5c,?,d).d). of macromolecular targeting vectors and small molecules, specifically high target specificity and short organ and tissue residence occasions, respectively.1,4 To achieve this, the targeting vector is administered first and allowed to GGTI298 Trifluoroacetate build up at the target site and clear GGTI298 Trifluoroacetate from off-target organs prior to the injection of a small effector molecule transporting the payload of interest (radionuclide; Fig. 1).4,5,9 In order to enable their recombination, both entities are equipped with complementary functionalities that enable an ligation reaction.10,11 Appropriate pairs of reaction partners that have been employed in pretargeting approaches include streptavidinbiotin12, complementary oligonucleotide strands13, and click chemistry-based reaction pairs.10,14,15 While strategies based on streptavidin-biotin have shown somewhat deflating outcomes in the clinic16, the use of a bispecific, CEA-targeting antibody in combination with a radiolabeled hapten peptide has shown very encouraging clinical results.17 Open in a separate window Determine 1. (a) Schematic illustration of the pretargeting approach: a macromolecular targeting vector in our case an antibody-TCO conjugate is usually injected first and allowed to reach the target site while clearing slowly from systemic blood circulation. After a specific accumulation time, the small molecule effector probe (in this case a radiolabeled tetrazine probe) is usually administered systemically and undergoes bioorthogonal click reaction with the TCO-groups KILLER of the immunoconjugate at the target site. (b) Modular chemistry approach for radioligand design: radioligands consisted of a Tz moiety for click chemistry, a linker for altering the biodistribution, and a chelator for the attachment of the positron-emitting metal ions 68Ga3+ and [18F]-AlF2+. One of the newer users of the click chemistry toolbox, the inverse electron-demand Diels-Alder (IEDDA) reaction between pretargeting.2C5,7,10,11 The IEDDA ligation is selective and bioorthogonal, but its principal advantage for pretargeting compared to other click reactions such as the Staudinger ligation18 or the strain-promoted alkyne-azide cycloaddition14 (SPAAC) lies in its velocity. To wit, the first-order rate constants of the Tz/TCO ligation lie in the realm of 104-105 M?1s?1, orders of magnitude faster than the rates of the Staudinger and SPAAC reactions (0.002 M?1s?1 and 0.07 M?1s?1, respectively).11 The feasibility of the IEDDA reaction between a TCO-modified monoclonal GGTI298 Trifluoroacetate antibody (mAb) and a radiolabeled Tz has been demonstrated by numerous groups using a wide range of antigen-targeting immunoconjugates and tetrazines labeled with an array of radionuclides for imaging (including 111In, 64Cu, 99mTc, 18F, 68Ga, and 11C)1,4,5,19C21 and therapy (177Lu).7 In terms of pretargeted positron emission tomography (PET) imaging, GGTI298 Trifluoroacetate Zeglis, overall performance of small molecule radioligands in pretargeting systems. Tracer library and SPR study design The synthesis of the radioligands library as the first step of this study was based on previously reported protocols.1,2,4 New reaction routes and radiolabeling procedures developed within this study are described in the supporting information (Supplementary Sections 2 and 3). Overall, the radioligands were designed to display structural variation in order to cover a broad spectrum of physicochemical properties, thereby enabling the study of the relationship between structure and behavior (Tab. 1). Each radioligand is composed of 3 different structural building blocks (Tab. 1; Fig. 2a). First, a Tz component (1C4) was selected for click chemistry. Second, a linker moiety consisting of polyethylene glycol GGTI298 Trifluoroacetate [PEG7 (5) or PEG11 (6)], amino acids (AA) [AA = lysine (K, 7), histidine (H, 8), aspartate (R, 9), and arginine (D, 10)], or a combination of both is usually attached. Finally, a bifunctional chelator, either 1,4,7-triazacyclononane-1,4-diacetic acid (NODA, 11,12) or 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA, 13,14), was launched, allowing for the installation of 18F and 68Ga radionuclides. Tz moieties 1C4 were selected based on their previously reported stability and reaction kinetics with TCO to ensure a wide range of properties.4,5,21,23 The use of PEG linkers to modulate PK of small molecules has previously been reported, including accelerated non-target organ clearance as well as increased renal clearance.1,2,24 We included them into our study in order to investigate their impact on radiotracer PK alone or in combination with AA linkers (which, to the best of our knowledge, have not yet been systematically reported in any SPR study). The amino acids lysine, arginine, histidine, and aspartate were regarded as useful structural components to significantly influence behavior and PK parameters. It was reasoned that their charged side chains should have a measureable effect on tracer PK, and.