Site-selective protein-modification chemistry for basic biology and drug development.

Nature has produced intricate machinery to covalently diversify the structure of proteins after their synthesis in the ribosome. In an attempt to mimic nature, chemists have developed a large set of reactions that enable post-expression modification of proteins at pre-determined sites. These reactions are now used to selectively install particular modifications on proteins for many biological and therapeutic applications. For example, they provide an opportunity to install post-translational modifications on proteins to determine their exact biological roles. Labelling of proteins in live cells with fluorescent dyes allows protein uptake and intracellular trafficking to be tracked and also enables physiological parameters to be measured optically. Through the conjugation of potent cytotoxicants to antibodies, novel anti-cancer drugs with improved efficacy and reduced side effects may be obtained. In this Perspective, we highlight the most exciting current and future applications of chemical site-selective protein modification and consider which hurdles still need to be overcome for more widespread use.We thank FCT Portugal (FCT Investigator to G.J.L.B.), the EU (Marie-Curie CIG to G.J.L.B. and Marie-Curie IEF to O.B.) and the EPSRC for funding. G.J.L.B. is a Royal Society University Research Fellow.This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/nchem.239

Tumor-Targeting Antibody–Anticalin Fusion Proteins for <i>in Vivo</i> Pretargeting Applications

Pretargeting approaches rely on the injection of bispecific antibodies capable of recognizing both an accessible disease marker and a small ligand, which is typically administered at a later stage and which serves as delivery vehicle for a payload for imaging or therapy applications. In the oncology field, pretargeting strategies have exhibited extremely promising biodistribution results and <i>in vivo</i> selectivity, but have often relied on the cumbersome preparation of multispecific antibodies by chemical conjugation techniques. Here, we describe the design, production, and characterization of a novel class of bispecific multivalent antibody products, which contain both antibody fragments and an anticalin moiety for the simultaneous recognition of tumor-associated antigens and a small organic molecule. Anticalins are derivatives of the naturally occurring binding proteins lipocalins, which have been engineered to recognize a target molecule with high affinity. In particular, we produced and compared <i>in vitro</i> and <i>in vivo</i> different fusion proteins, which contained the anticalin FluA that selectively recognizes various different fluorescein derivatives and the F8 antibody specific to the alternatively spliced EDA domain of fibronectin (a marker of tumor angiogenesis). The selective accumulation of the most promising fusion-protein scFv­(F8)-FluA-scFv­(F8) on solid tumors and simultaneous binding of fluorescein derivatives could be visualized <i>in vivo</i> using a fluorescein-near-infrared fluorescent dye conjugate, confirming the potential of antibody-anticalin fusion proteins for pretargeting applications

A Microdosing Study with 99mTc-PHC-102 for the SPECT/CT Imaging of Primary and Metastatic Lesions in Renal Cell Carcinoma Patients

99mTc-PHC-102 is a 99mTc-labeled derivative of acetazolamide, a high-affinity small organic ligand of carbonic anhydrase IX (CAIX). 99mTc-PHC-102 has previously shown favorable in vivo biodistribution properties in mouse models of CAIX-positive clear cell renal cell carcinoma (ccRCC) and colorectal cancer. In this study, we aimed to explore the targeting performance of 99mTc-PHC-102 in SPECT in patients with renal cell carcinoma while also assessing the safety and tolerability of the radiotracer. Methods: We studied 5 patients with localized or metastatic ccRCC in a microdosing regimen, after the administration of a 50-μg total of CAIX ligand and 600–800 MBq of 99mTc-PHC-102. Tissue distribution and residence time in normal organs and tumors were analyzed by serial SPECT/CT scans at 3 time points (30 min, 2 h, and 6 h) after intravenous administration. Results: In the 5 patients studied, 99mTc-PHC-102 was well tolerated and no study drug–related adverse events were recorded. In the stomach, kidneys, and gallbladder, the radiotracer showed a rapid initial uptake, which cleared over time. Localization of the study drug in primary tumors of 5 patients was observed, with favorable tumor-to-background ratios. 99mTc-PHC-102 SPECT/CT allowed the identification of 4 previously unknown lung and lymph node metastases in 2 patients. Conclusion: 99mTc-PHC-102 is a promising SPECT tracer for the imaging of patients with ccRCC. This tracer has the potential to identify primary and metastatic lesions in different anatomic locations. 99mTc-PHC-102 might also serve as a companion diagnostic agent for future CAIX-targeting therapeutics.ISSN:0097-9058ISSN:0022-3123ISSN:0161-5505ISSN:2159-662XISSN:1535-566

Image-based ex-vivo drug screening for patients with aggressive haematological malignancies: interim results from a single-arm, open-label, pilot study

ISSN:2352-302

Selective Inhibition of Bruton's Tyrosine Kinase by a Designed Covalent Ligand Leads to Potent Therapeutic Efficacy in Blood Cancers Relative to Clinically Used Inhibitors.

Bruton's tyrosine kinase (BTK) is a member of the TEC-family kinases and crucial for the proliferation and differentiation of B-cells. We evaluated the therapeutic potential of a covalent inhibitor (JS25) with nanomolar potency against BTK and with a more desirable selectivity and inhibitory profile compared to the FDA-approved BTK inhibitors ibrutinib and acalabrutinib. Structural prediction of the BTK/JS25 complex revealed sequestration of Tyr551 that leads to BTK's inactivation. JS25 also inhibited the proliferation of myeloid and lymphoid B-cell cancer cell lines. Its therapeutic potential was further tested against ibrutinib in preclinical models of B-cell cancers. JS25 treatment induced a more pronounced cell death in a murine xenograft model of Burkitt's lymphoma, causing a 30-40% reduction of the subcutaneous tumor and an overall reduction in the percentage of metastasis and secondary tumor formation. In a patient model of diffuse large B-cell lymphoma, the drug response of JS25 was higher than that of ibrutinib, leading to a 64% "on-target" efficacy. Finally, in zebrafish patient-derived xenografts of chronic lymphocytic leukemia, JS25 was faster and more effective in decreasing tumor burden, producing superior therapeutic effects compared to ibrutinib. We expect JS25 to become therapeutically relevant as a BTK inhibitor and to find applications in the treatment of hematological cancers and other pathologies with unmet clinical treatment

Combined chemosensitivity and chromatin profiling prioritizes drug combinations in CLL

ISSN:1552-4450ISSN:1552-446