Antibody Complementarity-determining Regions Research Articles

Antibody Complementarity-Determining Regions (CDRs): A Bridge between Adaptive and Innate Immunity

BackgroundIt has been documented that, independently from the specificity of the native antibody (Ab) for a given antigen (Ag), complementarity determining regions (CDR)-related peptides may display differential antimicrobial, antiviral and antitumor activities.Methodology/Principal FindingsIn this study we demonstrate that a synthetic peptide with sequence identical to VHCDR3 of a mouse monoclonal Ab (mAb) specific for difucosyl human blood group A is easily taken up by macrophages with subsequent stimulation of: i) proinflammatory cytokine production; ii) PI3K-Akt pathway and iii) TLR-4 expression. Significantly, VHCDR3 exerts therapeutic effect against systemic candidiasis without possessing direct candidacidal properties.Conclusions/SignificanceThese results open a new scenario about the possibility that, beyond the half life of immunoglobulins, Ab fragments may effectively influence the antiinfective cellular immune response in a way reminiscent of regulatory peptides of innate immunity.

A Human Germ Line Antibody Light Chain with Hydrolytic Properties Associated with Multimerization Status

Antibodies with nucleophilic or catalytic properties often have these characteristics encoded in their germ line genes. Because hydrolytic activity has been reported to be associated with light chain V regions, we have begun an analysis of germ line light chain proteins that could be the basis for affinity maturation into hydrolytic or other reactive antibodies. We produced the germ line A18b light chain and characterized its hydrolytic, nucleophilic, and tertiary structural activities. This light chain was purified to >99% purity and found to hydrolyze aminomethylcoumarin-peptide and larger protein substrates and bind a fluorophosphonate probe. Mutation of putative catalytic residues only resulted in loss of activity of a tetrameric but not dimeric form of the light chain. These biochemical properties provide a framework for understanding the structure-function relationships of germ line antibodies.

Correlations between Terasaki’s HLA class I epitopes and HLAMatchmaker‐defined eplets on HLA‐A, ‐B and ‐C antigens

Although the determination of human leukocyte antigen (HLA) antibody specificity has traditionally been directed toward HLA antigens, there is now increasing attention to structurally defined HLA epitopes. An understanding of the HLA epitope repertoire is important to acceptable mismatching for sensitized patients and to a new epitope-based matching algorithm aimed to reduce antibody-mediated rejection. There are two strategies to determine the HLA epitope repertoire. Terasaki's group has used an empirical method to analyze the reactivity of single allele Luminex panels with mouse monoclonal antibodies (mAbs) and absorbed/eluted alloantibodies with a computer program based on shared residues in the amino acid sequences of reactive alleles. HLAMatchmaker is a theoretical algorithm that predicts HLA epitopes on the HLA molecular surface from stereochemical modeling of epitope-paratope interfaces of antigen-antibody complexes. Our epitope repertoire is based on so-called 'eplets' representing 3-A patches of at least one polymorphic residue on the molecular surface. A comparative analysis has shown that 81/103 Terasaki's HLA class I epitopes are equivalent to individual eplets (n = 50) or pairs of eplets (n = 31) separated far enough to serve as potential contact sites for two complementarity-determining regions of antibody. An additional 12 Terasaki's epitopes (TerEps) correspond to eplets with permissible residue combinations that do not seem to affect epitope specificity. We could not identify corresponding eplets for the remaining 10 TerEps, including 8 that might be considered xeno-epitopes defined by mouse mAbs. Conversely, HLAMatchmaker has 38 additional eplets in well-exposed surface positions that do not have equivalent TerEps, and for many of them, we have found specific antibodies. These findings strengthen the concept that eplets are essential basic units of HLA epitopes and that they provide a better understanding of HLA immunogenicity (i.e. ability to induce an antibody response) and antigenicity (i.e. reactivity with specific antibody).

Antibody Complementarity-Determining Regions (CDRs) Can Display Differential Antimicrobial, Antiviral and Antitumor Activities

BackgroundComplementarity-determining regions (CDRs) are immunoglobulin (Ig) hypervariable domains that determine specific antibody (Ab) binding. We have shown that synthetic CDR-related peptides and many decapeptides spanning the variable region of a recombinant yeast killer toxin-like antiidiotypic Ab are candidacidal in vitro. An alanine-substituted decapeptide from the variable region of this Ab displayed increased cytotoxicity in vitro and/or therapeutic effects in vivo against various bacteria, fungi, protozoa and viruses. The possibility that isolated CDRs, represented by short synthetic peptides, may display antimicrobial, antiviral and antitumor activities irrespective of Ab specificity for a given antigen is addressed here.Methodology/Principal FindingsCDR-based synthetic peptides of murine and human monoclonal Abs directed to: a) a protein epitope of Candida albicans cell wall stress mannoprotein; b) a synthetic peptide containing well-characterized B-cell and T-cell epitopes; c) a carbohydrate blood group A substance, showed differential inhibitory activities in vitro, ex vivo and/or in vivo against C. albicans, HIV-1 and B16F10-Nex2 melanoma cells, conceivably involving different mechanisms of action. Antitumor activities involved peptide-induced caspase-dependent apoptosis. Engineered peptides, obtained by alanine substitution of Ig CDR sequences, and used as surrogates of natural point mutations, showed further differential increased/unaltered/decreased antimicrobial, antiviral and/or antitumor activities. The inhibitory effects observed were largely independent of the specificity of the native Ab and involved chiefly germline encoded CDR1 and CDR2 of light and heavy chains.Conclusions/SignificanceThe high frequency of bioactive peptides based on CDRs suggests that Ig molecules are sources of an unlimited number of sequences potentially active against infectious agents and tumor cells. The easy production and low cost of small sized synthetic peptides representing Ig CDRs and the possibility of peptide engineering and chemical optimization associated to new delivery mechanisms are expected to give rise to a new generation of therapeutic agents.

Investigating the Properties of Bacillus thuringiensis Cry Proteins with Novel Loop Replacements Created Using Combinatorial Molecular Biology

Cry proteins are a large family of crystalline toxins produced by Bacillus thuringiensis. Individually, the family members are highly specific, but collectively, they target a diverse range of insects and nematodes. Domain II of the toxins is important for target specificity, and three loops at its apex have been studied extensively. There is considerable interest in determining whether modifications in this region may lead to toxins with novel specificity or potency. In this work, we studied the effect of loop substitution on toxin stability and specificity. For this purpose, sequences derived from antibody complementarity-determining regions (CDR) were used to replace native domain II apical loops to create "Crybodies." Each apical loop was substituted either individually or in combination with a library of third heavy-chain CDR (CDR-H3) sequences to create seven distinct Crybody types. An analysis of variants from each library indicated that the Cry1Aa framework can tolerate considerable sequence diversity at all loop positions but that some sequence combinations negatively affect structural stability and protease sensitivity. CDR-H3 substitution showed that loop position was an important determinant of insect toxicity: loop 2 was essential for activity, whereas the effects of substitutions at loop 1 and loop 3 were sequence dependent. Unexpectedly, differences in toxicity did not correlate with binding to cadherins--a major class of toxin receptors--since all Crybodies retained binding specificity. Collectively, these results serve to better define the role of the domain II apical loops as determinants of specificity and establish guidelines for their modification.

Thermodynamic Consequences of Mutations in Vernier Zone Residues of a Humanized Anti-human Epidermal Growth Factor Receptor Murine Antibody, 528

To investigate the role of Vernier zone residues, which are comprised in the framework regions and underlie the complementarity-determining regions (CDRs) of antibodies, in the specific, high affinity interactions of antibodies with their targets, we focused on the variable domain fragment of murine anti-human epidermal growth factor receptor antibody 528 (m528Fv). Grafting of the CDRs of m528Fv onto a selected framework region of human antibodies, referred to as humanization, reduced the antibody's affinity for its target by a factor of 1/40. The reduction in affinity was due to a substantial reduction in the negative enthalpy change associated with binding. Crystal structures of the ligand-free antibody fragments showed no noteworthy conformational changes due to humanization, and the loop structures of the CDRs of the humanized antibodies were identical to those of the parent antibodies. Several mutants of the CDR-grafted (humanized) variable domain fragment (h528Fv), in which some of the Vernier zone residues in the heavy chain were replaced with the parental murine residues, were constructed and prepared using a bacterial expression system. Thermodynamic analyses of the interactions between the mutants and the soluble extracellular domain of epidermal growth factor receptor showed that several single mutations and a double mutation increased the negative enthalpy and heat capacity changes. Combination of these mutations, however, led to somewhat reduced negative enthalpy and heat capacity changes. The affinity of each mutant for the target was within the range for the wild-type h528Fv, and this similarity was due to enthalpy-entropy compensation. These results suggest that Vernier zone residues make enthalpic contributions to antigen binding and that the regulation of conformational entropy changes upon humanization of murine antibodies must be carefully considered and optimized.

Construction, Affinity Maturation, and Biological Characterization of an Anti-tumor-associated Glycoprotein-72 Humanized Antibody

The tumor-associated glycoprotein (TAG)-72 is expressed in the majority of human adenocarcinomas but is rarely expressed in most normal tissues, which makes it a potential target for the diagnosis and therapy of a variety of human cancers. Here we describe the construction, affinity maturation, and biological characterization of an anti-TAG-72 humanized antibody with minimum potential immunogenicity. The humanized antibody was constructed by grafting only the specificity-determining residues (SDRs) within the complementarity-determining regions (CDRs) onto homologous human immunoglobulin germ line segments while retaining two mouse heavy chain framework residues that support the conformation of the CDRs. The resulting humanized antibody (AKA) showed only about 2-fold lower affinity compared with the original murine monoclonal antibody CC49 and 27-fold lower reactivity to patient serum compared with the humanized antibody HuCC49 that was constructed by CDR grafting. The affinity of AKA was improved by random mutagenesis of the heavy chain CDR3 (HCDR3). The highest affinity variant (3E8) showed 22-fold higher affinity compared with AKA and retained the original epitope specificity. Mutational analysis of the HCDR3 residues revealed that the replacement of Asn(97) by isoleucine or valine was critical for the affinity maturation. The 3E8 labeled with (125)I or (131)I showed efficient tumor targeting or therapeutic effects, respectively, in athymic mice with human colon carcinoma xenografts, suggesting that 3E8 may be beneficial for the diagnosis and therapy of tumors expressing TAG-72.

Display of somatostatin-related peptides in the complementarity determining regions of an antibody light chain

Peptide display in antibody complementarity determining regions (CDRs) offers several advantages over other peptide display systems including the potential to graft heterologous peptide sequences into multiple positions in the same backbone molecule. Despite the presence of six CDRs in an antibody variable domain, the majority of insertions reported have been made in heavy chain CDR3 (h-CDR3) which may be explained in part by the highly variable length and sequence diversity found in h-CDR3 in native antibodies. The ability to graft peptide sequences into CDRs is restricted by amino acids in these loops that make structural contacts to framework regions or are oriented towards the hydrophobic interior and are important for the proper folding of the antibody. To identify such positions in human κ-light chain CDR1 (κ-CDR1) and CDR2 (κ-CDR2), we performed alignments of 1330 κ-light chain variable region amino acid sequences and 19 variable region X-ray crystal structures. From analyses of these alignments, we predict insertion points where sequences can be grafted into κ-CDR1 and κ-CDR2 to prepare synthetic antibody molecules. We then tested these predictions by inserting somatostatin and somatostatin-related sequences into κ-CDR1 and κ-CDR2, and analyzing the expression and ability of the modified antibodies to bind to membranes containing somatostatin receptor 5. These results expand the repertoire of CDRs that can be used for the display of heterologous peptides in the CDRs of antibodies.

SDR grafting—a new approach to antibody humanization

A major impediment to the clinical utility of the murine monoclonal antibodies is their potential to elicit human anti-murine antibody (HAMA) response in patients. To circumvent this problem, murine antibodies have been genetically manipulated to progressively replace their murine content with the amino acid residues present in their human counterparts. To that end, murine antibodies have been humanized by grafting their complementarity determining regions (CDRs) onto the variable light (V L) and variable heavy (V H) frameworks of human immunoglobulin molecules, while retaining those murine framework residues deemed essential for the integrity of the antigen-combining site. However, the xenogeneic CDRs of the humanized antibodies may evoke anti-idiotypic (anti-Id) response in patients. To minimize the anti-Id response, a procedure to humanize xenogeneic antibodies has been described that is based on grafting, onto the human frameworks, only the specificity determining residues (SDRs), the CDR residues that are most crucial in the antibody–ligand interaction. The SDRs are identified through the help of the database of the three-dimensional structures of the antigen–antibody complexes of known structures or by mutational analysis of the antibody-combining site. An alternative approach to humanization, which involves retention of more CDR residues, is based on grafting of the ‘abbreviated’ CDRs, the stretches of CDR residues that include all the SDRs. A procedure to assess the reactivity of the humanized antibody to sera from patients who had been administered the murine antibody has also been described.

Analysis of the Antigen Combining Site: Correlations Between Length and Sequence Composition of the Hypervariable Loops and the Nature of the Antigen

It has long been suggested that the overall shape of the antigen combining site (ACS) of antibodies is correlated with the nature of the antigen. For example, deep pockets are characteristic of antibodies that bind haptens, grooves indicate peptide binders, while antibodies that bind to proteins have relatively flat combining sites. In 1996, MacCallum, Martin and Thornton used a fractal shape descriptor and showed a strong correlation of the shape of the binding region with the general nature of the antigen. However, the shape of the ACS is determined primarily by the lengths of the six complementarity-determining regions (CDRs). Here, we make a direct correlation between the lengths of the CDRs and the nature of the antigen. In addition, we show significant differences in the residue composition of the CDRs of antibodies that bind to different antigen classes. As well as helping us to understand the process of antigen recognition, autoimmune disease and cross-reactivity, these results are of direct application in the design of antibody phage libraries and modification of affinity.

Structure of an anti-HIV monoclonal Fab antibody fragment specific to a gp120 C-4 region peptide.

Structure of an anti-HIV monoclonal Fab antibody fragment specific to a gp120 C-4 region peptide.

Monoclonal antibody therapy

Monoclonal antibody therapy (MAT) makes use of all the major features of the immune response. It involves vaccination/ immunization, albeit in experimental animals, to induce the desired specific immune response. It exploits the high specificity, selectivity, and affinity of the antibody complementarity-determining regions (CDRs) toward the target antigen to be recognized, highlighted, inactivated, or eliminated, using the characteristics of the Fc portion of an immunoglobulin to facilitate the means for such inactivation or elimination and for selection of appropriate effector mechanisms. In fact, some of these mAbs can be regarded as major breakthroughs in the treatment of such disorders as transplant rejection, coronary artery disease, rheumatoid arthritis, non-Hodgkin's lymphoma, and breast cancer. However, it is questionable if the precise specificity of the mAb therapeutic strategy will ever be surpassed, and the versatility of mAb engincering provides exciting new avenues for mAbbased therapeutic strategies.

Peptide Design Based on an Antibody Complementarity-Determining Region (CDR): Construction of Porphyrin-Binding Peptides and Their Affinity Maturation by a Combinatorial Method

Peptide Design Based on an Antibody Complementarity-Determining Region (CDR): Construction of Porphyrin-Binding Peptides and Their Affinity Maturation by a Combinatorial Method

Peptide design based on an antibody complementarity-determining region (CDR): construction of porphyrin-binding peptides and their affinity maturation by a combinatorial method.

We have utilized sequence information from an antiheme monoclonal antibody to develop novel porphyrin-binding peptides. Several peptides which have an intramolecular disulfide bond in different positions and different chain lengths were prepared. The affinities of peptides for meso-tetrakis(4-carboxyphenyl)porphyrin were increased by an appropriate conformational restraint using a disulfide bond. Detailed studies with a representative 12-peptide, 12C4, whose length was reduced from 20 residues of the complementarity-determining region (CDR), indicated that both the hydrophobic and electrostatic interactions were essential factors in the peptide-porphyrin binding. Moreover, two-dimensional 1H NMR spectroscopy revealed the conformation of the peptide and the critical residues for the porphyrin-binding. According to the obtained results, a further minimized 9-peptide, 9L, was successfully redesigned with a sequence capable of forming a beta-turn instead of a disulfide bond. Furthermore, affinity maturation studies of 9L were performed by using a combinatorial approach such as the spot-synthesis method. Peptides with an improved affinity for porphyrins were prepared by systematic amino acid replacement. Thus, the design of peptides targeted to porphyrins was demonstrated by the combination of antibody information and the rationally designed combinatorial method.

Three-dimensional structures of the free and antigen-bound Fab from monoclonal antilysozyme antibody HyHEL-63(,).

Antigen-antibody complexes provide useful models for studying the structure and energetics of protein-protein interactions. We report the cloning, bacterial expression, and crystallization of the antigen-binding fragment (Fab) of the anti-hen egg white lysozyme (HEL) antibody HyHEL-63 in both free and antigen-bound forms. The three-dimensional structure of Fab HyHEL-63 complexed with HEL was determined to 2.0 A resolution, while the structure of the unbound antibody was determined in two crystal forms, to 1.8 and 2.1 A resolution. In the complex, 19 HyHEL-63 residues from all six complementarity-determining regions (CDRs) of the antibody contact 21 HEL residues from three discontinuous polypeptide segments of the antigen. The interface also includes 11 bound water molecules, 3 of which are completely buried in the complex. Comparison of the structures of free and bound Fab HyHEL-63 reveals that several of the ordered water molecules in the free antibody-combining site are retained and that additional waters are added upon complex formation. The interface waters serve to increase shape and chemical complementarity by filling cavities between the interacting surfaces and by contributing to the hydrogen bonding network linking the antigen and antibody. Complementarity is further enhanced by small (<3 A) movements in the polypeptide backbones of certain antibody CDR loops, by rearrangements of side chains in the interface, and by a slight shift in the relative orientation of the V(L) and V(H) domains. The combining site residues of complexed Fab HyHEL-63 exhibit reduced temperature factors compared with those of the free Fab, suggesting a loss in conformational entropy upon binding. To probe the relative contribution of individual antigen residues to complex stabilization, single alanine substitutions were introduced in the epitope of HEL recognized by HyHEL-63, and their effects on antibody affinity were measured using surface plasmon resonance. In agreement with the crystal structure, HEL residues at the center of the interface that are buried in the complex contribute most to the binding energetics (DeltaG(mutant) - DeltaG(wild type) > 3.0 kcal/mol), whereas the apparent contributions of solvent-accessible residues at the periphery are much less pronounced (<1.5 kcal/mol). In the latter case, the mutations may be partially compensated by local rearrangements in solvent structure that help preserve shape complementarity and the interface hydrogen bonding network.

Importance of hydropathic complementarity for the binding of the neuropeptide substance P to a monoclonal antibody: Equilibrium and kinetic studies

In a previous study (Boquet et al., 1995, Molec. Immunol. 32, 303–308) we observed remarkable inversions of hydropathic profiles between complementarity determining regions (CDRs) of an anti-substance P monoclonal antibody (SP31) and the corresponding 5 amino acid C-terminal peptide epitope. Here we demonstrate the importance this hydropathic complementarity by measuring the immunoreactivity of SP-related peptides which have been modified in their C-terminal parts so that hydropathic profile has been conserved (by substituting amino acids in the epitope) or modified (by mixing the sequence of amino acids in the epitope). Experiments performed in equilibrium conditions using a competitive enzyme immunoassay showed that most of the peptides conserving the hydropathic profile of SP epitope were recognized by mAb SP31 (even if marked variations in affinity were observed), while those for which the hydropathic profile was modified exhibited very low or undetectable affinity. The kinetic parameters ( k a and k d) of peptide-antibody interactions were determined using Surface Plasmon Resonance technology (BIACORE 2000). These measurements showed that all peptides recognized by mAb SP31 had similar association rate constants (close to 2 × 10 5 M −1 s −1), differences in binding affinities essentially resulting from differences in dissociation rate constants (ranging from 1.61 × 10 −4 to 1.15 × 10 −1 s −1). From these results, it was concluded that hydropathic complementarity between the epitope and the paratope could be a necessary but not sufficient condition for establishing high-affinity binding. We hypothesize that hydropathic interactions may play a critical role during the first contacts between antibody CDRs and the peptide, possibly by favouring reorganization of water molecules at the antibody-peptide interface.

Proline-rich tandem repeats of antibody complementarity-determining regions bind and neutralize human immunodeficiency virus type 1 particles.

The proline-rich tandem repeat domain of human mucin MUC1 forms an extended structure containing large repeating loops that are crested by a turn. We show that the repeating-loop structure of MUC1 can be replaced by an antibody complementarity-determining region loop of a human immunodeficiency virus type 1 (HIV-1)-specific neutralizing antibody to create a chimeric, multivalent, mucin-like, anti-HIV-1 compound. We used 8 residues of an antibody molecule to replace 8 of 20 residues of the MUC1 tandem-repeat sequence. The antiviral peptide discussed here contains three copies of a 20-residue tandem repeat, (IYYDYEEDPAPGSTAPPAHG)3, for a total of 60 residues. We demonstrate that the mucin-antibody chimera retains the binding specificity of the parent antibody (monoclonal antibody F58), GPGR of the HIV-1 gp120 V3 neutralizing epitope, and the ability to neutralize virus particles. In inhibition enzyme-linked immunosorbent assay, the mucin-antibody chimeric peptide could inhibit 71 to 84% of binding to a V3 loop peptide by monoclonal antibodies known to be specific for GPGR in the V3 loop. The mucin-antibody chimeric peptide could also inhibit monoclonal antibody binding to native gp120 captured from virus particles. In addition, the chimeric peptide neutralized the homologous HIV-IIIB virus in a standard neutralization assay. The methods of antiviral peptide design and construction presented here are general and theoretically limited only by the size of the antibody repertoire. This approach could be used to synthesize peptides for a variety of therapeutic applications.

Isomerization of an aspartic acid residue in the complementarity-determining regions of a recombinant antibody to human IgE: identification and effect on binding affinity.

This report describes the effect on antigen binding of an isomerized aspartate residue located in the complementarity-determining regions (CDRs) of a recombinant monoclonal antibody. The antibody, which binds human IgE, contains two Asp-Gly sequences within its CDRs, but only one site was found to be labile to isomerization. Isolation and characterization of antibody fragments differing in the labile sequence were facilitated by using a technique involving hydrophobic interaction chromatography (HIC) that separates aspartyl, isoaspartyl, and cyclic imide variants to the residue located in CDR-L1. The variants were isolated for structural characterization and for determination of their relative antigen binding affinities. Mutants were constructed with altered residues to obviate the effects of isomerization and were evaluated for their ability to bind to IgE. Inspection of published crystal structures of CDRs of antibodies indicated that hydrogen binding of the Asp side chain of the unreactive residue may be the constraint that prevents isomerization. The strategy outlined here may prove to be of general utility in the biochemical and immunochemical characterization of recombinant antibodies.

Structural significance of sequence variability in antibody complementarity-determining regions

Structural significance of sequence variability in antibody complementarity-determining regions

Thermodynamic analysis of an antibody functional epitope.

We have probed the relative contribution of polar and nonpolar interactions to antibody-antigen interaction by measuring the effect of single amino acid substitutions in an humanized anti-p185HER2 antibody (hu4D5-5) on the thermodynamics of antigen binding. First we mapped the functional epitope by complete alanine-scan mutagenesis of the antibody complementarity-determining region (CDR). Four residues, H91 in VL and R50, W95, and Y100a in VH, make large contributions to the free energy of binding (delta delta G > 3 kcal mol-1) and have delta delta G > delta delta H. These residues are clustered in a shallow pocket on the antibody surface in the X-ray structure determined for hu4D5 Fv. The majority of other CDR residues make less energetically important contributions (delta delta G < 1 kcal mol-1) to binding but have delta delta H > delta delta G, suggesting that the wild-type side chain does contact antigen but the loss in entropy, perhaps through restriction of side-chain conformational freedom, offsets the favorable enthalpic term. Effects of Phe and Ala substitutions on the delta G and delta Cp of antigen binding indicate that the favorable contribution of antibody tyrosine residues to binding results primarily from burial of the aromatic ring in the interface with antigen. Burial of the phenyl ring has a favorable delta H at 25 degrees C but at least for one site (VL-Y92) is opposed by delta S. This latter feature is inconsistent with the thermodynamics predicted for the hydrophobic effect based on hydrocarbon-transfer experiments.(ABSTRACT TRUNCATED AT 250 WORDS)