Phase I Clinical Study Combining L19-IL2 With Stereotactic Ablative Body Radiotherapy in Patients With Oligometastatic Solid Tumor.

1. Tumor vasculature Profound differences exist between vascular endothelium and surrounding stroma of normal tissues and tumors. Tumor vasculature is extremely disorganized and tortuous. Vascular shunts are frequent, and distinguishing arterioles from venules can be challenging. Apart from the architecture, the blood flow itself is strikingly altered: it can be sluggish, sometimes stationary or even reverse. The endothelium in tumors proliferates rapidly and contributes to active angiogenesis. The direct contact of the tumor endothelium with the host's blood pool makes this site a unique target for selective drug delivery.
2. L19 Fibronectin (FN) is a broadly present soluble constituent of plasma and other body fluids. FN usually exists as a dimer formed by two nearly identical (approximately 250 kDa) subunits covalently linked near their C-terminus by a pair of disulfide bonds. Even though FN molecules are the product of a single gene, the resulting protein can exist in multiple forms that arise from alternative spicing of pre-mRNA that can generate up to 20 variants in humans. Splicing occurs in 3 regions of the FN gene, leading to inclusion or exclusion of either one of the two type III repeats, named EDB and EDA. The 91 amino acid sequence of EDB is identical in mice, rats, rabbits, dogs and humans. EDB-containing FN is dispensable during embryogenesis, but is thought to play a modulating role in the growth of connective tissues. In adults, EDB-FN is highly expressed in normal tissues during angiogenesis, but not in mature vessels. Furthermore, EDB-containing FN is abundantly found in solid tumors. It is mainly produced by tumor cells and deposited in the subendothelial extracellular matrix of solid tumors and hematological malignancies.

L19 is the single chain (scFv) human antibody that specifically targets EDB-FN. Antibody fragments in small scFv formats are useful and versatile tools with various advantages including rapid blood clearance and easy manipulation for antibody engineering. The L19 antibody has been shown to recognize and target EDB-FN in vivo both in animal models and patients. In past years, the L19 antibody has been conjugated with numerous agents, including therapeutic radionuclides, and cytokines. 3. Interleukin-2 Cytokines are a heterogeneous group of soluble small polypeptides or glycoproteins exerting pleiotropic or redundant effects promoting growth, differentiation, and activation of normal cells. Cytokines produced by immune cells may have pro- or anti-inflammatory and immune-modulatory activities. In malignant diseases, cytokine production and release can be affected by the tumor itself and/or therapeutic interventions. Cytokines may also display potent anticancer activities, but are frequently hampered by treatment-related toxicities prohibiting dose-escalation to therapeutically effective concentrations.

Interleukin-2 (IL2) plays an essential role in the activation phases of both specific and natural immune responses. Even though it has no direct cytotoxic effects on cancer cells, it can induce tumor regression by stimulating a potent cell-mediated response. As such, IL2 is one of the treatment options in metastatic renal cell carcinoma patients. 4. L19-IL2 In order to overcome toxicity while simultaneously delivering therapeutic doses of IL2 to the tumor issue, the elegant option of combining the anti-EDB scFv L19 antibody with IL2 was pursued. It has been shown that the L19-IL2 conjugate mediates the selective delivery and accumulation of IL2 at tumor endothelial cells, where the EDB antigen is expressed during angiogenesis, leading to a dramatic increase of the therapeutic efficacy of IL2. In the first preclinical study, 80% of the xenograft tumors (including teratocarcinoma and small cell lung cancer) tackled with L19-IL2 were subsequently composed of connective and necrotic tissue. At the same time an increase in the levels of interferon-gamma and of cytotoxic lymphocytes, macrophages and natural killer cells was found. These histological and therapeutic effects were underlined in a orthotopic pancreatic cancer model treated with the antibody-cytokine conjugate. One year later, this response percentage was repeated in Ramos lymphoma xenografts treated with L19-IL2 and the anti-CD20 antibody rituximab whereby a complete remission lasting for more than one year was found in 4 of the 5 mice treated.

In a recent phase I/II clinical trial, the use of L19-IL2 was proven safe in a variety of stage IV malignancies with a recommended dose of 22.5 Mio IU. Furthermore, it was safely combined with dacarbazine in stage IV melanoma patients maintaining the same recommended dose level. In the first study, the overall objective response rate was reported to be 51% after two cycles, and in the second this rate was 28% with one complete response still ongoing 21 months after treatment initiation.

At present, three phase I/II clinical trials on L19-IL2 alone or in combination with chemotherapy for patients with metastatic melanoma (ClinicalTrials.gov numbers: NCT01055522 and NCT01253096) and pancreatic cancer (ClinicalTrials.gov number: NCT01198522) are ongoing.