Peter J. Hudson

The Australian Imaging, Biomarkers and Lifestyle (AIBL) study of aging: methodology and baseline characteristics of 1112 individuals recruited for a longitudinal study of Alzheimer's disease

BACKGROUND The Australian Imaging, Biomarkers and Lifestyle (AIBL) flagship study of aging aimed to recruit 1000 individuals aged over 60 to assist with prospective research into Alzheimers disease (AD). This paper describes the recruitment of the cohort and gives information about the study methodology, baseline demography, diagnoses, medical comorbidities, medication use, and cognitive function of the participants. METHODS Volunteers underwent a screening interview, had comprehensive cognitive testing, gave 80 ml of blood, and completed health and lifestyle questionnaires. One quarter of the sample also underwent amyloid PET brain imaging with Pittsburgh compound B (PiB PET) and MRI brain imaging, and a subgroup of 10% had ActiGraph activity monitoring and body composition scanning. RESULTS A total of 1166 volunteers were recruited, 54 of whom were excluded from further study due to comorbid disorders which could affect cognition or because of withdrawal of consent. Participants with AD (211) had neuropsychological profiles which were consistent with AD, and were more impaired than participants with mild cognitive impairment (133) or healthy controls (768), who performed within expected norms for age on neuropsychological testing. PiB PET scans were performed on 287 participants, 100 had DEXA scans and 91 participated in ActiGraph monitoring. CONCLUSION The participants comprising the AIBL cohort represent a group of highly motivated and well-characterized individuals who represent a unique resource for the study of AD. They will be reassessed at 18-month intervals in order to determine the predictive utility of various biomarkers, cognitive parameters and lifestyle factors as indicators of AD, and as predictors of future cognitive decline.

Design and application of diabodies, triabodies and tetrabodies for cancer targeting

Multivalent recombinant antibody fragments provide high binding avidity and unique specificity to a wide range of target antigens and haptens. This review describes the design and expression of diabodies, triabodies and tetrabodies using examples of scFv molecules that target viruses (influenza neuraminidase) and cancer (Ep-CAM; epithelial cell adhesion molecule). We discuss the preferred choice of linker length between V-domains to direct the formation of either diabodies (60 kDa), triabodies (90 kDa) or tetrabodies (120 kDa), each with size, flexibility and valency suited to different applications for in vivo imaging and therapy. The increased binding valency of these scFv multimers results in high avidity (low off-rates). A particular advantage for tumour targeting is that molecules of 60-100 kDa have increased tumour penetration and fast clearance rates compared to the parent Ig (150 kDa). We highlight a number of cancer-targeting scFv multimers that have recently successfully undergone pre-clinical trials for in vivo stability and efficacy. We also review the design of multi-specific Fv modules suited to cross-link two or more different target antigens. These bi- and tri-specific multimers can be formed by association of different scFv molecules and, in the first examples, have been designed as cross-linking reagents for T-cell recruitment into tumours (immunotherapy), viral retargeting (gene therapy) and as red blood cell agglutination reagents (immunodiagnostics).

High avidity scFv multimers; diabodies and triabodies.

Multivalent recombinant antibody fragments provide high binding avidity and unique specificity to a wide range of target antigens and haptens. This review describes how careful choice of linker length between V-domains creates new types of Fv modules with size, flexibility and valency suited to in vivo imaging and therapy. Further, we review the design of multi-specific Fv modules suited to cross-linking target antigens for cell-recruitment, viral delivery and immunodiagnostics. Single chain Fv antibody fragments (scFvs) are predominantly monomeric when the V(H) and V(L) domains are joined by polypeptide linkers of at least 12 residues. An scFv molecule with a linker of 3 to 12 residues cannot fold into a functional Fv domain and instead associates with a second scFv molecule to form a bivalent dimer (diabody, approximately 60 kDa). Reducing the linker length below three residues can force scFv association into trimers (triabodies, approximately 90 kDa) or tetramers ( approximately 120 KDa) depending on linker length, composition and V-domain orientation. The increased binding valency in these scFv multimers results in high avidity (long off-rates). A particular advantage for tumor targeting is that molecules of approximately 60-100 kDa have increased tumor penetration and fast clearance rates compared to the parent Ig. A number of cancer-targeting scFv multimers have recently undergone pre-clinical evaluation for in vivo stability and efficacy. Bi- and tri-specific multimers can be formed by association of different scFv molecules and, in the first examples, have been designed as cross-linking reagents for T-cell recruitment into tumors (immunotherapy) and as red blood cell agglutination reagents (immunodiagnostics).

Dimeric and trimeric antibodies : high avidity scFvs for cancer targeting

Recombinant antibody fragments can be engineered to assemble into stable multimeric oligomers of high binding avidity and specificity to a wide range of target antigens and haptens. This review describes the design and expression of diabodies (dimers), triabodies (trimers) and tetrabodies (tetramers). In particular we discuss the role of linker length between V-domains and the orientation of the V-domains to direct the formation of either diabodies (60 kDa), triabodies (90 kDa) or tetrabodies (120 kDa), and how the size, flexibility and valency of each molecules is suited to different applications for in vivo imaging and therapy. Single chain Fv antibody fragments joined by polypeptide linkers of at least 12 residues irrespective of V-domains orientation predominantly form monomers with varying amounts of dimer and higher molecular mass oligomers in equilibrium. A scFv molecule with a linker of 3-12 residues cannot fold into a functional Fv domain and instead associates with a second scFv molecule to form a bivalent dimer (diabody, approximately 60 kDa). Reducing the linker length below three residues can force scFv association into trimers (triabodies, approximately 90 kDa) or tetramers ( approximately 120 kDa) depending on linker length, composition and V-domain orientation. A particular advantage for tumour targeting is that molecules of 60-100 kDa have increased tumour penetration and fast clearance rates compared with the parent Ig (150 kDa). We highlight a number of cancer-targeting scFv diabodies that have undergone successful pre-clinical trials for in vivo stability and efficacy. We also briefly review the design of multi-specific Fv modules suited to cross-link two or more different target antigens. Bi-specific diabodies formed by association of different scFv molecules have been designed as cross-linking reagents for T-cell recruitment into tumours (immunotherapy), viral retargeting (gene therapy) and as red blood cell agglutination reagents (immunodiagnostics). The more challenging trispecific multimers (triabodies) remain to be described.

Deletion mapping and expression in Escherichia coli of the large genomic segment of a birnavirus.

The large genomic segment of infectious bursal disease virus encodes a polyprotein in which the viral polypeptides are present in the following order: N-VP2-VP4-VP3-C. Expression in Escherichia coli of the large segment results in the processing of the polyprotein. The expression product reacts with a virus neutralizing and protective monoclonal antibody that recognizes a conformational epitope on the surface of the virus. Different regions of the large genomic segment were deleted at defined restriction sites and the truncated fragments were ligated to various expression vectors for high-level expression in E. coli. The expressed proteins were probed with three different monoclonal antibodies that recognize epitopes encoded by different regions of the large genomic segment. These deletion mapping studies suggest that VP4 is involved in the processing of the precursor polyprotein, and the conformational epitope recognized by the virus neutralizing monoclonal antibody is present within VP2.

Recombinant antibody constructs in cancer therapy.

Recombinant antibodies and their fragments now represent over 30% of all biological proteins undergoing clinical trials for diagnosis and therapy. The focus on antibodies as the ideal cancer-targeting reagents recently culminated in approval by the Food and Drugs Administration for the first engineered therapeutic antibodies. In the past year, important advances have been made in the design, selection and production of new types of engineered antibodies. Innovative selection methods have enabled the isolation of high-affinity cancer-targeting and antiviral antibodies, the latter capable of redirecting viruses for gene therapy applications. In other strategies for cancer diagnosis and therapy, recombinant antibody fragments have been fused to radioisotopes, drugs, toxins, enzymes and biosensor surfaces. Bispecific antibodies and related fusion proteins have been produced for cancer immunotherapy, effectively enhancing the human immune response in anticancer vaccines and T cell recruitment strategies.

Recombinant antibody fragments

Recombinant antibodies and their fragments now represent over 30% of all biological proteins undergoing clinical trials, which recently culminated in FDA approval for the first engineered cancer therapeutic antibody. Other successful applications include both the effective enhancement of the human immune response for cancer therapy and the reduction of unwanted immune rejections following transplantation and antibody therapy. Important advances have been made in the methods for design, selection and production of these new types of engineered antibodies. Innovative selection methods have enabled the isolation of catalytic antibodies and high-affinity viral-specific antibodies, the latter capable of redirecting viruses for gene therapy applications. In numerous practical applications, recombinant antibody fragments have been fused to radioisotopes, drugs, toxins, enzymes and biosensor surfaces.

Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries.

The new antigen receptor (NAR) from nurse sharks consists of an immunoglobulin variable domain attached to five constant domains, and is hypothesised to function as an antigen-binding antibody-like molecule. To determine whether the NAR is present in other species we have isolated a number of new antigen receptor variable domains from the spotted wobbegong shark (Orectolobus maculatus) and compared their structure to that of the nurse shark protein. To determine whether these wNARs can function as antigen-binding proteins, we have used them as scaffolds for the construction of protein libraries in which the CDR3 loop was randomised, and displayed the resulting recombinant domains on the surface of fd bacteriophages. On selection against several protein antigens, the highest affinity wNAR proteins were generated against the Gingipain K protease from Porphyromonas gingivalis. One wNAR protein bound Gingipain K specifically by ELISA and BIAcore analysis and, when expressed in E. coli and purified by affinity chromatography, eluted from an FPLC column as a single peak consistent with folding into a monomeric protein. Naturally occurring nurse shark and wobbegong NAR variable domains exhibit conserved cysteine residues within the CDR1 and CDR3 loops which potentially form disulphide linkages and enhance protein stability; proteins isolated from the in vitro NAR wobbegong library showed similar selection for such paired cysteine residues. Thus, the New Antigen Receptor represents a protein scaffold with possible stability advantages over conventional antibodies when used in in vitro molecular libraries.

Triabodies: single chain Fv fragments without a linker form trivalent trimers

A single chain Fv fragment (scFv) of the murine monoclonal antibody 11‐1G10 was constructed by directly joining the C‐terminal residue of the VH domain to the N‐terminal residue of VL. 11‐1G10 is an anti‐idiotype and competes with the antigen, influenza virus neuraminidase (NA), for binding to the NC41 antibody. The scFv formed stable trimers with three active antigen combining sites for NC41 Fab fragments. We propose that trimeric scFvs may be the preferred conformation for directly linked VH‐VL molecules, which contrasts the formation of scFv dimers (diabodies) when the VH and VL domains are joined by short flexible linkers of between 5–10 residues. BIAcore biosensor binding experiments showed that the trimeric scFv showed an expected increase in binding affinity, due to avidity, compared to the monomeric 15‐residue linked scFv. The increase in avidity of scFv trimers offers advantages for imaging and immunotherapy.

Recombinant antibodies for cancer diagnosis and therapy

Recombinant antibodies currently represent over 30% of biopharmaceuticals in clinical trials, highlighted by the recent Food and Drug Administration (FDA) approvals of Zevalin™ (ibritumomab-tiuxetan; IDEC Pharmaceuticals, San Dieago, CA, USA) for cancer radioimmunotherapy and Humira™ (adalimumab; Abbott Laboratories, IL, USA) for rheumatoid arthritis. Together, these FDA approvals have excited the biotechnology industry, particularly since sales of recombinant antibodies are increasing rapidly to a predicted US