Enhancement of the antitumor properties of interleukin-2 by its targeted delivery to the tumor blood vessel extracellular matrix

Abstract

Angiogenic processes depend on the precise coordination of different cell types and a complex exchange of signals, many of which derive from new specific components of the provisional, angiogenesisrelated, extracellular matrix (ECM). Angiogenesis-associated ECM components thus represent appealing targets for the selective delivery of therapeutic molecules to newly forming tumor vessels. Results of a previous study indicated that a high affinity recombinant antibody (L19) to ED-B, a domain contained in the angiogenesis-associated isoform of fibronectin (B-FN), selectively and efficiently targets tumor vessels. The present study shows that a fusion protein between L19 and interleukin 2 (L19-IL-2) mediates the selective delivery and concentration of IL-2 to tumor vasculature, thereby leading to a dramatic enhancement of the therapeutic properties of the cytokine. By contrast, IL-2 fused to an irrelevant recombinant antibody used as a control fusion protein showed neither accumulation in tumors nor therapeutic efficacy. Tumors in mice treated with L19-IL-2 were significantly smaller compared to those in animals treated with saline, the control fusion protein, or IL-2 alone (P ‫؍‬ .003, .003, and .002, respectively). Moreover, no significant differences in size were observed among the tumors from the different control groups (using the control fusion pro- IntroductionDuring tumor progression, the extracellular matrix (ECM) of the normal tissues in which the tumor grows is remodeled through 2 different processes: proteolytic degradation and neosynthesis of ECM components by both neoplastic and stromal cells. These processes generate a "tumoral ECM" that differs quantitatively and qualitatively from the normal tissue ECM and that apparently gives rise to a more suitable environment (inductive or instructive) for tumor progression. [1][2][3] In particular, ECM components modulate vascular cell behavior and angiogenic processes. 4,5 This observation is upheld by the recent report that the majority of messenger RNAs (mRNAs) newly expressed by tumoral endothelial cells encode for ECM proteins. 6 Thus, these provisional ECM components that appear during the angiogenic processes represent an appealing target for the selective delivery of therapeutic molecules to newly forming blood vessels. 7 Furthermore, because new vessel formation is common to all solid tumors, the angiogenesisassociated ECM components can be regarded as pan-tumoral antigens. [8][9][10][11][12] One such ECM component is a fibronectin (FN) isoform, B-FN, which contains an extra FN type III repeat of 91 amino acids, the domain B (ED-B). 13 Because the amino acid sequence of ED-B is identical in mouse, humans, and other mammals, antibodies to this domain react equally well with mouse, human, and other species B-FN. B-FN is detectable only in the stroma of fetal and neoplastic tissues and around newly forming blood vessels, but not in mature vessels. 14,15 Using a radioiodinated human recombinant single-chain Fv (scFv; L19) to the ED-B domain of FN, we demon...

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

“…Our group previously prepared a UG-free immunocytokine composed of IL2 and L19 and showed that its fusion with L19 enhanced the therapeutic index of the cytokine (11). This fusion protein is currently being investigated in a multicenter phase II clinical study in Italy and Germany (37).…”

“…The obtained sequence was inserted into HindIII/NotI digested vector pcDNA3.1. We obtained the sequence encoding for the linker and IL2 by PCR from the construct pcDNA3.1/L19-IL2 as described in Carnemolla et al (11) using the primers TI-58 and TI-59. The cDNA fragment was inserted into NotI/XbaI-digested pcDNA3.1/L19-mUG to generate pcDNA3.1/L19-mUG-IL2.…”

“…Treatment with purified L19-UG-IL2 fusion protein was performed as described by Carnemolla et al (11) in groups of six 129/SvHsd mice (Harlan Italy, Udine, Italy), each injected subcutaneously with 3 ϫ (15). Housing, treatment, and killing of animals followed national legislative provisions (Italian law no.…”

An engineered antibody–interleukin‐12 fusion protein with enhanced tumor vascular targeting properties

“…4,7 In all experimental series, the loss of body weight for the 2 fusion proteins was comparable, and was less than 5% at all time points (V. Gafner, data not shown).…”

Intralesional Treatment of Stage III Metastatic Melanoma Patients with L19–IL2 Results in Sustained Clinical and Systemic Immunologic Responses

“…L19 recognizes the alternatively spliced extra-domain B (EDB) of fibronectin (FN), a marker of angiogenesis (19). EDB-containing FN is present in the neovasculature of most solid tumors and hematologic malignancies (19) but absent from almost all healthy adult tissues (except tissues of the female reproductive cycle). Therefore, L19-IL2 represents a targeted form of IL2, which is able to accumulate at sites where angiogenesis occurs and the EDB-FN antigen is expressed.…”