Vector-mediated drug delivery to the brain.

As a consequence of the growing ageing population, many neurodegenerative diseases, cancer and infections of the brain will become more prevalent. Despite major advances in neuroscience, many potential therapeutic agents are denied access to the central nervous system (CNS) because of the existence of the blood-brain barrier (BBB). This barrier is formed by the endothelial cells of the brain capillaries and its primary characteristic is the impermeability of the capillary wall due to the presence of complex tight junctions and a low endocytic activity. The BBB behaves as a continuous lipid bilayer and prevents the passage of polar and lipid-insoluble substances. The BBB is, therefore, the major obstacle to drugs that are potentially useful for combating diseases affecting the CNS. Extensive efforts have been made to develop CNS drug delivery strategies in order to enhance delivery of therapeutic molecules across the BBB. The current challenge is to develop drug-delivery strategies that will allow the passage of therapeutic drugs through the BBB in a safe and effective manner. This review focuses specifically on the strategies developed to enhance drug delivery across the BBB with an emphasis on the vector-mediated strategy.

The hunchback and its neighbours: proline as an environmental modulator.

The unique ability of Pro or Pro-rich repeats to affect the stability and function of proteins has recently been highlighted by biophysical studies on fragments from prions, signalling domains and muscle proteins. Pro-rich regions have been observed to either occupy disordered states or adopt various helical structures; some are also able to undergo an environmental-dependent transformation between these states. Such a transformation could explain some of the inherent functional properties of the parent proteins and, additionally, can be efficiently exploited to generate novel temperature- and pH-switches in more conventional globular proteins.

Reduced antibody response to streptavidin through site-directed mutagenesis.

Streptavidin provides an effective receptor for biotinylated tumoricidal molecules, including radionuclides, when conjugated to an antitumor antibody and administered systemically. Ideally, one would like to administer this bacterial protein to patients repeatedly, so as to maximize the antitumor effect without eliciting an immune response. Therefore, we attempted to reduce the antigenicity of streptavidin by mutating surface residues capable of forming high energy ionic or hydrophobic interactions. A crystallographic image of streptavidin was examined to identify residues with solvent-exposed side chains and residues critical to streptavidin's structure or function, and to define loops. Mutations were incorporated cumulatively into the protein sequence. Mutants were screened for tetramer formation, biotin dissociation, and reduced immunoreactivity with pooled patient sera. Patient antisera recognized one minor continuous epitope with binding locus at residue E101 and one major discontinuous epitope involving amino acid residues E51 and Y83. Mutation of residues E51, Y83, R53, and E116 reduced reactivity with patient sera to <10% that of streptavidin, but these mutations were no less antigenic in rabbits. Mutant 37, with 10 amino acid substitutions, was only 20% as antigenic as streptavidin. Rabbits immunized with either streptavidin or mutant 37 failed to recognize the alternative antigen. Biotin dissociated from mutant 37 four to five times faster than from streptavidin. Residues were identified with previously undescribed impact on biotin binding and protein folding. Thus, substitution of charged, aromatic, or large hydrophobic residues on the surface of streptavidin with smaller neutral residues reduced the molecule's ability to elicit an immune response in rabbits.

Sodium sulphate reactivates a protein A minidomain with a short elastin beta-turn.

Elastin polymer sequences derived from muscle exhibit temperature and salt-induced reversible contractions and expansions. A folded and contracted beta-turn helical elastin structure is stabilised by increased intramolecular hydrophobic interactions. We have generated a switchable binding protein by inserting a typical elastin turn sequence, GVPGVG, between the two IgG-binding helices of an engineered globular minidomain from Protein A. This mutant showed increased binding of IgG-molecules compared to the wild-type sequence in the presence of sodium sulphate, as measured by surface plasmon resonance spectroscopy. Analysis by circular dichroism revealed a salt-induced folding of the mutant minidomain to a native type I beta-turn, likely stabilised as a result of the two interacting dehydrated valines across the beta-turn. Since sodium sulphate can be successfully used to regulate the folding/unfolding or binding/dissociation of this minidomain, this suggests an alternative protein purification method.

Recombinant antibodies as carrier proteins for sub-unit vaccines: influence of mode of fusion on protein production and T-cell activation.

A major objective in development of vaccines is the design of sub-unit vaccines with the ability to induce strong T-cell responses. For this purpose, T-cell epitopes have been genetically inserted into various carrier proteins. Ig molecules may be especially useful as vehicles for delivery of CD4(+) T-cell epitopes to antigen presenting cells (APC). We have previously replaced loop structures between beta-strands in the C(H)1 domain of human IgG3 with a defined 11 amino acids long, MHC class II-restricted T-cell epitope. In this report we have added the same T-cell epitope into loops in the C(H)1 domain of mouse IgG2b. The following major points can be made: (1) Loops can accommodate an elongation of at least 11 amino acids without disruption of the overall Ig structure and secretion. (2) The recombinant Ig molecules are processed by spleen APC and the epitopes that are released are presented to T-cells. (3) Site of integration influences efficiency of processing and presentation. (4) Elongation of two neighbouring loops reduces Ig secretion. Taken together, our present results indicate that IgG C(H)1 domains may be engineered to carry T-cell epitopes in loop structures between beta-strands, but not all loops may be equally suitable for this purpose.

Guided selection of a pan carcinoma specific antibody reveals similar binding characteristics yet structural divergence between the original murine antibody and its human equivalent.

Antibody engineering provides an excellent tool for the generation of human immunotherapeutics for the targeted treatment of solid tumours. We have engineered and selected a completely human antibody to epithelial glycoprotein-2 (EGP-2), a transmembrane glycoprotein present on virtually all human simple epithelia and abundantly expressed on a variety of human carcinomas. We chose to use the procedure of "guided selection" to rebuild a high-affinity murine antibody into a human antibody, using two consecutive rounds of variable domain shuffling and phage library selection. As a starting antibody, the murine antibody MOC-31 was used. After the first round of guided selection, where the V(H) of MOC-31 was combined in Fab format with a human V(L)C(L) library, a small panel of human light chains was identified, originating from a segment of the VkappaIII family, whereas the MOC-31 V(L) is more homologous to the VkappaII family. Nevertheless, one of the chimaeric Fabs, C3, displayed an off-rate similar to MOC-31 scFv. Combining the V(L) of C3 with a human V(H) library, while retaining the V(H) CDR3 of MOC-31, clones were selected using human V(H) genes originating from the rarely used V(H)7 family. The best clone, 9E, shows over 13 amino acid mutations from the germline sequence, has an off-rate comparable to the original antibody and specifically binds to the "MOC-31"-epitope on EGP-2 in specificity and competition ELISA, FACS analysis and immunohistochemistry. In both V(L) and V(H) of antibody 9E, three germline mutations were found creating the MOC-31 homologue residue. Structural modelling of both murine and human antibodies reveals that one of the germline mutations, 53Y in V(H) CDR2, is likely to be involved in antigen binding. We conclude that, although they may bind the same epitope and have similar binding affinity to the antigen as the original murine antibody, human antibodies derived by guided selection unlike CDR-grafted antibodies, may retain only some of the original key elements of the binding site chemistry. The selected human anti-EGP-2 antibody will be a suitable reagent for tumour targeting.

Trifluoroethanol may form a solvent matrix for assisted hydrophobic interactions between peptide side chains.

Several models for interactions between trifluoroethanol (TFE) and peptides and proteins have recently been proposed, but none have been able to rationalize the puzzling observations that on the one hand TFE can stabilize some hydrophobic interactions in secondary structures, but on the other can also melt the hydrophobic cores of globular proteins. The former is illustrated in this paper by the effect of TFE on a short elastin peptide, GVG(VPGVG)(3), which forms type II beta-turns stabilized by hydrophobic interactions between two intra-turn valine side chains. This folding, driven by increasing the entropy of bulk water, is stimulated in TFE-water mixtures and/or by raising the temperature. To explain these apparently contradictory observations, we propose a model in which TFE clusters locally assist the folding of secondary structures by first breaking down interfacial water molecules on the peptide and then providing a solvent matrix for further side chain--side chain interactions. This model also provides an explanation for TFE-induced transitions between secondary structures, in which the TFE clusters may redirect non-local to local interactions.

WAM: an improved algorithm for modelling antibodies on the WEB.

An improved antibody modelling algorithm has been developed which incorporates significant improvements to the earlier versions developed by Martin et al. (1989, 1991), Pedersen et al. (1992) and Rees et al. (1996) and known as AbM (Oxford Molecular). The new algorithm, WAM (for Web Antibody Modelling), has been launched as an online modelling service and is located at URL http://antibody.bath.ac.uk. Here we provide a summary only of the important features of WAM. Readers interested in further details are directed to the website, which gives extensive background information on the methods employed. A brief description of the rationale behind some of the newer methodology (specifically, the knowledge-based screens) is also given.

An engineered minidomain containing an elastin turn exhibits a reversible temperature-induced IgG binding.

A two-helix version of the triple alpha-helical staphylococcal Protein A, previously shown to retain the Fc binding properties of protein A, has been engineered to contain an elastin sequence, GVPGVG, within the inter-helix turn. The original type I beta-turn was replaced with a beta-turn from the muscle protein elastin, which has an inverse temperature-induced folding transition. These "elastin mutants" had lost their helical structure, as measured by circular dichroism (CD), and exhibited a lower stability than the wild-type domains (T(m) reduced by about 48 degrees C) in 30% trifluoroethanol. For the wild-type domains, the amount of alpha-helix and the binding affinity for Fc decreased as the temperature was increased. In contrast, although the starting affinity was lower for the disulfide elastin-turn mutant, it exhibited a 21-fold improvement in affinity over the same temperature range. The melting curve for the elastin-turn minidomain showed cooperative behavior, as measured by the increase in CD-amplitude at 222 nm. The observed CD behavior is consistent with the formation of a type I beta-turn, exhibiting similar DeltaH and DeltaS values to those seen previously for short elastin peptides [Reiersen, H., Clarke, A. R., and Rees, A. R. (1998) J. Mol. Biol. 283, 255-264], and accounting for the increase in on-rate. This demonstrates that, when inserted into a stable globular protein, short elastin sequences have the ability to modify local structure and activity, by operating as temperature modulated switches.

Molecular modeling and preclinical evaluation of the humanized NR-LU-13 antibody.

A mouse-human chimeric monoclonal antibody (chNR-LU-13), specific for the EGP40 pancarcinoma antigen, was humanized through three-dimensional molecular modeling. Humanization of the chNR-LU-13 antibody is expected to enhance its use for patients undergoing immunotherapy. On the basis of the observed amino acid sequence identity, chNR-LU-13 complementary determining regions (CDRs) of the V(L) and V(H) regions were grafted onto the human anti-DNA-associated idiotype immunoglobulin clone, R3.5H5G'CL. Ten amino acids residues within the humanized framework were back-mutated to their corresponding chNR-LU-13 sequence, because they were predicted to disrupt the canonical classification of the CDRs or were within 5 A of a CDR. Synthesis of the V(L) and V(H) regions was accomplished by recursive PCR, and the dual-chain expression vector p451.C4 was positioned under control of the CMV(P+E). We observed by competitive ELISA that the recombinant humanized NR-LU-13 (huNR-LU-13) IgG1 antibody exhibited an indistinguishable immunoreactivity profile when compared with the murine monoclonal antibody (muNR-LU-10). The huNR-LU-13 antibody was effective in mediating both antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity when assayed against either the breast carcinoma cell line, MCF-7, or the colon adenocarcinoma cell line, SW1222. Biodistribution studies using i.v. coinjected 131I-muNR-LU-10 and 125I-huNR-LU-13 confirmed that the huNR-LU-13 specifically targets to the tumor in athymic BALB/c mice bearing the SW1222 human tumor xenograft. Humanization of the chNR-LU-13 antibody is expected to eliminate an undesired human antimouse antibody response, allowing for repeated i.v. administration into humans.

Short elastin-like peptides exhibit the same temperature-induced structural transitions as elastin polymers: implications for protein engineering.

Elastin is a major protein component of the vascular wall and is responsible for its unusual elastic properties. Polymers of its repeating VPGVG sequences have been synthesised and shown to exhibit an inverse temperature transition where, as temperature rises, the polymer collapses from an extended chain to a beta-spiral structure with three VPGVG units per turn, each pentamer adopting a type II beta-turn conformation. These studies, however, have not established whether the temperature-driven conformational change is an intrinsic property of the individual pentameric sequences or a global, co-operative effect of many pentamers within the beta-spiral structure. Here, we examine by circular dichroism the behaviour of elastin-like peptides (VPGVG)n, where n varies between 1 and 5. Remarkably, we find that all lengths of peptide undergo an extended left and right arrow beta-turn transition with increasing temperature, suggesting that the induction of the beta-spiral occurs at the level of single pentameric units. The origin of this effect is a positive DeltaS term for the transition. At 35 degreesC, the average transition midpoint temperature, the value of TDeltaS is about 15 kcal mol-1. With larger oligomers (n=3), there is only a modest rise in DeltaS, suggesting that the dominant entropic effect resides within the monomer and that interactions between these units make only a small contribution to the energetics of the transition. Charges at the termini, and residue replacements or additions, regulate the transitions for the short peptides in a manner similar to that observed for the longer polymers. The behaviour of the same peptides in trifluoroethanol and SDS solutions is consistent with formation of the beta-turn being driven by interactions between non-polar groups. The significance of this behaviour for the rational design of temperature-induced responses in proteins is discussed.

Inhibition of activating transcription factor 1- and cAMP-responsive element-binding protein-activated transcription by an intracellular single chain Fv fragment.

Activating transcription factor 1 (ATF1) and cAMP-responsive element (CRE)-binding protein (CREB) activate transcription through CREs located in the promoters of cellular and viral genes. We previously described a monoclonal antibody (mAb41.4) that prevents ATF1 binding to DNA and reduces CRE-driven promoter activity in vitro (Orten, D. J., Strawhecker, J. M., Sanderson, S. D., Huang, D., Prytowsky, M. B. , and Hinrichs, S. H. (1994) J. Biol. Chem. 269, 32254-32263). A single chain Fv (scFv) fragment from the mAb41.4-expressing hybridoma was generated to provide a means to investigate transcription factor function via intracellular expression of the scFv fragment. The affinity of scFv4 (subgroup: VL kappa-III, VH miscellaneous) for ATF1 was similar to that of the parental mAb and the Fab fragment, but it demonstrated greater inhibitory activity and reacted with CREB. scFv4 disrupted the binding of both ATF1 and CREB in electrophoretic mobility shift assays and reduced expression of CRE-driven expression in vitro. Transient expression of scFv had no effect on the non-CRE-containing adenovirus major late promoter. The proliferating cell nuclear antigen promoter, containing two CREs, was significantly more sensitive to inhibition by scFv than the cytomegalovirus immediate-early promoter, containing five CREs. Cotransfection of either ATF1 or CREB in the presence of scFv restored basal levels of expression. The intracellular expression of scFv provides a unique means to investigate the roles of the transcription factors ATF1 and CREB.

A comparison of two murine monoclonal antibodies humanized by CDR-grafting and variable domain resurfacing.

The variable domain resurfacing and CDR-grafting approaches to antibody humanization were compared directly on the two murine monoclonal antibodies N901 (anti-CD56) and anti-B4 (anti-CD19). Resurfacing replaces the set of surface residues of a rodent variable region with a human set of surface residues. The method of CDR-grafting conceptually consists of transferring the CDRs from a rodent antibody onto the Fv framework of a human antibody. Computer-aided molecular modeling was used to design the initial CDR-grafted and resurfaced versions of these two antibodies. The initial versions of resurfaced N901 and resurfaced anti-B4 maintained the full binding affinity of the original murine parent antibodies and further refinements to these versions described herein generated five new resurfaced antibodies that contain fewer murine residues at surface positions, four of which also have the full parental binding affinity. A mutational study of three surface positions within 5 A of the CDRs of resurfaced anti-B4 revealed a remarkable ability of the resurfaced antibodies to maintain binding affinity despite dramatic changes of charges near their antigen recognition surfaces, suggesting that the resurfacing approach can be used with a high degree of confidence to design humanized antibodies that maintain the full parental binding affinity. By comparison CDR-grafted anti-B4 antibodies with parental affinity were produced only after seventeen versions were attempted using two different strategies for selecting the human acceptor frameworks. For both the CDR-grafted anti-B4 and N901 antibodies, full restoration of antigen binding affinity was achieved when the most identical human acceptor frameworks were selected. The CDR-grafted anti-B4 antibodies that maintained high affinity binding for CD19 had more murine residues at surface positions than any of the three versions of the resurfaced anti-B4 antibody. This observation suggests that the resurfacing approach can be used to produce humanized antibodies with reduced antigenic potential relative to their corresponding CDR-grafted versions.

Site-directed mutagenesis of proteolytic antibody light chain.

Amino acid residues in a proteolytic antibody light chain selected by molecular modeling were substituted with Ala by site-directed mutagenesis. Hydrolysis of vasoactive intestinal polypeptide (VIP), the immunogen employed to elicit the antibody light chain, was reduced by > 95% by replacement of Ser27a or His93 by Ala residues. Similar reductions in the activity were observed using synthetic protease substrates containing Arg-methylcoumarinamide (MCA) and Lys-MCA bonds. Turnover of the Ser27a and His93 mutants was lower than that of wild-type protein by about two orders of magnitude. The activity of the wild-type protein was inhibited selectively by diisopropylfluorophosphate (DFP), a serine protease inhibitor, but the residual activity of the Ser26 mutant was refractory to DFP. The affinity of the wild-type light chain for the substrate ground state was nearly unaffected by mutations at Ser27a and His93. In contrast, a Ser26 single mutant and a His27d/Asp28 double mutant displayed increased Km (by about tenfold) and increased turnover (by about tenfold) using VIP as substrate. The kinetic constants for these mutants and the wild type protein were essentially identical with Boc-Glu-Ala-Arg as substrate. Thus, two types of residues participating in catalysis by the light chain have been identified. Ser27a and His93 are essential for catalysis but not for initial high affinity complexation and substrate. Ser26 and His27d or Asp28 participate in VIP binding and limit turnover indirectly.

Mapping T cell recognition: the identification of a T cell receptor residue critical to the specific interaction with an influenza hemagglutinin peptide.

The fine specificity of T cell receptor (TCR) interaction with the influenza hemagglutinin peptide HA 307-319 in the context of the DR1 (DRA, DRB1 0101) and DR4 (DRA, DRB1 0404) was studied in two human T cell clones (HA1.7 and Cl-1) derived from different individuals. Sequencing of amplified TCR transcripts revealed that these two clones express highly related TCR alpha chains, with a conserved junctional motif, but very different TCR beta chains. Modeling studies led to the prediction that the conserved glutamic acid residue in the TCR alpha chain could interact with the lysine at position 316 in the peptide, a known TCR contact residue. HA1.7 TCR-CD3 zeta chimeric constructs were expressed in the rat basophil line (RBL) and shown to confer specific antigen recognition. In two TCR alpha chain mutants, with the conserved glutamic acid residue altered to alanine and lysine, respectively, peptide recognition was lost. Specific recognition was not rescued by altered peptide ligands. Furthermore, Jurkat derivatives expressing the related Jurkat TCR alpha chain paired with the HA 1.7 TCR beta chain did not recognize the HA 307-319/DR1 complex. These data provide evidence for the critical interaction of a TCR residue with antigenic peptide.

Physicochemical characterization of the cytoplasmic domain of the epidermal growth factor receptor and evidence for conformational changes associated with its activation by ammonium sulphate.

The physiochemical properties of the purified cytoplasmic domain of the epidermal growth factor (EGF) receptor, its self-phosphorylation and peptide phosphorylation activities, and its activation by ammonium sulphate have been studied. Highly efficient purification procedures for the isolation of the recombinant cytoplasmic domain (Met644-Ala1186) of the EGF receptor, expressed in the baculovirus/insect cell system, are described. Physicochemical characterization of the protein included investigation of its isoelectric and hydrodynamic properties, stability, oligomeric status, and secondary structure using far-u.v. circular dichroism. The recombinant protein was not recognized by anti-phosphotyrosine antibodies, unless first self-phosphorylated in vitro. Tryptic phosphopeptide maps of self-phosphorylated recombinant cytoplasmic domain and the EGF-stimulated A431-membrane receptor were very similar, suggesting that the recombinant had similar self-phosphorylation capacity and specificity. The preparations were characterized by high specific activity towards peptide tyrosine phosphorylation. Although the cytoplasmic domain was isolated as a homogeneously monomeric protein, storage at 4 degrees C led to slow, spontaneous aggregation with reduction in specific activity. Both high activity and monomeric state were maintained by storage below 0 degree C. The dependence of the initial rate of self-phosphorylation on protein concentration was consistent with cross-phosphorylation but not with the known oligomerization-induced activation of holoreceptor. The peptide phosphorylation activity was stimulated by Mn2+, Mg2+ and (NH4)2SO4 at high concentrations. The substrate specificity of (NH4)2SO4 activation was studied using synthetic peptides. Self-phosphorylation was inhibited by (NH4)2SO4 in the range 0-0.25 M but activated at 1.0-1.5 M, possibly as a result of ionic and hydrophobic protein interactions respectively. Phosphopeptide maps of cytoplasmic domain phosphorylated in the presence of high (NH4)2SO4 showed that the protein was more extensively phosphorylated than in the absence of salt, or than the native receptor. Far-u.v. circular-dichroism spectra of the cytoplasmic domain changed dramatically at 1 M (NH4)2SO4, raising the possibility that (NH4)2SO4 activates the kinase catalytic domain by inducing conformational changes.

Antibody design: beyond the natural limits.

Dissection of antibody-antigen interactions requires a knowledge of antibody structure, the ability to model accurately the conformation of antibody-combining sites, and an understanding of the energetic factors governing the interactions. When this understanding has reached the point where the molecular shape and chemical character of a combining site necessary to define a particular specificity and binding requirement can be designed, the antibody repertoire will have been extended 'beyond the natural limits'.

Antibody-combining sites. Extending the natural limits.

The antibody repertoire is very large with at least 10(9) different antibody specificities, yet there are currently only 800 variable-region sequences known and < 23 Fab structures deposited with the Brookhaven Protein Data Bank. To engineer the antibody-combining site rationally, we need to define the rules that govern antibody structure. To understand the process of antibody-antigen recognition, we need not only to predict complementary determining regions accurately, but to simulate accurately the interaction of antibody with antigen. We have made progress in the modeling of antibody-combining sites and in the simulation of antibody complex formation. The combination of these approaches will allow us to extend the natural limits of antibody-combining sites in a more rational manner.

Humanization of murine monoclonal antibodies through variable domain resurfacing.

Two murine monoclonal antibodies, N901 (anti-CD56) and anti-B4 (anti-CD19), were humanized by a process we call "resurfacing." A systematic analysis of known antibody structures has been used to determine the relative solvent accessibility distributions of amino acid residues in murine and human antibody variable (Fv) regions and has shown that the sequence alignment positions of surface amino acids for human and murine variable region heavy (VH) and light (VL) chains are conserved with 98% fidelity across species. While the amino acid usage at these surface positions creates surface residue patterns that are conserved within species, there are no identical patterns across species. However, surprisingly few amino acid changes need to be made to convert a murine Fv surface pattern to that characteristic of a human surface. Resurfacing was used to change the patterns of surface accessible residues in the Fv regions of the N901 and anti-B4 antibodies to resemble those found on the Fv regions of human antibody sequences. Two different procedures for selecting a human sequence were compared. For anti-B4, a data base of clonally derived human VL-VH sequence pairs was used, while for N901, sequences for VL and VH were independently selected from the Kabat et al. data base [Kabat, E. A., Wu, T. T., Reid-Miller, M., Perry, H. M. & Gottesman, K. S. (1991) Sequences of Proteins of Immunological Interest (DHHS, Washington, DC), 5th Ed.]. Resurfaced N901 and anti-B4 antibodies had apparent affinities for their cell surface ligands that were identical to those of their respective parent murine antibodies. These data provide evidence that, despite the differences in the surfaces of mouse and human Fv regions, it is possible to substitute one for the other while retaining full antigen binding affinity.