Marc Whitlow

Bio: Marc Whitlow is an academic researcher from Enzon Pharmaceuticals, Inc.. The author has contributed to research in topics: Factor Xa Inhibitor & Binding site. The author has an hindex of 42, co-authored 81 publications receiving 10429 citations. Previous affiliations of Marc Whitlow include Bayer & Boston College.

Single-chain antigen-binding proteins

Abstract: Single-chain antigen-binding proteins are novel recombinant polypeptides, composed of an antibody variable light-chain amino acid sequence (VL) tethered to a variable heavy-chain sequence (VH) by a designed peptide that links the carboxyl terminus of the VL sequence to the amino terminus of the VH sequence. These proteins have the same specificities and affinities for their antigens as the monoclonal antibodies whose VL and VH sequences were used to construct the recombinant genes that were expressed in Escherichia coli. Three of these proteins, one derived from the sequence for a monoclonal antibody to growth hormone and two derived from the sequences of two different monoclonal antibodies to fluorescein, were designed, constructed, synthesized, purified, and assayed. These proteins are expected to have significant advantages over monoclonal antibodies in a number of applications.

Multivalent antigen-binding proteins

Abstract: Compositions of, genetic constructions coding for, and methods for producing multivalent antigen-binding proteins are described and claimed. The methods include purification of compositions containing both monomeric and multivalent forms of single polypeptide chain molecules, and production of multivalent proteins from purified monomers. Production of multivalent proteins may occur by a concentration-dependent association of monomeric proteins, or by rearrangement of regions involving dissociation followed by reassociation of different regions. Bivalent proteins, including homobivalent and heterobivalent proteins, are made in the present invention. Genetic sequences coding for bivalent single-chain antigen-binding proteins are disclosed. Uses include all those appropriate for monoclonal and polyclonal antibodies and fragments thereof, including use as a bispecific antigen-binding molecule.

Rapid Tumor Penetration of a Single-Chain Fv and Comparison with Other Immunoglobulin Forms

Abstract: Single-chain antigen-binding proteins, or sFvs, represent potentially unique molecules for targeted delivery of drugs, toxins, or radionuclides to a tumor site. In previous studies (Cancer Res., 51:6363-6371, 1991) using a human colon carcinoma xenograft model, it was demonstrated that the sFv has an extremely rapid plasma and whole body clearance, as compared to intact IgG or Ig fragments. One potential consequence of the rapid sFv pharmacokinetic properties was the reduced percentage of injected dose/g of the radiolabeled sFv found in the tumor throughout a range of time points. The present study was designed to define the tumor penetration properties of a radiolabeled sFv in comparison with other Ig forms. 125I-labeled sFv, Fab', F(ab')2, and IgG forms of monoclonal antibody CC49, directed against the human pancarcinoma antigen TAG-72, were used to target the LS-174T human colon carcinoma xenograft in athymic mice. At various time points after systemic Ig administration, quantitative autoradiographic analyses of surgically removed tumors were used to define the rate and degree of penetration of the various Ig forms. These studies revealed that most of the intact IgG delivered to the tumor was concentrated in the region of or immediately adjacent to vessels, while the sFv was more evenly distributed throughout the tumor mass. The distributions of the Fab' and F(ab')2 fragments showed intermediate penetration in a size-related manner. The sFv demonstrated maximum tumor penetration at 0.5 h postinjection, while the intact IgG reached an equivalent degree of penetration at 48 to 96 h postinjection. These studies thus reveal a greater degree of uptake throughout the tumor for the sFv than would be expected by gross analyses of percentage injected dose/g and demonstrate an extremely rapid tumor penetration of the sFv. These studies should aid in the rational design of potential applications of drug-, toxin-, and radionuclide-conjugated sFvs in cancer therapy.

A novel, highly stable fold of the immunoglobulin binding domain of streptococcal protein G.

Abstract: The high-resolution three-dimensional structure of a single immunoglobulin binding domain (B1, which comprises 56 residues including the NH2-terminal Met) of protein G from group G Streptococcus has been determined in solution by nuclear magnetic resonance spectroscopy on the basis of 1058 experimental restraints. The average atomic root-mean-square distribution about the mean coordinate positions is 0.27 angstrom (A) for the backbone atoms, 0.65 A for all atoms, and 0.39 A for atoms excluding disordered surface side chains. The structure has no disulfide bridges and is composed of a four-stranded beta sheet, on top of which lies a long helix. The central two strands (beta 1 and beta 4), comprising the NH2- and COOH-termini, are parallel, and the outer two strands (beta 2 and beta 3) are connected by the helix in a +3x crossover. This novel topology (-1, +3x, -1), coupled with an extensive hydrogen-bonding network and a tightly packed and buried hydrophobic core, is probably responsible for the extreme thermal stability of this small domain (reversible melting at 87 degrees C).

Construction, Binding Properties, Metabolism, and Tumor Targeting of a Single-Chain Fv Derived from the Pancarcinoma Monoclonal Antibody CC49

Abstract: CC49 is a "second generation" monoclonal antibody to B72.3, which reacts with the pancarcinoma antigen TAG-72. CC49 has been shown to efficiently target human colon carcinoma xenografts and is currently being evaluated in both diagnostic and therapeutic clinical trials. We describe here the construction and characterization of a recombinant single-chain Fv (sFv) of CC49. The sFv was shown to be a Mr 27,000 homogeneous entity which could be efficiently radiolabeled with 125I or 131I. Comparative direct binding studies and competition radioimmunoassays using CC49 intact IgG, F(ab')2, Fab', and sFv revealed that the monomeric CC49 Fab' and sFv had relative binding affinities 8-fold lower than the dimeric F(ab')2 and intact IgG. Nonetheless, the 131I-labeled sFv was shown to bind biopsies of TAG-72-expressing tumors. Metabolism studies in mice, using radiolabeled CC49 IgG, F(ab')2, Fab', and sFv, demonstrated an extremely rapid plasma and whole body clearance for the sFv. CC49 sFv plasma pharmacokinetic studies in rhesus monkeys also showed a very rapid plasma clearance (T1/2 alpha of 3.9 min and T1/2 beta of 4.2 h). Tumor targeting studies with all four radiolabeled Ig CC49 forms, using the LS-174T human colon carcinoma xenograft model, revealed a much lower percentage injected dose/g tumor binding for the CC49 monomeric sFv and Fab' as compared to the dimeric F(ab')2 and intact IgG. However, tumor:normal tissue ratios (radiolocalization indices) for the sFv were comparable to or greater than those of the other Ig forms. High kidney uptake with 125I-labeled Fab' and F(ab')2 was not seen with 125I-sFv. Gamma scanning studies also showed that 131I-CC49 sFv could efficiently localize tumors. The CC49 sFv may thus have utility in diagnostic and perhaps therapeutic applications for a range of human carcinomas.

All-atom empirical potential for molecular modeling and dynamics studies of proteins.

Abstract: New protein parameters are reported for the all-atom empirical energy function in the CHARMM program. The parameter evaluation was based on a self-consistent approach designed to achieve a balance between the internal (bonding) and interaction (nonbonding) terms of the force field and among the solvent−solvent, solvent−solute, and solute−solute interactions. Optimization of the internal parameters used experimental gas-phase geometries, vibrational spectra, and torsional energy surfaces supplemented with ab initio results. The peptide backbone bonding parameters were optimized with respect to data for N-methylacetamide and the alanine dipeptide. The interaction parameters, particularly the atomic charges, were determined by fitting ab initio interaction energies and geometries of complexes between water and model compounds that represented the backbone and the various side chains. In addition, dipole moments, experimental heats and free energies of vaporization, solvation and sublimation, molecular volume...

The OPLS [optimized potentials for liquid simulations] potential functions for proteins, energy minimizations for crystals of cyclic peptides and crambin.

Abstract: A complete set of intermolecular potential functions has been developed for use in computer simulations of proteins in their native environment. Parameters are reported for 25 peptide residues as well as the common neutral and charged terminal groups. The potential functions have the simple Coulomb plus Lennard-Jones form and are compatible with the widely used models for water, TIP4P, TIP3P, and SPC. The parameters were obtained and tested primarily in conjunction with Monte Carlo statistical mechanics simulations of 36 pure organic liquids and numerous aqueous solutions of organic ions representative of subunits in the side chains and backbones of proteins. Bond stretch, angle bend, and torsional terms have been adopted from the AMBER united-atom force field. As reported here, further testing has involved studies of conformational energy surfaces and optimizations of the crystal structures for four cyclic hexapeptides and a cyclic pentapeptide. The average root-mean-square deviation from the X-ray structures of the crystals is only 0.17 A for the atomic positions and 3% for the unit cell volumes. A more critical test was then provided by performing energy minimizations for the complete crystal of the protein crambin, including 182 water molecules that were initially placed via a Monte Carlo simulation. The resultant root-mean-square deviation for the non-hydrogen atoms is still ca. 0.2 A and the variation in the errors for charged, polar, and nonpolar residues is small. Improvement is apparent over the AMBER united-atom force field which has previously been demonstrated to be superior to many alternatives. Computer simulations are undoubtedly destined to became an increasingly important means for investigating the structures and dynamics of biomolecular systems.' At the heart of such theoretical calculations are the force fields that describe the interatomic interactions and the mechanics of deformations of the molecules.* There is also little doubt that there will be a continual evolution in force fields with added complexity and improved performance paralleling the availability of computer resources. Our own efforts in this area over the last few years have resulted in the OPLS potential functions for proteins whose development and performance are summarized here. These potential functions have a simple form and they have been parametrized directly to reproduce experimental thermodynamic and structural data on fluids. Consequently, they are computationally efficient and their description of proteins in solution or crystalline environments should be superior to many alterantives that have been developed with limited condensed-phase data. The latter point is pursued here primarily through calculations on the crystal structures for four cyclic hexapeptides, a cyclic pentapeptide, and the protein crambin. Improvements are apparent in comparison to the AMBER united-atom force field3 which has previously been shown to be superior to many alternative^.^ (1) Beveridge, D. L., Jorgensen, W. L., Eds. Ann. N.Y. Acad. Sci. 1986, 482. ( 2 ) For reviews, see: (a) Levitt, M. Annu. Reu. Biophys. Eioeng. 1982, 11, 251. (b) McCammon, J. A. Rep. Prog. Phys. 1984, 47, 1. (3) Weiner, S. J.; Kollman, P. A.; Case, D. A,; Singh, U. C.; Ghio, C.; Alagona, G.; Profeta, S.; Weiner, P. J. Am. Chem. SOC. 1984, 106, 765. Parametrization The peptide residues of proteins contain readily identifiable organic subunits such as amides, hydrocarbons, alcohols, thioethers, etc. In view of this and since data are available on the corresponding pure organic liquids, our approach to developing a force field for proteins was to build it up from parameters demonstrated to yield good descriptions of organic liquids. U1timately, the force field would need to treat both intramolecular terms for bond stretches, angle bends, and torsions, as well as the intermolecular and intramolecular nonbonded interactions. The latter are generally accepted to be the most difficult part of the problem and have been our focus.3 A simple, computationally efficient form was chosen to represent the nonbonded interactions through Coulomb and Lennard-Jones terms interacting between sites centered on nuclei (eq 1). Thus, the intermolecular inter-

Nitric oxide synthases: structure, function and inhibition

Abstract: This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in our understanding of this enzyme family. There is now information on the enzyme structure at all levels from primary (amino acid sequence) to quaternary (dimerization, association with other proteins) structure. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) allow us to interpret other information in the context of this important part of the enzyme, with its binding sites for iron protoporphyrin IX (haem), biopterin, L-arginine, and the many inhibitors which interact with them. The exact nature of the NOS reaction, its mechanism and its products continue to be sources of controversy. The role of the biopterin cofactor is now becoming clearer, with emerging data implicating one-electron redox cycling as well as the multiple allosteric effects on enzyme activity. Regulation of the NOSs has been described at all levels from gene transcription to covalent modification and allosteric regulation of the enzyme itself. A wide range of NOS inhibitors have been discussed, interacting with the enzyme in diverse ways in terms of site and mechanism of inhibition, time-dependence and selectivity for individual isoforms, although there are many pitfalls and misunderstandings of these aspects. Highly selective inhibitors of iNOS versus eNOS and neuronal NOS have been identified and some of these have potential in the treatment of a range of inflammatory and other conditions in which iNOS has been implicated.

Nitric oxide synthases: regulation and function.

Abstract: Nitric oxide (NO), the smallest signalling molecule known, is produced by three isoforms of NO synthase (NOS; EC 1.14.13.39). They all utilize l-arginine and molecular oxygen as substrates and require the cofactors reduced nicotinamide-adenine-dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and (6R-)5,6,7,8-tetrahydrobiopterin (BH(4)). All NOS bind calmodulin and contain haem. Neuronal NOS (nNOS, NOS I) is constitutively expressed in central and peripheral neurons and some other cell types. Its functions include synaptic plasticity in the central nervous system (CNS), central regulation of blood pressure, smooth muscle relaxation, and vasodilatation via peripheral nitrergic nerves. Nitrergic nerves are of particular importance in the relaxation of corpus cavernosum and penile erection. Phosphodiesterase 5 inhibitors (sildenafil, vardenafil, and tadalafil) require at least a residual nNOS activity for their action. Inducible NOS (NOS II) can be expressed in many cell types in response to lipopolysaccharide, cytokines, or other agents. Inducible NOS generates large amounts of NO that have cytostatic effects on parasitic target cells. Inducible NOS contributes to the pathophysiology of inflammatory diseases and septic shock. Endothelial NOS (eNOS, NOS III) is mostly expressed in endothelial cells. It keeps blood vessels dilated, controls blood pressure, and has numerous other vasoprotective and anti-atherosclerotic effects. Many cardiovascular risk factors lead to oxidative stress, eNOS uncoupling, and endothelial dysfunction in the vasculature. Pharmacologically, vascular oxidative stress can be reduced and eNOS functionality restored with renin- and angiotensin-converting enzyme-inhibitors, with angiotensin receptor blockers, and with statins.

Methods for producing members of specific binding pairs

Abstract: A member of a specific binding pair (sbp) is identified by expressing DNA encoding a genetically diverse population of such sbp members in recombinant host cells in which the sbp members are displayed in functional form at the surface of a secreted recombinant genetic display package (rgdp) containing DNA encoding the sbp member or a polypeptide component thereof, by virtue of the sbp member or a polypeptide component thereof being expressed as a fusion with a capsid component of the rgdp. The displayed sbps may be selected by affinity with a complementary sbp member, and the DNA recovered from selected rgdps for expression of the selected sbp members. Antibody sbp members may be thus obtained, with the different chains thereof expressed, one fused to the capsid component and the other in free form for association with the fusion partner polypeptide. A phagemid may be used as an expression vector, with said capsid fusion helping to package the phagemid DNA. Using this method libraries of DNA encoding respective chains of such multimeric sbp members may be combined, thereby obtaining a much greater genetic diversity in the sbp members than could easily be obtained by conventional methods.