Reagents for Astatination of Biomolecules. 6. An Intact Antibody Conjugated with a Maleimido-closo-Decaborate(2-) Reagent via Sulfhydryl Groups Had Considerably Higher Kidney Concentrations than the Same Antibody Conjugated with an Isothiocyanato-closo-Decaborate(2-) Reagent via Lysine Amines

Abstract

We are investigating the use of an 211At-labeled anti-CD45 monoclonal antibody (mAb) as a replacement of total body irradiation in conditioning regimens designed to decrease the toxicity of hematopoietic cell transplantation (HCT). As part of that investigation, dose-escalation studies were conducted in dogs using 211At-labeled anti-canine CD45 mAb, CA12.10C12, conjugated with a maleimido-closo-decaborate(2-) derivative, 4. Unacceptable renal toxicity was noted in the dogs receiving doses in the 0.27 – 0.62 mCi/kg range. This result was not anticipated, as no toxicity had been noted in prior biodistribution and toxicity studies conducted in mice. Studies were conducted to understand the cause of the renal toxicity and to find a way to circumvent it. A dog biodistribution study was conducted with 123Ilabeled CA12.10C12 that had been conjugated with 4. The biodistribution data showed that 10-fold higher kidney concentrations were obtained with the maleimido-conjugate than had been obtained in a previous biodistribution study with 123I-labeled CA12.10C12 conjugated with an amine-reactive phenylisothiocyanato-CHX-A” derivative. The difference in kidney concentrations observed in dogs for the two conjugation approaches led to an investigation of the reagents. SE-HPLC analyses showed that the purity of the CA12.10C12 conjugated via reduced disulfides was lower than that obtained with amine-reactive conjugation reagents, and non-reducing SDS-PAGE analyses indicated protein fragments were present in the disulfide reduced conjugate. Although we had previously prepared closo-decaborate(2-) derivatives with amine-reactive functional groups (e.g. 6 & 8), a new easily synthesized, amine-reactive (phenylisothiocyanate) derivative, 10, was prepared for use in the current studies. A biodistribution was conducted with co-administered 125I- and 211At-labeled CA12.10C10 conjugated with 10. In that study, lower kidney concentrations were obtained for both radionuclides than had been obtained in the earlier study of the same antibody conjugated with 4 after reduction of disulfide bonds.

Anti-CD45 radioimmunotherapy using 211At with bone marrow transplantation prolongs survival in a disseminated murine leukemia model

“…12,21,29 In fact, even 211 At-labeled B10-mAb using a different B10 conjugation chemistry resulted in two cases of canine renal toxicity in a dose-escalation study. 22 In this study, 211 At was linked to the anti-CD45 mAb via a lysine amine reaction with B10-NCS; this 211 At-labeling method did not result in high absorbed doses to the kidney and did not produce renal dysfunction, even without the administration of renal protective agents. The renal safety profile of 211 At may be advantageous compared with that of other a emitters that may decay into shortlived nephrotoxic daughter a emitters such as 221 Fr, 217 At, and 213 Bi.…”

“…Isothiocyantophenethyl-ureido-closodecaborate(2-) (B10-NCS) at 10 mg/mL in dimethylsulfoxide was added to antibody solution, 22 and the reaction proceeded overnight with gentle tumbling at room temperature. The next morning, the mixture was split into two equal fractions and passed over two PD-10 columns and collected in phosphate-buffered saline.…”

“…12,14,29 Minimal renal uptake also supports the viability, stability, and targeted delivery of astatinated macromolecules via the conjugation chemistry of the B10-NCS reagent, corroborating earlier studies that showed comparable in vivo stability of 211 At-and 125 I-labeled mAb at 24 hours in canine biodistribution studies that used 211 At-labeled mAb via our B10 chemistry. 22 In these RIT studies, the delivery of 211 At-anti-CD45 RIT resulted in improved OS for leukemic mice that was most significant when combined with BMT, in which 40% of mice treated at the highest dose (ie, 24 mCi 211 At-B10-30F11) survived at least 180 days. Moreover, 211 At-anti-CD45 RIT was well tolerated, with relatively minimal leukopenia that resolved by 4 weeks after BMT.…”

“…22 The isolated 211 At solution was used directly in the labeling procedure. Briefly, a 250-mL solution of 500 mM sodium phosphate (pH 6.8) was combined with 250 mL B10-30F11 or B10-rat IgG. )…”

Antibody–drug conjugates: targeted weapons against cancer

“…33 There are examples where both lysine and cysteine conjugation were tested for the same antibody, with significantly different outcomes. 34 A glutamine tag can be introduced and exploited for binding, 35 but an engineered transglutaminase (mTgase) can catalyse direct conjugation of drug molecules using native antibody heavy chains as a substrate (Dophen Biomedical, West Sacramento, CA, USA). Alternatively, a new site-specific conjugation technology based on bacterial …”