Francesca Pretto

A Small‐Molecule Drug Conjugate for the Treatment of Carbonic Anhydrase IX Expressing Tumors

Antibody-drug conjugates are a very promising class of new anticancer agents, but the use of small-molecule ligands for the targeted delivery of cytotoxic drugs into solid tumors is less well established. Here, we describe the first small-molecule drug conjugates for the treatment of carbonic anhydrase IX expressing solid tumors. Using ligand-dye conjugates we demonstrate that such molecules can preferentially accumulate inside antigen-positive lesions, have fast targeting kinetics and good tumor-penetrating properties, and are easily accessible by total synthesis. A disulfide-linked drug conjugate with the maytansinoid DM1 as the cytotoxic payload and a derivative of acetazolamide as the targeting ligand exhibited a potent antitumor effect in SKRC52 renal cell carcinoma in vivo. It was furthermore superior to sunitinib and sorafenib, both small-molecule standard-of-care drugs for the treatment of kidney cancer.

Curative Properties of Noninternalizing Antibody–Drug Conjugates Based on Maytansinoids

It is generally thought that the anticancer efficacy of antibody-drug conjugates (ADC) relies on their internalization by cancer cells. However, recent work on an ADC that targets fibronectin in the tumor microenvironment suggests this may not be necessary. The alternatively spliced extra domains A and B (EDA and EDB) of fibronectin offer appealing targets for ADC development, because the antigen is strongly expressed in many solid human tumors and nearly undetectable in normal tissues except for the female reproductive system. In this study, we describe the properties of a set of ADCs based on an antibody targeting the alternatively spliced EDA of fibronectin coupled to one of a set of potent cytotoxic drugs (DM1 or one of two duocarmycin derivatives). The DM1 conjugate SIP(F8)-SS-DM1 mediated potent antitumor activity in mice bearing DM1-sensitive F9 tumors but not DM1-insensitive CT26 tumors. Quantitative biodistribution studies and microscopic analyses confirmed a preferential accumulation of SIP(F8)-SS-DM1 in the subendothelial extracellular matrix of tumors, similar to the pattern observed for unmodified antibody. Notably, we found that treatments were well tolerated at efficacious doses that were fully curative and compatible with pharmaceutical development. Our findings offer a preclinical proof-of-concept for curative ADC targeting the tumor microenvironment that do not rely upon antigen internalization.

Paclitaxel Enhances Therapeutic Efficacy of the F8-IL2 Immunocytokine to EDA-Fibronectin–Positive Metastatic Human Melanoma Xenografts

The selective delivery of bioactive agents to tumors reduces toxicity and enhances the efficacy of anticancer therapies. In this study, we show that the antibody F8, which recognizes perivascular and stromal EDA-fibronectin (EDA-Fn), when conjugated to interleukin-2 (F8-IL2) can effectively inhibit the growth of EDA-Fn-expressing melanomas in combination with paclitaxel. We obtained curative effects with paclitaxel administered before the immunocytokine. Coadministration of paclitaxel increased the uptake of F8 in xenografted melanomas, enhancing tumor perfusion and permeability. Paclitaxel also boosted the recruitment of F8-IL2-induced natural killer (NK) cells to the tumor, suggesting a host response as part of the observed therapeutic benefit. In support of this likelihood, NK cell depletion impaired the antitumor effect of paclitaxel plus F8-IL2. Importantly, this combination reduced both the tumor burden and the number of pulmonary metastatic nodules. The combination did not cause cumulative toxicity. Together, our findings offer a preclinical proof that by acting on the tumor stroma paclitaxel potentiates the antitumor activity elicited by a targeted delivery of IL2, thereby supporting the use of immunochemotherapy in the treatment of metastatic melanoma.

A chemically modified antibody mediates complete eradication of tumours by selective disruption of tumour blood vessels

Background:The possibility of eradicating cancer by selective destruction of tumour blood vessels may represent an attractive therapeutic avenue, but most pharmaceutical agents investigated so far did not achieve complete cures and are not completely specific. Antibody conjugates now allow us to evaluate the impact of selective vascular shutdown on tumour viability and to study mechanisms of action.Methods:We synthesised a novel porphyrin-based photosensitiser suitable for conjugation to antibodies and assessed anticancer properties of its conjugate with L19, a clinical-stage human monoclonal antibody specific to the alternatively spliced EDB domain of fibronectin, a marker of tumour angiogenesis.Results:Here we show in two mouse model of cancer (F9 and A431) that L19 is capable of highly selective in vivo localisation around tumour blood vessels and that its conjugate with a photosensitiser allows selective disruption of tumour vasculature upon irradiation, leading to complete and long-lasting cancer eradication. Furthermore, depletion experiments revealed that natural killer cells are essential for the induction of long-lasting complete responses.Conclusions:These results reinforce the concept that vascular shutdown can induce a curative avalanche of tumour cell death. Immuno-photodynamic therapy may be particularly indicated for squamous cell carcinoma of the skin, which we show to be strongly positive for markers of angiogenesis.

The Antibody-Based Delivery of Interleukin-12 to the Tumor Neovasculature Eradicates Murine Models of Cancer in Combination with Paclitaxel

Purpose: Interleukin-12 (IL12) is a potent proinflammatory cytokine with antitumor activity. Its heterodimeric nature makes it compatible with a large variety of different immunocytokine formats. Here we report the design, production, and characterization of a novel immunocytokine, based on the fusion of the F8 antibody (specific to the alternatively spliced EDA domain of fibronectin, a marker of tumor neovasculature) with IL12 (termed IL12-F8-F8). Experimental Design: We developed a novel immunocytokine based on the sequential fusion of interleukin-12 as a single polypeptide with two F8 antibodies in single-chain Fv (scFv) format. The fusion protein was characterized in vitro, and its targeting performance was assessed in vivo. The immunocytokine antitumor activity was studied as monotherapy as well as in combination therapies in three different murine tumor models. Moreover, depletion experiments and tumor analysis revealed a dominant role of natural killer cells for the mechanism of action. Results: IL12-F8-F8 can be produced in mammalian cells, yielding a product of good pharmaceutical quality, capable of selective localization on the tumor neovasculature in vivo, as judged by quantitative biodistribution analysis with radioiodinated protein preparations. The protein potently inhibited tumor growth in three different immunocompetent syngeneic models of cancer. The treatment was generally well tolerated. Moreover, the IL12-F8-F8 fusion protein could be produced both with murine IL12 (mIL12) and with human IL12 (hIL12). Conclusions: The potent antitumor activity of mIL12-F8-F8, studied alone or in combination with paclitaxel in different tumor models, paves the way to the clinical development of the fully human immunocytokine. Clin Cancer Res; 18(15); 4092–103. ©2012 AACR.

Determination of Paclitaxel Distribution in Solid Tumors by Nano-Particle Assisted Laser Desorption Ionization Mass Spectrometry Imaging

A sensitive, simple and reproducible protocol for nanoparticle-assisted laser desorption/ionization mass spectrometry imaging technique is described. The use of commercially available TiO2 nanoparticles abolishes heterogeneous crystallization, matrix background interferences and enhances signal detection, especially in the low mass range. Molecular image normalization was based on internal standard deposition on tissues, allowing direct comparison of drug penetration and distribution between different organs and tissues. The method was applied to analyze the distribution of the anticancer drug paclitaxel, inside normal and neoplastic mouse tissue sections. Spatial resolution was good, with a linear response between different in vivo treatments and molecular imaging intensity using therapeutic drug doses. This technique distinguishes the different intensity of paclitaxel distribution in control organs of mice, such as liver and kidney, in relation to the dose. Animals treated with 30 mg/kg of paclitaxel had half of the concentration of those treated with 60 mg/kg. We investigated the spatial distribution of paclitaxel in human melanoma mouse xenografts, following different dosage schedules and found a more homogeneous drug distribution in tumors of mice given repeated doses (5×8 mg/kg) plus a 60 mg/kg dose than in those assigned only a single 60 mg/kg dose. The protocol can be readily applied to investigate anticancer drug distribution in neoplastic lesions and to develop strategies to optimize and enhance drug penetration through different tumor tissues.

A bivalent small molecule-drug conjugate directed against carbonic anhydrase IX can elicit complete tumour regression in mice

There is a pressing need for the development of innovative chemical drug delivery strategies in oncology, since conventional chemotherapeutic agents typically do not localise to solid tumours in vivo. It is widely accepted that bivalent antibody formats accumulate in tumours more strongly than monovalent ones and that they should thus be preferred for antibody-based pharmacodelivery approaches. For small molecule-drug conjugates this is less clear. Here, we show that a bivalent ligand against the tumour marker carbonic anhydrase IX leads to an improved tumour-targeting performance compared with the corresponding monovalent counterpart in the SKRC52 model of constitutively CAIX-positive renal cell carcinoma. A bivalent disulfide-linked small drug conjugate with the potent cytotoxic maytansinoid DM1 as the payload can mediate complete eradication of the same tumours, which are resistant to standard-of-care therapeutics, in a proportion of treated mice. In the A375 melanoma model, which preferentially expresses CAIX at sites distant to blood vessels, no measurable tumour accumulation could be observed. Our results suggest that the use of bivalent small molecule ligand-drug conjugates against CAIX may represent an attractive chemical strategy for the treatment of constitutively CAIX-positive kidney cancer.

Site-Specific and Stoichiometric Conjugation of Cationic Porphyrins to Antiangiogenic Monoclonal Antibodies

Synthesis of three new cationic thiol-reactive maleimide-porphyrin derivatives and their use in site-specific conjugation to monoclonal antibodies is reported. The selective reactivity toward thiols is demonstrated using competition experiments, where both thiols and amines are present. This selectivity was used to successfully achieve specific conjugation of two porphyrins to cysteine residues present in the antiangiogenic antibody L19, expressed in small immunoprotein (SIP) format. The effect of length and hydrophilicity of the linkage between porphyrin and maleimide was also investigated, and maximum photocytotoxicity was achieved with the longest and most hydrophilic chain. Immunoreactivity and in vitro photocytotoxicity for these well-characterized porphyrin-antibody conjugates are described.

Angiogenesis Inhibitors: Implications for Combination with Conventional Therapies

Angiogenesis is associated with tumor development and malignancy and is a validated target for cancer treatment. Preclinical and clinical evidence substantiates the feasibility of combining angiogenesis inhibitors with conventional anticancer therapy. This review discusses recent progress in combining antiangiogenic drugs, mainly acting on the VEGF/VEGFR pathway, with chemotherapy and other conventional therapies. Strategies for the optimization of combination therapy and the selection of appropriate treatment regimens are examined. As new drugs are entering clinical trials, reliable biomarkers are needed to stratify patients for antiangiogenic therapy, to identify resistant patients and to monitor response to treatment.

A 99mTc-Labeled Ligand of Carbonic Anhydrase IX Selectively Targets Renal Cell Carcinoma In Vivo

Small organic ligands, selective for tumor-associated antigens, are increasingly being considered as alternatives to monoclonal antibodies for the targeted delivery of diagnostic and therapeutic payloads such as radionuclides and drugs into neoplastic masses. We have previously described a novel acetazolamide derivative, a carbonic anhydrase ligand with high affinity for the tumor-associated isoform IX (CAIX), which can transport highly potent cytotoxic drugs into CAIX-expressing solid tumors. The aim of the present study was to quantitatively investigate the biodistribution properties of said ligand and understand whether acetazolamide conjugates merit further development as drug carriers and radioimaging agents. Methods: The conjugate described in this study, consisting of a derivative of acetazolamide, a spacer, and a peptidic 99mTc chelator, was labeled using sodium pertechnetate under reducing conditions and injected intravenously into CAIX-expressing SKRC-52 xenograft–bearing mice. Animals were sacrificed, and organ uptake as percentage injected activity of radiolabeled ligand per gram of tissues (%IA/g) was evaluated between 10 min and 24 h. Additionally, postmortem imaging by SPECT was performed. Results: The acetazolamide conjugate described in this study could be labeled to high radiochemical purity (>95%, 2.2–4.5 MBq/nmol). Analysis of organ uptake at various time points revealed that the ligand displayed a maximal tumor accumulation 3 h after intravenous injection (22 %IA/g), with an excellent tumor-to-blood ratio of 70:1 at the same time point. The ligand accumulation in the tumor was more efficient than in any other organ, but a residual uptake in the kidney, lung, and stomach (9, 16, and 10 %IA/g, respectively) was also observed, in line with patterns of carbonic anhydrase isoform expression in those tissues. Interestingly, tumor-to-organ ratios improved on administration of higher doses of radiolabeled ligand, suggesting that certain binding sites in normal organs can be saturated in vivo. Conclusion: The 99mTc-labeled acetazolamide conjugate exhibits high tumor uptake and favorable tumor-to-kidney ratios of up to 3 that may allow imaging of tumors in the kidney and distant sites at earlier time points than commonly possible with antibody-based products. These data suggest that the described molecule merit further development as a radioimaging agent for CAIX-expressing renal cell carcinoma.