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Results for “Complementarity Determining Regions”

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PubMedDate not supplied

Replacing the complementarity-determining regions in a human antibody with those from a mouse.

The variable domains of an antibody consist of a beta-sheet framework with hypervariable regions (or complementarity-determining regions--CDRs) which fashion the antigen-binding site. Here we attempted to determine whether the antigen-binding site could be transplanted from one framework to another by grafting the CDRs. We substituted the CDRs from the heavy-chain variable region of mouse antibody B1-8, which binds the hapten NP-cap (4-hydroxy-3-nitrophenacetyl caproic acid; KNP-cap = 1.2 microM), for the corresponding CDRs of a human myeloma protein. We report that in combination with the B1-8 mouse light chain, the new antibody has acquired the hapten affinity of the B1-8 antibody (KNP-cap = 1.9 microM). Such 'CDR replacement' may offer a means of constructing human monoclonal antibodies from the corresponding mouse monoclonal antibodies.

Amino Acid Sequence↗

PubMed1989

Detection of minimal disease in hematopoietic malignancies of the B-cell lineage by using third-complementarity-determining region (CDR-III)-specific probes.

Approximately 80% of hematopoietic malignancies of the B-cell lineage carry only one or two immunoglobulin heavy chain gene rearrangements indicating their clonal origin. These rearrangements due to the recombination of various variable, diversity, and joining regions of the heavy-chain gene segments during B-cell commitment result in a region called complementarity-determining region III (CDR-III). This region, which encompasses the diversity region of the heavy-chain segment, because of extensive somatic mutations, provides a DNA-encoded signature specific for each B-cell clone. CDR-III sequences were obtained from DNA of pre-B-cell acute lymphoblastic leukemia by using suitable primers and the polymerase chain reaction. The sequences were used to generate diagnostic probes that hybridized only to the amplified CDR-III of leukemic cells from which the sequences were derived. With these probes, leukemic cells could be detected when diluted 1:10,000 with other cells. By cloning the amplified CDR-III into recombinant libraries residual leukemic cells were accurately quantitated in bone-marrow samples from repeated relapses and remissions in one case of acute lymphoblastic leukemia. During a clinical remission lasting greater than 7 mo, malignant cells were present in marrow at greater than 1 per 1000 cells. These findings indicate that custom-made diagnostic probes will be useful in accurate quantitation of malignant cells in acute lymphoblastic leukemia patients in clinical remission and will allow investigation of the biological significance of low or high numbers of residual leukemic cells in evolution of that disease.

Alleles↗

PubMed1984

Rearranged immunoglobulin heavy chain variable region (VH) pseudogene that deletes the second complementarity-determining region.

We have cloned two rearranged heavy chain variable region (VH) genes from the IgG-producing human cell line CESS. The VH gene, which is linked to the mu chain constant region (C mu) gene, has two deletions at residues 45-62 and 82A-90, the former of which corresponds closely to the second complementarity-determining region (CDR2). These results could indicate that translocation of CDR2 occurred and could give support to the argument that reassortment of the V mini-genes is involved in the generation of hypervariability during evolution. However, the rearranged pseudogene could have also arisen by fortuitous deletion. The other VH gene of CESS is an expressed form and is probably linked to the C gamma gene. The diversity region (D) segments used in these rearranged V genes are less than 38% homologous to known human germline D segments, indicating the presence of more unknown germline D segments.

Amino Acid Sequence↗

PubMed1995

Limited usage of T-cell receptor beta chains and sequences of the complementarity determining region 3 of lymphocytes infiltrating in the liver of autoimmune hepatitis.

To study the role of antigen-specific T lymphocytes in the pathogenesis of autoimmune hepatitis, messenger RNA of T-cell receptors (TCR) was analyzed in liver biopsy specimens from four patients with autoimmune hepatitis. Using the TCR beta-chain variable region family specific oligonucleotides, a remarkable bias for the usage of beta-chain variable region 3 was detected in all four patients. Therefore, nucleotide and amino acid sequences of the complementarity-determining region 3 rearranged to the beta-chain variable region 3, which is a putative contact site for peptide fragments from antigens bound in the groove of the human leukocyte antigen molecule, was further analyzed in randomly selected 10 clones from each patient. An Asp-Arg-Pro motif in the complementarity-determining region 3 was identified in three of four patients with human leukocyte antigen DR4, and this motif was always rearranged to the beta-chain junctional region 1.2. From these results, beta-chain variable region 3+, Asp-Arg-Pro+, beta-chain junctional region 1.2+ T-cell clones may be among the responsible lymphocytes involved in the liver damage in autoimmune hepatitis, especially in patients with human leukocyte antigen DR4. Thus, an analysis of the complementarity-determining region 3 may give us an important clue to clarify characteristics of target antigens included in autoimmune hepatitis.

Adult↗

PubMed1992

Refined crystal structure of a recombinant immunoglobulin domain and a complementarity-determining region 1-grafted mutant.

We report the solution of the crystal structure of a mutant of the immunoglobulin VL domain of the antibody McPC603, in which the complementarity-determining region 1 segment is replaced with that of a different antibody. The wild-type and mutant crystal structures have been refined to a crystallographic R-factor of 14.9% at a nominal resolution of 1.97 A. A detailed description of the structures is given. Crystal packing results in a dimeric association of domains, in a fashion closely resembling that of an Fv fragment. The comparison of this VL domain with the same domain in the Fab fragment of McPC603 shows that the structure of an immunoglobulin VL domain is largely independent of its mode of association, even in places where the inter-subunit contacts are not conserved between VL and VH. In all three complementarity-determining regions we observe conformations that would not have been predicted by the canonical structure hypothesis. Significant differences between the VL domain dimer and the Fab fragment in the third complementarity-determining region show that knowledge of the structure of the dimerization partner and its exact mode of association may be needed to predict the precise conformation of antigen-binding loops.

Amino Acid Sequence↗

PubMed1994

Heterogeneity of T-cell receptor alpha-chain complementarity-determining region 3 in myelin basic protein-specific T cells increases with severity of multiple sclerosis.

The pathogenesis of multiple sclerosis (MS) is thought to involve a T-cell-mediated autoimmune process. Experimental allergic encephalomyelitis (EAE), an animal model resembling MS, can be induced by immunization with myelin antigens such as myelin basic protein. The T-cell antigen receptor (TCR) usage in EAE is highly restricted in some strains of animals and experimental treatments targeting the TCR have been successful in EAE. Examination of the TCR beta-chain variable-region (V beta) usage of MBP-specific T-cell lines in MS patients has produced conflicting results. Our previous studies of TCR alpha-chain variable-region usage in monozygotic twins demonstrated a general skewing of the TCR repertoire in individuals with MS. This skewing became apparent only after stimulation with antigens; in peripheral blood lymphocyte preparations from individuals with MS V alpha 8-bearing T cells were preferentially selected by stimulation with myelin basic protein. In the present study we examined complementarity-determining region 3 of those V alpha 8-positive TCRs. Marked sequence heterogeneity was found in all individuals with severe MS. In contrast, restricted areas of complementarity-determining region 3 were found in healthy control individuals and in individuals with a mild form of MS. Sequences from tetanus toxoid-specific V alpha 8-positive T cells generated from the same individuals were relatively homogeneous within individuals regardless of disease activity and were distinct from the sequences of complementarity-determining region 3 in myelin basic protein-stimulated lines. These findings suggest that disease severity may be associated with increased heterogeneity of myelin antigen-specific T cells and could reflect an impaired ability of the immune system to down-regulate these anti-self responses.

Adult↗

PubMed1993

A complementarity-determining region synthetic peptide acts as a miniantibody and neutralizes human immunodeficiency virus type 1 in vitro.

A complementarity-determining region (CDR) of the mouse monoclonal antibody (mAb) F58 was constructed with specificity to a neutralization-inducing region of human immunodeficiency virus type 1 (HIV-1). The mAb has its major reactivity to the amino acid sequence I--GPGRA in the V3 viral envelope region. All CDRs including several framework amino acids were synthesized from the sequence deduced by cloning and sequencing mAb F58 heavy- and light-chain variable domains. Peptides derived from the third heavy-chain domain (CDR-H3) alone or in combination with the other CDR sequences competed with F58 mAb for the V3 region. The CDR-H3 peptide was chemically modified by cyclization and then inhibited HIV-1 replication as well as syncytium formation by infected cells. Both the homologous IIIB viral strain to which the F58 mAb was induced and the heterologous SF2 strain were inhibited. This synthetic peptide had unexpectedly potent antiviral activity and may be a potential tool for treatment of HIV-infected persons.

Antibodies, Anti-Idiotypic↗

PubMed1979

Structural evidence for independent joining region gene in immunoglobulin heavy chains from anti-galactan myeloma proteins and its potential role in generating diversity in complementarity-determining regions.

We have determined the variable region sequences of four heavy chains from beta(1-6)D-galactan-binding myeloma proteins. Two of these proteins are identical to position 100 which is located in the third complementarity-determining region (CDR-3). The remaining two differ at a total of 8 positions over the first 100 amino acids, and all of the differences can be explained by single-base mutations at the DNA level. When an assessment is made of the protein segment following CDR-3, which has been termed "J segment" or "FR4," a completely different pattern of variation is observed. The J segments from the four proteins can be divided into two sets. Members of each set share a series of linked amino acids not found in members of the alternative set. The two proteins identical to position 100 have J segments from the two different sets, suggesting that recombination has occurred between V and J genes. An examination of the CDR-3 sequences from the four heavy chains reveals substitutions at positions 100 and 105. Gly is found at 100 in two of the proteins and His in the remaining two. In the two proteins with Gly-100, the following J sequence is limited to one of the two sets of J segments defined by linked amino acids. Similarly, the two heavy chains with His-100 have J segments from the second set. Thus, at the protein level an apparent association is seen between CDR-3 and J segment. If CDR-3 should be found linked to J segment at the DNA level, a new mechanism would be introduced for increasing antibody diversity by recombining various CDR-3 plus J genes with genes coding for the remainder of the variable region. Alternatively, if CDR-3 were coded for by the V gene, then the recombination of V with J may provide an opportunity to introduce mutations in CDR-3. In this case the linkage of amino acids in CDR-3 and the J segments would suggest that recognition signals are used such that certain V genes only pair with a given J gene.

Amino Acid Sequence↗

PubMed1991

Malignant cell detection in Burkitt's lymphoma using third-complementarity-determining region (CDRIII), clone-specific probe developed by sequencing DNA from stored slides.

The DNA sequence of the third-complementarity-determining region (CDRIII) of the immunoglobulin heavy chain (IgH) gene in a case of Burkitt's lymphoma was determined by polymerase chain reaction (PCR) using template DNA extracted from a smear stored at room temperature for more than one year. The DNA sequence obtained from the stored slide was compared with that of DNA from a frozen lymph node biopsied at the initial presentation. The sequences were shown to be identical, implying that DNA from a smear on a stored slide can be used as a source of DNA for PCR amplification, sequencing, and development of a clone-specific probe. Using oligonucleotides generated from one of the CDRIII sequences of the IgH gene as molecular probes, a retrospective study for the malignant clone on the smears was carried out. Malignant cells were detectable in the peripheral blood at an early stage of bone marrow relapse but not in the peripheral blood or bone marrow at the initial presentation. No malignant clone was detected in the bone marrow when testicular infiltration was diagnosed by examination of a pathological specimen. Thus, the technique permits molecular analysis of hematologic malignancies of B-cell lineage in cases where fresh or frozen specimens are not available.

Base Sequence↗

PubMed1992

Characterization of the immunoglobulin heavy chain complementarity determining region (CDR)-III sequences from human B cell precursor acute lymphoblastic leukemia cells.

Sequence analysis of the immunoglobulin heavy chain complementarity determining region (CDR)-III of B-lineage cells at various stages has provided important insights concerning B cell maturation and selection. Knowledge of human CDR-III sequences has been relatively limited compared with that of the murine system. We analyzed the CDR-III sequences of B cell precursor acute lymphoblastic leukemia (pre-B ALL) cells in 23 newly diagnosed and 10 relapsed patients, in order to elucidate the organization of CDR-III in B cell precursors. We found a very low frequency of somatic mutations in D and JH regions, preferential use of DLR, DXP, DHQ52, and DN elements, and of 3' side JH segments, and no predominant usage of D coding frames. Unusual joinings such as VH-D-D-JH and VH-JH were observed in three, and one sequences, respectively. We compared the CDR-III sequences derived from 10 patients between diagnosis and relapse. Two of them had three spots of mutated nucleotides at relapse, all of which were found in the N region near the D segments. Our data showed the possibility of somatic mutation at relapse, in addition to developmentally regulated rearrangement of the immunoglobulin gene at the stage of B cell precursors.

Adolescent↗

PubMed1991

Stimulation of glycoprotein gp120 dissociation from the envelope glycoprotein complex of human immunodeficiency virus type 1 by soluble CD4 and CD4 peptide derivatives: implications for the role of the complementarity-determining region 3-like region in membrane fusion.

We have used a recombinant vaccinia virus vector encoding the envelope glycoprotein of human immunodeficiency virus type 1 to study receptor-induced structural changes related to membrane fusion. A truncated soluble form of human CD4 (sCD4) was found to stimulate dissociation of the external subunit (gp120) from the envelope glycoprotein complex of human immunodeficiency virus type 1 expressed at the cell surface. sCD4 stimulation of gp120 release was time- and concentration-dependent and was associated with specific binding of sCD4 to gp120. Synthetic peptide derivatives corresponding to residues 81-92 of human CD4 (overlapping the complementarity-determining region 3-like region) inhibited cell-cell fusion mediated by the interaction between recombinant vaccinia-encoded CD4 and human immunodeficiency virus envelope glycoprotein. These peptide derivatives also stimulated gp120 release from the envelope glycoprotein complex. An analogous peptide derivative from chimpanzee CD4 (containing a single Glu----Gly substitution at the position corresponding to CD4 residue 87) was considerably less active at inhibition of cell-cell fusion and stimulation of gp120 release, consistent with the known inhibitory effect of this substitution on the ability of membrane-associated CD4 to mediate cell fusion. These results suggest that the sCD4-induced release of gp120 reflects postbinding structural changes in the envelope glycoprotein complex involved in membrane fusion, with the complementarity-determining region 3-like region playing a critical role.

CD4 Antigens↗

PubMed1992

The His-probe method: effects of histidine residues introduced into the complementarity-determining regions of antibodies on antigen-antibody interactions at different pH values.

We examined the effects of histidine residues that were artificially introduced into complementarity-determining regions of antibodies on antigen-antibody interactions at different pH values. Using a monoclonal antibody specific for hen egg-white lysozyme and three mutant antibodies that contained a histidine residue, we measured binding constants for antibodies and lysozyme at different pH values (pH 5-8). No gross conformational changes were evident over this range of pH values, as determined by analysis of the spectra of circular dichroism. Since the charge on a histidine residue is the most likely factor that can vary over this range of pH values, differences on pH-dependent antigen-binding patterns observed between the wild-type and mutant antibodies should be due mainly to the effects of the charges on the histidine residues. The three mutant antibodies showed different and characteristic patterns of pH-dependent binding to lysozyme, which depended on the location of the artificially introduced histidine residues.

Amino Acid Sequence↗

PubMed1995

Conformational similarity and systematic displacement of complementarity determining region loops in high resolution antibody x-ray structures.

Comparison of seven high resolution x-ray structures shows that the conformations of canonical complementarity determining region (CDR) loops, which are shared by these antibodies, are very similar. However, large spatial displacements (up to 2.7 A) of the essentially identical CDR loops become evident when the antibody beta-sheet frameworks, to which the loops are attached, are least-squares superposed. The loop displacements follow, and amplify, small positional differences in framework/loop splice points. Intradomain structural variability and, to a lesser extent, domain-domain orientation appear to cause the observed loop divergences. The results suggest that the selection of framework regions for loop grafting procedures is more critical than previously thought.

Amino Acid Sequence↗

PubMed1991

Profound alteration in an alpha beta T-cell antigen receptor repertoire due to polymorphism in the first complementarity-determining region of the beta chain.

Amino acid residues that are critical in maintaining the framework structure of immunoglobulin heavy- and light-chain variable (V) regions are strongly conserved in the V alpha and V beta proteins of the alpha beta T-cell antigen receptor (TCR alpha beta). Consequently, it has been proposed that TCR alpha beta has a conformation similar to that of an immunoglobulin Fab fragment and that the regions of the TCR homologous to the three immunoglobulin complementarity-determining regions (CDRs 1, 2, and 3) bind to the peptide antigen-major histocompatibility complex (MHC) molecule ligand. A single amino acid substitution in the predicted CDR1 of the V beta 3 protein of certain mouse strains dramatically altered TCR alpha beta usage in an antigen-specific MHC-restricted immune response but did not abrogate V beta 3 specificity for the superantigens minor lymphocyte stimulatory locus (Mls)c and staphylococcal enterotoxin A (SEA). The results confirm the importance of the V beta CDR1 in antigen-MHC molecule recognition, supporting the Fab-like structural model of TCR alpha beta, and provide further evidence that conventional antigen-MHC recognition and superantigen recognition are mediated by distinct regions of the TCR beta chain. They also suggest that allelic polymorphism may be a significant source of diversity in the TCR repertoire.

Animals↗

PubMed1991

Immunoglobulin complementarity-determining region grafting by recombinant polymerase chain reaction to generate humanised monoclonal antibodies.

We describe an approach to rapidly generate humanised monoclonal antibodies by grafting rodent complementarity-determining regions onto human immunoglobulin frameworks using recombinant polymerase chain reaction (PCR) methodology. The approach was applied to grafting a rat complementarily-determining region onto a human framework and amplifying the entire humanised heavy chain. The terminal oligodeoxyribonucleotide primers incorporated restriction sites to allow forced cloning into plasmid vectors for sequencing and expression. No nucleotide errors were introduced into the 1463-bp sequence even after sequential applications of PCR.

Animals↗

PubMed1995

Specific binding of a synthetic peptide derived from an antibody complementarity determining region to phosphatidylserine.

We have established a series of monoclonal antibodies that bind to phosphatidylserine (PS). One mAb, PS4A7, showed a strict specificity for PS and distinguished the stereospecific configuration of its serine moiety. We determined the amino acid sequences of the heavy and light chain variable regions of PS4A7, and examined the reactivity of the synthetic peptides corresponding to the complementarity determining region (CDR) of the mAb with phospholipids. We found that a 12-amino acid synthetic peptide corresponding to the third CDR of the heavy chain (amino acid residues 93-102, referred to as CDR3-H) bound specifically to PS. Although the affinity of the peptide to PS was markedly lower, the peptide was shown to bind to 1,2-diacyl-sn-glycero-3-phospho-L-serine (PS), but not to 1,2-diacyl-sn-glycero-3-phospho-D-serine, showing a similar specificity to that of PS4A7. The specific binding of the CDR3-H peptide to PS was confirmed by ELISA and TLC-immunostaining assay. The interaction between the CDR3-H peptide and water-soluble PS-derivatives was investigated by inhibition of the ELISA. PS effectively inhibited the binding and phosphoserine showed a weak but significant inhibition, but no appreciable inhibition was observed with serine. These observations suggest that the CDR3-H peptide plays a major role in the interaction of PS4A7 with the phosphoserine residue of the PS molecule.

Amino Acid Sequence↗

PubMed1988

Studies of a human lambda-chain epitope related to a complementarity-determining region.

A tryptic non-adecapeptide representing the 24-42 sequence of the MCG lambda-type Bence-Jones protein, and which contains its entire complementarity-determining region-1, was isolated. The peptide was utilized in preparing an affinity column that was used to isolate an antibody having the reactivity of a previously employed idiotypic antibody to MCG. This antibody preparation, as well as 13 monoclonal mouse antibodies to human lambda-chains, was employed in an enzyme-linked immunoassay to detect other Bence-Jones proteins with this serologic specificity. The results obtained with two of the monoclonal antibodies suggest that the epitope in question is a noncontiguous one.

Amino Acids↗

PubMed1985

Possible involvement of human D minigenes in the first complementarity-determining region of kappa light chains.

The nucleotide sequences of the complementary strands of two human diversity region (D) minigenes, D2 and D4, show stretches of homology with two human variable region kappa chain (V kappa) genes, NG9 and HK101, respectively, in the first complementarity-determining region. In one V kappa sequence, the homology includes the 5' flanking region of D minigenes, which may comprise a recombinase recognition signal. It is thus conceivable that gene conversions involving D minigenes may contribute to V kappa diversity.

Antibody Diversity↗