PMID- 33233524 OWN - NLM STAT- PubMed-not-MEDLINE LR - 20201201 IS - 2072-6694 (Print) IS - 2072-6694 (Electronic) IS - 2072-6694 (Linking) VI - 12 IP - 11 DP - 2020 Nov 20 TI - Nimotuzumab Site-Specifically Labeled with (89)Zr and (225)Ac Using SpyTag/SpyCatcher for PET Imaging and Alpha Particle Radioimmunotherapy of Epidermal Growth Factor Receptor Positive Cancers. LID - 10.3390/cancers12113449 [doi] LID - 3449 AB - To develop imaging and therapeutic agents, antibodies are often conjugated randomly to a chelator/radioisotope or drug using a primary amine (NH(2)) of lysine or sulfhydryl (SH) of cysteine. Random conjugation to NH(2) or SH groups can require extreme conditions and may affect target recognition/binding and must therefore be tested. In the present study, nimotuzumab was site-specifically labeled using ∆N-SpyCatcher/SpyTag with different chelators and radiometals. Nimotuzumab is a well-tolerated anti-EGFR antibody with low skin toxicities. First, DeltaN-SpyCatcher was reduced using tris(2-carboxyethyl)phosphine (TCEP), which was followed by desferoxamine-maleimide (DFO-mal) conjugation to yield a reactive DeltaN-SpyCatcher-DFO. The DeltaN-SpyCatcher-DFO was reacted with nimotuzumab-SpyTag to obtain stable nimotuzumab-SpyTag-∆N-SpyCatcher-DFO. Radiolabeling was performed with (89)Zr, and the conjugate was used for the in vivo microPET imaging of EGFR-positive MDA-MB-468 xenografts. Similarly, ∆N-SpyCatcher was conjugated to an eighteen-membered macrocyclic chelator macropa-maleimide and used to radiolabel nimotuzumab-SpyTag with actinium-225 ((225)Ac) for in vivo radiotherapy studies. All constructs were characterized using biolayer interferometry, flow cytometry, radioligand binding assays, HPLC, and bioanalyzer. MicroPET/CT imaging showed a good tumor uptake of (89)Zr-nimotuzumab-SpyTag-∆N-SpyCatcher with 6.0 +/- 0.6%IA/cc (n = 3) at 48 h post injection. The EC(50) of (225)Ac-nimotuzumab-SpyTag-∆N-SpyCatcher and (225)Ac-control-IgG-SpyTag-∆N-SpyCatcher against an EGFR-positive cell-line (MDA-MB-468) was 3.7 +/- 3.3 Bq/mL (0.04 +/- 0.03 nM) and 18.5 +/- 4.4 Bq/mL (0.2 +/- 0.04 nM), respectively. In mice bearing MDA-MB-468 EGFR-positive xenografts, (225)Ac-nimotuzumab-SpyTag-∆N-SpyCatcher significantly (p = 0.0017) prolonged the survival of mice (64 days) compared to (225)Ac-control IgG (28.5 days), nimotuzumab (28.5 days), or PBS-treated mice (30 days). The results showed that the conjugation and labeling using SpyTag/∆N-SpyCatcher to nimotuzumab did not significantly (p > 0.05) alter the receptor binding of nimotuzumab compared with a non-specific conjugation approach. (225)Ac-nimotuzumab-SpyTag-∆N-SpyCatcher was effective in vitro and in an EGFR-positive triple negative breast cancer xenograft model. FAU - Solomon, Viswas Raja AU - Solomon VR AUID- ORCID: 0000-0002-9863-4452 AD - Department of Medical Imaging, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 0W8, Canada. FAU - Barreto, Kris AU - Barreto K AD - Department of Pathology and Laboratory Medicine, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada. FAU - Bernhard, Wendy AU - Bernhard W AD - Department of Pathology and Laboratory Medicine, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada. FAU - Alizadeh, Elahe AU - Alizadeh E AUID- ORCID: 0000-0003-4046-1226 AD - Department of Medical Imaging, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 0W8, Canada. FAU - Causey, Patrick AU - Causey P AD - Canadian Nuclear Laboratories, Chalk River, ON K0J 1J0, Canada. FAU - Perron, Randy AU - Perron R AD - Canadian Nuclear Laboratories, Chalk River, ON K0J 1J0, Canada. FAU - Gendron, Denise AU - Gendron D AD - Canadian Nuclear Laboratories, Chalk River, ON K0J 1J0, Canada. FAU - Alam, Md Kausar AU - Alam MK AD - Department of Pathology and Laboratory Medicine, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada. FAU - Carr, Adriana AU - Carr A AD - Research and Development Direction, Center of Molecular Immunology, 216 Street and 15 Avenue, Atabey, Playa, P.O. Box 16040, Havana 11600, Cuba. FAU - Geyer, C Ronald AU - Geyer CR AUID- ORCID: 0000-0003-1628-9665 AD - Department of Pathology and Laboratory Medicine, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada. FAU - Fonge, Humphrey AU - Fonge H AUID- ORCID: 0000-0001-9388-6872 AD - Department of Medical Imaging, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 0W8, Canada. AD - Department of Medical Imaging, Royal University Hospital, Saskatoon, SK S7N 0W8, Canada. LA - eng GR - J2018-0042/Sylvia Fedoruk Centre/ GR - 300030/Canadian Breast Cancer Foundation/ PT - Journal Article DEP - 20201120 PL - Switzerland TA - Cancers (Basel) JT - Cancers JID - 101526829 PMC - PMC7699480 OTO - NOTNLM OT - EGFR OT - SpyTag/∆N-SpyCatcher OT - diagnostic OT - immunoPET OT - radioimmunotherapy OT - site-specific labeling COIS- The authors declare no conflict of interest. EDAT- 2020/11/26 06:00 MHDA- 2020/11/26 06:01 PMCR- 2020/11/20 CRDT- 2020/11/25 01:03 PHST- 2020/08/26 00:00 [received] PHST- 2020/11/12 00:00 [revised] PHST- 2020/11/13 00:00 [accepted] PHST- 2020/11/25 01:03 [entrez] PHST- 2020/11/26 06:00 [pubmed] PHST- 2020/11/26 06:01 [medline] PHST- 2020/11/20 00:00 [pmc-release] AID - cancers12113449 [pii] AID - cancers-12-03449 [pii] AID - 10.3390/cancers12113449 [doi] PST - epublish SO - Cancers (Basel). 2020 Nov 20;12(11):3449. doi: 10.3390/cancers12113449.