Katharina Frey

Selection of Carbonic Anhydrase IX Inhibitors from One Million DNA-Encoded Compounds

DNA-encoded chemical libraries, i.e., collections of compounds individually coupled to distinctive DNA fragments serving as amplifiable identification barcodes, represent a new tool for the de novo discovery of small molecule ligands to target proteins of pharmaceutical interest. Here, we describe the design and synthesis of a novel DNA-encoded chemical library containing one million small molecules. The library was synthesized by combinatorial assembly of three sets of chemical building blocks using Diels-Alder cycloadditions and by the stepwise build-up of the DNA barcodes. Model selections were performed to test library performance and to develop a statistical method for the analysis of high-throughput sequencing data. A library selection against carbonic anhydrase IX revealed a new class of submicromolar bis(sulfonamide) inhibitors. One of these inhibitors was synthesized in the absence of the DNA-tag and showed accumulation in hypoxic tumor tissue sections in vitro and tumor targeting in vivo.

New Strategy for the Extension of the Serum Half-Life of Antibody Fragments

Antibody fragments can recognize their cognate antigen with high affinity and can be produced at high yields, but generally display rapid blood clearance profiles. For pharmaceutical applications, the serum half-life of antibody fragments is often extended by chemical modification with polymers or by genetic fusion to albumin or albumin-binding polypeptides. Here, we report that the site-specific chemical modification of a C-terminal cysteine residue in scFv antibody fragments with a small organic molecule capable of high-affinity binding to serum albumin substantially extends serum half-life in rodents. The strategy was implemented using the antibody fragment F8, specific to the alternatively spliced EDA domain of fibronectin, a tumor-associated antigen. The unmodified and chemically modified scFv-F8 antibody fragments were studied by biodistribution analysis in tumor-bearing mice, exhibiting a dramatic increase in tumor uptake for the albumin-binding antibody derivative. The data presented in this paper indicate that the chemical modification of the antibody fragment with the 2-(3-maleimidopropanamido)-6-(4-(4-iodophenyl)butanamido)hexanoate albumin-binding moiety may represent a general strategy for the extension of the serum half-life of antibody fragments and for the improvement of their in vivo targeting performance.

Antibody-Based Delivery of Interleukin-2 to Neovasculature Has Potent Activity Against Acute Myeloid Leukemia

Neovascular structures in acute myeloid leukemia can be targeted using a cognate antibody fused to human interleukin-2. The Blood Mobile For solid tumors, new blood vessel formation—angiogenesis—is thought to be critical to bring oxygen and nutrients to all parts of the tumors, and targeting these new vessels has long been a focus of cancer therapy development. However, angiogenesis-targeting therapy has been relatively neglected in the context of blood-borne tumors like leukemias. Now, Gutbrodt et al. find that targeting neovascular structures found in the bone marrow of patients with acute myeloid leukemia (AML) has activity against blood-borne cancers as well. The authors observed that AML patients have increased blood vessel density in the bone marrow, and that these vessels could be stained by clinical-stage human antibodies against tumor angiogenesis markers. They then took this observation into mouse models and found that if they fused interleukin-2 to the targeting antibody, they could inhibit AML progression. The effect was further enhanced by combination with the chemotherapeutic cytarabine. Indeed, this effect was long-lasting in immunocompetent mice and was mediated by both natural killer and CD8+ T cells. Early results from clinical trials support these results in human patients, although further trials are needed to confirm efficacy. Acute myeloid leukemia (AML) is a rapidly progressing disease that is accompanied by a strong increase in microvessel density in the bone marrow. This observation prompted us to stain biopsies of AML and acute lymphoid leukemia (ALL) patients with the clinical-stage human monoclonal antibodies F8, L19, and F16 directed against markers of tumor angiogenesis. The analysis revealed that the F8 and F16 antibodies strongly stained 70% of AML and 75% of ALL bone marrow specimens, whereas chloroma biopsies were stained with all three antibodies. Therapy experiments performed in immunocompromised mice bearing human NB4 leukemia with the immunocytokine F8-IL2 [consisting of the F8 antibody fused to human interleukin-2 (IL-2)] mediated a strong inhibition of AML progression. This effect was potentiated by the addition of cytarabine, promoting complete responses in 40% of treated animals. Experiments performed in immunocompetent mice bearing C1498 murine leukemia revealed long-lasting complete tumor eradication in all treated mice. The therapeutic effect of F8-IL2 was mediated by both natural killer cells and CD8+ T cells, whereas CD4+ T cells appeared to be dispensable, as determined in immunodepletion experiments. The treatment of an AML patient with disseminated extramedullary AML manifestations with F16-IL2 (consisting of the F16 antibody fused to human IL-2, currently being tested in phase 2 clinical trials in patients with solid tumors) and low-dose cytarabine showed significant reduction of AML lesions and underlines the translational potential of vascular tumor–targeting antibody-cytokine fusions for the treatment of patients with leukemia.

Antibody-based targeting of interferon-alpha to the tumor neovasculature: a critical evaluation

The antibody-mediated targeted delivery of cytokines, growth factors and immunomodulators offers great potential for the therapy of cancer and other serious conditions. Interferon-alpha has long been used in the clinic for the treatment of patients with certain malignancies or with viral disease. Promising anticancer activity has recently been reported for two fusion proteins consisting of immunoglobulins bearing the interferon-alpha polypeptide at the C-terminal end of the molecule. Here we describe the design, production and characterization of a novel immunocytokine, in which murine interferon-alpha2 was sequentially fused with the tumor-targeting antibody fragment scFv(F8), specific to the alternatively-spliced EDA domain of fibronectin. The resulting fusion protein (F8-IFNa) could be produced to homogeneity and was shown to retain both antigen binding activity and interferon-alpha activity. Biodistribution studies in tumor-bearing mice with radioiodinated protein preparations confirmed the ability of F8-IFNa to selectively localize at the tumor site. However, using two different murine models of cancer (F9 teratocarcinomas and Cloudman S91 melanomas in immunocompetent mice), we could not detect a striking superiority for the therapeutic performance of F8-IFNa as compared to KSF-IFNa, a fusion protein of irrelevant specificity in the mouse which was used as negative control. In the paper, we present hypotheses why the antibody-based pharmacodelivery of interferon-alpha fails to eradicate tumors, in contrast to the situation observed by our group for other immunocytokines, which benefit from a selective localization at the tumor site.

The Immunocytokine F8-IL2 Improves the Therapeutic Performance of Sunitinib in a Mouse Model of Renal Cell Carcinoma

PURPOSE We investigated the therapeutic action of F8-IL2, a fusion protein consisting of the F8 antibody specific to the alternatively spliced extradomain-A of fibronectin, in diabody format and of human interleukin-2 in the Caki-1 (ATCC®) model of human renal cell carcinoma grafted subcutaneously in nude mice. MATERIALS AND METHODS F8-IL2 was cloned, expressed in CHO cells and purified to homogeneity. This immunocytokine was administered alone or combined with 3 standard drugs commonly used as therapy for kidney cancer, including sunitinib, sorafenib and interferon-α, in 2 sets of doses and treatment schedules. RESULTS Neither F8-IL2 nor any other therapeutic agent cured tumor bearing mice when used as a single agent. The best therapeutic results were observed for the combination of sunitinib with F8-IL2 in a continuous administration schedule, which yielded a 28% cure rate and substantial tumor growth retardation. CONCLUSIONS Considering that recombinant interleukin-2 based immunocytokines are now being investigated in several clinical trials in patients with cancer alone or combined with chemotherapy our preclinical results provide a motivation to study F8-IL2 combined with sunitinib in clinical trials in patients with kidney cancer.

Comparative in vivo analysis of the atherosclerotic plaque targeting properties of eight human monoclonal antibodies

OBJECTIVE The selective in vivo localization of antibody derivatives in atherosclerotic plaques may open novel diagnostic and therapeutic applications. Here, we present a comparative in vivo localization analysis of eight radioiodinated human monoclonal antibodies in apolipoprotein E-deficient (ApoE(-/-)) mice. METHODS Animals were fed with a cholesterol-rich diet, followed by harvesting of the aorta 24h after intravenous antibody injection and investigated by autoradiographic analysis. Localization of F8 antibody on atherosclerotic plaque structures was further studied in three-color fluorescence microscopy. RESULTS The study revealed that the F8 antibody, specific to the alternatively spliced EDA domain of fibronectin, exhibited the highest plaque-targeting potential among the antibodies analyzed in this study, with an ability to preferentially localize to all plaques within the aorta. Targeting results were confirmed by injection of fluorescein-labeled F8 antibody, followed by three-color fluorescence microscopy analysis. CONCLUSION These findings open novel biomolecular avenues for the in vivo imaging of atherosclerotic plaques and for pharmacodelivery applications, since F8 had previously been reported by our group to strongly stain atherosclerotic plaques in human carotid arteries.

Different patterns of fibronectin and tenascin-C splice variants expression in primary and metastatic melanoma lesions.

Abstract:  We have investigated the staining patterns of primary and metastatic melanoma lesions using F8, L19 and F16. These three clinical‐stage antibodies are currently being studied in clinical trials for the pharmacodelivery of cytokines or therapeutic radionuclides to neoplastic sites in patients with cancer. Frozen sections of 24 primary and 29 metastatic melanoma lesions were stained, using immunofluorescence procedures, with biotinylated preparations of the F8, L19 and F16 antibodies, which are specific to the alternatively spliced extra domain A and extra domain B domains of fibronectin and A1 domain of tenascin‐C, respectively. Blood vessels were costained using von Willebrand factor–specific antibodies. In primary cutaneous melanoma lesions, F16 and F8 (but not L19) strongly stained the basal lamina at the interface between epidermis and dermis, with a strikingly complementary pattern. By contrast, metastatic melanoma lesions displayed a strong and diffuse pattern of immunoreactivity with all three antibodies. It was found that the extracellular matrix in melanoma undergoes extensive remodelling during the transition from primary to metastatic lesions. The intense staining of metastatic melanoma lesions by the F8, L19 and F16 antibodies provides a strong rationale for the use of these antibodies and their derivatives for the treatment of melanoma patients and possibly for the personalized choice of the best performing antibody in individual patients.

The targeted delivery of IL17 to the mouse tumor neo-vasculature enhances angiogenesis but does not reduce tumor growth rate

There has been a long controversy as to whether interleukin-17 (IL17) has an impact on tumor growth. In order to assess whether IL17 may affect tumor growth, it would be convenient to achieve high levels of this pro-inflammatory cytokine at the tumor neo-vasculature, since IL17 is known to promote angiogenesis. Here, we have generated and tested in vivo a fusion protein, consisting of the F8 antibody (specific to the alternatively spliced EDA domain of fibronectin, a marker of angiogenesis) and of murine IL17 (mIL17). The resulting immunocytokine (termed F8-mIL17) was shown to selectively localize at the tumor neo-vasculature and to vigorously promote tumor angiogenesis, without however reducing or enhancing tumor growth rate both in immunocompetent and in immunodeficient mice.

Antibody-Based Targeting of Tumor Vasculature and Stroma

Conventional cytotoxic therapies of cancer often suffer from a lack of specificity, leading to a poor therapeutic index and toxicities to normal organs. The targeted delivery of bioactive molecules (cytokines, radionuclides, drugs, photosensitizer, etc.) by means of an antibody specific to tumor-associated markers of the tumor stroma and endothelium offers an elegant way to overcome these drawbacks. In fact, tumor markers of the endothelium and extracellular matrix are particularly attractive, because of their accessibility for intravenously administered agents, their genomic stability and the various therapeutic options they offer, ranging from direct tumor cell killing, to tumor infarction and to the recruitment of immune cells.