Complementarity Determining Region Peptides Research Articles

Maximizing hydrophobic peptide recovery in proteomics and antibody development using a mass spectrometry compatible surfactant

Peptide loss due to surface absorption can happen at any step in a protein analysis workflow and is sometimes especially deleterious for hydrophobic peptides. In this study, we found the LC-MS compatible surfactant, n-Dodecyl-β-D-maltoside (DDM), can maximize hydrophobic peptide recovery in various samples including single cell digests, mAb clinical PK samples, and mAb peptide mapping samples. In HeLa single cell proteomics analysis, more than half of all unique peptides identified were found only in DDM prepared samples, most of which had significantly higher hydrophobicities compared to peptides in control samples. In clinical PK studies, DDM enhanced hydrophobic complementarity-determining region (CDR) peptide signals significantly. The fold change of CDR peptides’ intensity enhancement in DDM added samples compared to controls correlate with peptide retention time and hydrophobicity, providing guidance for surrogate peptide selection and peptide standard handling in PK studies. For peptide mapping analysis of mAbs, DDM can improve hydrophobic peptide signal and solution stability over 48 h in an autosampler at 4 °C, which can aid method qualification and transfer during drug development. Lastly, maximizing hydrophobic peptide recovery from samples dried in vacuo was achieved by DDM reconstitution, which provided higher signal for later eluting peaks and higher proteome coverage overall.

Design Strategy to Create Antibody Mimetics Harbouring Immobilised Complementarity Determining Region Peptides for Practical Use

Monoclonal antibodies (mAbs) are attractive therapeutics for treating a wide range of human disorders, and bind to the antigen through their complementarity-determining regions (CDRs). Small stable proteins containing structurally retained CDRs are promising alternatives to mAbs. In this report, we present a method to create such proteins, named fluctuation-regulated affinity proteins (FLAPs). Thirteen graft acceptor (GA) sites that efficiently immobilise the grafted peptide structure were initially selected from six small protein scaffolds by computational identification. Five CDR peptides extracted by binding energy calculations from mAbs against breast cancer marker human epithelial growth factor receptor type 2 (HER2) were then grafted to the selected scaffolds. The combination of five CDR peptides and 13 GA sites in six scaffolds revealed that three of the 65 combinations showed specific binding to HER2 with dissociation constants (KD) of 270–350 nM in biolayer interferometry and 24–65 nM in ELISA. The FLAPs specifically detected HER2-overexpressing cancer cells. Thus, the present strategy is a promising and practical method for developing small antibody mimetics.

Synthetic Polyclonal-Derived CDR Peptides as an Innovative Strategy in Glaucoma Therapy.

The pathogenesis of glaucoma is strongly associated with the occurrence of autoimmune-mediated loss of retinal ganglion cells (RGCs) and additionally, recent evidence shows that specific antibody-derived signature peptides are significantly differentially expressed in sera of primary-open angle glaucoma patients (POAG) compared to healthy controls. Synthetically antibody-derived peptides can modulate various effector functions of the immune system and act as antimicrobial or antiviral molecules. In an ex vivo adolescent glaucoma model, this study, for the first time, demonstrates that polyclonal-derived complementarity-determining regions (CDRs) can significantly increase the survival rate of RGCs (p = 0.013). We subsequently performed affinity capture experiments that verified the mitochondrial serine protease HTRA2 (gene name: HTRA2) as a high-affinity retinal epitope target of CDR1 sequence motif ASGYTFTNYGLSWVR. Quantitative proteomic analysis of the CDR-treated retinal explants revealed increased expression of various anti-apoptotic and anti-oxidative proteins (e.g., VDAC2 and TXN) compared to untreated controls (p < 0.05) as well as decreased expression levels of cellular stress response markers (e.g., HSPE1 and HSP90AA1). Mitochondrial dysfunction, the protein ubiquitination pathway and oxidative phosphorylation were annotated as the most significantly affected signaling pathways and possibly can be traced back to the CDR-induced inhibition or modulation of the master regulator HTRA2. These findings emphasize the great potential of synthetic polyclonal-derived CDR peptides as therapeutic agents in future glaucoma therapy and provide an excellent basis for affinity-based biomarker discovery purposes.

Development of a new monoclonal antibody specific to mouse Vγ6 chain.

There are seven Vγ gene segments in the TCR γ chain loci of mice. We developed monoclonal antibodies (mAbs) specific to the Vγ6 chain (Heilig & Tonegawa nomenclature). By immunizing Vγ4/6 KO mice with complementarity-determining region peptides in Vγ6 chains, we generated three hybridomas. These hybridomas produced mAbs capable of cell surface staining of Vγ6/Vδ1 gene-transfected T-cell line lacking TCR as well as of Vγ1- Vγ4- Vγ5- Vγ7- γδ T cells and the CD3high TCRδint γδ T cells in various organs. The location of Vγ6+ γδ T cells, which peaked in the newborn thymus, was associated with mTEC. In vivo administration of clone 1C10-1F7 mAb impaired protection against Klebsiella pneumoniae infection but ameliorated psoriasis-like dermatitis induced by imiquimod treatment. These new mAbs are useful to elucidate the development, location, and functions of Vγ6 γδ T cells in mice.

Anti-tumor activities of peptides corresponding to conserved complementary determining regions from different immunoglobulins

Short synthetic peptides corresponding to sequences of complementarity-determining regions (CDRs) from different immunoglobulin families have been shown to induce antimicrobial, antiviral and antitumor activities regardless of the specificity of the original monoclonal antibody (mAb). Presently, we studied the in vitro and in vivo antitumor activity of synthetic peptides derived from conserved CDR sequences of different immunoglobulins against human tumor cell lines and murine B16F10-Nex2 melanoma aiming at the discovery of candidate molecules for cancer therapy. Four light- and heavy-chain CDR peptide sequences from different antibodies (C36-L1, HA9-H2, 1-H2 and Mg16-H2) showed cytotoxic activity against murine melanoma and a panel of human tumor cell lineages in vitro. Importantly, they also exerted anti-metastatic activity using a syngeneic melanoma model in mice. Other peptides (D07-H3, MN20v1, MS2-H3) were also protective against metastatic melanoma, without showing significant cytotoxicity against tumor cells in vitro. In this case, we suggest that these peptides may act as immune adjuvants in vivo. As observed, peptides induced nitric oxide production in bone-marrow macrophages showing that innate immune cells can also be modulated by these CDR peptides. The present screening supports the search in immunoglobulins of rather frequent CDR sequences that are endowed with specific antitumor properties and may be candidates to be developed as anti-cancer drugs.

Identification of isomerization and racemization of aspartate in the Asp–Asp motifs of a therapeutic protein

A thermally stressed Fab molecule showed a significant increase of basic variants in imaged capillary isoelectric focusing (iCIEF) analysis. Mass analyses of the reduced protein found an increase in −18Da species from both light chain and heavy chain. A tryptic peptide map identified two isoAsp-containing peptides, both containing Asp–Asp motifs and located in complementarity-determining regions (CDRs) of light chains and heavy chains, respectively. The approaches of hydrolyzing succinimide in H218O followed by tryptic digestion were used to label and identify the sites of isomerization. This method enabled identification of the isomerization site by comparing the MS/MS spectra of isomerized peptides with and without 18O incorporation. The light chain peptide L2 VTITCITSTDID12DDMNWYQQKPGK underwent simultaneous isomerization and recemization at residue Asp-12 after thermal stress as evidenced by the coinjection of synthetic peptide L2 with l-Asp-12, l-isoAsp-12, d-Asp-12, and d-isoAsp-12, respectively. A thermal stress study of the synthetic peptide (l-)L2 showed that the isomerization and racemization did not occur, indicating that the Asp degradation in this Asp–Asp motif is more related to the protein conformation than the primary sequence. Another isomerization site was identified as Asp-24 in the heavy chain peptide H5 QAPGQGLEWMGWINTYTGETTYAD24DFK. No other isomerizations were detected in CDR peptides containing either Asp–Ser or Asp–Thr motifs.

An antibody-based biomarker discovery method by mass spectrometry sequencing of complementarity determining regions

Autoantibodies are increasingly used as biomarkers in the detection of autoimmune disorders and cancer. Disease specific antibodies are generally detected by their binding to specific antigens. As an alternative approach, we propose to identify specific complementarity determining regions (CDR) of IgG that relate to an autoimmune disorder or cancer instead of the specific antigen(s). In this manuscript, we tested the technical feasibility to detect and identify CDRs of specific antibodies by mass spectrometry. We used a commercial pooled IgG preparation as well as purified serum IgG fractions that were spiked with different amounts of a fully human monoclonal antibody (adalimumab). These samples were enzymatically digested and analyzed by nanoLC Orbitrap mass spectrometry. In these samples, we were able to identify peptides derived from the CDRs of adalimumab. These peptides could be detected at an amount of 110 attomole, 5 orders of magnitude lower than the total IgG concentration in these samples. Using higher energy collision induced dissociation (HCD) fragmentation and subsequent de novo sequencing, we could successfully identify 50% of the detectable CDR peptides of adalimumab. In addition, we demonstrated that an affinity purification with anti-dinitrophenol (DNP) monoclonal antibody enhanced anti-DNP derived CDR detection in a serum IgG background. In conclusion, specific CDR peptides could be detected and sequenced at relatively low levels (attomole-femtomole range) which should allow the detection of clinically relevant CDR peptides in patient samples.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-010-4361-9) contains supplementary material, which is available to authorized users.

Prediction of MHC Class I-Restricted Immunogenic Epitopes of B-Cell Receptors Indicates T Cell-Mediated Immunosurveillance of Malignant B Cell Clones.

Abstract 1949Poster Board I-972Tumor-specific immune responses can be induced in patients with indolent B cell lymphomas (iNHL) by active immunization against the individual B cell receptor (BCR) expressed by the malignant B cell clone, the so-called “idiotype” (Id). In subsequent trials of intradermal vaccination with recombinant lymphoma-derived Fab fragment in iNHL, we have studied the specificity of MHC class I-restricted anti-Id T cell responses by epitope mapping experiments with synthetic Id-derived peptides predicted to be presented by the respective patient's HLA complex. While such peptides exist in hypervariable and conserved Id regions, these assays have shown consistently that in vivo-induced T cell responses occur preferentially against individual Id epitopes located in complementarity-determining regions (CDR), whereas framework (FR) and constant region-derived epitopes are ignored (Bertinetti et al., Cancer Res. 2006; Navarrete et al., ASH 2008). These results contrast with in vitro studies showing that FR-derived peptides are excellent targets for cytotoxic T cells in iNHL patients (Trojan et al., Nat Med 2000). To gain further insight into the relative predominance and immunological role of MHC class I-restricted Id epitopes, we conducted a comprehensive reverse immunology study in follicular lymphoma (FL).Clonal and functional IgH chain transcript sequences were identified from tumor biopsies of 39 FL patients by A-PCR (Bertinetti et al., EJH 2006). The HLA-A and B haplotype of the patients was determined by conventional serological testing and high-resolution PCR genotyping. Potentially MHC-presentable peptides from all Id sequences and their corresponding germ-line (GL) VH genes were identified for the HLA haplotypes of all 39 patients by reverse immunology (bimas.cit.nih.gov). Identified peptides were ranked for each haplotype according to their predicted score, and the sum of the scores for the 20 highest ranking peptides was calculated. The sum score for any given Id was compared to the mean of the sum scores of the other 38 Ids on the respective patient's HLA haplotypes. Separate analyses were performed for CDR peptides (containing at least 2 AA in any CDR) versus non-CDR-peptides (allocated through imgt.cines.fr), Id versus GL sequences, and Id versus contaminating sporadic Ig sequences that represent bona fide normal B cells in the biopsies.72% of all peptides with BIMAS scores of ≥50 and ≥10, respectively, were located in FR, expecially in FR3. The ranked sum Id scores were lower for the patients' own tumor Id than for the mean of the allogeneic Ids (Table; Wilcoxon's matched pair test). This difference was mostly attributable to CDR-derived epitopes throughout all CDRs despite overall lower immunogenicity compared to FR. There was no evidence for differential immunogenicity between a hypermutated FL Id and the corresponding GL (p=0.58). Finally, a preliminary survey of IgH sequences from non-clonal B cells indicated similar immunogenicity compared to FL Id (p=0.31).These bioinformatic findings indicate T cell-mediated immunosurveillance against the BCR of malignant and perhaps nonmalignant B cells. T cell activity appears to be directed predominantly against individual CDR peptides despite their lesser predicted HLA binding capacity compared to FR peptides. Existing CDR epitopes are not generated during the hypermutation process of BCRs, raising the possibility that randomly generated, more immunogenic hypervariable peptides are not permitted by the immune system. In conjunction with the T cell activity observed in in vivo and in vitro studies cited above, these findings are consistent with strong peripheral tolerance to shared Id structures. On the other hand, T cell control of individual Id peptides may play a role in immunosurveillance of malignant B cells and may be exploited for active immunotherapy of lymphoma. In contrast, generic or pan-B-cell epitopes are predicted to be less effective in inducing anti-lymphoma T cell responses.Median (range) BIMASPatient IdMean of allogeneic IdscomparisonAll peptides213 (40-5920)369 (56-5520)p=0.0012FR peptides157 (20-5415)239 (18-3891)p=0.045CDR peptides74 (7-648)175 (21-1760)p<0.0001- CDR1 peptides21 (0.7-144)52 (1.9-630)p=0.0007- CDR2 peptides7.6 (0.2-345)30 (2.2-212)p=0.0089- CDR3 peptides16 (1.3-506)37 (6-980)p=0.0008 Disclosures:No relevant conflicts of interest to declare.

Antibodies as Crypts of Antiinfective and Antitumor Peptides

Antibodies (Abs), often associated with antimicrobial and antitumor agents, have emerged as an important class of novel drugs for antigen-driven therapeutic purposes in diverse clinical settings, including oncology and infectious diseases. Abs commonly give rise in the treated host to anti-Ab responses, which may induce adverse reactions and limit their therapeutic efficacy. Their modular domain architecture has been exploited to generate alternative reduced formats (Fabs, scFvs, dAbs, minibodies, multibodies), essentially devoid of the Fc region. The presence of complementarity determining regions (CDRs) ensures the maintenance of selective binding to antigens and supports their use for biotechnological and therapeutic applications. Paradigmatic Abs mimicking the wide-spectrum antimicrobial activity of a yeast killer toxin (killer Abs) have revealed the existence of a family of Abs exerting a direct in vitro and/or in vivo microbicidal activity. Based on the variable sequence of an antiidiotypic recombinant killer Ab, CDR-related peptides have been synthesized, engineered by alanine-scanning and selected according to antimicrobial, antiviral and immunomodulatory properties. Irrespective of the native Ab specificity, synthetic CDRs from unrelated murine and human monoclonal Abs, have shown to display differential in vitro, in vivo and/or ex vivo antifungal (Candida albicans), antiviral (HIV-1) and antitumor (melanoma cells) activities. Alanine substitution of single residues of synthetic CDR peptides resulted in further differential increased/unaltered/decreased biological activity. The intriguing potential of Abs as source of antiinfective and antitumor therapeutics will be discussed, in light of recent advances in peptide design, stability and delivery.

A peptide based on the CDR3 of an anti-DNA antibody of experimental SLE origin is also a dominant T-cell epitope in (NZBXNZW)F1 lupus-prone mice

A molecular homology has been demonstrated between sequences of the heavy chain variable regions of the anti-DNA, anti-cardiolipin monoclonal antibody, 2C4C2, isolated from C3H.SW mice with induced systemic lupus erythematosus, and sequences of the anti-DNA monoclonal antibody BW16 originating in the lupus-prone (NZBXNZW)F1 mice. It was of interest to determine whether these homologous sequences function also as immunodominant T-cell epitopes, in order to establish a connection between spontaneous and induced experimental models. Therefore, three peptides were designed and synthesized based on the complementarity determining region (CDR)1, CDR2 and CDR3 of the heavy chain of the monoclonal antibody 2C4C2. In the present study, we compare these peptides with the CDR1- and CDR3-based peptides of another murine anti-DNA antibody; namely, 5G12. The comparison was carried out by analyzing the ability of the peptides to induce T-cell activation in (NZBXNZW)F1 lupus-prone mice and in mouse strains susceptible to induction of experimental systemic lupus erythematosus. Immunization of (NZBXNZW)F1 mice with the 2C4C2 mAb or with its CDR-based peptides, as well as immunization with the 5G12-based CDR peptides, induced significant lymph node proliferation to the pCDR3 of the 5G12 mAb. Naive (NZBXNZW)F1 splenocytes exhibited activation to the same peptide. It is also shown that MHC class II molecules of (NZBXNZW)F1 macrophages bind preferentially the 5G12-based pCDR3. It is proposed that the CDR3-based peptide of 5G12 mAb of experimental lupus is also a dominant and relevant epitope in the (NZBXNZW)F1 lupus-prone mice.

T‐cell‐epitope mapping of the idiotypic monoclonal IgG heavy and light chains in multiple myeloma

The idiotypic structures of the myeloma protein might be regarded as tumor-specific antigens. The present study was designed to map T-cell epitopes of the idiotypic myeloma protein to prove the existence of naturally occurring major-histocompatibility-complex-dependent idiotype (peptide)-specific T cells in multiple myeloma. The fine specificity of idiotype-reactive, interferon-γ-producing blood T cells of a patient with multiple myeloma stage I was characterized by identification of idiotype (heavy and light chains)-derived MHC-restricted T-cell epitopes. T cells specifically reacting with peptides corresponding to each of the 3 complementarity-determining regions (CDRs) of the heavy-chain variable part (VH) of the autologous idiotype were found. In contrast, none of the peptides corresponding to the 3 CDRs of the light chain (VL) induced a specific T-cell response. The idiotype amino-acid sequence corresponding to the junction of the VH, diversity (D), and joining (J) gene segments of the VH appeared to be an important target for T cells, since the sequence expressed MHC-class-I- as well as MHC-class-II-restricted epitopes. The study provides further support for the existence of MHC-restricted idiotype-specific T cells, which may target immunogenic CDR peptides in multiple myeloma. Such T cells could be an important part of the specific anti-tumor immune responses induced in idiotype vaccination protocols. Int. J. Cancer 80:671–680, 1999. © 1999 Wiley-Liss, Inc.

Immunochemical Features of Complementarity Determining Region (CDR) Peptide in Anti Hemin Monoclonal Antibody

Messenger RNA purified from the anti hemin monoclonal antibody (1D3) secreting hybridoma was amplified by RT-PCR and the nueleotide and amino acid sequences of the antibody were determined. The role of complementarity determining regions (CDRs) in porphyrin recognition and its immunochemical feature of the antibody were investigated by using ELISA, fluorescence measurement and computational calculation of the conformation. All CDR peptides of the heavy chain of the antibody were synthesized and their affinity constants to porphyrins were determined. The value of CDR2 of heavy chain (CDRH2) of 1D3 was 1.5×105/M for protoporphyrin and 7×107/M for TCPP, respectively, while that of the whole antibody showed to be 1.2×107/M for TCPP. Though CDRH2 is a 17 meric peptide, it showed higher affinity than the whole antibody (1D3). Porphyrins can be considered to firmly bind with CDRH2, while CDRH3 is not involved in the antigen binding. CDR-1 may participate in the recognition with a small contribution. By the computational analysis of steric conformation, it was suggested that CDRH1 and CDRH2 co-operatively function in the recognition of porphyrin. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.

Immunochemical Features of Complementarity Determining Region (CDR) Peptide in Anti Hemin Monoclonal Antibody

Messenger RNA purified from the anti hemin monoclonal antibody (1D3) secreting hybridoma was amplified by RT-PCR and the nueleotide and amino acid sequences of the antibody were determined. The role of complementarity determining regions (CDRs) in porphyrin recognition and its immunochemical feature of the antibody were investigated by using ELISA, fluorescence measurement and computational calculation of the conformation. All CDR peptides of the heavy chain of the antibody were synthesized and their affinity constants to porphyrins were determined. The value of CDR2 of heavy chain (CDRH2) of 1D3 was 1.5×105/M for protoporphyrin and 7×107/M for TCPP, respectively, while that of the whole antibody showed to be 1.2×107/M for TCPP. Though CDRH2 is a 17 meric peptide, it showed higher affinity than the whole antibody (1D3). Porphyrins can be considered to firmly bind with CDRH2, while CDRH3 is not involved in the antigen binding. CDR-1 may participate in the recognition with a small contribution. By the computational analysis of steric conformation, it was suggested that CDRH1 and CDRH2 co-operatively function in the recognition of porphyrin. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.

Peptides Derived from the Complementarity-determining Regions of Anti-Mac-1 Antibodies Block Intercellular Adhesion Molecule-1 Interaction with Mac-1

Peptides or small molecules that can block the interaction of the integrin Mac-1 with its receptor, intercellular adhesion molecule-1 (ICAM-1), have not previously been developed. We studied this interaction by measuring the adherence of ICAM-1-expressing Chinese hamster ovary (CHO) cells to immobilized, purified Mac-1. Nucleotide sequence information was obtained for the complementarity determining regions (CDRs) of three antibodies (44aacb, MY904, and 118.1) shown to block Mac-1-mediated cell adherence. Peptides were synthesized based on the predicted amino acid sequences of the CDRs and tested for the ability to block cell adhesion to Mac-1. Peptides derived from CDR1 of 44aacb, CDR2 of 118.1, and CDRs 1 and 3 of MY904 heavy chains were found to possess blocking activity at 10-100 muM. This may indicate that one or two CDRs contribute disproportionately to the antibody binding affinity. The binding of ligands to Mac-1 has been shown to require a region of the alpha-chain known as the I- or A-domain. We have recombinantly produced Mac-1 I-domain, and show that it is also capable of supporting the adherence of ICAM-1-expressing CHO cells. The adherence of ICAM-1-CHO cells to the I-domain is inhibited by 44aacb and 118.1 and by the CDR peptides from 44aacb and 118.1. By using phage display of peptide libraries based on the 118.1 CDR peptide with five residues randomized, we were able to identify a novel peptide inhibitor of Mac-1 with substitutions at all five positions. These peptides provide lead structures for development of Mac-1 antagonists.

Fv structure of monoclonal antibody II-481 against herpes simplex virus Fc gamma-binding glycoprotein gE contains immunodominant complementarity determining region epitopes that react with human immunoglobulin M rheumatoid factors.

Human immunoglobulin M (IgM) rheumatoid factors (RFs) show primary direct enzyme-linked immunosorbent assay (ELISA) reactivity with Fab rather than Fc fragments of monoclonal antibody (mAb) II-481 directed against the Fc gamma-binding site of herpes simplex virus glycoprotein gE. This preferential anti-Fab specificity suggests that RFs react with antigen-binding portions of mAb II-481 as anti-idiotypic antibodies directed at the combining site regions of mAb reacting with the Fc gamma-binding region of gE. Analysis of this idiotype-anti-idiotype reaction employed polymerase chain reaction amplification and sequencing of the variable heavy and light (VH and VL) regions of mAb II-481. When VH and VL regions of mAb II-481 were synthesized as overlapping 7-mer peptides on polypropylene pins, a panel of 10 polyclonal and 6 monoclonal human IgM RFs reacted primarily with epitopes within the three solvent-exposed mAb II-481 complementarity determining regions (CDRs). Preincubation of single CDR heptamer peptides with IgM RFs in free solution, resulted in 63-100% inhibition of RF binding to mAb II-481 on the ELISA plate, confirming the antigenic importance of linear CDR regions for RF reactivity. Combinations of two or three CDR peptides frequently produced 94-100% inhibition of RF binding to whole mAb II-481. Control peptides, singly or in combination, showed no inhibition. Computer modeling suggested that the RF-reactive mAb II-481 Fv region and a previously demonstrated RF-reactive CH3 epitope displayed considerable three-dimensional similarities in conformation. These studies may provide insight into limited shape homologies possibly involved in an RF anti-idiotypic reaction.

Interaction of lysozyme with synthetic anti-lysozyme D1.3 antibody fragments studied by affinity chromatography and surface plasmon resonance

Synthetic antibody fragments of monoclonal anti-lysozyme antibody D1.3 have been tested on binding with hen egg white lysozyme using immunoaffinity chromatography and surface plasmon resonance. Upon immunoaffinity chromatography, peptides containing one or two complementarity determining regions (CDRs) of D1.3 show interaction with lysozyme. Surface plasmon resonance with immobilized CDR peptides showed that this interaction is not based on the antigen-antibody interaction. Nevertheless, these peptides could be useful as ligands for the purification of lysozyme from a mixture of proteins.