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Results for “Complementarity Determining Regions”

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At least 19 recordsLinked to original sources

PubMed2000

Characterization and role in experimental systemic lupus erythematosus of T-cell lines specific to peptides based on complementarity-determining region-1 and complementarity-determining region-3 of a pathogenic anti-DNA monoclonal antibody.

Peptides based on the complementarity-determining region 1 (CDR1) and CDR3 of an anti-DNA monoclonal antibody (mAb) carrying the 16/6 idiotype (Id) were shown to induce experimental systemic lupus erythematosus (SLE) in susceptible mouse strains. In the present study, T-cell lines specific to the pCDR1 and pCDR3 peptides were established in BALB/c and in SJL mice, respectively. The T-cell lines were characterized and analysed for their pathogenicity upon administration to syngeneic mouse strains. Both T-cell lines expressed the alphabeta T-cell receptor (TCR) and the CD4+ CD8- phenotype. Additionally, both cell lines secreted interleukin (IL)-4 and IL-10 upon stimulation with their specific peptide, thus belonged to the T helper 2 (Th2) subset. Upon immunization, the pCDR3-specific T-cell line induced experimental SLE in SJL mice. The animals produced high levels of autoimmune anti-DNA and antinuclear protein antibodies, as well as anti-16/6 Id antibodies (Abs). Furthermore, the mice developed clinical manifestations, including leukopenia, proteinuria and accumulation of immune complex deposits in their kidneys. The pCDR1-specific T-cell line failed to induce SLE when injected into BALB/c mice. It is thus suggested that pCDR3 is an immunodominant epitope in experimental SLE and that pCDR3-specific T cells initiate autoimmunity, leading to SLE, probably via epitope spreading.

Animals↗

PubMed2003

Analysis of T cell receptor variable regions and complementarity determining region 3 of infiltrating T lymphocytes in the liver of patients with chronic type B hepatitis.

OBJECTIVES: T cell receptor (TCR) variable regions and complementarity determining regions 3 (CDR3) of infiltrating T cells in the livers of patients with chronic hepatitis B were examined in detail. METHODS: TCR usage was examined by the inverse polymerase chain reaction in 2 hepatitis B e antigen-positive patients, one at an early stage and the other at an advanced stage. RESULTS: In 61 productively rearranged clones bearing TCRalpha genes in the liver of the patient with early stage chronic hepatitis B, the Valpha 7.2 gene segment was most frequently used (16.4%), while the other Valpha gene segments were used less frequently. All but one of the clones with the Valpha7.2 gene were joined with the Jalpha33 gene, and the CDR3 regions of these clones were the same length and similar in amino acid sequence. In contrast, in 68 productively rearranged clones from the liver of the patient with advanced stage disease, all Valpha gene segments were used less frequently, and combinations of the Valpha and Jalpha gene segments varied. CONCLUSIONS: Our data suggest a relatively restricted usage of TCR in the liver in early stage chronic hepatitis B.

Adult↗

PubMed1994

Conversion of an anti-single-stranded DNA active site to an anti-fluorescein active site through heavy chain complementarity determining region transplantation.

Complementarity determining region (CDR) transplant studies were conducted between two monoclonal antibodies of distinctly different specificities (anti-fluorescein monoclonal antibody (mAb) 4-4-20 and anti-single-stranded DNA (ssDNA) mAb 04-01) which possessed nearly identical light chains but dissimilar heavy chains. The variations in binding specificities between the two immunoglobulins suggested that the active-site features of anti-fluorescein antibodies were dictated by characteristics intrinsic to the heavy chain (H-chain). To identify specific regions of the H-chain which influence the structure and function of an anti-fluorescein active site, CDR transplantation was systematically employed to convert the anti-ssDNA 04-01 antibody active site to an active site with anti-fluorescein activity. Each mAb 4-4-20 H-chain CDR (HCDR) was transplanted into the H-chain of a single-chain derivative of the 04-01 molecule. A fluorescence polarization ligand binding assay was utilized to determine the equilibrium dissociation constant, Kd, of hybrid transplant single-chain antibody HCDR1-HCDR2-HCDR3(4-4-20) for fluorescein (3.8 x 10(-7) M, indicating successful conversion of an anti-ssDNA active site to an anti-fluorescein binding site. A similar Kd (6.3 x 10(-7) M) was determined using a fluorescein fluorescence quenching assay. The transplantation results are discussed in terms of the relative contribution of each HCDR to a successful conversion in antibody specificity.

Amino Acid Sequence↗

PubMed1996

The I binding specificity of human VH 4-34 (VH 4-21) encoded antibodies is determined by both VH framework region 1 and complementarity determining region 3.

Essentially all cold agglutinins (CA) with red blood cell I/i specificity isolated from patients with CA disease stemming from lymphoproliferative disorders utilize the VH 4-34 (VH 4-21) gene segment. This near universality of the restricted use of a single gene segment is substantially greater than that demonstrated for other autoantibodies. The monoclonal antibody 9G4 exclusively binds VH 4-34 encoded antibodies and serves as a marker for the VH 4-34 gene segment. Previous studies form our laboratory localized the 9G4 reactive area to framework region 1 (FR1). In the present study, the relative roles of VH FR1, heavy (H) chain complementarity determining region 3 (CDRH 3) and the light (L) chain in I antigen binding were investigated. Mutants containing FR1 sequences from the other VH families, CDRH 3 exchanges, and combinatorial antibodies involving L chain interchanges were produced in the baculovirus system and tested in an I binding assay. The data indicate that FR1 of the VH 4-34 gene segment and the CDRH 3 are essential for the interaction between CA and the I antigen, with the CDRH 3 being fundamental in determining the fine specificity of antigen binding (I versus i). Mutants with substantially altered CDRH 1 and CDRH 2 regions bind I as long as the FR1 is VH 4-34 encoded and the CDRH 3 has a permissive sequence. Light chain swaps indicate that even though antigen binding is predominantly mediated by the H chain, the association with antigen can be abrogated by an incompatible L chain. The necessity for VH 4-34 FR1 explains the almost exclusive use of the VH 4-34 gene segment in cold agglutinins. We hypothesize that, as a general phenomenon, the H chain FR1 of many antibodies may be important in providing the contact required for the close association of antibody with antigen, while the CDRH 3 dictates the fine specificity and strenght of binding.

Agglutinins↗

PubMed1998

Improved quantitation of minimal residual disease in multiple myeloma using real-time polymerase chain reaction and plasmid-DNA complementarity determining region III standards.

The complementarity determining region III of the rearranged immunoglobulin heavy chain gene has been the target for tumor-specific PCR assays for the detection and follow-up of B-cell malignancies. Previously, these assays have relied on gel-based end point data collection methods (i.e., band densitometry) and, thus, have provided at best a semiquantitative assessment of tumor levels. We show the development of a novel, real-time TaqMan PCR assay to quantitate residual multiple myeloma cells in clinical samples after high-dose chemotherapy and autologous stem cell transplantation. We provide evidence that real-time PCR is reproducible, sensitive, and quantitative. In a 40-replicate PCR experiment targeting the beta-actin gene, the coefficient of variation for threshold cycle data was 1.6%, whereas it increased to 13.6% and 31%, respectively, for end point fluorescence and gel densitometry. Moreover, in an experiment directly comparing standard curves obtained from band densitometry and threshold cycle data, the standard curve constructed from threshold cycle data had a multiple R2 value of 1.00 and demonstrated a dynamic range >4 logs, compared with the 2-log linear range of gel densitometry. Finally, we show that when a complementarity determining region III-specific PCR primer is used in conjunction with a consensus primer for the immunoglobulin heavy chain joining gene, plasmid DNA can be used as a readily available and effective substitute for clonal plasma-cell genomic DNA when preparing standards. By applying real-time PCR to the analysis of clinical samples, we are able to quantitate levels of tumor involvement with unparalleled reproducibility and statistical confidence. Real-time PCR technology may well provide the accuracy and reliability necessary for minimal residual disease detection to have real prognostic significance.

Base Sequence↗

PubMed1996

Staphylococcal protein A simultaneously interacts with framework region 1, complementarity-determining region 2, and framework region 3 on human VH3-encoded Igs.

Staphylococcal protein A (SPA) is a B cell superantigen that binds to human VH3-encoded Igs independently of the D- and JH-encoded regions and light chain sequences. The SPA-binding structure formed by VH3-encoded Igs remains controversial. We localized the regions in a VH3-encoded Ab required for SPA binding by producing mutant Abs in the baculovirus expression system in which regions of a human-derived Ab known to bind SPA were exchanged with those from a mouse Ab of the J558 family, a family not associated with SPA binding. The pattern of SPA binding indicates not only that residues in FR1, CDR2, and FR3 are involved but also that the three regions are required to interact simultaneously with SPA for binding to occur. When any one of the three regions was replaced with the corresponding region from the nonbinding Ab, SPA binding was severely disrupted. These data indicate that SPA requires simultaneous interaction with three distinct regions of a VH3 structure, which together in three-dimensional space form an extended solvent-exposed surface. These studies more precisely define the genetic requirements for VH3-encoded Ig binding to SPA.

Animals↗

PubMed1999

Different properties of T-cell epitopes within complementarity-determining regions 1 and 2 of idiotypic VH in B-lymphoma.

The idiotypic protein expressed by B-lymphoma cells can evoke an autologous idiotype-specific T-cell response in both animal studies and lymphoma patients. However, the locations within the idiotypic protein of the immune epitopes remains unclear. In this study, we determined the nature of any immune responses generated by peptides corresponding to the complementarity-determining region 1 (CDR1) and complementarity-determining region 2 (CDR2) sequences of idiotypic heavy chain variable region (VH) to identify the clinical potential of the peptides for immunotherapy of lymphoma. We found that a synthetic peptide corresponding to CDR1 sequence contains a functional T-cell epitope able to evoke a major histocompatibility complex (MHC) class II-dependent T-cell proliferative response and cytokine release without direct cytotoxicity for peptide-pulsed target or fresh autologous lymphoma cells. Although a peptide corresponding to CDR2 was able to generate MHC class I and class II dependent T-cell proliferative responses and cytokine release, T-cell epitopes within the peptide were cryptic in the context of intact idiotypic protein. Our results therefore suggest that the CDR1 sequence could be used for the immune targeting of B-lymphoma.

Amino Acid Sequence↗

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↗

PubMed2003

The function of conserved amino acids in or near the complementarity determining regions for related antibodies to Cryptococcus neoformans glucuronoxylomannan.

Most monoclonal antibodies (mAbs) to Cryptococcus neoformans glucuronoxylomannan (GXM) antigen (Ag) are grouped as Class II based on usage of V(H)7183, Vkappa5.1, J(H)2, and Jkappa1 gene elements. Comparative analysis of 43 Class II mAbs revealed conservation of I51, G54, and D61 in heavy-chain variable region (V(H)) complementarity determining region 2 (CDR2), and R95 and D96 in CDR3. Furthermore, position 100b (Kabat numbering scheme) in CDR3 always had an aromatic amino acid (aa) and F was found at position 100c in 96% of mAbs. The function of these conserved residues for binding to GXM and peptide mimetics, and idiotype (Id) structure, was investigated using site-directed mutagenesis. In addition, we mutated W36 and V37 in the second framework. Mutations W36A, Y100bA, and F100cA interfered with antibody (Ab) secretion, but not assembly, and cytoplasmic Ab bound to GXM and Id mAbs. In contrast, mutations V37A, I51A, G54A, and D61A did not affect assembly, secretion, or binding to GXM. Mutating the R95-D96 motif in CDR3 to DR, DD, RR, RA, AD, KD, HD, RE, RN or AA revealed that the positive charge at position 95 was essential for binding GXM, whereas the negative charge at position 96 could be substituted for a non-charged aa. Our results: (1) extend the concept that CDR3 diversity is essential for Ag and Id specificity to a polysaccharide-binding Ab; (2) show that aa conservation in CDRs does not imply a requirement for Ag binding; (3) establish a role for W36 in secretion; and (4) demonstrate that aa motifs used for binding GXM and peptide mimetics can differ.

Antibodies↗

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↗

PubMed2005

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 kappa-light chain CDR1 (kappa-CDR1) and CDR2 (kappa-CDR2), we performed alignments of 1330 kappa-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 kappa-CDR1 and kappa-CDR2 to prepare synthetic antibody molecules. We then tested these predictions by inserting somatostatin and somatostatin-related sequences into kappa-CDR1 and kappa-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.

Amino Acid Sequence↗

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↗

PubMed2001

Improvement in affinity and HIV-1 neutralization by somatic mutation in the heavy chain first complementarity-determining region of antibodies triggered by HIV-1 infection.

We assessed the impact of somatic hypermutation in the framework region 1 (FR1) and complementarity-determining region 1 (CDR1) of three clonally-related heavy chains from the human monovalent antigen-binding fragments Fab S19, S8 and S20 on gp120 binding and HIV-1 neutralization capacity. Nucleotide changes were introduced in the heavy chains to revert single and multiple amino acid residues, and two Fab libraries were constructed with the same light chain to express equivalent amounts of parental and reverted phage Fab. We studied the contribution of each amino acid replacement to antigen binding by calculating the frequency of phage Fab retrieval after competitive library selection on gp120. Whereas mutations in FR1 had no effect on antigen binding, somatic replacements in the CDR1 of the heavy chain (HCDR1) appeared to produce significant changes. In S19 HCDR1, somatic mutation of residue 32 reduced gp120 binding. In Fab S20, the Arg(30) and Asp(31) somatically replaced residues in HCDR1 improved antigen binding. Both of these residues are necessary to increase Fab binding to gp120; reversion of either residue alone results in a decrease in binding. The impact of these two replacements was confirmed by the greater neutralization capacity of S20 compared to the other Fab. Molecular modeling of S20 HCDR1 suggests that Arg(30) and Asp(31) are the main interaction sites for gp120, increasing antibody affinity and promoting the enhanced neutralization ability of S20. These findings are consistent with a gp120-driven process, supporting a role for affinity maturation and intraclonal evolution of HIV-1 neutralizing antibodies.

Amino Acid Sequence↗

PubMed1997

Site-specific mutagenesis of a recombinant anti-single-stranded DNA Fab. Role of heavy chain complementarity-determining region 3 residues in antigen interaction.

The heavy chain complementarity-determining region 3 (HCDR3) of the anti-oligo(dT) recombinant antibody fragment, DNA-1, contributes significantly to antigen binding (Komissarov, A. A., Calcutt, M. J., Marchbank, M. T., Peletskaya, E. N., and Deutscher, S. L. (1996) J. Biol. Chem. 271, 12241-12246). In the present study, the role of separate HCDR3 residues of DNA-1 in interaction with oligo(dT) was elucidated. Based on a molecular model of the combining site, residues at the base (Arg98 and Asp108) and in the middle (Tyr101-Arg-Pro-Tyr-Tyr105) of HCDR3 were predicted to support the loop conformation and directly contact the ligand, respectively. Twenty-five site-specific mutants were produced as hexahistidine-tagged proteins, purified, and examined for binding to (dT)15 using two independent methods. All mutations in the middle of HCDR3 led to either abolished or diminished affinity. Tyr101 likely participates in hydrogen bonding, while Tyr104 and Tyr105 may be involved in aromatic-aromatic interactions with the ligand. The residues Arg102 and Pro103 were not as critical as the tyrosines. It is speculated that HCDR3 interacts with the thymines, rather than the phosphates, of the ligand. A 3-fold increase in affinity was observed by mutation of Asp108 to alanine. The highly conserved Arg98 and Asp108 do not appear to form a salt bridge.

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↗

PubMed2002

"Superhumanized" antibodies: reduction of immunogenic potential by complementarity-determining region grafting with human germline sequences: application to an anti-CD28.

Humanized Abs are created by combining, at the genetic level, the complementarity-determining regions of a murine mAb with the framework sequences of a human Ab variable domain. This leads to a functional Ab with reduced immunogenic side effects in human therapy. In this study, we report a new approach to humanizing murine mAbs that may reduce immunogenicity even further. This method is applied to humanize the murine anti-human CD28 Ab, 9.3. The canonical structures of the hypervariable loops of murine 9.3 were matched to human genomic V gene sequences whose hypervariable loops had identical or similar canonical structures. Framework sequences for those human V genes were then used, unmodified, with the 9.3 complementarity-determining regions to construct a humanized version of 9.3. The humanized 9.3 and a chimeric 9.3 control were expressed in Escherichia coli as Fab. The humanized Fab showed a moderate loss in avidity in a direct binding ELISA with immobilized CD28-Ig fusion protein (CD28-Ig). Humanized 9.3 blocked ligation of CD28-Ig to cells expressing the CD28 receptor CD80. Lastly, the humanized 9.3 showed biological activity as an immunosuppressant by inhibiting a MLR.

Amino Acid Sequence↗

PubMed1993

The anti-idiotypic response by cynomolgus monkeys to humanized anti-Tac is primarily directed to complementarity-determining regions H1, H2, and L3.

The anti-ld response developed by cynomolgus monkeys to the humanized anti-Tac antibody was analyzed by using 12 humanized anti-Tac variants differing in V region structure. The majority of the monkey response was directed against idiotopes composed wholly or in part of complementarity-determining regions H1, H2, and L3. There was no detectable response directed solely to five single complementarity-determining regions examined or solely to the modified human V region framework.

Animals↗