Towards the definition of a chimpanzee and human conserved CD6 domain 1 epitope recognized by T1 monoclonal antibody.

Scavenger receptor cysteine-rich (SRCR) domains are evolutionally conserved modules that display complex structures stabilized by key amino acids, while some other residues have evolved with a relative independence, thus allowing the functional diversity of these receptors. CD6, a highly glycosylated membrane protein predominantly expressed on lymphocytes, contains three SRCR domains. The lack of CD6 domain crystal structure has limited the characterization of the binding sites for the interacting molecules. The interaction between CD6 and its ligand, activated leukocyte-cell adhesion molecule (ALCAM)/CD166, through the membrane-proximal SRCR3 domain, has low affinity and involves conserved sites in both molecules mediating a cross-species binding. The CD6-ALCAM interaction has been involved in cell adhesion, maturation, regulation of activation, and survival processes, suggesting the potential relevance of this target for therapeutic interventions. Several anti-CD6 monoclonal antibodies (MAb) have been described but their affinity and epitope definition remain unclear. We found the murine and humanized T1 MAb versions have similar CD6 recognition profiles and affinity constants of about 6 x 10(8). These antibodies do not block the CD6-ALCAM interaction and recognize a conformational epitope independent of the CD6 N-glycosylation. This epitope was additionally found in the chimpanzee and contains an RXE/Q consensus motif located in the membrane-distal SRCR1. These results, together with the therapeutic evidence previously obtained with these MAbs, suggest a differential contribution of CD6 domains to lymphocyte biology. Potential mechanisms for T1 MAb therapeutic effect different from CD6-CD166 interaction blocking would be dissected.

CIGB-300, a novel proapoptotic peptide that impairs the CK2 phosphorylation and exhibits anticancer properties both in vitro and in vivo.

Protein Kinase CK2 is a serine-threonine kinase frequently deregulated in many human tumors. Here, we hypothesized that a peptide binder to the CK2 phosphoacceptor site could exhibit anticancer properties in vitro, in tumor animal models, and in cancer patients. By screening a random cyclic peptide phage display library, we identified the CIGB-300 (formerly P15-Tat), a cyclic peptide which abrogates the CK2 phosphorylation by blocking recombinant substrates in vitro. Interestingly, synthetic CIGB-300 led to a dose-dependent antiproliferative effect in a variety of tumor cell lines and induced apoptosis as evidenced by rapid caspase activation. Importantly, CIGB-300 elicited significant antitumor effect both by local and systemic administration in murine syngenic tumors and human tumors xenografted in nude mice. Finally, we performed a First-in-Man trial with CIGB 300 in patients with cervical malignancies. The peptide was found to be safe and well tolerated in the dose range studied. Likewise, signs of clinical benefit were clearly identified after the CIGB-300 treatment as evidenced by significant decrease of the tumor lesion area and histological examination. Our results provide an early proof-of-principle of clinical benefit by using an anti-CK2 approach in cancer. Furthermore, this is the first clinical trial where an investigational drug has been used to target the CK2 phosphorylation domain.

Gangliosides, Ab1 and Ab2 antibodies I. Towards a molecular dissection of an idiotype-anti-idiotype system.

This report is focused on the molecular basis for the interaction of a monoclonal antibody (mAb) and its anti-idiotypic mAb. P3 mAb (Ab1) recognizes N-glycolyl-gangliosides, and 1E10 mAb is one of its anti-idiotypic mAbs (Ab2). Chimeric versions of both antibodies retained their specificity. Charged residues in their H-CDRs, particularly H-CDR3, were considered to play a major role in their binding and immunogenic properties. P3 mAb has the unusual property of generating a strong antibody response in syngeneic mice, even when it is administered in saline. We selected phagotopes from a 12mer peptide library displayed on filamentous phage to characterize amino acid motifs recognized by these antibodies. The peptides were enriched in charged amino acids similar to those present in P3 and 1E10 mAb H-CDR3. We also report the construction of four mutants of the P3 antibody, where arginine residues in the heavy chain CDRs were substituted by serine residues, and the characterization of their interaction with 1E10 mAb and GM3(NeuGc) ganglioside, as well as their immunogenic properties in Balb/c mice. H-CDR1 R31 residue appears to have a central role in P3 mAb reactivity and antigenicity. H-CDR3 R100a residue seems to be more involved in the immunogenicity of the P3 idiotype.

Gangliosides, Ab1 and Ab2 antibodies II. Light versus heavy chain: An idiotype-anti-idiotype case study.

The antibody heavy chain is generally more important than the light chain for the interaction with the antigen, although many reports demonstrate the influence of the light chain in the antibody binding properties. The heavy chains of anti-N-glycolyl-ganglioside P3 mAb and anti-idiotypic 1E10 mAb display complementary charged residues in their H-CDRs, particularly in H-CDR3. A basic residue in P3 mAb H-CDR1 was shown to be crucial for the interaction with the antigen and 1E10 mAb. The immunogenetic features of three other P3 mAb anti-idiotypic mAbs are now analyzed. One of them bears the same heavy chain as 1E10 mAb and a different light chain, but differs in its binding to P3 mAb mutants where H-CDR basic residues were replaced and in the binding to 1E10-specific phagotopes. Chimeric hybrid antibodies with P3 and 1E10 mAb heavy chains and unrelated light chains were obtained to further determine the importance of heavy chains in P3 and 1E10 mAb binding properties. One of the P3 heavy chain hybrid antibodies retained the specificity of P3 mAb with slight affinity differences. The heavy chains appear to play the main role in these mAb interactions, with the light chains modulating the affinity to their ligands.

Intramyocardial gene transfer of vascular endothelial growth factor 121 improves myocardial perfusion and function in the ischemic porcine heart

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that is angiogenic in vitro and in vivo. Several studies report on gene transfer of VEGF121 to promote angiogenesis in the ischemic myocardium of animals and patients. We hypothesized that intramyocardial administration of naked plasmid DNA encoding VEGF121 could improve myocardial perfusion and function in a porcine model of myocardial ischemia. Yorkshire swine underwent thoracotomy and placement of an ameroid constrictor on the circumflex coronary artery. Four weeks later, pVEGF121 plasmid was administered into the ischemic myocardium. Four weeks after gene transfer, SPECT imaging demonstrated significant reduction in the ischemic area in pVEGF121-treated animals compared with controls. In the pVEGF121 group, most of the animals evolved from light ischemia to a normal perfusion. In contrast, control animals exhibited similar or impaired ischemic conditions. Our results indicate that intramyocardial gene transfer of VEGF121 as naked plasmid DNA results in significant improvement in myocardial perfusion and function.

Antitumor effect of a novel proapoptotic peptide that impairs the phosphorylation by the protein kinase 2 (casein kinase 2).

Protein Kinase (casein kinase 2, CK2) is a serine-threonine kinase that is frequently dysregulated in many human tumors. Therefore we hypothesized that peptides capable of binding to the CK2 acidic domain may exhibit potential anticancer properties. By screening a random cyclic peptide phage display library, we have identified a novel peptide, P15, that abrogated CK2 phosphorylation by blocking the substrate in vitro. To verify its potential antineoplastic effect, P15 was fused to the cell-penetrating peptide derived from the HIV-Tat protein. Interestingly, P15-Tat induced apoptosis as evidenced by rapid caspase activation and cellular cytotoxicity in a variety of tumor cell lines. Furthermore, direct injection of P15-Tat into C57BL6 mice bearing day 7-established solid tumors, resulted in substantial regression of the tumor mass. Our findings describe a new proapoptotic cyclic peptide that blocks the CK2 phosphorylation and exhibits antitumor effect in vivo, indicating that the P15 peptide may potentially be used clinically to treat solid tumors or as an adjuvant for cancer therapy.