Novel human monoclonal antibodies specific to the alternatively spliced domain D of Tenascin C efficiently target tumorsin vivo

Abstract

Antibody-based delivery of bioactive molecules represents a promising strategy for the improvement of cancer immunotherapy. Here, we describe the generation and characterization of R6N, a novel fully human antibody specific to the alternatively spliced domain D of Tenascin C, which is highly expressed in the stroma of primary tumors and metastasis. The R6N antibody recognized its cognate tumor-associated antigen with identical specificity in mouse and human specimens. Moreover, the antibody was able to selectively localize to solid tumors in vivo as evidenced by immunofluorescence-based biodistribution analysis. Encouraged by these results, we developed a novel fusion protein (termed mIL12-R6N) consisting of the murine interleukin 12 fused to the R6N antibody in homodimeric tandem single-chain variable fragment arrangement. mIL12-R6N exhibited potent antitumor activity in immunodeficient mice bearing SKRC52 renal cell carcinoma, as well as in immunocompetent mice bearing SMA-497 glioma. The experiments presented in this work provide a rationale for possible future applications for the R6N antibody for the treatment of cancer patients.

Isolation of a Natural Killer Group 2D Small‐Molecule Ligand from DNA‐Encoded Chemical Libraries

“…Size exclusion chromatography profile showed the prevalent formation of a homo‐dimer in solution ( N2 ; Figure 1c). The recombinant NKG2D protein contained an AviTag [21] and a site‐specific biotinylation was performed in vitro using BirA, biotin and ATP, to enable the immobilization of the protein on streptavidin‐coated beads and affinity‐capture selections [Figure 1d, e and Supporting Information Figure S1d, e] [23] …”

“…MBP‐BirA production . MBP‐BirA in pET28a (Kanamycin resistant) was produced in BL21(DE3) with IPTG induction and purified with NiNTA‐Resin as previously reported [21] …”

“…[20a,b] Those libraries were interrogated against recombinant NKG2D, which had been biotinylated at a site-specific position and immobilized on streptavidin-coated magnetic beads for affinity capture procedures. [21] The screening campaign, performed in duplicate, allowed the reproducible and preferential enrichment of a combination of building blocks and revealed a structure-activity relationship associated with NKG2D recognition. The most enriched DEL compound [ (3-(2-(1-(tert-butoxycarbonyl)-5-methyl-1H-indol-2-yl)phenyl)-2-(4sulfamoylbenzamido)propanoic acid)] was resynthesized and labelled with a fluorescein tag to enable affinity measurements against the target protein.…”

“…In this article, we describe the discovery of a novel small organic NKG2D ligand, achieved by screening two compact single‐stranded single‐pharmacophore DELs (NF_DEL and GB_DEL) [20a,b] . Those libraries were interrogated against recombinant NKG2D, which had been biotinylated at a site‐specific position and immobilized on streptavidin‐coated magnetic beads for affinity capture procedures [21] . The screening campaign, performed in duplicate, allowed the reproducible and preferential enrichment of a combination of building blocks and revealed a structure‐activity relationship associated with NKG2D recognition.…”

Matricellular protein tenascin C: Implications in glioma progression, gliomagenesis, and treatment

“…Another application is as a candidate for targeted therapy, depending on its characteristic expression pattern. Many ligands targeting TNC have been developed, including F16 (204), G11 (205), R6N (206), PL1 (207), PL3 (208), and Ft (209). The new Ft peptide was synthesized to target glioma-associated TNC and neuropilin-1 synergistically in neovasculature for the specific penetration of nanoparticles in anti-GBM therapy (209).…”