Epitope characterization of an anti‐PD‐L1 antibody using orthogonal approaches

The binding of programmed death ligand 1 protein (PD‐L1) to its receptor programmed death protein 1 (PD‐1) mediates immunoevasion in cancer and chronic viral infections, presenting an important target for therapeutic intervention. Several monoclonal antibodies targeting the PD‐L1/PD‐1 signaling axis are undergoing clinical trials; however, the epitopes of these antibodies have not been described. We have combined orthogonal approaches to localize and characterize the epitope of a monoclonal antibody directed against PD‐L1 at good resolution and with high confidence. Limited proteolysis and mass spectrometry were applied to reveal that the epitope resides in the first immunoglobulin domain of PD‐L1. Hydrogen–deuterium exchange mass spectrometry (HDX‐MS) was used to identify a conformational epitope comprised of discontinuous strands that fold to form a beta sheet in the native structure. This beta sheet presents an epitope surface that significantly overlaps with the PD‐1 binding interface, consistent with a desired PD‐1 competitive mechanism of action for the antibody. Surface plasmon resonance screening of mutant PD‐L1 variants confirmed that the region identified by HDX‐MS is critical for the antibody interaction and further defined specific residues contributing to the binding energy. Taken together, the results are consistent with the observed inhibitory activity of the antibody on PD‐L1‐mediated immune evasion. This is the first report of an epitope for any antibody targeting PD‐L1 and demonstrates the power of combining orthogonal epitope mapping techniques. Copyright © 2015 John Wiley & Sons, Ltd.     

A highly potent CD73 biparatopic antibody blocks organization of the enzyme active site through dual mechanisms

The dimeric ectonucleotidase CD73 catalyzes the hydrolysis of AMP at the cell surface to form adenosine, a potent suppressor of the immune response. Blocking CD73 activity in the tumor microenvironment can have a beneficial effect on tumor eradication and is a promising approach for cancer therapy. Biparatopic antibodies binding different regions of CD73 may be a means to antagonize its enzymatic activity. A panel of biparatopic antibodies representing the pairwise combination of 11 parental monoclonal antibodies against CD73 was generated by Fab-arm exchange. Nine variants vastly exceeded the potency of their parental antibodies with ≥90% inhibition of activity and subnanomolar EC₅₀ values. Pairing the Fabs of parents with nonoverlapping epitopes was both sufficient and necessary whereas monovalent antibodies were poor inhibitors. Some parental antibodies yielded potent biparatopics with multiple partners, one of which (TB19) producing the most potent. The structure of the TB19 Fab with CD73 reveals that it blocks alignment of the N- and C-terminal CD73 domains necessary for catalysis. A separate structure of CD73 with a Fab (TB38) which complements TB19 in a particularly potent biparatopic shows its binding to a nonoverlapping site on the CD73 N-terminal domain. Structural modeling demonstrates a TB19/TB38 biparatopic antibody would be unable to bind the CD73 dimer in a bivalent manner, implicating crosslinking of separate CD73 dimers in its mechanism of action. This ability of a biparatopic antibody to both crosslink CD73 dimers and fix them in an inactive conformation thus represents a highly effective mechanism for the inhibition of CD73 activity.     

Epitope characterization of anti-JAM-A antibodies using orthogonal mass spectrometry and surface plasmon resonance approaches

Junctional adhesion molecule-A (JAM-A) is an adherens and tight junction protein expressed by endothelial and epithelial cells and associated with cancer progression. We present here the extensive characterization of immune complexes involving JAM-A antigen and three monoclonal antibodies (mAbs), including hz6F4-2, a humanized version of anti-tumoral 6F4 mAb identified by a functional and proteomic approach in our laboratory. A specific workflow that combines orthogonal approaches has been designed to determine binding stoichiometries along with JAM-A epitope mapping determination at high resolution for these three mAbs. Native mass spectrometry experiments revealed different binding stoichiometries and affinities, with two molecules of JAM-A being able to bind to hz6F4-2 and F11 Fab, while only one JAM-A was bound to J10.4. Surface plasmon resonance indirect competitive binding assays suggested epitopes located in close proximity for hz6F4-2 and F11. Finally, hydrogen-deuterium exchange mass spectrometry was used to precisely identify epitopes for all mAbs. The results obtained by orthogonal biophysical approaches showed a clear correlation between the determined epitopes and JAM-A binding characteristics, allowing the basis for molecular recognition of JAM-A by hz6F4-2 to be definitively established for the first time. Taken together, our results highlight the power of MS-based structural approaches for epitope mapping and mAb conformational characterization.     

埃博拉病毒NP蛋白的单克隆抗体制备及抗原肽的定位

埃博拉病毒(Ebola virus,EBOV)是一种能导致人类及脊椎动物出血热的致死性病毒,对公共卫生具有较严重的危害。EBOV的NP蛋白在病毒复制中具有重要作用,也是诊断该病重要的靶蛋白。文中原核表达重组扎伊尔型EBOV的NP蛋白,重组蛋白免疫bal/c小鼠,制备了一株小鼠抗EBOV-NP的单克隆抗体。利用Western blotting方法,该抗体能特异识别真核表达和原核表达的重组EBOV-NP,并能同莱斯顿型(RestonEbola virus,REBOV)、科特迪瓦型(Cote-d’Ivoire Ebola virus,CIEBOV)和本迪布焦型(Bundibugyo Ebola virus,BEBOV)埃博拉病毒产生交叉反应,而不与苏丹型(the Sudan Ebola virus,SEBOV)和马堡型(the Marburgvirus,MARV)埃博拉病毒产生反应。利用突变PCR和Western blotting方法,定位了该抗体识别的抗原决定簇序列,该序列(PPLESD)位于EBOV-NP蛋白的C端583-588aa。生物信息学研究表明,该序列在已经公布的ZEBOV、CIEBOV、BEBOV共16个型和REBOV的4个型中高度保守。研究结果为建立以上各型埃博拉病毒的检测方法提供了工具,也为研究埃博拉病毒复制及致病机理提供了基础。     

Hydrogen/deuterium exchange mass spectrometry and computational modeling reveal a discontinuous epitope of an antibody/TL1A Interaction

TL1A, a tumor necrosis factor-like cytokine, is a ligand for the death domain receptor DR3. TL1A, upon binding to DR3, can stimulate lymphocytes and trigger secretion of proinflammatory cytokines. Therefore, blockade of TL1A/DR3 interaction may be a potential therapeutic strategy for autoimmune and inflammatory diseases. Recently, the anti-TL1A monoclonal antibody 1 (mAb1) with a strong potency in blocking the TL1A/DR3 interaction was identified. Here, we report on the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to obtain molecular-level details of mAb1′s binding epitope on TL1A. HDX coupled with electron-transfer dissociation MS provided residue-level epitope information. The HDX dataset, in combination with solvent accessible surface area (SASA) analysis and computational modeling, revealed a discontinuous epitope within the predicted interaction interface of TL1A and DR3. The epitope regions span a distance within the approximate size of the variable domains of mAb1′s heavy and light chains, indicating it uses a unique mechanism of action to block the TL1A/DR3 interaction.     

Molecular basis for the mechanism of action of an anti-TACE antibody

Inhibitors of tumor necrosis factor-α converting enzyme (TACE) have potential as therapeutics for various diseases. Many small molecule inhibitors, however, exhibit poor specificity profiles because they target the highly conserved catalytic cleft of TACE. We report for the first time the molecular interaction of a highly specific anti-TACE antagonistic antibody (MEDI3622). We characterized the binding of MEDI3622 using mutagenesis, as well as structural modeling and docking approaches. We show that MEDI3622 recognizes a unique surface loop of sIVa-sIVb β-hairpin on TACE M-domain, but does not interact with the conserved catalytic cleft or its nearby regions. The exquisite specificity of MEDI3622 is mediated by this distinct structural feature on the TACE M-domain. These findings may aid the design of antibody therapies against TACE.     

Epitope mapping of a monoclonal antibody against human thrombin by H/D-exchange mass spectrometry reveals selection of a diverse sequence in a highly conserved protein

The epitope of a monoclonal antibody raised against human thrombin has been determined by hydrogen/deuterium exchange coupled to MALDI mass spectrometry. The antibody epitope was identified as the surface of thrombin that retained deuterium in the presence of the monoclonal antibody compared to control experiments in its absence. Covalent attachment of the antibody to protein G beads and efficient elution of the antigen after deuterium exchange afforded the analysis of all possible epitopes in a single MALDI mass spectrum. The epitope, which was discontinuous, consisting of two peptides close to anion-binding exosite I, was readily identified. The epitope overlapped with, but was not identical to, the thrombomodulin binding site, consistent with inhibition studies. The antibody bound specifically to human thrombin and not to murine or bovine thrombin, although these proteins share 86% identity with the human protein. Interestingly, the epitope turned out to be the more structured of two surface regions in which higher sequence variation between the three species is seen.     

Resolving self-association of a therapeutic antibody by formulation optimization and molecular approaches

A common challenge encountered during development of high concentration monoclonal antibody formulations is preventing self-association. Depending on the antibody and its formulation, self-association can be seen as aggregation, precipitation, opalescence or phase separation. Here we report on an unusual manifestation of self-association, formation of a semi-solid gel or “gelation." Therapeutic monoclonal antibody C4 was isolated from human B cells based on its strong potency in neutralizing bacterial toxin in animal models. The purified antibody possessed the unusual property of forming a firm, opaque white gel when it was formulated at concentrations >30 mg/mL and the temperature was <6°C. Gel formation was reversible with temperature. Gelation was affected by salt concentration or pH, suggesting an electrostatic interaction between IgG monomers. A comparison of the C4 amino acid sequences to consensus germline sequences revealed differences in framework regions. A C4 variant in which the framework sequence was restored to the consensus germline sequence did not gel at 100 mg/mL at temperatures as low as 1°C. Additional genetic analysis was used to predict the key residue(s) involved in the gelation. Strikingly, a single substitution in the native antibody, replacing heavy chain glutamate 23 with lysine (E23K), was sufficient to prevent gelation. These results indicate that the framework region is involved in intermolecular interactions. The temperature dependence of gelation may be related to conformational changes near glutamate 23 or the regions it interacts with. Molecular engineering of the framework can be an effective approach to resolve the solubility issues of therapeutic antibodies.     

Inhibition of insulin receptor function by a human,allosteric monoclonal antibody

Novel therapies are needed for the treatment of hypoglycemia resulting from both endogenous and exogenous hyperinsulinema. To provide a potential new treatment option, we identified XMetD, an allosteric monoclonal antibody to the insulin receptor (INSR) that was isolated from a human antibody phage display library. To selectively obtain antibodies directed at allosteric sites, panning of the phage display library was conducted using the insulin-INSR complex. Studies indicated that XMetD bound to the INSR with nanomolar affinity. Addition of insulin reduced the affinity of XMetD to the INSR by 3-fold, and XMetD reduced the affinity of the INSR for insulin 3-fold. In addition to inhibiting INSR binding, XMetD also inhibited insulin-induced INSR signaling by 20- to 100-fold. These signaling functions included INSR autophosphorylation, Akt activation and glucose transport. These data indicated that XMetD was an allosteric antagonist of the INSR because, in addition to inhibiting the INSR via modulation of binding affinity, it also inhibited the INSR via modulation of signaling efficacy. Intraperitoneal injection of XMetD at 10 mg/kg twice weekly into normal mice induced insulin resistance. When sustained-release insulin implants were placed into normal mice, they developed fasting hypoglycemia in the range of 50 mg/dl. This hypoglycemia was reversed by XMetD treatment. These studies demonstrate that allosteric monoclonal antibodies, such as XMetD, can antagonize INSR signaling both in vitro and in vivo. They also suggest that this class of allosteric monoclonal antibodies has the potential to treat hyperinsulinemic hypoglycemia resulting from conditions such as insulinoma, congenital hyperinsulinism and insulin overdose.     

Resolving self-association of a therapeutic antibody by formulation optimization and molecular approaches

A common challenge encountered during development of high concentration monoclonal antibody formulations is preventing self-association. Depending on the antibody and its formulation, self-association can be seen as aggregation, precipitation, opalescence or phase separation. Here we report on an unusual manifestation of self-association, formation of a semi-solid gel or “gelation." Therapeutic monoclonal antibody C4 was isolated from human B cells based on its strong potency in neutralizing bacterial toxin in animal models. The purified antibody possessed the unusual property of forming a firm, opaque white gel when it was formulated at concentrations >30 mg/mL and the temperature was <6°C. Gel formation was reversible with temperature. Gelation was affected by salt concentration or pH, suggesting an electrostatic interaction between IgG monomers. A comparison of the C4 amino acid sequences to consensus germline sequences revealed differences in framework regions. A C4 variant in which the framework sequence was restored to the consensus germline sequence did not gel at 100 mg/mL at temperatures as low as 1°C. Additional genetic analysis was used to predict the key residue(s) involved in the gelation. Strikingly, a single substitution in the native antibody, replacing heavy chain glutamate 23 with lysine (E23K), was sufficient to prevent gelation. These results indicate that the framework region is involved in intermolecular interactions. The temperature dependence of gelation may be related to conformational changes near glutamate 23 or the regions it interacts with. Molecular engineering of the framework can be an effective approach to resolve the solubility issues of therapeutic antibodies.     

KIM127, an Antibody that Promotes Adhesion, Maps to a Region of CD18 that Includes Cysteine-Rich Repeats

A series of fusion proteins have been generated between human and mouse CD18. These proteins have been used to carry out preliminary mapping studies on a number of anti-CD18 antibodies including KIM127 an antibody that promotes CD18-dependent adhesion. This antibody maps to a region of the CD18 molecule between amino acids 406 and 570 in a region containing cysteine-rich repeats.     

Inhibition of attachment and growth of tumor cells on collagen by a monoclonal antibody

Summary A murine monoclonal antibody, VM-1, which binds to basal cells of normal human epidermis, reduces the ability of human squamous cell carcinoma cells (SCL-1) derived from the skin to attach and spread on collagen by about 50% and causes cell rounding. Similar effects have been previously shown using normal human keratinocytes. The attachment of cell lines derived from human lung squamous cell carcinomas (SW1271 and SW900), melanoma A375, glioblastoma 126, and fibrosarcoma HT1080 is also inhibited by this antibody. VM-1 antibody does not bind to normal human fibroblasts, benign nevus cells, or the human B-cell-derived line 8866. VM-1 antibody inhibits the growth of SCL-1 cells in vitro as measured by cell numbers and [³H]thymidine ([³H]TdR) incorporation. It is not cytolytic in the presence of complement as measured by⁵¹Cr release. Repeated treatment of SCL-1 cells with VM-1 antibody significantly reduces the proportion of SCL-1 cells that attach to collagen. In addition, after treatment of SCL-1 cells with VM-1 antibody, several proteins can no longer be demonstrated by gel electrophoresis of the cell-free supernatant. The VM-1 antibody effect on attachment and spreading is partially reversed by pretreatment of the collagen surface with laminin and fibronectin, but not with the carbohydrates chondroitin-6-sulfate or hyaluronic acid or with the protein lysozyme. By fluorescence staining, the antigen recognized by VM-1 antibody is membrane-bound and Triton X-100 extractable. The VM-1 antigen is excluded from Bio-Sil TSK-400 and sediments at about 10.5 S. It has a covalent molecular weight on the order of 10⁶. Proteinase K digestion produces VM-1 antibody reactive fragments, assumed to be polysaccharides, with a polydisperse molecular weight distribution in the range 5000 to 30 000. The VM-1 antigen is partially lost from solution on boiling and is no longer detectable in the aqueous or organic phase after chloroform-methanol extraction. The properties of the VM-1 antigen are consistent with those of a proteoglycan involved in attachment and spreading of kerationcytes and certain tumor cells on collagen. This research was supported by a grant from the Elsa U. Pardee Foundation, a Training Grant from the National Institutes of Health, Bethesda, MD, and the Psoriasis Research Institute. Part of this work has appeared as an abstract in Fed. Proc. 43:1929, Abst. #2994, 1984.     

KIM127, an Antibody that Promotes Adhesion,Maps to a Region of CD18 that Includes Cysteine-Rich Repeats

A series of fusion proteins have been generated between human and mouse CD18. These proteins have been used to carry out preliminary mapping studies on a number of anti-CD18 antibodies including KIM127 an antibody that promotes CD18-dependent adhesion. This antibody maps to a region of the CD18 molecule between amino acids 406 and 570 in a region containing cysteine-rich repeats.     

Conformational characterization of the charge variants of a human IgG1 monoclonal antibody using H/D exchange mass spectrometry

MAb1, a human IgG1 monoclonal antibody produced in a NS0 cell line, exhibits charge heterogeneity because of the presence of variants formed by processes such as N-terminal glutamate cyclization, C-terminal lysine truncation, deamidation, aspartate isomerization and sialylation in the carbohydrate moiety. Four major charge variants of MAb1 were isolated and the conformations of these charge variants were studied using hydrogen/deuterium exchange mass spectrometry, including the H/D exchange time course (HX-MS) and the stability of unpurified proteins from rates of H/D exchange (SUPREX) techniques. HX-MS was used to evaluate the conformation and solution dynamics of MAb1 charge variants by measuring their deuterium buildup over time at the peptide level. The SUPREX technique evaluated the unfolding profile and relative stability of the charge variants by measuring the exchange properties of globally protected amide protons in the presence of a chemical denaturant. The H/D exchange profiles from both techniques were compared among the four charge variants of MAb1. The two techniques together offered extensive understanding about the local and subglobal/global unfolding of the charge variants of MAb1. Our results demonstrated that all four charge variants of MAb1 were not significantly different in conformation, solution dynamics and chemical denaturant-induced unfolding profile and stability, which aids in understanding the biofunctions of the molecules. The analytical strategy used for conformational characterization may also be applicable to comparability studies done for antibody therapeutics.     

Mapping of the low pH-sensitive conformational epitope of rabies virus glycoprotein recognized by a monoclonal antibody #1-30-44

Monoclonal antibody (mAb) #1-30-44 recognized an acid-sensitive conformational epitope of rabies virus glycoprotein (G). The antigenicity of G protein exposed on the cell surface was lost when the infected cells were exposed to pH 5.8. By comparing the deduced amino acid sequence of G protein between the HEP-Flury strain and the epitope-negative CVS strain as well as the mAb-resistant escape mutants, two distant sites that contained Lys-202 and Asn-336 were shown to be involved in the epitope formation. Lys-202 is located in the so-called neurotoxin-like sequence, while Asn-336 is included in antigenic site III and is very near the amino acid at position 333, which is known to affect greatly the neuropathogenicity of rabies virus when changed. Consistent with this finding, antigenicity of a neurovirulent revertant of the HEP-Flury strain, in which Gln-333 of G protein was replaced by Arg, was also affected as shown by its greatly decreased reactivity with mAb #1-30-44 compared to that of the original avirulent HEP virus. Based on these results, we hypothesize that the neurotoxin-like domain and some amino acids in antigenic site III come into contact with each other to form a conformational epitope for mAb #1-30-44, and such a configuration would be lost when exposed to acidic conditions to perform a certain low pH-dependent function of G protein.     

一株抗蓝舌病病毒8型VP2蛋白单克隆抗体识别的线性抗原表位的鉴定

为研究本实验室制备的一株抗蓝舌病病毒8型(BTV-8)VP2蛋白的单克隆抗体(MAb)3G11识别的B细胞抗原表位,利用噬菌体肽库展示技术对3G11识别的抗原表位进行筛选并鉴定。经过4轮淘选后挑取蓝斑测序,测序结果经分析后获得KLLAT序列,与BTV-8 VP2蛋白氨基酸序列比对后获得共同的短肽序列为283LL284;合成4种短肽序列:KLLAA、KALAT、KLAAT和KLLAT,与3G11细胞上清和腹水分别进行间接ELISA鉴定,结果表明,短肽KLLAA和KLLAT与3G11细胞上清及腹水具有较强的结合能力;与24种BTV标准阳性血清反应结果表明,这两种短肽都可与BTV-8阳性血清发生特异性反应;序列分析结果可见,该表位的氨基酸序列283LL284在不同来源的BTV-8毒株间保守,确定283LL284为MAb3G11识别抗原表位的关键氨基酸。本研究为建立8型BTV特异性的免疫学检测方法和相关病毒蛋白的功能研究奠定了基础。     

In vivo andin vitro antitumor activity of mitomycin C conjugates at 7-N position through a linker containing thiocarbamate bond with CD10 monoclonal antibody

Through a linker containing thiocarbomate bound to the 7-N position of mitomycin C (MMC), conjugates with a monoclonal antibody to CD10 (NL-1) were prepared, and their antitumor activities were examined. All five conjugates, except one, showedin vitro cytotoxity to two CD10⁺ lymphoid cell lines superior to MMC. The conjugate displaying the highest cytotoxicity was selected and further tested against three CD10⁺ and two CD10 lymphoid cell linesin vitro. The conjugate with NL-1 antibody demonstrated higher cytotoxic activity against CD10+ tumor cells than the control conjugate with normal immunoglobulin, while there was no significant difference, when tested against CD10^– tumors. The cytotoxic activity of the NL-1 conjugate to CD10+ tumors was significantly blocked by NL-1 antibody. In vivo antitumor activity of the NL-1 conjugate was then tested against a CD10⁺ tumor transplanted to nude mice, and side effects were recorded. The NL-1 conjugate (4 mg/kg) showed anin vivo antitumor effect similar to MMC (2 mg/kg), which is at nearly maximal tolerable dose; the latter induced decreases in numbers of leukocytes and platelets, while the former did not, suggesting less side effect by the NL-1 conjugate. Since MMC demonstrates a broad spectrum of antitumor activity, the conjugate, as such, may be applicable for the treatment of cancer patients.     

Neutralization of NGF-TrkA receptor interaction by the novel antagonistic anti-TrkA monoclonal antibody MNAC13: a structural insight

MNAC13, a mouse monoclonal antibody, recognizes with high affinity and specificity the neurotrophin receptor TrkA and displays a neutralizing activity toward the NGF/TrkA interaction. Detailed knowledge of the molecular basis determining the specificity of this antibody is of importance because of its potential use as a modulator of the TrkA-mediated NGF activity. Here, we report a full biochemical and structural characterization of the MNAC13 antibody. Epitope mapping studies, by serial deletion mutants and by phage display, reveal a conformational epitope that is localized on the carboxy-terminal region of the first immunoglobulin-like domain (d4) of TrkA. The X-ray crystal structure of the MNAC13 Fab fragment has been determined and refined to 1.8 A resolution. The antigen-binding site is characterized by a crevice, surrounded by hydrophilic-charged residues on either side, dipping deep toward three mainly hydrophobic subsites. Remarkably an isopropanol molecule has been found to bind in one of the hydrophobic crevices. Overall, the surface topology (shape and electrostatic potential) of the combining site is consistent with the binding data on TrkA ECD serial deletions mutants. The structure of the MNAC13 Fab fragment may assist in the rational structure-based design of high affinity humanized forms of MNAC13, appropriate for therapeutic approaches in neuropathy and inflammatory pain states.     

Detection of guinea pig macrophages by a new CD68 monoclonal antibody, PM-1K

A new monoclonal antibody, PM-1K, was raised against 24-h cultured human peritoneal macrophages. In immunohistochemical assays, PM-1K recognized freshly isolated blood monocytes and most tissue macrophages as well as myeloid dendritic cells such as Langerhans cells and interdigitating cells. The molecular size of the antigen recognized by PM-1K was determined to be 110 kD by means of immunoaffinity purification. Because this affinity-purified antigen recognized by PM-1K was also recognized by anti-CD68 antibodies, it is believed to be one of the heterogeneous molecules of the CD68 antigen. Analysis showed interspecies reactivity of PM-1K with macrophages from guinea pigs, pigs, bovine species, and monkeys. Among these macrophages, those of the guinea pig reacted strongly with PM-1K. Patterns of PM-1K immunostaining in guinea pig tissues were similar to those found in human tissues. Studies with the immunoelectron microscope revealed reaction products of PM-1K in the cytoplasm, especially around endosomes. Since only a few antibodies are available to label guinea pig macrophages, PM-1K is considered to be one of the most suitable antibodies to examine macrophages in experimental guinea pig models.     

Molecular analysis of cross-reactive human monoclonal antibody AE6F4 generated by in vitro immunization: Epitope mapping of AE6F4 antibody on 14-3-3 family proteins and cytokeratin 8

We reported previously that adenocarcinoma-reactive human monoclonal antibody AE6F4, which had been generated by in vitro immunization method, recognizes both 14-3-3protein and cytokeratin 8 (CK8). In this study, to analyze the cross-reactivity of AE6F4 antibody, epitopes of AE6F4 antibody on 14-3-3 proteins and CK8 were studied by using synthetic linear peptide scanning technology. To determine the locations of B cell epitope, 48 and 95 of decapeptides covering the entire 14-3-3 proteins and CK8, respectively,were synthesized and binding to AE6F4 antibody was examined by ELISA. The AE6F4 antibody was strongly reactive to peptides containing amino acid sequences TLWTSDTQGD in 14-3-3 proteins and INFLRQLYEE in CK8. These results indicate that AE6F4 antibody can recognize the different peptide sequences in 14-3-3 proteins and CK8. This revised version was published online in July 2006 with corrections to the Cover Date.