Engineered humanized diabodies for microPET imaging of prostate stem cell antigen-expressing tumors

Abstract

We have previously demonstrated preclinical in vivo targeting of prostate stem cell antigen (PSCA) using a humanized anti-PSCA 2B3 monoclonal antibody (mAb). However, humanization resulted in 5-fold loss of apparent affinity relative to the parental mAb (1 nM). In this study, diabodies (scFv dimers of 55 kDa) were generated from 2B3 including variants with different linker lengths as well as back-mutations to original murine residues to improve affinity. Parental 2B3 (p2B3) and back-mutated 2B3 (bm2B3) diabodies (Dbs) with five-or eight-amino acid linkers (p2B3-Db5, p2B3-Db8, bm2B3-Db5 and bm2B3-Db8) were evaluated for binding to PSCA by flow cytometry and affinities were determined by surface plasmon resonance. Back-mutation restored the affinity from 5.4 to 1.9 nM. Stability, evaluated by size exclusion, revealed that diabodies with eight-residue linkers existed as a mixture of dimeric and monomeric species at low concentrations (1 mg/ml). Shortening the linker from eight to five residues improved dimer stability, notably in the bm2B3-Db8 compared with bm2B3-Db5. Both p2B3-Db8 and bm2B3-Db8 were radioiodinated with 124 I and evaluated by serial micro-positron emission tomography imaging in mice bearing LAPC-9 human prostate cancer xenografts. Localization in LAPC-9 xenografts was seen at 4 h, whereas at 20 h most of the activity had cleared from the tumor. Highest tumor-to-background contrast ratios and best images were obtained at 12 h. Although the higher affinity bm2B3-Db8 demonstrated improved tumor retention at later time points (20 h), it did not improve tumor targeting or imaging compared with p2B3-Db8 at 12 h.

A Novel Chimeric Antigen Receptor Against Prostate Stem Cell Antigen Mediates Tumor Destruction in a Humanized Mouse Model of Pancreatic Cancer

“…We demonstrate that Ha1-4.117-based CARs presented superior surface expression in human T cells and conferred higher target recognition capabilities than a CAR based on bm2b3 (a variant of 2b3 with enhanced affinity) (Leyton et al, 2009), without compromising antigen specificity. This design is expected to prevent the development of an immune response directed to the remaining murine immunoglobulin regions present in 2b3-based CARs.…”

“…In addition, we generated an anti-PSCA CAR derived from humanized mouse antibody bm2b3 (Fig. 2a) (Leyton et al, 2008(Leyton et al, , 2009). These CAR sequences were inserted into retroviral vectors and used to transduce human PBL.…”

Human monoclonal antibodies targeting carbonic anhydrase IX for the molecular imaging of hypoxic regions in solid tumours

“…In this study, we have used antibodies in SIP format (Borsi et al, 2002;Villa et al, 2008), as this format and similar mini-antibody formats have extensively been shown to offer distinctive advantages both for imaging applications (Leyton et al, 2009);von Lukowicz et al, 2007;Wei et al, 2008) and for radioimmunotherapy of cancer (Berndorff et al, 2005;Tijink et al, 2006;Kenanova et al, 2007;Sauer et al, 2009). Recent publications suggest that smaller high-affinity ligands (MW o 2000 Da) may enjoy a much more rapid tissue distribution compared with antibodies and antibody fragments (Low et al, 2008).…”

ImmunoPET imaging of B-cell lymphoma using 124I-anti-CD20 scFv dimers (diabodies)

“…The scFv were all designed in the V L -V H orientation and the linker lengths were one, three, five and eight residues. Although linker lengths of ,12 residues will generally force dimerization of the scFv fragments, we have observed that linkers of eight residues can produce a mixture of monomers and dimers and that the ratio between the two species was concentration-dependent (Leyton et al, 2009). For this reason, we decided to engineer another anti-CD20 scFv fragment with the more commonly used linker length of five amino acids.…”