Complementarity-determining Region 3 Sequences Research Articles

Cytogenetic Heterogeneity in Clonal Plasma Cell Disorders: A Study in Highly Purified Aberrant Plasma Cells

AbstractAbstract 3945 Background:Recurrent immunoglobulin heavy chain (IGH) translocations - t(14q32) - such as t(4;14) or t(11;14) are considered to be primary cytogenetic events which play an important role in the pathogenesis of clonal plasma cell (PC) disorders. However, previous cytogenetic studies in these diseases have mainly been done in whole bone marrow or CD138+ microbead-enriched PC; the latter technique has several pitfalls such as coexistence of CD138+ and CD138− aberrant PC or apoptotic loss of CD138. Design and Methods:Overall, highly-purified aberrant PC (purity ≥98%) from 217 patients with either multiple myeloma (MM; n=155) or monoclonal gammopathy of undetermined significance (MGUS; n=62) were analyzed for the presence of different cytogenetic alterations such as del(13q14), del(17p13) and t(14q32) by multicolor interphase fluorescence in situ hybridization (iFISH). Purity of sorted PC populations was further confirmed by iFISH studies of residual normal - i.e. polyclonal - PC fractions in a subgroup of 10 exemplarily cases with clonal PC disorder. Additionally, analysis of IGH gene arrangements and complementarity determining region 3 (CDR3) sequencing was carried out in a subset of patients (n=9) and simultaneous application of iFISH and immunofluorescent protein staining (FICTION) was performed in selected cases to confirm the clonal relationship between different fractions of tumor PC from individual patients and specific cytogenetic findings. Results:At diagnosis, 96% of all MM vs. 77% of MGUS cases (p<0.001) showed ≥1 cytogenetic alteration and/or hyperdiploidy. Interestingly, among cases with t(14q32), in 24% of MM vs. 62% of MGUS patients (p=0.02) aberrant PC with and without IGH translocation - e.g. t(4;14) or t(11;14) - coexisted in the same patient. Furthermore, longitudinal cytogenetic studies in a subset of MM patients at different stages of the disease showed that an aberrant PC subclone lacking a recurrent t(14q32) might even expand during disease evolution. PCR and sequencing studies confirmed the existence of only one unique CDR3 sequence - in the absence of an oligo-/polyclonal background - in all tested samples indicating clonal relationship of cytogenetically-defined aberrant PC subclones. These observations together with FICTION analyses performed in selected cases, led further to assume that our findings are not due to contaminating residual normal PC. Finally, MM and MGUS cases frequently showed different but related cytogenetic profiles when other cytogenetic alterations which were presumably sequentially acquired by the PC, such as deletions/gains of IGH, FGFR3, CCND1 and MAF, as well as the DNA ploidy status determined by multiparameter flow cytometry were additionally considered. Conclusions:Our findings using highly-purified aberrant PC suggest that also recurrent IGH translocations might evolve secondarily during disease evolution in a significant fraction of MGUS and MM cases, which raises the question about their precise role in the pathogenesis of clonal PC disorders. Our data further supports the existence of a high intratumoral cytogenetic heterogeneity in MM and MGUS, challenging individualized treatment approaches in these diseases. Disclosures:No relevant conflicts of interest to declare.

Molecular and Functional Analysis of Peripheral Lymphocytes in Churg Strauss Syndrome Reveals Several Monoclonal Expansions of CD8+ Cells with a Th1/Proinflammatory Profile

AbstractAbstract 1051Churg Strauss Syndrome (CSS) is a systemic small-vessel necrotizing vasculitis characterized by asthma, chronic rhinosinusitis, pulmonary infiltrations, eosinophil-rich inflammation, vascular and extra-vascular granulomas. We recently documented in the peripheral blood of CSS patients the presence of one or two numerically expanded V-Beta (V-B) families of CD8+ lymphocytes with an effector memory phenotype and with a monoclonal or oligoclonal T-cell receptor (TCR), as shown by TCR-γ rearrangement analysis.To define the characteristics of numerically expanded families at a molecular level, we purified the expanded V-B family of two CSS patients and cloned purified T-cells by limiting dilution. Ten clones from each patient were randomly selected for molecular analysis and the Complementarity-Determining Region 3 (CDR3) of their TCR alpha e beta chain was sequenced. In each patient, 9/10 clones had an identical CDR3 sequence. We also amplified by PCR the cDNA obtained from whole PBMC with specific primers for the expanded V-B family of the same patients (i.e. V-B1 and V-B8) and sequenced the PCR product. The sequence obtained from the entire V-B family (as detected by PCR amplification from whole PBMC) was identical to that obtained from each T-cell clone. These data indicate the presence of a dominant clonotype within a numerically expanded V-B family, which is detectable by molecular analysis not only of single cells but also of whole PBMC.To compare the TCR V-B diversity of CSS patients with healthy donors (HD), we performed TCR-CDR3 length analysis by spectratyping of purified PBMC, CD4 and CD8 cells in CSS patients and in age-matched HD. Analysis was performed by determining the complexity score of 22 V-B families in 8 patients and 8 HD. The TCR repertoire of CSS circulating T-cells was skewed (i.e. with a significantly lower complexity score than HD) and this skewing was found exclusively within the CD8+ subset. Therefore, the CD8+ repertoire of CSS patients is composed by one or two TCR V-B families that are numerically expanded and by several other V-B families that have a skewed spectrum, but are not numerically expanded.In one patient, we also analyzed the cDNA of 11 different V-B families with a low complexity score on a polyacrylamide gel. Five discrete bands were detected, that were purified and sequenced. Sequences from 2/5 PCR-amplified V-B families were readable and representative of a productive and clonal VDJ rearrangement of the CDR3 region. This confirmed that a skewed V-B family at spectratyping is very often the result of an antigen-driven proliferation of a single cell with a distinct TCR.These molecular data revealed that alterations of the T-cell repertoire in CSS patients are more profound than initially expected, involving many CD8+ V-B families. We hypothesized that functional analysis of the whole CD8+ fraction could reveal the main functional characteristics of the several clonal expansions observed within this subset. We therefore analyzed cytokines and chemokine receptor expression of CD8+ and CD4+ cells from the same CSS patients and HD. A significantly higher percentage of both CD8+ and CD4+ cells producing INFγ and TNFα was observed in CCS compared to HS. IL1β, IL2, IL4, IL6, IL13, IL17, GM-CSF, and TGF-β in CD4+ and CD8+ cells were not different between CSS patients and controls, even if a trend toward a higher expression of IL-13 was observed. CD4+ cells, but not CD8+ cells, from CSS patients had a significantly higher production of IL5 and IL10 than HS. These data suggested that patients’ CD8+ cells have a Th1/proinflammatory profile, while the functional profile of CD4+ T-cells is less clearly polarized. Expression of CCR5 and, to a lesser extent, of CXCR3 by total and Vβ-expanded CD8+ cells was significantly higher in CSS than in HS. Expression of CRTH2, but not of CCR4, was significantly higher in CD4+ cells of CSS patients than in controls.In conclusion, the molecular profiling of T-cells revealed that monoclonal expansion of CD8+ lymphocytes in CSS patients are frequent and involve several V-B families. CD8+ cells of patients have a Th1/proinflammatory profile, potentially involved in vasculitic damage with the cooperation of CD4+/Th1 cells. CD4+/Th2 cells (but not CD8+ cells) are functionally apt to mediate eosinophil-rich inflammation and asthma. Disclosures:No relevant conflicts of interest to declare.

Construction of a rationally designed antibody platform for sequencing-assisted selection

Antibody discovery platforms have become an important source of both therapeutic biomolecules and research reagents. Massively parallel DNA sequencing can be used to assist antibody selection by comprehensively monitoring libraries during selection, thus greatly expanding the power of these systems. We have therefore constructed a rationally designed, fully defined single-chain variable fragment (scFv) library and analysis platform optimized for analysis with short-read deep sequencing. Sequence-defined oligonucleotide libraries encoding three complementarity-determining regions (L3 from the light chain, H2 and H3 from the heavy chain) were synthesized on a programmable microarray and combinatorially cloned into a single scFv framework for molecular display. Our unique complementarity-determining region sequence design optimizes for protein binding by utilizing a hidden Markov model that was trained on all antibody-antigen cocrystal structures in the Protein Data Bank. The resultant ~10(12)-member library was produced in ribosome-display format, and comprehensively analyzed over four rounds of antigen selections by multiplex paired-end Illumina sequencing. The hidden Markov model scFv library generated multiple binders against an emerging cancer antigen and is the basis for a next-generation antibody production platform.

Aggregation-resistant domain antibodies engineered with charged mutations near the edges of the complementarity-determining regions

Antibodies commonly contain hydrophobic residues within their complementarity-determining regions (CDRs) that mediate binding to target antigens. Unfortunately, hydrophobic CDRs can also promote antibody aggregation, which is especially concerning for therapeutic antibodies due to the immunogenicity of antibody aggregates. Here we investigate how the sequences of CDRs within single-domain (V(H)) antibodies specific for the Alzheimer's amyloid β peptide can be engineered to resist aggregation without reducing binding affinity. We find that domain antibodies containing clusters of hydrophobic residues within their third CDR (CDR3) are prone to aggregate within days at 25°C and minutes above 70°C. However, inserting two or more negatively charged residues at each edge of CDR3 potently suppresses antibody aggregation without altering binding affinity. We also find that inserting charged mutations at one edge of CDR3 (N- or C-terminal) prevents aggregation, but only if such mutations are located at the edge closest to most hydrophobic portion of CDR3. In contrast, charged mutations outside of CDR3 fail to suppress aggregation. Our findings demonstrate that the sequence of CDR loops can be engineered in a systematic manner to improve antibody solubility without altering binding affinity or specificity.

Rationalization and design of the complementarity determining region sequences in an antibody-antigen recognition interface.

Protein-protein interactions are critical determinants in biological systems. Engineered proteins binding to specific areas on protein surfaces could lead to therapeutics or diagnostics for treating diseases in humans. But designing epitope-specific protein-protein interactions with computational atomistic interaction free energy remains a difficult challenge. Here we show that, with the antibody-VEGF (vascular endothelial growth factor) interaction as a model system, the experimentally observed amino acid preferences in the antibody-antigen interface can be rationalized with 3-dimensional distributions of interacting atoms derived from the database of protein structures. Machine learning models established on the rationalization can be generalized to design amino acid preferences in antibody-antigen interfaces, for which the experimental validations are tractable with current high throughput synthetic antibody display technologies. Leave-one-out cross validation on the benchmark system yielded the accuracy, precision, recall (sensitivity) and specificity of the overall binary predictions to be 0.69, 0.45, 0.63, and 0.71 respectively, and the overall Matthews correlation coefficient of the 20 amino acid types in the 24 interface CDR positions was 0.312. The structure-based computational antibody design methodology was further tested with other antibodies binding to VEGF. The results indicate that the methodology could provide alternatives to the current antibody technologies based on animal immune systems in engineering therapeutic and diagnostic antibodies against predetermined antigen epitopes.

Mass spectrometry analyses of κ and λ fractions result in increased number of complementarity‐determining region identifications

Sera from lung cancer patients contain antibodies against tumor-associated antigens. Specific amino acid sequences of the complementarity-determining regions (CDRs) in the antigen-binding fragment (Fab) of these antibodies have potential as lung cancer biomarkers. Detection and identification of CDRs by mass spectrometry can significantly be improved by reduction of the complexity of the immunoglobulin molecule. Our aim was to molecular dissect IgG into κ and λ fragments to reduce the complexity and thereby identify substantially more CDRs than by just total Fab isolation. We purified Fab, Fab-κ, Fab-λ, κ and λ light chains from serum from 10 stage I lung adenocarcinoma patients and 10 matched controls from the current and former smokers. After purification, the immunoglobulin fragments were enzymatically digested and measured by high-resolution mass spectrometry. Finally, we compared the number of CDRs identified in these immunoglobulin fragments with that in the Fab fragments. Twice as many CDRs were identified when Fab-κ, Fab-λ, κ and λ (3330) were combined than in the Fab fraction (1663) alone. The number of CDRs and κ:λ ratio was statistically similar in both cases and controls. Molecular dissection of IgG identifies significantly more CDRs, which increases the likelihood of finding lung cancer-related CDR sequences.

Next Generation Sequencing Spectratyping (NGS-S) Comprehensively Monitors T Cell Receptor Diversity in Children with T Cell Abnormalities

Abstract▪2173▪This icon denotes a clinically relevant abstractThe adaptive arm of the immune system - the T-cell compartment – may become compromised by inherited or acquired defects resulting in cancer, autoimmunity, or increased susceptibility to microbial infectious agents. A normal polyclonal T cell compartment comprises an estimated number of 2.5 × 10E7 individual T cell clones each expressing a unique antigen recognizing T cell receptor (TCR). The functionality of the T cell compartment is thus – at least partly - reflected by TCR sequence diversity. There is a medical need for rapid and robust diagnostic approaches that accurately monitor TCR diversity in patient samples, e.g. after bone marrow transplantation (BMT).Previously, complementarity determining region 3 (CDR3) size spectratyping in TCR β-chain subfamilies (Vβ), an immunoscopic technique, was employed for the analysis of T cell diversity. However, spectratyping is limited to the analysis of CDR3 length polymorphisms only. Underlying diversity of TCR Vβ sequences of equal length remain undetected. Furthermore, spectratyping is time consuming and consequently data can only be interpreted with a delay of weeks.To determine TCR diversity fast and accurately we developed next-generation-sequencing spectratyping (NGS-S), which employs high coverage, massive parallel Roche/454-sequencing of TCR Vβ-chain amplicons.Three different sample groups were analyzed in parallel by spectratyping and NGS-S: T cells from (1) healthy children (n=6), (2) children at diagnosis of severe aplastic anemia (n=7), and (3) children who underwent haploidentical BMT (n=7). In brief, RNA was extracted from bone marrow derived CD8+ cells and transcribed to cDNA. Amplicon libraries were generated by PCR employing two degenerated wobble primers (VP1, VP2), designed to cover most of the known TCR Vβ gene segments and a universal reverse primer (CP1) located in the conserved TCR region (Figure 1). The mean overall coverage of the CDR3 region achieved was 23.133 per patient. [Display omitted] For simultaneous characterization of these individual amplicons we generated the TCR Profiler (Figure 2). This new software tool automatically preprocessed raw sequencing data using a threshold quality value (q=30) to trim the 3’ end of the TCR β-chain sequences. Rearranged germline TCR Vβ and Jβ genes were identified by Smith-Waterman local alignment against each human TCR β-chain germline gene of the IMGT/GENE-DB reference directory. Base call reliability was assured by incorporating phred-like quality values provided by the sequencer into the Smith-Waterman local alignment routine. A quality value, q, was computed for each base as the log-transformation of the probability p of a base being incorrectly called, q = − 10 x log10(p). Transformed into a reliability measure, r = 1- (1/10^(q/10)), q-values indicated the probability for each base to be correctly called. CDR3 regions were delimited using specific flanking amino acid sequence motifs. The 5’ end motif varies dependently on the rearranged TCR Vβ gene and was identified using the IMGT/GENE-DB reference directory sequence set, whereas the 3’ end motif [W/F]GXG (IUPAC code) is conserved in all TCR β-chains. CDR3 length was calculated including all amino acids between the two motifs. Screening for in-frame stop codons was done to exclude non-functional TCR β-chain transcripts. [Display omitted] The TCR profiler identified on average 16165 of the input sequences as unique CDR3 sequences. Of these a mean of 9840 were predicted to be functionally rearranged.Whereas spectratyping gave a rough estimate of CDR3 size and allowed T cell deficient samples to be identified, NGS-S determined the exact length and sequence composition of the CDR3, identified the rearranged TCR Vβ and Jβ genes and the specific recombination (Figure 3A). [Display omitted] Utilization of specific genes, the resulting amino acid composition of the CDR3 region as well as its length and overall diversity were integrated into a novel NGS-S score.This score reliably differentiated not only between normal and T cell deficient samples, but also between the different T cell deficient groups (SAA and BMT) (Figure 3B). We conclude that NGS-S allows rapid and precise determination of TCR diversity in clinical samples. Disclosures:No relevant conflicts of interest to declare.

Development of Diffuse Large B-Cell Lymphoma in a Patient with Waldenström’s Macroglobulinemia/Lymphoplasmacytic Lymphoma: Clonal Identity between Two B-Cell Neoplasms

Waldenström's macroglobulinemia (WM)/ lymphoplasmacytic lymphoma (LPL) is an indolent mature B-cell neoplasm. In rare cases of WM/LPL, diffuse large B-cell lymphoma (DLBCL) develops as a result of histologic transformation. In this report, we present a case of DLBCL developing in a patient with WM/LPL. Combination chemotherapy for DLBCL was effective and complete remission was eventually achieved. We attempted to determine the clonal relatedness between WM/LPL and DLBCL in the patient by analyzing complementarity-determining region 3 (CDR3) in the immunoglobulin heavy chain gene. A common CDR3 sequence was found in tumor cells of DLBCL and those of WM/LPL, indicating that tumor cells of DLBCL are clonally identical to those of WM/LPL. Therefore, in the present case, DLBCL is developed from WM/LPL cells by clonal evolution.

Construction and molecular characterization of mouse single-chain variable fragment antibodies against Burkholderia mallei and Burkholderia pseudomallei

We have selected two lipopolysaccharide (LPS) specific Burkholderia mallei mouse monoclonal antibodies (mAbs) and four anti-capsular B. pseudomallei-specific mAbs to generate mouse single-chain variable fragment (scFv) antibodies. This selection was made through extensive in vitro and in vivo assay from our library of mAbs against B. mallei and B. pseudomallei. We initially generated the mouse immunoglobulin variable heavy chain (VH) and light chain (VL) regions from each of these six selected mAbs using a phage display scFv technology. We determined the coding sequences of the VH and VL regions and successfully constructed two B. mallei-specific scFv phage antibodies consisting of two different VH (VH1 and VH2) and one Vλ1 families. Four scFvs constructed against B. pseudomallei had two VH (VH1 and VH6) and two VL (Vκ4/5 and Vκ21) families. All of six scFv antibodies constructed demonstrated good binding activity without any rounds of biopanning against B. mallei (M5D and M18F were 0.425 and 0.480 at OD405nm) and B. pseudomallei (P1E7, P2I67, P7C6, and P7F4 were 0.523, 0.859, 0.775, and 0.449 at OD405nm) by ELISA, respectively. A comparison of the immunoglobulin gene segments revealed that the gene sequences in complementarity-determining regions (CDRs) of three out of four B. pseudomallei-specific scFvs are highly conserved. We determined that the two B. mallei-specific scFvs have different CDRs in the VH, but the amino acid sequences of CDRs in the VL are conserved. This high sequence homology found in CDRs of VH or VL of these mAbs contributes to our better understanding and determination of binding to the specific antigenic epitope(s). The scFv phage display technology may be a valuable tool to develop and engineer mAbs with improved antigen-binding affinity.

Analysis of the TCR β-chain gene rearrangement and CDR3 repertoire in patients with breast cancer.

Objective To analyze the clonal gene rearrangement and complementarity determining region 3 (CDR3) repertoire of TCR β-chain in fine needle aspiration biopsy (FNAB) specimens of lymph node metastasis in patients with breast cancer. Methods The TCR CDR3 region genes of 24 TCR Vβ subfamilies were amplified by utilizing RT-PCR technology, and the CDR3 lengths of TCR β-chain were analyzed with gene scan technology for 2 cases with lymph node reactive hyperplasia and 3 patients with lymph node metastasis of breast cancer. The clonality of T cells presumed by spectra typing was further confirmed by CDR3 sequencing. Results TCR β-chain presented specific repertoire skewing in metastatic lymph node,and only 3-5 TCR Vβ subfamily of T cells were identified, respectively. Clonal expanded T cells, including oligoclonal, polyclonal patterns, in one or more Vβ subfamilies were found in all cases. The oligoclonal expanded T cells had different CDR3 amino acid sequences. Conclusion There are characteristic T cells cloning proliferation and selected usage of TCR Vβ subfamily T cells could be found in metastatic lymph node.The sequences of CDR3 in different TCR clone proliferation are mostly different. Key words: Breast neoplasms; Gene rearrangement; Complementarity determining regions; Gene scan

Characterization of conserved CDR3 sequence of TCR α- and β-chain genes in peripheral blood T-cells from patients with diffuse large B-cell lymphoma

T-cell immunodeficiency is a common feature in cancer patients, but clonally expanded T cells which were considered to have the specific anti-tumor cytotoxicity can be identified in most cancer patients, including those with hematological malignancies. We previously reported that there were skewed usages of T-cell receptor (TCR) Vα and TCR Vβ subfamily in clonally expanded T cells in the peripheral blood (PB) of patients with diffuse large B-cell lymphoma (DLBCL). In order to investigate the characteristics of the complementarity determining region 3 (CDR3) of TCR α- and β-chain genes in clonally expanded T cells, the sequences of TCR-CDR3 region of clonally expanded TCR Vα and TCR Vβ subfamily T cells were analyzed. Conserved amino acid motifs of TCR-CDR3 region were identified in different clonally expanded T cells. RSxYNTDKLI, SxGG, GGS, and DSxY motifs were found in the TCR Vα subfamily T-cell clones, while SxGTG, SRG, and GTxD motifs existed in the TCR Vβ subfamily T-cell clones. Moreover, the TCR harboring conserved motif in CDR3 region of some T-cell clones had high homology in the three-dimensional spatial structures. These findings revealed the mono/oligoclonally expanded T cells were derived from the stimulation of some antigens in the PB of patients with DLBCL, and may be recognizing an identical antigenic epitope.

Development and characterization of a fully functional small anti-HER2 antibody

The penetrating of monoclonal antibodies (mAbs) into solid tumor may be hampered by their large size. The antibody mimetics, composed of two complementarity-determining regions (CDRs) through a cognate framework region (FR), have been demonstrated to have the capacity to penetrate tumors superior to its parental intact IgG. In this study, we used CDR and FR sequences from the humanized anti-HER2 monoclonal antibody trastuzumab to design four antibody mimetics. Then these antibody mimetics were fused to human IgG Fc to generate mimetics-Fc small antibodies. One of the four mimetics-Fc antibodies binds well to HER2-overexpressing SK-BR3 cells and effectively inhibits the binding of trastuzumab. This mimetics-Fc, denoted as HMTI-Fc, was shown to be effective in mediating antibody-dependent cellular cytotoxicity and exhibit an antiproliferative effect in SK-BR3 cells. To our knowledge, the HMTI-Fc antibody shown here is the smallest fully functional antibody and may have a potential for treatment of cancer.

Analyses of the TCR β-chain gene rearrangement and CDR3 repertoire in patients with peripheral T-cell lymphoma

Objective To analyze the elonal gene rearrangement and complementarity determining region 3 (CDR3) Repertoire of TCR β-chain in fine needle aspiration biopsy (FNAB) specimens of peripheral T-cell lymphoma. Methods The TCR CDR3 region genes of 24 TCR Vβ subfamilies were amplified by utilizing RT-PCR technology, and the CDR3 size lengths of TCR β-chain were analyzed with genesean technology for 4 healthy individuals and 4 patients with peripheral T-cell lymphoma. The clonality of T cells presumed by spectratyping was further confirmed by CDR3 sequencing. Results TCR β-chain presented specific repertoire skewing in 4 cases with peripheral T-cell lymphoma, and only 1-4 TCR Vβ subfamily T cells were identified, respectively. Clonal expanded T cells, including mono, bioclonal and oligoclonal trend patterns, in one or more Vβ subfamilies were found in all cases. The mono expanded T cells have different CDR3 amino acid sequences. Conclusion Characteristic T cells cloning proliferation and selected usage of TCR Vβ subfamily T cells were found in 4 cases with peripheral T-cell lymphoma. The sequences of CDR3 in different TCR clone proliferation are different. Key words: Gene rearrangement; Complementarity determining regions; Lymphoma; T-cell; Genescan

Automated Identification of Complementarity Determining Regions (CDRs) Reveals Peculiar Characteristics of CDRs and B Cell Epitopes

Exact identification of complementarity determining regions (CDRs) is crucial for understanding and manipulating antigenic interactions. One way to do this is by marking residues on the antibody that interact with B cell epitopes on the antigen. This, of course, requires identification of B cell epitopes, which could be done by marking residues on the antigen that bind to CDRs, thus requiring identification of CDRs. To circumvent this vicious circle, existing tools for identifying CDRs are based on sequence analysis or general biophysical principles. Often, these tools, which are based on partial data, fail to agree on the boundaries of the CDRs. Herein we present an automated procedure for identifying CDRs and B cell epitopes using consensus structural regions that interact with the antigens in all known antibody-protein complexes. Consequently, we provide the first comprehensive analysis of all CDR-epitope complexes of known three-dimensional structure. The CDRs we identify only partially overlap with the regions suggested by existing methods. We found that the general physicochemical properties of both CDRs and B cell epitopes are rather peculiar. In particular, only four amino acids account for most of the sequence of CDRs, and several types of amino acids almost never appear in them. The secondary structure content and the conservation of B cell epitopes are found to be different than previously thought. These characteristics of CDRs and epitopes may be instrumental in choosing which residues to mutate in experimental search for epitopes. They may also assist in computational design of antibodies and in predicting B cell epitopes.

Antibody Complementarity-Determining Regions (CDRs) Can Display Differential Antimicrobial, Antiviral and Antitumor Activities

BackgroundComplementarity-determining regions (CDRs) are immunoglobulin (Ig) hypervariable domains that determine specific antibody (Ab) binding. We have shown that synthetic CDR-related peptides and many decapeptides spanning the variable region of a recombinant yeast killer toxin-like antiidiotypic Ab are candidacidal in vitro. An alanine-substituted decapeptide from the variable region of this Ab displayed increased cytotoxicity in vitro and/or therapeutic effects in vivo against various bacteria, fungi, protozoa and viruses. The possibility that isolated CDRs, represented by short synthetic peptides, may display antimicrobial, antiviral and antitumor activities irrespective of Ab specificity for a given antigen is addressed here.Methodology/Principal FindingsCDR-based synthetic peptides of murine and human monoclonal Abs directed to: a) a protein epitope of Candida albicans cell wall stress mannoprotein; b) a synthetic peptide containing well-characterized B-cell and T-cell epitopes; c) a carbohydrate blood group A substance, showed differential inhibitory activities in vitro, ex vivo and/or in vivo against C. albicans, HIV-1 and B16F10-Nex2 melanoma cells, conceivably involving different mechanisms of action. Antitumor activities involved peptide-induced caspase-dependent apoptosis. Engineered peptides, obtained by alanine substitution of Ig CDR sequences, and used as surrogates of natural point mutations, showed further differential increased/unaltered/decreased antimicrobial, antiviral and/or antitumor activities. The inhibitory effects observed were largely independent of the specificity of the native Ab and involved chiefly germline encoded CDR1 and CDR2 of light and heavy chains.Conclusions/SignificanceThe high frequency of bioactive peptides based on CDRs suggests that Ig molecules are sources of an unlimited number of sequences potentially active against infectious agents and tumor cells. The easy production and low cost of small sized synthetic peptides representing Ig CDRs and the possibility of peptide engineering and chemical optimization associated to new delivery mechanisms are expected to give rise to a new generation of therapeutic agents.

Investigating the Properties of Bacillus thuringiensis Cry Proteins with Novel Loop Replacements Created Using Combinatorial Molecular Biology

Cry proteins are a large family of crystalline toxins produced by Bacillus thuringiensis. Individually, the family members are highly specific, but collectively, they target a diverse range of insects and nematodes. Domain II of the toxins is important for target specificity, and three loops at its apex have been studied extensively. There is considerable interest in determining whether modifications in this region may lead to toxins with novel specificity or potency. In this work, we studied the effect of loop substitution on toxin stability and specificity. For this purpose, sequences derived from antibody complementarity-determining regions (CDR) were used to replace native domain II apical loops to create "Crybodies." Each apical loop was substituted either individually or in combination with a library of third heavy-chain CDR (CDR-H3) sequences to create seven distinct Crybody types. An analysis of variants from each library indicated that the Cry1Aa framework can tolerate considerable sequence diversity at all loop positions but that some sequence combinations negatively affect structural stability and protease sensitivity. CDR-H3 substitution showed that loop position was an important determinant of insect toxicity: loop 2 was essential for activity, whereas the effects of substitutions at loop 1 and loop 3 were sequence dependent. Unexpectedly, differences in toxicity did not correlate with binding to cadherins--a major class of toxin receptors--since all Crybodies retained binding specificity. Collectively, these results serve to better define the role of the domain II apical loops as determinants of specificity and establish guidelines for their modification.

Clonal Focusing of Epitope-Specific CD8+T Lymphocytes in Rhesus Monkeys following Vaccination and Simian-Human Immunodeficiency Virus Challenge

To afford the greatest possible immune protection, candidate human immunodeficiency virus (HIV) vaccines must generate diverse and long-lasting CD8(+) T lymphocyte responses. In the present study, we evaluate T-cell receptor Vbeta (variable region beta) gene usage and a CDR3 (complementarity-determining region 3) sequence to assess the clonality of epitope-specific CD8(+) T lymphocytes generated in rhesus monkeys following vaccination and simian-human immunodeficiency virus (SHIV) challenge. We found that vaccine-elicited epitope-specific CD8(+) T lymphocytes have a clonal diversity comparable to those cells generated in response to SHIV infection. Moreover, we show that the clonal diversity of vaccine-elicited CD8(+) T-lymphocyte responses is dictated by the epitope sequence and is not affected by the mode of antigen delivery to the immune system. Clonal CD8(+) T-lymphocyte populations persisted following boosting with different vectors, and these clonal cell populations could be detected for as long as 4 years after SHIV challenge. Finally, we show that the breadth of these epitope-specific T lymphocytes transiently focuses in response to intense SHIV replication. These observations demonstrate the importance of the initial immune response to SHIV, induced by vaccination or generated during primary infection, in determining the clonal diversity of cell-mediated immune responses and highlight the focusing of this clonal diversity in the setting of high viral loads. Circumventing this restricted CD8(+) T-lymphocyte clonal diversity may present a significant challenge in the development of an effective HIV vaccine strategy.

High-throughput Generation of Synthetic Antibodies from Highly Functional Minimalist Phage-displayed Libraries

We have previously established a minimalist approach to antibody engineering by using a phage-displayed framework to support complementarity determining region (CDR) diversity restricted to a binary code of tyrosine and serine. Here, we systematically augmented the original binary library with additional levels of diversity and examined the effects. The diversity of the simplest library, in which only heavy chain CDR positions were randomized by the binary code, was expanded in a stepwise manner by adding diversity to the light chain, by diversifying non-paratope residues that may influence CDR conformations, and by adding additional chemical diversity to CDR-H3. The additional diversity incrementally improved the affinities of antibodies raised against human vascular endoethelial growth factor and the structure of an antibody–antigen complex showed that tyrosine side-chains are sufficient to mediate most of the interactions with antigen, but a glycine residue in CDR-H3 was critical for providing a conformation suitable for high-affinity binding. Using new high-throughput procedures and the most complex library, we produced multiple high-affinity antibodies with dissociation constants in the single-digit nanomolar range against a wide variety of protein antigens. Thus, this fully synthetic, minimalist library has essentially recapitulated the capacity of the natural immune system to generate high-affinity antibodies. Libraries of this type should be highly useful for proteomic applications, as they minimize inherent complexities of natural antibodies that have hindered the establishment of high-throughput procedures. Furthermore, analysis of a large number of antibodies derived from these well-defined and simplistic libraries allowed us to uncover statistically significant trends in CDR sequences, which provide valuable insights into antibody library design and into factors governing protein–protein interactions.

Diversity in the Complementarity-Determining Region 3 (CDR3) of Antibodies from Mice with Evolving Anti-Thyroid-Stimulating Hormone Receptor Antibody Responses

In a mouse model of autoimmune Graves' disease, stimulatory anti-TSH receptor (TSHR) antibodies (TSAbs) slowly evolve upon repeated immunization with TSHR and lead to hyperthyroidism. Although all immunized mice developed high levels of TSH-binding inhibitory Ig (TBII), only a subset of these mice become hyperthyroid, suggesting that the generation of pathogenic antibodies (Abs) may require affinity maturation. We analyzed the complementarity-determining region 3 (CDR3) of IGHV1 and IGHV5 heavy chains from mice at different stages of disease development. Subcloned CDR3 PCR products were amplified from RNA isolated from enriched splenic B/plasma cells of a control mouse, and mice with low TBII and normal T(4) levels (LTNT(4)), high TBII and normal T(4) levels (HTNT(4)), and high TBII and high T(4) levels (HTHT(4)). Using statistical analyses, we correlated usage of D and J genes and the amino acid composition and length of and mutations within the CDR3 with different outcomes after TSHR immunization. CDR3 sequences from TSHR-immunized mice contained a higher frequency of D gene SP2.9 relative to control, whereas sequences from HTHT(4) contained a higher frequency of D gene Q52 compared with sequences from LTNT(4). Furthermore, HTHT(4) sequences also contained higher CDR3 replacement mutations, relative to LTNT(4) and HTNT(4) mice, that are indicative of somatic hypermutation. Collectively, our results suggest that higher somatic mutations within the CDR3 may correlate with pathogenic antibodies against the TSHR.

Single-chain Antibodies to the EWS NH2 Terminus Structurally Discriminate between Intact and Chimeric EWS in Ewing's Sarcoma and Interfere with the Transcriptional Activity of EWS In vivo

The chimeric protein EWS-FLI1, arising from chromosomal translocation in Ewing's sarcoma family tumors (ESFT), acts as an aberrant tumorigenic transcription factor. The transforming activity of EWS-FLI1 minimally requires an ETS DNA binding domain and the EWS NH(2) terminus. Proteins interacting with the EWS portion differ between germ-line and chimeric EWS despite their sharing identical sequences in this domain. We explored the use of the phage display technology to isolate anti-EWS-FLI1 specific single-chain antibody fragments (scFvs). Using recombinant EWS-FLI1 as bait, 16 independent specific antibody clones were isolated from combinatorial phage display libraries, of which six were characterized in detail. Despite differing in their complementarity-determining region sequences, all six scFvs bound to the same epitope spanning residues 51 to 75 within the shared minimal transforming EWS domain. Whereas all six scFvs bound efficiently to cellular EWS, reactivity with ESFT-expressed EWS-FLI1 was weak and restricted to denatured protein. One scFv, scFv-I85, when expressed as an intrabody, efficiently suppressed EWS-dependent coactivation of hepatocyte nuclear factor 4- and OCT4-mediated transcription in vivo but no effect on known EWS-FLI1 target genes was observed. These data suggest that a prominent EWS epitope exposed on recombinant EWS-FLI1 structurally differs between germ-line and chimeric EWS in mammalian cells and that this region is functionally involved in the transcriptional activity of EWS. Thus, we have generated a tool that will prove useful to specifically differentiate between normal and rearranged EWS in functional studies.