In vivo tumor targeting of a recombinant single-chain antigen-binding protein.

We describe here the first in vivo targeting of tumors with a single-chain antigen-binding protein. The molecule, which was constructed and expressed in Escherichia coli, is a novel recombinant protein composed of a variable light-chain (VL), amino acid sequence of an immunoglobulin tethered to a variable heavy-chain (VH) sequence by a designed peptide. We show that this protein, derived from the DNA sequence of the variable regions of the antitumor monoclonal antibody B6.2, has the same in vitro antigen-binding properties as the B6.2 Fab' fragment. Comparative pharmacokinetic studies in athymic mice demonstrate much more rapid alpha and beta phases of plasma clearance for the single-chain antigen-binding protein than for the Fab' fragment, as well as an extremely rapid whole-body clearance. Half-life values for alpha and beta phases of single-chain antigen-binding protein clearance were 2.4 minutes and 2.8 hours, respectively, versus 14.8 minutes and 7.5 hours for Fab'. Furthermore, the single-chain antigen-binding protein molecule did not show accumulation in the kidney as did the Fab' molecule or, as previously shown, the F(ab')2 molecule. Despite its rapid clearance, the single-chain antigen-binding protein showed uptake in a human tumor xenograft comparable to that of the Fab' fragment, resulting in tumor to normal tissue ratios comparable to or greater than those obtained with the Fab' fragment. These studies thus demonstrate the in vivo stability of recombinant single-chain antigen-binding proteins and their potential in some diagnostic and therapeutic clinical applications in cancer and other diseases.

Construction, binding properties, metabolism, and tumor targeting of a single-chain Fv derived from the pancarcinoma monoclonal antibody CC49.

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.

Physicochemical basis for the rapid time-action of LysB28ProB29-insulin: dissociation of a protein-ligand complex.

The rate-limiting step for the absorption of insulin solutions after subcutaneous injection is considered to be the dissociation of self-associated hexamers to monomers. To accelerate this absorption process, insulin analogues have been designed that possess full biological activity and yet have greatly diminished tendencies to self-associate. Sedimentation velocity and static light scattering results show that the presence of zinc and phenolic ligands (m-cresol and/or phenol) cause one such insulin analogue, LysB28ProB29-human insulin (LysPro), to associate into a hexameric complex. Most importantly, this ligand-bound hexamer retains its rapid-acting pharmacokinetics and pharmacodynamics. The dissociation of the stabilized hexameric analogue has been studied in vitro using static light scattering as well as in vivo using a female pig pharmacodynamic model. Retention of rapid time-action is hypothesized to be due to altered subunit packing within the hexamer. Evidence for modified monomer-monomer interactions has been observed in the X-ray crystal structure of a zinc LysPro hexamer (Ciszak E et al., 1995, Structure 3:615-622). The solution state behavior of LysPro, reported here, has been interpreted with respect to the crystal structure results. In addition, the phenolic ligand binding differences between LysPro and insulin have been compared using isothermal titrating calorimetry and visible absorption spectroscopy of cobalt-containing hexamers. These studies establish that rapid-acting insulin analogues of this type can be stabilized in solution via the formation of hexamer complexes with altered dissociation properties.

Composite membrane deformation on the mesoscopic length scale.

The physics of soft materials can be investigated using nuclear spin-lattice relaxation, which depends on the spectral densities of motion in the MHz range. For the first time, NMR relaxation has been used to study influences of the acyl length, polar head groups, a cosurfactant, and cholesterol on the viscoelastic properties of membrane lipids. The results imply the concept of elastic deformation is relevant on lengths approximately equal to the bilayer thickness and less, involving a broad spectrum of collective modes which contribute to the forces between lipid bilayers.

Molecular areas of phospholipids as determined by 2H NMR spectroscopy. Comparison of phosphatidylethanolamines and phosphatidylcholines.

The role of lipid diversity in biomembranes is one of the major unsolved problems in biochemistry. One parameter of possible importance is the mean cross-sectional area occupied per lipid molecule, which may be related to formation of nonbilayer structures and membrane protein function. We have used 2H NMR spectroscopy to compare the properties of 1,2-diperdeuteriopalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-d62) and 1,2-diperdeuteriopalmitoyl-sn-glycero-3-phosphocholine (DPPC-d62) in the L alpha phase. We find that DPPE has greater segmental order than DPPC, and that this increase in order is related to the smaller area per acyl chain found for DPPE. Values of the mean cross-sectional chain area are calculated using a simple diamond lattice model for the acyl chain configurational statistics, together with dilatometry data. The results obtained for the mean area per molecule are comparable with those from low angle x-ray diffraction studies.

Area per lipid and acyl length distributions in fluid phosphatidylcholines determined by (2)H NMR spectroscopy.

Deuterium ((2)H) NMR spectroscopy provides detailed information regarding the structural fluctuations of lipid bilayers, including both the equilibrium properties and dynamics. Experimental (2)H NMR measurements for the homologous series of 1, 2-diacyl-sn-glycero-3-phosphocholines with perdeuterated saturated chains (from C12:0 to C18:0) have been performed on randomly oriented, fully hydrated multilamellar samples. For each lipid, the C-D bond order parameters have been calculated from de-Paked (2)H NMR spectra as a function of temperature. The experimental order parameters were analyzed using a mean-torque potential model for the acyl chain segment distributions, and comparison was made with the conventional diamond lattice approach. Statistical mechanical principles were used to relate the measured order parameters to the lipid bilayer structural parameters: the hydrocarbon thickness and the mean interfacial area per lipid. At fixed temperature, the area decreases with increasing acyl length, indicating increased van der Waals attraction for longer lipid chains. However, the main effect of increasing the acyl chain length is on the hydrocarbon thickness rather than on the area per lipid. Expansion coefficients of the structural parameters are reported and interpreted using an empirical free energy function that describes the force balance in fluid bilayers. At the same absolute temperature, the phosphatidylcholine (PC) series exhibits a universal chain packing profile that differs from that of phosphatidylethanolamines (PE). Hence, the lateral packing of phospholipids is more sensitive to the headgroup methylation than to the acyl chain length. A fit to the area per lipid for the PC series using the empirical free energy function shows that the PE area represents a limiting value for the packing of fluid acyl chains.

Hierarchical modeling of phenolic ligand binding to 2Zn--insulin hexamers.

Phenolic ligands, e.g., phenol and m-cresol, bind to 2Zn(II)-insulin hexamers and induce a conformational change at the N-terminus of the B-chain for each monomer. The binding of these phenolic ligands to 2Zn(II)-insulin hexamers has been studied by isothermal titrating calorimetry (ITC). The binding isotherms were modeled and thermodynamic parameters were quantified using a novel, flexible algorithm that permitted the development of a hierarchical series of physical models. With the insulin hexamer represented as a dimer of trimers, the modeling demonstrated that ligand binding is highly cooperative in nature, both intra- and inter-trimer. The isotropic inter-trimer cooperativity was dominant and negative in every system studied, with initial binding constants typically an order of magnitude greater for the binding of ligands to the first trimer relative to the second. The inter-trimer cooperatively estimated from the modeling of solution calorimetry data is consistent with a T6 <--> T3R3 <--> R6 equilibrium first proposed from crystallographic investigations. Intra-trimer cooperatively was present only in the enthalpy coefficient space, not in the equilibrium coefficient space, and therefore, less of a factor. The order of binding affinity for the ligands studied in resorcinol >> phenol > or = m-cresol as determined from their overall free energies of binding to the 2Zn(II)-insulin hexamer (-26.6, -23.4, and -23.4 kcal/mol, respectively) and their intrinsic binding constants (8780, 5040, and 3370 L/mol, respectively) at 14 degrees C. The temperature dependence of phenol binding to 2Zn(II)-insulin hexamer was modeled. Increasing temperature decreased the magnitude of both the intrinsic binding constant and the inter-trimer was cooperatively. The second phase of the ITC binding profile was also found to be highly temperature dependent. At lower temperatures the second phase is endothermic but gradually decreases with increasing temperature and subsequently becomes exothermic. This effect is attributed to loss of water from the hydration shell of the insulin hexamer with increasing temperature and consequently reduces the entropic contributions to the T <--> R transition in the phenol/2Zn(II)-insulin hexamer system.

Conformational stability, folding, and ligand-binding affinity of single-chain Fv immunoglobulin fragments expressed in Escherichia coli.

A fluorescein-binding single-chain Fv (scFv) was chosen as a model for the study of the physicochemical parameters associated with synthetic IgG fragments. Three such scFv proteins were designed from the primary sequences of one anti-fluorescyl monoclonal antibody (Mab 4.4.20). These were constructed with varying-length interdomain peptide linkers of between 12 and 25 residues, expressed in Escherichia coli, and the protein folding, stability, and antigen-binding characteristics were assessed. Efficient renaturation could be accomplished in vitro to yield approximately 26 mg of active scFv/L of fermentation. Scatchard analysis for fluorescein ligand binding revealed that the scFv designs come within 2-fold of the Ka = 1.99 (+/- 0.18) x 10(9) observed for the parental 4.4.20 Fab and have identical stoichiometries (n approximately 0.99). Reversible solvent denaturation studies demonstrated that the unfolding/refolding equilibria for the scFv proteins can be fit to a simple two-state model and that two of the scFv designs were found to be slightly more stable than single IgG domains (VL and CL) when assessed in terms of the free energy of unfolding, delta Gon-u, or nearly identical to other multiple domain immunoglobulin proteins such as light chains and Fab's when relative transition midpoints, Cm, are compared. Linkers which conferred conformational flexibility beyond the minimally required length of 12 residues were found to have a stabilizing effect. By these criteria of ligand-binding function and protein stability, the scFv proteins were found to be bona fide minimal replicas of their parental IgG molecules.

Construction and characterization of a single-chain catalytic antibody.

The antigen-binding (Fab) fragment of the catalytic monoclonal antibody NPN43C9 has recently been cloned by using bacteriophage lambda. By inserting the variable regions of this Fab coding sequence into a (NH2)-VL-linker-VH-(COOH) construct (where VL and VH represent the heavy and light chain variable regions), we have assembled a recombinant gene encoding a catalytic single-chain antigen-binding protein. This protein has been expressed in Escherichia coli and exhibits the same catalytic parameters as the parent monoclonal antibody NPN43C9. Single-chain forms of catalytic antibodies may prove valuable for structural and site-directed mutagenesis studies as well as for large-scale applications of catalytic antibodies.

Production of engineered IgM-binding single-chain antibodies in Escherichia coli.

Two single-chain antibodies were engineered and tested as novel binding proteins with specificity for immunoglobulin M. Genes for the two single-chain Fv proteins were assembled from the variable light chain cDNA and variable heavy chain cDNA of monoclonal antibodies DA4.4 and Bet 2, which specifically bind human IgM and mouse IgM, respectively. Both single-chain Fv proteins were designed with a 14-amino acid linker which bridged the variable light chain and variable heavy chain domains. The two proteins were expressed in Escherichia coli, purified and assayed for IgM-binding activity. Both proteins demonstrate a binding specificity for their corresponding IgM which is similar to the monoclonal antibodies from which they were derived. These small IgM-binding proteins may have applications in the investigation of the immune response and in the detection and purification of monoclonal antibodies, cell-associated antibodies, and IgM from serum.

Reversible adsorption of soluble hexameric insulin onto the surface of insulin crystals cocrystallized with protamine: an electrostatic interaction.

Mixing pharmaceutical preparations of soluble neutral regular insulin solution (NRI) and neutral protamine Hagedorn (NPH) crystalline insulin suspension leads to a reduction in the measurable amount of soluble insulin in the formulation supernatant. However in spite of the loss in soluble insulin, the time-actions of these components have been shown, in clinical trials, to be unaffected. The interaction between these different physical forms of insulin has been studied using reversed-phase HPLC, isothermal titrating calorimetry, and Doppler electrophoretic light scattering analysis. Sorbent surface and solution perturbation studies revealed that the NRI adsorbs to the surface of the NPH crystal with an equilibrium constant ranging from 10(4) M-1 to 10(7) M-1, depending on the protamine concentration, pH, ionic strength, and temperature. This adsorption behavior suggests that the binding is mediated by electrostatic interactions arising between the positively-charged NPH crystal and the negatively-charged NRI hexamer. Doppler electrophoretic light scattering results, used to probe the pH-dependent surface charge of NPH and soluble insulin hexamer, support the conclusion that electrostatic interactions mediate the adsorption process. Adsorption studies under physiological conditions indicate that the elevated temperature and ionic strength, in a subcutaneous depot, are sufficient to lead to the dissociation of the NRI/NPH complex that exists in these NPH mixture formulations.

Lipid bilayer dynamics and rhodopsin-lipid interactions: new approach using high-resolution solid-state 13C NMR.

High-resolution, solid-state 13C NMR spectra have been obtained for unsonicated multilamellar dispersions of 1,2-dilauryl-sn-glycero-3-phosphocholine (DLPC), recombinant membranes containing DLPC and rhodopsin, and native retinal rod disk membranes. The roles of 1H dipolar decoupling, 1H-13C cross-polarization, and magic-angle sample spinning have been investigated. Rotating-frame 13C relaxation times have been measured and are discussed in terms of lipid bilayer dynamics and rhodopsin-lipid interactions.

Large increases in general stability for subtilisin BPN' through incremental changes in the free energy of unfolding.

Six individual amino acid substitutions at separate positions in the tertiary structure of subtilisin BPN' (EC 3.4.21.14) were found to increase the stability of this enzyme, as judged by differential scanning calorimetry and decreased rates of thermal inactivation. These stabilizing changes, N218S, G169A, Y217K, M50F, Q206C, and N76D, were discovered through the use of five different investigative approaches: (1) random mutagenesis; (2) design of buried hydrophobic side groups; (3) design of electrostatic interactions at Ca2+ binding sites; (4) sequence homology consensus; and (5) serendipity. Individually, the six amino acid substitutions increase the delta G of unfolding between 0.3 and 1.3 kcal/mol at 58.5 degrees C. The combination of these six individual stabilizing mutations together into one subtilisin BPN' molecule was found to result in approximately independent and additive increases in the delta G of unfolding to give a net increase of 3.8 kcal/mol (58.5 degrees C). Thermodynamic stability was also shown to be related to resistance to irreversible inactivation, which included elevated temperatures (65 degrees C) or extreme alkalinity (pH 12.0). Under these denaturing conditions, the rate of inactivation of the combination variant is approximately 300 times slower than that of the wild-type subtilisin BPN'. A comparison of the 1.8-A-resolution crystal structures of mutant and wild-type enzymes revealed only independent and localized structural changes around the site of the amino acid side group substitutions.(ABSTRACT TRUNCATED AT 250 WORDS)