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.

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