Investigador Principal:

LAURA SANZ ALCOBER (2014-today)

LUIS ÁLVAREZ-VALLINA (2012-2013)

Research Areas

Tumor angiogenesis

New blood vessels are formed by one of two related but distinct mechanisms: vasculogenesis (the development of blood vessels from precursor cells during embryogenesis) and angiogenesis (the outgrowth of vasculature from pre-existing blood vessels). In the normal adult, the vasculature is quiescent (except for processes such as wound healing and the female reproductive cycle), but can become activated in response to particular stimuli. This event is confined to pathological situations, such as tumour growth, and has become a major research topic. Tumour vessels can grow by sprouting, intussusception or incorporation of endothelial precursors, with sprouting from pre-existing vessels being the best characterised (Figure. 1).

 

Although the responsible mechanisms are not fully understood, the sequence of events that leads to new vessel formation is well known (Figure. 1). Angiogenic growth factors are released, which bind to specific receptors on the endothelial cells (ECs) of nearby pre-existing blood vessels, triggering signal transduction cascades. These angiogenic signals initiate the proliferation and invasion phase of angiogenesis, characterised by the secretion of extracellular matrix (ECM) components and proteolytic enzymes, which are involved in remodeling the tumour microenvironment. Dissolution of the basement membrane (BM) enables ECs to invade the surrounding ECM and proliferate at the leading edge of a migrating column, thereby forming a sprout. These events are accompanied by changes in the integrin expression pattern on ECs, enabling them to respond and interact with the remodeled ECM. The vascular sprouts begin to mature as EC differentiation and lumen formation occurs. Eventually, the new vessel secretes BM components that induce and maintain ECs in a differentiated and quiescent state.

 

The idea that tumours could be starved to death by inhibiting their neovascularization was first proposed more than thirty years ago by Judah Folkman. Many anti-angiogenic agents are now in clinical trials, and the main strategies include:

• Blocking growth factor activity.

• Inhibiting matrix proteinases.

• Directly targeting ECs.

• Targeting and/or blocking ECM active sites.

• Upregulating endogenous inhibitors.

 

Current ECM-directed anti-angiogenic strategies have focused on finding potential markers for the targeted delivery of therapeutic or diagnostic agents to newly formed blood vessels, but the use of the ECM itself as a target remains an unexplored area for angiogenesis inhibition. BM deposition is known to be crucial in the functional organisation and maturation of newly formed vessels and, recently, it has proved to be essential for vasculogenic mimicry (VM) by cancer cells. The morphogenetic action of BM depends on its capacity to bind specific cell-surface receptors and to resist mechanical loads applied to those receptors (Figure 1). We propose that the disruption of these crucial interactions could be a novel therapeutic strategy.

 

For this approach to be successful, several challenges need to be met (Figure 1):

1. Identifying Therapeutic Targets (ECM components that play a key role in the morphological differentiation of human ECs).

2. Obtaining Therapeutic Agents (with enough affinity to disrupt BM–EC interactions).

3. Delivering blocking agents efficiently to generate locally non-permissive BM.

 

Recombinant antibodies (rAbs) directed against functionally active ECM domains, accessible in vivo, could serve as valuable tools for therapy. Using a human phagemid single-chain Fv (scFv) antibody library we generated several laminin-specific Ab fragments, and a functionally active rAb was identified by biological testing in vitro (Figure 1). An anti-laminin blocking antibody inhibited angiogenesis in vivo and prevented the establishment and growth of subcutaneous tumours in mice.

 

Antibodies and gene therapy

To circumvent limitations associated with systemic administration of Ab molecules (difficulties in the production and in the achievement of effective and sustained levels in situ); we have developed Ab-based gene therapy approaches (Figure 2). Moreover, genetic approaches provide Ab molecules with new functions in unexpected scenarios: expression of Ab domains in precise intracellular locations (Intrabodies), and grafting of new binding activities to engineered cells (Chimeric immune receptors).

 

Figure 1. Schematic representation of the angiogenic process. (B) Schematic representation of the in vitro angiogenesis assay. Human macro or microvascular endothelial cells are allowed to differentiate into capillary-like structures on reconstituted basement membrane components (Matrigel) for 12-14 hours. (C) Panning and screening for antigen-specific binders from phage-antibody libraries.

 

Figure 2. Antibody-based cancer gene therapy strategies. Tumor cells, bystander cells and progenitor or terminal differentiated effector cells can be genetically modified with appropriate constructs to express different types of antibody-based molecules in different cellular locations. These strategies include the production of soluble antibodies (blue box): monospecific antibody fragments against cell surface antigens, growth factors or extracellular matrix proteins; or bispecific antibody fragments capable of redirecting or enhancing effector activity towards tumor cells by binding to cell activation molecules with one domain and to tumor-associated antigens with the other. Intrabodies (red box) designed with the appropriate trafficking signals (striped squares) can be directed to different intracellular compartments to bind their cognate antigen. Surface-bound, antibody-containing molecules (green box) comprise chimeric immune receptors (CIRs) and artificial legends (ALs). CIRs comprise a recognition unit (the antibody fragment) attached to the transmembrane and intracytoplasmic sequences of a signaling molecule. ALs contain antibody fragments specific for individual T cell receptors and a short motif to attach them to the cell surface.