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Molecular-targeted imaging probes can be used with a variety of imaging modalities to detect diseased tissues and guide their removal. EGFR is a useful biomarker for a variety of cancers, because it is expressed at high levels relative to normal tissues. Previously, we showed the anti-EGFR antibody nimotuzumab can be used as a positron emission tomography and fluorescent imaging probe for EGFR positive cancers in mice. These imaging probes are currently in clinical trials for PET imaging and image-guided surgery, respectively. One issue with using antibody probes for imaging is their long circulation time and slow tissue penetration, which requires patients to wait a few days after injection before imaging or surgery, multiple visits and longer radiation exposure. Here, we generated a Fab2 fragment of nimotuzumab, by pepsin digestion and labeled it with IRDye800CW to evaluate its optical imaging properties. The Fab2 had faster tumor accumulation and clearance in mice relative to the nimotuzumab IgG. The fluorescent signal peaked at 2 h post injection and remained high until 6 h post injection. The properties of the Fab2 allow a higher signal to background to be obtained in a shorter time frame, reducing the wait time for imaging after probe infusion. [ABSTRACT FROM AUTHOR]

Simple Summary: Abundance of certain proteins such as epidermal growth factor receptor (EGFR) and their growth factors on cancer cells is in part responsible for their uncontrolled growth. Compounds that selectively bind to such proteins have diagnostic and/or therapeutic implications. EGFR has four binding domains (I-IV). Most anti-EGFR therapeutic antibodies bind to domain III. Compounds that bind to other domains have implications not only for diagnosis but also for monitoring therapy response. We describe the development of a diagnostic agent to be used with positron emission tomography (PET) that binds to domain II of EGFR. We developed 89Zr-8709-scFv-Fc antibody PET agent and evaluated its binding characteristics in cancer cells and mouse models. The presence of a domain III-binding antibody such as nimotuzumab did not inhibit the binding of 89Zr-8709-scFv-Fc, and vice versa. Therefore, 89Zr-8709-scFv-Fc PET/CT can be used for diagnosis and monitoring therapy response in the presence of a domain III-binding agent. Epidermal growth factor receptor I (EGFR) is overexpressed in many cancers. The extracellular domain of EGFR has four binding epitopes (domains I- IV). All clinically approved anti-EGFR antibodies bind to domain III. Imaging agents that bind to domains other than domain III of EGFR are needed for accurate quantification of EGFR, patient selection for anti-EGFR therapeutics and monitoring of response to therapies. We recently developed a domain II-specific antibody fragment 8709. In this study, we have evaluated the in vitro and in vivo properties of 89Zr-8709-scFv-Fc (105 kDa). We conjugated 8709-scFv-Fc with the deferoxamine (DFO) chelator and radiolabeled the DFO-8970-scFv with 89Zr. We evaluated the binding of 89Zr-DFO-8709-scFv-Fc in EGFR positive and negative cell lines DLD-1, MDA-MB-231 and MDA-MB-435, respectively, and in mouse xenograft models. Simultaneously, we have compared the binding of 89Zr-8709-scFv-Fc with 111In-nimotuzumab, a domain III anti-EGFR antibody. DFO-8709-scFv-Fc displayed similar cell binding specificity as 8709-scFv-Fc. Saturation cell binding assay and immunoreactive fraction showed that radiolabeling did not alter the binding of 8709-scFv-Fc. Biodistribution and microPET showed good uptake of 89Zr-8709-scFv-Fc in xenografts after 120 h post injection (p.i). and was domain-specific to EGFR domain II. 89Zr-8709-scFv-Fc did not compete for binding in vitro and in vivo with a known domain III binder nimotuzumab. The results show that 89Zr-8709-scFv-Fc is specific to domain II of EGFR making it favorable for quantification of EGFR in vivo, hence, patient selection and monitoring of response to treatment with anti-EGFR antibodies. [ABSTRACT FROM AUTHOR]

Simple Summary: Monoclonal antibodies (IgG) are excellent probes for targeting cell surface receptors for imaging and therapeutic applications. These theranostic agents are often developed by randomly conjugating radioisotopes/drugs/chelators to the primary amine of lysine or the sulfhydryl groups of cysteine on the antibody. Random conjugation often alters the properties of the antibody. We have site-specifically radiolabeled nimotuzumab an anti-epidermal growth factor receptor (EGFR) monoclonal antibody with 89Zr and 225Ac using SpyTag: ∆N-SpyCatcher for positron emission tomography (PET) imaging and alpha particle radiotherapy, and evaluated these agents in a model of EGFR-positive triple negative breast cancer (TNBC). Nimotuzumab-SpyTag-∆N-SpyCatcher constructs showed improved binding in vitro compared with randomly conjugated constructs. 89Zr-nimotuzumab-SpyTag-∆N-SpyCatcher specifically delineated EGFR-positive xenograft in vivo using microPET/CT imaging. Compared with control treatment groups, 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher more than doubled the survival of mice bearing EGFR-positive MDA-MB-231 TNBC xenograft. This work highlights a facile method to site-specifically radiolabel antibodies using SpyTag: ∆N-SpyCatcher. To develop imaging and therapeutic agents, antibodies are often conjugated randomly to a chelator/radioisotope or drug using a primary amine (NH2) of lysine or sulfhydryl (SH) of cysteine. Random conjugation to NH2 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, ΔN-SpyCatcher was reduced using tris(2-carboxyethyl)phosphine (TCEP), which was followed by desferoxamine-maleimide (DFO-mal) conjugation to yield a reactive ΔN-SpyCatcher-DFO. The ΔN-SpyCatcher-DFO was reacted with nimotuzumab-SpyTag to obtain stable nimotuzumab-SpyTag-∆N-SpyCatcher-DFO. Radiolabeling was performed with 89Zr, 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 (225Ac) 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 89Zr-nimotuzumab-SpyTag-∆N-SpyCatcher with 6.0 ± 0.6%IA/cc (n = 3) at 48 h post injection. The EC50 of 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher and 225Ac-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, 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher significantly (p = 0.0017) prolonged the survival of mice (64 days) compared to 225Ac-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. 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher was effective in vitro and in an EGFR-positive triple negative breast cancer xenograft model. [ABSTRACT FROM AUTHOR]

β - -emitting 177 Lu-octreotate is an approved somatostatin receptor subtype 2 (SSTR2)-directed peptide receptor radionuclide therapy for the treatment of gastroenteropancreatic neuroendocrine tumors (NETs). However, 177 Lu-octreotate has fast pharmacokinetics, requiring up to 4 treatment doses. Moreover, 177 Lu is less than ideal for theranostics because of the low branching ratio of its γ-emissions, which limits its SPECT imaging capability. Compared with 177 Lu, 67 Cu has better decay properties for use as a theranostic. Here, we report the preclinical evaluation of a long-lived somatostatin analog, [ 67 Cu]Cu-DOTA-Evans blue-TATE (EB-TATE), against SSTR2-positive NETs. Methods: The in vitro cytotoxicity of [ 67 Cu]Cu-EB-TATE was investigated on 2-dimensional cells and 3-dimensional spheroids. In vivo pharmacokinetics and dosimetry were studied in healthy BALB/c mice, whereas ex vivo biodistribution, micro-SPECT/CT imaging, and therapy studies were done on athymic nude mice bearing QGP1.SSTR2 and BON1.SSTR2 xenografts. Therapeutic efficacy was compared with [ 177 Lu]Lu-EB-TATE. Results: Projected human effective doses of [ 67 Cu]Cu-EB-TATE for male (0.066 mSv/MBq) and female (0.085 mSv/MBq) patients are tolerable. In vivo micro-SPECT/CT imaging of SSTR2-positive xenografts with [ 67 Cu]Cu-EB-TATE showed tumor-specific uptake and prolonged accumulation. Biodistribution showed tumor accumulation, with concurrent clearance from major organs over a period of 72 h. [ 67 Cu]Cu-EB-TATE was more effective (60%) at eliminating tumors that were smaller than 50 mm 3 within the first 15 d of therapy than was [ 177 Lu]Lu-EB-TATE (20%) after treatment with 2 doses of 15 MBq administered 10 d apart. Mean survival of [ 67 Cu]Cu-EB-TATE-treated groups was 90 d and more than 90 d, whereas that of [ 177 Lu]Lu-EB-TATE was more than 90 d and 89 d against vehicle control groups (26 d and 53 d), for QGP1.SSTR2 and BON1.SSTR2 xenografts, respectively. Conclusion: [ 67 Cu]Cu-EB-TATE exhibited high SSTR2-positive NET uptake and retention, with favorable dosimetry and SPECT/CT imaging capabilities. The antitumor efficacy of [ 67 Cu]Cu-EB-TATE is comparable to that of [ 177 Lu]Lu-EB-TATE, with [ 67 Cu]Cu-EB-TATE being slightly more effective than [ 177 Lu]Lu-EB-TATE for complete remission of small tumors. [ 67 Cu]Cu-EB-TATE therefore warrants clinical development., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

Eighty percent of colorectal cancers (CRCs) overexpress epidermal growth factor receptor (EGFR). Kirsten rat sarcoma viral oncogene (KRAS) mutations are present in 40% of CRCs and drive de novo resistance to anti-EGFR drugs. BRAF oncogene is mutated in 7%-10% of CRCs, with even worse prognosis. We have evaluated the effectiveness of [ 225 Ac]Ac-macropa-nimotuzumab in KRAS mutant and in KRAS wild-type and BRAF V600E mutant EGFR-positive CRC cells in vitro and in vivo. Anti-CD20 [ 225 Ac]Ac-macropa-rituximab was developed and used as a nonspecific radioimmunoconjugate. Methods: Anti-EGFR antibody nimotuzumab was radiolabeled with 225 Ac via an 18-membered macrocyclic chelator p -SCN-macropa. The immunoconjugate was characterized using flow cytometry, radioligand binding assay, and high-performance liquid chromatography, and internalization was studied using live-cell imaging. In vitro cytotoxicity was evaluated in 2-dimensional monolayer EGFR-positive KRAS mutant DLD-1, SW620, and SNU-C2B; in KRAS wild-type and BRAF V600E mutant HT-29 CRC cell lines; and in 3-dimensional spheroids. Dosimetry was studied in healthy mice. The in vivo efficacy of [ 225 Ac]Ac-macropa-nimotuzumab was evaluated in mice bearing DLD-1, SW620, and HT-29 xenografts after treatment with 3 doses of 13 kBq/dose administered 10 d apart. Results: In all cell lines, in vitro studies showed enhanced cytotoxicity of [ 225 Ac]Ac-macropa-nimotuzumab compared with nimotuzumab and controls. The inhibitory concentration of 50% in the DLD-1 cell line was 1.8 nM for [ 225 Ac]Ac-macropa-nimotuzumab versus 84.1 nM for nimotuzumab. Similarly, the inhibitory concentration of 50% was up to 79-fold lower for [ 225 Ac]Ac-macropa-nimotuzumab than for nimotuzumab in KRAS mutant SNU-C2B and SW620 and in KRAS wild-type and BRAF V600E mutant HT-29 CRC cell lines. A similar trend was observed for 3-dimensional spheroids. Internalization peaked 24-48 h after incubation and depended on EGFR expression. In the [ 225 Ac]Ac-macropa-nimotuzumab group, 3 of 7 mice bearing DLD-1 tumors had complete remission. Median survival was 40 and 34 d for mice treated with phosphate-buffered saline and [ 225 Ac]Ac-macropa-rituximab (control), respectively, whereas it was not reached for the [ 225 Ac]Ac-macropa-nimotuzumab group (>90 d). Similarly, median survival of mice bearing HT-29 xenografts was 16 and 12.5 d for those treated with [ 225 Ac]Ac-macropa-rituximab and phosphate-buffered saline, respectively, and was not reached for those treated with [ 225 Ac]Ac-macropa-nimotuzumab (>90 d). One of 7 mice bearing HT-29 xenografts and treated using [ 225 Ac]Ac-macropa-nimotuzumab had complete remission. Compared with untreated mice, [ 225 Ac]Ac-macropa-nimotuzumab more than doubled (16 vs. 41 d) the median survival of mice bearing SW620 xenografts. Conclusion: [ 225 Ac]Ac-macropa-nimotuzumab is effective against KRAS mutant and BRAF V600E mutant CRC models., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

Molecular-targeted imaging probes can be used with a variety of imaging modalities to detect diseased tissues and guide their removal. EGFR is a useful biomarker for a variety of cancers, because it is expressed at high levels relative to normal tissues. Previously, we showed the anti-EGFR antibody nimotuzumab can be used as a positron emission tomography and fluorescent imaging probe for EGFR positive cancers in mice. These imaging probes are currently in clinical trials for PET imaging and image-guided surgery, respectively. One issue with using antibody probes for imaging is their long circulation time and slow tissue penetration, which requires patients to wait a few days after injection before imaging or surgery, multiple visits and longer radiation exposure. Here, we generated a Fab 2 fragment of nimotuzumab, by pepsin digestion and labeled it with IRDye800CW to evaluate its optical imaging properties. The Fab 2 had faster tumor accumulation and clearance in mice relative to the nimotuzumab IgG. The fluorescent signal peaked at 2 h post injection and remained high until 6 h post injection. The properties of the Fab 2 allow a higher signal to background to be obtained in a shorter time frame, reducing the wait time for imaging after probe infusion., (© 2023. The Author(s).)