Antibody PEGylation in bioorthogonal pretargeting with trans-cyclooctene/tetrazine cycloaddition: in vitro and in vivo evaluation in colorectal cancer models

Abstract

Bioorthogonal chemistry represents a challenging approach in pretargeted radioimmunotherapy (PRIT). We focus here on mAb modifications by grafting an increase amount of trans-cyclooctene (TCO) derivatives (0 to 30 equivalents with respect to mAb) bearing different polyethylene glycol (PEG) linkers between mAb and TCO (i.e. PEG0 (1), PEG4 (2) and PEG12 (3)) and assessing their functionality. We used colorectal xenograft (HT29/Ts29.2) and peritoneal carcinomatosis (A431-CEA-Luc/35A7) as tumor cells/mAbs models and fluorescent tetrazines (TZ). MALDI-TOF MS shows that grafting with 2,3 increases significantly the number of TCO per mAb compared with no PEG. In vitro immunofluorescence showed that Ts29.2 and 35A7 labeling intensity is correlated with the number of TCO when using 1,3 while signals reach a maximum at 10 equivalents when using 2. Under 10 equivalents conditions, the capacity of resulting mAbs-1–3 for antigen recognition is similar when reported per grafted TCO and comparable to mAbs without TCO. In vivo, on both models, pretargeting with mAbs-2,3 followed by TZ injection induced a fluorescent signal two times lower than with mAbs-1. These findings suggest that while PEG linkers allow a better accessibility for TCO grafting, it might decrease the number of reactive TCO. In conclusion, mAb-1 represents the best candidate for PRIT.

Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging

“…46 Note that use of PEG spacers for TCO conjugation to mAbs has been evaluated elsewhere. 55 Conjugation of 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide (i.e., “DOTA-maleimide”, product #B-272, Macrocyclics, Dallas, TX) to 7C2-LC:K149C and 7C2-LC:K149C+HC:L177C+HC:Y376C (giving DOTA-to-antibody ratio of ~2 and ~6) were also prepared as controls for direct labeling methods used for tissue distribution and imaging, via 111 In-DOTA 2 -7C2 and 111 In-DOTA 6 -7C2, respectively.…”

Harnessing 64 Cu/ 67 Cu for a theranostic approach to pretargeted radioimmunotherapy

“…Broadly speaking, this methodology consists of four steps: 1) the administration of a monoclonal antibody (mAb)-TCO immunoconjugate; 2) an interval period during which the antibody accumulates at the tumor and clears from the blood; 3) the injection of a small molecule Tz-based radioligand; and 4) the in vivo click ligation between two components, followed by the rapid clearance of any excess radioligand. This strategy has proven highly effective for pretargeted PET imaging (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49) as well as pretargeted radioimmunotherapy (PRIT) using beta-emitting ( 177 Lu) and alpha-emitting ( 225 Ac) radionuclides (50)(51)(52)(53)(54)(55). Yet as the field stands poised to make the jump from the laboratory to the clinic, it stands to reason that the development of a theranostic approach to Tz/TCO pretargeting could prove instrumental in ensuring the future success of this modality.…”

Effect of Modulating FcRn Binding on Direct and Pretargeted Tumor Uptake of Full-length Antibodies

“…Anti-HER2 THIOMAB™ antibodies (clone 7C2, which binds a different epitope than trastuzumab or pertuzumab; see Supplementary Table 1 for 7C2 sequence as previously reported in (23)) were engineered to introduce cysteine residues at three positions (24) for conjugation to the click-reactive TCO-PEG 3 -maleimide (PEG = polyethylene glycol) linker (25,26), the metal chelator DOTA-maleimide (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7triyl)triacetic acid) for imaging via 111 In-DOTA 6 -mAbs, and the capping reagent N-ethyl maleimide (NEM) as previously described (15) to give final conjugates with ~6 TCO, DOTA, or NEM groups per antibody (Supplementary Table 2). These cysteine mutations were engineered alone or in combination with additional mutations -H310A/H435Qto ablate binding to FcRn (20).…”