Ligand binding to a humanized anti-cocaine mAb detected by non-reducing SDS-PAGE

Monoclonal antibodies are very useful tools in experimental biology, as well as being valuable and effective therapeutic drugs. They can be targeted against proteins with varied functions, or against small molecules of interest to both researchers and clinicians, such as drugs of abuse, including cocaine. Since there is no currently FDA approved pharmacological treatment for cocaine abuse, our laboratory has developed an anti-cocaine mAb for the treatment of cocaine use disorders. This humanized anti-cocaine antibody, named h2E2, has been thoroughly characterized both functionally and structurally, in preparation for the start of clinical development. We previously showed that this mAb could be characterized by sequential thermal unfolding of antibody domains using non-reducing SDS-PAGE. We also demonstrated that ligand-induced protein stabilization can be used to quantitatively measure cocaine and cocaine metabolite binding to the h2E2 mAb, utilizing differential scanning fluorimetry. Here, we demonstrate the utility of non-reducing SDS-PAGE for the qualitative assessment of binding of cocaine and some of its metabolites, both to the intact mAb, as well as to fragments containing the antigen binding site (Fab and F(ab’)₂ fragments). These results clearly show a ligand concentration dependence of the stabilization of the cocaine binding domain in non-reducing SDS-PAGE, as well as visually differentiating the relative binding affinities of various cocaine metabolites. Thus, non-reducing SDS-PAGE is a simple and widely available technique that is useful as a measure of binding of cocaine and its metabolites to the h2E2 mAb, and it is likely that this technique will also be applicable to other small molecule-directed mAbs.     

Oxidation of specific tryptophan residues inhibits high-affinity binding of cocaine and its metabolites to a humanized anticocaine mAb

Cocaine addiction remains a serious problem lacking an effective pharmacological treatment. Thus, we have developed a high-affinity anti-cocaine monoclonal antibody (mAb), h2E2, for the treatment of cocaine use disorders. We show that selective tryptophan (Trp) oxidation by 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) resulted in a loss of high-affinity binding of cocaine to this mAb. The newly developed use of excess methionine (Met) to protect mAb met residues from AAPH oxidation did not substantially attenuate the effects of oxidation on cocaine binding but greatly decreased the modification of met residues in the mAb. Similar large decreases in ligand affinity (5000–10,000-fold) upon oxidation were observed using cocaine and two cocaine metabolites, cocaethylene and benzoylecgonine, which also bind with nanomolar affinity to this h2E2 mAb. The decrease in binding affinity was accompanied by a decrease of approximately 50% in Trp fluorescence, and increases in mAb 310 to 370 nm absorbance were consistent with the presence of oxidized forms of Trp. Finally, mass spectral analysis of peptides derived from control and AAPH-oxidized mAb indicated that excess free met did effectively protect mAb met residues from oxidation, and that AAPH-oxidized mAb heavy-chain Trp33 and light-chain Trp91 residues are important for cocaine binding, consistent with a recently derived h2E2 Fab fragment crystal structure containing bound benzoylecgonine. Thus, protection of the anti-cocaine h2E2 mAb from Trp oxidation prior to its clinical administration is critical for its proposed therapeutic use in the treatment of cocaine use disorders.     

A human recombinant monoclonal antibody to cocaine: Preparation,characterization and behavioral studies

Cocaine abuse remains prevalent worldwide and continues to be a major health concern; nonetheless, there is no effective therapy. Immunopharmacotherapy has emerged as a promising treatment strategy by which anti-cocaine antibodies bind to the drug blunting its effects. Previous passive immunization studies using our human monoclonal antibody, GNCgzk, resulted in protection against cocaine overdose and acute toxicity. To further realize the clinical potential of this antibody, a recombinant IgG form of the antibody has been produced in mammalian cells. This antibody displayed a high binding affinity for cocaine (low nanomolar) in line with the superior attributes of the GNCgzk antibody and reduced cocaine-induced ataxia in a cocaine overdose model.     

Crystal structure of a cocaine-binding antibody

Murine monoclonal antibody GNC92H2 was elicited by active immunization with a cocaine immunoconjugate and binds free cocaine with excellent specificity and moderate affinity. Improvement of affinity, as well as humanization of GNC92H2, would be advantageous in immunopharmacotherapy for cocaine addiction, and for emergency cases of drug overdose. Toward this end, the crystal structure of an engineered murine-human chimeric Fab of GNC92H2 complexed with cocaine was determined at 2.3 A resolution. Structural analysis reveals a binding pocket with high shape and charge complementarity to the cocaine framework, which explains the specificity for cocaine, as opposed to the pharmacologically inactive cocaine metabolites. Importantly, the structure provides a foundation for mutagenesis to enhance the binding affinity for cocaine and potent cocaine derivatives, such as cocaethylene, and for additional humanization of the antibody.     

Tyrosine nitration of a humanized anti-cocaine mAb differentially affects ligand binding of cocaine and its metabolites

Tetranitromethane was used to selectively modify tyrosine residues of a humanized anti-cocaine mAb (h2E2), under development for the treatment of cocaine use disorders. The effect of mild tyrosine nitration on the affinity of cocaine and two high affinity cocaine metabolites, cocaethylene and benzoylecgonine, was assessed using differential scanning fluorimetry to measure ligand affinities via ligand-induced thermal stabilization of the mAb antigen binding region. Nitrated tyrosine residues were identified by mass spectral analysis of thermolysin peptides. One objective was to understand the binding affinity differences observed for these three ligands, which are not explained by the published crystal structure of the h2E2 mAb Fab fragment co-crystalized with benzoylecgonine, since the carboxylic acid of benzoylecgonine that is esterified to form cocaine and cocaethylene is not in contact with the mAb. Importantly, the binding affinity of the cocaine metabolite benzoylecgonine was not decreased by mild nitration, whereas the binding affinities of cocaine and cocaethylene were decreased about two-fold. These ligands differ only in the substituent attached to the carboxylate moiety of the compound, with benzoylecgonine having an unesterified carboxylate, and cocaine and cocaethylene having methyl and ethyl esters, respectively, at this position. The results are consistent with nitration of light chain tyrosine residue 34, resulting in a less favorable interaction with cocaine and cocaethylene carboxylate esters, while not affecting binding of benzoylecgonine. Thus, light chain Tyr34 residue may have molecular interactions with cocaine and cocaethylene not present for benzoylecgonine, leading to the observed affinity differences for these three ligands.     

Expression and characterization of a humanized cocaine-binding antibody

The murine immunoglobulin G (IgG) cocaine-binding monoclonal antibody (mAb), GNC92H2, is notable for its exquisite specificity for cocaine, as opposed to chemically-related cocaine metabolites, and for its moderately high affinity (K(d) approximately 200 nM) for cocaine. Recently, we described the crystal structure of a mouse/human chimeric Fab construct at 2.3 A resolution. Herein, we report the successful framework humanization of a single-chain Fv (scFv) GNC92H2 construct without loss of affinity for cocaine. In brief, we compared the mAb GNC92H2 sequence to human antibody sequences, and used structure-based design to incorporate mutations (total = 49) that would humanize the framework region without affecting the overall shape of the binding pocket or the key cocaine-contact residues. The codons of the rationally designed sequence were optimized for E. coli expression, and the gene was synthesized by a de novo PCR reaction using 14 overlapping primers. Expression of the scFv construct was significantly improved in E. coli by fusion to thioredoxin. Intriguingly, this construct apparently refolds to form soluble active antibody in the reducing environment of the cytoplasm. Competitive ELISA and equilibrium dialysis demonstrated comparable binding activity between the humanized scFv and the whole IgG. The successful humanization of mAb GNC92H2 should enhance its potential therapeutic value by reducing its overall. immunogenicity.     

Intracellular antibodies and cancer: New technologies offer therapeutic opportunities

Since the realisation that the antigen‐binding regions of antibodies, the variable (V) regions, can be uncoupled from the rest of the molecule to create fragments that recognise and abrogate particular protein functions in cells, the use of antibody fragments inside cells has become an important tool in bioscience. Diverse libraries of antibody fragments plus in vivo screening can be used to isolate single chain variable fragments comprising VH and VL segments or single V‐region domains. Some of these are interfering antibody fragments that compete with protein‐protein interactions, providing lead molecules for drug interactions that until now have been considered difficult or undruggable. It may be possible to deliver or express antibody fragments in target cells as macrodrugs per se. In future incarnations of intracellular antibodies, however, the structural information of the interaction interface of target and antibody fragment should facilitate development of binding site mimics as small drug‐like molecules. This is a new dawn for intracellular antibody fragments both as macrodrugs and as precursors of drugs to treat human diseases and should finally lead to the removal of the epithet of the ‘undruggable’ protein‐protein interactions.     

Augmenting the efficacy of anti-cocaine catalytic antibodies through chimeric hapten design and combinatorial vaccination

Given the need for further improvements in anti-cocaine vaccination strategies, a chimeric hapten (GNET) was developed that combines chemically-stable structural features from steady-state haptens with the hydrolytic functionality present in transition-state mimetic haptens. Additionally, as a further investigation into the generation of an improved bifunctional antibody pool, sequential vaccination with steady-state and transition-state mimetic haptens was undertaken. While GNET induced the formation of catalytically-active antibodies, it did not improve overall behavioral efficacy. In contrast, the resulting pool of antibodies from GNE/GNT co-administration demonstrated intermediate efficacy as compared to antibodies developed from either hapten alone. Overall, improved antibody catalytic efficiency appears necessary to achieve the synergistic benefits of combining cocaine hydrolysis with peripheral sequestration.     

Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3

The crystal structures of the Fab' fragment of the anti-progesterone monoclonal antibody DB3 and its complexes with steroid haptens have shown that the D-JH junctional residue TrpH100 is a key contributor to binding site interactions with ligands. The indole group of TrpH100 also undergoes a significant conformational change between the bound and unliganded states, effectively opening and closing the combining site pocket. In order to explore the effect of substitutions at this position on steroid recognition, we have carried out mutagenesis on a construct encoding a three-domain single-chain fragment (VH/K) of DB3 expressed in Escherichia coli. TrpH100 was replaced by 13 different amino acids or deleted, and the functional and antigenic properties of the mutated fragments were analyzed. Most substitutions, including small, hydrophobic, hydrophilic, neutral, and negatively charged side chains, were reduced or abolished binding to free progesterone, although binding to progesterone-BSA was partially retained. The reduction in antigen binding was paralleled by alteration of the idiotype associated with the DB3 combining site. In contrast, the replacement of TrpH100 by Arg produced a mutant that retained wild-type antibody affinity and idiotype, but with altered specificity. Significant changes in this mutant included increased relative affinities of 10(4)-fold for progesterone-3-carboxymethyloxime and 10-fold for aetiocholanolone. Our results demonstrate an essential role for the junctional residue H100 in determining steroid-binding specificity and combining site idiotype and show that these properties can be changed by a single amino acid substitution at this position.     

Epitope‐specificity of recombinant antibodies reveals promiscuous peptide‐binding properties

Protein–peptide interactions are a common occurrence and essential for numerous cellular processes, and frequently explored in broad applications within biology, medicine, and proteomics. Therefore, understanding the molecular mechanism(s) of protein–peptide recognition, specificity, and binding interactions will be essential. In this study, we report the first detailed analysis of antibody–peptide interaction characteristics, by combining large‐scale experimental peptide binding data with the structural analysis of eight human recombinant antibodies and numerous peptides, targeting tryptic mammalian and eukaryote proteomes. The results consistently revealed that promiscuous peptide‐binding interactions, that is, both specific and degenerate binding, were exhibited by all antibodies, and the discovery was corroborated by orthogonal data, indicating that this might be a general phenomenon for low‐affinity antibody–peptide interactions. The molecular mechanism for the degenerate peptide‐binding specificity appeared to be executed through the use of 2–3 semi‐conserved anchor residues in the C‐terminal part of the peptides, in analogue to the mechanism utilized by the major histocompatibility complex–peptide complexes. In the long‐term, this knowledge will be instrumental for advancing our fundamental understanding of protein–peptide interactions, as well as for designing, generating, and applying peptide specific antibodies, or peptide‐binding proteins in general, in various biotechnical and medical applications.     

Combined cocaine hydrolase gene transfer and anti-cocaine vaccine synergistically block cocaine-induced locomotion

Mice and rats were tested for reduced sensitivity to cocaine-induced hyper-locomotion after pretreatment with anti-cocaine antibody or cocaine hydrolase (CocH) derived from human butyrylcholinesterase (BChE). In Balb/c mice, direct i.p. injection of CocH protein (1 mg/kg) had no effect on spontaneous locomotion, but it suppressed responses to i.p. cocaine up to 80 mg/kg. When CocH was injected i.p. along with a murine cocaine antiserum that also did not affect spontaneous locomotion, there was no response to any cocaine dose. This suppression of locomotor activity required active enzyme, as it was lost after pretreatment with iso-OMPA, a selective BChE inhibitor. Comparable results were obtained in rats that developed high levels of CocH by gene transfer with helper-dependent adenoviral vector, and/or high levels of anti-cocaine antibody by vaccination with norcocaine hapten conjugated to keyhole limpet hemocyanin (KLH). After these treatments, rats were subjected to a locomotor sensitization paradigm involving a "training phase" with an initial i.p. saline injection on day 1 followed by 8 days of repeated cocaine injections (10 mg/kg, i.p.). A 15-day rest period then ensued, followed by a final "challenge" cocaine injection. As in mice, the individual treatment interventions reduced cocaine-stimulated hyperactivity to a modest extent, while combined treatment produced a greater reduction during all phases of testing compared to control rats (with only saline pretreatment). Overall, the present results strongly support the view that anti-cocaine vaccine and cocaine hydrolase vector treatments together provide enhanced protection against the stimulatory actions of cocaine in rodents. A similar combination therapy in human cocaine users might provide a robust therapy to help maintain abstinence.     

Crystal structure to 2.45 A resolution of a monoclonal Fab specific for the Brucella A cell wall polysaccharide antigen.

The atomic structure of an antibody antigen-binding fragment (Fab) at 2.45 A resolution shows that polysaccharide antigen conformation and Fab structure dictated by combinatorial diversity and domain association are responsible for the fine specificity of the Brucella-specific antibody, YsT9.1. It discriminates the Brucella abortus A antigen from the nearly identical Brucella melitensis M antigen by forming a groove-type binding site, lined with tyrosine residues, that accommodates the rodlike A antigen but excludes the kinked structure of the M antigen, as envisioned by a model of the antigen built into the combining site. The variable-heavy (VH) and variable-light (VL) domains are derived from genes closely related to two used in previously solved structures, M603 and R19.9, respectively. These genes combine in YsT9.1 to form an antibody of totally different specificity. Comparison of this X-ray structure with a previously built model of the YsT9.1 combining site based on these homologies highlights the importance of VL:VH association as a determinant of specificity and suggests that small changes at the VL:VH interface, unanticipated in modeling, may cause significant modulation of binding-site properties.     

Affinity and Specificity of Levamlodipine-Human Serum Albumin Interactions: Insights into Its Carrier Function

The affinity and specificity of drugs with human serum albumin (HSA) are crucial factors influencing the bioactivity of drugs. To gain insight into the carrier function of HSA, the binding of levamlodipine with HSA has been investigated as a model system by a combined experimental and theoretical/computational approach. The fluorescence properties of HSA and the binding parameters of levamlodipine indicate that the binding is characterized by one binding site with static quenching mechanism, which is related to the energy transfer. As indicated by the thermodynamic analysis, hydrophobic interaction is the predominant force in levamlodipine-HSA complex, which is in agreement with the computational results. And the hydrogen bonds can be confirmed by computational approach between levamlodipine and HSA. Compared to predicted binding energies and binding energy spectra at seven sites on HSA, levamlodipine binding HSA at site I has a high affinity regime and the highest specificity characterized by the largest intrinsic specificity ratio (ISR). The binding characteristics at site I guarantee that drugs can be carried and released from HSA to carry out their specific bioactivity. Our concept and quantification of specificity is general and can be applied to other drug-target binding as well as molecular recognition of peptide-protein, protein-protein, and protein-DNA interactions.     

Structure of an anti-Lewis X Fab fragment in complex with its Lewis X antigen

The Lewis X trisaccharide is pivotal in mediating specific cell-cell interactions. Monoclonal antibody 291-2G3-A, which was generated from mice infected with schistosomes, has been shown to recognize the Lewis X trisaccharide. Here we describe the structure of the Fab fragment of 291-2G3-A, with Lewis X, to 1.8 A resolution. The crystallographic analysis revealed that the antigen binding site is a rather shallow binding pocket, and residues from all six complementary determining regions of the antibody contact all sugar residues. The high specificity of the binding pocket does not result in high affinity; the K(D) determined by isothermal calorimetry is 11 microM. However, this affinity is in the same range as for other sugar-antibody complexes. The detailed understanding of the antibody-Lewis X interaction revealed by the crystal structure may be helpful in the design of better diagnostic tools for schistosomiasis and for studying Lewis X-mediated cell-cell interactions by antibody interference.     

Predicting molecular interactions and inducible complementarity: Fragment docking of fab-peptide complexes

Antibody-antigen interactions are representative of a broad class of receptor-ligand interactions involving both specificity and potential inducible complementarity. To test possible mechanisms of antigenantibody recognition and specificity computationally, we have used a Metropolis Monte Carlo algorithm to dock fragments of the epitope Glu-Val-Val-Pro-His-Lys-Lys to the X-ray structures of both the free and the complexed Fab of the antibody B13I2 (raised against the C-helix of myohemerythri). The fragments Pro-His and Val-Pro-His, which contain residues experimentally identified as important for binding, docked correctly to both structures, but all tetrapeptide and larger fragments docked correctly only to the complexed Fab, even when torsional flexibility was added to the ligand. However, only tetrapeptide and larger fragments showed significantly more favorable energies when docked to the complexed Fab coordinates than when docked to either the free Fab or a non-specific site remote from the combining site. Comparison of the free and complexed B13I2 structures revealed that atoms within 5 Å of Val-Pro-His showed little movement upon peptide binding, but atoms within 5 Å of the other four epitope residues showed greater movements. These results computationally distinguish recognition and binding processes with practical implications for drug design strategies. Overall, this new fragment docking approach establishes distinct roles for the “lock-and-key” (recognition) and the “handshake” (binding) paradigms in antibody-antigen interaction, suggests an incremental approach to incorporating flexibility in computational docking, and identifies critical regions within receptor binding sites for ligand recognition. © 1994 Wiley-Liss, Inc.     

Three-dimensional structure-activity relationship modeling of cocaine binding to two monoclonal antibodies by comparative molecular field analysis

Successful immunotherapy of cocaine addiction and overdoses requires cocaine-binding antibodies with specific properties, such as high affinity and selectivity for cocaine. We have determined the affinities of two cocaine-binding murine monoclonal antibodies (mAb: clones 3P1A6 and MM0240PA) for cocaine and its metabolites by [3H]-radioligand binding assays. mAb 3P1A6 (K(d) = 0.22 nM) displayed a 50-fold higher affinity for cocaine than mAb MM0240PA (K(d) = 11 nM) and also had a greater specificity for cocaine. For the systematic exploration of both antibodies' binding specificities, we used a set of approximately 35 cocaine analogues as structural probes by determining their relative binding affinities (RBAs) using an enzyme-linked immunosorbent competition assay. Three-dimensional quantitative structure-activity relationship (3D-QSAR) models on the basis of comparative molecular field analysis (CoMFA) techniques correlated the binding data with structural features of the ligands. The analysis indicated that despite the mAbs' differing specificities for cocaine, the relative contributions of the steric (approximately 80%) and electrostatic (approximately 20%) field interactions to ligand-binding were similar. Generated three-dimensional CoMFA contour plots then located the specific regions about cocaine where the ligand/receptor interactions occurred. While the overall binding patterns of the two mAbs had many features in common, distinct differences were observed about the phenyl ring and the methylester group of cocaine. Furthermore, using previously published data, a 3D-QSAR model was developed for cocaine binding to the dopamine reuptake transporter (DAT) that was compared to the mAb models. Although the relative steric and electrostatic field contributions were similar to those of the mAbs, the DAT cocaine-binding site showed a preference for negatively charged ligands. Besides establishing molecular level insight into the interactions that govern cocaine binding specificity by biopolymers, the three-dimensional images obtained reflect the properties of the mAbs binding pockets and provide the initial information needed for the possible design of novel antibodies with properties optimized for immunotherapy.     

Functional mapping of conserved, surface-exposed charges of antibody variable domains

Surface-exposed charges can affect protein structure, stability and solubility as well as the kinetics of both the folding process and interaction with binding partners. We have investigated the influence on kinetic interaction parameters of 14 conserved, surface-exposed charges located away from the paratope in the variable domains of two antibodies of different specificity. We found that conserved, surface-exposed, charged framework residues are asymmetrically distributed on opposite faces of both VH and VL domains. Some of the charges play a critical role in protein folding and stability. While electrostatic forces within or close to the binding interface can be used to optimize the association rate, we confirmed the predicted minor effects of charge modifications remote from the binding site. They had no effect on the dissociation rate parameter. Our study demonstrates the role of residues remote from the interaction site in the recognition function as well as the limited effect of surface charge modifications in antibody fragments on kinetic interaction parameters.     

Contribution of a single heavy chain residue to specificity of an anti-digoxin monoclonal antibody.

Two distinct spontaneous variants of the murine anti-digoxin hybridoma 26-10 were isolated by fluorescence-activated cell sorting for reduced affinity of surface antibody for antigen. Nucleotide and partial amino acid sequencing of the variant antibody variable regions revealed that 1 variant had a single amino acid substitution: Lys for Asn at heavy chain position 35. The second variant antibody had 2 heavy chain substitutions: Tyr for Asn at position 35, and Met for Arg at position 38. Mutagenesis experiments confirmed that the position 35 substitutions were solely responsible for the markedly reduced affinity of both variant antibodies. Several mutants with more conservative position 35 substitutions were engineered to ascertain the contribution of Asn 35 to the binding of digoxin to antibody 26-10. Replacement of Asn with Gln reduced affinity for digoxin 10-fold relative to the wild-type antibody, but maintained wild-type fine specificity for cardiac glycoside analogues. All other substitutions (Val, Thr, Leu, Ala, and Asp) reduced affinity by at least 90-fold and caused distinct shifts in fine specificity. The Ala mutant demonstrated greatly increased relative affinities for 16-acetylated haptens and haptens with a saturated lactone. The X-ray crystal structure of the 26-10 Fab in complex with digoxin (Jeffrey PD et al., 1993, Proc Natl Acad Sci USA 90:10310-10314) reveals that the position 35 Asn contacts hapten and forms hydrogen bonds with 2 other contact residues. The reductions in affinity of the position 35 mutants for digoxin are greater than expected based upon the small hapten contact area provided by the wild-type Asn. We therefore performed molecular modeling experiments which suggested that substitution of Gln or Asp can maintain these hydrogen bonds whereas the other substituted side chains cannot. The altered binding of the Asp mutant may be due to the introduction of a negative charge. The similarities in binding of the wild-type and Gln-mutant antibodies, however, suggest that these hydrogen bonds are important for maintaining the architecture of the binding site and therefore the affinity and specificity of this antibody. The Ala mutant eliminates the wild-type hydrogen bonding, and molecular modeling suggests that the reduced side-chain volume also provides space that can accommodate a congener with a 16-acetyl group or saturated lactone, accounting for the altered fine specificity of this antibody.     

Complement subcomponent C1q secreted by cultured human monocytes has subunit structure identical with that of serum C1q.

Rat alpha-foetoprotein (alpha-FP) strongly binds the drugs warfarin and phenylbutazone, as does albumin; however, the binding sites for the two drugs seemed to be different. This possibility and the specificity of this/these drug-binding site(s) of rat alpha-FP were investigated by competitive protein-binding experiments with a variety of drugs, representing different pharmacological groups, and biomolecules that are strongly bound by the foetal protein and that are suspected to play a specific role during foetal development. The binding mechanisms were further investigated by using comparisons between computer-derived theoretical displacement curves and experimental points in order to distinguish different possible binding models. The results indicate: that warfarin and phenylbutazone are bound at two distinct sites on rat alpha-FP and that a negative modulatory effect is exerted between the two sites; that the degree of specificity of these two drug-binding sites is different, since the warfarin-binding site appears to be specific for the binding of coumarinic and anthranilic drugs whereas that for phenylbutazone is able to bind substances of very varied chemical structure and is more hydrophobic; that the phenylbutazone-binding site is the site that binds oestrogens that thyroid hormones and, probably, fatty acids and bilirubin are bound at (an)other site(s) but exert negative modulatory effects on phenylbutazone binding. The nature of the different binding areas of rat alpha-FP is compared with that of those already proposed for albumin. The potential risks of toxicity of such interactions between drugs and/or biomolecules on foetal development are also discussed.     

Crystal structure of an anti-interleukin-2 monoclonal antibody Fab complexed with an antigenic nonapeptide

The three-dimensional structure of the Fab fragment of a monoclonal antibody (LNKB-2) to human interleukin-2 (IL-2) complexed with a synthetic antigenic nonapeptide, Ac-Lys-Pro-Leu-Glu-Glu-Val-Leu-Asn-Leu-OMe, has been determined at 3.0 A resolution. In the structure, four out of the six hypervariable loops of the Fab (complementarity determining regions [CDRs] L1, H1, H2, and H3) are involved in peptide association through hydrogen bonding, salt bridge formation, and hydrophobic interactions. The Tyr residues in the Fab antigen binding site play a major role in antigen-antibody recognition. The structures of the complexed and uncomplexed Fab were compared. In the antigen binding site the CDR-L1 loop of the antibody shows the largest structural changes upon peptide binding. The peptide adopts a mostly alpha-helical conformation similar to that in the epitope fragment 64-72 of the IL-2 antigen. The side chains of residues Leu 66, Val 69, and Leu 70, which are shielded internally in the IL-2 structure, are involved in interactions with the Fab in the complex studied. This indicates that antibody-antigen complexation involves a significant rearrangement of the epitope-containing region of the IL-2 with retention of the alpha-helical character of the epitope fragment.