BACKGROUND Different strategies can be used to improve the tumor:non‐tumor ratio of radiolabeled antibodies in immunotargeting. One approach is to use secondary antibodies to clear out redundant, circulating primary antibodies. In the current study, the in vitro complex formation and in vivo clearing capabilities and metabolism of the monoclonal antibody TS1 and its monoclonal anti‐idiotype, αTS1, were studied. METHODS Complex formation studies were performed using polyacrylamide gel electrophoresis (PAGE), gel permeation chromatography, and electron microscopy. The clearance and metabolism of the complexes were studied in nude mice. RESULTS PAGE and gel permeation chromatography showed that more than 70% of the antibodies formed complexes. The electron microscopy studies revealed that the complexes formed between TS1 and αTS1 are mainly ring‐shaped (66.6–73.4%), comprising 4 to > 8 antibodies. These rings consist of equal numbers of idiotype and anti‐idiotype. The most commonly observed complexes were tetrameric rings (26.8–40.5%), hexameric rings (10.7–11.9%), and rings containing more than eight monoclonal antibodies (6.6–14‐4%). The in vivo study illustrated that within 24 hours 80% of the total nuclide content had been degraded and excreted via the urine, compared with 25% for similarly treated mice that did not receive any anti‐idiotype. CONCLUSIONS Interestingly, the electron microscopy study demonstrated that dimers were rare (0.4–1.2%), probably reflecting a location of epitopes incompatible with tight, sterically constrained dimeric interactions; insufficient flexibility of the immunoglobulin G1 subtype hinge regions; or both. The anti‐idiotypic clearing mechanisms proved efficient in nude mice. In vivo metabolic studies indicate that the accumulation and degradation of TS1/αTS1 immune complexes, to a large extent, take place in the liver, where a substantial amount was detected as soon as 1 hour after anti‐idiotype injection. Cancer 2002;94:1306–13. © 2002 American Cancer Society. DOI 10.1002/cncr.10301
The monoclonal antibody TS1 against cytokeratin 8 and its antiidiotype alphaTS1 have been used for immunotargeting and therapy of carcinomas in experimental tumor model systems. The interaction surfaces between mab TS1, the cytokeratin 8 epitope, and its anti-idiotypic antibody, alphaTS1, were studied in detail in order to make future veneering of the interactions possible. The V-genes of TS1 and alphaTS1 were cloned and sequenced and the CDRs and the framework residues were identified. Amino acids participating in the interactions were identified following chemical modifications of residues in non-protected and protected molecules of cytokeratin 8, alphaTS1 and TS1. From the sequences, the three-dimensional structures were generated using computer modelling of the antibody variable regions. Several charged amino acid, histidine and tyrosine residues were displayed in the antibody surfaces implicated in the interactions and chemical modification confirmed the importance of these amino acids. The cytokeratin 8 epitope has previously been identified by Johansson et al. and it displays negatively charged amino acid residues which could be identified in the chemical modification. It was also revealed that the TS1 binding to cytokeratin 8 and alphaTS1 respectively are partly overlapping; a histidine identified in TS1 is probably involved only in the interaction with alphaTS1. Furthermore, the chemical modification demonstrated that exchanging aspartic-glutamic acids to asparagine-glutamine residues in TS1 increased the binding of TS1 to cytokeratin 8, indicating that there is at least one acidic amino acid that is an obstacle in the TS1-CK8 binding. The detailed assembly of the interaction surfaces will facilitate the future use of site directed mutagenesis to improve the TS1-CK8 association rate and the clearing of TS1 with alphaTS1 in vivo.
We have used a number of in vitro and in vivo techniques to identify the molecules that can bind to the cytoplasmic tail of the Ly49A receptor. Affinity chromatography using peptides corresponding to the N-terminal 18 amino acids of Ly49A allowed the recovery of a number of proteins that bound preferentially to the tyrosine-phosphorylated peptide, including SH2-containing phosphatase-1 (SHP1) and the SH2-containing inositol 5' phosphatase (SHIP). In another approach, using the entire cytoplasmic domain of the Ly49A receptor, we found that SHP2 also interacted with the tyrosine-phosphorylated form of the Ly49A cytoplasmic tail. Using BIACORE(R)2000 analysis, we determined that both SHP1 and SHP2 bound to the tyrosine-phosphorylated cytoplasmic tail of Ly49A with affinities in the nanomolar range, whilst SHIP showed no binding. Mutation of tyrosine-36 to phenylalanine did not significantly affect the affinities of these proteins for the tyrosine-phosphorylated cytoplasmic tail of Ly49A. In addition, using a whole-cell system with T-cell lymphoma cell lines that expressed the Ly49A receptor or its H-2Dd ligand, we determined that engagement of Ly49A by its major histocompatibility complex (MHC) ligand leads to tyrosine-phosphorylation events and recruitment of SHP1. Recruitment of SHP1 was rapid and transient, reaching a maximum after 5 min. These data suggest that mechanisms for the inhibitory signal are generated following receptor engagement. They also provide direct evidence that ligand engagement of the Ly49A receptor is responsible for recruitment of downstream signalling molecules.
First trial to report safety and activity of the microtubule inhibitor vinflunine plus the tyrosine kinase inhibitor sorafenib in post-platinum metastatic urothelial cancer (mUC) patients.• A recommended phase II dose was identified for the treatment combination of vinflunine plus sorafenib, with main adverse events including fatigue, febrile neutropenia, neutropenia, hypertension, and hyponatremia. • An overall response rate of 41% to second-line vinflunine plus sorafenib treatment in patients with platinum-resistant mUC was confirmed.
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