Significance VEGF is the pivotal growth factor for angiogenesis, and its inhibition by passive immunotherapy results in improved survival in patients with several types of cancer. We believe that the clinical benefit could be increased by inducing a humoral immune response against VEGF through active immunization with VEGF-based peptides. In this study, we describe that correct peptide design is vital for success. We show that only 3D-structured peptides perfectly mimicking the crucial β5–turn–β6 loop of endogenous VEGF are able to induce neutralizing antibodies. We developed a vaccine with potent in vitro and in vivo VEGF-neutralizing activities, as shown in passive and active immunization tumor models. The VEGF vaccination strategy has the potential to outperform current clinical anti-VEGF treatment strategies.

Making peptide-based molecules that mimic functional interaction sites on proteins remains a challenge in biomedical sciences. Here, we present a robust technology for the covalent assembly of highly constrained and discontinuous binding site mimics, the potential of which is exemplified for structurally complex binding sites on the "Cys-knot" proteins hFSH and hCG. Peptidic structures were assembled by Ar(CH2Br)(2)-promoted peptide cyclizations, combined with oxime ligation and disulfide formation. The technology allows unprotected side chain groups and is applicable to peptides of different lengths and nature. A tetracyclic FSH mimic was constructed, showing >600-fold improved binding compared to linear or monocyclic controls. Binding of a tricyclic hCG mimic to anti-hCG mAb 8G5 was identical to hCG itself (IC50= 260 vs. 470 pm), whereas this mimic displayed an IC50 value of 149 nm for mAb 3468, an hCG-neutralizing antibody with undetectable binding to either linear or monocyclic controls.

The cover picture shows the X-ray crystal structure of hCGβ (the “pregnancy hormone”, in blue) in complex with a monoclonal antibody (mAb3468, in green) that binds with sub-nanomolar affinity (PDB ID: 1QFW). The structure illustrates the molecular complexity of the discontinuous and conformational β1/β3 binding site on hCGβ, and further suggests that the binding surfaces of hCGβ and mAb3468 only interact efficiently when they adopt the correct secondary and tertiary structures. On p. 91 ff., P. Timmerman et al. present a new synthetic methodology for manufacturing small (3.5–5 kDa), peptide-based mimics of the highly discontinuous β1/β3 epitope on hCGβ that retain both the secondary and tertiary structures as well as the strong binding to mAb3468, while neglecting 75 % of the total protein. Synthesis of these mimics involves the use of multiple constraints, including cyclization with a synthetic scaffold and connecting the loops both via oxime linkages and natural disulfide bonds. These constraints were shown to be essential for the binding properties of these mimics.

This paper describes immunization studies with CLIPS-constrained peptides covering only the major part (beta3-loop) of a structurally complex antigenic site on human Follicle Stimulating Hormone beta-subunit (FSH-beta). In cases where linear and SS-constrained peptides fail, the CLIPS-constrained peptides generate polyclonal antibodies with high neutralizing activity for hFSH. The sera were shown to be specific for hFSH over human Luteinizing Hormone (hLH) and human Chorionic Gonadotropin (hCG). ELISA-competition studies and circular dichroism (CD)-measurements illustrate clearly that activity of the peptides in antibody binding and generation relates directly to precise and appropriate fixation of the peptide conformation. Design of the CLIPS-peptides was entirely based on epitope mapping studies with two neutralizing anti-hFSH mAbs. Both mAbs were shown to bind to a conformational epitope located at the top of the beta1-beta3-loop covering the amino acid sequences Y58-P77 (beta3-loop). The results described in this paper show that CLIPS-constrained peptides covering the Y58-P77 sequence provide the minimally required structural entity necessary to generate reproducibly sera with high hFSH-neutralizing activity.

This paper describes the application of two novel screening technologies, i.e. Domain Scan™ (24- and 30-mer peptides) and Matrix Scan™ (24-mer peptides)technology, in the mapping of a discontinuous epitope on FSH-β for a series of 20 monoclonal antibodies. 11 out of 20 mAb's, mapping of which was not successful by conventional Pepscan™ technology (12-merpeptides), showed selective binding to peptide-constructs corresponding to the β3-loop of FSH in the Domain™ and/or Matrix Scan™. Systematic replacement analysis studies with peptide-construct 57VYETVRVPGCAC-SAc-ADSLYTYPVATQ81 revealed that for most mAb's the amino acids R62, A70,D71, and L73 form the core of the epitope. A DomainScan™ performed in the C-O format showed highly selective binding for mAb's 1 and 2 with only three β1-β3 peptide-constructs covering the residues 60TVRVPGCAHHADSLY74 in combination with 10IAIEKEECRFAI21, while for mAb 10 binding was observed with peptide-constructs containing the C-terminal residues97RGLGPSYCSFGEMKE114 in combination with the residues 10IAIEKEECRFAI21. A Matrix Scan™ of mAb 17 showed that peptides from four different regions on FSH (1st strand β3-loop, α1-loop, longα2-loop, det. loop) showed enhanced binding in combination with several 70ADSL73-containing peptides. BIACORE measurements with mAb's 1, 2, 13, and 17 using a set of 21 different peptide(-construct)s partially confirmed the Domain and MatrixScan™ screening results. Only 24- and 33-mer peptides covering both the 1st and 2nd strand of the β3-loop showed measurable binding. Cyclic β3-loop peptide mimics were found to bind significantly stronger (Kd∼ 5 μM) than the lineair analogues, in agreement with the fact that the discontinuous epitope is part of a loop structure. Coupling of the lineair β1-peptide 10IAIEKEECRFAI21to the linear β3-peptide*52TFKELVYETVRVPGCAHHADSLYTYPVATQAH83# via disulfide bond formation showed a 2–3 fold increase in Kd, thus conforming participation of the β 1-loop in antibody binding for these mAb's.

The majority of protein-interaction sites are discontinuous, this means that various loops form the three-dimensional binding pocket. For proteins with unknown sequence and structure random libraries can be used to define the interaction site [1]. Proteins with a defined sequence and/or structure can be scanned with overlapping peptides against, for instance, monoclonal antibodies. However, testing monoclonal antibodies in ELISA against all overlapping 12- to 15-mer peptides from these proteins is not always successful. Scanning against 25- to 30-mer peptides [2] reveals more information, since these peptides cover a larger area of the protein surface. However, to discover complete discontinuous interaction sites that contain different parts of the epitope — which are (far) apart in the primary sequence, but are brought close spatially in the protein — we developed the matrix scan. The method consists of making all possible peptides that comprise two or more different regions from a protein. The scan includes also branched combinations. Screening of these peptides provides clear insight into the nature and composition of the discontinuous epitopes as demonstrated for FSH (Follicle Stimulating Hormone).

Two small random peptide libraries, one composed of 4550 dodecapeptides and one of 8000 tripeptides, were synthesized in newly developed credit-card format miniPEPSCAN cards (miniPEPSCAN libraries). Each peptide was synthesized in a discrete well (455 peptides/card). The two miniPEPSCAN libraries were screened with three different monoclonal antibodies (Mabs). Two other random peptide libraries, expressed on the wall of bacteria (recombinant libraries) and composed of 10(7) hexa- and octapeptides, were screened with the same three Mabs. The aim of this study was to compare the amino acid sequence of peptides selected from small and large pools of random peptides and, in this way, investigate the potential of small random peptide libraries. The screening of the two miniPEPSCAN libraries resulted in the identification of a surprisingly large number of antibody-binding peptides, while the screening of the large recombinant libraries, using the same Mabs, resulted in the identification of only a small number of peptides. The large number of peptides derived from the small random peptide libraries allowed the determination of consensus sequences. These consensus sequences could be related to small linear and nonlinear parts of the respective epitopes. The small number of peptides derived from the large random peptide libraries could only be related to linear epitopes that were previously mapped using small libraries of overlapping peptides covering the antigenic protein. Thus, with respect to the cost and speed of identifying peptides that resemble linear and nonlinear parts of epitopes, small diversity libraries based on synthetic peptides appear to be superior to large diversity libraries based on expression systems.

Recently, we developed a panel of monoclonal antibodies (MoAbs) to rat IL-IP and found that MoAbs binding to the aminoacid sequences 66-85 and 123-143 of mature rlL-1 beta inhibited the binding of rlL-1 beta to murine EL4 cells. Here we study whether MoAbs to these and other domains of IL-1 interfere with the biological effects of rlL-1 beta in adult male rats in vivo. Administration of rlL-1 beta (1 or 5 mu g/kg i,v.) enhanced the plasma concentrations of ACTH, corticosterone (CORT) and of IL-6 in a time- (0.5-4 h) and dose-dependent manner. Because 2 h after 5 mu g/kg i.v,, all three parameters were consistently elevated, this dose and time interval was used for further studies, Prior to injection, rlL-1 beta was incubated alone or in the presence of a MoAb (10 mg/kg) for 30 min at 37 degrees C or at 4 degrees C. Plasma ACTH, CORT and IL-6 responses to these mixtures are compared to those obtained after preincubation of rlL-1 beta with a non-IL-l binding MoAb (PEN7). SILK 3, a MoAb that binds to the 66-85 domain of rIL-1 beta, reduced the ACTH and IL-6 responses by 48 and 45% respectively, In contrast, a MoAb to the 123-143 domain (SILK 5) and SILK 16, which binds to the 106-124 domain did not affect any of the IL-l induced responses, whereas a MoAb directed to domain 78-97 of rlL-1 beta (SILK 20) enhanced the ACTH and CORT responses by 51 and 41% respectively, but not the IL-6 response. These observations lead us to conclude that the domain 66-85, harbours sequences that are important for receptor binding and for the biological actions of rIL-1 beta in the rat and that this biologically active domain is located at the 'closed side' rather than at the 'open side' of the IL-1 beta molecule where domains involved in receptor binding and biological activity are considered to be located.

AbstractRecently, we developed a panel of monoclonal antibodies (MoAbs) to rat IL‐1β and found that MoAbs binding to the aminoacid sequences 66–85 and 123–143 of mature rIL‐1β inhibited the binding of rIL‐1β to murine EL4 cells. Here we study whether MoAbs to these and other domains of IL‐1 interfere with the biological effects of rIL‐1β in adult male rats in vivo. Administration of rIL‐1β (1 or 5 μg/kg i.v.) enhanced the plasma concentrations of ACTH, corticosterone (CORT) and of IL‐6 in a time‐ (0.5–4 h) and dose‐dependent manner. Because 2 h after 5μg/kg i.v., all three parameters were consistently elevated, this dose and time interval was used for further studies. Prior to injection, rIL‐1β was incubated alone or in the presence of a MoAb (10 mg/kg) for 30 min at 37°C or at 4°C. Plasma ACTH, CORT and IL‐6 responses to these mixtures are compared to those obtained after preincubation of rIL‐1β with a non‐IL‐1 binding MoAb (PEN7). SILK 3, a MoAb that binds to the 66–85 domain of rIL‐1β, reduced the ACTH and IL‐6 responses by 48 and 45% respectively. In contrast, a MoAb to the 123–143 domain (SILK 5) and SILK 16, which binds to the 106–124 domain did not affect any of the IL‐1 induced responses, whereas a MoAb directed to domain 78–97 of rIL‐1β (SILK 20) enhanced the ACTH and CORT responses by 51 and 41% respectively, but not the IL‐6 response.These observations lead us to conclude that the domain 66–85, harbours sequences that are important for receptor binding and for the biological actions of rIL‐1β in the rat and that this biologically active domain is located at the ‘closed side’ rather than at the ‘open side’ of the IL‐1β molecule where domains involved in receptor binding and biological activity are considered to be located.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTDifferent effects of substitution of the near-invariant glutamine-4 on the properties of porcine and bovine pancreatic phospholipases A2Gustaaf J. M. Van Scharrenburg, Wouter C. Puijk, Peter R. Seeger, Gerard H. De Haas, and Arend J. SlotboomCite this: Biochemistry 1984, 23, 6, 1256–1263Publication Date (Print):March 13, 1984Publication History Published online1 May 2002Published inissue 13 March 1984https://pubs.acs.org/doi/10.1021/bi00301a037https://doi.org/10.1021/bi00301a037research-articleACS PublicationsRequest reuse permissionsArticle Views14Altmetric-Citations4LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTEffects of substitution of the absolutely invariant Gln-4 and Phe-5 in bovine pancreatic phospholipase A2 on enzymic activity and substrate binding propertiesGustaaf J. M. Van Scharrenburg, Wouter C. Puijk, Maarten R. Egmond, Peter Van der Schaft, Gerard H. De Haas, and Arend J. SlotboomCite this: Biochemistry 1982, 21, 6, 1345–1352Publication Date (Print):March 16, 1982Publication History Published online1 May 2002Published inissue 16 March 1982https://pubs.acs.org/doi/10.1021/bi00535a037https://doi.org/10.1021/bi00535a037research-articleACS PublicationsRequest reuse permissionsArticle Views29Altmetric-Citations21LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-AlertscloseSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSemisynthesis of phospholipase A2. Preparation and properties of arginine-6 bovine pancreatic phospholipase A2Gustaaf J. M. Van Scharrenburg, Wouter C. Puijk, Maarten R. Egmond, Gerard H. De Haas, and Arend J. SlotboomCite this: Biochemistry 1981, 20, 6, 1584–1591Publication Date (Print):March 1, 1981Publication History Published online1 May 2002Published inissue 1 March 1981https://pubs.acs.org/doi/10.1021/bi00509a027https://doi.org/10.1021/bi00509a027research-articleACS PublicationsRequest reuse permissionsArticle Views40Altmetric-Citations26LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-AlertscloseSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts

The phosphatidylcholine exchange protein from bovine liver consists of a single polypeptide chain and has a blocked N terminus. The protein contains an estimated 244 amino acid residues in accordance with a determined molecular weight of 28000. The protease from mouse submaxillaris gland cleaved the citraconylated and S‐carboxymethylated derivative of the exchange protein at one specific site (Arg14‐Glu15) close to the N terminus. Analysis of the two resulting peptides showed that N‐acetyl‐methionine was the N‐terminal residue and gave the sequence of the first 41 residues.The modified protein was also fragmented with the protease from Staphylococcus aureus. The peptides isolated represented 88% of the protein; their sequences were determined by manual and automated Edman degradation. Alignment of a number of these peptides gave the complete sequence of the N‐terminal half up to position 122.

FEBS LettersVolume 92, Issue 2 p. 361-364 Full-length articleFree Access High enzymatic activity of specifically iodinated bovine phospholipase A2 Arend J. Slotboom, Arend J. Slotboom Laboratory of Biochemistry, State University of Utrecht, Transitorium 3, University Centre ‘De Uithof’, Padualaan 8, Utrecht, The NetherlandsSearch for more papers by this authorHubertus M. Verheij, Hubertus M. Verheij Laboratory of Biochemistry, State University of Utrecht, Transitorium 3, University Centre ‘De Uithof’, Padualaan 8, Utrecht, The NetherlandsSearch for more papers by this authorWouter C. Puijk, Wouter C. Puijk Laboratory of Biochemistry, State University of Utrecht, Transitorium 3, University Centre ‘De Uithof’, Padualaan 8, Utrecht, The NetherlandsSearch for more papers by this authorAlain G.R. Dedieu, Alain G.R. Dedieu Laboratory of Biochemistry, State University of Utrecht, Transitorium 3, University Centre ‘De Uithof’, Padualaan 8, Utrecht, The NetherlandsSearch for more papers by this authorGerard H. De Haas, Gerard H. De Haas Laboratory of Biochemistry, State University of Utrecht, Transitorium 3, University Centre ‘De Uithof’, Padualaan 8, Utrecht, The NetherlandsSearch for more papers by this author Arend J. Slotboom, Arend J. Slotboom Laboratory of Biochemistry, State University of Utrecht, Transitorium 3, University Centre ‘De Uithof’, Padualaan 8, Utrecht, The NetherlandsSearch for more papers by this authorHubertus M. Verheij, Hubertus M. Verheij Laboratory of Biochemistry, State University of Utrecht, Transitorium 3, University Centre ‘De Uithof’, Padualaan 8, Utrecht, The NetherlandsSearch for more papers by this authorWouter C. Puijk, Wouter C. Puijk Laboratory of Biochemistry, State University of Utrecht, Transitorium 3, University Centre ‘De Uithof’, Padualaan 8, Utrecht, The NetherlandsSearch for more papers by this authorAlain G.R. Dedieu, Alain G.R. Dedieu Laboratory of Biochemistry, State University of Utrecht, Transitorium 3, University Centre ‘De Uithof’, Padualaan 8, Utrecht, The NetherlandsSearch for more papers by this authorGerard H. De Haas, Gerard H. De Haas Laboratory of Biochemistry, State University of Utrecht, Transitorium 3, University Centre ‘De Uithof’, Padualaan 8, Utrecht, The NetherlandsSearch for more papers by this author First published: August 15, 1978 https://doi.org/10.1016/0014-5793(78)80787-XCitations: 15AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat References 1 G.H. De Haas, N.M. Postema, W. Nieuwenhuizen, L.L.M. Van Deenen, Biochim. Biophys. Acta, 159, (1968), 103– 117. 2 W.A. Pieterson, J.C. Vidal, J.J. Volwerk, G.H. De Haas, Biochemistry, 13, (1974), 1455– 1460. 3 R. Verger, M.C.E. Mieras, G.H. De Haas, J. Biol. Chem., 248, (1973), 4023– 4034. 4 C.E. Dutilh, P.J. Van Doren, F.E.A.M. Verheul, G.H. De Haas, Eur. J. Biochem., 53, (1975), 91– 97. 5 W. Nieuwenhuizen, H. Kunze, G.H. De Haas, Methods Enzymol., 32B, (1974), 147– 154. 6 G.H. De Haas, P.P.M. Bonsen, W.A. Pieterson, L.L.M. Van Deenen, Biochim. Biophys. Acta, 239, (1971), 252– 266. 7 A.J. Aarsman, L.L.M. Van Deenen, H. Van Den Bosch, Bioorg. Chem., 5, (1976), 241– 253. 8 A. Evenberg, H. Meyer, W. Gaastra, H.M. Verheij, G.H. De Haas, J. Biol. Chem., 252, (1977), 1189– 1196. 9 E.A.M. Fleer, H.M. Verheij, G.H. De Haas, Eur. J. Biochem., 82, (1978), 261– 269. 10 R.L. Misorowski, M. Wells, Biochemistry, 13, (1974), 4921– 4927. 11 R.L. Perlman, H. Edelhoch, J. Biol. Chem., 242, (1967), 2416– 2422. 12 O.A. Roholt, D. Pressman, Methods Enzymol., XXV, (1972), 438– 449. 13 A.J. Slotboom, M.C.E. Van Dam-Mieras, G.H. De Haas, J. Biol. Chem., 252, (1977), 2948– 2951. 14 F.M. Van Wezel, A.J. Slotboom, G.H. De Haas, Biochim. Biophys. Acta, 452, (1976), 101– 111. 15 R. Verger, J. Rietsch, M.C.E. Van Dam-Mieras, G.H. De Haas, J. Biol. Chem., 251, (1976), 3128– 3133. Citing Literature Volume92, Issue2August 15, 1978Pages 361-364 ReferencesRelatedInformation