Anthony R. Rees

Antibody-antigen interactions

Abstract The structural origins of antibody diversity are well understood as a result of X-ray crystallographic and molecular modelling studies of Fab fragments. A similar understanding of antibody specificity is beginning to emerge from an analysis of structures of hapten, peptide and protein antibody complexes. While the nature of the antibody-antigen interface, and any conformational changes that occur on complex formation, can be described in structural terms, a full explanation of the thermodynamic and mechanistic basis of affinity is less accessible from structure alone. A number of physiochemical studies carried out on wild type and mutant antibodies have raised questions about the nature of the energetics of the interaction, and the possibility of a key role for water molecules has been discussed. The possibility of ‘induced fit’ as a common mechanism for antigen-antibody interactions has been raised, and a molecular basis for hapten and protein cross-reactivity proposed. These recent contributions to the field, as well as providing partial solutions to old problems, have provided exciting new insights.

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 streptavidins 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 molecules ability to elicit an immune response in rabbits.

Brain drug delivery technologies: novel approaches for transporting therapeutics

The blood-brain barrier (BBB) denies many therapeutic agents access to brain tumours and other diseases of the central nervous system (CNS). Despite remarkable advances in our understanding of the mechanisms involved in the development of the brain diseases and the actions of neuroactive agents, drug delivery to the brain remains a challenge. For more than 20 years, extensive efforts have been made to enhance delivery of therapeutic molecules across vascular barriers of the CNS. The current challenge is to develop drug-delivery strategies that will allow the passage of drug molecules through the BBB in a safe and effective manner, and this review will provide an insight into some of the strategies developed to enhance drug delivery across the BBB.

A strategy for mapping and neutralizing conformational immunogenic sites on protein therapeutics

Antibodies are highly specific recognition molecules which are increasingly being applied to target therapy in patients. One type of developmental antibody‐based therapy is antibody directed enzyme prodrug therapy (ADEPT) for the treatment of cancer. In ADEPT, an antibody specific to a tumor marker protein delivers a drug‐activating enzyme to the cancer. Subsequent intravenous administration of an inactive prodrug results in drug activation and cytotoxicity only within the locale of the tumor. Pilot clinical trials with chemical conjugates of the prodrug activating enzyme carboxypeptidase G2 (CPG2) chemically conjugated with an antibody to and carcinoembryonic antigen (CEA), have shown that CPG2‐mediated ADEPT is effective but limited by formation of human antibodies to CPG2 (HACA). We have developed a recombinant fusion protein (termed MFE‐CP) of CPG2 with an anti‐CEA single chain Fv antibody fragment and we have developed methods to address the immunogenicity of this therapeutic. A HACA‐reactive discontinuous epitope on MFE‐CP was identified using the crystal structure of CPG2, filamentous phage technology and surface enhanced laser desorption/ionization affinity mass spectrometry. This information was used to create a functional mutant of MFE‐CP with a significant reduction (range 19.2 to 62.5%, median 38.5%) in reactivity with the sera of 11 patients with post‐therapy HACA. The techniques described here are valuable tools for identifying and adapting undesirable immunogenic sites on protein therapeutics.

Molecular Modeling of Antibody-Combining Sites

Each of the six CDRs of Gloop2 is shown with the modeled structure in. Overall, the results obtained using the combined algorithm are similar in accuracy to those achieved using the canonical method of Chothia et al. However, the canonical method is limited to those loops where the key residues identified by Chothia are present. With the number of antibody structures currently available, it is not possible to classify CDR-H3 into canonical ensembles. Additionally, a small percentage of examples in the remaining CDRs do not match the current canonical classifications and the protein engineer may well wish to mutate the key residues, precluding the use of Chothias method for modeling the resulting conformation. Thus the best approach appears to be to use Chothias method (at least to model the backbone conformation) when the loop to be modeled is represented in the database of canonical structures. Any other loops, either unrepresented among the known canonicals (including CDR-H3), or where mutations have been made to the key residues, may then be modeled by the combined algorithm presented here.

Peptide Delivery to the Brain via Adsorptive-Mediated Endocytosis: Advances With SynB Vectors

Biological membranes normally restrict the passage of hydrophilic molecules. This impairs the use of a wide variety of drugs for biomedical applications. To overcome this problem, researchers have developed strategies that involve conjugating the molecule of interest to one of a number of peptide entities that are efficiently transported across the cell membranes. In the past decade, a number of different peptide families with the ability to cross the cell membranes have been identified. Certain of these families enter the cells by a receptor-independent mechanism, are short (10–27 amino acid residues), and can deliver successfully various cargoes across the cell membrane into the cytoplasm or nucleus. Surprisingly, some of these vectors, the SynB vectors, have also shown the ability to deliver hydrophilic molecules across the blood-brain barrier, one of the major obstacles to the development of drugs to combat diseases affecting the CNS.

Antibody modeling: Beyond homology

The immune system is capable of producing at least 109 different antibody specificities. There are currently about 800 variable-region sequences known, whereas there are only 16 deposited Fab structures. Thus, the rate of sequence acquisition far outstrips the rate of structure determination. While for many protein families the presence of 16 highly homologous structures would enable accurate modeling of new family members by homology, the hypervariability of combining site sequences and structures precludes this. To solve this problem we have developed an algorithm (CAMAL) that exploits the knowledge base of protein structures but adds to it the power of conformational search and the subtlety of energy screening. The accuracy that this algorithm is able to achieve leads to the possibility that, with further development, extensive use of X-ray crystallography for antibody structure determination will become unnecessary.

Induction of antigen-specific CTL responses using antigens conjugated to short peptide vectors.

Linear peptides (SynB vectors) with specific sequence motifs have been identified that are capable of enhancing the transport of a wide range of molecules into cells. These peptide vectors have been used to deliver exogenous peptides and protein Ags across the cell membrane and into the cytoplasm of cells. Specifically, in vitro analysis indicated that these SynB peptides enhanced the uptake of two 9-mer peptide Ags, NP147–155 and Mtb250–258 (T cell epitopes of influenza nucleoprotein and Mycobacterium tuberculosis, respectively) and the M. tuberculosis Ag Mtb8.4 protein, into K562 cells when covalently linked to the respective Ags. Furthermore, selected SynB vectors, when conjugated to these same Ags and used as immunogens, resulted in considerably enhanced Ag-specific CTL responses. Several SynB vectors were tested and resulted in varying levels of cellular uptake. The efficiency of uptake correlated with the ability of the SynB construct to deliver each epitope in vivo and induce specific CTL responses in mice. These data suggest that peptide vectors, such as SynB that transport target Ags across the cell membrane in a highly efficient manner, have significant potential for vaccine delivery.

Antibody-combining sites. Extending the natural limits.

The antibody repertoire is very large with at least 109 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.