ß-Amyloid Antibodies with Specific Proteolytic Activity

A BetaPassive immunotherapy with anti-amyloid beta peptide antibodies [anti-Aβ Abs] offers the benefits but not the toxicity of active immunization with Aβ peptide. The purpose of collaborative project is to develop and test proteolytic anti-Aβ Abs that not only bind Aβ peptide but also cleave specifically the Aβ peptide.

Three approaches will be used to develop proteolytic anti-Aβ Abs: [1] selection of Ab fragments (single chain Fv fragments) from a phage library expressing the human Ab repertoire based on coordinated noncovalent recognition and irreversible binding of covalently reactive Aβ analogs (CRAs) containing chemical groups reactive with the nucleophiles in the active site of serine proteases. This approach has been validated for its capacity to identify Abs that combine specific antigen recognition with the ability to hydrolyze peptide bonds. [2] Improvement in anti-Aβ specificity by: (a) Randomizing the sequence of the heavy chain variable (VH) domain CDR3, a region governing Ab specificity, combined with covalent affinity selection using the CRAs; and (b) Pairing of light chain V domains expressing promiscuous proteolytic activity with VH domains derived from specific anti-Aβ Abs.

In each case, the scFv clones will be tested for their capacity to neutralize Aβ peptide-mediated neurotoxicity in vitro. [3] Induction of proteolytic monoclonal anti-Aβ Abs by immunization with Aβ CRA, a strategy designed to permit clonal selection of B cells producing Abs with enhanced and specific anti-Aβ proteolytic activity will be studied. Determination of the Aβ cleavage site(s) will identify Ab clones capable of cleaving the determinant (residues 25-35) and oligomeric peptide states thought to be important in Aβ peptide toxicity using proteolytic and non-proteolytic Abs.

The most active Abs will be tested in vivo for their capacity to inhibit cerebral amyloid deposits and cognitive decline in APP/PS1-transgenic mice. A RAG-1-deficient line of APP/PS1-transgenic mice will be used to test human proteolytic Abs, thus avoiding confounding murine immune responses to xenogeneic Abs. Proteolytic anti-Aβ Abs capable of preventing the deposition of Aβ and cognitive decline would be candidates for passive immunotherapy of humans with AD. (PI: Sudhir Paul)