Biomedical subjects

Todd M Corneillie

Publications and source records attributed to Todd M Corneillie.

7 recordsLinked to original sources

PubMed2006

Irreversibly binding anti-metal chelate antibodies: Artificial receptors for pretargeting.

Antibodies against metal chelates may potentially be used in biomedical applications such as targeted imaging and therapy of cancer. Highly specific monoclonal antibodies can be developed, but their binding strength needs to be maximized for them to be of practical use. In general, the half-life for dissociation of an antibody-ligand complex is more than an order of magnitude lower than the half-lifetimes for decay of medically useful radiometal ions. Practically speaking, the metal chelate-based ligand will not be bound to its receptor long enough for all of the bound radiometal to decay. A novel approach to this problem is a combination of synthetic chemistry and site-directed mutagenesis, to position a mildly reactive group on the metal chelate adjacent to a complementary reactive group on the antibody when the complex is formed. The partners are chosen to be sufficiently unreactive so that they coexist with other molecules in living systems without undergoing reaction. When the antibody-chelate complex is formed the effective local concentrations of the two groups can be non-physically large, so that a permanent link is formed in the complex even though no reaction occurs when the partners are free in solution.

Antibodies↗

PubMed2003

Crystal structures of two complexes of the rare-earth-DOTA-binding antibody 2D12.5: ligand generality from a chiral system.

We report the crystal structures of antibody 2D12.5 Fab bound to an yttrium-DOTA analogue and separately to a gadolinium-DOTA analogue. The rare earth elements have many useful properties as probes, and 2D12.5 binds the DOTA (1,4,7,10-tetraazacyclododecane-N,N',N' ',N' "-tetraacetic acid) complexes of all of them (Corneillie et al. J. Am. Chem. Soc. 2003, 125, 3436-3437). The structures show that there are no direct protein-metal interactions: a bridging water acts as a link between the protein and metal, with the chelate present as the M isomer in each case. DOTA forms an amphipathic cylinder with the charged carboxylate groups toward the face of the chelate near the metal ion, while nonpolar methylene groups from the macrocycle and the carboxymethyl groups occupy the rear and sides of the molecule. The orientation of the metal-DOTA in the 2D12.5 complex places most of the methylene carbon atoms of DOTA in hydrophobic contact with aromatic protein side chains. Other binding interactions between the metal complex and the antibody include a bidentate salt bridge, four direct H-bonds, and four to five water-mediated H-bonds. We find that 2D12.5 exhibits enantiomeric binding generality, binding yttrium chelates in both Lambda(deltadeltadeltadelta) and Delta(lambdalambdalambdalambda) configurations with modestly different affinities. This develops from the symmetrical nature of the DOTA chelate, which places heteroatoms and hydrophobic atoms in approximately the same relative positions regardless of the helicity of DOTA.

Binding Sites↗

PubMed2003

A rare earth-DOTA-binding antibody: probe properties and binding affinity across the lanthanide series.

An antibody that binds rare earth complexes selectively could be used as a docking station for a set of probe molecules, of particular interest for medical imaging and therapy. The rare earths are rich in probe properties, such as the paramagnetism of Gd, the luminescence of Tb and Eu, and the nuclear properties of Lu and Y. We find that antibody 2D12.5, initially developed to bind analogues of Y-DOTA (1,4,7,10-tetraazacyclododecane-N,N',N' ',N' ''-tetraacetic acid) for radiotherapy, binds not only Y-DOTA analogues but also analogous DOTA complexes of all of the lanthanides. Surprisingly, chelates of some metals such as Gd3+ bind more tightly than the original Y3+ complex. When the shape of the complex is perturbed by either increasing or decreasing the radius of the lanthanide ion, the thermodynamic stability of the protein-ligand complex changes in a regular fashion. The behavior of DeltaDeltaG as a function of ionic radius fits a parabola, as might be expected for a system that behaves in a thermodynamically elastic way. The broad specificity and high affinity of this antibody for all rare earth-DOTA complexes make it particularly interesting for applications that take advantage of the unique characteristics of lanthanides. For example, UV excitation of the Tb-DOTA-2D12.5 complex leads to energy transfer from aromatic side chains of the antibody to bound Tb-DOTA, enhancing green terbium luminescence >104 relative to unbound Tb-DOTA.

Antibodies, Monoclonal↗

PubMedDate not supplied

Molecular tools for targeted imaging and therapy of cancer.

Here we review an approach to the design and production of antibody/ligand pairs for use in cell targeting procedures, to achieve functional affinity far greater than avidin/biotin. Using fundamental chemical principles, we have developed antibody/ligand pairs that retain the binding specificity of the antibody, but do not dissociate. By eliminating the dissociation of the ligand from the antibody, we have made the affinity functionally infinite. This methodology is applicable to other biological binding pairs.

Animals↗

PubMedDate not supplied

Element-coded affinity tags for peptides and proteins.

Isotope-coded affinity tags (ICAT) represent an important new tool for the analysis of complex mixtures of proteins in living systems [Aebersold, R., and Mann, M. (2003) Nature, 422, 198-207]. We envisage an alternative protein-labeling technique based on tagging with different element-coded metal chelates, which affords affinity chromatography, quantification, and identification of a tagged peptide from a complex mixture. As proof of concept, a synthetic peptide was modified at a cysteine side chain with either a carboxymethyl group or acetamidobenzyl-1,4,7,10-tetraazacyclododecane-N,N',N' ',N' "-tetraacetic acid (AcBD) chelates of terbium or yttrium. A mixture of the three modified peptides in a mole ratio of 100:1.0:0.83 carboxymethyl:AcBD-Tb:AcBD-Y was trypsinized, purified on a new affinity column that binds rare-earth DOTA chelates, and analyzed by LC-MS/MS. Chelate-tagged tryptic peptides eluted cleanly from the affinity column; the tagged peptides chromatographically coeluted during LC-MS analysis, were present in the expected ratio as indicated by MS ion intensity, and were sequence-identified by tandem mass spectrometry. DOTA-rare earth chelates have exceptional properties for use as affinity tags. They are highly polar and water-soluble. Many of the rare earth elements are naturally monoisotopic, providing a variety of simple choices for preparing mass tags. Further, the rare earths are heavy elements, whose mass defects give the masses of tagged peptides exact values not normally shared by molecules that contain only light elements.

Affinity Labels↗

PubMedDate not supplied

Irreversible engineering of the multielement-binding antibody 2D12.5 and its complementary ligands.

Engineering the permanent formation of a receptor-ligand complex has a number of potential applications in chemistry and biology, including targeted medical imaging and therapy. Starting from the crystal structure of the rare-earth-DOTA binding antibody 2D12.5 (Corneillie, T. M., Fisher, A. J., and Meares, C. F. (2003) J. Am. Chem. Soc. 125, 15039-15048), we used the site-directed incorporation of cysteine nucleophiles at the periphery of the antibody's binding site, paired with the chemical design of a weakly electrophilic ligand, to produce a receptor-ligand pair that associates efficiently and permanently. Protein residues proximal to the ligand's side chain were identified for engineering cysteine mutants. Fab fragments incorporating a cysteine at position 54, 55, or 56 of the heavy chain (complementarity determining region 2) were designed from the structure and then cloned, expressed in Drosophila S2 cells, and tested for reactivity with mildly electrophilic DOTA-yttrium ligands. All showed permanent binding activity, indicating that there is some tolerance for the location of the reactive mutant on the protein surface near the binding site. The G54C Fab mutant displayed the highest expression levels and permanent binding activity in initial experiments and was produced in high yield for further study. Upon examining the behavior of the G54C mutant with a small set of electrophilic ligands, differences in reactivity were observed which indicated that the substituents near the electrophilic atom can be important determinants of permanent binding. The G54C mutant permanently attaches to Y(3+) complexes of (S)-2-(4-acrylamidobenzyl)-DOTA with a half-time of approximately 13 min at 37 degrees C, making it potentially useful for in vivo pretargeting applications.

Animals↗

PubMedDate not supplied

Converting weak binders into infinite binders.

Monoclonal antibody 2D12.5 binds DOTA chelates of all the rare earths with K(d) approximately 10(-)(8) M, making it useful for the capture of probe molecules with a variety of properties. To make 2D12.5 even more useful for biological applications, we have engineered a single cysteine residue at position 54 of the heavy chain, a site proximal to the protein's binding site, so that weakly electrophilic metal complexes of (S)-2-(4-acrylamidobenzyl)-DOTA (AABD) may bind and form permanent linkages. At 37 degrees C, pH 7.5, all of the rare earth-AABD complexes bind permanently to the 2D12.5 G54C mutant within 5 min, in yields that correlate with their relative binding affinities. Surprisingly, indium-AABD also binds permanently in >50% yield within 5 min, despite the fact that changing the metal to indium reduces the affinity approximately 100x; even copper-AABD, which has approximately 10 000x lower binding affinity than the rare earths, binds permanently in >70% yield within 2 h. However, acrylamido compounds with no measurable affinity do not bind permanently. The important practical implication is that the G54C mutant of 2D12.5 may be used for applications that include not only the rare earths, but also an unexpected range of other elements as well. This infinite binding system can exhibit selective and permanent attachment with a remarkable range of structurally related ligands, albeit at slower rates as affinities decrease.

Antibodies, Monoclonal↗