WO2006049498A1

WO2006049498A1 - Means and methods for isolating and/or identifying a target molecule

Info

Publication number: WO2006049498A1
Application number: PCT/NL2005/000778
Authority: WO
Inventors: Jozef Maria Hendrik Raats; Daniëlle HOF
Original assignee: Modiquest B.V.
Priority date: 2004-11-05
Filing date: 2005-11-04
Publication date: 2006-05-11

Abstract

The invention provides a method for identifying a target molecule present in a first sample but not, or to a significantly lesser extent, present in a second sample, comprising: - providing a library of binding molecules; - incubating at least part of said library with at least a part of said second sample; - essentially removing at least one binding molecule of said library which is bound to a component of said second sample; - incubating at least part of the remaining library with at least a part of said first sample; and - isolating at least one binding molecule from said remaining library which is bound to a target molecule of said first sample; wherein said binding molecule is used for identifying said target molecule. Preferably, said target molecule is isolated from a sample by affinity purification with a binding molecule capable of selectively binding said target molecule. Alternatively, said binding molecule is used to screen said first sample in order to detect native targets. In one embodiment a phage display library is used.

Description

Title: Means and methods for isolating and/or identifying a target molecule

The invention relates to the field of biotechnology. More specifically, the invention relates to the field of proteomics.

Recombinant DNA technology has provided insight into mechanisms of many biological processes. The functioning and malfunctioning of various kinds of organisms has been investigated. Such investigations sometimes result in successful methods of diagnosis and/or treatment of disease. However, many processes are still not (fully) understood and many diseases are still not always cured. Therefore, a lot of investigation still needs to be done. Next to genomic or translational analyses, detailed proteomic analyses are extremely important for studying biological processes. Development of techniques to detect specific components that differ between two proteomes is invaluable for unravelling biological processes such as for instance cancer, inflammation, autoimmunity, and/or cellular differentiation.

In the current proteomics era, the sparse amount of tools to analyse proteomes and, even more important, differences between proteomes, is hampering the research in various important medical fields such as cancer, infection and autoimmunity. In order to obtain more insight, various methods employing phage display technology for generating monoclonal antibody fragments against cell-surface proteins that differ between two proteomes have been explored.

Since the introduction of phage display in 1985 the technology has been extensively used to obtain binding molecules against various antigens. Antibody phage display technology is based on the expression of a library of binding molecules, such as for instance antibodies, single chain antibodies, single domain antibodies, peptides or other binding scaffolds, on phage particles. These binding molecules are for instance derived from (immunised) animals, from humans suffering from disease or from healthy human or animal donors. Depending on the method of production and the source of the antibody repertoire, there are nowadays three main types of antibody phage display libraries; immune (immunised animal or human patient derived) libraries, naϊve (healthy donor derived) libraries, and synthetic or semi synthetic libraries derived of germline gene antibody sequences in which a repertoire of complemetarity determining (CDR) hypervariable synthetic regions is introduced.

The applicability of the various types of libraries varies. Because in vivo antibody affinity maturation has taken place in immunised animals or human patients, immune libraries contain a high percentage of high affinity antibodies directed to a limited amount of target antigens (immunogens). Therefore, libraries with a relatively small complexity are capable of generating high affinity binders for specific antigens. Usually, for each antigen or patient group studied, a separate library has to be constructed. Since, for obvious ethical reasons, active immunisation of humans is not possible, the generation of human antibodies to most targets is not feasible via this approach. The antibody repertoire present in naϊve and synthetic libraries did not evolve through antigen driven antibody maturation. Consequently, the complexity of these libraries has to be very high (>10¹⁰), to ensure a sufficient statistical chance that some of the antibodies in the libraries are capable of binding to any given target with reasonable or high affinity. The same is true for, for instance, synthetic libraries of peptides, other binding scaffolds, and/or single domain antibodies (or nanobodies). The advantage of naϊve and synthetic libraries is that they are suitable for use as a single pot library to obtain specific binding molecules to any given immunogenic, non- immunogenic, or toxic target.

The initial focus of interest of various methods in the art employing phage display technology was the identification of cell-surface markers specific for certain tumours, pathogens, cell- or tissue-types. Various whole-cell based selection approaches have been used successfully to identify such cell-surface markers (Cai and Garen, 1995; Cai and Garen, 1996; De Greeff et al., 2000; de Kruif et al., 1995; Ditzel et al., 2000; Hansen et al., 2001; Hegmans et al., 2002; Lekkerkerker and Logtenberg, 1999; Li et al., 2001; Noronha et al., 1998; Owens et al., 2000; Palmer et al., 1997; Shadidi and Sioud, 2001; Siegel et al., 1997; van der Vuurst de Vries and Logtenberg, 1999; van der Vuurst De Vries and Logtenberg, 1999; Van Ewijk et al., 1997; Zhou et al., 2002; and patent applications WO 01/48485 and WO 02/18948). These approaches are relatively straightforward when targets are abundantly expressed but become significantly more difficult when target concentrations are low.

Although the identification of cell-surface markers is important, cell surface components represent only a very small fraction of the proteomic complexity of a cell. To enable identification of several kinds of proteomic differences (including also other than cell-surface markers), another approach is required. Several methods to identify differences between proteomes have been tried with varying results. Analysis of two proteomes on silver stained two-dimensional gels sometimes revealed differentially occurring epitopes. Subsequent transfer onto PVDF membrane, excision of the area comprising the differentially occurring protein, and the use of these excised filter pieces in subsequent phage selections, allowed for selection of recombinant antibodies from synthetic phage display libraries specific for the protein targets contained on the excised filter parts (Liu et al., 2002; Liu and Marks, 2000; Nakamura et al., 2001). The drawback of this approach is that only abundant proteomic targets can be detected on 2D analysis. In another approach (Pini et al., 1998) biotinylated antigen was separated on two-dimensional gel, visualised by silver staining, eluted from the 2D gel, and subsequently used for panning in solution using streptavidin magnetic beads. Similar to the previous approach, the bottle neck here is the requirement for relatively large amounts of target, to enable the initial detection of the target spot on the 2D gel. Alternatively, instead of first identifying the differentially occurring proteomic targets on 2D gel before applying the phage display selection method, direct subtractive phage display selection methods have been developed to enable the selection of antibodies against differentially occurring proteomic targets being unknown beforehand. The use of complete cell extracts, derived from cells infected with similar but not identical virus strains coated to immuno-tubes, in a sequential subtractive selection method yielded virus strain specific antibodies from an immune library. An immune phage library was incubated with HSV-2 antigen-coated ELISA wells before being exposed to HSV-I antigen-coated wells. After this step, recovered phages were subjected to immunoaffinity enrichment against HSV-I antigens (Burioni et al., 1998). Recently, another example of a subtractive selection approach using complex proteomes in combination with a synthetic antibody phage display library was described (Stausbol-Gron et al., 2001). In this paper, a combination of subtraction selection, epitope masking (Sanna et al., 1995), and pre-elution (Bruggeman et al., 1995) was used for the identification of a cell type specific target. Although these selection methods were successful, they only succeeded in selecting antibodies against relatively abundant proteomic targets.

Yet another approach uses a combination of phage display selection on immobilised antigen or complex antigen mixtures such as cell extracts, followed by antibody arrays for high throughput screening of antigen-antibody interaction (de Wildt et al., 2000). This approach shows promising results towards the identification of multiple antibodies specific for multiple differentially occurring targets. The use of a trypsin sensitive helper phage KM 13 allows for a faster enrichment of phages expressing functional scFvs and a reduction of the background. Unfortunately, also this approach is currently only applicable for relative abundant proteomic targets, because the detection of the differentially occurring targets requires high concentrations of these target antigens. Hence, disadvantages of methods described in the art for identifying proteomic targets are the relative high amount of antigen required, possible epitope masking by a coating procedure and, since most selection methods described are based on immobilised antigens coated either on immunotubes or on nitrocellulose- or PVDF-membrabes, denaturation of protein antigens.

The present invention provides an alternative method for isolating and/or identifying a target molecule, such as for instance an antigen. In a method of the invention, at least one binding molecule, capable of specifically binding a target molecule, is obtained which binding molecule is used for isolation and/or identification of said target molecule. Said target molecule preferably comprises a marker and/or causative agent of disease. A method of the invention has the advantage that very low amounts of target molecules are detectable. Besides, several embodiments of the invention enable selection of binding molecules such as for instance (part of) antibodies, peptides or other binding scaffolds, that recognize the native state of a target molecule. A method of the invention is very suitable for isolation and characterization of antigenic determinants that differ between proteomes.

The invention provides a method for isolating and/or identifying a target molecule present in a first sample but not, or to a significantly lesser extent, present in a second sample, comprising: a) providing a library of binding molecules; b) incubating at least part of said library with at least part of said second sample; c) essentially removing at least one binding molecule of said library which is bound to a component of said second sample; d) incubating at least part of the remaining library with at least part of said first sample; and e) isolating at least one binding molecule from said remaining library capable of binding to a target molecule of said first sample; wherein said binding molecule is used for isolating and/or identifying said target molecule.

Surprisingly, with a method of the invention it is possible to detect small amounts of target molecules. Moreover, false positive results are, at least in part, avoided. A method of the invention is therefore very suitable for comparing proteomes from different cells and/or individuals, which often comprise very small amounts of a certain target molecule. A method of the invention is particularly suitable for detecting and/or identifying unknown target molecules. Most methods in the art are dedicated to a search for new and/or improved antibodies or functional parts thereof, or peptides or other binding scaffolds, directed against known antigens. Although a method of the present invention also provides this possibility, the present invention is especially suitable for detection, identification and use of new target molecules such as antigens. For instance, a method of the invention allows for isolation and/or identification of target molecules involved with disease, and use thereof as a diagnostic and/or therapeutic tool.

It is possible to carry out a method of the invention in various ways. For instance, several methods to provide a library of binding molecules are known in the art. As one option, a peptide library or other binding scaffold library is used. Preferably, however, a phage display library is generated comprising phages displaying a proteinaceous molecule such as an antibody, scFv, Fab fragment and/or single domain antibody at their surface. In yet another preferred embodiment, a ribosome display library is used. Any library of binding molecules is suitable as long as said binding molecules are at least in part available for binding during incubation with a sample. A (part of a) library is for instance incubated with a (part of a) sample by administration of (part of) said sample to a batch comprising (part of) said library. Preferably, care is taken to retain molecules of said sample, such as for instance proteinaceous molecules, in their native state. For instance, protein denaturing conditions are preferably avoided. Step c enriches said library for binding molecules capable of binding a target molecule present in said first sample but not, or to a significantly lesser extent, in said second sample. Preferably, said binding molecules are capable of specifically binding a target molecule present in said first sample but not, or to a significantly lesser extent, in said second sample. A binding molecule capable of (specifically) binding a component of said second sample is essentially removed. This diminishes false positive end-results. By essentially removing at least one binding molecule of said library which is bound to a component of said second sample is meant herein that said binding molecule or binding molecule-second sample component complex is separated from the incubation mixture. Said binding molecule/complex is either removed from the incubation mixture, or immobilized or bound to another component to such extent that said binding molecule/complex is less capable of reacting with at least part of said first sample in step d). Preferably, after separation, the remaining incubation mixture does not comprise a detectable level of (free) binding molecule -second sample component complexes.

A part of a sample is defined herein as a part comprising at least one component of said sample. Preferably, said component is capable of specifically binding a binding molecule of said library. A part of said first sample and/or a part of said second sample is preferably a representative part. In terms of the invention, a representative part is defined as a part which is indicative for said sample. Preferably, said representative part comprises essentially the same contents as said sample in kind, though not necessarily in amount. A representative part of said first sample is for instance obtained by stirring said sample such that the contents are equally divided within said sample and meanwhile, or directly afterwards, pipetting a certain volume. Binding molecule -second sample component complexes are for instance removed by an affinity column capable of binding said second sample component. However, many alternative methods are known in the art, such as (capture) ELISA and binding of streptavidin magnetic beads to said second sample component. In the latter case, said component is preferably biotinylated before a complex comprising said component is removed from the incubation mixture.

In step e, a binding molecule bound to a target molecule of said first sample is isolated, for instance using the above mentioned methods such as an affinity column and/or (capture) ELISA. In one embodiment, said first sample is biotinylated before incubation with the remaining (enriched) library. Binding molecule— target molecule complexes are isolated with streptavidin magnetic beads after incubation of said first sample with said remaining library. In one embodiment, steps b through e are at least once repeated. Preferably, steps b through e are at least two times repeated. More preferably, steps b through e are at least three times repeated. If steps b through e are repeated several times, false-positive results are avoided.

Once a binding molecule of the invention, capable of specifically binding a target molecule, has been isolated, it is used for identifying said target molecule. This is for instance performed by incubating a sample with said binding molecule and subsequently isolating and analysing bound target molecule.

In one aspect of the invention a method of the invention further comprises the following steps: f) separating at least two components of said first sample; g) incubating at least one of said components with at least one binding molecule obtained in step e); h) detecting a target molecule bound by said binding molecule; and i) identifying said target molecule.

In step f, at least two components of said first sample are separated. Of course, preferably most components of interest, and/or essentially all components, are separated in order to enhance the chance of identifying an unknown target molecule. Step f is preferably performed with another batch of the same kind of sample that was used in step d. In one embodiment, said components comprise proteinaceous molecules. Proteinaceous molecules are for instance separated by means of gel electrophoresis. In one embodiment, one- dimensional gel electrophoresis is applied. Preferably however, two- dimensional electrophoresis is applied. In one embodiment, separated proteinaceous molecules are subsequently transferred to a membrane, such as a nitrocellulose membrane. Preferably, however, a poly(vinyliden fluoride) membrane is used, because such membrane significantly reduces background binding of proteinaceous molecules and phages.

Alternatively, other methods for separating (proteinaceous) molecules, known in the art, are applied. One example of an alternative method for separating potential target molecules, known in the art, comprises for instance chromatography. Subsequently, separated components are preferably incubated with at least one binding molecule obtained in step e. Preferably, most or essentially all of the binding molecules obtained in step e are used. If a phage library is used, it is preferred to culture the phages that are obtained in step e before step g is performed, so that a higher amount of phages capable of specifically binding a target molecule of said first sample is obtained. Several (monoclonal) cultures are for instance used. In one embodiment, steps b through e are at least once repeated, while the phages that are obtained in step e of each round are cultured. Preferably, steps b through e are at least two times repeated. More preferably, steps b through e are at least three times repeated. If steps b through e are repeated several times, false-positive results are avoided. In one aspect of the invention, phages comprising said binding molecules at their surface are used to incubate said separated components. In another aspect, binding molecules are used. Said binding molecules are preferably soluble. Binding molecules are for instance obtained by infecting bacteria with a phage capable of inducing said bacteria to produce said binding molecules. Alternatively, binding molecules are derived from a library of binding molecules and isolated in step e).

Phages and/or binding molecules are in one embodiment administered to a gel or membrane comprising separated (proteinaceous) molecules from sample 1. As a control, another gel or membrane comprising separated (proteinaceous) molecules from sample 2 is preferably incubated with said phages or binding molecules as well. After incubation with said phages or binding molecules, it is preferably determined to which (proteinaceous) molecule(s) said phages and/or binding molecules have specifically bound. This is for instance done with labelled anti-phage antibodies. In one embodiment TMB staining or chemiluminescence is applied. In case said phages or binding molecules specifically bind to a (proteinaceous) molecule present on a gel or membrane containing sample 1 components, which (proteinaceous) molecule is not present on a gel or membrane containing sample 2 components, said

(proteinaceous) molecule is considered a target molecule of the invention. In this embodiment of the invention, a target molecule of the invention is detected and identified in step h and i. In this embodiment, separated components of sample 1 (and, preferably, sample 2) are incubated with at least one binding molecule obtained in step e. After this, bound target molecules are identified. This is for instance done by removing bound binding molecules from the target molecules, subsequently isolating said target molecules and/or identifying them, for instance by mass spectrometric identification. If components of sample 1 (and, preferably, sample 2) are separated by gel electrophoresis, a target molecule is preferably isolated from the gel. Alternatively, said target molecule is transferred to a membrane before isolation. Said membrane preferably comprises a poly(vinyliden fluoride) membrane ("PVDF membrane"). Since in this embodiment a target molecule is isolated from the membrane and subsequently used for identification, it is especially important that background binding of binding molecules and/or phages is avoided. Other membranes, such as nitrocellulose membranes, involve many non-specifically bound molecules. For instance, phage particles often bind non-specifically. A PVDF membrane, however, is resistant to protein binding in aqueous solutions and contaminations are therefore significantly reduced.

In another aspect of the invention a method of the invention further comprises the following steps: f) separating at least two components of said first sample; g) incubating at least one of said components with at least one binding molecule obtained in step e); h) detecting at least one binding molecule bound to a target molecule; i) providing said at least one binding molecule; j) at least in part isolating said target molecule from a sample with help of said binding molecule; and k) identifying said target molecule.

Steps f) - h) have been outlined above. In step i), a binding molecule capable of binding a target molecule is obtained. In case of phages and/or binding molecules bound to a target molecule on a gel or membrane, gel or membrane spots are preferably excised and bound phages and/or binding molecules are eluted, for instance with TEA. In a preferred embodiment eluted phages are cultured again, for instance by infecting Escherichia coli. Monoclonal phages are subsequently used in this embodiment to screen another (part of a) gel or membrane containing (proteinaceous) molecules from sample 1 and, preferably, another (part of a) gel or membrane containing (proteinaceous) molecules from sample 2.

After a binding molecule capable of (specifically) binding a target molecule has been identified, it is used for isolating and/or identifying said target molecule from a sample. In one embodiment, said binding molecule is at least in part isolated and incubated with a sample comprising said target molecule. Preferably, said first sample, or the same kind of sample, is used, although this is not necessary. For instance, a sample is used that is obtained from the same individual that provided said first sample. In the art, many methods are known for isolating a target molecule once a binding molecule specific for said target molecule is known. For instance, affinity purification, a (capture) ELISA assay and/or capture on a microarray and/or a microchip are suitable. Once a target molecule has been isolated, it is purified and/or identified using common methods in the art. For instance, said target molecule is identified by mass spectrometric identification.

Of course, it is recognized by a person skilled in the art that a target molecule is also detected, obtained and identified with alternative embodiments, which are within the scope of the present invention.

In a preferred embodiment a binding molecule capable of (specifically) binding a target molecule is used for isolating and/or identifying a target molecule from a sample, wherein said target molecule has been subjected to gel electrophoresis. In this embodiment a method of the invention is carried out as follows: at least two components of said first sample are separated in step f), preferably by gel electrophoresis, after which said separated components are incubated with at least one binding molecule obtained in step e). Subsequently, a bound binding molecule is detected and provided. Said binding molecule is for instance provided by isolating said binding molecule, or by (artificially) generating the same kind of binding molecules. Then, at least two components from said first sample, or from a sample comparable to said first sample (for instance a sample obtained from the same individual who provided said first sample) are separated by gel electrophoresis. Preferably, ID or 2D gel electrophoresis is applied. Said binding molecule is subsequently incubated with a gel, or membrane, comprising said separated components. Binding molecules specifically bound to a target molecule of sample 1 are subsequently used for isolation and/or identification of said target molecule.

Figures 6A - 6F schematically depict a preferred embodiment of the present invention.

In yet another aspect of the invention a method of the invention further comprises the following steps: f) amplifying said binding molecule; g) incubating at least one amplified binding molecule with at least a representative part of said first sample and at least a representative part of said second sample; h) obtaining a binding molecule capable of binding a target molecule present in a first sample but not, or to a significantly lesser extent, present in a second sample; i) at least in part isolating said target molecule from a sample with help of said binding molecule; and j) identifying said target molecule.

This embodiment of the present invention is particularly suitable for identifying native targets. In this embodiment, a binding molecule obtained in step e is amplified. This is preferably done by culturing phages comprising said binding molecule or culturing bacteria producing said binding molecule, preferably a soluble binding molecule. Subsequently, at least one amplified binding molecule, preferably free, soluble and/or phage-bound, is incubated with at least a part, preferably a representative part, of said first sample and at least a part, preferably a representative part, of said second sample. This is for instance performed by coating biotinylated or non-biotinylated extracts of said first and, preferably, said second sample to microtiter wells or to a membrane and subsequently incubating said wells or membrane with said at least one amplified binding molecule. If said extract is biotinylated, said wells preferably contain streptavidin and/or said membrane is preferably coated with streptavidin. After incubation, first sample -containing wells and/or a (part of a) membrane comprising an extract of said first sample are compared with second sample -containing wells and/or a (part of a) membrane comprising an extract of said second sample in order to estimate whether a binding molecule is capable of binding a target molecule present in a first sample but not, or to a significantly lesser extent, present in a second sample. Such binding molecule is isolated and used for at least in part isolating and/or identifying said target molecule from a sample, for instance by affinity purification or (capture) ELISA, as described above. A target molecule is preferably purified and/or identified using known methods in the art, for instance mass spectrometric identification.

Instead of microtiter wells or a membrane it is also possible to use lipid vesicles comprising membrane-bound molecules, such as for instance proteins or functional parts, derivatives and/or analogues thereof. Such vesicles are for instance derived from different populations of cells, bacteria, parasites, etc. A distinction is for instance made between vesicles comprising cell membranes and vesicles comprising membranes derived from membrane -containing organelles such as mitochondria, nuclear envelope and endoplasmatic reticulum. In one embodiment said vesicles are incubated with (amplified) binding molecules obtained in step e. This way binding molecules capable of binding a membrane-bound target molecule are obtained in order to isolate said target molecule. Such target molecule for instance comprises a surface marker. After a binding molecule has been obtained that is capable of specifically binding a certain target molecule, said target molecule is isolated from said vesicles with help of said binding molecule. Alternatively, said target molecule is isolated from another sample using said binding molecule.

It is possible to distinguish vesicles derived from different cell types and/or organelle types with different markers. In one embodiment, said markers are capable of binding a cell type-specific or organelle type-specific membrane-bound protein or functional part thereof. In another embodiment, each kind of vesicle is provided with a specific kind of antigen against which a known binding molecule is available. In yet another aspect of the invention, said vesicles are provided with biotinylated proteins or functional parts, derivatives and/or analogues thereof. After incubation of said vesicles with candidate binding molecules, vesicles from different cell types and/or organelle types are separated by affinity purification using the above-mentioned known binding molecules, or using magnetic beads if said vesicles are provided with biotinylated proteins or functional parts, derivatives and/or analogues thereof. In one embodiment vesicles are provided with fluorochromes that can be detected and isolated in a FACS device.

With a method of the invention it is possible to screen high amounts of potential target molecule -binding molecule complexes in a high throughput screening assay. For instance, 1000 — 100000 potential binding molecules are screened in one run. This allows fast and sensitive assays for obtaining binding molecules that are suitable for comparing proteomes, and/or detecting unknown antigens.

A method of the invention is particularly suitable for comparing different proteomes. Since proteomes of different cells/individuals often comprise many proteins in common, many binding molecules of said library which are capable of binding proteins both present in sample 1 and sample 2 are removed in step c. Preferably, steps b) through e) are at least once repeated with the at least one proteinaceous molecule obtained in step e). With several selection rounds, the sensitivity and specificity of a method of the invention is improved.

In a preferred embodiment, step d) is performed in the presence of at least one component of said second sample. In such competition reaction competitor molecules of sample 2 are capable of binding to binding molecules of said library, which are capable of binding components both present in said first and second sample. Such binding molecule — sample 2 component complexes are subsequently removed.

Preferably, said library comprises a phage display library. A phage display library allows for very efficient amplification of a binding molecule present in said library. Once a binding molecule is identified which is capable of specifically binding a certain target molecule, the phage comprising said binding molecule is preferably cultured. This allows amplification of said binding molecule to high levels. In one embodiment, at least one of said binding molecules comprises an antibody, a single domain antibody, a single chain antibody and/or a FAB fragment, or a functional part, derivative and/or analogue thereof, because they are well capable of binding a proteinaceous molecule of a proteome, such as an antigen.

With a functional part of an antibody, a single domain antibody, a single chain antibody and/or a FAB fragment is meant a part which has essentially the same properties in kind, not necessarily in amount. Such functional part is for instance also capable of specifically binding an antigen of said antibody, single domain antibody, single chain antibody and/or a FAB fragment. For instance, such functional part comprises a CDR domain. A functional derivative of an antibody, a single domain antibody, a single chain antibody and/or a FAB fragment is defined as a molecule which has been altered such that the properties of said molecule are essentially the same in kind, not necessarily in amount. A derivative can be provided in many ways, for instance through conservative amino acid substitution. An analogous compound of an antibody, a single domain antibody, a single chain antibody and/or a FAB fragment is for instance generated through screening of a peptide library. Such an analogue has essentially the same properties of said antibody, single domain antibody, single chain antibody and/or FAB fragment in kind, not necessarily in amount. In one embodiment, a hyperphage or complete phage system is used. An hyperphage or complete phage system has an enhanced display level of binding molecules at the surface of the phages as compared to conventional phage display libraries. Said enhanced display level enhances the avidity of the phages.

A target molecule is any molecule of interest which is in principle capable of binding to the kind of binding molecules present in said library. Preferably, said target molecule comprises an antigen. Screening samples from different cells and/or individuals, said samples preferably comprising at least a representative part of the proteomes of said cells and/or individuals, for the presence of differentially occurring antigens is a useful tool for detecting a diagnostic marker and/or causative agent of a disease. For instance, a proteome of a healthy individual is compared with a proteome of a diseased individual to investigate whether said diseased individual comprises a target molecule not normally present in healthy individuals. Such target molecule is a diagnostic marker, and/or allows for further investigating the cause of such disease and/or development of therapeutic and/or prophylactic applications.

Any type of phage display libraries, such as immunised animal, human naϊve or synthetic, autoimmune patient phage antibody display libraries, or peptide phage display libraries is suitable for use in a method of the invention. Several factors, such as subtraction efficacy and affinity of the binders (for instance antibodies or peptides) determine the sensitivity of the method. An improvement of the subtraction efficacy is obtained using various additional steps involving for example epitope masking (Sanna et al., 1995) and pre- elution (Bruggeman et al., 1995) techniques. An increase in the number of high affinity binders is for instance achieved by using either very large high quality naϊve or synthetic libraries, or libraries that contain in vivo affinity matured antibodies (e.g. (autoimmune-, pathogen infected-, or cancer-) patient or immunised animal derived phage display libraries). In the case of immune (immunised animal, patient) libraries a strong bias towards certain immunogenic components in the immunisation cocktail occurs which is less suitable for the selection of general differential marker antibodies. Although the use of large high quality naϊve or synthetic libraries, eventually in combination with additional epitope masking and pre-elution methods, is a more generally applicable method to identify differences between any two given proteomes, the use of patient derived libraries yields differentially occurring targets that have a direct link to the original patients disease, such as autoimmune disease. Preferably, high display levels are used, thereby increasing the avidity of binding phages as compared to phage systems and phagemid systems. High display levels enhance the applicability of phage display libraries (especially the naϊve and synthetic) for detecting low abundant differentially displayed targets (O'Connell et al., 2002). Alternatively, display levels on phage are increased using the hyperphage system, known in the art.

In one preferred embodiment a method of the invention is provided wherein said phage display library comprises an immune library. Autoimmune diseases pose a significant problem for the world population (1-2% of the population is affected), by causing physical disablement for many individuals as well as high costs for communities confronted with these diseases. Autoimmune diseases are considered acquired diseases, and are often characterised by the development of autoantibodies to intracellular antigens, important for biosynthetic functions such as transcription, precursor mRNA splicing, DNA replication, and/or protein synthesis. Although several hypotheses have been put forward to explain why autoimmune reactions are preferentially targeted to cellular components participating in such important biological functions, at the present time the aetiology of autoimmunity is still unclear. One hypothesis states that during abnormal apoptotic events the (apoptotically-modified) antigens become exposed to the immune system, leading to a primary immune response. This response can, in time and in genetically susceptible individuals, evolve via epitope spreading, into a complex full-blown autoimmune response. An increasing number of papers support the above described hypothesis. One paper describes a mouse model in which a defect in a gene involved in apoptosis led to a phenotype similar to systemic lupus erythematosus (SLE). Other recent papers suggest a link between defects or abnormalities in apoptotic pathways and autoimmune diseases. It has also been shown that autoantigens are grouped together in different apoptotic blebs in apoptotic cells. There is accumulating evidence that apoptotic (or necrotic) autoantigen modifications do occur. Within the group of the present inventors, several in vivo apoptotic modifications of autoantigens could be demonstrated (dephosphorylation and partial cleavage of La (SSB) protein, degradation of the cytoplasmic Y-RNAs, cleavage of the Ul-RNA, dephosphorylation of ribosomal P proteins). Very interesting in this respect is our observation that an apoptotically modified amino acid is an essential determinant of epitopes frequently recognised by sera from Rheumatoid Arthritis patients. For an extensive overview see (Rodenburg et al., 2000).

Papers by Casciola-Rosen, and Andrade and co-workers (Andrade et al., 1998; Casciola-Rosen et al., 1999), indicate that Granzyme B induced apoptosis might be of particular interest for autoimmunity. Several autoantigens that are substrates for Caspases are also cleaved by Granzyme B. Actually, it has been proposed that cleavage by Granzyme B can predict autoantigenic status of an antigen, because non-autoantigenic proteins (except for Caspases) do not seem to be cleaved by Granzyme B. Even more important in this respect is the fact that, although Granzyme B is a protease related to Caspases, its substrate specificity is different, thereby giving rise to unique proteolytic fragments not generated by any other protease. Granzyme B is present in the cytoplasmic granules of cytotoxic T lymphocytes (CTLs) that kill malignant or virus infected cells and that are involved in the immune responses that give rise to graft rejection. Thus CTL induced apoptosis generates both Caspase and Granzyme B specific proteolytic fragments, either of which might induce an autoimmune response.

Since the above described hypothesis states that (apoptotic) modifications of antigens are the triggering events that initiate autoimmunity, autoantibodies recognising such (apoptotically) modified antigenic determinants would, according to this hypothesis, be the first antibodies to appear in individuals developing an autoimmune disease. It is extremely important to identify and analyse such early autoantigens in autoimmune patients. Detailed characterisation of early autoantigenic determinants lead to improved serological tests. The availability of a test using a method of the present invention for detection of autoantibodies in a very early stage of a disease will be important and helpful for designing earlier and better treatments for patients, thereby at least in part avoiding or delaying serious physical disablement of individuals developing an autoimmune disease. Hence, in one preferred embodiment a method of the invention is provided wherein said phage display library comprises an immune library.

In another embodiment, said first and/or second sample comprises a cell-free solution and/or a purified cell extract. This way target molecules normally present within cells are optimally available for said library of binding molecules. By a cell-free solution is meant a solution wherein the structure of more than 50% of the cells has been disrupted. Preferably, the structure of more than 70% of the cells has been disrupted. More preferably, the structure of more than 80% of the cells has been disrupted. More preferably, the structure of more than 90% of the cells has been disrupted. Most preferably, the structure of essentially all cells has been disrupted. In a preferred embodiment, also the structure of essentially all organelles have been disrupted, so that target molecules are optimally available. Most preferably a cell-free solution essentially comprises no intact cells. A purified cell extract is defined herein as an extract comprising components of a cell which are essentially not present in their natural cellular environment. For instance, such extract preferably comprises cell-derived proteins in solution.

In one embodiment a method of the invention is provided wherein said second sample comprises biotinylated components in step b, and/or wherein said first sample comprises biotinylated components in step d. As has been explained above, biotinylated components are readily removed and, optionally, isolated from a sample using streptavidin magnetic beads. Hence, a method of the invention is provided wherein removing said binding molecule in step c) comprises binding of streptavidin magnetic beads to a biotinylated component - binding molecule complex. A method of the invention wherein isolating said binding molecule in step e) comprises binding of streptavidin magnetic beads to a biotinylated antigen - binding molecule complex is also herewith provided. If a competition reaction is performed comprising incubating a library of binding molecules with said first sample in the presence of at least one component of said second sample, it is preferred that said component of said second sample is essentially not biotinylated. This enables direct isolation, with streptavidin magnetic beads, of binding molecules bound to target molecules of said first sample with little - if at all - contaminating complexes comprising a component of said second sample.

Target molecules are obtained by using a binding molecule capable of binding said target molecule for isolation of target molecules from a sample. This is for instance performed by using such binding molecule for affinity purification. One embodiment therefore provides a method of the invention further comprising:

- isolating said bound binding molecule detected in step h;

- culturing phages comprising said binding molecule; - producing said binding molecules; and

- isolating said target molecules by way of affinity purification using said binding molecules.

In one embodiment, affinity purification is performed with phages comprising said binding molecule. In another embodiment, free and/or soluble binding molecules such as antibodies, single domain antibodies, scFvs, FAB fragments or functional parts, derivatives and/or analogues thereof are used. Preferably, said free and/or soluble binding molecules are produced by bacteria infected with a phage that was detected in step h.

In one preferred embodiment, a method of the invention comprises initial subtraction selection against biotinylated competitor antigens of sample 2. In this embodiment, competitor-antigen binders, which form part of a phage display library, are removed by streptavidin magnetic beads. Remaining phages are then panned against biotinylated target antigens, preferably in the presence of excess of non-bio tiny late d competitor-antigen. Preferably, about 500 times excess of non-biotinylated competitor-antigen is present. After each selection round, polyclonal phage-pools are preferably differentially screened on native or (partially) denatured competitor and target antigens. In this embodiment, differential native antigen screening of individual clones (phages or for instance scFv) is for instance performed on captured biotinylated competitor and target antigens (e.g. in a high throughput ELISA format (applying a colony picking robot)) using streptavidin coated plates on which the biotinylated antigens are captured, in an array format as described by (de Wildt et al., 2000) where the detection filter carries biotinylated antigen captured on streptavidin, and/or in a protein chip format where individual binding molecules are spotted on a chip (e.g. via metal- His or anti tag or anti- phage binding) and binding to labeled competitor and target antigens is detected using differently labelled competitor and target antigens.

In yet another embodiment, differential denatured antigen screening is performed using western blots of two-dimensional IEF/SDS-PAGE containing competitor and target antigens. After incubation with, and detection of binding (polyclonal) phages, differentially occurring spots, containing their cognate (phage) binding molecules, are excised accurately and phages are eluted from the membrane spots and used to infect bacteria. Individual colonies are subsequently analysed in this embodiment by fingerprinting and clones displaying different fingerprints are screened as monoclonal phage antibodies, or functional parts, derivatives and/or analogues thereof, for binding to differentially occurring targets. Differential screening of monoclonal phages for instance occurs on biotinylated or non biotinylated native competitor and target antigens captured in (streptavidin) ELISA plates, or on (strep tavidin) PVDF or nitrocellulose filters, and/or on ID or 2D western blots containing denatured competitor and target antigens. Subsequently, cDNAs encoding antibodies that recognise differentially occurring epitopes are preferably cloned into a vector containing a label such as a strep-tag sequence (Skerra and Schmidt, 20000), allowing an efficient and low background-affinity purification of said target molecule via a streptavidin column. Finally, said target molecule is purified in this embodiment via PAGE, preferably 2 dimensional PAGE, and identified by mass spectrometry.

Of course, as will be recognized by the person skilled in the art, many steps of this preferred embodiment can be carried out in alternative ways.

An isolated target molecule obtainable by a method of the invention is useful for diagnosis. A target molecule known to be present in a proteome of a diseased individual, and known to be essentially absent in healthy individuals, is useful as a diagnostic marker for such disease. Individuals are preferably screened for the presence of such diagnostic marker. The presence of such diagnostic marker is indicative of (a risk of) a disease. Likewise, it is possible to identify diagnostic markers for activated versus non-activated T-cells with a method of the present invention, by comparing the proteomes of activated and non-activated T-cells. Alternatively, a proteome of a cell before a certain treatment is compared with a proteome of a cell after such treatment. This way, the effect of a treatment upon a certain disease, or possible side-effects, is monitored by detecting whether an increase, or decrease, of a certain target molecule occurs. The invention therefore also provides an isolated target molecule obtainable by a method of the invention.

An isolated target molecule of the invention is useful as a diagnostic marker of a disease. Said target molecule is also useful as a marker for a specific tissue or cell type, or as a marker for healthy versus diseased tissue. With a method of the invention a target molecule is identified that is present in an aberrant cell but less, preferably not (or below detection level), present in a healthy cell. Subsequently, a sample is screened for the presence of such target molecule. If said target molecule appears to be present, it is indicative of the presence of at least one aberrant cell. This way, the presence of for instance a tumour cell is determined. Alternatively, a target molecule that is specific for certain strains of parasites, bacteria and/or viruses is identified with a method of the invention. Subsequently, such strains are detected in a sample by determining whether said strain-specific target molecule is present. This is useful for diagnosis of an infection, and/or for identifying a pathologic organism. An isolated and/or identified target molecule of the invention is furthermore suitable for developing therapeutic means and methods for at least in part preventing and/or counteracting a disease and/or infection associated with the presence of said target molecule. For instance, said target molecule is used for (passive) immunisation. Alternatively, preferably in case of an autoimmune disease, a molecule capable of at least in part inhibiting the presence and/or action of antibodies specifically directed against a target molecule of the invention is used for therapeutic and/or prophylactic purposes.

Once a target molecule of the invention and/or a binding molecule capable of specifically binding said target molecule has been identified, it is possible to generate an anti-idiotype antibody or functional part, derivative and/or analogue thereof. Such anti-idiotype antibody or functional part, derivative and/or analogue thereof is also herewith provided. An anti-idiotype antibody is an antibody specifically directed against at least part of an antigen-specific part of a sequence of an antibody and/or T cell receptor. Anti¬ idiotype antibodies mostly have a structure that is comparable to an antigen of said antibody and/or T-cell receptor. Said antigen is referred to as a related antigen of said anti-idiotype antibody. Anti-idiotype antibodies are for instance generated using DNA encoding a binding molecule capable of specifically binding said related antigen. Anti-idiotype antibodies are useful for diagnosis. It is for instance determined whether a sample comprises antibodies capable of specifically binding said anti-idiotype antibody. If a sample from an individual appears to comprise antibodies against such anti-idiotype antibody, it indicates the presence of a related antigen in said individual. This for instance implies that said individual is suffering from, or at risk of suffering from, a disease involving the presence of said antigen, such as an autoimmune disease. The presence of such related antigen alternatively implies an infection, the presence of a tumor, etcetera.

An anti-idiotype antibody is also suitable for vaccination purposes. For instance, an individual is vaccinated with anti-idiotype antibodies mimicking a parasite, bacterial and/or viral antigen. Vaccination provides protection against such organisms, since an immune response against a related antigen is enhanced by administration of said anti-idiotype antibody. In yet another aspect of the invention, an anti-idiotype antibody is used for treatment of a disease and/or infection. Administration of said anti-idiotype antibody evokes and/or enhances an immune response of the host against a certain disease or pathogen.

If anti-idiotype antibodies are used, it is not necessary anymore to purify and/or identify the related antigen. This saves laborious, time consuming and costly purification and/or isolation procedures. The use of anti-idiotype antibodies, or functional parts, derivatives and/or analogues thereof, is therefore especially preferred if a certain target molecule is not easily isolated and/or identified. This is for instance the case if such target molecule is very unstable.

Additionally, the invention provides a method for identifying a diagnostic marker for a disease, comprising:

- identifying a target molecule which is present in a proteome of an individual suffering from said disease, but which is not or to a significantly lesser extent present in a proteome of an individual not suffering from said disease, by a method of the invention.

Preferably said diagnostic marker is subsequently obtained using well known methods in the art.

A use of a diagnostic marker obtainable by a method of the invention for diagnosis of a disease is also herewith provided. The invention provides new markers for autoimmune disease. This is shown in the examples. According to the present invention, polypyrimidine tract binding protein-associated splicing factor (PSF) and nuclear RNA and DNA binding protein p54^nrb are markers for autoimmune disease. One embodiment of the invention therefore provides a use of polypyrimidine tract binding protein-associated splicing factor (PSF) and/or nuclear RNA and DNA binding protein p54^nrb, or a functional part, derivative and/or analogue thereof, or a modified form thereof such as for instance a posttranslationally modified form thereof, for diagnosis of autoimmune disease. According to the invention, the markers polypyrimidine tract binding protein-associated splicing factor (PSF), nuclear RNA and DNA binding protein p54^nrb, heterogeneous nuclear ribonucleoprotein Cl and heterogeneous nuclear ribonucleoprotein C2 are indicative for systemic lupus erythematosus, systemic sclerosis and/or rheumatoid arthritis. The invention therefore furthermore provides a use of polypyrimidine tract binding protein- associated splicing factor (PSF), nuclear RNA and DNA binding protein p54^m"^b, heterogeneous nuclear ribonucleoprotein Cl and/or heterogeneous nuclear ribonucleoprotein C2, or a functional part, derivative and/or analogue thereof, or a modified form thereof such as for instance a post-translationally modified form, for diagnosis of systemic lupus erythematosus, systemic sclerosis and/or rheumatoid arthritis.

In yet another aspect the invention provides a method for diagnosing whether an individual is suffering from, or is at risk of suffering from, a disease comprising:

- determining whether a sample of said individual comprises a diagnostic marker identifiable by a method according to the invention, or a modified form thereof, such as for instance a post-translationally modified form thereof.

The invention also provides a method for diagnosing whether an individual is suffering from, or is at risk of suffering from, a disease comprising: - determining whether a sample of said individual comprises a binding molecule capable of specifically binding a diagnostic marker identifiable by a method according to the invention, and/or a modified form thereof, such as for instance a post-translationally modified form thereof.

Now that the invention has provided new markers for autoimmune disease, they are used in a method for diagnosing disease. The invention thus provides a method for diagnosing whether an individual is suffering from, or at risk of suffering from, an autoimmune disease, comprising:

- determining whether a sample of said individual comprises polypyrimidine tract binding protein-associated splicing factor (PSF), nuclear RNA and DNA binding protein p54^nrb, heterogeneous nuclear ribonucleoprotein Cl and/or heterogeneous nuclear ribonucleoprotein C2, or a functional part, derivative and/or analogue thereof, or a modified form thereof such as for instance a post- translationally modified form thereof. A preferred embodiment provides a method of the invention wherein the presence of a modified form of a marker, preferably a marker which has been posttranslationally modified, in a sample is investigated.

A preferred embodiment of the invention provides a method for diagnosing whether an individual is suffering from, or at risk of suffering from, an autoimmune disease, comprising: - determining whether a sample of said individual comprises a binding molecule capable of specifically binding polypyrimidine tract binding protein- associated splicing factor (PSF), nuclear RNA and DNA binding protein p54^nrb, heterogeneous nuclear ribonucleoprotein Cl and/or heterogeneous nuclear ribonucleoprotein C2, or a functional part, derivative and/or analogue thereof or a modified form thereof, such as for instance a post-translationally modified form thereof. Said binding molecule preferably comprises an antibody. The presence of antibodies specifically directed against a marker, which marker is not, or to a significantly lesser extent, present in a healthy individual, is indicative for disease. Moreover, the presence of antibodies specifically directed against a modified form of a marker, which modified form is not, or to a significantly lesser extent, present in a healthy individual, is particularly indicative for disease, especially autoimmune disease.

Preferably, said autoimmune disease comprises systemic lupus erythematosus, systemic sclerosis and/or rheumatoid arthritis. Said sample preferably comprises a body fluid. More preferably, said sample comprises blood, serum, plasma, liquor and/or synovial fluid.

Now that markers involved with autoimmune disease have been provided by the present invention, it has become possible to at least in part prevent and/or treat said autoimmune disease. This is possible by at least in part counteracting said markers. Preferably however, antibodies capable of specifically binding said markers are counteracted. The invention thus provides a method for at least in part treating an autoimmune disease comprising counteracting the presence and/or activity of polypyrimidine tract binding protein-associated splicing factor (PSF), nuclear RNA and DNA binding protein p54^nrb, heterogeneous nuclear ribonucleoprotein Cl and/or heterogeneous nuclear ribonucleoprotein C2, or a functional part, derivative and/or analogue thereof, or a modified form thereof, such as for instance a post-translationally modified form. A preferred embodiment provides a method for at least in part treating an autoimmune disease comprising counteracting the presence and/or activity of an antibody capable of specifically binding polypyrimidine tract binding protein-associated splicing factor (PSF), nuclear RNA and DNA binding protein p54^nrb, heterogeneous nuclear ribonucleoprotein Cl and/or heterogeneous nuclear ribonucleoprotein C2, or a functional part, derivative and/or analogue thereof, or a modified form thereof, such as for instance a post-translationally modified form. Said autoimmune disease preferably comprises systemic lupus erythematosus, systemic sclerosis and/or rheumatoid arthritis.

In one embodiment, a method or a use of the invention is provided, wherein said disease comprises an infectious disease (preferably malaria).

In case of malaria, diagnostics is designed based on differentially expressed target molecules present at the various stages of the parasite infection. Such differentially expressed target molecules are identified by comparing proteomes of samples derived at various stages of infection with each other, and/or with a proteome of a sample from a non-infected individual. Subsequently, it is determined whether a sample from an individual comprises such target molecule. This way it is not only possible to detect the occurrence of an infection, but also to determine which stage of infection is involved. This facilitates an efficient treatment of an individual. In addition, infection is, at least partly, avoided by using anti-idiotypic antibodies, as described above, as a vaccine and/or a medicament. An individual is preferably provided with anti-idiotypic antibodies that mimic an original epitope of a parasite. Preferably, anti-idiotype antibodies mimicking a differentially expressed target molecule of a specific stage of infection are provided.

The invention also provides a kit for performing a method according to the invention. Such kit comprises suitable means for performing at least one step of a method of the invention. A person skilled in the art is well capable of determining which means are suitable for each individual step of a method of the invention. A diagnostic kit comprising a diagnostic marker obtainable by a method of the invention is also provided herewith. A preferred embodiment of the invention provides a diagnostic kit comprising polypyrimidine tract binding protein-associated splicing factor (PSF), nuclear RNA and DNA binding protein p54^nrb, heterogeneous nuclear ribonucleoprotein Cl and/or heterogeneous nuclear ribonucleoprotein C2, or a functional part, derivative and/or analogue thereof, or a modified form thereof, such as for instance a post-translationally modified form. A binding molecule capable of specifically binding polypyrimidine tract binding protein-associated splicing factor (PSF), nuclear RNA and DNA binding protein p54^nrb, heterogeneous nuclear ribonucleoprotein Cl and/or heterogeneous nuclear ribonucleoprotein C2, or a functional part, derivative and/or analogue thereof, or a modified form thereof, such as for instance a post-translationally modified form, is also suitable for diagnosis. Another preferred embodiment therefore provides a diagnostic kit comprising at least one binding molecule capable of specifically binding polypyrimidine tract binding protein-associated splicing factor (PSF), nuclear RNA and DNA binding protein p54^nrb, heterogeneous nuclear ribonucleoprotein Cl and/or heterogeneous nuclear ribonucleoprotein C2, or a functional part, derivative and/or analogue thereof, or a modified form thereof, such as for instance a post-translationally modified form thereof. More preferably, the invention provides a diagnostic kit comprising polypyrimidine tract binding protein-associated splicing factor (PSF) and/or nuclear RNA and DNA binding protein p54^nrb, or a functional part, derivative and/or analogue thereof or a modified form thereof such as for instance a post-translationally modified form, and/or a binding molecule capable of specifically binding said polypyrimidine tract binding protein-associated splicing factor (PSF) and/or nuclear RNA and DNA binding protein p54^nrb or functional part, derivative and/or analogue thereof or (posttranslationally) modified form thereof. Said diagnostic kit preferably furthermore comprises suitable means for detecting antigen-binding molecule complexes. Another embodiment provides a diagnostic kit comprising a molecule capable of being recognized by a binding molecule capable of specifically binding polypyrimidine tract binding protein- associated splicing factor (PSF), nuclear RNA and DNA binding protein p54^nrb, heterogeneous nuclear ribonucleoprotein Cl and/or heterogeneous nuclear ribonucleoprotein C2, or a modified form thereof, such as for instance a post¬ translationally modified form. Preferably, said molecule comprises an anti- idiotype antibody mimicking an epitope of said polypyrimidine tract binding protein-associated splicing factor (PSF), nuclear RNA and DNA binding protein p54^nrb, heterogeneous nuclear ribonucleoprotein Cl and/or heterogeneous nuclear ribonucleoprotein C2, or a modified form thereof, such as for instance a post-translationally modified form. In a preferred embodiment said molecule comprises a mimotope of said polypyrimidine tract binding protein-associated splicing factor (PSF), nuclear RNA and DNA binding protein p54^nrb, heterogeneous nuclear ribonucleoprotein Cl and/or heterogeneous nuclear ribonucleoprotein C2, or a modified form thereof such as for instance a post-translationally modified form.

The invention furthermore provides a method for at least in part treating an autoimmune disease comprising counteracting the presence and/or activity of an antibody capable of specifically binding polypyrimidine tract binding protein-associated splicing factor (PSF), nuclear RNA and DNA binding protein p54^nrb, heterogeneous nuclear ribonucleoprotein Cl and/or heterogeneous nuclear ribonucleoprotein C2, or a functional part, derivative and/or analogue thereof or a (post-translationally) modified form thereof

A molecule capable of counteracting the presence and/or activity of an antibody which antibody is capable of specifically binding polypyrimidine tract binding protein-associated splicing factor (PSF), nuclear RNA and DNA binding protein p54^nrb, heterogeneous nuclear ribonucleoprotein Cl and/or heterogeneous nuclear ribonucleoprotein C2, or a functional part, derivative and/or analogue thereof or a (post-translationally) modified form thereof, for use as a medicament is also herewith provided. A preferred embodiment provides a use of a molecule capable of counteracting the presence and/or activity of an antibody which antibody is capable of specifically binding polypyrimidine tract binding protein-associated splicing factor (PSF), nuclear RNA and DNA binding protein p54^nrb, heterogeneous nuclear ribonucleoprotein Cl and/or heterogeneous nuclear ribonucleoprotein C2 or a functional part, derivative and/or analogue thereof or a (post-translationally) modified form thereof, for the preparation of a medicament for treatment of an autoimmune disease. Said autoimmune disease preferably comprises systemic lupus erythematosus, systemic sclerosis and/or rheumatoid arthritis.

The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention. Many alternative embodiments can be carried out, which are within the scope of the present invention.

All references cited in this application are incorporated herein by reference. EXAMPLES

Index

Materials and Methods

1. Libraries and bacteria

2. Trypsin sensitive helper phage

3. Cell lines, induction of cell death and preparation of cell extracts

4. Protein electrophoresis and western blotting 5. Test selections

5.1 Test selections against normal HeLa SlOO cytoplasmic cell extract coated on Immunotubes

5.2 Test selections using biotinylated normal HeLa SlOO cytoplasmic cell extract and magnetic streptavidin-coated beads

5.3 Polyclonal phage ELISA

6. Subtractive selection against biotinylated apoptotic HeLa cell extract followed by phage selection on western blots 7. Screening and sequencing

8. Antigen identification by immunoprecipitation and subsequent mass spectrometry

9. Western blot analysis

10. Immunofluorescence analysis 11. Ul snRNP immunoprecipitations Results

1. Test selections of anti-La and anti-Ro52 phages against normal cell extract

2. Subtractive selection of recombinant antibodies against apoptotic cell extract components 3. Selected scFv recognize proteins that are differentially expressed between apoptotic and non-apoptotic cell extracts 3.a PSF/p54^b 3.b hnRNP C1/C2 3.c U1-70K

4. Identified proteins are recognized by autoimmune patient sera

Materials and Methods

1 Libraries and bacteria Autoimmune patient libraries of systemic lupus erythematosus (SLE) (patients D5, D18, D181, Oil and Z5), systemic sclerosis (SSc) (patients B92, H248, J70, S185 and T5) and rheumatoid arthritis (RA) (patients Du, He and Wy) patients, and a naive IgM library were available (Degen et al., 2000; Hoet et al., 1998; Hoet et al., 1993; Raats et al., 2003; Zampieri et al., 2003), and used for selection. A semi-synthetic library was kindly provided by Nissim (Nissim et al., 1994). E.coli TGl was grown in 2xTY (16 g/1 pepton (Gibco-BRL), 10 g/1 yeast extract (Gibco-BRL) and 5 g/1 NaCl).

2 Trypsin sensitive helper phage

To reduce the background of phages that do not express a functional antibody - gene III fusion protein, and to reduce the number of selection rounds needed, a trypsin sensitive helper phage (Kristensen and Winter, 1998) was used in combination with the pHen based phagemid systems for phage amplification.

3 Cell lines, induction of cell death and preparation of cell extracts Jurkat (human T cell leukemia) suspension cells were grown in RPMI 1640 medium (Gibco-BRL), supplemented with 1 mM sodium pyruvate, 1 mM penicillin, 1 mM streptomycin and 10% heat-inactivated fetal calf serum (FCS; Gibco-BRL), in a humidified 37⁰C incubator containing 5% CO2. Cells were maintained at a concentration of IxIO⁶ cells/ml. HeLa cells were either purchased from Computer Cell Culture Centre (Mons, Belgium), or HeLa suspension cells were grown in S-MEM medium with Joklik modification (Oxoid), supplemented with non-essential amino acids (Gibco-BRL), 5% new born serum (NBS, Gibco-BRL), 2 mM L-glutamine (Gibco-BRL), 1 mM penicillin and 1 mM streptomycin. Cells were grown at a constant concentration of IxIO⁶ cells/ml. Apoptosis was induced in Jurkat and in HeLa cells by addition of 10 μg/ml anisomycin. Eight hours after induction, apoptotic cells were harvested by centrifugation at 80Og for 10 minutes and washed with PBS. Non-apoptotic HeLa SlOO cytoplasmic extract was prepared as described (Fouraux et al., 2002). Extracts of apoptotic HeLa cells were prepared by freezing and thawing the apoptotic cell pellet in PBS. Apoptotic and non- apoptotic Jurkat total cell extracts were prepared by sonication. Cells were harvested and resuspended in ice-cold lysis buffer (50 mM Tris-HCl pH 7.6, 100 mM KCl, 0.5% NP-40, 1 mM EDTA, 1 mM DTE and 0.5 mM PMSF) at a concentration of 5xlO⁷ cells/ml and lysed by sonication. After cell lysis, extracts were centrifuged at 12,00Og and 4⁰C for 30 minutes, and clear lysates were stored at -70⁰C. Cell extracts were biotinylated in 50 mM NaHCOs pH 8.3 with a 10-fold molar excess of NHS-LC-biotin (Pierce) and dialyzed against 0.5 mM DTE and 20% glycerol in PBS. The protein concentration of the biotinylated cell extract was measured using a bicinchoninic acid (BCA) protein assay (Pierce) with bovine serum albumin (BSA) as a standard, and the amount of magnetic streptavidin beads (DYNAL Biotech) needed to completely capture a given amount of biotinylated cell extract was established empirically. 4 Protein electrophoresis and western blotting Prior to western blotting, proteins were separated by one- or two-dimensional gel electrophoresis. Two-dimensional gel electrophoresis was performed as described previously (O'Farrell, 1975), with minor adjustments. In the first dimension glass capillaries with a length of 7 cm and a diameter of 1.0 mm (BIO-RAD) (small gels) or custom made glass capillaries with a length of 14 cm and a diameter of 3.0 mm (large gels) were used. Isoelectric focussing (IEF) gels with a non-linear pH gradient (range pH 3-10) contained 9.2 M urea, 4 % acrylamide, 2 % NP-40, 0.54 % carrier ampholytes pH 3-10 and 1.5 % carrier ampholytes pH 5-8. After pre-focussing of carrier ampholytes, proteins were applied and focussed at 400 Volts for five hours (small gels) or for sixteen hours (large gels). An SDS-PAGE 12.5% (w/v) poly acrylamide gel was used in a second dimension. Proteins were stained with colloidal Coomassie (SERVA), or proteins were transferred to a nitrocellulose (Schleicher&Schuell) or PVDF (Millipore) membrane by semi-dry electroblotting in transfer buffer containing 25 inM Tris base, 192 mM glycine, 0.05% SDS and 20% methanol.

5 Test selections

S.I Test selections against normal HeLa SlOO cytoplasmic cell extract coated on Immunotubes

To test whether selection against cell extract coated on Immunotubes was feasible, anti-La, anti-Ro52 and anti-Ribo P phages, previously selected from human autoimmune patient derived libraries, were diluted at three different ratios in a naϊve IgM library (complexity 1.5xlO⁸) at a l:10⁴ ratio, at a l:10⁶ ratio and at a l:10⁸ ratio. For example for a l:10⁴ ratio, 10⁷ anti-La phages, 10⁷ anti-Ro52 phages and 10⁷ anti-Ribo P phages were mixed with 10¹¹ naϊve IgM library phages. 750 μg non-apoptotic HeLa SlOO cytoplasmic cell extract was coated on Immunotubes (NUNC) in 2 ml 50 mM NaHCO₃ pH 9.3, at 4⁰C overnight. Phages were pre-blocked with 1% BSA, 1% New Born Serum (NBS) and 5% non-fat dried milk powder in PBS (MPBS), and subsequently panned against HeLa SlOO cytoplasmic cell extract for two hours at room temperature, using an end-over-end rotator. Tubes were washed several times with PBST and twice with PBS and bound phages were eluted by incubating the tube with 2 ml trypsin (10 g/1 in PBS, pH 7.4 and pre-warmed at 37⁰C) for 30 minutes at room temperature. Eluted phages were incubated with 0.5 ml 10% BSA for 5 minutes at room temperature and immediately used to infect exponentially growing E.coli TGl. Phages were amplified by super-infection with M13 trypsin sensitive helper phage and overnight growth at 3O⁰C with shaking, and purified from the medium by PEG precipitation (Marks et al., 1991). 5.2 Test selections using biotinylated normal HeLa SlOO cytoplasmic cell extract and magnetic streptavidin-coated beads To test whether selection against biotinylated cell extract was promising, anti- La and anti-Ro52 phages previously selected from human autoimmune patient derived libraries, were diluted in a semi-synthetic phagemid library at two different ratios, namely a l:10⁶ ratio and a l:10⁹ ratio. For example, for a l:10⁶ ratio 10⁷ anti-La and 10⁷ anti-Ro52 were mixed with 10¹³ phages of the Nissim semi-synthetic library. All panning steps were performed using an end-over- end rotator at room temperature. First, the phages were incubated with 1 mg magnetic streptavidin beads, pre-blocked in MPBS, for one hour. Beads were magnetically separated from the solution and discarded, while the solution containing the remaining phages was transferred to a clean tube. Subsequently, phages were incubated with 1.5 μg biotinylated normal HeLa SlOO cytoplasmic cell extract for one hour, 1 mg pre-blocked magnetic streptavidin beads were added and the mixture was rotated for 15 minutes. Beads were magnetically harvested from the solution and washed several times with PBST and twice with PBS. Bound phages were eluted by incubating the washed beads in 1 ml trypsin (10 g/1 in PBS, pH 7.4 and pre- warmed at 37⁰C) for 30 minutes at room temperature. Beads were harvested and discarded, whereas the solution containing the eluted phages was transferred to a clean tube and incubated with 1 ml of NBS for 5 minutes. Phages were then amplified and purified as described above. 5.3 Polyclonal phage ELISA

Polyclonal phage pools of each round were screened in ELISA for reactivity with La and Ro52, and against BSA as a control. Antigens were coated on 96 wells maxisorb microtitre plates (NUNC) at a concentration of 0.1 μg per well in 100 μl 50 mM NaHCO₃ pH 9.3, at 4⁰C overnight. Plates were blocked with 400 μl MPBS, containing 0.1% Tween-20 (MPBST) per well at room temperature for two hours. Phages were diluted in MPBST at several concentrations and 100 μl was added at room temperature for one hour. The plates were washed six times with PBST and then horse radish peroxidase (HRP)-conjugated anti-M13 moAb (Pharmacia) was added at a 5,000-fold dilution in MPBST at room temperature for one hour. Plates were washed six times with PBST and twice with PBS. Bound HRP-conjugated antibodies were detected by 3,3',5,5'-tetramethylbenzidine (TMB, NEOGEN). Reactions were stopped with 1 M H2SO4 and the absorbance at 450 nm was measured. 6 Subtractive selection against biotinylated apoptotic HeLa cell extract followed by phage selection on western blots

Phages recognizing apoptosis-specific components were selected in a two-step manner. First, phages were subtractively selected against biotinylated apoptotic cell extract components. Second, enriched phage pools were subsequently selected on western blots containing proteins separated by two- dimensional electrophoresis. Phages recognizing protein spots specifically present on blot containing apoptotic material were selected and reamplified in this second selection step.

Subtractive selections against apoptotic HeLa cell extract components were performed using autoimmune patient derived pHenIX phagemid libraries. Libraries of RA, SLE and SSc patients were equally mixed and used for selection. All panning steps were performed using an end-over-end rotator at room temperature. First, the phages were incubated with 1 mg magnetic streptavidin beads, pre-blocked in MPBS, for one hour. Beads were magnetically separated from the solution and discarded, while the solution containing the remaining phages was transferred to a clean tube. To subtract phages recognizing normal cell extract components, phages were incubated with 1.5 μg biotinylated non-apoptotic HeLa SlOO cytoplasmic cell extract for one hour, 1 mg pre-blocked magnetic streptavidin beads were added and the mixture was rotated for 15 minutes. Beads were harvested and discarded, and the remaining phage solution was transferred to a clean tube. This subtraction step was repeated with another 1.5 μg of biotinylated normal cell extract and 1 nig pre-blocked beads. Remaining phages were again transferred to a clean tube. Subsequently, phages specifically recognizing apoptotic cell extract components were selected by panning against 1.5 μg biotinylated apoptotic HeLa cell extract. 1 mg pre-blocked magnetic streptavidin beads were added and the mixture was rotated for 15 minutes. Beads were harvested, washed several times with PBST and twice with PBS, and bound phages were eluted by incubating the beads in 1 ml trypsin (10 g/1 in PBS, pH 7.4). Beads were discarded and eluted phages were incubated with 1 ml NBS, and immediately used to infect exponentially growing E.coli TGl. Phages were amplified using trypsin sensitive helper phage and purified from the medium by polyethylene glycol (PEG) precipitation.

After three selection rounds, polyclonal phage pools of each round were analyzed on western blots of small two-dimensional gels containing apoptotic and non-apoptotic cell extracts. Membranes were blocked for one hour with 1% gelatin in PBS containing 0.1% Tween-20 (GPBST). Alternatively, PVDF membranes were not blocked, but treated as described by Marks et al. (Liu et al., 2002). The membrane was incubated with PEG purified phages, diluted in GPBST at a concentration of 10¹¹ cfu/ml, at room temperature for one hour. After extensive washing with GPBST, the membrane was incubated with HRP-conjugated anti-M13 moAb, 5,000-fold diluted in GPBST at room temperature for one hour. The membrane was washed extensively with GPBST and twice with PBS, and bound antibodies were detected by TMB staining (Koch et al., 1985) or enhanced chemilurαinescence (ECL). The phage stock from the third round showed highest signals and most differences between apoptotic and non-apoptotic extracts. To specifically select phages recognizing a protein spot that was differentially present in apoptotic versus non-apoptotic cell extracts, western blot membranes of large two-dimensional gels were incubated with the phage stock from the third selection round as described above. Directly after visualization of bound phages, the membrane was covered with PBS and membrane spots of interest were accurately excised, using a clean razor blade for each spot. TMB staining resulted in a blue precipitate on the membrane, allowing highly accurate excision of spots. For ECL detection, the film was placed on a light box and exactly overlaid with the membrane. Bound phages were eluted from the excised spots by incubating the blot piece in 100 mM TEA , and neutralized with 1 M Tris-HCl pH 7.4. TEA eluted, pHenIX derived phages were treated with trypsin as described above to reduce background. Subsequently, phages were used to infect exponentially growing E.coli TGl and plated on square 2xTY agar plates containing 100 μg/ml ampicillin and 2% glucose.

7 Screening and sequencing

Single colonies were analyzed for VSV-G-expression by a dot blot assay, and for the presence of full length antibody fragment cDNA by PCR, using primers LMB3 (CAG GAA ACA GCT ATG ACC ATG) and FdSeql (GTA ACG ATC TAA AGT TTT GTC G), and were subsequently fingerprinted with the restriction enzyme BsiNI. From all unique clones with good expression, phages were produced in 2 ml-deepwell microtiter plates. In short, colonies were picked in 96-wells microtiter plates containing 100 μl 2xTY supplemented with 100 μg/ml ampicillin and 2% glucose and grown overnight at 37⁰C and 200 rpm. The next day, 20 μl of overnight culture was transferred to 1.5 ml 2xTY supplemented with 100 μg/ml ampicillin and 0.05% glucose in a 96 wells 2 ml- deepwell microtiter plate, and grown for 2 hours at 37⁰C and 200 rpm. Cultures were then super-infected with trypsin sensitive helper phage for 30 minutes at 37⁰C and kanamycin was added at a final concentration of 70 μg/ml. After overnight growth at 30⁰C and 200 rpm, the deepwell microtiter plate was centrifuged at 3,000 rpm for 10 minutes and culture supernatants were stored in a clean plate for further analysis. Undiluted culture supernatants, containing phage, were screened on western blots of one- dimensional 12 or 15% polyacrylamide SDS-PAGE gels containing normal and apoptotic cell extracts (2xlO⁵ cells per 3 mm blot strip). Phages were detected on blot as described above. Several phages recognized proteins that were differentially present between non-apoptotic and apoptotic cell extracts. Of these clones scFvs BlHIl, GlGlO and R3A4 were chosen for further analysis. The cDNAs of clones of interest were sequenced essentially as described (McCafferty et al., 1990).

8 Antigen identification by immunoprecipitation and subsequent mass spectrometry

ScFvs were C-terminally tagged with a 6xHis-tag by cloning the cDNA in a pUCH9 vector via compatible Ncol and Noil digestion. Recombinant scFv antibody fragments were isolated from E.coli periplasm as previously described (Raats et al., 2003) and dialyzed against IPP500 buffer (50 mM phosphate buffer pH 8.0, containing 500 mM NaCl). All steps were performed at 4⁰C using an end-over-en rotator. 10 ml concentrated scFv solution was incubated with 250 μl Ni-NTA agarose beads (QIAGEN) for 2 hours. Subsequently, beads were washed three times with 15 ml IPP 500 and three times with 15 ml IPP150 (50 mM phosphate buffer pH 8.0, containing 150 mM NaCl), and incubated with 2.5xlO⁸ cells, at a concentration of 5xlO⁷ cells/ml for 2 hours. Beads were washed three times with IPP150. To remove non- specifically bound proteins, beads were incubated with 10 ml 5 mM imidazole for 10 minutes and the supernatant was discarded. Beads were transferred to a clean Eppendorf tube and incubated with 150 μl 100 mM triethylamine for 10 minutes. Supernatant was transferred to a clean Eppendorf tube, and elution was repeated with 150 μl 100 mM triethylamine. Supernatant was pooled with the first supernatant and neutralized with 100 μl IM Tris buffer pH 7.4. The proteins were separated on a large 12.5% SDS-PAGE, as described above, and the gel was stained with colloidal Coomassie. Protein bands were cut out, and digested in-gel with trypsin. Peptides were then eluted from the gel slice and measured via mass spectrometric analysis.

9 Western blot analysis All incubation steps were carried out at room temperature on a shaking table. Western blots were prepared from one-dimensional SDS-PAGE gels containing non-apoptotic and apoptotic Jurkat cell extracts as described above, and pre- blocked with MPBST for one hour. Subsequently, membranes were incubated with scFv, 10 to 100-fold diluted in MPBST, for one hour. After washing with MPBST membranes were incubated with mouse anti-VSV-G monoclonal antibody, 5, 000-fold diluted in MPBST, for one hour. Membranes were washed with MPBST. Then, membranes were incubated with HRP-conjugated rabbit anti-mouse antibody (DAKO), 2,500-fold diluted in MPBST. Membranes were washed with PBST and PBS and bound antibodies were detected by ECL.

10 Immunofluorescence analysis

HeLa cells were grown directly on glass slides, whereas apoptotic and non- apoptotic Jurkat cells were spotted on glass slides by cytospin. Cells were fixed using methanol/acetone (Raats et al., 1991) or p-formaldehyde (Jones et al., 2000). ScFvs were purified from periplasmic fractions as described (Raats et al., 2003) and incubated with the cells. Bound scFvs were detected via their VSV-G-tag using anti-VSV-G mouse monoclonal antibody, used at a 500-fold dilution in PBS, followed by FITC-labeled rabbit anti-mouse antibody, 50-fold diluted in PBS. Finally, cells were embedded in mowiol and analyzed using a fluorescence microscope.

11 Ul snRNP immunoprecipitations

Apoptotic and non-apoptotic Jurkat cells were resuspended in Nonidet-P40 (NP40) lysis buffer (25 mM Tris-HCl pH 7.6, 1% NP40, 100 mM KCl, 10 mM MgCk, 0.25 mM dithioerythritol (DTE), containing "complete protease inhibitor cocktail (Roche)) at a concentration of IxIO⁸ cells/ml and lysed on ice for 30 minutes. Cell lysates were centrifuged for 30 min at 12,00Og and 4⁰C and supernatants were used immediately or stored at -70⁰C. AU incubations were performed in IPP150 (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% NP40) at 4⁰C using an end-over-end rotator. Between incubations, beads were washed three times with IPP150. Protein A-agarose beads (20 μl of 50% slurry, Kem-En-Tec) were pre-coated with 12.5 μl rabbit anti-mouse IgG (Dako) for 2 hours and subsequently coated with 50 μl mouse anti- VSV-G mAb for 2 hours. Then, beads were incubated with 5 ml concentrated solution of VSV-G-tagged recombinant scFv antibody fragments, isolated from E.coli periplasm as described above. Finally, beads were incubated with 60 μl of IxIO⁸ cells/ml Jurkat apoptotic or non-apoptotic cell extract for 2 hours. Mouse moAb 2.73, directed against U1-70K protein was used as a positive control. Anti-BSA scFv, previously selected from an SLE patient library, was used as a negative control. Finally, beads were resuspended in 100 μl IPP150/0.5% SDS and co- immunoprecipitated RNA was isolated by addition of TRIzol reagent (Gibco- BRL) according to the manufacturer's protocol. Isolated RNA was subsequently size -fractionated on 6% polyacrylamide/8M urea gels and transferred to a nylon membrane (Hybond™-N, Amersham Pharmacia Biotech) by Northern blotting. Finally, Ul RNA was detected using a ³²P- labeled Ul RNA specific anti-sense probe, transcribed from vector pGEM3-Ul, and analyzed by autoradiography.

Results

1 Test selections of anti-La and anti-R0S2 phages against normal cell extract Successful selections against complex mixtures of antigens, such as a cellular extracts, depend both on the amount and concentration of antigen that is present during panning, and on the affinity of the antibody fragments. Different selection conditions were tested by means of mock selections, in which monoclonal phages recognizing the autoantigens La and Ro52 (and Ribo P), previously selected from SLE derived libraries ((Raats et al., 2003), unpublished results), were mixed with a naϊve IgM or a semi-synthetic library at several ratios. The l:10⁹ ratio approximated the complexity of the libraries used in following experiments. It was estimated that approximately 10¹⁰ molecules La and 10⁸ molecules Ro52 were present in 1.5 μg of soluble protein obtained from cell extracts. After several selection rounds, the polyclonal phage pools were tested in ELISA for reactivity against La and Ro52, and against BSA as a control antigen. First, panning against 750 μg cell extract immobilized on Immunotubes was investigated, but titres of eluted phages were low and no amplification was noticed between selection rounds. Then, panning in solution against 1.5 μg biotinylated cell extract was examined. As shown in Figure 1, at a l:10⁶ ratio anti-La phages were recovered after no more than one selection round, whereas at a l:10⁹ ratio anti-La phages were retrieved after two rounds of panning. At a l:10⁶ ratio the polyclonal phage stock showed slight reactivity with Ro52, whereas at a l:10⁹ ratio no reactivity against Ro52 was observed after two selection rounds. Reactivity with control antigen BSA was not observed. These results demonstrated that it is possible to select phage antibodies from libraries derived of autoimmune patients using only small amounts of biotinylated cell extract. Furthermore, it also indicates that if sufficiently high affinity antibodies are present in naϊve/synthetic immune libraries these will also be applicable in this selection system. 2 Subtractive selection of recombinant antibodies against apoptotic cell extract components

Phages recognizing apoptosis-specific components were selected in a two-step manner. In the first step, phages were subtractively panned against biotinylated apoptotic HeLa cell extract during a number of selection rounds. In the second step, a polyclonal phage pool, obtained after several selection rounds and enriched for specific phages, was subsequently selected on western blot. Phages recognizing apoptosis-specific protein spots were eluted from these spots and amplified.

Patient libraries of RA, SLE and SSc patients were equally mixed and subtractively panned against biotinylated apoptotic cell extract. Prior to panning on biotinylated apoptotic HeLa cell extract, phages recognizing normal cell extract components were removed by subtractive panning on biotinylated non-apoptotic HeLa SlOO cytoplasmic cell extract. The polyclonal phage pools of the first three selection rounds were analyzed on western blots of small two-dimensional IEF/SDS-PAGE gels containing non-apoptotic and apoptotic cell extracts. The first round phage pool showed weak signals and only a few differences between the apoptotic extract and the non-apoptotic extract. The phage pool from the second selection round showed even lower signals than the phage pool from the first round. A decrease in phage amplification between the first and the second selection round was observed as well. Each selection round started with approximately 10¹³ phages, and after the first panning 1.2xlO⁵ phages eluted, whereas after the second panning only 1.5xlO⁴ phages were retrieved. In the third selection round a strong amplification was observed: in this selection round 4.OxIO⁸ phages eluted. As shown in Figure 2, the polyclonal phage stock obtained after three selection rounds showed strong reactivity on western blot, and was chosen for further analysis. Several phages reacted specifically with protein spots present on the western blot containing apoptotic extract, although some phages reacted with protein spots present on both the western blots with non-apoptotic and apoptotic extract. Several of these specific spots were cut out and bound phages were eluted. It was tested whether the detection method influenced the infectivity of the phages, but the amount of phages eluted from ECL detected spots was equal to the amount of phages eluted from TMB stained spots. In average, 200 cfu eluted from one spot (approximately 1 mm x 2 mm). To reduce the amount of colonies to be screened, 24 single colonies were first analyzed by DNA fingerprinting of the scFv encoding genes, and by scFv expression. Per spot, clones were grouped based on fingerprinting data. In average, six to eleven groups of unique clones were eluted per spot, varying from one to five clones per group. From each group, one clone showing relatively high scFv expression was tested as phage for binding to target proteins on western blot strips of one-dimensional gels of apoptotic and non-apoptotic cell extracts. Finally, three clones were chosen for further analysis: BlHIl, GlGlO and R3A4.

3 Selected scFv recognize proteins that are differentially expressed between apoptotic and non-apoptotic cell extracts

3.a PSF andp54^nrb

On western blots of non-apoptotic and apoptotic Jurkat cell extract, scFv BlHIl recognized protein bands of 100, 55 and 45 kD, and an additional protein band of approximately 50 kD specifically present in apoptotic cell extract, as demonstrated in Figure 3a. Via immunoprecipitation of the antigens from Jurkat cell extract, followed by mass spectrometric analysis, the identity of the 100 kD was established as polypyrimidine tract binding protein- associated splicing factor (PSF), and the identity of the 55 kD protein as nuclear RNA- and DNA binding protein of 54 kD (p54^nrb). Polypyrimidine tract binding protein (PTB)-associated splicing factor (PSF) and nuclear RNA and DNA binding protein p54^nrb are found in the nucleus as a heterodimer complex. These multi-functional proteins are involved in mRNA splicing, transcription and DNA replication.

The 45 kD protein band is considered as a breakdown product of p54^nrb. Both PSF and p54^nrb are modified during apoptosis: PSF is hyperphosphorylated [86], and p54^Mb is cleaved to a 50 kD product [87], which can be noticed as the extra 50 kD protein band specifically recognized in apoptotic cell extract by scFv BlHlI (Figure 3a). The identity of PSF was confirmed by probing western blots, containing the immunoprecipitated antigens, with anti-PSF specific mouse monoclonal B92 [86]. Moreover, scFv BlHIl reacted with recombinant PSF and p54^nrb in ELISA and on western blots. Figure 3d shows that in immunofluorescence analysis, scFv BlHIl gave a nuclear staining with a speckled pattern, which is consistent with the fact that PSF and p54^nrb are nuclear proteins. It is also consistent with the fact that PSF is described as paraspeckle protein [86]. 3. b hnRNP C1/C2

On western blots of non-apoptotic Jurkat cell extract, scFv GlGlO recognized a protein band of approximately 35 kD, which is demonstrated in Figure 3b. The molecular weight of the protein band on western blots of apoptotic Jurkat cell extract appeared to be slightly smaller. The identity of the antigen was established through immunoprecipitation of the antigen from Jurkat cell extract followed by mass spectrometric analysis, and was found to be heterogeneous nuclear ribonucleoprotein Cl, or it splice variant C2 (hnRNP C1/C2). During transcription, pre-mRNA is immediately associated with a set of specific proteins, called heterogeneous ribonucleoprotein (hnRNP). HnRNP A, B and C comprise the majority of the mass of mammalian 4OS hnRNP particles. HnRNP C protein exists as a tetramer of two splice variants, called Cl and C2, which differ in size by 13 amino acids. Three Cl monomers and one C2 monomer form the C protein tetramer. The reactivity of scFv GlGlO and the reactivity of an anti-hnRNP C1/C2- specific patient serum [88] was analyzed on western blots containing non- apoptotic and apoptotic cell extracts, and both the scFv and the patients antibodies gave the same pattern, since they both recognized protein bands of the same molecular weights. On western blots of cell extracts separated on a 10% poly aery lamide gel, the antigen can be visualized as two separate protein bands (data not shown). The identity of the antigen as hnRNP C1/C2 was further confirmed by immunoprecipitation of the antigen using the anti- hnRNP Cl/C2-specific patient serum, followed detection of the precipitated antigen by scFv GlGlO on western blot. As can be seen in Figure 3f, in immunofluorescence analysis on non-apoptotic HeLa cells, scFv GlGlO gave a nuclear staining, which is consistent with the fact that hnRNP C1/C2 is a nuclear protein. Furthermore, in immunofluorescence analysis of non- apoptotic and apoptotic Jurkat cells, it was observed that hnRNP C1/C2 was present in the nucleus of non-apoptotic cells (Figure 3g), but translocated to the apoptotic blebs or apoptotic bodies (i.e. the vesicles that appear at the apoptotic cell surface) on apoptotic cells (Figure 3h). In addition, it is known that hnRNP C1/C2 is cleaved during apoptosis. During this process a small part of both proteins are removed, which is consistent with the finding that the molecular weights are somewhat smaller in apoptotic cell extracts than in non- apoptotic cell extracts.

3.c U1-70K

ScFv R3A4 reacted with a protein band of approximately 68 kD on western blots containing non-apoptotic cell extracts, whereas it reacted with a protein band of approximately 40 kD on western blots containing apoptotic cell extracts (Figure 3c). From this finding the idea rose that the antigen might be U1-70K. U1-70K is a component of the Ul small nuclear ribonucleoprotein particle (snRNP), and is cleaved from a 70 kD protein to a 40 kD apoptotic product during apoptosis. Moreover, U1-70K is a known autoantigen in SLE overlap syndrome. The cDNA encoding scFv R3A4 was sequenced and was almost identical to cDNAs encoding anti-Ul-70K scFv, previously selected from SLE libraries (Degen et al., 2000). In immunofluoresence analysis of non- apoptotic HeLa cells, scFv R3A4 gave a nuclear staining (Figure 3e), which is consistent with the fact that U1-70K is a nuclear protein.

Moreover, we analyzed the reactivity of scFv R3A4 with intact U1-70K compared to its reactivity with the 40 kD apoptotic form of U1-70K. Ul snRNP complexes were immunoprecipitated from non-apoptotic and apoptotic Jurkat cell extracts by anti-Ul-70K scFvs 4, 6 and 7, previously selected from SLE derived libraries (Degen et al., 2000), and scFv R3A4. As a positive control, mouse monoclonal antibody 2.73 was used. As a negative control, an anti-BSA scFv selected from an SLE derived library (unpublished results), with no reactivity with one of the components of the Ul snRNP, was used. The amount of precipitated particles was measured by analyzing the amount of Ul RNA on northern blots. As shown in Figure 4, it was observed that scFv R3A4, together with scFv 6 and 7 precipitated more Ul snRNP from apoptotic cell extracts than from non-apoptotic cell extracts, whereas scFv 4 precipitated equal amounts of Ul snRNP from non-apoptotic and apoptotic cell extracts. Mouse monoclonal antibody 2.73 precipitated more Ul snRNP from non-apoptotic cell extracts than Ul snRNP from apoptotic cell extracts, which is consistent with the observation that 2.73 reacts stronger with intact U1-70K than with its apoptotic 40 kD product on western blots. The un-related anti-BSA scFv precipitated small amounts of Ul-complexes from the non-apoptotic extract, which was considered to be background. These finding indicate that scFv R3A4, together with scFv 6 and 7, has a higher affinity for the apoptotic form of U1-70K than for non-apoptotic U1-70K.

4 Identified proteins are recognized by autoimmune patient sera U1-70K is a well-known autoantigen specifically recognized by patients suffering from mixed connective tissue disease (MCTD), also called SLE overlap syndrome [89]. Autoantibodies directed against U1-70K were found in the serum of SLE patients Oil and Z5, whose bone marrow was used to generate the libraries used in this experiment. In the past, scFvs recognizing U1-70K have been successfully isolated from these SLE libraries (Degen et al., 2000).

HnRNP C1/C2 has been described as an autoantigen in a few patients with a broad range of diseases, including a patient suffering from a rare combination of systemic sclerosis and psoriatic arthritis. The sera from patients whose bone marrow was used for library generation were analyzed on western blots containing relatively small amounts of immunoprecipitated antigen, but none of the library sera was reactive. This might be due to low signals in combination with high background. Recombinant expression of hnRNP C1/C2, and analysis of patients sera is still in progress.

PSF and p54^nrb have not been described as autoantigens. We found that one of the sera from patients whose bone marrow was used for library generation, reacted with recombinant p54^nrb on western blots, although titers were relatively low. This serum derived from SLE patient D181.

Brief description of the drawings

Figure 1. Reactivity of polyclonal phage stocks from mock selections, tested in ELISA on La, Ro52 and BSA. Anti-La and anti-Ro52 monoclonal phages were mixed with a semi-synthetic library at two different ratios. For a l:10⁹ ratio (A) 10⁴ anti-La phages and 10⁴ anti-Ro52 phages were mixed with 10¹³ semi-synthetic phages. For a l:10⁶ ratio (B) 10⁷ anti-La phages and 10⁷ anti-Ro52 phages were mixed with 10¹³ semi-synthetic phages. Both mixtures were panned against 1.5 μg biotinylated non-apoptotic HeLa SlOO cytoplasmic cell extract in combination with magnetic streptavidin-coated beads, during two selection rounds. 10¹⁰ cfu of each phage stock, including the un-selected phage mixtures (selection round 0), were analyzed for reactivity with La, Ro52 and BSA. Bound phages were colorimetrically detected using HRP-labeled anti-Ml3 moAb at 450 nm. At a l:10⁹ ratio (A), reactivity with La was observed in the phage pool obtained after two selection rounds, whereas the un-selected phages and the phages obtained after one selection round did not react with the antigen. Reactivity with Ro52 and BSA was not observed. At a ratio of l:10⁶ (B), reactivity to La was observed in the phage pools obtained after the first and the second round, whereas the un-selected phages did not react with La. Reactivities to Ro52 were low, although higher than reactivities observed in phage pools obtained from l:10⁹ ratio mock selections. Reactivity against control antigen BSA was not observed.

Figure 2. Analysis of phages, obtained after three subtractive selection rounds on biotinylated apoptotic HeLa cell extract, on western blots of two-dimensional IEF/SDS-PAGE gels containing non- apoptotic and apoptotic cell extracts.

100 μg (2a) or 40 μg (2b) soluble protein of non-apoptotic HeLa SlOO cytoplasmic cell extract (2a; A & C and 2b; A) or apoptotic HeLa cell extract (2a; B & D and 2b;B) were separated on large two-dimensional gels and blotted onto a nitrocellulose membrane. The membranes were incubated with IxIO¹¹ phages obtained after three subtractive selection rounds on biotinylated apoptotic HeLa cell extract, and bound phages were detected using HRP- labeled anti-M13 moAb followed by TMB staining (2a) which gave a blue precipitate on the membrane.

Alternatively, the membranes were incubated with 5xlOⁿ cfu/ml, and detected with HRP-labeled anti-M13 mAb and ECL (2b;A & B). Several phages reacted specifically with protein spots present on the membranes containing apoptotic cell extract (2a;B or 2b;B), although a number of selected phages reacted with proteins spots present on both membranes (A and B). Apoptosis- specific spots were accurately excised from the membrane (D) and bound phages were eluted and amplified.

Figure 3. Western blot and immunofluorescence analyses of scFvs BlHIl, GlGlO and R3A4. Non-apoptotic (n) and apoptotic (a) Jurkat cell extracts were separated on 12.5% polyacrylamide gels and separated proteins were transferred to a nitrocellulose membrane. Bound scFvs were detected via their VSV-G-tags using anti-VSV-G moAb and HRP-labeled rabbit anti-mouse antibodies, and visualized by ECL. ScFv BlHIl recognizes the 100 kD protein PSF and the 54 kD protein p54^nrb (A). A 45 kD protein band is recognized as well, which might be a breakdown product. Besides, BlHlI reacts with a 50 kD protein band specifically present in the apoptotic cell extract. ScFv GlGlO recognizes hnRNP C1/C2 (B), two splice variants of approximately 35 kD, which are cleaved during apoptosis. ScFv R3A4 is directed to U1-70K, which is cleaved to a 40 kD product during apoptosis. In non-apoptotic cell extract a protein band of 70 kD is recognized, whereas in apoptotic cell extract a 40 kD protein band is recognized (C). For immunofluorescence analysis, HeLa or Jurkat cells were fixed and incubated with the scFv. Bound scFv were detected using anti-VSV-G antibody and FITC-labeled anti-mouse antibodies. Anti- PSF/p54^nrb scFv BlHIl gives a nuclear staining (D) with a speckled pattern. Anti-hnRNP C1/C2 scFv GlGlO gives a nuclear staining in HeLa cells (F). In non-apoptotic Jurkat cells GlGlO gives a nuclear staining (G), whereas in apoptotic Jurkat cells GlGlO stains the apoptotic blebs and/or apoptotic bodies (H) on the cell surface. Anti-Ul-70K scFv R3A4 also gives a nuclear staining (E). All staining patterns are consistent with the sub-cellular localization of the involved proteins.

Figure 4. Immunoprecipitations of Ul snRNP particles from non- apoptotic and apoptotic cell extracts using several anti-Ul-70K scFv.

Figure 4A) Anti-Ul-70K scFvs 4, 6, 7 and R3A4 were coupled to beads and incubated with non-apoptotic (n) and apoptotic (a) Jurkat cell extracts. The amount of isolated particles was evaluated by analyzing the amount of Ul RNA present in both samples. RNA was isolated from immunoprecipitated particles and analyzed on Northern blots using a ³²P-labeled Ul RNA-specific antisense probe. As a positive control mouse moAb 2.73 was used, which has higher reactivity with non-apoptotic U1-70K than with the 4OkD apoptotic form of U1-70K. As a negative control, an un-related scFv directed to BSA was used. RNA isolated from apoptotic (lanes 2, 21 and 23) and non-apoptotic (lanes 1, 20 and 22) Jurkat cells was run on the same gels (input). ScFv 6 precipitated more apoptotic particles (lanes 3-5) than non-apoptotic particles (lanes 6-8). ScFv R3A4 precipitated more particles from apoptotic cell extract (lanes 9-10) than from non-apoptotic (lanes 11-13) cell extract. ScFv 7 also isolated more apoptotic (lanes 14-16) than non-apoptotic (lanes 17-19) Ul snRNP particles. ScFv 4, on the other hand, precipitated equal amounts of Ul snRNP complexes from apoptotic (lanes 30-31) and non-apoptotic (lanes 32-34) cells extracts. Mouse monoclonal antibody 2.73, with higher reactivity with intact U1-70K than with its apoptotic 40 kD form, precipitated more Ul snRNP particles from non-apoptotic (lane 36) than from apoptotic (lane 35) cell extracts. The un-related anti-BSA scFv precipitated only small amounts of Ul snRNP particles from non-apoptotic (lanes 27-29) cell extracts, but did not precipitate detectable amounts of Ul snRNP particles from apoptotic cell extract (lanes 24-25).

Figure 4B) Graphic representation of Ul snRNP immunoprecipitation described under Figure 4A. The signals on the northern blots shown in Figure 4A were quantified, and the percentage of precipitated Ul snRNA was determined.

Figure 5. Schematic overview of the subtractive selection method. The selection method consists of two parts. In the first part, phages are depleted of non-wanted binders to (bio tiny late d) proteome A, subsequently they are selected on biotinylated proteome B in combination with a capturing step using magnetic streptavidin-coated beads (A and B). In the second part of the selection procedure, enriched phage pools are selected on western blots containing proteins separated by two-dimensional electrophoresis. Phages recognizing protein spots specifically present on western blots containing proteome B are selected and amplified ©. In short, phages recognizing proteome A (non-apoptosis-specific epitopes) are removed from the phage pool by panning proteome A (A). The remaining phages, which do not bind to components present in proteome A, are subsequently panned on proteome B (apoptotic cell extract) (B). After several selection rounds, polyclonal phage pools are analyzed on western blots of two- dimensional gels containing proteome A and B (C). Spots that are recognized specifically in proteome B are excised and bound phages are eluted and amplified (C). Single colonies are subsequently analyzed for scFv expression and their cDNAs are fingerprinted. Unique clones with good expression are finally analyzed for reactivity on western blot strips containing proteome A and B. Figure 6A-6F. Schematic overview of a preferred embodiment of the present invention.

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Claims

1. A method for isolating and/or identifying a target molecule present in a first sample but not, or to a significantly lesser extent, present in a second sample, comprising: a) providing a library of binding molecules; b) incubating at least part of said library with at least a part of said second sample; c) essentially removing at least one binding molecule of said library which is bound to a component of said second sample; d) incubating at least part of the remaining library with at least a part of said first sample; and e) isolating at least one binding molecule from said remaining library capable of specifically binding a target molecule of said first sample; wherein said binding molecule is used for isolating and/or identifying said target molecule.

2. A method according to claim 1, further comprising the following steps: f) separating at least two components of said first sample; g) incubating at least one of said components with at least one binding molecule obtained in step e); h) detecting a target molecule bound by said binding molecule; and i) identifying said target molecule.

3. A method according to claim 1, further comprising the following steps: f) separating at least two components of said first sample; g) incubating at least one of said components with at least one binding molecule obtained in step e); h) detecting at least one binding molecule bound to a target molecule; i) providing said at least one binding molecule; j) at least in part isolating said target molecule from a sample with help of said binding molecule; and k) identifying said target molecule.

4. A method according to claim 1, further comprising the following steps: f) amplifying said binding molecule; g) incubating at least one amplified binding molecule with at least a representative part of said first sample and at least a representative part of said second sample; h) obtaining a binding molecule capable of binding a target molecule present in a first sample but not, or to a significantly lesser extent, present in a second sample; i) at least in part isolating said target molecule from a sample with help of said binding molecule; and j) identifying said target molecule.

5. A method according to any one of claims 1-4, wherein steps b) through e) are at least once repeated with the at least one binding molecule obtained in step e).

6. A method according to any one of claims 1-5, wherein step d) is performed in the presence of at least one component of said second sample.

7. A method according to any one of claims 1-6, wherein said library comprises a phage display library.

8. A method according to claim 7, wherein said phage display library comprises an immune library.

9. A method according to any one of claims 1-8, wherein at least one of said binding molecules comprises a single chain antibody and/or a FAB fragment, or a functional part, derivative and/or analogue thereof.

10. A method according to any one of claims 1-9, wherein said target molecule comprises an antigen.

11. A method according to any one of claims 1-10, wherein said first and second sample are representatives of a proteome.

12. A method according to any one of claims 1-11, wherein said first and/or second sample comprises a cell-free solution and/or a purified cell extract.

13. A method according to any one of claims 1-12, wherein said second sample comprises biotinylated antigens in step b).

14. A method according to any one of claims 1-13, wherein said first sample comprises biotinylated antigens in step d).

15. A method according to any one of claims 1-14, wherein said at least one component of said second sample is essentially not biotinylated.

16. A method according to any one of claims 1-15, wherein removing said binding molecule in step c) comprises binding of streptavidin magnetic beads to a biotinylated antigen - binding molecule complex.

17. A method according to any one of claims 1-16, wherein isolating said binding molecule in step e) comprises binding of streptavidin magnetic beads to a biotinylated antigen - binding molecule complex.

18. A method according to any one of claims 2-17, wherein said components are separated from each other in step f) by gel electrophoresis.

19. A method according to claim 18, wherein said gel electrophoresis comprises two dimensional gel electrophoresis.

20. A method according to claim 18 or 19, wherein said separated components of step f) are transferred to a poly(vinylidene fluoride) membrane.

21. An isolated target molecule obtainable by a method according to any one of claims 1-20.

22. A method for identifying a diagnostic marker for a disease, comprising:

- identifying a target molecule which is present in a proteome of an individual suffering from said disease, but which is not or to a significantly lesser extent present in a proteome of an individual not suffering from said disease, by a method according to any one of claims 1-20.

23. A method for providing a diagnostic marker for a disease, comprising:

- identifying a diagnostic marker for a disease with a method according to claim 22, and

- providing said diagnostic marker.

24. A diagnostic marker obtainable by a method according to claim 23.

25. Use of a diagnostic marker obtainable by claim 23 for diagnosis of a disease.

26. Use of polypyrimidine tract binding protein-associated splicing factor and nuclear RNA and DNA binding protein p54^nrb, or a functional part, derivative and/or analogue thereof, or a (post-translationally) modified form thereof, for diagnosis of autoimmune disease.

27. Use of polypyrimidine tract binding protein-associated splicing factor (PSF), nuclear RNA and DNA binding protein p54^nrb, heterogeneous nuclear ribonucleoprotein Cl and/or heterogeneous nuclear ribonucleoprotein C2, or a functional part, derivative and/or analogue thereof, or a (post-translationally) modified form thereof, for diagnosis of systemic lupus erythematosus, systemic sclerosis and/or rheumatoid arthritis.

28. A method for diagnosing whether an individual is suffering from, or at risk of suffering from, a disease, comprising:

- determining whether a sample from said individual comprises a diagnostic marker according to claim 24, or a (post-translationally) modified form thereof.

29. A method for diagnosing whether an individual is suffering from, or at risk of suffering from, an autoimmune disease, comprising:

- determining whether a sample from said individual comprises polypyrimidine tract binding protein-associated splicing factor (PSF), nuclear RNA and DNA binding protein p54^nrb, heterogeneous nuclear ribonucleoprotein Cl and/or heterogeneous nuclear ribonucleoprotein C2, or a functional part, derivative and/or analogue thereof, or a (post- translationally) modified form thereof.

30. A method according to claim 22, 23 or 28, or a use according to claim 25, wherein said disease comprises an autoimmune disease or malaria.

31. A kit for performing a method according to any one of claims 1-20, 22, 23, 26 or 27.

32. A diagnostic kit comprising a diagnostic marker according to claim 24.

33. A diagnostic kit comprising polypyrimidine tract binding protein- associated splicing factor (PSF), nuclear RNA and DNA binding protein p54^nrb, heterogeneous nuclear ribonucleoprotein Cl and/or heterogeneous nuclear ribonucleoprotein C2, or a functional part, derivative and/or analogue thereof or a (post-translationally) modified form thereof, and/or at least one binding molecule capable of specifically binding polypyrimidine tract binding protein-associated splicing factor (PSF), nuclear RNA and DNA binding protein p54^nrb, heterogeneous nuclear ribonucleoprotein Cl and/or heterogeneous nuclear ribonucleoprotein C2 or a functional part, derivative and/or analogue thereof or a (post-translationally) modified form thereof.

34. A method for at least in part treating an autoimmune disease comprising counteracting the presence and/or activity of an antibody capable of specifically binding polypyrimidine tract binding protein-associated splicing factor (PSF), nuclear RNA and DNA binding protein p54^nrb, heterogeneous nuclear ribonucleoprotein Cl and/or heterogeneous nuclear ribonucleoprotein C2, or a functional part, derivative and/or analogue thereof or a (post-translationally) modified form thereof.

35. A molecule capable of counteracting the presence and/or activity of an antibody capable of specifically binding polypyrimidine tract binding protein-associated splicing factor (PSF), nuclear RNA and DNA binding protein p54^nrb, heterogeneous nuclear ribonucleoprotein Cl and/or heterogeneous nuclear ribonucleoprotein C2, or a functional part, derivative and/or analogue thereof or a (post-translationally) modified form thereof, for use as a medicament.