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. 2023 Mar 8;18(3):443–448. doi: 10.1021/acschembio.2c00634

Genetic Code Expansion for Site-Specific Labeling of Antibodies with Radioisotopes

Christine Koehler †,‡, Paul F Sauter †,‡, Benedikt Klasen §, Christopher Waldmann ∥, Stefanie Pektor ∥, Nicole Bausbacher ∥, Edward A Lemke ⊥,#,*, Matthias Miederer ∥,*
PMCID: PMC10029752  PMID: 36889678

Abstract

graphic file with name cb2c00634_0004.jpg

Due to their target specificity, antibody–drug conjugates—monoclonal antibodies conjugated to a cytotoxic moiety—are efficient therapeutics that can kill malignant cells overexpressing a target gene. Linking an antibody with radioisotopes (radioimmunoconjugates) enables powerful diagnostics and/or closely related therapeutic applications, depending on the isotope. To generate site-specific radioimmunoconjugates, we utilized genetic code expansion and subsequent conjugation by inverse electron-demand Diels–Alder cycloaddition reactions. We show that, using this approach, site-specific labeling of trastuzumab with either zirconium-89 (89Zr) for diagnostics or lutetium-177 (177Lu) for therapeutics yields efficient radioimmunoconjugates. Positron emission tomography imaging revealed a high accumulation of site-specifically 89Zr-labeled trastuzumab in tumors after 24 h and low accumulation in other organs. The corresponding 177Lu-trastuzumab radioimmunoconjugates were comparably distributed in vivo.

Introduction

Monoclonal antibodies (mAbs) are versatile biologics used as targeted treatments for a broad range of illnesses, for example, autoimmune diseases and various types of cancer, with over 80 mAbs currently approved for clinical application.1,2 Beyond their therapeutic use as targeted blockers of molecular function, owing to their extraordinarily high antigen specificity, they are being increasingly deployed in a drug delivery role as carrier molecules in antibody–drug conjugates (ADCs). In recent years, a growing number of drugs in this respective class have been clinically approved. Examples are trastuzumab deruxtecan (Enhertu) and trastuzumab emtansine (Kadcyla) for treating metastasized breast cancer, highlighting the potential of mAb-based targeted therapy.3,4 Another attractive strategy is to deliver radioactive isotopes instead of cytotoxic drugs to the site of disease. In cancer radioimmunotherapy, mAbs labeled with suitable radioisotopes deliver their radioactive payload to the tumor site. Although there have been only two approvals of radioimmunoconjugates (RICs) to date, interest in this class of therapeutics is growing, and currently there are 31 active clinical trials, one of which is investigating lutetium-177 (177Lu) lilotomab satetraxetan (Betalutin) for the treatment of non-Hodgkin lymphoma.5 The conjugation of radioisotopes to mAbs also affords the opportunity to replace the therapeutic radioisotopes, often beta minus or alpha emitters, with diagnostic isotopes, commonly positron emitters for positron emission tomography (PET), to image disease-associated targets of interest. This concept of combining therapeutic and diagnostic capabilities in one molecule has developed into a highly dynamic field within nuclear medicine referred to as “theranostics”.

The most widely used methods for conjugating functional molecules to antibodies are based on the stochastic coupling to native lysine or cysteine residues. These methods lead to mixtures of conjugates having varying drug-to-antibody ratios (DARs), which can influence the properties of an ADC, such as its pharmacokinetics, stability, and efficacy.6,7 In contrast, site-specific conjugation methods result in homogeneous ADCs and improvements in the aforementioned properties.7,8 Site-specific labeling has been shown to improve RIC properties such as stability, immunoreactivity, and biodistribution.9 Site-specific modification of an antibody can be achieved in several ways, for example, by utilizing engineered cysteine residues, enzymatic coupling to amino acid tags or glycans, or the incorporation of noncanonical amino acids (ncAAs) using genetic code expansion (GCE).7,9−12 In the latter, custom-designed ncAAs contain chemical moieties that can undergo specific chemical reactions—often click chemistry—for coupling a payload (Figure 1A).

Figure 1.

Figure 1

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General scheme of antibody labeling via SPIEDAC and chemical structures of compounds 1–3. (A) Principle of SPIEDAC of site-specifically introduced trans-cyclooctene amino acid in an antibody with tetrazine payload. (B) N6-({[(S,E)-Cyclooct-2-en-1-yl]oxy}carbonyl)-l-lysine (TCO*A, 1). (C) DFO-tetrazine (2). (D) DOTA-PEG9-tetrazine (3).

Here we report the use of trans-cyclooctene (TCO)-based ncAAs for RIC. TCOs can react with 1,2,4,5-tetrazines by a strain-promoted inverse electron-demand Diels–Alder cycloaddition (SPIEDAC) reaction, which is one of the fastest bioorthogonal click reactions known.13−16 For this study, we chose the axial trans-cyclooct-2-ene-modified ncAA (TCO*-A, 1, Figure 1B), which reacts with fast kinetics (k > 10 000 M–1 s–1) compared to other ncAAs.16 The incorporation of TCO*A has previously been shown useful for several other applications, including the attachment of fluorophores to proteins.15,17 TCO*A was site-specifically introduced into our mAb using an insect cell expression system. The use of Spodoptera frugiperda (Sf21) combined with baculovirus transduction, developed previously,18 is simple and cost-effective compared to other expression hosts and is, furthermore, capable of producing complex proteins such as antibodies also intracellularly without any glycosylation.18,19

The use of radioisotopes for therapeutic studies has particularly high prerequisites for purity and stability in a biological system over several hours to days. With a half-life of 3.3 days, the positron emitter zirconium-89 (89Zr) is well-suited for antibody-derived PET imaging because it is compatible with the biological half-lives of full antibodies. Proteins can be labeled with 89Zr via desferrioxamine (DFO) chelating moieties, which chelate 89Zr at 37 °C. For therapeutic purposes, the most commonly used isotopes are beta-emitting isotopes such as 177Lu, which has a half-life of 6.7 days. Peptides or proteins can be labeled with a 177Lu-containing macrocycle based on a tetraazacyclododecane tetraacetic acid (DOTA) ligand; such chelates form in high-yielding coordination reactions and are highly stable.

In this study, we site-specifically labeled trastuzumab with the radioisotopes 89Zr and 177Lu for diagnostic and therapeutic purposes. Trastuzumab is a clinically used antibody indicated mainly for the treatment of breast cancer. HER2 is highly expressed in breast cancer cell lines, such as BT-474, and its binding is well-characterized, making it a good model for studying radioisotopes for imaging and therapy.20,21 Using our approach, the amino acid at which the antibody is labeled can be chosen with residue specificity, which results in a high degree of freedom for labeling compared to other technologies.22 After reacting a tetrazine-DFO moiety to trastuzumab substituted with TCO*A at position A121 and subsequent complexation of 89Zr, we showed a temporal accumulation of 89Zr-trastuzumabA121DFO-Tet at the tumor site in mice xenografts. The ex vivo biodistribution confirmed a high accumulation of 89Zr in the tumor. Furthermore, we observed very low accumulation in other organs and thus a high contrast between tumor and nontumor tissues, which we also found for another RIC we prepared analogously, 177Lu-trastuzumabA121PEG9-DOTA. 177Lu is well-known for its tumor-killing potential in radiotherapeutic applications.23

Results and Discussion

To generate a site-specifically radiolabeled antibody, we utilized the MultiBacTAG system to prepare a trastuzumab mutant carrying the ncAA TCO*A (1) at position A121, with an average yield of about 1 mg/L expression.18 This corresponds to approximately 30–50% of the expression yield of wild-type trastuzumab under the same expression conditions. The purified antibody was characterized by size-exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and mass spectrometry analysis (Supporting Information Table S1, Figures S1–S4).

For diagnostic PET imaging, we prepared DFO-conjugated trastuzumabA121DFO-Tet for radiolabeling with 89Zr as described in detail in the Supporting Information. In brief, the synthesis of DFO-tetrazine (2, Figure 1C) for site-specifically modifying trastuzumabA121TCO*A was achieved by amide coupling of the DFO mesylate salt with 4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzoic acid. TrastuzumabA121TCO*A was reacted with 2 at 37 °C, and the click product was purified on a size-exclusion column. The purified trastuzumabA121DFO-Tet was then radiolabeled using a tetravalent zirconium-89 oxalate salt. Finally, 89Zr-trastuzumabA121DFO-Tet was purified using size-exclusion chromatography (SEC) (Supporting Information Figure S5). Radiochemical purity was analyzed by thin-layer chromatography (TLC) and was typically above 97% (Supporting Information Figure S6). Contrary to stochastic labeling, here the maximum number of radioisotopes per mAb is limited to two.

Mice bearing subcutaneous BT-474 xenograft tumors at their shoulders were injected with 89Zr-trastuzumabA121DFO-Tet, and PET and magnetic resonance imaging (MRI) were performed after 24, 72, and 168 h (Figure 2). After 24 h, 89Zr-trastuzumabA121DFO-Tet could be readily visualized at the tumor site, with increasing contrast versus background at later time points. In particular, compartments known for antibody retention, such as blood and liver, exhibited only low uptake (Figure 2B and Supporting Information Figure S7). This low nonspecific background can probably be attributed to lack of glycosylation and lower Fc receptor interaction, which is known to reduce accumulation of radiolabeled antibodies in liver.22 After 7 days, the mice were sacrificed, and an ex vivo biodistribution analysis showed an uptake of 34% injected dose per gram of tissue (ID/g) in the tumor, 10% ID/g in the bones, and less than 5% in the liver, spleen, and kidneys, matching well the quantitative analysis obtained from PET imaging (Figure 2B,C, Supporting Information Figure S7). This compares favorably with a study adopting a random labeling approach, which demonstrated accumulation of less than 25% at the tumor site.24 In addition, quantification of in vivo tumor uptake from PET images (in a segmented region of 40% of a local maximum) showed a slow decline from 44% ID/mL at 24 h to 34% ID/mL and 33% ID/mL at 72 and 168 h, respectively, which corresponds to standard uptake values (SUVs) of 8.2 at 24 h and 6.3 at 72 and 168 h (Supporting Information Figure S7C). Thus, targeting of the PET imaging probe to the tumor and its retention was demonstrated as expected, with minimal release of free 89Zr from the DFO chelate that is typically observed.25 In particular, a high contrast of tumor versus other organs at later time points is a main prerequisite for delivering therapeutic radioisotopes. It indicates the suitability of site-specifically labeled trastuzumabA121TCO*A for this purpose. This result also demonstrates the relatively rapid targeting by the antibody and stable retention within the tumor over several days. Long retention is important for clinical applications of both ADCs and therapeutic RICs.

Figure 2.

Figure 2

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Pharmacokinetics and PET imaging of 89Zr-trastuzumabA121DFO-Tet. (A) PET/MR imaging at 24, 72, and 168 h after injection of 4 MBq 89Zr-trastuzumabA121DFO-Tet. Shown are transaxial, sagittal, and coronal fused images and the maximum intensity projection of the PET. (B) Ex vivo biodistribution at 168 h (n = 4). (C) Uptake values (%ID/cc) in tumor, blood, and liver after 24, 72, and 168 h.

For radiolabeling trastuzumabA121TCO*A with 177Lu, we first formed the coordination complex between 177Lu and DOTA-PEG9-tetrazine (3, Figure 1D) and used an excess of the crude mixture for bioconjugation by click chemistry (see Supporting Information for more details). This simple methodology proved feasible, because complexation of 177Lu with DOTA-based ligands is highly efficient, and the robust SPIEDAC labeling reaction can be achieved conveniently in one pot without loss of yield. Furthermore, this strategy spared the temperature-sensitive antibody from the harsh reaction conditions required to chelate the 177Lu before being reacted under biocompatible SPIEDAC conditions. The required 3 was synthesized by reacting succinimidyl 4-(1,2,4,5-tetrazin-3-yl)benzoate with Boc-N-amido-PEG9-amine, deprotecting the amine, then coupling with DOTA-NHS ester. After radiolabeling 3 with 177Lu at 90 °C, the crude mixture was incubated at 37 °C with trastuzumabA121TCO*A, and the resulting 177Lu-trastuzumabA121PEG9-DOTA was purified by SEC on a PD10 column. The radiochemical purity was analyzed using thin-layer chromatography (Supporting Information Figure S8A,B).

We next investigated the biodistribution of 177Lu-trastuzumabA121PEG9-DOTA in BT-474 mice xenografts. Ex vivo biodistribution was determined after 48 h. For the tumor, we measured an accumulation of ∼55% ID/g, which corresponds well to the uptake as measured by PET imaging (using spherical volumes of interest placed with help of coregistered MRI; Figure 3). The accumulation in liver and spleen of around 20% ID/g tissue is consistent with the literature23 and can be attributed to both the typical pharmacokinetic properties of mAbs and accumulation of free 177Lu. Because free 89Zr distributes in bone, PET imaging of 89Zr-trastuzumabA121DFO-Tet showed an even lower background within liver, diminishing over time (Figure 2B,C).

Figure 3.

Figure 3

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Biodistribution of 177Lu-trastuzumabA121PEG9-DOTA. Ex vivo biodistribution at 48 h (n = 3); one control animal received 177Lu-DOTA-PEG9-tetrazine.

The fact that 177Lu is a β-emitter has made it a popular radioisotope for therapies, as local enrichment correlates with its cytotoxic potential. While a therapeutic analysis of 177Lu-trastuzumabA121PEG9-DOTA goes beyond our study, some mice were not sacrificed after 48 h for the ex vivo biodistribution analysis described above, but instead, we measured the size of subcutaneous BT-474 xenograft tumors 30 days after injection. Although the group sizes were very small (n = 4 and 5) and not all animals showed tumor growth, total ex vivo tumor masses at the end of the observation period differed substantially between treatment and growth control groups (mean of treated group = 0.07 ± 0.05 (SEM, standard error of the mean), mean of growth control group = 0.23 ± 0.10, Supporting Information Figure S8C,D). For translation into clinical application and to better understand treatment strategies, studies involving larger group sizes, systematic investigation of tumor targeting at different specific activities (ratio of radioisotope to trastuzumab), and application schemes with more than one application are needed. At an activity of 7 MBq 177Lu per animal, as used here, no toxic side effects are expected, and continued normal weight gain of the animals was observed (Supporting Information Figure S8D). This activity is in the lowest range for therapeutically active RICs.26

In summary, we demonstrated that both a long-lasting effect and a tumor-preventing effect can be expected from this approach to therapy. This is in contrast to a similar approach involving a small molecule labeled with 177Lu, with which tumor regrowth after a single treatment was observed.27 Indeed, the slow pharmacokinetics of mAbs might complement the 6.7-day half-life of 177Lu, and HER2 seems to be a promising target for 177Lu-labeled mAbs.28 Moreover, the diagnostic/therapeutic pair of 89Zr/177Lu for mAbs is typically highly similar in its targeting of tumors.29 Conversely, the pharmacokinetics properties of small molecules cleared by renal elimination might confer advantages over full-sized mAbs in regard to lower background and radiation dose to healthy tissue, if these agents are used as radioisotope carriers.30 Nevertheless, for smaller molecules, the approach of site-specific conjugation might even be more important. Given that radiation has a distinct mechanism of action, this approach is expected to introduce a new modality to the existing therapeutic options. In particular, it is hypothesized that taking such a therapeutic approach to advanced-stage breast cancer would be associated with fewer side effects and longer therapeutic duration than current therapies. To adequately treat patients efficiently, a theranostic approach, i.e., a combination of diagnostic and therapeutic strategies consisting of PET imaging and radioimmunotherapy (RIT), is highly promising. Site-specific labeling in combination with versatile and fast click chemistry is ideally suited for further optimizing, for example, the use of other radioisotopes or different ratios of radioisotope to antibody. In addition, this combination might also afford the possibility to separate the pharmacokinetics of a carrying antibody from those of a (radioactive) payload by transferring the click reaction to an in vivo compartment such as the bloodstream.

Acknowledgments

E.A.L. acknowledges funding by ERC POC RadioClick. M.M. received research grant support from VERAXA Biotech GmbH. We thank the Proteomics core facility at EMBL Heidelberg as well as the Proteomics core facility at IMB, JGU Mainz, for the mass spectrometry analysis.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.2c00634.

  • A detailed description of the chemicals, analytical methods, and animal studies used (PDF)

Author Contributions

∇ C.K. and P.F.S. contributed equally to this work.

The authors declare the following competing financial interest(s): Koehler, Sauter, Lemke and VERAXA Biotech GmbH holds patents regarding the site specific modification of antibodies.

Supplementary Material

cb2c00634_si_001.pdf (1.6MB, pdf)

References

  1. Elgundi Z.; Reslan M.; Cruz E.; Sifniotis V.; Kayser V. The State-of-Play and Future of Antibody Therapeutics. Adv. Drug Delivery Rev. 2017, 122, 2–19. 10.1016/j.addr.2016.11.004. [DOI] [PubMed] [Google Scholar]
  2. Houen G.Therapeutic Antibodies. In Therapeutic Antibodies: An Overview ;Houen G., Ed.; Methods in Molecular Biology Book Series; Humana, 2022; Vol. 2313, p 1. 10.1007/978-1-0716-1450-1_1. [DOI] [PubMed] [Google Scholar]
  3. Modi S.; Saura C.; Yamashita T.; Park Y. H.; Kim S.-B.; Tamura K.; Andre F.; Iwata H.; Ito Y.; Tsurutani J.; Sohn J.; Denduluri N.; Perrin C.; Aogi K.; Tokunaga E.; Im S.-A.; Lee K. S.; Hurvitz S. A.; Cortes J.; Lee C.; Chen S.; Zhang L.; Shahidi J.; Yver A.; Krop I. Trastuzumab Deruxtecan in Previously Treated HER2-Positive Breast Cancer. https://doi.org/10.1056/NEJMoa1914510 2020, 382 (7), 610–621. 10.1056/NEJMoa1914510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Verma S.; Miles D.; Gianni L.; Krop I. E.; Welslau M.; Baselga J.; Pegram M.; Oh D.-Y.; Diéras V.; Guardino E.; Fang L.; Lu M. W.; Olsen S.; Blackwell K. Trastuzumab Emtansine for HER2-Positive Advanced Breast Cancer. N. Engl. J. Med. 2012, 367 (19), 1783–1791. 10.1056/NEJMoa1209124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kolstad A.; Illidge T.; Bolstad N.; Spetalen S.; Madsbu U.; Stokke C.; Blakkisrud J.; Løndalen A.; O’Rourke N.; Beasley M.; Jurczak W.; Fagerli U. M.; Kaščák M.; Bayne M.; Obr A.; Dahle J.; Rojkjaer L.; Pascal V.; Holte H. Phase 1/2a Study of 177Lu-Lilotomab Satetraxetan in Relapsed/Refractory Indolent Non-Hodgkin Lymphoma. Blood Adv. 2020, 4 (17), 4091–4101. 10.1182/bloodadvances.2020002583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Zhou Q. Site-Specific Antibody Conjugation for ADC and Beyond. Biomed. 2017, 5 (4), 64. 10.3390/biomedicines5040064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Junutula J. R.; Raab H.; Clark S.; Bhakta S.; Leipold D. D.; Weir S.; Chen Y.; Simpson M.; Tsai S. P.; Dennis M. S.; Lu Y.; Meng Y. G.; Ng C.; Yang J.; Lee C. C.; Duenas E.; Gorrell J.; Katta V.; Kim A.; McDorman K.; Flagella K.; Venook R.; Ross S.; Spencer S. D.; Lee Wong W.; Lowman H. B.; Vandlen R.; Sliwkowski M. X.; Scheller R. H.; Polakis P.; Mallet W. Site-Specific Conjugation of a Cytotoxic Drug to an Antibody Improves the Therapeutic Index. Nat. Biotechnol. 2008, 26 (8), 925–932. 10.1038/nbt.1480. [DOI] [PubMed] [Google Scholar]
  8. Sievers E. L.; Senter P. D. Antibody-Drug Conjugates in Cancer Therapy. Annu. Rev. Med. 2013, 64 (1), 15–29. 10.1146/annurev-med-050311-201823. [DOI] [PubMed] [Google Scholar]
  9. Kristensen L. K.; Christensen C.; Jensen M. M.; Agnew B. J.; Schjöth-Frydendahl C.; Kjaer A.; Nielsen C. H. Site-Specifically Labeled 89Zr-DFO-Trastuzumab Improves Immuno-Reactivity and Tumor Uptake for Immuno-PET in a Subcutaneous HER2-Positive Xenograft Mouse Model. Theranostics 2019, 9 (15), 4409–4420. 10.7150/thno.32883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Axup J. Y.; Bajjuri K. M.; Ritland M.; Hutchins B. M.; Kim C. H.; Kazane S. A.; Halder R.; Forsyth J. S.; Santidrian A. F.; Stafin K.; Lu Y.; Tran H.; Seller A. J.; Biroc S. L.; Szydlik A.; Pinkstaff J. K.; Tian F.; Sinha S. C.; Felding-Habermann B.; Smider V. V.; Schultz P. G. Synthesis of Site-Specific Antibody-Drug Conjugates Using Unnatural Amino Acids. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (40), 16101–16106. 10.1073/pnas.1211023109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Yao H.; Jiang F.; Lu A.; Zhang G. Methods to Design and Synthesize Antibody-Drug Conjugates (ADCs). Int. J. Mol. Sci. 2016, 17 (2), 194. 10.3390/ijms17020194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chen P. R.; Groff D.; Guo J.; Ou W.; Cellitti S.; Geierstanger B. H.; Schultz P. G. A Facile System for Encoding Unnatural Amino Acids in Mammalian Cells. Angew. Chemie Int. Ed. 2009, 48 (22), 4052–4055. 10.1002/anie.200900683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Blackman M. L.; Royzen M.; Fox J. M. Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity. J. Am. Chem. Soc. 2008, 130 (41), 13518–13519. 10.1021/ja8053805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jang H. S.; Jana S.; Blizzard R. J.; Meeuwsen J. C.; Mehl R. A. Access to Faster Eukaryotic Cell Labeling with Encoded Tetrazine Amino Acids. J. Am. Chem. Soc. 2020, 142 (16), 7245–7249. 10.1021/jacs.9b11520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Plass T.; Milles S.; Koehler C.; Szymański J.; Mueller R.; Wiessler M.; Schultz C.; Lemke E. A. Amino Acids for Diels-Alder Reactions in Living Cells. Angew. Chem., Int. Ed. Engl. 2012, 51 (17), 4166–4170. 10.1002/anie.201108231. [DOI] [PubMed] [Google Scholar]
  16. Reinkemeier C. D.; Koehler C.; Sauter P. F.; Shymanska N. V.; Echalier C.; Rutkowska A.; Will D. W.; Schultz C.; Lemke E. A. Synthesis and Evaluation of Novel Ring-Strained Noncanonical Amino Acids for Residue-Specific Bioorthogonal Reactions in Living Cells. Chem. - A Eur. J. 2021, 27, 6094. 10.1002/chem.202100322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hoffmann J. E.; Plass T.; Nikic I.; Aramburu I. V.; Koehler C.; Gillandt H.; Lemke E. A.; Schultz C. Highly Stable Trans-Cyclooctene Amino Acids for Live-Cell Labeling. Chem. - A Eur. J. 2015, 21 (35), 12266–12270. 10.1002/chem.201501647. [DOI] [PubMed] [Google Scholar]
  18. Koehler C.; Sauter P. F.; Wawryszyn M.; Girona G. E.; Gupta K.; Landry J. J. M.; Fritz M. H.-Y.; Radic K.; Hoffmann J.-E.; Chen Z. A.; Zou J.; Tan P. S.; Galik B.; Junttila S.; Stolt-Bergner P.; Pruneri G.; Gyenesei A.; Schultz C.; Biskup M. B.; Besir H.; Benes V.; Rappsilber J.; Jechlinger M.; Korbel J. O.; Berger I.; Braese S.; Lemke E. A. Genetic Code Expansion for Multiprotein Complex Engineering. Nat. Methods 2016, 13 (12), 997–1000. 10.1038/nmeth.4032. [DOI] [PubMed] [Google Scholar]
  19. Koehler C.; Lemke E. A.. MultiBacTAG-Genetic Code Expansion Using the Baculovirus Expression System in Sf21 Cells. In Noncanonical Amino Acids ,Lemke E., Ed.; Methods in Molecular Biology Book Series; Humana Press Inc., 2018; Vol. 1728, pp 297–311. 10.1007/978-1-4939-7574-7_19. [DOI] [PubMed] [Google Scholar]
  20. Li H. K.; Morokoshi Y.; Kodaira S.; Kusumoto T.; Minegishi K.; Kanda H.; Nagatsu K.; Hasegawa S. Utility of 211At-Trastuzumab for the Treatment of Metastatic Gastric Cancer in the Liver: Evaluation of a Preclinical α-Radioimmunotherapy Approach in a Clinically Relevant Mouse Model. J. Nucl. Med. 2021, 62 (10), 1468–1474. 10.2967/jnumed.120.249300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Woo S. K.; Jang S. J.; Seo M. J.; Park J. H.; Kim B. S.; Kim E. J.; Lee Y. J.; Lee T. S.; An G. Il; Song I. H.; Seo Y.; Kim K. Il; Kang J. H. Development of 64Cu-NOTA-Trastuzumab for HER2 Targeting: Radiopharmaceutical with Improved Pharmacokinetics for Human Study. J. Nucl. Med. 2019, 60 (1), 26–33. 10.2967/jnumed.118.210294. [DOI] [PubMed] [Google Scholar]
  22. Ahn S. H.; Vaughn B. A.; Solis W. A.; Lupher M. L.; Hallam T. J.; Boros E. Site-Specific 89Zr- And 111In-Radiolabeling and in Vivo Evaluation of Glycan-Free Antibodies by Azide-Alkyne Cycloaddition with a Non-Natural Amino Acid. Bioconjugate Chem. 2020, 31 (4), 1177–1187. 10.1021/acs.bioconjchem.0c00100. [DOI] [PubMed] [Google Scholar]
  23. Rasaneh S.; Rajabi H.; Akhlaghpoor S.; Sheybani S. Radioimmunotherapy of Mice Bearing Breast Tumors with 177Lu- Labeled Trastuzumab. Turkish J. Med. Sci. 2012, 42, 1292–1298. 10.3906/sag-1105-29. [DOI] [Google Scholar]
  24. Lee W.; Sarkar S.; Pal R.; Kim J. Y.; Park H.; Huynh P. T.; Bhise A.; Bobba K. N.; Kim K. Il; Ha Y. S.; Soni N.; Kim W.; Lee K.; Jung J. M.; Rajkumar S.; Lee K. C.; Yoo J. Successful Application of CuAAC Click Reaction in Constructing 64 Cu-Labeled Antibody Conjugates for Immuno-PET Imaging. ACS Appl. bio Mater. 2021, 4 (3), 2544–2557. 10.1021/acsabm.0c01555. [DOI] [PubMed] [Google Scholar]
  25. Chomet M.; Schreurs M.; Bolijn M. J.; Verlaan M.; Beaino W.; Brown K.; Poot A. J.; Windhorst A. D.; Gill H.; Marik J.; Williams S.; Cowell J.; Gasser G.; Mindt T. L.; van Dongen G. A. M. S.; Vugts D. J. Head-to-Head Comparison of DFO* and DFO Chelators: Selection of the Best Candidate for Clinical 89Zr-Immuno-PET. Eur. J. Nucl. Med. Mol. Imaging 2021, 48 (3), 694–707. 10.1007/s00259-020-05002-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Korsen J. A.; Gutierrez J. A.; Tully K. M.; Carter L. M.; Samuels Z. V.; Khitrov S.; Poirier J. T.; Rudin C. M.; Chen Y.; Morris M. J.; Bodei L.; Pillarsetty N.; Lewis J. S.. Delta-like Ligand 3-Targeted Radioimmunotherapy for Neuroendocrine Prostate Cancer. Proc. Natl. Acad. Sci. U. S. A. 2022, 119 ( (27), ). 10.1073/pnas.2203820119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Graf F.; Fahrer J.; Maus S.; Morgenstern A.; Bruchertseifer F.; Venkatachalam S.; Fottner C.; Weber M. M.; Huelsenbeck J.; Schreckenberger M.; Kaina B.; Miederer M. DNA Double Strand Breaks as Predictor of Efficacy of the Alpha-Particle Emitter Ac-225 and the Electron Emitter Lu-177 for Somatostatin Receptor Targeted Radiotherapy. PLoS One 2014, 9 (2), e88239. 10.1371/journal.pone.0088239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kang L.; Li C.; Rosenkrans Z. T.; Huo N.; Chen Z.; Ehlerding E. B.; Huo Y.; Ferreira C. A.; Barnhart T. E.; Engle J. W.; Wang R.; Jiang D.; Xu X.; Cai W. CD38-Targeted Theranostics of Lymphoma with 89Zr/177Lu-Labeled Daratumumab. Adv. Sci. 2021, 8 (10), 384–402. 10.1002/advs.202001879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kang L.; Li C.; Rosenkrans Z. T.; Huo N.; Chen Z.; Ehlerding E. B.; Huo Y.; Ferreira C. A.; Barnhart T. E.; Engle J. W.; Wang R.; Jiang D.; Xu X.; Cai W. CD38-Targeted Theranostics of Lymphoma with 89Zr/177Lu-Labeled Daratumumab. Adv. Sci. 2021, 8 (10), 2001879. 10.1002/advs.202001879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Altunay B.; Morgenroth A.; Beheshti M.; Vogg A.; Wong N. C. L.; Ting H. H.; Biersack H. J.; Stickeler E.; Mottaghy F. M. HER2-Directed Antibodies, Affibodies and Nanobodies as Drug-Delivery Vehicles in Breast Cancer with a Specific Focus on Radioimmunotherapy and Radioimmunoimaging. Eur. J. Nucl. Med. Mol. Imaging 2021, 48 (5), 1371–1389. 10.1007/s00259-020-05094-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

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