Extra-domain B Of Fibronectin Research Articles

Intralesional administration of L19IL2/L19TNF in high-risk locally advanced basal cell carcinoma or cutaneous squamous cell carcinoma.

TPS9617 Background: Basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC) account for 99% of skin neoplasms, with a rising incidence due to increased sun exposure and growing life expectancy. Approximately 0.8-5% of patients (pts) develop high-risk locally advanced (LA) disease, which is treated with surgery, radiotherapy, sonic hedgehog inhibitors, PD1-based immune checkpoint blockade, or chemotherapy. While surgery offers high cure rates, factors such as location, functional and cosmetic impairment, or patient factors including age, comorbidities, and personal preferences may preclude surgery. Intralesional treatment with immunostimulatory drugs represents an attractive therapeutic approach to treat LA BCC and LA cSCC. Here, we investigate a combination of two immunocytokines (Bifikafusp alfa (L19IL2) and Onfekafusp alfa (L19TNF)) targeting the extradomain B of fibronectin (EDB) for the treatment of high-risk locally advanced BCC and cSCC. EDB is an oncofetal antigen that is undetectable in virtually all healthy adult tissues but becomes highly expressed in tumors, including BCC and cSCC. Methods: In a single-arm phase II study (NCT04362722), pts with locally advanced, non-metastatic, node-negative, single, or multifocal BCC or cSCC not eligible for surgery or radiotherapy are treated with four weekly intratumoral administrations of L19IL2/L19TNF. The study is divided into two parts: a first exploratory part with a total of 40 pts with laBCC or lacSCC, and a second part where the laBCC cohort has been expanded to up to 72 pts not eligible for surgery or radiotherapy. The first part of the trial has been completed with 28 laBCC pts and 12 lacSCC pts evaluable for safety and efficacy. The pre-specified activity goal for the first stage of accrual was met, and the second stage of accrual began in September 2023, with a total of 39 BCC and 18 cSCC pts enrolled so far. Clinical trial information: NCT04362722 .

CAR-T Therapy Targets Extra Domain B of Fibronectin Positive Solid Tumor Cells

ABSTRACT Background CAR-T cell immunotherapy has achieved remarkable success in malignant B-cell malignancies, but progress in solid tumors is slow, and one of the key reasons is the lack of ideal targets. Cancer-specific extra domain B of fibronectin (EDB-FN) is widely upregulated in solid tumors and expressed at low levels in normal tissues. Many imaging and targeted cancer therapies based on EDB-FN targets have been developed and tested in clinical trials, making EDB-FN an ideal target for immunotherapy. Methods We constructed two EDB-FN-targeted CAR-Ts based on the peptide APT0 and the single-chain antibody CGS2 in a lentiviral infection manner for the first time. Luciferase cytotoxicity assay to assess CAR-T killing of tumor cells. An enzyme-linked immunosorbent assay was used to detect the release of the cytokine IFN-γ. Fluorescence imaging to evaluate the dynamics of CAR-T cell and tumor cell coculture. Knockdown assays were used to validate the target specificity of CAR-T cells. Results In this research, two CAR-Ts targeting EDB-FN, APT0 CAR-T, and CGS2 CAR-T, were constructed. In vitro, both CAR-T cells produced broad-spectrum killing of multiple EDB-FN-positive solid tumor cell lines and were accompanied by cytokine IFN-γ release. Regarding safety, the two CAR-T cells did not affect T cells’ normal growth and proliferation and were not toxic to HEK-293T human embryonic kidney epithelial cells. Conclusion APT0 CAR-T and CGS2 CAR-T cells are two new CAR-Ts targeting EDB-FN. Both CAR-T cells can successfully identify and specifically kill various EDB-FN-positive solid tumor cells with potential clinical applications.

EDB-FN targeted probes for the surgical navigation, radionuclide imaging, and therapy of thyroid cancer.

Extradomain B of fibronectin (EDB-FN) is a promising diagnostic and therapeutic biomarker for thyroid cancer (TC). Here, we identified a high-affinity EDB-FN targeted peptide named EDBp (AVRTSAD) and developed three EDBp-based probes, Cy5-PEG4-EDBp(Cy5-EDBp), [18F]-NOTA-PEG4-EDBp([18F]-EDBp), and [177Lu]-DOTA-PEG4-EDBp ([177Lu]-EDBp), for the surgical navigation, radionuclide imaging, and therapy of TC. Based on the previously identified EDB-FN targeted peptide ZD2, the optimized EDB-FN targeted peptide EDBp was identified by using the alanine scan strategy. Three EDBp-based probes, Cy5-EDBp, [18F]-EDBp, and [177Lu]-EDBp, were developed for fluorescence imaging, positron emission tomography (PET) imaging, and radiotherapy in TC tumor-bearing mice, respectively. Additionally, [18F]-EDBp was evaluated in two TC patients. The binding affinity of EDBp to the EDB fragment protein (Kd = 14.4 ± 1.4nM, n = 3) was approximately 336-fold greater than that of the ZD2 (Kd = 4839.7 ± 361.7nM, n = 3). Fluorescence imaging with Cy5-EDBp facilitated the complete removal of TC tumors. [18F]-EDBp PET imaging clearly delineated TC tumors, with high tumor uptake (16.43 ± 1.008%ID/g, n = 6, at 1-h postinjection). Radiotherapy with [177Lu]-EDBp inhibited tumor growth and prolonged survival in TC tumor-bearing mice (survival time of different treatment groups: saline vs. EDBp vs. ABRAXANE vs. [177Lu]-EDBp = 8.00 d vs. 8.00 d vs. 11.67 d vs. 22.33 d, ***p < 0.001). Importantly, the first-in-human evaluation of [18F]-EDBp demonstrated that it had specific targeting properties (SUVmax value of 3.6) and safety. Cy5-EDBp, [18F]-EDBp, and [177Lu]-EDBp are promising candidates for the surgical navigation, radionuclide imaging, and radionuclide therapy of TC, respectively.

An Engineered IFNγ-Antibody Fusion Protein with Improved Tumor-Homing Properties.

Interferon-gamma (IFNγ) is one of the central cytokines produced by the innate and adaptive immune systems. IFNγ directly favors tumor growth control by enhancing the immunogenicity of tumor cells, induces IP-10 secretion facilitating (CXCR3+) immune cell infiltration, and can prime macrophages to an M1-like phenotype inducing proinflammatory cytokine release. We had previously reported that the targeted delivery of IFNγ to neoplastic lesions may be limited by the trapping of IFNγ-based products by cognate receptors found in different organs. Here we describe a novel fusion protein consisting of the L19 antibody, specific to the alternatively spliced extra-domain B of fibronectin (EDB), fused to a variant of IFNγ with reduced affinity to its cognate receptor. The product (named L19-IFNγ KRG) selectively localized to tumors in mice, showed favorable pharmacokinetic profiles in monkeys and regained biological activity upon antigen binding. The fusion protein was investigated in two murine models of cancer, both as monotherapy and in combination with therapeutic modalities which are frequently used for cancer therapy. L19-IFNγ KRG induced tumor growth retardation and increased the intratumoral concentration of T cells and NK cells in combination with anti-PD-1.

Effective Combination Immunotherapy through Vessel Normalization Using a Cancer‐Targeting Antiangiogenic Peptide–Antibody Hybrid

AbstractAlthough cancer immunotherapy using immune checkpoint blockade (ICB) has changed the paradigm for treating patients with certain cancers, its therapeutic benefits are limited to approximately one‐fourth of patients, highlighting the potential for combining immunotherapy with another therapeutic modality. Here, a treatment regimen that combines a cancer‐targeting antiangiogenic agent that inhibits angiogenesis within the tumor and ICB to improve therapeutic outcome is reported. The cancer‐targeting antiangiogenic modality is constructed as a hybrid complex, designated HyPEPEDB‐VEGF, comprising a cotinine‐labeled bispecific peptide targeting both extra domain B of fibronectin (EDB) and vascular endothelial growth factor (VEGF) and an anticotinine antibody (Abcot). The resulting HyPEPEDB‐VEGF specifically bound to EDB‐overexpressing CT26 murine colorectal cancer cells and inhibited VEGF‐induced proliferation of human umbilical vascular endothelial cells. Upon intraperitoneal injection, HyPEPEDB‐VEGF preferentially accumulates in CT26 syngeneic tumors and inhibits tumor growth in a dose‐dependent manner. Furthermore, the combination of HyPEPEDB‐VEGF with an anti‐PD‐1 antibody (αPD‐1) in conjunction with dose optimization of the two modalities leads to substantial inhibition of tumor growth without loss of body weight due to vascular normalization within the tumor. These findings suggest that the combination of cancer‐specific antiangiogenic therapy using HyPEPEDB‐VEGF together with ICB may be a feasible approach for effective cancer therapy.

Targeted Delivery of Cabazitaxel Using Cyclic Cell-Penetrating Peptide and Biomarkers of Extracellular Matrix for Prostate and Breast Cancer Therapy

Targeted drug delivery for cancer therapy is an emerging area of research. Cancer cells overexpress certain biomarkers that can be exploited for their targeted therapy. Cyclic cell-penetrating peptides (cCPP) are increasingly assessed for intracellular cargo delivery in cancer cells. In this study, we have conjugated cabazitaxel (CBT) to the cCPP via an ester bond to assist CBT release in the tumor's acidic environment. Integrin targeting (RGDC, TP1) and extra domain B of fibronectin (EDB-Fn) targeting (CTVRTSAD, TP2) peptides were linked to the peptide-drug conjugate (cCPP-CBT) via a disulfide bond to provide targeting ability to the conjugates until they reach the tumor site. Conjugate 11 (TP1-cCPP-CBT) and conjugate 16 (TP2-cCPP-CBT) showed approximately 3-4-fold less antiproliferative activity on integrin and EDB-FN overexpressing cancer cell lines as compared to the CBT analogue used for comparison (CBT-GA, 5). Conjugates (11 and 16) were less toxic (31-34-fold less antiproliferative activity) to the normal human embryonic kidney (HEK-293) cells as compared to CBT. The flow cytometry and quantitative confocal microscopy data further confirm the selective efficacy of conjugates (TP1-cCPP-FAM (10) and TP1-cCPP-FAM (15)) toward biomarker overexpressing cancer cells. Furthermore, the stability and release studies of conjugate 11 revealed its therapeutic potential under different conditions, such as human plasma, different pHs, and redox conditions. This conjugation strategy was proven to enhance chemotherapeutics agents' efficacy and targeting and can be applied to other chemotherapeutic agents.

Extra-domain B of fibronectin as an alternative target for drug delivery and a cancer diagnostic and prognostic biomarker for malignant glioma.

Extra-domain B of fibronectin (EDB-FN) is an alternatively spliced form of fibronectin with high expression in the extracellular matrix of neovascularized tissues and malignant cancer cells. In this study, we evaluated the practicality of using EDB-FN as a biomarker and therapeutic target for malignant gliomas (MGs), representative intractable diseases involving brain tumors.Methods: The microarray- and sequence-based patient transcriptomic database 'Oncopression' and tissue microarray of MG patient tissue samples were analyzed. EDB-FN data were extracted and evaluated from 23,344 patient samples of 17 types of cancer to assess its effectiveness and selectivity as a molecular target. To strengthen the results of the patient data analysis, the utility of EDB-FN as a molecular marker and target for MG was verified using active EDB-FN-targeting ultrasmall lipidic micellar nanoparticles (~12 nm), which had a high drug-loading capacity and were efficiently internalized by MG cells in vitro and in vivo.Results: Brain tumors had a 1.42-fold cancer-to-normal ratio (p < 0.0001), the second highest among 17 cancers after head and neck cancer. Patient tissue microarray analysis showed that the EDB-FN high-expression group had a 5.5-fold higher risk of progression than the EDB-FN low-expression group (p < 0.03). By labeling docetaxel-containing ultrasmall micelles with a bipodal aptide targeting EDB-FN (termed APTEDB-DSPE-DTX), we generated micelles that could specifically bind to MG cells, leading to superior antitumor efficacy of EDB-FN-targeting nanoparticles compared to nontargeting controls.Conclusions: Taken together, these results show that EDB-FN can be an effective drug delivery target and biomarker for MG.

FluoroCalins: engineered lipocalins with novel binding functions fused to a fluorescent protein for applications in biomolecular imaging and detection.

FluoroCalins represent novel bifunctional protein reagents derived from engineered lipocalins fused to a fluorescent reporter protein, here the enhanced green fluorescent protein (eGFP). We demonstrate the construction, facile bacterial production and broad applicability of FluoroCalins using two Anticalin® molecules directed against the tumor vasculature-associated extra domain B of fibronectin (ED-B) and the vascular endothelial growth factor receptor 3, a marker of tumor and lymphangiogenesis. FluoroCalins were prepared with two different spacers: (i) a short Ser3Ala linker and (ii) a long hydrophilic and conformationally unstructured PASylation® polypeptide comprising 200 Pro, Ala and Ser residues. These FluoroCalins were applied for direct target quantification in enzyme-linked immunosorbent assay as well as target detection by flow cytometry and fluorescence microscopy of live and fixed cells, respectively, demonstrating high specificity and signal-to-noise ratio. Hence, FluoroCalins offer a promising alternative to antibody-based reagents for state of the art fluorescent in vitro detection and biomolecular imaging.

EDB-FN Targeted Peptide-Drug Conjugates for Use against Prostate Cancer.

Prostate cancer (PCa) is the most common malignancy in men and is the leading cause of cancer-related male mortality. A disulfide cyclic peptide ligand [CTVRTSADC] 1 has been previously found to target extra domain B of fibronectin (EDB-FN) in the extracellular matrix that can differentiate aggressive PCa from benign prostatic hyperplasia. We synthesized and optimized the stability of ligand 1 by amide cyclization to obtain [KTVRTSADE] 8 using Fmoc/tBu solid-phase chemistry. Optimized targeting ligand 8 was found to be stable in phosphate buffered saline (PBS, pH 6.5, 7.0, and 7.5) and under redox conditions, with a half-life longer than 8 h. Confocal microscopy studies demonstrated increased binding of ligand 8 to EDB-FN compared to ligand 1. Therefore, we hypothesized that the EDB-FN targeted peptides (1 and 8) conjugated with an anticancer drug via a hydrolyzable linker would provide selective cytotoxicity to the cancer cells. To test our hypothesis, we selected both the normal prostate cell line, RWPE-1, and the cancerous prostate cell lines, PC3, DU-145, LNCaP, and C4-2, to evaluate the anticancer activity of synthesized peptide–drug conjugates. Docetaxel (Doce) and doxorubicin (Dox) were used as anticancer drugs. Dox conjugate 13 containing disulfide linkage showed comparable cytotoxicity versus Dox after 72 h incubation in all the cancer cell lines, whereas it was found to be less cytotoxic on RWPE-1, suggesting that it can act as a Dox prodrug. Doce conjugate 14 was found to be less cytotoxic in all the cell lines as compared to drug alone.

Magnetic Resonance Imaging-Guided Drug Delivery to Breast Cancer Stem-Like Cells.

The feasibility of detecting breast cancer stem-like cells (BCSCs) with magnetic resonance imaging using extradomain-B of fibronectin (EDB-FN)-specific peptide (APTEDB )-conjugated thermally cross-linked superparamagnetic iron oxide nanoparticles (APTEDB -TCL-SPIONs) is previously demonstrated. Here, doxorubicin (Dox)-loaded APTEDB -TCL-SPIONs (Dox@APTEDB -TCL-SPIONs) are generated and their theranostic ability in a BCSC xenograft mouse model is assessed. The Dox@APTEDB -TCL-SPIONs enable more efficient delivery of Dox to tumors than nontargeted Dox@TCL-SPIONs. Much greater inhibition of BCSC tumor growth is observed after treatment with the Dox@APTEDB -TCL-SPIONs than with either Dox@TCL-SPIONs or free Dox. Hypointense signals are observed in the majority of the mice in postcontrast but not precontrast T2*-weighted MR images of tumors 7 days after treatment with Dox@APTEDB -TCL-SPIONs. An inverse correlation is observed between signal intensity and both EDB-FN expression and response to chemotherapy. The data indicate Dox@APTEDB -TCL-SPIONs can detect BCSCs within tumors by targeting EDB-FN-expressing cells. These nanoparticles thus have theranostic potential in breast cancer.

Abstract 4552: Tumor-restricted immune modulation by multispecific molecules from the DARPin toolbox

Abstract During the last years, immune-modulating drugs became an important cornerstone in the treatment of cancer patients. In particular, the PD1/PDL-1 and CTLA-4 antagonists revolutionized the field. However, only a limited number of patients benefit from these antagonistic molecules and more combination therapies are on the way to increase the number of patients benefiting from these novel therapies. Among the combinations, drugs that are T-cell or myeloid cell agonists belonging to the TNFR-superfamily show first promising clinical results. However, systemic immune activation bears the risk of severe side effects that will not allow using these powerful drugs at an effective dose. We have developed a new class of DARPin molecules that enable tumor-restricted immune cell activation of TNFR-superfamily agonists in the tumor only, thereby preventing systemic immune-activation. We generated DARPin molecules that bind with high affinity to TNFR-superfamily members (CD134, CD137 and CD40) and DARPin molecules that bind to tumor-specific antigens such as HER2 and EGFR or targets restricted to the tumor stroma compartment like FAP and extra-domain B of fibronectin (ED-B). Using these building blocks from our DARPin toolbox, we constructed a variety of multi-specific molecules consisting of a TNFR-superfamily receptor targeting DARPin molecule and a tumor-localizing DARPin molecule. In reporter cell assays the multi-specific DARPin molecules activate the respective TNF-superfamily receptor only in the presence of a cell expressing the tumor-localizer; e.g. the FAP-CD134 molecule activates CD134 only in the presence of a stromal cell expressing FAP and not in its absence. Moreover, activation of CD134 was dependent on the density of FAP expression on cells, showing that CD134 only becomes activated if a certain level of the tumor-localizing target is expressed. These finding were confirmed in experiments with primary immune cells where we see immune-cell activation only upon binding to the tumor-localizing target. This could be shown for multiple combinations (&amp;gt;8) and supports the concept that multi-specific DARPin molecules are powerful drug-candidates allowing tumor-restricted immune cell activation. Citation Format: Ulrike Fiedler, Christian Reichen, Joanna Taylor, Patricia Schildknecht, Sophie Barsin, Clara Metz, Anja Schlegel, Simon Fontaine, Denis Villemagne, Julia Ahlskog, Yvonne Kaufmann, Alexander Link, Nicolo Rigamonti, Julia Hepp, Michael T. Stumpp. Tumor-restricted immune modulation by multispecific molecules from the DARPin toolbox [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 4552.

Targeted Therapy for Breast Cancer Stem Cells by Liposomal Delivery of siRNA against Fibronectin EDB.

Targeted therapy for breast cancer stem cell (BCSC): A novel liposomal system (APTEDB -LS-siRNA(EDB) ) that enables simultaneous targeting and knockdown of extra domain B of fibronectin (EDB-FN) shows potent therapeutic efficacy in the BCSC-derived tumors in vivo.

Potentiating the activity of rituximab against mantle cell lymphoma in mice by targeting interleukin-2 to the neovasculature

There is increasing interest in the site-directed pharmacodelivery of therapeutic payloads to the tumor site using antibodies as transport vehicles. Here, we investigated the efficacy of L19-IL2, an antibody–cytokine fusion protein that specifically delivers IL-2 to the tumor site by homing to the extra-domain B of fibronectin (EDB-Fn) expressed on tumor-associated blood vessels, against mantle cell lymphoma (MCL) in mice. L19-IL2 was shown to selectively localize at lymphoma lesions in vivo and to mediate significant lymphoma growth retardation, which was potentiated by co-administration of the anti-CD20 antibody rituximab. When co-injected with rituximab, L19-IL2 induced complete remissions of localized MCL xenografts in 6/8 mice (75%), whereas the combination of rituximab and equivalent doses of non-targeted IL-2 only slightly delayed tumor growth. In disseminated MCL, combination therapy with L19-IL2 and rituximab exhibited a significant survival benefit over treatment with IL-2 and rituximab and completely eradicated the disease in 2/7 cases (28.6%). Mechanistically, histological analyses of post-therapeutic lymphoma tissues revealed a strong intratumoral accumulation of macrophages and natural killer cells after a single dose of the immunocytokine, whereas L19-IL2 had no significant impact on microvessel density or on tissue penetration of co-injected rituximab. Collectively, these results provide the scientific rationale for the clinical evaluation of L19-IL2 in combination with anti-CD20 immunotherapy in patients with MCL.

MRI of breast tumor initiating cells using the extra domain-B of fibronectin targeting nanoparticles.

The identification of breast tumor initiating cells (BTICs) is important for the diagnosis and therapy of breast cancers. This study was undertaken to evaluate whether the extra domain-B of fibronectin (EDB-FN) could be used as a new biomarker for BTICs and whether EDB-FN targeting superparamagnetic iron oxide nanoparticles (SPIONs) could be used as a magnetic resonance imaging (MRI) contrast agent for BTIC imaging in vitro and in vivo. BTICs (NDY-1) exhibited high EDB-FN expression, whereas non-BTICs (MCF-7, BT-474, SUM-225, MDA-MB-231) did not exhibit EDB-FN expression. Furthermore, Cy3.3-labeled EDB-FN specific peptides (APTEDB) showed preferential binding to the targeted NDY-1 cells. To construct an EDB-FN targeted imaging probe, APTEDB was covalently attached to a thermally cross-linked SPION (TCL-SPION) to yield APTEDB-TCL-SPION. In the in vitro MRI of cell phantoms, selective binding of APTEDB-TCL-SPION to NDY-1 cells was evident, but little binding was observed in MCF-7 cells. After the intravenous injection of APTEDB-TCL-SPION into the NDY-1 mouse tumor xenograft model, a significant decrease in the signal within the tumor was observed in the T2*-weighted images; however, there was only a marginal change in the signal of non-targeting SPIONs such as APTscramble-TCL-SPION or TCL-SPION. Taken together, we report for the first time that EDB-FN was abundantly expressed in BTICs and may therefore be useful as a new biomarker for identifying BTICs. Our study also suggests that APTEDB-TCL-SPION could be used as an MRI contrast agent for BTIC imaging.

Abundant in vitro expression of the oncofetal ED-B-containing fibronectin translates into selective pharmacodelivery of 131I-L19SIP in a prostate cancer patient

The extradomain B of fibronectin (ED-B) is a promising vascular target for selective pharmacodelivery in cancer patients. We analyzed a large series of prostatectomies from patients with prostate cancer, hyperplastic prostate disease, and normal prostates to study extent and tumor-selectivity of ED-B expression. Using immunohistology, 68 adenocarcinomas of the prostate or prostate cancer-inflicted lymph nodes, 4 samples of benign prostatic hyperplasia, and 6 normal prostate glands were studied for ED-B expressing newly formed blood vessels. Further, we treated an advanced prostate cancer patient with the anti-ED-B antibody (131)I-L19SIP to study in vivo target accessibility. ED-B-positive blood vessels were found significantly more frequent in prostate cancers as compared with peritumoral prostate tissues or normal prostate glands, independent of tumor differentiation. The ED-B-positive blood vessels' density was 97 (±23), 65 (±9), and 59 (±9)/mm(2) in G3, G2, and G1 prostate cancers, respectively, and 7 (±5)/mm(2) in normal prostate glands. In high-grade (G3) prostate cancers, also the peritumoral tissue showed a higher density of ED-B vessels than normal prostate glands. Similar results were obtained when ED-B-positive vessel density was expressed as a fraction of CD34-positive vessel density. Finally, selective uptake of ED-B-binding (131)I-L19SIP to tumor lesions was found in an advanced prostate cancer patient by whole-body planar scintigraphy. ED-B-positive blood vessels were found to a large extent in prostate cancer tissues, but only rarely in normal prostates or benign prostatic hyperplasia. Whole-body planar scintigraphy in a prostate cancer patient confirmed selective uptake of (131)I-L19SIP in the prostate cancer tissues, qualifying ED-B as a promising target for selective pharmacodelivery of anticancer agents in prostate cancer.

Targeting Interleukin-2 to the Neovasculature Potentiates Rituximab‘s Activity Against Mantle Cell Lymphoma in Mice

AbstractAbstract 3716 Introduction:Antibody-based tumor targeting strategies promise to specifically target anti-neoplastic agents (e.g. radionuclides, drugs, cytokines) to the tumor site while sparing healthy tissues. In this context, the selective delivery of bioactive molecules to the vasculature seems particularly attractive, considering the fact that antigens expressed in blood vessels are the most accessible structures within a tumor mass and that a vigorous neo-vasculature development is a common feature of virtually all solid and hematologic malignancies. Vascular targeting antibodies functionalized with different payloads are currently being investigated in patients with solid tumors and are increasingly being considered also for the treatment of hematological malignancies. In the present work, we evaluated the efficacy of the fusion protein L19-IL2, comprising the human monoclonal antibody fragment scFv(L19) linked to the cytokine Interleukin-2 (IL-2), against Mantle Cell Lymphoma (MCL), a disease for which existing therapeutic approaches still remain highly unsatisfactory. Methods:Expression of the extra-domain B of fibronectin (EDB-Fn), a well-characterized marker of angiogenic blood vessels, was analyzed in Granta-519 and HBL-2 MCL xenografts and human MCL specimens using immunohistochemistry and dual-color immunofluorescence. The in vivo targeting performance of the EDB-Fn targeting immunocytokine L19-IL2 was investigated using ex vivo immunofluorescence analyses. SCID mice xenografted subcutaneously with Granta-519 or intravenously with HBL-2 MCL cells were used for mono- and combination therapy experiments, featuring i.v. injections of L19-IL2 (30 μg, corresponding to 9.9 μg or 177000 IU rIL-2 equivalents), unconjugated rIL-2 (9.9 μg), the anti-CD20 antibody rituximab (200 μg), rituximab plus IL-2, rituximab plus L19-IL2 or saline. Results:In vivo, L19-IL2 selectively homed to EDB-Fn expressing angiogenic blood vessels, thereby mediating the accumulation of IL-2 in MCL lesions. In mono-therapy experiments, L19-IL2 showed a superior anti-lymphoma activity compared to equimolar amounts of non-targeted recombinant IL-2. In combination with rituximab, L19-IL2 induced complete remissions (CR) of established subcutaneous Granta-519 tumors in 75 % (6/8) of the cases, whereas rituximab alone or in combination with free IL-2 only delayed lymphoma progression. Mice that experienced lymphoma relapse after having achieved a CR responded a second time to L19-IL2 and rituximab treatment. In the systemic HBL-2 MCL model, the combination of L19-IL2 and rituximab almost doubled survival of mice as compared to IL-2 and rituximab (median survival 63 vs. 33 days). Conclusions:In summary, we show that the vascular targeting immunocytokine L19-IL2 promotes a potent anti-lymphoma response in combination with anti-CD20 immunotherapy. The clinical evaluation of this combination is facilitated by the fact that L19-IL2 is entirely human and already being studied in Phase II clinical trials in patients with solid tumors. Disclosures:Menssen:Philogen: Employment. Neri:Philogen: Dario Neri is a co-founder and shareholder of Philogen, the biotech company which has licensed the L19 antibody from the ETH Zurich Other, Equity Ownership.

Local delivery of IL-2 reduces atherosclerosis via expansion of regulatory T cells

Objective Recent studies indicate that regulatory T cells (Tregs) attenuate murine atherosclerosis. Since interleukin (IL)-2 induces Tregs proliferation, we tested the impact of L19-IL2, a fusion antibody specific to extra-domain B of fibronectin (ED-B) containing an active human IL-2 molecule, in experimental atherosclerosis. Methods and results L19-IL2 or appropriate controls were given intravenously to 6 month old Western diet-fed apoE −/− mice on day 1, 3, and 5. Human IL-2 was detected on day 7 within atherosclerotic plaques of L19-IL2-treated mice, and magnetic resonance imaging of the plaques showed a significant adventitial gadolinium enhancement on day 7 and 13, suggesting microvascular leakage as a result of the pharmacodynamic activity of L19-IL2. Treatment with L19-IL2 significantly reduced the size of pre-established atherosclerotic plaques at the thoracic aorta (Sudan III stained area) and in the aortic root area (microscopic, morphometric analysis) on day 7 as compared to controls (L19, D1.3-IL2, NaCl) as well as compared to baseline (day 0). Tregs markers Foxp3 and CTLA4 were highly increased in plaques after L19-IL2 treatment compared to controls ( p < 0.01), whereas the macrophage marker Mac3 was significantly reduced ( p < 0.03). Co-treatment with IL-2-receptor blocking antibody PC61 abrogated L19-IL2-induced plaque reduction compared with IgG control ( p < 0.03). Conclusion L19-IL2 delivers functional IL-2 to pre-established atherosclerotic plaques of WD-fed apoE −/− mice resulting in significant plaque size reduction mediated by local Tregs.

Radioimmunotherapy (RIT) with 131l-L19SIP in solid cancers (SC) and lymphoproliferative diseases: Final results of the first human trial.

2523 Background: The extradomain B of fibronectin (EDBF) is a promising target for cancer therapies due to its selective expression in newly formed blood vessels of SC and lymphomas. L19 is a human...

Tumor-targeting human L19IL2 monoclonal antibody-cytokine fusion protein in combination with DTIC in chemotherapy-naïve stage IV melanoma patients.

8555 Background: L19IL2 is a tumor targeted immunocytokine constituted by a single chain Fragment variable (scFv) format directed against the extradomain-B of fibronectin and the human cytokine interleukin-2 (IL2). The recommended dose for monotherapy of advanced solid cancer patients was established to be 22.5 Mio IU L19IL2 on days 1, 3, and 5 of a 21-day cycle in a previous dose escalation study. Methods: Chemotherapy-naive patients with advanced melanoma and normal LDH were treated with L19IL2 as an i.v. infusion at a dosage of 22.5 Mio IU L19IL2 on days 1, 3 and 5 every 21 days in combination with DTIC 1,000 mg/m2 on day 1 for up to 6 treatment cycles. In the subsequent maintenance phase 22.5 Mio IU L19IL2 was applied biweekly, while DTIC was omitted. Tumor assessment was performed every 6 weeks. Data on safety and efficacy were evaluated using CTC v3.0 and RECIST criteria, respectively. Results: 22 patients were evaluable. Median age was 54.5 years (20-83). Treatment was well tolerated. There were no treatment related deaths; details of CTC evaluation will be presented. Median number of applied induction cycles was 6 (range 2-6), 6/22 patients entered the maintenance phase. Several RECIST-confirmed tumor responses and durable disease stabilizations were observed. Updated efficacy data will be available for presentation. Conclusions: L19IL2 at a dose of 22.5 Mio IU L19IL2 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. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Philogen Philogen, Bayer Philogen

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

We report a novel strategy to engineer and express stable and soluble human recombinant polyvalent/polyspecific fusion proteins. The procedure is based on the use of a central skeleton of uteroglobin, a small and very soluble covalently linked homodimeric protein that is very resistant to proteolytic enzymes and to pH variations. Using a human recombinant antibody (scFv) specific for the angiogenesis marker domain B of fibronectin, interleukin 2, and an scFv able to neutralize tumor necrosis factor-alpha, we expressed various biologically active uteroglobin fusion proteins. The results demonstrate the possibility to generate monospecific divalent and tetravalent antibodies, immunocytokines, and dual specificity tetravalent antibodies. Furthermore, compared with similar fusion proteins in which uteroglobin was not used, the use of uteroglobin improved properties of solubility and stability. Indeed, in the reported cases it was possible to vacuum dry and reconstitute the proteins without any aggregation or loss in protein and biological activity.