Alessandra Leprini

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.

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.

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.

THE HUMAN TENASCIN-R GENE

The human tenascin-R gene encodes a multidomain protein belonging to the tenascin family, until now detected only in the central nervous system. During embryo development, tenascin-R is presumed to play a pivotal role in axonal path finding through its adhesive and repulsive properties. Recently, the primary structure of human tenascin-R has been elucidated (Carnemolla, B., Leprini, A., Borsi, L., Querzé, G., Urbini, S., and Zardi, L. (1996) J. Biol. Chem. 271, 8157-8160). As a further step to investigate the role of human tenascin-R, we defined the structure of its gene. The gene, which spans a region of chromosome 1 approximately 85 kilobases in length, consists of 21 exons, ranging in size from 90 to >670 base pairs. The sequence analysis of intron splice donor and acceptor sites revealed that the position of introns in human tenascin-R are precisely conserved in the other two tenascin family members, tenascin-C and tenascin-X. The determination of intronic sequences flanking the exon boundaries will allow investigation of whether mutations may be responsible for altered function of the gene product(s) leading to central nervous system development defects.

Procedure for the purification of the fibronection proteolytic fragments containing the ED-B oncofetal domain

Fibronectin (FN) is the blend of structurally different molecules (isoforms) whose makeup varies depending on the FN sources. Fibronectin polymorphism is caused by three sequences (called ED-A, ED-B, and IIICS) which may be included or excluded from the FN molecule depending on the alternative splicing patterns of a single primary transcript. The sequence ED-B, which is a complete type III repeat of 91 amino acids, presents some interesting peculiarities: it is the most conserved FN region and, in normal adult tissues, the ED-B-containing FN has an extremely restricted distribution while having a much greater expression in fetal and tumor tissues (Carnemolla et al., 1989, J. Cell Biol. 108, 1139-1148), suggesting that the ED-B sequence may confer to the FN molecules specific biological activities required during ontogenesis and oncogenetic processes. Here we describe a detailed procedure to purify fibronectin fragments containing the ED-B sequence. These purified fragments are useful reagents in the study of the biological function(s) of the ED-B-containing FN molecules.