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Protein catalysed capture agents for molecular imaging

Figure 1. Assembly of multi-peptide PCC agents by protein-templated in situ click chemistry. An anchor peptide (red circles) is identified by screening a one-bead-one-compound (OBOC) peptide library (grey sphere) against a target protein. Bi-ligands are assembled by screening an OBOC library consisting of >1 million alkyne-functionalized secondary peptides (blue circles) against the target in the presence of soluble azide-functionalized anchor peptide. Repeating this process generates a tri-ligand by conjugation of the OBOC tertiary peptides (green circles) and bi-ligand on the protein surface.
Figure 2. Functionalization of PCC Agents. Examples of the types of labels (= X) that can be site-specifically conjugated to linear or branched PCC agent tri-ligands.

There is a growing need for affinity agents for targeted molecular imaging of disease biomarkers. Protein catalysed capture agents (PCC agents) are assembled through target-guided in situ click chemistry and display low nanomolar affinity and specificity. The iterative design process, architecture, and composition of the resulting multi-ligands make this a promising technology for imaging agent development.

by Dr Steven W. Millward,  Dr Heather D. Agnew, Dr Suresh Pitram,  Dr Bert T. Lai, Dr Rosemary D. Rohde and Dr Norman Hardman

 

Personalized medicine is increasingly becoming a major component            of cancer treatment and management. Many of the advances in personalized medicine have come from high throughput genomic sequencing and multiplexed proteomic analysis of tumour and tissue specimens. While these technologies have dramatically increased our understanding of cancer and cancer heterogeneity at the molecular level, translation of this knowledge into clinical imaging agents to non-invasively characterize tumour phenotypes in vivo has lagged behind.  While small molecules (e.g. FDG) have proven very useful for clinical measurement of metabolic activity, molecular imaging of cell surface and secreted biomarkers overexpressed in cancer (e.g. IGFR, EGFR, etc.) is largely confined to the preclinical and early clinical trial setting.

While groundbreaking work to determine the biological and therapeutic roles of these proteins has produced a wealth of high affinity antibodies, antibody fragments, and peptides, attempts to translate them into radiotracers for molecular imaging have produced mixed results. Antibody-based imaging agents suffer from three disadvantages in this setting:

1) Their long serum half-lives, while advantageous for therapeutic applications, result in high background signal in perfused tissue and lengthen the time between agent administration and image acquisition.
2) Antibody-based radiotracers often show accumulation and metabolic processing in the liver, increasing nonspecific signal and potentially obscuring critical tumour-specific signal in the abdominal cavity. 
3) Antibodies are biologicals, and as such their high production costs and regulatory burdens are significant economic barriers to commercialization and clinical translation. Linear peptides derived from phage display represent a second class of potential imaging agents, however their rapid degradation in vivo typically results in nonspecific background signal and poor tumour uptake. An ideal targeted molecular imaging agent would combine the affinity and specificity of antibodies with the high bio-stability, tumour uptake, and clearance of small molecules.

PCC agent design
Protein catalysed capture (PCC) agent technology represents a novel approach to rapidly design ligands with high affinity and specificity that can be potentially adapted to a variety of research and in vivo



Agnew and co-workers were the first to demonstrate and characterize multi-ligand design by in situ click chemistry by assembling a tri-ligand for bovine carbonic anhydrase II (bCAII) [2]. Importantly, each component of the multi-ligand was selected from million-member OBOC libraries containing all-D-amino acid peptides, biasing the resulting ligand to proteolytic and potentially metabolic stability. The authors found that the addition of each peptide component resulted in approximately 100-fold improvement in target affinity with the final tri-ligand exhibiting a mid-nanomolar dissociation constant (KD = 45 nM). This affinity was sufficient for use of the tri-ligand as an antibody replacement for nanogram-level detection of bCAII in serum by immunoblotting.

Millward and co-workers extended this approach to design a branched tri-ligand for the Akt kinase [3]. In this study, the OBOC peptide screen was modified to explicitly select sequences that bound to the target and participated in the in situ click reaction leading to a more rapid selection of peptide components that enhanced both the affinity and specificity of the multi-ligand [Figure 1]. The resulting branched Akt-specific tri-ligand displayed mid-nanomolar affinity (KD = 200 nM) for Akt1 and functioned as an allosteric inhibitor of the enzyme. When conjugated to fluorescein and incubated with fixed, permeabilized cells, the fluorescent tri-ligand was successfully used to image the plasma membrane localization of Akt in an ovarian cancer cell line stimulated through the PI3K pathway. The authors observed that the image quality and signal obtained with the tri-ligand were significantly better than a commercially available antibody raised against the intact protein. While it is difficult to directly compare the antibody and tri-ligand, this effect may arise from the small size and increased affinity of the tri-ligand compared to the monoclonal antibody, which also performed poorly in cell lysate immunoprecipitation from the same line. Alternatively, the non-specific conjugation of the fluorophore to the antibody, in contrast to the specific and single-site labelling of the tri-ligand during chemical synthesis, may have attenuated its affinity and/or specificity for the target.

PCC agents and imaging
This brief review of PCC technology highlights some of the potential advantages of peptide multi-ligands over antibodies as affinity agents for molecular imaging. One of the distinguishing features of PCC development is the iterative selection and elaboration of the anchor peptide. This approach enables affinity and/or specificity to be improved in a modular fashion by adding additional peptide components that have been screened for the desired property. For example, in the Akt screens, the first elaboration of the anchor peptide resulted in a 100-fold increase in affinity but at the cost of off-target kinase binding. In the second elaboration, affinity was essentially unchanged but selectivity was improved by introducing destabilizing interactions with the major off-target kinase.

This fortuitous effect illustrates the potential benefits to be gained by iteratively increasing the size of the multi-ligand while enforcing the requirement that the target protein be able to simultaneously bind and assemble each of the component peptides through the in situ click reaction. In a manner analogous to antibodies, multi-ligand affinity and specificity for the target protein are achieved through multiple interactions of varying strength to create a large binding interface with the target surface [4].

The individual components of the PCC agent can be selected from chemical libraries containing non-natural amino acids for increased resistance to proteolytic degradation and metabolic modification. This is essential for increasing the stability of multi-ligands for in vivo applications. Linear peptides containing only natural amino acids normally undergo rapid degradation in vivo and have biological half-lives of less than 10 minutes [5]. This is unsuitable for most imaging applications where degradation and loss of label can result in high background and poor tumour visualization. The use of branched and non-linear architectures can potentially provide a second route to improving bio-stability. Indeed, cyclic peptides such as the clinical imaging agent 111In-DPTA-octreotide (OctreoScan), often display excellent bio-stability. Incorporation of these structural modifications into the in situ screens can potentially enhance the resulting multi-ligand stability without the need for costly and time-consuming efforts to improve compound stability, possibly at the expense of biological activity.

As a result of their size and biological origin, antibodies and antibody fragments often display batch-to-batch variability as well as chemical and thermal lability, potentially resulting in erratic affinity and specificity. Antibodies are also difficult to selectively modify uniformly with imaging functionalities with a defined stoichiometry, further complicating their use in clinical imaging applications where a precise measurement of label incorporation is needed. In translational and clinical imaging applications, variable biological activity and label incorporation can pose significant challenges for reproducible measurements of specific tumour uptake and biodistribution (% Injected Dose), critical metrics for clinical diagnostic imaging.



Future directions
PCC agents are highly flexible and can be integrated as synthetic replacements for antibodies in research and clinical applications. PCC agent multi-ligands have been immobilized via biotin to immunoprecipitate target proteins from complex mixtures and functionalized with fluorescent dyes to image protein distribution within the cell by fluorescence microscopy. We anticipate that these agents can be readily linked to chelating functionalities and radiotracers with minimal impact on biological activity to image cancer biomarkers in vivo. The iterative PCC design process and the ease of modification make PCC agents a potentially powerful tool for both basic science and clinical molecular imaging.

References
1. Lewis WG, Green LG, Grynszpan F, Radić Z, Carlier PR, Taylor P, Finn MG, Sharpless KB. Click chemistry in situ: acetylcholinesterase as a reaction vessel for the selective assembly of a femtomolar inhibitor from an array of building blocks. Angew. Chem. Int. Ed. 2002; 41 (6): 875-875.
2. Agnew HD, Rohde RD, Millward SW, Nag A, Yeo W-S, Hein JE, Pitram SM, Tariq AA, Burns VM, Krom RJ, Fokin VV, Sharpless KB, Heath JR. Iterative in situ click chemistry creates antibody-like protein-capture agents. Angew. Chem. Int. Ed. 2009; 48 (27): 4944-4948.
3. Millward SW, Henning RK, Kwong GA, Pitram S, Agnew HD, Deyle KM, Nag A, Hein J, Lee SS, Lim J, Pfeilsticker JA, Sharpless KB, Heath JR. Iterative in situ click chemistry assembles a branched capture agent and allosteric inhibitor for Akt1. J. Am. Chem. Soc. 2011; 133 (45): 18280-18288.
4. Millward SW, Agnew HD, Lai B, Lee SS, Lim J, Nag A, Pitram S, Rohde R, Heath JR. In situ click chemistry: from small molecule discovery to synthetic antibodies. Integr. Biol. 2012, Advance Article.
5. Okarvi SM. Peptide-based radiopharmaceuticals: Future tools for diagnostic imaging of cancers and other diseases. Medicinal Research Reviews 2004; 24 (3): 357-397.

The authors
Steven W. Millward PhD1*, Heather D. Agnew PhD2, Suresh Pitram PhD2, Bert T. Lai PhD2, Rosemary D. Rohde PhD2 and Norman Hardman PhD2

1Department of Experimental Diagnostic Imaging, University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA
2Integrated Diagnostics, Culver City, USA

* Corresponding author


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