The incidence of brain metastasis is rising and poses a severe clinical problem, as we lack effective therapies and knowledge of mechanisms that control metastatic growth in the brain. Here we demonstrate a crucial role for high-affinity tumor cell integrin ␣v␤3 in brain metastatic growth and recruitment of blood vessels. Although ␣v␤3 is frequently up-regulated in primary brain tumors and metastatic lesions of brain homing cancers, we show that it is the ␣v␤3 activation state that is critical for brain lesion growth. Activated, but not non-activated, tumor cell ␣v␤3 supports efficient brain metastatic growth through continuous up-regulation of vascular endothelial growth factor (VEGF) protein under normoxic conditions. In metastatic brain lesions carrying activated ␣v␤3, VEGF expression is controlled at the post-transcriptional level and involves phosphorylation and inhibition of translational respressor 4E-binding protein (4E-BP1). In contrast, tumor cells with nonactivated ␣v␤3 depend on hypoxia for VEGF induction, resulting in reduced angiogenesis, tumor cell apoptosis, and inefficient intracranial growth. Importantly, the microenvironment critically influences the effects that activated tumor cell ␣v␤3 exerts on tumor cell growth. Although it strongly promoted intracranial growth, the activation state of the receptor did not influence tumor growth in the mammary fat pad as a primary site. Thus, we identified a mechanism by which metastatic cells thrive in the brain microenvironment and use the high-affinity form of an adhesion receptor to grow and secure host support for proliferation. Targeting this molecular mechanism could prove valuable for the inhibition of brain metastasis.angiogenesis ͉ brain metastasis ͉ integrin activation ͉ 4E-BP1 B rain metastases are diagnosed in 10% to 40% of patients with progressing cancer, and the incidence is rising as patients live longer and extracranial metastases respond to improved treatments. However, brain metastases still cannot be treated effectively, and mechanisms controlling brain metastatic growth are largely unknown (1-3).Here, we demonstrate that the high-affinity state of tumor cell adhesion receptor integrin ␣ v ␤ 3 critically promotes metastatic growth and recruitment of supporting blood vessels within the brain microenvironment. Integrins are cell surface receptors composed of non-covalently linked ␣ and ␤ subunits that mediate cell-matrix and cell-cell interactions and transduce signals that have impacts on cell survival, proliferation, adhesion, migration, and invasion. Integrin signals can also originate inside cells, affect receptor affinity, and thereby control ligand binding, cross talk with other receptors, and alter cell adhesion and proliferation (4-6). Integrin ␣ v ␤ 3 also plays a role on sprouting endothelial cells and contributes to angiogenesis (7). In several tumor types, including glioma, breast cancer, and melanoma, expression of ␣ v ␤ 3 supports invasion and metastasis (8-11). Notably, these tumors either originate in the brain or freque...

Immunoconjugates and multispecific antibodies are rapidly emerging as highly potent experimental therapeutics against cancer. We have developed a method to incorporate an unnatural amino acid, p-acetylphenylalanine (pAcPhe) into an antibody antigen binding fragment (Fab) targeting HER2 (human epidermal growth factor receptor 2), allowing site-specific labeling without disrupting antigen binding. Expression levels of the pAcPhe-containing proteins were comparable to that of wild-type protein in shake-flask and fermentation preparations. The pAcPhe–Fabs were labeled by reaction with hydroxylamine dye and biotin species to produce well–defined, singly conjugated Fabs. We then coupled a hydroxylamine biotin to the pAcPhe–Fab and demonstrated controlled assembly of Fabs in the presence of the tetrameric biotin-binding protein, NeutrAvidin. The position of Fab biotinylation dictates the geometry of multimer assembly, producing unique multimeric Fab structures. These assembled Fab multimers differentially attenuate Her2 phosphorylation in breast cancer cells that overexpress the Her2 receptor. Thus, an encoded unnatural amino acid produces a chemical “handle” by which immunoconjugates and multimers can be engineered.

ABSTRACT

Summary We report a new strategy for the generation of heterodimeric protein conjugates using an unnatural amino acid with orthogonal reactivity. This paper addresses the challenges of site-specificity and homogeneity with respect to the synthesis of bivalent proteins and antibody-drug conjugates. There are numerous antibody-drug conjugates in preclinical and clinical development, yet these are based either on nonspecific lysine coupling chemistry or on disulfide modification made difficult by the large number of cysteines in antibodies. Here we describe a recombinant approach that can be used to rapidly generate a variety of constructs with defined conjugation sites. Moreover, this methodology results in homogeneous antibody conjugates whose biological, physical and pharmacological properties can be quantitatively assessed and subsequently optimized. As proof of concept, we have generated anti-Her2 Fab-Saporin conjugates that demonstrate excellent potency in vitro.

No abstract

These data demonstrate a lack of tolerability of PLGA microspheres upon intravitreal injection, and suggest that the size, shape, and/or surface area of PLGA depots are critical attributes in determining ocular toxicity.

Metastasis is the main cause of death in cancer patients, and understanding mechanisms that control tumor cell dissemination may lead to improved therapy. Tumor cell adhesion receptors contribute to cancer spreading. We noted earlier that tumor cells can expressing the adhesion receptor integrin αvβ3 in distinct states of activation, and found that cells which metastasize from the blood stream express it in a constitutively high affinity form. Here, we analyzed steps of the metastatic cascade in vivo and asked, when and how the affinity state of integrin αvβ3 confers a critical advantage to cancer spreading. Following tumor cells by real time PCR, non-invasive bioluminescence imaging, intravital microscopy and histology allowed us to identify tumor cell extravasation from the blood stream as a rate-limiting step supported by high affinity αvβ3. Successful transendothelial migration depended on cooperation between tumor cells and platelets involving the high affinity tumor cell integrin and release of platelet granules. Thus, this study identifies the high affinity conformer of integrin αvβ3 and its interaction with platelets as critical for early steps during hematogenous metastasis and target for prevention of metastatic disease.

Gliomas are the most common primary brain tumors and offer a poor prognosis in patients because of their infiltrative and treatment-resistant nature. The median survival time after diagnosis is approximately 11-12 months. There is a strong need for novel treatment modalities in targeting gliomas, and recent advances use neural progenitor cells as delivery systems for different therapeutic strategies. In this study, we show that rat embryonic neural progenitor cell (NPC) lines, transplanted at a distant site from a 3-day-preestablished glioma in the striatum, were able to migrate toward and colocalize with tumor isles without general spread into the brain parenchyma. Upon encounter with tumor, neural progenitor cells changed phenotype and became vimentin positive. These results demonstrate that transplanted neural progenitor cells respond to queues from a tumor and home to and exert an antitumor effect on the preestablished glioma, significantly decreasing the tumor volume with approximately 67% compared with control tumors after 1-2 weeks. Moreover, these early effects could be translated into increased survival times of animals treated with neural progenitor cell grafts 3 days after intrastriatal tumor inoculation. In contrast, there was no activation or migration of endogenous subventricular zone (SVZ) neuroblasts in response to an intrastriatal syngeneic tumor. In conclusion, NPC possess the ability to influence tumor growth as well as respond to queues from the tumor or tumor microenvironment, demonstrating a cross-talk between the cells.