Monique Cosman

Pharmacokinetic Characterization in Xenografted Mice of a Series of First-Generation Mimics for HLA-DR Antibody, Lym-1, as Carrier Molecules to Image and Treat Lymphoma

Despite their large size, antibodies (Abs) are suitable carriers to deliver systemic radiotherapy, often molecular image–based, for lymphoma and leukemia. Lym-1 Ab has proven to be an effective radioisotope carrier, even in small amounts, for targeting human leukocyte antigen DR (HLA-DR), a surface membrane protein overexpressed on B-cell lymphoma. Pairs of molecules (referred to as ligands), shown by computational and experimental methods to bind to each of 2 sites within the Lym-1 epitopic region, have been linked to generate small (<2 kDa) molecules (referred to as selective high-affinity ligands [SHALs]) to mimic the targeting properties of Lym-1 Ab. Methods: A lysine-polyethylene glycol (PEG) backbone was used to synthetically link 2 of the following ligands: deoxycholate, 5-leuenkephalin, triiodothyronine, thyronine, dabsyl-l-valine, and N-benzoyl-l-arginyl-4-amino-benzoic acid to generate a series of 13 bidentate SHALs with a biotin or 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA) chelate attached to the linker. These SHALs have been assessed for their selectivity in binding to HLA-DR10–expressing cells and for their pharmacokinetics and tissue biodistribution in mice. Biotinylated versions of these SHALs discriminated cell lines positive for HLA-DR10 expression with near-nanomolar affinity. The DOTA versions of 4 SHALs were labeled with 111In for pharmacokinetic studies in mice with HLA-DR10–expressing malignant Raji xenografts. Results: The bidentate, biotinylated, and DOTA-SHALs were synthesized in high-purity, multimilligram amounts. Mean radiochemical and product yields and purities were 90%, 75%, and 90% at mean specific activities of 3.9 MBq/μg (105 μCi/μg) for the 111In-labeled SHALs. As expected, rapid blood clearance and tumor targeting were observed. The pharmacokinetics of the SHALs was influenced by the component ligands. Biliary clearance, kidney localization, and serum receptor binding contributed to less favorable tumor targeting. Conclusion: A series of SHALs was readily synthesized in multimilligram amounts and showed the expected selective binding in vitro. Better selection of the SHAL components should provide second-generation SHALs with improved properties to fulfill the substantial potential of these novel molecular carriers for targeting.

Laser-Lathe Lithography—a Novel Method for Manufacturing Nuclear Magnetic Resonance Microcoils

A novel 3-dimensional laser-lathe process for manufacturing magnetic resonance microcoils is presented. The process has been used to print coils on a variety of materials, including glass and Teflon. The dimensions of these coils can be varied easily to allow any number of different coil designs, including solenoids and saddle coils. In our fabrication process, capillary tubes sputter-coated with a thin titanium-copper multilayer are plated with a positive electrodeposited photoresist. The resist is exposed with a computer-controlled laser-lathe apparatus consisting of an argon-ion laser, an acousto-optic modulator, a movable aperture, a lead screw stage and a spindle stage. After exposure and development, copper is electrolytically deposited through the resist mask. Following copper deposition the resist mask is removed and the sputtered copper and titanium are etched away, leaving a microcoil firmly adhered to the capillary. The resistivity of the laser-lathe copper windings is 7.6% higher than the resistivity of hand-wound coils (1.85 μΩ-cm for laser-lathe copper compared with 1.72 μΩ-cm for bulk annealed copper). For laser-lathe and hand-wound microcoils of similar size and geometry, the coil quality factor, Q, of the laser patterned coils would be 7.6% lower than the hand-wound coils. Examples of 13C NMR spectra obtained using laser-lathe coils are shown, and a relative improvement of 68 in the NMR sensitivity is calculated for a laser-lathe microcoil compared with a conventional 5 mm NMR sample tube.

Solution structure of the 2-amino-1- methyl-6-phenylimidazo[4,5-b]pyridine C8-deoxyguanosine adduct in duplex DNA

The carcinogenic heterocyclic amine (HA) 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed during the cooking of various meats. To enable structure/activity studies aimed at understanding how DNA damaged by a member of the HA class of compounds can ultimately lead to cancer, we have determined the first solution structure of an 11-mer duplex containing the C8-dG adduct formed by reaction with N-acetoxy-PhIP. A slow conformational exchange is observed in which the PhIP ligand either intercalates into the DNA helix by denaturing and displacing the modified base pair (main form) or is located outside the helix in a minimally perturbed B-DNA duplex (minor form). In the main base-displaced intercalation structure, the minor groove is widened, and the major groove is compressed at the lesion site because of the location of the bulky PhIP-N-methyl and phenyl ring in the minor groove; this distortion causes significant bending of the helix. The PhIP phenyl ring interacts with the phosphodiester-sugar ring backbone of the complementary strand and its fast rotation with respect to the intercalated imidazopyridine ring causes substantial distortions at this site, such as unwinding and bulging-out of the strand. The glycosidic torsion angle of the [PhIP]dG residue is syn, and the displaced guanine base is directed toward the 3′ end of the modified strand. This study contributes, to our knowledge, the first structural information on the biologically relevant HA class to a growing body of knowledge about how conformational similarities and differences for a variety of types of lesions can influence protein interactions and ultimately biological outcome.

An empirical relationship between rotational correlation time and solvent accessible surface area

Structure–dynamics interrelationships are important in understanding protein function. We have explored the empirical relationship between rotational correlation times (τc and the solvent accessible surface areas (SASA) of 75 proteins with known structures. The theoretical correlation between SASA and τc through the equation SASA = Krτc(2/3) is also considered. SASA was determined from the structure, τccalc was determined from diffusion tensor calculations, and τcexpt was determined from NMR backbone13 C or 15N relaxation rate measurements. The theoretical and experimental values of τc correlate with SASA with regression analyses values of Kr as 1696 and 1896 m2s-(2/3), respectively, and with corresponding correlation coefficients of 0.92 and 0.70.

An Empirical Correlation between Secondary Structure Content and Averaged Chemical Shifts in Proteins

It is shown that the averaged chemical shift (ACS) of a particular nucleus in the protein backbone empirically correlates well to its secondary structure content (SSC). Chemical shift values of more than 200 proteins obtained from the Biological Magnetic Resonance Bank are used to calculate ACS values, and the SSC is estimated from the corresponding three-dimensional coordinates obtained from the Protein Data Bank. ACS values of (1)H(alpha) show the highest correlation to helical and sheet structure content (correlation coefficient of 0.80 and 0.75, respectively); (1)H(N) exhibits less reliability (0.65 for both sheet and helix), whereas such correlations are poor for the heteronuclei. SSC estimated using this correlation shows a good agreement with the conventional chemical shift index-based approach for a set of proteins that only have chemical shift information but no NMR or x-ray determined three-dimensional structure. These results suggest that even chemical shifts averaged over the entire protein retain significant information about the secondary structure. Thus, the correlation between ACS and SSC can be used to estimate secondary structure content and to monitor large-scale secondary structural changes in protein, as in folding studies.

Selective High-Affinity Ligand Antibody Mimics for Cancer Diagnosis and Therapy: Initial Application to Lymphoma/Leukemia

Purpose: More than two decades of research and clinical trials have shown radioimmunotherapy to be a promising approach for treating various forms of cancer. Lym-1 antibody, which binds selectively to HLA-DR10 on malignant B-cell lymphocytes, has proved to be effective in delivering radionuclides to non–Hodgkins lymphoma and leukemia. Using a new approach to create small synthetic molecules that mimic the targeting properties of the Lym-1 antibody, a prototype, selective high-affinity ligand (SHAL), has been developed to bind to a unique region located within the Lym-1 epitope on HLA-DR10. Experimental Design: Computer docking methods were used to predict two sets of small molecules that bind to neighboring cavities on the β subunit of HLA-DR10 surrounding a critical amino acid in the epitope, and the ligands were confirmed to bind to the protein by nuclear magnetic resonance spectroscopy. Pairs of these molecules were then chemically linked together to produce a series of bidentate and bisbidentate SHALs. Results: These SHALs bind with nanomolar to picomolar Kds only to cell lines expressing HLA-DR10. Analyses of biopsy sections obtained from patients also confirmed that SHAL bound to both small and large cell non–Hodgkins lymphomas mimicking the selectivity of Lym-1. Conclusions: These results show that synthetic molecules less than 1/50th the mass of an antibody can be designed to exhibit strong binding to subtle structural features on cell surface proteins similar to those recognized by antibodies. This approach offers great potential for developing small molecule therapeutics that target other types of cancer and disease.

Marmoset Fine B Cell and T Cell Epitope Specificities Mapped onto a Homology Model of the Extracellular Domain of Human Myelin Oligodendrocyte Glycoprotein

Aberrant association of autoantibodies with myelin oligodendrocyte glycoprotein (MOG), an integral membrane protein of the central nervous system (CNS) myelin, has been implicated in the pathogenesis of multiple sclerosis (MS). Sensitization of nonhuman primates (Callithrix jacchus marmosets) against the nonglycosylated, recombinant N-terminal domain of rat MOG (residues 1-125) reproduces an MS-like disease in which MOG-specific autoantibodies directly mediate demyelination. To assess the interrelationship between MOG structure and the induction of autoimmune CNS diseases and to enable structure-based rational design of therapeutics, a homology model of human MOG(2-120) was constructed based on consensus residues found in immunoglobulin superfamily variable-type proteins having known structures. Possible sites for posttranslational modifications and dimerization have also been identified and analyzed. The B cell and T cell epitopes have been identified in rat MOG-immunized marmosets, and these sequences are observed to map primarily onto accessible regions in the model, which may explain their ability to generate potent antibody responses.

An improved experimental scheme to measure self-diffusion coefficients of biomolecules with an advantageous use of radiation damping

Abstract An improved nuclear magnetic resonance (NMR) experimental scheme to measure self-diffusion coefficients of biomolecules in 95% H2O using pulsed-magnetic-field gradients is presented. The radiation damping of water, which deleteriously effects the quality of the spectra in high-field NMR, is advantageously used in reducing the water signal. The enhancements of the new sequence, designated BPP-SED, a novel combination of BPP-LED (bipolar-gradient-pulse pair longitudinal eddy current delay) and SED (selective echo dephasing) is demonstrated by measuring self-diffusion coefficients of dilute protein samples (0.5 mM) in limited volumes (250 μl).

Novel relaxation compensated method to measure proton exchange rates in biomolecules based on decorrelation of heteronuclear two-spin order

A combined experiment based on decorrelation of heteronuclear two‐spin order and a new analysis method is presented for measuring accurate rapid amide proton exchange rates (kexHH = kex) in 15N‐labeled biomolecules, such as proteins or nucleic acids, in water. The term ‘decorrelation’ is defined as the loss of the initial correlation between a labile biomolecule proton and its coupled nitrogen when they are separated by intermolecular chemical exchange with water. The NMR pulse sequence [DECORrelated EXchange SpectroscopY (DECOREXSY)] measures the decay of the heteronuclear two‐spin order terms with minimal interference effects from relaxation processes and solvent‐induced artifacts. The new analysis protocol based on backbone relaxation measurements is introduced to compensate for relaxation contributions to the exchange rates that are otherwise inseparable. This simple and straightforward scheme has several potential applications in protein folding and biomolecular recognition and binding studies, and fills the need for a sensitive experiment to measure absolute fast‐amide proton exchange rates predominantly on the sub‐millisecond time‐scale. Copyright

Identification of a Thermo-Regulated Glutamine-Binding Protein from Yersinia pestis

Here we present modeling and NMR spectroscopic evidence that the function of a Yersinia pestis pMT1 plasmid protein, designated as orf38, is most likely a glutamine binding protein. The modeling was homology-based at a very low level of sequence identity ( approximately 16%) and involved structural comparison of multiple templates, as well as template-substrate interaction analyses. Transferred nuclear Overhauser and saturation transfer difference experiments were used to characterize the binding of sugars and amino acids to orf38. The identification and characterization of an unknown protein function using the strategy presented here has applicability to a variety of research areas, including functional genomics and proteomics efforts.