Synthetic immunoglobulin domains with binding properties engineered in regions of the molecule different from the complementarity determining regions

[0001] The present invention relates to a method of constructing and producing a modified immunoglobulin.

[0002] Generally, the field of the invention is the construction of proteins to give them specific binding properties. More specifically, the proteins constructed herein are immunoglobulins (antibodies), and more specifically, single domains, pairs or combinations of single immunoglobulin domains.

immunoglobulin binding are features because they control interaction with other molecules, such as antigens, enabling the use of immunoglobulins for diagnostic and therapeutic purposes.

Specific properties of significant importance. [0003] A discussion of the basic structure of an antibody will be made here by the example of an intact IgG1 immunoglobulin molecule.

[0004] By combining two identical heavy (H) chains and two identical light (L) chains, a characteristic form of the Y-shaped antibody molecule is formed. Each heavy chain has four domains. Amino-terminated (VH) variable domains are located at the apex of the "arms" of the letter Y. Behind them are three constant domains: CH1, CH2 and a carboxyl-terminated domain of CH3 at the base of the "leg" of the letter Y. The combination of heavy chain constant regions with the variable region provides a short stretch known as a "linker". Whereas the so-called the hinge connects the CH2 and CH3 domains (Fc fragment) with the rest of the antibody (Fab fragments). By subjecting the hinge region of the intact antibody to proteolysis, one Fc fragment and two identical Fab fragments are obtained. Light chains are made up of two domains, one variable (VL) and one constant (CL), separated by a link.

[0005] Both heavy chains are joined in the hinge region by disulfide bonds. Additional disulfide bonds provide a combination of light and heavy chains. Carboxy fragments are attached via asparagine at various sites of the constant domains, depending on the immunoglobulin class.

In the case of IgG1, heavy chains are connected by two disulfide bonds between cysteine ​​pairs at positions 235 and 238 located in the hinge region. The light chains are attached to heavy chains by two additional disulfide bonds, between cysteine ​​molecules at position 229s in the CH1 domains and at position 214 in the CL domains. The carbohydrate fragments are attached to asparagine at position 306 in each CH2 domain, forming a characteristic bulge on the leg of the letter Y.

[0006] The described features entail important functional properties. The variable regions of both heavy (VH) and light (VL) chains are located at the "tops" of the arms of the letter Y to react with the antigen there. This tip of the molecule is the place where the N-terminus of the amino acid sequence is located. In contrast, the "foot" of the letter effectively mediates in the implementation of effector functions such as complement activation and interaction with receptors for Fc, or ADCC and ADCP. The bulges of the CH2 and CH3 domains facilitate interaction with effector proteins. The C-terminus of the amino acid sequence is located at the opposite end, which could be referred to as the "base" of the letter Y. The structure of intact IgG1 immunoglobulin is shown in Figure 1a.

[0007] Antibodies have two types of light chain chains, referred to as lambda (λ) and kappa (κ). A given immunoglobulin has either two λ chains or two κ chains, but never one of each type. So far, no functional differences have been found between antibodies containing λ light chains and antibodies with κ chains.

[0008] Figure 1b shows the organizational structure of monomers representing major classes of human immunoglobulins. The classes differ in the composition of the heavy chains and their sequence. IgM and IgE immunoglobulins do not have a hinge region, but each has an additional domain (CH4) in the heavy chain. The number and location of disulfide bonds (lines) connecting the chains are different in different isotypes. Isotypes also differ in the arrangement of N-mediated carbohydrate groups, symbolically marked in the form of circles.

[0009] Each domain in the antibody molecule has a similar structure in the form of two highly mutually packed "β-sheets" forming a compressed anti-parallel "β-barrel". This conservative structure is called an immunoglobulin fold. An immunoglobulin fold formed from constant domains contains a sheet of paper

3-thread, packed in a 4-thread card. The immunoglobulin fold is stabilized by: hydrogen bonding between the β-threads of each page, a hydrophobic bond between the residues of opposite pages in the inner part, and a disulfide bond between pages. The 3-strand card contains threads C, F and G, and the 4-strand card contains threads A, B, E and D. Letters from A to G denote successive positions of β-threads along the amino acid sequence of the immunoglobulin fold.

[0010] An immunoglobulin fold made of variable domains has 9 β-strands forming two pages, consisting of 4 and 5 strands, respectively. The 5-strand card is structurally homologous to the 3-strand card of the constant domains, but contains additional C 'and C' threads. The remaining strands (A, B, C, D, E, F, G) have the same topology and similar structure as their counterparts in immunoglobulin folds from constant domains. As in the constant domains, the disulfide bond joins the B and F strands in opposite sheets. FIG. 2 shows an immunoglobulin fold for an immunoglobulin variable and constant domain, respectively.

[0011] The variable domains of both heavy and immunoglobulin light chains contain three hypervariable loops, called complementarity (so-called V domains (CDR1, CDR2, also regions determining the CDR regions). Three CDR regions CDR3) form a clot at one end β-barrel. The CDRs are loops that connect BC, C'-C "and FG immunoglobulin folds. Residues in the CDR regions are different in different immunoglobulins, giving each antibody specific antigen specificity.

[0012] The VL and VH domains located on the tops of the arms of the antibody molecules are tightly packed in such a way that 6 CDR regions (3 per domain) together form a plane (or cavity) ensuring specific antigen binding. The natural antigen binding site of the antibody molecule is therefore made of loops that connect the BC, C'-C "and FG strands of the light chain variable domain and the BC, C'-C" and FG strands of the heavy chain variable domain.

[0013] Using the three-dimensional structure of the protein as a design aid, randomized (i.e. randomly selected) amino acid residues occurring on the surface of many proteins were used, using the core protein structure as a skeleton. Examples of this strategy are described or cited in the reference works listed below: Nygren PA, Uhlen M., Curr Opin Strukt Biol. (1997) 7: 463-9; Binz HK, Amstutz P, Kohl A, Stumpp MT, Briand C, Forrer P, Grutter MG, Pluckthun A. Nat. Biotechnol. (2004) 22: 575-82; Vogt M, Skerra A. Chembiochem. (2004) 5: 191-9; US 6, 562,617.

[0014] The basic principle of this technique is based on the observation that many proteins have a stable core, formed by the special arrangement of elements of the secondary structure, such as β-sheets or α-helix, which are connected with each other by structures such as loops, bends or random coils. Typically, the last three structural elements are less important for the overall structure of the protein, and the amino acid residues found in these structural elements can be exchanged without prejudice to the overall folding of the protein. Antibodies to CDRs are naturally occurring examples of this design principle. Examples of artificial solutions are lipocalins, ankirins and other protein skeletons.

[0015] Loops that are not CDR loops in native immunoglobulin, do not have the specificity of antigen or epitope binding, nevertheless contribute to the correct folding of the entire immunoglobulin molecule and / or to its effector functions or other functions, therefore they are determined for the purposes of the present invention called structural loops.

[0016] US Patent No. 6,294,654 demonstrates that it is possible to obtain modified antibodies by introducing a peptide antigen into a non-CDR antibody loop (Ab) and placing it in the CH1 region between the hinge and variable region, thereby resulting in the antibody ( Ab) can be adopted by APC (antigen presenting cell), as a result of which the peptide antigen is presented on the surface of APC in the context of MHCII, thus being able to elicit an immune response. The peptides thus inserted play the role of epitopes, and the overall structure of the carrier molecule is not important. It has been shown that it is possible to place the RAS peptide on a (non-CDR region) immunoglobulin loop and the given immunoglobulin will continue to be secreted. The cells have a rigorous "quality control" that prevents the secretion of the immunoglobulin if it is not folded properly, so a change in the sequence of amino acids within the loop may cause the molecule to adopt a structure that the cell recognizes as abnormal and will break down. Therefore, despite the presented examples, it was found that it would be difficult to introduce more far-reaching changes in the immunoglobulin structural loops without changing its basic character at the same time.

[0017]

U.S. Patent Application Number

2004/0101905 describes binding molecules that contain a target binding site and a peptide The Fc effector peptide is an Fc effector peptide that interacts with an effector molecule.

The method of insertion of the effector peptide into a non-CDR located immunoglobulin loop is shown.

CH1 domain of the fragment [0018] Fc effector peptides are structures naturally occurring in antibody loops that are not CDR loops, so they are not expected to interfere with the antibody structure when implanted in various equivalent positions in the immunoglobulin molecule.

[0019] On the other hand, any peptide introduced into the non-CDR loop of this disclosure will most likely be inactive due to the different structural environment that has been selected.

[0020] In both of the above-mentioned previous documents in the field, it was found that it is difficult to introduce peptides into a loop that is to retain its structure and function, since the key element is not to disturb the folded structure of the immunoglobulin, since proper functioning and secretion depend on it.

[0021] US Patent Applications Numbers

2004/0132101 and 2005/0244403 describe mutated immunoglobulins with changed affinity ligand affinity, which are natural ligands for antibody structural loops. This document describes a number of mutations in various regions within an immunoglobulin molecule that affect the effector function of the antibody as a whole.

[0022] International Application No. WO 01/83525 relates to proteins containing Fc domains fused to biologically active peptides, said peptides being attached to the N-terminus or C-terminus of the Fc domains.

[0023] US Patent No. 2002/0106370 describes chimeric polypeptides consisting of a binding fragment that has specific binding affinity for the target surface of a eukaryotic cell, and an effector fragment.

[0024] Patent number WO 02/32925 discloses proteins capable of performing antibody functions. To this end, loop structures corresponding to the structure and location of the CDR loops are introduced into said proteins.

[0025] International Application No. WO 2006/036834 discloses molecules and methods that allow the introduction of biologically active peptides into a loop region in the Fc domain. After the biologically active peptide with the desired biological activity has been selected, it is then introduced into the Fc domains, either by linking said peptide to a protein molecule, or by introducing the relevant nucleic acid into the nucleic acid encoding the Fc domain.

[0026] Other prior documents in the art indicate that up to now the immunoglobulin-like skeleton has been used to manipulate within an existing antigen binding site to obtain new binding properties. Thus, until now, reconstructions modifying antigen binding have been introduced only within the CDR regions, in other words, in order to change the binding affinity or specificity of the immunoglobulin fold, only the natural antigen binding sites have been modified. There is extensive literature describing various formats of immunoglobulins modified in this way, often expressed in the form of single-chain Fv fragments (scFv) or Fab fragments, either displayed on the surface of phage particles, or expressed in soluble form in various prokaryotic or eukaryotic expression systems. Greg Winter is one of the leading authors in this field,

Andreas P ^ ckthun and Hennie Hoogenboom.

[0027] The object of the present invention is to provide immunoglobulins in which new antigen binding sites have been introduced, and methods for constructing and producing said immunoglobulins.

[0028] The present invention therefore relates to an immunoglobulin constant domain or part thereof as defined in claims 1 to 7, and nucleic acid molecules encoding said immunoglobulin as defined in claim 7.

[0029] A method of constructing an immunoglobulin comprising at least one modification in the structural loop region of said immunoglobulin and determining the binding of said immunoglobulin to an antigen epitope, wherein the unmodified immunoglobulin does not show significant binding to said epitope, comprises the following:

- providing a nucleic acid encoding an immunoglobulin comprising at least one structural loop region,

- modifying at least one nucleotide residue in at least one of said structural loop regions,

- transfer of said modified nucleic acid in the expression system,

- causing expression of said immunoglobulin,

- contacting the modified resulting immunoglobulin with an epitope, and

- determining whether the modified immunoglobulin binds to a given epitope.

In particular, said construction method relates to an immunoglobulin that specifically binds to an epitope of an antigen selected from the group consisting of allergens, tumor associated antigens, autoantigens, enzymes, bacterial antigens, fungal antigens, protozoal antigens and viral antigens. By modifying the structural loop region, an epitope-binding immunoglobulin can be constructed. In a preferred embodiment, the immunoglobulin binds specifically to at least two such epitopes that differ from each other and belong to the same or different antigens.

[0031] For example, this method relates to the construction of an immunoglobulin that specifically binds to at least one first epitope and comprising at least one modification in at least one structural loop region of said immunoglobulin, and determining the specific binding of said at least one loop region to at least one second epitope, wherein the epitope is selected from the group of above-mentioned antigens, wherein the unmodified structural loop region (non-CDR region) does not specifically bind to said at least one second epitope by the following steps:

- providing a nucleic acid encoding an immunoglobulin that specifically binds to at least one first epitope, comprising at least one structural loop region

- modifying at least one nucleotide residue relating to at least one of said loop regions encoded by said nucleic acid

- transfer of said modified nucleic acid in an expression system

- causing expression of said modified immunoglobulin

- contacting the modified resulting immunoglobulin with said at least one second epitope, and

- determining whether said modified immunoglobulin binds specifically to a second epitope.

[0032] The method preferably relates to at least one modification in at least one structural loop region of said immunoglobulin and the determination of specific binding of said at least one loop region to at least one antigen selected from the group consisting of allergens, tumor associated antigens, autoantigens, enzymes, antigens bacterial, fungal antigens, viral antigens and protozoal antigens, whereas an immunoglobulin containing an unmodified loop structural region does not specifically bind to said at least one antigen.

of the present (the terms here are mono- or [0033] The term "immunoglobulins", according to the invention to be modified "immunoglobulin" and "antibody" interchangeably), may exhibit characteristics of a multispecific or multivalent binding, at least two, and preferably at least three sites of specific binding of epitopes belonging to e.g. antigens, effector molecules / proteins. Immunoglobulins of the invention are also functional fragments adopted in the art, such as Fc, Fab, scFv, single chain dimers from CH / CL, Fv domains or other derivatives or combinations of immunoglobulins, heavy and light chain variable domain domains (such as Fd, V1 , Vk, Vh) and the constant region of an intact antibody, such as CH1, CH2,

CH3, CH4, Cl and Ck, as well as mini-domains consisting of two β-strands of the immunoglobulin domain connected by a structural loop.

[0034] It is understood that the terms "immunoglobulin", "modified immunoglobulin" and "immunoglobulin according to the invention" also encompass immunoglobulin derivatives. A derivative is any combination of one or more immunoglobulins of the invention and / or a fusion protein in which any domain or minidomain of the immunoglobulin of the invention can be introduced anywhere in one or more proteins (such as other immunoglobulins, ligands, skeletal proteins) , enzyme toxins, etc.). The immunoglobulin derivative of the invention can also be obtained by combining it with other substances by various chemical techniques, such as covalent coupling, electrostatic interaction, disulfide bridges, etc.

[0035] Other immunoglobulin-bound substances may be lipids, carbohydrates, nucleic acids, organic and inorganic molecules, as well as any combinations thereof (e.g., PEG, prodrugs and drugs). The derivative is also an immunoglobulin with the same amino acid sequence but formed entirely or partly from unnatural or chemically modified amino acids.

[0036] Immunoglobulins constructed according to the invention will be useful both as single proteins and as fused or derived proteins, usually obtained by fusion in such a way that they become part of larger immunoglobulin structures or complete antibody molecules, or parts thereof, e.g. Fab fragments, Fc fragments, Fv fragments and others. Proteins constructed in this way can be used to produce molecules that will be monospecific, bispecific, and trispecific, and perhaps even have many specificities at the same time, it will be possible to control and pre-bind the binding in accordance with the requirements of the use of such molecules.

simultaneously select the intended [0037] According to the present invention, antigen binding regions or antigen binding sites for all kinds of allergens, tumor associated antigens, autoantigens, enzymes, bacterial antigens, fungal antigens, protozoal antigens and viral antigens can be introduced into the structural loop of a given antibody structure. .

[0038] The term "antigen" according to the present invention means molecules or structures known to interact or are capable of interacting with the CDR loop region of immunoglobulins. Structural loop regions of the previous type do not interact with antigens, but rather contribute to the overall structure and / or to binding to effector molecules.

[0039] The term "allergens, tumor associated antigens, autoantigens, enzymes, bacterial antigens, fungal antigens, protozoal antigens and viral antigens" according to the present invention includes all allergens and antigens that can be recognized by the structure of the antibody, and fragments of such molecules ( particularly substructures generally referred to as "epitopes" (e.g., B cell epitopes)) as long as they are immunologically significant, i.e. also recognized by natural or monoclonal antibodies.

[0040] The term "epitope" according to the present invention means a molecular structure which can be entirely constituted by a binding partner or be part of a binding partner specifically binding to the binding domain or immunoglobulin object of the present invention.

[0041] Chemically, the epitope may consist of a carbohydrate, a peptide, a fatty acid, an inorganic substance, derivatives or combinations thereof. If the epitope is a polypeptide, it will usually contain at least 3 amino acids, preferably 8 to 50 amino acids, and preferably about 10 to 20 amino acids in the peptide. There is no critical upper limit to the length of the peptide, which may include almost the full length of the polypeptide sequence. Epitopes may be linear or conformational epitopes. The linear epitope is made up of a single segment of the primary sequence of the polypeptide chain. Linear epitopes may be contiguous or overlapping. Conformational epitopes are made up of amino acids clustered by folding the polypeptide that forms the quaternary structure, with the amino acids themselves not necessarily adjacent to each other in a linear sequence.

[0042] In particular, epitopes are at least part of diagnostically important molecules, i.e. the absence or presence of an epitope in a sample is qualitatively or quantitatively correlated with either the disease or the state of health or the state of the manufacturing process or the state of the environment and food. Epitopes can also be at least part of the therapeutically relevant molecules, i.e. molecules that can be targeted by the specifically binding domain, which changes the course of the disease.

[0043] Preferred "allergens, tumor associated antigens, autoantigens, enzymes, bacterial antigens, fungal antigens, protozoal antigens and viral antigens" are those allergens or antigens that have been proven to be or may be immunologically or therapeutically important, in particular those for which clinical efficacy tests have been carried out.

[0044] On the other hand, in accordance with another aspect of the present invention, other binding abilities can also be introduced into the structural loop regions, e.g. the ability to bind small molecules, such as drugs or enzymes, catalytic sites for enzymes or for enzymatic substrates or an analogous transition state to the enzyme substrate.

[0045] It is preferred that the new antigen binding site in the structural loops is foreign to unmodified immunoglobulin. Therefore, according to the invention, it is preferred to exclude targets such as effector molecules or Fc receptors from the binding molecules and from the range of immunoglobulin specificity.

[0046] It is preferred that the new antigen binding sites in the structural loops are introduced by substitution (substitution), deletion (deletion) and / or insertion (insertion) within the immunoglobulin encoded by the selected nucleic acid.

[0047] According to another preferred embodiment of the present invention, modification of the at least one nucleotide results in substitution, deletion and / or insertion of an immunoglobulin encoded by said nucleic acid.

[0048] The result of modification of at least one loop region may be a substitution, deletion and / or insertion of one or more amino acids, preferably a point mutation, replacement of whole loops, and better a change of at least 2, 3, 4, 5, 6, 7, 8 , 9, 10, up to 30 amino acids.

[0049] Site-specific random mutation is also preferred. This method allows to replace one or more specific amino acid residues in the structural loop, or to introduce randomly generated inserts into such structural loops. Another preferred alternative is a combinatorial approach.

[0050] Mutation or modification of at least one loop region using methods such as random, semi-random mutagenesis and, in particular, site-directed random mutagenesis is preferred. These methods can be used to modify amino acids at selected positions of the immunoglobulin object of the present invention. In such cases, items are selected randomly or amino acid changes are made using simplified rules. For example, all residues can be mutated to alanine, which is referred to as alanine scanning. These types of methods can be combined with a more sophisticated engineering approach, using selection methods to view higher levels of sequence diversity. A preferred method of the invention relates to a randomly modified nucleic acid molecule that comprises at least one nucleotide repeating unit with the sequence 5'-NNS-3 ', 5'-NNN-3' or 5'-NNK-3 '.

[0051] The randomly modified nucleic acid molecule may contain repeating units as defined above that encode all naturally occurring amino acids.

[0052] As is well known in the art, there are a number of different selection techniques that can be used to identify and isolate proteins with specific binding properties and affinities, including, for example, display techniques such as phage expression, ribosomal expression, cell surface expression, and similar as described below. Methods for producing and screening (i.e. screening) various antibody variants are well known in the art. General methods regarding antibody molecular biology, expression, purification and screening are described in the following papers: Antibody Engineering, published by Duebel & Kontermann, Springer-Verlag, Heidelberg 2001; and Hayhurst & Georgiou, 2001, Curr Opin Chem Biol 5: 683-689; Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2: 339-76.

[0053] The term "structural loop" or "non-CDR loop" according to the present invention should be understood as follows: immunoglobulins are built from domains having so-called immunoglobulin fold. Basically, anti-parallel β-sheets are connected by loops, thus forming a compressed anti-parallel β-barrel. In the variable region, some of the domain loops contribute significantly to the specificity of the antibody, i.e., antigen binding. These loops are called CDR loops. All other loops falling within the domain domain rather co-create the structure of the molecule and / or are responsible for effector functions. These loops are defined here as structural loops or non-loops

CDR.

[0054] Nucleic acid molecules encoding modified immunoglobulins (and always included throughout the following description: immunoglobulin fragments) can be cloned into host cells, expressed and tested for binding properties. These practices are carried out by means of well-known procedures, and a number of methods that may be used in the present invention are described in Molecular Cloning - A Laboratory Manual, third edition revised and supplemented (Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001) and Current Protocols in Molecular Biology (John Wiley & Sons). Nucleic acids encoding the modified immunoglobulins of the present invention can be introduced into an expression vector to express said immunoglobulins. Expression vectors usually contain operatively linked immunoglobulin, placed in a functional relationship, with control or regulatory sequences, selectable markers, any fusion partners, and / or additional elements. The modified immunoglobulins of the present invention can be produced by culturing a host cell transformed with a nucleic acid, preferably an expression vector, containing a nucleic acid encoding the modified immunoglobulins, under appropriate conditions to induce or cause expression of the modified immunoglobulins. Methods for introducing exogenous nucleic acid molecules into a host cell are well known in the art and their selection depends on the type of host. Of course, cell-free or extracellular expression systems can also be used to express modified immunoglobulins.

[0055] In a preferred embodiment of the present invention, the modified immunoglobulins are purified or isolated after expression. Modified immunoglobulins can be isolated or purified in a variety of ways well known to those skilled in the art. Standard purification methods include chromatographic, electrophoretic, immunological, precipitation, dialysis, filtration, concentration and chromatographic focusing techniques. Cleansing is often possible thanks to the use of a special fusion partner. For example, antibodies can be purified with glutathione resin if a GST fusion is used, using Ni affinity chromatography⁺²if a 'polyhistidine tail' (His tag) is used, or by using an immobilized anti-flag antibody if a 'flag tail' (flag tag) is used. For general familiarization with the relevant purification techniques, see Antibody Purification: Principles and Practices, 3rd Edition revised and supplemented, Scopes, Springer-Verlag, NY, 1994. Expression of the modified immunoglobulins of the present invention is of course also possible on the surface of the host, in particular on the surface of a bacterial cell, insect cell or yeast cell, or on the surface of phages or viruses.

[0056] Modified immunoglobulins can be screened by a variety of methods, including, but not limited to, methods using in vitro tests, in vivo tests and cell-based assays, and selection techniques. Automated and high performance screening technologies can be used as part of screening procedures. Screening may also require the use of a fusion partner or label, e.g. an enzyme, immuno tag, isotope tag, or small molecular tag such as a fluorescent or colorimetric dye, or a light-emitting molecule.

[0057] In a preferred embodiment, the functional and / or biophysical features of immunoglobulins are screened as part of an in vitro test. In a preferred embodiment, the antibody is screened for its functionality, e.g., the ability to catalyze the reaction or binding affinity for its target.

[0058] Assays may use various detection methods, including, but not limited to, chromogenic, fluorescent, luminescent or isotope labeling.

[0059] As is known in the art of the subject, methods that select preferred library members are a subgroup of screening methods. These methods are referred to herein as "selection methods" and these methods find use in the present invention for screening modified immunoglobulins. By screening immunoglobulin libraries using a given selection method, only preferred library members are selected, i.e. meeting specific selection criteria, subjecting them to multiplication, isolation and / or observation because only the most variants are observed, such methods then theirs. As it turns out, useful enable screening of libraries larger than those that can be screened by methods testing the usefulness of library members individually. Selection is enabled by any method, technique or fusion partner that binds, either covalently or non-covalently, an immunoglobulin phenotype to its genotype, i.e. the function of the antibody with the nucleic acid that encodes them. For example, the use of phage expression as a selection method is possible due to the fusion of library members with the III gene protein. In this way, the selection or isolation of modified immunoglobulins that meet specific criteria, e.g. having binding affinity for an immunoglobulin target, it also selects for or isolates the nucleic acid that encodes them. After isolation, the gene or genes encoding modified immunoglobulins can be amplified. This isolation and amplification process, known as panning, can be repeated, enriching the collection of preferred antibody variants in the library. Definition of the attached nucleic acid sequence ultimately allows gene identification.

[0060] A number of selection methods are known in the art that may find use in the present invention for screening immunoglobulin libraries. These methods include, but are not limited to, phage display (Phage display of peptides and antibodies: a laboratory manual, Kay et al., 1996, Academic Press, San Diego, California, 1996; Lowman et al., 1991, Biochemistry 30: 10832 -10838; Smith, 1985, Science 228: 1315-1317) and its derivative forms such as selective phage infection (Malmborg et al., 1997, J Mol Biol 273: 544-551), the method of selectively infective phage (Krebber et al., 1997 , J Mol Biol 268: 619-630) and delayed infectivity panning (DIP) (Benhar et al., 2000, J Mol Biol 301: 893-904), cell surface presentation (Wittrup, 2001, Curr Opin

Biotechnol., 12: 395-399) such as presentation on the surface of bacterial cells (Georgiou et al., 1997, Nat Biotechnol 15: 29-34; Georgiou et al., 1993, Trends Biotechnol 11: 6-10; Lee et al ., 2000, Nat Biotechnol 18: 645-648; Jun et al., 1998, Nat Biotechnol 16: 576-80), yeast cells (Boder & Wittrup, 2000, Methods Enzymol 328: 430-44; Boder & Wittrup, 1997, Nat Biotechnol 15: 553-557) and mammalian cells (Whitehorn et al., 1995, Bio / technology 13: 1215-1219) as well as in vitro expression techniques (Amstutz et al., 2001, Curr Opin Biotechnol 12: 400-405) such as polysomal presentation (Mattheakis et al., 1994, Proc Natl Acad Sci USA 91: 9022-9026), ribosomal presentation (Hanes et al., 1997, Proc Natl Acad Sci USA 94: 4937-4942 ), presentation on mRNA (Roberts & Szostak, 1997, Proc Natl Acad Sci USA 94: 12297-12302; Nemoto et al., 1997, FEBS Lett 414: 405-408), and the ribosomal inactivation expression system (Zhou et al., 2002, J Am Chem Soc 124, 538543).

[0061] Other selection methods that may find use in the present invention include non-presentation based methods such as in vivo methods, including but not limited to periplasmic expression and cytometric screening (Chen et al., 2001, Nat Biotechnol 19: 537-542), antibody fragment complementation assay (Johnsson & Varshavsky, 1994, Proc Natl Acad Sci USA 91: 10340-10344; Pelletier et al. 1998, Proc Natl Acad Sci USA 95: 12141-12146) and a two-hybrid yeast system (Fields & Song, 1989, Nature, 340: 245-246) used in selection mode (Visintin et al., 1999, Proc Natl Acad Sci USA 96: 11723 -11,728). In an alternative embodiment, the selection is enabled by a fusion partner that binds to a specific expression vector sequence, covalently or non-covalently linking the fusion partner and associated member library of Fc variants with the nucleic acid encoding them. For example, PCT WO 00/22906; PCT WO 01/49058; PCT WO 02/04852; PCT WO 02/04853; PCT WO 02/08023; PCT WO 01/28702; and PCT WO 02/07466 describe such a fusion partner and technique that may find use in the present invention. In an alternative embodiment, in vivo selection is possible if the expression of the antibody brings the cell specific benefits in terms of growth, reproduction or survival.

[0062] A subgroup of selection methods referred to as "directed evolution" methods are methods involving mating or propagation of preferred sequences during selection; sometimes in conjunction with the inclusion of new mutations. According to those skilled in the art of the subject, controlled evolution methods may enable identification of the most preferred sequences available in the library, as well as may increase the variety of screened sequences. Various methods of directed evolution are known in the art that may find use in the present invention for screening various antibody variants, including, but not limited to, DNA shuffling (PCT WO 00/42561 A3; PCT WO 01/70947 A3), exon shuffling ( U.S. Patent No. 6,365,377; Kolkman & Stemmer, 2001, Nat Biotechnol 19: 423428), family shuffling (Crameri et al., 1998, Nature 391: 288-291; U.S. Patent Document No. 6,376,246), the RACHITT.TM method. (random chimerogenesis on transient matrices) (Coco et al., 2001, Nat Biotechnol 19: 354-359; PCT WO 02/06469), STEP and random attachment of primers in in vitro recombination (Zhao et al., 1998, Nat Biotechnol 16 : 258-261; Shao et al.,

1998, Nucleic Acids Res

26: 681-683), gene arrangement mediated by endonuclease (US Patent Document No. 6,352,842; US Patent Document 6,361,974), Gene Site Saturation Mutagenesis.TM (Gene Locagen Mutagenesis) (US Patent No. 6,358,709) , Gene Reassembly.TM. (gene rearrangement) (US Patent Document No. 6,358,709), SCRATCHY (Lutz et al., 2001, Proc Natl Acad Sci USA 98: 11248-11253), DNA fragmentation methods (Kikuchi et al., Gene 236: 159-167) , single chain DNA shuffling (Kikuchi et al., 2000, Gene 243: 133-137), and AMEsystem.TM. guided based antibody engineering technology

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Patent Document No. 5,723,323).

[0063] One or more cell-based or in vivo assays can be used to screen for different antibody variants. These types of tests typically involve the addition of purified or non-purified, modified immunoglobulins from the outside, so that the cells are exposed to individual immunoglobulins or immunoglobulin pools belonging to the library. These tests usually, though not always, the functioning of immunoglobulin; ie.

antibodies to bind to its target structure and mediate certain biochemical phenomena, such as, for example, effector function, inhibition of ligand / receptor binding, apoptosis, etc. like for example. cell survival, cell death, change in cell morphology, as well as the appearance of transcriptional activation in the form of cellular expression of a natural gene or reporter gene. For example, these tests can measure the ability of different antibody variants to elicit ADCC, ADCP or CDC. Some tests may require additional cells or components to be added to target cells, such as serum complement or effector cells such as peripheral blood mononuclear cells (PBMCs), NK cells, macrophages etc. Such additional cells may be from any organism, preferably from humans, mice, rats, rabbits and monkeys. Immunoglobulins may cause apoptosis of certain cell lines that express the target structure, or may mediate the attack of immune cells added to the assay on target cells. In the subject field, methods for monitoring cell death or viability are known and are based on the use of dyes, including immunochemical, cytochemical and radioactive reagents. For example, tests based on caspase staining may enable the measurement of apoptosis, and the uptake or release of radioactive substrates or fluorescent dyes, such as the Alamar blue test, may allow monitoring of cell growth or activation. In a preferred embodiment, a cytotoxicity assay based on DELFIA.RTM.EuTDA (Perkin Elmer, MA) can be used. Alternatively, cell death or damage can also be monitored by measuring the release of one or more natural intracellular compounds, such as lactate dehydrogenase. The transcriptional activation used in cell-based assays can also be used to test the function. In this case, monitoring of the response may consist of testing natural genes or immunoglobulins that may be elevated, e.g. the release of specific interleukins may be measured, or the reading may be carried out using a reporter construct. Cell-based assays may also include measuring morphological changes in cells as a response to the presence of modified immunoglobulins. Prokaryotic or eukaryotic cells can be used for these tests, a number of cell lines are known in the art that can be used for this purpose. A possible solution is also the creation of cell screens using cells that have been transformed or transfected with nucleic acids encoding the variants. In other words, antibody variants are not added to external cells. For example, in one embodiment, the cell screen utilizes cell surface presentation. You can then use a fusion partner that will allow presentation of the modified immunoglobulins on the cell surface (Wittrup, 2001, Curr Opin Biotechnol, 12: 395-399).

[0064] In a preferred embodiment, the immunogenicity of the modified immunoglobulins can be determined experimentally using one or more cell-based assays. In a preferred embodiment, ex vivo T cell activation assays are used to experimentally determine immunogenicity. In this method, cells presenting antigens and virgin T cells from selected donors are confronted, once or several times, with a given peptide or whole antibody. Detection of T cell activation can then be carried out using a variety of methods, e.g., monitoring cytokine production or measuring uptake of tritiated thymidine. The most preferred implementation involves monitoring the production of interferon γ using Elispot tests (Schmittel et al., 2000, J. Immunol. Meth.

24:17-24).

[0065] The biological properties of the modified immunoglobulins of the present invention can be determined by means of experiments on cells, tissues and whole organisms. As is known in the art, drug research is often conducted on animals, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs and monkeys, and the purpose of these studies is to measure the effectiveness of the drug in the treatment of a given disease or disease model, or measurement of pharmacokinetics, toxicity and other drug properties. Animals are sometimes referred to as disease models. Therapeutic agents are often tested in mice, including, but not limited to, nude mice, SCID mice, xenograft mice, and transgenic mice (including knock-in and knock-out mice). Such experiments can provide valuable data to determine the potential of a given antibody as a possible therapeutic agent. All organisms can be used for testing, mammals are preferred. For example, due to genetic similarity to human beings, monkeys may be a particularly useful therapeutic model, which may be used to test the efficacy, toxicity, pharmacokinetics and other properties of the modified immunoglobulins of the present invention. A necessary condition for the authorization of active substances for use as medicines is their testing on humans, so such experiments are anticipated. Therefore, the modified immunoglobulins of the present invention can be tested in humans to determine their therapeutic efficacy, toxicity, immunogenicity, pharmacokinetics and / or other clinical properties.

[0066] The modified immunoglobulins of the present invention may find use in a variety of products of the present therapeutic preparative one of the subject antibody-based agent. The antibody variant of the invention is used either prophylactically or analytically, as a diagnostic, as an industrial compound or as a scientific reagent, a therapeutic agent is preferred. The antibody variant may find use in a monoclonal or polyclonal antibody composition. A preferred embodiment envisages the use of the modified antibodies of the present invention to kill cells carrying the target antigen, e.g., cancer cells. In another preferred embodiment, the modified immunoglobulins of the present invention serve as blockers, antagonists or agonists of the target antigen, e.g.

antagonizing the cytokine or cytokine receptor. In another preferred embodiment, the modified immunoglobulins of the present invention serve as blockers, antagonists or agonists of the target antigen, killing the target cells carrying the target antigen. In another preferred embodiment, the modified immunoglobulins of the present invention serve as blockers, antagonists or agonists of growth factors or growth factor receptors, killing target cells that are carrier or require a target antigen. In another preferred embodiment, the modified immunoglobulins of the present invention serve as blockers, antagonists or agonists of enzymes and enzyme substrates [0067] The modified immunoglobulins of the present invention can be used for a variety of therapeutic purposes. In a preferred embodiment, the antibody containing the modified immunoglobulins is administered to a patient to treat a specific disorder. For the purposes of the present invention, the term "patient" includes human beings and other animals, primarily mammals and humans. By "specific disorder" is meant a disorder that can be corrected by administering the particular pharmacological composition containing the modified immunoglobulin object of the present invention.

[0068] In one embodiment, the modified immunoglobulin object of the present invention is the only therapeutically active agent administered to a patient. Alternatively, the modified immunoglobulins of the invention are administered in combination with one or more other therapeutic agents, including but not limited to cytotoxic agents, chemotherapeutic agents, cytokines, growth inhibitors, antihormonal agents, kinase inhibitors, anti-angiogenic factors, cardioprotective agents and other factors therapeutic. Modified immunoglobulins may be administered along with one or more other therapeutic regimens. For example, the antibody variant of the present invention may be administered to a patient in combination with chemotherapy, radiation therapy, or also concomitant chemotherapy and radiation therapy. In one embodiment, the modified immunoglobulins of the present invention may be administered in combination with one or more antibodies that may or may not contain the antibody variant of the present invention. According to another embodiment of the invention, it is envisaged to use the modified immunoglobulins of the present invention in combination with one or more other anti-tumor therapies to combat cancer cells ex vivo. It is anticipated that this type of ex vivo treatment could be useful in bone marrow transplants, in particular in autologous bone marrow transplants. Of course, it is also envisaged that the antibodies of the invention may be used in combination with other therapeutic techniques, including surgical procedures.

[0069] There are a number of other therapeutic agents that can be administered in combination with the modified immunoglobulins of the present invention. In one embodiment, the modified immunoglobulin is administered in conjunction with an anti-angiogenic agent, which is a blocking compound or to some extent interferes with the formation of blood vessels. The anti-angiogenic agent may be a small molecule or protein, e.g. an antibody, Fc fusion, or cytokine that binds to a growth factor or growth factor receptor that supports the angiogenesis process. A preferred anti-angiogenic factor is an antibody that binds to vascular endothelial growth factor (VEGF). An alternative embodiment provides for the administration of the modified immunoglobulin in combination with a therapeutic agent that induces or enhances the adaptive immune response, e.g., a CTLA-4 targeted antibody. In another embodiment, the modified immunoglobulin is administered in combination with a tyrosine kinase inhibitor, which is a molecule that inhibits to some extent tyrosine kinase activity. Another alternative embodiment is the administration of the modified immunoglobulins of the present invention in combination with a cytokine. By "cytokine" is meant here a generic term denoting proteins released by a given population of cells, acting on other cells as intercellular mediators, including chemokines.

[0070] Therapeutic compositions are envisaged in which the modified immunoglobulins of the present invention and one or more active therapeutic agents are used in one formulation. Preparation of the subject antibody variants for storage involves mixing immunoglobulins with the required level of purification with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed., 1980) in the form of lyophilized formulas or aqueous solutions . It is preferred that the formulations intended for in vivo administration are sterile. This condition is not difficult to fulfill by using filtration through sterile filtration membranes or other methods. The modified immunoglobulins and other therapeutically active agents presented herein may also have an immunoliposome formula and / or be encapsulated in myocapsules.

[0071] The pharmaceutical composition containing the modified immunoglobulin of the present invention, preferably in the form of a sterile aqueous solution, can be administered by a variety of methods, including, but not limited to, orally, subcutaneous, intravenous, intranasal, intracutaneous, transdermal, topical (e.g., gels, ointments, lotions, creams etc.), intraperitoneal, intramuscular, pulmonary (e.g. AERx inhalation technique)^TM offered commercially by Aradigm, or using the Inhance pulmonary application system^TM offered commercially by Inhale Therapeutics), vaginally, enterally, rectally or intraocularly.

[0072] The term "specific binding" as used herein refers to a binding reaction that allows the emerging of a related ligand from a heterogeneous population of molecules. Under the conditions indicated (e.g., for immunoglobulins, these will be the conditions of the immunoassay), the specific antibody binds to its specific "target", but does not bind in significant amounts to other molecules present in the sample. The modified structural loop regions, comparable to the CDRs of antibodies, are protein fragments that bind to antigens or molecules, but are not antigens as such.

[0073] The term "expression system" refers to nucleic acid molecules containing the desired coding sequence and control sequences in operative linkage that, when introduced into a "host" by transformation or transfection, will render it capable of producing the proteins they encode. To effect transformation, a given expression system is inserted into the vector; however, in such a situation, the given DNA can be integrated into the host chromosome.

[0074] The expression system may comprise a vector. Any expression vector known in the art may be used for this purpose.

[0075] Expression of the modified immunoglobulin in a host is preferred, preferably in a bacterial cell, yeast cell, plant cell, animal cell, plant or animal.

[0076] A variety of host cells can be used to express the modified immunoglobulin, including, but not limited to, mammalian cells (animal cells), plant cells, bacteria (e.g., Bacillus subtilis,

Escherichia coli), insect cells and yeast cells (e.g., Pichia pastoris, Saccharomyces cerevisiae). For example, a number of cell lines that may find use in the present invention are described in the ATCC cell line catalog available in the American Type Culture Collection. In addition, according to the present invention, plant and animal cells can also serve as hosts for the expression of immunoglobulins. The selection of cassettes or expression or transfection vectors should be made according to the selection of the host.

[0077] Of course, cell-free or extracellular protein expression systems can also be used. In vitro transcription / translation protein expression platforms that produce sufficient amounts of protein offer many benefits of cell-free protein expression, eliminating the need for tedious upstream or downstream steps for major surgery (e.g. transformation of host cells, cell cultures, lysis), which usually accompany cell-based expression systems.

[0078] The immunoglobulin or pharmaceutical preparation containing it, which includes at least one modification in the structural loop region of said immunoglobulin, and determining whether said immunoglobulin binds to an antigen epitope, wherein unmodified immunoglobulins do not bind to said epitope to a significant extent, perform as follows:

- providing a nucleic acid encoding an immunoglobulin comprising at least one structural loop region,

- modifying at least one nucleotide residue in at least one of said loop regions,

- transfer of said modified nucleic acid in the expression system,

- expression of said modified immunoglobulin,

- contacting the expressed modified immunoglobulin with an epitope,

- determining whether said modified immunoglobulin binds to said epitope, and

- providing a modified immunoglobulin binding to said epitope and, optionally, processing it into a pharmaceutical preparation.

In particular, a multispecific immunoglobulin that specifically binds to at least one first molecule, or a pharmaceutical preparation thereof, comprising at least one modification in at least one structural loop region of said immunoglobulin, and determining the specific binding of said at least one region loop with at least one second molecule selected from the group consisting of allergens, tumor associated antigens, autoantigens, enzymes, bacterial antigens, fungal antigens, protozoal antigens and viral antigens, wherein the immunoglobulin containing the unmodified loop structural region does not specifically bind to said at least one second molecule can be made as follows:

- providing a nucleic acid encoding an immunoglobulin that specifically binds to at least one first molecule comprising at least one structural loop region,

- modifying at least one nucleotide residue in at least one of said loop regions encoded by said nucleic acid,

- transferring said modified nucleic acid into the expression system

- expression of said modified immunoglobulin

- contacting the expressed modified immunoglobulin with said at least one second molecule, and

- determining whether said modified immunoglobulin binds specifically to the second molecule and

- providing the modified immunoglobulin specifically binding to said at least one second molecule and, optionally, processing it into a pharmaceutical preparation.

[0080] It is preferred to construct within a member of the specifically binding pair more than one specific binding site (Kufer at al., (2004) Trends in Biotechnology, vol. 22 pages 238-244).

[0081] Several attempts have been made to produce multispecific, e.g. bispecific, monoclonal antibodies or antibody fragments. One of the problems in producing bispecific antibodies built from two different polypeptide chains (heavy and light chains) is the need to express four different chains (two heavy and two light) in one cell, resulting in a number of different combinations of molecules that need to be separated from the desired bispecific molecules in a given mixture. Because of the similarity of these particles, their separation is difficult and expensive. In order to minimize the occurrence of such undesirable vapors, a number of useful techniques have been developed and implemented (Carter (2001) Journal of Immunological Methods, vol. 248, pages 7-15).

[0082] A possible solution to a given problem is to produce one polypeptide chain with two specific binding sites, such as two scFvs fragments linked together, or to produce so-called "Diabodies". These types of molecules have been shown to deviate significantly from the folding of a natural molecule and are invariably difficult to produce (LeGall et al. (2004) Protein Engineering, Design &amp; Selection, vol. 17 pages 357366).

[0083] Another problem accompanying the current construction of bispecific antibodies is the fact that even if the parental antibodies bind bivalently to their respective partners (e.g., IgG), the resulting bispecific antibody is monovalent to each of its binding partners.

[0084] The preferred multispecific molecules of the present invention solve these problems:

[0085] Expression of the bispecific molecule as one polypeptide chain is possible (modified Ig domain with two specific binding sites, see chapter on examples), which is easier to achieve than expression of two antibody polypeptide chains (Cabilly et al. Proc. Natl. Acad. Sci. USA 81: 3273-3277 (1984)).

[0086] It can also be produced as an antibody-like molecule (e.g., composed of two polypeptide chains); due to the fact that the second specific binding site is located in the invariant region of the molecule, there is no need to provide two different heavy chains or two different light chains. Thus, it is not possible to mistake the chains in pairs.

[0087] The antibody of the present invention may consist of a heavy chain and a light chain, forming together a variable region that binds to a specific binding partner, wherein the second specific binding site may be formed by a modified loop being a heavy or light chain structural loop . The binding site can also be formed by several non-CDR loops whose structures may be adjacent (either on the heavy or light chain, or on both chains).

[0088] The modified antibody or derivative thereof may be a complete antibody or antibody fragment (e.g. Fab, CH1-CH2, CH2-CH3).

<td>[0089] It can happen</td><td colspan="2">it associate with</td><td>mono binding partner</td><td>be</td>

<td>wielowalentnie,</td><td>or</td><td>even</td><td colspan="2">with different valences from</td>

<td>each construction.</td><td colspan="2">partners</td><td>bindings, depending on</td><td>from</td>

<td>[0090] Because</td><td>is</td><td>number</td><td>various loops that</td><td>can</td>

to select and construct a specific binding site in non-CDR regions located in heavy and light chains, antibody derivative forms with even more than two specificities can be constructed without encountering the above-mentioned problems.

[0091] Specific binding domains located within one polypeptide chain can be linked to each other with or without the aid of a polypeptide linker.

[0092] Some classes of antibodies can be regarded as multispecific, in particular bispecific, in nature: they bind to an antigen (which is usually e.g. either a foreign structure or a tumor associated structure) by means of a variable region and bind to Fc effector molecules using an Fc fragment (e.g. Fc receptors on different cells of the immune system or a complementary protein), thus enabling effects like ADCC, ADCP or CDC.

[0093] Fc effector molecules are bound by the Fc fragment of an immunoglobulin molecule (in the case of IgG1 it consists of the CH2 and CH3 domains), with a number of methods described to optimize effector function by improving the binding of the Fc fragment of the antibody molecule or by glycoengineering techniques (US Patent Document No. 6,602,684), or protein engineering techniques used directly within Fc (US Patent Document No. 2005/0054832) or indirectly outside of the Fc fragment (US Patent Document 2005/02444403). Using these techniques, binding of the Fc region to the Fc receptor and / or binding to complementary proteins of the Cq 1 type was modified. Usually, the affinity of this type of Fc effector molecule is expected to improve because it correlates with improved effector function.

[0094] The present invention allows the construction of an antibody that binds to an Fc effector molecule outside of the natural Fc binding region. Modified domain loops that are not loops involved in the "natural" binding process of the Fc effector molecule can be selected from a library or constructed to bind to one or more Fc effector molecules. An antibody equipped with such additional Fc effector molecule binding sites would have stronger avidity to either a particular Fc effector molecule or to an effector cell displaying an Fc effector molecule, and could thus interact more strongly than antibodies obtained by glycoengineering or Fc regions improved by other methods. However, in some embodiments of the present invention, the effector properties of the antibody to be modified should not be changed directly, but should remain intact by modifying the structural loop of the present invention.

[0095] Antibody fragments have some advantages over whole antibodies. The fragments are usually characterized by good biodistribution and are easier to produce. However, most constructed antibody fragments have no effector function and have a short in vivo half-life (Hollinger P. et al., Nat Biotechnol. (2005) 23: 1126-36).

[0096] Neither the CH1 domains nor the Ck and CA domains mediate effector functions, which explains why Fab fragments do not show ADCC, ADCP and CDC. Patent application number WO 02/44215 describes binding molecules that consist of an antibody antigen binding site and Fc binding peptide effector molecules. In this way, an antibody fragment exhibiting effector functions can be constructed. The peptide is introduced into the binding molecule in a place that does not interfere with antigen binding or the ability of the peptide to bind to the Fc effector molecule.

[0097] In accordance with the present invention, binding to Fc effector molecules can be provided by modified immunoglobulin domains previously selected for binding to the Fc effector molecule from library resources collecting random loops within the established immunoglobulin domain skeleton. It is therefore possible to select specific loop sequences that will not bind to Fc effector molecules outside of the immunoglobulin domain backbone. Preferred polypeptides resulting from the present invention should therefore consist of over one hundred amino acids.

[0098] In order to select for the potential effector function of such domains of the present invention, libraries that collect mutant CH1, Ck or CA domains should be selected for binding to Fc receptors and / or complementary factors such as C1q.

[0099] To increase the half-life of a molecule constructed from or containing such a domain (e.g. CH1, CH2, CH3, CH4, Ck or CA), selection for binding to FcRn can be made from mutant-accumulating libraries, e.g. CH1-, CH2-, CH3-, CH4-, Ck- or CA- according to the present invention.

[0100] FcRn receptors for selection can be obtained either on the surface of cells in which expression of such receptors occurs naturally, or by expression and purification of the extracellular portion of a given receptor. For the purposes of the present invention, the first FcRn screening allows the selection of mutated domains that can then be subjected to further in vitro testing and even further characterization in FACS experiments by binding to FcRn receptor expressing cells. Further characterization may consist in determining the binding affinity ranking with various recombinant FcRn, isoforms and allotypes e.g. by surface plasmon resonance techniques.

[0101] The immunoglobulin object of the present invention is an immunoglobulin of human origin.

[0102] Because the modified immunoglobulin can be used for a variety of purposes, in particular in pharmaceutical compositions, it is preferred that it be an immunoglobulin of human origin. The modified immunoglobulin may of course also be a chimeric immunoglobulin.

[0103] According to another preferred embodiment of the present invention, the human immunoglobulin is from the group of immunoglobulins type G, in particular from IgG1, IgG2,

IgG3 or IgG4.

[0104] The modified immunoglobulin may be derived from one of the above-mentioned classes of immunoglobulins.

[0105] An immunoglobulin comprising one heavy and / or one immunoglobulin light chain or part thereof is preferred.

[0106] The modified immunoglobulin may contain one heavy and / or one light chain, at least one variable and / or constant domain.

[0107] According to the present invention, the immunoglobulin comprises at least one or part of the immunoglobulin constant domain, including the minidomain.

[0108] The constant domain is an immunoglobulin fold unit belonging to the constant fragment of an immunoglobulin molecule, also called a constant region domain (e.g. CH1, CH2, CH3, CH4, Ck, Cl).

[0109] A preferred immunoglobulin according to the present invention consists of a constant domain selected from the group consisting of CH1, CH2, CH3, CH4, Igk-C, Igl-C, or parts thereof, including a minidomain having at least one loop region, and characterized by in that said at least one loop region comprises at least one amino acid modification forming at least one modified loop region, wherein said at least one modified loop region binds specifically to at least one antigen epitope.

[0110] The constant domain is selected from the group consisting of CH1, CH2, CH3 or CH4 domains, CL domain, domain

Ck, C \ domain, Fab fragment or Fc fragment or combinations thereof.

[0111] The modified immunoglobulin according to the present invention may contain one or more constant domains (e.g., at least two, three, four, five, six, ten). If more than one domain is present in the modified immunoglobulin, these domains may be of the same type or of different types (e.g. CH1-CH1-CH2, CH3-CH3). Also, the order of individual domains can be any (e.g. CH1CH3-CH2, CH4-CH1-CH3-CH2).

[0112] All amino acid sequence numbers in immunoglobulins are consistent with the IMGT numbering system (IMGT, ImMunoGeneTics international information system @ imgt.cines.fr; <a href="http://imgt.cines.fr">http://imgt.cines.fr</a>; Lefranc et al., 1999, Nucleic Acids Res. 27: 209-212; Ruiz et al., 2000 Nucleic Acids Res. 28: 219-221; Lefranc et al., 2001, Nucleic Acids Res. 29: 207-209; Lefranc et al., 2003, Nucleic Acids Res. 31: 307-310; Lefranc et al., 2005, Dev Comp Immunol 29: 185203).

[0113] According to another preferred embodiment of the present invention, the modified loop regions of the CH1, CH2, CH3 and CH4 domains contain amino acids 7 to 21, amino acids 25 to 39, amino acids 41 to 81, amino acids 83 to 85, amino acids 89 to 103 and amino acids 106 to 117.

[0114] It is preferred that the Igk-C and Igl-C loop regions of human origin contain amino acids 8 to 18, amino acids 27 to 35, amino acids 42 to 78, amino acids 83 to 85, amino acids 92 to 100, amino acids 108 to 117 and amino acids 123 to 126.

[0115] It is preferred that the structural loop regions of the human immunoglobulin variable domain contain amino acids 8 to 20, amino acids 44 to 50, amino acids 67 to 76 and amino acids 89 to 101.

[0116] The above-mentioned amino acid regions of the respective immunoglobulins comprise loop regions that are subject to modification.

[0117] Specific binding of a given modified immunoglobulin to a molecule is determined by a binding assay selected from the group consisting of immunoassays, preferably enzyme-linked immunosorbent assays (ELISAs), surface plasmon resonance tests, nuclear magnetic resonance spectroscopy examining differences in saturation transfer, magnetic resonance spectroscopy nuclear research NOE transfer (tr NOE), competitive tests, tissue binding assays, tests

<td>bindings to phones.</td><td>the living</td><td>cell</td><td>and tests</td><td>extracts</td>

<td>[0118] Tests</td><td>bond</td><td>can on</td><td>carry</td><td>using</td>

<td>different methods</td><td colspan="2">known in</td><td>present</td><td>the field,</td>

including, but not limited to, FRET (fluorescence resonance energy transfer) and BRET (bioluminescence resonance energy transfer) tests, AlphaSkrin.TM. (homogeneous amplified luminescence proximity test), proximation-scintillation test, ELISA (enzyme-linked immunosorbent assay), SPR (surface plasmon resonance, also known as BIACORE.RTM), isothermal calorimetric titration, differential gel filtration calorimetry and electrophoresis, These and other methods may use some fusion partners or labels.

[0119] Modified immunoglobulin conjugated to a tag selected from the group consisting of organic molecules, enzyme tags, radioactive tags, fluorescent tags, stained tags, chromogenic tags, luminescent tags, haptens, digoxygenin, biotin, metal complexes, metals, colloidal gold and mixtures is preferred these.

[0120] The modified immunoglobulin can be conjugated to other molecules that allow simple detection of said conjugate, e.g. in binding assays (e.g. ELISA) and binding assays.

[0121] According to a particularly preferred embodiment of the present invention, the immunoglobulin consists of a constant domain selected from the group consisting of the CH1, CH2, CH3, CH4 domains, Igk-C, Igl-C, or parts thereof, including minidomains, or combinations thereof at least one loop region, and characterized in that said at least one loop region comprises at least one amino acid modification forming at least one modified loop region, wherein said at least one modified loop region binds specifically to at least one antigen epitope.

molecule fragment [0122] It is preferred that the molecular binding of at least one modified antibody domain (= binding to a specific partner via invariant sequences or structural loops) to at least one other binding which may be an antibody, antibody, soluble receptor, ligand or other modified antibody domain.

[0123] The molecule is selected from the group consisting of proteinaceous molecules, nucleic acids and carbohydrates.

[0124] Loop regions of modified immunoglobulins can bind specifically to binding molecules of any kind, in particular to proteinaceous molecules, proteins, peptides, polypeptides, nucleic acids, glycans, carbohydrates, lipids, small organic molecules, and inorganic molecules. Modified immunoglobulins may, of course, contain at least two loop regions, each of which loop regions may specifically bind to other molecules or epitopes.

[0125] According to a preferred embodiment of the present invention, the molecule that binds to the modified structural loop region is selected from the group consisting of tumor associated antigens, in particular

EpCAM, tumors glycoprotein 72 (TAG-72), tumor associated CA 125 antigen, prostate cell membrane antigen (PSMA), high molecular weight melanome associated antigen (HMW-MAA), tumor associated carbohydrate-expressing antigen associated with Lewis Y, antigen carcinogenesis (CEA), CEACAM5, HMFG PEM, mucin MUC1, MUC18 and tumor-associated cytokeratin antigen, bacterial antigens, viral antigens, allergens, fluorescein, lysozyme, toll-like receptor 9, erythropoietin, CD2, CD3, CD3E, CD4, CD11, CD11a, CD14, CD18, CD19, CD20, CD22, CD23, CD25, CD28, CD29, CD30, CD33 (p67 protein), CD38, CD40, CD40L, CD52, CD54, CD56, CD80, CD147, GD3, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-5, IL6, IL-6R, IL-8, IL-12, IL-15, IL-18, IL-23, interferon alpha, interferon beta, interferon gamma; TNF-alpha, TNFbeta2, TNF.alfa, TNFalfabeta, TNF-R1, TNF-RII, FasL, CD27L, CD30L, 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEG1, OX40L, RECEPTOR 1 TRAIL, adenosine A1 receptor, beta lymphotoxin receptor, TACI, BAFF-R, EPO; LFA-3, ICAM-1, ICAM-3, beta1 integrin, beta2 integrin, alpha4 / beta7 integrin, alpha2 integrin, alpha3 integrin, alpha4 integrin, alpha5 integrin, alpha6 integrin, alfav integrin, alpha Vbeta3 integrin, FGFR-3 growth factor keratocytes, VLA-1, VLA-4, L-selectin, anti-Id, E-selectin, HLA, HLADR, CTLA-4, T cell receptor, B7-1, B7-2, VNR integrin, TGFbeta1, TGFbeta2, eotaxin1, BLyS (B-lymphocyte stimulator), complementary factor C5, IgE, factor VII, CD64, CBL, NCA 90, EGFR (ErbB-1), Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB4), tissue factor, VEGF, VEG-FR, endothelin receptor, VLA-4, carbohydrates such as blood group antigens and related carbohydrates, galyl glycosylation, gastrin, gastrin receptors, tumor carbohydrates, hapten NP-cap or NIP-cap, T alpha / beta cell receptor, E selectin, digoxin, placental alkaline phosphatase (PLAP) and PLAP-like alkaline nuclear phosphatase, transferrin receptor, heparanase, human myosin glycoprotein IIb / IIIa (GPIIb / IIIa), glycoprotein gH of the human cytomegalovirus (HCMV) envelope, HIV gp120, HCMV, respiratory syncytial virus RSV F, RSVF Fgp, integrin VNR, Hep B gp120, CMV, gpIIbIIIa, loop HIV III V3, Fgp respiratory syncytial virus (RSV), herpes simplex virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV envelope gB glycoprotein, Clostridium perfringens toxin and fragments thereof.

[0126] A preferred immunoglobulin according to the present invention may bind to one of the above-mentioned molecules. These molecules also include antigens.

[0127] According to another preferred embodiment of the present invention, amino acid residues at positions 17, 29 to 34, 85.4 to 85.3, 92 to 94, 97 to 98 and / or 108 to 110 in the CH3 domain are modified.

[0128] A preferred modification of the immunoglobulin according to the invention consists in deletion, substitution or insertion.

[0129] According to the present invention, at least 1, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10 and 15 amino acids should be removed, replaced by other amino acids (including modified amino acids) or introduced into the immunoglobulin loop region. However, the maximum number of amino acids inserted into the immunoglobulin loop region must not exceed 30, preferably 25, and preferably 20. Random amino acid substitution and insertion by methods known in the art and as described in this patent application is preferred.

[0130] The immunoglobulin according to the invention is characterized according to a specific embodiment in that the CH3 region comprises

SEQ ID No. 16 or SEQ ID No. 18 when EpCam binds to said immunoglobulin, SEQ ID No. 20 when fluorescein binds to said immunoglobulin, SEQ ID No. 22, 24, 26, 28, 30 or 32, if said immunoglobulin binds to lysozyme, SEQ ID No. 34, 36, 38 or 40 if TLR9 binds to said immunoglobulin, and SEQ ID No. 42 if lysozyme and / or erythropoietin binds to said immunoglobulin.

[0131] According to a particular embodiment of the invention, the immunoglobulin is characterized in that it comprises the sequence SEQ ID No. 44 or SEQ ID No. 46 if lysozyme and gp41 bind to said immunoglobulin.

[0132] Preferred is a modified immunoglobulin that is conjugated to a tag or reporter molecule selected from the group consisting of organic molecules, enzyme tags, radioactive tags, stained tags, fluorescent tags, chromogenic tags, luminescent tags, haptens, digoxygenin, biotin, metal complexes , metals, colloidal gold and mixtures thereof.

[0133] Modified immunoglobulins conjugated to the above-mentioned labels can be used, e.g., in diagnostic methods.

[0134] A further aspect of the present invention relates to the use of the immunoglobulin according to the present invention or obtainable by the method according to the present invention for the preparation of a vaccine for active immunization. In this way, the immunoglobulin can either serve as an antigenic drug substance for formulation of the vaccine, or for retrieval or capture of antigenic structures for later use in the vaccine formulation.

[0135] A further aspect of the present invention relates to the use of the immunoglobulin according to the present invention or obtainable by the method according to the present invention for the preparation of an immunoglobulin protein library.

[0136] The immunoglobulin object of the invention can be used in the method of specific binding and / or detection of a molecule, with the following steps:

present to the applicant

a) contacting the modified immunoglobulin according to the present invention or the modified immunoglobulin which can be obtained by the method according to the present invention with a test sample in which said molecule is suspected, and

b) detecting possible formation of a specific immunoglobulin / molecule complex.

[0137] The immunoglobulin object of the present invention can be used in a method for specifically isolating a molecule, consisting of the following steps:

a) contacting the modified immunoglobulin according to the present invention or the modified immunoglobulin which can be obtained by the method according to the present invention with a sample containing said molecule,

b) isolating the resulting specific immunoglobulin / molecule complex, and

c) optionally isolating the molecule from said complex.

[0138] The immunoglobulins of the present invention can be used to specifically isolate molecules present in a sample. The use of multispecific immunoglobulins allows to isolate more than one molecule from a sample. The use of modified immunoglobulins in such methods is particularly advantageous, since it allows, e.g., to generate a matrix with a homogeneous surface on which a defined number of partners is placed (i.e. modified immunoglobulins) capable of binding selected molecules. However, when using monospecific binding partners, a homogeneous matrix cannot be generated because the single binding partners do not bind with the same efficiency to the matrix.

[0139] The immunoglobulin object of the present invention can be used in a method for delivery to a target compound, comprising the following steps:

a) contacting the modified immunoglobulin according to the present invention or the modified immunoglobulin which can be obtained by the method according to the present invention capable of binding specifically to said compound,

b) delivering the immunoglobulin / compound complex to the target.

[0140] The modified immunoglobulins of the present invention may serve to deliver at least one compound associated with the CDR regions and / or modified loop regions to a target.

[0141] The immunoglobulin according to the present invention or the immunoglobulin obtainable according to the present invention may be part of a protein library.

[0142] Preferred methods for creating said library are set out above and in the examples. The library of the present invention can be used to identify immunoglobulins that bind to a specific molecule.

[0143] A protein library comprising the immunoglobulin according to the present invention or which can be obtained by the method according to the present invention can be used to construct immunoglobulin derivative forms.

[0144] Modification of an existing immunoglobulin, consisting of the introduction of antigen binding sites into any domain or minidomain, is possible by using a protein library of a given domain containing at least 10, better 100, better 1000, better 10000, better 100,000, preferably over 1,000,000 domain variants having at least one modified loop. The library is then screened for binding to a specific antigen. After molecular confirmation of the desired properties, the selected domain or minidomain is cloned, combined with its introduction into the original immunoglobulin by genetic engineering methods to replace the wild-type region.

Alternatively, DNA alone encoding loops or encoding mutated amino acids can be mentioned to obtain an immunoglobulin equipped with an additional antigen binding site.

[0145] The choice of site for a mutant, structural loop specific for a particular antigen depends on the structure of the original immunoglobulin and the purpose of the additional binding site to be used. For example, if the original molecule is a complete immunoglobulin to which an additional antigen binding site needs to be inserted without interfering with its effector function, the loops to be modified will be selected from domains located relatively far away from the CH2 and CH3 domains, which are natural binding partners with Fc effector molecules. If the original immunoglobulin is a Fab fragment, then it is possible to modify loops belonging to the constant domains of light chains or light chains, or to the respective variable domains. To generate a library, libraries of mutant original molecules containing mutations in one or more structural loops belonging to one or more domains can be prepared. Selection on complete, mutated original molecules may have specific benefits, as selection for antigen binding using a modified structural loop should provide modifications that will be sterically most beneficial if tested for other properties that the modified immunoglobulin is also tested for. should have.

[0146] The requirement for the size of the protein library (i.e.

number of variant proteins) for a mutated domain or minidomain or a fusion-linking protein depends on the task given. In general, a larger library will be needed to generate a de novo antigen binding site than a library for further modification of an existing engineering-generated antigen binding site from a modified structural loop (e.g., to enhance affinity or a slight change in antigen specificity).

[0147] The immunoglobulin library or nucleic acid library contains a variety of different immunoglobulins, i.e. a constant domain, minidomain and / or at least one structural loop region contained in the minidomain, or nucleic acid molecules encoding them. The library groups members with different modifications, with many members being defined by modifications contained in at least one structural loop region. A preferred library of nucleic acids contains at least 10 different members (resulting in the exchange of one amino acid), preferably at least 100 members, better 1000 or 10,000 different members (e.g.

constructed for randomization strategies or combinatorial techniques). Even more diverse numbers of individual members, such as at least 1,000,000 or 1,000,000, are also preferred.

[0148] A combination of two different domains or minidomains selected from at least two libraries of the present invention can also be used to generate multispecific immunoglobulins. Specific immunoglobulins selected in this way can be combined with each other and with other molecules in a manner reminiscent of building blocks to construct the optimal arrangement of domains or minidomains to obtain the required characteristics.

[0149] Furthermore, according to the present invention, it is possible to introduce one or more modified immunoglobulins at some or all possible different places of a protein molecule without disturbing its structure. This kind of "domain shuffling" technique allows you to create new libraries, among which you can then select for the desired properties.

[0150] The library may contain the immunoglobulins of the present invention selected from the group consisting of immunoglobulin domains, minidomains or derivatives thereof.

[0151] A preferred embodiment of the present invention is an antigen binding molecule (antigen binding molecule) consisting of at least one immunoglobulin domain and a structural loop region modified according to the present invention to bind to the antigen, said binding molecule having no variable domains antibodies. Instead, it may contain other fragments useful for antibody activity (e.g. in the type of natural or modified effector regions (sequences); however, it does not have a "natural" binding region characterized by antibodies, i.e. variable domains at their natural occurrence. Such antigen binding molecules of the present invention have the advantages described above for the present molecules, with the exception of specific binding activity characteristic of the antibodies; instead, they exhibit specific binding activity, which is a new feature introduced into the structural loop region.

[0152] Antigen binding molecules of the present invention that contain the domains CH1, CH2, CH3, CH4, Igk-C, Igl-C or combinations thereof are preferred; said combinations should contain at least two, preferably at least four, especially at least six constant domains and at least one structural loop region modified according to the present invention. Structural loop regions joined by a structured loop region modified according to the present invention or naturally occurring structural loops between two constant domains of this type are preferred. The implementation of such antigen binding molecules of the present invention consists of the Fc region of the antibody with at least one modification within the structural loop in accordance with the present invention. With respect to antigen binding molecules according to the present invention, it is preferred that new antigen binding sites are introduced into structural loops by random selection techniques, i.e. by exchanging one or more amino acid residues in the loop by randomization techniques or by introducing for randomly generated structural insert loops.

The use of combinatorial methods is a preferred alternative.

[0153] Modified immunoglobulin may have an antigen binding site that is foreign to unmodified immunoglobulin and that has been introduced into one or more structural loops. The term "foreign" means that the antigen binding site is not created naturally by a specific immunoglobulin region, and the partner does not naturally bind to the given immunoglobulin but the foreign partner. And this means that a foreign antigen binding site for unmodified immunoglobulin is not expected to bind to a binding partner like the Fc receptor or immune effector molecule.

[0154] It is preferred to select an antigen from the group consisting of pathogenic antigen, tumor associated antigen, enzyme, substrate, autoantigen, organic molecule or allergen. More preferred are antigens selected from the group consisting of viral antigens, bacterial antigens or antigens derived from pathogens of eukaryotic cells or phages. Preferred viral antigens include: antigens of HAV, HBV, HCV, HIV I, HIV II, parvoviruses, influenza viruses, HSV viruses, hepatitis viruses, flaviviruses, West Nile virus, Ebola virus, smallpox virus, smallpox virus, measles virus, herpes virus, adenovirus, papillomavirus , polyoma virus, parvovirus, rhinitis virus, Coxsackie virus, anterior spinal cord virus (polio), ECHO virus, Japanese encephalitis virus, dengue virus, tick-borne encephalitis virus, yellow fever virus, coronavirus, respiratory syncytial virus (RSV), Newcastle disease virus, La Crosse virus, Lass virus, rabies virus and rotavirus; preferred bacterial antigens include the antigens of the following bacteria: from the genus Pseudomonas, Mycobacterium, Staphylococcus, Salmonella, meningococci, from the genus Borelia, Listeria, Neisseria, Clostridium, Escherichia, Legionella, Bacillus, Lactobacillus, Streptococcus, Enterococcus, Cardnebaxoccus,

Crucella, Campylobacter, Cardiobacterium, Francisella,

Helicobacter, Haemophilius, Klebsiella, Sgihella, Yersinia,

Vibrio, Chlamydia, Leptospira, Rickettsia, Mycobacterium, Treponema, Bartonella. Preferred eukaryotic antigens of pathogenic eukaryotic cells include the genera: Giardia, Toxoplasma, Cyclospora, Cryptosporidium, Trichinella, Yeast, Candida, Aspergillus, Cryptococcus, Blastomyces, Histoplasma, Coccidioides.

[0155] Preferred immunoglobulins of the present invention comprise at least two antigen binding sites, wherein the first binding site binds to the first epitope and the second site binds to the second epitope.

[0156] According to a preferred embodiment, the present immunoglobulin comprises at least two loop regions, wherein the first loop region binds to the first epitope while the second loop region binds to the second epitope. At least the first, or at least the second, or both loop regions may comprise a structural loop. The immunoglobulins of the present invention comprise fragments thereof, known in the art as functional fragments, comprising the key elements of the present invention: i.e. the structural loop region modified according to the present invention.

[0157] A preferred immunoglobulin according to the present invention consists of at least two immunoglobulin domains, or a fragment thereof including a minidomain, and each domain contains at least one antigen binding site.

[0158] The immunoglobulin according to the present invention may contain at least one domain in the constant region, or part thereof containing a minidomain. Thus, one preferred embodiment is a variable domain, which is e.g. modified in the C-terminal region, or a variable domain linked to a modified CH1 region, e.g. a modified CH1 minidomain.

[0159] A preferred immunoglobulin according to the present invention comprises a domain having at least 50% homology with the unmodified domain.

[0160] The term "homology" indicates that the polypeptides have the same or conserved amino acid residues at the appropriate position within the primary, secondary and tertiary structure. The term also includes two or more nucleotide sequences encoding homologous polypeptides.

[0161] "Homologous immunoglobulin domain" means the immunoglobulin domain of the present invention showing at least 50% amino acid sequence compatibility with the full native sequence immunoglobulin domain sequence or any other fragment of the full immunoglobulin domain sequence as described herein. A homologous immunoglobulin domain having at least 50% amino acid sequence compatibility, preferably at least 55%, is preferred

<td>compliance</td><td>sequence</td><td>amino acid,</td><td>better</td><td>What</td><td>least</td><td> 60%</td>

<td>compliance</td><td>sequence</td><td>amino acid,</td><td>better</td><td>What</td><td>least</td><td> 65%</td>

<td>compliance</td><td>sequence</td><td>amino acid,</td><td>better</td><td>What</td><td>least</td><td> 70%</td>

<td>compliance</td><td>sequence</td><td>amino acid,</td><td>better</td><td>What</td><td>least</td><td> 75%</td>

<td>compliance</td><td>sequence</td><td>amino acid,</td><td>better</td><td>What</td><td>least</td><td> 80%</td>

amino acid sequence match, better at least 85% amino acid sequence match, better at least 90% amino acid sequence match, better at least 95% amino acid sequence match with native immunoglobulin domain sequence or any other specific portion of the full immunoglobulin domain sequence as described herein.

nucleotide sequence correspondence "in the domain sequences described herein are defined as the percentage of residues within the candidate amino acid residues as defined [0162]" Percent (relative to the immunoglobulin amino acid sequences identical to those forming the specific sequence of the immunglobulin domain, after alignment of the sequences and introduction of gaps, if this is the case necessary to achieve maximum percentage compliance, and when excluding conservative substitutions as part of sequence compatibility. Sequence alignment to determine percent amino acid sequence alignment can be accomplished in a number of ways known in the art using, for example, commonly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Persons skilled in the art of the subject can determine the appropriate parameters for measuring fit, including any algorithms necessary to achieve maximum fit over the entire length of the compared sequence.

[0163] Percentage compliance of the amino acid sequence can be obtained as described below using the computer program WU-BLAST-2 (Altschul et al., Methods in Enzymology 266: 460-480 (1996)). Most search parameters in the WU-BLAST-2 program are set as default values. Those parameters that are not set as default values, i.e. adjustable parameters, set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11 and scoring matrix = BLOSUM26. Using the WU-BLAST-2 program, the percentage of amino acid sequence correspondence is calculated by dividing a) the number of immunoglobulin amino acid residues of interest from the sequence of the native immunoglobulin derived domain that is identical to the given compared amino acid sequence (i.e. domain of interest, sequence, to which the immunoglobulin being compared, which is the unmodified sequence of the immunoglobulin domain), the object can be determined using the WU-BLAST-2 program, by b) the total number of immunoglobulin amino acid residues occurring in fragments that were not subjected to random selection. For example, in the statement "a polypeptide having an amino acid sequence A that exhibits at least 80 percent amino acid sequence compatibility with the amino acid sequence B", the amino acid sequence A is the amino acid sequence being compared, and the amino acid sequence B is the amino acid sequence of the immunoglobulin domain of interest.

[0164] The immunoglobulin of the present invention may be used for any purpose known in the art with respect to immunoglobulins, but it also allows applications that depend on the combination of specificities introduced by the present invention. Accordingly, it is preferred that the immunoglobulins of the present invention are used for preventive and therapeutic purposes (e.g. in the form of active or passive immunotherapy); for preparative and analytical purposes and for diagnostic purposes.

[0165] The immunoglobulin object of the present invention may find use as a set of binding partners, comprising:

(a) a modified immunoglobulin having an antigen binding site foreign to the immunoglobulin introduced into one or more structural loops, and (b) a binding molecule comprising an epitope of said antigen.

[0166] Such a binding molecule, which is part of the kit of the present invention, can be used to identify the binding specificity of a given modified immunoglobulin according to the present invention. Using the binding molecule of the kit of the present invention, the potential of the modified immunoglobulins of the present invention can be determined.

[0167] Potential, as defined herein, means the ability of a modified molecule to bind to its antigen. Binding can be quantified and / or qualitatively in terms of specificity and / or affinity and / or avidity as practiced for quality control purposes.

[0168] Furthermore, the binding molecule of the kit of the present invention may be used to select the modified immunoglobulin of the present invention from a library comprising at least

10, preferably at least 100, preferably at least 1000, preferably at least 10000, especially at least

100000 immunoglobulins with various modifications in structural loops.

[0169] According to the present invention, one of the key features of the present invention is that the construction of immunoglobulin domains takes place in regions that do not normally participate in antigen binding, in other words in regions that are not the CDR regions of the antibody. It has been noted that specific folding of the immunoglobulin domains allows the introduction of random mutations in regions that are structurally analogous to the CDR regions but differ in positioning in sequence. The regions identified by the present invention are, like the CDR regions, loop regions connecting β-strands of the immunoglobulin fold.

[0170] More specifically, it is described herein that by introducing random mutations into the AB and EF β-strand connecting loops in the CH3 domain of human IgG1 immunoglobulin, mutant CH3 domains that specifically bind to either Toll-like receptor 9 (TLR) peptide have been selected. -9), or with chicken egg lysozyme, which are peptide and protein, respectively, that are not normally recognized and bound by the human CH3 IgG1 immunoglobulin domain. The mutations introduced by us include mutations in which selected amino acid residues in the wild-type sequence have been replaced by randomly selected residues, as well as contain additional amino acid residues introduced into the above-mentioned loops.

[0171] Similarly, immunoglobulin domains derived from any class immunoglobulin and from any species derived immunoglobulin are engineered by this type of engineering. Furthermore, manipulations are not only possible within the specific loops targeted by the present invention, but similarly manipulations can be made within any β-strand connecting loop in the immunoglobulin domains.

[0172] The constructed immunoglobulin domains derived from any organism and from immunoglobulins belonging to any class can be used according to the present invention either as such (i.e. as single domains) or as part of a larger molecule. For example, they may be part of an intact immunoglobulin, which consequently should have a "normal" antigen binding region formed by 6 CDR loops, as well as a new, constructed antigen binding region. In this way, a multispecific, e.g. bispecific, immunoglobulin can be generated. Properly constructed immunoglobulin domains can also be part of any fusion protein. The use of immunoglobulins thus constructed falls within the main field of application of immunoglobulins.

[0173] As immunoglobulin domains are meant here the domains of the following immunoglobulins:

for IgG, IgD and IgA: VL, CL, VH, CH1, CH2, CH3 for IgM and IgE: VL, CL, VH, CH1, CH2, CH3, CH4.

1. Individual immunoglobulin domains randomized on one side, i.e. either within the loops connecting BC, DE or FG β-strands ("apex", but excluding variable domains covered by numerous patents), or AB, CD β-strands (CC 'and C' '- D for variable domains) or EF (' base '). Randomization can involve single loops or any combination of loops. It is possible to change amino acid residues, delete or introduce additional residues.

2. Individual immunoglobulin domains randomized on both sides, i.e. at the apex and at the base.

3. Any protein containing one of the single randomized domains, such as

a) dimers from "single chain CH3 domains" (scCH3), scCH2, scCH1 / CL, randomized on one or both sides

b) single-chain Fv fragments randomized at the "base", opposite the CDR loop

c) Fab fragments randomized at the "base", ie at the C-terminus of the CH1 domain and the CL domain

d) Fc fragments (i.e. proteins consisting of CH2-CH3 domains) randomized on one or both sides

e) complete immunoglobulins randomized at the bottom of the Fc fragment

f) other eligible domains.

[0174] Leading advantages of single domains: are very similar to all those arguments that are used to promote camel VH molecules ("nanobodies", see also <a href="http://www.ablynx.com">www.ablynx.com</a>). Randomized immunoglobulin domains are very small proteins (molecular weight is around 12-15 kDa, depending on the amount of amino acid residues introduced), therefore, compared to conventional antibodies or antibody fragments such as scFv and Fab, they will have the following advantages: hidden epitopes, binding to wells or active sites of protein targets, ease of production, and more. In the case of a randomized immunoglobulin on both sides, a bivalent or bispecific. The main advantage of single domains as elements of fusion proteins is the ability to generate additional binding properties on any other protein.

[0175] It is anticipated that any expression system can be used to produce proteins. Some analogy to the single domains described herein can be seen in camel antibodies that have only the VH domain but not the VL domain. In these proteins, only 3 CDR loops are responsible for antigen binding (instead of 6, as in "normal" antibodies).

[0176] The following reference patent documents are reference documents that should be considered as cited in its entirety:

US Patent Document No. 6,294,654 Modified Antigen Binding Immunoglobulin Molecule In Non-CDR Loop Region US Patent Document No. 5,844,094 Target Binding Polypeptide US Patent Document No. 5,395,750 Methods for Producing Proteins Binding to Predetermined Antigens and multispecific high avid American patent document No. 2004/0018508 Replacement Antibodies and Methods for their Production and Use US Patent Document No. 2003/0157091 Multifunctional Proteins US Patent Document No. 2003/0148372 Searching Method of Phage Expression Libraries Using Different Ligands US Patent Document No. 2002/0103345 Bispecific, immunoglobulin-like antigen-binding proteins and methods of their production American Patent Document No. 2004/0097711 Immunoglobulin-like proteins American Patent Document No. 2004/0082508 Secretory proteins American Patent Document No. 2004/0063924 Secretion proteins American Patent Document No. 2004/0043424 Immunoglobulin-like proteins American Document Patent No. 5,892,019 Production of immunoglobulin encoded by a single gene US Patent No. 5,844,094 Target binding polypeptide The present invention is further illustrated by the following drawings and examples, but is not limited thereto.

used primers

Fig. 1a shows the structure of an intact IgG1 immunoglobulin. The arrows indicate domains.

Figure 1b shows the structural organization of monomers of basic human immunoglobulin isotypes. The lines symbolize disulfide bridges, carbohydrate groups connected to N are marked with circles.

Fig. 2 shows the immunoglobulin fold of the constant domain (left) and variable domain (right). The arrows indicate β-threads.

Figure 3 shows a molecular model of the CH3 domain constructed according to the present invention, with a randomized fragment indicated by the surface available for solvent. The surface is marked with a circle.

Fig. 4 is a schematic of the PCR primers used to produce fragments for constructing the mutated CH3 domain. Arrows indicate primers and 5'-3 'orientation, vertical lines indicate the approximate location of the introduced restriction sites that to construct the mutated gene. The following restriction sites for ligation of PCR fragments are located: CH3LNCO: Ncol; CH3LSAC and CH3CSAC: Sacl; CH3CHIN and CH3RHIN: HindIII; CH3RNOT: Notl.

shows several examples of immunoglobulin domains of the present application.

marked with an asterisk. Specificities in the randomized regions of one molecule may be identical or different.

Fig. 6 is a design diagram of a bispecific constructed CH3 domain. The names of the primers are given in

Fig. 5 the use of randomized subject possible

Framed regions, arrows indicate the direction in which primers are elongated. Sections marked with oblique lines indicate the relative position of the regions that are randomized in this construct, sections marked with the vertical position of the regions, generating clone lines indicate the relative positions that were introduced to

C24, restriction sites used in the cloning procedure are data.

Fig. 7 is a design schematic of the bispecific constructed CH3 domain. The nucleotide sequence and its translation are shown in the basic bispecific project of constructed CH3. Red sequences indicate randomized regions to generate a bispecific construct, green segments correspond to randomized regions to generate a clone

C24.

Fig. 8 shows the list of sequences disclosed herein.

DESCRIPTION OF PARTICULAR EXAMPLES:

Example 1: Construction of the CH3 library and display on the surface of the phage [0177] The crystal structure of the IgG1 immunoglobulin Fc fragment, published in the Brookhaven database as entry 1OQO.pbd, was used to design the mutated CH3 domain.

[0178] The sequence that was used as the basis for constructing the CH3 library is given in SEQ ID No. 1. In this sequence, the first amino acid corresponds to proline 343 in chain A described in entry 1oqo.pdb in the Brookhaven database. The last residue contained in entry 1oqo.pdb is serine 102 of SEQ ID No. 1. After careful analysis of the 1oqo.pdb structure and visual assessment of the residues forming the β-strand connecting loops, it was decided to randomize residues 17, 18 and 19, which are part of the β-strand connecting loop, as well as residues 71, 72, 73, 76, and

77, being part of the loop connecting β-strand EF SEQ ID No. 1. The molecular model of the thus constructed CH3 domain, with a random fragment indicated by the surface available for the solvent, is shown in Fig. 3. The constructed gene was the result of a series of PCR reactions terminated by ligation of PCR products . To facilitate ligation, some codons of the nucleotide sequence encoding SEQ ID No. 1 were modified to create restriction sites without altering the amino acid sequence (silent mutations). For insertion into the cloning vector pHEN1 (Nucleic Acids Res., August 11, 1991; 19 (15): 4133-7. Multisubunit proteins present on the surface of filamentous phages; methodologies for presenting heavy and light fragments (Fab) of the antibody. Hoogenboom HR, Griffiths AD, Johnson KS, Chiswell DJ, Hudson P, Winter G) in a frame with pelB secretion signal, additional 5 'nucleotide residues encoding Met-Ala are added at the 5' end of the sequence to create an Ncol restriction site. For randomized residues, the NNS codon (IUPAC code where S is C or G) was chosen that encodes all 20 naturally occurring amino acids, but bypasses 2 of 3 stop codons. The sequence thus constructed is given as the nucleotide sequence in SEQ ID No. 2 and as the amino acid sequence in SEQ ID No. 3. The letter X in SEQ ID No. 3 means randomized amino acid residues. The sequences of the PCR primers used to assemble the mutated CH3 domain are given in SEQ ID NO: 4 to 9. SEQ ID NO: 4

PCR generated to construct the mutated gene, and primers used for this purpose.

[0179] As a template for PCR, cDNA was used for the heavy chain of the human monoclonal antibody 3D6 (Felgenhauer M, Kohl J, Rϋker F. The nucleotide sequences of cDNAs encoding the V regions of the H and L chains of a human monoclonal antibody with specificity for HIV1gp41. Nucleic Acids Res. August 25, 1990; 18 (16): 4927). The 3 PCR products were digested with SacI and / or HindIII, respectively, and ligated. The ligation product was further digested with Ncol and Notl and then ligated into the phagemid expression vector previously digested with Ncol and Notl. A number of selected clones were checked by restriction analysis and DNA sequencing, stating that they contain the desired insert, including correctly introduced randomized sequences. The subsequent phage preparation steps are carried out based on standard protocols. Briefly, the ligation mix transformed the E. coli TG1 strain using electroporation. Subsequently, phage particles were recovered from E. coli TG1 cells using M13-KO7 helper phage, then precipitated from culture supernatant with PEG / NaCl in a two-step procedure, dissolved in water, and used for panning or frozen at -80 ° C for storage.

Example 2: Construction of the CH3 + 3 library [0180] This library was constructed and cloned in the same way as the CH3 library. The amino acid sequence of the constructed molecule is given in SEQ ID No. 10; the appropriate nucleotide sequence is given in SEQ ID No. 11, and the primers used for construction were SEQ ID No. 4 to 7, SEQ ID No. 9 and SEQ ID No. 12.

Example 3: Construction of the CH3 + library [0181] This library was constructed and cloned in the same way as the CH3 library. The amino acid sequence of the constructed molecule is given in SEQ ID No. 13; the appropriate nucleotide sequence is given in SEQ ID No. 14, and the primers used for construction were SEQ ID No. 4 to 7, SEQ ID No. 9 and SEQ ID No. 15.

Example 4: Panning of the CH3 phage library on TLR-9 peptide [0182] Three panning rounds were performed according to standard protocols. In short, the following method was used. Maxisorp 96-well plates (Nunc) were coated with a synthetic peptide representing a fragment of the Toll-like 9 receptor sequence (TLR-9). 200 pl of the following solution was added to each well: 0.1 M sodium carbonate buffer, pH = 9.6, and the following concentrations of dissolved peptide:

<td> 1.</td><td>round</td><td>panning:</td><td>peptide</td><td>TLR-9</td><td>in</td><td>concentration</td><td colspan="2">1mg / ml</td>

<td> 2.</td><td>round</td><td>panning:</td><td>peptide</td><td>TLR-9</td><td>in</td><td>concentration</td><td> 500</td><td>pg / ml</td>

<td> 3.</td><td>round</td><td>panning:</td><td>peptide</td><td>TLR-9</td><td>in</td><td>concentration</td><td> 100</td><td>pg / ml</td>

[0183] After a 1-hour incubation at 37 ° C, blockage was made with 2% powdered milk solution (M-PBS) at 200 µl per well for 1 hour at room temperature.

[0184] The expression phage library was then allowed to react with the bound peptide by adding 100 µl of phage suspension and 100 µl of 4% powdered milk solution (M-PBS), followed by incubation for 45 min on a shaker and for another 90 min without shaking at temperature room temperature.

[0185] Unbound phage particles were washed as follows: after the first panning round: 10 x 300 µL T-PBS, 5 x 300 µ PBS; after the second round of panning: 15 x 300 pl TPBS, 10 x 300 pl PBS; after the third panning round: 20 x 300 pl T-PBS, 20 x 300 pl PBS.

[0186] Bound phage particles were eluted by adding 200 µl of 0.1 M glycine to each well, pH = 2.2, followed by incubation on a shaker for 30 minutes at room temperature. The phage suspension was then neutralized by the addition of 60 µl of 2M TRIS stock solution, then infected with E. coli TG1 strain cells by mixing 10 ml of exponentially growing culture with 0.5 ml of phage eluate, incubating the whole for 30 min at 37 ° C. Finally, infected bacterial cells were plated on TYE medium with 1% glucose and 100 pg / ml ampicillin, and incubated overnight at 30 ° C.

Table 1: Panning results of the CH3 phage library on TLR-9 peptide (phage titers)

<td>Round</td><td>TLR-9 concentration</td><td>worth it</td><td>worth it</td>

<td>panning</td><td>during</td><td>initial</td><td>final</td>

<td></td><td>panning</td><td>(number</td><td>(number</td>

<td></td><td></td><td>phages / ml)</td><td>phages / ml)</td>

<td> 1.</td><td>1 mg / ml</td><td>6 x 10¹⁸</td><td>2 x 10¹⁰</td>

<td> 2.</td><td>0.5 mg / ml</td><td>4 x 10¹⁸</td><td>2 x 10¹⁰</td>

<td> 3.</td><td>0.1 mg / ml</td><td>4 x 10²²</td><td>6 x 10¹⁰</td>

Example 5: Cloning of selected clones of CH3 mutants selected against TLR-9 for expression of soluble form [0187] Phagemid DNA from phage selected in three panning rounds, was isolated using Midi-Prep. DNA encoding the mutated CH3 regions were periodically amplified by PCR and cloned with Ncol-Notl into the pNOTBAD / Myc-His vector, which is in E cells. Coli expression vector pBAD / Myc-His (Invitrogen) with an inserted NotI restriction site to facilitate cloning. The ligated constructs were transformed into E. Coli cells of strain LMG194 (Invitrogen) using electroporation, then placed on TYE medium supplemented with 1% glucose and ampicillin and left overnight at 30 ° C for expansion. Selected clones were inoculated into 200 μl of 2xYT medium with ampicillin, left overnight at 30 ° C for expansion, and then induced by the addition of L-arabinose to a final concentration of 0.1%. After overnight expression at 16 ° C, the cells were centrifuged and after adding 100 μl sodium borate buffer, pH = 8.0, incubated overnight at 4 ° C to prepare periplasmic extracts. 50 μl of periplasmic extracts were used in the ELISA (see lower).

Example 6: ELISA assay of CH3 mutants selected for TLR-9 [0188] Selected clones were tested by ELISA for specific binding to TLR-4 peptide

Coating: microtiter plate (NUNC, Maxisorp), 100 pl per well, 20 pg TLR-9 peptide / ml 0.1 M sodium carbonate buffer, pH = 9.6, for 1 h at

37 ° C

Washing: 3 x 200 pl PBS

Blocking: 1% BSA-PBS, for 1 h at room temperature Washing: 3 x 200 pl PBS

Periplasmic extract binding: 50 pl periplasmic extract in 50 pl 2% BSA-PBS, at room temperature overnight

Washing: 3 x 200 pl PBS

1. antibody: anti-His4 (Qiagen), 1: 1000 in 1% BSA-PBS, for 90 min at room temperature, 100 pl per well

Washing: 3 x 200 pl PBS

2. antibody: goat anti-mouse * HRP (SIGMA), 1: 1000 in 1% BSA-PBS, for 90 min at room temperature, 100 pl per well

Washing: 3 x 200 pl PBS

Detection: 3 mg / ml OPD (orthophenyldiamine) in sodium citrate / phosphate buffer, pH = 4.5, 0.4 pl 30% H2O2 Reaction stop: 100 ml 3M H2SO4

Absorption reading: 492/620 nm [0189] Clones giving a high signal in the first, preliminary ELISA were then grown in a volume of 20 ml under the same conditions as described above. Their periplasmic extracts were then isolated in 1/20 of the culture volume as described above, after which they were tested by ELISA (as described above) to confirm the results.

Table 2: Results of the confirmatory ELISA test

<td></td><td>with antigen</td><td>no antigen</td>

<td>clone</td><td>^AND492/620 4 readings</td><td>^AND492/620 1 reading</td>

<td>A67</td><td> 0,0435</td><td> 0, 019</td>

<td>B54</td><td> 0,0937</td><td> 0, 051</td>

<td>C67</td><td> 0,0295</td><td> 0, 013</td>

<td>Background (sam</td><td>antigen) (</td><td>2 parallel</td>

<td>readings):</td><td> 0,0115</td><td></td>

Example 7: Panning of CH3 and CH3 + 5 phage libraries on chicken egg lysozyme [0190] Three panning rounds were performed. Maxisorp 96-well plates (Nunc) were coated with chicken egg lysozyme, adding 200 pl of each solution to each well:

PBS, with the following concentrations of dissolved chicken egg lysozyme (HEL):

<td> 1.</td><td>round</td><td>panning:</td><td>HELIUM</td><td>in</td><td>concentration</td><td>2mg / ml</td>

<td> 2.</td><td>round</td><td>panning:</td><td>HELIUM</td><td>in</td><td>concentration</td><td>1 mg / ml</td>

<td> 3.</td><td>round</td><td>panning:</td><td>HELIUM</td><td>in</td><td>concentration</td><td>1 mg / ml</td>

After a 1-hour incubation at 37 ° C, blockage was made by adding 200 μΐ 2% powdered milk (M-PBS) to each well, followed by incubation for 1 hour at room temperature.

[0191] To allow the expression phage library to react with bound chicken egg lysozyme, 100 μΐ phage suspension and 100 μΐ 4% powdered milk solution (M-PBS) were added, followed by incubation for 45 min on a shaker and for another 90 min without shaking in room temperature.

[0192] Unbound phage particles were washed away as follows:

<td> 1.</td><td>round</td><td>panning:</td><td> 10</td><td>x</td><td> 300</td><td>ml</td><td>T-PBS.</td><td> 5</td><td>x 300</td><td>ml</td><td>PBS</td>

<td> 2.</td><td>round</td><td>panning:</td><td> 15</td><td>x</td><td> 300</td><td>ml</td><td>T-PBS.</td><td> 10</td><td>x 300</td><td>ml</td><td>PBS</td>

<td> 3.</td><td>round</td><td>panning:</td><td> 20</td><td>x</td><td> 300</td><td>ml</td><td>T-PBS.</td><td> 20</td><td>x 300</td><td>ml</td><td>PBS</td>

[0193] Bound phage particles were eluted by adding 200 μΐ 0.1 M glycine to each well, pH = 2.2, followed by incubation on a shaker for 30 minutes at room temperature. The phage suspension was then neutralized by the addition of 60 μΐ 2M TRIS stock solution, then infected with E. coli TG1 strain cells by mixing 10 ml of exponentially growing culture with 0.5 ml of phage eluate, incubating the whole for 30 min at 37 ° C. Finally, infected bacterial cells were plated on TYE medium with 1% glucose and 100 μg / ml ampicillin, and incubated overnight at 30 ° C.

Table 3: Panning results of the CH3 phage library on chicken egg lysozyme (phage titers)

<td>Round panning</td><td>concentration lysozyme during panning</td><td>initial value (number of phages / ml)</td><td>final value (number of phages / ml)</td>

<td> 1.</td><td>2 mg / ml</td><td></td><td>4.7 x 10¹⁰</td>

<td> 2.</td><td>1 mg / ml</td><td>1.29 x 10²²</td><td>8.0 x 10⁹</td>

<td> 3.</td><td>1 mg / ml</td><td>5.71 x 10²⁰</td><td>4.8 x 10¹⁰</td>

Table 4: Panning results of the CH3 + 5 phage library on chicken egg lysozyme (HEL) (phage titers)

<td>Round panning</td><td>concentration lysozyme during panning</td><td>initial value (number of phages / ml)</td><td>final value (number of phages / ml)</td>

<td> 1.</td><td>2 mg / ml</td><td>8.3 x 10¹⁶</td><td>2.9 x 10⁹</td>

<td> 2.</td><td>1 mg / ml</td><td>2.1 x 10¹⁹</td><td>2.6 x 10⁹</td>

<td> 3.</td><td>1 mg / ml</td><td>5.4 x 10¹⁹</td><td>1.2 x 10¹⁰</td>

Example 8: Cloning of selected clones from Example 7 for soluble expression [0194] Cloning of selected clones for soluble expression was performed as described above for TL3-9 selected CH3 mutants.

Example 9: Expression of the soluble form of selected clones from Example 7 [0195] Expression of the soluble form of selected clones was performed as described above for TL3-9 selected CH3 mutants. Periplasmic extracts were examined by preliminary ELISA (protocol see example 10).

[0196] High signal clones in the first, preliminary ELISA were cultured in 20 ml volume under the same conditions as described above. Their periplasmic extracts were then isolated in 1/20 of the culture volume as described above, after which they were tested by ELISA (as described in Example 10) to confirm the result.

Example 10: ELISA assay of CH3 mutants selected against hen egg lysozyme [0197]

Coating: microtiter plate (NUNC, Maxisorp),

<td>100 pl on</td><td>well, 100</td><td>pg</td><td>lysozyme with eggs</td>

<td>chicken / ml in</td><td>PBS, for 1 hour</td><td>temp.</td><td>37 ° C</td>

<td>Wash:</td><td>3 x 200 pl PBS</td><td></td><td></td>

<td>Blocking:</td><td>1% BSA-PBS, through</td><td>1 hw</td><td>room temperature</td>

<td>Wash:</td><td>3 x 200 pl PBS</td><td></td><td></td>

Periplasmic extract binding: 50 pl periplasmic extract in 50 pl 2% BSA-PBS, at room temperature overnight

Washing: 3 x 200 pl PBS

1. antibody: anti-His4 (Qiagen), 1: 1000 in 1% BSA-PBS, for 90 min at room temperature, 100 pl per well

Washing: 3 x 200 pl PBS

2. antibody: goat anti-mouse * HRP (SIGMA), 1: 1000 in 1% BSA-PBS, for 90 min at room temperature, 100 pl per well

Washing: 3 x 200 pl PBS

Detection: 3 mg / ml OPD in sodium citrate / phosphate buffer, pH = 4.5, 0.4 pl 30% H₂ABOUT₂

Stopping the reaction: 100 ml 3M H2SO4. Absorption reading: 492/620 nm

Table 5: Results of the confirmatory ELISA for CH3 mutants selected for chicken egg lysozyme

<td></td><td>with antigen</td><td>no antigen</td>

<td>clone</td><td>^AND492/620 4 readings</td><td>^AND492/620 1 reading</td>

<td>B12</td><td> 0,396</td><td> 0, 012</td>

<td>D10</td><td> 0,415</td><td> 0, 026</td>

<td>D46</td><td> 0,398</td><td> 0, 011</td>

<td>Background about (sam</td><td>antigen) (</td><td>2 parallel</td>

<td>readings):</td><td> 0,1763</td><td></td>

Table 6: Results of the confirmatory ELISA using different dilutions of the antigen for CH3 mutants selected for chicken lysozyme

<td>c (pg / ml) clone</td><td> 200</td><td> 100</td><td> 50</td><td> 25</td><td> 12,5</td><td> 6,25</td><td> 3,125</td><td> 1,55</td><td> 0,78</td><td> 0,39</td>

<td>B12</td><td> 0,707</td><td> 0,532</td><td> 0,432</td><td> 0,297</td><td> 0, 192</td><td> 0, 150</td><td> 0, 148</td><td> 0, 049</td><td> 0, 034</td><td> 0, 015</td>

<td>D46</td><td> 0,713</td><td> 0,561</td><td> 0,342</td><td> 0,220</td><td> 0, 133</td><td> 0, 088</td><td> 0, 047</td><td> 0, 032</td><td> 0, 021</td><td> 0, 010</td>

<td>D10</td><td> 0,715</td><td> 0, 685</td><td> 0,571</td><td> 0,368</td><td> 0,231</td><td> 0, 175</td><td> 0, 171</td><td> 0, 068</td><td> 0, 047</td><td> 0, 026</td>

<td>- (nc)</td><td> 0,449</td><td> 0,360</td><td> 0, 165</td><td> 0, 072</td><td> 0, 038</td><td> 0, 023</td><td> 0, 017</td><td> 0, 013</td><td> 0, 009</td><td> 0, 007</td>

<td>N / A: without c</td><td>odatku</td><td colspan="5">periplasmic extract</td><td></td><td></td><td></td><td></td>

[0198] It should be noted that chicken egg lysozyme reacts with the anti-his4 antibody, therefore a relatively high background is observed.

Table 7: Results of confirmatory ELISA for CH3 + 5 mutants selected for chicken egg lysozyme

<td></td><td>with antigen</td><td>no antigen</td>

<td>clone</td><td>^AND492/620 4 readings</td><td>^AND492/620 1 reading</td>

<td>A13</td><td> 0, 197</td><td> 0, 016</td>

<td>A6 6</td><td> 0,461</td><td> 0, 019</td>

<td>B18</td><td>0.533 (5 readings)</td><td>not done</td>

<td>B20</td><td> 0, 184</td><td> 0, 016</td>

<td>B68</td><td> 0,535</td><td> 0, 019</td>

<td>B40</td><td> 0,706</td><td> 0, 051</td>

<td>C24</td><td> 0,352</td><td> 0, 072</td>

<td>D22</td><td> 0, 147</td><td> 0, 019</td>

<td>C22</td><td> 0,439</td><td> 0, 017</td>

<td>D37</td><td> 0,360</td><td> 0, 026</td>

<td>D40</td><td> 0,559</td><td> 0, 034</td>

<td>D56</td><td> 0,369</td><td> 0, 019</td>

<td colspan="3">Background (antigen only) (12 parallel readings): 0.1334 Note: chicken egg lysozyme reacts with the anti-his4 antibody, therefore</td>

relatively high background was observed

Example 11: CL library [0199] Visual control of the crystal structure of the Fab fragment (the structure of the Fab fragment of the human monoclonal antibody 3D6 is used: RSCB protein database (<a href="http://www.rcsb.org/pdb">http://www.rcsb.org/pdb</a>) entry 1DFB.PDB (He XM et al. Proc Natl Acad Sci USA, August 1, 1992; 89 (15): 7154-8) and computer analysis (e.g. Protein Explorer (you can use<a href="http://molvis.sdsc.edu/protexpl/fmtdoor.htm">http://molvis.sdsc.edu/protexpl/fmtdoor.htm</a>) the secondary and tertiary structure of this protein allow to identify residues located in the loop regions that connect the β-strand of the CL domain skeleton. These residues include amino acids 8 to 18, amino acids 27 to 35, amino acids 42 to 78, amino acids 83 to 85, amino acids 92 to 100, amino acids 108 to 117 and amino acids 123 to 126 (numbering according to the IMGT numbering system (Lefranc MP, et al ., Nucleic Acids Res. 2005, January 1, 2005, 33 (database number): D593-7; Lefranc MP, et al. Dev Comp Immunol. , 2005; 2005; 29 (3): 185-203)).

[0200] More specifically, residues 11, 12, 14-18 and 92-95 are randomized within the human CL domain (SEQ ID No. 48). Randomization is achieved by PCR amplification of coding sequences using PCR primers in which the positions of the respective codons are encoded by the 5'-NNS-3 'nucleotide sequence, which potentially encodes all 20 amino acids, avoiding 2 out of 3 retaining codons. The library insert is amplified in two separate PCR reactions, followed by ligating both PCR fragments via the HpyCH4IV restriction site introduced by PCR primers as silent mutation. The primers also provide restriction endonuclease sites, Ncol and NotI, respectively, for cloning into the pHEN phage expression vector (Hoogenboom HR, et al. Nucleic Acids Res., August 11, 1991; 19 (15): 4133-7). The C-terminal cysteine ​​of the CL domain is not phage-expressed, but can be added later when the modified CL clone is used, e.g., to construct a Fab fragment.

[0201] As a template for PCR amplification, a plasmid like pRcCMV-3D6LC (R (ker F, et al Ann NY Acad Sci., 27/12/1991; 646: 212-9), containing the complete human monoclonal antibody light chain, is used.

[0202] For the CL + 3 (SEQ ID No. 50, 51) and CL + 5 (SEQ ID No. 52, 53) libraries containing additional residues inserted between positions 92 and 95 of the CL domain, the CLRHPY3 primer is used instead of the CLRHPY primer, respectively and CLRHPY5.

[0203] The following is the nucleotide and amino acid sequence of the final PCR reaction and ligation product, cloned to the Ncol site of the pHEN1 vector, which leads to attachment of the pelB leader sequence to the N-terminus of the constructed molecule (SEQ ID No. 48, 49).

<td> + 3</td><td>Μ KY</td><td>LLPT</td><td>AAA</td><td>GLL</td><td>LLAA</td>

<td> 1</td><td colspan="2">ATGAAATACC TATTGCCTAC</td><td colspan="3">GGCAGCCGCT GGATTGTTAT TACTCGCGGC</td>

<td> + 3</td><td colspan="2">NcoI Q Ρ Α Μ AV</td><td>AAPS</td><td>VFI</td><td>FPP</td>

<td> 51</td><td>CCAGCCGGCC</td><td>ATGGCCGTGG</td><td>CTGCACCATC</td><td>TGTCTTCATC</td><td>TTCCCGCCAT</td>

<td> + 3 101</td><td>SQ CTNNSNNSCA</td><td>GNNSNNSNNS</td><td>AND NNSNNSGCCT</td><td>SVVC CTGTTGTGTG</td><td>LLN CCTGCTGAAT</td>

<td> + 3</td><td>nfy</td><td>PREA</td><td>KVQ</td><td>WKV</td><td>Dnal</td>

<td> 151</td><td>AACTTCTATC</td><td>CCAGAGAGGC</td><td>CAAAGTACAG</td><td>TGGAAGGTGG</td><td>ATAACGCCCT</td>

<td> + 3</td><td>QSG</td><td>NSQE</td><td>; SVT</td><td>EQD</td><td>SKD</td>

<td> 201</td><td>CCAATCGGGT</td><td>AACTCCCAGG</td><td>AGAGTGTCAC</td><td>AGAGCAGGAC</td><td>AGCAAGGACA</td>

HpyCH4IV

<td> + 3</td><td>JAN · S</td><td>LSS</td><td>TLTL</td><td>YE</td>

<td> 251</td><td>GCACCTACAG</td><td>CCTCAGCAGC</td><td>ACCCTGACGT TGNNSNNSNN</td><td>SNNSTACGAG</td>

<td> + 3</td><td>K Η K</td><td>VYAC</td><td>Ε V Τ HQG</td><td>LSSP</td>

<td> 301</td><td>AAACACAAAG</td><td>TCTACGCCTG</td><td>CGAAGTCACC CATCAGGGCC NotI</td><td>TGAGCTCGCC</td>

<td> + 3</td><td>VTK</td><td>SFN</td><td>RGEA AA</td><td></td>

<td> 351</td><td>CGTCACAAAG</td><td>AGCTTCAACA</td><td>GGGGAGAGGC GGCCGCh</td><td></td>

List of primers for the CL library:

[0204] cllnco: 5'-cttaccatgg ccgtggctgc accatctgtc ttcatcttcc cgc catctnn snnscagnns nnsnnsnnsn nsgcctctgt tgtgtgc-3 '(SEQ ID No. 56) cllhpy: 5'-tggggcctcct '-tcagaacgtt gnnsnnsnns nnstacgaga aacacaaagt c-3' (SEQ ID No. 58) clrhpy3: 5'-tcagaacgtt gnnsnnsnns nnsnnsnnsn nstacgagaa acacaaagtc-3 '(SEQ ID No. 59) clrhtcnnnnnns '(SEQ ID No. 60) clmot: 5'-catcgcggcc gcctctcccc tgttgaagct c-3 '(SEQ ID No. 61) [0205] A series of selected library clones (mutated CL domains cloned into the pHEN1 phagemid vector) are checked by restriction analysis and DNA sequencing to confirm that they contain the intended insert, including correctly inserted randomized sequences. The subsequent stages of phage preparation are carried out according to standard protocols. Briefly, the ligation mix is ​​transformed into the E. coli TG1 strain using electroporation. Then, using M13-KO7 helper phage, phage particles are recovered from E. Coli TG1 cells, then precipitated from culture supernatant with PEG / NaCl in a two-step procedure, dissolved in water and used for panning selection or frozen at -80 ° C for storage.

Example 12: CH1 Library [0206] Visual control of the crystal structure of the Fab fragment (the structure of the Fab fragment of the human monoclonal antibody 3D6 is used: protein data bank

RSCB, entry 1DFB.PDB) and computer analysis (Protein Explorer is used) of the secondary and tertiary structure of this protein allow to identify residues located in the loop regions that connect the β-strand of the CH1 domain skeleton. These residues include amino acids 7 to 21, amino acids 25 to 39, amino acids 41 to 81, amino acids 83 to 85, amino acids 89 to 103 and amino acids 106 to 117 (numbering according to the IMGT numbering system).

[0207] More specifically, residues 12-19 and 93-100 are randomized within the human CH1 domain (SEQ ID No. 54, 55). Randomization is achieved by PCR amplification of coding sequences using PCR primers in which the positions of the respective codons are encoded by the 5'-NNS-3 'nucleotide sequence, which potentially encodes all 20 amino acids, avoiding 2 out of 3 retaining codons. The library insert is amplified in two separate PCR reactions, followed by ligating both PCR fragments via a BstEII restriction site naturally occurring in the CH1 domain. The primers also provide sites for Ncol and NotI restriction endonuclease, respectively, for cloning into the pHEN phage expression vector (Hoogenboom HR, et al. Nucleic Acids Res., 11 August 1991; 19 (15): 4133-7). The C-terminal cysteine ​​of the CL domain is not phage-expressed, but can be added later when the modified CH1 clone is used, e.g., to construct a Fab fragment.

[0208] A plasmid like pRcCMV-3D6HC, containing the complete heavy chain of a human monoclonal antibody as the insert, is used as a template for PCR amplification.

[0209] The following is the nucleotide and amino acid sequence of the final PCR reaction and ligation product, cloned to the Ncol site of the pHEN1 vector, which leads to attachment of the pelB leader sequence to the N-terminus of the constructed molecule (SEQ ID No. 54, 55):

<td> + 3</td><td>Μ Κ. Υ</td><td>LL Ρ Τ</td><td>AA Α</td><td>GLL</td><td>LLAA</td>

<td> 1</td><td>ATGAAATACC</td><td>TATTGCCTAC</td><td>GGCAGCCGCT</td><td>GGATTGTTAT</td><td>TACTCGCGGC</td>

<td> + 3</td><td colspan="2">NcoI Q Ρ Α Μ A Α</td><td>S Τ Κ G</td><td>Ρ SV</td><td>FPL</td>

<td> 51</td><td>CCAGCCGGCC</td><td>ATGGCCGCCT</td><td>CCACCAAGGG</td><td>CCCATCGGTC</td><td>TTCCCCCTGG</td>

<td> + 3 101</td><td>Α Ρ SS CACCCTCCTC</td><td>CNNSNNSNNS</td><td>NNSNNSNNSN</td><td>AL NSNNSGCCCT</td><td>GCL GGGCTGCCTG</td>

<td> + 3</td><td>V Κ D</td><td>Υ F Ρ Ε</td><td>Ρ V Τ</td><td>VSW</td><td>NSGA</td>

<td> 151</td><td>GTCAAGGACT</td><td>ACTTCCCCGA</td><td>ACCGGTGACG</td><td>GTGTCGTGGA</td><td>ACTCAGGCGC</td>

<td> + 3</td><td>L Τ S.</td><td>GV Η</td><td>Τ F Ρ Α</td><td>Vi Q</td><td>SSG</td>

<td> 201</td><td>CCTGACCAGC</td><td>GGCGTGCACA</td><td>CCTTCCCGGC</td><td>TGTCCTACAG</td><td>TCCTCAGGAC</td>

BstEII

<td> +3</td><td>Lysle</td><td>SSV</td><td>VTV</td><td>P</td><td></td>

<td> 251</td><td>TCTACTCCCT</td><td>CAGCAGCGTG</td><td>GTGACCGTGC</td><td>CCNNSNNSNN</td><td>SNNSNNSNNS</td>

<td> + 3</td><td>YOU</td><td>ICNV</td><td>N Η K</td><td>PSN</td><td>Τ Κ VD</td>

<td> 301</td><td>NNSACCTACA</td><td>TCTGCAACGT</td><td>GAATCACAAG</td><td>CCCAGCAACA</td><td>CCAAGGTGGA</td>

<td></td><td></td><td></td><td>NotI</td><td></td><td></td>

<td> + 3</td><td>KKV</td><td>Ε Ρ K</td><td>SAAA</td><td></td><td></td>

<td> 351</td><td>CAAGAAAGTT</td><td>GAGCCCAAAT</td><td>CTGCGGCCGC</td><td>AND</td><td></td>

List of primers for the CH1 library [0210]

CH1LNC0: 5'-acgtccatgg ccgcctccac caagggccca tcggtcttcc ccctggcacc ctcctccnns nnsnnsnnsn nsnnsnnsnn sgccctgggc tgcctggtc-3 '(SEQ ID No. 62)

CH1LBST: 5'-ggcacggtca ccacgctgct gag-3 '(SEQ ID No. 63)

CH1RBST: 5'-agcgtggtga ccgtgcccnn snnsnnsnns nnsnnsnnsa cctacatctg caacgtgaat c-3 '(SEQ ID No. 64) [0211] CH1RNOT: 5'-catagcggcc gcagatttgg gctcaacttt ctgtctse mutant CH1 domains cloned into the phagemid vector (pHEN1) are controlled by restriction analysis and DNA sequencing to confirm that they contain the intended insert, including correctly inserted randomized sequences. The subsequent stages of phage preparation are carried out according to standard protocols. Briefly, the ligation mix is ​​transformed into the E. coli TG1 strain using electroporation technology. Then, using M13-KO7 helper phage, phage particles are recovered from E. Coli TG1 cells, then precipitated from culture supernatant with PEG / NaCl in a two-step procedure, dissolved in water and used for panning selection or frozen at -80 ° C for storage.

Example 13: Panning of the CH1 phage library on chicken egg lysozyme (HEL) [0213] Three panning rounds should be performed using the CH1 phage library (see example 12). Maxisorp 96-well plates (Nunc) are coated with chicken egg lysozyme, adding to each well 200 pl of the following solution: PBS, with the following concentrations of dissolved chicken egg lysozyme (HEL):

<td> 1.</td><td>round</td><td>panning:</td><td>HELIUM</td><td>in</td><td>concentration</td><td>2mg / ml</td>

<td> 2.</td><td>round</td><td>panning:</td><td>HELIUM</td><td>in</td><td>concentration</td><td>1 mg / ml</td>

<td> 3.</td><td>round</td><td>panning:</td><td>HELIUM</td><td>in</td><td>concentration</td><td>1 mg / ml</td>

[0214] After 1 hour incubation at 37 ° C, blockage is made by adding 200 µl of 2% powdered milk (M-PBS) to each well followed by incubation for 1 hour at room temperature.

[0215] To allow the expression phage library to react with the associated chicken egg lysozyme, 100 µl of phage suspension and 100 µl of a 4% powdered milk solution (M-PBS) are added, followed by incubation for 45 min on a shaker and for another 90 min without shaking at room temperature.

[0216] Unbound phage particles are washed away as follows:

<td> 1.</td><td>round</td><td>panning:</td><td> 10</td><td>x</td><td> 300</td><td>en</td><td>T-PBS.</td><td> 5</td><td>x 300 pl</td><td>PBS</td>

<td> 2.</td><td>round</td><td>panning:</td><td> 15</td><td>x</td><td> 300</td><td>en</td><td>T-PBS.</td><td> 10</td><td>x 300 pl</td><td>PBS</td>

<td> 3.</td><td>round</td><td>panning:</td><td> 20</td><td>x</td><td> 300</td><td>en</td><td>T-PBS.</td><td> 20</td><td>x 300 pl</td><td>PBS</td>

[0217] Elution of bound phage particles is performed by adding 200 µl of 0.1 M glycine to each well, pH = 2.2, followed by incubation on a shaker for 30 minutes at room neutralizing temperature. The phage suspension is then added by adding 60 µl of a 2M TRIS stock solution, followed by infection of E. coli TG1 strain cells by mixing 10 ml of the exponentially growing culture with 0.5 ml of phage eluate, incubating the whole for 30 min at 37 ° C. Finally, infected bacterial cells are plated on TYE medium with 1% glucose and 100 pg / ml ampicillin and incubated overnight at 30 ° C.

[0218] Cloning of selected clones of CH1 mutants selected against lysozyme for expression of soluble form [0219] Phagmid DNA derived from phage selected in three rounds of panning is isolated using Midi-Prep. DNA encoding mutated CH1 regions are periodically amplified by PCR and cloned with Ncol-Notl into the pNOTBAD / Myc-His vector, which is E. coli expression vector pBAD / Myc-His (Invitrogen) with inserted Notl restriction site to facilitate cloning . The ligated constructs are transformed into E cells. Coli strain LMG194 (Invitrogen), using electroporation, then places on TYE medium with 1% glucose and ampicillin and left overnight at 30 ° C for multiplication. Selected clones are inoculated into 200 µl 2xYT medium with ampicillin, left overnight at 30 ° C for expansion, and then induced by adding L-arabinose to a final concentration of 0.1%. After overnight expression at 16 ° C, the cells should be centrifuged, and after adding 100 µl of sodium borate buffer, pH = 8.0, incubate overnight at 4 ° C to prepare periplasmic extracts. 50 pl of periplasmic extracts are then used in the ELISA test.

[0220] High signal clones in the first, preliminary ELISA are then grown in a volume of 20 ml under the same conditions as described above

Their periplasmic extracts are then isolated in 1/20 of the culture volume as described above, then controlled by ELISA (as described above) to confirm the result.

[0221] ELISA assay for CH1 mutants selected for chicken egg lysozyme

Coating: microtiter plate (NUNC, Maxisorp),

<td>100 pl on</td><td>well, 100</td><td>pg</td><td>lysozyme with eggs</td>

<td>chicken / ml in</td><td>PBS, for 1 hour</td><td>temp.</td><td>37 ° C</td>

<td>Wash:</td><td>3 x 200 pl PBS</td><td></td><td></td>

<td>Blocking:</td><td>1% BSA-PBS, through</td><td>1 hw</td><td>room temperature</td>

<td>Wash:</td><td>3 x 200 pl PBS</td><td></td><td></td>

Periplasmic extract binding: 50 pl periplasmic extract in 50 pl 2% BSA-PBS, at room temperature overnight

Washing: 3 x 200 pl PBS

1. antibody: anti-His4 (Qiagen), 1: 1000 in 1% BSAPBS, for 90 min at room temperature, 100 pl per well

Washing: 3 x 200 pl PBS

2. antibody: goat anti-mouse * HRP (SIGMA), 1: 1000 in 1% BSA-PBS, for 90 min at room temperature, 100 pl per well

Washing: 3 x 200 pl PBS

Detection: 3 mg / ml OPD in sodium citrate / phosphate buffer, pH = 4.5, 0.4 pl 30% H2O2 Reaction stop: 100 ml 3M H2SO4 Absorption reading: 492/620 nm [0222] Clones are interpreted as positive if their ELISA signal is at least 3 times higher than the background signal.

Example 14: Panning of the CL phage library on chicken egg lysozyme (HEL) [0223] Three panning rounds were performed using the CL phage library (see example 11). Maxisorp 96-well plates (Nunc) are coated with chicken egg lysozyme, adding to each well 200 pl of the following solution: PBS, with the following concentrations of dissolved chicken egg lysozyme (HEL):

<td> 1.</td><td>round</td><td>panning:</td><td>HELIUM</td><td>in</td><td>concentration</td><td>2mg / ml</td>

<td> 2.</td><td>round</td><td>panning:</td><td>HELIUM</td><td>in</td><td>concentration</td><td>1 mg / ml</td>

<td> 3.</td><td>round</td><td>panning:</td><td>HELIUM</td><td>in</td><td>concentration</td><td>1 mg / ml</td>

[0224] After 1 hour incubation at 37 ° C, blockage is made by adding 200 µl of 2% powdered milk (M-PBS) to each well followed by incubation for 1 hour at room temperature.

[0225] To allow the expression phage library to react with bound chicken egg lysozyme, 100 µl of phage suspension and 100 µl of a 4% powdered milk solution (M-PBS) are added, followed by incubation for 45 min on a shaker and another 90 min without shaking at room temperature.

[0226] Unbound phage particles are washed away as follows:

<td> 1.</td><td>round</td><td>panning:</td><td> 10</td><td>x</td><td> 300</td><td>μ!</td><td>T-PBS.</td><td> 5</td><td>x 300</td><td>μ!</td><td>PBS</td>

<td> 2.</td><td>round</td><td>panning:</td><td> 15</td><td>x</td><td> 300</td><td>μ!</td><td>T-PBS.</td><td> 10</td><td>x 300</td><td>μ!</td><td>PBS</td>

<td> 3.</td><td>round</td><td>panning:</td><td> 20</td><td>x</td><td> 300</td><td>μ!</td><td>T-PBS.</td><td> 20</td><td>x 300</td><td>μ!</td><td>PBS</td>

[0227] Elution of bound phage particles is performed by adding 200 μl of 0.1 M glycine to each well, pH = 2.2, followed by incubation on a shaker for 30 minutes at room temperature. The phage suspension is then neutralized by the addition of 60 μl of a 2M TRIS stock solution, followed by infection of E. coli TG1 cells by mixing 10 ml of exponentially growing culture with 0.5 ml of phage eluate, incubating the whole for 30 min at 37 ° C . Finally, infected bacterial cells are plated on TYE medium with 1% glucose and 100 μg / ml ampicillin, and incubated overnight at 30 ° C.

[0228] Cloning of selected clones of CL mutants cloned relative to lysozyme for expression of soluble form [0229] Phagmid DNA from phage selected in three rounds of panning is isolated using Midi-Prep. DNA encoding mutated CL regions is periodically amplified by PCR and cloned with Ncol-Notl into the pNOTBAD / Myc-His vector, which is the pBAD / Myc-His expression vector used in E.Coli cells with an inserted Notl restriction site to facilitate cloning.

The ligated constructs are transformed into E. Coli cells of the LMG194 strain (Invitrogen) using electroporation, then placed on TYE medium with 1% glucose and ampicillin and left overnight at 30 ° C for propagation. Selected clones are inoculated into 200 μl of 2xYT medium with ampicillin, left overnight at 30 ° C for expansion and then induced by the addition of L-arabinose to a final concentration of 0.1%. After overnight expression at 16 ° C, the cells should be centrifuged, and after adding 100 μl sodium borate buffer pH 8.0, incubate overnight at 4 ° C to prepare periplasmic extracts. 50 μl of periplasmic extracts is then used in the ELISA test.

[0230] Clones that give high ELISA are cultured under the same conditions, periplasmic culture volume extracts are consistently controlled by ELISA (to confirm the result.

[0231] ELISA assay of chicken lysozyme mutants signal first, then pre-filled in a volume of 20 ml as described above. They are then isolated in 1/20 with the above description, followed by the above-described method) in order

CL selected relative to

Coating 100 μl per chicken / ml in Washing: Blocking: Washing:

: microtiter plate (NUNC, Maxisorp), well, 100 μg egg lysozyme

PBS, incubation for 1 h at 37 ° C 3 x 200 μl PBS

1% BSA-PBS, for 1 h at room temperature 3 x 200 μl PBS

Periplasmic extract binding: 50 pl periplasmic extract in 50 pl 2% BSA-PBS, at room temperature overnight

Washing: 3 x 200 pl PBS

1. antibody: anti-His4 (Qiagen), 1: 1000 in 1% BSAPBS, for 90 min at room temperature, 100 pl per well

Washing: 3 x 200 pl PBS

2. antibody: goat anti-mouse * HRP (SIGMA), 1: 1000 in 1% BSA-PBS, for 90 min at room temperature, 100 pl per well

Washing: 3 x 200 pl PBS

Detection: 3 mg / ml OPD in sodium citrate / phosphate buffer, pH = 4.5, 0.4 pl 30% H2O2

Stopping the reaction: 100 ml 3M H2SO4. Absorption reading: 492/620 nm [0232] Clones are interpreted as positive if their ELISA signal is at least 3 times higher than the background signal.

Example 15: Construction of a randomized immunoglobulin domain on both sides (bispecific constructed CH3 domain) [0233] This example describes a constructed immunoglobulin domain with two specific binding sites.

[0234] The design of the present constructed immunoglobulin domain is based on the following strategy:

• the constructed CH3 domain, clone C24, was used as a starting point (see example 10), derived from the CH3 + 5 library, binding specifically to lysozyme • in this modified CH3 domain, residues intended for randomization have been identified, connecting β-strands of the immunoglobulin fold and located on the other side of the domain compared to the residues, which were mutated during C24 clone generation • PCR primers were designed to randomize these residues and synthesize said constructed immunoglobulin domain following a procedure similar to that described above for CH3, CH3 + 3 and CH3 + libraries [0235] 4 PCR reaction products containing randomized sites were ligated and full-size inserts were amplified by PCR. They were then cloned into the pHEN1 vector using Ncol-Notl sites and transformed into E. coli strain TG-1 cells to

10⁸ constructing a library of randomly selected colonies was sequenced, stating that the randomized sites were independently mutated. No "wild-type" (C24) sequence was observed. The library was generated according to standard protocols, achieving a phage titer of 6.32 x 10¹⁰ TU / ml.

okay.

colonies [0236] To test bispecificity, recombinant human erythropoietin (rhEPO) was chosen as the second antigen, expecting that the engineered molecule retained the originally constructed chicken egg lysozyme specificity. RhEPO reactive phage was selected in four rounds of panning. In order to preserve the population of C24 clones, which should still bind to chicken egg lysozyme after mutagenesis, after the first round of selection on rhEPO a panning round of phage population on chicken egg lysozyme (1 mg / ml in PBS) was performed. A volume of 200 µl rhEPO was coated on 5 wells of a microtiter plate (Maxisorp, Nunc) in 0.1 M sodium carbonate buffer, pH = 9.6, in decreasing concentrations during subsequent panning rounds (see Table below). After blocking with 2% M-PBS, the phage in the blocking agent was allowed to bind for 2 h at room temperature. After 20 washes with T-PBS and 20 washes with PBS, the phage was eluted with 0.1 glycine, pH = 2.2, then neutralized by the addition of 2M TRIS. The eluted phage was used immediately to infect exponentially proliferating TG-1 cells. Infected cells were selected on ampicillin supplemented medium. Phage particles were recovered from the culture supernatant by superinfection from the helper M13-KO7, concentrated for use in the next panning round. After each round of panning, the initial and final number of phages were determined, expressed as the number of transforming units (TU) of E.Coli (Table 8).

using the phage using PEG and

Table 8:

<td>round panning</td><td>antigen</td><td>initial number of phages (TU / ml)</td><td>final number phages (TU / ml)</td>

<td> 1</td><td>rhEPO, 500 pg / ml</td><td>6.32 x 10¹⁰</td><td>1.9 x 10⁵</td>

<td> 2</td><td>lysozyme, 1 mg / ml</td><td>6.16 x 10¹⁵</td><td>4.53 x 10¹⁰</td>

<td> 3</td><td>rhEPO, 100</td><td>6.07 x 10¹⁵</td><td>6.78 x 10¹⁰</td>

<td></td><td>mg / ml</td><td></td><td></td>

<td> 4</td><td>rhEPO, 50 mg / ml</td><td>8.42 x 10¹⁵</td><td>3.0 x 10¹¹</td>

<td> 5</td><td>rhEPO, 50 mg / ml</td><td>5.12 x 10¹⁵</td><td>4.28 x 10¹⁰</td>

[0237] The resulting colonies were scraped off the plates, grown in 2xYT with ampicillin, and their plasmid DNA was isolated using Midi-Prep. The inserts were amplified by PCR, cloned into the pNOTBAD vector and used to transform E. coli strain E104 cells. 4x72 colonies were cultured in 200 μl 2xYT with ampicillin and induced with 0.1% L-arabinose the next day. After 24-hour expression at 16 ° C, the cells were lysed in 200 μl sodium borate buffer, pH = 8.0, for 6h at 4 ° C, after which the obtained periplasmic extract was used in the ELISA.

[0238] For ELISA purposes, Maxisorp plates were coated with chicken egg lysozyme in PBS (20Pg / ml) or rhEPO in 0.1 M sodium carbonate buffer, pH = 9.6, for 1 hour at 37 ° C. After blocking with 1% BSA-PBS, the periplasmic extract in the same blocking agent was allowed to bind overnight. Binding was detected with an anti-His- (4) antibody and a goat anti-mouse IgG antibody conjugated to HRP (for detection of chicken egg lysozyme) or AP (for detection of rhEPO). A reading at 492/620 nm was made for the colored reaction indicating OPD conversion (HRP), after stopping the reaction by adding 1.25 M H2SO4, while for pNPP (AP) conversion a reading was made at 405/620 nm. 14 clones with promising absorbance values ​​were selected for expression on a 20 ml scale. After 24100 hours of arabinose induction at 16 ° C, the cells were harvested and then lysed overnight in 1 ml of sodium borate buffer at 4 ° C, and the obtained lysate was used in the ELISA. Four parallel ELISA tests were performed as described above, and wells without periplasmic extract and no antigen were used as negative controls. The results (shown in Table 9) were obtained using a clone according to SEQ ID No. 42, 43.

Table 9:

<td>antigen</td><td></td><td>absorbance for bond</td><td>no extract periplasmic</td><td>without antigen</td>

<td>lysosis</td><td>A 492 / 620nm</td><td> 0,299</td><td> 0, 110</td><td> 0, 018</td>

<td>rhEPO</td><td>A 405 / 620nm</td><td> 0,258</td><td> 0, 095</td><td> 0, 090</td>

Example 16: Constructed CH3 domains provide bispecificity in a Fab-like format [0239] In the construct used in this example, the VL and VH chains of the antibody are fused to form the constructed CH3 domain.

[0240] As a fusion partner for the constructed CH3 domain, clone C24, which specifically binds to chicken egg lysozyme, the VL and VH region of the human monoclonal antibody 3D6 (He XM, et al. Proc Natl Acad Sci USA. 1992 89: 7154-8 .; Kohl J, et al. Ann NY Acad Sci. 1991 646: 106-14 .; Felgenhauer M, et al. Nucleic Acids Res. 1990 18: 4927), which recognizes an epitope on HIV-1 gp41.

101 [0241] To promote VL-CH3 / VH-CH3 dimer formation via a disulfide bridge, Ser-Cys residues were added to the C-terminus of the C24 sequence.

[0242] The nucleotide and amino acid sequences of both chains, 3D6VL-C24 and 3D6VH-C24, respectively, are given in SEQ ID Nos. 47, 46 and SEQ ID Nos. 45.44.

[0243] Primers were designed that allow the amplification of coding regions while introducing restriction sites (silent mutations) that were used to ligate the coding regions. The expression system of Pichia pastoris was selected for gene expression. The constructs were cloned into the appropriate Pichia pastoris expression vectors: 3D6VL-C24 was cloned into pPIC9K (final name: pPIC9K3LC), while 3D6VH-C24 (final name: pPICZ3HC) was cloned into pPICZalphaA. The pPICZ3HC construct was developed into a line with BgIII, transformed into GS115 Pichia pastoris, and the transformants obtained were selected in solid medium with the addition of zeocin. One of the transformants was then used as a host cell for the pPIC9K3LC construct linearized with Sal-I. Double transformants were selected on RDB medium.

[0244] Clones were inoculated into 30 ml YPG medium, where they were multiplied to reach OD600 = 10, then induced by the addition of 1% methanol in BMMY medium. The induction was continued for 36 hours at 16 ° C. The supernatant was removed by centrifugation and then concentrated about 10 times. The presence of the recombinant protein was confirmed by Western blot using the antiHis (4) antigen, and its concentration was estimated at about 50-100 pg / l of the initial culture.

102 [0245] The first functional tests were performed using 10x concentrated supernatant. To start with, Maxisorp plate wells were coated with chicken egg lysozyme at 20 pg / ml in PBS or epitope 3D6 at 20 pg / ml in 0.1 M sodium carbonate buffer, pH = 9.6, for 1h at 37 ° C. The 3D6 epitope was used as a recombinant GST fusion protein. After blocking with 1% BSA-PBS, concentrated supernatants were allowed to bind overnight in the same blocking agent. Anti-His (4) and goat anti-mouse antibodies conjugated to HRP were used to reveal binding, visualizing the binding process as a colored reaction resulting from OPD conversion at 492/620 nm (Table 10).

Table 10:

<td>antigen</td><td>ELISA signal^(AND492/620)</td><td>background (no antigen)</td><td>background (none supernatant)</td>

<td>lysosis</td><td> 0, 198</td><td> 0, 003</td><td> 0, 043</td>

<td>3D6 epitope</td><td> 0,061</td><td> 0,001</td><td> 0,007</td>

103

LIST OF SEQUENCES [0246] <110> Ruker, Florian <120> SYNTHETIC IMMUNOGLOBULIN DOMAINS WITH BINDING PROPERTIES CONSTRUCTED IN REGIONS OF MOLECULES DIFFERENT FROM REGIONS DETERMINING COMPLETERS <> <> 170> <0170 US 60/641144 <151> 2005-01-05 <160> 65 <170> Patent version 3. 3 <210> 1 <211> 108 <212> PRT <213> Homo sapiens <400> 1

Pro Arq Q u Pro Gl n Val Tyr Thr Leu Pro Pro Ser Arg Asp Gu Leu 15 10 15

Thr Lys Asn Qn Val Ser Leu Thr Cys Leu Val Lys Gy Phe Tyr Pro 20 25 30

Asp II e Al a Val G u Trp G u Cheese Asn G y G n Pro G u Asn Asn 35 40 45

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp G y Ser Phe Phe Leu 50 55 60

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gn Qn Gy Asn Val

70 75 80

Phe Ser Cys Ser Val Nfet H s G u Al a Leu H ​​s Asn H s Tyr Thr G n

90 95

Lys Cheese Leu Cheese Leu Cheese Pro Gy Lys Ala Ala Ala 100 105

104 <210> 2 <211> 332 <212> DNA <213> Artificial <220>

<223> Artificial sequence <220>

ro <221> misc_feature <222> (57) .. (58) <223> η is a, c, g, or t <220>

T5 <221> misc_feature <222> (60) .. (61) <223> n is a, c, g, or t <220>

<221> misc_feature <222> (63) .. (64) <223> n is a, c, g, or t <220>

<221> miscjeature <222> (219) .. (220) <223> n is a, c, g, or t <220>

<221> miscjeature <222> (222) .. (223) <223> n is a, c, g, or t <220>

<221> misc_feature <222> (225) .. (226) <223> n is a, c, g, or t * 0 <220>

<221> misc_feature <222> (234) .. (235) <223> n is a, c, g, or t <220>

<221> misc_feature <222> (23η .. (238) <223> n is a, c, g, or t <400> 2

105 CCAT ggcccc nsnnscaggt agt gggagag ccgacggct c ggaacgt ct l GCCL ct CCCT ccgagaacca cagcctgacc CAAT gggcag ct t ct t cct c ct cat GCT cc gt ct ccgggt caggt gt aca tgcctggtca ccggagaaca t acagcaagc gt grade gcat g aaagcggccg CCCT gccccc aaggct t ct and act acaagac 11 accgt gnn aggct ct gca ca at cccgggat tcccagcgac cacgcct ccc snnsnnsagg caaccact ac gagct cnnsn at cgccgt gg gtgctggact t ggnnsnnsg acacagaaga

120

180

240

300

332 <21O> 3 <211> 110 <212> PRT <213> Artificial <220>

<223> Artificial sequence <220>

<221> miscjeature <222> (19) .. (21) <223> Xaa can be any amino acid found in nature <220>

<221> miscjeature <222> (73) .. (75) <223> Xaa can be any amino acid found in nature 30 <220>

<221> misatureature <222> (78) .. (79)

106

<td rowspan="3"> 5</td><td colspan="2" rowspan="2"> &lt;223&gt; &lt;400&gt;</td><td colspan="8">Xaa can be any naturally occurring arri no</td><td colspan="4">aci d</td>

<td colspan="4"> 3</td><td rowspan="2">Tyr</td><td rowspan="2">Thr 10</td><td rowspan="2">Leu</td><td rowspan="2">Pr</td><td rowspan="2">Pr</td><td rowspan="2">Cheese</td><td rowspan="2">Arg 15</td><td rowspan="2">asp</td>

<td>Nfet 1</td><td>Al a</td><td>Pr</td><td>Arg</td><td>gu 5</td><td>Pr o G n Val</td>

<td></td><td>α u</td><td>Leu</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>G n Val Ser</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Leu</td><td>val</td><td>lys</td><td>gy</td><td>phe</td>

<td></td><td></td><td></td><td></td><td> 20</td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>ΪΟ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>Tyr</td><td>Pr</td><td>Cheese</td><td>asp</td><td>11 e</td><td>Al a Val G u</td><td>Trp</td><td>G u</td><td>Cheese</td><td>own</td><td>gy</td><td>G n</td><td>Pro</td><td>G u</td>

<td></td><td></td><td></td><td> 35</td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>T5</td><td>own</td><td>own</td><td>Tyr</td><td>lys</td><td>Thr</td><td>Thr Pro Pro</td><td>val</td><td>Leu</td><td>asp</td><td>Cheese</td><td>asp</td><td>G y</td><td>Cheese</td><td>phe</td>

<td></td><td></td><td> 50</td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td></td><td>phe</td><td>Leu</td><td>Tyr</td><td>Cheese</td><td>lys</td><td>Leu Thr -Val</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Arg</td><td>IRP</td><td>Xaa</td><td>Xaa</td><td>&lt;3 y</td>

<td></td><td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td> 20</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>own</td><td>val</td><td>phe</td><td>Cheese</td><td>Cys</td><td>Val Nfet cheese</td><td>H p</td><td>G u</td><td>Al a</td><td>Leu</td><td>HS</td><td>own</td><td>H p</td><td>Tyr</td>

<td></td><td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td> 25</td><td>Thr</td><td>α n</td><td>lys</td><td>Cheese</td><td>Leu</td><td>Leu Cheese Cheese</td><td>Pr</td><td>pregnancy</td><td>lys</td><td>Al a</td><td>Al a</td><td>Al a</td><td></td><td></td>

100 105 110 <210> 4 <211> 33 <212> DNA <213> Artificial <220>

?; <223> Artificial sequence <400> 4 ct t gccat gg ccccccgaga accacaggt gt ac 33 <0 <210> 5 <211> 30 <212> DNA <213> Artificial

J5 <220>

<223> Artificial sequence <400> 5 agt cgagct c gt cacgggat gggggcaggg 30 <210> 6 <211> 41 <212> DNA <213> Artificial <220>

<223> artificial sequence

107 <220>

<221> misc_feature <222> (11) .. (12) <223> η is a, c, g, or t <220>

<221> misc_feature <222> (14) .. (15) <223> n is a, c, g, or t <220>

<221> misc_feature <222> (17) .. (18) <223> n is a, c, g, or t <400> 6 gt acgagct c nnsnnsnnsc aagt cagcct gacct gcct gg <210> 7 <211> 32 < 212> DNA <213> Artificial <220>

<223> Artificial sequence <400> 7 tgccaagctt gctgtagagg aagaaggagc cg 32 <210> 8 <211> 59 <212> DNA <213> Artificial <220>

<223> Artificial sequence <220>

<221> misc_feature <222> (17) .. (18) <223> n is a, c, g, or t <220>

<221> misc_feature <222> (20) .. (21) <223> n is a, c, g, or t <220>

<221> misc_feature <222> (23) .. (24) <223> n is a, c, g, or t <220>

<221> misc_feature <222> (32) .. (33) <223> n is a, c, g, or t <220>

108 <221> miscjeature <222> (35) .. (36) <223> n is a, c, g, or t <400> 8 t gccaagct t accgt gnnsn nsnnsaggt g gnnsnnsggg aacgt ct t ct cat gct ccg <210> 9 <211> 33 and <<122> DNA <213> Artificial <220>

<223> Artificial sequence <400> 9 agttgcggcc gctttacccg gagacaggga gag 33 <210> 10 <211> 113 <212> PRT <213> Artificial <220>

<223> Artificial sequence <220>

<221> miscjeature <222> (19) .. (21) <223> Xaa can be any amino acid found in nature <220>

<221> miscjeature <222> (73) .. (78) <223> Xaa can be any amino acid found in nature <220>

<221> miscjeature <222> (81) .. (82) <223> Xaa can be any amino acid found in nature <400> 10

109

<td></td><td>Nfet 1</td><td>Al a</td><td>Pro</td><td>Arg</td><td>&lt;3 u 5</td><td>Pr</td><td colspan="2">α n Val</td><td>Tyr</td><td>Thr 10</td><td>Leu</td><td>Pro</td><td>Pro</td><td>Cheese</td><td>Arg 15</td><td>asp</td>

<td> 5</td><td>3 u</td><td>Leu</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>(3 n</td><td>val</td><td>Cheese</td><td>Leu</td><td>Thr</td><td rowspan="2">Cys</td><td>Leu</td><td>val</td><td>lys</td><td>qy</td><td>phe</td>

<td></td><td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td></td><td rowspan="2">Tyr</td><td>Pro</td><td>Cheese</td><td rowspan="2">asp</td><td>lle</td><td>Al a</td><td>val</td><td>Q u</td><td>Trp</td><td>3 u</td><td>Cheese</td><td>own</td><td>. y</td><td>α n</td><td>Pro</td><td>3 u</td>

<td>ΪΟ</td><td></td><td> 35</td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td></td><td>own</td><td>own</td><td rowspan="2">Tyr</td><td rowspan="2">lys</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Pro</td><td>vai</td><td>Leu</td><td>asp</td><td>Cheese</td><td>asp</td><td>3 y</td><td>Cheese</td><td>phe</td>

<td></td><td></td><td> 50</td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>T5</td><td>phe</td><td>Leu</td><td rowspan="2">Tyr</td><td>Cheese</td><td rowspan="2">lys</td><td>Leu</td><td>Thr</td><td>val</td><td>xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Arg</td><td>Trp</td>

<td></td><td> 65</td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td></td><td>Xaa</td><td>Xaa</td><td rowspan="2">qy</td><td>own</td><td>val</td><td>phe</td><td>Cheese</td><td rowspan="2">Cys</td><td>Cheese</td><td>val</td><td>fvtet</td><td>H p</td><td>Q u</td><td>Al a</td><td>Leu</td><td>H p</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td> 20</td><td>own</td><td>H p</td><td rowspan="2">Tyr</td><td>Thr</td><td>□ n</td><td rowspan="2">lys</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Pr</td><td>GI y</td><td>lys</td><td>Al a</td><td>Al a</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<210> 11 <211> 341 <212> DNA <213> Artificial <220>

<223> Artificial sequence <220>

<221> miscjeature <222> (57) .. (58) <223> η is a, c, g, or t <220>

<221> miscjeature <222> (60) .. (61) <223> n is a, c, g, or t <220>

<221> miscjeature <222> (63) .. (64) <223> n is a, c, g, or t <220>

<221> miscjeature <222> (219) .. (220) <223> n is a, c, g, or t <220>

<221> misatureature <222> (222) .. (223)

110 <223> η is a, c, g, or t <220>

<221> miscjeature 5 <222> (225) .. (226) <223> n is a, c, g, or t <220>

<221> miscjeature 10 <222> (228) .. (229) <223> n is a, c, g, or t <220>

<221> missions> 5 <222> (231) .. (232) <223> n is a, c, g, or t <220>

<221> misatureature 20 <222> (234) .. (235) <223> n is a, c, g, or t <220>

<221> misatureature 25 <222> (243) .. (244) <223> n is a, c, g, or t <220>

<221> missions 30 <222> (246) .. (247) <223> n is a, c, g, or t <400> 11 ccat ggcccc nsnnscaagt agt gggagag ccgacggct c ggnnsnnsgg cacagaagag ccgagaacca cagcct gacc caat gtgcc tc cct ccct g caggt gt aca t gcct ggt ca ccggagaaca t acagcaagc tcat gctccg tct ccgggt a ccct gccccc aaggct tct a act acaagac ttaccgtgnn t gat gcccgc gcc gcc gcc gcc gcc gcc

gagct cnnsn at cgccgt gg gt gct ggact nnsnnsaggt aaccact aca

120

180

240

300

341 <21o> 12 <211> 68 <212> DNA <213> Artificial <220>

<223> Artificial sequence <220>

<221> miscjeature <222> (17) .. (18) <223> n is a, c, g, or t

111 <220>

<221> miscjeature <222> (20) .. (21) <223> η is a, c, g, or t <220>

<221> miscjeature <222> (23) .. (24) <223> n is a, c, g, or t <220>

<221> miscjeature <222> (26) .. (27) <223> n is a, c, g, or t <220>

<221> miscjeature <222> (29) .. (30) <223> n is a, c, g, or t <220>

<221> miscjeature <222> (32) .. (33) <223> n is a, c, g, or t <220>

<221> miscjeature <222> (41) .. (42) <223> n is a, c, g, or t <220>

<221> miscjeature <222> (44) .. (45) <223> n is a, c, g, or t <400> 12 tgccaagctt accgtgnnsn nsnnsnnsnn snnsaggtgg nnsnnsggga acgtcttctc at gct ccg <210> 13 <211> 115 < 212> PRT <213> Artificial <220>

<223> Artificial sequence <220>

<221> miscjeature <222> (19) .. (21) <223> Xaa can be any amino acid found in nature <220>

<221> misatureature <222> (73) .. (80)

112 <223> Xaa can be any naturally occurring amino acid <220>

<221> miscjeature <222> (83) .. (84) <223> Xaa can be any amino acid found in nature <400> 13

<td>Wfet</td><td>Al a</td><td>Pro</td><td rowspan="2">Arg</td><td>au</td><td>Pro</td><td>Q n</td><td>val</td><td rowspan="2">Tyr</td><td>Thr</td><td>Leu</td><td>Pro</td><td>Pr</td><td>Cheese</td><td>Arg</td><td>asp</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>α u</td><td>Leu</td><td>Xaa</td><td>xaa</td><td>Xaa</td><td>G n</td><td>val</td><td>Cheese</td><td>Leu</td><td>Thr</td><td rowspan="2">Cys</td><td>Leu</td><td>val</td><td>lys</td><td>qy</td><td>phe</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td rowspan="2">Tyr</td><td>Pr</td><td>Cheese</td><td rowspan="2">asp</td><td>11 e</td><td>Al a</td><td>val</td><td>α u</td><td>Trp</td><td>au</td><td>Cheese</td><td>own</td><td>qy</td><td>□ n</td><td>Pro</td><td>au</td>

<td></td><td> 35</td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>own</td><td>own</td><td rowspan="2">Tyr</td><td rowspan="2">lys</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Pro</td><td>vai</td><td>Leu</td><td>asp</td><td>Cheese</td><td>asp</td><td>G y</td><td>Cheese</td><td>phe</td>

<td></td><td> 50</td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>phe</td><td>Leu</td><td rowspan="2">Tyr</td><td>Cheese</td><td rowspan="2">lys</td><td>Leu</td><td>Thr</td><td>val</td><td>Xaa</td><td>Xaa</td><td>xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa</td>

<td> 65</td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Arg</td><td>Trp</td><td>Xaa</td><td>Xaa</td><td>oy 35</td><td>own</td><td>val</td><td>phe</td><td>Cheese</td><td>Cys 90</td><td>Cheese</td><td>val</td><td>Myth</td><td>H p</td><td>0 u 95</td><td>Al a</td>

<td>Leu</td><td>HS</td><td>own</td><td>H p</td><td rowspan="2">Tyr</td><td>Thr</td><td>and π</td><td rowspan="2">lys</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Pr</td><td>ay</td><td>lys</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

Al a Al a Al a 115 <210> 14 <211> 347 <212> DNA <213> Artificial <220>

<223> Artificial sequence <220>

<221> miscjeature <222> (57) .. (58) <223> n is a, c, g, or t <220>

<221> miscjeature <222> (60) .. (61) <223> n is a, c, g, or t <220>

<221> misatureature <222> (63) .. (64)

113 <223> η is a, c, g, or t <220>

<221> miscjeature 5 <222> (219) .. (220) <223> n is a, c, g, or t <220>

<221> miscjeature 10 <222> (222) .. (223) <223> n is a, c, g, or t <220>

<221> missions> 5 <222> (225) .. (226) <223> n is a, c, g, or t <220>

<221> miscjeature 20 <222> (228) .. (229) <223> n is a, c, g, or t <220>

<221> misatureature 25 <222> (231) .. (232) <223> n is a, c, g, or t <220>

<221> miscjeature 30 <222> (234) .. (235) <223> n is a, c, g, or t <220>

<221> misatureature 35 <222> (237) .. (238) <223> n is a, c, g, or t <220>

<221> miscjeature 10 <222> (240) .. (241) <223> n is a, c, g, or t <220>

<221> miscjeature - <5 <222> (249) .. (250) <223> n is a, c, g, or t <220>

<221> misatureature 50 <222> (252) .. (253) <223> n is a, c, g, or t <400> 14

114 CCAT ggcccc nsnnscaagt agt gggagag ccgacggctc nsaggt ggnn act acacaca ccgagaacca cagcct Gacc CAAT gggcag ct t ct t cct c snnsgggaac gaagagcctc caggt gt aca tgcctggtca ccggagaaca t acagcaagc gt ct t ct cat tccctgt ctc ccctgccccc aaggct t ct and act acaagac 11 accgt GNN GCT CCGT gat cgggt aaagc at cccgt gac t cccagcgac cacgcct ccc snnsnnsnns gcat gaggct ggccgca gagct cnnsn at cgccgt gg gt gct ggact nnsnnsnnsn ct gcacaacc

120

180

240

300

347 <210> 15 <211> 74> 5 <212> DNA <213> Artificial <220>

<223> Artificial sequence <220>

<221> miscjeature <222> (17) .. (18) <223> n is a, c, g, or t <220>

<221> miscjeature <222> (20) .. (21) <223> n is a, c. G, or t <220>

<221> miscjeature <222> (23) .. (24) <223> n is a, c, g, or t

SS <220>

<221> miscjeature <222> (26) .. (27) <223> n is a, c, g, or t «0 <220>

<221> miscjeature <222> (29) .. (30) <223> n is a, c, g, or t <220>

<221> miscjeature <222> (32) .. (33) <223> n is a, c, g, or t <220>

<221> miscjeature <222> (35) .. (36) <223> n is a, c, g, or t <220>

<221> misatureature <222> (38) .. (39)

115 <223> η is a, c, g, or t <220>

<221> miscjeature <222> (47) .. (48) <223> n is a, c, g, or t <220>

<221> missions <222> (50) .. (51) <223> n is a, c, g, or t <400> 15 tgccaagctt accgtgnnsn nsnnsnnsnn snnsnnsnns aggtggnnsn nsgggaacgt cttctcatgc t ccg <210> 16 <211> 110 <211> 212> PRT <213> Artificial <220>

<223> Artificial sequence <400> 16

Pro Arg Gu Pro Gn Val Tyr Thr Leu Pro Pro Ser Arg Asp Gu Leu 15 10 15

<td rowspan="2">qy</td><td rowspan="2">Trp</td><td>Pro</td><td>α n</td><td>val</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Leu</td><td>val</td><td>lys</td><td>G y</td><td>phe</td><td>Tyr</td><td>Pro</td>

<td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Cheese</td><td rowspan="2">asp</td><td>II e</td><td>Al a</td><td>val</td><td>Q u</td><td rowspan="2">Trp</td><td>au</td><td>Cheese</td><td>own</td><td rowspan="2">&lt;3y</td><td>G n</td><td>Pro</td><td>G u</td><td>own</td><td>own</td>

<td></td><td> 35</td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td rowspan="2">Tyr</td><td>lys</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Pr</td><td>vai</td><td>Leu</td><td rowspan="2">asp</td><td>Cheese</td><td>asp</td><td>Q y</td><td>Cheese</td><td>phe</td><td>phe</td><td>Leu</td>

<td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Tyr</td><td>Cheese</td><td rowspan="2">lys</td><td>Leu</td><td>Thr</td><td>val</td><td>Pr</td><td rowspan="2">lys</td><td>Arg</td><td>Trp</td><td>Cys</td><td>val</td><td>Cheese</td><td>vai</td><td>Arg</td><td>Trp</td>

<td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Pro</td><td>Pr</td><td rowspan="2">&lt;3y</td><td>own</td><td>val</td><td>phe</td><td>Cheese</td><td rowspan="2">Cys</td><td>Cheese</td><td>val</td><td>futet</td><td>H p</td><td>α u</td><td>Al a</td><td>Leu</td><td>H p</td>

<td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>own</td><td>HS</td><td rowspan="2">Tyr</td><td>Thr</td><td>α n</td><td rowspan="2">lys</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Pro</td><td>qy</td><td>lys</td><td></td><td></td>

<td></td><td></td><td> 100</td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<210> 17 <211 > 330

116 <212> DNA <213> Artificial <220>

<223> Artificial sequence <400> 17 ccccgagaac gt cagcct aa agcaat gggc tccttcttcc cccccgggga cagaagagcc cacaggt gt a cct gcct ggt agccggagaa t ct acagcaa acgg tc ccccctcct at cccagcg accacgcct c cccaagcggt at gcat gagg acgagct cgg acat egeegt ccgt get gga ggt gcgt gag ct ct gcacaa ct ggccgcaa ggagt gggag ctccgacgg2 <20> cgg <2> cgg

<223> artificial sequence

<td> &lt;400&gt; 18</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Pro</td><td rowspan="2">Arg</td><td>G u</td><td>Pr</td><td>G n</td><td>val</td><td rowspan="2">Tyr</td><td>Thr</td><td>Leu</td><td>Pro</td><td>Pro</td><td>Cheese</td><td>Arg</td><td>asp</td><td>G u</td><td>Leu</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Cheese</td><td>val</td><td>Cheese</td><td>G n</td><td>val</td><td>Cheese</td><td>Pro</td><td>Thr</td><td>Cys</td><td>Leu</td><td>val</td><td>lys</td><td>G y</td><td>phe</td><td>Tyr</td><td>Pro</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Cheese</td><td rowspan="2">asp</td><td>1 1 e</td><td>AND! and</td><td>val</td><td>G u</td><td>Trp</td><td>G u</td><td>Cheese</td><td>own</td><td>gy</td><td>G n</td><td>Pro</td><td>G u</td><td>own</td><td>own</td>

<td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Tyr</td><td>lys 50</td><td>Thr</td><td>Thr</td><td>Pr</td><td>Pro</td><td>val 55</td><td>Leu</td>

<td>Tyr 65</td><td>Cheese</td><td>lys</td><td>Leu</td><td>Thr</td><td>val 70</td><td>11 e</td><td>Pro</td>

<td>Trp</td><td>11 e</td><td>Q y</td><td>own</td><td>val 85</td><td>phe</td><td>Cheese</td><td>Cys</td>

<td>own</td><td>HS</td><td>Tyr</td><td>Thr</td><td>G n</td><td>lys</td><td>Cheese</td><td>Leu</td>

100

<td>asp</td><td>Cheese</td><td>asp</td><td>gy 60</td><td>Cheese</td><td>phe</td><td>phe</td><td>Leu</td>

<td>phe</td><td>Cys</td><td>Arg 75</td><td>Nfet</td><td>Cheese</td><td>Pr</td><td>Arg</td><td>Trp 80</td>

<td>Cheese</td><td>val 90</td><td>mst</td><td>HS</td><td>G u</td><td>And I</td><td>Leu 95</td><td>4 p</td>

<td>Cheese 105</td><td>Leu</td><td>Cheese</td><td>Pr</td><td>¢ 3 y</td><td>lys 110</td><td></td><td></td>

<210> 19

117 <211> 330 <212> DNA <213> Artificial <220>

<223> Artificial sequence <400> 19

<td>ccccgagaac</td><td>cacaggt gt a</td><td>caccct gccc</td><td>ccat cccgt g-</td><td>acgagct ct</td><td>ggt gt egeaa</td><td> 60</td>

<td>gt cagcccga</td><td>cct gcct ggt</td><td>caaaggct tc</td><td>t at cccagcg</td><td>acat egeagt</td><td>ggagt gggag</td><td> 120</td>

<td>agcaat gggc</td><td>agccggagaa</td><td>caact acaag</td><td>accacgcct c</td><td>ccgt gct gga</td><td>ct ccgacggc</td><td> 180</td>

<td>tccttcttcc</td><td>t ct acagcaa</td><td>gettaccgtg</td><td>at cccct t ct</td><td>gcaggatgag</td><td>ccccaggtgg</td><td> 240</td>

<td>t ggat cggga</td><td>acgt ct t ct c</td><td>at gct ccgt g</td><td>at gcat gagg</td><td>ct ct gcacaa</td><td>ccact acaca</td><td> 300</td>

<td>cagaagagcc</td><td>t ct ccct gt c</td><td>t ccgggt aaa</td><td></td><td></td><td></td><td> 330</td>

<td> &lt;210&gt;20</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;211&gt; 105</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>&lt;212&gt; PRT</td><td></td><td></td><td></td><td></td><td></td><td></td>

<213> Artificial <220>

<223> Artificial sequence <400> 20

<td>Pr 1</td><td>Arg</td><td>Q u</td><td>Pro</td><td>3 n 5</td><td>val</td><td>Tyr</td><td>Thr</td><td>Leu</td><td colspan="3">Pro Pro Ser 10</td><td>Arg</td><td colspan="3">Asp 3 u Leu 15</td>

<td>3 u</td><td>Al a</td><td>Leu</td><td>3 rt</td><td>Val</td><td>Ser</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Leu</td><td>vai</td><td rowspan="2">Lys</td><td>3 y</td><td>Phe</td><td>Tyr</td><td>Pr o</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Ser</td><td rowspan="2">Asp</td><td>11 e</td><td>Al a</td><td>Val</td><td>3 u</td><td rowspan="2">Tr p</td><td>3 u</td><td>Ser</td><td>Asn</td><td rowspan="2">3y</td><td>3 n</td><td>Pr o</td><td>3 u</td><td>Asn</td><td>Asn</td>

<td></td><td> 35</td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td rowspan="2">Tyr</td><td>Lys</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Pr o</td><td>Val</td><td>Leu</td><td>Asp</td><td>Ser</td><td>Asp</td><td>3y</td><td>Ser</td><td>Phe</td><td>Phe</td><td>Leu</td>

<td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Tyr</td><td>Ser</td><td rowspan="2">Lys</td><td>Leu</td><td>Thr</td><td>Val</td><td rowspan="2">Arg</td><td rowspan="2">Arg</td><td>Asn</td><td rowspan="2">Arg</td><td>Trp</td><td>Ser</td><td>Trp</td><td>3 y</td><td>Asn</td><td>Val</td>

<td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Phe</td><td>Ser</td><td rowspan="2">Cys</td><td>Ser</td><td>Val</td><td>Mit</td><td>hi s</td><td> 3 □</td><td>Al a</td><td>Leu</td><td>H S</td><td>Asn</td><td>4 s</td><td>Tyr</td><td>Thr</td><td>3 n</td>

<td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

Lys Ser Leu Ser Leu Ser Pro Gy Lys 100 105 <21o> 21

118 <211> 315 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 21 cct cgagaac gt cagcct ga agcaat gggc t cct t ct t cc t tct cat gct ctgtctccgg cacaggt gt a cct gcct ggt agccggagaa t ct acagcaa ccgt gat gca gt aaa caccct gccc caaaggct t c caact acaag gettaccgtg t gaggct ct g ccat cccgt g t at cccagcg accacgcct c cggcgcaaca cacaaccact acgagct ega acat cgccgt ccgt gctgga ggt ggt cct g acacacagaa ggcgctgcaa ggagt gggag ct ccgacggc ggggaacgt c gagcct ct cc <210>22 <211> 108 <212> PRT <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 22

<td>Pro 1</td><td colspan="2">Arg G u</td><td>Pro</td><td>G n 5</td><td>Val</td><td>Tyr</td><td>Thr</td><td>Leu</td><td>Pro 10</td><td colspan="2">Pro Ser</td><td colspan="2">Arg Asp</td><td>G u 15</td><td>Leu</td>

<td>G n</td><td>Gy</td><td>Ser</td><td>G n 20</td><td>Val</td><td>Ser</td><td>Leu</td><td>Thr</td><td>Cys 25</td><td>Leu</td><td>Val</td><td>Lys</td><td>G y</td><td>Phe 30</td><td>Tyr</td><td>Pro</td>

<td>Ser</td><td>Asp</td><td>11 e 35</td><td>Al a</td><td>Val</td><td>G u</td><td>Trp</td><td>G u 40</td><td>Ser</td><td>Asn</td><td>Gy</td><td>G n</td><td>Pro 45</td><td>G u</td><td>Asn</td><td>Asn</td>

<td>Tyr</td><td>Lys 50</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Pro</td><td>vai 55</td><td>Leu</td><td>Asp</td><td>Ser</td><td>Asp</td><td>Gy 60</td><td>Ser</td><td>Phe</td><td>Phe</td><td>Leu</td>

<td>Tyr 65</td><td>Ser</td><td>Lys</td><td>Leu</td><td>Thr</td><td>Val 70</td><td>Lys</td><td>Ser</td><td>Arg</td><td>Al a</td><td>Thr 75</td><td>Arg</td><td>Arg</td><td>Trp</td><td>Val</td><td>Val 80</td>

<td>Qy</td><td>Asn</td><td>Val</td><td>Phe</td><td>Ser 85</td><td>Cys</td><td>Ser</td><td>Val</td><td>lyfet</td><td>H S 90</td><td>G o</td><td>Al a</td><td>Leu</td><td>H s</td><td>Asn 95</td><td>H s</td>

<td>Tyr</td><td>Thr</td><td>G n</td><td>Lys 100</td><td>Asn</td><td>Leu</td><td>Ser</td><td>Leu</td><td>Ser 105</td><td>Pro</td><td>Gy</td><td>Lys</td><td></td><td></td><td></td><td></td>

<210>23 <211 >324

119 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 23 ccccgagaac gt cagcct ga agcaat gggc t cct t ct t cc gggaacgt ct aacct ct ccc cacaggtgta caccctgccc cct gcct ggt caaaggcttc agccggagaa caact acaag tctacagcaa gcttaccgtg tltcttgctc cgtgatgcat t gt ct ccggg t aaa ccat cccgt g t at cccagcg accacgcct c aagt cgcgcg gaggctctgc acgagct cca acat cgccgt ccgt gctgga ccacccggag acaaccact a ggggagccaa ggagt gggag ct ccgacggc gt gggt ggt g cacacagaag

120

180

240

300

324 <210> 24 <211> 107 <212> PRT <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 24

<td>Pro</td><td rowspan="2">Arg</td><td>α u</td><td>Pro</td><td>G n</td><td>Val</td><td rowspan="2">Tyr</td><td>Thr</td><td>Leu</td><td>Pro</td><td>Pr o</td><td>Ser</td><td>Arg</td><td>Asp</td><td>G u</td><td>Leu</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Al a</td><td>11 e</td><td>α y</td><td>α n</td><td>Val</td><td>Ser</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Leu</td><td>Val</td><td rowspan="2">Lys</td><td>G y</td><td>Phe</td><td>Tyr</td><td>Pro</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Ser</td><td rowspan="2">Asp</td><td>I I e</td><td>Al a</td><td>Val</td><td>G u</td><td rowspan="2">Trp</td><td>G u</td><td>Ser</td><td>Asn</td><td>Gy</td><td>G n</td><td>Pr o</td><td>G u</td><td>Asn</td><td>Asn</td>

<td></td><td> 35</td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td rowspan="2">Tyr</td><td>Lys</td><td>Thr</td><td>Thr</td><td>Pr o</td><td>Pro</td><td>Val</td><td>Leu</td><td>Asp</td><td>Ser</td><td>Asp</td><td>G y</td><td>Ser</td><td>Phe</td><td>Phe</td><td>Leu</td>

<td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Tyr</td><td>Ser</td><td rowspan="2">Lys</td><td>Leu</td><td>Thr</td><td>Val</td><td rowspan="2">Arg</td><td>Ser</td><td>Thr</td><td rowspan="2">Ar g</td><td>Asp</td><td>Asn</td><td>Arg</td><td>Trp</td><td>Leu</td><td>Val</td>

<td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td rowspan="2">ety</td><td>Asn</td><td>Val</td><td>Phe</td><td>Ser</td><td rowspan="2">Cys</td><td>Ser</td><td>Val</td><td>wet</td><td>H s</td><td>G u</td><td>Al a</td><td>Leu</td><td>H S</td><td>Asn</td><td>H S</td>

<td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td rowspan="2">Tyr</td><td>Thr</td><td>¢3 n</td><td>Lys</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Pr o</td><td>G y</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<210>25 <211 >324 <212> DNA

120 <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 25 ccccgagaac cacaggtgla caccctgccc ccatcccgtg acgagctcgc gatcggccaa 60 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagt gggag 120 agcaalgggc agccggagaa caact acaag accacgcctc ccgtgctgga ctccgacggc 180 . tccttcttcc tctacagcaa gcttaccgtg cgctcgacga gggacaacag gtggctggtg 240 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacacagaag 300

<td> 20</td><td>agcct ct ccc</td><td colspan="3">tgtctccggg t</td><td>aaa</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;210&gt; 26 &lt;211&gt; 110</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 25</td><td>&lt;212&gt; PRT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td colspan="2">&lt;213&gt; Sztuczna</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 30</td><td colspan="4">&lt;223&gt; Sekwencja sztuczna</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;400&gt; 26</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 35</td><td>Pr o Ar g 1</td><td>α u</td><td>Pro</td><td>G n 5</td><td>Val</td><td>Tyr</td><td>Thr</td><td>Leu</td><td>Pr o 10</td><td>Pro</td><td>Ser</td><td>Arg</td><td>Asp</td><td>G u 15</td><td>Leu</td>

<td></td><td>Ser Qy</td><td>Al a</td><td>G n 20</td><td>Val</td><td>Ser</td><td>Leu</td><td>Thr</td><td>Cys 25</td><td>Leu</td><td>Val</td><td>Lys</td><td>Gy</td><td>Phe 30</td><td>Tyr</td><td>Pr o</td>

<td> 40</td><td>Ser Asp</td><td>11 e 35</td><td>Al a</td><td>Val</td><td>Q u</td><td>Trp</td><td>G u 40</td><td>Ser</td><td>Asn</td><td>Gy</td><td>G n</td><td>Pr o 45</td><td>G u</td><td>Asn</td><td>Asn</td>

<td> 45</td><td>Tyr Lys 50</td><td>Thr</td><td>Thr</td><td>Pr o</td><td>Pr o</td><td>Val 55</td><td>Leu</td><td>Asp</td><td>Ser</td><td>Asp</td><td>Gy 60</td><td>Ser</td><td>Phe</td><td>Phe</td><td>Leu</td>

<td></td><td>Tyr Ser 65</td><td>Lys</td><td>Leu</td><td>Thr</td><td>Val 70</td><td>Trp</td><td>Phe</td><td>Arg</td><td>G n</td><td>G u 75</td><td>Q y</td><td>Gy</td><td>lyfet</td><td>Arg</td><td>Trp 80</td>

<td> 50</td><td>Phe Al a</td><td>Gy</td><td>Asn</td><td>Val 85</td><td>Phe</td><td>Ser</td><td>Cys</td><td>Ser</td><td>Val 90</td><td>lUfet</td><td>H s</td><td>G u</td><td>Al a</td><td>Leu 95</td><td>H S</td>

<td> 55</td><td>Asn H s</td><td>Tyr</td><td>Thr 100</td><td>G n</td><td>Lys</td><td>Ser</td><td>Leu</td><td>Ser 105</td><td>Leu</td><td>Ser</td><td>Pro</td><td>Gy</td><td>Lys 110</td><td></td><td></td>

<21o> 27

121 <211 >330 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 27 ccccgagaac gt cagcct ga agcaat gggc i cct t ct t cc ttcgcgggga cagaagagcc cacaggt gt a cct gcct ggt agccggagaa t ct acagcaa acgt ct t ct c t ct ccct gt c caccct gccc caaaggct t c caact acaag gettaccgtg at gct ccgt g t ccgggt aaa ccat cccgt g t at cccagcg accacgcct οι ggt t caggc at gcat gagg acgagct cag acat egeegt ccgt gct gga aggagggcgg ct ct gcacaa cggggcgcaa ggagt gggag et ccgacggc catgaggtgg ccact acaca <21o> 28 <211> 110 <212> PRT <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 28

<td>Pro</td><td rowspan="2">Arg</td><td>G u</td><td>Pr o</td><td>G n</td><td>Val</td><td rowspan="2">Tyr</td><td>Thr</td><td>Leu</td><td>Pr o</td><td>Pr o</td><td>Ser</td><td>Arg</td><td>Asp</td><td>G u</td><td>Leu</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Val</td><td>Leu</td><td>Gy</td><td>G n</td><td>Val</td><td>Ser</td><td>Pr o</td><td>Thr</td><td>Cys</td><td>Leu</td><td>Val</td><td rowspan="2">Lys</td><td rowspan="2">G y</td><td>Phe</td><td>Tyr</td><td>Pr o</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Ser</td><td rowspan="2">Asp</td><td>11 e</td><td>Al a</td><td>Val</td><td>G u</td><td rowspan="2">Trp</td><td>G u</td><td>Ser</td><td>Asn</td><td rowspan="2">Gy</td><td>G n</td><td>Pro</td><td>3 u</td><td>Asn</td><td>Asn</td>

<td></td><td> 35</td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td rowspan="2">Tyr</td><td>Lys</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Pro</td><td>vai</td><td>Leu</td><td>Asp</td><td>Ser</td><td>Asp</td><td>G y</td><td>Ser</td><td>Phe</td><td>Phe</td><td>Leu</td>

<td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Tyr</td><td rowspan="2">3y</td><td rowspan="2">Lys</td><td>Leu</td><td>Thr</td><td>Val</td><td>Pro</td><td>Pro</td><td rowspan="2">Arg</td><td>Leu</td><td>Lys</td><td>ay</td><td>Trp</td><td>Pr o</td><td>Arg</td><td>Trp</td>

<td> 65</td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td rowspan="2">Cly</td><td rowspan="2">Trp</td><td rowspan="2">Gy</td><td>Asn</td><td>vai</td><td>Phe</td><td>Ser</td><td rowspan="2">Cys</td><td>Ser</td><td>Val</td><td>Nfet</td><td>H s</td><td>G u</td><td>Al a</td><td>Leu</td><td>H s</td>

<td></td><td> 85</td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Asn</td><td>4 s</td><td rowspan="2">Tyr</td><td>Thr</td><td>G n</td><td rowspan="2">Lys</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Pro</td><td rowspan="2">Gy</td><td>Lys</td><td></td><td></td>

<td></td><td></td><td> 100</td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<210> 29 <211 >330

122 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 29

<td>ΐ0</td><td>ccccgagaac</td><td>cacaggt gt a</td><td>caccct gccc</td><td>ccat cccgt g</td><td>acgagct cgt</td><td>cttggggcaa</td><td> 60</td>

<td></td><td>gt cagcccga</td><td>cct gcct ggt</td><td>caaaggct t c</td><td>t at cccagcg</td><td>acat cgccgt</td><td>ggagt gggag</td><td> 120</td>

<td></td><td>agcaat gggc</td><td>agccggagaa</td><td>caact acaag</td><td>accacgcct c</td><td>ccgt gct gga</td><td>ct ccgacggc</td><td> 180</td>

<td>ΐ5</td><td>t cct t ct t cc</td><td>t ct acggcaa</td><td>gettaccgtg</td><td>cccccgcggt</td><td>t gaagggct g</td><td>gccgaggt gg</td><td> 240</td>

<td></td><td>ggct ggggga</td><td>acgtcttctc</td><td>at gct ccgt g</td><td>at gcat gagg</td><td>ct ct gcacaa</td><td>ccact acaca</td><td> 300</td>

<td></td><td>cagaagagcc</td><td>t ct ccct gt c</td><td>t ccgggt aaa</td><td></td><td></td><td></td><td> 330</td>

<210>30 <211> 105 <212> PRT <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 30

<td colspan="2">Pro Arg G u Pro G n Val</td><td>Tyr Thr Leu Pro Pro Ser</td><td>Ar g Asp (3 u Leu</td>

<td> 1</td><td> 5</td><td> 10</td><td> 15</td>

<td>Leu</td><td>Al a</td><td rowspan="2">Tyr</td><td>G n</td><td>Val</td><td>Ser</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Leu</td><td>Val</td><td>Lys</td><td>Gy</td><td>Phe</td><td>Tyr</td><td>Pr o</td>

<td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Ser</td><td rowspan="2">Asp</td><td>11 e</td><td>Al a</td><td>Val</td><td>G u</td><td>Trp</td><td>G u</td><td>Ser</td><td>Asn</td><td>Gy</td><td>G n</td><td>Pr o</td><td>G u</td><td>Asn</td><td>Asn</td>

<td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td rowspan="2">Tyr</td><td>Lys</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Pr o</td><td>Val</td><td>Leu</td><td>Asp</td><td>Ser</td><td>Asp</td><td>Gy</td><td>Ser</td><td>Phe</td><td>Phe</td><td>Leu</td>

<td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Tyr</td><td>Ser</td><td rowspan="2">Lys</td><td>Leu</td><td>Thr</td><td>Val</td><td>Val</td><td>Al a</td><td rowspan="2">Gy</td><td rowspan="2">Arg</td><td>Trp</td><td>Thr</td><td>Cys</td><td>G y</td><td>Asn</td><td>Val</td>

<td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Phe</td><td>Ser</td><td rowspan="2">Cys</td><td>Ser</td><td>Val</td><td>Nfet</td><td>H s</td><td>G u</td><td>Al a</td><td>Leu</td><td>H S</td><td>Asn</td><td>H S</td><td>Tyr</td><td>Thr</td><td>G n</td>

<td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Lys</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Pro</td><td>Gy</td><td>Lys</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

100 105 <210> 31

123 <211 > 315 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 31 ccccgagaac gt cagcct ga agcaat gggc t cct t ct t cc ct cat gct ctgtctccgg cacaggt gt a cct gcctggt agccggagaa t ct acagcaa ccgt gat gca gt aaa caccct gccc caaaggct t c caact acaag gcttaccgtg tgaggctctg ccat cccgt g t at cccagcg accacgcct c gt ggccggca cacaaccact acgagct cct acat cgccgt ccgt gctgga ggt ggacgt g acacacagaa ggcgt accaa ggagt gggag ct ccgacggc cgggaacgtc gagcct ct cc <210> 32 <211> 110 <212> PRT <213> Sztuczna <220>

<223> Sekwencja sztuczna

<td> &lt;400&gt; 3</td><td>I2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Pr o</td><td rowspan="2">Arg</td><td>α u</td><td>Pr o</td><td>G n</td><td>Val</td><td rowspan="2">Tyr</td><td>Thr</td><td>Leu</td><td>Pro</td><td>Pr o</td><td>Ser</td><td>Arg</td><td>Asp</td><td>G u</td><td>Leu</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td rowspan="2">Cys</td><td>Val</td><td>Pro</td><td>Q n</td><td>Val</td><td>Ser</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Leu</td><td>Val</td><td>Lys</td><td>Gy</td><td>Phe</td><td>Tyr</td><td>Pro</td>

<td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Ser</td><td rowspan="2">Asp</td><td>I I e</td><td>Al a</td><td>Val</td><td>G u</td><td rowspan="2">Trp</td><td>&lt;3 u</td><td>Ser</td><td>Asn</td><td>Gy</td><td>Q n</td><td>Pr o</td><td>Q u</td><td>Asn</td><td>Asn</td>

<td></td><td> 35</td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td rowspan="2">Tyr</td><td>Lys</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Pro</td><td>Val</td><td>Leu</td><td rowspan="2">Asp</td><td>Ser</td><td>Asp</td><td>G</td><td>y</td><td>Se</td><td>;r</td><td>Phe</td><td>Phe</td><td>Leu</td>

<td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td colspan="2"> 60</td><td></td><td></td><td></td><td></td><td></td>

<td>Tyr</td><td>Ser</td><td rowspan="2">Lys</td><td>Leu</td><td>Thr</td><td>Val</td><td>Val</td><td>Leu</td><td rowspan="2">Lys</td><td>Val</td><td>Val</td><td>G</td><td>n</td><td>Al</td><td>a</td><td>Arg</td><td>Arg</td><td>Trp</td>

<td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 80</td>

<td>G u</td><td>Val</td><td rowspan="2">Gy</td><td>Asn</td><td>Val</td><td>Phe</td><td>Ser</td><td rowspan="2">cys</td><td>Ser</td><td>Val</td><td>lyfet</td><td>H</td><td>s</td><td>G</td><td>u</td><td>Al a</td><td>Leu</td><td>H s</td>

<td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Asn</td><td>H s</td><td rowspan="2">Tyr</td><td>Thr</td><td>G n</td><td rowspan="2">Lys</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Pr</td><td> 0</td><td>G</td><td>y</td><td>Lys</td><td></td><td></td>

<td></td><td></td><td> 100</td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

124 <210> 33 <211 >330 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 33 ccccgagaac gt cagcct ga agcaat gggc t cct t ct t cc gaggt gggga cagaagagcc cacaggt gt a cct gcct ggt agccggagaa t ct acagcaa acgt ct t ct c t ct ccct gt c caccct gccc caaaggct t c caact acaag gcttaccgtg at gct ccgt g t ccgggt aaa ccat cccgt g t at cccagcg accacgcct c gtgctcaagg at gcat gagg acgagct ct g acat cgccgt ccgt gctgga t cgt gcaggc ct ct gcacaa cgt cccgcaa ggagt gggag ct ccgacggc gcgcaggt gg ccact acaca <210> 34 <211> 105 <212> PRT <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 34

<td>Pro</td><td rowspan="2">Arg</td><td>G u</td><td>Pr o</td><td>G n</td><td>Val</td><td rowspan="2">Tyr</td><td>Thr</td><td>Leu</td><td>Pro</td><td>Pr o</td><td>Ser</td><td rowspan="2">Arg</td><td rowspan="2">Asp</td><td>G u</td><td>Leu</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td> 15</td><td></td>

<td rowspan="2">Gy</td><td>I I e</td><td>Al a</td><td>G n</td><td>Val</td><td>Ser</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Leu</td><td>Val</td><td rowspan="2">Lys</td><td rowspan="2">Gly</td><td>Phe</td><td>Tyr</td><td>Pro</td>

<td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Ser</td><td rowspan="2">Asp</td><td>I I e</td><td>Al a</td><td>Val</td><td>G u</td><td rowspan="2">Trp</td><td>G u</td><td>Ser</td><td>Asn</td><td rowspan="2">Gy</td><td>G n</td><td>Pr o</td><td>G u</td><td>Asn</td><td>Asn</td>

<td></td><td> 35</td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td rowspan="2">Tyr</td><td>Lys</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Pro</td><td>Val</td><td>Leu</td><td>Asp</td><td>Ser</td><td>Asp</td><td>ay</td><td>Ser</td><td>Phe</td><td>Phe</td><td>Leu</td>

<td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Tyr</td><td>Ser</td><td rowspan="2">Lys</td><td>Leu</td><td>Thr</td><td>Val</td><td>Leu</td><td rowspan="2">Gy</td><td rowspan="2">Arg</td><td rowspan="2">Arg</td><td>Trp</td><td>Thr</td><td>Leu</td><td rowspan="2">Gly</td><td>Asn</td><td>Val</td>

<td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td> 75</td><td></td><td></td><td></td><td> 80</td>

<td>Phe</td><td>Ser</td><td>Cys</td><td>Ser</td><td>Val</td><td>rvfet</td><td>Hi s</td><td>G u</td><td>Al a</td><td>Leu</td><td>H S</td><td>Asn</td><td>H s</td><td>Tyr</td><td>Thr</td><td>G n</td>

90 95

Lys Ser Leu Ser Leu Ser Pro Gy Lys 100 105

125 <210> 35 <211 > 315 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 35 ccccgagaac gt cagcct ga agcaacgggc t ct 11 ct t cc 11 ct cat gct ctgtctccgg cacaggt gt a cct gcct ggt agccggagaa t ct acagcaa ccgt gat gca gt aaa caccct gccc caaaggct t c caact acaag gettaccgtg t gaggct ct g ccat cccggg t at cccagcg accacgcct c 11 gggeegea cacaaccact acgagct cgg acat egeegt ccgt gct gga ggtggaccct acacacagaa cat egegeaa ggagt gggag ct ccgacggc ggggaacgt c gagcct ct cc <210> 36 <211> 105 <212> PRT <213> Sztuczna <220>

<223> Sekwencja sztuczna

<td colspan="2"> &lt;400&gt; 36</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Pr o</td><td rowspan="2">Arg</td><td>G u</td><td>Pr o</td><td>G n</td><td>Val</td><td rowspan="2">Tyr</td><td>Thr</td><td>Leu</td><td>Pr o</td><td>Pr o</td><td>Ser</td><td rowspan="2">Arg</td><td>Asp</td><td>G u</td><td>Leu</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td> 15</td><td></td>

<td rowspan="2">Gy</td><td>11 e</td><td>Al a</td><td>G n</td><td>Val</td><td>Ser</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Leu</td><td>Val</td><td rowspan="2">Lys</td><td>Gy</td><td>Phe</td><td>Tyr</td><td>Pr o</td>

<td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Ser</td><td rowspan="2">Asp</td><td>I I e</td><td>Al a</td><td>Val</td><td>G u</td><td rowspan="2">Trp</td><td>G u</td><td>Ser</td><td>Asn</td><td>ay</td><td>G n</td><td>Pro</td><td>G u</td><td>Asn</td><td>Asn</td>

<td></td><td> 35</td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td rowspan="2">Tyr</td><td>Lys</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Pr o</td><td>Val</td><td>Leu</td><td rowspan="2">Asp</td><td>Ser</td><td rowspan="2">Asp</td><td>ay</td><td>Ser</td><td>Phe</td><td>Phe</td><td>Leu</td>

<td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Tyr</td><td>Ser</td><td rowspan="2">Lys</td><td>Leu</td><td>Thr</td><td>Val</td><td>Leu</td><td>G y</td><td rowspan="2">Arg</td><td rowspan="2">Arg</td><td>Trp</td><td>Thr</td><td>Leu</td><td>α y</td><td>Asn</td><td>Val</td>

<td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Phe</td><td>Ser</td><td rowspan="2">cys</td><td>Ser</td><td>Val</td><td>fvfet</td><td>H s</td><td>G u</td><td>Al a</td><td>Leu</td><td>Hi s</td><td>Asn</td><td>H s</td><td>Tyr</td><td>Thr</td><td>G n</td>

<td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Lys</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Pr o</td><td>Gy</td><td>Lys</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

100 105 <210> 37

126 <211 > 315 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 37 ccccgagaac gtcagcttga agcaacgggc t cct t ct t cc 11 ct cat gct ctgtctccgg cacaggt gt a cct gcct ggt agccggagaa t ct acagcaa ccgt gat gca gt aaa caccct gccc caaaggct 11 caact acaag gettaccgtg t gaggct ctg ccat cccgt g t at cccagcg accacgcct c 11 gggeegea cacaaccact.

acgagctcgg acat cgccgt ccgtgctgga ggt ggaccct acacacagaa cat egegeaa ggagt gggag et ccgacggc ggggaacgtc gagcct ct cc <210> 38 <211> 105 <212> PRT <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 38

<td>Pro 1</td><td>Arg</td><td>α u</td><td>Pr o</td><td>Gl n 5</td><td>Val</td><td>Tyr</td><td>Thr</td><td>Leu</td><td>Pro 10</td><td>Pro</td><td>Ser</td><td>Arg</td><td>Asp</td><td>G u 15</td><td>Leu</td>

<td>Leu</td><td>Pro</td><td>Cys</td><td>Gl n 20</td><td>Val</td><td>Ser</td><td>Leu</td><td>Thr</td><td>cys 25</td><td>Leu</td><td>Val</td><td>Lys</td><td>Gly</td><td>Phe 30</td><td>Tyr</td><td>Pr o</td>

<td>Ser</td><td>Asp</td><td>I I e 35</td><td>Al a</td><td>Val</td><td>Gl u</td><td>Trp</td><td>Gl u 40</td><td>Ser</td><td>Asn</td><td>Qy</td><td>Gl n</td><td>Pro 45</td><td>Gl u</td><td>Asn</td><td>Asn</td>

<td>Tyr</td><td>Lys 50</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Pro</td><td>Val 55</td><td>Leu</td><td>Asp</td><td>Ser</td><td>Asp</td><td>ay 60</td><td>Ser</td><td>Phe</td><td>Phe</td><td>Leu</td>

<td>Tvr 65</td><td>•Ser</td><td>I VQ — J</td><td>Leu</td><td>Thr</td><td>Va! 70</td><td>Phe</td><td>cys</td><td>Pr o</td><td>Ar π • “ 55</td><td>Tr n r^- 75</td><td>Leu</td><td>Gl v ~~ J</td><td>Gl v J</td><td>.Asn</td><td>Val 80</td>

<td>Phe</td><td>Ser</td><td>cys</td><td>Ser</td><td>Val 85</td><td>Nbt</td><td>H s</td><td>α u</td><td>Al a</td><td>Leu 90</td><td>Hi s</td><td>Asn</td><td>H s</td><td>Tyr</td><td>Thr 95</td><td>G n</td>

<td>Lys</td><td>Ser</td><td>Leu</td><td>Ser 100</td><td>Leu</td><td>Ser</td><td>Pr o</td><td>Qy</td><td>Lys 105</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<210> 39 <211 > 315

127 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 39 ccccgagaac cacaggtgta caccctgccc ccatcccgtg acgagctctt gccct gccaa gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagt gggag agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ct ccgacggc tctttcttcc tctacagcaa gcttaccgtg ttctgcccca ggt ggct ggg ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacacagaa gagcctctcc ctgtctccgg gtaaa <210> 40 <211> 105 <212> PRT <213> Sztuczna <220>

<223> Sekwencja sztuczna

<td> &lt;400:</td><td> &gt;40</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Pr o</td><td rowspan="2">Arg</td><td>G u</td><td>Pro</td><td>G n</td><td>Val</td><td rowspan="2">Tyr</td><td>Thr</td><td>Leu</td><td>Pro</td><td>Pr o</td><td>Ser</td><td rowspan="2">Arg</td><td>Asp</td><td>G u</td><td>Leu</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Thr</td><td rowspan="2">Lys</td><td>Asn</td><td>G n</td><td>Val</td><td>Ser</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Leu</td><td>Val</td><td rowspan="2">Lys</td><td>gi y</td><td>Phe</td><td rowspan="2">Tyr</td><td>Pr o</td>

<td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td> 30</td><td></td>

<td>Ser</td><td rowspan="2">Asp</td><td>11 e</td><td>Al a</td><td>Val</td><td>G u</td><td rowspan="2">Trp</td><td>G u</td><td>Ser</td><td>Asn</td><td rowspan="2">Gy</td><td>G n</td><td>Pr o</td><td>G u</td><td>Asn</td><td>Asn</td>

<td></td><td> 35</td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td rowspan="2">Tyr</td><td>Lys</td><td>Thr</td><td>Thr</td><td>Pr o</td><td>Pr o</td><td>Val</td><td>Leu</td><td rowspan="2">Asp</td><td>Ser</td><td rowspan="2">Asp</td><td>ay</td><td>Ser</td><td>Phe</td><td>Phe</td><td>Leu</td>

<td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Tyr</td><td>Ser</td><td rowspan="2">Lys</td><td>Leu</td><td>Thr</td><td>Val</td><td>Pr o</td><td rowspan="2">Cys</td><td>rvbt</td><td rowspan="2">Arg</td><td>Trp</td><td rowspan="2">Trp</td><td rowspan="2">ay</td><td rowspan="2">Gly</td><td>Asn</td><td>Val</td>

<td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td> 75</td><td></td><td> 80</td>

<td>Phe</td><td>Ser</td><td rowspan="2">Cys</td><td>Ser</td><td>Val</td><td>lyfet</td><td>H s</td><td>G u</td><td>Al a</td><td>Leu</td><td>H s</td><td>Asn</td><td>H s</td><td rowspan="2">Tyr</td><td>Thr</td><td>G n</td>

<td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Lys</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Pr o</td><td>Gy</td><td>Lys</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

100 105 <210> 41

128 <211 > 315 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 41 ccccgagaac gt cagcct ga agcaat gggc t cct t ct t cc 11 ct cat gct ct gt ct ccgg cacaggt gt a cctgcctggt agccggagaa t ct acagcaa ccgt gat gca gt aaa caccct gccc caaaggct t c caact acaag gett accgtg tgaggctctg ccat cccggg t at cccagcg accacgcct c ccct gcat ga cacaaccact at gaget gac acat cgccgt ccgt gct gga ggt ggt gggg acacacagaa caagaaccag ggagt gggag ct ccgacggc cgggaacgtc gagcct ct cc <210> 42 <211> 115 <212> PRT <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 42

129

<td>Arg</td><td rowspan="2">Arg</td><td>G u</td><td>Pr o</td><td>G n</td><td>Val</td><td rowspan="2">Tyr</td><td>Thr</td><td>Leu</td><td>Pr o</td><td>Pr o</td><td>Ser</td><td>Arg</td><td>Asp</td><td>G u</td><td>Leu</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Val</td><td>Leu</td><td>G y</td><td>G n</td><td>Val</td><td>Ser</td><td>Leu</td><td>Al a</td><td>Cys</td><td>Leu</td><td>Val</td><td>Lys</td><td>Qy</td><td>Phe</td><td>Val</td><td>Val</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td rowspan="2">Arg</td><td>Leu</td><td>I I e</td><td>Al a</td><td>Val</td><td>G u</td><td rowspan="2">Trp</td><td>G u</td><td>Ser</td><td>Asn</td><td rowspan="2">Gy</td><td>G n</td><td>Pr o</td><td>G u</td><td>Asn</td><td>Asn</td>

<td></td><td> 35</td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td rowspan="2">Tyr</td><td>Lys</td><td>Thr</td><td>Thr</td><td>Pr o</td><td>Pr o</td><td>Val</td><td>Leu</td><td>Asp</td><td>Ser</td><td>Asp</td><td>Qy</td><td>Arg</td><td>G n</td><td>Leu</td><td>Al a</td>

<td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Asp</td><td>Ser</td><td>Phe</td><td>Phe</td><td>Leu</td><td>Tyr</td><td>Ser</td><td rowspan="2">Lys</td><td>Leu</td><td>Thr</td><td>Val</td><td>Pr o</td><td>Pr o</td><td>Arg</td><td>Leu</td><td>Lys</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td rowspan="2">Gy</td><td rowspan="2">Trp</td><td>Pr o</td><td rowspan="2">Arg</td><td>Trp</td><td rowspan="2">Qy</td><td rowspan="2">Trp</td><td>G y</td><td>Asn</td><td>Val</td><td>Phe</td><td>Ser</td><td>Cys</td><td>Ser</td><td>Val</td><td>IVfet</td>

<td></td><td> 85</td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Phe</td><td>Leu</td><td>Al a</td><td>Leu</td><td>hi s</td><td>Asn</td><td>H s</td><td rowspan="2">Tyr</td><td>Thr</td><td>G n</td><td rowspan="2">Lys</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Leu</td><td>Ser</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

Pro G y Lys 115 <210> 43 <211> 345 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 43 cggcgagaac gt cagcct gg agcaat gggc cggcagt t gg nnrtnnrnna cacaaccact cacaggt gt a cct gcct cgt agccggagaa cggact cct t nnłnnnnntn

55*· 55 5555 55* ' 55 acacacagaa caccct gccc gaaaggct t c caact acaag ct t cct ct ac nnfiriaarnt n

53 53 5553 * ~ gagcct ct cc ccat cccgt g gt ggt ccggt accacgcct c agcaagct t a 11 ct cat gca ctgtctccgg acgagct cgt t gat cgccgt ccgt t ct aga ccgt gccccc gt gt gat gt t gt aaa cttggggcaa ggagt gggag ct ccgacggc gcggt t gaag cct ggcgct g

120

180

240

300

345 <210> 44 <211 >238 <212> PRT <213> Sztuczna <220>

<223> Sekwencja sztuczna

130

<td colspan="16"> &lt;400&gt; 44</td>

<td>G u</td><td>Val</td><td>G n</td><td>Leu</td><td>Val</td><td>Q u</td><td>Ser</td><td>Gy</td><td>ay</td><td>gi y</td><td>Leu</td><td>Val</td><td>G n</td><td>Pr o</td><td>Giy</td><td>Arg</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Ser</td><td>Leu</td><td>Ar g</td><td>Leu</td><td>Ser</td><td>Cys</td><td>Al a</td><td>Al a</td><td>Ser</td><td>Gi y</td><td>Phe</td><td>Thr</td><td>Phe</td><td>Asn</td><td>Asp</td><td>Tyr</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Al a</td><td>Nfet</td><td>hi s</td><td>Trp</td><td>Val</td><td>Arg</td><td>G n</td><td>Al a</td><td>Pr o</td><td>Gly</td><td>Lys</td><td>Giy</td><td>Leu</td><td>G u</td><td>Trp</td><td>Val</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Ser</td><td>Gy</td><td>I I e</td><td>Ser</td><td>Trp</td><td>Asp</td><td>Ser</td><td>Ser</td><td>Ser</td><td>11 e</td><td>Gly</td><td>Tyr</td><td>Al a</td><td>Asp</td><td>Ser</td><td>Val</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Lys</td><td>Gy</td><td>Arg</td><td>Phe</td><td>Thr</td><td>I I e</td><td>Ser</td><td>Arg</td><td>Asp</td><td>Asn</td><td>Al a</td><td>Lys</td><td>Asn</td><td>Ser</td><td>Leu</td><td>Tyr</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Leu</td><td>G n</td><td>Mit</td><td>Asn</td><td>Ser</td><td>Leu</td><td>Arg</td><td>Al a</td><td>G u</td><td>Asp</td><td>Mit</td><td>Al a</td><td>Leu</td><td>Tyr</td><td>Tyr</td><td>cys</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Val</td><td>Lys</td><td>Gy</td><td>Arg</td><td>Asp</td><td>Tyr</td><td>Tyr</td><td>Asp</td><td>Ser</td><td>ay</td><td>Gly</td><td>Tyr</td><td>Phe</td><td>Thr</td><td>Val</td><td>Al a</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Phe</td><td>Asp</td><td>I I e</td><td>Trp</td><td>Gy</td><td>G n</td><td>Q y</td><td>Thr</td><td>Mit</td><td>Val</td><td>Thr</td><td>Val</td><td>Ser</td><td>Ser</td><td>Al a</td><td>Ser</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Thr</td><td>Lys</td><td>Gy</td><td>Pr o</td><td>G n</td><td>Val</td><td>Tyr</td><td>Thr</td><td>Leu</td><td>Pro</td><td>Pro</td><td>Ser</td><td>Arg</td><td>Asp</td><td>G u</td><td>Leu</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Val</td><td>Leu</td><td>Gy</td><td>G n</td><td>Val</td><td>Ser</td><td>Pr o</td><td>Thr</td><td>Cys</td><td>Leu</td><td>Val</td><td>Lys</td><td>Giy</td><td>Phe</td><td>Tyr</td><td>Pro</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Ser</td><td>Asp</td><td>I I e</td><td>Al a</td><td>Val</td><td>G u</td><td>Trp</td><td>G u</td><td>Ser</td><td>Asn</td><td>GI y</td><td>G n</td><td>Pr o</td><td>G u</td><td>Asn</td><td>Asn</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Tyr</td><td>Lys</td><td>Thr</td><td>Thr</td><td>Pr o</td><td>Pr o</td><td>Val</td><td>Leu</td><td>Asp</td><td>Ser</td><td>Asp</td><td>Gly</td><td>Ser</td><td>Phe</td><td>Phe</td><td>Leu</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Tyr</td><td>Gy</td><td>Lys</td><td>Leu</td><td>Thr</td><td>Val</td><td>Pr o</td><td>Pr o</td><td>Arg</td><td>Leu</td><td>Lys</td><td>Gy</td><td>Trp</td><td>Pr o</td><td>Arg</td><td>Trp</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Gy</td><td>Trp</td><td>Gy</td><td>Asn</td><td>Val</td><td>Phe</td><td>Ser</td><td>Cys</td><td>Ser</td><td>Val</td><td>Mit</td><td>hi s</td><td>G u</td><td>Al a</td><td>Leu</td><td>H s</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Asn</td><td>hi s</td><td>Tyr</td><td>Thr</td><td>G n</td><td>Lys</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Pro</td><td>Gly</td><td>Lys</td><td></td><td></td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td></td>

<210> 45 <211> 714 <212> DNA <213> Sztuczna

131 <220>

<223> Sekwencja sztuczna <400> 45 gaagt gcagc tcct gt gcag ccagggaagg gcggactctg ct gcaaat ga gat t act at g at ggt caccg cgtgacgagc agcgacat cg cct cccgt gc cggt t gaagg gaggct ct gc t ggt ggagt c cct ct ggat t gcct ggagt g t gaagggccg acagt ct gag at agt ggt gg t ct ct t cagc t cgt ct t ggg ccgt ggagt g t ggact ccga gct ggccgag acaaccact a tgggggaggc cacct 11 aat ggtctcaggt at t caccat c agct gaggac 11 at 11 cacg ct ccaccaag gcaagt cagc ggagagcaat cggct cct t c gt ggggct gg cacacagaag ttggtacagc gat t at gcca at aagt t ggg t ccagagaca at ggcct t at gttgcttttg ggcccacagg ccgacct gcc gggcagccgg 11 cct ct acg gggaacgtct agcct ct ccc ctggcaggtc tgcactgggt at agt agt ag acgccaagaa at t act gt gt at at ctgggg t gt acaccct t ggt caaagg agaacaact a gcaagct t ac t ct cat gct c t gt ct ccggg cct gagact c ccggcaagct t at aggct at ct ccct gt at aaaaggcaga ccaagggaca gcccccat cc ct t ct at ccc caagaccacg cgt gcccccg cgt gat gcat t aaa <210> 46 <211 >217 <212> PRT <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 46

132

<td>Asp 1</td><td>11 e</td><td>G n</td><td>IVfet</td><td>Thr 5</td><td colspan="2">G n Ser</td><td colspan="2">Pro Ser</td><td>Thr 10</td><td>Leu</td><td>Ser</td><td>Al a</td><td>Ser</td><td>Val 15</td><td>ay</td>

<td rowspan="2">Asp</td><td rowspan="2">Arg</td><td>Val</td><td>Thr</td><td>I I e</td><td>Thr</td><td rowspan="2">cys</td><td rowspan="2">Arg</td><td>Al a</td><td>Ser</td><td>G n</td><td>Ser</td><td>I I e</td><td>Ser</td><td>Arg</td><td>Trp</td>

<td></td><td> 20</td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Leu</td><td>Al a</td><td>Trp</td><td rowspan="2">Tyr</td><td>G n</td><td>G n</td><td rowspan="2">Lys</td><td>Pro</td><td>ay</td><td>Lys</td><td>Val</td><td>Pro</td><td>Lys</td><td>Leu</td><td>Leu</td><td>11 e</td>

<td></td><td></td><td> 35</td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td rowspan="2">Tyr</td><td>Lys</td><td>Al a</td><td>Ser</td><td>Ser</td><td>Leu</td><td>G u</td><td>Ser</td><td rowspan="2">ay</td><td>Val</td><td>Pro</td><td>Ser</td><td>Arg</td><td>Phe</td><td>Ser</td><td>ay</td>

<td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Ser</td><td rowspan="2">α y</td><td>Ser</td><td>a y</td><td>Thr</td><td>G u</td><td>Phe</td><td>Thr</td><td>Leu</td><td>Thr</td><td>11 e</td><td>Ser</td><td>Ser</td><td>Leu</td><td>G n</td><td>Pr o</td>

<td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td rowspan="2">Asp</td><td rowspan="2">Asp</td><td>Phe</td><td>Al a</td><td>Thr</td><td rowspan="2">Tyr</td><td>Tyr</td><td>cys</td><td>G n</td><td>G n</td><td>Tyr</td><td>Asn</td><td>Ser</td><td>Tyr</td><td>Ser</td><td>Phe</td>

<td></td><td></td><td> 85</td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td rowspan="2">ay</td><td>Pr o</td><td rowspan="2">ay</td><td>Thr</td><td rowspan="2">Lys</td><td>Val</td><td rowspan="2">Asp</td><td>11 e</td><td>Lys</td><td rowspan="2">Arg</td><td>Thr</td><td>Val</td><td>Al a</td><td>G u</td><td>Pr o</td><td>G n</td>

<td></td><td> 100</td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Val</td><td rowspan="2">Tyr</td><td>Thr</td><td>Leu</td><td>Pro</td><td>Pr o</td><td>Ser</td><td>Arg</td><td rowspan="2">Asp</td><td>G u</td><td>Leu</td><td>Val</td><td>Leu</td><td>ay</td><td>G n</td><td>Val</td>

<td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Ser</td><td>Pr o</td><td>Thr</td><td rowspan="2">Cys</td><td>Leu</td><td>Val</td><td>Lys</td><td>ay</td><td>Phe</td><td rowspan="2">Tyr</td><td>Pr o</td><td>Ser</td><td>Asp</td><td>I I e</td><td>Al a</td><td>Val</td>

<td></td><td> 130</td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>G u</td><td rowspan="2">Trp</td><td>G u</td><td>Ser</td><td>Asn</td><td>Qy</td><td>G</td><td>n</td><td>Pro</td><td>G u</td><td>Asn</td><td>Asn</td><td rowspan="2">Tyr</td><td rowspan="2">Lys</td><td>Thr</td><td>Thr</td><td>Pr o</td>

<td> 145</td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td> 160</td>

<td>Pro</td><td>Val</td><td>Leu</td><td rowspan="2">Asp</td><td>Ser</td><td rowspan="2">Asp</td><td>a</td><td>y</td><td>Ser</td><td>Phe</td><td>Phe</td><td>Leu</td><td rowspan="2">Tyr</td><td rowspan="2">ay</td><td rowspan="2">Lys</td><td>Leu</td><td>Thr</td>

<td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td> 175</td><td></td>

<td>Val</td><td>Pr o</td><td>Pro</td><td>Arg</td><td>Leu</td><td rowspan="2">Lys</td><td>a</td><td>y</td><td rowspan="2">Trp</td><td>Pr o</td><td rowspan="2">Arg</td><td rowspan="2">Trp</td><td>ay</td><td rowspan="2">Trp</td><td>ay</td><td>Asn</td><td>Val</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td> 185</td><td></td><td> 190</td><td></td><td></td>

<td>Phe</td><td>Ser</td><td>cys</td><td>Ser</td><td>Val</td><td>IVbt</td><td>H</td><td>s</td><td>a u</td><td>Al a</td><td>Leu</td><td>H S</td><td>Asn</td><td>H s</td><td rowspan="2">Tyr</td><td>Thr</td><td>G n</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td>

<td>Lys</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Pr</td><td> 0</td><td>ay</td><td>Lys</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

210 215 <210> 47 <211 > 651 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna

133 <400> 47 gacat ccaga at cact t gcc gggaaagt cc aggttcagcg gatgattttg aaagt ggat a gacgagct cg yaCai CyCCy cccgt gctgg 11 gaagggct gct ct gcaca t gacccagt c gggccagt ca ct aagct cct gcagt ggat c caact t at t a t caaacgaac tcttggggca 199θ9^{999^aact ccgacgg ggccgaggt g accact acac t cct t ccacc gagt at t agt gat ct at aag tgggacagaa ct gccaacag tgtggctgaa agt cagcccg gagcaatggg ct cct t ct t c gggct ggggg acagaagagc ct gt ct gcat aggt ggt t gg gcat ct agt t 11 cact ct ca t at aat agt t ccacaggt gt acct gcct gg

CayCCyyaya ct ct acggca aacgt ct t ct ct ct ccct gt ct gt aggaga cct ggt at ca t agaaagt gg ccat cagcag at t ct 11 cgg acaccct gcc t caaaggct t aCaaCt 3C33 agct t accgt cat gct ccgt ct ccgggt aa cagagt cacc gcagaaacca ggt cccat ca cct gcagcct ccct gggacc cccat cccgt ct at cccagc yaCCaCyCCi gcccccgcgg gat gcat gag a

<210> 48 <211> 129 <212> PRT <213> Sztuczna <220>

<223> Sekwencja sztuczna <220>

<221 > misc_feature <222> (35)..(36) <223> Xaa mo that it can be any amino acid found in nature <220>

<221> misc_feature

134 <222> (38) .. (42) <223> Xaa can be any amino acid found in nature <220>

<221> rri sc_f eat ur e <222> (95) .. (98) <223> Xaa can be any amino acid found in nature <400> 48

<td>h / fet 1</td><td>lys</td><td>Tyr</td><td>Leu</td><td>Leu 5</td><td>Pr</td><td>Thr</td><td>Al a</td><td>Al a</td><td>Al a 10</td><td>gy</td><td>Leu</td><td>Leu</td><td>Leu</td><td>Leu 15</td><td>Al a</td>

<td>Al a</td><td>G n</td><td>Pr</td><td>Al a 20</td><td>IVfet</td><td>Al a</td><td>val</td><td>Al a</td><td>Al a 25</td><td>Pro</td><td>Cheese</td><td>val</td><td>phe</td><td>11 e 30</td><td>phe</td><td>Pr</td>

<td>Pr</td><td>Cheese</td><td>Xaa 35</td><td>Xaa</td><td>G n</td><td>Xaa</td><td>Xaa</td><td>Xaa 40</td><td>Xaa</td><td>Xaa</td><td>Al a</td><td>Cheese</td><td>val 45</td><td>val</td><td>cys</td><td>Leu</td>

<td>Leu</td><td>own 50</td><td>own</td><td>phe</td><td>Tyr</td><td>Pr</td><td>Arg 55</td><td>G u</td><td>Al a</td><td>lys</td><td>val</td><td>G n 60</td><td>Trp</td><td>lys</td><td>val</td><td>Asp</td>

<td>own 65</td><td>Al a</td><td>Leu</td><td>G n</td><td>Cheese</td><td>gy 70</td><td>own</td><td>Cheese</td><td>G n</td><td>G u</td><td>Cheese 75</td><td>val</td><td>Thr</td><td>G u</td><td>G n</td><td>Asp 80</td>

<td>Cheese</td><td>lys</td><td>Asp</td><td>Cheese</td><td>Thr 85</td><td>Tyr</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Ser 90</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Leu</td><td>Xaa 95</td><td>Xaa</td>

<td>Xaa</td><td>Xaa</td><td>Tyr</td><td>G u 100</td><td>Lys</td><td>H s</td><td>Lys</td><td>Val</td><td>Tyr 105</td><td>Al a</td><td>cys</td><td>G u</td><td>Val</td><td>Thr 110</td><td>H s</td><td>G n</td>

<td colspan="5">G y Leu Ser Ser Pr o 115 Al a &lt;210&gt; 49 &lt;211 &gt;387 &lt;212&gt; DNA &lt;213&gt; Sztuczna &lt;220&gt; &lt;223&gt; Sekwencja sztuczna</td><td>Val</td><td>Thr</td><td>Lys 120</td><td>Ser</td><td>Phe</td><td>Asn</td><td>Arg</td><td>Qy 125</td><td>G u</td><td>Al a</td><td>Al a</td>

<220>

<221 > misc_feature <222> (103)..(104) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (106)..(107) <223> n is a, c, g, ort <220>

135 <221 > misc_feature <222> (112)..(113) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (115)..(116) <223> n i s a, c, g, ort <220>

<221 > misc_feature <222> (118)..(119) <223> n i s a, c, g, ort <220>

<221 > misc_feature <222> (121)..(122) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (124)..(125) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (283)..(284) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (286)..(287) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (289)..(290) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (292)..(293) <223> n is a, c, g, ort <400> 49 at gaaat acc at ggccgtgg nnsnnsgcct tggaaggt gg agcaaggaca aaacacaaag agct t caaca t at t gcct ac ct gcaccat c ctgttgt gtg at aacgccct gcacct acag t ct acgcct g ggggagaggc ggcagccgct t gt ct t cat c cct gct gaat ccaat cgggt cct cagcagc cgaagt cacc ggccgca ggat t gt t at 11 cccgccat aact t ct at c aact cccagg accct gacgt cat cagggcc t act cgcggc ct nnsnnsca ccagagaggc agagt gt cac t gnnsnnsnn t gagct cgcc ccagccggcc gnnsnnsnns caaagt acag agagcaggac snnst acgag cgt cacaaag

120

180

240

300

360

387

136 <210> 50 <211> 132 <212> PRT <213> Sztuczna <220>

<223> Sekwencja sztuczna <220>

<221 > misc_feature <222> (35).. (36)

<td colspan="15">&lt;223&gt; Xaa mo that any amino acid found in nature</td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2">&lt;221&gt; misc_</td><td>feature</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="3"> &lt;222&gt; (38)..(42)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="12">&lt;223&gt; Xaa can be any naturally occurring amino acid</td><td></td><td></td><td></td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2">&lt;221&gt; misc_</td><td>feature</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="3"> &lt;222&gt; (95)..(101)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="12">&lt;223&gt; Xaa can be any naturally occurring amino acid</td><td></td><td></td><td></td>

<td colspan="2"> &lt;400&gt; 50</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Myth</td><td rowspan="2">lys</td><td rowspan="2">Tyr</td><td>Leu</td><td>Leu</td><td>Pro</td><td>Thr</td><td>Al a</td><td>Al a</td><td>Al a G y</td><td>Leu</td><td>Leu</td><td>Leu</td><td>Leu</td><td>Al a</td>

<td> 1</td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Al a</td><td>G n</td><td>Pr</td><td>Al a</td><td>Myth</td><td>Al a</td><td>val</td><td>Al a</td><td>Al a</td><td>Pro Ser</td><td>val</td><td>phe</td><td>II e</td><td>phe</td><td>Pro</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Pr</td><td>Cheese</td><td>Xaa</td><td>Xaa</td><td>G n</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa Al a</td><td>Cheese</td><td>val</td><td>val</td><td rowspan="2">Cys</td><td>Leu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td> 45</td><td></td><td></td>

<td>Leu</td><td>own</td><td>own</td><td>phe</td><td rowspan="2">Tyr</td><td>Pr</td><td>Arg</td><td>G u</td><td>Al a</td><td>Lys Val</td><td>G n</td><td rowspan="2">Trp</td><td rowspan="2">lys</td><td>val</td><td rowspan="2">Asp</td>

<td></td><td> 50</td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td> 60</td><td></td>

<td>own</td><td>Al a</td><td>Leu</td><td>G n</td><td>Cheese</td><td>Gly</td><td>own</td><td>Cheese</td><td>G n</td><td>G u Ser</td><td>val</td><td>Thr</td><td>G u</td><td>G n</td><td>Asp</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Cheese</td><td rowspan="2">lys</td><td rowspan="2">Asp</td><td>Cheese</td><td>Thr</td><td rowspan="2">Tyr</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Ser Thr</td><td>Leu</td><td>Thr</td><td>Leu</td><td>Xaa</td><td>Xaa</td>

<td></td><td></td><td> 85</td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Xaa</td><td>Xaa</td><td rowspan="2">Xaa</td><td>Xaa</td><td>Xaa</td><td>Tyr</td><td>G u</td><td rowspan="2">1 . . Λ i_yo</td><td>i· 5</td><td rowspan="2">1 , . Λ \ /« 1 Lyo ναι</td><td>Tyr</td><td rowspan="2">Al a</td><td>Cy 3</td><td rowspan="2">G u</td><td rowspan="2">Val</td>

<td></td><td></td><td> 100</td><td></td><td></td><td></td><td> 105</td><td></td><td> 110</td>

<td>Thr</td><td>H s</td><td>G n</td><td rowspan="2">ay</td><td>Leu</td><td>Ser</td><td>Ser</td><td>Pr o</td><td>Val</td><td rowspan="2">Thr Lys</td><td>Ser</td><td>Phe</td><td>Asn</td><td rowspan="2">Arg</td><td rowspan="2">Gly</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td> 125</td><td></td>

<td>G u</td><td>Al a</td><td>Al a</td><td>Al a</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<210> 51 <211 >396

130

137 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <220>

<221 > misc_feature <222> (103)..(104) <223> η is a, c, g, ort <220>

<221 > misc_feature <222> (106)..(107) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (112)..(113) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (115)..(116) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (118)..(119) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (121)..(122) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (124)..(125) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (283)..(284) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (286)..(287) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (289)..(290) <223> n is a, c, g, ort <220>

<221 > misc_feature

138 <222> (292)..(293) <223> η is a, c, g, ort <220>

<221 > misc_feature <222> (295)..(296) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (298)..(299) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (301)..(302) <223> n is a, c, g, ort <400> 51 at gaaat acc at ggccgt gg nnsnnsgcct tggaaggtgg agcaaggaca nnst acgaga gt cacaaaga t at t gcct ac ct gcaccat c ct gt t gt gt g at aacgccct gcacct acag aacacaaagt gct t caacag ggcagccgct t gt ct t cat c cct gct gaat ccaat cgggt cct cagcagc ct acgcct gc ggat t gt t at 11 cccgccat aact t ct at c aact cccagg accct gacgt gaagt caccc gggagaggcg gccgca t act cgcggc ct nnsnnsca ccagagaggc agagt gt cac t gnnsnnsnn at cagggcct ccagccggcc gnnsnnsnns caaagt acag agagcaggac snnsnnsnns gagct cgccc <210> 52 <211> 134 <212> PRT <213> Sztuczna <220>

<223> Sekwencja sztuczna <220>

<221 > misc_feature <222> (35).. (36) <223> Xaa mo that it can be any amino acid found in nature <220>

<221> misc_feature <222> (38) .. (42) <223> Xaa can be any amino acid found in nature <220>

<221> misc_feature <222> (95) .. (103) <223> Xaa can be any amino acid found in nature

139 <400> 52

<td>fyfet 1</td><td>lys</td><td>Tyr</td><td>Leu</td><td>Leu 5</td><td>Pr</td><td>Thr</td><td>Al a</td><td>Al a</td><td>Al a 10</td><td>gy</td><td>Leu</td><td>Leu</td><td>Leu</td><td>Leu 15</td><td>Al a</td>

<td>AND! 3</td><td>Q n</td><td>Pr</td><td>AND! a 20 "</td><td></td><td>AND! and</td><td>Va!</td><td>AND! and</td><td>Al a 25 "</td><td>Pr</td><td>Cheese</td><td>Va!</td><td>phe</td><td>2nd floor</td><td>phe</td><td>Pr</td>

<td>Pr</td><td>Cheese</td><td>Xaa 35</td><td>Xaa</td><td>G n</td><td>Xaa</td><td>Xaa</td><td>Xaa 40</td><td>Xaa</td><td>Xaa</td><td>Al a</td><td>Cheese</td><td>val 45</td><td>val</td><td>cys</td><td>Leu</td>

<td>Leu</td><td>own 50</td><td>own</td><td>phe</td><td>Tyr</td><td>Pr</td><td>Arg 55</td><td>G u</td><td>Al a</td><td>lys</td><td>val</td><td>G n 60</td><td>Trp</td><td>lys</td><td>val</td><td>Asp</td>

<td>own 65</td><td>Al a</td><td>Leu</td><td>G n</td><td>Cheese</td><td>gy 70</td><td>own</td><td>Cheese</td><td>G n</td><td>G u</td><td>Cheese 75</td><td>val</td><td>Thr</td><td>G u</td><td>G n</td><td>Asp 80</td>

<td>Cheese</td><td>lys</td><td>Asp</td><td>Cheese</td><td>Thr 85</td><td>Tyr</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Ser 90</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Leu</td><td>Xaa 95</td><td>Xaa</td>

<td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa 100</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Tyr</td><td>G u 105</td><td>Lys</td><td>H S</td><td>Lys</td><td>Val</td><td>Tyr 110</td><td>Al a</td><td>cys</td>

<td>G u</td><td>Val</td><td>Thr 115</td><td>H s</td><td>G n</td><td>Gy</td><td>Leu</td><td>Ser 120</td><td>Ser</td><td>Pr o</td><td>Val</td><td>Thr</td><td>Lys 125</td><td>Ser</td><td>Phe</td><td>Asn</td>

<td>Arg</td><td>Gy 130</td><td>G u</td><td>Al a</td><td>Al a</td><td>Al a</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<210> 53 <211 >402 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <220>

<221 > misc_feature <222> (103)..(104) <223> η is a, c, g, ort <220>

<221 > misc_feature <222> (106)..(107) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (112)..(113) <223> n is a, c, g, ort <220>

140 <220>

<221 > misc_feature <222> (307)..(308) <223> η is a, c, g, ort <400> 53

<td>at gaaat acc</td><td>t at t gcct ac</td><td>ggcagccgct</td><td>ggat t gt t at</td><td>t act cgcggc</td><td>ccagccggcc</td><td> 60</td>

<td>at ggccgt gg</td><td>ct gcaccat c</td><td>t gt ct t cat c</td><td>11 cccgccat</td><td>ct nnsnnsca</td><td>gnnsnnsnns</td><td> 120</td>

<td>nnsnnsgcct</td><td>ctgttgt gtg</td><td>cct gct gaat</td><td>aact t ct at c</td><td>ccagagaggc</td><td>caaagt acag</td><td> 180</td>

<td>t ggaaggt gg</td><td>at aacgccct</td><td>ccaat cgggt</td><td>aact cccagg</td><td>agagt gt cac</td><td>agagcaggac</td><td> 240</td>

<td>agcaaggaca</td><td>gcacct acag</td><td>cct cagcagc</td><td>accct gacgt</td><td>t gnnsnnsnn</td><td>snnsnnsnns</td><td> 300</td>

<td>nnsnnsnnst</td><td>acgagaaaca</td><td>caaagt ct ac</td><td>gcct gcgaag</td><td>t cacccat ca</td><td>gggcct gagc</td><td> 360</td>

<td>t cgcccgt ca</td><td>caaagagct t</td><td>caacagggga</td><td>gaggcggccg</td><td>ca</td><td></td><td> 402</td>

<td> &lt;210&gt; 54</td><td></td><td></td><td></td><td></td><td></td><td></td>

<211>127 <212> PRT <213> Sztuczna <220>

<223> Sekwencja sztuczna <220>

<221 > misc_feature <222> (38)..(45) <223> Xaa może być dowolnym aminokwasem występującym w naturze <220>

<221 > misc_feature <222> (95).. (101) <223> Xaa może być dowolnym aminokwasem występującym w naturze <400> 54

<td>fyfet 1</td><td>Lys</td><td>Tyr</td><td>Leu</td><td>Leu 5</td><td>Pr o</td><td>Thr</td><td>Al a</td><td>Al a</td><td>Al a 10</td><td>Gly</td><td>Leu</td><td>Leu</td><td>Leu</td><td>Leu 15</td><td>Al a</td>

<td>Al a</td><td>Gl n</td><td>Pr o</td><td>Al a 20</td><td>tvbt</td><td>Al a</td><td>Al a</td><td>Ser</td><td>Thr 25</td><td>Lys</td><td>ay</td><td>Pro</td><td>Ser</td><td>Val 30</td><td>Phe</td><td>Pro</td>

<td>Leu</td><td>Al a</td><td>Pr o 35</td><td>Ser</td><td>Ser</td><td>Xaa</td><td>Xaa</td><td>Xaa 40</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa 45</td><td>Al a</td><td>Leu</td><td>Gy</td>

<td>Cys</td><td>Leu 50</td><td>Val</td><td>Lys</td><td>Asp</td><td>Tyr</td><td>Phe 55</td><td>Pr o</td><td>G u</td><td>Pr o</td><td>Val</td><td>Thr 60</td><td>Val</td><td>Ser</td><td>Trp</td><td>Asn</td>

<td>Ser 65</td><td>Gy</td><td>Al a</td><td>Leu</td><td>Thr</td><td>Ser 70</td><td>ay</td><td>Val</td><td>H s</td><td>Thr</td><td>Phe 75</td><td>Pro</td><td>Al a</td><td>Val</td><td>Leu</td><td>G n 80</td>

141

<td>Ser</td><td>Ser</td><td>Qy</td><td>Leu</td><td>Tyr 85</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Ser</td><td>Val 90</td><td>Val</td><td>Thr</td><td>Val</td><td>Pr o</td><td>Xaa 95</td><td>Xaa</td>

<td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Thr</td><td rowspan="2">Tyr</td><td>I I e</td><td>cys</td><td>Asn</td><td>Val</td><td>Asn</td><td>H s</td><td>Lys</td><td>Pr o</td><td>Ser</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Asn</td><td>Thr</td><td>Lys</td><td>Val</td><td>Asp</td><td>Lys</td><td>Lys</td><td>Val</td><td>G u</td><td>Pr o</td><td>Lys</td><td>Ser</td><td>Al a</td><td>Al a</td><td>Al a</td><td></td>

115 120 125 <210> 55 <211 >381 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <220>

<221 > misc_feature <222> (112)..(113) <223> η is a, c, g, ort <220>

<221 > misc_feature <222> (115)..(116) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (118)..(119) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (121)..(122) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (124)..(125) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (127)..(128) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (130)..(131) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (133)..(134) <223> n is a, c, g, ort

142 <220>

<221 > misc_feature <222> (283)..(284) <223> η is a, c, g, ort <220>

<221 > misc_feature <222> (286) .. (287) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (289)..(290) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (292)..(293) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (295)..(296) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (298)..(299) <223> n is a, c, g, ort <220>

<221 > misc_feature

<td></td><td> &lt;222&gt; (301)..</td><td> (302)</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>&lt;223&gt; n is a,</td><td>c, g, ort</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;400&gt; 55</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>at gaaat acc</td><td>t at t gcct ac</td><td>ggcagccgct</td><td>ggat t gt t at</td><td>t act cgcggc</td><td>ccagccggcc</td><td> 60</td>

<td> 40</td><td>at ggccgcct</td><td>ccaccaaggg</td><td>cccat cggt c</td><td>11 ccccct gg</td><td>caccct cct c</td><td>cnnsnnsnns</td><td> 120</td>

<td></td><td>nnsnnsnnsn</td><td>nsnnsgccct</td><td>gggct gcct g</td><td>gt caaggact</td><td>act t ccccga</td><td>accggt gacg</td><td> 180</td>

<td></td><td>gt gt cgt gga</td><td>act caggcgc</td><td>cct gaccagc</td><td>ggcgt gcaca</td><td>cct t cccggc</td><td>t gt cct acag</td><td> 240</td>

<td> 45</td><td>t cct caggac</td><td>t ct act ccct</td><td>cagcagcgt g</td><td>gtgaccgt gc</td><td>ccnnsnnsnn</td><td>snnsnnsnns</td><td> 300</td>

<td></td><td></td><td></td><td></td><td>/"»/&gt;/&gt; O &lt;1 o o /* o</td><td>nnoonnł nno</td><td>noonooonf ł</td><td> 360</td>

<td></td><td>nnsacciaca</td><td>iCiyCaSCyi</td><td>gaatcacaag</td><td></td><td>uuaayyi</td><td></td><td></td>

<td></td><td>gagcccaaat</td><td>ct gcggccgc</td><td>a</td><td></td><td></td><td></td><td> 381</td>

<210> 56 <211> 87 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna

143 <220>

<221 > misc_feature <222> (49)..(50) <223> η is a, c, g, ort <220>

<221 > misc_feature <222> (52)..(53) <223> n i s a, c, g, ort <220>

<221 > misc_feature <222> (58).. (59) <223> n i s a, c, g, ort <220>

<221 > misc_feature <222> (61).. (62) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (64).. (65) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (67)..(68) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (70)..(71) <223> n is a, c, g, ort <400> 56 cttaccatgg ccgt ggct gc accatctgtc ttcatcttcc cgccatctnn snnscagnns 60 nnsnnsnnsn nsgcctctgt tgtgtgc 87 <210> 57 <211 >26 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 57 tgacaacgtc agggt gct gc t gaggc 26 <210> 58 <211 >41 <212> DNA <213> Sztuczna

144 <220>

<223> Sekwencja sztuczna <220>

<221 > misc_feature <222> (12)..(13) <223> η is a, c, g, ort <220>

<221 > misc_feature <222> (15)..(16) <223> n is a, c, g, or t <220>

<221 > misc fssturs <222> (18)..(19) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (21).. (22) <223> n is a, c, g, ort <400> 58 tcagaacgtt gnnsnnsnns nnst acgaga aacacaaagt c <210> 59 <211 >50 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <220>

<221 > misc_feature <222> (12)..(13) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (15)..(16) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (18)..(19) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (21).. (22) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (24).. (25) <223> n is a, c, g, ort

145 <220>

<221 > misc_feature <222> (27) .. (28) <223> η is a, c, g, ort <220>

<221 > misc_feature <222> (30) .. (31) <223> n is a, c, g, ort <400> 59 t cagaacgt t gnnsnnsnns nnsnnsnnsn nst acgagaa acacaaagt c <210> 60 <211 >56 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <220>

<221 > misc_feature <222> (12)..(13) <223> n is a, c, g, ort <220>

<221 > > misc_feature <222> (15)..(16) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (18)..(19) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (21)..(22) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (24)..(25) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (27).. (28) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (30).. (31) <223> n is a, c, g, ort <220>

<221 > misc_feature

146 <222> (33) .. (34) <223> η is a, c, g, ort <220>

<221 > misc_feature <222> (36) .. (37) <223> n is a, c, g, ort <400> 60 tcagaacgtt t gnnsnnsnns nnsnnsnnsn nsnnsnnsta cgagaaacac aaagt c <210> 61 <211 >31 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 61 cat cgcggcc gcct ct cccc t gt t gaagct c 31 <210> 62 <211> 99 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <220>

<221 > misc_feature <222> (58) .. (59) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (61).. (62) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (64) .. (65) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (67) .. (68) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (73).. (74) <220>

<221 > misc_feature <222> (70).. (71) <223> n is a, c, g, ort

147 <223> η is a, c, g, ort <220>

<221 > misc_feature <222> (76) .. (77) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (79).. (80) <223> n is a, c, g, ort <400> 62 acgtccatgg ccgcctccac caagggccca tcggtcttcc ccct ggcacc ctcctccnns nnsnnsnnsn nsnnsnnsnn sgccctgggc tgcctggtc <210> 63 <211 >23 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 63 ggcacggt ca ccacgct gct gag 23 <210> 64 <211> 61 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <220>

<221 > misc_feature <222> (19).. (20) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (22) .. (23) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (25) .. (26) <223> n is a, c, g, ort <220>

<221 > misc_feature <222> (28) .. (29) <223> n is a, c, g, ort

148 <220>

<221> misc_feature <222> (31).. (32) <223> n is a, c, g, or t <220>

<221 > miscjeature <222> (34).. (35) <223> n is a, c, g, or t <220>

<221 > miscjeature <222> (37)..(38) <223> n is a. c. g, or t <400> 64 agcgtggtga ccgtgcccnn snnsnnsnns nnsnnsnnsa cctacalctg caacgt gaat 60 c · 61 <210> 65 <211 >36 <212> DNA <213> Sztuczna <220>

<223> Sekwencja sztuczna <400> 65 cat agcggcc gcagatttgg gctcaacttt cttgtc c 36

F-star Biotechnologische Forschungsund Entwicklungsges.m.b.H., Austria

Pe łnomocnik

149

EP 1 752 471 B9 Z-5712/09