Afuco™ Anti-Human TNC ADCC Recombinant Antibody (F16-131I), ADCC Enhanced (CAT#: AFC-600CL)

Figure 1 Immunohistochemistry on human glioblastoma specimens and on mouse U87MG xenografts

Immunohistochemical analysis of U87MG human glioblastoma xenografts and of human glioblastoma surgical specimens using the F16 antibody, specific to the extradomain A1 of tenascin-C, and the L19 antibody, specific to the extradomain B of fibronectin (serial tissue sections). Both antibodies stained tumour perivascular structures considerably. In negative controls (NC), the primary antibody was omitted. Scale bars indicate 100 μm.

Pedretti, M., Verpelli, C., Mårlind, J., Bertani, G., Sala, C., Neri, D., & Bello, L. (2010). Combination of temozolomide with immunocytokine F16–IL2 for the treatment of glioblastoma.British journal of cancer, 103(6), 827.

Figure 2 Therapeutic activity of F16-IL2 combined with temozolomide in subcutaneous and intracranial glioblastoma xenografts

(A) Preclinical therapy study with subcutaneous U87MG human glioblastoma xenografts. The treatment regimen consisted of five total administrations, every third day, of temozolomide (0.525 mg, corresponding to 75 mg m−2) in a saline 10% dimethyl sulfoxide (DMSO) solution, F16-IL2 (20 μg) in phosphate-buffered saline, a combination of F16-IL2 and temozolomide (same doses), or saline 10% DMSO solution. The combination therapy group exhibited the highest therapeutic benefit with a complete remission of the animals, which remained tumour free for over 160 days. (B) Preclinical therapy study, using intracranial U87MG human glioblastoma xenografts. The same therapeutic schedule of the subcutaneous study was used. The combination of F16-IL2 with temozolomide exhibited the highest therapeutic benefit. Pairwise comparisons between the combination therapy group and temozolomide alone (P=0.009), F16-IL2 alone (P=0.001), and the control group (P<0.001) were calculated using the Student's t-test and showed significant results. (I) Photograph of a mouse hemisphere with tumour, imaged from two sides. (II) Tumour volumes at day 25 from the start of treatment (13 days after the last drug administration), expressed as average mean±s.d. (C) Survival study using intracranial U87MG human glioblastoma xenografts, with the same therapeutic schedule of the previous subcutaneous and intracranial studies. Results indicate a longer survival for the combination treatment group (combo vs TMZ: P<0.002; combo vs F16-IL2: P<0.002; combo vs control: P<0.0001).

Pedretti, M., Verpelli, C., Mårlind, J., Bertani, G., Sala, C., Neri, D., & Bello, L. (2010). Combination of temozolomide with immunocytokine F16–IL2 for the treatment of glioblastoma.British journal of cancer, 103(6), 827.

Figure 3 Immunofluorescence analysis of tumour-infiltrating immune cells and of microvascular density in the subcutaneous (A) and intracranial (B) glioblastoma models, 24 h after the third injection of therapeutic agents.

The F16-IL2+temozolomide treatment groups show the largest increase in the infiltration of leukocytes and in particular of natural killer cells and macrophages (serial tissue sections). Scale bars indicate 100 μm.

Pedretti, M., Verpelli, C., Mårlind, J., Bertani, G., Sala, C., Neri, D., & Bello, L. (2010). Combination of temozolomide with immunocytokine F16–IL2 for the treatment of glioblastoma.British journal of cancer, 103(6), 827.

Figure 4 Immunofluorescence analysis of F16-IL2 fusion protein localisation in subcutaneous (A) and intracranial (B) glioblastoma xenografts, 24 h after the third injection of therapeutic agents (serial tissue sections).

Scale bars indicate 100 μm.

Pedretti, M., Verpelli, C., Mårlind, J., Bertani, G., Sala, C., Neri, D., & Bello, L. (2010). Combination of temozolomide with immunocytokine F16–IL2 for the treatment of glioblastoma.British journal of cancer, 103(6), 827.

Figure 5 Immunofluorescence analysis of apoptosis and proliferation in subcutaneous glioblastoma xenografts, 24 h after the third injection of therapeutic agents.

Results show a clear increase in apoptosis and the complete suppression of proliferation in the combination F16-IL2+temozolomide treatment group. Scale bars indicate 50 μm.

Pedretti, M., Verpelli, C., Mårlind, J., Bertani, G., Sala, C., Neri, D., & Bello, L. (2010). Combination of temozolomide with immunocytokine F16–IL2 for the treatment of glioblastoma.British journal of cancer, 103(6), 827.

Figure 6 BIAcore sensograms of anti-tenascin-C antibodies at different concentrations.

A, monomeric fractions of scFv(D5) (top) and affinity-matured scFv(F16) (bottom) on human TnC-A1. B, scFv(D5) on mouse TnC-A1 shows no binding. C, SIP(F16) on human TnC-A1. D, monomeric fractions of scFv(F4) (top) and affinity-matured antibodies scFv(D11) (middle) and scFv(P12) (bottom) on human TnC-D. E, monomeric fractions of scFv(P12) on mouse TnC-D. F, SIP(P12) on human TnC-D.

Brack, S. S., Silacci, M., Birchler, M., & Neri, D. (2006). Tumor-targeting properties of novel antibodies specific to the large isoform of tenascin-C. Clinical Cancer Research, 12(10), 3200-3208.

Figure 7 Immunohistochemistry with relevant antibodies on different mouse cancer models.

Left, U87 human glioblastoma; middle, A375 human melanoma; right, F9 murine teratocarcinoma. The sections were stained for EDB with L19, for TnC-D with F4 and P12, and for TnC-A1 with D5 and F16. As expected, D5 and F16 did not give any staining on murine F9 teratocarcinoma (data not shown). Bar, 100 μm.

Brack, S. S., Silacci, M., Birchler, M., & Neri, D. (2006). Tumor-targeting properties of novel antibodies specific to the large isoform of tenascin-C. Clinical Cancer Research, 12(10), 3200-3208.

Figure 8 iodistribution of SIP(F16) and SIP(P12) in U87-bearing nude mice at four different time points after i.v. injection.

A, biodistribution of ¹²⁵I-labeled SIP(F16). Each time point corresponds to an average of three animals. B, biodistribution of ¹²⁵I-labeled SIP(P12). Each time point corresponds to an average of four animals. Results are expressed as %ID/g tissue.

Brack, S. S., Silacci, M., Birchler, M., & Neri, D. (2006). Tumor-targeting properties of novel antibodies specific to the large isoform of tenascin-C. Clinical Cancer Research, 12(10), 3200-3208.

Figure 9 Immunofluorescence staining of head and neck cancer samples.

The expression patterns and intensities of the splice isoforms extra domain A (EDA) and extradomain B (EDB) of fibronectin and the A1 domain of tenascin C were detected with the antibodies F8, L19 and F16, respectively (shown in green). A co-staining with an anti-von Willebrand factor antibody that stains blood vessels (red) and Dapi (blue) was performed. Abbreviations: SCC: squamous cell carcinoma; PA: pleomorphic adenoma.

Schwager, K., Villa, A., Rösli, C., Neri, D., Rösli-Khabas, M., & Moser, G. (2011). A comparative immunofluorescence analysis of three clinical-stage antibodies in head and neck cancer. Head & neck oncology, 3(1), 25.

Figure 10 Analysis of staining intensities.

Comparison of staining intensities obtained with the antibodies F8, L19 and F16 in A) primary tumours and metastasis tumour samples and in B) primary benign and primary maligant tumour specimens. C) Average staining intensities of tumours of the oral cavity, the pharynx or the larynx. Standard Errors of the Mean of scoring values are displayed.

Schwager, K., Villa, A., Rösli, C., Neri, D., Rösli-Khabas, M., & Moser, G. (2011). A comparative immunofluorescence analysis of three clinical-stage antibodies in head and neck cancer. Head & neck oncology, 3(1), 25.