Synthesis and biodistribution studies of iodine‐131 D‐amino acid YYK peptide as a potential therapeutic agent for labeling an anti‐CD20 antibody

Abstract

A major drawback of conventionally radioiodinated monoclonal antibodies for radioimmunotherapy is in vivo dehalogenation of iodine as a result of deiodinase recognition. To solve this problem we have synthesized a YYK tri-peptide consisting of non-metabolizable D-amino acids modified with the N-succinimidyl (N-Succ) function. The chemical purity of the synthesized peptide as assessed by analytical high performance liquid chromatography was 95%. Labeling of the Fmoc-D-Tyr( t Bu)-D-Tyr( t Bu)-D-Lys(Boc)-N-Succ was performed using the chloramine-T method and the conventional extraction, resulting in a radiochemical yield of 50-71% and a radiochemical purity of 495%. Radioiodination of the peptide was followed by conjugation to anti-CD20 antibody with 65-75% labeling efficiency and 90% radiochemical purity. The effect of radioiodinated peptide on the biological behavior of the conjugate was evaluated through biodistribution studies in normal Lewis rats. Thyroid and stomach levels from Rituximab labeled with [131 I]-YYK-peptide were two-to fourfold less than those with directly labeled [ 131 I]-Rituximab, suggesting low recognition of its D-iodotyrosine residue by endogenous deiodinases. The favorable in vitro/in vivo stability and biodistribution profiles suggest that this radioiodinelabeled YYK peptide is a good candidate for further exploration of its potential clinical application.

Radioimmunotherapy of MCF7 breast cancer cell line with 131I-PR81 monoclonal antibody against MUC1: Comparison of direct and indirect radioiodination methods

“…In this way, PR81, which exhibited high affinity (2.19 × 10 8 M −1 ) towards MUC1 antigen and some MUC1 positive cell line (MCF-7, BT-20 and T-47D) may be suitable for in vivo tumor targeting, imaging and therapy [26]. Sadri et al documented the advantage of residualizing 131 I-YYK-peptide -Rituximab over directly labeled 131 I-Rituximab in an animal model of non Hodgkin lymphoma [27]. The promising clinical results obtained with directly radioiodinated mAb [35,36] prompted us to develop a residualizing 131 I-based approach to further increase the achievable tumor dose.…”

“…Recognition of the factors contributing to reduced tumor retention of radioiodine from directly radioiodinated mAbs has spawned the development of several intracellularly trapped forms of 131 I (residualizing 131 I) [19][20][21][22][23][24][25]. Here we describe such an approach for the labeling of an anti-MUC1 mAb, PR81 [26] with a residualizing 131 I label, 131 I-YYK-peptide [27], to safely and reliably generate multi-gigabecquerel levels of the pure product. YYK-peptideis Fmoc-D-Tyr (tBu)-D-Tyr (tBu)-D-Lys (Boc)-OH was attached to Nhydroxysuccinimide as a linker.…”

Recombinant fragment of an antibody tailored for direct radioiodination

“…Both variable domains are connected in the scFv polypeptide by a flexible linker (Gly 4 Ser) 3 in the orientation V L -linker-V H . Finally, the scFv fragment contains at its C-terminus a Myc epitope (amino acid sequence: EQKLISEEDL), which enables detection by an anti-Myc antibody.…”

“…The TU20 scFv was assembled with cassette insertions of the re-amplified TU20 V H and V L sequences into a previously constructed plasmid harboring another scFv with (Gly 4 Ser) 3 linker, using the above-mentioned restriction sites. The TU20 scFv module [EcoRV(V L )XhoI]-linker-[PstI(V H )BstEII] was completed with myc and His 5 tags at its carboxy terminus, and the completed module was shuttled into a pET22-like T7-promoter-based expression vector, encoding also the signal peptide.…”

“…2 In another example of the improvement of radioiodination results, two tyrosine residues of D-chirality were attached to therapeutic antibody rituximab by conjugation of an activated pre-radioiodinated YYK peptide. 3 A straightforward introduction of additional tyrosine residues into antibodies intended for imaging use can also be expected to render further useful properties, such as increased radioiodination propensity. We show here that a recombinant single chain variable fragment (scFv) of an anti-b-III-tubulin antibody, 4 having a potential use in imaging neural lesions or astrocytoma and certain nonneuronal tumors, [5][6][7] can be modified in a similar manner.…”