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Purpose Fibroblast activation protein (FAP) is a membrane-bound protease that has limited expression in normal adult tissues but is highly expressed in the tumor microenvironment of many solid cancers. FAP-2286 is a FAP-binding peptide coupled to a radionuclide chelator that is currently being investigated in patients as an imaging and therapeutic agent. The potency, selectivity, and efficacy of FAP-2286 were evaluated in preclinical studies. Methods FAP expression analysis was performed by immunohistochemistry and autoradiography on primary human cancer specimens. FAP-2286 was assessed in biochemical and cellular assays and in in vivo imaging and efficacy studies, and was further evaluated against FAPI-46, a small molecule–based FAP-targeting agent. Results Immunohistochemistry confirmed elevated levels of FAP expression in multiple tumor types including pancreatic, breast, and sarcoma, which correlated with FAP binding by FAP-2286 autoradiography. FAP-2286 and its metal complexes demonstrated high affinity to FAP recombinant protein and cell surface FAP expressed on fibroblasts. Biodistribution studies in mice showed rapid and persistent uptake of 68Ga-FAP-2286, 111In-FAP-2286, and 177Lu-FAP-2286 in FAP-positive tumors, with renal clearance and minimal uptake in normal tissues. 177Lu-FAP-2286 exhibited antitumor activity in FAP-expressing HEK293 tumors and sarcoma patient-derived xenografts, with no significant weight loss. In addition, FAP-2286 maintained longer tumor retention and suppression in comparison to FAPI-46. Conclusion In preclinical models, radiolabeled FAP-2286 demonstrated high tumor uptake and retention, as well as potent efficacy in FAP-positive tumors. These results support clinical development of 68Ga-FAP-2286 for imaging and 177Lu-FAP-2286 for therapeutic use in a broad spectrum of FAP-positive tumors.

Fibroblast activation protein (FAP) is a promising target for diagnosis and therapy of numerous malignant tumors. FAP-2286 is the conjugate of a FAP-binding peptide, which can be labeled with radionuclides for theranostic applications. We present the first-in-human results using 177Lu-FAP-2286 for peptide-targeted radionuclide therapy (PTRT). Methods: PTRT using 177Lu-FAP-2286 was performed in 11 patients with advanced adenocarcinomas of pancreas, breast, rectum and ovary after prior confirmation of uptake on 68Ga-FAP-2286/-FAPI-04- PET/CT. Results: Administration of 177Lu-FAP-2286 (5.8 ± 2.0 GBq; range, 2.4–9.9 GBq) was well tolerated, with no adverse symptoms or clinically detectable pharmacologic effects being noticed or reported in any of the patients. The whole-body effective doses were 0.07 ± 0.02 Gy/GBq (range 0.04 – 0.1). The mean absorbed doses for kidneys and red marrow were 1.0 ± 0.6 Gy/GBq (range 0.4 – 2.0) and 0.05 ± 0.02 Gy/GBq (range 0.03 – 0.09), respectively. Significant uptake and long tumor retention of 177Lu-FAP-2286 resulted in high absorbed tumor doses, e.g., 3.0 ± 2.7 Gy/GBq (range 0.5 – 10.6) in bone metastases. No grade (G) 4 adverse events were observed. G3 events occurred in 3 patients – 1 pancytopenia, 1 leukocytopenia and 1 pain flare-up; 3 patients reported pain-response. Conclusion:177Lu-FAP-2286 PTRT, applied in a broad spectrum of cancers, was relatively well-tolerated with acceptable side effects and demonstrated long retention of the radiopeptide. Prospective clinical studies are warranted.

Fibroblast activation protein (FAP) is a promising target for diagnosis and therapy of numerous malignant tumors. FAP-2286 is the conjugate of a FAP-binding peptide, which can be labeled with radionuclides for theranostic applications. We present the first-in-humans results using

Background: FAP is a membrane-bound protease with limited expression in normal tissues but high expression on cancer-associated fibroblasts abundant in the stroma of most tumors. FAP-2286 is a potent and selective FAP-targeted peptide linked to the chelator DOTA that allows for attachment of radionuclides for therapeutic and imaging applications. Assessing patterns of FAP expression in different tumor types and correlating expression with FAP-2286 uptake can help guide tumor selection for FAP-2286 therapy. Methods: FAP immunohistochemistry (IHC) was performed on FFPE tissue specimens from 16 tumor types using the SP325 antibody. Overall and tumor/stroma-specific H-scores were calculated using Visiopharm and HALO analysis, respectively. Autoradiography with 111In-FAP-2286 was performed on a subset of matched frozen tissue sections. Results: Gene expression analysis across The Cancer Genome Atlas data set revealed elevated FAP mRNA expression in multiple tumor types. A pan-tumor IHC screen confirmed that ≥30% of samples in various indications (eg, sarcoma, pancreatic adenocarcinoma, mesothelioma, head and neck squamous cell carcinoma) had high FAP expression (H-score ≥30). While in most tumor types FAP was predominantly localized to the stroma, FAP expression was also observed in tumor cells, especially in tumors of mesenchymal origin, eg, sarcoma and mesothelioma. High FAP expression was independent of tumor stage or grade and detected in both primary and metastatic samples. Multiple sarcoma and mesothelioma subtypes demonstrated high FAP H-scores, suggesting that FAP expression is not limited to a specific subtype. There was significant correlation between FAP expression observed by IHC and FAP-2286 binding as assessed by autoradiography in matched frozen tissues (Pearson r=0.73; p Citation Format: Tanya T. Kwan, Minh Nguyen, Dirk Zboralski, Anne Schumann, Anne Bredenbeck, Matthias Paschke, Christian Haase, Aileen Hoehne, Ulrich Reineke, Christiane Smerling, Frank Osterkamp, Jim Xiao, Andrew D. Simmons, Thomas C. Harding, Kevin L. Lin. Pan-cancer analysis of fibroblast activation protein alpha (FAP) expression to guide tumor selection for the peptide-targeted radionuclide therapy FAP-2286 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA032.

Neurotensin receptor 1 (NTR1) is overexpressed in ductal pancreatic adenocarcinoma, which is still one of the deadliest cancers, with a very poor prognosis. Eligible patients were offered salvage radiopharmaceutical therapy with the novel NTR1 antagonist 177Lu-3BP-227. Methods: Six patients with confirmed ductal pancreatic adenocarcinoma who had exhausted all other treatment options received 177Lu-3BP-227 for evaluation of NTR1 expression in vivo. Three patients received treatment activities of 5.1-7.5 GBq. Results: Administration of 177Lu-3BP-227 was well tolerated by all patients. The kidneys were identified as the dose-limiting organ. The most severe adverse event was reversible grade 2 anemia. One patient achieved a partial response and experienced significant improvement of symptoms and quality of life. This patient survived 13 mo from diagnosis and 11 mo from the start of 177Lu-3BP-227 therapy. Conclusion: This initial report provides clinical evidence of the feasibility of treatment of ductal pancreatic adenocarcinoma using 177Lu-3BP-227.

Increased expression of neurotensin receptor 1 (NTR1) has been shown in a large number of tumor entities such as pancreatic or colon carcinoma. Hence, this receptor is a promising target for diagnostic imaging and radioligand therapy. Using the favorable biodistribution data of the NTR1-targeting agent 111In-3BP-227, we investigated the therapeutic effect of its 177Lu-labeled analog on the tumor growth of NTR1-positive HT29 colon carcinoma xenografts. Methods: 3BP-227 was labeled with 177Lu. To assess its biodistribution properties, SPECT and CT scans of HT29-xenografted nude mice injected with 177Lu-3BP-227 were acquired, and ex vivo tissue activity was determined. To evaluate therapeutic efficacy, 2 groups of mice received the radiopharmaceutical in a median dose of either 165 MBq (129-232 MBq, n = 10) or 110 MBq (82-116 MBq, n = 10), whereas control mice were injected with vehicle (n = 10). Tumor sizes and body weights were monitored for up to 49 d. Renal function and histologic morphology were evaluated. Results: Whole-body SPECT/CT images allowed clear tumor visualization with low background activity and high tumor-to-kidney and -liver ratios. Ex vivo biodistribution data confirmed high and persistent uptake of 177Lu-3BP-227 in HT29 tumors (19.0 ± 3.6 vs. 2.7 ± 1.6 percentage injected dose per gram at 3 and 69 h after injection, respectively). The application of 177Lu-3BP-227 resulted in a distinct delay of tumor growth. Median tumor doubling time for controls was 5.5 d (interquartile range [IQR], 2.8-7.0), compared with 17.5 d (IQR, 5.5-22.5 d) for the 110-MBq and 41.0 d (IQR, 27.5-55.0) for the 165-MBg group. Compared with controls, median relative tumor volume at day 23 after injection was reduced by 55% (P = 0.034) in the 110-MBq and by 88% (P < 0.01) in the 165-MBq group. Renal histology and clinical chemistry results did not differ between radiotherapy groups and controls, suggesting absence of therapy-induced acute renal damage. Conclusion: These data demonstrate that the novel NTR1-targeting theranostic agent 3BP-227 is an effective and promising candidate for radioligand therapy, with a favorable preliminary safety profile and high potential for clinical translation.

Neurotensin receptor-1 (NTR1) is a promising target for diagnostic imaging and targeted radionuclide therapy. The aim of this study was to evaluate the biodistribution profiles of a series of newly developed diarylpyrazole-based NTR1 antagonists regarding their suitability as diagnostic and potentially radiotherapeutic agents. Methods: 3BP-227, 3BP-228, and 3BP-483 were labeled with 111In and injected intravenously into NTR1-positive HT29 xenograft–bearing nude mice. At 3, 6, 12, and 24 h after administration, SPECT/CT images were acquired or mice were sacrificed for ex vivo determination of tissue-associated radioactivity. Results: High-contrast tumor visualization in SPECT/CT images was achieved using the 3 compounds of this study. Ex vivo biodistribution studies confirmed a high and persistent tumor uptake, peaking at 6 h after injection for 111In-3BP-227 (8.4 ± 3.1 percentage injected dose per gram [%ID/g]) and at 3 h after injection for 111In-3BP-228 (10.2 ± 5.3 %ID/g) and 111In-3BP-483 (1.9 ± 0.8 %ID/g). Tumor–to–normal-tissue ratios obtained with 111In-3BP-227 and 111In-3BP-228 were consistently greater than 1. Conclusion: On the basis of the superior biodistribution profile compared with previously reported radiolabeled NTR1 ligands, 111In-3BP-227 is an ideal candidate for further development as a theranostic tracer.

A short peptide as mimic for the hemopoietic growth factor erythropoietin (containing 165 amino acids) could be identified with the aid of peptide libraries on phage surfaces (phage display). The crystal structure of a peptide dimer complexed with two erythropoietin receptors (shown on the right) provides an insight into the molecular basis of this protein mimicry.

Background: Tissue factor pathway inhibitor (TFPI) inhibits coagulation factors Xa and VIIa. Results: A de novo synthesized 20-mer peptide that binds to TFPI was structurally and functionally characterized. Conclusion: The peptide binds to the Kunitz domain 1 of TFPI and blocks inhibition of factor Xa and factor VIIa by TFPI. Significance: The peptide can potentially prevent bleeding in hemophilia patients. Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits activated factor X (FXa) via a slow-tight binding mechanism and tissue factor-activated FVII (TF-FVIIa) via formation of a quaternary FXa-TFPI-TF-FVIIa complex. Inhibition of TFPI enhances coagulation in hemophilia models. Using a library approach, we selected and subsequently optimized peptides that bind TFPI and block its anticoagulant activity. One peptide (termed compound 3), bound with high affinity to the Kunitz-1 (K1) domain of TFPI (K-d approximate to 1 nm). We solved the crystal structure of this peptide in complex with the K1 of TFPI at 2.55- resolution. The structure of compound 3 can be segmented into a N-terminal anchor; an -shaped loop; an intermediate segment; a tight glycine-loop; and a C-terminal -helix that is anchored to K1 at its reactive center loop and two-stranded -sheet. The contact surface has an overall hydrophobic character with some charged hot spots. In a model system, compound 3 blocked FXa inhibition by TFPI (EC50 = 11 nm) and inhibition of TF-FVIIa-catalyzed FX activation by TFPI (EC50 = 2 nm). The peptide prevented transition from the loose to the tight FXa-TFPI complex, but did not affect formation of the loose FXa-TFPI complex. The K1 domain of TFPI binds and inhibits FVIIa and the K2 domain similarly inhibits FXa. Because compound 3 binds to K1, our data show that K1 is not only important for FVIIa inhibition but also for FXa inhibition, i.e. for the transition of the loose to the tight FXa-TFPI complex. This mode of action translates into normalization of coagulation of hemophilia plasmas. Compound 3 thus bears potential to prevent bleeding in hemophilia patients.

Increased expression of neurotensin receptor 1 (NTR1) has been shown in a large number of tumor entities such as pancreatic or colon carcinoma. Hence, this receptor is a promising target for diagnostic imaging and radioligand therapy. Using the favorable biodistribution data of the NTR1-targeting agent

Nogo-A is a potent inhibitor of axonal outgrowth in the central nervous system of adult mammals, where it is expressed as a membrane protein on oligodendrocytes and in myelin. Here we describe an attempt to identify linear peptide epitopes in its sequence that are responsible for the interaction either with the Nogo receptor (NgR) or with the neutralizing monoclonal antibody IN-1. Analysis of an array of immobilized overlapping 15 mer peptides covering the entire amino acid sequence of human Nogo-A (1192 residues) revealed a single epitope with prominent binding activity both towards the recombinant NgR and the IN-1 Fab fragment. Further truncation and substitution analysis yielded the minimal epitope sequence 'IKxLRRL' (x ≠ P), which occurs within the so-called Nogo66 region (residues 1054–1120) of Nogo-A. The bacterially produced Nogo66 fragment exhibited binding activity both for the recombinant NgR and for the IN-1 Fab fragment on the Western blot as well as in ELISA. Unexpectedly, the synthetic epitope peptide and the recombinant Nogo66 showed cross-reactivity with the 8-18C5 Fab fragment, which is directed against myelin oligodendrocyte glycoprotein (MOG) as a structurally unrelated target. On the other hand, the recombinant N-terminal domain of Nogo-A (residues 334–966) was shown to specifically interact on the Western blot and in an ELISA with the IN-1 Fab fragment but not with the recombinant NgR, which is in agreement with previous results. Hence, our data suggest that there is a distinct binding site for the Nogo receptor in the Nogo66 region of Nogo-A, whereas its interaction with NgR is less specific than anticipated before. Although there probably exists a non-linear epitope for the neutralizing antibody IN-1 in the N-terminal region of Nogo-A, which is likely to be accessible from outside the cell, a previously postulated second binding site for NgR in this region (called Nogo-A-24) remains elusive. Copyright © 2007 John Wiley & Sons, Ltd.

We report the stepwise transformation of a linear peptide epitope recognized by the anti-transforming growth factor alpha monoclonal antibody Tab2 into peptomers and finally into peptoid analogs. The key experiment in this study is the substitution analysis in which each position of the peptide is exchanged by a set of different peptoid building blocks resulting in a peptidomimetic array. After probing the array toward antibody binding, the best binding peptomer spots were selected and subjected to a successive transformation. The best peptoid found in this study has a K(D) of 200 nM when binding to Tab2, which is only 8-fold higher than the starting peptide. Moreover, this approach permits to ask directly questions about the transformation of peptide lead structures into non-peptidic compounds in the context of protein recognition.

Objective: Prostate cancer cells secrete the unique protease human glandular kallikrein 2 (hK2) that represents a target for proteolytic activation of cytotoxic prodrugs. The objective of this study was to identify hK2-selective peptide substrates that could be coupled to a cytotoxic analogue of thapsigargin, a potent inhibitor of the sarcoplasmic/endoplasmic reticulum calcium ATPase pump that induces cell proliferation–independent apoptosis through dysregulation of intracellular calcium levels. Methods: To identify peptide sequence requirements for hK2, a combination of membrane-bound peptides (SPOT analysis) and combinatorial chemistry using fluorescence-quenched peptide substrates was used. Peptide substrates were then coupled to 8-O-(12[l-leucinoylamino]dodecanoyl)-8-O-debutanoylthapsigargin (L12ADT), a potent analogue of thapsigargin, to produce a prodrug that was then characterized for hK2 hydrolysis, plasma stability, and in vitro cytotoxicity. Results: Both techniques indicated that a peptide with two arginines NH2-terminal of the scissile bond produced the highest rates of hydrolysis. A lead peptide substrate with the sequence Gly-Lys-Ala-Phe-Arg-Arg (GKAFRR) was hydrolyzed by hK2 with a Km of 26.5 μmol/L, kcat of 1.09 s−1, and a kcat/Km ratio of 41,132 s−1 mol/L−1. The GKAFRR-L12ADT prodrug was rapidly hydrolyzed by hK2 and was stable in plasma, whereas the GKAFRR-L peptide substrate was unstable in human plasma. The hK2-activated thapsigargin prodrug was not activated by cathepsin B, cathepsin D, and urokinase but was an excellent substrate for plasmin. The GKAFRR-L12ADT was selectively cytotoxic in vitro to cancer cells in the presence of enzymatically active hK2. Conclusion: The hK2-activated thapsigargin prodrug represents potential novel targeted therapy for prostate cancer.

Rapid synthesis and screening of compound libraries enables the accelerated identification of novel protein ligands in order to support processes like analysis of protein interactions, drug target discovery or lead structure discovery. SPOT synthesis—a well established method for the rapid preparation of peptide arrays—has recently been extended to the field of nonpeptides. In this contribution we report on the systematic evaluation of the SPOT technique for the assembly of N -alkylglycine (peptoid) library arrays. In the course of this investigation bromoacetic acid 2,4-dinitrophenylester ( 1a ) was identified to be the most suited agent for bromoacetylation in terms of yield and N -selectivity enabling straightforward submonomer synthesis on hydroxy-group rich cellulose membranes. The potential of this method for the rapid identification of novel nonpeptidic protein ligands was demonstrated by synthesis and screening of a library consisting of 8000 peptoids and peptomers (i.e. their hybrids with α-substituted amino acids) allowing the identification of micromolar ligands for the monoclonal antibody Tab-2.

A kinase-anchoring proteins (AKAPs) coordinate cAMP-mediated signaling by binding and localizing cAMP-dependent protein kinase (PKA), using an amphipathic helical docking motif. Peptide disruptors of PKA localization that mimic this helix have been used successfully to assess the involvement of PKA in specific signaling pathways. However, these peptides were developed as disruptors for the type II regulatory subunit (RII) even though both RI and RII isoforms can bind to AKAPs and have discrete functions. To evaluate the effects of each localized isoform, we designed peptides that specifically bind to either RI or RII. Using a peptide array, we have defined the minimal binding sequence of dual specific-AKAP 2 ( d -AKAP2), which binds tightly to both RI and RII. Side-chain requirements for affinity and isoform specificity were evaluated by using a peptide substitution array where each position along the A kinase binding domain of d -AKAP2 was substituted by the other 19 l -amino acids. This array comprises 513 single-site substitution analogs of the d -AKAP2 sequence. Peptides containing single and multiple mutations were evaluated in a quantitative fluorescence binding assay and a cell-based colocalization assay. This strategy has allowed us to design peptides with high affinity ( K D = 1–2 nM) and high specificity for RIα versus RIIα. These isoform-specific peptides will be invaluable tools to evaluate functional differences between localized RI and RII PKA and are RIα-specific disruptors. This array-based analysis also provides a foundation for biophysical analysis of this docking motif.

T helper cell-recognized epitopes were determined in chitinase of Onchocerca volvulus, a vaccine candidate protein. The proliferation of splenic T cells of mice immunized with recombinant protein was tested with a library of chitinase-peptides of 16 amino acids with termini overlapping by 12 amino acids, and a library of "designer peptides", i.e. sequences identified with three epitope-predicting algorithms. Fourteen epitope-bearing stretches were identified with the peptides of the overlapping library. Testing of the designer peptides partially confirmed these data and revealed additional epitopes. Five clusters of epitopes were identified for the creation of peptide or minigene DNA vaccines with good potency and potential range of MHC allele presentation.

The growing range of applications for peptide arrays synthesized on coherent membranes by the SPOT-synthesis method proves they have emerged as a powerful proteomics technique to study molecular recognition events and identify biologically active peptides. Several developments, such as the introduction of novel polymeric surfaces, linkers, synthesis/cleavage strategies and detection methods, are facilitating an increasing spectrum of accessible compounds and applications in biological or pharmaceutical research.

The mechanism of recognition of proteins and peptides by antibodies and the factors determining binding affinity and specificity are mediated by essentially the same features. However, additional effects of the usually unfolded and flexible solution structure of peptide ligands have to be considered. In an earlier study we designed and optimized six peptides (pepI to pepVI) mimicking the discontinuous binding site of interleukin-10 for the anti-interleukin-10 monoclonal antibody (mab) CB/RS/1. Three of them were selected for analysis of their solution conformation by circular dichroism measurements. The peptides differ in the content of alpha-helices and in the inducibility of helical secondary structures by trifluoroethanol. These properties, however, do not correlate with the binding affinity. PepVI, a 32-mer cyclic epitope mimic, has the highest affinity to mab CB/RS/1 identified to date. CD difference spectroscopy suggests an increase of the alpha-helix content of pepVI with complex formation. Binding of pepVI to mab CB/RS/1 is characterized by a large negative, favorable binding enthalpy and a smaller unfavorable loss of entropy (DeltaH degrees = -16.4 kcal x mol(-1), TDeltaS degrees = -6.9 kcal x mol(-1)) resulting in DeltaG degrees = -9.5 kcal x mol(-1) at 25 degrees C as determined by isothermal titration calorimetry. Binding of pepVI is enthalpically driven over the entire temperature range studied (10-35 degrees C). Complex formation is not accompanied by proton uptake or release. A negative heat capacity change DeltaC(p) of -0.354 kcal x mol(-1) x K(-1) was determined from the temperature dependence of DeltaH degrees. The selection of protein mimics with the observed thermodynamic properties is promoted by the applied identification and iterative optimization procedure.

Effective spatially addressed parallel assembly of trisamino- and amino-oxy-1,3,5-triazines was achieved by applying the SPOT-synthesis technique on cellulose and polypropylene membranes. In addition to developing a suitable linker strategy and employing amines and phenolate ions as building blocks, a highly effective microwave-assisted nucleophilic substitution procedure at membrane-bound monochlorotriazines was developed. The 1,3, 5-triazines obtained could be cleaved in parallel from the solid support by TFA vapor to give compounds adsorbed on the membrane surface in a conserved spatially addressed format for analysis and screening. The reaction conditions developed were employed for the synthesis of 8000 cellulose-bound 1,3,5-triazines which were probed in parallel for binding to the anti-transforming growth factor-alpha monoclonal antibody Tab2 in order to identify epitope mimics.

Positionally addressable syntheses of peptides on continuous cellulose membranes (spot synthesis) have often been reported in detail, but important questions dealing with synthesis quality, reproducibility and subsequent binding assays have largely been under-emphasized. In this report we have investigated some of these problems. The most important results were: (i) the signal intensity of ligate binding to cellulose-bound peptides and the affinity of the corresponding soluble peptides show good correlation, illustrated by three different ligate binding assays; (ii) reducing peptide density on the cellulose avoids the ‘ring spot’ effect, i.e. where less binding is observed in the spot-center compared to the rim. We recommend a peptide density of 10 nmol/cm2 as a reasonable starting point for further optimization; (iii) statistical analysis of binding assay reproducibility with more than 15 000 peptides resulted in a mean standard signal deviation of 0.18; and (iv) optimization of side-chain deprotection revealed that a 30-min pretreatment of the cellulose with 90% trifluoroacetic acid followed by the standard deprotection protocol resulted in higher purity of the synthesized products.

We synthetically reconstructed a discontinuous binding site on interleukin-10 (IL-10) that recognizes the neutralizing anti-IL-10 antibody CB/RS/1. To design the 32-mer IL-10 mimic, a discontinuous interaction site on IL-10 was mapped, and binding studies with epitope-derived peptides led to specific replacement of several amino acids. Both parts of the interaction site were combined by addition of a linker molecule. Systematic analoging of the combined molecule then led to introduction of several additional substitutions in both regions and the linker. All possible disulfide bridge-containing variants of the 32-mer were tested by binding studies. Parallel syntheses were performed on continuous cellulose membranes by spot synthesis. As a result, a conformationally stabilized IL-10-derived molecule was obtained that both binds to and neutralizes the biological activity of CB/RS/1 in the low nanomolar range. This synthetic approach is a powerful alternative to phage display methods for the design of protein mimics.

The discontinuous interleukin-10(IL-10)/interleukin-10 receptor (IL-10R) combining site was mapped using sets of overlapping peptides derived from both binding partners bound to continuous cellulose membranes. Low affinity binding of single regions of the discontinuous contact sites on IL-10 and IL-10R could be identified due to (1) high peptide density on the membrane support, (2) incubation with high protein concentrations, (3) indirect immunodetection of the ligates after electrotransfer onto polyvinylene difluoride membranes, and (4) use of highly overlapping peptide scans of different length (6-mers and 15-mers). The single binding regions identified for each protein species are separated in the protein sequences, but form continuous areas on the surface of IL-10 (X-ray structure) and IL-10R (computer model). Furthermore, four epitopes of neutralizing anti-IL-10 and anti-IL-10R antibodies were mapped and overlap with these binding regions. Soluble peptides (15- to 19-mers) each spanning one of the three identified IL-10-derived receptor binding regions displayed no significant affinity to IL-10R as expected, whereas a peptide (35-mer) comprising two of these regions had considerably higher binding activity. The data are consistent with a previously published computer model of the IL-10/IL-10R complex. This approach should be generally applicable for the mapping of non-linear protein-protein contact sites.

We have characterized the interaction of two monoclonal antibodies with their respective antigens using cellulose-bound sets of overlapping peptides (peptide scans). Both antibodies CB/RS/5 and CB/MT/1 recognize discontinuous epitopes present in human interleukin-10 (IL-10) and tumor necrosis factor alpha (TNF-alpha). In addition, the interaction between TNF-alpha and its 55-kDa receptor (TNF-R) was investigated by the same approach. Both antibodies, as well as TNF-alpha, interacted with two or more regions of the peptide scans. Antibody-binding competition studies between the native antigens and peptides, covering single parts of the binding regions, enabled us to distinguish between binding to the paratope or other regions of the antibody. The combination of these experimental approaches allowed the identification of short antigen-derived sequences that are separated on the primary sequence but close in space on the surface of IL-10 and TNF-alpha, thus representing putative discontinuous epitopes. In the case of the TNF-R-derived peptide scans, two of the identified regions interact with the structurally similar TNF-beta in the TNF-beta-TNF-R complex. These data indicate that this approach should be generally applicable for mapping nonlinear protein-protein contact sites.

Monoclonal antibody (MAb) 2c, specific for glycoprotein B of herpes simplex virus (HSV), had been shown to mediate clearance of infection from the mucous membranes of mice, thereby completely inhibiting mucocutaneous inflammation and lethality, even in mice depleted of both CD4(+) and CD8(+) cells. Additionally, ganglionic infection was highly restricted. In vitro, MAb 2c exhibits a potent complement-independent neutralising activity against HSV type 1 and 2, completely inhibits the viral cell-to-cell spread as well as the syncytium formation induced by syncytial HSV strains (Eis-Hübinger et al. in Intervirology 32:351-360, 1991; Eis-Hübinger et al. in J Gen Virol 74:379-385, 1993). Here, we describe the mapping of the epitope for MAb 2c. The antibody was found to recognise a discontinuous epitope comprised of the HSV type 1 glycoprotein B residues 299 to 305 and one or more additional discontinuous regions that can be mimicked by the sequence FEDF. Identification of the epitope was confirmed by loss of antibody binding to mutated glycoprotein B with replacement of the epitopic key residues, expressed in COS-1 cells. Similarly, MAb 2c was not able to neutralise HSV mutants with altered key residues, and MAb 2c was ineffective in mice inoculated with such mutants. Interestingly, identification and fine-mapping of the discontinuous epitope was not achieved by binding studies with truncated glycoprotein B variants expressed in COS cells but by peptide scanning with synthetic overlapping peptides and peptide key motif analysis. Reactivity of MAb 2c was immensely increased towards a peptide composed of the glycoprotein B residues 299 to 305, a glycine linker, and a C-terminal FEDF motif. If it could be demonstrated that antibodies of the specificity and bioactivity of MAb 2c can be induced by the epitope or a peptide mimicking the epitope, strategies for active immunisation might be conceivable.

A short peptide as mimic for the hemopoietic growth factor erythropoietin (containing 165 amino acids) could be identified with the aid of peptide libraries on phage surfaces (phage display). The crystal structure of a peptide dimer complexed with two erythropoietin receptors (shown on the right) provides an insight into the molecular basis of this protein mimicry.

Information at the amino acid level about the epitopes of proteins recognized by antibodies or antibody fragments is important for their use as biological and diagnostic tools, therapeutic molecules, and for understanding molecular recognition events in general. The use of chemically prepared arrays of short peptides has emerged as a powerful tool to identify and characterize antibody epitopes. In this chapter the SPOT synthesis technique is described in detail. In addition, three different types of peptide libraries and their applications are described: protein sequence-derived scans of overlapping peptides (peptide scans) used to locate epitopes within the protein sequence, truncation libraries used to identify the minimal peptide length required for antibody binding, and complete substitutional analyses to identify the key residues important for antibody binding.

Identification of antibody binding peptides may be based on the primary structure of the protein antigens used to raise the antibodies (knowledge- or sequence-based approach). This involves scanning the entire sequence of the antigen with overlapping peptides (peptide scan), which are then probed for binding to the respective antibody. If a natural protein binding partner is not known, one has to use combinatorial synthetic libraries with peptide mixtures, randomly generated chemically synthesized libraries of single individual sequences, or biologically produced libraries (e.g., phage display libraries, see Chapter "Epitope Mapping Using Phage Display Peptide Libraries"). This chapter describes chemically synthesized combinatorial, as well as randomly generated peptide libraries, collectively called de novo approaches, and their application for antibody epitope mapping.

Identification of antibody binding peptides may be based on the primary structure of the protein antigens used to raise the antibodies (knowledge- or sequence-based approach). This involves scanning the entire sequence of the antigen with overlapping peptides (peptide scan), which are then probed for binding to the respective antibody. If a natural protein binding partner is not known, one has to use combinatorial synthetic libraries with peptide mixtures, randomly generated chemically synthesized libraries of single individual sequences, or biologically produced libraries (e.g., phage display libraries, see Chapter "Epitope Mapping Using Phage Display Peptide Libraries"). This chapter describes chemically synthesized combinatorial, as well as randomly generated peptide libraries, collectively called de novo approaches, and their application for antibody epitope mapping.

Information at the amino acid level about the epitopes of proteins recognized by antibodies or antibody fragments is important for their use as biological and diagnostic tools, therapeutic molecules, and for understanding molecular recognition events in general. The use of chemically prepared arrays of short peptides has emerged as a powerful tool to identify and characterize antibody epitopes. In this chapter the SPOT synthesis technique is described in detail. In addition, three different types of peptide libraries and their applications are described: protein sequence-derived scans of overlapping peptides (peptide scans) used to locate epitopes within the protein sequence, truncation libraries used to identify the minimal peptide length required for antibody binding, and complete substitutional analyses to identify the key residues important for antibody binding.

Array technology has become a powerful tool for today’s high throughput approaches in biology and chemistry. These large-scale technologies emerged mainly from the genomics field driven by the human genome project and other species sequencing efforts. The associated development of many new technologies led to the initiation of several additional “omics” fields such as proteomics, lipidomics, glycomics and others. Scientists in these fields subsequently demanded tools that allow rapid and reliable characterization of large numbers of molecules of very different natures including nucleic acids, proteins, carbohydrates, peptides, and small molecules with very small amounts of biological or synthetic material.

Protein domains or small molecules that mimic protein-protein contact sites are playing an increasing role in drug discovery, diagnostics and biotechnology. The identification of such protein mimics is based on two different strategies: (1) biologically as well as chemically prepared combinatorial libraries that are utilized for the de novo generation of novel sequences; and (2) approaches that are based on three-dimensional structures of endogenous protein ligands. Reduction of their size by truncation or deletion of amino acids which are not involved in the interaction and subsequent optimization by site-directed mutagenesis may also involve and library techniques. In contrast, this study describes an approach based on the primary structure of a protein ligand. One important technique for the mapping of protein-protein contact sites is the use of scans of overlapping peptides derived from a protein sequence (peptide scan, Fig. 1 left) which are subsequently tested for binding of the respective interaction partner [1]. This technique is the method of choice for the identification of linear binding sites where most of the important residues for the interaction are located within one stretch of the primary structure. In contrast, the key interacting residues of discontinuous binding sites are distributed over two or more binding regions which are separated in the protein sequence and only form the composite high affinty epitope upon protein folding. The mapping of discontinuous binding sites with peptide scans is very difficult, if not impossible, since peptides comprising single binding regions characteristically have very low affinities for the binding partner. Hence, we introduced a novel type of peptide library, called a duotope scan. The rationale of this scan is that a discontinuous binding site can only be mimicked adequately if two or more binding regions are connected in one molecule by a linker moiety resembling their spacing in the three-dimensional structure. Therefore, all possible combinations of two overlapping peptides from a conventional peptide scan are synthesized as one linear molecule i. E. combinatorial chemistry with peptides as second level building blocks (Fig. 1 right). The duotope scans are synthesized on continuous cellulose membranes by SPOT-synthesis [2], a highly parallel positionally addressable synthesis technique.

Introduction Tissue factor pathway inhibitor (TFPI) is an important inhibitor of the extrinsic coagulation pathway as it inhibits factor Xa (FXa) and the tissue factor (TF) – FVIIa complex. Inhibition of TFPI with blocking antibodies, aptamers, or peptide inhibitors improves hemostasis and may become an option to treat patients with hemophilia including those with inhibitors. We developed a TFPI-antagonistic fusion peptide (FP) consisting of a linear and a cyclic peptide connected by a linker. The two peptide entities bind to different epitopes on TFPI and together synergistically inhibit TFPI. The FP was further improved by half-life extending (HL) non-covalent albumin binding. HL-FP was characterized for in vitroinhibition of TFPI, pharmacokinetics, and improvement of coagulation in animal models of hemophilia. Methods HL-FP was characterized in a set of in vitro assays for binding to and inhibition of TFPI. Interaction with immobilized TFPI was studied by BiaCore. Functional inhibition was analyzed in model assay systems such as inhibition of FXa and FX activation by TF/FVIIa and plasma assays according to the calibrated automated thrombography (CAT) protocol at low TF in hemophilia plasma. Addition of TFPI simulated conditions of potentially elevated TFPI plasma concentrations. In a single dose PK study, mice (n=6 per time point) received 400 nmol/kg of the HL-FP intravenously (i.v.) or subcutaneously (s.c.). Plasma was sampled up to 38 h after dosing and HL-FP level quantified by a compound specific LC-MS protocol. To provide an ex vivo activity measure, FVIII inhibitory antibodies were added to mouse plasma to mimic a hemophilic condition and then analyzed by calibrated automated thrombography (CAT). A 2-week repeated i.v. dose study in mice investigated TFPI accumulation due to HL-FP. HL-FP was dosed at 40, 400, and 2000 nmol/kg and mouse plasma TFPI levels determined by ELISA. The efficacy of the HL-FP was studied in a hemophilia A mouse tail cut model and in a marmoset monkey model of ex vivoimprovement of coagulation. FVIII knockout mice (n=16 per group) were dosed i.v. with 12-400 nmol/kg HL-FP in the presence of a sub-therapeutic level of recombinant FVIII (10 U/kg) and blood loss (mg) was assessed. Marmoset monkeys (N=4) received 400 nmol/kg HL-FP i.v. and plasma samples obtained 1 h after dosing were analyzed by CAT in the presence of FVIII inhibitory antibodies. Results HL-FP bound to and efficiently inhibited TFPI as demonstrated in several in vitro test systems. Binding affinity of < 1nM correlated well with functional inhibition of TFPI in model assays, resulting in IC50s of ~0.7nM. The HL-fusion peptide (HL-FP) efficiently inhibited plasma TFPI, which resulted in an improvement of all thrombin generation parameters in plasma of hemophilia A and B patients, with EC50s ranging from 6 to 20nM. HL-FP increased peak thrombin levels of hemophilia plasma to or slightly above a range established for individual normal plasma. Non-covalent binding to albumin substantially increased the half-life to ~4 h with ~ 50% s.c. bioavailability in mice. The ex vivo procoagulant activity determined by CAT correlated well with HL-FP plasma concentrations. In the repeated dose study, the HL-FP was well tolerated and did not accumulate TFPI, which strongly indicates that HL-FP did not interfere with TFPI clearance receptor interactions. HL-FP significantly reduced bleeding in the hemophilia mouse tail cut bleeding model at a dose as low as 40 nmol/kg. In marmoset monkeys, HL-FP efficiently improved ex vivo plasma thrombin generation, even at low peptide plasma concentrations (25- 55 nM). Summary We developed a TFPI inhibitor composed of two TFPI antagonistic peptides that completely inhibits TFPI. Introduction of an entity non-covalently bound to albumin provides intermediate half-life extension and s.c. bioavailability. This HL-FP improved coagulation and hemostasis in animal models of hemophilia and did not interfere with TFPI clearance receptor interactions. TFPI-antagonistic peptides with a prolonged half-life, resistance to elevated TFPI, and minimal interference with TFPI clearance. Our HL-FP appears to be useful in preventing bleeding in hemophilia and provides a FVIII and FIX independent approach for non-i.v. treatment. Disclosures Dockal: Baxter Innovations GmbH, Vienna, Austria: Employment. Hartmann:Baxter Innovations GmbH, Vienna, Austria: Employment. Polakowski:3B Pharmaceuticals GmbH, Berlin, Germany: Employment. Panholzer:Baxter Innovations GmbH, Vienna, Austria: Employment. Kammlander:Baxter Innovations GmbH, Vienna, Austria: Employment. Osterkamp:3B Pharmaceuticals, Berlin, Germany: Employment. Reineke:3B Pharmaceuticals GmbH, Berlin, Germany: Employment. Schiviz:Baxter Innovations GmbH, Vienna, Austria: Employment. Hoellriegl:Baxter Innovations GmbH, Vienna, Austria: Employment. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.

Introduction Tissue factor pathway inhibitor (TFPI) is a three-Kunitz domain (KD1-3) protease inhibitor that downregulates the extrinsic coagulation pathway. TFPI has a double inhibitory effect; it inactivates factor Xa (FXa) by 1:1 binding via its KD2, and it prevents further FX activation by binding the tissue factor (TF) – factor VIIa (FVIIa) complex via its KD1 and the formation of a quaternary complex. Recently, we demonstrated the crystal structure of a linear TFPI inhibitory peptide composed of 20 amino acids, bound to a TFPI protein composed of N-terminus and KD1. On the other hand, a cyclic TFPI inhibitory peptide of 23 amino acids was shown to co-crystallize with TFPI KD1-KD2. Molecular fusion of the linear and cyclic peptide by an optimized linker sequence would thus target two independent epitopes and combine the antagonistic properties of the two peptides. Methods The binding properties of simultaneous interaction of the linear and cyclic peptide with TFPI were studied in Biacore experiments using immobilized human TFPI 1-160 on a CM5 chip. Measurements with the linear or cyclic peptide were done with and without prior saturation of TFPI with the linear peptide and the fusion peptide. The results were confirmed by native-PAGE analysis of peptide/KD1-KD2 mixtures, where the TFPI fragment KD1-KD2 had been incubated with either linear or cyclic peptide or both. The TFPI inhibitory effect of the linear, cyclic, and fusion peptide was assessed in several TFPI sensitive assays including inhibition of FXa, FX activation by TF/FVIIa, and thrombin generation. Calibrated automated thrombography (CAT) was performed in human hemophilia plasma triggered with low tissue factor. To model a situation of elevated plasma levels of TFPI, the assay was carried out at TFPI concentrations up to 10 nM, which is 40-fold higher than the physiological TFPI plasma concentration. Results Biacore binding studies demonstrated that binding kinetics of the cyclic peptide to TFPI 1-160 were not influenced by prior saturation of immobilized TFPI with the linear peptide and vice versa. Prior saturation of immobilized TFPI with the fusion peptide prohibited the linear and cyclic peptide from binding to TFPI, clearly demonstrating the independent binding of the two peptides to different epitopes. By native-PAGE, the linear peptide shifted the KD1-KD2 band completely, whereas the cyclic peptide shifted it only partially. In the presence of both peptides, KD1-KD2 shifted to the highest MW to charge ratio, indicating the formation of a ternary complex consisting of K1-K2, cyclic, and linear peptide. Although the linear and cyclic peptide inhibited TFPI in functional assays, fusion of the two molecular entities provided the most efficient inhibition of TFPI. This was most evident in assays involving multiple epitopes of TFPI to provide functions such as inhibition of extrinsic FX activation complex and thrombin generation, or at high TFPI concentrations. Thrombin generation assays using of 5- to 40-fold elevated TFPI showed that, separately, the two monomeric peptides are only partial inhibitors, and that a mixture of these peptides led to an improved response. However, molecular fusion of the two entities resulted in the most efficient TFPI neutralization. Thus, a synergistic effect is achieved by linking both peptides. Importantly, thrombin generation compromised by a 40-fold of normal TFPI level is normalized by fusion peptide concentrations as low as 50 nM. Summary Based on structural information, we developed a peptide inhibitor composed of two TFPI inhibitory entities. Binding studies support an independent binding mode to non-overlapping binding sites without allosteric cross-talk between binding sites. This introduces synergistic improvement of binding and functional inhibition by bivalent interaction with TFPI. This optimized fusion peptide facilitates efficient TFPI neutralization and resistance to highly increased TFPI levels. Our results further support the use of a fusion peptide in the development of subcutaneous treatment for patients with hemophilia including those with inhibitors. Disclosures Dockal: Baxter Innovations GmbH, Vienna, Austria: Employment. Hartmann:Baxter Innovations GmbH, Vienna, Austria: Employment. Polakowski:3B Pharmaceuticals GmbH, Berlin, Germany: Employment. Redl:Baxter Innovations GmbH, Vienna, Austria: Employment. Panholzer:Baxter Innovations GmbH, Vienna, Austria: Employment. Kammlander:Baxter Innovations GmbH: Employment. Osterkamp:3B Pharmaceuticals, Berlin, Germany: Employment. Reineke:3B Pharmaceuticals GmbH, Berlin, Germany: Employment. Brandstetter:Department of Molecular Biology, University of Salzburg, Salzburg, Austria: Research Funding. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.

Interleukin(IL)-10 and IL-22 are structurally related cytokines. Their heterodimeric receptors consist of the cytokine-specific chains IL-10R1 and IL-22R1, respectively, and the common chain IL-10R2. This study focused on the question of whether IL-10 modulates IL-22 effects and vice versa. This question is important because IL-10 and IL-22 exert anti- and proinflammatory effects, respectively, and, as we show here, are simultaneously present in both systemic and local inflammation. The revealed lacking concomitance of IL-10R1 and IL-22R1 on identical cells excluded any possible interaction between IL-10 and IL-22 apart from the competition for IL-10R2. To study this competition, monocytes and hepatocytes were chosen. The dependence of the cytokine action on IL-10R2 was verified. Interestingly, no influence of IL-22 on IL-10 effects was observed. The same was true when IL-22 was used in complex with IL-22-binding protein. Similarly, no influence of IL-10 was found on IL-22 action. This missing competition seemed to be due to a lack of binding between IL-10R2 and the native cytokines in the absence of their corresponding R1 chain. However, IL-10R2 interacted with defined IL-10- and IL-22-derived peptides supporting the hypothesis that cytokine binding to its corresponding R1 chain creates a binding site on this cytokine for IL-10R2.

Tumor necrosis factor (TNF)-alpha converting enzyme (TACE) is responsible for the ectodomain release of various membrane proteins by proteolytic cleavage in close proximity to the cell membrane. Despite the wide spectrum of possible substrates, selective cleavage can be achieved by substrate cross-linking. To explore the underlying mechanism, we studied the TACE-mediated shedding of CD30. Whereas the constitutive release of the soluble ectodomain of CD30 (sCD30) from the lymphoma cell line Karpas 299 was enhanced by most anti-CD30 antibodies, it was inhibited by antibodies Ber-H2 and Ki-4. On the basis of the recognized epitopes, shedding seemed to depend on the availability of the cysteine-rich domains (CRD) 2 and 5 of the CD30 ectodomain. CRD2 and 5 have almost identical amino acid sequences and are localized distant from the TACE-targeted cleavage site. Soluble CD30, the product of this enzyme reaction, did not inhibit, but on the contrary, it stimulated CD30 shedding in a CRD2/5-dependent manner. This process could also be induced by CRD2/5-derived peptides but not by a CRD1-derived control peptide. This example of a product-activation was CD30 selective since other TACE substrates such as TNFR1 or TNF-alpha were not affected. These data suggest that CD30 shedding is stimulated by an elevated local availability of CRD2 or 5, possibly by forming a docking station for the releasing enzyme through substrate aggregation.

Over the past decade of proteome research peptide arrays have become a widespread and powerful tool to study molecular recognition events and to identify biologically active peptides. A variety of applications such as epitope mapping, characterisation of protein–protein interactions, enzyme–substrate or inhibitor interactions, and many more, have been published. Today's technologies for array production, inspired by DNA chips, have recently turned to the miniaturisation of peptide arrays. These advances open up an expanding spectrum of applications and the information obtained will be well-suited to developing substrates and inhibitors for diagnostic and therapeutic purposes.

The ectodomain of the human transferrin receptor (TfR) is released as soluble TfR into the blood by cleavage within a stalk. The major cleavage site is located C-terminally of Arg-100; alternative cleavage sites are also present. Since the cleavage process is still unclear, we looked for proteases involved in TfR ectodomain release. In the supernatant of U937 histiocytic cells we detected alternatively cleaved TfR (at Glu-110). In membrane fractions of these cells we identified two distinct proteolytic activities responsible for TfR cleavage within the stalk at either Val-108 or Lys-95. Both activities could be inhibited by serine protease inhibitors, but not by inhibitors of any other class of proteases. Protein purification yielded a 28 kDa protein that generated the Val-108 terminus. The protease activity could be ascribed to neutrophil elastase according to the substrate specificity determined by amino acid substitution analysis of synthetic peptides, an inhibitor profile, the size of the protease and the use of specific antibodies. The results of analogous experiments suggest that the second activity is represented by another serine protease, cathepsin G. Thus, membrane-associated forms of neutrophil elastase and cathepsin G may be involved in alternative TfR shedding in U937 cells.

We used a relatively small library of 5520 randomly generated single 15-mer peptides prepared by SPOT synthesis as an array of 28.5x19.0 cm to identify epitopes for three distinct monoclonal antibodies, namely anti-p24 (human immunodeficiency virus (HIV)-1) monoclonal anibody (mab) CB4-1, anti-interleukin-10 (IL-10) mab CB/RS/13, and anti-transforming growth factor alpha (TGFalpha) mab Tab2. Initially identified peptide ligands mostly had very low affinities for the antibodies with dissociation constants around 10(-4) M. Subsequent identification of residues critical for the antibody interactions involved complete L-amino acid substitutional analyses. Several substitutions resulted in analogs with dissociation constants in the low micromolar and high nanomolar range. Specifically binding peptides with key residue patterns matching the wild-type epitopes were identified for all three antibodies. In addition, for antibody CB4-1 mimotopes that showed no homology to the known epitope were selected. Our results suggest that a very limited library diversity, although far from covering the entire sequence repertoire, can suffice to rapidly and economically select peptidic antibody epitopes and mimotopes.

Since its invention the SPOT-synthesis methodology has become one of the most efficient strategies for the miniaturized assembly of large numbers of peptides. The combination of a facile synthetic method with high throughput solid- and solution-phase screening assays qualifies the SPOT-technique as a valuable tool in biomedical research. Recent developments such as the introduction of novel polymeric surfaces, new linker and cleavage strategies as well as automated robot systems extended the scope of practical chemical reactions that can be accommodated as well as the numbers of compounds obtainable by this technique. Thus, highly complex spatially addressed compound arrays have become accessible. Together with the introduction of novel screening assays, the method is excellently suited to elucidate recognition events on the molecular level.

Information about the epitopes of proteins recognized by antibodies or antibody fragments is important for their use as biological or diagnostic tools as well as for understanding molecular recognition events. Several techniques for mapping epitopes have been described, among them X-ray crystallography of antigen-antibody fragment complexes, a method leading to the most detailed results but usually being obtained with expensive and time consuming procedures (Davies 1996). For many purposes, information about the antigen-antibody interaction at the amino acid level, but not necessarily at the atomic level is needed. Protein digestion and chemical or enzymatic protein modifications in protein footprinting experiments, site-directed mutagenesis, binding assays with naturally occurring antigen variants such as species or tissue specific proteins or viral escape mutants and the screening of biological protein and peptide libraries are only some methods to map epitopes at the amino acid level (Morris 1996). Regarding the time required for the experiments and resolution of the data obtained, the use of synthetic peptides derived from the antigen sequence is a recommended strategy (Reineke 1999a).

Since its invention the SPOT-synthesis methodology has become one of the most efficient strategies for the miniaturized assembly of large numbers of peptides. The combination of a facile synthetic method with high throughput solid- and solution-phase screening assays qualifies the SPOT-technique as a valuable tool in biomedical research. Recent developments such as the introduction of novel polymeric surfaces, new linker and cleavage strategies as well as automated robot systems extended the scope of practical chemical reactions that can be accommodated as well as the numbers of compounds obtainable by this technique. Thus, highly complex spatially addressed compound arrays have become accessible. Together with the introduction of novel screening assays, the method is excellently suited to elucidate recognition events on the molecular level.

Combinatorial chemistry is well suited for the rapid and systematic optimization of molecular properties. The approach is commonly used for the generation of large numbers of compounds which can be applied for the examination of novel materials, catalysts or receptors. However, most frequently assembly of compound libaries is directed to the identification and optimization of novel therapeutic leads to accelerate the drug discovery process. The majority of combinatorial libraries assembled so far was synthesized on solid supports. The advantages of this method are the opportunity to use excesses of reagents driving reactions to completion and the fact that several technologies were invented to automatize and miniaturize these reactions. Beside high throughput parallel synthesis on resin beads, polymeric pins and chips [1], SPOT-synthesis using continuous membranes has been described to be an efficient synthetic approach [2]. The major feature of the latter method is the positionally addressed delivery of small volumes of liquids to a membrane surface. The droplets dispensed by a robot form separate spots which can be considered as micro-reactors provided that a non-volatile solvent system is used (Fig. 1).