Dataset Information

Nimotuzumab Site-Specifically Labeled with ⁸⁹Zr and ²²⁵Ac Using SpyTag/SpyCatcher for PET Imaging and Alpha Particle Radioimmunotherapy of Epidermal Growth Factor Receptor Positive Cancers.

ABSTRACT: 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, ?N-SpyCatcher was reduced using tris(2-carboxyethyl)phosphine (TCEP), which was followed by desferoxamine-maleimide (DFO-mal) conjugation to yield a reactive ?N-SpyCatcher-DFO. The ?N-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.

SUBMITTER: Solomon VR

PROVIDER: S-EPMC7699480 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Publications

Nimotuzumab Site-Specifically Labeled with 89Zr and 225Ac Using SpyTag/SpyCatcher for PET Imaging and Alpha Particle Radioimmunotherapy of Epidermal Growth Factor Receptor Positive Cancers.

Solomon Viswas Raja VR Barreto Kris K Bernhard Wendy W Alizadeh Elahe E Causey Patrick P Perron Randy R Gendron Denise D Alam Md Kausar MK Carr Adriana A Geyer C Ronald CR Fonge Humphrey H

Cancers 20201120 11

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 ...[more]

PMID: 33233524

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Project description:Targeted ?-particle radiation using the radioisotope (225)Ac is a promising form of therapy for various types of cancer. Historic obstacles to the use of (225)Ac have been the difficulty in finding suitable chelators to stably attach it to targeting vehicles such as peptides and monoclonal antibodies, the low specific activities of the products, and the lack of cost-effective radiolabeling procedures. We initially solved the first problem with a procedure involving 2 chemical steps that has been used as a standard in preclinical and clinical studies. However, this procedure involves the loss of 90% of the input (225)Ac. A more efficient, economical process is needed to facilitate the more widespread use of (225)Ac.We conjugated representative antibodies with 2 forms of DOTA as well as other chelators as controls. We developed conditions to radiolabel these constructs in 1 chemical step and characterized their stability, immunoreactivity, biodistribution, and therapeutic efficacy in healthy and tumor-bearing mice.DOTA-antibody constructs were labeled to a wide range of specific activities in 1 chemical step at 37°C. Radiochemical yields were approximately 10-fold higher, and specific activities were up to 30-fold higher than with the previous approach. The products retained immunoreactivity and were stable to serum challenge in vitro and in mice. Labeling kinetics of DOTA-antibody constructs linked through a benzyl isothiocyanate linkage were more favorable than those linked through an N-hydroxysuccinimide linkage. Tissue distribution was similar but not identical between the constructs. The constructs produced specific therapeutic responses in a mouse model of acute myeloid leukemia.We have characterized an efficient, 1-step radiolabeling method that produces stable, therapeutically active conjugates of antibodies with (225)Ac at high specific activity. We propose that this technology greatly expands the possible clinical applications of (225)Ac monoclonal antibodies.

Project description:We report a simple strategy for the creation of lentiviral vectors specific to any desired target cells. SpyTag is inserted into an engineered Sindbis virus envelope protein and displayed on the lentivirus surface to create Sindbis virus-SpyTag pseudoparticles (Sind-SpyTag-pp). The SpyTag serves as the covalent anchoring site for a target-cell-specific cell-binding protein (CBP) that is fused to a truncated SpyCatcher (SpyCatcher?). Target-cell-specific lentiviruses are created by mixing the Sind-SpyTag-pp and CBP-SpyCatcher? in vitro We first used a HER2-binding designed ankyrin repeat protein (DARPin.9.26) as the model CBP. The DARPin-conjugated lentivirus transduced HER2+ SKOV3 cells with an infectious titer of 5.2 × 106 IU/ml, >500-fold higher than the unfunctionalized "naked" virions (<104 IU/ml). The ability of the DARPin-conjugated lentivirus to transduce HER2+ cells correlated with the surface expression level of HER2. Furthermore, these lentiviruses preferentially transduced HER2+ cells in cocultures containing HER2+ and HER2- cells. To enable the use of commercially available monoclonal antibodies (MAbs) as the CBP, we developed a convenient click chemistry-based approach to conjugate MAb-derived Fab fragments to a variant SpyCatcher? protein containing a nonnatural amino acid, 4-azido-l-phenylalanine (AzF). Using the HER2-binding trastuzumab as a model cell-specific MAb, we created Fab-conjugated lentiviral vectors that transduced HER2+ SKOV3 cells with an infectious titer of 2.8 × 106 IU/ml, on par with the result achieved using the DARPin-SpyCatcher? fusion protein. The ability to create cell-specific lentiviral vectors through chemical conjugation of a CBP should make this approach generalizable to any antibody, giving it broad utility for a wide range of research and clinical applications.IMPORTANCE Lentiviral vectors hold great potential in gene therapy. However, it remains a major hurdle to robustly engineer cell-specific lentiviral vectors. This article reports a simple and effective strategy to functionalize lentiviral vectors with cell-binding proteins, thus retargeting these viruses to cells expressing the binding partner of the CBP. The CBP is genetically or chemically linked to the SpyCatcher. The SpyTag is displayed on the virion surface as a fusion to an engineered Sindbis virus envelope protein and is used as the anchorage site for SpyCatcher-linked CBP. Using this strategy, we created lentiviral vectors highly infectious toward HER2+ cancer cells. The ability to rapidly create cell-specific lentiviral vectors targeting a wide range of cell types should accelerate the development of custom lentiviral vectors for many research and clinical applications.

Dataset Information

Nimotuzumab Site-Specifically Labeled with ⁸⁹Zr and ²²⁵Ac Using SpyTag/SpyCatcher for PET Imaging and Alpha Particle Radioimmunotherapy of Epidermal Growth Factor Receptor Positive Cancers.

Publications

Nimotuzumab Site-Specifically Labeled with <sup>89</sup>Zr and <sup>225</sup>Ac Using SpyTag/SpyCatcher for PET Imaging and Alpha Particle Radioimmunotherapy of Epidermal Growth Factor Receptor Positive Cancers.

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