Site-Specifically Labeled Immunoconjugates for Molecular Imaging--Part 1: Cysteine Residues and Glycans.

Pierre Adumeau,Colleen Brent,Brian M Zeglis,Sai Kiran Sharma

doi:10.1007/s11307-015-0919-4

Abstract

Due to their remarkable selectivity and specificity for cancer biomarkers, immunoconjugates have emerged as extremely promising vectors for the delivery of diagnostic radioisotopes and fluorophores to malignant tissues. Paradoxically, however, these tools for precision medicine are synthesized in a remarkably imprecise way. Indeed, the vast majority of immunoconjugates are created via the random conjugation of bifunctional probes (e.g., DOTA-NCS) to amino acids within the antibody (e.g., lysines). Yet antibodies have multiple copies of these residues throughout their macromolecular structure, making control over the location of the conjugation reaction impossible. This lack of site specificity can lead to the formation of poorly defined, heterogeneous immunoconjugates with suboptimal in vivo behavior. Over the past decade, interest in the synthesis and development of site-specifically labeled immunoconjugates—both antibody-drug conjugates as well as constructs for in vivo imaging—has increased dramatically, and a number of reports have suggested that these better defined, more homogeneous constructs exhibit improved performance in vivo compared to their randomly modified cousins. In this two-part review, we seek to provide an overview of the various methods that have been developed to create site-specifically modified immunoconjugates for positron emission tomography, single photon emission computed tomography, and fluorescence imaging. We will begin with an introduction to the structure of antibodies and antibody fragments. This is followed by the core of the work: sections detailing the four different approaches to site-specific modification strategies based on cysteine residues, glycans, peptide tags, and unnatural amino acids. These discussions will be divided into two installments: cysteine residues and glycans will be detailed in Part 1 of the review, while peptide tags and unnatural amino acids will be addressed in Part 2. Ultimately, we sincerely hope that this review fosters interest and enthusiasm for site-specific immunoconjugates within the nuclear medicine and molecular imaging communities.

Highlights

Over the last three decades, medical imaging has revolutionized cancer care, providing clinicians with the means to noninvasively acquire anatomical, functional, and biological information about tumors
Antibody conjugates bearing a wide range of reporters—ranging from Zr-89 for positron emission tomography (PET) to near-infrared fluorophores for optical imaging (OI)—have been successfully developed and translated to the clinic [3, 4]
In a small variation on this approach, Li et al employed DO3A-PEGn constructs (n = 12, 24, and 48) bearing a vinyl sulfone moiety to site- modify an anti-TAG-72 diabody with a C-terminal cysteine (Fig. 3 and 5a) [65]. After radiolabeling these conjugates with Cu-64, PET imaging was performed using mice bearing LS174T xenografts, and the authors found that while all of the diabodies proved able to target the tumor, the background activity levels in the blood and kidneys were highly dependent on the length of the PEG chain

Summary

Introduction

Over the last three decades, medical imaging has revolutionized cancer care, providing clinicians with the means to noninvasively acquire anatomical, functional, and biological information about tumors. In a small variation on this approach, Li et al employed DO3A-PEGn constructs (n = 12, 24, and 48) bearing a vinyl sulfone moiety to site- modify an anti-TAG-72 diabody with a C-terminal cysteine (Fig. 3 and 5a) [65] After radiolabeling these conjugates with Cu-64, PET imaging was performed using mice bearing LS174T xenografts, and the authors found that while all of the diabodies proved able to target the tumor, the background activity levels in the blood and kidneys were highly dependent on the length of the PEG chain. Subsequent SPECT imaging experiments using mice bearing subcutaneous, bilateral MDAMB-361 (HER2-positive) and MDA-MB-231 (HER2negative) breast cancer xenografts illustrated that the sitespecifically labeled [111In]DOTA-trastuzumab targeted the HER2-expressing xenografts but did not offer a significant improvement over an [111In]DOTA-trastuzumab construct synthesized using a traditional, nonsite-specific conjugation method [90]

Glycoengineering Methods

Conclusion

Conflict of Interest