Laura Borsi

Selective targeting of tumoral vasculature: Comparison of different formats of an antibody (L19) to the ED-B domain of fibronectin

We recently demonstrated that a human recombinant scFv, L19, reacting with the ED‐B domain of fibronectin, a marker of angiogenesis, selectively targets tumoral vasculature in vivo. Using the variable regions of L19, we constructed and expressed a human “small immunoprotein” (SIP) and a complete human IgG1 and performed biodistribution studies in tumor‐bearing mice to compare the blood clearance rate, in vivo stability and performance in tumor targeting of the 3 L19 formats [dimeric scFv (scFv)2, SIP and IgG1]. The accumulation of the different antibody formats in the tumors studied was a consequence of the clearance rate and in vivo stability of the molecules. Using the SIP, the %ID/g in tumors was 2–5 times higher than that of the (scFv)2, reaching a maximum 4–6 hr after injection. By contrast, the accumulation of IgG1 in tumors constantly rose during the experiments. However, due to its slow clearance, the tumor‐blood ratio of the %ID/g after 144 hr was only about 3 compared to a ratio of 10 for the (scFv)2 and 70 for the SIP after the same period of time. The different in vivo behavior of these 3 completely human L19 formats could be exploited for different diagnostic and/or therapeutic purposes, depending on clinical needs and disease. Furthermore, the fact that ED‐B is 100% homologous in human and mouse, which ensures that L19 reacts equally well with the human and the murine antigen, should expedite the transfer of these reagents to clinical trials.

A high-affinity human monoclonal antibody specific to the alternatively spliced EDA domain of fibronectin efficiently targets tumor neo-vasculature in vivo

The alternatively spliced extra‐domain B of fibronectin is one of the best characterized markers of tumor angiogenesis. Similarly, the extra‐domain A (EDA), which can also be inserted in the fibronectin transcript by a mechanism of alternative splicing, has been shown to preferentially accumulate around new blood vessels in certain tumors, but this antigen has not been investigated so far as a target for antibody‐based biomolecular intervention. We here describe the generation of 3 human monoclonal antibodies (named F8, B7 and D5), which recognize the same epitope of EDA, but which differ in terms of their dissociation constant to the human antigen (KD = 3.1, 16 and 17 nM, measured for monomeric preparations of the F8, B7 and D5 antibodies, respectively, in recombinant scFv format). When the 3 antibody fragments were cloned and expressed with a 5 amino acid linker, the 3 resulting homodimeric antibody preparations displayed comparable tumor: organ ratios in quantitative biodistribution studies, performed in immunocompetent 129SvEv mice, bearing subcutaneous syngeneic F9 murine tumors. The percent injected dose per gram (%ID/g) values in tumors 24 hr after intravenous injection were 9.3, 10.2 and 13 for F8, B7 and D5, respectively. The F8 antibody may serve as useful building block for the development of antibody‐based targeted anti‐cancer therapeutics. Preclinical and clinical investigations are facilitated by the fact that F8 recognizes the human and mouse antigen with comparable affinity, and by the observation that EDA over‐expression is detectable not only in solid tumors, but also in hematological malignancies.

Transforming growth factor β regulates the levels of different fibronectin isoforms in normal human cultured fibroblasts

Fibronectin (FN) polymorphism is caused by alternative splicing patterns in at least three regions of the primary transcript of a single gene. Using a monoclonal antibody (Mab) specific for an FN segment (ED‐A), that can be included or omitted from the molecule depending on the pattern of splicing, we have examined whether transforming growth factor β (TGF‐β) and dexamethasone, which are both known to increase the level of total FN, regulate the levels of different FN isoforms. We found that, while dexamethasone does not significantly change the ratio between the total FN and the ED‐A containing FN, TGF‐β preferentially increases the expression of the FN isoform containing the ED‐A sequence.

Differentiation between high- and low-grade astrocytoma using a human recombinant antibody to the extra domain-B of fibronectin.

Different fibronectin (FN) isoforms are generated by the alternative splicing of the primary FN transcript. We previously demonstrated that the isoform containing the extra domain B sequence of fibronectin (B-FN), a complete type-III-homology repeat, is a marker of angiogenesis that accumulates around neovasculature only during angiogenic processes. We produced a single-chain human recombinant antibody (scFv), L19, which reacts specifically with B-FN and selectively targets tumor vasculature in vivo. We used this scFv and an antibody against a pan-endothelial marker (Factor VIII) in a double-staining procedure on specimens of low- and high-grade astrocytomas to determine the percentage of B-FN-positive vessels, (denominating the resulting value angiogenic index [AI]). Compared to vascular density and proliferative activity (evaluated using antibodies to Factor VIII and Ki67, respectively), AI correlated better with tumor grade (1.6 +/- 2.6% and 92.0 +/- 8.7% of B-FN-positive vessels in low- and high-grade astrocytomas, respectively) and was a more precise diagnostic tool than either of the two conventional methods. In fact, discriminating analysis using these three parameters showed that only AI accurately classified 100% of the cases studied, compared to 64% and 89% correctly diagnosed by vascular density and of proliferating cells, respectively.

Transforming growth factor‐β regulates the splicing pattern of fibronectin messenger RNA precursor

Fibronectin (FN) polymorphism is caused by alternative splicing patterns in at least three regions (ED‐A, ED‐B and IIICS) of the primary transcript of a single gene. Using monoclonal antibodies, we previously demonstrated that transforming growth factor‐β (TGF‐β) preferentially increases the accumulation of the FN isoforms containing the ED‐A sequence in cultured normal human fibroblasts [Balza et al., (1988) FEBS Lett. 228, 42‐44]. To determine the basis of this effect, we have examined through S1 nuclease analysis, the levels of ED‐A‐ and ED‐B‐containing mRNAs in cultured normal human skin flbroblasts before and after TGF‐β treatment. These experiments have shown that TGF‐β increases the relative amount of m‐RNA for ED‐A‐ and ED‐B‐containing FN isoforms. These data demonstrate that a growth factor may regulate the splicing pattern of a pre‐mRNA.

Identification of a Glioblastoma-Associated Tenascin-C Isoform by a High Affinity Recombinant Antibody

Tenascin-C exists in several polymorphic isoforms due to alternative splicing of nine fibronectin-like type III repeats. Large Tenascin-C isoforms are present in almost all normal adult tissues but are upregulated in fetal, regenerating, and neoplastic tissues. Here, we report a human antibody fragment, TN11, derived from a phage library with high affinity for the spliced repeat C and demonstrate that this repeat is undetectable in normal adult tissues, barely detectable or undetectable in breast, lung and gastric carcinomas, meningioma, and low grade astrocytoma, but extremely abundant in high grade astrocytoma (grade III and glioblastoma), especially around vascular structures and proliferating cells. The antibody appears to have potential for development of a therapeutic agent for patients with high grade astrocytoma.

Selective targeting of tumour neovasculature by a radiohalogenated human antibody fragment specific for the ED-B domain of fibronectin.

Abstract. Angiogenesis is a characteristic feature of many aggressive tumours and other disorders. Antibodies capable of binding to new blood vessels, but not to mature vessels, could be used as selective targeting agents for immunoscintigraphic and radioimmunotherapeutic applications. Here we show that scFv(L19), a recombinant human antibody fragment with sub-nanomolar affinity for the ED-B domain of fibronectin, a marker of angiogenesis, can be stably labelled with iodine-125 and astatine-211 with full retention of immunoreactivity, using a trimethyl-stannyl benzoate bifunctional derivative. Biodistribution studies in mice bearing two different types of tumour grafted subcutaneously, followed by ex vivo micro-autoradiographic analysis, revealed that scFv(L19) rapidly localises around tumour blood vessels, but not around normal vessels. Four hours after intravenous injection of the stably radioiodinated scFv(L19), tumour to blood ratios were 6:1 in mice bearing the F9 murine teratocarcinoma and 9:1 in mice bearing an FE8 rat sarcoma. As expected, all other organs (including kidney) contained significantly less radioactivity than the tumour. Since the ED-B domain of fibronectin has an identical sequence in mouse and man, scFv(L19) is a pan-species antibody and the results presented here suggest clinical utility of radiolabelled scFv(L19) for the scintigraphic detection of angiogenesis in vivo. Furthermore, it should now be possible to investigate scFv(L19) for the selective delivery of 211At to the tumour neovasculature, causing the selective death of tumour endothelial cells and tumour collapse.

Localization of the cellular-fibronectin-specific epitope recognized by the monoclonal antibody IST-9 using fusion proteins expressed in E. coli

Here we report on a monoclonal antibody (IST‐9) which distinguishes between human cellular and plasma fibronectin. Using β‐galactosidase‐fibronectin fusion proteins expressed in E. coli we have demonstrated that this monoclonal antibody is specific for a fibronectin segment (ED) which can be included or omitted from the molecule depending on the pattern of splicing of the mRNA precursors. Furthermore, using the same fusion proteins we have been able to localize precisely the epitopes of two other monoclonal antibodies (IST‐1 and IST‐2), specific for the heparin‐binding domain 5 of fibronectin.

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.

Synthesis and protein distribution of the unspliced large tenascin‐C isoform in oral squamous cell carcinoma

The inclusion or omission of the alternatively spliced region in the tenascin‐C (Tn‐C) mRNA gives rise to the large (Tn‐CL) or small (Tn‐CS) variant, respectively. Tn‐CL is thought to be a typical component of provisional extracellular matrices (ECMs) and is expressed during tumour stroma remodelling. Tn‐CL synthesis has been studied using RNA/RNA in situ hybridization, and Tn‐CL protein distribution, using immunohistochemistry (clone BC‐2), in 18 oral squamous cell carcinomas (OSCCs) of different grades of malignancy. While the Tn‐CL protein was demonstrated within the whole stromal compartment regardless of grade of malignancy, the majority of the Tn‐CL mRNA signal‐bearing cells were carcinoma cells. Only a few stromal myofibroblasts were able to synthesize Tn‐CL, as revealed by α‐smooth muscle actin double staining. In well‐differentiated carcinomas (G1), the Tn‐CL synthesizing carcinoma cells were localized as a single positive cell layer in the tumour stroma interface, particularly in invasive areas. A higher grade of malignancy (G2/G3) is associated with a significantly increased number of Tn‐CL synthesizing carcinoma cells randomly distributed within the invading tumour areas. Double‐staining experiments (Tn‐CL mRNA ISH/BC‐2 immunohistochemistry) indicate that these cells are capable of organizing and depositing a three‐dimensional Tn‐CL matrix. Even though an instructive and/or inductive role of the carcinoma cells in tumour stroma formation cannot be excluded, these results demonstrate that carcinoma cells can directly produce the ECM components of tumour stroma. Copyright