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Results for “Complementarity Determining Regions”

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PubMed1994

Control of autoantibody affinity by selection against amino acid replacements in the complementarity-determining regions.

Rheumatoid factor (RF) autoantibodies can be produced in healthy individuals after infections or immunizations and thus escape normal tolerization mechanisms. It has not been clear whether such autoantibodies can undergo somatic hypermutation and affinity maturation similar to antibodies to exogenous antigens. We have investigated how these autoantibodies are regulated in normal individuals by analyzing the sequences of monoclonal IgM RFs obtained as hybridomas from donors after immunization. The variable regions undergo extensive hypermutation, but in contrast to antibodies against exogenous antigens, there is a strong selection against mutations that result in replacement of amino acids in the hypervariable, or complementarity-determining, regions. Furthermore, we found no increase in affinity of these RFs with the accumulation of mutations. This suggests that high-affinity variants are tolerized during the hypermutation process and there is a peripheral mechanism operating on certain autoreactive B cells that, while not deleting or anergizing all autoreactive cells, prevents the generation of high-affinity autoantibodies. Comparison of RFs by using the VH1 DP-10 heavy chain variable region segment from both normal individuals and rheumatoid arthritis (RA) patients suggests that RF from RA patients may not be subject to such a controlling mechanism.

Antibody Affinity↗

PubMed1998

Prophylactic administration of a complementarity-determining region derived from a neutralizing monoclonal antibody is effective against respiratory syncytial virus infection in BALB/c mice.

Immunotherapy with antibodies against respiratory syncytial virus (RSV) is a treatment option given the absence of any vaccine or other available satisfactory treatment. We selected one of our monoclonal antibodies, RS-348, that is highly neutralizing. We showed that a single peptide (PEP3H) derived from complementarity-determining region 3 (CDR3) of its heavy chain was capable of neutralizing the virus in vitro. When intranasally administered 24 h before challenge, this peptide protected BALB/c mice against RSV lung infection. These results indicate that a single CDR can be effective against RSV infection.

Amino Acid Sequence↗

PubMed1999

TCR reactivity in human nickel allergy indicates contacts with complementarity-determining region 3 but excludes superantigen-like recognition.

Nickel is the most common inducer of contact sensitivity in humans. We previously found that overrepresentation of the TCRBV17 element in Ni-induced CD4+ T cell lines of Ni-allergic patients relates to the severity of the disease. Amino acid sequences of these beta-chains suggested hypothetical contact points for Ni2+ ions in complementarity-determining region (CDR) 1 and CDR3. To specifically address the molecular requirements for Ni recognition by TCR, human TCR alpha- and beta-chains of VB17+ Ni-reactive T cell clones were functionally expressed together with the human CD4 coreceptor in a mouse T cell hybridoma. Loss of CD4 revealed complete CD4 independence for one of the TCR studied. Putative TCR/Ni contact points were tested by pairing of TCR chains from different clones, also with different specificity. TCRBV17 chains with different J regions, but similar CDR3 regions, could be functionally exchanged. Larger differences in the CDR3 region were not tolerated. Specific combinations of alpha- and beta-chains were required, excluding a superantigen-like activation by Ni. Mutation of amino acids in CDR1 of TCRBV17 did not affect Ag recognition, superantigen activation, or HLA restriction. In contrast, mutation of Arg95 or Asp96, conserved in many CDR3B sequences of Ni-specific, VB17+ TCR, abrogated Ni recognition. These results define specific amino acids in the CDR3B region of a VB17+ TCR to be crucial for human nickel recognition. CD4 independence implies a high affinity of such receptor types for the Ni/MHC complex. This may point to a dominant role of T cells bearing such receptors in the pathology of contact dermatitis.

Amino Acid Sequence↗

PubMed1997

Modulation of murine systemic lupus erythematosus with peptides based on complementarity determining regions of a pathogenic anti-DNA monoclonal antibody.

Experimental systemic lupus erythematosus (SLE) can be induced in naive mice by immunization with a murine monoclonal anti-DNA antibody (mAb), 5G12, that bears a major idiotype designated 16/6 Id. Strain-dependent differences were observed in the proliferative responses of lymph node cells of mice immunized with two peptides based on the sequences of the complementarity determining region (CDR) 1 and 3 of mAb 5G12. The capacity of the peptides to bind to major histocompatibility complex class II molecules correlated with the proliferative responses. Immunization of high responder strains with the CDR-based peptides led to production of autoantibodies and clinical manifestations characteristic to experimental SLE. The CDR-based peptides could prevent autoantibody production in neonatal mice that were immunized later either with the peptide or with the pathogenic autoantibody. Furthermore, the peptides inhibited specific proliferation of lymph node cells of mice immunized with the same peptide, with mAb 5G12 or with the human mAb anti-DNA, 16/6 Id. Thus, the CDR-based peptides are potential candidates for therapy of SLE.

Amino Acid Sequence↗

PubMed1997

A complementarity-determining region peptide of an anti-desmosome autoantibody may interact with the desmosomal plaque through molecular mimicry with a cytoplasmic desmoglein 1 sequence.

The sera of patients with pemphigus, a group of autoimmune blistering skin diseases, contain autoantibodies directed against components of adhering junctions termed desmosomes. F12, a human monoclonal antibody derived from a pemphigus patient, recognizes an unknown polypeptide of the desmosomal and hemidesmosomal plaques. The third complementarity-determining region of the F12 heavy chain (VH-CDR3) was shown to share a four-amino-acid sequence (GSSG) with the intracellular domains of desmoglein 1 and bullous pemphigoid antigen 2 which interact with components of, respectively, the desmosomal and hemidesmosomal plaques. Computer modeling of F12 showed that the GSSG sequence protudes inside the antigen-combining site and thus might be involved in antigen interactions. The GSSG sequence is essential to F12 function, since a peptide containing the VH-CDR3 inhibited its binding to target antigens while VH-CDR3 peptides with specific modifications of the GSSG sequence did not. These data allow us to hypothesize that certain autoantibodies produced during the course of an autoimmune disease can behave as adhesion molecules, through the molecular mimicry of the motif involved in protein/protein adhesion, and to propose a new self-antigen binding mechanism for some autoantibodies.

Amino Acid Sequence↗

PubMed1996

Third complementarity determining region sequence analysis of low-grade bronchus-associated lymphoid tissue lymphoma: genotypic analysis reveals heterogeneity in maturation.

It has recently been suggested that autoimmunity may play a role in the pathogenesis of low-grade mucosa-associated lymphoid tissue lymphomas, but precise genotypic analysis of antigen binding sites of low-grade bronchus-associated lymphoid tissue (BALT) lymphomas has not been reported. We analyzed the third complementarity determining region (CDR3) in eight cases of low-grade BALT lymphoma by 2-step PCR and sequencing analysis. All cases showed a distinct monoclonal band (about 100 base pairs) on electrophoresis. In seven cases, a single major CDR3 sequence was identified with five to nine clones among 10 vector clones being identical; and in the remaining case, two major sequences were obtained, with four clones among nine vector clones being identical. Findings suggestive of the autoimmunity of low-grade BALT lymphoma were obtained: (1) VHDJH rearrangements in seven of the eight lymphoma cell clones were potentially functional; (2) genotypically, two lymphoma cell clones showed 60 to 74% homology with G6 positive lymphocyte clones; and (3) five lymphoma cell clones showed 61 to 71 % homology with lymphocyte clones derived from fetal liver or cord blood. In one case, the N-D-N length of the neoplastic clone was very short and lacked N nucleotides at the D-JH junction. Therefore, our study demonstrates genotypic heterogeneity in maturation in low-grade BALT lymphoma.

Adult↗

PubMed1998

Peptides derived from the complementarity-determining regions of anti-Mac-1 antibodies block intercellular adhesion molecule-1 interaction with Mac-1.

Peptides or small molecules that can block the interaction of the integrin Mac-1 with its receptor, intercellular adhesion molecule-1 (ICAM-1), have not previously been developed. We studied this interaction by measuring the adherence of ICAM-1-expressing Chinese hamster ovary (CHO) cells to immobilized, purified Mac-1. Nucleotide sequence information was obtained for the complementarity determining regions (CDRs) of three antibodies (44aacb, MY904, and 118.1) shown to block Mac-1-mediated cell adherence. Peptides were synthesized based on the predicted amino acid sequences of the CDRs and tested for the ability to block cell adhesion to Mac-1. Peptides derived from CDR1 of 44aacb, CDR2 of 118.1, and CDRs 1 and 3 of MY904 heavy chains were found to possess blocking activity at 10-100 muM. This may indicate that one or two CDRs contribute disproportionately to the antibody binding affinity. The binding of ligands to Mac-1 has been shown to require a region of the alpha-chain known as the I- or A-domain. We have recombinantly produced Mac-1 I-domain, and show that it is also capable of supporting the adherence of ICAM-1-expressing CHO cells. The adherence of ICAM-1-CHO cells to the I-domain is inhibited by 44aacb and 118.1 and by the CDR peptides from 44aacb and 118.1. By using phage display of peptide libraries based on the 118.1 CDR peptide with five residues randomized, we were able to identify a novel peptide inhibitor of Mac-1 with substitutions at all five positions. These peptides provide lead structures for development of Mac-1 antagonists.

Amino Acid Sequence↗

PubMed1998

Changes in the specificity of antibodies against steroid antigens by introduction of mutations into complementarity-determining regions of the V(H) domain.

An attempt was made to change the specificity of antibodies (Abs) by introduction of mutations. A monoclonal Ab specific for 17alpha-hydroxyprogesterone (17-OHP) was used as the starting Ab. On the basis of a model that was generated by a computer-driven model-building system, we constructed a phage-display library of Abs in which 16 residues were mutated in three complementarity-determining regions of the heavy chain that appeared to form the steroid-binding pocket. We screened the library with 17-OHP and cortisol that had been conjugated with bovine serum albumin, and we isolated many clones that had retained 17-OHP-binding ability as well as clones with the newly developed ability to bind cortisol in addition to 17-OHP. We compared the amino acid sequences between 17-OHP-specific and cortisol-binding Abs, and then constructed several additional Abs. Our results indicated that a change in specificity could be achieved by changing only a single, critical amino acid residue. Models of the 17-OHP-and cortisol-binding pockets formed by the mutated Abs could explain these observations.

17-alpha-Hydroxyprogesterone↗

PubMed1993

Complementarity-determining region 2 is implicated in the binding of staphylococcal protein A to human immunoglobulin VHIII variable regions.

Staphylococcal protein A (SPA) has two distinct binding sites on human immunoglobulins. In addition to binding to the Fc region of most IgG molecules, an "alternative" binding site has been localized to the Fab region of human immunoglobulins encoded by heavy chain variable gene segments belonging to the VHIII family. Comparison of amino acid sequences of closely related SPA-binding and -non-binding proteins suggested that VHIII-specific residues in the second complementarity-determining region (CDR2) were likely responsible for SPA binding activity. Site-directed mutagenesis of a single amino acid residue in CDR2 converted an IgM rheumatoid factor which did not bind SPA to an SPA binder. These findings, therefore, locate a critical site involved in SPA binding to the CDR2 of human immunoglobulins encoded by VHIII family gene segments.

Amino Acid Sequence↗

PubMed1995

Predicted complementarity determining regions of the T cell antigen receptor determine antigen specificity.

The antigen receptor on T cells (TCR) has been predicted to have a structure similar to a membrane-anchored form of an immunoglobulin F(ab) fragment. Virtually all of the conserved amino acids that are important for inter- and intramolecular interactions in the VH-VL pair are also conserved in the TCR V alpha and V beta chains. A molecular model of the TCR has been constructed by homology and we have used the information from this, as well as the earlier structural predictions of others, to study the basis for specificity. Specifically, regions of a TCR cloned from an antigen-specific T cell were stitched into the corresponding framework of a second TCR. Results indicate that the substitution of amino acid sequences corresponding to the complementarity determining regions (CDRs) of immunoglobulin can convey the specificity for antigen and major histocompatibility complex molecules. These data are consistent with a role, but not an exclusive role, for CDR3 in antigen peptide recognition.

Amino Acid Sequence↗

PubMed1995

Comparable profiles of the immunoglobulin heavy chain complementarity determining region (CDR)-3 in CD5+ and CD5- human cord blood B lymphocytes.

It has been suggested that CD5+ B cells are closely related to the pathogenesis of autoimmune disease or chronic lymphocytic leukaemia, whereas the origin and physiological role of CD5+ B cells remain controversial. To study the molecular differences between CD5+ and CD5- B cells in terms of immunoglobulin gene structure, we sorted both subsets from new-born cord blood and analysed the complementarity determining region (CDR)-3 profiles of the immunoglobulin heavy chain (IgH) gene. The CDR-3 sequences from both CD5+ and CD5- B cells represented the same incidence and length of N-region, and the same usage of D and JH segments. When translated into amino acids, 24 of 32 clones (75%) and 25 of 37 clones (65.8%) from their respective subset were productive, and the composition of the deduced amino acids were similar between both subsets. These data suggest that CD5+ B cells are not a distinct lineage presenting a biased immunoglobulin repertoire in B cells.

Amino Acid Sequence↗

PubMed1997

T cells recognize the VH complementarity-determining region 3 of the idiotypic protein of B cell non-Hodgkin's lymphoma.

The idiotypic protein expressed by B lymphoma cells is a clone-specific tumor antigen which may be suitable for immune targeting by T cells. In this study, we cloned the immunoglobulin heavy chain variable gene (VH) of the idiotypic protein from a patient with B cell lymphoma and used a synthetic peptide of 22 amino acids corresponding to the VH complementarity-determining region (CDR)-3 of the idiotypic protein to investigate whether autologous T cells could recognize this unique peptide. We demonstrated that autologous T cells possessing both CD4+ and CD8+ phenotypes could be propagated. The T cells were able to proliferate, secrete cytokines, and lyse autologous cells presensitized with the specific peptide in a major histocompatibility complex-dependent manner. Moreover, these CDR3 peptide-primed T cells were also able to kill autologous lymphoma cells. Our results therefore offer new perspectives for specific therapeutic vaccination for the treatment of B cell lymphoma.

Amino Acid Sequence↗

PubMed1991

Analysis of T cell receptor beta chains in Lewis rats with experimental allergic encephalomyelitis: conserved complementarity determining region 3.

This study explores the usage of T cell antigen receptor (TCR) beta chain elements in Lewis rats with experimentally induced allergic encephalomyelitis (EAE). TCRs from 15 different T cell clones and hybridomas derived from animals immunized with myelin basic protein (MBP), and all having specificity for the 21-mer encephalitogenic fragment MBP 68-88, utilized V beta 8.2. In addition, there was a marked conservation of the first two amino acid residues of the junctional complementarity determining region 3 (CDR3) associated with the V beta 8.2 receptors. 12 of 15 contained an aspartic acid followed by serine regardless of the associated J beta element. At the nucleotide level, this conservation of AspSer residues was accomplished with few or no nongermline-encoded nucleotide (N) additions. A similar pattern of AspSer usage and N region nucleotide additions was observed in a number of V beta 8.2 isolates derived from MBP-immunized lymph nodes. In contrast, V beta 8.2 polymerase chain reaction amplified isolates from Lewis T cells activated with concanavalin A or from lymph nodes of complete Freund's adjuvant-immunized animals showed no AspSer utilization (0/31) in the CDR3, and four to nine N region nucleotide additions. We conclude from this finding that AspSer residues in the CDR3, limited N region nucleotide additions, along with V beta 8.2 sequences, contribute to TCR specificity for MBP 68-88. This raises the possibility that encephalitogenic, disease-causing T cells either represent a population that derives from late fetal life or alternatively, that they are rare cells with this particular TCR phenotype contributed to the T cell pool throughout adulthood and are selected by antigen. In either case, the CDR3 AspSer sequences as well as V beta 8.2 sequences are candidates for the receptor target structures recognized by regulator T cells in recovery from and resistance to active EAE. In this respect, a preliminary analysis of TCR utilization in three T cell clones specific for MBP 68-88 isolated from animals recovered from active EAE indicates that while all three use V beta 8.2, only one contains AspSer in the CDR3.

Amino Acid Sequence↗

PubMed1999

Effects of complementarity determining region mutations on the affinity of an alpha/beta T cell receptor: measuring the energy associated with CD4/CD8 repertoire skewing.

It has been proposed that the generally low affinities of T cell receptors (TCRs) for their peptide-major histocompatibility complex (pMHC) ligands (Kd approximately 10(-4) to 10(-7) M) are the result of biological selection rather than an intrinsic affinity limitation imposed by the TCR framework. Using a soluble version of the 2C TCR, we have used complementarity determining region (CDR)-directed mutagenesis to investigate whether the affinity of this receptor for its allogeneic pMHC ligand can be improved upon. We report that several mutants at positions lying within CDR3alpha and CDR2beta showed increased affinities for pMHC compared with the wild-type receptor. Additionally, we have investigated whether Valpha mutations that have been implicated in the phenomenon of CD8(+) repertoire skewing achieve this skewing by means of generalized increases in affinity for MHC-I molecules. Two mutants (S27F and S51P), which each promote skewing toward a CD8(+) phenotype, exhibited significantly reduced affinity for pMHC-I, consistent with a quantitative-instructional model of CD4/CD8 lineage commitment. This model predicts that CD8 is downregulated on thymocytes that have TCR-ligand interactions above a minimal energy threshold. Together, the results (a) demonstrate that engineering higher affinity TCRs is feasible, and (b) provide TCR-pMHC energy values associated with CD4/CD8 repertoire skewing.

Alanine↗

PubMedDate not supplied

Breast cancer imaging with radiolabelled peptide from complementarity-determining region of antitumour antibody.

Specific tumour imaging with radiolabelled monoclonal antibodies has been extensively investigated. Although some success has been reported, there are many limitations due to the slow kinetics, poor extravasation, catabolism by the reticuloendothelial system, and non-specific uptake of macromolecules such as antibodies. We have tried to overcome some of the problems associated with monoclonal antibodies while retaining their specificity by using an antibody-derived synthetic peptide. A synthetic pentadecapeptide (alpha M2) derived from the third heavy-chain complementarity-determining region (CDR-3H) of a tumour-associated monoclonal antibody was produced and shown to retain its specificity against the pan-carcinoma cell-surface antigen, polymorphic epithelial mucin, detected by the parent antibody. The peptide was radiolabelled with technetium-99m and injected intravenously to image malignant lesions in 26 women with primary, recurrent, or metastatic breast cancer. Visualisation of breast tumours and their metastases was obtained shortly after administration of alpha M2, and was optimum by 3 h. Overall, 57 (77%) of 74 sites were visualised. Successful imaging was achieved in 14 of 15 primary tumour sites and all of eight local recurrences. Five of six metastases in the opposite breast, eight of 15 metastatic axillary lymph nodes, and all of six metastatic supraclavicular lymph nodes were imaged. Metastatic sites in the lungs, mediastinum, chest wall, and liver were poorly visualised because of background cardiac blood pool. alpha M2 detected small lesions ( < 2 cm) as efficiently as larger ones. The peptide was rapidly (3 h) cleared from the circulation. No acute or chronic adverse reactions due to the alpha M2 were observed. Specific tumour targeting with the radiolabelled anticancer peptide alpha M2 offers new opportunities for breast cancer imaging and possibly therapy.

Adult↗

PubMed1994

Fv structure of monoclonal antibody II-481 against herpes simplex virus Fc gamma-binding glycoprotein gE contains immunodominant complementarity determining region epitopes that react with human immunoglobulin M rheumatoid factors.

Human immunoglobulin M (IgM) rheumatoid factors (RFs) show primary direct enzyme-linked immunosorbent assay (ELISA) reactivity with Fab rather than Fc fragments of monoclonal antibody (mAb) II-481 directed against the Fc gamma-binding site of herpes simplex virus glycoprotein gE. This preferential anti-Fab specificity suggests that RFs react with antigen-binding portions of mAb II-481 as anti-idiotypic antibodies directed at the combining site regions of mAb reacting with the Fc gamma-binding region of gE. Analysis of this idiotype-anti-idiotype reaction employed polymerase chain reaction amplification and sequencing of the variable heavy and light (VH and VL) regions of mAb II-481. When VH and VL regions of mAb II-481 were synthesized as overlapping 7-mer peptides on polypropylene pins, a panel of 10 polyclonal and 6 monoclonal human IgM RFs reacted primarily with epitopes within the three solvent-exposed mAb II-481 complementarity determining regions (CDRs). Preincubation of single CDR heptamer peptides with IgM RFs in free solution, resulted in 63-100% inhibition of RF binding to mAb II-481 on the ELISA plate, confirming the antigenic importance of linear CDR regions for RF reactivity. Combinations of two or three CDR peptides frequently produced 94-100% inhibition of RF binding to whole mAb II-481. Control peptides, singly or in combination, showed no inhibition. Computer modeling suggested that the RF-reactive mAb II-481 Fv region and a previously demonstrated RF-reactive CH3 epitope displayed considerable three-dimensional similarities in conformation. These studies may provide insight into limited shape homologies possibly involved in an RF anti-idiotypic reaction.

Amino Acid Sequence↗

PubMed2000

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.

Amino Acid Sequence↗

PubMed1999

Conserved T cell receptor complementarity-determining region 3 of ocular T cells in mice with experimental autoimmune uveoretinitis.

PURPOSE: To characterize T cells infiltrating into the ocular tissues of mice with experimental autoimmune uveoretinitis (EAU) immunized with interphotoreceptor retinoid-binding protein (IRBP). METHODS: The T cell receptor (TCR) repertoire on T cells obtained from ocular lesions of EAU mice was analyzed by reverse transcriptase-polymerase chain reaction. The clonotype of the T cells was examined by the single-strand conformation polymorphism (SSCP) method, followed by sequence analysis of the TCR beta-chain complementarity-determining region 3 (CDR3). RESULTS: The repertoire of the TCR BV gene in T cells from inflamed lesions was heterogeneous. SSCP analysis showed accumulation of multiple T cells specifically in ocular tissues of EAU mice, suggesting that these cells were expanded by an antigen-driven stimulation. Junctional sequence analysis demonstrated the presence of highly conserved amino acid sequence motifs (AGTGG, AGD) in CDR3 of BV2-positive T cells. CONCLUSIONS: Our findings suggest that T cells infiltrating into ocular lesions of EAU mice recognize restricted T cell epitopes of IRBP, resulting in autoimmune uveoretinitis.

Amino Acid Sequence↗