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Results for “Complementarity Determining Regions”

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PubMed1997

Complementarity determining region-III is a useful molecular marker for the evaluation of minimal residual disease in mantle cell lymphoma.

Bone marrow (BM) and peripheral blood (PB) involvement in 10 patients with mantle cell lymphoma (MCL) was analysed by a polymerase chain reaction (PCR)-mediated RNase protection assay. The complementarity determining regions (CDR)-III of all 10 MCLs examined was amplified efficiently with consensus V(H) and J(H) primers by PCR. and BM and/or PB involvement was evaluated by RNase protection assay in all 10 patients examined. Our assay showed BM and/or PB of the entire group to have neoplastic cells at presentation, despite the fact that eight patients were found to have BM and/or PB involvement on the basis of morphological examination and/or surface marker analysis. We also examined minimal residual disease (MRD) after conventional chemotherapy, and detected MRD in a patient in complete remission (CR). Although previous studies have shown that t(11;14) breakpoint amplification by PCR was only applicable to about 30-40% of cases, the present study indicates that CDR-III is a useful molecular marker and the PCR-mediated RNase protection assay is a good tool for the evaluation of MRD in MCL. It is suggested that BM and PB of MCL patients are quite frequently involved at presentation and even after conventional chemotherapy at the molecular level.

Aged↗

PubMed2003

Conservation of nonpeptide antigen recognition by rhesus monkey V gamma 2V delta 2 T cells.

We have previously found that monkey Vgamma2Vdelta2(+) T cells mount adaptive immune responses in response to Mycobacterium bovis bacillus Calmette-Guérin infections. We have now analyzed rhesus monkey gammadelta T cell responses to nonpeptide Ags and superantigens. Like human Vgamma2Vdelta2(+) T cells, rhesus monkey gammadelta T cells are stimulated when exposed to prenyl pyrophosphate, bisphosphonate, and alkylamine Ags. Responsiveness was limited to gammadelta T cells expressing Vgamma2Vdelta2 TCRs. Rhesus monkey Vgamma2Vdelta2(+) T cells also responded to the superantigen, staphyloccocal enterotoxin A. Sequencing of the rhesus monkey Vgamma2Vdelta2 TCR revealed a strong sequence homology to human Vgamma2Vdelta2 TCR that preserves important sequence motifs. Moreover, chimeric TCRs that pair human Vgamma2 with monkey Vdelta2 and monkey Vgamma2 with human Vdelta2 retain reactivity to nonpeptide Ags and B cell lymphomas. A molecular model of the rhesus monkey Vgamma2Vdelta2 TCR has a basic region in the complementarity-determining region 3 binding groove that is similar to that seen in the human Vgamma2Vdelta2 TCR and preserves the topology of the complementarity-determining region loops. Thus, recognition of nonpeptide prenyl pyrophosphate, bisphosphonate, and alkylamine Ags is conserved in primates suggesting that primates can provide an animal model for human gammadelta T cell Ag responses.

Amino Acid Sequence↗

PubMed1995

Evaluation of a complementarity-determining region-grafted (humanized) anti-carcinoembryonic antigen monoclonal antibody in preclinical and clinical studies.

A complementarity-determining region-grafted (humanized) version of MN-14 (hMN-14), a high-affinity, anti-carcinoembryonic antigen (CEA) murine monoclonal antibody (mMAb), was selected from several clones that differed slightly in their framework composition. One clone was selected based on its similar binding affinity to CEA as that observed with mMN-14 MAb and its production yields. Targeting studies, using 131I-labeled humanized MN-14 (hMN-14)/125I-labeled mMN-14 IgG in GW-39 tumor-bearing nude mice, showed excellent tumor uptake and tumor: nontumor ratios, similar to the mMN-14. A pilot clinical imaging trial was initiated to determine the targeting, pharmacokinetics, and dosimetry for 131I-labeled hMN-14 IgG. Nineteen patients with advanced CEA-producing tumors were given 8 to 30 mCi (0.5 to 20.0 mg). Eleven patients also received 131I-labeled mMN-14 IgG for comparison. The biodistribution, tumor targeting, and pharmacokinetic behavior of the hMN-14 was similar to that seen with the mMN-14. The average time required to clear 50% of the radiolabeled hMN-14 from the blood and total body was 32.9 +/- 25.6 h and 109 +/- 73 h, respectively. Patients with elevated plasma CEA (i.e., > 200 ng/ml) had more than 30% of the labeled antibody complexed within 1 h after injection. In some of these patients, increased complexation resulted in enhanced metabolism of the antibody with more rapid clearance from the blood than that seen in patients with lower plasma CEA. The average radiation absorbed dose measured in 20 tumors (average weight, 204 +/- 205 g) in 14 patients was 7.6 +/- 5.3 cGy/mCi. Tumor: nontumor dose ratios were 2.5 +/- 1.6, 9.5 +/- 5.8, and 2.6 +/- 1.8 for the red marrow, total body, and liver, respectively. One patient, with a highly elevated human anti-mouse antibody response from a prior OncoScint study (murine B72.3 IgG), received 3 injections of the hMN-14 without an adverse experience, and showed no evidence of altered biodistribution characteristic of mMAb-human anti-mouse antibody interactions. An antibody response to hMN-14 (HAhMN14) was not detected in patients who received only the hMN-14 (as many as three injections), but in three patients who received two injections of the mMN-14, a HAhMN14 response was detected. With similar, excellent targeting properties as the mMN-14 and the potential for reduced immunogenicity, hMN-14 is an attractive candidate for further clinical imaging and therapy applications.

Adult↗

PubMed1994

Investigation of shape variations in the antibody binding site by molecular dynamics computer simulation.

Molecular dynamics simulations have been used to investigate the flexibility and variations in the shape of the binding site of an antibody against human Rhinovirus serotype 2 (HRV2) and its complex with a 15 amino acid oligopeptide, the structure of which has been recently determined by X-ray crystallography. During the simulation of the unbound antibody the binding site, defined in terms of the hypervariable regions or complementarity determining regions (CDRs), shows significant fluctuations in shape. For the complex such variations in the shape of the binding site were reduced. The largest fluctuations in the unbound antibody occurred within the CDR-H3. The largest differences between the bound and unbound crystal structures are also associated with CDR-H3. The relative displacements of the loops have been analysed in terms of internal distortions, rigid body motions of the loops and changes with respect to the framework regions. The degree to which the motions of the loops are correlated and the variation in the volume of the binding pocket during the simulation have also been examined.

Amino Acid Sequence↗

PubMed2004

Remarkably similar antigen receptors among a subset of patients with chronic lymphocytic leukemia.

Studies of B cell antigen receptors (BCRs) expressed by leukemic lymphocytes from patients with B cell chronic lymphocytic leukemia (B-CLL) suggest that B lymphocytes with some level of BCR structural restriction become transformed. While analyzing rearranged V(H)DJ(H) and V(L)J(L) genes of 25 non-IgM-producing B-CLL cases, we found five IgG(+) cases that display strikingly similar BCRs (use of the same H- and L-chain V gene segments with unique, shared heavy chain third complementarity-determining region [HCDR3] and light chain third complementarity-determining region [LCDR3] motifs). These H- and L-chain characteristics were not identified in other B-CLL cases or in normal B lymphocytes whose sequences are available in the public databases. Three-dimensional modeling studies suggest that these BCRs could bind the same antigenic epitope. The structural features of the B-CLL BCRs resemble those of mAb's reactive with carbohydrate determinants of bacterial capsules or viral coats and with certain autoantigens. These findings suggest that the B lymphocytes that gave rise to these IgG(+) B-CLL cells were selected for this unique BCR structure. This selection could have occurred because the precursors of the B-CLL cells were chosen for their antigen-binding capabilities by antigen(s) of restricted nature and structure, or because the precursors derived from a B cell subpopulation with limited BCR heterogeneity, or both.

B-Lymphocytes↗

PubMed2007

Molecular analysis of TCR and peptide/MHC interaction using P18-I10-derived peptides with a single D-amino acid substitution.

For the structural analysis of T-cell receptor (TCR) and peptide/MHC interaction, a series of peptides with a single amino acid substitution by a corresponding D-amino acid, having the same weight, size, and charge, within P18-I10 (aa318-327: RGPGRAFVTI), an immunodominant epitope of HIV-1 IIIB envelope glycoprotein, restricted by the H-2Dd class I MHC molecule, has been synthesized. Using those peptides, we have observed that the replacement at positions 324F, 325V, 326T, and 327I with each corresponding D-amino acid induced marked reduction of the potency to sensitize targets for P18-I10-specific murine CD8+ cytotoxic T lymphocytes (CTLs), LINE-IIIB, recognition. To analyze further the role of amino acid at position 325, the most critical site for determining epitope specificity, we have developed a CTL line [LINE-IIIB(325D)] and its offspring clones specific for the epitope I-10(325v) having a D-valine (v) at position 325. Taking advantage of two distinct sets of CD8+ CTLs restricted by the same Dd, three-dimensional structural analysis on TCR and peptide/MHC complexes by molecular modeling was performed, which indicates that the critical amino acids within the TCRs for interacting with 325V or 325v appear to belong to the complementarity-determining region 1 but not to the complementarity-determining region 3 of Vbeta chain.

Amino Acid Substitution↗

PubMed1997

A Shannon entropy analysis of immunoglobulin and T cell receptor.

In 1970, before any antigen-bound immunoglobulin structure had been solved, Elvin Kabat proposed that regions of high amino acid diversity would be the antigen binding sites of immunoglobulin (Kabat, 1970). Conversely, sites of low variability were proposed to be structural, framework regions. This variability was defined by Wu and Kabat as the number of different amino acids found at a site divided by the relative frequency of the most common amino acid at that site (Wu and Kabat, 1970). Several groups have subsequently devised improvements of Kabat-Wu variability analysis (Litwin and Jores, 1992). While these methods are somewhat better than Kabat-Wu, they still suffer from Kabat-Wu's basic limitation: they account for only the most common one or two amino acids in estimating diversity. This leads to underestimates of low diversities and exaggerations of high diversities. Shannon information analysis eliminates serious bias and is more stable than Kabat-Wu and second generation measures of diversity (Jores et al. 1990; Wu and Kabat, 1970). Statistical reliability can be measured using Shannon analysis, and Shannon measurements can be provided with error estimates. Here we use Shannon's method to analyze the amino acid diversity at each site of T cell receptor Valpha and Vbeta to identify complementarity determining regions and framework sites. Our results reveal that the T cell receptor is significantly more diverse than immunoglobulin-suggesting T cell receptor has more than the previously-discovered four complementarity determining regions. These new complementarity determining regions may represent a larger antigen combining site, additional combining sites, or an evolutionary strategy to avoid inappropriate interaction with other molecules.

Animals↗

PubMed1997

Evidence for cooperation between TCR V region and junctional sequences in determining a dominant cytotoxic T lymphocyte response to herpes simplex virus glycoprotein B.

TCR repertoire availability has the potential to influence the immune response to foreign antigens. Here we have analysed how changes in V region availability influence the H-2b-restricted cytotoxic T lymphocyte (CTL) response to a dominant peptide determinant derived from the herpes simplex virus glycoprotein B (gB). We have previously shown that C57BL/6 mice mount a gB-specific, Kb-restricted CTL response which is dominated by a TCRBV10+ population and a TCRBV8S1+ subpopulation, both containing highly conserved CDR3 elements. We find that this dominant gB-specific CTL pool is lost in C57/L mice which have a different TCRBV haplotype. A population of CTL with diverse TCRBV and junctional sequence usage, which otherwise represents a minor subset in the gB-specific response, appears to emerge as a consequence of this TCRBV gene variation. The loss of preferential V region-encoded complementarity determining regions (CDR) 1- and/or CDR2-ligand interactions in this emerging population also results in a change in CDR3 sequence usage and a corresponding focusing of an otherwise promiscuous pattern of cross-reactivity with a panel of gB498-505 substitution analogues. This suggests that the difference between the two distinct TCR populations is the relative contributions of the CDR towards ligand recognition. Therefore, preferential V region-ligand interaction, at the expense of CDR3 peptide recognition, appears to control the dominant TCR selection in the C57BL/6 response to this peptide determinant.

Amino Acid Sequence↗

PubMed2006

A synthetic camel anti-lysozyme peptide antibody (peptibody) with flexible loop structure identified by high-resolution affinity mass spectrometry.

We describe the synthesis and characterisation of the fully functional molecular recognition structure of a 26-amino acid residue peptide antibody, referred to as peptibody, designed from a monoclonal single-domain antibody fragment derived from a camel heavy-chain antibody. The CDR3 region (CDR = complementarity determining region) of the cAbLys3 camel antibody fragment, which binds to the active site of hen eggwhite lysozyme (HEL) and acts as a potent enzyme inhibitor by mimicking an oligosaccharide substrate, was prepared by solid-phase peptide synthesis. To obtain a closed loop-like structure resembling that in the crystal structure, N- and C-terminal cysteine residues were added to the linear peptide and oxidised to a cyclic disulfide-bridged peptide by using dimethylsulfoxide. A further, internal cysteine-12 residue was acetamidomethyl-protected to prevent possible oxidative byproducts. Affinity separation on a lysozyme microcolumn combined with MALDI-TOF mass spectrometry revealed that the peptide resumed high affinity to lysozyme only after deprotection of Cys-12, suggesting the importance of this paratope sequence for epitope recognition. The complex of lysozyme and active peptibody was characterised directly by conducting high-resolution ESI-FTICR mass spectrometry, which provided a molecular comparison of affinities for linear and cyclic peptibodies.

Amino Acid Sequence↗

PubMed1994

A skewed distribution of amino acids at recognition sites of the hypervariable region of immunoglobulins.

Antibody binding site are formed by six hypervariable regions or complementarity determining regions (CDRs). The CDRs, three from the heavy chain and three from the light chain, are known as hypervariable segments and provide a surface complementary to that of the epitope. In recent work it was found that the amino acids in these positions fulfill different functions: Some play a structural role and others are involved in the specificity-determining function. It is reported here that the frequency of amino acids at hypervariable sites is skewed. By means of an informational algorithm, key physicochemical attributes of the dominant residues were identified for some of those sites. The results for about 1,500 antibodies suggest that approximately 35% of sites involved in the recognition process require only general properties such as composition, volume, and bulk or hydrogen bonding which are satisfied by a small set of amino acids instead of any one particular complementary amino acid.

Algorithms↗

PubMed2000

Construction and analysis of three-dimensional graphic model of single-chain Fv derived from an anti-human placental acidic isoferritin monoclonal antibody by computer.

A three-dimensional (3D) graphic model of a single-chain Fv (scFv) which was derived from an anti-human placental acidic isoferritin (PAF) monoclonal antibody (MAb) was constructed by a homologous protein-predicting computer algorithm on Silicon graphic computer station. The structure, surface static electricity and hydrophobicity of scFv were investigated. Computer graphic modelling indicated that all regions of scFv including the inker, variable regions of the heavy (VH) and light (VL) chains were suitable. The VH region and the VL region were involved in composing the "hydrophobic pocket". The linker was drifted away VH and VL regions. The complementarity determining regions (CDRs) of VH and VL regions surrounded the "hydrophobic pocket". This study provides a theory basis for improving antibody affinity, investigating antibody structure and analyzing the functions of VH and VL regions in antibody activity.

Amino Acid Sequence↗

PubMed2005

The arginine-rich carboxy-terminal domain of the hepatitis B virus core protein mediates attachment of nucleocapsids to cell-surface-expressed heparan sulfate.

Binding of hepatitis B virus nucleocapsids to mouse B cells leads to production of nucleocapsid-specific antibodies, class II presentation of peptides and the generation of T helper-1 immunity. This T-cell-independent activation of B cells is thought to result from cross-linking of cell-surface immunoglobulin molecules, if these contain a specific motif in the framework region 1-complementarity determining region 1 junction. In the present study, it was observed that nucleocapsids bound to different B-cell lines, an interaction that was not dependent on cell-surface-expressed immunoglobulins. Furthermore, binding to several non-B-cell lines was observed. Capsids that lacked the carboxy-terminal protamine-like domains did not bind to cells. Treatment of nucleocapsids with ribonucleases enhanced the attachment of nucleocapsids to cells. Various soluble glycosaminoglycans inhibited attachment of nucleocapsids, while treatment of cells with heparinase I also reduced binding. These observations demonstrated that the arginine-rich protamine-like regions of the core proteins are responsible for the attachment of nucleocapsids to glycosaminoglycans expressed on the plasma membranes of cells.

Animals↗

PubMed1990

[Production of antibodies with the aid of genetic technology].

During the past decade we have witnessed a rapid development of methods for the production of monoclonal antibodies with predefined specificity. These antibodies are now regularly being used both for diagnostic and therapeutic purposes. The first antibodies thus produced were mainly from cell lines generated by fusion of a mouse B lymphocyte with a given specificity with a mouse myeloma line. Efforts have lately been made also to make cell lines producing human antibodies either by immortalizing human cells or by "humanizing" mouse antibodies. The latter has recently been accomplished by genetic engineering. In these experiments the entire variable region of a mouse antibody or the hypervariable regions only (complementarity determining regions) have been grafted onto human constant region genes thus creating chimeric antibodies. Fragments of antibodies with retained binding properties have also been generated and some of these have been effectively produced in bacteria, allowing large scale production. Bifunctional antibodies have also been created with success and have been applied in different systems. The ease with which antibodies with desired specificities can now be designed and produced will form the beginning of a new era in immunology with a potential that we can not as yet foresee.

Animals↗

PubMed2000

Crystal structure of an antibody bound to an immunodominant peptide epitope: novel features in peptide-antibody recognition.

The crystal structure of Fab of an Ab PC283 complexed with its corresponding peptide Ag, PS1 (HQLDPAFGANSTNPD), derived from the hepatitis B virus surface Ag was determined. The PS1 stretch Gln2P to Phe7P is present in the Ag binding site of the Ab, while the next three residues of the peptide are raised above the binding groove. The residues Ser11P, Thr12P, and Asn13P then loop back onto the Ag-binding site of the Ab. The last two residues, Pro14P and Asp15P, extend outside the binding site without forming any contacts with the Ab. The PC283-PS1 complex is among the few examples where the light chain complementarity-determining regions show more interactions than the heavy chain complementarity-determining regions, and a distal framework residue is involved in Ag binding. As seen from the crystal structure, most of the contacts between peptide and Ab are through the five residues, Leu3-Asp4-Pro5-Ala6-Phe7, of PS1. The paratope is predominantly hydrophobic with aromatic residues lining the binding pocket, although a salt bridge also contributes to stabilizing the Ag-Ab interaction. The molecular surface area buried upon PS1 binding is 756 A(2) for the peptide and 625 A(2) for the Fab, which is higher than what has been seen to date for Ab-peptide complexes. A comparison between PC283 structure and a homology model of its germline ancestor suggests that paratope optimization for PS1 occurs by improving both charge and shape complementarity.

Amino Acid Sequence↗

PubMed2000

Immunoglobulin variable-region diversity in the zebrafish.

As part of an investigation of the immune system in the developing zebrafish, Danio rerio, we cloned and characterized a genomic V(H) segment and a number of cDNAs encoding IgM heavy chains. The genomic V(H) has the characteristic features of V(H) in other vertebrates, including a leader segment interrupted by a short intron, and regulatory and recombination signal sequences. The V(H) sequences of 75 cDNA clones fell into four distinct groups or families. All of these families cluster most closely with other teleost V(H); one of the families, V(H)3, is also closely related to other vertebrate V(H). Analysis by Southern blotting suggests that there are a number of germline genes corresponding to each family. Most variability within the V(H)1 family is confined to codons 30-32, at the end of the first framework region and in the beginning of the first complementarity-determining region. The nucleotide sequence of the genomic V(H) in this region contains motifs associated with a relatively high frequency of somatic mutation. There is much variability in segments of the clones corresponding to the third complementarity-determining region. The cDNA sequences encoding the fourth framework region are consistent with the contribution of four distinct J(H) segments.

Amino Acid Sequence↗

PubMed2002

The complementarity-determining region-like loops of CD8 alpha interact differently with beta 2-microglobulin of the class I molecules H-2Kb and thymic leukemia antigen, while similarly with their alpha 3 domains.

The murine CD8 glycoprotein interacts with both classical MHC class I molecules and some nonclassical molecules, including the thymic leukemia Ag (TL). TL binds preferentially to CD8alphaalpha homodimers with a 10-fold higher affinity than H-2K(b) class I molecules. To understand the molecular basis for this difference, we created a panel of CD8alpha mutants and tested the ability of the CD8alphaalpha homodimers to bind to H-2K(b) tetramers and TL tetramers. Mutations in three CD8 residues located on the complementarity-determining region-like loops contacting the negatively charged loop in the alpha3 domain of MHC class I greatly reduced binding to both tetramers. Because TL and H-2K(b) class I sequences are highly conserved in the alpha3 domain of MHC class I, this suggests that CD8 contacts the alpha3 domain of TL and H-2K(b) in a similar manner. In contrast, mutations in residues on the A and B beta strands of CD8 that are involved in contact with beta(2)-microglobulin affected interaction with the H-2K(b) tetramer, but not the TL tetramer. Therefore, the orientation of interaction of TL with CD8 appears to be different from that of H-2K(b). The unique high affinity binding of TL with CD8alphaalpha is most likely a result of amino acid differences in the alpha3 domain between TL and H-2K(b), particularly at positions 198 (K to D) and 228 (M to T), which are contact residues in the CD8alphaalpha-H-2K(b) cocrystal.

Amino Acid Sequence↗

PubMed2005

Unconventional topology of self peptide-major histocompatibility complex binding by a human autoimmune T cell receptor.

Autoimmune diseases are caused by self-reactive lymphocytes that have escaped deletion. Here we have determined the structure of the trimolecular complex for a T cell receptor (TCR) from a patient with multiple sclerosis that causes autoimmunity in transgenic mice. The structure showed a TCR topology notably different from that of antimicrobial TCRs. Rather than being centered on the peptide-major histocompatibility complex, this TCR contacted only the N-terminal peptide segment and made asymmetrical interactions with the major histocompatibility complex helices. The interaction was dominated by the hypervariable complementarity-determining region 3 loops, indicating that unconventional topologies are possible because of the unique complementarity-determining region 3 sequences created during rearrangement. This topology reduces the interaction surface with peptide and alters the geometry for CD4 association. We propose that unusual TCR-binding properties can permit autoreactive T cells to escape deletion.

Amino Acid Sequence↗

PubMed1990

Structural characterization of idiotopes by using antibody variants generated by site-directed mutagenesis.

Four anti-idiotopic mAB, 107, MB, AI, and AD8, react with mouse hybridoma protein 36-65 specific for the hapten p-azophenylarsonate. The four antiidiotypic antibodies do not react with hybridoma protein 36-71, a somatically mutated variant of 36-65 whose H and L chain V region sequence differs at 19 amino acid positions. To determine which regions of 36-65 are important for the interaction with each of the four anti-idiotypic antibodies, variants of 36-65 containing one or more of the 36-71 substitutions were generated by oligonucleotide-directed mutagenesis of the rearranged 36-65 H chain V region gene, followed by expression of mutant proteins containing either the 36-65 or the 36-71 L chain in transfected hybridoma cells. Idiotypic characterization of the mutant proteins showed that reactivity correlates with the 36-65 H chain, but some contributions from the 36-65 L chain come into play. In the 36-65 H chain V region, idiotopes were mapped to the first and third complementarity-determining regions for anti-idiotypic antibodies 107, MB, and AI, and to all three complementarity-determining regions for anti-idiotypic antibody AD8. The binding of all four anti-idiotypic antibodies to hybridoma protein 36-65 was hapten inhibitable. However, a comparison between the effect of individual 36-71 substitutions on idiotope expression and their effect on Ag-binding affinity suggests that none of the four anti-idiotypic antibodies bodies mimics the structure of Ag.

Amino Acid Sequence↗