Barbara Carnemolla

Identification of PVR (CD155) and Nectin-2 (CD112) as Cell Surface Ligands for the Human DNAM-1 (CD226) Activating Molecule

Human natural killer (NK) cells express a series of activating receptors and coreceptors that are involved in recognition and killing of target cells. In this study, in an attempt to identify the cellular ligands for such triggering surface molecules, mice were immunized with NK-susceptible target cells. On the basis of a functional screening, four mAbs were selected that induced a partial down-regulation of the NK-mediated cytotoxicity against the immunizing target cells. As revealed by biochemical analysis, three of such mAbs recognized molecules of ∼70 kD. The other mAb reacted with two distinct molecules of ∼65 and 60 kD, respectively. Protein purification followed by tryptic digestion and mass spectra analysis, allowed the identification of the 70 kD and the 65/60 kD molecules as PVR (CD155) and Nectin-2 δ/α (CD112), respectively. PVR-Fc and Nectin-2-Fc soluble hybrid molecules brightly stained COS-7 cells transfected with the DNAM-1 (CD226) construct, thus providing direct evidence that both PVR and Nectin-2 represent specific ligands for the DNAM-1 triggering receptor. Finally, the surface expression of PVR or Nectin-2 in cell transfectants resulted in DNAM-1–dependent enhancement of NK-mediated lysis of these target cells. This lysis was inhibited or even virtually abrogated upon mAb-mediated masking of DNAM-1 (on NK cells) or PVR or Nectin-2 ligands (on cell transfectants).

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.

Phage antibodies with pan‐species recognition of the oncofoetal angiogenesis marker fibronectin ED‐B domain

Fibronectin (FN) exists in several polymorphic forms due to alternative splicing. The B‐FN isoform (with ED‐B domain inserted by splicing) is present in the stroma of foetal and neoplastic tissues and in adult and neoplastic blood vessels during angiogenesis but is undetectable in mature vessels. This isoform, therefore, represents a promising marker for angiogenesis, as already shown using the mouse monoclonal antibody (MAb) BC‐I directed against an epitope on human B‐FN. However, this MAb does not directly recognise the human ED‐B domain nor does it recognise B‐FN of other species; therefore, it cannot be used as a marker of angiogenesis in animal models. In principle, antibodies directed against the human ED‐B domain should provide pan‐species markers for angiogenesis as the sequence of this domain is highly conserved in different species (and identical in humans and mice). As it has proved difficult to obtain such antibodies by hybridoma technology, we used phage display technology. Here, we describe the isolation of human antibody fragments against the human ED‐B domain that bind to human, mouse and chicken B‐FN. As shown by immunohistochemistry, the antibody fragments stain human neoplastic tissues and the human, mouse and chicken neovasculature.

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.

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.

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.

Fibronectin Type III5 Repeat Contains a Novel Cell Adhesion Sequence, KLDAPT, Which Binds Activated α4β1 and α4β7 Integrins

The region of fibronectin encompassing type III repeats 4–6 contains a low affinity heparin binding domain, but its physiological significance is not clear. We have studied whether this domain is able to interact with cells as already shown for other heparin binding domains of fibronectin. A computer search based on homologies with known active sites in fibronectin revealed the sequence KLDAPT located in FN-III5. A synthetic peptide containing this sequence induced lymphoid cell adhesion upon treatment with the activating anti-β1 monoclonal antibody (mAb) TS2/16 or with Mn2+, indicating that KLDAPT was binding to an integrin. A recombinant fragment containing repeat III5 (FN-III5) also mediated adhesion of TS2/16/Mn2+-treated cells while the FN-III6 fragment did not. Soluble KLDAPT peptide inhibited cell adhesion to FN-III5 as well as to a 38-kDa fibronectin fragment and VCAM-1, two previously known ligands for α4β1 integrin. KLDAPT also competed with the binding of soluble alkaline phosphatase-coupled VCAM-Ig to Mn2+-treated α4β1. Furthermore, mAbs anti-α4 and anti-α4β7, but not mAbs to other integrins, inhibited cell adhesion to FN-III5 and KLDAPT. These results therefore establish a cell adhesive function for the FN-III5 repeat and show that KLDAPT is a novel fibronectin ligand for activated α4 integrins.

Fibronectin: a chromatin-associated protein?

We have previously reported that chromatin preparations from human cultured fibroblasts contain a single homologous serum protein. In this paper we present evidence, based on immunological identity and physicochemical properties, that this serum protein is fibronectin. Furthermore, using a radioimmunoassay system, we have estimated that fibronectin represents about 0.7% of the total protein in both chromatin preparations and whole fibroblasts. Using a nitrocellulose filter assay system, we also show that fibronectin is a DNA-binding protein having an equilibrium constant of 4.6 x 10(-6) M. Equilibrium competition experiments have demonstrated that fibronectin has the ability to differentiate among nucleotides, indicating that fibronectin-DNA interaction is at least partially specific, and that a minimum polymer length of 12-18 nucleotides is required for effective binding to occur. Fibronectin has been isolated readily from plasma using DNA-affinity chromatography. We do not have direct evidence that fibronectin is an actual nonhistone chromosomal protein, but fibronectin is a DNA-binding protein (at least under in vitro assay conditions) and appears to be a normal constituent of chromatin as chromatin is currently isolated from cell nuclei.

A simplified procedure for the preparation of antibodies to serum fibronectin

In the present paper we describe in detail a simple procedure for the preparation of monospecific antisera to human and mouse serum fibronectin. A similar procedure could also be used to prepare antibodies to fibronectin from other species. The procedure, based on the recently reported affinity of fibronectin for gelatin, essentially consists of two steps (1) Immunization of rabbits with fibronectin purified from serum by affinity chromatography using gelatin coupled to CNBr-activated Sepharose 4B. (2) Absorption of the antiserum obtained by an immunoabsorbent prepared using fibronectin-free serum proteins that remained after absorbing serum with gelatin-Sepharose. The antisera obtained were monospecific, as determined by immunoelectrophoresis and did not show any difference with respect to antisera prepared by different procedures.