C-terminus anti-Shh antibodies an attractive therapeutic strategy.

Ex vivo analyses of A549 xenograft tumors from mice treated with the C-terminus Shh antibody Ab 1C11-2G4 provided further support that Shh signal transduction is modestly down-regulated after treatment, as evidenced by suppressed transcript and protein levels of Shh downstream target GLI. Activation of the pathway occurs when Shh binds to its receptor Patched but signaling is inhibited in a small population of cells if full-length Shh binds instead to the blocking antibody 2G4, a potential therapeutic, so that the protein cannot transmit downstream signals.

Another reason for the modest efficacy the research team observed could be that the antibody also binds cleaved C-terminus Shh produced by a majority of the cells, which has no known Shh signaling function, and which may deplete the amount of antibody available for therapeutic binding to cell-surface expressed full-length Shh.

The researchers said "Administration of Ab 1C11-2G4 not only reduced cell viability in 7 cancer cell lines but also significantly inhibited tumor growth in a xenograft model of A549 lung cancer cells."

When ectopically expressed, Shh triggers tumor formation in preclinical models, lending credence to the idea that ligand-dependent Shh pathway activation promotes tumor initiation and progression.

Targeting ligand-dependent Shh signaling with Smoothened antagonists or Shh neutralizing antibodies inhibits the growth of several tumor cell lines in vitro and in vivo.

While blocking the Shh pathway is an attractive anti-cancer strategy, no therapeutic antibody raised against the carboxy -terminal of the Shh protein has been described.

The researchers recently reported the presence of uncleaved, membrane-bound, full-length Shh on fresh primary lung tumors collected from patients and in cancer cell lines and have reported initial validation of this full-length protein as a novel marker of cancer stem cells (CSC) in NSCLC. These findings led them to hypothesize that antibodies generated against the C-terminus Shh epitope can bind and neutralize full-length Shh found exclusively on the CSC population, while leaving the cleaved N-terminus Shh, important for physiologic Shh signaling, unperturbed

More information: Bhairavi Tolani et al. Preclinical characterization of therapeutic antibodies targeted at the carboxy-terminus of Sonic hedgehog, Oncotarget (2018). DOI: 10.18632/oncotarget.24510

Journal information: Oncotarget 

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