Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary

Definitions

• the invention relates to methods and means for providing binding molecules with improved properties, be it in binding or other properties, as well as the novel binding molecules themselves.
• the invention further relates to methods applying these molecules in all their versatility.
• Proteinaceous binding molecules have been applied in purification of substances from mixtures, in diagnostic assays for a wide array of substances, as well as in the preparation of pharmaceuticals, etc.
• Naturally occurring proteinaceous molecules such as immunoglobulins (or other members of the immunoglobulin superfamily) as well as receptors and enzymes have been used.
• peptides derived from such molecules have been used.
• the present invention provides a structural context that is designed based on a common structural element (called a core structure) that has been identified herein to occur in numerous binding proteins.
• This so called common core has now been produced as a novel proteinaceous molecule that can be provided with one or more desired affinity regions.
• This proteinaceous structure does not rely on any amino acid sequence, but only on common structural elements. It can be adapted by providing different amino acid sequences and/or amino acid residues in sequences for the intended application. It can also be adapted to the needs of the particular affinity region to be displayed.
• the invention thus also provides libraries of both structural contexts and affinity regions to be combined to obtain an optimal proteinaceous binding molecule for a desired purpose.
• the invention provides a synthetic or recombinant proteinaceous molecule comprising a binding peptide and a core, said core comprising a ⁇ - barrel comprising at least 5 strands, wherein said ⁇ -barrel comprises at least two ⁇ -sheets, wherein at least one of said ⁇ -sheets comprises three of said strands and wherein said binding peptide is a peptide connecting two strands in said ⁇ -barrel and wherein said binding peptide is outside its natural context.
• This core structure in many proteins, ranging from galactosidase to human (and e.g. camel ) antibodies with all kinds of molecules in between. Nature has apparently designed this structural element for presenting desired peptide sequences.
• the structure comprising one affinity region (desired peptide sequence) and two ⁇ -sheets forming one ⁇ -barrel is the most basic form of the invented proteinaceous binding molecules, (proteinaceous means that they are in essence amino acid sequences, but that side chains and/or groups of all kinds may be present; it is of course possible, since the amino acid sequence is of less relevance for the structure to design other molecule of non proteinaceous nature that have the same orientation is space and can present peptidic affintiy regions; the orientation in space is the important parameter).
• the invention also discloses optimised core structures in which less stable amino acids are replaced by more stable residues (or vice versa) according to the desired purpose.
• optimised core structures in which less stable amino acids are replaced by more stable residues (or vice versa) according to the desired purpose.
• other substitutions or even amino acid sequences completely unrelated to existing structures are included, since, once again, the important parameter is the orientation of the molecule in space.
• the invention preferably provides a proteinaceous molecule according the invention wherein said ⁇ -barrel comprises at least 5 strands, wherein at least of said sheets comprises 3 of said strands, more preferably a proteinaceous molecule according to the invention, wherein said ⁇ -barrel comprises at least 6 strands, wherein at least two of said sheets comprises 3 of said strands, ⁇ - barrels wherein each of said sheets comprises at least 3 strands are sufficiently stable while at the same time providing sufficient variation possibilities to adapt the core/affinity region (binding peptide) to particular purposes. Though suitable characteristic can also be found with cores that comprise less strands per sheet. Thus variations wherein one sheet comprises only two strands are within the scope of the present invention.
• the invention provides a proteinaceous molecule according to the invention wherein said ⁇ -barrel comprises at least 7 strands, wherein at least one of said sheets comprises 4 of said strands.
• the invention provides aproteinaceous molecule according to the invention, wherein said beta-barrel comprises at least 8 strands, wherein at least one of said sheets comprisesa 4 of said strands.
• a proteinaceous molecule according to the invention wherein said ⁇ -barrel comprises at least 9 strands, wherein at least one of said sheets comprises 4 of said strands is provided.
• the core structure there is a more open side where nature displays affinity regions and a more closed side, where connecting sequences are present.
• at least one affinity region is located at said more open side.
• the invention provides a proteinaceous molecule according to the invention, wherein said binding peptide connects two strands of said ⁇ -barrel on the open side of said barrel.
• the location of the desired peptide sequence may be anywhere between two strands, it is preferred that the desired peptide sequence connects the two sheets of the barrel.
• the invention provides a proteinaceous molecule according to the invention, wherein said binding peptide connects said at least two ⁇ -sheets of said barrel.
• one affinity region may suffice it is preferred that more affinity regions are present to arrive at a better binding molecule.
• these regions are arranged such that they can cooperate in binding (e.g. both on the open side of the barrel).
• the invention provides a proteinaceous molecule according to the invention, which comprises at least one further binding peptide.
• a successful core element in nature is the one having three affinity regions and three connecting regions.
• This core in its isolated form is a preferred embodiment of the present invention.
• the connecting sequences on the less open side of the barrel can be used as affinity regions as well. This way a very small bispecific binding molecule is obtained.
• the invention provides a proteinaceous molecule according the invention, which comprises at least 4 binding peptides. Bispecific herein means that the binding molecule has the possibility to bind to two target molecules (the same or different).
• the various strands in the core are preferably encoded by a single open reading frame.
• the loops connecting the various strands may have any type of configuration. So as not to unduly limit the versatility of the core it is preferred that loops connect strands on the same side of the core, i.e. and N- terminus of strand (a) connects to a C-terminus of strand (b) on either the closed side or the open side of the core. Loops may connect strands in the same ⁇ -sheet or cross-over to the opposing ⁇ -sheet.
• a preferred arrangement for connecting the various strands in the core are given in the examples and the figures, and in particular figure 1.
• Strands in the core may be in any orientation (parallel or antiparallel) with respect to each other. Preferably the strands are in the configuration as depicted in figure 1.
• optimise binding molecules both in the binding properties and the structural properties such as stability under different circumstances (temperature,pH, etc), the antigenicity, etc.). This is done, according tot the invention by taking at least one nucleic acid according to the invention (encoding a proteinaceous binding molecule according to the invention) and mutating either the encoded structural regions or the affinity regions or both and testing whether a molecule with desired binding properties and structural properties has been obtained.
• the invention provides a method for identifying a proteinaceous molecule with an altered binding property, comprising introducing an alteration in the core of proteinaceous molecules according to the invention, and selecting from said proteinaceous molecules, a proteinaceous molecule with an altered binding property, as well as a method for identifying a proteinaceous molecule with an altered structural property, comprising introducing an alteration in the core of proteinaceous molecules according to the invention, and selecting from said proteinaceous molecules, a proteinaceous molecule with an altered structural property.
• These alterations can vary in kind, an example being a post- translational modification. The person skilled in the art can design other relevant mutations.
• the mutation would typically be made by mutating the encoding nucleic acid and expressing said nucleic acid in a suitable system, which may be bacterial, eukaryotic or even cell-free. Once selected one can of course use other systems than the selection system.
• the invention also provides methods for producing nucleic acids encoding proteinaceous binding molecules according to the invention, such as a method for producing a nucleic acid encoding a proteinaceous molecule capable of displaying at least one desired peptide sequence comprising providing a nucleic acid sequence encoding at least a first and second structural region separated by a nucleic acid sequence encoding said desired peptide sequence or a region where such a sequence can be inserted and mutating said nucleic acid encoding said first and second structural regions to obtain a desired nucleic acid encoding said proteinaceous molecule capable of displaying at least one desired peptide sequence and preferably a method for displaying a desired peptide sequence, providing a nucleic acid encoding at least a two ⁇ -sheets, said , said ⁇ -sheets forming a ⁇ -barrel, said nucleic acid comprising a region for inserting a sequence encoding said desired peptide sequence, inserting a nucleic acid sequence compris
• the invention further provides the application of the novel binding molecules in all fields where binding molecules have been envisaged until today, such as separation of substances from mixtures, typically complex biological mixtures, such as body fluids or secretion fluids, such as blood or milk, or serum or whey.
• the gene delivery vehicle can also encode a binding molecule according to the invention to be delivered to a target, possibly fused to a toxic moiety.
• Conjugates of toxic moieties to binding molecules are also well known in the art and are included for the novel binding molecules of the invention.
• the present invention relates to the design, construction, production, screening and use of proteins that contain one or more regions that may be involved in molecular binding.
• the invention also relates to naturally occurring proteins provided with artificial binding domains, re-modelled natural occurring proteins provided with extra structural components and provided with one or more artificial binding sites, re-modelled natural occurring proteins disposed of some elements (structural or others) provided with one or more artificial binding sites, artificial proteins containing a standardized core structure motif provided with one or more binding sites. All such proteins are called VAPs (Versatile Affinity Proteins) herein.
• the invention further relates to novel VAPs identified according to the methods of the invention and the transfer of binding sites on naturally occurring proteins that contain a similar core structure. 3D modelling or mutagenesis of such natural occurring proteins can be desired before transfer in order to restore or ensure antigen binding capabilities by the affinity regions present on the selected NAP. Further, the invention relates to processes that use selected NAPs, as described in the invention, for purification, removal, masking, liberation, inhibition, stimulation, capturing, etc.of the chosen ligand capable of being bound by the selected VAP(s). LIGAND BINDING PROTEINS
• the scaffold structures ensures a stable 3 dimensional conformation for the whole protein, and act as a steppingstone for the actual recognition region.
• the invariable ligand binding proteins contain a fixed number, a fixed composition and an invariable sequence of amino acids in the binding pocket in a cell of that species.
• examples of such proteins are all cell adhesion molecules, e.g. N-CAM and V- CAM, the enzyme families, e.g. kinases and proteases and the family of growth receptors,e.g EGF-R, bFGF-R.
• the genetically variable class of ligand binding proteins is under control of an active genetic shuffling-, mutational or rearrangement mechanism enabling an organism or cell to change the number, composition and sequence of amino acids in, and possibly around, the binding pocket.
• these are all types of light and heavy chain of antibodies, B-cell receptor light and heavy chains and T-cell receptor alfa, beta, gamma and delta chains.
• the molecular constitution of wild type scaffolds can vary to a large extent.
• Zinc finger containing DNA binding molecules contain a totally different scaffold (looking at the amino acid composition and structure) than antibodies although both proteins are able to bind to a specific target.
• the class of ligand binding proteins that express variable (putative) antigen binding domains has been shown to be of great value in the search for ligand binding proteins.
• the classical approach to generate ligand binding proteins makes use of the animal immune system. This system is involved in the protection of an organism against foreign substances. One way of recognizing, binding and clearing the organism of such foreign highly diverse substances is the generation of antibodies against these molecules. The immune system is able to select and multiply antibody producing cells that recognize an antigen. This process can also be mimicked by means of active immunizations. After a series of immunizations antibodies may be formed that recognize and bind the antigen. The possible number of antibodies with different affinity regions that can be formed due to genetic rearrangements arid mutations, exceeds the number of 10 40 .
• Immunization belongs to a painful and stressful operation and must be prevented as much as possible.
• immunizations do not always produce antibodies or do not always produce antibodies that contain required features such as binding strength, antigen specificity, etc. The reason for this can be multiple: the immune system missed by co-incidence such a putative antibody; the initially formed antibody appeared to be toxic or harmful; the initially formed antibody also recognizes animal specific molecules and consequently the cells that produce such antibodies will be destroyed; or the epitope cannot be mapped by the immune system (this can have several reasons).
• scaffolds Although most energy and effort is put in the development and optimization of natural derived or copied human antibody derived libraries, other scaffolds have also been described as successful scaffolds as carriers for one or more ligand binding domains.
• Examples of scaffolds based on natural occurring antibodies encompass minibodies (Pessi et al., 1993), Camelidae VHH proteins (Davies and Riechmann, 1994; Hamers-Casterman et al., 1993) and soluble VH variants (Dimasi et al., 1997; Lauwereys et al., 1998).
• T-cell receptor chains Two other natural occurring proteins that have been used for affinity region insertions are also member of the immunoglobulin superfamily: the T-cell receptor chains (Kranz et al., WO Patent 0148145) and fibronectin domain-3 regions (Koide US Patent 6,462,189; Koide et al., 1998).
• the two T-cell receptor chains can each hold three affinity regions according to the inventors while for the fibronectin region the investigators described only two regions.
• non-immunoglobulin domain containing scaffolds have been investigated. All proteins investigated contain only one protein chain and one to four affinity related regions. Smith and his colleagues (1998) reported the use of knottins (a group of small disulfide bonded proteins) as a scaffold. They successfully created a library based on knottins that had 7 mutational amino acids. Although the stability and length of the proteins are excellent, the low number of amino acids that can be randomized and the singularity of the affinity region make knottin proteins not very powerful. Ku and Schultz (1995) successfully introduced two randomized regions in the four-helix-bundle structure of cytochrome b ⁇ 62.
• binders were shown to bind with micromolar Kd values instead of the required nanomolar or even better range.
• Another alternate framework that has been used belongs to the tendamistat family of proteins. McConnell and Hoess (1995) demonstrated that alpha-amylase inhibitor (74 amino acid beta-sheet protein) from Streptomyces tendae could serve as a scaffold for ligand binding libraries. Two domains were shown to accept degenerated regions and function in ligand binding. The size and properties of the binders showed that tendamistats could function very well as ligand mimickers, called mimetopes. This option has now been exploited.
• Lipocalin proteins have also been shown to be successful scaffolds for a maximum of four affinity regions (Beste et al, 1999; Skerra, 2000 BBA; Skerra, 2001 RMB). Lipocalins are involved in the binding of small molecules like retinoids, arachidonic acid and several different steroids. Each lipocalin has a specialized region that recognizes and binds one or more specific ligands. Skerra (2001) used the lipocalin RBP and lipocalin BBP to introduce variable regions at the site of the ligand binding domain. After the construction of a library and successive screening, the investigators were able to isolate and characterize several unique binders with nanomolar specificity for the chosen ligands. It is currently not known how effective lipocalins can be produced in bacteria or fungal cells. The size of lipocalins (about 170 amino acids) is pretty large in relation to VHH chains (about 100 amino acids), which might be too large for industrial applications.
• VHH antibodies are very stable, can have high specificities and are relatively small.
• the scaffold has evolutionarily been optimised for an immune dependent function but not for industrial applications.
• the highly diverse pool of framework regions that are present in one pool of antibodies prevents the use of modular optimisation methods. Therefore a new scaffold was designed based on the favourable stability of V HH proteins. 3D -modelling and comparative modelling software was used to design a scaffold that meets the requirements of versatile affinity proteins (VAPs).
• VAPs versatile affinity proteins
• ig-like fold containing proteins are: V-CAM, immunoglobulin heavy chain variable domains, immunoglobulin light chain variable domains, constant regions of immunglobulines, T-cell receptors, fibronectin, reovirus coat protein, beta-galactosidase, integrins, EPO-receptor, CD58, ribulose carboxylase, desulphoferrodoxine, superoxide likes, biotin decarboxylase and P53 core DNA binding protein.
• a classification of most ig-like folds can be obtained from the SCOP database (Murzin A.
• SCOP classifies these folds as: all beta proteins, with an immunoglobulin-like beta- sandwich in which the sandwich contains 7 strands in 2 sheets although some members that contain the fold have additional strands.
• CATH classifies these folds as: Mainly beta proteins with an architecture like a sandwich in an immunoglobulin-like fold designated with code 2.60.40.
• the core structure is therefore designated as the far most important domain within these proteins.
• the number of beta- elements that form the core can vary between 7 and 9 although 6 stranded core structures might also be of importance. All beta- elements of the core are arranged in two beta-sheets. Each beta-sheet is build of anti-parallel oriented beta-elements. The minimum number of beta- elements in one beta-sheet that was observed was 3 elements. The maximum number of beta-element in one sheet that was observed was 5 elements, although it can not be excluded that higher number of beta-elements might be possible. Connecting loops connect the beta-elements on one side of the barrel.
• Some connections cross the beta-sheets while others connect beta-elements that are located within one beta-sheet.
• the loops that are indicated as L2, L4, L6 and L8 are used in nature for ligand binding and are therefore preferred sites for the introduction or modification of binding pep tide/affinity regions.
• the high variety in length, structure, sequences and amino acid compositions of the LI, L3, L5 and L7 loops clearly indicates that these loops can also be used for ligand binding, at least in an artificial format.
• Amino acid side chains in the beta-elements that form the actual core that are projected towards the interior of the core, and thus fill the space in the centre of the core, can interact with each other via H-bonds, covalent bonds (cysteine bridges) and other forces, and determine the stability of the fold. Because amino acid composition and sequence of the residues of the beta-element parts that line up the interior were found to be extremely variable, it was concluded that many other sequence formats and can be created.
• beta element 1 or 9 can be omitted but also elements 5 or 6 can be omitted.
• an eight stranded core preferably comprises elements 2-8, and either 1 or 9.
• Another preferred eight stranded core comprises elements 1-4, 7-9, and either strand 5 or strand 6.
• 2 beta-elements can be removed among which combinations of element 1 and 9, 1 and 5, 6 and 9, 9 and 5 and, elements 4 and 5. The exclusion of elements 4 and 5 is preferred because of spatial constrains.
• Six stranded structures lack preferably element 1, 4 and 5 or 4, 5 and 9 but also other formats were analyzed with Insight and Modeller and shown to be reliable enough for engineering purposes.
• the constructed primary scaffolds are subjected to a mild mutational process by PCR amplification that includes error-prone PCR, such as unequimolar dNTP concentration, addition of manganese or other additives, or the addition of nucleotide analogues, such as dITP (Spee et al., 1993) or dPTP (Zaccolo et al., 1996) in the reaction mixture which can ultimately change the amino acid compositions and amino acid sequences of the primary scaffolds. This way new (secondary) scaffolds are generated.
• error-prone PCR such as unequimolar dNTP concentration, addition of manganese or other additives, or the addition of nucleotide analogues, such as dITP (Spee et al., 1993) or dPTP (Zaccolo et al., 1996) in the reaction mixture which can ultimately change the amino acid compositions and amino acid sequences of the primary scaffolds. This way new (secondary) scaffolds are generated.
• a set of known affinity regions such as 1MEL for binding lysozyme and 1BZQ for binding RNase were inserted in the primary modularly constructed scaffolds.
• Functionality, heat and chemical stability of the constructed VAPs were determined by measuring unfolding conditions. Functionality after chemical or heat treatment was determined by binding assays (ELISA), while temperature induced unfolding was measured using a circular dichroism (CD) polarimeter. Phage display techniques were used to select desired scaffolds or for optimisation of scaffolds.
• affinity regions can be obtained from natural sources, degenerated primers or stacked DNA triplets. AU of these sources have certain important limitations as described above.
• affinity regions can be used in modular systems, are extremely flexible in use and optimization, are fast and easy to generate and modulate, have a low percentage of stop codons, have an extremely low percentage of frameshifts and wherein important structural features will be conserved in a large fraction of the new formed clones and new structural elements can be introduced.
• the major important affinity region (CDR3) in both light and heavy chain in normal antibodies has a average length between 11 (mouse) and 13 (human) amino acids. Because in such antibodies the CDR3 in light and heavy chain cooperatively function as antigen binder, the strength of such a binding is a result of both regions together. In contrast, the binding of antigens by VHH antibodies (Camelidae) is a result of one CDR3 region due to the absence of a light chain. With an estimated average length of 16 amino acids these CDR3 regions are significantly longer than regular CDR3 regions (Mol. Immunol. Bang Vu et al., 1997, 34, 1121-1131).
• CDR3 regions have potentially more interaction sites with the ligand and can therefore be more specific and bind with more strength.
• Another exception are the CDR3 regions found in cow (Bos taurus) (Berens et al., 1997). Although the antibodies in cow consists of a light and a heavy chain, their CDR3 regions are much longer than found in mouse and humans and are comparable in length found for camelidae CDR3 regions.
• Average lengths of the major affinity region(s) should preferably be about 16 amino acids. In order to cover as much as possible potentially functional CDR lengths the major affinity region can vary between 1 and 50 or even more amino acids.
• CDR3 regions were amplified from mRNA coding for VHH antibodies originating from various animals of the camelidae group or from various other animals containing long CDR3 regions by means of PCR techniques.
• this pool of about 10 8 different CDR3 regions, which differ in the coding for amino acid composition, amino acid sequence, putative structural classes and length, is subjected to a mutational process by PCR as described above.
• the result is that most products will differ from the original templates and thus contain coding regions that potentially have different affinity regions.
• Other very important consequences are that the products keep their length, the pool keeps their length distribution, a significant part will keep structural important information while others might form non-natural classes of structures, the products do not or only rarely contain frame shifts and the majority of the products will lack stop codons.
• MAST Modular Affinity and Scaffold Transfer technology
• a method for producing a library comprising artificial binding peptides comprising providing at least one nucleic acid template wherein said templates encode different specific binding peptides, producing a collection of nucleic acid derivatives of said templates through mutation thereof and providing said collection or a part thereof to a peptide synthesis system to produce said library comprising artificial binding peptides.
• the complexity of the library increases with increasing number of different templates used to generate the library. In this way an increasing number of different structures used.
• at least two nucleic acid templates, and better at least 10 nucleic acid templates are provided. Mutations can be introduced using various means and methods.
• the method introduces mutations by changing bases in the nucleic acid template or derivative thereof.
• Suitable modification strategies include amplification strategies such as PCR strategies encompass for example unbalanced concentrations of dNTPs (Cadwell et al., (1992); Leung et al., (1989) 1; Kuipers, (1996) , the addition of dITP (Xu et al (1999); Spee et al (1993; Kuipers, (1996), dPTP (Zaccolo et al., 5 (1996)), 8-oxo-dG (Zaccolo et al., (1996)), Mn 2 + (Cadwell et al., (1992); Leung et al., (1989) 1, , Xu et al., (1999)), polymerases with high misincorporation levels (Mutagene ®, Stratagene).
• PCR strategies encompass for example unbalanced concentrations of dNTPs (Cadwell et al., (1992); Leung et al., (1989) 1;
• degenerate primers can be used thus also provided is a method wherein at least one non- degenerate primer further comprises a degenerate region.
• the methods for generating libraries of binding peptides is especially suited for the generation of the above mentioned preferred larger affinity regions. In these a larger number of changes can be introduced while maintaining the same of similar structure.
• at least one template encodes a specific binding peptide having an affinity region comprising at least 14 amino acids and preferably at least 16 amino acids. Though non consecutive regions can be used in this embodiment of the invention it is preferred that the region comprises at least 14 consecutive amino acids. When multiple templates are used it is preferred that the regions comprise an average length of 24 amino acids.
• Method for generating a library of binding peptides may favourable be combined with core regions of the invention and method for the generation thereof. For instance, once a suitable binding region is selected a core may be designed or selected to accommodate the particular use envisaged. However, it is also possible to select a particular core region, for reasons of the intended use of the binding peptide. Subsequently libraries having the core and the mentioned library of binding peptides may be generated. Uses of such libraries are of course manifold. Alternatively, combinations of strategies may be used to generate a library of binding peptides having a library of cores. Complexities of the respective libraries can of course be controlled to adapt the combination library to the particular use.
• At least one of said templates encodes a proteinaceous molecule according to the invention.
• the mentioned peptide, core and combination libraries may be used to select proteinaceous molecules of the invention, thus herein is further provided a method comprising providing a potential binding partner for a peptide in said library of artificial peptides and selecting a peptide capable of specifically binding to said binding partner from said library.
• a selected proteinaceous molecule obtained using said method is of course also provided.
• at least the core and the binding peptide is displayed on a replicative package comprising nucleic acid encoding the displayed core/peptide proteinaceous molecule.
• the replicative package comprises a phage, such as used in phage display strategies.
• a phage display library comprising at least one proteinaceous molecule of the invention.
• the method for generating a library of binding peptides can advantageously be adapted for core regions.
• a method for producing a library comprising artificial cores comprising providing at least one nucleic acid template wherein said templates encode different specific cores, producing a collection of nucleic acid derivatives of said templates through mutation thereof and providing said collection or a part thereof to a peptide synthesis system to produce said library of artificial cores.
• Preferred binding peptides libraries are derived from templates comprising CDR3 regions from cow (Bos Taurus) or camelidae (preferred lama pacos and lama glama).
• Protein-ligand interactions are one of the basic principles of life. All protein- ligand mediated interactions in nature either between proteins, proteins and nucleic acids, proteins and sugars or proteins and other types of molecules are mediated through an interface present at the surface of a protein and the molecular nature of the ligand surface. The very most of protein surfaces that are involved in protein-ligand interactions are conserved throughout the life cycle of an organism. Proteins that belong to these classes are for example receptor proteins, enzymes and structural proteins. The interactive surface area for a certain specific ligand is usually constant. However, some protein classes can modulate their nature of the exposed surface area through e.g. mutations, recombinations or other types of natural genetic engineering programs.
• Proteins that belong to such classes are e.g. antibodies, B-cell receptors and T-cell receptor proteins. Although there is in principle no difference between both classes of proteins, the speed of surface changes for both classes differ.
• the first class is mainly sensitive to evolutionary forces
• Binding specificity and affinity between receptors and ligands is mediated by an interaction between exposed interfaces of both molecules. Protein surfaces are dominated by the type of amino acids present at that location. The 20 different amino acids common in nature each have their own side chain with their own chemical and physical properties. It is the accumulated effect of all amino acids in a certain exposed surface area that is responsible for the possibility to interact with other molecules. Electrostatic forces, hydrophobicity, H-bridges, covalent coupling and other types of properties determine the type, specificity and strength of binding with ligands.
• the most sophisticated class of proteins involved in protein-ligand interactions is those of antibodies.
• An ingenious system has been evolved that controls the location and level mutations, recombinations and other genetic changes within the genes that can code for such proteins.
• Genetic changing forces are mainly focussed to these regions that form the exposed surface area of antibodies that are involved in the binding of putative ligands.
• the enormous numbers of different antibodies that can be formed indicate the power of antibodies. For example: if the number of amino acids that are directly involved in ligand binding in both the light and heavy chains of antibodies are assumed to be 8 amino acids for each chains (and this is certainly not optimistic) then 20 2 * 8 which approximates 10 20 (20 amino acids types, 2 chains, 8 residues) different antibodies can be formed.
• Natural derived antibodies and their affinity regions have been optimized to certain degree, during immune selection procedures. These selections are based upon the action of such molecules in an immune system. Antibody applications outside immune systems can be hindered due to the nature and limitations of the immune selection criteria. Therefore, industrial, cosmetic, research and other applications demand often different properties of ligand binding proteins.
• the environment in which the binding molecules may be applied can be very harsh for antibody structures, e.g. extreme pH conditions, salt conditions, odd temperatures, etc.
• CDRs might or might not be transplanted from natural antibodies on to a scaffold. For at least some application unusual affinity regions will be required. Thus, artificial constructed and carefully selected scaffolds and affinity regions will be required for other applications.
• Affinity regions present on artificial scaffolds can be obtained from several origins.
• First, natural affinity regions can be used. CDRs of cDNAs coding for antibody fragments can be isolated using PCR and inserted into the scaffold at the correct position. The source for such regions can be of immunized or non- immunized animals.
• Second, fully synthetic AR's can be constructed using degenerated primers.
• Third, semi-synthetic AR's can be constructed in which only some regions are degenerated.
• triplets coding for selected amino acids (monospecific or mixtures) can be fused together in a predetermined fashion.
• Fifth, natural derived affinity regions (either from immunized or naive animals) which are being mutated during amplification procedures e.g.
• NASBA or PCR by introducing mutational conditions (e.g. manganese ions) or agents (e.g. dITP) during the reaction.
• mutational conditions e.g. manganese ions
• agents e.g. dITP
• immunization based CDRs can be successful but the majority of ligands or ligand domains will not be immunogenic.
• Artificial affinity regions in combinations with powerful selection and optimization strategies become more and more important if not inevitable.
• Primer based strategies are not very powerful due to high levels of stop codons, frameshifts, difficult sequences, too large randomizations, relative small number of mutational spots (maximum of about 8 spots) and short randomization stretches (no more than 8 amino acids).
• the power of non- natural derived AR's depends also on the percentage of AR's that putatively folds correctly, i.e. being able to be presented on the scaffold without folding problems of the AR's or even the scaffold. Hardly any information is currently available about structures and regions that are present in AR's. Therefore the percentage of correctly folded and presented artificial AR's constructed via randomizations, especially long AR's, will be reciprocal with the length of constructed ARs. Insight in CDR and AR structures will most likely be available in the future, but is not available yet.
• Single scaffold proteins which are used in applications that require high affinity and high specificity in general require at least one long affinity region or multiple medium length ARs in order to have sufficient exposed amino acid side chains for ligand interactions.
• Synthetic constructed highly functional long ARs, using primer or triplet fusion strategies, will not be very efficient for reasons as discussed above. Libraries containing such synthetic ARs would either be too low in functionality or too large to handle.
• the only available source for long ARs is those that can be obtained from animal sources (most often CDR3s in heavy chains of antibodies). Especially cow-derived and camelidae-derived CDR3 regions of respectively Vh chains and Vhh chains are unusual long. The length of these regions is in average above 13 amino acids but 30 amino acids or even more are no exceptions.
• CDR regions especially CDR3 regions
• PCR PCR-specific primers
• the introduction of minor, mild, medium level or high level random mutations via nucleic acid amplification techniques like for example PCR will generate new types of affinity regions.
• the benefits of such AR pools are that length distributions of such generated regions will be conserved.
• stop codon introductions and frame shifts will be prevented to a large degree due to the relatively low number of mutations if compared with random primers based methods.
• a significant part or even the majority of the products will code for peptide sequences that exhibit structural information identical or at least partly identical to their original template sequence present in the animal.
• binding properties can be altered in respect to the original template not only in strength but also in specificity and selectivity. This way libraries of long AR regions can be generated with strongly reduced technical or physical problems as mentioned above if compared with synthetic, semi synthetic and natural obtained ARs.
• PCR strategies encompass for example unbalanced concentrations of dNTPs (Cadwell et al., (1992) 2,; Leung et al., (1989),; Kuipers, (1996)), the addition of dITP (Xu et al., (1999); Spee et al., (1993); Kuipers 57 (1996)), dPTP (Zaccolo et al., (1996)), 8-oxo-dG (Zaccolo et al., (1996)), Mn2+ (Cadwell et al, (1992); Leung et al (1989) ;Xu et al., (1999)), polymerases with high misincorp oration levels (Mutagene ®, Stratagene).
• VAP coding regions or parts thereof can be the subject of a mutational program as described above due to its modular nature.
• the whole VAP or VAPs can be used as a template.
• Third framework regions can be mutated.
• Fourth, fragments throughout the VAP can be used as a template.
• itterative processes can be applied to change more regions. The average number of mutations can be varied by changing PCR conditions.
• the VAPs of the invention can be used in an enormous variety of applications, from therapeutics to antibiotics, from detection reagents to purification modules, etc. etc.
• the environment and the downstream applications determines the features that a ligand binding protein should have, e.g. temperature stability, protease resistance, tags, etc.
• scaffolds contain a modular design in order to be able to mutate, remove, insert and swap regions easily and quick. Modularity makes it possible to optimize for required properties via standardized procedures and it allows domain exchange programs, e.g. exchange of pre-made cassettes.
• optimal modular scaffold genes should meet certain features, they have to be designed and synthetically constructed while it is very unlikely that natural retrieved genes contains such features.
• the specific binding agent must also be capable of being linked to a solid phase such as a carrier material in a column, an insoluble bead, a plastic, metal or paper surface or any other useful surface. Ideally, this linkage is achievable without any adverse effects on the specific binding activity. Therefore the linkage is preferably accomplished with regions in the VAP molecule that are relatively remote from the specific affinity regions.
• An important embodiment of the invention is an affinity- absorb ant material comprising a specific binding agent immobilised on a porous silica or the like, the specific binding agent comprising a selection of VAP molecules.
• a particularly important embodiment of the invention is an affinity-absorbant material comprising a special binding agent immobilised on a porous carrier material, such as silica or an inert, rigid polymer or the like, having a pore size of at least 30A but not greater than 1000A, wherein the specific binding agent comprises a selection of VAP molecules.
• the carrier has a pore size of at least 60A.
• the pore size is not greater than 500A, and more preferably, not greater than 300A.
• polymers as support or carrier material for VAPs include, but are not limited to nylon, vinylpolymers, polyethylene, polypropylene, polystyrene, polymethylmethacrylate, polyvinylacetate, polytetrafluoroethylene. poly inylidenefluoride, cellulose, chitin, chitosan , agarose, proteins.
• Activated (i.e. ready for protein coupling) support materials are commercially available or can be chemcially activated by a person skilled in the art.
• the pore size of a carrier medium such as silica or inert polymers can be determined using e.g. standard size exclusion techniques or other published methods.
• the pore volume and surface area can be determined by standard nitrogen absorption methods.
• VAPs in which VAPs can be applied in a way that leaves the VAPs present up to, and also including, the end product, have examples from a very wide range of products. But also in processes where the VAPs are immobilized and preferably can be regenerated for recycled use, the major advantage of VAPs is fully exploited, i.e. the relative low cost of VAPs that makes them especially suitable for large scale applications, for which large quantities of the affinity bodies need to be used. The list below is given to indicate the scope of applications and is in no way limiting. Product or process examples with possible applications in brackets are; (1) industrial food processing such as the processing of whey, tomato pomace, citrus fruits, etc.
• VAPs can be used in line with existing processing steps and the VAPs do not end up in the final product as a result of their irreversible immobilisation to support-materials, they are exceptionally suited for the large scale industrial environments that are customary in agro-foodprocessing industries.
• the whey fraction that is the result of the cheese manufacturing processes contain a relatively large number of low- abundant proteins that have important biological functions, e.g. during the development of neonates, as natural antibiotics, food-additives etc.
• proteins are lactoferrin, lactoperoxidase, lysozyme, angiogenine, insulin-like growth factors (IGF), insulin receptor, IGF- binding proteins, transforming growth factors (TGF), bound- and soluble TGF-receptors, epidermal growth factor (EGF), EGF-receptor ligands, interleukine-1 receptor antagonist.
• Another subclass of valuable compounds that can be recovered from whey are the immunoregulatory peptides that are present in milk or colostrum. Also specific VAPs can be selected for the recovery of hormones from whey. Examples of hormones that are present in milk are; prolactin, somatostatin, oxytocin, luteinizing hormone-releasing hormone, thyroid- stimulating hormone, thyroxine, calcitonin, estrogen, progesterone
• non-food applications such as printing inks, glues, paints, paper, hygiene tissues etc. (surface-specific inks, glues, paints etcetera for surfaces that are otherwise difficult to print e.g. polyofines -plastic bottles or containers, or for surfaces where highly specific binding is required, e.g. lithographic processes in electronic chip manufacturing, authentication of value papers,)
• environmental protection processes such as water purification, bioremediation, clean-up of process waters, concentration of contaminants (removal of microorganisms, viruses, organic pollutants in water purification plants or e.g. green-house water recycling systems, removal of biological hazards from air-ventilation ducts)
• VAPs are superior to conventional antibodies or fragments thereof.
• Coventional antibodies can only discriminate the infectious form under denatured conditions, while the small and exposed AR's of VAPs are able to recognize more inwardly placed peptide sequences.
• VAPs can be applied, as is illustrated by the following categories that form a matrix in combination with the applications:
• VAPs are immobilized on an appropriate support e.g. in chromatography columns that can be used in line, in series or in carroussel configurations for fully continuous operation. Also pipes, tubes, in line filters etc. can be lined with immobilized VAPs.
• the support material on which the VAPs can be immobilized can be chosen to fit the process requirements in terms of compression stability, flow characteristics, chemical inertness, temperature-, pH- and solvent stability etc.
• Relatively incompressible carriers are preferred, especially silica or rigid inert polymers.
• insoluble beads are a different form of affinity chromatography where the support material on which VAPs are immobilized are not fixed in position but are available as beads from for example , silica, metal, magnetic particles, resins and the like, can be mixed in process streams to bind specific ligands in e.g. fluidised beds or stirred tanks, after which the beads can be seperated from the process stream in simple procedures using gravity, magnetism, filters etc.
• VAPs should be bivalent, i.e. at least two AR's must be constructed on either side of the scaffold.
• the two AR's can have the same molecular target but wo different molecular targets are preferred to provide the cross-linking or coaggulation effects.
• masking of specific molecules to protect sensitive motives during processing steps to increase the stability of the target ligand for adverse pH, temperature or solvent conditions, or to increase the resistance against deteriorating or degrading enzymes.
• Other functional effects of molecular masking can be the masking of volatile molecules to alter the sensory perception of such molecules.
• the slow and conditional release of such molecules from VAPs can be invisaged in more down-stream processing steps, during consumption or digestion or after targeting the
• VAPs-ligand complex to appropriate sites for biomedical or research applications. Also molecular mimics of volatile compounds using VAPs with specific receptor binding capacity can be used to mask odours from consumer products. 5. coating of insoluble materials with VAPs to provide highly specific surface affinity properties or to bind VAPs or potential fusion products (i.e. products that are chemically bound to the VAPs or, in case of protein, are co-translated along with the VAPs in such manner that the specicifity of the VAPs remains unchanged) to specific surfaces. Examples are the use of VAPs to immobilize specific molecules to e.g. tissues, on plates etc. to increase detection levels, localize specific compounds on a fixed surface, fix tracer molecules in position etc.
• the invention further provides a proteinaceous molecule, method therefore, therewith or use thereof, wherein said proteinaceous molecule comprises a molecule as depicted in table 2, 3, 10, 13 or 16.
• Immunoglobulin-like (ig-like) folds are very common throughout nature.many proteins, especially in the animal kingdom, have a fold region within the protein that belongs to this class. Reviewing the function of the proteins that contain an ig-like fold and reviewing the function of this ig-like fold within that specific protein, it is apparent thatmost of these domains, if not all, are involved in ligand binding.
• ig-like fold containing proteins are: V-CAM, immunoglobulin heavy chain variable domains, immunoglobulin light chain variable domains, constant regions of immunglobulines, T-cell receptors, fibronectin, reovirus coat protein, beta-galactosidase, integrins, EPO-receptor, CD58, ribulose carboxylase, desulphoferrodoxine, superoxide likes, biotin decarboxylase and P53 core DNA binding protein.
• a classification ofmost ig-like folds can be obtained from the SCOP database (Murzin A.
• SCOP classifies these folds as: all beta proteins, with an immunoglobulin-like beta- sandwich in which the sandwich contains 7 strands in 2 sheets although somemembers that contain the fold have additional strands.
• CATH classifies these folds as:mainly beta proteins with an architecture like a sandwich in an immunoglobulin-like fold designated with code 2.60.40.
• connecting loops On both sides of the core, extremely variable sub-domains were present that are called connecting loops. These connecting loops can vary in amino acid content, sequence, length and configuration.
• the core structure is therefore designated as the farmost important domain within these proteins.
• the number of beta- elements that form core can vary between 7 and 9 although 6 stranded core structure smight also be of importance.
• the beta-elements are all arranged in two beta-sheets. Each beta-sheet is build of anti-parallel beta-element orientations. Theminimum number of beta-elements in one beta-sheet that was observed was 3 elements. Themaximum number of beta-element in one sheet that was observed was 5 elements. Higher number of beta- elementsmight be possible.
• Connecting loops connect the beta-elements on one side of the barrel. Some connections cross the beta-sheets while others connect beta-elements that are located within one beta-sheet. Especially the loops that are indicated as L2, L4, L6 and L8 are used in nature for ligand binding. The high variety in length, structure, sequences and amino acid compositions of the LI, L3, L5 and L7 loops clearly indicates that these loops can also be used for ligand binding, at least in an artificial format.
• Amino acid side chains in the beta-elements that form the actual core that are projected towards the interior of the core and thus fill the space in the centre of the core, can interact with each other via H-bonds, covalent bonds (cysteine bridges) and other forces, to stabilize the fold. Because amino acid composition and sequence of the residues of the beta-element parts that hne up the interior were found to be extremely variable it was concluded thatmany other formats and can also be created.
• Protein folding depends on interaction between amino acid backbone atoms and atoms present in the side chains of amino acids.
• Beta sheets depend on both types of interactions while interactions between two beta sheets, for example in the abovementioned structures, aremainlymediated via amino acid side chain interactions of opposing residues.
• Spatial constrains, physical and chemical properties of amino acid side chains limit the possibilities for specific structures and folds and thus the types of amino acids that can be used at a certain location in a fold or structure.
• 3D analysis software Modeller, Prosa, Insigthll, What if and Procheck
• amino acid residues that provoked incompatibilities are exchanged by an amino acid that exhibits amore accurate and reliable fit.
• amino acid sequences aligning the interior of correctly folded double beta-sheet structures thatmeet criteria as described above and also in example 1, were obtained by submitting PDB files for structural alignments in e.g. VAST
• affinity loops that recognize known ligands can be transplanted on the core structure. Because anti-chicken lysozyme (structure known as 1MEL) is well documented, and the features of these affinity regions (called CDR's in antibodies) are well described, these loops were inserted at the correct position on core sequences obtained via themethod described in the first phase. Correct positions were determined via structural alignments, i.e. overlap projections of the already obtained folds with the file that describes the 3D structure of 1MEL (PDB file; example). Similar projections and subsequent loop transplantations were carried out for the bovine RNase A binding affinity region that were extracted from the structure described by IBZQ (PDB).
• PDB file 3D structure of 1MEL
• the transplanted affinity loops connect one end of the beta elements with one other.
• Affinity region 1 connects beta-element 2 with 3 (L2)
• AR2 connects beta element 4 and 5 (L4)
• AR3 connects beta elements 6 and 7 (L6)
• AR4 connects beta elements 8 and 9 (L8).
• the other end of each of the beta elements was connected by loops that connect element 1 with 2 (LI), 3 with 4 (L3), 5 with 6 (L5) and 7 with 8 (L7) respectively (see schematic projection in figure 3A).
• all kinds of loops can be used to connect the beta elements.
• Sources of loop sequences and loop lengths encompass for example loops obtained via loopmodeling (software) and from available data from natural occurring loops that have been described in the indicated classes of for example SCOP and CATH.
• C-alpha backbones of loops representing loops 1 (LI), 3 (L3), 5 (L5) and 7 (L7; figure 3A) were selected from structures like for example 1NEU, 1EPF-B, 1QHP-A, 1CWV-A, 1EJ6-A, 1E50-C, 1MEL, IBZQ and 1F2X, but many others could have been used with similar results.
• 3D-aligments of the core structures obtained in the first phase as described above, together with loop positions obtained from structural information that is present in the PDB files of the example structures 1EPF, 1NEU, lCWV, 1F2X, 1QHP, 1E50 and 1EJ6 were realized using powerful computers and Cn3D,modeller and/or Insight software of.
• Corresponding loops were inserted at the correct position in the first phasemodels. Loops did not have to fit exactly on to the core because a certain degree of energy and/or spatial freedom can be present. The type of amino acids that actually will form the loops and the position of these amino acids within the loop determine this energy freedom of the loops. Loops from different sources can be used to shuffle loop regions.
• Glycine residues can be introduced at locations that have extreme spatial constrains. These residues do not have side chains and are thusmore or less neutral in activity. However, the extreme flexibility and lack of interactive side chains of glycine residues can lead to destabilization and therefore glycine residues were not commonly used.
• themodels were assessed usingmodeller.modeller was programmed to accept cysteine-cysteine bridges when appropriate. Next all predicted protein structures were assessed with Prosall (http://www.came.sbg.ac.at/Services/prosa.html), Procheck and What if(http://www.cmbi.kun.nl/What if).
• Prosall zp-comb scores of less then -4.71 were assumed to indicate protein sequences thatmight fold in vivo into the desired betamotif.
• the seven protein sequences depicted in table 1 represent a collection of acceptable solutions meeting all criteria mentioned above. Procheck and What ifassessments also indicated that these sequencesmight fit into themodels and thus as being reliable (e.g. pG values larger than 0.80; Sanchez et al., 1998)
• Synthetic VAPs were designed on basis of their, predicted, three dimensional structure.
• the amino acid sequence (Table 3) was back translated into DNA sequence (Table 4) using the preferred codon usage for enteric bacterial gene expression (Informax Vector Nti).
• the obtained DNA sequence was checked for undesired restriction sites that could interfere with future cloning steps. Such sites were removed by changing the DNA sequence without changing the amino acid codons.
• the DNA sequence was adapted to create a Ndel site at the 5' end to introduce the ATG start codon and at the 3' end a Sfi I site, both required for unidirectional cloning purposes.
• PCR assembly consists of four steps: oligo primer design (ordered at Operon' s), gene assembly, gene amplification, and cloning.
• the scaffolds were assembled in the following manner: first both plus and minus strands of the DNA sequence were divided into oligonucleotide primers of approximately 35 bp and the oligonucleotide primer pairs that code for opposite strands were designed in such a way that they have complementary overlaps of approximately 16 - 17 bases.
• PCR assembly product 5 ⁇ l was used in a standard PCR amplification reaction using, both outside primers of the synthetic scaffold, 1 Unit Taq polymerase, 1 x PCR buffer +mgCl2, and O.lmM dNTP in a final volume of 50 ⁇ l, 25 cycles; 30 sec. 92°C, 30 sec. 55°C, Imin.
• PCR products were analyzed by agarose gel electrophoresis, PCR products of the correct size were digested with Ndel and Sfil and ligated into vector p CM 126 linearized with Ndel and Sfil. Ligation products were transformed into TOP10 competent cells (InVitrogen) grown overnight at 37°C on 2xTY plates containing lOOmicrogram/ml ampicillin and 2% glucose. Single colonies were grown in liquidmedium containing 100 ⁇ g ampicillin, plasmid DNA was isolated and used for sequence analysis.
• CM126 A vector for efficient protein expression (CM126; see figure 4A) based on pET- 12a (Novagen) was constructed.
• the signal peptide OmpT was omitted from pET- 12a.
• iMablOO was PCR amplified using forward primer 129 (see table 5) that contains a 5' Ndel overhanging sequence and a very long reverse oligonucleotide primer 306 (see table 5) containing all linkers and tag sequences and a BamHI overhanging sequence.
• PCR product and pET-12a were digested with Ndel and BamHI. After gel purification products were purified via the Qiagen gel-elution system according tomanufactures procedures. The vector and PCR fragment were ligated and transformed by electroporation in E.coli TOP10 cells. Correct clones were selected and verified for their sequence by sequencing. This vector including the dummy VAP acted as the basic vector for expression analysis of other VAPs. Insertion of other VAPs was performed by amplification with primers 129 and 51 (see table 5), digestion with Ndel and Sfil and ligation into Ndel and Sfil digested CM126.
• E. coli BL21 (DE3) (Novagen) was transformed with expression vector CM 126- iMablOO.
• Cells were grown in 250ml shaker flasks containing 50ml 2*TYmedium (16 g/1 tryptone, 10 g/1 yeast extract, 5 g/1 NaCI (Merck)) supplemented with ampicillin (200 microgram/ml) and agitated at 30°C.
• Isopropylthio- ⁇ -galactoside (IPTG) was added at a final concentration of 0.2mM to initiate protein expression when OD (600 nm) reached one.
• iMablOO Protein from inclusion bodies using heat.
• IMablOO was expressed in E. coli BL21 (CM 126-iMablOO) as described in example 5. most of the expressed iMablOO was deposited in inclusionbodies. This is demonstrated in Figure X lane 3, which represents soluble proteins of E. coli BL21 (CM126) after lysis (French press) and subsequent centrifugation (12.000g, 15min).
• Inclusion bodies were purified as follows. Cell pellets (from a 50ml culture) were resuspended in 5ml PBS pH 8 up to 20 g cdw/1 and lysed by 2 passages through a cold French pressure cell (Sim-Aminco).
• Inclusion bodies were collected by centrifugation (12.000 g, 15min) and resuspended in PBS containing 1 % Tween-20 (ICN) in order to solubilize and remove me mbrane- bound proteins. After centrifugation (12.000 g, 15min), pellet (containing inclusion bodies) was washed 2 times with PBS. The isolated inclusion bodies were resuspended in PBS pH 8 + 1% Tween-20 and incubated at 60°C for lOminutes. This resulted in nearly complete solubilization of iMablOO as is demonstrated in Figure 5. Lane 1 represents isolated inclusion bodies of iMablOO.
• Lane 2 represents solubilized iMablOO after incubation of the isolated inclusion bodies in PBS pH 8 + 1% Tween-20 at 60 °C for lOminutes.
• the supernatant was loaded on a Nickel-Nitrilotriacetic acid (Ni-NTA) superfLow column and purified according to a standard protocol as described by Qiagen (The QIAexpressionistTM, fifth edition, 2001).
• the binding of the thus purified iMablOO to chicken lysozyme was analyzed by ELISA (according to example 8) and is summarized in Table 6.
• iMablOO was solubilized from inclusion bodies using 8m urea and purified into an active form bymatrix assisted refolding.
• Inclusion bodies were prepared as described in example 6 and solubilized in 1ml PBS pH 8 + 8m urea.
• the solubilized proteins were clarified from insolublematerial by centrifugation (12.000 g, 30min.) and subsequently loaded on a Ni-NTA super- flow column (Qiagen) equilibrated with PBS pH 8 + 8M urea. Aspecific proteins were released by washing the column with 4 volumes PBS pH 6.2 + 8M urea.
• the bound His-tagged iMablOO was allowed to refold on the column by a stepwise reduction of the urea concentration in PBS pH 8 at room temperature.
• the column was washed with 2 volumes of PBS + 4M urea, followed by 2 volumes of PBS + 2M urea, 2 volumes of PBS + IM urea and 2 volumes of PBS without urea.
• IMablOO was eluted with PBS pH 8 containing 250mM imidazole.
• the released iMablOO was dialyzed overnight against PBS pH 8 (4°C), concentrated by freeze drying and characterized for binding and structuremeasurements.
• ELISA Enzyme Linked Immuno Sorption Assay
• Bound iMab proteins or phages were detected by the standard ELISA protocol using anti-VSV-hrp conjugate (Roche) or anti-Ml3-hrp conjugate (Pharmacia), respectively. Colorimetric assays were performed using Turbo-TMB (3, 3', 5, 5' -tetramethylbenzidine, Pierce) as a substrate. Binding of iMablOO to chicken lysozyme was assayed as follows.
• iMablOO Purified iMablOO ( ⁇ 50 ng) in 100 ⁇ l was added to a microtiter plate well coated with either ELK (control) or lysozyme (+ ELK as a blocking agent) and incubated for 1 hour at room temperature on a table shaker (300 rpm). Themicrotiter plate was excessively washed with PBS (3 times), PBS + 0.1% Tween-20 (3 times) and PBS (3 times). Bound iMablOO was detected by incubating the wells with 100 ⁇ l ELK containing anti-VSV-HRP conjugate (Roche) for 1 hour at room temperature.
• ELK control
• lysozyme (+ ELK as a blocking agent
• the purified iMablOO was analyzed formolecular weight distribution using a
• Example 11 iMablOO stability over time at 20 °C iMablOO stability was determined over a period of 50 days at 20 °C.
• iMablOO O.lmilligram/milliliter
• iMablOO O.lmilligram/milliliter
• Samples were diluted 200 times in PBS. Lysozyme binding of these samples was tested by ELISA measurements using 1:2000 in PBS diluted anti-VSV-hrp (Roche). TMB-ultra (Pierce) was used as a substrate for hrp enzyme levels ( Figure 8).
• iMablOO was very stable at room temperture. Activity of iMablOO hardly decreased over time, and thus it can be concluded that the iMab scaffold and its affinity regions are extremely stable.
• Example 12 iMablOO size determination, resistance against pH 4.8 environment, testing by gel and Purified iMablOO (as described in example 6) was brought to pH 4.8 using postassium acetate (final concentration of 50 mM) which resulted in precipitation of the protein.
• the precipitate was collected by centrifugation (12000 g, 30 minutes), redissolved in PBS pH 7.5 and subsequently filtered through a 0.45 micrometer filter to remove residual precipitates.
• the samples fore and after pH shock were analyzed by SDS-PAGE, western blotting and characterized for binding using ELISA (example 8). It was demonstrates that all iMablOO was precipitated at pH 4.8 and could also be completely recovered after redissolving in PBS pH 7.5 and filtering. ELISA measurements demonstrated that precipitation and subsequent resolubilization did nort result in a loss of activity (Table 7). It was confirmed that the VSV-tag is not lost during purification and precipitation and that no degradation products are formed.
• iMablOO The structure of iMablOO was analyzed and compared with another structure using a circular dichroism polarimeter (CD).
• CD circular dichroism polarimeter
• iMablOO and VhhlO-2/271102 were prepared with a purity of 98% in PBS pH 7.5 and OD280 ⁇ 1.0.
• Sample was loaded in a 0.1 cm quartz cuvette and the CD spectrummeasured with a computer controlled JASCO Corporation J-715 spectropolarimeter software (Spectramanager version 1.53.00, JASCO Corporation). Baseline corrections were obtained bymeausring the spectrum of PBS. The obtained PBS signal was substracted from allmeasurement to correct for solvent and salt effects.
• AR4 in iMablOO which was retreived from 1MEL, can be classified as random coil-like. Also, AR4 present in iMablOO is about 10 amino acids longer than the CDR3 of the Vhh protein.
• the temperature stability of the iMablOO protein was determined in a similar way using the CD -meter except that the temperature at which themeasurements were performed were adjusted.
• Example 14 E. coli BL21 (DE3) (Novagen) was transformed with expression vector CM126 containing various VAP inserts for iMabl302, iMabl602, iMabl202 and iMab 122 all containing 9 ⁇ -strands. Growth and expression was similar as described in example 5.
• iMab proteins All 9-stranded iMab proteins were purified bymatrix assisted refolding similar as is described in example 7.
• the purified fractions of iMabl302, iMabl602, iMab 1202 and iMab 122 were analysed by SDS-PAGE as is demonstrated in
• Figure 10 laneslO, 9, 8 and 7 respectively.
• iMabl302( ⁇ 50 ng), iMab 1602( ⁇ 50 ng), iMabl202( ⁇ 50 ng) and iMab 122 ( ⁇ 50 ng) were analyzed for binding to either ELK (control) or lysozyme (+ ELK as a blocking agent) similar as is described in Example 8.
• ELISA confirmed specific binding of purified iMabl302, iMab 1602, iMabl202 and iMab 122 to chicken lysozyme as is demonstrated in Table 6
• Example 16 CD spectra of various 9 stranded iMab iMablOO, iMab 1202, Imabl302 and iMab 1602 were purified as described in example 14 and analyzed for CD spectra as described in example 13.
• the spectra of iMab 1202, iMabl302 and iMab 1602 were measured at 20°C, 95°C and back at 20°C to test scaffold stability and refolding characteristics. The corresponding spectra are demonstrated in Figure 9D, 9E and 9F respectively.
• the spectra measured at 20°C were compared with the spectrum of iMablOO at 20°C to determine the degree of similarity of the secondary structure (see Figure 9J). It can be concluded that all different 9 strand scaffolds behave identical. This indicates that the basic structure of these scaffolds is identical. The data obtained after successive 20-95-20 degrees Celsius treatments clearly show that all scaffolds return to their original conformation.
• beta-elements were connected with one other by affinity region retrieved from anti-chicken lysozyme binding region obtained from the structure 1MEL or the bovine RNase A binding regions of IBZQ (L2, L6 and L8).
• beta-elements were connected with C-alpha backbone trace loops obtained from several different origins (1E50, lCWV, 1QHP, 1NEU, 1EPF, lF2x or 1E 6). The procedure for the attachment and fit of the loops is described in detail in example 2.
• amino acid side chains that determine the solubility of the proteins located in the core and loops 1, 3, 7 were determined as described in example 2.
• Example 18 E. coli BL21 (DE3) (Novagen) was transformed with expression vector CM126 containing various VAP inserts for iMab 1300, iMab 1200, iMab 101 and iMab900 all containing 7 beta-strands. Growth and expression was similar as described in example 5.
• iMabl300 ( ⁇ 50 ng), iMabl200( ⁇ 5 ng), iMabl01( ⁇ 20 ng) and iMab900 ( ⁇ 10 ng) were analyzed for binding to either ELK (control) or lysozyme (+ ELK as a blocking agent) similar as is described in example 8.
• ELK control
• lysozyme (+ ELK as a blocking agent
• ELISA confirmed specific binding of purified iMabl300, iMabl200, iMablOl and iMab900 to chicken lysozyme as is demonstrated in table 6.
• IMabl200 and iMablOl were purified as described in example 18 and analyzed for CD spectra as described in examplel3.
• the spectra of iMab 1200 and iMablOl were measured at 20°C, 95°C and back at 20°C to test scaffold stability and refolding characteristics. The corresonding spectra are demonstrated in Figure 9H and 9G respectively.
• the spectra of iMab 1200 and iMablOl measured at 20°C were compared with each other to determine the degree of similarity of the secondary structure (see Figure 9K). It can be concluded that the different 7 strand scaffolds behave identical. This indicates that the basic structure of these scaffolds is identical.
• beta-elements were connected with one other by affinity region retrieved from anti-chicken lysozyme binding region obtained from the structure 1MEL or the bovine RNase A binding regions of IBZQ (L2, L6 and L8).
• beta- elements were connected with C-alpha backbone trace loops obtained from several different origins (1E50, lCWV, 1QHP, 1NEU, 1EPF, lF2x or 1EJ6). The procedure for the attachment and fit of the loops is described in detail in example 2 and 3.
• amino acid side chains that determine the solubility of the proteins located in the core and loops LI, L3, L7 were determined as described in example 2 and 3.
• themodels were assessed usingmodeller. modeller was programmed to accept cysteine-cysteine bridges when appropriate.
• Next all predicted protein structures were assessed with Prosall, Procheck and WHAT IF.
• Prosall zp-comb scores were determined (table 12) to indicate if the created protein sequences might fold in vivo into the desired ig-like beta motif fold.
• Procheck and What if assessments were applied to check whether sequences might fit into the models (table 13).
• E. coli BL21 (DE3) (Novagen) was transformed with expression vector CM126 containing an VAP insert for iMab701 containing 6 beta-strands. Growth and expression was similar as described in example 5.
• iMab701 proteins were purified bymatrix assisted refolding similar as is described in example 7.
• the purified fraction of iMab701 was analysed by SDS-PAGE as is demonstrated in Figure 6 lane 4.
• IMab701 was purified as described in example 22 and analyzed for CD spectra as described in example 13.
• the spectra of iMab701 was measured at 20°C, 95°C and again at 20°C to test scaffold stability and refolding characteristics. The corresponding spectra are demonstrated in Figure 91. It can be concluded that the 6 strand scaffold behaves identical to the 7 strand scaffolds as described in example 20. This indicates that the basic structure of this scaffold is identical to the structure of the 7 strand containing scaffolds. Even more, as the obtained signals form the 9 stranded scaffolds (example 16) are similar to the signals obeserved for this 6 strand scaffold as presented here, it can also be concluded that the both types of scaffolds have an similar conformations. The data obtained after successive 20-95-20 degrees Celsius treatments clearly show that all scaffolds stay in their original conformation.
• Example 25 The data obtained after successive 20-95-20 degrees Celsius treatments clearly show that all scaffolds stay in their original conformation.
• a minimal scaffold is designed according to the requirements and features as described in example 1. However now only four and five beta-elements are used in the scaffold (see figure 1). In the case of 5 beta-elements amino acids side chains of beta-elements 2, 3, 6, 7 and 8 that are forming themantle of the new scaffold need to be adjusted for a watery environment.
• the immunoglobulin killer receptor 2dl2 (VAST code 2D LI) is used as a template for comparativemodelling to design a new small scaffold consisting of 5 beta- elements.
• Lysine residues contain chemical active amino-groups that are convenient in for example covalent coupling procedures of VAPs. Covalent coupling can be used for immobilization of proteins on surfaces or irreversible coupling of othermolecules to the target.
• the spatial position of lysine residues within the VAP determines the positioning of the VAP on the surface after immobilization. Wrong positioning can easily happen with odd located lysine residues exposed on the surface of VAPs. Therefore itmay be required for some VAP structures to remove lysine residues from certain locations, especially from those locations that can result in diminished availability of affinity regions.
• iMablOO outer surface lysine residues were changed. 3D imaging indicated that all lysine residues present in iMablOO are actually located on the outer surface. 3Dmodelling and analysis software (Insightll) determined the spatial consequence of such replacements.
• Modeller software was programmed in such a way that either cysteine bridge formation between the beta-sheets was taken into account or the cysteine bridges were neglected in analyses. All retrievedmodels were build with Prosall software formore or less objective result ranking. The zp-comb parameter of Prosall indicated the reliability of themodels. Results showed that virtually all types of amino acids could replace lysine residues. However, surface exposed amino acid side chains determine the solubility of a protein. Therefore only amino acids that will solubilize the proteins were taken into account andmarked with an X (see table 14).
• N- glycosylation can interfere strongly with protein functions if the glycosylation site is for example present in a putative ligand-binding site.
• iMablOO proteins were shown to be glycosylated in Pichia pastoris cells and unable to bind to the ligand. Analysis showed that there is a putative N- glycosylation site in AR3. Inspection of the iMablOO structure using template- modeling strategies withmodeller software revealed that this site is potentially blocking ligand binding due to obstruction by glycosylation. This site could be removed in two different ways, by removing the residue being glycosylated or by changing the recognitionmotif for N-glycosylation.
• glycosylation site itself (..RDNAS..) was removed. All residues could be used to replace the amino acid, after which Prosall, What ifand Procheck could be used to check the reliability of each individual amino acid.
• some amino acids could introduce chemical or physical properties that are unfavorable. Cysteine for example couldmake the proteins susceptible to covalent dimerization with proteins that also bear a free cysteine group. Also non-hydrophilic amino acids could disturb the folding process and were omitted.
• methionine is coded by ATG, which can introduce aberrant start sites in DNA sequences. The introduction of ATG sequencesmight result in alternative protein products due to potential alternative start sites.methionine residues were only assessed if no other amino acids would fit. All other amino acid residues were assessed with Prosall, What ifand Procheck. Replacement of N with Q was considered to be feasible and reliable, protein sequence from iMab with glycosylation site:
• iMablOO in Pichia pastoris was performed by amplification of 10 ng of CMll4-iMablOO DNA in a lOOmicroliter PCR reactionmix comprising 2 units Taq polymerase (Roche), 200 micromilor of each dNTP (Roche), buffers (Roche Taq buffer system), 2.5 micromolar of primer 107 and 108 in a Primus96 PCRmachine (MWG) with the following program 25 times [94°C 20", 55°C 25", 72°C 30"], digestion with EcoRI and Notl and ligation in EcoRI and Notl digested pPIC9 (InVitrogen). Constructs were checked by sequencing and showed all the correct iMablOO sequence.
• MWG Primus96 PCRmachine
• Transformation of Pichia pastoris was performed by electroporation according to themanufacturers protocol. Growth and induction of protein expression bymethanol was performed according to themanufacturers protocol. Expression of iMablOO resulted in the production of a protein that on a SDS-PAGE showed a size of 50 kD, while expressed in E.coli the size of iMablOO is 21 kD. This difference ismost likely due to glycosylation of the putative N-glycosylation site present in iMablOO as described above. Therefore this glycosylation site was removed by exchange of the asparagine (N) for a glutamine (Q) in a similar way as described in example 26 except that primer 136 (table 5) was used.
• iMab 115 This resulted in iMab 115.
• Expression of iMabll ⁇ in E. coli resulted in the production of a 21 kD protein.
• ELISA experiments confirmed specificity of this iMab for lysozyme.
• ARs in iMabll ⁇ were positioned correctly and,more specifically, replacement of the asparagine with glutamine in AR3 did not alter AR3 properties.
• Changing amino acids in the interior of the core removal of cysteine residues. Obtained sequences that fold in an ig-like structure, can be used for the retrieveal of similarly folded structures but aberant amino acid seqeunces. Amino acids can be exchanged with other amino acids and thereby putatively changing the physical and chemical properties of the new protein if compared with the template protein. Changes on the out side of the protein structure were shown to be rather straightforward. Here we changed amino acids that are lining up with the interior of the core. Spatial constrains of neighboring amino acid side chains and the spatial constrains of the core structure itself determine and limit the types of side chains that can be present at these locations. In addition, chemical properties of neighboring side chains can also influence the outcome of the replacements.
• CM126-iMabll6 was selected and used for further testing.
• E. coli BL21 (DE3) (Novagen) was transformed with expression vector CM126 containing an VAP insert for iMab 116 containing 9 beta-strands and potentially lacking a cysteine bridge in the core (as described in example 27).
• IMab 116 was purified bymatrix assisted refolding similar as is described in example 7.
• the purified fraction of iMabll ⁇ was analysed by SDS-PAGE as is demonstrated in Figure 6 lane 11.
• iMabll ⁇ Specific binding of iMabll ⁇ to chicken lysozyme (ELISA) Purified iMabll6( ⁇ 50 ng) was analyzed for binding to either ELK (control) and lysozyme (+ ELK as a blocking agent) similar as is described in Example 8.
• IMab 116 was purified as described in example 28 and analyzed for CD spectra 10 as described in example 13. The spectrum of iMabll ⁇ was measured at 20°C,
• cysteine bridge Chemical bonding of two cysteine residues in a proteins structure (cysteine bridge) can dramatically stabilize a protein structure at temperatures below about 70 degrees Celsius. Above this temperature cysteine bridges can be ⁇ broken. Some applications demand proteins that aremore stable than the original protein.
• the spatial constrains of the core of beta strand folds as referred to in example 1, enables cysteine bridges. This conclusion is based on the observation that in some natural occurring proteins with the referred fold a cysteine bridge is present in the center of the core (e.g. all heavy chain variable domains in antibodies). The distance between C-alpha backbone atoms of such cysteines ismost often found to be between 6.3 and 7.4 angstrom.
• iMablOO derivative that contains two extra cysteines in the core
• iMab 111 An oligo nucleotidemediated site directed mutagenesis method was used to construct an iMablOO derivative, named iMab 111 (table 3), that received two extra cysteine residues.
• CMll4-iMabl00 was used as a template for the PCR reactions together with oligo nucleotides pr33, pr3 ⁇ , pr82, pr83 (see table ⁇ ).
• primers pr82 and pr83 were used to generate a 401 bp
• PCR fragment In this PCR fragment a glutamine and a glycine coding residue were changed into cysteine coding sequences.
• This PCR fragment is used as a template in two parallel PCR reaction: In one reaction the obtained PCR fragment, CM114-iMabl00 template and pr33 were used, while in the other reaction the obtained PCR fragment, CM114-iMabl00 template and primers l ⁇ 3 ⁇ were used. The firstmentioned reaction gave a ⁇ 84 bp product while the second one produced a 531 bp fragment. Both PCR fragments were isolated via agarose gel separation and isolation (Qiagen gel extraction kit). The products weremixed in an equimolar relation and an fragment overlap-PCR reaction with primers pr33 and pr3 ⁇ resulted in a 714 bp fragment.
• This PCR fragment were isolated via agarose gel separation and isolation (Qiagen gel extraction kit). The products weremixed in an equimolar relation and an fragment overlap-PCR reaction with primers
• Example 34 Expression of iMab 111 iMablll DNA was subcloned in CM126 as described in example 28.
• CM126- iMablll transformed BL21(DE3) cells were induced with IPTG and protein was isolated as described in example 7. Protein extracts were analysed on 15% SDS-PAGE gels and showed a strong induction of a 21 KD protein.
• the expected length of iMablll including tags is also about 21 kD indicating high production levels of this clone.
• E. coli BL21 (DE3) (Novagen) was transformed with expression vector CM126 containing an VAP inserts for iMablll containing 9 beta-strands potentially containing an extra cysteine bridge (as described in example 32 and 33). 10 Growth and expression was similar as described in example 5 and 34. iMablll was purified by matrix assisted refolding similar as is described in example 7. The purified fraction of iMablll was analysed by SDS-PAGE as is demonstrated in Figure 6 lane 12.
• IMablll was purified as described in example 32 and analyzed for CD spectra as described in example 13.
• the spectrum of iMabll ⁇ was measured at 20°C, 9 ⁇ °C and again at 20°C to test scaffold stability and refolding characteristics. The corresponding spectra are demonstrated in Figure 9C.
• the spectra are demonstrated in Figure 9C.
• the properties of a scaffold need to be optimized.
• example heat stability, acid tolerance or proteolytic stability can be advantageous or even required in certain environments in order to function well.
• Amutation and re-selection program can be applied to create a new scaffold with similar binding properties but with improved properties.
• a selected binding protein is improved to resist proteolytic l ⁇ degradation in a proteolytic environment.
• New scaffolds can be tested for proteolytic resistance by a treatment with amixture of proteases or alternatively a cascade treatment with specific protease.
• new scaffolds can be tested for resistance by introducing the scaffolds in the environment of the future application. In orde to obtain proteolytic restant
• the gene(s) that codes for the scaffold(s) is (are)mutated usingmutagenesismethods.
• a phage display library is build from themutated PCR products so that the new scaffolds are expressed on the outside of phages as fusion proteins with a coat protein.
• the phages are added to a the desired proteolytic active environment for a certain time at the desired
• Intact phages can be used in a standard panning procedure as described. After extensive washing bound phages are eluted, infected in E.coli cells that bear F-pili and grown overnight on a agar plate that contains appropriate antibiotics. Individual clones are re-checked for their new properties and sequenced. The process ofmutation introduction and selection can be repeated several times or other selection conditions can be applied in further optimization rounds.
• Primers annealing just 3 prime and ⁇ prime of the desired region are used for amplification in the presence of dITP or dPTP as described. Thesemutated fragments are amplified in a second PCR reaction with primers having the identical sequence
• CM114-iMabl00 A vector for efficient phage display (CM114-iMabl00; see figure 4B) was constructed using part of the backbone of a pBAD (InVitrogen). The required
• Cysteine bridges between AR4 and other affinity regions can be involved in certain types of structures and stabilities that are not very likely without cysteine bridge formations. Not only can AR1 be used as an attachment for cysteines present in some affinity regions 4, but also AR2 and AR3 are obvious stabilizing sites for cysteine bridge formation. Because AR2 is an attractive alternative location for cysteine bridge formation with
• an expression vector is constructed which is 100% identical to CM114- iMablOO with the exception of the locations of a cysteine codon in AR2 and the lack of such in ARl.
• 3D-modelling analysis revealed that the best suitable location for cysteine in AR2 is at the location originally determined as a threonine (.VATIN.. into ..VACIN..).
• the original cysteine in ARl was replaced by a serine that turned out to be a suitable replacement according to 3Dmodelling analysis (table 3).
• the new determined sequence, named iMabll3, (table 4) was constructed
• An expression vector lacking cysteines in ARl, 2 and 3 was constructed. This vector is 100% identical to CM114 with the exception that the cysteine in ARl (..PYCMG..) has been changed to a serine (..PMSMG..; see table 3).
• the new determined sequence, named iMab 114, (table 4) was constructed according to the gene construction procedure as described above (example 3) and inserted in CM114 replacing iMablOO.
• Lama pacos and Lama glama blood lymphocytes were isolated according to standard procedures as described in Spinelli et al. (Biochemistry 39 (2000) 1217-1222). RNA from these cells was isolated via Qiagen RNeasymethods according tomanufactures protocol. cDNA was generated usingmuMLv or AMV (New England Biolabs) according tomanufactures procedure.
• CDR3 regions from Vhh cDNA were amplified (see figure 10) using 1 ⁇ l cDNA reaction in lOOmicroliter PCR reactionmix comprising 2 units Taq polymerase (Roche), 200 ⁇ M of each dNTP (Roche), buffers (Roche Taq buffer system), 2. ⁇ ⁇ M of forward and reverse primers in a Primus96 PCRmachine (MWG) with the following program 3 ⁇ times [94°C 20", ⁇ 0°C 2 ⁇ ", 72°C 30"].
• MWG Primus96 PCRmachine
• primer 16 (table ⁇ ) was used as reverse primer.
• Products were separated on a 1% Agarose gel and products of the correct length ( ⁇ 2 ⁇ 0 bp) were isolated and purified using Qiagen gel extraction kit. 5 ⁇ l of these products were used in a next round of PCR similar as described above in which primer 8 (table 5) and primer 9 (table ⁇ ) were used to amplify CDR3 regions.
• Products were separated on a 2% Agarose gel and products of the correct length ( ⁇ 80 - 150 bp) were isolated and purified using Qiagen gel extraction kit.
• Cow (Bos taurus) blood lymphocytes were isolated according to standard procedures as described in Spinelli et al. (Biochemistry 39 (2000) 1217-1222).
• RNA from these cells was isolated via Qiagen RNeasymethods according tomanufactures protocol.
• cDNA was generated usingmuMLv or AMV (New England Biolabs) according tomanufactures procedure.
• CDR3 regions from Vh cDNA was amplified using 1 ⁇ l cDNA reaction in lOOmicroliter PCR reactionmix comprising 2 units Taq polymerase (Roche), 200 ⁇ M of each dNTP l ⁇ (Roche), buffers (Roche Taq buffer system), 2. ⁇ ⁇ M of primer 299 (table ⁇ ) and 300 (table 5) in a Primus96 PCRmachine (MWG) with the following program 35 times [94°C 20", ⁇ 0°C 25", 72°C 30"]. Products were separated on a 2% Agarose gel and products of the correct length were isolated and purified using Qiagen gel extraction kit. The length distribution of the PCR products
• Isolated and purified products can be used to adapt the sequences around the actual CDR3/AR4 location in a way that the coding regions of the frameworks are gradually adapted via several PCRmodifications rounds similarly as described for lama derived ARs (see example 43).
• Example 45 Libraries containing loop variegations in AR4 by insertion of amplified CDR3 regions
• a nucleic acid phage display library having variegations in AR4 was prepared by the followingmethod.
• Amplified CDR3 regions from lama's immunized with lactoperoxidase and lactoferrin was obtained as described in example 43 and were digested with Pstl and Kpnl and ligated with T4 DNA ligase into the Pst ⁇ and Kpnl digested and alkaline phosphatase treated vector CM114- iMabll3 or CM114-iMabll4. Cysteine containing CDR3s were cloned into CMll4-iMabll4 while CDR3s without cysteines were cloned into vector CM114-iMabll3.
• the libraries were constructed by electroporation into E. coli TGI electrocompetent cells by using a BTX electrocellmanipulator ECM 630. Cells were recovered in SOB and grown on plates that contained 4% glucose, lOOmicrogram ampicillin permilliliter in 2*TY-agar. After overnight culture at 37 °C, cells were harvested in 2*TYmedium and stored in 50% glycerol as concentrated dispersions at -80°C. Typically, ⁇ x 10 8 transformants were obtained with 1 ⁇ g DNA and a library contained about 10 9 independent clones.
• CDR3 regions A nucleic acid phage display hbrary having variegations in AR4 by insertion of randomized CDR3 regions was prepared by the followingmethod.
• CDR3 regions from non-immunized and immunized lama's were amplified as described in example 28 except that in the second PCR round dITP according to Spee et al. (1993) or dPTP according to Zaccolo et al. (1996) were included as described in example 35.
• Preparation of the library was performed as described in example 28. With dITP amutation rate of 2 % was achieved while with dPTP included in the PCR amutation rate of over 20% was obtained.
• I ⁇ was filtered through a 0.45micrometer PVDF filtermembrane.
• Poly-ethylene- glycol and NaCI were added to the flow through with final concentrations of respectively 4% and 0.5M.
• phages precipitated on ice and were pelleted by centrifugation at 6000g.
• the phage pellet was solved in 50% glycerol/ ⁇ 0% PBS and stored at -20°C.
• a targetmolecule (antigen) was immobilized in an immunotube (Nunc) ormicrotiter plate (Nunc) in 0. lm sodium carbonate buffer (pH 9.4) at 4 °C o/n. After the removal of this solution, the tubes were blocked with a 3% skimmilk powder solution (ELK) in PBS or a similar blocking agent
• phagemid library solution containing approximately 10 12 - 10 13 colony forming units (cfu), which was preblocked with blocking buffer for 1 hour at room temperature, was added in blocking buffer. Incubation was performed on a slow rotating platform for 1 hour at room temperature.
• Recovered phages were amplified as described above employing E.coli XLI- Blue (Stratagene) or ToplOF' (InVitrogen) cells as the host. The selection process was repeated several times to concentrate positive clones. After the final round, individual clones were picked and their binding affinities and DNA sequences were determined.
• VAPs The binding affinities of VAPs were determined by ELISA as described in example 6, either as gill-fusion protein on the phage particles or after subcloning as a Ndel-Sfil into the expression vector CM126 as described in example 4
• E.coli BL21(DE3) or Origami(DE3) (Novagen) were transformed by electroporation as described in example 5 and transformants were grown in 2x TYmedium supplemented with Ampicillin (100 ⁇ g/ml). When the cell cultures reached an OD600 ⁇ 1 protein expression was induced by adding IPTG (0.2mM). After 4 hours at 37 °C cells were harvested by centrifugation. Proteins were isolated as described in example ⁇
• Lactoferrin (LF) was supplied by DMV-Campina.
• a phage display library with variegations in AR4 as described in example 45 was used to select LF binding VAPs.
• LF (10 microgram in 1ml sodium bicarbonate buffer (0.1m, pH 9.4)) was immobilized in an immunotube (Nunc) followed by blocking with 3% chicken serum in PBS. Panning was performed as described in example 32. 10 13 phages were used as input. After the 1 st round of panning about 10000 colonies were formed. After the 2 nd panning round 500 to 1000 colonies were formed. Individual clones were grown and VAPs were produced and checked by ELISA as described in example 6. Enrichment was found for clones with the following AR4: CAAQTGGPPAPYYCTEYGSPDSW
• LP Purified Lactoperoxidase
• LP 10 microgram in 1ml sodium
• RNase A binder constructionmaturation and panning.
• CM114-iMabl30 Chimeric phages with iMabl30 as a fusion protein with the g3 coat protein were produced under conditions as described for library amplification procedure in example 32 Panning with these chimeric phages against RNase A coated immunotubes (see example 32 for panning procedure) failed to show RNase A specific binding of iMab 130. Functional positioning of
• the iMabl30 coding region wasmutated using the followingmethod: iMab 130 present in vector CM114 wasmutagenised using either dITP or dPTP during amplification of the scaffold with primers 120and
• 121(table)mutagenizing concentrations of 1.7mM dITP or 300 ⁇ M, 75 ⁇ M or 10 ⁇ M dPTP were used.
• Resulting PCR products were isolated from an agarose gel via Qiagen's gel elution system according tomanufactures procedures. Isolated products were amplified in the presence of lOO ⁇ M of dNTPs (Roche) in order to generate dITP and dPTP free products. After purification via Qiagen's PCR clean up kit, these PCR fragments were digested with Notl and Sfil (NEB) and ligated into Notl and Sfil linearized CM114.
• Precipitated and 70% ethanol washed ligation products were transformed into TGI bymeans of electroporation and grown in 2xTYmedium containing lOO ⁇ g/ml ampicillin and 2% glucose and subsequently infected with VCSM13 helper phage (Stratagene) for chimeric phage production as described in example 32. Part of the transformation was plated on 2xTY plates containing 2% glucose and lOOmicrogram/ml ampicillin to determine transformation frequency: These phage libraries were used in RNase A panning experiments as described in example 32 RNase A was immobilized in immunotubes and panning was performed.
• phages were eluted and used for infection of TOP10 F' (InVitrogen), and grown overnight at 37°C on 2xTY plates containing 2% glucose and lOO ⁇ g/ml ampicillin and 25microgram/ml tetracycline. The number of retrieved colonies is indicated in table 17. As can be concluded from the number of colonies obtained after panning with phage libraries derived from differentmutagenesis levels of iMabl30, a significant increase of binders can be observed from the library with a mild mutagenesis level, being dITP (table 17)
• Example 51 Immobilisation procedure lg of epoxy activated Sepharose 6B (manufacturer Amersham Biosciences) was packed in a column and washed with 10 bed volumes coupling buffer (200mM potassium phosphate, pH 7). The protein to be coupled was dissolved in coupling buffer at a concentration of lmg/ml and passed over the column at a flow rate of O.lml/min. After passing 20 bed volumes of protein solution, the column was washed with coupling buffer. Passing 10 bed volumes of 0.2M ethanolamine/ 200mM potassium phosphate pH 7 blocked the unreacted epoxy groups. The resin was then washed with 20 bed volumes of ⁇ O M potassium phosphate pH 7 after which it was ready for use.
• coupling buffer 200mM potassium phosphate, pH 7
• Lysozyme was immobilized on Eupergit, an activated epoxy-resin from Rohm and used in a column.
• a solution containing iMablOO was passed on the column and the concentration wasmeasured in a direct bypass and the flow through from the column (A280 nm). The difference indicated the amount of iMablOO that was bound to the column.
• the bound iMablOO could be released with a CAPS buffer pHll. Control experiments with BSA indicated that the binding of iMablOO to immobilized lysozyme was specific.
• Lysozyme purification via iMablOO immobihzed beads iMablOO was immobilized on Eupergit and used in a column.
• a solution containing Lysozyme was passed on the column and the concentration wasmeasured and in a direct bypass and the flow through from the column (A280 nm). The difference indicated the amount of Lysozyme that was bound to the column.
• the bound Lysozyme could be released with a CAPS buffer pHll. Control experiments with BSA indicated that the binding of Lysozyme to immobilized iMablOO was specific.
• iMablOO The stability of iMablOO in several milk fractions was measured by lysozyme ⁇ coated plates via ELISA methods (example 8). If the tags, scaffold regions or affinity regions were proteolytically degraded, a decreased anti-lysozyme activity would be observed.
• iMablOO was diluted in several different solution: lxPBS as a control, ion-exchange fraction from cheese-whey, gouda-cheese- whey and low pasteurised undermilk, 1.4 ⁇ m filtered to a final concentration of 0 40 ⁇ g/ml. All fractions were stored at 8 °C, samples were taken after:0, 2 and ⁇ hours and after 1, 2, 3, 4, 5 and 7 days.
• iMabs were purified using urea and subsequent matrix assisted refolding 5 (example 7), except for iMablOO which was additionally also purified by heat- induced solubilization of inclusion bodies (example 6).
• PROSAII results (zp-comp) and values for the objective function from MODELLER for 7-stranded iMab proteins. Lower values correpond to iMab proteins which are more likely to fold correctly.
• PROSAII results (zp-comp) from iMablOO derivatives of which lysine was replaced at either position 3, 7, 19 and 6 ⁇ with all other possible amino acid 40 residues. Models were made with and without native cysteine bridges. The more favourable derivatives (which are hydrophilic) are denoted with X. Table l ⁇
• PROSAII results (zp-comp) from iMablOO derivatives of which cysteine at position 96 was replaced with all other possible amino acid residues.
• the basic core beta elements are the nominated in example A.
• This basic structure contains 9 beta-elements positioned in two plates.
• One beta-sheets contains elements 1, 2, 6 and 7 and the contains elements 3, 4, ⁇ , 8 and 9.
• beta-elements are also depicted.
• Bold lines are connecting loops between beta- elements that are in top position while dashed lines indicate connecting loops that are located in bottom position.
• a connection that starts dashed and ends solid indicates a connection between a bottom and top part of beta-elements.
• the numbers of the beta-elements depicted in the diagram correspond to the
• D 7b beta element topology: for example E-cadherin domain (IFF ⁇ )
• F 6b beta element topology: for example Fc epsilon receptor type alpha (1J88)
• G 6c beta element topology: for example interleukin- 1 receptor type-1 (IGOY) 0 H: ⁇ beta element topology
• IGOY interleukin- 1 receptor type-1
• Putative antigen binding proteins that contain a core structure as described 5 here can be used for transfer operations.
• individual or multiple elements or regions of the scaffold or core structures can also be used for transfer actions.
• the transfer operation can occur between structural identical or comparable scaffolds or cores that differ in amino acid composition.
• Putative affinity regions can be transferred from one scaffold or core to another scaffold 0 or core by for example PCR, restriction digestions, DNA synthesis or other molecular techniques. The results of such transfers is depicted here in a schematic diagram.
• the putative (coding) binding regions from molecule A (top part, affinity regions) and the scaffold (coding) region of molecule B (bottom part, framework regions) can be isolated by molecular means. After 5 recombination of both elements a new molecule appears (hybrid structure) that has binding properties of molecule A and scaffold properties of scaffold B.
• the diagram represents the topologies of protein structures consisting of respectively 9, 7 and 6 beta-elements (indicated 1-9 from N-terminal to C- terminal). Beta elements 1,2, 6 and 7 and elements 3, 4, ⁇ , 8 and 9 form two beta-sheets. Eight loops (L1-L8) are responsible for the connection of all beta-elements. ⁇ Loop 2, 4, 6 and 8 are located at the top site of the diagram and this represents the physical location of these loops in example proteins. The function of loops 2,4 and 8 in light and antibody variable domains is to bind antigens, known as CDR regions. The position of L6 (also marked with a patterned region) also allows antigen binding activity, but has not been indicated as a binding region. 0 L2, L4, L6, L8 are determined as affinity regionl (ARl), AR2, AR3 and AR4 respectively. Loops 1, 3, ⁇ and 7 are located at the opposite site of the proteins.
• D. iMab 1202 far UV spectrum determined at 20 °C, (partially) denatured at 95 °C, and refolded at 20 °C, compared to the iMablOO spectrum at 20 °C.
• E. iMabl302 far UV spectrum determined at 20 °C, (partially) denatured at 95 °C, and refolded at 20 °C, compared to the iMablOO spectrum at 20 °C.
• iMabl602 far UV spectrum determined at 20 °C, (partially) denatured at 95 °C, and refolded at 20 °C, compared to the iMablOO spectrum at 20 °C.
• I iMab701, far UV spectrum determined at 20 °C, (partially) denatured at 9 ⁇ °C, and refolded at 20 °C
• J Overlay of native (undenatured) 9 strand iMab scaffolds.
• K Overlay of native (undenatured) 7 strand iMab scaffolds.
• L Far UV CD spectra of iMablOO and a V HH (courtesy Kwaaitaal M, Wageningen University and Research, Wageningen, the Netherlands).
• FIG 10 Schematic overview of PCR isolation of CDR3 for MAST.
• Lane 1. 1 microgram Llama cDNA cyst+, PCR amplified with primers 8 and 9.
• Lane 2 1 microgram Llama cDNA cyst-, PCR amplified with primers 8 and 9.
• Lane 5. 1.5 microgram Cow cDNA PCR amplified with primers 299 and 300.
• Lane 6. 0.75 microgram Cow cDNA PCR amplified with primers 299 and 301.
• Lane 7. 1.5 microgram Cow cDNA PCR amplified with primers 299 and 301.
• Figure 12 Lysozyme binding activity measured with ELISA of iMablOO.
• Test samples were diluted 100 times in figures A) and C) while samples were 1000 times diluted in figures B) and D).
• A) and B) show lysozyme activity while C) and D) show background activity.
• ATOM 110 CA GLY A 44 -17.378 11.364 7.293 1.00 26.93 c
• ATOM 162 CA GLY A 33 -1.574 -7.581 1.983 1.00 28.77 c
• ATOM 171 CA GLY A 43 -3.890 4.861 -12.131 1.00 14.44 c
• ATOM 180 CA GLY A 52 -11.032 -17.051 6.076 1.00 24.55 c
• ATOM 181 CA GLY A 53 -13.512 -18.935 8.234 1.00 17.82 c
• ATOM 204 CA GLY A 91 -10.820 -10.288 -2.524 1.00 17.13 c ATOM 205 CA GLY A 92 -11.033 -6.489 -2.552 1.00 12.84 C
• ATOM 231 CA GLY A 126 -2.079 7.899 -0.493 1.00 10.84 c
• ATOM 236 CA GLY C 3 -10.267 -7.502 10.101 1.00 30.20 c
• ATOM 272 CA GLY C 58 -19.057 ⁇ -12.667 -7.642 1.00 24.08 c ATOM 273 CA GLY 59 -20.627 -9.992 444 1.00 22.25 C ATOM 274 CA GLY 60 -21.579 -6.311 553 1.00 22.96 C ATOM 275 CA GLY 61 -23.676 -7.234 605 1.00 28.83 C ATOM 276 CA GLY 62 -25.740 -4.088 210 1.00 32.00 C ATOM 277 CA GLY 63 -22.747 -1.772 -7 854 .00 30.87 C ATOM 278 CA GLY 66 -17.371 -2.468 -7 103 .00 23.17 C ATOM 279 CA GLY 67 -16.897 -6.161 -7 488 .00 22.56 C ATOM 280 CA GLY 68 -15.405 -9.022 -5.517 .00 21.60 c ATOM 281 CA GLY 69 -15.3
• NVKLVE KGG-NFVEN—DDD —KLTCRAEGYTI GPYCMGWFRQ ⁇ APNDDSTNVATIN GGGITYYGDSVKERFDIRRDNASNTVTLSMDDLQP
• NVKVVT KRE-NFGE —GSDV—KLTCRASGYTI GPICFG FYQ
• VAPs amino acid sequences amino acid sequences:
• iMab702 20 AVKSVFKVSTNFIENDGTMDSKLTFRASGYTIGPQCLGFFQQGVPDDSTNV ATINMGGGITYYGDSVKSI
• CTGAAGGTTA 81 CACCATTGGC CCGTACTGCA TGGGTTGGTT CCGTCAGGCG CCGAACGACG ACAGTACTAA CGTGGCCACG
• ATCAACATGG 161 GTGGCGGTAT TACGTACTAC GGTGACTCCG TCAAAGAGCG CTTCGATATC CGTCGCGACA ACGCGTCCAA
• CTGAAGGTTA 81 CACCATTGGC CCGTACTCCA TGGGTTGGTT CCGTCAGGCG CCGAACGACG ACAGTACTAA CGTGGCCACG l ⁇ ATCAACATGG 161 GTGGCGGTAT TACGTACTAC GGTGACTCCG TCAAAGAGCG CTTCGATATC CGTCGCGACA ACGCGTCCAA
• CTGAAGGTTA 81 CACCATTGGC CCGTACTGCA TGGGTTGGTT CCGTCAGGCG CCGAACGACG ACAGTACTAA CGTGGCCACG
• ATCAACATGG 161 GTGGCGGTAT TACGTACTAC GGTGACTCCG TCAAAGAGCG CTTCGATATC CGTCGCGACC AGGCGTCCAA
• ACGCGTCCAA 241 CACCGTTACC TTATCGATGG ACGATCTGCA ACCGGAAGAC TCTGCAGAAT ACAATGGTGC AGGTGATTCT
• CTGAAGGTTA 81 CACCATTGGC CCGTACTGCA TGGGTTGGTT CCGTCAGGCG CCGAACGACG ACAGTACTAA CGTGGCCACG
• AACGTTGCTA 161 CCATCTCCGA AACCGGTGGT GACATCGTTT ACACCAACTA CGGTGACTCC GTTAAAGAAC GTTTCGACAT
• CTGGTTCCGT 321 TTACGGTTCC GGTTGGCGTC CGGACCGTTA CGACTACCGT GGTCAGGGTA CCGACGTTAC CGTTTCCTCG
• AACGTTGCTA 161 CCATCCGTTG GAACGGTGGT TCCACCTACT ACACCAACTA CGGTGACTCC GTTAAAGAAC GTTTCGACAT
• CCGTGTTGAC 241 CAGGCTTCCA ACACCGTTAC CCTGTCCATG GACGACCTGC AGCCGGAAGA CTCCGCTGAA TACAACTGCG l ⁇ CTGGTACCGA 321 CATCGGTGAC GGTTGGTCCG GTCGTTACGA CTACCGTGGT CAGGGTACCG ACGTTACCGT TTCCTCGGCC
• AACGTTGCTA 161 CCATCACCTG GTCCGGTCGT CACACCCGTT ACGGTGACTC CGTTAAAGAA CGTTTCGACA TCCGTCGTGA
• CCAGGCTTCC 241 AACACCGTTA CCCTGTCCAT GGACGACCTG CAGCCGGAAG ACTCCGCTGA ATACAACTGC GCTGGTGAAG
• CTACCGTGGT 241 CAGGGTACCG ACGTTACCGT TTCCTCGGCC AGCTCGGCC 0 iMab D125
• GTGGCCACCA 161 TCGACTCGGG TGGCGGCGGT ACCCTGTACG GTGACTCCGT CAAAGAGCGC TTCGATATCC GTCGCGACAA
• ATCGGTGAAA 161 TCAACATGGG TACCGCTTAC GTTGACCTGT CCTCCCTGAC CTCCGAAGAC TCCGCTGTTT ACTACTGCGC
• TGCTGACTCC 241 ACCATCTACG CTTCCTACTA CGAATGCGGT CACGGTATCT CCACCGGTGG TTACGGTTAC GACTACTGGG GTCAGGGTAC
• CGGTATCTCC 241 ACCGGTGGTT ACGGTTACGA CTACTGGGGT CAGGGTACCA CCCTGACCGT TTCCTCGGCC AGCTCGGCC iMab D400
• TGCTGACTCC 241 ACCATCTACG CTTCCTACTA CGAATGCGGT CACGGTATCT CCACCGGTGG TTACGGTTAC GACGTTTGGG GTCAGGGTAC
• GACTCCACCA 161 ACGTTGCTAC CATCAACATG GGTGGTGGTA TCACCTACTA CGGTGACTCC GTTAAACTGC GTTTCGACAT CCGTCGTGAC
• TGCTGACTCC 241 ACCATCTACG CTTCCTACTA CGAATGCGGT CACGGTATCT CCACCGGTGG TTACGGTTAC TCCGAATGGT CCGAACCGGT
• GGTACCTACT 161 CCGCTCCGCT
• GACCTCCACC ACCCTGGGTG TTGCTACCGT TACCTGCGCT GCTGACTCCA
• TTCCTACTAC 241 GAATGCGGTC ACGGTATCTC CACCGGTGGT TACGGTTACG CTGCTTTCTC CGTTCCGTCC GTTACCGTTA
• CTTTCGACAC 81 CACCCTGGGT AACAACATGG GTACCTACTC CGCTCCGCTG ACCTCCACCA CCCTGGGTGT TGCTACCGTT
• ACCTGCGCTG 161 CTGACTCCAC CATCTACGCT TCCTACTACG AATGCGGTCA CGGTATCTCC ACCGGTGGTT ACGGTTACGC
• TGCTTTCTCC 241 GTTCCGTCCG TTACCGTTAA CTTCACCGCG GCCAGCTCGG CC iMab D702
• GACTCCACCA 161 ACGTTGCTAC CATCAACATG GGTGGTGGTA TCACCTACTA CGGTGACTCC GTTAAATCCA TCTTCGACAT
• CAGACTCCAC 321 CATCTACGCT TCCTACTACG AATGCGGTCA CGGTCTGTCC ACCGGTGGTT ACGGTTACGA CCTGATCCTG CGTACCCTGC
• ATCTCCCCGA 161 TCAACATGGG TTCCTACACC GCTACCGTTG TTGGTAACTC CGTTGGTGAC GTTACCATCA CCTGCGCTGC
• TGACTCCACC 241 ATCTACGCTT CCTACTACGA ATGCGGTCAC GGTATCTCCA CCGGTGGTTA CGGTTACACC CTGATCCTGT
• AACAAAATCA 161 CCGAAAACAT GGGTGTTGCT CGTATCGCTC TGACCAACAC CACCGACGGT GTTACCGTTG TTACCTGCGC TGCTGACTCC
• GACGCTTCCA 161 AAGTTACCTG CGCTGCTGAC TCCACCATCT ACGCTTCCTA CTACGAATGC GGTCACGGTA TCTCCACCGG
• TGGTTACGGT 241 TACCACTGGA AAGCTGAAAA CTCGGCCAGC TCGGCC iMab D1001 1 CATA TGGTTGACGG TGGTTACCTG TGCAAAGCTT CCGGTTACAC CATCGGTCCG GAATGCATCG
• GCTGCTGACT 161 CCACCATCTA CGCTTCCTAC TACGAATGCG GTCACGGTAT CTCCACCGGT GGTTACGGTT ACCACTGGAA
• TGCTGACTCC 241 ACCATCTACG CTTCCTACTA CGAATGCGGT CACGGTATCT CCACCGGTGG TTACGGTTAC TCCGCTTGGT
• CTACGCTTCC 241 TACTACGAAT GCGGTCACGG TATCTCCACC GGTGGTTACG GTTACAACCC GGTTTCCCTG GGTTCCTTCG
• TAAAGGCACC 241 TGGACCCTGT CCATGGACTT CCAGCCGGAA GGTATCTACG AAATGCAGTG CGCTGCAGAC TCCACCATCT ⁇ ACGCTTCCTA
• TTCCTACTAC 241 GAATGCGGTC ACGGTATCTC CACCGGTGGT TACGGTTACC AGTCCGAAGC TACCGTTAAC GTTAAAATCT
• TGACAACGCT 241 AAAGACACCT CCACCCTGTC CATCGACGAC GCTCAGCCGG AAGACGCTGG TATCTACAAA TGCGCTGCAG
• ACTCCACCAT 321 CTACGCTTCC TACTACGAAT GCGGTCACGG TCTGTCCACC GGTGGTTACG GTTACGACTC CGAAGCTACC

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