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Results for “Complementarity Determining Regions”

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PubMed2000

Immune response of SLE patients to peptides based on the complementarity determining regions of a pathogenic anti-DNA monoclonal antibody.

We have examined the humoral and cellular responses of SLE patients to peptides based on the complementarity-determining regions (CDR) of a monoclonal anti-DNA antibody with a major idiotype- 16/6 Id, in comparison to their responses to the whole 16/6 Id-bearing antibody. Sera of 63% of the SLE patients had antibodies that bound the 16/6 Id, 80% had antibodies to one of the CDR-based peptides, and 40% of the patients reacted with both CDRs. Sera of only a few controls reacted with either the 16/6 Id (6%) or the CDR based peptides (4%) (P < 0.01). Peripheral blood lymphocytes (PBL) of 39% of the patients proliferated in response to the 16/6 Id or to one of the CDR-based peptides (37%), while in the control group the proliferation rates were 66% to the 16/6 Id and 59% to one of the CDR-based peptides (P < 0.05). The correlation between (both) the humoral and cellular immune responses to the CDR-based peptides and to the 16/6 Id suggests the relevance of these peptides to the 16/6 Id and provides additional information on the pathogenic moiety of the latter antibody.

Amino Acid Sequence↗

PubMed2004

Amelioration of lupus manifestations by a peptide based on the complementarity determining region 1 of an autoantibody in severe combined immunodeficient (SCID) mice engrafted with peripheral blood lymphocytes of systemic lupus erythematosus (SLE) patients.

A peptide based on the complementarity determining region (CDR)1 of a human monoclonal anti-DNA autoantibody (hCDR1) was shown to either prevent or treat an already established murine lupus in systemic lupus erythematosus (SLE)-prone mice or in mice with induced experimental SLE. The present study was undertaken to determine the therapeutic potential of hCDR1 in a model of lupus in severe combined immunodeficient (SCID) mice engrafted with peripheral blood lymphocytes (PBL) of patients with SLE. To this end, PBL obtained from lupus patients were injected intraperitoneally into two equal groups of SCID mice that were treated either with the hCDR1 (50 micro g/mouse) once a week for 8 weeks, or with a control peptide. Mice were tested for human IgG levels, anti-dsDNA autoantibodies, anti-tetanus toxoid antibodies and proteinuria. At sacrifice, the kidneys of the successfully engrafted mice were assessed for human IgG and murine complement C3 deposits. Of the 58 mice transplanted with PBL of SLE patients, 38 (66%) were engrafted successfully. The mice that were treated with the control peptide developed human dsDNA-specific antibodies. Treatment with hCDR1 down-regulated the latter significantly. No significant effect of the treatment on the levels of anti-tetanus toxoid antibodies could be observed. Treatment with hCDR1 resulted in a significant amelioration of the clinical features manifested by proteinuria, human IgG complex deposits as well as deposits of murine complement C3. Thus, the hCDR1 peptide is a potential candidate for a novel specific treatment of SLE patients.

Adoptive Transfer↗

PubMed2002

Inhibition of hepatitis C virus NS3 protease by peptides derived from complementarity-determining regions (CDRs) of the monoclonal antibody 8D4: tolerance of a CDR peptide to conformational changes of a target.

We have synthesized and characterized peptides derived from complementarity-determining regions (CDRs) of 8D4, a mouse monoclonal antibody against NS3 protease domain of hepatitis C virus. 8D4 inhibits enzymatic activity without its cofactor, NS4A peptide. One of the synthetic peptides derived from CDRs, CDR1 of the heavy-chain (CDR-H1) peptide strongly inhibited NS3 protease activity competitively in the absence of NS4A and non-competitively in the presence of NS4A. Moreover, cyclic CDR-H1 peptides bridged by disulfide inhibited NS3 protease more potently. The chain length of the CDR-H1 peptide is critical for strong inhibition, even when the peptide is circularized. This finding suggests the importance of peptide conformation. In contrast to a cognate antibody molecule, CDR-derived peptides may provide good ligands for target molecules by having a tolerance to conformational changes of the targets caused by cofactor binding or mutation.

Amino Acid Sequence↗

PubMed2004

B-cell repertoire reconstitution after hematopoietic stem cell transplantation in children evaluated by immunoglobulin heavy chain third complementarity determining region fingerprinting.

Immunodeficiency after hematopoietic stem cell transplantation leads to a high risk of opportunistic infections. We evaluated B-lymphocyte reconstitution in 36 children by heavy chain third complementarity determining region (CDR3)-fingerprinting and immunophenotypic analysis. The time necessary to return to the normal immunoglobulin heavy chain-CDR3 polyclonal situation was basically related to the type of transplant and this process did not recapitulate fetal ontogenesis.

Adolescent↗

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↗

PubMed2005

Amelioration of SLE-like manifestations in (NZBxNZW)F1 mice following treatment with a peptide based on the complementarity determining region 1 of an autoantibody is associated with a down-regulation of apoptosis and of the pro-apoptotic factor JNK kinase.

Treatment with peptides based on the complementarity determining regions (CDR) of murine and human monoclonal anti-DNA antibodies that bear the common idiotype, 16/6 Id, ameliorates disease manifestations of mice with either induced or spontaneous SLE. Aberrant expression and function of the p21Ras/MAP kinase pathway are associated with active SLE. Therefore, we examined the effect of treatment with a CDR1-based peptide of a human autoantibody (hCDR1) on the p21Ras pathway and SLE manifestations of SLE-prone (NZBxNZW)F1 mice. Untreated SLE-afflicted mice demonstrated increased expression of p21Ras and the phosphorylated active form of its down-stream element JNK kinase in conjunction with reduced hSOS and unchanged p120GAP, as compared to healthy controls. Amelioration of SLE manifestations following treatment with hCDR1 was associated with a diminished expression of phosphorylated JNK kinase, mainly in the T cell population that also exhibited reduced rates of apoptosis. Thus, hCDR1 therapy ameliorates SLE, at least in part, via down-regulation of the activity of the pro-apoptotic JNK kinase.

Animals↗

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↗

PubMed1998

Inhibition of HIV-1 replication by a monoclonal antibody directed toward the complementarity determining region 3-like domain of CD4 in CD45 expressing and CD45-deficient cells.

Monoclonal antibodies directed toward the complementarity determining region (CDR)3-like loop of the aminoterminal domain of CD4 have been shown to inhibit the replication of human immunodeficiency virus (HIV) in CD4 positive T cells. The mechanism of action of these antibodies is not yet elucidated, although several observations suggest that they inhibit viral transcription by signal transduction through the CD4 molecule, potentially implicating the activation of a protein tyrosine kinase (PTK) cascade. Since CD45 is the major protein tyrosine phosphatase associated to the plasma membrane in T cells, and has been shown to regulate the activity of several PTK, we postulated that CD45 may be necessary for the inhibitory action of the CDR3-like specific anti-CD4 antibodies. Therefore we tested the effect of one such anti-CD4 monoclonal antibody, 13B8.2, in repressing HIV replication in CD45 positive cell lines and CD45 deficient variants. Our data show that cells respond to 13B8.2 postinfection treatment regardless of CD45 expression, indicating that neither CD45 nor PTK regulated by CD45 are implicated in the mechanism of action of this antibody.

Antibodies, Monoclonal↗

PubMed2004

Characterization of the human Ig heavy chain antigen binding complementarity determining region 3 using a newly developed software algorithm, JOINSOLVER.

We analyzed 77 nonproductive and 574 productive human V(H)DJ(H) rearrangements with a newly developed program, JOINSOLVER. In the productive repertoire, the H chain complementarity determining region 3 (CDR3(H)) was significantly shorter (46.7 +/- 0.5 nucleotides) than in the nonproductive repertoire (53.8 +/- 1.9 nucleotides) because of the tendency to select rearrangements with less TdT activity and shorter D segments. Using criteria established by Monte Carlo simulations, D segments could be identified in 71.4% of nonproductive and 64.4% of productive rearrangements, with a mean of 17.6 +/- 0.7 and 14.6 +/- 0.2 retained germline nucleotides, respectively. Eight of 27 D segments were used more frequently than expected in the nonproductive repertoire, whereas 3 D segments were positively selected and 3 were negatively selected, indicating that both molecular mechanisms and selection biased the D segment usage. There was no bias for D segment reading frame (RF) use in the nonproductive repertoire, whereas negative selection of the RFs encoding stop codons and positive selection of RF2 that frequently encodes hydrophilic amino acids were noted in the productive repertoire. Except for serine, there was no consistent selection or expression of hydrophilic amino acids. A bias toward the pairing of 5' D segments with 3' J(H) segments was observed in the nonproductive but not the productive repertoire, whereas V(H) usage was random. Rearrangements using inverted D segments, DIR family segments, chromosome 15 D segments and multiple D segments were found infrequently. Analysis of the human CDR3(H) with JOINSOLVER has provided comprehensive information on the influences that shape this important Ag binding region of V(H) chains.

Algorithms↗

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↗

PubMed1996

Analysis of antigen binding and idiotypic expression by antibodies with polyglycine-replaced complementarity-determining regions.

We investigated the feasibility and usefulness, for structure-function studies, of removing the side chains of entire complementarity-determining regions (CDRs) of Abs by replacement with polyglycine. The CDRs of a murine Ab specific for p-azophenylarsonate (Ars) were replaced with polyglycine, one CDR at a time and in combinations, by oligonucleotide-directed mutagenesis of the V region genes. Mutant Abs were expressed in transfected hybridoma cells and analyzed for Ars binding and for idiotypic expression. The results suggest that, except for the longest CDRs, polyglycine replacement does not alter the general structure of the Ab molecule. However, for analysis of functional contributions of a CDR, the polyglycine replacement method appears to be most useful for CDRs with extended structures whose replacement by polyglycine does not affect the structure of other parts of the variable regions. In the current studies, such CDRs were CDR1 of the heavy chain (H1) and CDR2 of the light chain (L2). The polyglycine replacement of L2, which does not contain an Ag-contacting residue, allowed the formation of an Ars binding Ab. Furthermore, this mutant Ab revealed previously uncharacterized contributions of L2 to idiotypic expression. Polyglycine replacement of H1 abolished Ars binding as expected, because H1 contains an Ag-contacting residue. However, introduction of the contacting residue (Asn) on the polyglycine-replaced H1 background restored the ability of the Ab to bind Ars. The results suggest that polyglycine replacement of CDRs can provide structural information that complements and extends the information obtained by other methods.

Amino Acid Sequence↗

PubMed2000

Antibody repertoire development in fetal and neonatal piglets. II. Characterization of heavy chain complementarity-determining region 3 diversity in the developing fetus.

Since the actual combinatorial diversity in the V(H) repertoire in fetal piglets represents <1% of the potential in mice and humans, we wondered whether 1) complementarity-determining region 3 (CDR3) diversity was also restricted; 2) CDR3 diversity changed with fetal age; and 3) to what extent CDR3 contributed to the preimmune VDJ repertoire. CDR3 spectratyping and sequence analyses of 213 CDR3s recovered from >30 fetal animals of different ages showed that >95% of VDJ diversity resulted from junctional diversity. Unlike sheep and cattle, somatic hypermutation does not contribute to the repertoire. These studies also revealed that 1) N region additions are as extensive in VDJ rearrangements recovered at 30 days as those in late term fetuses, suggesting that TdT is fully active at the onset of VDJ rearrangement; 2) nearly 90% of all rearrangement are in-frame until late gestation; 3) the oligoclonal CDR3 spectratype of 30-day fetal liver becomes polyclonal by 50 days, while this change occurs much later in spleen; 4) there is little evidence of individual variation in CDR3 spectratype or differences in spectratype among lymphoid tissues with the exception of the thymus; and 4) there is a tendency for usage of the most J(H) proximal D(H) segment (D(H)B) to decrease in older fetuses and for the longer D(H) segment to be trimmed to the same length as the shorter D(H) when used in CDR3. These findings suggest that in the fetal piglet, highly restricted combinatorial diversity and the lack of somatic mutation are compensated by early onset of TdT activity and other mechanisms that contribute to CDR3 junctional diversity.

Animals↗

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↗

PubMed1997

Somatic mutation in VH complementarity-determining region 2 and framework region 2: differential effects on antigen binding and Ig secretion.

The extent to which somatic mutation impairs the Ig complementarity-determining region (CDR) and framework region (FRW) structure/function is not clear. Previously, we found that the VH CDR2 of the murine T15 Ab is highly sensitive to mutation; 56% (26 of 46) of Abs mutated in vitro had reduced or no Ag binding capability, and 9% were secretion impaired. Here we test whether the T15 VH CDR2 structure is unique by mutating the VH CDR2 of the anti-PC-protein murine Ab, PCG1-1. PCG1-1 VH is encoded by the M141 gene and is unrelated in sequence or structure to that of T15 VH1. The majority (54%, 20 of 37) of PCG1-1 mutants carrying one to five mutations in VH CDR2 had reduced or abolished Ag binding, while 10% were secretion impaired. Taken together, mutational analysis of the VH1 and VH M141 genes demonstrates that impaired binding and secretion may be common outcomes of CDR2 somatic mutation. We also tested the tolerance of the VH FRW2 of T15 to mutation, expecting this sequence-conserved region to be highly sensitive to alterations. However, FRW2 accommodated many nonconservative changes, and only 12% (3 of 25) of secreted mutants had impaired Ag binding. Moreover, mutations in FRW2 caused secretion defects in 24% (8 of 33), a frequency twice that of VH CDR2 mutants. A total of 16 unique secretion mutants have now been identified. These findings suggest that B cell losses from somatic mutation may be extensive and due to varied causes not all related to Ag binding.

Animals↗

PubMed2001

The mechanism by which a peptide based on complementarity-determining region-1 of a pathogenic anti-DNA auto-Ab ameliorates experimental systemic lupus erythematosus.

A peptide based on complementarity-determining region (CDR)-1 of a monoclonal murine anti-DNA Ab that bears the common idiotype, 16/6Id, was synthesized and characterized. The peptide, designated pCDR1, was found to be an immunodominant T-cell epitope in BALB/c mice. The CDR1-based peptide was shown to be capable of inhibiting the in vivo priming of BALB/c mice immunized with the peptide or with the whole anti-DNA 16/6Id(+) mAbs of either mouse or human origin. We show here that administration of pCDR1 (weekly, i.v., 100 microgram/mouse) in aqueous solution for 5 weeks starting at the time of disease induction with the human 16/6Id prevented the development of clinical manifestations of experimental systemic lupus erythematosus (SLE). Further, 10 weekly injections of pCDR1 to BALB/c mice with an established experimental SLE down-regulated clinical manifestations of SLE (e.g., anti-DNA auto-Abs, leukopenia, proteinuria, immune complex deposits in the kidneys) in the treated mice. Prevention of SLE induction was shown to be associated mainly with a decrease in the levels of IL-2, INFgamma, and the proinflammatory cytokine TNFalpha. On the other hand, the secretion of the immunosuppressive cytokine TGFbeta was elevated. Amelioration of the clinical manifestations of an already established experimental SLE correlated with a dramatic decrease in TNFalpha secretion, elevated levels of TGFbeta, and immunomodulation of the Th1 and Th2 type cytokines to levels close to those observed in healthy mice.

Amino Acid Sequence↗

PubMed1998

Immunity to T cell receptor peptides in multiple sclerosis. III. Preferential immunogenicity of complementarity-determining region 2 peptides from disease-associated T cell receptor BV genes.

Vaccination with synthetic TCR peptides from the BV5S2 complementarity-determining region 2 (CDR2) can boost significantly the frequency of circulating CD4+ peptide-specific Th2 cells in multiple sclerosis (MS) patients, with an associated decrease in the frequency of myelin basic protein (MBP)-reactive Th1 cells and possible clinical benefit. To evaluate the immunogenicity of CDR2 vs other regions of the TCR, we vaccinated seven MS patients with overlapping BV5S2 peptides spanning amino acids 1-94. Six patients responded to at least one of three overlapping or substituted CDR2 peptides possessing a core epitope of residues 44-52, and one patient also responded to a CDR1 peptide. Of the CDR2 peptides, the substituted (Y49T)BV5S2-38-58 peptide was the most immunogenic but cross-reacted with the native sequence and had the strongest binding affinity for MS-associated HLA-DR2 alleles, suggesting that position 49 is an MHC rather than a TCR contact residue. Two MS patients who did not respond to BV5S2 peptides were immunized successfully with CDR2 peptides from different BV gene families overexpressed by their MBP-specific T cells. Taken together, these results suggest that a widely active vaccine for MS might well involve a limited set of slightly modified CDR2 peptides from BV genes involved in T cell recognition of MBP.

Adult↗

PubMed2002

Tracking T cell clonotypes in complex T lymphocyte populations by real-time quantitative PCR using fluorogenic complementarity-determining region-3-specific probes.

The T cell receptor (TCR) alpha and beta chains are encoded by a series of stochastic rearrangements between variable (V), diversity (D) for TCR beta chain only, and joining (J) gene segments, creating hypervariable complementarity-determining region 3 (CDR3) regions that contact the peptide/MHC complex and confer specificity. In the present paper, we applied the recently developed real-time quantitative RT-PCR technique to the detection of rearranged TCR beta chain mRNA transcripts. We designed BV- and BJ-specific primers together with TaqMan probes specific for the CDR3 regions of the clones of interest. As an external reference, we used plasmids containing the entire TCR beta chains, making it possible to normalize the number of specific rearranged BV-J mRNA copies among the total number of TCR beta chains. Here, we present data validating this fluorogenic PCR-based method for the quantification of several TCR clonotypes characteristic of the CD4 T cell response to hen egg white lysozyme (HEL) in mice of the H-2d haplotype. This accurate and sensitive procedure permits the precise determination of T cell clone frequencies ranging from 10(-2) to less than 10(-5) in normal biological samples; it may provide an alternative approach when frequencies are too low to be assessed by flow cytometry.

Animals↗