EDB Domain Of Fibronectin Research Articles

Abstract 5553: A novel immunocytokine for the treatment of cancer

Abstract Interleukin-12 (IL12) is an immunomodulatory cytokine, which offers unique opportunities for cancer therapy due to its stimulatory function on cell-mediated immunity and its anti-angiogenic activity. IL12 was shown to polarize CD4+ T cells into a TH1 type and to be a key activator of NK cells and CD8+ T cells. It also induces the production of interferon-gamma by T cells and NK cells and subsequently of the anti-angiogenic chemokines CXCL10/IP-10, and CXCL9/Mig. However the potent anti-tumor IL12 activity, which has been reported in mice, has not yet been successfully translated into clinical development, mainly because of early reports of severe toxicity and low response rates in human. Following our pioneering work which started in 2002 with the first description of an IL12 based antibody-cytokine fusion protein (i.e. Immunocytokine), we have extensively explored and perfected alternative molecular formats, with the aim to further improve biodistribution properties and therapeutic activity of IL12-based immunocytokines. Here we describe the development and evaluation of new targeted variants of both murine and human IL-12 with enhanced therapeutic efficacy and improved safety profile. To this end we combined the immunomodulatory properties of the IL12 payload with the tumor-homing activity of the L19 antibody. L19 is a clinical grade fully human antibody, which recognizes with identical affinity in both mouse and human, the alternatively spliced EDB domain of fibronectin. EDB represent an optimal target for anti-cancer pharmacodelivery, due to its pan-tumoral over-expression nature combined with very low expression levels in normal tissues. This has also been confirmed by extensive Nuclear Medicine work in which radiolabeled L19 has been administered to more than 150 patients, making L19 one of the best validated tumor-targeting agent. Our novel immunocytokine, termed IL12-L19-L19, relies on the L19 antibody in tandem diabody format, with a monomeric IL12 moiety expressed as single-chain polypeptide at the N-terminal extremity. Recombinant IL12-L19-L19 proteins based either on human or murine IL12, were efficiently expressed in CHO cells and purified to high quality. The tumor-targeting properties of both variants were validated in tumor-bearing mice, using radioiodinated protein preparations. In preclinical therapy studies IL12-L19-L19 showed potent anti-cancer activity when used either as single agent or in combination with other anticancer agents. PK studies in Cynomolgus Monkey using the fully human IL12-L19-IL19 product, revealed a favorable profile, which is compatible with other clinical-stage immunocytokines based on antibody-fragments. These results strongly support the further development of the fully human IL12-L19-L19 product for future clinical investigations. Citation Format: Mattia Matasci, Emanuele Puca, Tiziano Ongaro, Sarah Wulhfard, Alessandra Villa, Dario Neri. A novel immunocytokine for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5553.

Abstract 5563: Kinetically-controlled release of blocking molecules for the targeted enhancement of therapeutic activity in antibody-cytokine fusion proteins

Abstract Cytokines are able to mediate a potent anticancer activity but have significant side effects that prevent dose escalation to therapeutically-active regimens. Targeting the cytokines to the site of disease helps to improve their therapeutic index while decreasing associated toxicities. However, peak concentrations in blood are still associated with side-effects. We have developed a novel technology (termed “CORK Technology”), which consists in masking the undesired activity of a cytokine in circulation. The “cork” can be either an antibody fragment or a small molecule, which specifically binds to the cytokine thus preventing the interaction with its receptor. Once the complex accumulates in the tumor, the cork dissociate from the cytokine allowing it to exert its function. Capitalizing on the fact that the kinetic dissociation constant koff of the complex is identical in different species, this technology can be easily translated from mouse to man with comparable results. In this poster we describe the experimental implementation of CORK Technology to improve the therapeutic index of the clinical stage L19-IL2 (L19 is an antibody fragment specific to the alternatively-spliced EDB domain of fibronectin, a marker of tumor angiogenesis). Two classes of IL2 inhibitors were discovered and characterized: (i) a fully human scFv fragment, and (ii) a small organic ligand, featuring a modified methylindole moiety. Both types of agents reduced IL2 activity in vitro, were able to dissociate from the cognate cytokine in a kinetically-tuned process and allowed the administration of the L19-IL2 fusion protein at higher doses, both in normal and in tumor-bearing mice. The technology is applicable to other cytokine payloads and facilitates the development of targeted cytokine products with “activity on demand” (i.e., with increased activity at the tumor site, promoting an activation and proliferation of tumor-resident lymphocytes). Albeit still in its infancy, this elegant approach provides promising results and a strong rationale to further investigate this therapeutic power of the CORK Technology. Citation Format: Tiziano Ongaro, Martina Bigatti, Filippo Sladojevich, Etienne Donckele, Alessandra Micaela Villa, Dario Neri. Kinetically-controlled release of blocking molecules for the targeted enhancement of therapeutic activity in antibody-cytokine fusion proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5563.

The Multicompartmental p32/gClqR as a New Target for Antibody-based Tumor Targeting Strategies

Tumor-associated cell surface antigens and tumor-associated vascular markers have been used as a target for cancer intervention strategies. However, both types of targets have limitations due to accessibility, low and/or heterogeneous expression, and presence of tumor-associated serum antigen. It has been previously reported that a mitochondrial/cell surface protein, p32/gC1qR, is the receptor for a tumor-homing peptide, LyP-1, which specifically recognizes an epitope in tumor cells, tumor lymphatics, and tumor-associated macrophages/myeloid cells. Using antibody phage technology, we have generated an anti-p32 human monoclonal antibody (2.15). The 2.15 antibody, expressed in single-chain fragment variable and in trimerbody format, was then characterized in vivo using mice grafted subcutaneously with MDA-MB-231 human breast cancers cells, revealing a highly selective tumor uptake. The intratumoral distribution of the antibody was consistent with the expression pattern of p32 in the surface of some clusters of cells. These results demonstrate the potential of p32 for antibody-based tumor targeting strategies and the utility of the 2.15 antibody as targeting moiety for the selective delivery of imaging and therapeutic agents to tumors.

Abstract B55: Phase I/II study of the tumor-targeting human L19TNF-monoclonal antibody-cytokine fusion protein in patients with advanced solid tumors

Abstract Background: L19TNF-α is a tumor targeted immunocytokine composed of a single chain variable fragment (scFv) directed against the ED-B domain of fibronectin and the human recombinant cytokine tumor necrosis factor-α. It was created to selectively localize TNF-α to tumor tissues and to minimize systemic toxicity. We evaluated safety, early signs of clinical activity and pharmacokinetic properties of L19TNF-α in patients with refractory solid tumors. Patients and Methods: Six cohorts of patients with refractory solid tumors were planned to receive escalating doses of L19TNF-α (60 minute-intravenous infusion of 1.3, 2.6, 5.2, 7.8, 10.4, e 13 g/kg) on days 1, 3 and 5 of a 21-days treatment cycle (maximum 6 cycles). Serum samples were taken for pharmacokinetic assessment and the analysis of 5-hydroxyindoleacetic acid (5-HIAA; surrogate marker of vascular disruption) during the first treatment cycle. Immunophenotyping (marker panels: CD71/CD8/CD3/CD4 for T-cell; CD71/CD19/CD3/CD56 for NK/B; CD25/CD122/CD3/CD4 for IL-2 expression on T-cell) on blood mononuclear cells was performed on days 1, 5, and 15 of each cycle for the first consecutive patients. The formation of human antifusion protein antibodies to L19 (HAFA) was analyzed at screening and on day 1 of each cycle. Data on safety and activity were collected. Results: Eightheen patients with solid tumors were recruited into the trial between January 2008 and September 2009. Patient's median age was 59 years (range 35–74), 8 were male and 9 female. Eight patients had colorectal cancer, 3 cholangiocarcinoma, 2 thymoma, 1 gastric carcinoma, 1 papilla Vater adenocarcinoma, and 1 parotid adenoidocystic carcinoma. A maximum tolerated dose was not yet reached. At the highest dose level achieved so far (10.4 g/kg) severe lumbar pain occurred in one patient during the L19TNF-infusion constituting a DLT. Most common (> 20%) toxicities included G1 Chills (100%), G2 Chills (41%), febricula (47%), G1 fever (70%), G1 asthenia (41%), headache (41%), G1 Nausea (52%), G1 vomiting (52%), G1 constipation (30%), herpes simplex recurrence (30%), G1 pain (58%), G2 pain (30%). Eight of the 18 patients achieved confirmed disease stabilization, lasting for 5 months (range: 1.5 to 8 months). All other patients progressed. Baseline and post-treatment 5′HIAA values were measured in patients of the first two cohorts (6 patients). A significant increase of 5′-HIAA levels on day 1 of the treatment (within 12 hours after end of infusion) as compared to baseline was found in most patients. Immunophenotyping, pharmacokinetic, HAFA and 5′HIAA analyses will be presented at the meeting. Conclusions: L19TNF- up to a dose of 10.4 g/kg was found safe when intravenously administered to patients with advanced progressive solid tumors in an outpatient setting. The observed toxicity profile was mild and reversible. Encouraging long-lasting disease stabilization was found in a larger fraction of patients, in particular in patients suffering from upper tract gastrointestinal adenocarcinomas. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B55.

Phase I/II study of the tumor-targeting human L19-IL2 monoclonal antibody-cytokine fusion protein in combination with DTIC in metastatic melanoma patients

9064 Background:L19-IL2 is a tumor targeted immunocytokine constituted by a single chain Fragment variable (scFv) format directed against the ED-B domain of fibronectin and the human cytokine interleukin-2 (IL2). The recommended dose (RD) for monotherapy of advanced solid cancer patients was established to be 22.5 Mio IU IL2 equivalent on day 1, 3, and 5 of a 21-day cycle. Here we report clinical results of the dose escalation part of a phase I/II study assessing L19-IL2 in combination with DTIC chemotherapy in metastatic melanoma patients. Methods: L19-IL2 was administered as an i.v. infusion at doses of 10 (n=3), 15 (n=3) and 22.5 MioIU IL2 equivalent dose (n=4) on days 1, 3 and 5 every 21 days in combination with DTIC 1000 mg/m2 on Day 1 for up to 6 treatment cycles. Serum samples for PK evaluation and induction of human antifusion antibodies to L19 (HAFA) were collected. Flow cytometry (T and NK/B cell panels) was performed. Data on safety and activity were evaluated using CTC v3.0 and RECIST criteria, respectively. Results: All 10 patients had progressive metastatic melanoma and the majority had already received prior systemic therapy. Median age at start of treatment was 62 years (52–74). There were no treatment related deaths and the treatment was well tolerated, details of CTC evaluation will be presented. The dose of 22.5 Mio IU day 1, 3, and 5 in combination with 1000 mg DTIC/m2 on day 1, repeated on day 22 was defined as the RD for up to 6 treatment cycles. 10 patients were evaluable for response. We observed 1 partial remission at the 15 Mio IL2 dose level after 4 treatment cycles. Immunophenotyping analysis showed transient stimulation of NK cells, T4 cells, and CTLs. Pharmacokinetic data will be presented. Conclusions: L19-IL2 at a dose of 22.5 Mio IU IL2 equivalent on days 1, 3, and 5 of a 3-weekly schedule can be safely combined with standard DTIC in metastatic melanoma patients. Toxicity was manageable and reversible. Preliminary evaluation suggests clinical activity of the L19IL2/DTIC regimen in metastatic melanoma patients. [Table: see text]

Phase I/II study of the tumor-targeting human L19-IL2 monoclonal antibody-cytokine fusion protein in patients with advanced renal cell carcinoma

16032 Background: L19-IL2 is a tumor targeted immunocytokine constituted by a single chain Fragment variable (scFv) format directed against the ED-B domain of fibronectin and the human cytokine interleukin-2 (IL2). We carried out a phase I/II study in patients with solid tumors to assess safety, pharmacokinetics (PK), and early signs of activity of L19-IL2 monotherapy. Methods: L19-IL2 was administered as an i.v. infusion at doses of 5 (n=1), 10 (n=1), 22.5 (n=15) and 30 MioIU IL2 equivalent dose (n=1) on days 1, 3 and 5 every 21 days. Serum samples for PK assessment were collected on day 1, 3, 5 and 10 of each cycle. Flow cytometry (marker panels: T-cells CD71/CD8/CD3/CD4; NK/B-cells CD71/CD19/CD3/CD56; IL-2R-positive T-cells CD25/-CD122/CD3/CD4) and assessment of human antifusion protein antibodies to L19 (HAFA) were performed at screening and on day 1, 3, 5 and 10 of each cycle. Data on safety and activity were evaluated using CTC v3.0 and RECIST criteria, respectively. Results: Among the total 33 patients,18 had progressive advanced renal clear-cell carcinoma (RCC). Of these, 6 were treated in the phase I and a further 12 planned patients with RCC in the phase II part of the study. The 30 MiU IL2 equivalent dose was considered toxic. The 22.5 MiU dose was determined to be the MTD, which was selected to be the RD. Total/median number of cycles was 62/4 (2–6). Median age was 62 years (52–74). 15 of 18 patients had received prior systemic therapy. We recorded three grade 3 toxicities: 1 hypertension and hypoxia, 1 dyspnoea, and 1 renal failure. All other toxicities were moderate and transient. There were no treatment-related deaths. 16 patients are evaluable for response. We observed stable disease in 13 patients (81%) and progressive disease in 3 (19%) after six infusions of L19-IL2. Immunophenotyping analysis showed stimulation of NK cells being by far the strongest, followed by T4 cells and CTLs. Pharmacokinetic analysis and HAFA development data will be shown. Conclusions: The part II of this study confirms that L19-IL2 can be safely administered repeatedly in patients with advanced RCC at 22.5 Mio IU IL2 equivalent dose. Preliminary evaluation suggests clinical activity in these mostly pretreated patients. Toxicity was manageable and reversible. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Bayer, Philogen, Philogen SPA Philogen SPA Bayer, Schering AG Philogen SPA

A dose finding pharmacokinetic study of the tumor-targeting human L19-IL2 monoclonal antibody-cytokine fusion protein in patients with advanced solid tumors

3057 Background: L19-IL2 is a tumor targeting immunocytokine constituted by a single chain Fragment variable format directed against ED-B domain of fibronectin and the human cytokine interleukin-2 (IL-2). It has striking anticancer activity in preclinical models. We evaluated safety, pharmacokinetic profile (PK) and activity of L19-IL2 in advanced cancer patients. Patients and Methods: Five cohorts of patients with recurrent/refractory solid tumors received a dose escalation intravenous infusion of L19-IL2 (5, 10, 15, 22.5 e 30 Mio IU IL2 equivalent dose) on days 1, 3 and 5 every 21 days. Serum samples for PK assessment, immunophenotyping and assays for detection of human antifusion protein antibodies to L19 (HAFA) were collected at screening and on day 1, 3, 5 and 10 of each cycle. Results: Eighteen solid cancer patients were enrolled from 11/05 to 9/06 (6 colorectal and 3 renal cell cancer (RCC) patients, 3 with melanoma, and 1 neuroendocrine tumor, thymic carcinoma, biliary tract carcinoma, breast cancer, peritoneal mesothelioma and parotid gland carcinoma). Median age was 49 years (range 35–69), 14 patients were male and 4 female. All patients were evaluable for safety and activity. The maximum tolerated dose was determined to be 22.5 Mio IU IL-2 equivalent. Drug-related dose-limiting toxicities at 30 Mio IU L19-IL2 were hypotension requiring vaso-pressor support and acute renal failure. Treatment-related deaths did not occur, and all toxicities were reversible. Five patients achieved stable disease (RCC, n=3; biliary tract carcinoma, n=1; peritoneal mesothelioma, n=1). All other patients progressed. Immunophenotyping disclosed IL2-typical transient activation of T-cell subsets. Very preliminary data showed a weak induction of HAFA at day 10 in 5 patients. These data could not be confirmed by competition ELISA experiments. Conclusions: Up to 22.5 Mio IU IL2 equivalent of L19-IL2 can be safely administered to advanced solid cancer patients in an outpatient setting. Some evidence of clinical activity in patients susceptible to immunomodulatory therapy (RCC) was found. All observed toxicity was mild and reversible. An expanded study at recommended dose in RCC patients is currently ongoing. [Table: see text]

The immunological basis of tumor therapy by targeted delivery of TNFa to tumor vessels

L19mTNFa is a fusion protein constituted by the scFv L19 specific for the oncofetal ED-B domain of fibronectin and TNFa. Treatment with L19mTNFa, 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 challenges with syngeneic tumor cells of different histological origin. In adoptive immunity transfer experiments the splenocytes from C51-cured mice protected 100% of naive mice both from C51 colon carcinoma and from WEHI-164 fibrosarcoma. The splenocytes from WEHI-164-cured mice protected 100% of mice from the fibrosarcoma and 80% from the C51 colon carcinoma. Similar results were also obtained in adoptive immunity transfer experiments using severely immunodepressed SCID mice. Experiments using depleted splenocytes showed that T cells play a major role in tumor rejection. These data demonstrate that the selective targeting of mTNFa 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 TNFa to tumor vasculature.

Vascular and Cellular Targeting for Photodynamic Therapy

Photodynamic therapy (PDT) involves the combination of photosensitizers (PS) with light as a treatment, and has been an established medical practice for about 10 years. Current primary applications of PDT are age-related macular degeneration (AMD) and several types of cancer and precancer. Tumor vasculature and parenchyma cells are both potential targets of PDT damage. The preference of vascular versus cellular targeting is highly dependent upon the relative distribution of photosensitizers in each compartment, which is governed by the photosensitizer pharmacokinetic properties and can be effectively manipulated by the photosensitizer drug administration and light illumination interval (drug-light interval) during PDT treatment, or by the modification of photosensitizer molecular structure. PDT using shorter PS-light intervals mainly targets tumor vasculature by confining photosensitizer localization within blood vessels, whereas if the sensitizer has a reasonably long pharmacokinetic lifetime, then PDT at longer PS-light intervals can induce more tumor cellular damage, because the photosensitizer has then distributed into the tumor cellular compartment. This passive targeting mechanism is regulated by the innate photosensitizer physicochemical properties. In addition to the passive targeting approach, active targeting of various tumor endothelial and cellular markers has been studied extensively. The tumor cellular markers that have been explored for active photodynamic targeting are mainly tumor surface markers, including growth factor receptors, low-density lipoprotein (LDL) receptors, transferrin receptors, folic acid receptors, glucose transporters, integrin receptors, and insulin receptors. In addition to tumor surface proteins, nuclear receptors are targeted, as well. A limited number of studies have been performed to actively target tumor endothelial markers (ED-B domain of fibronectin, VEGF receptor-2, and neuropilin-1). Intracellular targeting is a challenge due to the difficulty in achieving sufficient penetration into the target cell, but significant progress has been made in this area. In this review, we summarize current studies of vascular and cellular targeting of PDT after more than 30 years of intensive efforts.

Tumor Targeting

Cancer chemotherapy relies on the expectation that anti-cancer drugs will preferentially kill rapidly dividing tumor cells, rather than normal cells. Since a large portion of the tumor cells has to be killed in order to obtain and maintain a complete remission, large doses of drugs are typically used, with significant toxicity towards proliferating non-malignant cells. Our research focuses on the targeted delivery of therapeutic agents to the tumor environment by means of specific ligands (antibodies or small organic binding molecules) to tumor-associated antigens. In most cases, we target accessible antigens (such as the EDB domain of fibronectin or the C domain of tenascin-C) which are abundantly expressed around tumor blood vessels, but virtually undetectable in normal tissues.

Targeting the Vascular Endothelium – Novel Approaches

Department of Chemistry and Applied Biosciences Swiss Federal Institute of Technology Irchel Y36 M14, Winterthurerstr. 190, CH–8057 Zurich (Switzerland) Tel. 41 1 6356063, Fax 41 1 6356886 E-Mail neri@pharma.ethz.ch Angiogenesis, i.e., the proliferation of new blood vessels from pre-existing ones, is an underlying process in many human diseases, including cancer, blinding ocular disorders and rheumatoid arthritis. The ability to selectively target and occlude neovasculature will be potentially useful in diagnosis and treatment of angiogenesis-related diseases. A good-quality marker for both tumoural and nontumoural neovasculature is the extra-domain B (ED-B) of fibronectin, a sequence of 91 aminoacids that can be inserted into the fibronectin molecule by a mechanism of alternative splicing. To date, the production of monoclonal antibodies directly recognising the ED-B domain has not been possible using hybridoma technology, because of tolerance. In collaboration with Prof. Dr. Luciano Zardi (Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy), we have overcome this problem using large synthetic antibody phage libraries, generating several high-affinity antibody fragments specific for fibronectin containing the ED-B domain, and mapping their epitope onto the three-dimensional structure of the antigen. These antibodies stain vascular structures in tumour sections and selectively target tumour neovasculature, as shown in tumour-bearing mice using infrared fluorescence and radioactive techniques. Increased binding affinity leads to improved targeting of tumoural angiogenesis, as demonstrated by biodistributions studies performed using the L19 antibody fragment with affinity for the ED-B domain in the picomolar range and L19 mutants with reduced affinity. The ability of radiolabelled L19 to target tumours in patients with cancer has recently been demonstrated using scintigraphic detection methods. A number of derivatives of scFv(L19), with a therapeutic potential, have been studied in animal models. The antibody, coupled to a photosensitizer, was shown to be able to mediate a complete and selective photo-occlusion of new blood vessels in a rabbit model of ocular angiogenesis. Indeed, the EDB domain of fibronectin is strongly expressed in angiogenesis-related ocular disorders. A fusion of scFv(L19) with truncated tissue factor (a pro-coagulant protein) mediated the rapid intraluminal blood coagulation in tumour blood vessels, but not in normal blood vessels. As a result, a dramatic collapse of the tumour mass could be observed, particularly for large tumour masses. Systemic administration of cytokines, such as IL-2, TNF, GM-CSF or IL-12 can render some non-immunogenic tumours immunogenic, activating a protective immunity. However, systemic administration of cytokines is often associated with severe toxic side-effects, preventing the administration of a curative dose. A possible way of increasing the therapeutic index of cytokines consists of fusing them to antibodies, which mediate a preferential accumulation of the cytokine at the tumour site. Indeed, in the past decade, several groups have reported different antibody-cytokine fusions for different tumour associated Targeting the Vascular Endothelium – Novel Approaches

Discovery and investigation of lead compounds as binders to the Extra‐Domain B of the angiogenesis marker, fibronectin

AbstractAngiogenesis, the growth of new blood vessels from preexisting vessels, is a rare event in the adult (except in the case of the female reproductive cycle), but is a characteristic feature of diseases such as cancer, blinding ocular disorders (e.g., retinopathies), and rheumatoid arthritis. The ED‐B domain of fibronectin, a domain of 91 amino acids, inserted by a mechanism of alternative splicing of the primary transcript into the fibronectin molecule, is a high‐quality marker of angiogenesis and a target for molecular intervention, characterized by a high number of acidic residues on its surface, as well as some solvent‐exposed hydrophobic residues. A library of 113 low molecular‐weight organic compounds, containing both an aromatic moiety and at least one positive charge, was screened for binding to the ED‐B domain, using two‐dimensional heteronuclear NMR spectroscopy. One lead compound, 2,2‐diphenylethylamine, was found that binds specifically to the ED‐B domain, albeit with a dissociation constant in the millimolar range. Chemical modification of this scaffold revealed structural determinants required for binding, as well as amino acid residues in the ED‐B domain responsible for the interaction. The results presented represent the basis for the development of high‐affinity, low molecular‐weight binders by using a linked‐fragment approach or elongating the scaffold by means of combinatorial chemistry. Drug Dev. Res. 58:268–282, 2003. © 2003 Wiley‐Liss, Inc.

Strategies for vascular targeting in tumors.

Strategies for vascular targeting in tumors.

A High-Affinity Human Antibody That Targets Tumoral Blood Vessels

Angiogenesis is a characteristic feature of many aggressive tumors and of other relevant disorders. Molecules capable of specifically binding to new-forming blood vessels, but not to mature vessels, could be used as selective vehicles and would, therefore, open diagnostic and therapeutic opportunities. We have studied the distribution of the ED-B oncofetal domain of fibronectin, a marker of angiogenesis, in four different tumor animal models: the F9 murine teratocarcinoma, SKMEL-28 human melanoma, N592 human small cell lung carcinoma, and C51 human colon carcinoma. In all of these experimental models we observed accumulation of the fibronectin isoform containing the ED-B domain around neovascular structures when the tumors were in the exponentially growing phase, but not in the slow-growing phase. Then we performed biodistribution studies in mice bearing a subcutaneously implanted F9 murine teratocarcinoma, using a high-affinity human antibody fragment (L19) directed against the ED-B domain of fibronectin. Radiolabeled L19, but not an irrelevant anti-lysozyme antibody fragment (D1.3), efficiently localizes in the tumoral vessels. The maximal dose of L19 accumulated in the tumor was observed 3 hours after injection (8.2% injected dose per gram). By virtue of the rapid clearance of the antibody fragment from the circulation, tumor-to-blood ratios of 1.9, 3.7, and 11.8 were obtained at 3, 5, and 24 hours, respectively. The tumor-targeting performance of L19 was not dose-dependent in the 0.7 to 10 microg range of injected antibody. The integral of the radioactivity localized in tumoral vessels over 24 hours was greater than 70-fold higher than the integral of the radioactivity in blood over the same time period, normalized per gram of tissue or fluid. These findings quantitatively show that new-forming blood vessels can selectively be targeted in vivo using specific antibodies, and suggest that L19 may be of clinical utility for the immunoscintigraphic detection of angiogenesis in patients.