Identification of phage-displayed peptides which bind to the human HnRNPA1 protein-specific monoclonal antibody.

We have screened a peptide phage display library to examine if monoclonal antibody-binding phages could be isolated from the library and thereby predict the antigenic epitopes of the antibodies from the isolated phages. The library was screened for high-avidity binding to monoclonal antibodies by an affinity purification technique called biopanning. Among the monoclonal antibodies examined, the human hnRNPA1 protein-specific monoclonal antibody 9H10 showed selective binding of phages. After two rounds of the biopanning, twelve clones of high-avidity-binding phages were chosen and their inserts were sequenced. Nucleotide sequence comparison of the 12 clones showed that there were 5 different species, with two species containing four members, implying that they were predominantly selected by the biopanning. The amino acid sequences of the inserts of the 12 clones were compared with that of the human hnRNPA1 protein in order to find the putative epitope of the human hnRNPA1 protein for 9H10. The C-terminal region of the human hnRNPA1 protein shows significant homology with the peptide sequences of the selected phage clones. These results show that this peptide phage display library can be useful in defining the epitope of some monoclonal antibodies.     

Identification of peptides that bind to the constant region of a humanized IgG1 monoclonal antibody using phage display

The pFc' fragments of a humanized IgG1 monoclonal antibody were generated by digestion with immobilized pepsin. These pFc' fragments were separated from F(ab')2 fragments by affinity chromatography. The pFc' fragments corresponding to the constant region of the humanized IgG1 monoclonal antibody were used as targets for phage display using variable-length peptide libraries. Interacting phage-displayed peptides were selected by repetitious cycles of target screening and phage amplification. Peptide sequences, deduced by sequencing DNA from isolated phage, were aligned and analyzed for amino acid motifs against each other and protein A. These results indicated that an amino acid motif has been identified using phage display technology that is sufficient for pFc' binding. Furthermore, the peptides derived from this study may prove useful in the development of peptidomimetic alternatives to protein A for use in affinity chromatography.     

A monoclonal antibody to the trichothecene mycotoxin diacetoxyscirpenol

A monoclonal antibody to the trichothecene mycotoxin diacetoxyscirpenol (DAS) was produced by a hybridoma, designated 2E5. It secreted antibody of the IgGl subclass and had a detection limit for DAS of 16 ng/ml with a direct enzyme immunoassay on a double antibody solid phase. The relative cross-reactivities with 3α-acetyl-DAS, diacetylverrucarol, neosolaniol, T-2 tetraol tetraacetate, fusarenon X, T-2 toxin, and HT-2 were 2224·5, 53·7, 13·9, 9·2, 6·4, 1·7, 0·6, and 0·35%, respectively.     

Identification and polymorphism of Plasmodium vivax RBP-1 peptides which bind specifically to reticulocytes

Plasmodium vivax merozoite preferentially invades reticulocytes probably using PvRBP-1 as ligand. One hundred and ninety-five, 15-mer peptides has been synthesised from PvRBP-1 sequence; tested in reticulocyte- or erythrocyte-binding assays. Twenty-five peptides (K_d=76–380 nM) specifically defined four reticulocyte-binding regions. It has been reported that a highly conserved Region-I recombinant fragment binds specifically to reticulocytes. HABP-critical residues for reticulocyte-binding were highly conserved in 20 Colombian P. vivax clinical isolates, suggesting an important biological function. There were six overlapping reticulocyte-binding sites for these peptides according to enzyme sensitivity and mutual competition-binding assays; located on 26- and 41-kDa reticulocyte membrane surface proteins.     

Production and characterization of a monoclonal antibody to the trichothecene mycotoxin diacetoxyscirpenol

A monoclonal antibody was obtained by the fusion of mouse myeloma cells with splenocytes isolated from Balb/c mice, which had been immunized with diacetoxyscirpenol-hemiglutarate (DAS-hemiglutarate) and verrucarol-hemiglutarates covalently bound to ethylenediamine-modified bovine serum albumin. The anti-DAS-antibody that could be induced was of the IgM type with kappa-chains. The titer of the monoclonal anti-DAS-antibody in ascites fluid obtained from mice injected the selected cell line was much higher than those of conventional antisera. An enzyme-linked immunosorbent assay based on the competitive binding principle in which the antibody was applied had a sensitivity of 1 ng DAS per assay. The relative cross-reactivity of the monoclonal antibody in the CI-ELISA with the related trichothecenes such as triacetoxyscirpenol, 15-monoacetoxyscirpenol, diacetylverrucarol, 4-monoacetoxyscirpenol and scirpentriol were found to be 1.8, 0.8, 0.15, 0.02 and less than 0.001, respectively. The trichothecenes verrucarol, T-2 toxin, T-2 tetraol, deoxynivalenol, 3-acetyldeoxynivalenol and trichothecin showed no cross-reactivity.     

Use of a peptide mimotope to guide the humanization of MRK-16, an anti-P-glycoprotein monoclonal antibody.

A mimotope-guided strategy for engineering antibodies directed against orphan targets or antigens that are difficult to purify was developed and used to humanize the murine MRK-16 monoclonal antibody (mAb). MRK-16 recognizes a conformational epitope of a 170-kDa membrane protein, termed P-glycoprotein (P-gp). Elevated expression of P-gp on tumor cells is associated with resistance to cytotoxic drugs, a major obstacle in chemotherapy. Murine MRK-16 was used to enrich and screen a phage-displayed peptide library to identify reactive mimotopes. One peptide, termed ALR1, was enriched to a greater extent than others and subsequently was expressed as a fusion protein with glutathione S-transferase. ALR1 fusion protein bound MRK-16 specifically and inhibited binding of MRK-16 to cells expressing elevated levels of P-gp. To humanize MRK-16, the murine complementarity determining regions were grafted onto homologous human heavy and light chain variable region frameworks. Framework residues that differed between the murine MRK-16 and the homologous human templates were analyzed and subsequently, five framework positions potentially important for maintaining the specificity and affinity of MRK-16 were identified. A combinatorial library consisting of 32 variants encoding all possible combinations of murine and human residues at the five differing framework positions was expressed in a phage system. In the absence of purified P-gp, ALR1 fusion protein was used as surrogate antigen to screen the antibody library to identify the framework combination that most preserved the binding activity of the mAb. On the basis of the initial screening against the mimotope four antibody variants were selected for further characterization. The binding affinity of these variants for the ALR1 fusion protein correlated with their binding to cells expressing elevated levels of P-gp. Thus, peptide mimotopes which can be identified for virtually any antibody including those that recognize conformational or carbohydrate epitopes, can serve as antigen templates for antibody engineering.     

Anti-idiotypes against a monoclonal anti-haloperidol antibody bind to dopamine receptor

Anti-idiotypic antibodies were raised in rabbits by immunization with a monoclonal anti-haloperidol antibody. Some of these anti-idiotypic antibodies bind in a concentration dependent manner to bovine striatal membranes. Following affinity purification, these antibodies inhibit haloperidol binding to striatal membranes and deplete [3H]-spiperone binding sites from a solubilized preparation of striatal membranes. It is thus concluded that these anti-idiotypic antibodies are an internal image of haloperidol and as such can interact with D2-dopamine receptors.     

Serine repeat antigen peptides which bind specifically to red blood cells

It has been reported that serine repeat antigen (SERA) binds directly to human erythrocyte membranes, inside-out vesicles and intact mouse erythrocytes. Similarly, mAbs specific against SERA are effective in blocking red blood cell (RBC) invasion by P. falciparum merozoites. Furthermore, the N-terminal recombinant SERA fragment inhibits the merozoite invasion of erythrocyte. In this study of 49 non-overlapping 20-residue-long peptides encompassing the whole SERA protein FCR3 strain, seven peptides having high RBC binding activity were found. Six of these peptides (three from the SERA N-terminal domain) are located in conserved regions and show affinity constants between 150 and 1100 nM, Hill coefficients between 1.5 and 3.0 and 30000-120000 binding sites per cell. Some of these peptides inhibited in vitro merozoite invasion of erythrocyte and intra-erythrocytic development. Residues which are critical in the binding to erythrocytes (in bold face), i.e. 6725 (YLKETNNAISFESNSGSLEKK), 6733 (YALGSDIPEKCDTLASNCFLS), 6737 (YDNILVKMFKTNENNDKSELI), 6746 (DQGNCDTSWIFASKYHLETI), 6754 (YKKVQNLCGDDTADHAVNIVG) and 6762 (NEVSERVHVYHILKHIKDGK), were determined by means of competition assays with high-binding peptide glycine analogues. The identification of peptides which bind to erythrocyte membrane is important in understanding the process of RBC invasion by P. falciparum merozoites.     

Selection of phage-displayed peptides that bind to a particular ligand-bound antibody

Phage-displayed peptides that selectively bind to aldolase catalytic antibody 93F3 when bound to a particular 1,3-diketone hapten derivative have been developed using designed selection strategies with libraries containing 7-12 randomized amino acid residues. These phage-displayed peptides discriminated the particular 93F3-diketone complex from ligand-free 93F3 and from 93F3 bound to other 1,3-diketone hapten derivatives. By altering the selection procedures, phage-displayed peptides that bind to antibody 93F3 in the absence of 1,3-diketone hapten derivatives have also been developed. With using these phage-displayed peptides, ligand-bound states of the antibody were distinguished from each other. A docking model of one of the peptides bound to the antibody 93F3-diketone complex was created using a sequential divide-and-conquer peptide docking strategy; the model suggests that the peptide interacts with both the antibody and the ligand through a delicate hydrogen bonding network.     

Epitope and mimotope for an antibody to the Na, K-ATPase.

The epitope of a monoclonal antibody specific for the alpha 2 isoform of the Na,K-ATPase was determined and its accessibility in native enzyme was examined. Protein fragmentation with N-chlorosuccinimide, formic acid, trypsin, and leucine aminopeptidase indicated binding near the Na,K-ATPase N-terminus but did not unambiguously delineate the extent of the epitope. The ability of the antibody to bind to denatured enzyme made it a good candidate for screening a random peptide library displayed on M13 phage, but the consensus sequence that emerged was not found in the Na,K-ATPase, Full-length cDNA for the Na,K-ATPase was randomly fragmented and cloned into beta-galactosidase to create a lambda gt11 expression library; screening with the antibody yielded a set of overlaps spanning 23 amino acids at the N-terminus. Chimeras of Na,K-ATPase alpha 1 and alpha 2 narrowed down the epitope to 14-19 amino acids. The antibody did not recognize fusion proteins constructed with shorter segments of this epitope. It did recognize a fusion protein containing the M13 library consensus sequence, however, indicating that this sequence, which is rich in proline and hydrophobic amino acids (FPPNFLFPPPP), was a mimotope. The natural epitope, unique to the Na,K-ATPase alpha 2 isoform, was GREYSPAATTAENG. Reconstitution of antibody binding in a foreign context such as M13 PIII protein or beta-galactosidase thus required a relatively large number of amino acids, indicating that antibody mapping approaches must allow for epitopes of significant size. The epitope was accessible in native enzyme and exposed on the cytoplasmic side, documenting the surface exposure of a stretch of amino acids at the N-terminus, where the Na,K-ATPase isoforms differ most.     

A monoclonal antibody to aromatic amino acid hydroxylases. Identification of the epitope.

PH8 monoclonal antibody has previously been shown to react with all three aromatic amino acid hydroxylases, being particularly useful for immunohistochemical staining of brain tissue [Haan, Jennings, Cuello, Nakata, Chow, Kushinsky, Brittingham & Cotton (1987) Brain Res. 426, 19-27]. Western-blot analysis of liver extracts showed that PH8 reacted with phenylalanine hydroxylase from a wide range of vertebrate species. The epitope for antibody PH8 has been localized to the human phenylalanine hydroxylase sequence between amino acid residues 139 and 155. This highly conserved region of the aromatic amino acid hydroxylases has 11 out of 17 amino acids identical in phenylalanine hydroxylase, tyrosine hydroxylase and tryptophan hydroxylase.     

Identification of the region including the epitope for a monoclonal antibody which can neutralize human parvovirus B19.

In this study, we identified a region in the human parvovirus structural protein which involves the neutralization of the virus by a monoclonal antibody and site-specific synthetic peptides. A newly established monoclonal antibody reacted with both viral capsid proteins VP1 and VP2. The epitope was found in six strains of independently isolated human parvovirus B19. The monoclonal antibody could protect colony-forming unit erythroid in human bone marrow cell culture from injury by the virus. The monoclonal antibody reacted with only 1 of 12 peptides that were synthesized according to a predicted amino acid sequence based on nucleotide sequences of the coding region for the structural protein of B19 virus. The sequence recognized by the antibody was a site corresponding to amino acids 328 to 344 from the amino-terminal portion of VP2. This evidence suggests that the epitope of the viral capsid protein is located on the surface of the virus and may be recognized by virus-neutralizing antibodies.     

In vivo efficacy of monoclonal antibody — drug conjugates of three different subisotypes which bind the human tumor — associated antigen defined by the KS1/4 monoclonal antibody

Summary Monoclonal antibodies (mAbs) BW 625 and BW 704, of the IgG₃ isotype, bound to immunochemically indistinguishable epitopes on ganglioside II³(NeuAc)₂-GgOse₃-Cer. Despite this fact the mAbs showed a differential binding pattern on human glioma cell lines i.e. immunohistochemical data indicate that the detected epitopes are not identical. Furthermore, either mAb is able to mediate the antibody-dependent cellular cytotoxicity reaction (ADCC) and the human-complement-dependent cytotoxicity reaction (CDC) with epitope-expressing tumor cells. All cryopreserved tissue specimens from gliomas and neuroblastomas were immunohistochemically stained, whereas the other small round cell tumors of childhood, as well as melanomas and small-cell lung carcinomas, were essentially negative. Positive staining of normal cryopreserved tissues was restricted to amyelinic axons, Hassal's bodies and some connective tissue fibers in thymus and the tegumentary epithelium of skin. The high selectivity of mAb BW 704 for gliomas and neuroblastomas, the lack of cross-reactivity with major tissues and the strong ADCC and CDC potential argue for the use of mAb BW 704 in immunotherapy of neuroblastomas and gliomas.     

Immunotherapy of murine sarcomas with auto-anti-idiotypic monoclonal antibodies which bind to tumor-specific T cells

Hybridomas producing monoclonal antibodies (mAb) were obtained from BALB/c mice immunized against either of two transplanted, chemically induced syngeneic sarcomas, MCA-1490 or MCA-1511. Two mAb, 4.72 and 5.96, were obtained, one from each immunization. They were found to have apparent anti-idiotypic specificity in that they, when injected s.c., primed naive BALB/c mice for delayed-type hypersensitivity that was specific for the immunizing tumor and required homology at genes linked to the Igh-1 allotype locus. Neither mAb bound tumor antigen. When mice with established transplants of MCA-1490 or MCA-1511 were treated by repeated i.p. injections of the appropriate anti-idiotypic mAb (4.72 and 5.96, respectively), a significant reduction in tumor growth was observed in those mice that had received the appropriate mAb. The idiotope defined by mAb 4.72 was expressed by T cells in mice responding to MCA-1490. mAb 4.72 bound to T cell suppressor factors that were specific for MCA-1490 and were derived from T cell hybridomas or sera of mice bearing MCA-1490. mAb 4.72 also bound to cells from lymph nodes draining the area of a growing MCA-1490 tumor. It was used, in combination with cell sorting, to establish a T cell line, which mediated delayed-type hypersensitivity to MCA-1490 and inhibited the outgrowth of MCA-1490 in BALB/c mice. Thus, mAb specific for idiotopes on T cells responding to syngeneic tumor antigen had both direct immunotherapeutic activity and could be used to establish cultures of tumor-reactive T cells.     

Identification of plasma haptoglobin forms which loosely bind hemoglobin

Haptoglobin (Hpt) is known to capture circulating free hemoglobin (Hb) and bind apolipoprotein (Apo) A-I or E. Here, we report that Hb can be tightly bound by most of Hpt molecules (TB-Hpt, 80%), whereas loosely bound by a minor part of them (LB-Hpt, 20%). LB-Hpt amount was significantly increased (over 60%) in patients with acute coronary syndrome. LB-Hpt bound ApoA-I and ApoE less efficiently than TB-Hpt (8- and 4-fold less, respectively) and did not affect their activity of stimulating the enzyme lecithin-cholesterol acyltransferase. LB-Hpt and TB-Hpt displayed comparable levels of nitrotyrosine residues, but differences in glycan chains. Changes in LB-Hpt level might be associated with changes in Hpt functions.     

Isolation of a monoclonal antibody which blocks vaccinia virus infection.

We have isolated a monoclonal antibody, B2, that neutralizes vaccinia virus infection. B2 reacts with a trypsin-sensitive cell surface epitope. B2 does not neutralize infection of herpes simplex virus, suggesting that the B2-reactive epitope is specifically involved in vaccinia virus entry. A survey of 12 different cell lines reveals a correlation between B2 reactivity and susceptibility to vaccinia virus infection. In addition, B2 interferes with vaccinia virus adsorption to target cells. Taken together, the B2-reactive epitope is part of a receptor that appears important for vaccinia virus entry.     

Enhancement of monoclonal antibody production by lysine-containing peptides

In the search for peptides that could effectively enhance the monoclonal antibody production of a model hybridoma, the performance of five lysine-containing peptides was compared. The capacity of the peptides to enhance the monoclonal antibody yield correlated with their growth-suppressing activity. No correlation of the production-enhancing activity with the character of the distribution of cell-cycle phases could be found. All of the tested peptides, including the negative control peptide Gly-Phe-Gly, altered the cell-cycle phases distribution in favor of the proportion of the S phase.The peptides added to the hybridoma culture were found to be gradually decomposed into dipeptides and free amino acids. Among the set of tested lysine-containing di- to pentapeptides, the best results were obtained with the tripeptide Gly-Lys-Gly. The growth-suppressing and production-enhancing capacity of this peptide supplement was obviously associated with the temporary presence of the intact peptide molecule in the culture media, because the addition of a mixture of free amino acids constituting this peptide, i.e., glycine and lysine, displayed a different effect-a slight promotion of cell growth.     

Cryptic epitope explains the failure of a monoclonal antibody to bind to certain isolates of Trypanosoma cruzi

A mouse monoclonal antibody, WIC 29.26 Ab, has previously been characterized as recognizing a carbohydrate epitope on a 72,000 m.w. glycoprotein (GP72) expressed on the surface of Trypanosoma cruzi epimastigotes and metacyclic trypomastigotes. This molecule has been implicated as a receptor in the control of parasite transformation, and when used as an immunogen in mice, partially protects against T. cruzi infection. In previous experiments in which a radioimmunoassay was used, WIC 29.26 Ab was found to react with approximately 50% of T. cruzi strains and clones derived from a variety of sources. In this study, we attempted to determine whether the WIC 29.26 Ab-nonreactive isolates lack the entire GP72 or merely lack the epitope recognized by this monoclonal antibody. WIC 226.4 Ab, a monoclonal antibody raised against periodate-treated GP72, reacted in an immunofluorescence assay with all strains and clones studied, including those which had not reacted with WIC 29.26 Ab. Likewise, two polyvalent rabbit sera, directed specifically against GP72, bound to all T. cruzi isolates tested. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of detergent lysates of surface-labeled epimastigotes immunoprecipitated with WIC 29.26 Ab showed that the epitope bound by this antibody was present in all but one of the parasites that were surface-nonreactive, as well as in all those that were surface-reactive. WIC 29.26 Ab precipitated a single 72K Mr band from most strains and clones, but in several cases 79K Mr and 66K Mr bands were seen. Isolates from both the surface-reactive and the surface-nonreactive groups showed the latter pattern. These results demonstrate that GP72, or similar electrophoretic variants--and with one exception, the carbohydrate epitope bound by WIC 29.26 Ab--are present in the surface membrane of all strains and clones tested. This observation suggests that in intact epimastigotes of the surface-nonreactive isolates, the epitope is not accessible because of structural changes in the molecule itself or because of differences in the membrane environment of GP72.     

Identification of novel non-phosphorylated ligands, which bind selectively to the SH2 domain of Grb7.

Grb7 is an adapter-type signaling protein, which is recruited via its SH2 domain to a variety of receptor tyrosine kinases (RTKs), including ErbB2 and ErbB3. It is overexpressed in breast, esophageal, and gastric cancers, and may contribute to the invasive potential of cancer cells. Molecular interactions involving Grb7 therefore provide attractive targets for therapeutic intervention. We have utilized phage display random peptide libraries as a source of small peptide ligands to the SH2 domain of Grb7. Screening these libraries against purified Grb7 SH2 resulted in the identification of Grb7-binding peptide phage clones that contained a non-phosphorylated Tyr-X-Asn (YXN) motif. The tyrosine-phosphorylated form of this motif is characteristic of Grb7 SH2 domain binding sites identified in RTKs and other signaling proteins such as Shc. Peptides that are non-phosphorylated have greater potential in the development of therapeutics because of the instability of a phosphate group in vivo. Using a biased library approach with this conserved YXN motif, we identified seven different peptide phage clones, which bind specifically to the SH2 domain of Grb7. These peptides did not bind to the SH2 domain of Grb2 (which also selects for Asn at pY(+2)) or Grb14, a closely related family member. The cyclic structure of the peptides was required to bind to the Grb7 SH2 domain. Importantly, the synthetic Grb7-binding peptide G7-18 in cell lysates was able to specifically inhibit the association of Grb7 with the ErbB family of RTKs, in particular ErbB3, in a dose-dependent manner. These peptides will be useful in the development of targeted molecular therapeutics for cancers overexpressing Grb7 and in the development of Grb7-specific inhibitors to gain a complete understanding of the physiological role of Grb7.