Wouter Cornelis Puijk

Rapid and Quantitative Cyclization of Multiple Peptide Loops onto Synthetic Scaffolds for Structural Mimicry of Protein Surfaces

Structure-based design of synthetic peptide-based molecules that mimic the functional site of natural proteins, plays an important role in drug discovery nowadays. Their application is widespread, ranging from synthetic antiviral, 6] antifertility, 2, 7] or antitumor 7, 8] agents to therapeutic agents that are able to mimic or disrupt 10] protein–protein interactions. A variety of structural mimics exist for a-helices, 12] b-turns or hairpins, 13] and b-sheets. 14] However, more complex topologies, like four-helix bundles, are often needed in order to mimic protein function adequately. The total synthesis of such complex structures is generally demanding; this limits their application and emphasizes the need for high-efficiency synthetic strategies. In this communication, we describe a onestep procedure for the immobilization of (multiple) peptide loops onto a synthetic scaffold (Scheme 1) starting from a linear peptide. The reaction is extremely fast and clean and runs very well with linear peptides that are 2–30 amino acids long (>30 not tested). It is compatible with all possible unprotected side-chain functionalities (except for free cysteine). It therefore avoids the need for complex synthetic strategies and this makes the reaction highly versatile with a very wide scope. As part of our research program on the mapping and reconstruction of the discontinuous epitope of follicle-stimulating hormone (FSH), which is a heterodimeric member of the cysteine-knot protein family, we recently discovered the fast and quantitative cyclization of dicysteine-containing peptides upon their treatment with a,a’-dibromoxylenes (T2). In organic solvents such as ACN, the reaction is rather slow and unselective, but it becomes unusually fast and entirely selective for cysteines when performed in aqueous solutions. For example, treatment of a 0.5 mm solution of the peptide *CRVPGDAHHADSLC# (1 a, where * = acetyl and # = amide) with 1.05 equiv of m-T2 in a 1:7 mixture of ACN/NH4HCO3 (20 mm, pH 7.8) gives the corresponding monocyclic product 2 a with >80 % yield in less than 15 min at RT (see Table 1). The corresponding intramolecular SS-dimer 3 is not formed (<5 %) as oxidative cyclization is not competitive under these conditions. There is no doubt that the reaction takes place exclusively at the free sulfhydryl groups, since corresponding peptides without sulfhydryl groups do not react at all with T2 scaffolds in the solvent system used. The difference in reactivity amongst various dicysteine-containing peptides that we have studied is negligible. The half-lives of peptides 1 a–g in the reaction with m-T2 vary only slightly (t1/2 = 1.4– 3.0 min, see Table 1), despite the fact that their length (14–42) and the number of amino acids that separate the two cysteines (0–22) are very different. In sharp contrast to this, there is a large difference in reactivity amongst different scaffolds. o-T2 (average t1/2 = 1.4 min) is slightly more reactive than mT2 (average t1/2 = 2.2 min), but both react much faster than p-T2 (average t1/2 = 8.6 min). Even with a fivefold excess of m-T2 the reaction predominantly gives the monocyclic product 2, whereas the 1:2 product 3 (Scheme 2) is formed with <10 % yield. Cyclization of dicysteine-containing peptides is a two-steps process: initial formation of linear intermediate 4 (Scheme 2) followed by intramolecular cyclization to give cyclic peptide 2. All intermediates (o-4, m-4, and p-4) are highly reactive and cyclize rapidly at low concentration, but their reactivity does not follow that of the template itself. For example, intermediates o-4 and p-4 are the most reactive by far and can only be observed by HPLC for larger ring sizes (e.g. , peptide 1 f), whereas m-4 is significantly more stable and was observed in significant amounts (= 15 %) for most peptides (see Table 1). Most likely, intermediates o-4 and p-4 are activated by a stabilizing resonance-effect in which the sulfur of the first thioether bond activates the second bromomethyl group for nucleophilic attack. Scheme 1. Schematic representation of the one-step synthesis of single-, double-, and tripleloop peptide constructs by treating di-, tri-, and tetracysteine containing peptides with bis-,

Structural aspects of antibody-antigen interaction revealed through small random peptide libraries

SummaryTwo 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 107 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.

Mimotopes: realization of an unlikely concept

Theoretically it seems highly unlikely that relatively small peptides could mimic functionally discontinuous epitopes of antigens. Nevertheless various recent reports show this to be the case. Peptide mimics of protein‐, polysaccharide‐ and DNA‐epitopes have been shown to be able to replace the native epitope. Moreover, some of them are able to induce, when used in a vaccine, antibodies with the same activity as that of the antibody used as a template. These mimics, called mimotopes, can be used in vaccines and diagnostics and can be developed more or less systematically using solely antibodies and random, semi‐random and dedicated peptide arrays or libraries. Furthermore, the mimotope concept which seems to have proven itself for antibody and antigen interaction can be applied equally well to many receptor ligand interactions and thus may form a new generic approach to the development of drugs. Copyright

Identification of linear epitopes on Semliki forest virus E2 membrane protein and their effectiveness as a synthetic peptide vaccine

Semliki Forest virus (SFV) infection of mice was used as a model to study the applicability of synthetic peptides containing only linear epitopes as viral vaccines. The identification of linear epitopes with vaccine potential on the E2 membrane protein of SFV was based on the binding of SFV-specific antibodies to a set of overlapping synthetic hexapeptides (Pepscan) representing the whole E2 amino acid sequence. The 14 available E2-specific monoclonal antibodies which were protective in vivo proved to be unsuitable for the identification of linear epitopes because they recognized only conformational epitopes, as indicated by their lack of reactivity with unfolded, reduced E2 protein on immunoblots. Three epitopes were detected with polyclonal anti-SFV serum at amino acid positions 135 to 141, 177 to 185 and 240 to 246 of the E2 protein. Synthetic peptides containing these epitopes were coupled to a carrier protein and tested as a vaccine. Mice immunized with the peptide containing amino acids 240 to 255 of protein E2 were protected against a challenge with virulent SFV but protection of mice immunized with the peptides containing amino acids 126 to 141 or 178 to 186 was only marginally better than that of controls. The prechallenge sera of most peptide-immunized mice reacted with SFV-infected cells but none of these sera neutralized the virus in vitro. However, protection of mice correlated well with SFV-specific antibody titre, suggesting antibody-mediated protection.

Mapping of a discontinuous and highly conformational binding site on follicle stimulating hormone subunit-β (FSH-β) using domain Scan™ and Matrix Scan™ technology

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 mAbs, 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 mAbs 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 mAbs 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 mAbs 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 mAbs.

Targeted vaccination against the bevacizumab binding site on VEGF using 3D-structured peptides elicits efficient antitumor activity

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. Therapeutic targeting of the VEGF signaling axis by the VEGF-neutralizing monoclonal antibody bevacizumab has clearly demonstrated clinical benefit in cancer patients. To improve this strategy using a polyclonal approach, we developed a vaccine targeting VEGF using 3D-structured peptides that mimic the bevacizumab binding site. An in-depth study on peptide optimization showed that the antigen’s 3D structure is essential to achieve neutralizing antibody responses. Peptide 1 adopts a clear secondary, native-like structure, including the typical cysteine-knot fold, as evidenced by CD spectroscopy. Binding and competition studies with bevacizumab in ELISA and surface plasmon resonance analysis revealed that peptide 1 represents the complete bevacizumab binding site, including the hairpin loop (β5–turn–β6) and the structure-supporting β2–α2–β3 loop. Vaccination with peptide 1 elicited high titers of cross-reactive antibodies to VEGF, with potent neutralizing activity. Moreover, vaccination-induced antisera displayed strong angiostatic and tumor-growth-inhibiting properties in a preclinical mouse model for colorectal carcinoma, whereas antibodies raised with peptides exclusively encompassing the β5–turn–β6 loop (peptides 15 and 20) did not. Immunization with peptide 1 or 7 (murine analog of 1) in combination with the potent adjuvant raffinose fatty acid sulfate ester (RFASE) showed significant inhibition of tumor growth in the B16F10 murine melanoma model. Based on these data, we conclude that this vaccination technology, which is currently being investigated in a phase I clinical trial (NCT02237638), can potentially outperform currently applied anti-VEGF therapeutics.

Functional reconstruction of structurally complex epitopes using CLIPS™ technology

This review summarizes an illustrative set of data from our research on the reconstruction of structurally complex protein surfaces, i.e. those that fail to be mimicked properly by linear peptides. In the past 5 years, our newly developed CLIPS TM technology has proven an extremely valuable tool for 1) binding site mapping of therapeutically relevant mAbs, 2) generating hyperimmune sera via immunization with CLIPS peptides, and 3) the generation of monoclonal antibodies (mAbs) via the use of hybridoma technology. We currently have data available for more than 50 therapeutic targets. The examples described in this review illustrate the potential of this powerful new technology.

Effect of monoclonal antibodies to specific epitopes of rat interleukin-1Beta (IL-1ß) on IL-1ß-induced ACTH, corticosterone and IL-6 responses in rats

Recently, 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.

Synthetic peptides as putative therapeutic agents in transplantation medicine: application of PEPSCAN to the identification of functional sequences in the extracellular domain of the interleukin-2 receptor beta chain (IL-2Rβ)

A desired treatment strategy in transplantation medicine is the selective targeting of alloreactive T cells without impairing antileukemic and antiviral activities. One approach is the synthesis of peptides that interfere with the binding of interleukin-2 (IL-2) to its high affinity receptor (IL-2R). This blocks the activation and proliferation of the antigen-activated T cells and the secretion of IL-2. The latter binds to its receptor, via the extracellular domain of the IL-2Rbeta chain, while its cytoplasmic domain is required for intracellular signal transduction. In this study, the PEPSCAN method was applied in order to identify antigenic sequences (epitopes) in the extracellular domain of the IL-2Rbeta. Based on the primary amino acid (aa) sequence of the IL-2Rbeta, a total of 239 overlapping dodecapeptides, spanning the entire sequence of IL-2Rbeta, were synthesized by PEPSCAN and their immunoreactivity was tested by ELISA using monoclonal antibodies (mAbs) specific for IL-2Rbeta such as TU11, Mikbeta1, HuMikbeta1 and TU27. TU11 recognized a linear epitope located in the region 85R-Q(96). None of the 239 synthetic peptides was recognized by TU27. Mikbeta1 (and HuMikbeta1) recognized a discontinuous epitope formed by aa located in the IL-2Rbeta domains L(106) to P(148) and E(170) to A(202). Subsequently, synthetic peptides corresponding to the identified putative epitopic sequences were prepared by solid phase synthesis and their immunogenicity in vivo was assessed by raising polyclonal antibodies. Given that Mikbeta1 and HuMikbeta1 inhibit binding of IL-2 on the IL-2Rbeta, we addressed the question of whether the identified antigenic sequences serve as putative IL-2 binding domains. Synthetic peptides corresponding to these sequences were tested for their ability to compete with IL-2 for binding and, thereby, inhibit IL-2-induced proliferation of mitogen-stimulated human peripheral blood T cells. Sequences 107M-E(118) and 178Y-Q(199) probably represent functional IL-2 binding domains on IL-2Rbeta, since these synthetic peptides significantly inhibited the proliferation of activated T cells and secretion of IL-2.

Selecting highly structure-specific antibodies using structured synthetic mimics of the cystine knot protein sclerostin

Antibodies directed against specific regions of a protein have traditionally been raised against full proteins, protein domains or simple unstructured peptides, containing contiguous stretches of primary sequence. We have used a new approach of selecting antibodies against restrained peptides mimicking defined epitopes of the bone modulator protein sclerostin, which has been identified as a negative regulator of the Wnt pathway. For a fast exploration of activity defining epitopes, we produced a set of synthetic peptide constructs mimicking native sclerostin, in which intervening loops from the cystine-knot protein sclerostin were truncated and whose sequences were optimized for fast and productive refolding. We found that the second loop within the cystine knot could be replaced by unnatural sequences, both speeding up folding, and increasing yield. Subsequently, we used these constructs to pan the HuCAL phage display library for antibodies capable of binding the native protein, thereby restricting recognition to the desired epitope regions. It is shown that the antibodies that were obtained recognize a complex epitope in the protein that cannot be mimicked with linear peptides. Antibodies selected against peptides show similar recognition specificity and potency as compared with antibodies obtained from full-length recombinant protein.