Chain Complementarity-determining Region Research Articles

An autoreactive antibody from an SLE/HIV-1 individual broadly neutralizes HIV-1

Broadly HIV-1-neutralizing antibodies (BnAbs) display one or more unusual traits, including a long heavy chain complementarity-determining region 3 (HCDR3), polyreactivity, and high levels of somatic mutations. These shared characteristics suggest that BnAb development might be limited by immune tolerance controls. It has been postulated that HIV-1-infected individuals with autoimmune disease and defective immune tolerance mechanisms may produce BnAbs more readily than those without autoimmune diseases. In this study, we identified an HIV-1-infected individual with SLE who exhibited controlled viral load (<5,000 copies/ml) in the absence of controlling HLA phenotypes and developed plasma HIV-1 neutralization breadth. We collected memory B cells from this individual and isolated a BnAb, CH98, that targets the CD4 binding site (CD4bs) of HIV-1 envelope glycoprotein 120 (gp120). CH98 bound to human antigens including dsDNA, which is specifically associated with SLE. Anti-dsDNA reactivity was also present in the patient's plasma. CH98 had a mutation frequency of 25% and 15% nt somatic mutations in the heavy and light chain variable domains, respectively, a long HCDR3, and a deletion in the light chain CDR1. The occurrence of anti-dsDNA reactivity by a HIV-1 CD4bs BnAb in an individual with SLE raises the possibility that some BnAbs and SLE-associated autoantibodies arise from similar pools of B cells.

An immunodominant La/SSB autoantibody proteome derives from public clonotypes

The La/SSB autoantigen is a major target of long-term humoral autoimmunity in primary Sjögren's Syndrome (SS) and systemic lupus erythematosus. A majority of patients with linked anti-Ro60/Ro52/La responses target an NH2-terminal epitope designated LaA that is expressed on Ro/La ribonucleoprotein complexes and the surface membrane of apoptotic cells. In this study, we used high-resolution Orbitrap mass spectrometry to determine the clonality, isotype and V-region sequences of LaA-specific autoantibodies in seven patients with primary SS. Anti-LaA immunoglobulin (Ig)Gs purified from polyclonal sera by epitope-specific affinity chromatography were analysed by combined database and de-novo mass spectrometric sequencing. Autoantibody responses comprised two heavily mutated IgG1 kappa-restricted monoclonal species that were shared (public) across unrelated patients; one clonotype was specified by an IGHV3-30 heavy chain paired with IGKV3-15 light chain and the second by an IGHV3-43/IGKV3-20 pairing. Shared amino acid replacement mutations were also seen within heavy and light chain complementarity-determining regions, consistent with a common breach of B cell tolerance followed by antigen-driven clonal selection. The discovery of public clonotypic autoantibodies directed against an immunodominant epitope on La, taken together with recent findings for the linked Ro52 and Ro60 autoantigens, supports a model of systemic autoimmunity in which humoral responses against protein-RNA complexes are mediated by public sets of autoreactive B cell clonotypes.

BCR CDR3 length distributions differ between blood and spleen and between old and young patients, and TCR distributions can be used to detect myelodysplastic syndrome

Complementarity-determining region 3 (CDR3) is the most hyper-variable region in B cell receptor (BCR) and T cell receptor (TCR) genes, and the most critical structure in antigen recognition and thereby in determining the fates of developing and responding lymphocytes. There are millions of different TCR Vβ chain or BCR heavy chain CDR3 sequences in human blood. Even now, when high-throughput sequencing becomes widely used, CDR3 length distributions (also called spectratypes) are still a much quicker and cheaper method of assessing repertoire diversity. However, distribution complexity and the large amount of information per sample (e.g. 32 distributions of the TCRα chain, and 24 of TCRβ) calls for the use of machine learning tools for full exploration. We have examined the ability of supervised machine learning, which uses computational models to find hidden patterns in predefined biological groups, to analyze CDR3 length distributions from various sources, and distinguish between experimental groups. We found that (a) splenic BCR CDR3 length distributions are characterized by low standard deviations and few local maxima, compared to peripheral blood distributions; (b) healthy elderly people's BCR CDR3 length distributions can be distinguished from those of the young; and (c) a machine learning model based on TCR CDR3 distribution features can detect myelodysplastic syndrome with approximately 93% accuracy. Overall, we demonstrate that using supervised machine learning methods can contribute to our understanding of lymphocyte repertoire diversity.

Humanization of Antibodies Using Heavy Chain Complementarity-determining Region 3 Grafting Coupled with in Vitro Somatic Hypermutation

A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hβNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of hβNGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods.

Mantle cell lymphoma cell lines show no evident immunoglobulin heavy chain stereotypy but frequent light chain stereotypy

Mantle cell lymphoma shows a peculiar immunogenetic profile, but the functional consequences of this fact are unknown. We have determined the precise sequences of rearranged heavy and light chain genes in several mantle cell lymphoma cell lines and investigated the presence of heavy and light chain stereotypy. These cell lines use IGHV and IGLV genes that are known to be preferentially rearranged in mantle cell lymphoma, but we found no evidence of heavy chain stereotypy. In contrast, one cell line (Mino) showed a nearly identical light chain complementarity-determining region 3 when compared to the only published light chain cluster. Two cell line couples (Jeko-1/UPN-2 and JVM-2/JVM-13) showed a highly similar light chain that satisfied the criteria for stereotypy. Our data show that mantle cell lymphoma cell lines resemble the IGHV and IGLV usage of mantle cell lymphoma, and foster the hypothesis that light chain stereotypy might be under-recognized.

Crystal Structure and Computational Modeling of the Fab Fragment from a Protective Anti-Ricin Monoclonal Antibody

BackgroundMany antibody crystal structures have been solved. Structural modeling programs have been developed that utilize this information to predict 3-D structures of an antibody based upon its sequence. Because of the problem of self-reference, the accuracy and utility of these predictions can only be tested when a new structure has not yet been deposited in the Protein Data Bank.MethodsWe have solved the crystal structure of the Fab fragment of RAC18, a protective anti-ricin mAb, to 1.9 Å resolution. We have also modeled the Fv structure of RAC18 using publicly available Ab modeling tools Prediction of Immunoglobulin Structures (PIGS), RosettaAntibody, and Web Antibody Modeling (WAM). The model structures underwent energy minimization. We compared results to the crystal structure on the basis of root-mean-square deviation (RMSD), template modeling score (TM-score), Z-score, and MolProbity analysis.FindingsThe crystal structure showed a pocket formed mainly by AA residues in each of the heavy chain complementarity determining regions (CDRs). There were differences between the crystal structure and structures predicted by the modeling tools, particularly in the CDRs. There were also differences among the predicted models, although the differences were small and within experimental error. No one modeling program was clearly superior to the others. In some cases, choosing structures based only on sequence homology to the crystallized Ab yielded RMSDs comparable to the models.ConclusionsMolecular modeling programs accurately predict the structure of most regions of antibody variable domains of RAC18. The hypervariable CDRs proved most difficult to model, particularly H chain CDR3. Because CDR3 is most often involved in contact with antigen, this defect must be considered when using models to identify potential contacts between antibody and antigen. Because this study represents only a single case, the results cannot be generalized. Rather they highlight the utility and limitations of modeling programs.

β-Actin-binding Complementarity-determining Region 2 of Variable Heavy Chain from Monoclonal Antibody C7 Induces Apoptosis in Several Human Tumor Cells and Is Protective against Metastatic Melanoma

Complementarity-determining regions (CDRs) from monoclonal antibodies tested as synthetic peptides display anti-infective and antitumor activities, independent of the specificity of the native antibody. Previously, we have shown that the synthetic peptide C7H2, based on the heavy chain CDR 2 from monoclonal antibody C7, a mAb directed to a mannoprotein of Candida albicans, significantly reduced B16F10 melanoma growth and lung colony formation by triggering tumor apoptosis. The mechanism, however, by which C7H2 induced apoptosis in tumor cells remained unknown. Here, we demonstrate that C7H2 interacts with components of the tumor cells cytoskeleton, being rapidly internalized after binding to the tumor cell surface. Mass spectrometry analysis and in vitro validation revealed that β-actin is the receptor of C7H2 in the tumor cells. C7H2 induces β-actin polymerization and F-actin stabilization, linked with abundant generation of superoxide anions and apoptosis. Major phenotypes following peptide binding were chromatin condensation, DNA fragmentation, annexin V binding, lamin disruption, caspase 8 and 3 activation, and organelle alterations. Finally, we evaluated the cytotoxic efficacy of C7H2 in a panel of human tumor cell lines. All tumor cell lines studied were equally susceptible to C7H2 in vitro. The C7H2 amide without further derivatization significantly reduced lung metastasis of mice endovenously challenged with B16F10-Nex2 melanoma cells. No significant cytotoxicity was observed toward nontumorigenic cell lines on short incubation in vitro or in naïve mice injected with a high dose of the peptide. We believe that C7H2 is a promising peptide to be developed as an anticancer drug.

Structural basis of anthrax edema factor neutralization by a neutralizing antibody

Fine epitope mapping of EF13D, a highly potent neutralizing monoclonal antibody specific for the anthrax edema factor (EF), was accomplished through random mutagenesis and yeast surface display. A yeast-displayed library of single point mutants of an EF domain III (DIII), comprising amino acids 624–800, was constructed by random mutagenesis and screened for reduced binding to EF13D. With this method, residues Leu 667, Ser 668, Arg 671, and Arg 672 were identified as key residues important for EF13D binding. They form a contiguous patch on a solvent-exposed surface at one end of the four-helix bundle of DIII. Computational protein–protein docking experiments between anEF13D model and a crystal structure of EF indicate that the EF13D heavy chain complementarity-determining region 3 (HCDR3) is deeply buried within a hydrophobic cleft between two helices of DIII and interacts directly with residues Leu 667, Ser 668, Arg 671 and Arg 672, providing an explanation for the high binding affinity. In addition, they show that the HCDR3 binding site overlaps with the binding site of the N-terminal lobe of calmodulin (CaM), an EF enzymatic activator, consistent with a previous finding showing direct competition with CaM that results in neutralization of EF. Identifying the neutralization epitope of EF13D on EF improves our understanding of the neutralization mechanism and has implications for vaccine development.

Antibody mechanics on a membrane-bound HIV segment essential for GP41-targeted viral neutralization.

Broadly neutralizing antibodies such as 2F5 are directed against the membrane-proximal external region (MPER) of HIV-1 GP41 and recognize well-defined linear core sequences. These epitopes can be engrafted onto protein scaffolds to serve as immunogens with high structural fidelity. Although antibodies that bind to this core GP41 epitope can be elicited, they lack neutralizing activity. To understand this paradox, we used biophysical methods to investigate the binding of human 2F5 to the MPER in a membrane environment, where it resides in vivo. Recognition is stepwise, through a paratope more extensive than core binding site contacts alone, and dynamic rearrangement through an apparent scoop-like movement of heavy chain complementarity-determining region 3 (CDRH3) is essential for MPER extraction from the viral membrane. Core-epitope recognition on the virus requires the induction of conformational changes in both the MPER and the paratope. Hence, target neutralization through this lipid-embedded viral segment places stringent requirements on the plasticity of the antibody combining site.

Mutation Pattern of Paired Immunoglobulin Heavy and Light Variable Domains in Chronic Lymphocytic Leukemia B Cells

B-cell chronic lymphocytic leukemia (CLL) patients display leukemic clones bearing either germline or somatically mutated immunoglobulin heavy variable (IGHV ) genes. Most information on CLL immunoglobulins (Igs), such as the definition of stereotyped B-cell receptors (BCRs), was derived from germline unmutated Igs. In particular, detailed studies on the distribution and nature of mutations in paired heavy- and light-chain domains of CLL clones bearing mutated Igs are lacking. To address the somatic hyper-mutation dynamics of CLL Igs, we analyzed the mutation pattern of paired IGHV-diversity-joining (IGHV-D-J ) and immunoglobulin kappa/lambda variable-joining (IGK/LV-J ) rearrangements of 193 leukemic clones that displayed ≥ 2% mutations in at least one of the two immunoglobulin variable (IGV ) genes (IGHV and/or IGK/LV ). The relationship between the mutation frequency in IGHV and IGK/LV complementarity determining regions (CDRs) and framework regions (FRs) was evaluated by correlation analysis. Replacement (R) mutation frequency within IGK/LV chain CDRs correlated significantly with mutation frequency of paired IGHV CDRs in λ but not κ isotype CLL clones. CDRs of IGKV-J rearrangements displayed a lower percentage of R mutations than IGHVs. The frequency/pattern of mutations in kappa CLL Igs differed also from that in κ-expressing normal B cells described in the literature. Instead, the mutation frequency within the FRs of IGHV and either IGKV or IGLV was correlated. Notably, the amount of diversity introduced by replaced amino acids was comparable between IGHVs and IGKVs. The data indicate a different mutation pattern between κ and λ isotype CLL clones and suggest an antigenic selection that, in κ samples, operates against CDR variation.

HIC resolution of an IgG1 with an oxidized Trp in a complementarity determining region

Susceptibility of tryptophan (Trp) in a complementarity-determining region (CDR) to oxidation is a significant issue for recombinant monoclonal antibody (mAb) therapeutics due to the clinical efficacy and stability concerns. Here we present a case study using hydrophobic interaction chromatography (HIC) to separate an oxidized Trp containing population of an IgG1. The best separation was achieved using dual Dionex ProPac HIC-10 columns, and the oxidized Trp population was isolated as a separated pre-peak. Peptide map analysis revealed that the oxidized Trp is located in a heavy chain CDR. In addition, the HIC method was capable of monitoring the oxidation status of the CDR Trp, as the oxidation rate of the CDR Trp measured by HIC directly correlated with the results of the peptide maps. The same method conditions were also capable of separating oxidized methionine (Met) and isomerization/deamidation products, which co-elute as another pre-peak at a different retention time from the oxidized Trp species. These observations indicate that the HIC procedure can be utilized to monitor the oxidative status of the CDR Trp in the IgG1.

Stereotypical Chronic Lymphocytic Leukemia B-Cell Receptors Recognize Survival Promoting Antigens on Stromal Cells

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. Survival of CLL cells depends on their close contact with stromal cells in lymphatic tissues, bone marrow and blood. This microenvironmental regulation of CLL cell survival involves the stromal secretion of chemo- and cytokines as well as the expression of adhesion molecules. Since CLL survival may also be driven by antigenic stimulation through the B-cell antigen receptor (BCR), we explored the hypothesis that these processes may be linked to each other. We tested if stromal cells could serve as an antigen reservoir for CLL cells, thus promoting CLL cell survival by stimulation through the BCR. As a proof of principle, we found that two CLL BCRs with a common stereotyped heavy chain complementarity-determining region 3 (previously characterized as “subset 1”) recognize antigens highly expressed in stromal cells – vimentin and calreticulin. Both antigens are well-documented targets of autoantibodies in autoimmune disorders. We demonstrated that vimentin is displayed on the surface of viable stromal cells and that it is present and bound by the stereotyped CLL BCR in CLL-stroma co-culture supernatant. Blocking the vimentin antigen by recombinant soluble CLL BCR under CLL-stromal cell co-culture conditions reduces stroma-mediated anti-apoptotic effects by 20–45%. We therefore conclude that CLL BCR stimulation by stroma-derived antigens can contribute to the protective effect that the stroma exerts on CLL cells. This finding sheds a new light on the understanding of the pathobiology of this so far mostly incurable disease.

Research on the Characteristics of IGHV Gene In Chronic Lymphocytic Leukemia of China.

AbstractAbstract 4609 ObjectiveThe usage, mutation status and prognostic impact of immunoglobulin heavy chain variable (IGHV) gene in Chinese patients with chronic lymphocytic leukemia (CLL) is unclear. We set out to define the characteristics of IGHV gene and its relevance to clinical and biological parameter in our patients. MethodsIGHV gene mutations were detected by multiplex PCR in 202 Chinese CLL patients and the purified PCR amplification products were sequenced. IGHV somatic hypermutation status and gene usage were analyzed by IMGT/V-QUEST software. The association analysis between IGHV somatic mutation status and the clinical and biological features, including Binet staging, immunophenotype, cytogenetic aberrant, were also emphasized in this study. ResultsThe results showed that 129 patients had mutated (M) IGHV, and the remaining 73 patients had unmutated (UM) IGHV according to the cutoff value of accordance rate 98%. The most frequent VH gene family was found to be VH3 (47.5%), followed by VH4 (34.7%), VH1 (11.4%), VH2 (2.5%), VH5(1.5%), VH7(1.5%) and VH6(0.9%) gene families, which was similar to other Asian populations. The overall survival (OS) time of UM IGHV group was significant shorter than M IGHV group (P=0.025). Significance was found in the expression of CD38 and ZAP-70 between patients with and without IGHV mutations (P<0.0001 and P=0.015, respectively). Binet staging was significantly different with IGHV mutation status (P<0.001). “Unmutated” sequences had significantly longer heavy chain complementarity-determining region 3 (HCDR3). Seven of these patients used VH1-69, which was similar to other Asia countries, but in striking contrast to those in Western countries, where VH1-69 was one of the most frequently used genes. FISH was performed in 117 cases, del(11q22) was considered as high risk factors, and 10(10/42, 23.8%) cases with UM IGHV gene. On the other hand, there were 7(7/75, 9.3%) cases with M IGHV gene (P=0.033). No significance was found in del(17p),del(13q),del(6q),add(12),IGH translocation between IGHV mutation and unmuatioan patients. A total of five stereotyped BCR were identified, IGHV3-21/IGHD3-9/IGHJ6, IGHV4-34/IGHD2-15/IGHJ6, IGHV1-3/IGHD6-19/IGHJ4, IGHV4-59/IGHD3-22/IGHJ6 and IGHV4-39/IGHD6-13/IGHJ5. ConclusionsThe usage of IGHV gene families indicates significant difference in Chinese CLL patients compared with Western patients, suggesting involvement of ethnic and/or environmental factors in CLL disease initiation. In the development course of CLL, BCR play an important role in the immunological recognition and selection. There are intimate relationships between mutation status of IGHV gene and prognosis. The usage of IGHV provides enlightment for the occurrence mechanism of CLL. Disclosures:No relevant conflicts of interest to declare.

Homologous regions of autoantibody heavy chain complementarity-determining region 3 (H-CDR3) in patients with pemphigus cause pathogenicity

Pemphigus is a life-threatening autoimmune disease in which antibodies specific for desmogleins (Dsgs) cause loss of keratinocyte cell adhesion and blisters. In order to understand how antibodies cause pathogenicity and whether there are commonalities among antibodies in different patients that could ultimately be used to target specific therapy against these antibodies, we characterized Dsg-specific mAbs cloned by phage display from 3 patients with pemphigus vulgaris and 2 with pemphigus foliaceus. Variable heavy chain gene usage was restricted, but similar genes were used for both pathogenic and nonpathogenic mAbs. However, the heavy chain complementarity-determining region 3 (H-CDR3) of most pathogenic, but not nonpathogenic, mAbs shared an amino acid consensus sequence. Randomization of the H-CDR3 and site-directed mutagenesis indicated that changes in this sequence could block pathogenicity but not necessarily binding. In addition, for 2 antibodies with longer H-CDR3s, a tryptophan was critical for pathogenicity but not binding, a result that is consistent with blocking the tryptophan acceptor site that is thought to be necessary for Dsg-mediated adhesion. These studies indicate that H-CDR3 is critical for pathogenicity of a human autoantibody, that a small region (even 1 amino acid) can mediate pathogenicity, and that pathogenicity can be uncoupled from binding in these antibodies.

Targeting B cell leukemia with highly specific allogeneic T cells with a public recognition motif

The possibility that allogeneic T cells may be targeted to leukemia has important therapeutic implications. As most tumor antigens represent self-proteins, high-avidity tumor-specific T cells are largely deleted from the repertoire of the patient. In contrast, T cells from major histocompatibility complex (MHC)-mismatched donors provide naïve repertoires wherein such cells have not been systematically eliminated. Yet, evidence for peptide degeneracy or poly-specificity warrants caution in the use of foreign human leukocyte antigen (HLA) or peptide complexes as therapeutic targets. Here, we cocultured HLA-A(*)0201-negative T cells with autologous dendritic cells engineered to present HLA-A(*)0201 complexed with a peptide from the B cell antigen CD20 (CD20p). HLA-A(*)0201/CD20p pentamer-reactive CD8(+) T cells were readily obtained from all donors. The polyclonal cells showed exquisite peptide and MHC specificity, and efficiently killed HLA-A(*)0201-positive B cells, including primary chronic lymphocytic leukemia cells. The T cell receptor (TCR) sequences displayed a novel type of conservation, with extensive homology in the TCR β chain complementarity-determining region 3 and in J, but not V, region. This is surprising, as the donors were HLA disparate and their TCR repertoires are expected to show little overlap. The results demonstrate the first public recognition motif for an allogeneic HLA/peptide complex. The allo-restricted T cells or TCRs could provide graft-versus-leukemia in the absence of graft-versus-host disease.

Contribution of Light Chain Residues to High Affinity Binding in an HIV-1 Antibody Explored by Combinatorial Scanning Mutagenesis

Detailed analysis of factors governing high affinity antibody-antigen interactions yields important insight into molecular recognition and facilitates the design of functional antibody libraries. Here we describe comprehensive mutagenesis of the light chain complementarity determining regions (CDRs) of HIV-1 antibody D5 (which binds its target, "5-Helix", with a reported K(D) of 50 pM). Combinatorial scanning mutagenesis libraries were prepared in which CDR residues on the D5 light chain were varied among WT side chain identity or alanine. Selection of these libraries against 5-Helix and then sequence analysis of the resulting population were used to quantify energetic consequences of mutation from wild-type to alanine (DeltaDeltaG(Ala-WT)) at each position. This analysis revealed several hotspot residues (DeltaDeltaG(Ala-WT) >or= 1 kcal/mol) that formed combining site features critical to the affinity of the interaction. Tolerance of D5 light chain residues to alternative mutations was explored with a second library. We found that light chain residues located at the center and at the periphery of the D5 combining site contribute to shape complementarity and electrostatic characteristics. Thus, the affinity of D5 for 5-Helix arises from extended interactions involving both the heavy and light chains of D5. These results provide significant insight for future antibody engineering efforts.

T cell receptor CDR3 loops influence αβ pairing

T cell receptor transfer is an attractive strategy for the generation of antigen specific T cells to target infection and malignancy. Cross pairing of the transduced and endogenous TCR chains produces new and potentially auto-reactive specificities and dilutes the therapeutic TCR. This is further complicated as the efficiency of pairing for each αβ pair is unpredictable and the factors which influence it are not well characterized. Complementarity determining region 3 (CDR3) loops are the main sources of TCR alpha and beta diversity due to nucleotide insertion and deletion at V(D)J junctions. Given the variability in composition and length of these non-germ line encoded structures, it is likely that structural strain may occur during formation of some TCR hetero-dimers contributing to the observed pairing restrictions. The beta chain of the HY specific T cell receptor C6 is such an example. Despite pairing efficiently with the C6 alpha chain, it pairs poorly with many other alpha chains. To investigate whether the long, C6 beta CDR3 region underlies this effect, it was replaced with a short, artificial CDR3 region that restored efficient pairing with the endogenous alpha chain repertoire. Molecular modelling is consistent with the beta chain CDR3 region causing steric incompatibility. Despite poor pairing and low surface expression, the WT C6 beta chain mediates positive selection in retrogenic mice.

Abstract CN07-01: Two-in-one antibody: Herceptin variants that target HER2 and VEGF

Abstract CN07-01: Two-in-one antibody: Herceptin variants that target HER2 and VEGF

Chronic Lymphocytic Leukemia Subset Expressing Mutated IGHV3-23 Has Peculiar Clinical and Biological Features.

Abstract 1256Poster Board I-278 IntroductionIn the last years, the B cell receptor (BCR) has become a key molecule in chronic lymphocytic leukemia (CLL), given the correlation between mutational status of immunoglobulin heavy chain variable (IGHV) genes and disease prognosis. Recently, a fraction of CLL has been shown to preferentially express specific IGHV genes, often in a non-random combination with homologous heavy chain complementarity-determining region-3 (HCDR3) and peculiar light chains. Some of these stereotyped BCR mark CLL subsets with peculiar clinical behavior regardless of IGHV mutations. These data suggest a role for BCR in defining the clinical and biological features of CLL, also beyond the mutational status of IGHV genes. Patients and MethodsA HCDR3-driven clustering of 1,426 IG sequences (1,398 patients) was performed using ClustalX(1.83). Time to treatment (TTT) intervals, Rai staging, IGHV mutational status, CD38, ZAP-70, and karyotype abnormalities evaluated by FISH were available for 617 patients. Gene expression profiling (GEP) and quantitative real-time PCR experiments (QRT-PCR) were performed on purified CLL cells. ResultsIGHV3-23 was totally absent in 71 identified stereotyped clusters despite being the second most frequently used IGHV gene, such distribution was significantly skewed (p<0.0001), compared with the distribution of IGHV genes belonging to stereotyped BCR clusters observed in our series. Although 109/134 IGHV3-23 were mutated (M), alignment of IGHV sequences revealed a high degree of conservation in the context of the 13 AA positions involved in superantigen binding by IGHV3 subgroup genes, suggesting that the majority of M IGHV3-23 cases maintained the capacity to mediate superantigen recognition and binding. Median TTT (73 months) of 43 M IGHV3-23 CLL was significantly shorter than median TTT (253 months, p=0.0153) of 333 M CLL, as well as of 326 M CLL in which 7 cases belonging to the bad prognosis IGHV3-21/IGLV3-21 cluster were excluded (253 months, p=0.0082). Multivariate Cox proportional hazard analyses selected IGHV3-23 usage (p=0.029), Rai stage (p<0.0001) and FISH group (p<0.0001) as independent markers of disease progression for 376 M CLL, and for the cohort in which 7 M CLL from the IGHV3-21/IGLV3-21 cluster were excluded. Comparing 5 M IGHV3-23 and 22 M non-IGHV3-23 CLL for their differential GEP, 212 genes were selected, 108 up-regulated and 104 down-regulated in M IGHV3-23 CLL. Using the “Gene-Ontology Tree Machine” platform, a set of growth/tumor suppressor genes (PDCD4, TIA1, RASSF5), all down-regulated in M IGHV3-23 CLL, was constantly found in several gene-ontology categories related to apoptosis. QRT-PCR confirmed a significant down-regulation of these genes in 15 M IGHV3-23 compared to 35 M non-IGHV3-23 CLL. Given the notion that PDCD4 and TIA1 are among the genes under control of miR-15a and miR-16-1 a “Gene Set Enrichment Analysis” carried out on the 212 differentially expressed genes, confirmed that M IGHV3-23 samples were significantly deprived in genes whose expression is under control of miR-15a and miR-16-1. Accordingly, QRT-PCR experiments performed on 15 M IGHV3-23 and 35 M non-IGHV3-23 CLL revealed significant higher levels of both miR-15a (p=0.0007) and miR-16-1 (p=0.0031) in M IGHV3-23 cases. No difference was found in the distribution of patients with 13q14 deletion between M IGHV3-23 CLL and M non-IGHV3-23 CLL (p=0.19). Considering the cases used for microRNA expression experiments (data available in 47/50 cases), 8/15 M IGHV3-23 CLL bore the 13q14 deletion in more than 20% of nuclei, against 19/32 cases in the group of M non-IGHV3-23 CLL (p=0.94). ConclusionExpression of IGHV3-23 marks a subset of M CLL with a worse prognosis; such a peculiar clinical behavior may be related to superantigen stimulation combined with down-regulation of specific growth/tumor suppressor genes and up-regulation of miR-15a and miR-16-1. DisclosuresNo relevant conflicts of interest to declare.

A large human domain antibody library combining heavy and light chain CDR3 diversity

Domain antibodies (dAbs) are promising candidate therapeutics and diagnostics. Efficient selection of novel potent dAbs with potential for clinical utility is critically dependent on the library diversity and size, and the scaffold stability. We have previously constructed a large (size ∼2.5 × 10 10) dAb library by grafting human antibody heavy chain complementarity determining regions (CDRs) 2 and 3 (H2s, H3s) into their cognate positions in a human heavy chain variable domain (VH) scaffold and mutagenizing the CDR1 (H1). High-affinity binders against some antigens were selected from this library but panning against others was not very successful likely due to limited diversity. We have hypothesized that by grafting highly variable, both in length and composition, human CDRs into non-cognate positions, the dAb library diversity could be significantly increased and the library would allow for more efficient selection of high-affinity antibodies against some targets. To test this hypothesis we designed a novel type of dAb library containing CDRs in non-cognate positions. It is based on our previous library where H1 was replaced by a library of human light chain CDR3s (L3s) thus combining three most diversified fragments (L3, H3 and H2) in one VH scaffold. This large (size ∼10 10) phage-displayed library was highly diversified as determined by analyzing the sequences of 126 randomly selected clones. Novel high-affinity dAbs against components of the human insulin-like growth factor (IGF) system were selected from the new library that could not be selected from the previously constructed one. Most of the newly identified dAbs were highly soluble, expressible, monomeric and may have potential as candidate cancer therapeutics. The new library could be used not only for the selection of such dAbs thus complementing existing libraries but also as a research tool for the exploration of the mechanisms determining folding and stability of human antibody domains.