Cell-penetrating Alphabody protein scaffolds for intracellular drug targeting

The therapeutic scope of antibody and nonantibody protein scaffolds is still prohibitively limited against intracellular drug targets. Here, we demonstrate that the Alphabody scaffold can be engineered into a cell-penetrating protein antagonist against induced myeloid leukemia cell differentiation protein MCL-1, an intracellular target in cancer, by grafting the critical B-cell lymphoma 2 homology 3 helix of MCL-1 onto the Alphabody and tagging the scaffold’s termini with designed cell-penetration polypeptides. Introduction of an albumin-binding moiety extended the serum half-life of the engineered Alphabody to therapeutically relevant levels, and administration thereof in mouse tumor xenografts based on myeloma cell lines reduced tumor burden. Crystal structures of such a designed Alphabody in complex with MCL-1 and serum albumin provided the structural blueprint of the applied design principles. Collectively, we provide proof of concept for the use of Alphabodies against intracellular disease mediators, which, to date, have remained in the realm of small-molecule therapeutics

An Osteoblast-Derived Proteinase Controls Tumor Cell Survival via TGF-beta Activation in the Bone Microenvironment

Breast to bone metastases frequently induce a "vicious cycle" in which osteoclast mediated bone resorption and proteolysis results in the release of bone matrix sequestered factors that drive tumor growth. While osteoclasts express numerous proteinases, analysis of human breast to bone metastases unexpectedly revealed that bone forming osteoblasts were consistently positive for the proteinase, MMP-2. Given the role of MMP-2 in extracellular matrix degradation and growth factor/cytokine processing, we tested whether osteoblast derived MMP-2 contributed to the vicious cycle of tumor progression in the bone microenvironment.To test our hypothesis, we utilized murine models of the osteolytic tumor-bone microenvironment in immunocompetent wild type and MMP-2 null mice. In longitudinal studies, we found that host MMP-2 significantly contributed to tumor progression in bone by protecting against apoptosis and promoting cancer cell survival (caspase-3; immunohistochemistry). Our data also indicate that host MMP-2 contributes to tumor induced osteolysis (μCT, histomorphometry). Further ex vivo/in vitro experiments with wild type and MMP-2 null osteoclast and osteoblast cultures identified that 1) the absence of MMP-2 did not have a deleterious effect on osteoclast function (cd11B isolation, osteoclast differentiation, transwell migration and dentin resorption assay); and 2) that osteoblast derived MMP-2 promoted tumor survival by regulating the bioavailability of TGFβ, a factor critical for cell-cell communication in the bone (ELISA, immunoblot assay, clonal and soft agar assays).Collectively, these studies identify a novel "mini-vicious cycle" between the osteoblast and metastatic cancer cells that is key for initial tumor survival in the bone microenvironment. In conclusion, the findings of our study suggest that the targeted inhibition of MMP-2 and/or TGFβ would be beneficial for the treatment of bone metastases

Host-derived MMP-2 impacts mammary tumor growth in the bone microenvironment.

<p><b>A</b>, Representative timeline of PyMT-Luc luciferase expression in FVB wild type (WT; n = 10) and MMP-2 null (MMP-2^−/−; n = 10) mice. Hotter colors indicate areas of increased luciferase activity. <b>B</b>, Graph represents PyMT-Luc tumor growth over time as assessed by luciferase activity in WT and MMP-2^−/− animals. <b>C</b>, PyMT-Luc fail to grow in the MMP-2^−/− bone microenvironment as assessed over a 25 day period. PyMT-Luc were injected intratibially into syngeneic FVB wild type (WT; n = 10) or MMP-2 null (MMP-2^−/−; n = 10) mice. WT mice were euthanized on day 15 due to tumor size. <b>D</b>, 17L3C-Luc, an unrelated cell line derived from the PyMT model of mammary gland tumorigenesis, was intratibially injected into wild type (WT; n = 5) or MMP-2 null (MMP-2^−/−; n = 5) mice. Luciferase activity was assessed as a measure of tumor growth over a 7-day period. Data are mean ± SD; Asterisk denotes statistical significance (p<0.05).</p

Tumor induced osteolysis is controlled by host MMP-2.

<p><b>A</b>, Representative μCT scans of trabecular bone from tumor bearing and sham injected limbs of wild type (WT) and MMP-2 null (MMP-2^−/−) mice at day 9. Graph illustrates ratio of bone volume to total volume (BV/TV). <b>B</b>, Representative H&E stained photomicrographs of tumor bearing tibias from WT and MMP-2^−/− mice. Scale bars represent 1 mm. Graph represents the BV/TV ratio. <b>C</b>, Representative radiographic images from tumor injected WT and MMP-2^−/− animals at day 9. Arrow indicates lytic tumor lesions in the wild type group. Graph represents the tumor volume (TuV) over total volume (TV) ratio. <b>D</b>, Representative TRAcP stained photomicrographs of tumor injected WT and MMP-2^−/− animals at day 9. Scale bar represents 50 µm. Graph indicates the number of osteoclasts/mm of trabecular bone. Data are mean ± SD. Asterisk denotes that p<0.05.</p

Host-derived MMP-2 impacts tumor survival in the bone microenvironment.

<p><b>A</b>, Representative photomicrograph of MCM2 positive proliferating (brown) cells in the wild type (WT) and MMP-2 null (MMP-2^−/−) tumor-bone microenvironment. <b>B</b>, Proliferation in the tumor-bone microenvironment as a function of total cell number was assessed by staining for Mcm2 in tumor bearing tibias of WT and MMP-2 null mice at day 3 and day 6 post-surgery. <b>C</b>, Representative photomicrograph of caspase-3 positive apoptotic (brown) cells in the wild type (WT) and MMP-2 null (MMP-2^−/−) tumor-bone microenvironment. <b>D</b>, Apoptosis in the tumor-bone microenvironment as a function of total cell number was assessed by staining for cleaved caspase-3 at day 3 and day 6 post surgery. Data are mean ± SD; n.s. implies a non-significant p value (p>0.05).</p

MMP-2 mediates tumor survival in a TGFβ dependent manner.

<p><b>A</b>, Tumor survival in the presence of 1 ng/ml recombinant TGFβ was assessed by soft agar colony formation assay. <b>B</b>, The number of PyMT-Luc colonies formed in response to 10 days of treatment with control media (5% serum containing αMEM) or conditioned media derived from wild type or MMP-2 null osteoblasts was analyzed in a 2D (plastic) assay (non-treated group). Under similar conditions the impact of TGFβ inhibition was also determined (matched isotype antibody was added to control wells). Differences are significant as assessed by ANOVA. <b>C</b>, The effects of blocking TGFβ in the CM derived from WT and MMP-2^−/− osteoblasts was determined by soft agar colony formation assay. Data are mean ± SD. Asterisk indicates p<0.05 and n.s indicates non significance. Double asterisk denotes that the addition of recombinant MMP-2 to MMP-2^−/− CM significantly enhances tumor survival compared to MMP-2^−/− CM alone. <b>D</b>, Osteoblast-derived MMP-2 and TGF-β mediate tumor survival without impacting colony size. Using a soft agar colony formation assay, no difference in colony size was detected between the various treatment conditions. Data are mean ± SD.</p

Absence of host MMP-2 does not impair osteoclast precursor function.

<p><b>A</b>, Quantitative analysis of isolated wild type (WT) and MMP-2 null (MMP-2^−/−) CD11b^+ve osteoclast precursor migration to 10% serum or control serum free media. <b>B</b>, Analysis of the osteoclastogenic capability of WT or MMP-2^−/− osteoclast precursors. Arrows indicate TRAcP positive (red) multinucleated (blue) osteoclasts. Scale bars represent 50 µm. <b>C</b>, Analysis of the number of WT and MMP-2^−/− mature osteoclasts formed/well in each group (n≥3/group). <b>D</b>, Pit formation assay on dentin discs to test the functionality of mature WT and MMP-2^−/− osteoclasts. Asterisk denotes that p<0.05 while p = n.s. denotes non-significant p values.</p