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PubMed1999

Systematic mutagenesis of TCR complementarity-determining region 3 residues: a single conservative substitution dramatically improves response to both multiple HLA-DR alleles and peptide variants.

To define the relative contributions of HLA and peptide contacts with TCR complementarity-determining region (CDR) 3 residues in T cell recognition, systematic mutagenesis and domain swapping was conducted on two highly similar TCRs that both respond to the influenza hemagglutinin (HA) peptide, HA307-319, but with different HLA restrictions. Despite the primary sequence similarity of these TCRs, exchange of as little as two CDR3 residues between them completely abrogated responsiveness. At position 95 within CDR3alpha, various substitutions still allowed for some degree of recognition. One modest substitution, alanine for glycine (essentially the addition of a methyl group), significantly broadened the specificity of the TCR. Transfectants expressing this mutant TCR responded strongly in the context of multiple HLA-DR alleles and to HA peptide variants with substitutions at each TCR contact residue. These results suggest that the conformations of CDR3 loops are crucial to TCR specificity and that it may not be reliable to extrapolate from primary sequence similarities in TCRs to similarities in specificity. The ease with which a broad specificity is induced in this mutant TCR has implications for the mechanisms and frequency of alloreactivity and promiscuity in T cell responses.

Adenine↗
PubMed1992

Third complementarity determining region (CDR III) sequence analysis in childhood B-lineage acute lymphoblastic leukaemia: implications for the design of oligonucleotide probes for use in monitoring minimal residual disease.

The genetic sequence of the third complementarity determining region (CDR III) of the immunoglobulin heavy chain (IgH) gene was analysed in 55 rearranged alleles from 36 children presenting with B-lineage acute lymphoblastic leukaemia (ALL). This confirmed the unique nature of these rearrangements. However, contrary to the hypothesis that the CDR III is produced by a random process of rearrangement, biased utilisation of diversity (D) segments and of joining (J) regions 4, 5 and 6 was demonstrated. Moreover, preferred sequence boundaries were seen in J regions 1 to 5 and were suggested at the 3'-end of certain D regions, notably D21/9 and DK1. Similar patterns of rearrangement have been noted in normal B-lymphocyte clones. Together with relatively limited N nucleotide addition, these factors may restrict the potential sequence variability at the D-N-J junction. The occurrence of clonal progression by secondary gene rearrangements, such as V-V replacement, favours the use of this site when designing clone-specific oligonucleotide probes for use in monitoring minimal residual disease (MRD). In cases where biased features of D-J rearrangement are shared by both the leukaemic and normal B-lymphocyte clones this could reduce the sensitivity of these probes in detecting low levels of residual disease.

Base Sequence↗
PubMed2004

Contribution of Complementarity-Determining Region 3 of the T-Cell Receptor Vδ2 Chain to the Recognition of Aminobisphosphonates by Human γδ T-Cells.

Human gammadelta T-lymphocytes expressing Vgamma2Vdelta2 T-cell receptors (TCRs) can be stimulated by aminobisphosphonates and can kill certain tumor cells. Although germline-encoded lysine residues on the Jgamma1.2 segment have been demonstrated to be essential for the recognition of nonpeptide antigens by human gammadelta T-cells, the role of the junctional sequences of the TCR delta chain in the recognition of aminobisphosphonates by Vgamma2Vdelta2+ T-cells remains unknown. We examined the structure of complementarity-determining region 3 (CDR3) of Vdelta2 chains expressed by aminobisphosphonate-stimulated human gammadelta T-cells. CDR3 size-spectratyping analysis of Vdelta2 chains revealed that risedronate did not induce a CDR3 size distribution pattern of Vdelta2 cells that was distinct from that of Vdelta2 cells cultured without risedronate. The clonality of risedronate-expanded Vdelta2 T-cells was also determined by sequencing analysis, with the result that no particular consensus sequences were observed. However, most Vdelta2 T-cells freshly isolated from peripheral blood carried a distinctive junctional motif consisting of a hydrophobic amino acid residue (valine, leucine, or isoleucine [Val/Leu/Ile]) at conserved position 97, and this feature was not altered by risedronate stimulation. A significant proportion of Vdelta1 T-cells from the same individual had Leu at position 97, but Vdelta1 T-cells did not expand in response to risedronate. Moreover, Vdelta2 T-cells from the nonresponder against risedronate also carried a Val/Leu/Ile amino acid residue at position 97. These results suggest that the presence of a hydrophobic amino acid residue at position 97 in CDR3 of the TCR delta chain is not sufficient to account for the recognition of aminobisphosphonate by human gammadelta T-cells.

Amino Acid Sequence↗
PubMed2002

Fine specificity of TCR complementarity-determining region residues and lipid antigen hydrophilic moieties in the recognition of a CD1-lipid complex.

alphabeta TCR can recognize peptides presented by MHC molecules or lipids and glycolipids presented by CD1 proteins. Whereas the structural basis for peptide/MHC recognition is now clearly understood, it is not known how the TCR can interact with such disparate molecules as lipids. Recently, we demonstrated that the alphabeta TCR confers specificity for both the lipid Ag and CD1 isoform restriction, indicating that the TCR is likely to recognize a lipid/CD1 complex. We hypothesized that lipids may bind to CD1 via their hydrophobic alkyl and acyl chains, exposing the hydrophilic sugar, phosphate, and other polar functions for interaction with the TCR complementarity-determining regions (CDRs). To test this model, we mutated the residues in the CDR3 region of the DN1 TCR beta-chain that were predicted to project between the CD1b alpha helices in a model of the TCR/CD1 complex. In addition, we tested the requirement for the negatively charged and polar functions of mycolic acid for Ag recognition. Our findings indicate that the CDR loops of the TCR form the Ag recognition domain of CD1-restricted TCRs and suggest that the hydrophilic domains of a lipid Ag can form a combinatorial epitope recognized by the TCR.

Amino Acid Sequence↗
PubMed1999

The pH-dependent structural variation of complementarity-determining region H3 in the crystal structures of the Fv fragment from an anti-dansyl monoclonal antibody.

The Fv fragment from an anti-dansyl antibody was optimally crystallized into two crystal forms having slightly different lattice dimensions at pH 5.25 and 6.75. The two crystal structures were determined and refined at high resolution at 112 K (at 1.45 A for the crystal at pH 5.25 and at 1.55 A for that at pH 6.75). In the two crystal structures, marked differences were identified in the first half of CDRH3 s having an amino acid sequence of Ile95H-Tyr96H-Tyr97H-His98H-Tyr99H-Pro1 00H-Trp100aH-Phe100bH-Ala101H- Tyr102H. NMR pH titration experiments revealed the p Kavalues of four histidine residues (His27dL, His93L, His55H and His98H) exposed to solvent. Only His98H (p Ka=6.3) completely changed its protonation state between the two crystallization conditions. In addition, the environmental structures including hydration water molecules around the four histidine residues were carefully compared. While the hydration structures around His27dL, His93L and His55H were almost invariant between the two crystal structures, those around His98Hs showed great difference in spite of the small conformational difference of His98H between the two crystal structures. These spectroscopic and crystallographic findings suggested that the change in the protonation state in His98H was responsible for the structural differences between pH 5.25 and 6.75. In addition, the most plausible binding site of the dansyl group was mapped into the present structural models with our previous NMR experimental results. The complementarity-determining regions H1, H3 and the N-terminal region in the VH domain formed the site. The side-chain of Tyr96H occupied the site and interacted with Phe27H of H1, giving a clue for the binding mode of the dansyl group in the site.

Antibodies, Monoclonal↗
PubMed1999

Complementarity-determining region (CDR) implantation: a theme of recombination.

A novel technology in the area of antibody engineering has been developed which allows for the creation of new types of antibody molecules. It is called complementarity-determining region (CDR) implantation and permits the random combination of CDR sequences formed in vivo into a single master framework. Thus, totally new gene combinations can be produced and used in selection processes. The result is a genetic variability which is extremely large, even exceeding the natural variability found in the immune system. In this commentary, CDR implantation is presented and the technology is discussed.

Animals↗
PubMed1994

Defective secretion of an immunoglobulin caused by mutations in the heavy chain complementarity determining region 2.

We have investigated four secretion-deficient antibodies (Abs) derived from a panel of 46 mutant T15 anti-phosphocholine Abs, all of which have point mutations in the heavy chain (H) complementarity determining region 2 (CDR2). The level of secretion for these four Abs was < 10% of wild type when expressed together with the T15 light chain (L) in either SP2/0 or P3X63Ag8.653 myeloma cells although normal levels of H and L chain mRNA were produced. Moreover, abundant intracellular H and L chain proteins were detected. Three of the four mutants had little or no assembled H and L complexes intracellularly whereas one had a significant amount of intracellular immunoglobulin (Ig) which was shown to be capable of binding Ag. Thus, we demonstrate for the first time that point mutations confined to CDR2 of the H chain variable (V) region can impede Ab assembly and secretion. We then introduced the same CDR2 mutations into a related H chain which is encoded by the same T15 VH gene but different diversity (D) and joining (J) genes. When these H chains were expressed with a non-T15 L chain, the resulting Abs were secreted normally. The results thus suggest that the effects of the CDR2 mutations on Ab secretion are dependent on their interactions with L and/or H chain D-J sequences. These results also reveal a novel mechanism that could contribute to B cell wastage.

Amino Acid Sequence↗
PubMed2004

Alteration of amino acid residues at the L-chain N-terminus and in complementarity-determining region 3 increases affinity of a recombinant F(ab) for the human N blood group antigen.

BACKGROUND: To examine the fine specificity of glycopeptide-specific antibodies, this study focused on the human MN blood group system. F(ab) phage display methods were previously used to construct an F(ab) family in which the H-chain Fd fragment was held constant whereas the L chains were "shuffled." This yielded two related F(ab), N92 and NNA7, with low and high affinity for N, respectively. Although their L-chain sequences are very similar, sharing 92 percent amino acid identity, there are intriguing differences at the N-terminus and in complementarity-determining region 3 (CDR3) at positions 89, 91, 92, and 96. STUDY DESIGN AND METHODS: Site-directed mutagenesis, ELISA, and hemagglutination were used to examine the contributions of these variations to antibody affinity. RESULTS: Studies with the N92-S91G and NNA7-G91S mutants demonstrated that the Gly at position 91 was critically important for ensuring high affinity. Indeed, the affinity of N92-S91G was almost as high as N92TM, in which all four CDR3 residues were changed to match NNA7. N-terminal L-chain differences were surprisingly important in determining affinity. For example, when the N-terminus of N92 was changed to match that of NNA7, affinity increased approximately 30-fold. CONCLUSION: Specific residues at the L-chain N-terminus and in CDR3 significantly affected F(ab) affinity for N. Future structural studies of these F(ab), alone and complexed with this glycopeptide antigen, will provide further insights into these phenomena.

Animals↗
PubMed1993

Transfer of putative complementarity-determining region loops of T cell receptor V domains confers toxin reactivity but not peptide/MHC specificity.

We have used multiple-amino acid replacement mutagenesis to examine the roles of the TCR homologues of Ig complementarity-determining regions (CDR) and framework sequences in Ag-MHC and Staphylococcus aureus enterotoxin reactivity. In the three cases examined, transplantation of Ig CDR3 homologues between I-Ek-restricted TCR that recognize distinct peptides did not result in transfer of peptide reactivity. Thus the structural context of the CDR3 loops, e.g., both neighboring CDR and the V beta structure, must play a crucial, albeit supporting, role in ligand recognition. The extreme lability of this context was also shown by the fact that transplantation of the CDR1, -2, and -3 loops from the beta chain of 5C.C7 onto a V beta 1 framework failed to transfer MHC-peptide specificity even when the TCR-alpha chains were identical. In contrast, superantigen reactivity was readily transferred in several cases, with CDR2 transplants conferring strong staphylococcal enterotoxin B and A reactivity and CDR1 transplants yielding weak reactivities. This suggests that bacterial (and perhaps other) superantigens bind to many of the same regions of the TCR V beta that are believed to interact with MHC molecules. These regions of V beta may be ideal targets for superantigen binding precisely because they interact with MHC molecules and thus may be relatively conserved.

Animals↗
PubMed2005

Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity.

Combinatorial libraries of rearranged hypervariable V(H) and V(L) sequences from nonimmunized human donors contain antigen specificities, including anti-self reactivities, created by random pairing of V(H)s and V(L)s. Somatic hypermutation of immunoglobulin genes, however, is critical in the generation of high-affinity antibodies in vivo and occurs only after immunization. Thus, in combinatorial phage display libraries from nonimmunized donors, high-affinity antibodies are rarely found. Lengthy in vitro affinity maturation is often needed to improve antibodies from such libraries. We report the construction of human Fab libraries having a unique combination of immunoglobulin sequences captured from human donors and synthetic diversity in key antigen contact sites in heavy-chain complementarity-determining regions 1 and 2. The success of this strategy is demonstrated by identifying many monovalent Fabs against multiple therapeutic targets that show higher affinities than approved therapeutic antibodies. This very often circumvents the need for affinity maturation, accelerating discovery of antibody drug candidates.

Antibody Affinity↗
PubMedDate not supplied

Length of the antibody heavy chain complementarity determining region 3 as a specificity-determining factor.

The antigen binding site of an antibody is made up of residues residing in six hypervariable loops of the heavy and light chains. In most cases several or all of these loops are required for the establishment of the antigen-binding surface. Five of these loops display a limited diversity in length and sequence while the third complementarity determining region (CDR) of the heavy chain is highly different between antibodies not only with respect to sequence but also with respect to length. Its extensive diversity is a key component in the establishment of binding sites allowing for the recognition of essentially any antigen by humoral immunity. The relative importance of its sequence vs its length diversity in this context is however, not very well established. To investigate this matter further we have used an approach employing combinatorial antibody libraries and antigen-specific selection in the search for CDRH3 length and sequence diversity compatible with a given antigen specificity, the major antigenic determinant on the tumour-associated antigen mucin-1. In this way we have now defined heavy chain CDR3 length as a critical parameter in the creation of an antigen-specific binding site. We also propose that this may reflect a dependence of a particular structure of this hypervariable loop, the major carrier of diversity in the binding site, for establishment of a given specificity.

Antibody Specificity↗
PubMed2001

A role of the third complementarity-determining region in the affinity maturation of an antibody.

We recently found that there are two distinct antibody maturation pathways for the immune response of C57BL/6 mice to (4-hydroxy-3-nitrophenyl) acetyl and that a junctional amino acid introduced at a point far in advance of somatic hypermutation determined which pathway of affinity maturation was used. We describe here the structural basis for this aspect of maturation using recently developed H3 rules, which allow for reliable identification of the conformation of the third complementarity-determining region of the heavy chain (CDR-H3) from the primary amino acid sequences only. By the application of these rules, the anti-(4-hydroxy-3-nitrophenyl) acetyl antibodies examined here were classified into two major groups on the basis of their CDR-H3 structure, and these groups were found to be consistent with the maturation pathways. In addition, circular dichroism measurements revealed that the versatile nature of the antigen binding of the antibodies was significantly influenced by the pathway employed. We postulated in this study that flexibility in the CDR-H3 structure in the antigen-combining site could facilitate efficient antibody maturation supported by a plurality of possible antigen binding modes.

Animals↗
PubMed2002

Primary structure and complementarity-determining region (CDR) 3 spectratyping of rainbow trout TCRbeta transcripts identify ten Vbeta families with Vbeta6 displaying unusual CDR2 and differently spliced forms.

VDJ rearrangement at the teleost TCRbeta locus leads to a highly diverse repertoire of junctions for each VbetaJbeta combination. From a rainbow trout 5' RACE library of TCRbeta transcripts, 47 clones encompassing a full Vbeta-Dbeta-Jbeta-Cbeta sequence were selected and analyzed. A similarity analysis of the sequences evidenced 10 Vbeta families, of which 6 were not previously described. Immunoscope and sequence analysis of the Vbeta-Dbeta-Jbeta junctions of the new families confirmed that they create a polyclonal and diverse repertoire. Multiple alignments showed that rainbow trout Vbetas possess most of the conserved residues typical of Vbeta segments. However, this study revealed a high complementarity-determining region 2 (CDR2) and CDR1 length diversity among rainbow trout Vbeta families, suggesting that the spatial orientation of the TCR could fluctuate in the TCR/peptide/MHC complex, depending on the Vbeta expressed. Among the new Vbeta families, Vbeta6 displayed the strongest deviance from typical hypervariable CDR1 and CDR2 loops, with an unusually short CDR2. Moreover, the Vbeta6 sequence is overall divergent from typical Vbeta sequence, raising the question of its functional relevance. Immunoscope experiments identified a Vbeta6-Jbeta3 junction, which was amplified during the response against viral hemorrhagic septicemia virus, a fish rhabdovirus. Vbeta6 seems therefore to be expressed functionally in a selected TCR. However, the shorter Vbeta6 transcripts produced through an alternative splicing lack the C', C", D, and E strands of the Vbeta domain and are probably nonfunctional.

Alternative Splicing↗
PubMed2002

Interaction among human leucocyte antigen-peptide-T cell receptor complexes in cow's milk allergy: the significance of human leucocyte antigen and T cell receptor-complementarity determining region 3 loops.

BACKGROUND: Allergic individuals respond to only a few specific antigens, therefore allergic diseases are characterized by antigen specificity. Clarification of the mechanism of antigen specificity will lead to progress in the therapy of allergic diseases. OBJECTIVES: The purpose of this study is to determine the specific association among T cell epitopes, antigen-presenting molecules and T cell receptor (TCR), and to determine the TCR usage in the pathogenesis of allergies using antigen-specific T cell clones (TCCs). The results can clarify the mechanism of the antigen specificity of allergic diseases, and provide new therapeutic possibilities using analogue peptides. METHODS: Short-term T cell clones specific to beta-lactoglobulin (BLG) were established from peripheral blood mononuclear cells (PBMCs) collected from five patients allergic to cow's milk. We then identified the T cell epitopes and antigen-presenting molecules, and examined TCR usage. We also determined the sequence of the TCR-complementarity-determining region 3 (CDR3). RESULTS: Six TCCs established from the five patients recognized three different peptides, and BLGp97-117 was recognized by four of the six TCCs. BLGp101-112 (KYLLFCMENSAE) was the core sequence in the fragment. Sequence analysis of TCR by the RT-PCR method revealed a marked heterogeneity in TCR usage, and similar amino acid sequences were recognized in the CDR3 region. Four of the six TCCs recognized BLG in association with human leucocyte antigen (HLA)-DRB1*0405 as antigen-presenting molecules. CONCLUSION: We proposed the motif of the interaction between the HLA-DRB1*0405 allele and antigen peptide, and suggested that HLA-DRB1*0405 is an immunoregulatory gene product for T cell responses to BLG.

Alleles↗
PubMed2002

CD4 T cells selected by antigen under Th2 polarizing conditions favor an elongated TCR alpha chain complementarity-determining region 3.

The affinity of the MHC/peptide/TCR interaction is thought to be one factor determining the differentiation of CD4+ T cells into Th1 or Th2 phenotypes. To study whether CD4+ cells generated under conditions favoring Th1 or Th2 responses select structurally different TCRs, Th1 and Th2 clones and lines were generated from nonobese diabetic and nonobese diabetic H2-E transgenic mice against the peptides proteolipoprotein 56-70, glutamic acid decarboxylase(65) 524-543, and heat shock protein-60 peptides 168-186 and 248-264. Th1/Th2 polarization allowed the generation of clones and lines with fixed peptide specificity and class II restriction but differing in Th1/Th2 phenotype in which the impact on TCR selection and structure could be studied. The Th2 clones tended to use longer TCR complementarity-determining region (CDR)3alpha loops than their Th1 counterparts. This trend was confirmed by analyzing TCRalpha transcripts from Th1 and Th2 polarized, bulk populations. Molecular modeling of Th1- and Th2-derived TCRs demonstrated that Th2 CDR3alpha comprised larger side chain residues than Th1 TCRs. The elongated, bulky Th2 CDR3alpha loops may be accommodated at the expense of less optimal interactions between the MHC class II/peptide and other CDR loops of the TCR. We propose that CD4+ T cells selected from the available repertoire under Th2 polarizing conditions tend to have elongated TCR CDR3alpha loops predicted to alter TCR binding, reducing contact at other interfaces and potentially leading to impeded TCR triggering.

Amino Acid Sequence↗
PubMed2005

Evidence for structural plasticity of heavy chain complementarity-determining region 3 in antibody-ssDNA recognition.

Anti-DNA antibodies play important roles in the pathogenesis of autoimmune diseases. They also represent a unique and relatively unexplored class of DNA-binding protein. Here, we present a study of conformational changes induced by DNA binding to an anti-ssDNA Fab known as DNA-1. Three crystal structures are reported: a complex of DNA-1 bound to dT3, and two structures of the ligand-free Fab. One of the ligand-free structures was determined from crystals exhibiting perfect hemihedral twinning, and the details of structure determination are provided. Unexpectedly, five residues (H97-H100A) in the apex of heavy chain complementarity-determining region 3 (HCDR3) are disordered in both ligand-free structures. Ligand binding also caused a 2-4A shift of the backbone of Tyr L92 and ordering of the L92 side-chain. In contrast, these residues are highly ordered in the Fab/dT3 complex, where Tyr H100 and Tyr H100A form intimate stacking interactions with DNA bases, and L92 forms the 5' end of the binding site. The structures suggest that HCDR3 is very flexible and adopts multiple conformations in the ligand-free state. These results are discussed in terms of induced fit and pre-existing equilibrium theories of ligand binding. Our results allow new interpretations of existing thermodynamic and mutagenesis data in terms of conformational entropy and the volume of conformational space accessible to HCDR3 in the ligand-free state. In the context of autoimmune disease, plasticity of the ligand-free antibody could provide a mechanism by which anti-DNA antibodies bind diverse host ligands, and thereby contribute to pathogenicity.

Antibodies, Antinuclear↗
PubMed1993

Conformation of complementarity determining region L1 loop in murine IgG lambda light chain extends the repertoire of canonical forms.

The refined structure of Se155-4 Fab fragment, the first murine antibody with the lambda light chain, reveals a novel conformation of the light chain complementarity determining region L1. This conformation extends the repertoire of canonical structures. The main determinant of this conformation is the packing of the Val27c side-chain into a hydrophobic pocket formed by the side-chains of Ala33, Leu66, Ala71 and Leu90. The framework L-FR3 loop, encompassing residues 66 to 72, which packs next to the L1 loop, bends significantly more toward the exterior of the molecule than in other Fab fragments. Sequence analysis suggests that the conformations of the L1 and L-FR3 loops observed in Se155-4 are adopted by a majority of murine lambda light chains.

Amino Acid Sequence↗