Elinborg Ostermann

Effective Immunoconjugate Therapy in Cancer Models Targeting a Serine Protease of Tumor Fibroblasts

Purpose: Invasion and metastasis of malignant epithelial cells into normal tissues is accompanied by adaptive changes in the mesenchyme-derived supporting stroma of the target organs. Altered gene expression in these nontransformed stromal cells provides potential targets for therapy. The present study was undertaken to determine the antitumor effects of an antibody-conjugate against fibroblast activation protein-α, a cell surface protease of activated tumor fibroblasts. Experimental Design: A novel antibody-maytansinoid conjugate, monoclonal antibody (mAb) FAP5-DM1, was developed to target a shared epitope of human, mouse, and cynomolgus monkey fibroblast activation protein-α, enabling preclinical efficacy and tolerability assessments. We have used stroma-rich models in immunodeficient mice, which recapitulate the histotypic arrangement found in human epithelial cancers. Results: Treatment with mAb FAP5-DM1 induced long-lasting inhibition of tumor growth and complete regressions in xenograft models of lung, pancreas, and head and neck cancers with no signs of intolerability. Analysis of chemically distinct conjugates, resistance models, and biomarkers implicates a unique mode of action, with mitotic arrest and apoptosis of malignant epithelial cells coupled to disruption of fibroblastic and vascular structures. Conclusions: We show that mAb FAP5-DM1 combines excellent efficacy and tolerability and provides a first assessment of the mode of action of a novel drug candidate for tumor stroma targeting, thus encouraging further development toward clinical testing of this treatment paradigm.

A novel Fc-engineered monoclonal antibody to CD37 with enhanced ADCC and high proapoptotic activity for treatment of B-cell malignancies

The tetraspanin CD37 is widely expressed in B-cell malignancies and represents an attractive target for immunotherapy with mAbs. We have chimerized a high-affinity mouse Ab to CD37 and engineered the CH2 domain for improved binding to human Fcγ receptors. The resulting mAb 37.1 showed high intrinsic proapoptotic activity on malignant B cells accompanied by homotypic aggregation. Furthermore, the Ab-mediated high Ab-dependent cell-mediated cytotoxicity (ADCC) on lymphoma and primary CLL cells. mAb 37.1 strongly depleted normal B cells as well as spiked B-lymphoma cells in blood samples from healthy donors as well as malignant B cells in blood from CLL patients. In all assays, mAb 37.1 was superior to rituximab in terms of potency and maximal cell lysis. A single dose of mAb CD37.1 administered to human CD37-transgenic mice resulted in a reversible, dose-dependent reduction of peripheral B cells. In a Ramos mouse model of human B-cell lymphoma, administration of mAb 37.1 strongly suppressed tumor growth. Finally, a surrogate Fc-engineered Ab to macaque CD37, with in vitro proapoptotic and ADCC activities very similar to those of mAb 37.1, induced dose-dependent, reversible B-cell depletion in cynomolgus monkeys. In conclusion, the remarkable preclinical pharmacodynamic and antitumor effects of mAb 37.1 warrant clinical development for B-cell malignancies.

Tumor-targeted gene delivery of tumor necrosis factor-|[alpha]| induces tumor necrosis and tumor regression without systemic toxicity

We have recently developed surface-shielded transferrin–polyethylenimine (Tf–PEI)/DNA delivery systems that target reporter gene expression to distant tumors after systemic application. In the present study, we used surface-shielded Tf–PEI/DNA complexes for delivering the gene for a highly potent cytokine, tumor necrosis factor-α (TNFα). TNFα is known for its ability to induce hemorrhagic tumor necrosis and tumor regression. However, the therapeutic application of TNFα is hampered by its high systemic toxicity dictating the need to target TNFα activity to the tumor. Systemic application of surface-shielded Tf–PEI complexes with the TNFα gene resulted in preferential expression of TNFα in the tumor without detectable TNFα serum levels, in contrast to the application of nontargeted complexes. Tumor-targeted TNFα gene delivery induced pronounced hemorrhagic tumor necrosis and inhibition of tumor growth in three murine tumor models of different tissue origins, Neuro2a neuroblastoma, MethA fibrosarcoma, and M-3 melanoma, with complete tumor regressions observed in the MethA model. No systemic TNF-related toxicity was observed due to the localization of the TNFα activity to the tumor. Targeted gene therapy may be an attractive strategy applicable to highly active, yet toxic, molecules such as TNFα.

Characterization of a high-affinity monoclonal antibody specific for CD44v6 as candidate for immunotherapy of squamous cell carcinomas.

Abstract Variant isoforms of CD44, a family of cell-surface glycoproteins generated by alternative splicing and post-translational modifications, are expressed in a variety of human tumors and play important roles in tumor progression and metastasis formation. The murine monoclonal IgG1 antibody VFF18, specific for an epitope encoded by human CD44 variant exon 6, binds with high affinity to the recombinant protein (Kd = 1.7×10–10 M) as well as to tumor cell lines in vitro, and is suitable for immunohistochemical analysis of human tumors. Screening of more than 500 tumor samples of different histogenesis showed that VFF18 most strongly and uniformly reacts with squamous cell carcinomas (SCC). Detailed analysis of 185 SCC (head and neck, lung, skin) confirmed reactivity of the antibody with 99% of the samples, with intense and homogeneous staining of the tumor cells in the majority of cases, whereas reactivity of VFF18 with normal tissues is limited to certain epithelia and activated lymphocytes. When radiolabelled VFF18 was administered to nude mice bearing human epidermoid carcinoma (A-431) xenograft, fast and selective tumor uptake of the radioimmunoconjugate with a maximum of 18% of the injected dose per gram of tissue was observed. Taken together, these data suggest that mAb VFF18 is a promising targeting vehicle for radioimmunotherapy of squamous cell carcinomas in humans.

Tumor targeting properties of monoclonal antibodies with different affinity for target antigen CD44V6 in nude mice bearing head-and-neck cancer xenografts.

The CD44 protein family consists of isoforms with tissue‐specific expression, which are encoded by standard exons and up to 9 alternatively spliced variant exons (v2–v10) of the same gene. The murine MAbs U36 and BIWA‐1, directed against overlapping epitopes within the v6 region of CD44, have previously been shown to efficiently target HNSCC. We herein report on the construction of 1 chimeric (BIWA‐2) and 2 humanized (BIWA‐4 and BIWA‐8) derivatives of BIWA‐1. Together with U36 and BIWA‐1, these new antibodies were evaluated for affinity to the antigen in vitro as well as for biodistribution and efficacy in RIT using nude mice bearing the HNSCC xenograft line HNX‐OE. As determined by surface plasmon resonance, the MAbs bound to CD44v6 with an up to 46‐fold difference in affinity (Kd ranging from 1.1 × 10−8 to 2.4 × 10−10 M) with the following ranking: mMAb U36 < hMAb BIWA‐4 < hMAb BIWA‐8 < mMAb BIWA‐1 ∼ cMAb BIWA‐2. To evaluate their in vivo tumor‐targeting properties, 2 MAbs with identical murine or human isotype were labeled with either 131I or 125I and administered simultaneously (50 μg/10 μCi each) as pairs showing a stepwise decrease in the difference in affinity: U36 vs. BIWA‐1 (35.0‐fold difference), BIWA‐4 vs. BIWA‐2 (14.0‐fold) and BIWA‐4 vs. BIWA‐8 (4.0‐fold). Biodistribution was assessed at 1, 2, 3 or 4 and 7 days after injection. Remarkably, for all 3 MAb pairs tested, the lower‐affinity MAb showed a higher degree and specificity of tumor localization. The difference in tumor localization was more pronounced when the difference in affinity was larger. For example, 3 days after injection, the lower‐affinity mMAb U36 showed a 50% higher tumor uptake than the higher‐affinity mMAb BIWA‐1, while blood levels and uptake in organs were similar. After labeling with 186Re (300 or 400 μCi), the same MAb pairs showed RIT efficacy consistent with the biodistribution data: 186Re‐U36 was more effective than 186Re‐BIWA‐1, 186Re‐BIWA‐4 was slightly more effective than 186Re‐BIWA‐2 and 186Re‐BIWA‐4 and 186Re‐BIWA‐8 demonstrated similar efficacy. Based on these data, we conclude that antibodies with markedly lower affinity to a given target antigen (e.g., U36, BIWA‐4) may show superior tumor targeting in comparison with higher‐affinity versions of these antibodies.

Splice variants of the cell surface glycoprotein CD44 associated with metastatic tumour cells are expressed in normal tissues of humans and cynomolgus monkeys.

Certain isoforms of the CD44 glycoprotein family play an essential role in the metastatic spread of tumour cells. Protein expression of such CD44 isoforms has also been observed in a variety of human malignancies. In this study, we compared the expression of exon v5- and v6-containing CD44 isoforms in normal human and cynomolgus monkey (Macacca fasciculata) tissues. Cloning and sequencing of cynomolgus CD44 exons v5 and v6 revealed a homology of 97% and 95%, respectively, between man and monkey. Two monoclonal antibodies (MAbs) directed against an epitope encoded by human exon v5 (VFF8) and an epitope encoded by exon v6 (VFF18) were used to determine expression of CD44 isoforms in man and monkey. Immunohistochemical screening of a representative profile of normal human and cynomolgus tissues revealed that expression of exon v5- and v6-containing CD44 isoforms was almost identical in the two species. Exon v6 staining was observed only in a subset of epithelial tissues, whereas v5 staining was additionally detected on certain non-epithelial tissues. These data suggest that cynomolgus monkey could serve as a system to test the usefulness of antivariant CD44 MAbs with regard to antibody-based tumour therapy.

Pharmacodynamic and Antineoplastic Activity of BI 836845, a Fully Human IGF Ligand-Neutralizing Antibody, and Mechanistic Rationale for Combination with Rapamycin

Insulin-like growth factor (IGF) signaling is thought to play a role in the development and progression of multiple cancer types. To date, therapeutic strategies aimed at disrupting IGF signaling have largely focused on antibodies that target the IGF-I receptor (IGF-IR). Here, we describe the pharmacologic profile of BI 836845, a fully human monoclonal antibody that utilizes an alternative approach to IGF signaling inhibition by selectively neutralizing the bioactivity of IGF ligands. Biochemical analyses of BI 836845 demonstrated high affinity to human IGF-I and IGF-II, resulting in effective inhibition of IGF-induced activation of both IGF-IR and IR-A in vitro. Cross-reactivity to rodent IGFs has enabled rigorous assessment of the pharmacologic activity of BI 836845 in preclinical models. Pharmacodynamic studies in rats showed potent reduction of serum IGF bioactivity in the absence of metabolic adverse effects, leading to growth inhibition as evidenced by reduced body weight gain and tail length. Moreover, BI 836845 reduced the proliferation of human cell lines derived from different cancer types and enhanced the antitumor efficacy of rapamycin by blocking a rapamycin-induced increase in upstream signaling in vitro as well as in human tumor xenograft models in nude mice. Our data suggest that BI 836845 represents a potentially more effective and tolerable approach to the inhibition of IGF signaling compared with agents that target the IGF-I receptor directly, with potential for rational combinations with other targeted agents in clinical studies. Mol Cancer Ther; 13(2); 399–409. ©2013 AACR.

Abstract A208: Pharmacodynamic properties and antitumor efficacy of BI 836845, a fully human IGF ligand neutralizing antibody.

Insulin-like growth factor (IGF) signaling is thought to play an important role in the development of tumors and resistance to existing cancer therapies. The proliferative and pro-survival signals driven by the IGF ligands, IGF-1 and IGF-2, are transmitted through their binding to the IGF-1 receptor (IGF-1R). In addition, IGF-2 activates the insulin receptor variant A (IR-A) that is expressed during embryonic development as well as in many cancers. To target IGF signaling for cancer therapy we have developed BI 836845, a fully human IgG1 antibody derived from an antibody phage display library that can bind to and neutralize the functions of both IGF-1 and IGF-2. Surface plasmon resonance (Biacore) analysis demonstrated that BI 836845 has an affinity (K D ) of 0.07 nM and 0.8 nM for human IGF-1 and IGF-2, respectively. BI 836845 also shows comparable affinities to mouse as well as rat IGF-1 and IGF-2, allowing a comprehensive pre-clinical characterization of the pharmacodynamic properties of the antibody in these species. Cell-based ELISA assays were used to demonstrate that BI 836845 potently neutralizes the ability of IGF-1 and IGF-2 to phosphorylate the human IGF-1R. Similarly, BI 836845 inhibits phosphorylation of IR-A driven by IGF-2. BI 836845 was also shown to completely and potently inhibit IGF bioactivity in human serum samples ex vivo. Pharmacodynamic effects of BI 836845 were studied in growing rats treated intravenously once weekly with 20, 60, or 200 mg/kg antibody. Inhibition of IGF bioactivity as determined by ex vivo IGF-1R phosphorylation potential in the plasma was observed at all dose levels, despite a corresponding increase in total IGF-1 levels. An increase in total IGF-1 levels has also been seen with IGF-1R targeted antibodies in clinical studies and is thought to be due to blockade of a growth hormone mediated physiological feedback mechanism. Treatment with BI 836845 resulted in a marked dose dependent reduction of rat body weight gain at all dose levels, consistent with the known role for IGFs in normal growth and development. BI 836845 potently inhibits the in vitro proliferation of cell lines derived from various cancers, with mesenchymal-derived cancer cell lines being particularly sensitive (EC 50 values in the low nanomolar range). In a model of Ewing s sarcoma, using immunodeficient nude mice subcutaneously transplanted with human RD-ES cells, twice-weekly treatment with BI 836845 resulted in partial inhibition of tumor growth. Treatment in combination with rapamycin resulted in improved efficacy at tolerated doses. In conclusion, BI 836845 is a potent IGF ligand neutralizing antibody whose pharmacological profile warrants further investigation in clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A208.