Francesca Sassi

Targeted Delivery of Tumor Necrosis Factor-α to Tumor Vessels Induces a Therapeutic T Cell–Mediated Immune Response that Protects the Host Against Syngeneic Tumors of Different Histologic Origin

Purpose: We sought to demonstrate that a single systemic administration of L19mTNFα (a fusion protein constituted by the scFv L19 specific for the oncofetal ED-B domain of fibronectin and tumor necrosis factor α, TNFα) in combination with melphalan induced complete and long-lasting tumor eradication in tumor-bearing mice and triggered the generation of a specific T cell–based immune response that protects the animals from a second tumor challenge, as well as from challenges with syngeneic tumor cells of different histologic origin. Experimental Design and Results: Treatment with L19mTNFα, in combination with melphalan, induced complete tumor regression in 83% of BALB/c mice with WEHI-164 fibrosarcoma and 33% of animals with C51 colon carcinoma. All cured mice rejected challenges with the same tumor cells and, in a very high percentage of animals, also rejected challenges with syngeneic tumor cells of different histologic origin. In adoptive immunity transfer experiments, the splenocytes from tumor-cured mice protected naive mice both from C51 colon carcinoma and from WEHI-164 fibrosarcoma. Similar results were also obtained in adoptive immunity transfer experiments using severely immunodepressed mice. Experiments using depleted splenocytes showed that T cells play a major role in tumor rejection. Conclusions: The results show that the selective targeting of mTNFα to the tumor enhances its immunostimulatory properties to the point of generating a therapeutic immune response against different histologically unrelated syngeneic tumors. These findings predicate treatment approaches for cancer patients based on the targeted delivery of TNFα to the tumor vasculature.

Therapy‐induced antitumor vaccination by targeting tumor necrosis factor‐α to tumor vessels in combination with melphalan

Treatment of tumor‐bearing mice with mouse (m)TNF‐α, targeted to tumor vasculature by the anti‐ED‐B fibronectin domain antibody L19(scFv) and combined with melphalan, induces a therapeutic immune response. Upon treatment, a highly efficient priming of CD4+ T cells and consequent activation and maturation of CD8+ CTL effectors is generated, as demonstrated by in vivo depletion and adoptive cell transfer experiments. Immunohistochemical analysis of the tumor tissue demonstrated massive infiltration of CD4+ and CD8+ T cells 6 days after treatment and much earlier in the anamnestic response to tumor challenge in cured mice. In fact, the curative treatment with L19mTNF‐α and melphalan resulted in long‐lasting antitumor immune memory, accompanied by a mixed Th1/Th2‐type response and significant in vitro tumor‐specific cytolytic activity. Finally, the combined treatment reduced the percentage and absolute number of CD4+CD25+ regulatory T cells in the tumor‐draining lymph nodes of mice responding to therapy, and this was associated with the establishment of protective immunity. These findings pave the way for alternative therapeutic strategies based on the targeted delivery of biological and pharmacological cytotoxic compounds that not only kill most of the tumor cells but, more importantly, trigger an effective and long‐lasting antitumor adaptive immune response.

A novel human fibronectin cryptic sequence unmasked by the insertion of the angiogenesis‐associated extra type III domain B

The angiogenesis‐associated extra‐domain B (EDB) of fibronectin (FN) is a complete type III repeat of 91 amino acids. Its expression is modulated by the alternative splicing pattern of the FN pre‐mRNA. FN containing the EDB (B‐FN) is undetectable in tissues of healthy adults, with rare exceptions such as the female reproductive system where tissue remodeling and angiogenesis are recurrent physiological processes. On the contrary, B‐FN is expressed at high levels in neoplastic tissues and during angiogenesis; consequently, it is considered an excellent marker of angiogenesis. Here, we report on a novel FN cryptic sequence, localized on the FN type III repeat 8 (immediately downstream of the EDB) that is unmasked by the insertion of the EDB. This sequence is specifically recognized by the high‐affinity monoclonal antibody, C6, that selectively recognizes B‐FN by means of ELISA, immunohistochemical and Western blot assays. The variable regions of C6 were cloned and a divalent covalently linked mini‐antibody was generated. Biodistribution studies using the radioiodinated C6 mini‐antibody on tumor‐bearing mice demonstrated an efficient tumor targeting. This antibody represents a new tool for the study of the potential biological functions of hindered sequences that the inclusion of the EDB renders accessible, and likewise makes its epitope an additional angiogenesis target.

Alternative splicing of the angiogenesis associated extra-domain B of fibronectin regulates the accessibility of the B-C loop of the type III repeat 8.

Background Fibronectin (FN) is a multi-domain molecule involved in many cellular processes, including tissue repair, embryogenesis, blood clotting, and cell migration/adhesion. The biological activities of FN are mediated by exposed loops located mainly at the interdomain interfaces that interact with various molecules such as, but not only, integrins. Different FN isoforms arise from the alternative splicing of the pre-mRNA. In malignancies, the splicing pattern of FN pre-mRNA is altered; in particular, the FN isoform containing the extra-domain B (ED-B), a complete FN type III repeat constituted by 91 residues, is undetectable in normal adult tissues, but exhibits a much greater expression in fetal and tumor tissues, and is accumulated around neovasculature during angiogenic processes, thus making ED-B one of the best markers and targets of angiogenesis. The functions of ED-B are still unclear; however, it has been postulated that the insertion of an extra-domain such as ED-B modifies the domain-domain interface and may unmask loops that are otherwise cryptic, thus giving FN new potential activities. Methodology We used the mAb C6, which reacts with ED-B containing FN, but not with ED-B-free FN and various recombinant FN fragments containing mutations, to precisely localize the epitopes recognized by the mAb C6. Conclusion We formally demonstrated that the inclusion of the alternatively spliced angiogenesis-associated ED-B leads to the unmasking of the FNIII 8 B-C loop that is cryptic in FN molecules lacking ED-B. Thus, the mAb C6, in addition to providing a new reagent for angiogenesis targeting, represents a new tool for the study of the potential biological functions of the B-C loop of the repeat FNIII 8 that is unmasked during angiogenic processes.

Use of Uteroglobin for the Engineering of Polyvalent, Polyspecific Fusion Proteins

We report a novel strategy to engineer and express stable and soluble human recombinant polyvalent/polyspecific fusion proteins. The procedure is based on the use of a central skeleton of uteroglobin, a small and very soluble covalently linked homodimeric protein that is very resistant to proteolytic enzymes and to pH variations. Using a human recombinant antibody (scFv) specific for the angiogenesis marker domain B of fibronectin, interleukin 2, and an scFv able to neutralize tumor necrosis factor-α, we expressed various biologically active uteroglobin fusion proteins. The results demonstrate the possibility to generate monospecific divalent and tetravalent antibodies, immunocytokines, and dual specificity tetravalent antibodies. Furthermore, compared with similar fusion proteins in which uteroglobin was not used, the use of uteroglobin improved properties of solubility and stability. Indeed, in the reported cases it was possible to vacuum dry and reconstitute the proteins without any aggregation or loss in protein and biological activity.

The immunological basis of tumor therapy by targeted delivery of TNFa to tumor vessels.

L19mTNFa is a fusion protein constituted by the scFv L19 specific for the oncofetal ED-B domain of fibronectin and TNFa. Treatment with L19mTNFa, in combination with melphalan, induced complete tumor regression in 83% of BALB/c mice with WEHI-164 fibrosarcoma and 33% of animals with C51 colon carcinoma. All cured mice rejected challenges with the same tumor cells and, in a very high percentage of animals, also challenges with syngeneic tumor cells of different histological origin. In adoptive immunity transfer experiments the splenocytes from C51-cured mice protected 100% of naive mice both from C51 colon carcinoma and from WEHI-164 fibrosarcoma. The splenocytes from WEHI-164-cured mice protected 100% of mice from the fibrosarcoma and 80% from the C51 colon carcinoma. Similar results were also obtained in adoptive immunity transfer experiments using severely immunodepressed SCID mice. Experiments using depleted splenocytes showed that T cells play a major role in tumor rejection. These data demonstrate that the selective targeting of mTNFa to the tumor enhances its immunostimulatory properties to the point of generating a therapeutic immune response against different histologically unrelated syngeneic tumors. These findings predicate treatment approaches for cancer patients based on the targeted delivery of TNFa to tumor vasculature.